Self-immolative carboxamide linkers for use with targeted drug conjugates

Abstract

The invention relates to novel self-immolative carboxamide linkers for the conjugation and conditional release of payloads for use in targeted drug conjugates in the treatment and prophylaxis of hyperproliferative disorders such as cancer, and to drug conjugates comprising said linkers, compositions containing said drug conjugates and processes for their preparation.

Description

[0001] SELF-IMMOLATIVE CARBOXAMIDE LINKERS FOR USE WITH TARGETED DRUG CONJUGATES

[0002] FIELD OF THE INVENTION

[0003] The invention relates to novel self-immolative carboxamide linkers for the conjugation and conditional release of payloads for use in targeted drug conjugates in the treatment and prophylaxis of hyperproliferative disorders such as cancer, and to drug conjugates comprising said linkers, compositions containing said drug conjugates and processes for their preparation.

[0004] BACKGROUND OF THE INVENTION

[0005] 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.

[0006] 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).

[0007] 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 ofwell-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).

[0008] 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 could significantly improve the scope and patient benefit of ADC-based cancer therapies.

[0009] The majority of clinically approved ADCs to-date contain payloads that are amenable to antibody conjugation via a cleavable linker or spacer moiety utilising only a small handful of covalent attachment options. The currently approved cleavable linker chemistries are limited to acid-cleavable hydrazones (as used in ozogamicin), and “traceless” carbamates, carbonates or hemiaminals, which self-immolate when used in conjunction with cathepsin-mediated trigger moieties (as used in vedotin, govitecan and deruxtecan, for example). There is therefore a need to expand the existing options for linker chemistry in order to facilitate the incorporation of more diverse ADC payloads, i.e. those that are not amenable to the limited selection of chemistries currently available.

[0010] In particular, there is a need to facilitate the conjugation and conditional release of payloads containing functional groups that are poor leaving groups, for example as a result of high pKa, such as aliphatic alcohols, nitrogen heterocycles, and certain phenolic payloads. Progress has been made in this area, in particular for payloads comprising aliphatic alcohol leaving groups, with the introduction of the methylene alkoxy carbamate (MAC) (Kolakowski, (2016) Angew. Chem. Int. Ed. 55, 7948) and ethylene diamine self-immolative spacers (Jeffrey (2010) ACS Med. Chem. Lett. 1, 277; Dal Corso (2020) Angew. Chem. Int. Ed. 59, 4205; Dal Corso (2022) ChemMedChem. 17, e202200279). However, these technologies still rely on the payload being amenable to either carbamate or hemiaminal formation.

[0011] There is therefore a need to provide alternative linker technologies of ADC payload conjugation which provide optimum release of payloads which contain poor leaving groups, such as phthalazinones.SUMMARY OF THE INVENTION

[0012] According to a first aspect of the invention, there is provided a bivalent linker for releasing a phthalazinone containing payload from a tumour cell binding moiety, wherein said bivalent linker comprises a compound of formula (I):

[0013] R2

[0014]

[0015] wherein:

[0016] represents the attachment position to the phthalazinone containing payload;

[0017] ''' represents the attachment position to the tumour cell binding moiety;

[0018] X represents C2-4 alkylene;

[0019] R1represents hydrogen, C1.6 alkyl, -(CH2)m-O-R3, or -(CH2)m-R4;

[0020] m represents an integer selected from 2 to 4;

[0021] R3represents C1.6 alkanol or heterocyclyl wherein said heterocyclyl group may be optionally substituted by one or more C1.6 alkyl groups;

[0022] R4represents -SO2NRxRyor -SO2-C1.6 alkyl;

[0023] Rxand Ryindependently represent hydrogen or C1.6 alkyl;

[0024] R2represents hydrogen or C1.6 alkyl;

[0025] Y represents a self-immolative trigger moiety; and

[0026] Z represents a spacer moiety.

[0027] According to a second aspect of the invention, there is provided a drug conjugate comprising a compound of formula (II):

[0028] R2

[0029]

[0030] (II)wherein:

[0031] P1represents a phthalazinone containing payload;

[0032] X represents C2-4 alkylene;

[0033] R1represents hydrogen, C1.6 alkyl, -(CH2)m-O-R3, or -(CH2)m-R4;

[0034] m represents an integer selected from 2 to 4;

[0035] R3represents C1.6 alkanol or heterocyclyl wherein said heterocyclyl group may be optionally substituted by one or more C1.6 alkyl groups;

[0036] R4represents -SO2NRxRyor -SO2-C1.6 alkyl;

[0037] Rxand Ryindependently represent hydrogen or C1.6 alkyl;

[0038] R2represents hydrogen or C1.6 alkyl;

[0039] Y represents a self-immolative trigger moiety;

[0040] Z represents a spacer moiety;

[0041] T1represents a tumour cell binding moiety; and

[0042] p represents an integer selected from 1 to 10.

[0043] BRIEF DESCRIPTION OF THE FIGURES

[0044] 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), Example A3 (Figs. 1E & 1F), olaparib (Figs.

[0045] 1G & 1H), talazoparib (Figs. 11 & 1 J), saruparib (Figs. 1K & 1L), and Pip-6 (Figs. 1M & 1N).

[0046] Figures 2A to 2G: Dose-response plots showing chromatin retention of PARP1 and PARP2 by immunofluorescence in response to differing concentrations of Example A1 (Fig.

[0047] 2A), Example A2 (Fig. 2B), Example A3 (Fig. 2C), olaparib (Fig. 2D), talazoparib (Fig. 2E), saruparib (Fig. 2F), and Pip-6 (Fig. 2G).

[0048] Figures 2H to 2N: Dose-response plots showing inhibition of PARylation by immunofluorescence in response to differing concentrations of Example A1 (Fig. 2H), Example A2 (Fig.2I), ExampleA3 (Fig.2J), olaparib (Fig.2K), talazoparib (Fig.2L), saruparib (Fig. 2M), and Pip-6 (Fig. 2N).

[0049] Figure 3: Tumour growth inhibition and body weight plots from a DLD-1 BRCA2- / -mouse xenograft model showing the effect of treatment with differing doses of Example C1 (Figs. 3A & 3B), Examples C4 & C5 (Figs. 3C & 3D), Example C6 (Figs. 3E & 3F), Example C8 (Figs. 3G & 3H), and Example C17 (Figs. 3I & 3J).

[0050] Figure 4: Tumour growth inhibition and body weight plots from an NCI-N87 mouse xenograft model showing the effect of treatment with Examples C2 & C3 (Figs. 4A & 4B), Example C6 (Figs. 4C & 4D), Example C8 (Figs. 4E & 4F), and Example C17 (Figs. 4G &Figure 5: Plot showing release of payload over time in the presence and absence of recombinant β-glucuronidase for Examples B2, B4, B5, B6 & B7 (Fig. 5A), Examples B8, B16 & B18 (Fig. 5B), and Examples B13, B14 & B15 (Fig. 5C).

[0051] Figure 6: Tumour growth inhibition and body weight plots from a SUM149PT mouse xenograft model showing the effect of treatment with Example C17 (Figs. 6A & 6B).

[0052] Figure 7: Native mass spectra showing mass ions and DAR distribution of Sacituzumab (Fig. 7A), Example C8 (Fig. 7B), Reference Example C9 (Fig. 7C), Examples C10 (Fig. 7D), C11 (Fig. 7E) and C17 (Fig. 7F).

[0053] Figure 8: Dose-response plot showing differential sensitivity of hTERT RPE-1 WT, PARP1 KO, and PARP1 / 2 dual KO cells in response to treatment with Example A2 by CTG.

[0054] DETAILED DESCRIPTION OF THE INVENTION

[0055] Bivalent Linkers

[0056] According to a first aspect of the invention, there is provided a bivalent linker for releasing a phthalazinone containing payload from a tumour cell binding moiety, wherein said bivalent linker comprises a compound of formula (I):

[0057]

[0058] wherein:

[0059] represents the attachment position to the phthalazinone containing payload;v(b)

[0060] '' represents the attachment position to the tumour cell binding moiety;

[0061] X represents C2-4 alkylene;

[0062] R1represents hydrogen, C1.6 alkyl, -(CH2)m-O-R3, or -(CH2)m-R4;

[0063] m represents an integer selected from 2 to 4;

[0064] R3represents C1.6 alkanol or heterocyclyl wherein said heterocyclyl group may be optionally substituted by one or more C1.6 alkyl groups;

[0065] R4represents -SO2NRxRyor -SO2-C1.6 alkyl;

[0066] Rxand Ryindependently represent hydrogen or C1.6 alkyl;

[0067] R2represents hydrogen or C1.6 alkyl;

[0068] Y represents a self-immolative trigger moiety; and

[0069] Z represents a spacer moiety.Definitions

[0070] 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.

[0071] The term ‘C1.4 alkylene’ as used herein as a group or part of a group refers to a -(CH2)n-group where n represents an integer selected from 1 to 4.

[0072] The term “Ci-e alkanol” 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 optionally substituted by one or more hydroxy (i.e. -OH) groups.

[0073] The term ‘heterocyclyl’ as used herein refers to a monocyclic or bicyclic non-aromatic, partially saturated or fully saturated ring system containing for example 3 to 12 ring members. Each ring may contain up to five heteroatoms typically selected from nitrogen, sulfur and oxygen.

[0074] Particular examples of ‘heterocyclyl’ include morpholine, piperidine (e.g. piperidin-1-yl, piperidin-2-yl, piperidin-3-yl and piperidin-4-yl), piperidinone, pyrrolidine (e.g. pyrrolidin-1 -yl, pyrrolidin-2-yl and pyrrolidin-3-yl), pyrrolidone, azetidine, pyran (2H-pyran or4H-pyran), dihydrothiophene, oxetanyl, dihydropyran, dihydrofuran, dihydrothiazole, tetrahydrofuran, tetrahydrothiophene, dioxane, tetrahydropyran (e.g. tetrahydropyran-4-yl), imidazoline, imidazolidinone, oxazoline, thiazoline, pyrazolin-2-yl, pyrazolidine, piperazinone and piperazine.

[0075] It will be appreciated that the term ‘heterocyclyl” includes reference to spiro and bridged heterocyclic derivatives. Examples of such spiro and bridged heterocyclic derivatives include: 1-azaspiro[3.3]heptyl, 5-azaspiro[2.4]heptyl, 5-azaspiro[3.4]octyl, 8-azabicyclo[3.2.1]octyl, 3-azabicyclo[3.1.0]hexyl, octahydrocyclopenta[c]pyrrolyl, 2-azaspiro[3.3]heptyl, 3-azabicyclo[3.2.1]octyl, 6-azaspiro[3.4]octyl, 5-azaspiro[2.5]octyl or 2-oxa-6-azaspiro[3.4]octyl, hexahydropyrrolo[2,3-c]pyrrolidinyl, oxaspiro[3.3]heptanyl, diazaspiro[3.4]octanyl, diazaspiro[4.4]nonyl, oxa-azaspiro[3.4]octanyl, oxa-azaspiro[4.4]nonyl, tetrahydrofuro[3,4-c]pyrrolidinyl, oxa-azaspiro[3.3]heptyl, diazaspiro[4.5]decanyl, diazaspiro[3.4]octanyl, octahydro-naphthyridinyl, tetrahydropyrazino-oxazinyl, oxadiazospiro[5.5]undecanyl and oxabicyclo[2.2.1]heptanyl.The term ‘optionally substituted’ as used herein refers to a group which may be substituted or unsubstituted by a substituent as defined herein. Common substituents include halogen, hydroxy or hydroxyl, Ci-e alkyl, Ci-e alkoxy, cyano, nitro, amino, formyl, carboxyl and the like, as defined herein.

[0076] The self-immolative carboxamide linkers of the invention have surprisingly been found to facilitate the conjugation and selective release of payloads that are poor leaving groups, such as phthalazinones. In particular, the invention provides a novel method of conjugating phthalazinone-containing PARP1 inhibitors, such as compounds A1 to A4 described herein, among others, to a tumour-targeting moiety, such as an ADC. Furthermore, the self-immolative nature of the novel linkers enables “traceless” cleavage when used in conjunction with a suitable tumour-specific release mechanism, such as a β-glucuronidase trigger, for example, meaning that the phthalazinone-containing PARP1 inhibitor can be conjugated directly to the tumour targeting moiety without further modification of its structure or properties.

[0077] The bivalent linkers of the present invention are bifunctional or multifunctional moieties which can be used to attach one or more phthalazinone containing payloads to the tumour cell binding moiety to form the drug conjugates of the present invention. The connecting group attaching the bivalent linker of the present invention to the phthalazinone containing payload consists of a self-immolative carboxamide linker attached to a self-immolative trigger moiety. Attachment of the linker to the tumour cell binding moiety will typically comprise a spacer moiety which comprises 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 within the spacer moiety, such as a maleimide, dibromopyridazinedione or haloacetamide. The bivalent linker of the present invention may be further functionalised with one or more groups that modulate properties including solubility; lipophilicity; and aggregation.

[0078] The bivalent linkers of the present invention 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 bivalent linker of the present invention may be cleaved by an enzyme, such as a lysosomal enzyme; hydrolysis; catabolism; or other enzymatic process to release the one or more phthalazinone containing payloads.

[0079] Embodiments

[0080] In one embodiment, X represents C2-3 alkylene. In a yet further embodiment, X represents ethylene.In one embodiment, R1represents Ci-e alkyl, -(CH2)m-O-R3or -(CH2)m-4.

[0081] In a further embodiment, R1represents Ci-e alkyl. In a yet further embodiment, R1represents methyl.

[0082] In an alternative embodiment, R1represents -(CH2)m-O-R3.

[0083] In an alternative embodiment, R1represents -(CH2)m-R4.

[0084] In a further embodiment, R1represents methyl, -(CH2)2-O-(CH2)2-OH, -(CH2)2-SO2NH2, -(CH2)2-SO2-Me, or -(CH2)2-O-piperidinyl substituted by a methyl group (such as an N-methyl group).

[0085] In one embodiment, m represents an integer selected from 2 or 3. In a further embodiment, m represents an integer which is 2.

[0086] In one embodiment, R3represents Ci-e alkanol (such as -(CH2)2-OH) or heterocyclyl (such as a 6 membered heterocyclyl ring) optionally substituted by one or more Ci-e alkyl (such as methyl) groups.

[0087] In one embodiment, R3represents Ci-e alkanol (such as -(CH2)2-OH) or heterocyclyl (such as a 6 membered nitrogen containing heterocyclyl ring) optionally substituted by one or more Ci-6 alkyl (such as methyl) groups.

[0088] In one embodiment, R3represents Ci-e alkanol (such as -(CH2)2-OH) or heterocyclyl (such as piperidinyl) optionally substituted by one or more Ci-e alkyl (such as methyl) groups.

[0089] In one embodiment, R4represents -SO2NRxRy(such as -SO2NH2) or -SO2-C1.6 alkyl (such as -SO2-Me).

[0090] In one embodiment, Rxand Ryindependently represent hydrogen or methyl. In a further embodiment, Rxand Ryboth represent hydrogen.

[0091] In one embodiment, R2represents hydrogen or methyl. In a further embodiment, R2represents methyl.In one embodiment, the bivalent linker is a compound of formula (l)a:

[0092]

[0093] O

[0094] (l)a

[0095] wherein R1, (a), (b), Y and Z are as defined herein.

[0096] Self-lmmolative Trigger Moieties

[0097] References herein to the term “self-immolative trigger moiety” include a specific type of moiety which contains a chemical component which provides a mechanism whereby the moiety is designed to break down and release an attached, adjacent component upon exposure to an external stimulus. Such self-immolation is typically achieved through a cascade of reactions and / or fragmentations that dismantle the moiety in a controlled manner, releasing the attached, adjacent component.

[0098] It will be appreciated that the external stimulus can be via a variety of factors, such as changes in pH or temperature or exposure to light, enzymes or chemical reactions.

[0099] It will also be appreciated that the term “self-immolative trigger moiety” is a term well known in the art and the design and construction of such a moiety would be immediately apparent to the skilled person in the field of drug conjugation.

[0100] 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 conseguent release of the attached, adjacent component, i.e. the phthalazinone containing payload.

[0101] In a further embodiment, the self-immolative trigger moiety comprises a glucuronide containing moiety which has the following structure of formula (Y)a:®'X^V(O)

[0102] OA-'OH

[0103] O

[0104]

[0105] <Y O^H O£H>*OH

[0106] (Y)a

[0107] wherein “(O)” represents the O atom of the bivalent linker of formula (I) and “(Z)” represents the Z variable of the bivalent linker of formula (I).

[0108] Moieties

[0109] References herein to the term “spacer moiety” include any suitable moiety for convenient spacing and attachment of the tumour cell binding moiety to the self-immolative trigger moiety of variable Y.

[0110] It will be appreciated that the term “spacer moiety” is a term well known in the art and the design and construction of such a moiety would be immediately apparent to the skilled person in the field of drug conjugation.

[0111] In one embodiment, the spacer moiety Z comprises one or more polyethylene glycol (PEG) components. In a further embodiment, the spacer moiety Z comprises multiple (i.e. at least 2) polyethylene glycol (PEG) components. This embodiment has the advantage of optimal spacing between the self-immolative trigger moiety of variable Y and the tumour cell binding moiety.

[0112] In one embodiment, the spacer moiety Z additionally comprises a haloacetamide, maleimide, dibromopyridazinedione or dibromopyridazinotriazepinyl moiety. This embodiment has the advantage of providing an optimal attachment point for the tumour cell binding moiety, in particular when said moiety is a monoclonal antibody. For example, when the spacer moiety comprises a haloacetamide, maleimide, dibromopyridazinedione or dibromopyridazinotriazepinyl moiety, it forms a covalent bond to a cysteine thiol of the monoclonal antibody. In a further embodiment, the spacer moiety Z additionally comprises a maleimide moiety.

[0113] In a further embodiment, the spacer moiety Z is selected from a compound of any one of formula (Z)a, (Z)b, (Z)c, (Z)d, (Z)e, (Z)f, or (Z)9;JO ij(z)

[0114] jg o X

[0115] o o H!8(Z) o O N^XfA)

[0116] jg

[0117] ZHN

[0118] NH

[0119] O' O

[0120]

[0121] o(Z)g;

[0122] wherein “(Y)” represents the Y variable of the bivalent linker of formula (I).

[0123] Drug Conjugates

[0124] According to a second aspect of the invention, there is provided a drug conjugate comprising a compound of formula (II):

[0125]

[0126] (II)

[0127] wherein:

[0128] P1represents a phthalazinone containing payload;

[0129] X represents C2-4 alkylene;

[0130] R1represents hydrogen, C1.6 alkyl, -(CH2)m-O-R3, or -(CH2)m-R4;

[0131] m represents an integer selected from 2 to 4;

[0132] R3represents C1.6 alkanol or heterocyclyl wherein said heterocyclyl group may be optionally substituted by one or more C1.6 alkyl groups;

[0133] R4represents -SO2NRxRyor -SO2-C1.6 alkyl;

[0134] Rxand Ryindependently represent hydrogen or C1.6 alkyl;

[0135] R2represents hydrogen or C1.6 alkyl;

[0136] Y represents a self-immolative trigger moiety;

[0137] Z represents a spacer moiety;

[0138] T1represents a tumour cell binding moiety; and

[0139] p represents an integer selected from 1 to 10.

[0140] Embodiments

[0141] It will be appreciated that the embodiments for variables X, R1, m, R2, R3, R4, Rx, Ry, Y and Z defined hereinbefore with respect to the bivalent linkers of the first aspect of the invention apply equally to the drug conjugates of the second aspect of the invention and for brevity will not be repeated within this section.In one embodiment, p represents an integer selected from 4 or 8. In a further embodiment, p represents an integer which is 4. In an alternative embodiment, p represents an integer which is 8.

[0142] Phthalazinone containing payloads

[0143] It will be appreciated that the invention finds utility where any suitable payload contains a phthalazinone moiety.

[0144] In one embodiment, the phthalazinone containing payload is linked to the carboxamide group via a nitrogen atom.

[0145] In a further embodiment, the phthalazinone containing payload comprises the following moiety:

[0146] 0

[0147]

[0148] wherein (d) represents the attachment point to the remaining portion of the payload; and (e) represents the attachment point to the carbonyl moiety of the bivalent linker of formula (I).

[0149] In one embodiment, the phthalazinone containing payload comprises a PARP inhibitor. It will be appreciated that phthalazinone containing PARP inhibitors are well known to the skilled person and their preparation is also known. Examples of such phthalazinone containing PARP inhibitors are described in GB Patent Application Nos. 2502430.8, 2505006.3, 2505009.7 and 2508102.7, the PARP inhibitors of which are herein incorporated by reference. In a further embodiment, the PARP inhibitor is selected from any one of compounds A1 to A4 as described herein. In a further alternative embodiment, the PARP inhibitor is selected from olaparib, fluzoparib, simmiparib, thioparib, Pip-6, or talazoparib. In a further alternative embodiment, the PARP inhibitor is selected from olaparib, talazoparib, or Pip-6.

[0150] Tumour Cell Binding Moieties

[0151] 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. Suitableexamples include peptides (i.e. bicyclic peptides), antibodies (i.e. monoclonal antibodies (mAbs)) or antibody fragments.

[0152] 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).

[0153] 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.

[0154] 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.

[0155] 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 are 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.

[0156] 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).

[0157] 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). 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).

[0158] 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).

[0159] As used herein the term “bispecific antibodies” refers to antibodies that bind to two (or more) different antigens.

[0160] 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 et al. Methods Enzymology, 121:663-69, Academic Press, 1986.

[0161] 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 other 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.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.

[0162] 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).

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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 fragmentsspecifically bind to a tumour-associated antigen whose cell surface expression on a tumour cell is different to its expression on a healthy cell.

[0168] 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.

[0169] 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).

[0170] In a still yet preferred embodiment, the antibody or antigen binding fragment binds to a target on a tumour (i.e. cancer) cell selected from ErbB2 / HER2 and Trop-2.

[0171] 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.

[0172] 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).

[0173] In one embodiment, the antibody or antigen binding fragment is sacituzumab.In one embodiment, the invention provides a drug conjugate which is a compound of any one of Examples C1 to C8, C10 to C17 or C19 to C23.

[0174] Processes

[0175] It will be appreciated that the bivalent linkers may be prepared by the skilled person using standard procedures.

[0176] Thus, according to a further aspect of the invention there is provided a process for preparing a bivalent linker of formula (I) as defined herein, which comprises reacting a compound of formula (IV):

[0177] O O

[0178] (a)'' N N O

[0179] I I

[0180] R1R2

[0181]

[0182] (IV)

[0183] wherein (a), R1, X, R2and Y are as defined herein, with a compound of formula Z-(b), wherein Z and (b) are as defined herein. An example of such a process is described herein in Intermediate 7a inter alia.

[0184] Thus, according to a further aspect of the invention, there is provided a bivalent linker (such as a bivalent linker of a compound of formula (I) as defined herein), prepared by a process as defined herein.

[0185] It will also be appreciated that the drug conjugates may be prepared by the skilled person using standard procedures.

[0186] In particular, the process will comprise attaching the bivalent linker of formula (I) (as defined herein) to the phthalazinone containing payload (as defined herein, i.e. a PARP inhibitor), followed by attachment of the tumour cell binding moiety (as defined herein, i.e. a peptide, antibody or antibody fragment).

[0187] According to a further aspect of the invention there is provided a process for preparing the drug conjugate of a compound of formula (II) as defined herein which comprises reacting one or more molar equivalents of a compound of formula (III):o o

[0188]

[0189] wherein P1, R1, X, R2, Y and Z are as defined herein, with a tumour cell binding moiety.

[0190] The process of the invention typically comprises reduction of the tumour cell binding moiety with a suitable reducing agent, such as TCEP followed by reaction with the compound of formula (III) in a suitable solvent, such as PBS containing DMSO.

[0191] Thus, according to a further aspect of the invention, there is provided a drug conjugate (such as a drug conjugate of a compound of formula (II) as defined herein), prepared by a process as defined herein. More particularly, there is provided a drug conjugate of any one of Examples C1 to C23 prepared by a process as defined herein. Most particularly, there is provided a drug conjugate of any one of C1 to C8, C10 to C17 or C19 to C23 prepared by a process as defined herein.

[0192] Examples of suitable processes are described in Examples C1 to C23.

[0193] In one embodiment, the compound of formula (III) is selected from a compound of formula (lll)a, (lll)b, (lll)c, (lll)d, (lll)e, (III)f, or (lll)g;

[0194]

[0195] -p(lll)

[0196] 9 HO HO

[0197]

[0198] Br

[0199] Br

[0200]

[0201] (lll)g;

[0202] wherein P1, R1, X and R2are as defined herein.

[0203] 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.Thus, according to a further aspect of the invention there is provided an intermediate selected from a compound of formula (III), (lll)a, (lll)b, (lll)c, (lll)d, (lll)e, (III)f, (III)9, or B1 to B20.

[0204] Pharmaceutical Compositions

[0205] While it is possible for the 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.

[0206] 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 drug conjugate, together with one or more pharmaceutically acceptable excipients and optionally other therapeutic or prophylactic agents, as described herein.

[0207] Thus, according to a further aspect of the invention, there is provided a pharmaceutical composition comprising the drug conjugate as defined herein in combination with one or more pharmaceutically acceptable excipients.

[0208] 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.

[0209] 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.

[0210] Pharmaceutical compositions containing drug conjugates can be formulated in accordance with known techniques, see for example, Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, USA.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.

[0211] 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).

[0212] 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.

[0213] The pharmaceutical formulation can be prepared by lyophilising the drug conjugate.

[0214] Lyophilisation refers to the procedure of freeze-drying a composition. Freeze-drying and lyophilisation are therefore used herein as synonyms.

[0215] Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

[0216] 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.

[0217] 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 ethyl 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.

[0218] 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.

[0219] 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.

[0220] In another particular embodiment, the pharmaceutical composition is in a form suitable for sub-cutaneous (s.c.) administration.

[0221] 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.

[0222] Thus, tablet compositions can contain a unit dosage of active drug conjugate 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 example 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.

[0223] 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.

[0224] 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.

[0225] 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 drug conjugate in the stomach or in the ileum, duodenum, jejenum or colon.

[0226] 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 drug conjugate 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 drug conjugate 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 drug conjugate can be formulated in a delivery system that provides osmotic control of the release of the drug conjugate. Osmotic release and other delayed release or sustained release formulations (forexample formulations based on ion exchange resins) may be prepared in accordance with methods well known to those skilled in the art.

[0227] The 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 Technology for Drug Delivery”, edited by Ram B Gupta and llday 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.

[0228] 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.

[0229] 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.

[0230] 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 thetablet or capsule typically contain 0-10% (w / w) polymers, 0-3% (w / w) pigments, and / or 0-2% (w / w) plasticizers.

[0231] 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).

[0232] Formulations for intramuscular depots may also contain 0-99% (w / w) oils.

[0233] 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.

[0234] The drug conjugates 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.

[0235] 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.

[0236] 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 packageinsert 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.

[0237] 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.

[0238] 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 drug conjugate. Solutions of the active drug conjugate may also be used for rectal administration.

[0239] 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 drug conjugate together with an inert solid powdered diluent such as lactose.

[0240] The drug conjugates will generally be presented in unit dosage form and, as such, will typically contain sufficient drug conjugate 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 subranges of drug conjugate 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).

[0241] 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 drug conjugate.

[0242] The active drug conjugate 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.

[0243] Anti-Cancer TreatmentThe payloads within the drug conjugates of the invention are inhibitors of PARP1 and or PARP2, and therefore confer upon the drug conjugate the ability to prevent or treat disease states or conditions described herein.

[0244] In addition, the drug conjugates of the invention, 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.

[0245] Thus, for example, it is envisaged that the drug conjugates of the invention will be useful in alleviating or reducing the incidence of cancer.

[0246] The drug conjugates are generally administered to a subject in need of such administration, for example a human or animal patient, particularly a human.

[0247] The drug conjugates of the present invention may be useful for the treatment of the adult population. The drug conjugates of the present invention may be useful for the treatment of the pediatric population.

[0248] According to a further aspect of the invention there is a provided a drug conjugate as defined herein, for use in the treatment of cancer.

[0249] According to a further aspect of the invention there is a provided a drug conjugate as defined herein, for use in the treatment of tumours which overexpress PARP1 and or PARP2.

[0250] A further aspect provides the use of 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.

[0251] 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 drug conjugate as defined herein.

[0252] 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 drug conjugate as defined herein.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.

[0253] 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 cell 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, adrenaltumours, 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).

[0254] 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. Drug conjugates of the invention may therefore be useful in preventing and disrupting initiation of tumour angiogenesis. In particular, the drug conjugates of the invention may be useful in the treatment of metastasis and metastatic cancers.

[0255] 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 drug conjugates 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.

[0256] Particular cancers include hepatocellular carcinoma, melanoma, oesophageal, renal, colon, colorectal, lung e.g. mesothelioma or lung adenocarcinoma, breast, bladder, gastrointestinal, ovarian and prostate cancers.

[0257] The drug conjugates 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.

[0258] The potency of the payloads within the drug conjugates 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 payloads within the drug conjugates of thepresent invention are compounds having an IC50 value of less than 1 M, more particularly less than 0.1 pM.

[0259] The drug conjugates 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 the drug conjugate may outweigh the disadvantages of any toxic effects or side effects, in which case it may be considered desirable to administer drug conjugates in amounts that are associated with a degree of toxicity.

[0260] The drug conjugates 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).

[0261] A typical daily dose of 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.

[0262] 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 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.

[0263] The drug conjugates 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 drug conjugate may be administered once or more than once each day. The drug conjugate can be administered continuously (i.e. taken every day without a break for the duration of the treatment regimen). Alternatively, the drug conjugate 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.In one particular dosing schedule, a patient will be given an infusion of 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.

[0264] More particularly, a patient may be given an infusion of the drug conjugate for periods of one hour daily for 5 days and the treatment repeated every three weeks.

[0265] 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.

[0266] 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.

[0267] In another particular dosing schedule, a patient is given the drug conjugate orally once a week.

[0268] In another particular dosing schedule, a patient is given the drug conjugate orally once-daily for between 7 and 28 days such as 7, 14 or 28 days.

[0269] In another particular dosing schedule, a patient is given the drug conjugate 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.

[0270] In another particular dosing schedule, a patient is given the drug conjugate orally once-daily for 2 weeks followed by 2 weeks off.

[0271] In another particular dosing schedule, a patient is given the drug conjugate orally once-daily for 2 weeks followed by 1 week off.

[0272] In another particular dosing schedule, a patient is given the drug conjugate orally once-daily for 1 week followed by 1 week off.Ultimately, however, the quantity of drug conjugate 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.

[0273] It will be appreciated that PARP1 and / or PARP2 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-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regulat 1984;22: 27-55.

[0274] The drug conjugates as defined herein can be administered as the sole therapeutic agent or they can be administered in combination therapy with one of more other drug conjugates (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 drug conjugates 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 drug conjugate include but are not limited to:

[0275] • Topoisomerase I inhibitors;

[0276] • Antimetabolites;

[0277] • Tubulin targeting agents;

[0278] • DNA binder and topoisomerase II inhibitors;

[0279] • Alkylating Agents;

[0280] • Monoclonal Antibodies;

[0281] • Anti-Hormones;

[0282] • Signal Transduction Inhibitors;

[0283] • Proteasome Inhibitors;

[0284] • DNA methyl transferase inhibitors;

[0285] • Cytokines and retinoids;

[0286] • Chromatin targeted therapies;

[0287] • Radiotherapy; and

[0288] • Other therapeutic or prophylactic agents.

[0289] 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;

[0290] (ii) Taxane compounds, for example paclitaxel, paclitaxel protein bound particles (Abraxane™), docetaxel, cabazitaxel or larotaxel;

[0291] (iii) Topoisomerase I inhibitors, for example camptothecin compounds, for example camptothecin, irinotecan(CPT11), SN-38, ortopotecan;

[0292] (iv) Topoisomerase II inhibitors, for example anti-tumour epipodophyllotoxins or podophyllotoxin derivatives for example etoposide, or teniposide;

[0293] (v) Vinca alkaloids, for example vinblastine, vincristine, liposomal vincristine (Onco-TCS), vinorelbine, vindesine, vinflunine or vinvesir;

[0294] (vi) Nucleoside derivatives, for example 5-fluorouracil (5-FU, optionally in combination with leucovorin), gemcitabine, capecitabine, tegafur, UFT, S1, cladribine, cytarabine (Ara-C, cytosine arabinoside), fludarabine, clofarabine, or nelarabine;

[0295] (vii) Antimetabolites, for example clofarabine, aminopterin, or methotrexate, azacitidine, cytarabine, floxuridine, pentostatin, thioguanine, thiopurine, 6-mercaptopurine, or hydroxyurea (hydroxycarbamide);

[0296] (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);

[0297] (ix) Anthracyclines, anthracenediones and related drugs, for example daunorubicin, doxorubicin (optionally in combination with dexrazoxane), liposomal formulations of doxorubicin (e.g. Caelyx™, Myocet™, Doxil™), idarubicin, mitoxantrone, epirubicin, amsacrine, or valrubicin;

[0298] (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;

[0299] (xi) DNA methyl transferase inhibitors, for example temozolomide, azacytidine or decitabine, or SGI-110;

[0300] (xii) Antifolates, for example methotrexate, pemetrexed disodium, or raltitrexed;

[0301] (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;(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- 113945, bardoxolone, BMS-066, BMS-345541, IMD-0354, IMD-2560, or IMD-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;

[0302] (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;

[0303] (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 andAP23573, 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);

[0304] (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), 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);

[0305] (xxi) Estrogen receptor antagonists or selective estrogen receptor modulators (SERMs) or inhibitors of estrogen synthesis, for example tamoxifen, fulvestrant, toremifene, droloxifene, faslodex, or raloxifene;

[0306] (xxii) Aromatase inhibitors and related drugs, such as exemestane, anastrozole, letrazole, testolactone aminoglutethimide, mitotane or vorozole;

[0307] (xxiii) Antiandrogens (i.e. androgen receptor antagonists) and related agents for example bicalutamide, nilutamide, flutamide, cyproterone, or ketoconazole;

[0308] (xxiv) Hormones and analogues thereof such as medroxyprogesterone, diethylstilbestrol (a.k.a. diethylstilboestrol) or octreotide;

[0309] (xxv) Steroids for example dromostanolone propionate, megestrol acetate, nandrolone (decanoate, phenpropionate), fluoxymestrone or gossypol,

[0310] (xxvi) Steroidal cytochrome P45017alpha-hydroxylase-17,20-lyase inhibitor (CYP17), e.g.

[0311] abiraterone;

[0312] (xxvii) Gonadotropin releasing hormone agonists or antagonists (GnRAs) for example abarelix, goserelin acetate, histrelin acetate, leuprolide acetate, triptorelin, buserelin, or deslorelin;

[0313] (xxviii) Glucocorticoids, for example prednisone, prednisolone, dexamethasone;

[0314] (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;

[0315] (xxx) Farnesyltransferase inhibitors for example tipifarnib;(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;

[0316] (xxxii) Proteasome Inhibitors for example bortezomib, carfilzomib, delanzomib (CEP- 18770), ixazomib (MLN -9708), oprozomib (ONX-0912) or marizomib;

[0317] (xxxiii) Photodynamic drugs for example porfimer sodium or temoporfin;

[0318] (xxxiv) Marine organism-derived anticancer agents such as trabectidin;

[0319] (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.

[0320] Bismuth-213 or Actinium-225) for example ibritumomab or Iodine tositumomab;

[0321] (xxxvi) Telomerase inhibitors for example telomestatin;

[0322] (xxxvii) Matrix metalloproteinase inhibitors for example batimastat, marimastat, prinostat or metastat;

[0323] (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;

[0324] (xxxix) Selective immunoresponse modulators for example thalidomide, or lenalidomide; (xl) Therapeutic Vaccines such as sipuleucel-T (Provenge) or OncoVex;

[0325] (xli) Cytokine-activating agents include Picibanil, Romurtide, Sizofiran, Virulizin, or Thymosin;

[0326] (xlii) Arsenic trioxide;

[0327] (xliii) Inhibitors of G-protein coupled receptors (GPCR) for example atrasentan;

[0328] (xliv) Enzymes such as L-asparaginase, pegaspargase, rasburicase, or pegademase;

[0329] (xlv) DNA repair inhibitors such as PARP inhibitors for example, olaparib, velaparib, iniparib, rucaparib (AG-014699 or PF-01367338), talazoparib or AG-014699;

[0330] (xlvi)DNA damage response inhibitors such as ATM inhibitors AZD0156 MS3541, ATR inhibitors AZD6738, M4344, M6620wee1 inhibitor AZD1775;

[0331] (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;(xlviii) Prophylactic agents (adjuncts); i.e. agents that reduce or alleviate some of the side effects associated with chemotherapy agents, for example

[0332] - anti-emetic agents,

[0333] - 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 factor (GM-CSF) (e.g. sargramostim), and granulocyte-colony stimulating factor (G-CSF) and analogues thereof (e.g. filgrastim, pegfilgrastim),

[0334] - agents that inhibit bone resorption such as denosumab or bisphosphonates e.g. zoledronate, zoledronic acid, pamidronate and ibandronate,

[0335] - agents that suppress inflammatory responses such as dexamethasone, prednisone, and prednisolone,

[0336] - 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,

[0337] - non-steroidal anti-inflammatory drugs (NSAID) such as COX-2 inhibitors for example celecoxib, etoricoxib and lumiracoxib,

[0338] - agents for mucositis e.g. palifermin,

[0339] - agents for the treatment of side-effects including anorexia, cachexia, oedema or thromoembolic episodes, such as megestrol acetate.

[0340] 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. tram eti nib).

[0341] 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, the drug conjugate 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.

[0342] 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.

[0343] The camptothecin compound is advantageously administered in a dosage of 0.1 to

[0344] 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.

[0345] 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.

[0346] The anti-tumour vinca alkaloid is advantageously administered in a dosage of 2 to

[0347] 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.

[0348] 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

[0349] 1500 mg / m2, particularly for 5-FU 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

[0350] 2500 mg / m2per course of treatment.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.

[0351] 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

[0352] 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.

[0353] 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.

[0354] 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.

[0355] 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.

[0356] Where 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 exampleover 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.

[0357] In one embodiment is provided the drug conjugate for the manufacture of a medicament for use in therapy wherein said compound is used in combination with one, two, three, or four other therapeutic agents. In another embodiment is provided a medicament for treating cancer which comprises 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 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.

[0358] 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.

[0359] The weight ratio of the drug conjugate 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 drug conjugate 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 drug conjugates of the instant invention. A particular weight ratio for 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.The drug conjugates of the invention may also be administered in conjunction with non-chemotherapeutic treatments such as radiotherapy, photodynamic therapy, gene therapy; surgery and controlled diets.

[0360] The drug conjugates of the present invention also have therapeutic applications in sensitising tumour cells for radiotherapy and chemotherapy. Hence the drug conjugates of the present invention can be used as "radiosensitizer" and / or “chemosensitizer” or can be given in combination with another "radiosensitizer" and / or “chemosensitizer”. In one embodiment the drug conjugate of the invention is for use as chemosensitiser.

[0361] 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.

[0362] 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.

[0363] In one embodiment the drug conjugate of the invention is administered with a "radiosensitizer" and / or “chemosensitizer”. In one embodiment the drug conjugate of the invention is administered with an "immune sensitizer".

[0364] 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.

[0365] 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 (I UdR), bromodeoxycytidine, fluorodeoxyuridine (FudR), hydroxyurea, cisplatin, and therapeutically effective analogs and derivatives of the same.

[0366] 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, tinetioporphyrin, pheoborbide-a, bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanine, and therapeutically effective analogs and derivatives of the same.

[0367] Radiosensitizers may be administered in conjunction with a therapeutically effective amount of one or more other compounds, including but not limited to: drug conjugates 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 target cells; chemotherapeutic agents which act on the tumour with or without additional radiation; or other therapeutically effective compounds for treating cancer or other diseases.

[0368] Chemosensitizers may be administered in conjunction with a therapeutically effective amount of one or more other compounds, including but not limited to: drug conjugates 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.

[0369] 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).

[0370] Immune sensitizers may be administered in conjunction with a therapeutically effective amount of one or more other compounds, including but not limited to: drug conjugates 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.For use in combination therapy with another chemotherapeutic agent, 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.

[0371] In one embodiment is provided a combination of 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.

[0372] In another embodiment is provided the drug conjugate in combination with one or more (e.g.

[0373] 1 or 2) other therapeutic agents (e.g. anticancer agents) for use in therapy, such as in the prophylaxis or treatment of cancer.

[0374] In one embodiment the pharmaceutical composition comprises the drug conjugate together with a pharmaceutically acceptable carrier and optionally one or more therapeutic agent(s).

[0375] 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.

[0376] In a further embodiment the invention relates to a product containing 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.

[0377] EXAMPLES

[0378] The invention will now be illustrated, but not limited, by reference to the specific embodiments described in the following examples.

[0379] Abbreviations

[0380] Ac Acetyl

[0381] ADC Antibody-drug conjugateaq. Aqueous

[0382] Boc terf-Butyloxycarbonyl

[0383] BTC Bis(trichloromethyl) carbonate

[0384] CMPI 2-Chloro-1 -methylpyridinium iodide

[0385] DAR Drug-antibody ratio

[0386] DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene

[0387] DCM Dichloromethane

[0388] DI PEA / V, / V-Diisopropylethylamine

[0389] DMAP 4-Dimethylaminopyridine

[0390] DMF Dimethylformamide

[0391] DMSO Dimethylsulfoxide

[0392] EEDQ ethyl 2-ethoxyquinoline-1(2 / 7)-carboxylate

[0393] Et Ethyl

[0394] EtOH Ethanol

[0395] EtOAc Ethyl acetate

[0396] FA Formic acid

[0397] FLR1 Flow Reactor 1

[0398] Fmoc ((9 / 7-fluoren-9-yl)methoxy)carbonyl

[0399] h Hour(s)

[0400] HATU 1-[Bis(dimethylamino)methylene]-1 / 7-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0401] HOBt Hydroxybenzotriazole

[0402] HPLC High-performance liquid chromatography

[0403] Me Methyl

[0404] MeCN Acetonitrile

[0405] MeOH Methanol

[0406] min Minutes

[0407] n-Bu n-Butyl

[0408] NIS / V-iodosuccinimide

[0409] NMR Nuclear magnetic resonance

[0410] PBS Phosphate-buffered saline

[0411] PE Petroleum ether

[0412] Ph Phenyl

[0413] Prep Preparative

[0414] PS80 Polysorbate-80

[0415] rt Room temperature or ambient temperature

[0416] s SecondsSFC Supercritical fluid chromatography

[0417] SS Stainless steel

[0418] sat. Saturated solution

[0419] TBS terf-Butyldiphenylsilyl

[0420] t-Bu terf-Butyl

[0421] TCEP 3,3',3"-Phosphanetriyltripropionic acid

[0422] TEA Triethylamine

[0423] TFA Trifluoroacetic acid

[0424] THF Tetrahydrofuran

[0425] TMEDA / V, / V, / \ / ’, / \ / ’-Tetramethylethane-1,2-diamine

[0426] Typical Preparative HPLC method

[0427] 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.

[0428] Typical SEC-HPLC method

[0429] 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.

[0430] Intermediate 1: tert-Butyldimethylsilyl dimethyl phosphite

[0431] ° a OTBS

[0432] P-OMe '

[0433] n

[0434]

[0435] OMe MeO OMe

[0436] 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.1H NMR (400 MHz, CDCl3) δ ppm 3.49 (s, 3H), 3.46 (s, 3H), 0.94 (s, 9H), 0.19 (s, 6H).

[0437] Intermediate 2: Dimethyl (6-oxo-6,8-dihydro-[1,3]dioxolo[4,5-e]isobenzofuran-8-yl)phosphonate

[0438]

[0439] Step a. To a mixture of benzo[d][1,3]dioxole-5-carboxylic acid (CAS: 94-53-1; 125 g, 752 mmol) in SOCl2(150 mL) was added DMF (5.50 g, 75.2 mmol) dropwise at 20 °C. The reaction mixture was stirred at 80 °C for 3 h. Upon completion, the reaction mixture was evaporated to give benzo[d][1,3]dioxole-5-carbonyl chloride (140 g, 95% yield) as a yellow solid which was used without further purification.

[0440] Step b. To a mixture of benzo[d][1,3]dioxole-5-carbonyl chloride (140 g, 758 mmol) in THF (1 L) was added diethylamine (166 g, 2.28 mol) dropwise at 20 °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 water (1 L) and extracted with EtOAc (2 x 800 mL). The combined organic layers were washed with 20% citric acid (800 mL), 5% NaOH (800 mL) and brine (2 x 600 mL), dried over Na2SO4 and evaporated to give / V, / V-diethylbenzo[c(][1,3]dioxole-5-carboxamide (160 g, 95% yield) as a yellow solid which was used without further purification, m / z ES+ [M+H]+222.0;1H NMR (400 MHz, CDCl3) δ ppm 6.92 - 6.86 (m, 2H), 6.84 - 6.76 (m, 1H), 5.99 (s, 2H), 3.41 (br s, 4H), 1.18 (br s, 6H).

[0441] Step c. To a mixture of / V, / V-diethylbenzo[c(][1,3]dioxole-5-carboxamide (30.0 g, 135 mmol) and TMEDA (31.5 g, 271 mmol) in THF (1 L) was added n-BuLi (2.5 M in hexane, 162 mL) at -65 °C. The reaction mixture was stirred at -65 °C for 10 min, after which a mixture of DMF (39.6 g, 542 mmol) in THF (100 mL) was added. The reaction mixture was stirred at -65 °C for an additional 30 min. Upon completion, the reaction mixture was poured into 20% NH4CI (1500 mL) at 0 °C, and the aqueous mixture was extracted with EtOAc (3 x 800 mL). The combined organic layers were washed with 10% citric acid (400 mL), brine (3 x 500 mL), dried over Na2SO4 and evaporated. The residue was triturated (PE / EtOAc = 20 / 1 (120 mL)) at 20 °C for 30 min to give / V, / V-diethyl-4-formylbenzo[c(][1,3]dioxole-5-carboxamide (31.0 g, 91% yield) as a black-brown solid.m / z ES+ [M+H]+250.1;1H NMR (400 MHz, CDCl3) δ ppm 10.11 (s, 1H), 7.00 (d, J = 7.6 Hz, 1H), 6.79 (d, J= 7.6 Hz, 1H), 6.18 (s, 2H), 3.59 (q, J = 7.2 Hz, 2H), 3.17 (q, J = 7.2 Hz, 2H), 1.30 (t, J= 7.2 Hz, 3H), 1.06 (t, J= 7.2 Hz, 3H).

[0442] Step d. A mixture of / V, / V-diethyl-4-formylbenzo[c(][1,3]dioxole-5-carboxamide (30.0 g, 120 mmol) and Intermediate 1 (62.0 g, 276 mmol) in THF (350 mL) was stirred at 70 °C for 16 h. Upon completion, the reaction mixture was evaporated to give dimethyl (((tert- butyldimethylsilyl)oxy)(5-(diethylcarbamoyl)benzo[c(][1,3]dioxol-4-yl)methyl)phosphonate (60.0 g, 80% yield) as a black-brown gum which was used without further purification. m / z ES+ [M+H]+474.2;1H NMR (400 MHz, CDCl3) δ ppm 6.78 - 6.73 (m, 1H), 6.71 - 6.66 (m, 1H), 6.07 (d, J = 1.2 Hz, 1H), 5.98 (d, J= 1.2 Hz, 1H), 5.72 - 5.38 (m, 1H), 3.84 (d, J = 10.4 Hz, 3H), 3.79 (d, J = 10.4 Hz, 3H), 3.73 - 3.64 (m, 1H), 3.42 - 3.26 (m, 2H), 3.14 - 3.01 (m, 1H), 1.23 (t, J= 7.2 Hz, 3H), 1.13 (t, J= 7.2 Hz, 3H), 0.90 (s, 9H), 0.19 (s, 6H).

[0443] Step e. Methanesulfonic acid (24.3 g, 253 mmol) was added to a solution of dimethyl (((tert- butyldimethylsilyl)oxy)(5-(diethylcarbamoyl)benzo[c(][1,3]dioxol-4-yl)methyl)phosphonate (60.0 g, 126 mmol) in MeOH (500 mL) at 20 °C. The reaction mixture was stirred at 20 °C for 16 h. Upon completion, the reaction mixture was evaporated, the residue was re-dissolved in EtOAc (200 mL) and the pH of the mixture was adjusted to pH 8 using sat. aq. NaHCC. The aqueous mixture was extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with 5% citric acid (50 mL), brine (3 x 50 mL), dried over Na2SO4 and evaporated. The residue was triturated (PE / EtOAc = 4 / 1 (150 mL)) at 20 °C for 30 min to give the title compound (16.0 g, 44% yield) as a black-brown solid.

[0444] m / z ES+ [M+H]+287.1;1H NMR (400 MHz, CDCl3) δ ppm 7.55 (d, J= 8.0 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.25 (d, J= 0.8 Hz, 1H), 6.15 (d, J= 0.8 Hz, 1H), 5.70 (d, J= 9.6 Hz, 1H), 3.93 (d, J= 10.8 Hz, 3H), 3.78 (d, J= 10.8 Hz, 3H).

[0445] Intermediate 3: (S)-6-Methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2- a]pyrazine hydrochloride

[0446]

[0447] 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.

[0448] 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).

[0449] 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 pumped 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.

[0450] 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).

[0451] 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.

[0452] m / z ES+ [M+H]+238.2;1H NMR (400 MHz, CDCI3) 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).

[0453] 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). Thecombined 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 ferf-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.

[0454] 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).

[0455] Step e. A mixture of terf-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 column chromatography (PE / EtOAc = 15 / 1) to give ferf-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) 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).

[0456] The enantiomers were separated by chiral SFC (column: Daicel ChiralPak AD (250x50 mm; 10 m); mobile phase: A: 80% Heptane; B: 20% EtOH (0.1% NH4OH), isocratic elution to afford the second eluting enantiomer as ferf-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.

[0457] Step f. A mixture of ferf-butyl (S)-6-methyl-3-(perfluoroethyl)-5,6-dihydroimidazo[1,2-a]pyrazine-7(8 / - / )-carboxylate (4.50 g, 12.67 mmol) in 2 M HCI in 1,4-dioxane (30 mL) was stirred at 40 °C for 1 h. Upon completion, the reaction mixture was evaporated to give the title compound (3.65 g, 99% yield) as a yellow solid.

[0458] m / z ES+ [M+H]+256.1;1H NMR (400 MHz, DMSO-cfe) 6 ppm 10.67 (br s, 2H), 7.66 (s, 1H), 4.54 - 4.47 (m, 1H), 4.42 - 4.38 (m, 2H), 4.20 - 4.07 (m, 1H), 3.90 (br s, 1H), 1.46 (d, J = 6.4 Hz, 3H).

[0459] Intermediate 4: tert-Butyl (S)-2-methyl-5-thioxopiperazine-1 -carboxylateo

[0460]

[0461] Step a. 2-Chloroacetyl chloride (3.55 g, 31.5 mmol) was added to a solution of tert-butyl (S)-(1-aminopropan-2-yl)carbamate (CAS: 146552-71-8; 2.74 g, 15.7 mmol) and 2 M aq. Na2COs (15.7 mL) in EtOAc (50 mL) at 0 °C. The reaction mixture was stirred at 25 °C for 12 h. Upon completion, the reaction mixture was extracted with EtOAc (2 x40 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4and evaporated to give tert-butyl (S)-(1-(2-chloroacetamido)propan-2-yl)carbamate (3.75 g, 95% yield) as a white solid. m / z ES+ [M+Na]+273.1;1H NMR (400 MHz, CDCh) 6 ppm 4.57 (br s, 1 H), 4.09 - 4.00 (m, 2H), 3.91 - 3.80 (m, 1H), 3.41 - 3.35 (m, 1H), 3.31 - 3.19 (m, 1H), 1.45 (s, 9H), 1.19 (d, J = 6.8 Hz, 3H).

[0462] Step b. To a solution of tert-butyl (S)-(1-(2-chloroacetamido)propan-2-yl)carbamate (3.75 g, 14.96 mmol) in DCM (9 mL) was added TFA (9.21 g, 80.8 mmol) at 0 °C. The reaction mixture was stirred at 25 °C for 3 h. Upon completion, the reaction mixture was evaporated and the residue was re-dissolved in MeCN (120 mL) and K2CO3 (12.4 g, 89.7 mmol) and KI (7.45 g, 44.9 mmol) were added. The reaction mixture was stirred at 85 °C for 12 h. Upon completion, the reaction mixture containing a solution of (S)-5-methylpiperazin-2-one was used in the next step without further purification.

[0463] Step c. To a solution of (S)-5-methylpiperazin-2-one (1.71 g, 14.98 mmol) in MeCN (120 mL) was added BOC2O (3.60 g, 16.5 mmol). The reaction mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was evaporated and extracted with EtOAc (3 x 40 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4and evaporated to give tert-butyl (S)-2-methyl-5-oxopiperazine-1 -carboxylate (3.20 g, 100% yield over 2 steps) as a white solid.

[0464] m / z ES+ [MrtBu+H]+159.2;1H NMR (400 MHz, CDCh) 6 ppm 6.61 (br s, 1H), 4.50 - 4.43 (m, 1H), 4.31 (d, J= 18.8 Hz, 1H), 3.76 (d, J = 18.8 Hz, 1H), 3.63 - 3.59 (m, 1H), 3.13 - 3.09 (m, 1 H), 1.47 (s, 9H), 1.26 (d, J = 6.8 Hz, 3H).

[0465] Step d. To a solution of tert-butyl (S)-2-methyl-5-oxopiperazine-1-carboxylate (1.50 g, 7.00 mmol) in THF (10 mL) was added Lawesson’s Reagent (2.83 g, 7.00 mmol). The reaction mixture was stirred at 25 °C for 12 h. Upon completion, the reaction mixture was extracted with EtOAc (3 x 30 mL). The combined organic layers were washed with brine (40 mL), driedover Na2SC>4, evaporated and purified by column chromatography (PE / EtOAc = 4 / 1) to give the title compound (1.00 g, 62% yield) as a green oil.

[0466] m / z ES+ [M-tBu+H]+175.2;1H NMR (400 MHz, CDCh) 6 ppm 8.77 (br s, 1H), 4.85 - 4.72 (m, 1 H), 4.45 - 4.35 (m, 1H), 4.30 - 4.17 (m, 1H), 3.60 - 3.63 (m, 1H), 3.21 (d, J = 12.8 Hz, 1H), 1.47 (s, 9H), 1.22 (d, J= 6.8 Hz, 3H).

[0467] Intermediate 5: (S)-6-Methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine hydrochloride

[0468]

[0469] Step a. Methyl 2,2,3,3,3-pentafluoropropanoate (CAS: 378-75-6; 60.0 g, 337 mmol) was added to a solution of hydrazine monohydrate (38.1 g, 747 mmol) in MeOH (600 mL) at 0 °C. The reaction mixture was stirred at 25 °C for 16 h. Upon completion, the mixture was quenched with sat. aq. NH4CI (100 mL). The aqueous mixture was evaporated and the resulting residue was purified by column chromatography (AI2O3, PE / EtOAc = 10 / 1 to 0 / 1) to give 2, 2, 3,3,3-pentafluoropropanehydrazide (50.0 g, 83% yield) as a white solid.

[0470] 1H NMR (400 MHz, CDCh) 6 ppm 5.96 (br s, 1H).

[0471] Step b. A mixture of 2,2,3,3,3-pentafluoropropanehydrazide (5.20 g, 29.2 mmol), Intermediate 4 (2.80 g, 12.2 mmol), silver(l) benzoate (5.96 g, 26.0 mmol) and AcOH (2.19 g, 36.5 mmol) in DCE (40 mL) was stirred at60°C for 12 h. Upon completion, the reaction mixture was extracted with DCM (3 x 60 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SC>4, evaporated and purified by column chromatography (PE / EtOAc = 4 / 1) to give tert-butyl (S)-6-methyl-3-(perfluoroethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8 / - / )-carboxylate (3.30 g, 76% yield) as a white solid.

[0472] m / z ES+ [M+H]+357.1;1H NMR (400 MHz, CDCh) 6 ppm 5.30 (d, J = 18.2 Hz, 1H), 4.95 -4.90 (m, 1H), 4.48 (d, J= 18.2 Hz, 1H), 4.28 - 4.04 (m, 2H), 1.51 (s, 9H), 1.23 (d, J = 7.2 Hz, 3H).

[0473] Step c. A solution of tert-butyl (S)-6-methyl-3-(perfluoroethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8 / - / )-carboxylate (3.80 g, 10.7 mmol) in 2 M HCI in EtOAc (40 mL) was stirred at40 °C for 1 h. Upon completion, the reaction mixture was evaporated to give the title compound (3.12 g, 100% yield) as a white solid.

[0474] m / z ES+ [M+ H]+257.0.

[0475] Intermediate 6a: (S)-4-Fluoro-3-(6-methyl-3-(perfluoroethyl)-5, 6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzaldehyde

[0476]

[0477] Step a. To a mixture of 2-fluoro-5-formylbenzoic acid (CAS: 550363-85-4; 1.8 g, 10.7 mmol), Intermediate 3 (3.43 g, 11.7 mmol) and DIPEA (6.92 g, 53.5 mmol) in DMF (30 mL) was added CMPI (3.56 g, 13.9 mmol). The reaction mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched with H2O (0.2 mL), evaporated and purified by reverse phase flash chromatography (H2O (0.1% FA) / MeCN) to give the title compound (3.95 g, 91% yield) as a light yellow solid.

[0478] m / z ES+ [M+H]+406.0;1H NMR (400 MHz, CDCh) 5 ppm 10.00 (s, 1H), 8.11 - 7.96 (m, 2H), 7.53 - 7.39 (m, 1 H), 7.35 (t, J = 8.4 Hz, 1 H), 5.81 - 5.46 (m, 1 H), 4.87 - 4.62 (m, 1 H), 4.58 -4.00 (m, 3H), 1.48 - 1.27 (m, 3H).

[0479] Intermediate 6b: (S)-4-Fluoro-3-(6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro- [1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzaldehyde

[0480]

[0481] Step a. To a solution of Intermediate 5 (100 mg, 0.34 mmol), 2-fluoro-5-formylbenzoic acid (CAS: 550363-85-4; 58 mg, 0.34 mmol), DIPEA (132 mg, 1.03 mmol) in DMF (2 mL) was added HATU (130 mg, 0.34 mmol). The reaction mixture was stirred at 25 °C for 30 min. Upon completion, the reaction mixture was directly purified by reverse-phase flash chromatography (H2O (0.1% FA) / MeCN) to give the title compound (100 mg, 72% yield) as a white solid. m / z ES+ [M+H]+407.1;1H NMR (400 MHz, DMSO-d6) 5 ppm 10.02 (s, 1H), 8.24 - 8.02 (m, 2H), 7.64 (t, J= 8.0 Hz, 1H), 5.73 - 5.26 (m, 1H), 4.94 - 4.57 (m, 1H), 4.43 - 4.17 (m, 2H), 4.16 - 4.02 (m, 1H), 1.32 - 1.18 (m, 3H).Intermediate 7a: (2S,3 / ?,4S,5S,6S)-2-(2-(3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0482]

[0483] 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. NaHCOs (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.

[0484] 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).

[0485] Step b. The following process was conducted in 4 batches.

[0486] To a solution of (2S,3 / ?,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 (FW / 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).

[0487] 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.

[0488] m / z ES+ [M+H]+863.3;1H NMR (300 MHz, DMSO-cfe) 68.64 (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).

[0489] 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-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3, 4, 5-triyl triacetate (2.78 g, 80% yield) as a white solid.

[0490] m / z ES+ [M+H]+749.2;1H NMR (300 MHz, DMSO-cfe) 58.72 (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).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 the title compound (1.94 g, 67% yield) as a white solid.

[0491] 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).

[0492] Intermediate 7b: (2S,3R,4S,5S,6S)-2-(2-(2-((((9H-Fluoren-9- yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-((((4- nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H- pyran-3,4,5-triyl triacetate

[0493]

[0494] Step a. This step was conducted under continuous flow conditions: Solution 1 ((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; 12 g, 24.7 mmol) in THF (150 mL) and MeOH (150 mL); 1.2 mL / min) was pumped through Flow Reactor 1 packed with granular 1% Pt / C catalyst (WXSC1017, 7.5 g) {FLR1, SS, fixed bed, 6.35 (1 / 4") mm, 4.0 mL, 60 °C, retention time 3.33 min}. The H2 back pressure regulator was adjusted to 1.5 MPa, with an H2 flow rate of 60 mL / min. Upon completion, the reaction mixture was evaporated to give(2S,3R,4S,5S,6S)-2-(2-amino-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (10.5 g, 93% yield) as a yellow solid.

[0495] m / z ES+ [M+H]+456.0;1H NMR (400 MHz, CDCh) 6 ppm 6.90 (d, J= 8.0 Hz, 1H), 6.73 (d, J = 2.0 Hz, 1 H), 6.66 (dd, J = 8.0, 2.0 Hz, 1 H), 5.38 - 5.28 (m, 3H), 5.02 (d, J = 7.2 Hz, 1 H), 4.55 (s, 2H), 4.16 (d, J = 9.2 Hz, 1 H), 3.86 (br s, 2H), 3.76 (s, 3H), 2.14 - 1.99 (m, 9H).

[0496] Step b. To a solution of (2S,3R,4S,5S,6S)-2-(2-amino-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (6 g, 13.2 mmol) in DCM (100 mL) was added EEDQ (6.52 g, 26.4 mmol) and Fmoc-ZV-methylglycine (CAS: 77128-70-2; 4.92 g, 15.8 mmol). The reaction mixture was stirred at 20 °C for 12 h. Upon completion, the reaction mixture was diluted with H2O (200 mL) and extracted with EtOAc (3 x 200 mL). The combined organic layers were washed with sat. brine (3 x 100 mL), dried over Na2SO4, evaporated and purified by column chromatography (hexane / EtOAc = 1 / 2) to give (2S,3R,4S,5S,6S)-2-(2-(2-((((9 / 7-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (9 g, 91% yield) as a yellow solid.

[0497] m / z ES+ [M+H]+749.2.1H NMR (400 MHz, CDCh) 6 ppm 8.37 (d, J= 1.2 Hz, 1H), 8.20 - 8.02 (m, 1 H), 7.82 - 7.69 (m, 2H), 7.66 (br d, J = 5.6 Hz, 1 H), 7.62 - 7.49 (m, 1 H), 7.45 - 7.38 (m, 1H), 7.36 - 7.28 (m, 2H), 7.23 - 7.06 (m, 2H), 6.96 (d, J = 8.4 Hz, 1H), 5.46 - 5.38 (m, 1H), 5.37 - 5.22 (m, 3H), 5.14 - 5.05 (m, 1H), 4.70 - 4.59 (m, 2H), 4.49 - 4.39 (m, 2H), 4.34 - 4.18 (m, 2H), 4.15 - 4.11 (m, 2H), 3.77 - 3.70 (m, 3H), 3.14 (s, 3H), 2.08 - 2.01 (m, 9H).

[0498] Step c. 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 (9 g, 12 mmol) in DCM (100 mL) was added pyridine (3.8 g, 48.1 mmol) and 4-nitrophenyl chloroformate (4.85 g, 24 mmol). The reaction mixture was stirred at 0 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O (200 mL) and extracted with DCM (3 x 200 mL). The combined organic layers were washed with sat. brine (3 x 100 mL), dried over Na2SC>4, evaporated and purified by column chromatography (PE / EtOAc = 1 / 1) to give the title compound (9.6 g, 87% yield) as a white solid.

[0499] m / z ES+ [M+H]+914.3;1H NMR (400 MHz, CDCh) 6 ppm 8.55 (d, J= 2.0 Hz, 1H), 8.26 (d, J = 8.4 Hz, 2H), 8.22 - 8.04 (m, 1H), 7.82 - 7.74 (m, 1H), 7.73 - 7.61 (m, 2H), 7.54 - 7.51 (m, 1H), 7.46 - 7.29 (m, 5H), 7.23 - 7.09 (m, 2H), 6.99 (d, J = 8.4 Hz, 1H), 5.48 - 5.38 (m, 1H), 5.37 - 5.20 (m, 4H), 5.13 (d, J= 7.2 Hz, 1H), 4.46 (d, J= 7.2 Hz, 2H), 4.34 - 4.29 (m, 1H), 4.21 (d, J= 9.6 Hz, 1H), 4.14 - 4.10 (m, 2H), 3.81 - 3.69 (m, 3H), 3.23 - 3.10 (m, 3H), 2.06 (d, J = 4.4 Hz, 9H).Intermediate 8a: 2-((1-Methylpiperidin-4-yl)oxy)ethan-1 -amine dihydrochloride

[0500] Boc Boc Boc

[0501] i i i

[0502]

[0503] Step a. To a solution of benzyl 4-hydroxypiperidine-1 -carboxylate (CAS: 95798-23-5; 14.0 g, 59.5 mmol) and tert-butyl 1,2,3-oxathiazolidine-3-carboxylate 2,2-dioxide (CAS: 459817-82-4; 15.9 g, 71.4 mmol) in DMF (210 mL) was added t-BuONa (11.4 g, 119 mmol) at 0 °C. The reaction mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was slowly poured into H2O (500 mL), and the pH of the aqueous mixture was adjusted to pH 6 using 4 M aq. HCI. The aqueous mixture was extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with sat. brine (2 x 80 mL), dried over Na2SO4, evaporated and purified by column chromatography (DCM / MeOH = 95 / 5) to give benzyl 4-(2-((tert-butoxycarbonyl)amino)ethoxy)piperidine-1-carboxylate (14.0 g, 62% yield) as a yellow oil.

[0504] 1H NMR (400 MHz, CDCI3) 5 ppm 7.41 - 7.31 (m, 5H), 5.13 (s, 2H), 4.89 - 4.80 (m, 1H), 3.88 - 3.73 (m, 2H), 3.57 - 3.44 (m, 3H), 3.30 (d, J = 5.2 Hz, 2H), 3.26 - 3.18 (m, 2H), 1.86 - 1.75 (m, 2H), 1.58 - 1.52 (m, 2H), 1.45 (s, 9H).

[0505] Step b. This step was conducted under continuous flow conditions: Solution 1 (benzyl 4-(2-((terf-butoxycarbonyl)amino)ethoxy)piperidine-1-carboxylate (14.0 g, 36.9 mmol) in MeOH (140 mL); 0.4 mL / min) was pumped through Flow Reactor 1 packed with granular 5% Pd / ALOs (3.3 g) {FLR1, SS, fixed bed, 6.35 (1 / 4") mm, 5.0 mL, 50 °C, retention time 12.5 min}. The H2 back pressure regulator was adjusted to 1.5 MPa, with an H2 flow rate of 12.6 mL / min. Upon completion, the reaction mixture was evaporated to give tert-butyl (2-(piperidin-4-yloxy)ethyl)carbamate (8.8 g, 97% yield) as a colourless oil.

[0506] 1H NMR (400 MHz, CDCh) 6 ppm 4.89 (br s, 1H), 3.55 - 3.50 (m, 2H), 3.46 - 3.39 (m, 1H), 3.35 - 3.24 (m, 2H), 3.18 - 3.06 (m, 2H), 2.76 - 2.63 (m, 2H), 1.98 - 1.89 (m, 2H), 1.58 - 1.48 (m, 2H), 1.46 (s, 9H).

[0507] Step c. A mixture of tert-butyl (2-(piperidin-4-yloxy)ethyl)carbamate (8.8 g, 36 mmol), paraformaldehyde (1.62 g, 54 mmol) and acetic acid (216 mg, 3.6 mmol) in MeOH (90 mL) was stirred at 20 °C for 30 min, after which NaBHsCN (2.72 g, 43.2 mmol) was added and the reaction mixture was stirred at 20 °C fora further 16 h. Upon completion, the pH of the reaction mixture was adjusted to pH 8 using sat. aq. Na2COs. The aqueous mixture was filtered, the filtrate was evaporated and the resulting residue was re-dissolved in H2O (150 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers were dried over Na2SO4,evaporated and purified by column chromatography (DCM / MeOH = 85 / 15) to give tert-butyl (2-((1-methylpiperidin-4-yl)oxy)ethyl)carbamate (4.4 g, 47% yield) as a yellow oil.

[0508] m / z ES+ [M+H]+259.2;1H NMR (400 MHz, CDCb) 6 ppm 4.88 (brs, 1H), 3.55 - 3.46 (m, 2H), 3.40 - 3.23 (m, 3H), 2.80 - 2.65 (m, 2H), 2.31 (s, 3H), 1.97 - 1.87 (m, 4H), 1.74 - 1.58 (m, 2H), 1.45 (s, 9H).

[0509] Step d. A solution of tert-butyl (2-((1-methylpiperidin-4-yl)oxy)ethyl)carbamate (4.4 g, 17 mmol) in 2 M HCI in1,4-dioxane (20 mL) was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was evaporated to give the title compound (3.7 g, 93% yield) as a white solid, m / z ES+ [M+H]+159.0;1H NMR (400 MHz, DMSO-cfe) 6 ppm 10.98 - 10.46 (m, 1H), 8.39 -7.91 (m, 3H), 3.74 - 3.55 (m, 3H), 3.54 - 3.47 (m, 1 H), 3.37 - 3.35 (m, 1 H), 3.31 - 3.24 (m, 1 H), 3.17 - 3.14 (m, 1H), 2.99 - 2.86 (m, 2H), 2.74 - 2.66 (m, 3H), 2.14 - 1.70 (m, 4H).

[0510] Intermediate 8b: 2-(2-Aminoethoxy)ethyl acetate hydrochloride

[0511]

[0512] Step a. A mixture of 2-(2-aminoethoxy)ethan-1-ol (CAS: 929-06-6; 5.0 g, 47.5 mmol) in 2 M HCI in MeOH (35.6 mL) was stirred at 20 °C for 10 min. The reaction mixture was evaporated, the residue was re-dissolved in DCM (50 mL) and acetyl chloride (4.69 g, 59.7 mmol) was added at 5 °C. The reaction mixture was stirred at 20 °C for 16 h. Upon completion, the reaction mixture was evaporated and the residue was triturated with EtOAc (200 mL), hexane (300 mL), filtered and dried to give the title compound (8.00 g, 88% yield) as a white solid.1H NMR (400 MHz, DMSO-cfe) 6 ppm 8.03 (br s, 3H), 4.23 - 4.06 (m, 2H), 3.71 - 3.58 (m, 4H), 3.01 - 2.90 (m, 2H), 2.03 (s, 3H).Intermediate 9a: (2S,3 / ?,4S,5S,6S)-2-(2-(2-((((9H-Fluoren-9- yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-(((methyl(2-((2-((1-methylpiperidin-4- yl)oxy)ethyl)amino)ethyl)carbamoyl)oxy)methyl)phenoxy)-6- (methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate trifluoroacetate

[0513] o

[0514] AcO'*

[0515] b

[0516] c

[0517]

[0518] Step a. To a solution of Intermediate 7b (1.2 g, 1.31 mmol) in DMF (15 mL) was added 2- (methylamino)ethan-l-ol (128 mg, 1.7 mmol). The reaction mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was quenched with sat. aq. NH4CI (20 mL), diluted with water (30 mL) and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with sat. brine (3 x 20 mL), dried over Na2SO4, evaporated and purified by reversephase flash chromatography (H2O (0.1% FA) / MeCN) to give (2S,3?,4S,5S,6S)-2-(2-(2-((((9H- fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-((((2- hydroxyethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7- pyran-3,4,5-triyl triacetate (1.07 g, 96% yield) as a yellow solid.

[0519] m / z ES+ [M+H]+850.3;1H NMR (400 MHz, CDCb) 5 ppm 8.45 (d, J = 12.4 Hz, 1H), 8.30 - 8.05 (m, 1 H), 7.81 - 7.76 (m, 1 H), 7.75 - 7.64 (m, 2H), 7.63 - 7.50 (m, 1 H), 7.44 - 7.38 (m, 1 H), 7.37 - 7.29 (m, 2H), 7.05 - 7.01 (m, 2H), 6.97 - 6.91 (m, 1H), 5.46 - 5.37 (m, 1H), 5.36 - 5.23 (m, 2H), 5.15 - 5.05 (m, 3H), 4.46 - 4.44 (m, 2H), 4.37 - 4.07 (m, 4H), 3.82 - 3.66 (m, 5H), 3.45 (s, 2H), 3.18 - 3.08 (m, 3H), 3.05 - 2.94 (m, 3H), 2.15 - 1.94 (m, 9H).Step b. To a solution of (2S,3?,4S,5S,6S)-2-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-((((2-hydroxyethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (300 mg, 0.35 mmol) in DCM (6 mL) was added DMP (300 mg, 0.71 mmol). The reaction mixture was stirred at 0 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O (30 mL) and extracted with DCM (3 x 30 mL). The combined organic layers were washed with brine (3 x 20 mL), dried over Na2SO4 and evaporated to give (2S,3F?,4S,5S,6S)-2-(2-(2-((((9 / 7-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (300 mg, crude) as a yellow solid which was used without further purification.

[0520] m / z ES+ [M+Na]+870.6.

[0521] Step c. To a mixture of (2S,3R,4S,5S,6S)-2-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (2.8 g, 3.3 mmol) and acetic acid (793 mg, 13.2 mmol) in MeOH (70 mL) was added Intermediate 8a (1.53 g, 6.6 mmol). The reaction mixture was stirred at 25 °C for 30 min, after which NaBHsCN (622 mg, 9.9 mmol) was added, and the reaction mixture was stirred at 25 °C for a further 2 h. Upon completion, the reaction mixture was evaporated and purified by reverse-phase flash chromatography (H2O (0.1% FA) / MeCN) followed by Prep-HPLC to give the title compound (2.05 g, 50% yield) as a white solid.

[0522] m / z ES+ [M+H]+990.6;1H NMR (400 MHz, DMSO-cfe) 6 ppm 9.92 - 9.49 (m, 1H), 9.04 - 8.87 (m, 1 H), 8.82 - 8.44 (m, 2H), 8.04 - 7.80 (m, 3H), 7.68 (d, J = 7.6 Hz, 1 H), 7.59 (d, J = 7.6 Hz, 1H), 7.42 (d, J= 7.6 Hz, 1H), 7.35 (t, J= 6.8 Hz, 2H), 7.16 (d, J= 8.8 Hz, 3H), 5.68 - 5.56 (m, 1H), 5.51 (t, J = 9.6 Hz, 1H), 5.27 - 5.14 (m, 1H), 5.10 - 4.97 (m, 3H), 4.73 (d, J = 10.0 Hz, 1 H), 4.41 - 4.25 (m, 2H), 4.25 - 4.13 (m, 2H), 4.08 (s, 1H), 3.63 (s, 3H), 3.61 (s, 3H), 3.52 (d, J = 6.4 Hz, 4H), 3.24 (s, 1H), 3.12 (s, 5H), 2.95 (s, 3H), 2.85 (d, J= 8.0 Hz, 2H), 2.75 (d, J = 2.8 Hz, 3H), 2.26 - 1.82 (m, 12H), 1.78 (t, J= 10.4 Hz, 1H), 1.64 - 1.45 (m, 1H).

[0523] Intermediate 9b: (2S,3 / ?,4S,5S,6S)-2-(2-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-(4-methyl-3,14-dioxo-2,10,13-trioxa-4,7-diazapentadecyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate trifluoroacetate.0

[0524] 'OAc

[0525]

[0526] The title compound was prepared in a similar manner to Intermediate 9a, using Intermediate 8b in step c.

[0527] m / z ES+ [M+H]+979.4;1H NMR (400 MHz, DMSO-cfe) 6 ppm 9.03 - 8.91 (m, 1H), 8.54 - 8.38 (m, 2H), 8.02 - 7.90 (m, 2H), 7.89 - 7.78 (m, 1 H), 7.72 - 7.66 (m, 1 H), 7.59 (d, J = 7.6 Hz, 1 H), 7.47 - 7.40 (m, 1H), 7.40 - 7.31 (m, 2H), 7.29 - 7.06 (m, 3H), 5.72 - 5.60 (m, 1H), 5.51 (t, J = 9.6 Hz, 1H), 5.24 - 5.17 (m, 1H), 5.11 - 5.05 (m, 1H), 5.05 - 4.97 (m, 2H), 4.72 (d, J= 9.6 Hz, 1 H), 4.34 - 4.30 (m, 1H), 4.28 - 4.18 (m, 2H), 4.16 - 4.06 (m, 3H), 3.64 (br s, 2H), 3.62 (d, J = 8.8 Hz, 3H), 3.54 - 3.46 (m, 2H), 3.45 - 3.37 (m, 4H), 3.20 - 3.02 (m, 4H), 2.95 - 2.81 (m, 5H), 2.19 - 1.82 (m, 12H).

[0528] Intermediate 9c: (2S,3 / ?,4S,5S,6S)-2-(2-(2-((((9H-Fluoren-9- yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-(((methyl(2-((2- (methylsulfonyl)ethyl)amino)ethyl)carbamoyl)oxy)methyl)phenoxy)-6- (methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate trifluoroacetate

[0529] Fmoc

[0530] A -TFA

[0531] O CT NH A AcO'' YYA I

[0532]

[0533] OAc OH

[0534] The title compound was prepared in a similar manner to Intermediate 9a, using 2- (methylsulfonyl)ethan-l -amine (CAS: 49773-20-8) in step c.

[0535] m / z ES+ [M+H]+955.3;1H NMR (400 MHz, DMSO-d6) 5 ppm 9.03 - 8.85 (m, 1 H), 8.84 - 8.50 (m, 2H), 8.07 - 7.80 (m, 3H), 7.74 - 7.64 (m, 1 H), 7.64 - 7.55 (m, 1 H), 7.48 - 7.30 (m, 3H), 7.23 - 7.04 (m, 3H), 5.74 - 5.57 (m, 1 H), 5.56 - 5.43 (m, 1 H), 5.28 - 5.15 (m, 1H), 5.11 - 4.92 (m, 3H), 4.80 - 4.68 (m, 1 H), 4.23 (br s, 3H), 4.20 - 4.05 (m, 2H), 3.72 - 3.59 (m, 4H), 3.39 (br s, 5H), 3.21 - 3.06 (m, 4H), 2.94 (br s, 3H), 2.90 - 2.75 (m, 4H), 2.13 - 1.90 (m, 9H).

[0536] Intermediate 9d: (2S,3 / ?,4S,5S,6S)-2-(2-(2-((((9H-Fluoren-9- yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-(((methyl(2-((2-sulfamoylethyl)amino)ethyl)carbamoyl)oxy)methyl)phenoxy)-6- (methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate hydrochloride

[0537] Fmoc

[0538]

[0539] The title compound was prepared in a similar manner to Intermediate 9a, using Taurinamide (CAS: 4378-70-5) in step c.

[0540] m / z ES+ [M+H]+956.3;1H NMR (400 MHz, DMSO-cfe) 6 ppm 9.02 - 8.86 (m, 1 H), 8.75 - 8.63 (m, 2H), 8.05 - 7.89 (m, 2H), 7.89 - 7.81 (m, 1 H), 7.68 (d, J = 7.2 Hz, 1 H), 7.63 - 7.56 (m, 1 H), 7.47 - 7.40 (m, 1H), 7.39 - 7.32 (m, 2H), 7.21 (s, 2H), 7.19 - 7.05 (m, 3H), 5.71 - 5.59 (m, 1H), 5.50 (t, J = 9.6 Hz, 1H), 5.25 - 5.15 (m, 1H), 5.11 - 4.98 (m, 3H), 4.72 (d, J = 10.0 Hz, 1H), 4.39 - 4.13 (m, 4H), 4.09 - 4.03 (m, 1H), 3.62 (d, J = 8.2 Hz, 3H), 3.55 - 3.47 (m, 2H), 3.34 - 3.23 (m, 4H), 3.20 - 3.11 (m, 2H), 2.95 (s, 3H), 2.89 - 2.82 (m, 3H), 2.05 - 1.92 (m, 9H).

[0541] Intermediate 9e: (2S,3 / ?,4S,5S,6S)-2-(2-(2-((((9H-Fluoren-9- yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-(((methyl(2- (methylamino)ethyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro- 2H-pyran-3,4,5-triyl triacetate trifluoroacetate

[0542]

[0543] Step a. To a mixture of Intermediate 7b (1.28 g, 1.4 mmol) and tert-butyl methyl(2- (methylamino)ethyl)carbamate (CAS: 112257-19-9; 320 mg, 1.68 mmol) in DMF (12 mL) was added DI PEA (360 mg, 2.8 mmol) dropwise. The reaction mixture 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-(2-((((9 / 7-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundecyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (1.08 g, 80%) as a white solid.

[0544] m / z ES+ [M+H]+963.3;1H NMR (300 MHz, DMSO-cfe) 69.00 - 8.83 (m, 1H), 7.98 - 7.80 (m, 3H), 7.74 - 7.54 (m, 2H), 7.47 - 7.30 (m, 3H), 7.24 - 7.00 (m, 3H), 5.70 - 5.59 (m, 1H), 5.56 -5.43 (m, 1H), 5.25 - 5.15 (m, 1H), 5.12 - 5.01 (m, 1H), 5.00 - 4.92 (m, 2H), 4.77 - 4.68 (m, 1H), 4.37 - 4.04 (m, 5H), 3.67 - 3.57 (m, 2H), 3.31 - 3.20 (m, 5H), 3.00 - 2.90 (m, 3H), 2.87 - 2.61 (m, 6H), 2.12 - 1.89 (m, 9H), 1.33 (s, 9H).

[0545] Step b. To a solution of (2S,3R,4S,5S,6S)-2-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundecyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (200 mg, 0.21 mmol) in DCM (2 mL) at 0 °C was added TFA (0.67 mL) dropwise. The reaction mixture was stirred at rt for 1 h. Upon completion, the reaction mixture was evaporated to give the title compound (880 mg, crude) as a white solid that was used without further purification.

[0546] m / z ES+ [M+H]+863.3.

[0547] Intermediate 10: (2S,3R,4S,5S,6S)-2-(2-(((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(((methyl(2-((2-((1-methylpiperidin-4-yl)oxy)ethyl)amino)ethyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate ditrifluoroacetate

[0548]

[0549] Fmoc Fmoc

[0550] NH NH

[0551]

[0552] Step a. To a mixture of 4-hydroxybenzaldehyde (CAS: 123-08-0; 80.0 g, 655 mmol) and 2-chloro- / V-(hydroxymethyl)acetamide (CAS: 2832-19-1; 75.5 g, 611 mmol) in AcOH (300 mL) was added H2SO4 (480 mL) dropwise at 25 °C. The reaction mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was poured into ice-water (4000 mL) and the aqueous mixture was extracted with EtOAc (5 x 500 mL). The combined organic layers were dried over Na2SO4 and evaporated to give 2-chloro- / V-(5-formyl-2-hydroxybenzyl)acetamide (180 g, 40% yield) as a black-brown solid that was used without further purification. m / z ES+ [M+H]+227.9;1H NMR (400 MHz, DMSO-cfe) 6 11.46 (br s, 1H), 10.37 (s, 1H), 9.25 (t, J= 5.6 Hz, 1H), 8.30 - 8.24 (m, 2H), 7.58 (d, J= 8.0 Hz, 1H), 4.88 (d, J= 6.0 Hz, 2H), 4.75 (s, 2H).

[0553] Step b. To a mixture of 2-chloro- / V-(5-formyl-2-hydroxybenzyl)acetamide (90.0 g, 395 mmol) in 1,4-dioxane (200 mL) was added cone. aq. HCI (12 M, 200 mL). The reaction mixture was stirred at 100 °C for 9 h. Upon completion, the reaction mixture was evaporated to give 3- (aminomethyl)-4-hydroxybenzaldehyde hydrochloride (100 g, 70% yield) as a black-brown gum that was used without further purification.

[0554] m / z ES+ [M-NH2]+135.1.

[0555] Step c. To a mixture of 3-(aminomethyl)-4-hydroxybenzaldehyde hydrochloride (100 g, 373 mmol) and TEA (113 g, 1.12 mol) in 1,4-dioxane (1600 mL) and H2O (800 mL) was added 9-fluorenylmethyl chloroformate (CAS: 28920-43-6; 115 g, 447 mmol) at 25 °C. The reactionmixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was poured into water (2.5 L), and the pH of the mixture was adjusted to pH 6 using 3 M aq. HCI. The aqueous mixture was extracted with EtOAc (3 x 800 mL). The combined organic layers were dried over Na2SC>4, and evaporated. The residue was re-dissolved in EtOAc (800 mL) and stirred at 25 °C for 16 h. The resulting precipitate was filtered and discarded. The filtrate was evaporated and purified by column chromatography (PE / DCM / EtOAc = 4 / 1 / 1) to give (9 / 7-fluoren-9-yl)methyl (5-formyl-2-hydroxybenzyl)carbamate (40.0 g, 28% yield) as a yellow solid. m / z ES+ [M+H]+374.1;1H NMR (400 MHz, DMSO-cfe) 6 10.82 (s, 1H), 9.77 (s, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.83 (t, J= 6.0 Hz, 1H), 7.73 (d, J= 7.6 Hz, 2H), 7.70 - 7.66 (m, 2H), 7.44 - 7.39 (m, 2H), 7.35 - 7.30 (m, 2H), 6.98 (d, J = 8.8 Hz, 1 H), 4.36 - 4.29 (m, 2H), 4.25 (d, J = 4.4 Hz, 1H), 4.21 - 4.15 (m, 2H).

[0556] Step d. To a mixture of (9 / 7-fluoren-9-yl)methyl (5-formyl-2-hydroxybenzyl)carbamate (40.0 g, 107 mmol) and (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (CAS: 21085-72-3; 40.4 g, 101 mmol) in MeCN (600 mL) was added Ag2O (45.1 g, 194 mmol) at 25 °C. The reaction mixture was stirred in the dark at 25 °C for 16 h. Upon completion, the reaction mixture was filtered and the filtrate was evaporated to give (2S,3R,4S,5S,6S)-2-(2-(((((9 / 7-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-formylphenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (60.0 g, 81% yield) as a black brown solution in MeCN (500 mL) which was used without further purification, m / z ES+ [M+ H]+690.2.

[0557] Step e. To a mixture of (2S,3R,4S,5S,6S)-2-(2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-formylphenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (45.0 g, 65.2 mmol) in MeCN (480 mL) and I PA (60 mL) was added NaBH4 (3.21 g, 84.8 mmol) portionwise at 0 °C. The reaction mixture was stirred at 0 °C for 30 min. Upon completion, the reaction mixture was poured into sat. aq NH4CI (1 L) at 10 °C. The aqueous mixture was evaporated and the resulting residue was re-dissolved in water (800 mL) and extracted with DCM (3 x 600 mL). The combined organic layers were dried over Na2SC>4, evaporated and purified by column chromatography (PE / DCM / EtOAc = 5 / 1 / 5) to give (2S,3R,4S,5S,6S)-2-(2-(((((9 / 7-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (27.0 g, 59% yield) as a light yellow solid.

[0558] m / z ES+ [M+H]+692.2;1H NMR (400 MHz, CDCI3) 5 ppm 7.76 (d, J= 7.6 Hz, 2H), 7.67 - 7.51 (m, 2H), 7.47 - 7.35 (m, 3H), 7.34 - 7.28 (m, 3H), 6.99 (br d, J = 8.4 Hz, 1 H), 5.89 - 5.64 (m, 1H), 5.41 - 5.30 (m, 3H), 5.16 (d, J = 7.2 Hz, 1H), 4.72 - 4.58 (m, 2H), 4.53 - 4.33 (m, 3H), 4.32 - 4.24 (m, 1H), 4.24 - 4.14 (m, 2H), 3.74 - 3.72 (m, 1H), 3.67 (s, 3H), 2.10 - 2.06 (m, 9H).

[0559] Step f. To a mixture of (2S,3R,4S,5S,6S)-2-(2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (7.00 g, 10.1 mmol) and pyridine (2.40 g, 30.3 mmol) in DCM (70 mL) was added 4-nitrophenyl chloroformate (CAS: 7693-46-1; 4.08 g, 20.2 mmol) at 0 °C. The reaction mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was evaporated and purified by column chromatography (PE / EtOAc = 1 / 1) to give (2S,3?,4S,5S,6S)-2-(2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (7.50 g, 86% yield) as a light yellow solid.

[0560] m / z ES+ [M+H]+857.2;1H NMR (400 MHz, CDC ) 6 ppm 8.25 (br d, J= 8.8 Hz, 2H), 7.75 (d, J = 7.6 Hz, 2H), 7.61 (br t, J = 7.6 Hz, 2H), 7.53 (d, J = 2.0 Hz, 1 H), 7.44 - 7.33 (m, 5H), 7.32 - 7.28 (m, 3H), 7.02 (d, J= 8.4 Hz, 1H), 5.85 - 5.57 (m, 1H), 5.46 - 5.31 (m, 3H), 5.24 (s, 2H), 5.20 (d, J = 7.2 Hz, 1H), 4.45 - 4.36 (m, 2H), 4.34 - 4.25 (m, 1H), 4.24 - 4.17 (m, 2H), 3.76 -3.66 (m, 3H), 2.09 - 2.04 (m, 9H).

[0561] Step g. A mixture of (2S,3?,4S,5S,6S)-2-(2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (2.00 g, 2.33 mmol) and 2-(methylamino)ethan-l-ol (CAS: 109-83-1; 192 mg, 2.57 mmol) in DMF (10 mL) was stirred at 25 °C for 30 min. Upon completion, the reaction mixture was quenched with H2O (3 mL) and the aqueous mixture was directly purified by reverse-phase flash chromatography (H2O (0.1% FA) / MeCN) to give (2S,3R,4S,5S,6S)-2-(2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-((((2-hydroxyethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (1.20 g, 64% yield) as a white solid.

[0562] m / z ES+ [M+H]+793.3;1H NMR (CDCh) 6 ppm 7.75 (d, J= 7.6 Hz, 2H), 7.67 - 7.55 (m, 2H), 7.54 - 7.35 (m, 3H), 7.34 - 7.28 (m, 2H), 7.26 - 7.16 (m, 1H), 7.03 - 6.85 (m, 1H), 6.09 - 5.69 (m, 1 H), 5.45 - 5.28 (m, 3H), 5.15 (br d, J = 7.2 Hz, 1 H), 5.10 (s, 2H), 4.50 - 4.31 (m, 3H), 4.27 (br dd, J= 14.8, 5.6 Hz, 1H), 4.23 - 4.18 (m, 1H), 4.15 (br d, J= 10.0 Hz, 1H), 3.87 - 3.70 (m, 2H), 3.67 (s, 3H), 3.43 (br d, J = 4.4 Hz, 2H), 2.98 (s, 3H), 2.07 (d, J = 7.2 Hz, 9H).

[0563] Step h. To a mixture of (2S,3R,4S,5S,6S)-2-(2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-((((2-hydroxyethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (1.00 g, 1.26 mmol) in DCM (50 mL) was added DMP (CAS: 87413-09-0; 1.34 g, 3.15 mmol) at 0 °C. The reaction mixture was stirred at 15 °C for 1 h. Upon completion, the reaction mixture was poured into H2O (200 mL), and the pH of the mixture was adjusted to pH 8 using sat. aq. NaHCCh. The aqueous mixture was extracted with DCM (3 x 80 mL). The combined organic layers were dried over Na2SC>4 and evaporated to give (2S,3R,4S,5S,6S)-2-(2-(((((9 / 7-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(((methyl(2-oxoethyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (1.10 g, 80% yield) as a white solid.

[0564] 1H NMR (400 MHz, CDC ) 6 ppm 9.74 - 9.48 (m, 1H), 7.76 (d, J= 7.2 Hz, 2H), 7.63 (t, J= 7.2 Hz, 2H), 7.44 - 7.36 (m, 3H), 7.34 - 7.28 (m, 3H), 7.26 - 7.19 (m, 1H), 6.98 (t, J= 9.6 Hz, 1H), 5.76 (t, J = 6.4 Hz, 1H), 5.40 - 5.32 (m, 3H), 5.18 - 5.14 (m, 1H), 5.11 (s, 2H), 4.42 - 4.34 (m, 3H), 4.29 - 4.20 (m, 2H), 4.16 (d, J = 9.2 Hz, 1H), 4.08 (s, 1H), 3.68 (s, 3H), 2.99 (d, J = 9.2 Hz, 3H), 2.08 - 2.05 (m, 9H).

[0565] Step i. To a mixture of (2S,3R,4S,5S,6S)-2-(2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-(((methyl(2-oxoethyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (1.10 g, 1.39 mmol) and AcOH (334 mg, 5.56 mmol) in MeOH (150 mL) was added Intermediate 8a (803 mg, 3.48 mmol) at 25 °C. The reaction mixture was stirred at 0 °C for 30 min, after which NaBHsCN (262 mg, 4.17 mmol) was added and the reaction mixture was stirred at 25 °C for 15 h 30 min. Upon completion, the reaction mixture was quenched with H2O (5 mL), evaporated and purified by reverse-phase flash chromatography (H2O (0.1% TFA) / MeCN) to give the title compound (1.0 g, 60% yield) as a white solid.

[0566] m / z ES+ [M+H]+933.5;1H NMR (400 MHz, DMSO-cfe) 6 ppm 9.57 - 9.38 (m, 1H), 8.51 (br s, 2H), 7.91 (d, J = 7.2 Hz, 2H), 7.71 (d, J = 7.2 Hz, 3H), 7.48 - 7.40 (m, 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.30 - 7.25 (m, 1H), 7.18 (br s, 1H), 7.04 (d, J = 8.8 Hz, 1H), 5.60 (d, J = 8.0 Hz, 1H), 5.51 (t, J = 9.6 Hz, 1H), 5.17 (dd, J = 9.2, 8.4 Hz, 1H), 5.08 (t, J = 9.6 Hz, 1H), 4.99 (s, 2H), 4.73 (d, J = 9.6 Hz, 1H), 4.37 - 4.29 (m, 2H), 4.28 - 4.22 (m, 1H), 4.14 - 4.05 (m, 2H), 3.73 -3.67 (m, 1H), 3.64 (s, 3H), 3.61 (br s, 1H), 3.55 (d, J= 4.8 Hz, 3H), 3.35 - 3.20 (m, 2H), 3.18 - 2.90 (m, 6H), 2.82 (d, J = 6.0 Hz, 3H), 2.76 (d, J = 4.4 Hz, 3H), 2.22 - 2.06 (m, 1H), 2.04 -1.98 (m, 9H), 1.97 - 1.89 (m, 1 H), 1.87 - 1.68 (m, 1 H), 1.59 - 1.42 (m, 1 H).

[0567] Intermediate 11: 2,5-Dioxopyrrolidin-1-yl (S)-3-(2-(2-(4-amino-2-(2,5-dioxo-2,5-dihydro- 1 H-pyrrol-1 -yl)-4-oxobutanamido)ethoxy)ethoxy)propanoate

[0568]

[0569]

[0570] Step a. To a solution of (((9 / 7-fluoren-9-yl)methoxy)carbonyl)-L-asparagine (CAS: 71989-16-7, 1.4 g, 4.0 mmol), terf-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate (CAS: 756525-95-8, 1.1 g, 4.7 mmol) and HATU (1.8 g, 4.7 mmol) in DMF (30 mL) at 0 °C was added DIPEA (1.53 g, 11.9 mmol) dropwise. 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 terf-butyl (S)-5-(2-amino-2-oxoethyl)-1-(9 / 7-fluoren-9-yl)-3,6-dioxo-2,10,13-trioxa-4,7-diazahexadecan-16-oate (1.2 g, 53% yield) as a white solid.

[0571] m / z ES+ [M+H]+570.3;1H NMR (400 MHz, DMSO- cf6) 67.93 - 7.86 (m, 2H), 7.85 - 7.78 (m, 1H), 7.76 - 7.63 (m, 2H), 7.52 - 7.46 (m, 1H), 7.45 - 7.38 (m, 2H), 7.36 - 7.30 (m, 2H), 7.27 (s, 1H), 6.90 (s, 1H), 4.37 - 4.12 (m, 4H), 3.61 - 3.53 (m, 2H), 3.50 - 3.44 (m, 4H), 3.41 - 3.35 (m, 2H), 3.27 - 3.09 (m, 2H), 2.48 - 2.34 (m, 4H), 1.38 (s, 9H).

[0572] Step b. To a solution of terf-butyl (S)-5-(2-amino-2-oxoethyl)-1-(9 / 7-fluoren-9-yl)-3,6-dioxo-2,10,13-trioxa-4,7-diazahexadecan-16-oate (1 g, 1.8 mmol) in DMF (10 mL) at 0 °C was added diethylamine (CAS: 109-89-7, 283 mg, 3.9 mmol) dropwise. 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 terf-butyl (S)-3-(2-(2-(2,4-diamino-4-oxobutanamido)ethoxy)ethoxy)propanoate (500 mg, 82% yield) as a white solid.m / z ES+ [M+H]+348.2;1H NMR (300 MHz, DMSO-d6) 58.10 - 8.00 (m, 1H), 7.43 (s, 1H), 6.91 (s, 1H), 6.01 (s, 2H), 3.63 - 3.53 (m, 3H), 3.53 - 3.46 (m, 4H), 3.45 - 3.36 (m, 2H), 3.28 - 3.14 (m, 2H), 2.47 - 2.36 (m, 3H), 2.30 - 2.19 (m, 1H), 1.40 (s, 9H).

[0573] Step c. A mixture of tert-butyl (S)-3-(2-(2-(2,4-diamino-4-oxobutanamido)ethoxy)ethoxy)propanoate (450 mg, 1.3 mmol) and maleic anhydride (CAS: 108-31-6, 128 mg, 1.3 mmol) in acetone (4 mL) was stirred at rt for 16 h. Upon completion, the reaction mixture was evaporated and the residue was dissolved in AC2O (5 mL), after which NaOAc (128 mg, 1.56 mmol) was added. The reaction mixture was stirred at 90 °C for 3 h. Upon completion, the reaction mixture was filtered, the filtrate was evaporated and the residue was purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give tertbuty (S)-3-(2-(2-(4-amino-2-(2,5-dioxo-2,5-dihydro-1 / 7-pyrrol-1-yl)-4-oxobutanamido)ethoxy)ethoxy)propanoate (256 mg, 46% yield) as a white solid.

[0574] m / z ES+ [M+H]+428.2;1H NMR (400 MHz, DMSO-d6) 58.12 - 8.03 (m, 1H), 7.39 (s, 1H), 7.03 (s, 2H), 6.82 (s, 1H), 4.90 - 4.79 (m, 1H), 3.62 - 3.54 (m, 2H), 3.52 - 3.43 (m, 4H), 3.42 - 3.34 (m, 2H), 3.23 - 3.10 (m, 2H), 2.97 - 2.87 (m, 1H), 2.73 - 2.64 (m, 1H), 2.45 - 2.38 (m, 2H), 1.39 (s, 9H).

[0575] Step d. To a solution of tert-butyl (S)-3-(2-(2-(4-amino-2-(2,5-dioxo-2,5-dihydro-1 / 7-pyrrol-1-yl)-4-oxobutanamido)ethoxy)ethoxy)propanoate (250 mg, 0.59 mmol) in DCM (2 mL) at 0 °C was added TFA (0.5 mL) dropwise. The reaction mixture was stirred at rt for 2 h. Upon completion, the reaction mixture was evaporated and the residue was purified by reversephase flash chromatography (H2O (0.01% FA) / MeCN) to give (S)-3-(2-(2-(4-amino-2-(2,5-dioxo-2,5-dihydro-1 / 7-pyrrol-1-yl)-4-oxobutanamido)ethoxy)ethoxy)propanoic acid (158 mg, 73% yield) as a yellow liquid.

[0576] m / z ES+ [M+H]+372.1;1H NMR (400 MHz, DMSO-d6) 5 12.18 (s, 1H), 8.12 - 8.04 (m, 1H), 7.39 (s, 1H), 7.03 (s, 2H), 6.82 (s, 1H), 4.91 - 4.78 (m, 1H), 3.64 - 3.56 (m, 2H), 3.51 - 3.44 (m, 4H), 3.40 - 3.30 (m, 2H), 3.22 - 3.11 (m, 2H), 2.96 - 2.88 (m, 1 H), 2.73 - 2.63 (m, 1 H), 2.47 - 2.41 (m, 2H).

[0577] Step e. To a mixture of (S)-3-(2-(2-(4-amino-2-(2,5-dioxo-2,5-dihydro-1 / 7-pyrrol-1-yl)-4-oxobutanamido)ethoxy)ethoxy)propanoic acid (150 mg, 0.40 mmol) and bis(2,5-dioxopyrrolidin-1-yl) carbonate (CAS: 74124-79-1, 119 mg, 0.46 mmol) in DMF (3 mL) at 0 °C was added DIPEA (156 mg, 1.2 mmol) dropwise. 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 (84 mg, 44% yield) as a white solid.

[0578] m / z ES+ [M+H]+469.1;1H NMR (400 MHz, DMSO-d6) 58.13- 8.03 (m, 1 H), 7.39 (s, 1 H), 7.03 (s, 2H), 6.87 - 6.77 (m, 1 H), 4.92 - 4.80 (m, 1 H), 3.76 - 3.66 (m, 2H), 3.54 - 3.47 (m, 4H), 3.42- 3.33 (m, 2H), 3.21 - 3.09 (m, 2H), 2.98 - 2.89 (m, 3H), 2.86 - 2.75 (m, 4H), 2.73 - 2.63 (m, 1H).

[0579] Intermediate 12: 2,5-Dioxopyrrolidin-1-yl 3-(4,5-dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)propanoate

[0580]

[0581] 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 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.

[0582] 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).

[0583] 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).

[0584] 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 / 7)-yl)propanoic acid (650 mg, 76% yield) as a white solid.

[0585] m / z ES+ [M+H]+354.8, 356.9, 358.9;1H NMR (400 MHz, DMSO-cfe) 6 12.52 (s, 1H), 4.33 -4.19 (m, 2H), 3.56 (s, 3H), 2.71 - 2.56 (m, 2H).

[0586] Step d. To a solution of 3-(4,5-dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2 / 7)-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.

[0587] 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).

[0588] Intermediate 13: 3-((8,9-Dibromo-7,10-dioxo-1,2,4,5,7,10-hexahydro-3H-pyridazino[1,2-a][1,2,5]triazepin-3-yl)sulfonyl)propanoic acid

[0589]

[0590] Step a. To a solution of di-tert-butyl hydrazine-1, 2-dicarboxylate (CAS: 16466-61-8; 2.0 g, 8.6 mmol) and benzyl bis(2-chloroethyl)carbamate (CAS: 72791-76-5; 2.37 g, 8.6 mmol) in toluene (30 mL) were added 50% aq. NaOH (9 mL) and tetraethylammonium bromide (271 mg, 1.3 mmol). The reaction mixture was stirred under reflux for 16 h. Upon completion, the reaction mixture was poured into sat. aq. NH4CI (40 mL) and the aqueous mixture was extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SC>4, evaporated and purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give 5-benzyl 1,2-di-tert-butyl 1,2,5-triazepane-1,2,5-tricarboxylate (1.47 g, 39% yield) as a light-yellow oil.

[0591] m / z ES+ [M+Na]+458.2;1H NMR (300 MHz, DMSO-cfe) 57.49 - 7.15 (m, 5H), 5.20 - 5.00 (m, 2H), 4.07 - 3.80 (m, 2H), 3.66 - 3.34 (m, 4H), 3.19 - 2.84 (m, 2H), 1.55 - 1.16 (m, 18H).

[0592] Step b. A mixture of 10% Pd / C (140 mg, 0.13 mmol), and 5-benzyl 1,2-di-tert-butyl 1,2,5-triazepane-1,2,5-tricarboxylate (1.4 g, 3.2 mmol) in MeOH (15 mL) was stirred at rt for 4 hunder a H2 atmosphere (1 atm). Upon completion, the reaction mixture was filtered through a Celite pad and the filtrate was evaporated to give the di-terf-butyl 1,2,5-triazepane-1,2-dicarboxylate (900 mg, crude) as a light-yellow liquid that was used without further purification, m / z ES+ [M+H]+302.1;1H NMR (300 MHz, DMSO-cfe) 63.86 - 3.71 (m, 2H), 3.12 - 2.73 (m, 4H), 2.66 - 2.51 (m, 2H), 1.48 - 1.30 (m, 18H).

[0593] Step c. Under an N2 atmosphere, methyl 3-(chlorosulfonyl)propanoate (CAS: 15441-07-3; 744 mg, 4.0 mmol) was added dropwise to a mixture of di-terf-butyl 1,2,5-triazepane-1,2-dicarboxylate (800 mg, 2.7 mmol) and TEA (538 mg, 5.3 mmol) in DCM (10 mL) at 0 °C. The reaction mixture was stirred at rt for 4 h. Upon completion, the reaction mixture was quenched with H2O (0.5 mL), evaporated and purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give di-terf-butyl 5-((3-methoxy-3-oxopropyl)sulfonyl)-1,2,5-triazepane-1,2-dicarboxylate (1.06 g, 88%) as a light-yellow solid.

[0594] m / z ES+ [M+Na]+474.1;1H NMR (300 MHz, DMSO-cfe) 64.00 - 3.84 (m, 2H), 3.79 - 3.63 (m, 7H), 3.31 - 2.97 (m, 4H), 2.57 - 2.52 (m, 2H), 1.48 - 1.34 (m, 18H).

[0595] Step d. To a solution of di-terf-butyl 1,2,5-triazepane-1,2-dicarboxylate di-terf-butyl 5-((3-methoxy-3-oxopropyl)sulfonyl)-1,2,5-triazepane-1,2-dicarboxylate (600 mg, 1.33 mmol) in THF (6 mL) at 0 °C was added 1 M aq. LiOH (2.66 mL, 2.66 mmol) dropwise. The reaction mixture was stirred at rt for 4 h. Upon completion, the reaction mixture was directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give 3-((1,2-bis(ferf-butoxycarbonyl)-1,2,5-triazepan-5-yl)sulfonyl)propanoic acid (228 mg, 39%) as a white solid, m / z ES+ [M+Na]+460.1;1H NMR (300 MHz, DMSO-cfe) 54.01 - 3.76 (m, 2H), 3.54 - 3.41 (m, 2H), 3.25 - 3.11 (m, 4H), 3.09 - 2.95 (m, 2H), 2.67 - 2.55 (m, 2H), 1.53 - 1.27 (m, 18H).

[0596] Step e. A mixture of 3-((1,2-bis(ferf-butoxycarbonyl)-1,2,5-triazepan-5-yl)sulfonyl)propanoic acid (50 mg, 0.11 mmol) and 3, 4-dibromofuran-2, 5-dione (CAS: 1122-12-9, 34 mg, 0.13 mmol) in AcOH (1 mL) was stirred at 120 °C for 4 h. Upon completion, the reaction mixture was directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give the title compound (19 mg, 35%) as a white solid.

[0597] m / z ES+ [M+H]+473.9, 475.9, 477.8;1H NMR (300 MHz, DMSO-cfe) 5 12.56 (s, 1H), 4.56 -4.39 (m, 4H), 3.69 - 3.54 (m, 4H), 3.37 - 3.31 (m, 2H), 2.61 (t, J= 7.4 Hz, 2H).

[0598] Example A1: (S)-9-(4-Fluoro-3-(6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-[1,3]dioxolo[4,5-f]phthalazin-6(7H)-oneo

[0599] o

[0600]

[0601] Step a. To a mixture of Intermediate 2 (2.67 g, 9.33 mmol) and Intermediate 6a (3.40 g, 8.40 mmol) in THF (150 mL) was added DBU (2.84 g, 18.6 mmol) at 0 °C. The reaction mixture was stirred at 20 °C for 16 h. Upon completion, the reaction mixture was poured into 5% citric acid (500 mL) at 0 °C and extracted with EtOAc (3 x 280 mL). The combined organic layers were washed with brine (3 x 150 mL), dried over Na2SO4 and evaporated to give (S)-8-(4-fluoro-3-(6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzylidene)-[1,3]dioxolo[4,5-e]isobenzofuran-6(8 / - / )-one (5.00 g, 94% yield) as a black-brown solid.

[0602] m / z ES+ [M+H]+566.0;1H NMR (400 MHz, CDCh) 6 ppm 8.03 - 7.96 (m, 1H), 7.96 - 7.90 (m, 1H), 7.61 (s, 1H), 7.59 (s, 1H), 7.48 (t, J= 8.8 Hz, 1H), 7.27 (d, J= 8.0 Hz, 1H), 6.45 (s, 1H), 6.43 (s, 2H), 5.43 - 5.29 (m, 1H), 4.60 - 4.46 (m, 1H), 4.36 - 4.15 (m, 2H), 4.14 - 4.03 (m, 1H), 1.26 - 1.17 (m, 3H).

[0603] Step b. Hydrazine hydrate (9.70 g, 193 mmol) was added to a solution of (S)-8-(4-fluoro-3-(6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzylidene)-[1,3]dioxolo[4,5-e]isobenzofuran-6(8 / - / )-one (4.90 g, 8.67 mmol) in EtOH (40 mL). The reaction mixture was stirred at 80 °C for 16 h under a N2 atmosphere. Upon completion, the reaction mixture was evaporated and purified by reverse phase flash chromatography (H2O (0.1% FA) / MeCN) followed by Prep-HPLC to give the title compound (3.32 g, 66% yield) as an off-white solid.

[0604] m / z ES+ [M+H]+580.1;1H NMR (400 MHz, CDCh) 6 ppm 10.14 - 9.90 (m, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.49 - 7.32 (m, 3H), 7.25 (d, J= 8.4 Hz, 1H), 7.07 (t, J= 9.2 Hz, 1H), 6.19 (s, 2H), 5.83 - 5.39 (m, 1H), 4.84 - 4.58 (m, 1H), 4.58 - 4.32 (m, 1H), 4.31 - 4.25 (m, 2H), 4.26 - 4.13 (m, 1H), 4.12 - 3.94 (m, 1H), 1.43 - 1.19 (m, 3H).Example A2: (S)-9-(4-Fluoro-3-(6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)-[1,3]dioxolo[4,5-f]phthalazin-6(7H)-one

[0605]

[0606] Step a. To a solution of Intermediate 6b (90 mg, 0.22 mmol) and Intermediate 2 (63 mg, 0.22 mmol) in THF (6 mL) was added DBU (41 mg, 0.27 mmol). The reaction mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was poured into sat. aq. NH4CI (20 mL), and the aqueous mixture was extracted with EtOAc (2 x 15 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4 and evaporated to give (S)-8-(4-fluoro-3-(6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzylidene)-[1,3]dioxolo[4,5-e]isobenzofuran-6(8 / - / )-one (100 mg, 80% yield) as a white solid.

[0607] m / z ES+ [M+H]+567.2.

[0608] Step b. Hydrazine hydrate (210 mg, 4.2 mmol) was added to a solution of (S)-8-(4-fluoro-3-(6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzylidene)-[1,3]dioxolo[4,5-e]isobenzofuran-6(8 / - / )-one (90 mg, 0.16 mmol) in EtOH (6 mL). The reaction mixture was stirred at 80 °C for 16 h. Upon completion, the reaction mixture was evaporated and directly purified by prep-HPLC to give the title compound (78 mg, 85% yield) as a white solid.

[0609] m / z ES+ [M+H]+581.2;1H NMR (400 MHz, DMSO-d6) 5 ppm 12.34 (s, 1H), 7.84 (d, J= 8.4 Hz, 1H), 7.45 (d, J= 8.4 Hz, 1H), 7.40 (dd, J= 2.0, 5.2 Hz, 1H), 7.35 (d, J= 4.0 Hz, 1H), 7.28 (t, J= 8.8 Hz, 1H), 6.24 (s, 2H), 5.72 - 5.15 (m, 1H), 4.94 - 4.52 (m, 1H), 4.28 (s, 2H), 4.26 - 3.90 (m, 3H), 1.35 - 0.98 (m, 3H).

[0610] Example A3: (S)-4-(4-Fluoro-3-(6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)phthalazin-1(2H)-oneo

[0611]

[0612] Step a. This step was conducted in a similar manner to Intermediate 6a, step a, using Intermediate 3 and 2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid (CAS: 763114-26-7).

[0613] m / z ES+ [M+H]+536.1;1H NMR (400 MHz, DMSO-d6) 5 ppm 12.59 (s, 1H), 8.44 - 8.17 (m, 1H), 8.00 - 7.91 (m, 1H), 7.91 - 7.75 (m, 2H), 7.62 - 7.54 (m, 1H), 7.46 (d, J = 6.4 Hz, 2H), 7.30 (t, J = 8.8 Hz, 1 H), 5.30 (d, J = 17.2 Hz, 1 H), 4.66 - 4.40 (m, 1 H), 4.34 (s, 2H), 4.26 - 4.00 (m, 2H), 4.00 - 3.62 (m, 1H), 1.27 - 1.04 (m, 3H).

[0614] Example A4: (S)-4-(3-(6-Methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)phthalazin-1(2H)-one

[0615]

[0616] The title compound was prepared in a similar manner to Example A3, using 3-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid (CAS: 420846-72-6).

[0617] m / z ES+ [M+H]+518.2;1H NMR (400 MHz, DMSO-cfe) 6 12.60 (s, 1H), 8.30 - 8.18 (m, 1H), 8.01 - 7.93 (m, 1H), 7.92 - 7.86 (m, 1H), 7.85 - 7.78 (m, 1H), 7.57 (s, 1H), 7.49 - 7.39 (m, 3H), 7.34 (d, J= 7.2 Hz, 1H), 5.30 - 4.79 (m, 1H), 4.69 - 4.22 (m, 4H), 4.22 - 4.14 (m, 1H), 4.02 - 3.85 (m, 1H), 1.10 (d, J = 2.0 Hz, 3H).

[0618] 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-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-A / -methyl-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0619]

[0620] OH OH

[0621]

[0622] Step a. To a solution of Example A1 (22 mg, 0.038 mmol), DIPEA (25 mg, 0.19 mmol) and DMAP (4.6 mg, 0.038 mmol) in DCM (1 mL) was added BTC (CAS: 32315-10-9, 11 mg, 0.038 mmol) at 0 °C under an Ar atmosphere. The reaction mixture was warmed to rt and stirred for 1 h, after which ferf-butyl methyl(2-(methylamino)ethyl)carbamate (CAS: 112257-19-9, 11.3 mg, 0.038 mmol) in DCM (0.5 mL) was added at 0 °C under an Ar atmosphere. The reaction mixture was warmed to rt and stirred for a further 3 h. Upon completion, the reaction mixture was quenched with sat. aq. NaHCCh (0.05 mL), evaporated and purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give terf-butyl (S)-(2-(9-(4-fluoro-3-(6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / \ / -methyl-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5- / ]phthalazine-7-carboxamido)ethyl)(methyl)carbamate (24 mg, 79% yield) as a white solid.

[0623] m / z ES+ [M+H]+794.2;1H NMR (400 MHz, DMSO-d6) δ 7.90 (d, J = 8.3 Hz, 1 H), 7.63 - 7.55 (m, 1 H), 7.51 (d, J = 8.4 Hz, 1 H), 7.45 - 7.35 (m, 2H), 7.33 - 7.22 (m, 1 H), 6.27 (s, 2H), 5.44 -5.20 (m, 1H), 4.54 - 4.38 (m, 1H), 4.32 (s, 2H), 4.25 - 4.06 (m, 2H), 4.04 - 3.90 (m, 1H), 3.64 - 3.53 (m, 1H), 3.44 - 3.37 (m, 1H), 3.31 - 3.18 (m, 2H), 3.09 - 3.04 (m, 1H), 2.89 - 2.72 (m, 4H), 2.63 - 2.57 (m, 1H), 1.44 - 1.07 (m, 12H).

[0624] Step b. To a stirred solution of terf-butyl (S)-(2-(9-(4-fluoro-3-(6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / V-methyl-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5- / ]phthalazine-7-carboxamido)ethyl)(methyl)carbamate (24 mg, 0.030 mmol) in DCM (0.6 mL) was added TFA (0.3 mL) dropwise at 0 °C. The resulting mixture was warmed to rt and stirred for 2 h. Upon completion, the reaction mixture was evaporated to give (S)-9-(4-fluoro-3-(6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / V-methyl- / \ / -(2-(methylamino)ethyl)-6-oxo-[1,3]dioxolo[4,5- / ]phthalazine-7(6 / - / )-carboxamide 2,2,2-trifluoroacetate (19 mg, crude) as a yellow semi-solid that was used without further purification.

[0625] m / z ES+ [M+H]+694.2.

[0626] Step c. To a solution of Intermediate 7a (15 mg, 0.017 mmol) in DMF (0.4 mL) were added (S)-9-(4-fluoro-3-(6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / V-methyl- / \ / -(2-(methylamino)ethyl)-6-oxo-[1,3]dioxolo[4,5- / ]phthalazine-7(6 / - / )-carboxamide 2,2,2-trifluoroacetate (17 mg, 0.024 mmol) in DMF (0.2 mL), HOBt (3 mg, 0.019 mmol) in DMF (0.2 mL) and DIPEA (7 mg, 0.051 mmol) in DMF (0.2 mL) at 0 °C. The reaction mixture was warmed to rt 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-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / \ / -methyl-6-oxo-6, 7-dihydro-[1,3]dioxolo[4,5- / ]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (15 mg, 62% yield) as a white solid.

[0627] m / z ES+ [M+H]+1468.5.

[0628] Step d. To a solution of (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / \ / -methyl-6-oxo-6, 7-dihydro-[1,3]dioxolo[4,5- / ]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (15 mg, 0.01 mmol) in THF (0.15 mL) and MeOH (0.15 mL) was added 1 M aq. LiOH (0.15 mL, 0.15 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,5R,6S)-6-(2-(3-aminopropanamido)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / \ / -methyl-6-oxo-6, 7-dihydro-[1, 3]dioxolo[4, 5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid (9 mg, 79% yield) as a white solid.

[0629] m / z ES+ [M+H]+1106.3.

[0630] Step e. To a mixture of (2S,3S,4S,5R,6S)-6-(2-(3-aminopropanamido)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / V-methyl-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid (9 mg, 0.008 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; 4 mg, 0.012 mmol) in DMF (0.8 mL) was added DIPEA (3.2 mg, 0.025 mmol) in DMF (0.1 mL) dropwise at 0 °C. The reaction mixture was warmed to rt and stirred for 2 h under an Ar atmosphere. Upon completion, the reaction mixture directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give the title compound (5 mg, 48% yield) as a white solid.

[0631] m / z ES+ [M+H]+1345.5.1H NMR (300 MHz, DMSO-d6) δ 9.17 - 9.09 (m, 1H), 8.22 - 8.11 (m, 1H), 8.00 - 7.88 (m, 2H), 7.62 - 7.46 (m, 2H), 7.43 - 7.34 (m, 3H), 7.30 - 7.22 (m, 1H), 7.07 -6.99 (m, 4H), 6.27 (s, 2H), 5.80 - 5.71 (m, 1H), 5.34 - 5.19 (m, 2H), 5.02 - 4.94 (m, 1H), 4.81 - 4.72 (m, 1H), 4.61 - 4.40 (m, 2H), 4.33 - 4.10 (m, 5H), 3.76 - 3.39 (m, 18H), 3.07 - 3.03 (m, 2H), 2.92 - 2.80 (m, 4H), 2.75 - 2.70 (m, 3H), 2.30 - 2.24 (m, 4H), 1.15 - 1.04 (m, 3H).

[0632] 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-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / V-methyl-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0633]

[0634] The title compound was prepared in a similar manner to Example B1, using Intermediate 7b in step c and 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1 / 7-pyrrol-1-yl)propanoate (CAS: 55750-62-4) in step e.

[0635] m / z ES+ [M+H]+1257.5;1H NMR (400 MHz, DMSO-d6) δ 9.44 - 8.96 (m, 1H), 8.26 - 8.06 (m, 1H), 7.96 - 7.71 (m, 1H), 7.66 - 7.19 (m, 5H), 7.15 - 6.91 (m, 4H), 6.37 - 6.12 (m, 2H), 5.87 -5.70 (m, 1H), 5.48 - 5.16 (m, 2H), 5.10 -4.93 (m, 1H), 4.90-4.76 (m, 1H), 4.70-4.53 (m, 1H), 4.51 - 4.37 (m, 1H), 4.39 - 4.03 (m, 6H), 4.02 - 3.76 (m, 2H), 3.71 - 3.40 (m, 8H), 3.11 - 2.94 (m, 4H), 2.92 - 2.76 (m, 5H), 2.74 - 2.61 (m, 3H), 2.57 - 2.53 (m, 2H), 1.25 - 1.05 (m, 3H).

[0636] Example B3: (2S,3S,4S,5 / ?,6S)-6-(2-((S)-17-Amino-15-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)-3-methyl-4,14,17-trioxo-7,10-dioxa-3,13-diazaheptadecanamido)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / V-methyl-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acidOH OH

[0637] N\

[0638]

[0639] The title compound was prepared in a similar manner to Example B1, using Intermediate 7b in step c and Intermediate 11 in step e.

[0640] m / z ES+ [M+H]+1459.9;1H NMR (400 MHz, DMSO-d6) δ 9.33 - 8.95 (m, 1H), 8.21 - 7.75 (m, 3H), 7.63 - 7.53 (m, 1H), 7.53 - 7.46 (m, 1H), 7.45 - 7.31 (m, 3H), 7.30 - 7.18 (m, 1H), 7.13 - 6.94 (m, 3H), 6.87 -6.74 (m, 1H), 6.34 -6.15 (m, 2H), 5.43- 5.22 (m, 2H), 5.06-4.94 (m, 2H), 4.92 - 4.77 (m, 3H), 4.69 - 4.53 (m, 2H), 4.51 - 4.37 (m, 2H), 4.36 - 4.22 (m, 3H), 4.20 - 4.05 (m, 4H), 4.02 - 3.83 (m, 2H), 3.68 - 3.55 (m, 3H), 3.53 - 3.33 (m, 10H), 3.24 - 3.11 (m, 2H), 3.10 - 2.99 (m, 3H), 2.98 - 2.74 (m, 6H), 2.74 - 2.58 (m, 3H), 2.49 - 2.30 (m, 2H), 1.30 - 0.99 (m, 3H).

[0641] Example B4: (2S,3S,4S,5R,6S)-6-(2-(2-((2-(2,5-Dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethyl)(methyl)amino)acetamido)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-A / -(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acidOH OH

[0642]

[0643] OH OH

[0644]

[0645] Step a. To a solution of Example A1 (160 mg, 0.28 mmol) in DCM (3 mL) at 0 °C were added triphosgene (CAS: 32315-10-9, 90 mg, 0.30 mmol), DIPEA (181 mg, 1.035 mmol) and DMAP (34 mg, 0.28 mmol). The reaction mixture was stirred at rt for 90 min, after which the mixture was cooled to 0 °C and a solution of Intermediate 9a (145 mg, 0.17 mmol) in DCM (2 mL) was added. The reaction mixture was stirred at rt for an additional 16 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 (2S,3R,4S,5S,6S)-2-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / \ / -(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (46 mg, 17% yield) as a white solid.

[0646] m / z ES+ [M+H]+1595.9.

[0647] Step b. To a solution of (2S,3R,4S,5S,6S)-2-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / \ / -(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (46 mg, 0.03 mmol) in THF (0.4 mL) and MeOH (0.4 mL) at 0 °C was added 1 M LiOH (0.4 mL, 0.4 mmol). The reaction mixture was stirred at rt for 1 h. 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-(4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / V-(2-((1 -methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5- / ]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-(2-(methylamino)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid (30 mg, 70% yield) as a white solid, m / z ES+ [M+H]+1233.6.

[0648] Step c. To a solution of (2S,3S,4S,5R,6S)-6-(4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / \ / -(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5- / ]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-(2-(methylamino)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid (30 mg, 0.02 mmol) in DMF (1.5 mL) at 0 °C were added 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1 / 7-pyrrol-1-yl)propanoate (CAS: 55750-62-4, 9.6 mg, 0.04 mmol), HOBt (4.9 mg, 0.04 mmol) and DIPEA (6.2 mg, 0.05 mmol). 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 (7 mg, 21% yield) as a white solid.

[0649] m / z ES+ [M+H]+1384.8;1H NMR (400 MHz, DMSO-d6) δ 9.51 - 9.05 (m, 2H), 8.21 - 8.05 (m, 1H), 7.97 - 7.76 (m, 1H), 7.68 - 7.19 (m, 5H), 7.17 - 6.91 (m, 4H), 6.34 - 6.14 (m, 2H), 5.46 -5.17 (m, 1H), 5.11 - 4.94 (m, 1H), 4.91 -4.72 (m, 1H), 4.66 - 4.41 (m, 2H), 4.35 - 4.05 (m, 6H), 4.01 - 3.77 (m, 3H), 3.40 - 3.07 (m, 16H), 3.06 - 2.58 (m, 15H), 2.22 - 1.31 (m, 4H), 1.17 - 0.99 (m, 3H).

[0650] Example B5: (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-(7-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carbonyl)-12-hydroxy-4-methyl-3-oxo-2,10-dioxa-4,7-diazadodecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0651]

[0652] The title compound was prepared in a similar manner to Example B4, using Intermediate 9b in step a.

[0653] m / z ES+ [M+H]+1331.4;1H NMR (400 MHz, DMSO-d6) δ 9.43 - 9.06 (m, 1H), 8.29 - 8.02 (m, 1H), 7.96 - 7.75 (m, 1H), 7.66 - 7.34 (m, 4H), 7.32 - 6.90 (m, 5H), 6.36 - 6.11 (m, 2H), 5.92 -5.63 (m, 1H), 5.49 - 5.21 (m, 2H), 5.12 - 4.93 (m, 1H), 4.89 - 4.69 (m, 1H), 4.69 - 4.40 (m, 3H), 4.38 - 3.98 (m, 6H), 3.84 - 3.71 (m, 1H), 3.70 - 3.55 (m, 5H), 3.55 - 3.38 (m, 6H), 3.34 - 3.20 (m, 7H), 3.08 - 2.95 (m, 2H), 2.93 - 2.76 (m, 3H), 2.76 - 2.60 (m, 3H), 2.60 - 2.51 (m, 2H), 1.21 - 0.96 (m, 3H).

[0654] Example B6: (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-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / V-(2-(methylsulfonyl)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acidOH OH

[0655]

[0656] The title compound was prepared in a similar manner to Example B4, using Intermediate 9c in step a.

[0657] m / z ES+ [M+H]+1349.2;1H NMR (400 MHz, DMSO-d6) δ 9.40 - 9.04 (m, 1H), 8.23 - 8.04 (m, 1H), 7.97 - 7.78 (m, 1H), 7.64 - 7.45 (m, 2H), 7.44 - 7.31 (m, 2H), 7.31 - 7.18 (m, 1H), 7.15 - 6.90 (m, 4H), 6.41 - 6.08 (m, 2H), 5.89 - 5.75 (m, 1H), 5.45 - 5.15 (m, 2H), 5.06 - 4.93 (m, 1H), 4.91 - 4.73 (m, 2H), 4.68 - 4.39 (m, 2H), 4.38 - 4.04 (m, 6H), 4.01 - 3.73 (m, 3H), 3.71 - 3.40 (m, 9H), 3.32 - 3.15 (m, 2H), 3.12 - 2.94 (m, 4H), 2.93 - 2.63 (m, 7H), 2.62 - 2.51 (m, 2H), 1.22 - 0.96 (m, 3H).

[0658] Example B7: (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-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-6-oxo- / V-(2-sulfamoylethyl)-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acidOH OH

[0659] NH2

[0660]

[0661] The title compound was prepared in a similar manner to Example B4, using Intermediate 9d in step a.

[0662] m / z ES+ [M+H]+1350.3;1H NMR (300 MHz, DMSO-d6) δ 9.50 - 8.99 (m, 1H), 8.18 - 8.04 (m, 1H), 7.98 - 7.76 (m, 1H), 7.67 - 7.46 (m, 2H), 7.47 - 7.18 (m, 4H), 7.15 - 6.84 (m, 6H), 6.37 - 6.14 (m, 2H), 5.53 - 5.18 (m, 2H), 5.09 -4.96 (m, 1H), 4.92 -4.71 (m, 2H), 4.64-4.40 (m, 2H), 4.37 - 4.05 (m, 8H), 4.05 - 3.43 (m, 9H), 3.34 - 3.17 (m, 3H), 3.13 - 2.96 (m, 2H), 2.94 - 2.62 (m, 6H), 2.59 - 2.49 (m, 2H), 1.19 - 0.96 (m, 3H).

[0663] Example B8: (2S,3S,4S,5 / ?,6S)-6-(2-(2-(3-(4,5-Dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)-A / -methylpropanamido)acetamido)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / V-(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acidOH OH

[0664]

[0665] OH OH

[0666]

[0667] Step a. The following process was conducted in 20 batches.

[0668] To a mixture of Example A2 (200 mg, 0.34 mmol) and triphosgene (CAS: 32315-10-9, 133 mg, 0.45 mmol) in DCM (3 mL) at 0 °C were added TEA (172 mg, 1.7 mmol) dropwise, followed by DMAP (55 mg, 0.45 mmol). The reaction mixture was stirred at rt for 3 h. Separately, a mixture of Intermediate 9a (0.32 mmol, 330 mg) and TEA (120 mg, 1.2 mmol) in DCM (3 mL) was stirred at rt for 30 min, and was then added dropwise to the first reaction mixture. The reaction mixture was stirred at rt for 16 h. Upon completion, the reaction mixtures from the 20 batches were combined, evaporated and the residue was directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give (2S,3 / ?,4S,5S,6S)-2-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / \ / -(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (2 g, crude) as a white solid.

[0669] m / z ES+ [M+H]+1596.5.

[0670] Step b. The following process was conducted in 4 batches.

[0671] To a solution of (2S,3R,4S,5S,6S)-2-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)acetamido)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / \ / -(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (500 mg, 0.31 mmol) in THF (4 mL) and MeOH (4 mL) at 0 °C was added 1 M LiOH (4 mL, 4 mmol) dropwise. The reaction mixture was stirred at rt for 20min. Upon completion, the reaction mixtures from the 4 batches were combined, quenched with FA (8 mL) and directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give (2S,3S,4S,5R,6S)-6-(4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / \ / -(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5- / ]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-(2-(methylamino)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid (820 mg, crude) as a white solid, m / z ES+ [M+H]+1234.4.

[0672] Step c. A solution of (2S,3S,4S,5F?,6S)-6-(4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / \ / -(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5- / ]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-(2-(methylamino)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid (820 mg, 0.66 mmol) and Intermediate 12 (1.2 g, 2.66 mmol) in DMF (21 mL) was stirred at rt for 1 h. Upon completion, the reaction mixture was directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) followed by prep-HPLC to give the title compound (335 mg, 32% yield) as a white solid. m / z ES+ [M+H]+1570.3, 1572.3, 1573.3, 1574.3;1H NMR (300 MHz, DMSO-d6) δ9.50 - 8.98 (m, 1H), 8.18 - 8.03 (m, 1H), 7.95 - 7.78 (m, 1H), 7.60 - 7.21 (m, 5H), 7.17 - 6.88 (m, 1H), 6.36 - 6.12 (m, 2H), 5.11 - 4.94 (m, 2H), 4.91 - 4.70 (m, 4H), 4.66 - 4.45 (m, 6H), 4.28 - 4.03 (m, 7H), 3.99 - 3.79 (m, 3H), 3.70 - 3.46 (m, 9H), 3.45 - 3.37 (m, 4H), 3.08 - 2.98 (m, 3H), 2.94 -2.72 (m, 8H), 2.72 - 2.65 (m, 3H), 2.20 - 1.92 (m, 2H), 1.87 - 1.61 (m, 2H), 1.29 - 1.07 (m, 3H).

[0673] Example B9: (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-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / V-(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acidOH OH

[0674]

[0675] Steps a-b. These two steps were conducted as described in Example B8, steps a-b.

[0676] Step c. A mixture of (2S,3S,4S,5R,6S)-6-(4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / \ / -(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-(2-(methylamino)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid (10 mg, 0.008 mmol), 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1 / 7-pyrrol-1-yl)propanoate (CAS: 55750-62-4; 7 mg, 0.024 mmol), HOBt (1 mg, 0.008 mmol) and DIPEA (4 mg, 0.032 mmol) in DMF (0.3 mL)was stirred at rt for 1 h. Upon completion, the reaction mixture was directly purified by reversephase flash chromatography (H2O (0.01% FA) / MeCN) to give the title compound (4.5 mg, 40% yield) as a white solid.

[0677] m / z ES+ [M+H]+1385.2.1H NMR (300 MHz, DMSO-d6) 59.67 - 9.09 (m, 1H), 8.49 - 7.77 (m, 3H), 7.63 - 6.85 (m, 7H), 6.45 - 6.11 (m, 2H), 5.81 - 5.27 (m, 3H), 5.12 - 4.46 (m, 5H), 4.40 -3.92 (m, 7H), 3.88 - 3.53 (m, 16H), 3.30 - 2.77 (m, 10H), 2.75 - 2.54 (m, 5H), 2.37 - 2.01 (m, 2H), 1.90 - 1.45 (m, 2H), 1.25 - 1.08 (m, 3H).

[0678] Example B10: (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-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / V-methyl-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0679]

[0680] The title compound was prepared in a similar manner to Example B9, using Example A2 and Intermediate 9e in step a.

[0681] m / z ES+ [M+H]+1258.3;1H NMR (300 MHz, DMSO-d6) δ9.51 - 9.02 (m, 1H), 8.28 - 8.00 (m, 1H), 7.96 - 7.76 (m, 1H), 7.59 - 6.90 (m, 8H), 6.45 - 6.13 (m, 2H), 5.96 - 5.18 (m, 3H), 5.07 -4.49 (m, 5H), 4.40 - 3.99 (m, 7H), 3.87 - 3.37 (m, 9H), 3.11 - 2.57 (m, 13H), 1.36 - 0.99 (m, 3H).Example B11: (2S,3S,4S,5 / ?,6S)-6-(2-(2-(3-(4,5-Dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)-A / -methylpropanamido)acetamido)-4-((((2-(4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / V-(2-((1 -methylpiperidin-4-yl)oxy)ethyl)-1 -oxo-1,2-dihydrophthalazine-2-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0682]

[0683] The title compound was prepared in a similar manner to Example B8, using Example A3 in step a.

[0684] m / z ES+ [M+H]+1525.2, 1527.2, 1528.2, 1529.2;1H NMR (400 MHz, DMSO-d6) δ9.63 - 9.02 (m, 2H), 8.41 - 8.21 (m, 1H), 8.19 - 7.77 (m, 3H), 7.66 - 7.41 (m, 2H), 7.36 - 7.23 (m, 1H), 7.19 - 6.83 (m, 2H), 5.98 - 5.60 (m, 1H), 5.54 - 5.19 (m, 2H), 5.13 - 4.71 (m, 2H), 4.57 - 4.01 (m, 6H), 3.97 - 3.84 (m, 2H), 3.82 - 3.47 (m, 13H), 3.42 - 3.11 (m, 11 H), 3.08-2.54 (m, 13H), 2.19 - 1.50 (m, 4H), 1.30 - 0.96 (m, 3H).

[0685] Example B12: (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-(((methyl(2-(4-(3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-A / -(2-(( 1 -methylpiperidin-4-yl)oxy)ethyl)-1 -oxo-1,2-dihydrophthalazine-2-carboxamido)ethyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0686]

[0687] The title compound was prepared in a similar manner to Example B8, using Example A4 in step a.

[0688] m / z ES+ [M+H]+1507.2, 1509.2, 1510.2, 1511.2;1H NMR (300 MHz, DMSO-d6) δ9.45 - 9.05 (m, 1H), 8.50 - 7.79 (m, 5H), 7.73 - 7.27 (m, 5H), 7.21 - 6.85 (m, 2H), 5.98 - 5.24 (m, 2H), 5.12 - 4.71 (m, 3H), 4.63 - 3.84 (m, 10H), 3.82 - 3.40 (m, 14H), 3.33 - 2.59 (m, 20H), 2.49 - 1.58 (m, 4H), 1.33 - 0.96 (m, 3H).

[0689] Example B13: (2S,3S,4S,5R,6S)-6-(4-((((2-(4-(3-((S)-3-(1,1-Difluoroethyl)-6-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)-4-fluorobenzyl)-A / -methyl-1 -oxo-1, 2-dihydrophthalazine-2-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-(2-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -y I )-N-methylpropanamido)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0690]

[0691] The title compound was prepared in a similar manner to Example B9, using Pip-6 (CAS: 2891957-85-8) and Intermediate 9e in step a.

[0692] m / z ES+ [M+H]+1160.3.

[0693] Example B14: (2S,3S,4S,5 / ?,6S)-6-(2-(2-(3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)- / V-methylpropanamido)acetamido)-4-((((2-((8S,9 / ?)-5-fluoro-8-(4-fluorophenyl)- / V-methyl-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-2,7,8,9-tetrahydro-3H-pyrido[4,3,2-ote]phthalazine-2-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acidOH OH

[0694]

[0695] The title compound was prepared in a similar manner to Example B9, using talazoparib (CAS: 1207456-01-6) and Intermediate 9e in step a.

[0696] m / z ES+ [M+H]+1058.3;1H NMR (300 MHz, DMSO-d6) δ9.33 - 9.05 (m, 1H), 8.18 - 7.71 (m, 3H), 7.56 - 7.39 (m, 2H), 7.36 - 6.90 (m, 9H), 5.89 - 5.58 (m, 1H), 5.54 - 5.23 (m, 1H), 5.15 - 4.74 (m, 5H), 4.34 - 4.05 (m, 2H), 3.97 - 3.83 (m, 1H), 3.74 - 3.46 (m, 7H), 3.30 - 3.09 (m, 4H), 3.04 - 2.87 (m, 4H), 2.85 - 2.52 (m, 9H).

[0697] Example B15: (2S,3S,4S,5R,6S)-6-(4-((((2-(4-Benzyl- / V-methyl-1 -oxo-1,2-dihydrophthalazine-2-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)- / V-methylpropanamido)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acidOH OH

[0698]

[0699] The title compound was prepared in a similar manner to Example B1, using 4-benzylphthalazin-1(2H)-one (CAS: 32003-14-8) in step a, Intermediate 7b in step c, and 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1 / 7-pyrrol-1-yl)propanoate (CAS: 55750-62-4) in step e.

[0700] m / z ES+ [M+H]+914.2;1H NMR (400 MHz, DMSO-d6) δ9.58 - 9.12 (m, 1H), 8.33 - 7.80 (m, 5H), 7.41 - 6.92 (m, 9H), 5.91 - 5.59 (m, 1H), 5.30 - 4.55 (m, 3H), 4.52 - 4.05 (m, 4H), 3.82 -3.42 (m, 7H), 3.32 - 3.20 (m, 5H), 3.15 - 2.92 (m, 5H), 2.92 - 2.77 (m, 3H), 2.75 - 2.52 (m, 4H).

[0701] Example B16: (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-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / V-(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0702]

[0703]

[0704] Step a. The following process was conducted in 30 batches.

[0705] To a mixture of Example A2 (200 mg, 0.34 mmol), triphosgene (CAS: 32315-10-9, 133 mg, 0.45 mmol) in DCM (3 mL) at 0 °C were added TEA (172 mg, 1.7 mmol) dropwise, followed by DMAP (55 mg, 0.45 mmol). The reaction mixture was stirred at rt for 3 h, after which a solution of Intermediate 10 (0.32 mmol, 335 mg) in DCM (3 mL) and a solution of TEA (120 mg, 1.2 mmol) in DCM (1 mL) were added dropwise. The reaction mixture was stirred at rt for 16 h. Upon completion, the reaction mixtures from the 30 batches were combined, evaporated and the residue was directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) to give (2S,3F?,4S,5S,6S)-2-(2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / \ / -(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f|phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (3 g, crude) as a white solid.

[0706] m / z ES+ [M+H]+1539.4.

[0707] Step b. The following process was conducted in 6 batches.

[0708] To a solution of (2S,3F?,4S,5S,6S)-2-(2-(((((9 / 7-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / V-(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5- / ]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2 / 7-pyran-3,4,5-triyl triacetate (500 mg, 0.35 mmol) in THF (4.6 mL) and MeOH (4.6 mL) at 0 °C was added 1 M LiOH (4.6 mL, 4.6 mmol). The reaction mixture was stirred at rt for 30 min. Upon completion, the reaction mixtures from the 6 batcheswere combined, quenched with FA (10 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-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / \ / -(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5- / ]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid (900 mg, crude) as a white solid, m / z ES+ [M+H]+1177.3.

[0709] Step c. A mixture of (2S,3S,4S,5F?,6S)-6-(2-(aminomethyl)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / V-(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5- / ]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylicacid (900 mg, 0.77 mmol) and Intermediate 12 (1.38 g, 3.06 mmol) in DMF (25 mL) was stirred at rt for 1 h. Upon completion, the reaction mixture was directly purified by reverse-phase flash chromatography (H2O (0.01% FA) / MeCN) followed by prep-HPLC to give the title compound (370 mg, 32% yield) as a white solid.

[0710] m / z ES+ [M+H]+1513.2, 1515.3, 1516.3, 1517.3;1H NMR (300 MHz, DMSO-cfe) 6 9.35 (s, 1H), 8.44 - 8.21 (m, 1H), 8.00 - 7.78 (m, 1H), 7.60 - 6.78 (m, 7H), 6.41 - 6.14 (m, 2H), 5.75 -5.26 (m, 2H), 5.09 - 4.90 (m, 3H), 4.86 - 4.46 (m, 8H), 4.35 - 4.08 (m, 9H), 3.96 - 3.85 (m, 1 H), 3.78 - 3.56 (m, 3H), 3.57 - 3.51 (m, 3H), 3.49 - 3.36 (m, 4H), 3.29 - 3.08 (m, 3H), 3.02 - 2.79 (m, 4H), 2.79 - 2.66 (m, 3H), 2.60 - 2.52 (m, 2H), 2.22 - 1.93 (m, 2H), 1.86 - 1.60 (m, 2H), 1.34 - 1.01 (m, 3H).

[0711] Example B17: (2S,3S,4S,5 / ?,6S)-6-(2-((3-((8,9-Dibromo-7,10-dioxo-1,2,4,5,7,10-hexahydro-3H-pyridazino[1,2-a][1,2,5]triazepin-3-yl)sulfonyl)propanamido)methyl)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / V-(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acidOH OH

[0712]

[0713] Steps a-b. These 2 steps were conducted as described in Example B16, steps a-b.

[0714] Step c. A solution of Intermediate 13 (20 mg, 0.042 mmol), / V-hydroxysuccinimide (CAS: 6066-82-6, 5 mg, 0.043 mmol) and DCC (9 mg, 0.043 mmol) in THF (0.5 mL) was stirred at rt for 1 h, after which (2S,3S,4S,5 / ?,6S)-6-(2-(aminomethyl)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / \ / -(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid (13 mg, 0.011 mmol) was added. The reaction mixture stirred at rt for2 h. Upon completion, the reaction mixture was directly purified by prep-HPLC to give the title compound (3 mg, 17%) as a white solid.

[0715] m / z ES+ [M+H]+1632.3, 1634.3, 1635.3, 1636.3.

[0716] Example B18: (2S,3S,4S,5 / ?,6S)-6-(2-((3-(2,5-Dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanamido)methyl)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / V-(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0717]

[0718] The title compound was prepared in a similar manner to Example B2, using Intermediate 10 in step a.

[0719] m / z ES+ [M+H]+1314.2;1H NMR (400 MHz, DMSO-cfe) 69.09 - 8.65 (m, 1H), 8.00 - 7.78 (m, 1H), 7.58 - 6.88 (m, 9H), 6.38 - 6.14 (m, 2H), 5.76 - 5.42 (m, 2H), 5.36 - 5.13 (m, 1H), 5.11 -4.76 (m, 3H), 4.69 - 4.47 (m, 2H), 4.47 - 3.99 (m, 10H), 3.82 - 3.59 (m, 8H), 3.36 - 3.07 (m, 8H), 2.96 - 2.82 (m, 3H), 2.73 - 2.55 (m, 4H), 2.03- 1.49 (m, 4H), 1.25 - 1.06 (m, 3H).

[0720] Example B19: (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-((((2-(4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-A / -(2-((1-methylpiperidin-4-yl)oxy)ethyl)-1 -oxo-1, 2-dihydrophthalazine-2-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0721] Br

[0722] Br

[0723]

[0724] The title compound was prepared in a similar manner to Example B16, using Example A3 in step a.

[0725] m / z ES+ [M+H]+1468.2, 1470.2, 1471.2, 1472.2;1H NMR (300 MHz, DMSO-cfe) 68.41 - 8.23 (m, 2H), 8.14 - 7.79 (m, 3H), 7.65 - 7.43 (m, 3H), 7.33 - 6.85 (m, 4H), 5.69 - 5.51 (m, 1H), 5.41 - 5.18 (m, 2H), 5.09 - 4.67 (m, 3H), 4.58 - 4.01 (m, 11 H), 3.97 - 3.41 (m, 13H), 3.30 - 3.15 (m, 9H), 3.03 - 2.58 (m, 9H), 1.32 - 0.90 (m, 5H).

[0726] Example B20: (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-(((methyl(2-(4-(3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-A / -(2-((1-methylpiperidin-4-yl)oxy)ethyl)-1 -oxo-1, 2-dihydrophthalazine-2-carboxamido)ethyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acidBr

[0727] Br

[0728]

[0729] The title compound was prepared in a similar manner to Example B16, using Example A4 in step a.

[0730] m / z ES+ [M+H]+1450.2, 1452.2, 1453.2, 1454.2.

[0731] Preparation of Example C1: Sacituzumab-PARPi ADC (DAR 8.0)

[0732]

[0733] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.Sacituzumab has the following sequence:

[0734] Heavy chain (HC) QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEP TYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 3).

[0735] Light chain (LC) DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRF SGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4).

[0736] Sacituzumab was purchased commercially and reconstituted with PBS pH 7.4 to yield a 6.0 mg / mL solution.

[0737] 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.

[0738] 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.

[0739] 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.

[0740] The title compound was sampled for QC testing, with the below specification:

[0741] Quantity 11 mg

[0742] Average DAR 7.9

[0743] Endotoxin < 1 EU / mL

[0744] Purity (SEC-HPLC) 97.0%

[0745] Concentration 3.0 mg / mL

[0746] Residual Example A1 < 2%

[0747]

[0748] Preparation of Example C2: Sacituzumab-PARPi ADC (DAR 8.0)

[0749] 0XN

[0750]

[0751] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0752] Sacituzumab was purchased commercially and reconstituted with PBS pH 7.4 to yield a 6.0 mg / mL solution.

[0753] 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.

[0754] DMSO was added to formulate the reduced antibody to 10% v / v DMSO, after which 12 molar equiv. of Example B2 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.

[0755] 24 molar equiv. of 10 mM aq. cysteine was added at rt to quench unreacted Example B2. 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.

[0756] The title compound was sampled for QC testing, with the below specification:

[0757] Quantity 17 mg

[0758] Average DAR 7.7

[0759] Endotoxin < 1 EU Z mL

[0760] Purity (SEC-HPLC) 97.4%

[0761] Concentration 2.8 mg / mL

[0762] Residual Example A1 < 2%

[0763]

[0764] Preparation of Example C3: Sacituzumab-PARPi ADC (DAR 8.0)

[0765] OH OH

[0766]

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

[0768] Sacituzumab was purchased commercially and reconstituted with PBS pH 7.4 to yield a 6.0 mg / mL solution.

[0769] 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.

[0770] DMSO was added to formulate the reduced antibody to 10% v / v DMSO, after which 16 molar equiv. of Example B3 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.

[0771] 32 molar equiv. of 10 mM aq. cysteine was added at rt to quench unreacted Example B3. 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.

[0772] The title compound was sampled for QC testing, with the below specification:

[0773] Quantity 13 mg

[0774] Average DAR 7.9

[0775] Endotoxin < 1 EU Z mL

[0776] Purity (SEC-HPLC) 97.2%

[0777] Concentration 3.0 mg / mL

[0778] Residual Example A1 < 2%

[0779]

[0780] Preparation of Example C4: Sacituzumab-PARPi ADC (DAR 8.0)

[0781]

[0782] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0783] Sacituzumab was purchased commercially and reconstituted with PBS pH 7.4 to yield a 6.0 mg / mL solution.

[0784] 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.

[0785] DMSO was added to formulate the reduced antibody to 10% v / v DMSO, after which 12 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.

[0786] 24 molar equiv. of 10 mM aq. cysteine was added at rt to quench unreacted Example B4. 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.

[0787] The title compound was sampled for QC testing, with the below specification:

[0788] Quantity 8 mg

[0789] Average DAR 7.9

[0790] Endotoxin < 1 EU Z mL

[0791] Purity (SEC-HPLC) 96.9%

[0792] Concentration 3.0 mg / mL

[0793] Residual Example A1 < 2%

[0794]

[0795] Preparation of Example C5: Sacituzumab-PARPi ADC (DAR 8.0)

[0796]

[0797] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0798] Example C5 was prepared in a similar manner to Example C4, using Example B5 as the payload-linker, and was sampled for QC testing, with the below specification:

[0799] Quantity 7 mg

[0800] Average DAR 8.0

[0801] Endotoxin < 1 EU / mL

[0802] Purity (SEC-HPLC) 98.4%

[0803] Concentration 2.9 mg / mL

[0804] Residual Example A1 < 2%

[0805]

[0806] Preparation of Example C6: Sacituzumab-PARPi ADC (DAR 8.0)

[0807]

[0808] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0809] Example C6 was prepared in a similar manner to Example C4, using Example B6 as the payload-linker, and was sampled for QC testing, with the below specification:

[0810] Quantity 7 mg

[0811] Average DAR 8.0

[0812] Endotoxin < 1 EU / mL

[0813] Purity (SEC-HPLC) 98.1%

[0814] Concentration 3.0 mg / mL

[0815] Residual Example A1 < 2%

[0816]

[0817] Preparation of Example C7: Sacituzumab-PARPi ADC (DAR 8.0)

[0818]

[0819] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0820] Example C7 was prepared in a similar manner to Example C4, using Example B7 as the payload-linker, and was sampled for QC testing, with the below specification:

[0821] Quantity 4 mg

[0822] Average DAR 7.9

[0823] Endotoxin < 1 EU / mL

[0824] Purity (SEC-HPLC) 97.8

[0825] Concentration 3.0 mg / mL

[0826] Residual Example A1 < 2%

[0827]

[0828] Preparation of Example C8: Sacituzumab-PARPi ADC (DAR 4.0)OH OH

[0829]

[0830] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0831] 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).

[0832] 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.

[0833] DMSO was added to formulate the reduced antibody to 10% v / v DMSO, after which 5.8 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.

[0834] 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.

[0835] The title compound was sampled for QC testing, with the below specification:

[0836] Quantity 90 mg

[0837] Average DAR 4.0

[0838] Endotoxin < 1 EU Z mL

[0839] Purity (SEC-HPLC) 98.9%

[0840] Concentration 3.0 mg / mL

[0841] Residual Example A2 < 2%

[0842]

[0843] Preparation of Reference Example C9: lgG1-kappa-PARPi ADC (DAR 4.0)

[0844]

[0845] wherein Ab represents non-targeting antibody, lgG1 -kappa and wherein “S” represents a sulfur atom within a cysteine residue of said lgG1 -kappa.

[0846] Reference Example C9 was prepared in a similar manner to Example C8, using lgG1 -kappa as the antibody, 4.9 molar equiv. of Example B8, and was sampled for QC testing, with the below specification:

[0847] Quantity 7 mg

[0848] Average DAR 4.0

[0849] Endotoxin < 1 EU / mL

[0850] Purity (SEC-HPLC) 98.2%

[0851] Concentration 2.9 mg / mL

[0852] Residual Example A2 < 2%

[0853]

[0854] Preparation of Example C10: Sacituzumab-PARPi ADC (DAR 8.0)

[0855]

[0856] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0857] Sacituzumab was produced according to the protocol described in Example C8.

[0858] 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.

[0859] DMSO was added to formulate the reduced antibody to 10% v / v DMSO, after which 12 molar equiv. of Example B9 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.

[0860] 24 molar equiv. of 10 mM aq. cysteine was added at rt to quench unreacted Example B9. 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 2.9 mg / mL by membrane centrifugation.

[0861] The title compound was sampled for QC testing, with the below specification:

[0862] Quantity 12 mg

[0863] Average DAR 7.8

[0864] Endotoxin < 1 EU / mL

[0865] Purity (SEC-HPLC) 98.8%

[0866] Concentration 2.9 mg / mL

[0867] Residual Example A2 < 2%

[0868]

[0869] Preparation of Example C11: Sacituzumab-PARPi ADC (DAR 4.0)

[0870]

[0871] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0872] Sacituzumab was produced according to the protocol described in Example C8.

[0873] 2.5 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.

[0874] DMSO was added to formulate the reduced antibody to 10% v / v DMSO, after which 6 molar equiv. of Example B9 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.

[0875] 12 molar equiv. of 10 mM aq. cysteine was added at rt to quench unreacted Example B9. 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 2.9 mg / mL by membrane centrifugation.

[0876] The title compound was sampled for QC testing, with the below specification:

[0877] Quantity 11 mg

[0878] Average DAR 3.9

[0879] Endotoxin < 1 EU / mL

[0880] Purity (SEC-HPLC) 98.6%

[0881] Concentration 2.9 mg / mL

[0882] Residual Example A2 < 2%

[0883]

[0884] Preparation of Example C12: Sacituzumab-PARPi ADC (DAR 8.0)

[0885]

[0886] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0887] Example C12 was prepared in a similar manner to Example C10, using 15 molar equiv. of Example B10, and was sampled for QC testing, with the below specification:

[0888] Quantity 6.6 mg

[0889] Average DAR 7.8

[0890] Endotoxin < 1 EU / mL

[0891] Purity (SEC-HPLC) 98.9%

[0892] Concentration 3.0 mg / mL

[0893] Residual Example A2 < 2%

[0894]

[0895] Preparation of Example C13: Sacituzumab-PARPi ADC (DAR 4.0)

[0896]

[0897] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0898] Example C13 was prepared in a similar manner to Example C8, using 8 molar equiv. of Example B11, and was sampled for QC testing, with the below specification:

[0899] Quantity 15 mg

[0900] Average DAR 3.9

[0901] Endotoxin < 1 EU / mL

[0902] Purity (SEC-HPLC) 98.0%

[0903] Concentration 3.0 mg / mL

[0904] Residual Example A3 < 2%

[0905]

[0906] Preparation of Example C14: Sacituzumab-PARPi ADC (DAR 4.0)

[0907]

[0908] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0909] Example C14 was prepared in a similar manner to Example C8, using 6.2 molar equiv. of Example B12, and was sampled for QC testing, with the below specification:

[0910] Quantity 15 mg

[0911] Average DAR 3.9

[0912] Endotoxin < 1 EU / mL

[0913] Purity (SEC-HPLC) 98.5%

[0914] Concentration 3.0 mg / mL

[0915] Residual Example A4 < 2%

[0916]

[0917] Preparation of Example C15: Sacituzumab-PARPi ADC (DAR 8.0)

[0918]

[0919] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0920] Example C15 was prepared in a similar manner to Example C10, using 16 molar equiv. of Example B13, and was sampled for QC testing, with the below specification:

[0921] Quantity 6.4 mg

[0922] Average DAR 7.8

[0923] Endotoxin < 1 EU / mL

[0924] Purity (SEC-HPLC) 99.0%

[0925] Concentration 3.0 mg / mL

[0926] Residual Pip-6 < 2%

[0927]

[0928] Preparation of Example C16: Sacituzumab-PARPi ADC (DAR 8.0)

[0929]

[0930] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0931] Example C16 was prepared in a similar manner to Example C10, using 14 molar equiv. of Example B14, and was sampled for QC testing, with the below specification:

[0932] Quantity 16 mg

[0933] Average DAR 7.9

[0934] Endotoxin < 1 EU / mL

[0935] Purity (SEC-HPLC) 98.6%

[0936] Concentration 2.8 mg / mL

[0937] Residual Talazoparib < 2%

[0938]

[0939] Preparation of Example C17: Sacituzumab-PARPi ADC (DAR 4.0)

[0940]

[0941] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0942] Sacituzumab was produced according to the protocol described in Example C8.

[0943] 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.

[0944] DMSO was added to formulate the reduced antibody to 10% v / v DMSO, after which 5.8 molar equiv. of Example B16 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.

[0945] 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.

[0946] The title compound was sampled for QC testing, with the below specification:

[0947] Quantity 78 mg

[0948] Average DAR 4.0

[0949] Endotoxin < 1 EU / mL

[0950] Purity (SEC-HPLC) 98.7%

[0951] Concentration 3.8 mg / mL

[0952] Residual Example A2 < 2%

[0953]

[0954] Preparation of Reference Example C18: lgG1-kappa-PARPi ADC (DAR 4.0)

[0955]

[0956] wherein Ab represents non-targeting antibody, lgG1 -kappa and wherein “S” represents a sulfur atom within a cysteine residue of said lgG1 -kappa.

[0957] Reference Example C18 was prepared in a similar manner to Example C17, using lgG1- kappa as the antibody, 5.2 molar equiv. of Example B16, and was sampled for QC testing, with the below specification:

[0958] Quantity 10 mg

[0959] Average DAR 4.0

[0960] Endotoxin < 1 EU / mL

[0961] Purity (SEC-HPLC) 98.6%

[0962] Concentration 2.9 mg / mL

[0963] Residual Example A2 < 2%

[0964]

[0965] Preparation of Example C19: Sacituzumab-PARPi ADC (DAR 4.0)

[0966]

[0967] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0968] Sacituzumab was purchased commercially and reconstituted with PBS pH 7.4 to yield a 6.0 mg / mL solution.

[0969] 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.

[0970] DMSO was added to formulate the reduced antibody to 10% v / v DMSO, after which 5.8 molar equiv. of Example B17 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.

[0971] 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 2.9 mg / mL by membrane centrifugation.

[0972] The title compound was sampled for QC testing, with the below specification:

[0973] Quantity 12 mg

[0974] Average DAR 4.0

[0975] Endotoxin < 1 EU Z mL

[0976] Purity (SEC-HPLC) 98.3%

[0977] Concentration 2.9 mg / mL

[0978] Residual Example A2 < 2%

[0979]

[0980] Preparation of Example C20: Sacituzumab-PARPi ADC (DAR 8.0)

[0981]

[0982] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0983] Example C20 was prepared in a similar manner to Example C10, using 13 molar equiv. of Example B18 as the payload-linker, and was sampled for QC testing, with the below specification:

[0984] Quantity 11 mg

[0985] Average DAR 7.9

[0986] Endotoxin < 1 EU / mL

[0987] Purity (SEC-HPLC) 98.9%

[0988] Concentration 2.9 mg / mL

[0989] Residual Example A2 < 2%

[0990]

[0991] Preparation of Example C21: Sacituzumab-PARPi ADC (DAR 4.0)

[0992]

[0993] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[0994] Example C8 was prepared in a similar manner to Example C11, using 6.5 molar equiv. of Example B18 as the payload-linker, and was sampled for QC testing, with the below specification:

[0995] Quantity 11 mg

[0996] Average DAR 3.9

[0997] Endotoxin < 1 EU / mL

[0998] Purity (SEC-HPLC) 98.8%

[0999] Concentration 2.9 mg / mL

[1000] Residual Example A2 < 2%

[1001]

[1002] Preparation of Example C22: Sacituzumab-PARPi ADC (DAR 4.0)

[1003]

[1004] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[1005] Example C22 was prepared in a similar manner to Example C17, using 6.7 molar equiv. of Example B19, and was sampled for QC testing, with the below specification:

[1006] Quantity 16 mg

[1007] Average DAR 3.9

[1008] Endotoxin < 1 EU / mL

[1009] Purity (SEC-HPLC) 98.5%

[1010] Concentration 2.8 mg / mL

[1011] Residual Example A3 < 2%

[1012]

[1013] Preparation of Example C23: Sacituzumab-PARPi ADC (DAR 4.0)

[1014]

[1015] wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

[1016] Example C23 was prepared in a similar manner to Example C17, using 5.3 molar equiv. of Example B20, and was sampled for QC testing, with the below specification:

[1017] Quantity 16 mg

[1018] Average DAR 4.0

[1019] Endotoxin < 1 EU / mL

[1020] Purity (SEC-HPLC) 98.4%

[1021] Concentration 2.5 mg / mL

[1022] Residual Example A4 < 2%

[1023]

[1024] BIOLOGICAL EXAMPLES

[1025] 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.

[1026] Biological Example 1: PARP1 and PARP2 FRET binding assays

[1027] FRET probe

[1028] 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.

[1029]

[1030] PARP

[1031] 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.

[1032] FRET labelling of PARP proteins

[1033] 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 buffer (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.

[1034] PARP1 dumbbell DNA preparation

[1035] 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.

[1036]

[1037] PARP FRETAll 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 was freshly prepared just prior to use.

[1038] 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).

[1039] 2x reaction mixtures of Tb labeled PARP1 (2.4 nM), PARP2 (2.4 nM), PARP1 + DNA (2.4 nM PARP1, 40 nM DNA) or PAPP2 + 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 Fisher 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.

[1040] Data analysis

[1041] 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.

[1042] 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.

[1043] Table 1: FRET probe Kps

[1044] Assay Probe KD(nM)

[1045] PARP1 2.6

[1046] PARP1 + DNA 1.5

[1047]

[1048] PARP2 3.9

[1049] PARP2 + DNA 8.5

[1050]

[1051] Results

[1052] The results of the PARP1 and PARP2 FRET binding assays are shown below in Table 2.

[1053] Table 2: Results of Biological Example 1

[1054] Example PARP1 KDPARP1 + PARP2 KD PARP2 +

[1055] (nM) DNA KD(nM) (nM) DNA KD (nM) Example A1 0.16 0.009 0.022 0.023

[1056] Example A2 0.12 0.003 0.032 0.022

[1057] Example A3 1.22 0.020 0.013 0.026

[1058] Example A4 - 0.003 - 0.040

[1059] Olaparib 0.47 14 0.096 5.0

[1060] Talazoparib 0.60 0.030 15 9.9

[1061] Saruparib 0.29 0.027 520 3,300

[1062] Pip-6 0.79 0.021 0.019 0.030

[1063]

[1064] 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. Certain compounds of the invention are more potent binders of PARP1 in the presence and absence 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.

[1065] Biological Example 2: PARP1 and PARP2 DNA strand exchange assays

[1066] 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.

[1067] Table 3: Oligos used in DNA strand exchange assays

[1068] PARP1 5'- probe GCTGAGC / X / TCTGGTGAAGCTCAGCTCGCGGCAGCTGGTGCTGCCGC GA-3' (SEQ ID NO: 2)

[1069]

[1070] PARP 5’- unlabelled GCTGAGCTTCTGGTGAAGCTCAGCTCGCGGCAGCTGGTGCTGCCGCG DNA A-3’ (SEQ ID NO: 1)

[1071] PARP2 5'- Probe pGCTGAGC / X / TCTGGTGAAGCTCAGCTCGCGGCAGCTGGTGCTGCCG CGA-3' (SEQ ID NO: 2)

[1072] PARP2 5’- unlabelled pGCTGAGCTTCTGGTGAAGCTCAGCTCGCGGCAGCTGGTGCTGCCGC DNA GA-3’ (SEQ ID NO: 1)

[1073]

[1074] wherein X represents a [18F]-3-fluoro-l-a-methyl-tyrosine (FAMT) label.

[1075] 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 KCI).

[1076] PARP1 strand exchange assay

[1077] 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).

[1078] 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).

[1079] PARP2 strand exchange assay

[1080] 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 (SPTLabtech), 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).

[1081] 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.

[1082] Data analysis

[1083] 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 residence time was calculated as the reciprocal of the dissociation rate. Residence time is reported relative to the DMSO control.

[1084] Results

[1085] 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.

[1086] Table 4: Results of Biological Example 2

[1087] Example PARP1 FC PARP2 FC

[1088] Example A1 40 1.3

[1089] Example A2 30 1.8

[1090] Example A3 40 0.9

[1091] Olaparib 0.9 1.1

[1092] Talazoparib 8.5 1.6

[1093] Saruparib <0.9 0.9

[1094] Pip-6 27 1.5

[1095]

[1096] 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 the 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 selected compounds of the invention is greater than that for Pip-6. Surprisingly, the compounds of the invention do not induce a similar prolongation of residence time of PARP2 on DNA, which suggests that the pro-retention behaviour of the compounds is specific to PARP1.

[1097] Biological Example 3: Immunofluorescence assay to measure chromatin retention of PARP1 and PARP2 and inhibition of PARylation

[1098] 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 and 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).

[1099] 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 forPoly / 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 (AUG), were generated with GeneData Software. The units of AUG are expressed as % activity x concentration in pM (%A*pM).

[1100] Results

[1101] The results of the immunofluorescence chromatin retention and PARylation assay are shown in Figure 2 and are summarised below in Table 5.

[1102] Table 5: Results of Biological Example 3

[1103] Example PARP1 PARP2 PARP1 PARP2 PARP1 PARylati Chromatin Chromatin Chromatin Chromatin Selectivity on EC50 retention retention retention retention (AUC) (nM) EC50(nM) EC50(nM) AUC AUC

[1104] (%A*pM) (%A*pM)

[1105] Example A1 1.1 3.8 260 210 1.3 0.56 Example A2 0.54 1.1 320 280 1.1 0.54 Example A3 0.55 2.5 328 234 1.4 0.51 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

[1106]

[1107] 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.

[1108] Biological Example 4: Cell viability assays with compounds of the invention in a BRCA2 DLD-1 isogenic model

[1109] 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.

[1110] 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 finalconcentration 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.

[1111] 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.

[1112] Results

[1113] The results of Biological Example 4 are shown below in Table 6.

[1114] Table 6: Results of Biological Example 4

[1115] Example DLD-1 WT EC50 (nM) DLD-1 BRCA2-Z- EC50 (nM)

[1116] Example A1 0.78 0.11

[1117] Example A2 0.32 0.053

[1118] Example A3 0.59 0.077

[1119] Example A4 1.83 0.17

[1120] Olaparib > 1,000 46

[1121] Talazoparib 27 0.64

[1122] Saruparib > 1,000 1.00

[1123] Pip-6 1.16 0.15

[1124]

[1125] 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, the 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.

[1126] Biological Example 5: Cell viability assays with compounds of the invention in a range of cancer cell linesCells 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.

[1127] Results

[1128] The results of Biological Example 5 are summarised below in Table 7.

[1129] Table 7: Results of Biological Example 5

[1130] Viability EC50(nM)

[1131] HR

[1132] Cell line Tissue Example Example status Saruparib Talazoparib Pip-6

[1133] A1 A2 MCF7 Breast HRP > 1000 13 0.41 0.39 0.20 NCI-N87 Stomach HRP > 1000 286 2.10 1.3 0.63 PEO1 BRCA2

[1134] Ovary 0.86 0.77 0.21 0.27 0.079 clone 10 null

[1135] BRCA1

[1136] SUM149PT Breast 6.31 1.58 0.16 0.16 0.063

[1137] A11q

[1138]

[1139] The results of Biological Example 5 show that the tested compounds of the invention are highly cytotoxic to a 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.

[1140] Biological Example 6: In vivo efficacy of ADCs of the invention in a DLD-1 BRCA2- / - mouse xenograft model

[1141] 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 8 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 8.TGI was calculated for each group using the formula: TGI (%) = [1 -(Ti-TO) / (Vi-VO)] *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 VO 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.

[1142] Results

[1143] The results of Biological Example 6 are shown in Figure 3 and are summarised below in Table 8. The data in Table 8 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.

[1144] Table 8: Dosing regimen and results for Biological Example 6

[1145] Study Group n Treatment Dose %TGI (day)

[1146] 1 6 Vehicle - - 1

[1147] 2 6 Example C1 3 mg / kg 39% (d25)

[1148] 3 6 Example C1 10 mg / kg 51% (d25)

[1149] 1 6 Vehicle - - 2 2 6 Example C4 10 mg / kg 72% (d21)

[1150] 3 6 Example 05 10 mg / kg 65% (d21)

[1151] 1 6 Vehicle - - 3

[1152] 2 6 Example 06 10 mg / kg 69% (d25)

[1153] 1 6 Vehicle - - 4 2 6 Example 08 3 mg / kg 46% (d21)

[1154] 3 6 Example 08 10 mg / kg 83% (d21)

[1155] 1 6 Vehicle - - 5 2 6 Example 017 3 mg / kg 42% (d17)

[1156] 3 6 Example 017 10 mg / kg 88% (d17)

[1157]

[1158] The results of Biological Example 6 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.

[1159] Biological Example 7: In vitro metabolic stability assessment of compounds of the invention

[1160] In vitro metabolic stability assessments were carried out in human microsomes and hepatocytes under industry standard conditions as detailed below.

[1161] Microsomal stability assay

[1162] 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:

[1163] 1. Empty ‘Incubation’ plates T60 and NCF60 were pre-warmed at 37°C for 10 min.

[1164] 2. Microsome working solution (445 pL) was transferred into pre-warmed ‘Incubation’ plates T60 and NCF60, followed by a 10 min incubation at 37°C.

[1165] 3. Microsome working solution (54 pL) was transferred to a Blank60 plate, followed by the addition of 6 pL NADPH cofactor and 180 pL of stop solution into each well.

[1166] 4. Compound working solution (5 pL) was added to the ‘incubation’ plates (T60 and NCF60) containing microsomes.

[1167] 5. For the ‘Incubation’ plate NCF60, 50 pL of PB buffer was added, the plate was incubated at 37°C for 60 min.

[1168] 6. Stop solution (180 pL) and NADPH working solution (6 pL) were added to the TO plate.

[1169] Then, a mixture (54 pL) was removed from the ‘Incubation’ plate T60 and transferred to the TO plate.

[1170] 7. For the ‘Incubation’ plate T60, NADPH working solution (44 pL) was added, followed by a 60 min incubation at 37°C.

[1171] 8. At 5, 15, 30, 45, and 60 min, 60 pL of each sample at each time point was transferred to a well containing 180 pL of stop solution, followed by mixing.

[1172] 9. All sampling plates were shaken for 10 min, then centrifuged at 3220 xg for 20 min at 4°C.

[1173] 10. The supernatant (80 pL) was transferred into 240 pL of pure water and mixed using a plate shaker for 10 min.

[1174] 11. Each bioanalysis plate was sealed and shaken for 10 min prior to LC-MS / MS analysis.

[1175] The reagents used for the microsomal stability assay were prepared as detailed in Table 9 below.Table 9: Reagents for the microsomal stability assay

[1176] Reagent Stock cone. Final cone. Test compounds 10 mM 1 pM

[1177] Positive controls 10 mM 1 pM

[1178] Human liver microsomes - 0.5 mg / mL NADPH - 1 mM

[1179]

[1180] 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 MgCh solution. The stop solution consisted of cold (4°C) MeCN containing 250 nM tolbutamide and 250 nM labetalol as internal standards (IS).

[1181] Data analysis

[1182] The percentage of remaining test compound after incubation was calculated using the following equation:

[1183] Peak area ratio of analyte to internal standard at each time point

[1184] %Remaining= — — - -:— - - - -:- - - - — - -::— x 100

[1185] Peak area ratio of analyte to internal standard at zero time point

[1186] The equation of first-order kinetics was used to calculate T1 / 2:

[1187] Ct= C0-e-k-‘

[1188] 1 T xs ■'s $ <

[1189]

[1190] C Lint (microsomes) IS CdICUldtOd 8S follOWS.

[1191] CLjnt(mic) = 0.693 / Ti / 2 / mg microsome protein per mL

[1192] Hepatocyte stability assay

[1193] Stability of compounds was assessed in human hepatocytes (BiolVT) using the following procedure:

[1194] 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.

[1195] 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.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.

[1196] 4. 198 pL of pre-warmed cell suspension was added in 96-well plates.

[1197] 5. 2 pL working solution was added to each well of 96-well plates.

[1198] 6. TO Sample was mixed for about 1 min to achieve a homogenous suspension. Then 25 pL of each sample was transferred into wells containing 125 pL of ice-cold stop solution followed by mixing.

[1199] 7. Incubation plates were incubated at 37°C in a 95% humidified incubator at 5% CO2 with constant shaking.

[1200] 8. At 15, 30, 60 and 90 min, samples were mixed, then 25 pL of each sample at each time point was transferred to wells containing 125 pL of ice-cold stop solution followed by further mixing.

[1201] 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.

[1202] 10. The plates were shaken immediately on a plate shaker, then all sample plates were centrifuged at 3220 x g for 20 min.

[1203] 11. After centrifugation, 80 pL / well of supernatant in the sample plate was transferred to another set of pre-labelled 96-well plates containing 240 pL of ultra-pure water.

[1204] 12. Analytical plates were sealed and stored at 4°C prior to LC-MS / MS analysis.

[1205] The reagents used for the hepatocyte stability assay were prepared as detailed in Table 10 below.

[1206] Table 10: Reagents for the hepatocyte stability assay

[1207] Reagent Final cone.

[1208] Test compounds 1 pM

[1209] Positive controls 3 pM

[1210] Human hepatocytes 0.5 x106cells / mL

[1211] MeCN 0.90 %

[1212] DMSO 0.10 %

[1213]

[1214] 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).

[1215] Data analysis

[1216] The percentage of remaining test compound after incubation was calculated using the following equation:

[1217] Peak area ratio of analyte to internal standard at each time point

[1218] %Remaining= — — - -:— - - - -:- - - - — - -::— x 100

[1219] Peak area ratio of analyte to internal standard at zero time point

[1220] The equation of first-order kinetics was used to calculate T1 / 2:

[1221] Ct= C0-e-k-‘

[1222] 1c_... L&2 0.693

[1223] tk58■■■ ~ -

[1224]

[1225] ' 2 ' "* K* Ks

[1226] C Ljnt (hepatocytes) IS Calculated aS follOWS.

[1227] CLint(hep) = 0.693 / Ti / 2 / million cells per mL

[1228] Results

[1229] The results of Biological Example 7 are shown below in Table 11.

[1230] Table 11: Results of Biological Example 7

[1231] Example CLintiviicrosomes CLinthepatocytes (pL / min / 10**

[1232] (pL / min / mg) cells)

[1233] Example A1 800 49

[1234] Example A2 118 13

[1235] Example A3 687 61

[1236] Talazoparib < 9.6 < 6.4

[1237] Saruparib < 9.6 < 6.4

[1238] Pip-6 16 < 6.4

[1239]

[1240] The results of Biological Example 7 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 payload related toxicity.

[1241] Biological Example 8: In vitro permeability assessment of compounds of the invention In vitro permeability assessments were carried out in an MDCK assay under industry standard conditions.MDCK assay

[1242] 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.

[1243] 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 pM bi-directionally in duplicate, while Nadolol and Metoprolol were tested at 2.00 pM 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.

[1244] Data analysis

[1245] The apparent permeability coefficient Papp(cm / s) was calculated using the equation:

[1246] Papp=(dCr / dt) X Vr / (A X Co)

[1247] 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.

[1248] The efflux ratio was calculated using the equation:

[1249] Efflux Ratio = Papp(B-A) I Papp(A-B)

[1250] Results

[1251] The results of Biological Example 8 are shown below in Table 12.

[1252] Table 12: Results of Biological Example 8Example Papp A-B (106Papp B-A (106Efflux Ratio

[1253] cm / s) cm / s)

[1254] Example A1 16 9.5 0.6

[1255] Example A2 8.3 15 1.8

[1256] Example A3 8.2 7.7 0.9

[1257] Pip-6 3.8 23 6.2

[1258]

[1259] The results of Biological Example 8 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. Interestingly, the tested compounds of the invention display higher passive permeability and reduced efflux compared to Pip-6.

[1260] Biological Example 9: Cell viability assays with ADCs of the invention

[1261] Cell viability assays were performed as described in Biological Example 4 using serial dilutions of the indicated ADCs. Expression levels of TROP2 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) antibody, or rabbit Alexa Fluor 647-conjugated IgG (1:100, Novus Biologicals, NBP2-24891AF647) isotype control 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.

[1262] Results

[1263] The results of the cell viability assays are shown below in Table 13.

[1264] Table 13: Results of Biological Example 9

[1265] DLD-1 PEO1

[1266] MCF7 SUM149PT BRCA2 KO clone 10

[1267] TROP2 levels Medium Low Very high Medium (% positive cells) (33.4%) (7.8%) (99.6%) (42.5%)

[1268] BRCA1 HR status HRP BRCA2 null BRCA2 null

[1269] A11q o Example C1 >10,000 3980 20 1250 < Q Q s

[1270] U g1-7

[1271] I o E Example C8 - 3980 25 3980

[1272]

[1273] Dato-Dxd

[1274] (Datopotamab 6750 6140 4.9 1280 deruxtecan)

[1275]

[1276] The results of Biological Example 9 show that the tested ADCs of the invention are cytotoxic to a variety of cancer cells of different lineage in vitro. Additionally, the results confirm that the magnitude of the effect of the ADCs is dependent on both the expression levels of the target antigen and the HR status of the cancer cell.

[1277] Biological Example 10: In vivo efficacy of ADCs of the invention in an NCI-N87 mouse xenograft model

[1278] 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 6. Body weight was measured twice per week and the study was ended on the day indicated in Table 14.

[1279] Results

[1280] The results of Biological Example 10 are shown in Figure 4 and are summarised below in Table 14. The data in Table 14 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.

[1281] Table 14: Dosing regimen and results for Biological Example 10

[1282] Study Group n Treatment Dose % TGI (day)

[1283] 1 6 Vehicle - - 1 2 6 Example C2 10 mg / kg 61% (d36)

[1284] 3 6 Example C3 10 mg / kg 63% (d36)

[1285] 1 6 Vehicle - - 2

[1286] 2 6 Example C6 10 mg / kg 63% (d25)

[1287] 3 1 6 Vehicle - -

[1288]

[1289] 2 6 Example C8 6 mg / kg 100% (d35)

[1290] 3 6 Example C8 10 mg / kg 45% regression (d35) 1 6 Vehicle - - 4 2 6 Example C17 6 mg / kg 98% (d35)

[1291] 3 6 Example C17 10 mg / kg 41% regression (d35)

[1292]

[1293] The results of Biological Example 10 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.

[1294] Biological Example 11: B-Glucuronidase release assay

[1295] The selective release of payload upon exposure to p-glucuronidase activity was assessed by LC-MS / MS analysis of samples of linker-payloads incubated in the presence and absence of recombinant p-glucuronidase. The general procedure for this assay is as follows:

[1296] 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.

[1297] 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.

[1298] 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.

[1299] 4. Preparation of p-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 p-glucuronidase was dissolved in 3.98 mL of the 100 mM NaOAc solution to give a concentration of 1 mg / mL.

[1300] 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 of1 mg / mL p-glucuronidase solution were added to low adsorption tubes to achieve a final compound concentration of 5 pM, and p-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.

[1301] 6. Procedure for sample analysis: The plate was placed into a well plate autosampler.

[1302] The samples were evaluated by LC-MS / MS analysis and metabolites were detected using HRMS.

[1303] Final working stock concentrations, volumes and ratios are shown in Table 15 below.

[1304] Table 15: Reagent concentrations and volumes for the P-glucuronidase release assay Reagent Stock Cone. Final cone.

[1305] Test compound 5 mM 5 pM

[1306] / V-Acetylcysteine 10 mM 10 pM

[1307] P-glucuronidase - 0.5 mg / mL

[1308] - -

[1309]

[1310] PBS

[1311] Data analysis

[1312] 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:

[1313] Peak area ratio of analyte to internal standard at each time point

[1314] %= - - - - - x 100

[1315] Peak area ratio of analyte to internal standard at zero time point

[1316] Results

[1317] The results of the p-glucuronidase release assay are shown in Figure 5 and are summarised below in Table 16.

[1318] Table 16: Results of Biological Example 11Linker-Pay load Payload Example Condition* Remaining (%) Released (%)

[1319] 0 h 1 h 3 h 5 h 7 h 0 h 1 h 3 h 5 h 7 h + Glue 100 0.0 0.0 0.0 0.0 0.0 31 76 100 100 B2

[1320] - Glue 100 100 83 91 89 0.0 0.0 0.0 0.0 0.0 + Glue 100 0.5 0.5 0.8 0.5 0.1 38 82 100 100 B4

[1321] - Glue 100 100 100 100 98 0.1 0.1 0.1 0.1 0.1 + Glue 100 0.2 0.1 0.1 0.0 0.1 31 72 95 99 B5

[1322] - Glue 100 99 97 100 100 0.0 0.0 0.0 0.0 0.0 + Glue 100 0.0 0.8 0.0 0.3 0.0 42 84 100 99 B6

[1323] - Glue 100 89 100 100 100 0.0 0.0 0.0 0.0 0.0 + Glue 100 0.5 0.5 0.4 0.4 0.7 55 87 98 98 B7

[1324] - Glue 100 100 100 100 100 0.0 0.0 0.2 0.0 0.0 + Glue 100 1.6 0.2 0.1 0.2 0.2 21 49 59 60 B8

[1325] - Glue 100 95 92 91 94 0.1 0.1 0.1 0.1 0.1 + Glue 100 0.2 0.1 0.0 0.2 0.4 65 81 79 84 B13

[1326] - Glue 100 87 95 94 80 0.1 0.1 0.1 0.1 0.1 + Glue 100 0.1 0.1 0.2 0.2 0.5 70 77 75 73 B14

[1327] - Glue 100 99 97 91 100 0.2 0.4 0.3 0.4 0.3 + Glue 100 1.1 0.9 0.8 1.0 4.9 74 100 100 100 B15

[1328] - Glue 100 98 85 93 93 0.0 0.0 0.0 0.0 0.1 + Glue 100 0.0 0.1 0.1 0.1 0.1 6.8 31 42 51 B16

[1329] - Glue 100 96 97 96 99 0.1 0.1 0.1 0.0 0.0 + Glue 100 0.4 0.2 0.2 0.4 0.1 10 41 57 65 B18

[1330]

[1331] - Glue 100 99 99 95 100 0.0 0.0 0.0 0.0 0.0 indicates whether the incubation was conducted in the presence (+) or absence (-) of recombinant p-glucuronidase enzyme.

[1332] The results of Biological Example 11 confirm that the tested compounds of the invention selectively undergo decomposition to release payload in the presence of p-glucuronidase enzyme. The data also show that the tested compounds of the invention are stable in the absence of p-glucuronidase enzyme and that no payload is released, suggesting that the linker-payloads are resistant to hydrolysis under these conditions.

[1333] Biological Example 12: In v / o efficacy of ADCs of the invention in a SUM149PT mouse xenograft model

[1334] 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 thevolume 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 6. Body weight was measured twice per week and the study was ended on the day indicated in table 17.

[1335] Results

[1336] The results of Biological Example 12 are shown in Figure 6 and are summarised below in Table 17.

[1337] Table 17:

[1338]

[1339] and results for Bi

[1340]

[1341] 12

[1342] Group n Treatment Dose % TGI (day)

[1343] 1 6 Vehicle - -

[1344]

[1345] 2 6 Example C17 10 mg / kg 15% regression (d28)

[1346] The results of Biological Example 12 show that the tested ADC of the invention is efficacious in a mouse xenograft model of breast cancer following a single dose. The bodyweight data confirm that the ADC is well-tolerated at the administered dose for the duration of the study.

[1347] Biological Example 13: Native mass spec analysis of ADCs of the invention

[1348] 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:

[1349] Column: AdvanceBio SEC 200A, 2.1 x 50 mm, 1.9pm Mobile phase: 100 mM NH4Ac in H2O

[1350] Run time: 5 min

[1351] Ion source: DualAJS ESI

[1352] Scan patterns: Positive

[1353] Mass deconvolution range: 2000-7000 Da

[1354] Theoretical mass analysis was performed using Agilent MassHunter Sequence Manager 10.0.

[1355] The DAR was calculated using the following equation:ZPeak heiqht x n

[1356] ^ Total peak height

[1357]

[1358] where n is the number of payloads attached to the mAb.

[1359] Results

[1360] The results of Biological Example 13 are shown in Figure 7 and are summarised below in Table 18.

[1361] Table 18: Results of Biological Example 13

[1362] Theoretical Observed

[1363] Example DAR Peak Height % Height Mass (Da)* Mass (Da)

[1364] Sacituzumab N / A 148131 148132 2.33E+04 100

[1365] 2 150954 150958 2.27E+02 0.89 3 152366 152372 2.89E+03 11.30 Example C8

[1366] 4 153778 153781 2.13E+04 83.15 5 155350 155352 1.19E+03 4.66 3 149572 149573 4.39E+02 2.38 Reference

[1367] 4 150984 150984 1.69E+04 91.59 Example C9

[1368] 5 152556 152556 1.11E+03 6.03 4 153671 153653 1.20E+02 1.52 6 156442 156446 1.57E+02 1.99 Example

[1369] 7 157827 157842 6.24E+02 7.89 C10

[1370] 8 159212 159222 6.86E+03 86.73 9 160597 160608 1.48E+02 1.88 0 148130 148135 1.42E+03 6.98 1 149515 149521 2.82E+02 1.39 2 150901 150905 5.18E+03 25.52 3 152286 152289 3.74E+02 1.84 Example

[1371] 4 153671 153679 7.19E+03 35.44 C11

[1372] 5 155056 155061 3.56E+02 1.76 6 156442 156452 3.90E+03 19.21 7 157827 157834 1.73E+02 0.85 8 159212 159223 1.29E+03 6.38

[1373]

[1374] 9 160597 160607 1.28E+02 0.63

[1375] 3 152196 152198 1.06E+03 3.77 Example

[1376] 4 153551 153553 2.59E+04 91.86 C17

[1377] 5 154906 155067 1.23E+03 4.37

[1378]

[1379] *The calculated mass of Sacituzumab corresponds 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.

[1380] Biological Example 14: Cell viability assays with compounds of the invention in RPE-1 WT, PARP1 KO and PARP1 / PARP2 dual KO lines

[1381] 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.

[1382] Results

[1383] The results of Biological Example 14 are shown in Figure 8 and are summarised below in Table 19.

[1384] Table 19: Results of Biological Example 14

[1385] Viability EC50(nM)

[1386] Cell Line

[1387] Saruparib Talazoparib Example A2 hTERT RPE-1 WT > 1000 910 0.82 hTERT RPE-1

[1388] > 1000 500 1.7 PARP1 KO clone G7

[1389] hTERT RPE-1

[1390] PARP1_PARP2 KO > 1000 > 1000 > 10 clone E6

[1391]

[1392] The results of Biological Example 14 indicate that the cytotoxicity of the tested compound of the invention is mediated by both PARP1 and PARP2. Loss of PARP1 induces partial resistance to the tested compound of the invention, loss of both PARP1 and PARP2 together completely ablates cytotoxicity.

Claims

AMENDED CLAIMSreceived by the International Bureau on 17 July 2026CLAIMS1. Use of a bivalent linker which comprises a compound of formula (I):O O / X ^(b)(a)' N N O Z'R1R2<IMG file=null he=null id=imgf000180_0001 img-content=null img-format=null inline=null orientation=null wi=null>(I)wherein:represents the attachment position to the phthalazinone containing payload;'' represents the attachment position to the tumour cell binding moiety;X represents C2-4 alkylene;R1represents hydrogen, C1-6 alkyl, -(CH2)m-O-R3, or -(CH2)m-R4;m represents an integer selected from 2 to 4;R3represents C1-6 alkanol or heterocyclyl wherein said heterocyclyl group may be optionally substituted by one or more C1-6 alkyl groups;R4represents -SO2NRxRyor -SO2-C1-6 alkyl;Rxand Ryindependently represent hydrogen or C1-6 alkyl;R2represents hydrogen or C1-6 alkyl;Y represents a self-immolative trigger moiety; andZ represents a spacer moiety,to release a phthalazinone containing payload from a tumour cell binding moiety.

2. The use as defined in claim 1, wherein X represents:(a) C2-3 alkylene; or(b) ethylene.

3. The use as defined in claim 1 or claim 2, wherein R1represents:(a) Ci-6alkyl, -(CH2)m-O-R3or -(CH2)m-R4; or(b) C1-6 alkyl; or(c) methyl; or(d) -(CH2)m-O-R3; or(e) -(CH2)m-R4; or(f) methyl, -(CH2)2-O-(CH2)2-OH, -(CH2)2-SO2NH2, -(CH2)2-SO2-Me, or -(CH2)2-O-piperidinyl substituted by a methyl group (such as an N-methyl group).

4. The use as defined in any one of claims 1 to 3, wherein m represents:(a) an integer selected from 2 or 3; or(b) an integer which is 2.

5. The use as defined in any one of claims 1 to 4, wherein R3represents:(a) -(CH2)2-OH or a 6 membered heterocyclyl ring optionally substituted by one or more C1-6 alkyl groups; or(b) -(CH2)2-OH or a 6 membered nitrogen containing heterocyclyl ring optionally substituted by one or more C1-6 alkyl groups; or(c) -(CH2)2-OH or piperidinyl optionally substituted by one or more C1-6 alkyl groups; or(d) -(CH2)2-OH or piperidinyl optionally substituted by one or more methyl groups.

6. The use as defined in any one of claims 1 to 5, wherein R4represents -SO2NH2or -SO2-Me.

7. The use as defined in any one of claims 1 to 6, wherein:(a) Rxand Ryindependently represent hydrogen or methyl; or(b) Rxand Ryboth represent hydrogen.

8. The use as defined in any one of claims 1 to 7, wherein R2represents:(a) hydrogen or methyl; or(b) methyl.

9. The use as defined in claim 1, which is a compound of formula (l)a:R1O(a),, Jk. / Y.xS(b)N O Z'<IMG file=null he=null id=imgf000181_0001 img-content=null img-format=null inline=null orientation=null wi=null>O I(l)awherein R1, (a), (b), Y and Z are as defined in claim 1.

10. The use as defined in any one of claims 1 to 9, wherein Y comprises:(a) a glucuronide containing moiety; or(b) a glucuronide containing moiety which has the following structure of formula (Y)a:<IMG file=null he=null id=imgf000182_0001 img-content=null img-format=null inline=null orientation=null wi=null>OH OH(Y)awherein “(O)” represents the O atom of the bivalent linker of formula (I) and “(Z)” represents the Z variable of the bivalent linker of formula (I).

11. The use as defined in any one of claims 1 to 10, wherein Z comprises:(a) one or more polyethylene glycol (PEG) components; or(b) at least 2 polyethylene glycol (PEG) components; and / or(c) a haloacetamide, maleimide, dibromopyridazinedione or dibromopyridazinotriazepinyl moiety; and / or(d) a compound of any one of formula (Z)a, (Z)b, (Z)c, (Z)d, (Z)e, (Z)f, or (Z)g:(Z)b;<IMG file=null he=null id=imgf000182_0002 img-content=null img-format=null inline=null orientation=null wi=null>(Z)f; or<IMG file=null he=null id=imgf000183_0001 img-content=null img-format=null inline=null orientation=null wi=null>wherein “(Y)” represents the Y variable of the bivalent linker of formula (I).

12. A process for preparing a bivalent linker of formula (I) as defined in any one of claims 1 to 11, which comprises reacting a compound of formula (IV):O O<IMG file=null he=null id=imgf000183_0002 img-content=null img-format=null inline=null orientation=null wi=null>(IV)wherein (a), R1, X, R2and Y are as defined in claim 1, with a compound of formula Z — (b), wherein Z and (b) are as defined in claim 1.

13. A drug conjugate comprising a compound of formula (II):o ol_ —I p<IMG file=null he=null id=imgf000184_0001 img-content=null img-format=null inline=null orientation=null wi=null>(II)wherein:P1represents a phthalazinone containing payload;X represents C2-4 alkylene;R1represents hydrogen, C1-6 alkyl, -(CH2)m-O-R3, or -(CH2)m-R4;m represents an integer selected from 2 to 4;R3represents C1-6 alkanol or heterocyclyl wherein said heterocyclyl group may be optionally substituted by one or more C1-6 alkyl groups;R4represents -SO2NRxRyor -SO2-C1-6 alkyl;Rxand Ryindependently represent hydrogen or C1-6 alkyl;R2represents hydrogen or C1-6 alkyl;Y represents a self-immolative trigger moiety;Z represents a spacer moiety;T1represents a tumour cell binding moiety; andp represents an integer selected from 1 to 10.

14. The drug conjugate as defined in claim 13, wherein X represents:(a) C2-3 alkylene; or(b) ethylene.

15. The drug conjugate as defined in claim 13 or claim 14, wherein R1represents:(a) Ci-6alkyl, -(CH2)m-O-R3or -(CH2)m-R4; or(b) C1-6 alkyl; or(c) methyl; or(d) -(CH2)m-O-R3; or(e) -(CH2)m-R4; or(f) methyl, -(CH2)2-O-(CH2)2-OH, -(CH2)2-SO2NH2, -(CH2)2-SO2-Me, or -(CH2)2-O-piperidinyl substituted by a methyl group (such as an N-methyl group).

16. The drug conjugate as defined in any one of claims 13 to 15, wherein m represents: (a) an integer selected from 2 or 3; or(b) an integer which is 2.

17. The drug conjugate as defined in any one of claims 13 to 16, wherein R3represents:(a) -(CH2)2-OH or piperidinyl optionally substituted by one or more C1-6 alkyl groups; or(b) -(CH2)2-OH or piperidinyl optionally substituted by one or more methyl groups.

18. The drug conjugate as defined in any one of claims 13 to 17, wherein R4represents -SO2NH2or -SO2-Me.

19. The drug conjugate as defined in any one of claims 13 to 18, wherein:(a) Rxand Ryindependently represent hydrogen or methyl; or(b) Rxand Ryboth represent hydrogen.

20. The drug conjugate as defined in any one of claims 13 to 19, wherein R2represents:(a) hydrogen or methyl; or(b) methyl.

21. The drug conjugate as defined in any one of claims 13 to 20, wherein Y comprises:(a) a glucuronide containing moiety; or(b) a glucuronide containing moiety which has the following structure of formula (Y)a:OX. OHOH OH OH<IMG file=null he=null id=imgf000185_0001 img-content=null img-format=null inline=null orientation=null wi=null>wherein “(O)” represents the O atom of the bivalent linker of formula (I) and “(Z)” represents the Z variable of the bivalent linker of formula (I).

22. The drug conjugate as defined in any one of claims 13 to 21, wherein Z comprises:(a) one or more polyethylene glycol (PEG) components; or(b) at least 2 polyethylene glycol (PEG) components; and / or (c) a haloacetamide, maleimide, dibromopyridazinedione or dibromopyridazinotriazepinyl moiety; and / or(d) a compound of any one of formula (Z)a, (Z)b, (Z)c, (Z)d, (Z)e, (Z)f, or (Z)9:<IMG file=null he=null id=imgf000186_0001 img-content=null img-format=null inline=null orientation=null wi=null>(Z)f; or<IMG file=null he=null id=imgf000187_0001 img-content=null img-format=null inline=null orientation=null wi=null>(Z)g;wherein “(Y)” represents the Y variable of the bivalent linker of formula (I).

23. The drug conjugate as defined in any one of claims 13 to 22, wherein p represents:(a) an integer selected from 4 to 8;(b) an integer which is 4; or(c) an integer which is 8.

24. The drug conjugate as defined in any one of claims 13 to 23, wherein the phthalazinone containing payload:(i) is linked to the carboxamide group via a nitrogen atom;(ii) comprises the following moiety:0<IMG file=null he=null id=imgf000187_0002 img-content=null img-format=null inline=null orientation=null wi=null>wherein (d) represents the attachment point to the remaining portion of the payload; and (e) represents the attachment point to the carbonyl moiety of the bivalent linker of formula (I).

25. The drug conjugate as defined in any one of claims 13 to 24, wherein the phthalazinone containing payload comprises:(a) a PARP inhibitor, such as olaparib, fluzoparib, simmiparib, thioparib, Pip-6, or talazoparib, in particular olaparib, talazoparib, or Pip-6; or(b) any one of compounds A1 to A4.

26. The drug conjugate as defined in any one of claims 13 to 25, wherein said tumour cell binding moiety is selected from a peptide, an antibody or an antigen binding fragment.

27. The drug conjugate as defined in claim 26, 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.

28. The drug conjugate as defined in claim 26 or claim 27, wherein said antibody or antigen binding fragment binds to a target on a tumour cell selected from: from ErbB2 / HER2 and Trop- 2, such as Trop-2.

29. The drug conjugate as defined in any one of claims 26 to 28, wherein said tumour cell binding moiety is an antibody.

30. The drug conjugate as defined in claim 29, wherein said antibody is a monoclonal antibody.

31. The drug conjugate as defined in claim 30, wherein said monoclonal antibody is selected from: brentuximab, enfortumab, gemtuzumab, inotuzumab, loncastuximab, mirvetuximab, moxetumomab, polatuzumab, sacituzumab, LALA-sacituzumab, tisotumab and trastuzumab.

32. The drug conjugate as defined in claim 30 or claim 31, wherein said monoclonal antibody is sacituzumab.

33. The drug conjugate as defined in claim 13, which is a compound of any one of Examples C1 to C8, C10 to C17 or C19 to C23:Example C1: Sacituzumab-PARPi ADC (DAR 8.0)<IMG file=null he=null id=imgf000189_0001 img-content=null img-format=null inline=null orientation=null wi=null>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)<IMG file=null he=null id=imgf000189_0002 img-content=null img-format=null inline=null orientation=null wi=null>wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C3: Sacituzumab-PARPi ADC (DAR 8.0)OH OH<IMG file=null he=null id=imgf000190_0001 img-content=null img-format=null inline=null orientation=null wi=null>wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C4: Sacituzumab-PARPi ADC (DAR 8.0)<IMG file=null he=null id=imgf000190_0002 img-content=null img-format=null inline=null orientation=null wi=null>wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C5: Sacituzumab-PARPi ADC (DAR 8.0)<IMG file=null he=null id=imgf000191_0001 img-content=null img-format=null inline=null orientation=null wi=null>wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C6: Sacituzumab-PARPi ADC (DAR 8.0)<IMG file=null he=null id=imgf000191_0002 img-content=null img-format=null inline=null orientation=null wi=null>wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C7: Sacituzumab-PARPi ADC (DAR 8.0)<IMG file=null he=null id=imgf000192_0001 img-content=null img-format=null inline=null orientation=null wi=null>wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C8: Sacituzumab-PARPi ADC (DAR 4.0)<IMG file=null he=null id=imgf000192_0002 img-content=null img-format=null inline=null orientation=null wi=null>wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C10: Sacituzumab-PARPi ADC (DAR 8.0)<IMG file=null he=null id=imgf000193_0001 img-content=null img-format=null inline=null orientation=null wi=null>wherein 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)<IMG file=null he=null id=imgf000193_0002 img-content=null img-format=null inline=null orientation=null wi=null>wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C12: Sacituzumab-PARPi ADC (DAR 8.0)<IMG file=null he=null id=imgf000194_0001 img-content=null img-format=null inline=null orientation=null wi=null>wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C13: Sacituzumab-PARPi ADC (DAR 4.0)<IMG file=null he=null id=imgf000194_0002 img-content=null img-format=null inline=null orientation=null wi=null>wherein 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)0NN0<IMG file=null he=null id=imgf000195_0001 img-content=null img-format=null inline=null orientation=null wi=null>wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C15: Sacituzumab-PARPi ADC (DAR 8.0)<IMG file=null he=null id=imgf000195_0002 img-content=null img-format=null inline=null orientation=null wi=null>wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C16: Sacituzumab-PARPi ADC (DAR 8.0)<IMG file=null he=null id=imgf000196_0001 img-content=null img-format=null inline=null orientation=null wi=null>wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C17: Sacituzumab-PARPi ADC (DAR 4.0)<IMG file=null he=null id=imgf000196_0002 img-content=null img-format=null inline=null orientation=null wi=null>wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C19: Sacituzumab-PARPi ADC (DAR 4.0)<IMG file=null he=null id=imgf000197_0001 img-content=null img-format=null inline=null orientation=null wi=null>wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C20: Sacituzumab-PARPi ADC (DAR 8.0)<IMG file=null he=null id=imgf000197_0002 img-content=null img-format=null inline=null orientation=null wi=null>wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C21: Sacituzumab-PARPi ADC (DAR 4.0)<IMG file=null he=null id=imgf000198_0001 img-content=null img-format=null inline=null orientation=null wi=null>wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab;Example C22: Sacituzumab-PARPi ADC (DAR 4.0)<IMG file=null he=null id=imgf000198_0002 img-content=null img-format=null inline=null orientation=null wi=null>wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab; orExample C23: Sacituzumab-PARPi ADC (DAR 4.0)OH OHoo<IMG file=null he=null id=imgf000199_0001 img-content=null img-format=null inline=null orientation=null wi=null>wherein Ab represents Sacituzumab and wherein “S” represents a sulfur atom within a cysteine residue of said Sacituzumab.

34. A process for preparing the drug conjugate of a compound of formula (II) as defined in claim 13, which comprises reacting one or more molar equivalents of a compound of formula (III):O OP1N N O ZI IR1R2<IMG file=null he=null id=imgf000199_0002 img-content=null img-format=null inline=null orientation=null wi=null>(III)wherein P1, R1, X, R2, Y and Z are as defined in claim 13, with a tumour cell binding moiety.

35. A drug conjugate prepared by a process as defined in claim 34.

36. An intermediate selected from a compound of formula (III), (III)a, (III)b, (III)c, (III)d, (III)e, (III)f, (III)g, or any one of Examples B1 to B20:O O<IMG file=null he=null id=imgf000199_0003 img-content=null img-format=null inline=null orientation=null wi=null>(III)OH OH(III)b; OH OH„N,, X R2<IMG file=null he=null id=imgf000200_0001 img-content=null img-format=null inline=null orientation=null wi=null>Br Br (III)d;OH OH<IMG file=null he=null id=imgf000201_0001 img-content=null img-format=null inline=null orientation=null wi=null>P1(III)f; or<IMG file=null he=null id=imgf000202_0001 img-content=null img-format=null inline=null orientation=null wi=null>(lll)g;wherein P1, R1, X, Y, Z, and R2are as defined in claim 13;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-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / V-methyl-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5- / ]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)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-1 H-pyrrol-1 -yl)- / V-methylpropanamido)acetamido)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)- 5.6.7.8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / V-methyl-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f|phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / - / -pyran-2-carboxylic acid;Example B3: (2S,3S,4S,5 / ?,6S)-6-(2-((S)-17-Amino-15-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-methyl-4, 14,17-trioxo-7, 10-dioxa-3, 13-diazaheptadecanamido)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-A / -methyl-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5- / ]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid;Example B4: (2S,3S,4S,5R,6S)-6-(2-(2-((2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)(methyl)amino)acetamido)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)- 5.6.7.8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / V-(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5- / ]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid;Example B5: (2S,3S,4S,5 / ?,6S)-6-(2-(2-(3-(2,5-Dioxo-2,5-dihydro-1 / - / -pyrrol- 1 -yl)- / V-methylpropanamido)acetamido)-4-(7-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5, 6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f|phthalazine-7-carbonyl)-12-hydroxy-4-methyl-3-oxo-2,10-dioxa-4,7-diazadodecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid;Example B6: (2S,3S,4S,5R,6S)-6-(2-(2-(3-(2,5-Dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-M-methylpropanamido)acetamido)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)- 5.6.7.8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / V-(2-(methylsulfonyl)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5- / ]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid;Example B7: (2S,3S,4S,5R,6S)-6-(2-(2-(3-(2,5-Dioxo-2,5-dihydro-1 / - / -pyrrol- 1 -yl)- / V-methylpropanamido)acetamido)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)- 5.6.7.8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-6-oxo- / V-(2-sulfamoylethyl)-6,7-dihydro-[1,3]dioxolo[4,5-f|phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid;Example B8: (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-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)-A / -(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5- / ]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / - / -pyran-2-carboxylic acid;Example B9: (2S,3S,4S,5 / ?,6S)-6-(2-(2-(3-(2,5-Dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)- / V-methylpropanamido)acetamido)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)- 5.6.7.8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / V-(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5- / ]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid;Example B10: (2S,3S,4S,5R,6S)-6-(2-(2-(3-(2,5-Dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-M-methylpropanamido)acetamido)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)- 5.6.7.8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)-A / -methyl-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f|phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid;Example B11: (2S,3S,4S,5R,6S)-6-(2-(2-(3-(4,5-Dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2A / )-yl)- / V-methylpropanamido)acetamido)-4-((((2-(4-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / V-(2-((1-methylpiperidin-4-yl)oxy)ethyl)-1 -oxo-1, 2-dihydrophthalazine-2-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid;Example B12: (2S,3S,4S,5R,6S)-6-(2-(2-(3-(4,5-Dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)- / V-methylpropanamido)acetamido)-4-(((methyl(2-(4-(3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-A / -(2-((1-methylpiperidin-4-yl)oxy)ethyl)-1-oxo-1,2-dihydrophthalazine-2-carboxamido)ethyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid;Example B13: (2S,3S,4S,5R,6S)-6-(4-((((2-(4-(3-((S)-3-(1,1-Difluoroethyl)-6-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)-4-fluorobenzyl)- / V-methyl-1-oxo-1,2-dihydrophthalazine-2-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)- / V-methylpropanamido)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2 / - / -pyran-2-carboxylic acid;Example B14: (2S,3S,4S,5R,6S)-6-(2-(2-(3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)- / V-methylpropanamido)acetamido)-4-((((2-((8S,9 / ?)-5-fluoro-8-(4-fluorophenyl)- / V-methyl-9-(1-methyl-1 / - / -1,2,4-triazol-5-yl)-3-oxo-2,7,8,9-tetrahydro-3 / - / -pyrido[4,3,2-cte]phthalazine-2-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid;Example B15: (2S,3S,4S,5 / ?,6S)-6-(4-((((2-(4-Benzyl- / V-methyl-1-oxo-1,2-dihydrophthalazine-2-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)- / V-methylpropanamido)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2 / - / -pyran-2-carboxylic acid;Example B16: (2S,3S,4S,5 / ?,6S)-6-(2-((3-(4,5-Dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)pi'opanamido)methyl)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / V-(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5- / ]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid;Example B17: (2S,3S,4S,5 / ?,6S)-6-(2-((3-((8,9-Dibromo-7, 10-dioxo-1,2, 4, 5, 7, 10-hexahydro-3H-pyridazino[1,2-a][1,2,5]triazepin-3-yl)sulfonyl)propanamido)methyl)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / V-(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f|phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / - / -pyran-2-carboxylic acid;Example B18: (2S,3S,4S,5R,6S)-6-(2-((3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)methyl)-4-((((2-(9-(4-fluoro-3-((S)-6-methyl-3-(perfluoroethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzyl)- / V-(2-((1-methylpiperidin-4-yl)oxy)ethyl)-6-oxo-6,7-dihydro-[1,3]dioxolo[4,5-f]phthalazine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / - / -pyran-2-carboxylic acid;Example B19: (2S,3S,4S,5R,6S)-6-(2-((3-(4,5-Dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)propanamido)methyl)-4-((((2-(4-(4-fluoro-3-((S)-6-methyl-3- (perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)-A / -(2-((1-methylpiperidin-4-yl)oxy)ethyl)-1-oxo-1,2-dihydrophthalazine-2-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2 / 7-pyran-2-carboxylic acid; orExample B20: (2S,3S,4S,5R,6S)-6-(2-((3-(4,5-Dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)pi'opanamido)methyl)-4-(((methyl(2-(4-(3-((S)-6-methyl-3- (perfluoroethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-7-carbonyl)benzyl)- / V-(2-((1-methylpiperidin-4-yl)oxy)ethyl)-1-oxo-1,2-dihydrophthalazine-2-carboxamido)ethyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid.

37. A pharmaceutical composition comprising the drug conjugate as defined in any one of claims 13 to 33 or 35 in combination with one or more pharmaceutically acceptable excipients.

38. A pharmaceutical composition comprising the drug conjugate as defined in any one of claims 13 to 33 or 35, in combination with one or more therapeutic agents.

39. The drug conjugate as defined in any one of claims 13 to 33 or 35 or the pharmaceutical composition as defined in claim 37 or claim 38, for use in therapy.

40. The drug conjugate as defined in any one of claims 13 to 33 or 35 or the pharmaceutical composition as defined in claim 37 or claim 38, for use in the prophylaxis or treatment of cancer.

41. A method of treating cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of a drug conjugate as defined in any one of claims 13 to 33 or 35 or the pharmaceutical composition as defined in claim 37 or claim 38.

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