Linkers and protein and peptide conjugates incorporating the same

Abstract

The present invention relates to a linker compound having a fused nitrogen-containing heteroaromatic ring and a vinyl substituent for conjugating to a protein or peptide. The present invention further relates to a conjugate comprising the linker and an active agent and to a conjugate comprising the linker, a protein or peptide, and an active agent, such as a drug or labelling moiety. More specifically, the conjugate may be an antibody conjugate, such as an antibody drug conjugate (ADC).

Description

[0001] LINKERS AND CONJUGATES INCORPORATING THE SAME

[0002] Field of the Invention

[0003] The present invention relates to linkers and protein and peptide conjugates incorporating the same. More especially, the present invention relates to a conjugate comprising a protein or peptide, linker and an active agent, for example a drug or labelling moiety. Additionally, the present invention provides an improved linker for use in conjugates and methods of introducing said linker into said conjugates. More specifically, the conjugate may be an antibody conjugate, such as an antibody drug conjugate (ADC). Compositions comprising the ADCs and methods of using the same, including for the treatment of cancer, are also provided.

[0004] Background of the Invention

[0005] Protein drug conjugates, in particular antibody drug conjugates (ADCs), are known to provide targeted delivery of highly potent drugs to specific tissue for treatment. More specifically, ADCs, which typically consist of an antibody linked via a chemical linker with labile bonds, to a biologically active cytotoxic drug (often referred to as the “drug moiety” or “payload”), are known for use in anticancer treatments. The targeted delivery offered by such protein drug conjugates results from the ability of the antibody or the like to sensitively discriminate between healthy and diseased tissue, thus ensuring safe delivery of the highly potent drug. Accordingly, ADCs have significant potential to improve the treatment and survival of patients suffering from diseases such as cancer.

[0006] Linkers used in ADCs should be stable when circulating in human plasma to prevent early release of drugs while also allowing for release of the active drug payload upon internalisation into the tumour cell to induce cell death. Current linkers for ADC production still have numerous shortcomings and greatly influence an ADC stability, drug-antibody ratio (DAR) and drug distribution. For example, the linkage formed via commonly utilized maleimide conjugation to cysteine residues is unstable in circulation leading to premature dissociation of the antibody payload. Such heterogeneous and / or unstable ADCs are associated with unreliable pharmacokinetic profiles and toxic side effects. The plasma stability of maleimide-based linkers has been increased by hydrolysis of the succinimide thioether ring through linker modifications or antibody engineering (Lyon 2014). However, an inherently stable linker is preferential.

[0007] Vinylpyridine-based linkers have been shown to react selectively with thiol groups on an antibody to form highly stable thioether bonds. However, these linkers are less reactive towards thiols than maleimide linkers.

[0008] Accordingly, there remains a need to provide novel linker formats for protein drug conjugates, such as ADCs, that possess high stability and reactivity in order to improve the drug efficacy and safety profiles.

[0009] Summary of Invention

[0010] The present invention relates to a novel conjugating linker structure which has a fused nitrogen-containing heteroaromatic ring and a vinyl substituent that is capable of specifically binding to a thiol group of a protein or peptide. In particular, the present invention provides a linker for use in conjugates, with utility in linking proteins or peptides and active agents, for example antibodies and cytotoxins to provide antibodydrug conjugates (ADCs). The linker combines improved reactivity and stability as compared to currently known linker molecules for use in such conjugates. Linker stability may improve the tolerability where the active agent exerts a biological activity, while improved reactivity is desirable for more efficient and effective conjugation.

[0011] In a first aspect, there is provided a linker-active agent conjugate comprising a linker and an active agent, wherein the linker is represented by Formula (la) or (lb): wherein:

[0012] Ar is selected from the group consisting of phenyl, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrazole or imidazole; at least one of Ai and A2 is N and the other is selected from N or CR, with the proviso that when the linker is represented by Formula (lb) A1 is N and A2 is CR; n is an integer from 1 to 4; at least one R group is a connecting unit covalently linked to the active agent; and at each other occurrence R is independently selected from a hydrogen atom or an electron-withdrawing or electron-donating substituent.

[0013] A feature of the present invention lies in that the reactivity of the linker may be tuned depending on the desired use by appropriately selecting the type and position of the substituent on the fused heterocyclic aromatic ring system that provides the core of the linker structure. In particular, it is possible to control the electron density around the nitrogen atoms in close proximity to vinyl group, which may in turn affect the reaction rate of the vinyl group with a thiol. This makes it possible to impart a desired rate of reactivity to the linker. For example, the inclusion of an electron-donating group adjacent to a nitrogen atom in the same ring structure as the vinyl group may increase the basicity of the nitrogen, which leads to greater protonation of the nitrogen under physiological conditions and results in the vinyl group of the linker structure having increased reactivity towards thiol conjugation. In contrast, the presence of an electronwithdrawing group in the same position may negatively affect the reactivity of the linker. Accordingly, in some embodiments, the R substituent adjacent to a nitrogen atom in the same aromatic ring as the vinyl group is not an electron-withdrawing group.

[0014] Any electron-withdrawing substituents and electron-donating substituents may be used to impart the desired rate of reactivity, and these may be used alone or in combination. Non-limiting examples of electron-withdrawing substituents include an acyl group, an alkoxycarbonyl group, a carbamoyl group, an acyloxy group, an acylamino group, an alkoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylsulfanyl group, an alkylsulfonyl group, an arylsulfonyl group, a nitro group, a trifluoromethyl group and a cyano group. Nonlimiting examples of electron-donating substituents are an alkyl group having 1 to 10 carbon atoms, and preferable examples thereof include a methyl group, an ethyl group, a propyl group, an isopropyl group and a t-butyl group.

[0015] In some embodiments, R is independently selected at each other occurrence from a hydrogen atom or a substituent selected from an optionally substituted C1-C10 alkyl group, -(CH2)mF,

[0016] -(CH2)mCI, -(CH2)mBr, or -(CH2)ml, -(CH2)mCN, -(CH2)mNO2, -(CH2)mCF3, -(CH2)mN3, -(CH2)mORa, -(CH2)mSRa, -(CH2)mOC(O)-Ra, -(CH2)mN(Ra)2, -(CH2)mC(O)Ra, -(CH2)mC(O)ORa, -(CH2)mC(O)SRa, -(CH2)mSC(O)Ra, -(CH2)mOC(O)ORa, -(CH2)mOC(O)N(Ra)2, -(CH2)mC(O)N(Ra)2, -(CH2)mN(Ra)C(O)ORa, -(CH2)mN(Ra)C(O)Ra, -(CH2)mN(Ra)C(O)N(Ra)2, -(CH2)mN(Ra)C(NRa)N(Ra)2, -(CH2)mN(Ra)S(O)tRa, -S(O)tRa, -S(O)tORa, -S(O)tN(Ra)2, -S(O)tN(Ra)C(O)Ra, and -B(ORa)2; wherein: m is an integer from 0 to 10; t is 1 or 2; and

[0017] Rais independently selected at each occurrence from hydrogen, an optionally substituted C1-C10 alkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, and an optionally substituted C1-C10 heteroalkyl.

[0018] In some embodiments, the connecting unit covalently linked to the active agent has the Formula:

[0019] - W- Y - A wherein:

[0020] W is a linking group;

[0021] Y is an optional stretcher unit comprising one or more of a cleavable group, a hydrophilic spacer group, and a self-immolative group; and

[0022] A is the active agent.

[0023] A linking group is any group providing covalent attachment between an aromatic ring of the linker and Y or A. Suitable linking groups for utilisation in the present invention will be readily apparent to those skilled in the art. For example, the linking group may comprise or consist of an amide coupling.

[0024] In some embodiments, W may be selected from: wherein R’ and R” are independently selected from H and C1-C10 alkyl, q is at each independent occurrence an integer between 0 and 10, and r is an integer from 1 to 10.

[0025] A cleavable group is a group that may be cleaved to release an active agent during use e.g., cleavage via hydrolysis, reduction, or enzymatic reaction. Suitable cleavable groups are well known in the art and include, but are not limited to, acid-labile groups, hydrolysis-labile groups, enzymatically cleavable groups, reduction labile groups. In some embodiments, the cleavable group may be a protease-cleavable peptide moiety, a glycosidase-cleavable sugar moiety, a pH sensitive moiety, ora hydrolysable moiety. Suitable cleavable groups also include, but are not limited to, peptides, glucuronides, hydrazones, mal-caproyl units, and dipeptide units. The dipeptide units may be cathepsin cleavable or legumain cleavage. For example, the cleavable group may be a dipeptide unit selected from -Valine-Citrulline- (-Val-Cit-), -Valine- Alanine-(-Val-Ala- ), -Valine-Lysine-(-Val-Lys-), -Valine-Arginine-(-Val-Arg-), -Phenylalanine Acid- Citrulline-(-Phe-Cit-), -Phenylalanine-Lysine-(-Phe-Lys-), -Phenylalanine-Arginine-(- Phe-Arg-), - Asparagine- Asparagine (-Asn-Asn-), -Asparagine-Phenylalanine Acid- (- Asn-Phe-), -Asparagine-Histidine- (-Asn-His-), -Asparagine-Alanine- (-Asn-Ala-), - Asparagine-Valine- (-Asn-Val-), -Asparagine-Lysine- (-Asn-Lys-), -Asparagine- Citrulline- (-Asn-Cit-), or -Asparagine-Tryptophan- (-Asn-Trp-).

[0026] Alternatively, the cleavable may be a p-D-glucuronide linker. p-Glucuronide linkers are a family of enzymatically cleavable linkers that mediate payload drug release via the linker hydrolysis by the lysosomal enzyme p-glucuronidase (GLISB) within tumor cells. In some embodiments, O-glucosyl, O-galactosyl or O-mannosyl linkers may be similarly used.

[0027] In some embodiments, the cleavable group has a structure selected from:

[0028] P-D-Glucuronide p-D-Glucoside P-D-Galactoside p-D-Mannosoide wherein:

[0029] * indicates covalent attachment site to the active agent; and

[0030] L indicates attachment to the linker compound, either directly or indirectly.

[0031] The presence of a hydrophilic spacer group in the stretcher unit may provide several benefits, such as i) higher water solubility of the final conjugate, ii) higher resistance towards aggregation in aqueous solutions, and iii) the ability to link a higher number of active agent molecules per molecule of linker. Suitable hydrophilic spacer groups are well known in the art and include, but are not limited to, a poly(ethylene glycol) (PEG) units such as PEG diamine (H2N-PEG-NH2), amine-PEG-hydroxyl (H2N-PEG- OH), amine-PEG-COOH (H2N-PEG-COOH), diethylene triamine, or a combination thereof. In some embodiments, PEG may be represented by -(CH2CH2O)o- wherein o may be an integer ranging from 1 to 20. Additionally or alternatively, the stretcher unit may comprise other hydrophilic moieties, such as lysine and / or glutamate amino acid residues. Poly(alkylene glycol) and amino acid hydrophilic spacer groups may act as non-cleavable connecting groups or be used in addition to a cleavable group in a stretcher unit.

[0032] A “self-immolative group” refers to a moiety that spaces and covalently links together two or more components and degrades spontaneously in response to specific stimuli. Suitable self-immolative groups are well known in the art and include paraaminobenzyl (PAB) units, such as para-aminobenzyloxycarbonyl (PABC). Additionally or alternatively, the self-immolative group may comprise an N,N- dialkylethylenediamine moiety, such as an / V, / V-dimethylethylenediamine carbamate group.

[0033] As will be appreciated by persons skilled in the art, the cleavable group, hydrophilic spacer group, and self-immolative group may be connected via any suitable coupling group, such as an amide group. Other types of coupling reactions are also well-known in the art.

[0034] In some embodiments, the stretcher unit may have the formula: wherein:

[0035] Pi is an optional hydrophillic spacer group;

[0036] Q is a cleavable group; and

[0037] P2 is an optional self-immolative group.

[0038] Optionally, Pi may be a poly(alkylene glycol) (PEG) or an amino acid residue.

[0039] Optionally, Q may be a peptide linker. For example, Q may be a dipeptide selected from -Valine-Citrulline- (-Val-Cit-), -Valine- Alanine-(-Val-Ala-), -Valine-Lysine-(-Val- Lys-), -Valine-Arginine-(-Val-Arg-), -Phenylalanine-Citrulline-(-Phe-Cit-), Phenylalanine-Lysine-(-Phe-Lys-), -Phenylalanine-Arginine-(-Phe-Arg-), Asparagine- Asparagine (-Asn-Asn-), -Asparagine-Phenylalanine Acid- (-Asn-Phe-), - Asparagine-Histidine- (-Asn-His-), -Asparagine-Alanine- (-Asn-Ala-), -Asparagine- Valine- (-Asn-Val-), -Asparagine-Lysine- (-Asn-Lys-), -Asparagine-Citrulline- (-Asn- Cit-), or -Asparagine-Tryptophan- (-Asn-Trp-).

[0040] Optionally, P2 comprises para-aminobenzyl or para-aminobenzyloxycarbonyl.

[0041] In some embodiments, Ar is phenyl

[0042] In some embodiments, the linker is represented by Formula (la) and wherein:

[0043] (i) A1 is N and A2 is CR; or

[0044] (ii) A1 is CR and A2 is N; or

[0045] (iii) A1 is N and A2 is N.

[0046] In some embodiments, CR may be CH.

[0047] The active agent may be a drug, which may be a cytotoxic payload or a therapeutic peptide or polypeptide. The drug is preferably a cytotoxin. The cytotoxin may be selected from the group comprising microtubule inhibitors (e.g. dolastatins, auristatins, maytansinoids, eribulin, hemiasterlin, tubulysins, cryptophycins), DNA- damaging agents (e.g. calicheamicins, duocarmycins, pyrrolobenzodiazepines, pyrridinobenzodiazepines) topoisomerase inhibitors (e.g. camptothecins, anthracyclines), RNA polymerase inhibitors (e.g. a-Amanitin), thymidylate synthase inhibitors (e.g. raltitrexed) and cyclopropabenzaindole analogues (e.g. a 1 ,2,9,9a- tetrahydrocyclopropa[c]benzo[e]indol-4-one (CBI) dimer).

[0048] In other embodiments, the active agent may be a labelling moiety. The labelling moiety may be a fluorophore. Suitable fluorophores include fluorescein or dansyl derivatives. The labelling moiety may also be a biotin tag, derived from biotin. In a second aspect, there is provided a conjugate comprising a protein or a peptide, a linker and an active agent, wherein the linker is represented by Formula (Ila) or (lib):

[0049] (Ha) (Hb) wherein:

[0050] Ar is selected from the group consisting of phenyl, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrazole or imidazole; at least one of Ai and A2 is N and the other is selected from N or CR, with the proviso that when the linker is represented by Formula (lib) A1 is N and A2 is CR; n is an integer from 1 to 4; at least one R group is a connecting unit covalently linked to the active agent; and at each other occurrence R is independently selected from a hydrogen atom or an electron-withdrawing or electron-donating substituent.

[0051] The wavy line indicates a point of attachment to protein or peptide. The linker may be directly bound to the thiol group of a cysteine residue in the peptide or protein, such as an antibody.

[0052] Cysteine-based conjugation methods in the production of ADCs offers greater control of drug loading, i.e. the drug-to-antibody ratio (DAR) and homogeneity, compared to lysine conjugation methods. Greater ADC homogeneity is known to be associated with improved pharmacokinetics and efficacy and reduced off-target toxicity. The covalent thioether bond may be formed using existing thiol groups or by introducing thiol groups in a precursor step, for example by reacting one or more functional groups of the antibody to produce a thiol group, or by introducing a thiol group or a precursor thereof into the antibody. By way of example, this may involve the step of introducing a cysteine residue into the antibody at a site where it is desired to bind the linker to the antibody. This may be useful in situations where a convenient cysteine residue for reaction according to the present invention is not present in a starting or wild-type antibody. Conveniently, this may be achieved using site directed mutagenesis of the antibody, the use of which is well established in the art. Advantageously, this may also enable attachment of a drug or other active agent to the antibody in a site-specific manner. This may allow for the preparation of homogeneous ADCs having a defined number of drugs, which is known to improve pharmacokinetics and efficacy and is more desirable from a regulatory perspective.

[0053] Advantageously, the protein drug conjugates obtained from the novel conjugating linker structure have improved structural stability. In ADCs, linker stability is a critical factor in determining the efficacy and toxicity of the conjugate. An unstable linker can release the cytotoxic payload during blood circulation before it reaches the target site, leading to undesirable systemic toxicities. For example, some linkers, such as the widely-used maleimide attachment method, can suffer from non-specific release of payloads in non-tumorous tissues, leading to off-target toxicity and a limited therapeutic window. Advantageously, linkers according to the present invention provide a stable connection between the antibody and the drug, thus affording antibody-drug conjugates with improved safety properties and consequently enhanced tolerability profiles.

[0054] In some embodiments, the connecting unit covalently linked to the active agent has the formula:

[0055] - W - Y - A where W is a linking group;

[0056] Y is an optional stretcher unit comprising one or more of a cleavable group, a hydrophilic spacer group, and a self-immolative group; and

[0057] A is the active agent. As described above, in embodiments where the stretcher unit is not present, the linking group W may be formed from the reaction of a reactive group of the linker and a functional group on the active agent. The linking group W may be any suitable linking group formed from coupling reactions known in the art. For example, the linking group may be an amide group formed between an amine group on the linker and a carboxylic acid group on the drug or vice versa.

[0058] In some embodiments, W may be defined as in the first aspect above.

[0059] In some embodiments, the stretcher unit may have the Formula: wherein:

[0060] P1 is an optional hydrophilic spacer unit;

[0061] Q is a cleavable unit;

[0062] P2 is an optional self-immolative unit; and

[0063] * indicates covalent attachment site to the active agent.

[0064] Optionally, Pi, Q, P2 may be as defined in the first aspect above.

[0065] In some embodiments, the connecting unit may be covalently linked to more than one active agent. Each active agent may be the same or different. In some embodiments, the connecting unit may be branched such that an active agent can be connected to each terminating group of the connecting unit. Additionally, or alternatively, more than one R group may be a connecting unit covalently linked to an active agent, wherein each active agent may be the same or different. Incorporating more than one active agent per linker may enable a higher ratio of active agent per protein or peptide to be achieved. This may be useful, for example, where the target protein or peptide has only one or two reactive cysteine residues available for conjugation. Additionally, or alternatively, where the active agent is a drug, the conjugation of different active agents via a single linker may allow for the conjugation of drug compounds having a different mechanism of action. In some embodiments, Ar may be phenyl

[0066] In some embodiments, the linker is represented by Formula (la) and wherein:

[0067] (i) Ai is N and A2 is CR; or

[0068] (ii) A1 is CR and A2 is N; or

[0069] (iii) A1 is N and A2 is N.

[0070] In some embodiments, CR may be CH.

[0071] Optionally, the active agent may be a drug or a labelling moiety. The drug may be a cytotoxic payload or a therapeutic peptide or polypeptide. In particular, where the protein is an antibody or a fragment thereof and the protein drug conjugate is an ADC, the drug is preferably a cytotoxic drug. Alternatively, where the protein is albumin, the drug may be a cytotoxin or a therapeutic peptide or polypeptide.

[0072] The cytotoxic drug may be selected from the group comprising microtubule inhibitors (e.g. dolastatins, auristatins, maytansinoids, eribulin, hemiasterlin, tubulysins, cryptophycins), DNA-damaging agents (e.g. calicheamicins, duocarmycins, pyrrolobenzodiazepines, pyrridinobenzodiazepines) topoisomerase inhibitors (e.g. camptothecins, anthracyclines), RNA polymerase inhibitors (e.g. a-Amanitin), thymidylate synthase inhibitors (e.g. raltitrexed) and cyclopropabenzaindole analogues (e.g. a 1 ,2,9,9a-tetrahydrocyclopropa[c]benzo[e]indol-4-one (CBI) dimer).

[0073] The labelling moiety may be a fluorophore. Suitable fluorophores include fluorescein or dansyl derivatives. The labelling moiety may also be a biotin tag, derived from biotin.

[0074] In a third aspect, there is provided a linker compound represented by Formula (la) or

[0075] (la) (lb) wherein:

[0076] Ar is selected from the group consisting of phenyl, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrazole or imidazole; at least one of Ai and A2 is N and the other is selected from N or CR with the proviso that when the linker is represented by Formula (lb) A1 is N and A2 is CR; n is an integer from 1 to 4; at least one R group is a connecting unit comprising a reactive group capable of binding to another moiety, such as an active agent (e.g. a drug); at each other occurrence R is independently selected from a hydrogen atom or an electron-withdrawing or electron-donating substituent; and wherein the connecting unit has the Formula:

[0077] - W- Y - Z wherein:

[0078] W is a linking group;

[0079] Y is stretcher unit comprising one or more of a cleavable group, a hydrophilic spacer group, and a self-immolative group; and

[0080] Z is the reactive group and is selected from an amine-reactive group, a hydroxyl-reactive group, a halide-reactive group, a carboxylic acid-reactive group, an ester-reactive group, an azide-reactive group, an alkyne-reactive group, a hydroxylamine-reactive group, an aldehyde-reactive group, or a ketone-reactive group. Accordingly, the connecting unit is configured to covalently couple the linker with another moiety, such as a cytotoxin. Specifically, the connecting unit includes a reactive group capable of binding to another moiety. Example amine-reactive groups include, but are not limited to, an A / - hydroxysuccinmide ester, p-nitrophenyl ester, dinitrophenyl ester, or pentafluorophenyl ester. Example carboxylic-reactive groups include, but are not limited to, alcohols or amines. Example azide-reactive groups include, but are not limited to, alkynes. The alkynyl group (-C=C-) may be present in a cyclic alkynyl derivative, for example, selected from di benzocyclooctyne (DBCO), bicyclononynes (BCN), cyclooctyne, and difluorinated cyclooctyne. Example alkynereactive groups include, but are not limited to, azides. Example aldehyde or ketonereactive groups include, but not limited to, hydroxylamines. Example hydroxylaminereactive groups include, but are not limited to, aldehydes or ketones.

[0081] In some embodiments, the reactive group be selected from the group comprising or consisting of -C= a dibenzocyclooctyne (DBCO) group, a bicyclononyne (BCN) group, a 4-dibenzocyclooctynol (DIBO) group, a Difluorooctyne (DIFO) group, -N3, -C(O)OH, -NH2, -NHRb; -NRb2, where Rbis C1-C5 alkyl group, -OH, Cl, Br, I, -C(O)X or-OX, where X is a leaving group. A leaving group may be defined as an atom or group (charged or uncharged) that becomes detached from an atom in what is considered to be the residual or main part of the substrate in a specified reaction. For example, common leaving groups include halides such as Cl", Br" and I", and sulfonate esters such as tosylate (TsO) or mesylate (MsO), / V-hydroxysuccinimide (NHS), water (H2O), alcohols (R-OH), and amines (R3N).

[0082] The hydrophilic group, the self-immolative group and the cleavable group may be as defined in the first aspect.

[0083] In a fourth aspect, there is provided a linker compound represented by Formula (la) or wherein:

[0084] Ar is selected from the group consisting of phenyl, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrazole or imidazole; at least one of Ai and A2 is N and the other is selected from N or CR, with the proviso that when the linker is represented by Formula (lb) A1 is N and A2 is CR; n is an integer from 1 to 4; at least one R group is a connecting unit comprising a reactive group capable of binding to another moiety, such as an active agent (e.g. a drug), and is selected from:

[0085] ■ (CH2)e-Zi,or

[0086] ■ (CH2)f-Z2, or

[0087] ■ (CH2)e-Q-(CH2)f-Zi, or

[0088] ■ (CH2)e-Q-(CH2)f-Z2, or

[0089] ■ (CH2)e- Q - (CH2)f- Q - (CH2)f- Z1 , or,

[0090] ■ (CH2)e-Q-(CH2)f-Q-(CH2)f-Z2, where:

[0091] • Z1 is independently selected from -C(O)OH, -OH, -C(O)X, -OX, or NHRb, where X is a leaving group and Rbis a C1-C5 alkyl group;

[0092] • Z2 is independently selected from -C=CH, a dibenzocyclooctyne (DBCO) group, a bicyclononyne (BCN) group, a 4- dibenzocyclooctynol (DI BO) group, a Difluorooctyne (DIFO) group, - N3, -NH2, -Cl, -Br, -I; • Q is independently selected from O, S, an amide group, NH, or NRb, where Rbis a C1-C5 alkyl group;

[0093] • e is an integer from 0 to 10;

[0094] • f is an integer from 1 to 10, and at each other occurrence R is independently selected from a hydrogen atom or an electron-withdrawing or electron-donating substituent.

[0095] In some embodiments, the connecting unit is selected from:

[0096] ■ (CH2)e- Zi, or

[0097] ■ (CH2)f - Z2.

[0098] In some embodiments, Ar is phenyl.

[0099] In some embodiments, the linker is represented by Formula (la) and wherein:

[0100] (i) A1 is N and A2is CR; or

[0101] (ii) A1 is CR and A2is N; or

[0102] (iii) A1 is N and A2is N.

[0103] In some embodiments, CR may be CH.

[0104] In any of the embodiments described above, the linker may be represented by the For example, the linker may be represented by the formula (llla-c):

[0105] The linker may also be represented by the formula (IVa-c): where Ri is the connecting unit. Optionally, the connecting unit may be selected from:

[0106] ■ (CH2)e- Zi, or

[0107] ■ (CH2)f - Z2.

[0108] In a further aspect, there is provided a pharmaceutical composition comprising a conjugate according to the second aspect; and one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0109] The pharmaceutical composition may be for use as a medicament. For example, where the medicament is for use in the treatment of cancer, the active agent is a cytotoxin.

[0110] In another aspect, there is provided a method of treating cancer in a subject in need thereof, comprising the step of administering a therapeutic amount of the pharmaceutical composition to the subject. Optionally, the cancer may be selected from the group consisting of ovarian cancer cell, lung cancer cell, uterine cancer cell, testicular choriocarcinoma cell, ependymoma cell, mesothelioma cell, breast cancer cell, colon cancer cell, or renal cell carcinoma.

[0111] In another aspect, there is provided a method of producing a conjugate according to the second aspect, wherein the method comprises contacting a protein or peptide with a linker-active agent conjugate according to the first aspect. Optionally, the method may further comprise an initial step of providing a thiol group at one or more desired positions on the antibody or antigen binding fragment thereof (Ab).

[0112] Embodiments of the present invention will now be described by way of example and not limitation with reference to the accompanying figures.

[0113] Brief Description of Figures

[0114] The accompanying drawings illustrate presently exemplary embodiments of the disclosure, and together with the general description given above and the detailed description of the embodiments given below, serve to explain, by way of example, the principles of the disclosure.

[0115] Figure 1 shows the results of glutathione (GSH) conjugation kinetics: (a) the kinetics traces for reaction with compounds 3, 7 and 8; (b) the observed (kObs) rate constants; and (c) the relative rates for compounds 3, 7 and 8 at pH 7.4;

[0116] Figure 2 shows (a) kinetic traces for reaction of GSH with compounds 13 and 14; (b) the relative rates of conjugation at pH 7.4 including compounds 3, 7 and 8 for comparison; and (c) the relative rates of conjugation at pH 5.4 including compounds 3, 7 and 8 for comparison;

[0117] Figure 3 shows thiol-selectivity studies performed with compound 13;

[0118] Figure 4 shows thiol-selective reaction of compound 13 with A / Ac-Cys-OMe (T1) in the presence of A / Ac-Lys-OMe (L1) and A / Ac-Glu-OMe (G1) residues at pH 7.4: (a) 13 with 10 eq. T1 ; (b) 13 with 10 eq. T1 in the presence of L1 (50 eq.); and (c) 13 with 10 eq. T1 in the presence of G1 (50 eq.);

[0119] Figure 5 shows (a) % composition plot for 13 during incubation with L1 and control reaction containing only 13, as determined by LCMS analysis and (b) % composition plot for 13 during incubation with G1 and control reaction containing only 13, as determined by LCMS analysis;

[0120] Figure 6A shows representative PLRP chromatograms (A214nm) showing conjugation efficiency of PNU04 during 20 h reaction to monoclonal antibody Rituximab, with 0, 1.5 or 2.5 eq. TCEP. Letters designate the light (L) or heavy (H) chain;

[0121] Figure 6B shows representative PLRP chromatograms (A214nm) showing conjugation efficiency of PNU02 during 20 h reaction to monoclonal antibody Rituximab, with 0, 1.5 or 2.5 eq. TCEP. Letters designate the light (L) or heavy (H) chain;

[0122] Figure 6C shows representative PLRP chromatograms (A214nm) for monoclonal antibody Rituximab. Letters designate the light (L) or heavy (H) chain;

[0123] Figure 7 shows DAR values for conjugates obtained from PNU02 and PNU04 after TCEP reduction, with three antibodies: Herceptin, Rituximab and O-CA242;

[0124] Figure 8A shows representative PLRP data showing conjugation of compound 13 with monoclonal antibody O-CA242 after 0 h, 2 h and 22 h incubation. Letters designate the light (L) or heavy (H) chain;

[0125] Figure 8B shows representative PLRP data showing conjugation of compound 14 with monoclonal antibody O-CA242 after 0 h, 2 h and 22 h incubation. Letters designate the light (L) or heavy (H) chain;

[0126] Figure 9 shows conjugation kinetics of compounds 13 / 14 (n = 2). Heterogenous DAR conjugation of compounds 13 / 14 to hinge region thiols of O-CA242 (pH 7.4);

[0127] Figure 10 shows murine plasma stability of a-CA242-Compound 13 and O-CA242- Compound 14;

[0128] Figure 11 shows the percentage conversion to glutathione conjugate after 1 h incubation of compounds 3, 7-10, 23-25, 34-36, 41 , 43, 47-50, 52-54 and 56-58 with 10 eq. glutathione;

[0129] Figure 12 shows the pseudo first-order rate constants for compounds 3, 7-9, 23, 34, 36, 41, 47-50, 52, 55 and 59-62 conjugation measured at pH 7.4. Rate constants were calculated as an average (n = 3) and error bars report the standard deviation; Figure 13 shows the stability of compounds in the presence of (a) A / Ac-Glu-OMe (G1) or (b) A / Ac-Lys-OMe (L1). The plots show the normalised absorption (integrated starting material peaks) as a function of time;

[0130] Figure 14 shows the DAR values obtained at different time points during the bioconjugation of MMAE29 and MMAE30 with Trastuzumab;

[0131] Figure 15 shows PLRP chromatograms of MMAE29 (A) and MMAE30 (B) after 18- hour incubation with Trastuzumab antibody in the absence of TCEP reducing agent, and the PLRP chromatogram of unreduced Trastuzumab (without linker-drug or TCEP) (C);

[0132] Figure 16 shows representative PLRP (A) and SEC (B) chromatograms for Trastuzumab-MMAE29 and Trastuzumab-MMAE30 conjugates; and

[0133] Figure 17 shows IC50 values for ADCs comprised of MMAE29 and MMAE30; where (T) denotes an IC50 based on the concentration of payload moiety, and (P) denotes the IC50 based on concentration of protein.

[0134] Detailed Description

[0135] Definitions and Abbreviations

[0136] Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings. When trade names are used herein, the trade name includes the product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product, unless otherwise indicated by context.

[0137] In the context of the present invention, the term “protein” should be construed to cover any protein which has targeting capabilities and so has the ability to deliver a payload to a specific target tissue. Accordingly, “proteins” include antibodies and fragments thereof, albumin and transferrin, as well as any other alternatives known for use in conjugates. Proteins suitable for use in the present invention may be globular proteins.

[0138] The protein or peptide may specifically bind to a target molecule. In some embodiments, they may be a fragment of an antibody that contains at least one target molecule-binding site, lymphokines, hormones, growth factors, or any other cell binding molecule or substance that can specifically bind to a target.

[0139] The term "antibody" means an immunoglobulin molecule that recognises and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. References to antibodies include immunoglobulins whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein comprising an antigen binding domain. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

[0140] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, nanobodies, single chain antibodies, bispecific and multispecific antibodies formed from antibody fragments.

[0141] An antibody "specifically binds" to an epitope or antigenic molecule, which means that the antibody interacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the foregoing to an epitope or antigenic molecule than alternative substances, including unrelated proteins. In specific embodiments, "specifically binds" means, for instance, that an antibody binds to a protein with a KD of approximately 0.1 mM or less, but more usually, less than about 1 pM. In specific embodiments, "specifically binds" means that an antibody binds to a protein at times with a KD of approximately 0.1 pM or less, and at other times, with a KD of approximately 0.01 pM or less.

[0142] The "drug-antibody ratio" (DAR) in an antibody-drug conjugate of the invention is defined as the molar ratio between the drug moieties in the conjugate and the antibodies in the conjugate. Where an antibody has more than one site of attachment, more than one drug moiety may be linked to each antibody. In some instances, a mixture is obtained comprising more than one antibody-drug conjugate (ADC) molecules. The drug-antibody ratios of the antibody-drug conjugates can be measured by analytical methods known in the art, for example, as described below. In some embodiments, the antibody-drug conjugates have an average DAR of about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1.5 to about 3.5, or about 2 to about 4. In compositions comprising a mixture of antibody-drug conjugate and unconjugated antibody, the DAR of the composition as a whole will be diluted according to the ratio of antibody-drug conjugate to unconjugated antibody.

[0143] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells are characterised by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukaemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, mesothelioma, melanoma, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, fallopian tube cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, various lymphoma and leukaemia cancers, and various types of head and neck cancers.

[0144] “Tumour" refers to any mass of tissue that results from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including precancerous lesions. The term "subject" refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0145] The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulation can be sterile.

[0146] An "effective amount" as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be determined empirically and in a routine manner, in relation to the stated purpose.

[0147] The term "therapeutically effective amount" refers to an amount of an ADC or other drug effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the tumour size; inhibit (i.e. , slow to some extent and in a certain embodiment, stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and in a certain embodiment, stop) tumour metastasis; inhibit, to some extent, tumour growth; and / or relieve to some extent one or more of the symptoms associated with the cancer. See the definition of "treating" below. To the extent the drug can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0148] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent and / or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. In certain embodiments, a subject is successfully "treated" for cancer according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumour size; inhibition of or an absence of cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibition of or an absence of tumour metastasis; inhibition or an absence of tumour growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumourigenicity, tumourigenic frequency, or tumourigenic capacity, of a tumour; reduction in the number or frequency of cancer stem cells in a tumour; differentiation of tumourigenic cells to a non-tumourigenic state; or some combination of effects.

[0149] “Self-immolative spacer” refers to a moiety that spaces and covalently links together two or more components and degrades spontaneously in response to specific stimuli.

[0150] “Electron-withdrawing group” refers to a group that draws electron density from neighbouring atoms towards itself, typically by resonance or inductive effects.

[0151] “Electron-donating group” refers to a group that donates some of its electron density into a conjugated TT system via resonance, typically by resonance or inductive effects.

[0152] The term "alkyl" by itself or as part of another term refers to a straight chain or branched, saturated hydrocarbon having the indicated number of carbon atoms (e.g., "Ci-Cs" alkyl refers to an alkyl group having from 1 to 8 carbon atoms). Alkyl groups typically comprise from 1 to 20 carbon atoms, preferably from 1 to 8 carbon atoms, and more preferably from 1 to 4 carbon atoms. When the number of carbon atoms is not indicated, the alkyl group has from 1 to 8 carbon atoms. Representative straight chain Ci-Cs alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n- pentyl, n-hexyl, n-heptyl and n-octyl; while branched Ci-Cs alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tent-butyl, -isopentyl, and -2-methylbutyl; unsaturated C 2 -C 8 alkyls include, but are not limited to, vinyl, allyl, 1 -butenyl, 2- butenyl, isobutylenyl, 1 -pentenyl, 2-pentenyl, 3-methyl-1 -butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1 -hexyl, 2-hexyl, 3-hexyl, acetylenyl, propynyl, 1 -butynyl, 2- butynyl, 1 -pentynyl, 2-pentynyl and 3-methyl-1 -butynyl. Reference to “alkyl” herein refers to unsubstituted and substituted moieties as described above. The term "alkylene," by itself or as part of another term, refers to a saturated, branched or straight chain or cyclic hydrocarbon radical of the stated number of carbon atoms, typically 1-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Examples include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, ocytylene, nonylene and decalene. Reference to “alkylene” herein refers to unsubstituted and substituted moieties as described above.

[0153] An "alkene" moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon double bond.

[0154] The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain hydrocarbon, or combinations thereof, fully saturated or containing from 1 to 3 degrees of unsaturation, consisting of the stated number of carbon atoms and from one to three heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N and S may be placed at any interior position of the heteroalkyl group. Up to two heteroatoms may be consecutive. Heteroalkyl groups typically comprise from 1 to 15 carbon atoms, preferably from 1 to 12 carbon atoms, more preferably from 1 to 8 carbon atoms, and most preferably from 1 to 4 carbon atoms. Reference to “heteroalkyl” herein refers to unsubstituted and substituted moieties as described above. Unless otherwise indicated, the term "heteroalkylene" by itself or as part of another substituent means a divalent group derived from heteroalkyl (as discussed above). For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini. Reference to “heteroalkylene” herein refers to unsubstituted and substituted moieties as described above.

[0155] An "alkyne" moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon triple bond. The alkyl moiety, whether saturated or unsaturated, may be branched, straight chain, or cyclic.

[0156] The term "aryl" or "aromatic ring" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 14 (e.g. 5 to 10, such as 5, 6 or 10) carbon atoms, more preferably 6 to 10 carbon atoms. These can be arranged in one ring, e.g. phenyl, or two or more condensed rings (e.g. naphthyl). Preferably aryl refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. In some embodiments, the aryl is unsubstituted in some embodiments the aryl is substituted.

[0157] The term "cycloalkyl" as used herein refers to saturated or unsaturated, non-aromatic cycloalkyl comprising 1 , 2 or more rings. Examples include cyclopropyl, cyclo- butyl, cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexonyl, etc.

[0158] The term "acyl" as used herein refers to a functional group with the general formula Rac-C(O)H, wherein Racrefers to an optionally substituted hydrocarbon radical, in particular, a hydrocarbon chain having Ci - Cs carbon atoms.

[0159] The term "alkylsulfonyl" or "arylsulfonyl" refer to alkyl or aryl groups containing a SO2 residue.

[0160] The term "Nitro" refers to the -NO2 radical.

[0161] The present invention relates to a novel conjugating linker structure which has a fused nitrogen-containing heteroaromatic ring and a vinyl substituent that is capable of specifically binding to a thiol group of a protein or peptide (e.g. after full / partial reduction of inter-chain disulfide bonds). The novel conjugating linker structures include a connecting unit for conjugating or coupling to an active agent, such as a pharmaceutically active agent or drug.

[0162] The term "connecting unit" described in the present disclosure may include a stretcher unit to alter solubility or immunogenic properties of the functionalising group. The stretcher unit may comprise a cleavable group or linker or a non-cleavable group or linker. The stretcher unit may be useful in a cleavable ADC linker system, which will be described in more detail below. Suitable stretcher units for utilisation in the present invention will be readily apparent to the skilled person in the art.

[0163] Cleavable linkers can be chemically labile and enzyme-labile linkers. Due to the high plasma stability and good intracellular cleaving selectivity and efficiency, enzyme- labile linkers are commonly selected as cleavable linker candidates, for example, in ADCs. In some embodiments, enzyme-labile linkers may include a peptide unit (-AAs- ) selected from a group consisting of -Valine-Citrulline- (-Val-Cit-), -Valine-Alanine- (- Val-Ala-), -Valine-Lysine- (-Val-Lys-), -Valine-Arginine- (-Val-Arg-), -Phenylalanine- Citrulline- (-Phe-Cit-), -Phenylalanine-Lysine- (-Phe-Lys-), and -Phenylalanine- Arginine- (-Phe-Arg-). Typical enzyme-labile linkers include -Val-Cit- and -Phe-Lys-, which can be recognised by cathepsin B.

[0164] Alternatively, peptides recognised by legumain may be used, such as -Asparagine- Asparagine- (-Asn-Asn-), -Asparagine-Phenylalanine- (-Asn-Phe-), -Asparagine- Histidine- (-Asn-His-), -Asparagine-Alanine- (-Asn-Ala-), -Asparagine-Valine- (-Asn- Val-), -Asparagine-Lysine- (-Asn-Lys-), -Asparagine-Citrulline- (-Asn-Cit-), or - Asparagine-T ryptophan- (-Asn-T rp-).

[0165] Alternatively, the cleavable group may be selected from a p-D-glucuronide linker, p- Glucuronide linkers are a family of enzymatically cleavable linkers that mediate payload drug release via the linker hydrolysis by the lysosomal enzyme p- glucuronidase (GLISB) within tumor cells. In other embodiments, O-glucosyl, O- galactosyl or O-mannosyl linkers may be similarly used. Example cleavable linkers in accordance with the present invention are: wherein

[0166] * indicates covalent attachment site to the active agent

[0167] L indicates attachment to the antibody linker directly or indirectly via an additional spacer group. In some embodiments, the stretcher unit may additional or alternatively comprise moieties that are capable of increasing the hydrophilicity of the resulting conjugate. In one embodiment, the stretcher unit may include one or more poly(alkylene glycol). For example, the poly(alkylene glycol) may be poly(ethylene glycol) (PEG), PEG diamine (H2N-PEG-NH2), amine-PEG-hydroxyl (H2N-PEG-OH), amine-PEG-COOH (H2N- PEG-COOH), diethylene triamine, or a combination thereof. In some embodiments, PEG may be represented by -(CF^CFWjo- wherein o may be an integer ranging from 1 to 20. It will also be appreciated by the skilled person that the techniques disclosed herein may be used in conjunction with other poly(alkylene glycol) molecules, such as polypropylene glycol or polyethylene-polypropylene glycol copolymers. Branched or multi-arm poly(alkylene glycol) molecules, including branched or multi-arm PEG molecules, may also be used. Poly(alkylene glycol) molecules that may be used in accordance with the present invention are well known in the art and publicly available, for example from commercially available sources such as Sigma Aldrich. Similarly, hydrophilic amino acid residues may also be incorporated, including lysine and / or glutamate amino acid residues.

[0168] Poly(alkylene glycol) and amino acid hydrophilic spacer groups may act as non- cleavable stretcher units or be used in addition to an cleavable group in a cleavable stretcher unit.

[0169] In some embodiments, the connecting unit may additional or alternatively comprise a self-immolative group. A self-immolative group may be defined as a bifunctional chemical moiety which is capable of covalently linking together two spaced chemical moieties, which can release one of the spaced chemical moieties by means of enzymatic cleavage; and following enzymatic cleavage, can spontaneously cleave from the remainder of the molecule to release the other of the spaced chemical moieties. The self-immolative group may comprise a p-aminobenzyl alcohol-based group. In certain embodiments, the self-immolative group comprises a benzyloxycarbonyl group. For example, the self-immolative group may be p- aminobenzyloxycarbonyl (PABC). Predominantly, the benzylic functional group consists of a carbamate moiety, which will release carbon dioxide upon triggering the 1 ,6-elimination mechanism, and a primary or secondary amino group. Additionally or alternatively, the self-immolative group may comprise an / V, / V-dialkylethylenediamine moiety, such as an / V, / V-dimethylethylenediamine carbamate group. Thus, the present invention provides linkers for use in protein drug conjugates, with utility in linking proteins and active agents, for example antibodies and cytotoxins to provide ADCs. Advantageously, the protein drug conjugates obtained from the novel conjugating linker structure have improved structural stability, thus affording protein drug conjugates with improved safety properties and consequently enhanced tolerability profiles. Moreover, a narrower DAR (drug to antibody) distribution may also be obtained compared to currently known linker molecules for use in ADCs.

[0170] The antibody used herein recognises an antigen that is natively expressed or overexpressed by target cells, e.g., cancer cells, and can function as a targeting agent to deliver drug moieties to cancer cells with a high degree of specificity. When the antibody binds to the antigen, the antigen-conjugate forms a complex, is internalized, and ultimately enters the lysosome, and the linker between the drug moiety and the antibody is cleaved to release the drug moiety and thereby providing a cytotoxic effect.

[0171] The antibody may be, for example, selected from the group consisting of an anti-folate receptor alpha antibody, an anti-B7H3 antibody, an anti-MSLN antibody, an anti-Trop2 antibody, an anti-5T4 antibody, an anti-CD20 antibody, an anti-PSMA antibody, an anti-EGFR antibody, an anti-CD70 antibody, an anti-DLL3 antibody, an anti-ROR1 antibody, an anti-c-MET antibody, an anti-Her3 antibody, an anti-EphA3 antibody, an anti-CD30 antibody, an ant-CD79 antibody, an anti-NaPi3 antibody, an anti-CD22 antibody, an anti-CA242 antibody, an anti-CD33 antibody, an anti-Her2 antibody, an anti-MUC16, but is not limited thereto.

[0172] According to an alternative embodiment of the present invention, the antibody may be substituted for albumin.

[0173] By “active agent” it is meant any substance that is capable of producing a specific and intended effect or action. The active agent may be a drug or a labelling moiety. The active agent needs to comprise a functional group that can be linked to the linker. Such a group may include an amino group, a carboxylic acid, an imine group or a hydroxy group. By "drug" it is meant any chemical substance which has a known biological effect on humans or animals. In particular, the drug may be a pharmaceutical drug which is used in the treatment, cure, prevention or diagnosis of disease or to otherwise enhance physical or mental well-being. As will be appreciated, the drug may be a known drug which has obtained the necessary marketing authorisation or a novel drug which has not yet undergone testing or achieved marketing authorisation.

[0174] The drug may be a cytotoxic payload or a therapeutic peptide or polypeptide. Preferably the cytotoxin is a biologically active cytotoxic material.

[0175] The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents a cellular function and / or causes cell death or destruction.

[0176] The cytotoxin may be selected from the group comprising microtubule inhibitors (e.g. auristatins, maytansinoids, eribulin, hemiasterlin, tubulysins, cryptophycins), DNA- damaging agents (e.g. calicheamicins, duocarmycins, pyrrolobenzodiazepines, pyrridinobenzodiazepines) topoisomerase inhibitors (e.g. camptothecins, anthracyclines), RNA polymerase inhibitors (e.g. a-Amanitin), thymidylate synthase inhibitors (e.g. raltitrexed) and cyclopropabenzaindole analogues (e.g. a 1 ,2,9,9a- tetrahydrocyclopropa[c]benzo[e]indol-4-one (CBI) dimer).

[0177] However, additionally or alternatively, the cytotoxin could also be selected from other known cytotoxins including ricin subunits and other peptide based cytotoxic materials.

[0178] As well as drug conjugates, the linking of labelling moieties such as fluorophores and biotin tags to proteins and peptides can be useful in techniques such as flow cytometry, Immunofluorescence staining and immunohistochemical staining.

[0179] The conjugates according to the present invention may be useful in a variety of applications. For example, the conjugates of the present invention where the active agent is a drug may be used in therapeutic treatment methods, such as the treatment of cancer.

[0180] Accordingly, the present invention provides methods of treating cancer comprising administering a therapeutically effective amount of the conjugate to a subject (e.g., a subject in need of treatment). In certain embodiments, the cancer is a cancer selected from the group consisting of lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, pancreatic cancer, biliary cancer, cervical cancer and uterine cancer. In certain embodiments, the cancer is pancreatic cancer. In certain embodiments, the cancer is colorectal cancer. In certain embodiments, the subject is a human.

[0181] A conjugate may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs; surgery; and radiation therapy. Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the active ingredient, i.e. a conjugate according to the first aspect of the invention, where the active agent is a drug, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.

[0182] The pharmaceutical compositions of the present invention can be administered in any number of ways for either local or systemic treatment. Administration can be pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal); oral; or parenteral including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (e.g., intrathecal or intraventricular) administration.

[0183] For the treatment of the disease, the appropriate dosage of an antibody or agent of the present invention depends on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the composition is administered for therapeutic or preventative purposes, previous therapy, patient's clinical history, and so on all at the discretion of the treating physician. The compositions can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected ora diminution of the disease state is achieved (e.g. reduction in tumour size). Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient and will vary depending on the relative potency of an individual antibody or agent. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates.

[0184] It will be appreciated by persons skilled in the art that the above embodiment has been described by way of example only and not in any limitative sense, and that various alterations and modifications are possible without departing from the scope of the invention as defined by the appended claims.

[0185] Experimental Data and Discussion

[0186] The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations to the scope of this invention as defined by the appended claims.

[0187] The following abbreviations are used herein and have the indicated definitions:

[0188]

[0189] The linkers used in the examples and their corresponding chemical structures are listed in Table 1. Compounds 53 and 54 were purchased from commercial sources, and all other analogues were synthesised according to procedures reported in herein.

[0190] Table 1 : Structures of compounds evaluated.

[0191] Example 1. Evaluation of Conjugation Kinetics of Compounds

[0192] To initially evaluate the vinylquinoline scaffold as a potential bioconjugation handle, the reactivity of compounds 3, 7, 8, 13, 14 towards glutathione (GSH, 10 eq) was investigated under pseudo-first order conditions. Glutathione contains a thiol group which is readily available for conjugation, and can provide a good model for proteins to assess the suitability of linkers for use in the present invention for utility in protein drug conjugates, such as ADCs.

[0193] Time-resolved LCMS analysis was performed to track the consumption of vinyl compounds (Figure 1a), detect formation of the thiol conjugate species, determine the pseudo first-order rate constants (kObs) and determine the relative rates (Figure 1 b-c).

[0194] Method: 5 pL of 20mM compound 3 / 7 / 8 / 13 / 14 in DMA was mixed with 95 pL DMA, 850 pL 10mM PBS solution (pH 5.4) and 50 pL of 20mM glutathione in PBS buffer solution. Control reactions were run in the absence of glutathione: 5 pL of 20mM Compound 3 / 7 / 8 / 13 / 14 in DMA was dissolved in 95 pL DMA and 900 pL of PBS buffer (pH 5.4) was used. The samples were analysed by LCMS at several timepoints, and LC chromatograms (abs 254nm) were integrated to obtain peak areas and monitor consumption of the vinyl starting material and detect formation thiol-conjugated product. The reactions were performed under pseudo first order conditions. The natural log plot of starting material concentration versus time afforded linear plots, from which kobs was determined. This method was repeated at pH 7.4 and pH 8.4 (PBS buffer) to evaluate the effect of pH upon the conjugation kinetics.

[0195] Results: The results are shown in Figures 1a-c. Surprisingly, vinylquinoline 7 was observed to react approximately 26-fold faster than vinylpyridine 3 (see Figure 1b), a conjugation handle previously reported in W02016067021 A1 . Methyl 4-vinylquinoline- 6-carboxylate (8) was tested to evaluate the impact of introducing an ester substituent as a mimic for a conjugation handle (e.g. for coupling an active agent, such as a drug). The electron-withdrawing ester (8) revealed marginally faster thiol conjugation kinetics than 7, and 8 showed rapid reaction with GSH, such that conjugation was complete within 60 minutes (Figure 1a, see data for pH 5.4), whereas 3 took > 18 hours. Table 2 summarises the observed rate constants for all compounds performed at pH 5.4, 7.4 and 8.4. To further develop vinylquinoline 8 as a bioconjugation platform and evaluate its conjugation properties, compounds 13 and 14 were synthesised as non-cytotoxic alternatives to ADC linker-payloads and evaluated for their thiol-reactivity.

[0196] As shown in Figure 2, vinylquinoline 13 showed rapid conjugation to GSH, and was approximately 20-fold faster than 14 at pH 7.4, with a marginally more pronounced difference in the rate at pH 5.4.

[0197] Table 2: Relative rates for GSH conjugation at different pH conditions.

[0198] Example 2. Studies to Demonstrate Thiol-Selectivity and Stability

[0199] Vinylquinoline 13 was subjected to thiol-selectivity studies to confirm whether a reaction with lysine (L1) residues (see Figure 3b) or glutamate (G1) residues (Figure 3c) would compete with thiol-conjugation (Figure 3a). Studies were performed at pH 7.4, to confirm vinylquinoline conjugation handle (13) is selective for thiol under the bioconjugation conditions. Additional stability studies (see Figure 5 for results) were performed to confirm that 13 is stable in the presence of 10 eq. L1 (as shown in Figure 3d) or 10 eq. G1 (Figure 3e) and does not undergo any reaction to form byproducts (13-L1 and 13-G1) during the 24 h assay period.

[0200] Method Thiol-Selectivity Studies (see Figure 3a-c):

[0201] Three different experiments were run to test the thiol-selectivity, one sample containing only thiol (T1) (Figure 3a), one containing T1 and L1 simultaneously (Figure 3b) and one containing T1 and G1 (Figure 3c). The first reaction (Figure 3a) contains 5 pL of 20mM compound 13 in DMA, 95pL DMA, 850 pL 10mM PBS solution (pH 7.4) and 50 pL of 20mM thiol (T1) in PBS buffer solution. The second reaction (Figure 3b) contains 5 pL of 20mM compound 13 in DMA, mixed with 95pL DMA, 800 pL 10mM PBS solution (pH 7.4), 50 pL of 20mM thiol (T1) in PBS buffer solution and 50 pL of 100mM A / Ac-Lys-OMe (L1) in PBS buffer solution. The third reaction (Figure 3c) contains 5 pL of 20mM compound 13 in DMA, mixed with 95pL DMA, 800 pL 10mM PBS solution (pH 7.4), 50 pL of 20mM thiol (T1) in PBS buffer solution and 50 pL of 100mM A / Ac-Glu-OMe (G1) in PBS buffer solution. The samples were analysed by LCMS at timepoints 0, 1 and 24 hours, so the LC chromatograms (abs 254nm) were integrated to obtain peak areas. Integrated peak areas were analysed to confirm if compound 13 conjugated selectively to thiol T1 .

[0202] Results: See Figures 4b-c for results. Compound 13 reacted rapidly and selectively with thiol T1 (Figure 3a) within 1 hour, with LCMS analysis revealing that 13-T1 was the major product and only trace quantities of 13 remained (Figure 4a). Performing T1 conjugation in the presence of 10 eq L1 or G1 (as shown in Figure 3b-c) did not influence the outcome of the reaction, and thiol conjugate was still formed selectively within 1 hour of incubation. LCMS analysis (results not shown) showed that lysine (L1) and glutamate (L1) did not conjugate to the vinylquinoline group or interfere with the efficiency of thiol conjugation.

[0203] Method for L1 / G1 Stability Studies (Figure 3d-e):

[0204] 5 pL of 20mM compound 13 in DMA was mixed with 95 pL DMA, 890 pL 10mM PBS solution (pH 7.4) and 10 pL of 100mM A / Ac-Lys-OMe (L1) (reaction as shown in Figure 3d) or A / Ac-Glu-OMe (G1) (reaction as shown in Figure 3e) in PBS buffer solution. The samples were analysed by LCMS at timepoints 0, 1 and 24 hours, so the LC chromatograms (abs 280nm) were integrated to obtain peak areas. Integrated peak areas were analysed to confirm if compound 13 showed any reaction with amine or carboxylic acid side chains present in L1 / G1 or if they were stable. Control reactions were performed in the absence L1 / G1 , where 5 pL of 20mM compound 13 in DMA was dissolved in 95 pL DMA and 900 pL of PBS buffer was used. The method of sample analysis was identical to the reactions containing G1 / L1.

[0205] Results: See Figure 5a-b for results. Incubation of compound 13 with 10 eq lysine (L1) or 10 eq glutamate (G1) did not result in the formation of byproducts. Compound 13 remained stable and LCMS analysis confirmed the vinylquinoline moiety was unreacted over the 24 h incubation.

[0206] Example 3. Bioconjugation of PNU02 and PNU04

[0207] To evaluate the performance of the vinylquinoline conjugation handle, two PNU- containing linker-drugs (PNU02 and PNU04, see Scheme 1) were synthesised and conjugated to the hinge region of three antibodies (Herceptin, Rituximab and a- CA242). PNU04 comprises a vinylquinoline linker, a cathepsin cleavable linker (valinecitrulline), a para-aminobenzylcarbamate self-immolative spacer and PNU. PNU is a highly potent secondary metabolite of nemorubicin belonging to the anthracycline class of natural products. The use of PNU in an ADC format has recently gained interest due to its extremely high potency. PNU02 is structurally equivalent to PNU04 with the exception that the linker is a vinylpyridine-based linker.

[0208] Scheme 1 : Structures of PNU02 and PNU04.

[0209] Method: Conjugation comparison of PNU04 and PNU02 linker-drugs

[0210] Monoclonal antibodies Herceptin, Rituximab and a-CA242 in PBS, 4mM EDTA, pH 7.4 were partially reduced with 0, 1 .5 and 2.5 molar equivalents of TCEP (25°C, 2 h, 300rpm agitation) and subsequently conjugated with 10 molar equivalents of either PNU04 or PNU02 (10% (v / v) DMA, 30°C, 300rpm agitation). Samples were taken at 3 and 20 h, and conjugation reactions were halted by Sephadex G-25 buffer exchange column to remove residual linker-toxins (PBS, pH 7.4). DARs were analysed by PLRP (as reported in Method B).

[0211] Results: Conjugation reactions were explored with a range of TCEP concentrations to control DAR (see Table 3). In all cases, PNU04 showed more efficient conjugation than PNU02. In the absence of TCEP reducing agent, no conjugation was observed for PNU04 (DAR 0), and PLRP chromatograms (representative PLRP data shown in Figure 6A) were indistinguishable from that of the unreduced antibody (Figure 6C), thus confirming that the vinylquinoline exhibits thiol-specific conjugation with antibody and no unspecific conjugation to other amino acid residues (e.g. lysine / glutamate / serine) was observed. Across all three antibodies screened in bioconjugation reactions, PNU04 consistently showed higher DAR values than PNU02 (see Figure 7 and Table 3), confirming that vinylquinoline provides higher conjugation efficiency than the previously reported vinylpyridine moiety.

[0212] PLRP analysis.

[0213] Example 4. Bioconjugation of compounds 13 and 14

[0214] Compounds 13 and 14 were conjugated to monoclonal antibody (anti-CA242) to demonstrate the utility of the vinylquinoline conjugation handle and provide a direct comparison to the conjugation kinetics of previously reported vinylpyridines (as exemplified by compound 14). Method A: Conjugation kinetics with a-CA242 a-CA242 monoclonal antibody was prepared in PBS, 4mM EDTA, pH 7.4 then was partially reduced with 2.5 molar equivalents of TCEP (25°C, 2 h, 300rpm agitation) and subsequently conjugated with 10 molar equivalents of either Compound 13 or Compound 14 (10% (v / v) DMA, 25°C, 300rpm agitation). Samples were taken at 0, 2, and 22 h and conjugation reactions were halted by Sephadex G-25 buffer exchange column to remove residual Compound 13 / 14 (PBS, pH 7.4).

[0215] Method B: Drug / dye-to-antibody ratio (DAR) determination

[0216] The dansyl moiety in compounds 13 and 14 provides a surrogate for the linker-drug and so the dye-to-antibody ratio was determined. Conjugate samples were reduced with 2mM DTT (37°C, 15min) and analysed by Polymer-Linked Reverse-Phase (PLRP) chromatography, resolving peaks corresponding to unconjugated or drug / dye- conjugated antibody light and heavy chains. DARs are determined from the relative proportions of unconjugated light- and heavy-chain (L0 and HO) and conjugated light- (L1) and heavy-chain (H1 , H2, H3)._Peak separation was performed on an Agilent PLRP-S (1000A, 2.1 x 50mm, 5pm) column, using the following procedure: mobile phase A (water + 0.1 % TFA) and mobile phase B (MeCN + 0.1 % TFA); elution gradient 25-50% mobile phase B over 25 min.

[0217] Results: Bioconjugation kinetics of compounds 13 / 14 with a-CA242 antibody

[0218] Conjugation kinetics experiments were performed with anti-CA242 monoclonal antibody (a-CA242) for direct comparison of compounds 13 and 14 and evaluate their ability to achieve target DAR 4.0 within a timeframe of 2 - 22 h. Representative PLRP chromatograms are shown in Figure 8, and kinetics traces are plotted in Figure 9. 13 showed rapid and efficient conjugation at pH 7.4, reaching target DAR > 4.0 within 4 hours (Figure 8A). 14 required longer incubation times, and lower DAR (~ 3.6) was achieved (Figure 8B), thus demonstrating that vinylquinoline conjugation handle offers improved conjugation efficiency over the previously reported vinylpyridine moiety.

[0219] Example 5. In vitro stability of conjugates in whole mouse plasma

[0220] Conjugates of compounds 13 and 14 were evaluated to confirm that vinylquinoline linkers offer comparable plasma stability to that of previously reported vinylpyridines. Method: The conjugates (a-CA242-com pound 13 and a-CA242-com pound 14) were incubated at 1 mg / mL in 80% v / v mouse plasma at 37 °C, 300 rpm over 5 days under sterile conditions. Samples were collected at 0, 24, 48, 96 and 168 h and stored at - 20 °C. Samples were enriched by affinity for goat anti-human biotinylated IgG charged streptavidin resin (bound and washed in 25 mM Tris, 150 mM NaCI, pH 7.5; eluted with 100 mM glycine-HCI buffer, pH 2.7; neutralized with 200mM Tris, pH 7.5); samples were stored at -20°C pending analysis. Sample DARs were analysed by PLRP (as described in Method B) and normalised to DARs at time 0. Plasma stability was monitored as the percentage change in average DAR for a-CA242-compound 13 and a-CA242-com pound 14 at different incubation time points in comparison to day 0.

[0221] Results: Conjugates of a-CA242 and compounds 13 / 14 (a-CA242-Compound 13 and a-CA242-Compound 14) were analysed by PLRP to track changes in DAR over a period of 0 - 168 h. The conjugates a-CA242-com pound 13 and a-CA242-compound 14 showed good stability both in mouse plasma (Figure 10), indicating that vinylquinoline moiety within 13 forms a durable linkage to antibody. Conjugates prepared from 13, were found to exhibit comparable plasma stability to 14, indicating both compounds feature similar stability than those previously reported in W02016067021A1.

[0222] Summary of Bioconjugation Results

[0223] Conjugation kinetics of compounds 13 / 14 and PNU02 / PNU04 revealed that the vinylquinoline conjugation handle shows superior conjugation efficiency than vinylpyridine, delivering higher DAR conjugates within shorter incubation times. The vinylquinoline conjugation moiety also showed excellent plasma stability, comparable to that of the previously reported vinylpyridine conjugation handle.

[0224] Evaluation of compounds for further improvements in reactivity

[0225] Compounds 3, 7 - 10, 23 - 25, 34 - 36, 41, 43, 47 - 50, 52 - 60 (structures shown in Table 1) were purchased or synthesised and evaluated in small molecule thiol- selectivity and reactivity assays to identify further improvements compared to compounds 3 / 8. Example 6. Initial Screening of Thiol Reactivity

[0226] Compounds 3, 7 - 10, 23 - 25, 34 - 36, 41 , 43, 47 - 50, 52 - 54, and 56 - 58 were conjugated with GSH at pH 7.4 to evaluate thiol-reactivity and identify analogues with the fastest kinetics. The reactivity of compounds was evaluated and reported as the % conversion to thiol conjugate (Figure 11) after 1 hour of incubation with GSH. Analogues were grouped by their order of reactivity, with high and moderate reactivity analogues being taken forward for in-depth kinetic analysis (Figure 12) to benchmark their conjugation kinetics relative to comparators 3 and 7 / 8.

[0227] Method for Initial Screening of Thiol Reactivity: Compounds 3, 7 - 10, 23 - 25, 34 - 36, 41 , 43, 47 - 50, 52 - 54, and 56 - 58 were evaluated for their reactivity with thiol. Reactions were carried out in a phosphate buffered saline (PBS) / DMA solution (ratio 9:1) buffered to the appropriate pH at room temperature. The linker compound (5 L of 20mM stock dissolved in DMA) was mixed with 95 L DMA, 850 pL 10mM PBS solution (pH 7.4) and 50 pL of 20mM glutathione in PBS buffer solution. The samples were analysed by LCMS (C18 LIPLC column, MeCN / H2O 10-95% gradient with 0.1 % (v / v) formic acid). LC chromatograms (abs 254nm) were integrated to obtain the kinetics of thiol conjugation, reported for each compound as the % conversion to thiol conjugate after 1 h incubation (Figure 11).

[0228] Results: See Figure 11 for results. 35, 47 - 49 were the highest reactivity analogues, closely followed by 35 and 36 that also showed rapid conjugation, approaching completion within 1 hour and revealing promising candidates for further development. 52, 41 and 23 appeared to be mostly consumed within 1 hour, whilst 50 and 34 showed more moderate reactivity, suggesting that the substitution of 6-position with electrondonating or electron-withdrawing groups does not have a pronounced impact upon the reactivity of the vinyl group. The configuration of the 6-amide group (i.e. 52 versus 23) also appears not to be an important determinant for thiol reactivity.

[0229] Compounds 56 - 58 showed no reaction with GSH, indicating that electron donating (-methyl or -OMe) or electron withdrawing (-F) substituents on the vinyl group prevent thiol-conjugation.

[0230] Although 47 / 48 / 49 / 36 / 52 / 41 appear to have similar % conversion after 1 hour incubation with glutathione, compounds 47 - 49 were found to have much higher reactivity, as their conjugation reactions were complete within 10 - 20 minutes. To more precisely evaluate rates of thiol-conjugation, kinetics experiments were repeated in triplicate (see in-depth kinetics, Figure 12) with a greater number of timepoints to afford a more accurate determination of the order rate constants. Whilst subtle changes in the order of reactivity were observed compared to Figure 11 , 47 - 49 were found to be consistently the most reactive compounds.

[0231] Example 7. In-Depth Evaluation of Small Molecule Conjugation Kinetics

[0232] Compounds 8, 9, 23, 34, 36, 41 , 47 - 50, 52, 55 were selected for further study to accurately benchmark their conjugation kinetics relative to comparator compounds 3, 7 and 59. A greater number of timepoints were taken for LCMS analysis such that conjugation kinetics could be determined and reported as the pseudo first-order rate constants (kObs) (Figure 12). Based on these results, additional linker compounds 60- 62 were also synthesised and evaluated for their conjugation kinetics.

[0233] Method for in-depth kinetics Compounds 3 7 - 9, 23, 34, 36, 41 47 50,

[0234] 52, 55, 59 - 62 (5 pL of 20mM stock solution in DMA) was mixed with 95 pL DMA, 850 pL 10mM PBS solution (pH 7.4) and 50 pL of 20mM glutathione in PBS buffer solution. Controls were run without any glutathione: Linker compound (5 pL of 20mM stock dissolved in DMA) was diluted with 95 pL DMA and 900 pL of PBS buffer was added. LCMS of controls were collected at 2 - 3 varying timepoints. The samples were analysed by LCMS, so the LC chromatograms (abs 254nm) were integrated to obtain peak areas. The reactions were performed under pseudo first order conditions and the natural log plot of vinyl starting material concentration versus time gave linear plots, and kobs was determined from the gradient.

[0235] Results Compound 48 reacted the fastest under these conditions (reaction completion in approximately 10 min), closely followed by 49 and 47 (completion in approximately 15 min). These results demonstrate that the exact positioning of the nitrogen atom relative to the vinyl group greatly increases the rate of conjugation. Compound 49 is faster than 47, which indicates that having two nitrogen atoms surprisingly does not improve the reactivity. Thus, while compound 48 does possess higher reactivity, this may be due to the presence of the electron donating methyl substituent. Significantly, all linkers having a nitrogen-containing fused heterocyclic scaffold were more reactive in these studies than a vinylpyridine analogue 3 and vinylquinoline analogue 9.

[0236] The glutathione conjugation of 36 was complete within 30 minutes and the reactions with 41 and 50 took approximately 1 - 2 hours to complete. 36 / 41 were both faster than compounds 7 / 8, therefore these analogues all appear to be improvements. Compound 55 was observed to have lower reactivity than 8 and thus was excluded from further study. Compounds 60 - 62 showed comparable reactivity to 36.

[0237] Example 8. Stability of Linker Compounds in the Presence of A / Ac-Glu-OMe (G1) and A / Ac-Lys-OMe (L1).

[0238] Compounds 3, 7, 8, 9, 23, 34, 36, 41 , 47 - 50, 52, 55 and 60 - 62 were subjected to stability studies to confirm whether the compounds are stable in the presence of lysine (L1) or glutamate (G1) at pH 7.4, or undergo reaction to form byproducts.

[0239] Reactivity of compounds with A / Ac-Lys-OMe (L1) and A / Ac-Glu-OMe (G1)

[0240] Method: Compounds 3, 7, 8, 9, 23, 34, 36, 41 , 47 - 50, 52, 55 and 60 - 62 (5 pL of 20mM stock solution dissolved in DMA) was mixed with 95 pL DMA, 850 pL 10mM PBS solution (pH 7.4) and 50 pL of 20mM A / Ac-Lys-OMe (L1) or A / Ac-Glu-OMe (G1) in PBS buffer solution. The samples were analysed by LCMS at timepoints 0, 4 and 24 hours, so the LC chromatograms (abs 254nm) were integrated to obtain peak areas. Integrated peak areas were analysed to monitor changes in composition of the mixture and determine if any reaction with amine (L1) or carboxylic acid (G1) was observed.

[0241] Results:

[0242] As shown in Figure 13, compounds 3, 7, 9, 23, 34, 36, 41 , 47 - 50, 52 and 55 and 60 - 62 were shown to be highly stable in the presence of lysine (L1) or glutamate (G1), demonstrating that the vinyl group selectively reacts with thiol, and does not react with carboxylic acids or amine groups present within amino acid side chains of peptides. No formation of lysine (L1) or glutamate (G1) adducts was observed by LCMS. Bioconjugation Experiments

[0243] The linker compounds 61 and 62 were used to prepare MMAE-containing linker-drugs MMAE29 and MMAE30, as shown below. MMAE29 comprises vinylquinoline linker 61 , a PEG4 spacer, a glucuronide linker and MMAE. MMAE, or Monomethyl auristatin E, is a potent tubulin inhibitor used in several FDA-approved ADCs, including Adcetris® (brentuximab vedotin). MMAE30 uses vinylquinoline linker 62 in an otherwise identical structure.

[0244] Example 9. Preparation of antibody-drug conjugates (ADCs)

[0245] Linker-drugs MMAE29 and MMAE30 were conjugated to Trastuzumab, as a representative example of monoclonal antibody, and the kinetics of conjugation were monitored by PLRP analysis.

[0246] Method:

[0247] Trastuzumab antibody solubilised in PBS, 4mM EDTA, pH 7.4 was partially reduced with 0 - 5.0 molar equivalents of TCEP (25°C, 2 h) and subsequently conjugated with 5.0 or 10.0 molar equivalents of MMAE29 or MMAE30 dissolved in PBS, 4mM EDTA, pH 7.4 with (5% (v / v) DMA co-solvent and incubated at 30°C. Samples were taken for analysis after 10 minutes, 2 hours or 18 hours, and conjugation reactions were halted by two consecutive Sephadex G-25 buffer exchange columns of increasing column volumes to remove residual linker-toxins (PBS, pH 7.4). DAR values were then analysed by PLRP. Method for PLRP analysis:

[0248] Conjugate samples were reduced with 2mM DTT (37°C, 15min) and analysed by PLRP chromatography, resolving peaks corresponding to unconjugated or drug / dye- conjugated antibody light and heavy chains. DARs are determined from the relative proportions of unconjugated light- and heavy-chain (L0 and HO) and conjugated light- (L1) and heavy-chain (H1 , H2, H3). Peak separation was performed on an Agilent PLRP-S (1000A, 2.1 x 50mm, 5pm) column, eluting with mobile phase A (water + 0.1 % TFA) and gradient (30 - 50%) of mobile phase B (MeCN + 0.1 % TFA).

[0249] Results:

[0250] MMAE29 and MMAE30 both showed rapid conjugation kinetics with Trastuzumab, reaching DAR 3.9 - 5.2 within 2 hours. MMAE29 showed more rapid conjugation than MMAE30, reaching DAR 3.5 after 10 minutes (Figure 14A) when 3 equivalents of TCEP and 5 equivalents of linker-drug were used for the reaction. Using 10 equivalents of MMAE29 (Figure 14B) afforded DAR 4.4 after 10-minute conjugation, whilst MMAE30 gave DAR 3.4 under the same conditions.

[0251] Incubating MMAE29 or MMAE30 with Trastuzumab in the absence of TCEP reducing agent showed no conjugation, with PLRP chromatogram being indistinguishable from the unconjugated antibody (Figure 15). These control experiments demonstrate that MMAE29 and MMAE30 both exhibit thiol-specific conjugation to antibody, with no modification of lysine, or other non-cysteine amino acid residues.

[0252] Example 10. PLRP and SEC analysis of MMAE29 and MMAE30 conjugates.

[0253] PLRP Method:

[0254] Column: PLRP-S 1000A 5pM.

[0255] Flow rate: 0.6 mL / min Injection vol.; 5 - 10 pL; Column temp; 80 °C.

[0256] Mobile phase: 30-50% gradient of buffer B (MeCN + 0.1 % TFA) in buffer A (water + 0.1 % TFA).

[0257] SEC Method:

[0258] Column: Thermo Scientific MabPacSEC-1 5pM

[0259] Flow Rate: 0.8mL / min. Injection Volume: 10pL. Mobile phase: Isocratic 20mM MES Sodium Salt, 150mM NaCI, 5% MeCN, pH 6.0. Results:

[0260] Figures 16A and 16B show representative PLRP and SEC chromatograms of Trastuzumab-MMAE29 and Trastuzumab-MMAE30. Both conjugates showed negligible aggregation content (> 99% monomeric) by size-exclusion chromatography, demonstrating that the conjugates have excellent biophysical properties.

[0261] Example 11. In vitro activity of Trastuzumab-MMAE29 or Trastuzumab-MMAE30 in N87 and SKBR-3 cell lines.

[0262] Trastuzumab conjugates of MMAE29 and MMAE30 were evaluated in vitro alongside Isotype Control-MMAE to confirm the ADCs showed specific cytotoxic activity. Isotype Control-MMAE is a Glucuronide-MMAE antibody-drug conjugate that does not bind to antigens expressed upon the surface of N87 or SKBR-3 cell lines.

[0263] Method for ADC generation

[0264] ADCs comprised of MMAE29 and MMAE30, and Isotype Control-MMAE were prepared and characterised according to the methods of Examples 9-10.

[0265] Method for cell-line maintenance

[0266] N87 and SKBR3 cells were obtained from ATCC. Cell-lines were maintained in complete RPMI-1640 medium with addition of 10% foetal bovine serum. Cells were cultured at 37 °C in a humidified 5 % CO2 incubator and passaged twice weekly.

[0267] Method for cell viability assay

[0268] N87 and SKBR3 cells were seeded to white, clear-bottom 96-well microplates for a final cell concentration of 3000 cells / well. Plates were incubated for 16-24 hours.

[0269] Trastuzumab-MMAE29, Trastuzumab-MMAE30 and Isotype Control-MMAE were tested in a 9-point dose-response assay with 5-fold serial dilution, starting from 10 nM ADC. Microplates were incubated for 120 h prior to Cell-Titre Gio (CTG, Promega) assay performed according to manufacturer’s instructions. Luminescence was measured using a plate reader (GloMax Discover Promega).

[0270] Luminescence readings were normalized to cells only controls (100 % viability) and media only controls (0% viability). The resulting data were analysed using GraphPad Prism. A non-linear regression ([inhibitor] vs. response - variable slope (four parameters) curve fit was applied to determine the ECso.

[0271] Results:

[0272] Trastuzumab conjugates of MMAE29, MMAE30 showed cell-killing activity (Figure 17), with comparable IC50 values (see Table 4) in both N87 and SKBR-3 cell lines. No activity was demonstrated with Isotype Control-MMAE in both N87 and SKBR-3 celllines.

[0273] Table 4: IC50 values for ADCs comprised of MMAE29 and MMAE30; where (T) denotes an IC50 based on the concentration of payload moiety, and (P) denotes the IC50 based on concentration of protein.

[0274] Chemical Synthesis of Compounds

[0275] The bioconjugation compounds, linker-drugs and conjugates of the invention can be made using the synthetic procedures outlined below. Unless stated otherwise, reactions were performed under nitrogen atmosphere. All solvents were anhydrous grade.

[0276] Nomenclature of the compounds is based on the following numbering system:

[0277] Scheme 2: Nomenclature of vinylquinoline analogues Synthesis of compound 7 / - r , ,

[0278] Compound 7

[0279] Scheme 3: Synthesis of compound 7.

[0280] To a solution of 4-bromoquinoline (253 mg, 1.22 mmol, 1.0 eq) and potassium vinyltrifluoroborate (243.8 mg, 1.82 mmol, 1.5 eq) in / -PrOH (10 mL) was added Et3N (185.18 mg, 1.82 mmol, 1.5 eq) and Pd(dppf)Ch (26.8 mg, 0.037 mmol, 0.03 eq). The mixture was stirred at 90 °C under nitrogen for 3 hours. The mixture was concentrated and purified by column chromatography (SiO2, petroleum ether / EtOAc, 2:1 , v / v) to afford 4-vinylquinoline (7) (95 mg, 50% yield) as a green oil. LCMS m / z = 156.2 [M+H]+.1H NMR (300 MHz, CDCh) 6 8.89 (d, J = 4.6 Hz, 1 H), 8.12 (m, J = 8.9 Hz, 2H), 7.73 (ddd, J = 8.3 Hz, 1 H), 7.58 (ddd, J = 8.2 Hz, 1 H), 7.49 - 7.39 (m, 2H), 6.02 (dd, J = 17.4 Hz, 1 H), 5.70 (dd, J = 11.1 Hz, 1 H).

[0281] Synthesis of compound 8 / -PrOH, 90 C, 3 h

[0282] Compound 8

[0283] Scheme 4: Synthesis of compound 8.

[0284] To a solution of methyl 4-bromoquinoline-6-carboxylate (300 mg, 1.13 mmol, 1.0 eq), in / -PrOH (10 mL) was added potassium vinyltrifluoroborate (166 mg, 1.24 mmol, 1.1 eq) and Pd(dppf)Ch (16.5 mg, 0.023 mmol, 0.02 eq), followed by dropwise addition of Et3N (172 mg, 1.70 mmol, 1.5 eq) at room temperature. The reaction vessel was degassed three times by nitrogen / vacuum flushes. The resulting reaction mixture was stirred at 90 °C for 3 hours, then cooled to 20 °C. The precipitate was filtered and the filtrate concentrated in vacuo with the addition of TEMPO (15 mg) to give a red solid. The crude product was purified by column chromatography (SiO2, petroleum ether / EtOAc, 4:1 to 1 :1 , v / v) to afford 8 (141 mg, 59% yield) as a pale green solid. LCMS m / z = 214.1 [M+H]+.1H NMR (300 MHz, CDCh) 6 8.95 (d, J = 6.0 Hz, 1 H), 8.86 (s, 1 H), 8.28 (m, 1 H), 8.13 (d, J = 9.0 Hz, 1 H), 7.51 (m, 1 H), 7.44 (m, 1 H), 6.03 (d, J = 18.0 Hz, 1 H), 5.73 (d, J = 12.0 Hz ,1 H), 3.99 (s, 3H). Synthesis of compound 9 / - r , ,

[0285] Compound 9

[0286] Scheme 5: Synthesis of compound 9.

[0287] To a solution of 4-bromo-6-methoxyquinoline (100 mg, 0.42 mmol, 1.0 eq) and potassium vinyltrifluoroborate (113 mg, 0.84 mmol, 2.0 eq) in / -PrOH (2 mL) was added Et3N (127.5 mg, 1.26 mmol, 3.0 eq) and Pd(dppf)Ch (30.7 mg, 0.04 mmol, 0.1 eq). The mixture was stirred at 90 °C under nitrogen for 4 hours. The mixture was concentrated and purified by column chromatography (SiO2, petroleum ether / EtOAc, 10:1 , v / v) to afford 9 (60 mg, 77% yield) as a solid. LCMS m / z = 186.1 [M+H]+.1H NMR (400 MHz, CDCh) 5 8.74 (d, = 4.6 Hz, 1 H), 8.05 (d, = 9.2 Hz, 1 H), 7.45 (d, = 4.6 Hz, 1 H), 7.41 - 7.29 (m, 3H), 5.98 (dd, = 17.3, 1.1 Hz, 1 H), 5.67 (dd, = 11.0, 1.1 Hz, 1 H), 3.96 (s, 3H).

[0288] Synthesis of compound 10

[0289] Scheme 6: Synthesis of compound 10.

[0290] To a solution of 2,4-dichloro-6-methylpyrimidine (123 mg, 0.75 mmol, 1.05 eq) in 1 ,2- dichloroethane / t-BuOH (v / v 1 :1 , 4 mL / 4 mL) was added ZnCh (117 mg, 0.86 mmol, 1 .2 eq) slowly. The reaction mixture was stirred at room temperature under nitrogen for 10 min. Then methyl 3-aminopropanoate hydrochloride (100 mg, 0.72 mmol, 1.0 eq) and Et3N (247 mg, 2.44 mmol, 3.4 eq) were added. The reaction mixture was stirred at room temperature under nitrogen for 16 hours. The mixture was concentrated and purified by column chromatography (SiC>2, petroleum ether / EtOAc, 6:1 , v / v) to afford methyl 3-((4-chloro-6-methylpyrimidin-2-yl) amino) propanoate (20 mg, 12% yield) as a white solid. LCMS m / z = 230.0 [M+H]+.1H NMR (400 MHz, CDCh) 5 6.49 (s, 1 H), 5.67 (s, br, 1 H), 3.80 - 3.73 (m, 2H), 3.73 (s, 3H), 2.66 (t, = 6.2 Hz, 2H), 2.33 (s, 3H). To a solution of methyl 3-((4-chloro-6-methylpyrimidin-2-yl) amino) propanoate (80 mg, 0.35 mmol, 1 .0 eq), potassium vinyltrifluoroborate (93 mg, 0.70 mmol, 2.0 eq) and Et3N (106 mg, 1.04 mmol, 3.0 eq) in / -PrOH (2 mL) was added Pd(dppf)Ch (25.5 mg, 0.035 mmol, 0.1 eq). The reaction mixture was stirred at 90 °C under nitrogen for 4 hours. The mixture was concentrated and purified by column chromatography (SiC>2, petroleum ether / EtOAc, 4:1 , v / v) to afford 10 (59 mg, 76% yield) as a white solid. LCMS m / z = 222.1 [M+H]+.1H NMR (400 MHz, CDC ) 6 6.53 (dd, J = 17.3, 10.5 Hz, 1 H), 6.41 (s, 1 H), 6.34 (dd, J = 17.3, 1.6 Hz, 1 H), 5.53 (dd, J = 10.5, 1.6 Hz, 1 H), 5.43 (s, br, 1 H), 3.75 (q, J = 6.3 Hz, 2H), 3.68 (s, 3H), 2.65 (t, J = 6.3 Hz, 2H), 2.31 (s, 3H).

[0291] Synthesis of compound 23

[0292] Compound 23

[0293] Scheme 7: Synthesis of compound 23.

[0294] To a solution of 4-bromoquinolin-6-amine (120 mg, 0.54 mmol, 1.0 eq) in CH2CI2 (2.5 mL) was added Et3N (328 mg, 3.24 mmol. 6.0 eq) and AC2O (276 mg, 2.70 mmol, 5.0 eq) slowly. The mixture was stirred at room temperature under nitrogen for 4 hours. The mixture was diluted with water (20 mL) and extracted with CH2CI2 (3 x 20 mL). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated to afford / V-(4-bromoquinolin-6-yl)acetamide (130 mg, quantitative yield) as an oil. LCMS m / z = 266.0 [M+H]+.1H NMR (400 MHz, DMSO-d6) 5 10.46 (s, 1 H), 8.63 (d, J = 2.3 Hz, 1 H), 8.59 (d, J = 4.7 Hz, 1 H), 8.01 (d, J = 9.1 Hz, 1 H), 7.92 (dd, J = 9.1 , 2.3 Hz, 1 H), 7.88 (d, J = 4.7 Hz, 1 H), 2.14 (s, 3H).

[0295] To a solution of / V-(4-bromoquinolin-6-yl)acetamide (130 mg, 0.49 mmol, 1.0 eq) and potassium vinyltrifluoroborate (132 mg, 0.98 mmol, 2.0 eq) in / -PrOH (3 mL) was added Et3N (149 mg, 1.48 mmol, 3.0 eq) and Pd(dppf)Ch (36 mg, 0.05 mmol, 0.1 eq). The mixture was stirred at 90 °C under nitrogen for 3 hours. The mixture was concentrated and purified by preparative TLC (SiO2, petroleum ether / EtOAc, 6:1 , v / v) to afford 23 (49 mg, 48% yield) as a solid. LCMS m / z = 213.1 [M+H]+.1H NMR (400 MHz, DMSO- d6) 6 10.31 (s, 1 H), 8.74 (d, J = 4.6 Hz, 1 H), 8.59 (d, J = 2.2 Hz, 1 H), 7.97 (d, J = 9.0 Hz, 1H), 7.84 (dd, J= 9.1, 2.3 Hz, 1H), 7.60 (d, J = 4.5 Hz, 1H), 7.35 (dd, J= 17.4, 11.1 Hz, 1H), 6.11 (d, J= 17.3 Hz, 1H), 5.73 (d, J= 10.8 Hz, 1H), 2.12 (s, 3H).

[0296] Synthesis of compound 24

[0297] Scheme 8: Synthesis of compound 24.

[0298] To a solution of 4-bromoquinoline-2-carboxylic acid (100 mg, 0.40 mmol, 1.0 eq) and MeNH2'HCI (54 mg, 0.80 mmol, 2.0 eq) in DMF (3 mL) was added DIPEA (205 mg, 1.59 mmol.4.0 eq) and HATU (226 mg, 0.60 mmol, 1.5 eq). The reaction mixture was stirred at room temperature under nitrogen overnight. The mixture was diluted with water (10 mL) and extracted with EtOAc (3 x 10 mL), The mixture was purified by column chromatography (SiC>2, petroleum ether / EtOAc, 5:1, v / v) to afford 4-bromo-A / - methylquinoline-2-carboxamide (80 mg, 75 % yield) as a yellow solid. LCMS m / z = 265.0 [M+H]+.1H NMR (400 MHz, DMSO-d6) 58.96 (s, 1H), 8.40 (s, 1H), 8.24-8.20 (m, 1H), 8.17 (d, J= 8.4 Hz, 1H), 7.98 (ddd, J= 8.4, 6.9, 1.4 Hz, 1H), 7.91 -7.85 (m, 1H), 2.89 (d, J = 4.8 Hz, 3H).

[0299] To a solution of 4-bromo- / V-methylquinoline-2-carboxamide (68.0 mg, 0.25 mmol, 1.0 eq) and potassium vinyltrifluoroborate (69 mg, 0.51 mmol, 2.0 eq) in / -PrOH (1 mL) was added Pd(dppf)Ch (19 mg, 0.02 mmol, 0.1 eq) and Et3N (78 mg, 0.77 mmol, 3.0 eq) at room temperature. The reaction mixture was stirred at 90 °C under nitrogen overnight. The mixture was concentrated and purified by column chromatography (SiC>2, CH2Cl2 / MeOH, 50:1, v / v) to afford 24 (36 mg, 68% yield) as a white solid. LCMS: m / z = 213.1 [M+H]+.1H NMR (400 MHz, DMSO-d6) 58.90 (s, 1 H), 8.36 - 8.31 (m, 1H), 8.25 (s, 1H), 8.17-8.09 (m, 1H), 7.88 (ddd, J= 8.4, 6.8, 1.4 Hz, 1H), 7.77-7.61 (m, 2H), 6.22 (dd, J= 17.2, 1.1 Hz, 1H), 5.79 (dd, J= 11.0, 1.1 Hz, 1H), 2.90 (d, J = 4.8 Hz, 3H). Synthesis of compound 25

[0300] Compound 25

[0301] Scheme 9: Synthesis of compound 25.

[0302] To a solution of lithium 4-vinylquinoline-8-carboxylate (65 mg, 0.32 mmol, 1.0 eq) and DIPEA (205 mg, 1.58 mmol, 5.0 eq) in DMF (1 mL) was added MeNH2'HCI (43 mg, 0.63 mmol, 2 eq) and T3P (50% solution in EtOAc, 403 mg, 0.63 mmol, 2.0 eq). The mixture was stirred at room temperature under nitrogen overnight. The mixture was diluted with water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic extracts were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue obtained was purified by preparative TLC (SiO2, CF^Ch / MeOH, 20:1 , v / v) to afford 25 (15 mg, 22% yield) as an oil. LCMS m / z = 213.1 [M+H]+.1H NMR (400 MHz, CDCI3) 6 11 .22 (s, 1 H), 8.92 - 8.86 (m, 2H), 8.25 (dd, J = 8.4, 1 .5 Hz, 1 H), 7.69 (dd, J = 8.4, 7.4 Hz, 1 H), 7.54 (d, J = 4.6 Hz, 1 H), 7.45 (dd, J = 17.3, 11.0 Hz, 1 H), 6.00 (dd, J = 17.3, 1.1 Hz, 1 H), 5.73 (dd, J = 11.0, 1.1 Hz, 1 H), 3.15 (d, J = 4.7 Hz, 3H).

[0303] Synthesis of compound 34

[0304] / -PrOH, 90 °C, 2.5 h Compound 34

[0305] Scheme 10: Synthesis of compound 34.

[0306] To a solution of 4-bromoquinolin-6-amine (200 mg, 0.90 mmol, 1.0 eq) in HFIP (4 mL) was added methyl trifluoromethanesulfonate (221 mg, 1.34 mmol, 1.5 eq). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. The mixture was concentrated and purified by column chromatography (SiC>2, CH2Cl2 / EtOAc, 5:1 to 4:1 , v / v) to afford 4-bromo- / V-methylquinolin-6-amine (140 mg, 66% yield) as a yellow solid. LCMS m / z = 237.0 [M+H]+.1H NMR (400 MHz, CDCI3) 6 8.36 (d, J = 4.7 Hz, 1 H), 7.87 (d, J = 9.1 Hz, 1 H), 7.58 (d, J = 4.7 Hz, 1 H), 7.12 (dd, J = 9.0, 2.7 Hz, 1 H), 6.97 (d, J = 2.6 Hz, 1 H), 4.22 (br s, 1 H, NH), 3.00 (s, 3H). To a solution of 4-bromo- / V-methylquinolin-6-amine (70 mg, 0.29 mmol, 1.0 eq), potassium vinyltrifluoroborate (79 mg, 0.59 mmol, 2.0 eq) and Et3N (89.6 mg, 0.88 mmol, 3.0 eq) in / -PrOH (1.4 mL) was added Pd(dppf)Ch (22 mg, 0.03 mmol, 0.1 eq). The reaction mixture was stirred at 90 °C under nitrogen for 2.5 hours. The mixture was concentrated and purified by preparative TLC (SiC>2, CH2CI2 / EtOAc, 1 :1 , v / v) to afford 34 (35 mg, 65% yield) as a yellow oil. LCMS m / z = 185.1 [M+H]+.1H NMR (400 MHz, DMSO-ds) 6 8.44 (d, J = 4.5 Hz, 1 H), 7.69 (s, 1 H), 7.51 - 7.38 (m, 2H), 7.18 (dd, J = 9.1 , 2.5 Hz, 1 H), 6.77 (d, J = 2.5 Hz, 1 H), 6.28 (d, J = 5.0 Hz, 1 H), 6.02 (dd, J = 17.4, 1.4 Hz, 1 H), 5.61 (dd, J = 11.0, 1.4 Hz, 1 H), 2.81 (d, J = 5.0 Hz, 3H).

[0307] Synthesis of compound 35

[0308] Compound 35

[0309] Scheme 11 : Synthesis of compound 35.

[0310] To a solution of methyl 4-bromoquinoline-8-carboxylate (300 mg, 1.13 mmol, 1.0 eq), 4,4,5,5-tetramethyl-2-vinyl-1 ,3,2-dioxaborolane (348 mg, 2.26 mmol, 2.0 eq) and K2CO3 (469 mg, 3.39 mmol, 3.0 eq) in DME / H2O (3 mL, 1 :1 , v / v) was added Pd(PPhs)4 (131 mg, 0.11 mmol, 0.1 eq). The reaction mixture was stirred at 80 °C under nitrogen overnight. The mixture was diluted with water and extracted with EtOAc. The combined organic extracts were dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, petroleum ether / EtOAc, 5:1 to 4:1 , v / v) to afford methyl 4-vinylquinoline-8-carboxylate (160 mg, 67% yield) as a yellow solid. LCMS m / z = 214.2 [M+H]+.1H NMR (400 MHz, CDCI3) 6 9.01 (d, J = 4.5 Hz, 1 H), 8.24 (d, J = 8.5 Hz, 1 H), 8.00 (d, J = 7.1 Hz, 1 H), 7.62 - 7.55 (m, 1 H), 7.52 (d, J = 4.5 Hz, 1 H), 7.42 (dd, J = 17.3, 11.0 Hz, 1 H), 6.00 (d, J = 17.3 Hz, 1 H), 5.71 (d, J = 11.1 Hz, 1 H), 4.06 (s, 3H).

[0311] To a solution of methyl 4-vinylquinoline-8-carboxylate (121 mg, 0.57 mmol, 1.0 eq) in THF / MeOH (0.6 mL, 1 :1 , v / v) was added LiOH H2O (31 mg, 0.74 mmol, 1.3 eq) in H2O (0.3 mL). The reaction mixture was stirred at room temperature for 3 hours. The mixture was concentrated under a vacuum and the crude was washed with Et20 to afford 35 (100 mg, 86% yield) as a white solid. LCMS m / z = 200.1 [M+H]+.1H NMR (400 MHz, D2O) 5 8.80 (d, J = 4.7 Hz, 1 H), 8.24 (dd, J = 8.5, 1.5 Hz, 1 H), 7.76 - 7.63 (m, 3H), 7.56 (dd, J = 17.4, 11.1 Hz, 1 H), 6.13 (dd, J = 17.4, 1.1 Hz, 1 H), 5.78 (dd, J = 11.0, 1.1 Hz, 1 H).

[0312] Synthesis of compound 36

[0313] Scheme 12: Synthesis of compound 36.

[0314] To a solution of 4-chloroquinolin-8-amine (400 mg, 2.24 mmol, 1.0 eq) in DMF (40 mL) was added K2CO3 (3.1 g, 22.39 mmol, 10 eq) and Mel (1.6 g, 11.20 mmol, 5.0 eq) at 0 °C. The reaction mixture was stirred at 80 °C under nitrogen for 16 hours. The mixture was diluted with water and extracted with EtOAc (5 x 20 mL). The combined organic layers were washed with water and brine, dried over Na2SO4 and concentrated in vacuo. The crude was purified by column chromatography (SiC>2, petroleum ether / EtOAc, 25:1 , v / v) to afford 4-chloro- / V, / V-dimethylquinolin-8 amine (395 mg, 86% yield) as a yellow oil. LCMS m / z = 207.1 [M+H]+.1H NMR (400 MHz, DMSO-d6) 5 8.74 (d, J = 4.6 Hz, 1 H), 7.71 (d, J = 4.6 Hz, 1 H), 7.67 (dd, J = 8.4, 1.3 Hz, 1 H), 7.58 (t, J = 8.0 Hz, 1 H), 7.15 (dd, J= 7.7, 1.3 Hz, 1 H), 3.05 (s, 6H).

[0315] To a solution of 4-chloro-A / , / V-dimethylquinolin-8 amine (150 mg, 0.73 mmol, 1.0 eq), potassium vinyltrifluoroborate (195 mg, 1.46 mmol, 2.0 eq) and K2CO3 (302 mg, 2.18 mmol, 3.0 eq) in DME / H2O (5 mL, 4:1 , v / v) was added Pd(PPh3)4 (84 mg, 0.07 mmol, 0.1 eq). The reaction mixture was stirred at 90 °C under nitrogen for 6 hours. The mixture was diluted with water and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated. The crude was purified by column chromatography (SiO2, petroleum ether / EtOAc, 20:1 , v / v) to afford 36 (55 mg, 38% yield) as a yellow oil. LCMS m / z = 199.2 [M+H]+.1H NMR (400 MHz, DMSO-ds) 6 8.79 (d, J = 4.5 Hz, 1 H), 7.70 (dd, J = 8.4, 1.2 Hz, 1 H), 7.61 (d, J = 4.5 Hz, 1 H), 7.53 (dd, J = 17.3, 11.0 Hz, 1 H), 7.46 (t, J = 8.0 Hz, 1 H), 7.08 (dd, J = 7.6, 1.2 Hz, 1 H), 6.07 (dd, J = 17.3, 1.3 Hz, 1 H), 5.66 (dd, J = 11.0, 1.3 Hz, 1 H), 3.03 (s, 6H). Synthesis of compound 41 / -PrOH, 90 °C, 4 h

[0316] Compound 41

[0317] Scheme 13: Synthesis of compound 41.

[0318] To a solution of 4-bromo-2-methylquinoline (100 mg, 0.45 mmol, 1.0 eq) and potassium vinyltrifluoroborate (121 mg, 0.90 mmol, 2.0 eq) in / -PrOH (2 mL) was added Et3N (137 mg, 1.35 mmol, 3.0 eq) and Pd(dppf)Ch (33 mg, 0.04 mmol, 0.1 eq). The mixture was stirred at 90 °C under nitrogen for 4 hours. The mixture was concentrated in vacuo and purified by column chromatography (SiO2, petroleum ether / EtOAc, 20:3, v / v) to afford 41 (49 mg, 64% yield) as a yellow oil. LCMS m / z = 170.1 [M+H]+.1H NMR (400 MHz, CDCh) 6 8.10 - 7.97 (m, 2H), 7.75 - 7.64 (m, 1 H), 7.55 - 7.47 (m, 1 H), 7.42 - 7.32 (m, 2H), 5.96 (dd, J = 17.4, 1.2 Hz, 1 H), 5.64 (dd, J = 11.0, 1.2 Hz, 1 H), 2.75 (s, 3H).

[0319] Synthesis of compound 43

[0320] Scheme 14: Synthesis of compound 43.

[0321] To a solution of 2,4-dichloroquinoline (1.55 g, 7.81 mmol, 1.0 eq) in EtOH (10 mL) was added MeNH2 in THF (2 M, 45.5 mL, 9.37 mmol, 1.2 eq). The mixture was stirred at 50 °C under nitrogen overnight. The mixture was concentrated in vacuo and purified by column chromatography (SiC>2, petroleum ether / EtOAc, 12:1 , v / v) to afford 4-chloro- A / -methylquinolin-2-amine (368 mg, 25% yield) as a solid.1H NMR (400 MHz, CDCh) 5 7.98 (d, J = 1.4 Hz, 1 H), 7.70 (d, J = 8.4 Hz, 1 H), 7.58 (ddd, J = 8.4, 6.9, 1.5 Hz, 1 H), 7.32 - 7.27 (m, 1 H), 6.77 (s, 1 H), 4.80 (br s, 1 H, NH), 3.08 (d, J = 5.0 Hz, 3H).

[0322] To a solution of 4-chloro- / V-methylquinolin-2-amine (100 mg, 0.52 mmol, 1.0 eq) and 4,4,5,5-tetramethyl-2-vinyl-1 ,3,2-dioxaborolane (240 mg, 1.56 mmol, 3.0 eq) in 1 ,4- dioxane / H2O (3.3 mL, 10:1 , v / v) was added K2CO3 (574 mg, 4.15 mmol, 8.0 eq) and Pd(dppf)Ch (380 mg, 0.52 mmol, 1.0 eq). The mixture was stirred at 110 °C under nitrogen for 24 hours. The mixture was diluted with water (30 mL) and extracted with EtOAc (3 x 30 mL). The combined organic layers were washed with brine, dried over Na2SC>4 and concentrated. The residue obtained was purified by preparative TLC (CH2Cl2 / MeOH / aqueous NH3, 20:1 :0.1 , v / v) to afford 43 (45 mg, 47% yield) as an oil. LCMS m / z = 185.1 [M+H]+.1H NMR (400 MHz, CDCh) 6 7.84 (dd, J = 8.2, 1.4 Hz, 1 H), 7.72 (dd, J = 8.4, 1.2 Hz, 1 H), 7.54 (ddd, J = 8.4, 6.8, 1.5 Hz, 1 H), 7.33 - 7.28 (m, 1 H), 7.26 - 7.21 (m, 1 H), 6.71 (s, 1 H), 5.86 (dd, J = 17.3, 1.4 Hz, 1 H), 5.58 (dd, J = 11.0, 1.4 Hz, 1 H), 4.91 (s, 1 H), 3.11 (d, J = 5.0 Hz, 3H).

[0323] Synthesis of compound 47 ompoun 47

[0324] Scheme 15: Synthesis of compound 47.

[0325] To a solution of 4-chloroquinazoline (200.0 mg, 1.22 mmol, 1.0 eq), potassium vinyltrifluoroborate (326 mg, 2.43 mmol, 2.0 eq) and K2CO3 (504 mg, 3.64 mmol, 3.0 eq) in DME / H2O (5 mL, 4:1 , v / v) was added Pd(PPhs)4 (140 mg, 0.12 mmol, 0.1 eq). The reaction mixture was stirred at 90 °C under nitrogen for 6 hours. The mixture was concentrated and purified by preparative TLC (Ch^Ch / EtOAc, 8:1 , v / v) to afford 47 (24 mg, 13% yield) as a yellow oil. LCMS m / z = 157.1 [M+H]+.1H NMR (400 MHz, DMSO- d6) 6 9.25 (s, 1 H), 8.50 (d, J = 8.4 Hz, 1 H), 8.07 - 7.97 (m, 2H), 7.85 (dd, J = 16.8, 10.6 Hz, 1 H), 7.77 (ddd, J = 8.3, 5.2, 2.9 Hz, 1 H), 6.81 (dd, J = 16.7, 2.2 Hz, 1 H), 5.94 (dd, J = 10.6, 2.2 Hz, 1 H).

[0326] Synthesis of compound 48

[0327] Compound 48

[0328] Scheme 16: Synthesis of compound 48.

[0329] To a solution of 4-chloro-2-methylquinazoline (100 mg, 0.56 mmol, 1.0 eq) and potassium vinyltrifluoroborate (150 mg, 1.12 mmol, 2.0 eq) in DME / H2O (2.5 mL, 4:1 , v / v) was added K2CO3 (232 mg, 1.68 mmol, 3.0 eq) and Pd(PPhs)4 (65 mg, 0.06 mmol, 0.1 eq). The mixture was stirred at 90 °C under nitrogen for 2 hours. The mixture was concentrated and purified by preparative TLC (SiC>2, petroleum ether / EtOAc, 6:1 , v / v) to afford 48 (68 mg, 71 % yield) as an oil. LCMS m / z = 171.1 [M+H]+.1H NMR (400 MHz, CDCh) 68.16 (d, J = 8.4 Hz, 1H), 7.95 (d, J= 8.5 Hz, 1H), 7.84 (ddd, J = 8.4, 6.8, 1.4 Hz, 1H), 7.60-7.51 (m, 2H), 6.78 (dd, J= 16.9, 1.8 Hz, 1H), 5.88 (dd, J = 10.7, 1.8 Hz, 1H), 2.89 (s, 3H).

[0330] Synthesis of compound 49 / -PrOH, 90 °C, 3 h Compound 49

[0331] Scheme 17: Synthesis of compound 49.

[0332] To a solution of 1-chloroisoquinoline (100 mg, 0.61 mmol, 1.0 eq) and potassium vinyltrifluoroborate (163 mg, 1.22 mmol, 2.0 eq) in / -PrOH (2 mL) was added Et3N (186 mg, 1.83 mmol, 3.0 eq) and Pd(dppf)Ch (45 mg, 0.06 mmol, 0.1 eq). The mixture was stirred at 90 °C under nitrogen for 3 hours. The mixture was concentrated and purified by column chromatography (SiO2, petroleum ether / EtOAc, 10:1, v / v) to afford 49 (37 mg, 39% yield) as an oil. LCMS m / z = 156.0 [M+H]+.1H NMR (400 MHz, CDCh) 68.53 (d, J= 5.6 Hz, 1H), 8.27 (d, J= 8.2 Hz, 1H), 7.83 (d, J= 8.0 Hz, 1H), 7.71 -7.56 (m, 4H), 6.52 (dd, J= 17.0, 3.9 Hz, 1H), 5.72 (dd, J= 11.1, 3.8 Hz, 1H).

[0333] Synthesis of compound 50 2,3

[0334] / -PrOH, 90 °C, 3 h Compound 50

[0335] Scheme 18: Synthesis of compound 50.

[0336] To a solution of 2-chloroquinoline (150 mg, 0.92 mmol, 1.0 eq) and potassium vinyltrifluoroborate (246 mg, 1.83 mmol, 2.0 eq) in / -PrOH (3 mL) was added Et3N (278 mg, 2.75 mmol, 3.0 eq) and Pd(dppf)Ch (67 mg, 0.09 mmol, 0.1 eq). The mixture was stirred at 90 °C under nitrogen for 3 hours. The mixture was concentrated in vacuo and purified by preparative TLC (SiO2, petroleum ether / EtOAc, 10:1, v / v) to afford 50 (80 mg, 56% yield) as an oil. LCMS m / z = 156.1 [M+H]+.1H NMR (400 MHz, CDCh) 6 8.10 (dd, J= 19.4, 8.5 Hz, 2H), 7.78 (dd, J= 8.1, 1.5 Hz, 1H), 7.70 (ddd, J= 8.5, 6.9, 1.5 Hz, 1H), 7.61 (d, J= 8.6 Hz, 1H), 7.50 (ddd, J= 8.1, 6.8, 1.3 Hz, 1H), 7.05 (dd, J = 17.7, 10.9 Hz, 1H), 6.28 (dd, J= 17.6, 1.0 Hz, 1H), 5.67 (dd, J= 10.9, 1.0 Hz, 1H) Synthesis of compound 51 ompoun

[0337] Scheme 19: Synthesis of compound 51.

[0338] To a solution of 2-chloroquinoxaline (200 mg, 1.21 mmol, 1.0 eq) and potassium vinyltrifluoroborate (326 mg, 2.43 mmol, 2.0 eq) in DME / H2O (5 mL, 4:1 , v / v) was added K2CO3(504 mg, 3.64 mmol, 3.0 eq) and Pd(PPhs)4 (140 mg, 0.12 mmol, 0.1 eq). The mixture was stirred at 80 °C under nitrogen for 2 hours. The mixture was concentrated in vacuo and purified by column chromatography (SiO2, petroleum ether / EtOAc, 10:1 , v / v) to afford 51 (110 mg, 58% yield) as an oil. LCMS m / z = 157.1 [M+H]+.1H NMR (400 MHz, DMSO-d6) 5 9.22 (s, 1 H), 8.06 (ddd, J = 9.2, 7.8, 1.7 Hz, 2H), 7.83 (pd, J = 7.0, 1.7 Hz, 2H), 7.11 - 7.01 (m, 1 H), 6.60 (dd, J = 17.8, 1.0 Hz, 1 H), 5.84 (dd, J = 11.1 , 1.0 Hz, 1 H).

[0339] Synthesis of compound 52

[0340] Compound 52

[0341] Scheme 20: Synthesis of compound 52.

[0342] To a solution of lithium 4-vinylquinoline-6-carboxylate (100 mg, 0.49 mmol, 1.0 eq) and DIPEA (378 mg, 2.92 mmol, 6.0 eq) in DMF (2 mL) was added MeNH2HCI (82.3 mg, 1.22 mmol, 2.5 eq) and T3P (50% solution in EtOAc, 620 mg, 0.97 mmol, 2.0 eq). The mixture was stirred at room temperature under nitrogen for 4 hours. The mixture was diluted with water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated. The residue obtained was purified by preparative TLC (SiO2, CH2CI2 / MeOH, 15:1 , v / v) to afford 52 (30 mg, 29% yield) as a solid. LCMS m / z = 213.1 [M+H]+.1H N MR (400 MHz, CDCh) 6 8.93 (dd, J = 4.6, 1.7 Hz, 1 H), 8.61 (d, J = 1.8 Hz, 1 H), 8.19 - 8.12 (m, 1 H), 8.03 - 7.97 (m, 1 H), 7.53 (d, J = 5.2 Hz, 1 H), 7.51 - 7.43 (m, 1 H), 6.38 (s, 1 H), 6.02

[0343] (d, J = 17.3 Hz, 1 H), 5.73 (d, J = 10.4 Hz, 1 H), 3.09 (dd, J = 4.8, 1.7 Hz, 3H). Synthesis of compound 55

[0344] Scheme 21 : Synthesis compound 55.

[0345] To a solution of 9-chloroacridine (100 mg, 0.47 mmol, 1.0 eq) and potassium vinyltrifluoroborate (125 mg, 0.94 mmol, 2.0 eq) in DME / H2O (2.5 mL, 4:1 , v / v) was added K2CO3 (194 mg, 1.40 mmol, 3.0 eq) and Pd(PPhs)4 (54 mg, 0.05 mmol, 0.1 eq). The mixture was stirred at 80 °C under nitrogen for 2 hours. The mixture was concentrated and purified by preparative TLC (SiC>2, petroleum ether / EtOAc, 6:1 , v / v) to afford 55 (35 mg, 36% yield) as a solid. LCMS m / z = 206.1 [M+H]+.1H NMR (400 MHz, CDCI3) 6 8.26 (dd, J= 17.7, 8.8 Hz, 4H), 7.78 (ddt, J= 8.9, 6.5, 1.4 Hz, 2H), 7.56 - 7.51 (m, 2H), 7.50 - 7.43 (m, 1 H), 6.12 (dd, J = 11.7, 1.6 Hz, 1 H), 5.74 (dd, J = 17.9, 1.6 Hz, 1 H).

[0346] Synthesis of compound 56 / -PrOH, 90 °C, 3 h

[0347] Compound 56

[0348] Scheme 22: Synthesis of compound 56.

[0349] To a solution of 4-bromoquinoline (100 mg, 0.48 mmol, 1.0 eq), trifluoro(prop-1-en-2- yl)-A4-borane, potassium salt (142 mg, 0.96 mmol, 2.0 eq) and Et3N (145.9 mg, 1.44 mmol, 3.0 eq) in / -PrOH (2 mL) was added Pd(dppf)Ch (35 mg, 0.05 mmol, 0.1 eq). The reaction mixture was stirred at 90 °C under nitrogen for 3 hours. The mixture was concentrated in vacuo and purified by column (SiC>2, petroleum ether / EtOAc, 12:1 , v / v) to afford 56 (46 mg, 57% yield) as a yellow oil. LCMS m / z = 170.1 [M+H]+.1H NMR (400 MHz, CDCI3) 6 8.86 (d, J = 4.4 Hz, 1 H), 8.15 (d, J = 8.5 Hz, 1 H), 8.07 (d, J = 8.5 Hz, 1 H), 7.75 - 7.69 (m, 1 H), 7.58 - 7.51 (m, 1 H), 7.23 (d, = 4.5 Hz, 1 H), 5.49 (q, J = 1.7 Hz, 1 H), 5.13 (s, 1 H), 2.22 (s, 3H). Synthesis of compound 57

[0350] Compound 57

[0351] Scheme 23: Synthesis of compound 57.

[0352] To a solution of 4-bromoquinoline (200 mg, 0.96 mmol, 1.0 eq), (1- fluorovinyl)(methyl)diphenylsilane (350 mg, 1.44 mmol, 1.5 eq) and CsF (351 mg, 2.31 mmol, 2.4 eq) in DMF (8 mL) was added Cui (9 mg, 0.05 mmol, 0.05 eq) and Pd(PPhs)4 (56 mg, 0.05 mmol, 0.05 eq). The reaction mixture was stirred at room temperature under nitrogen for 5 hours. The mixture was diluted with water and extracted with EtOAc (4 x 30 mL). The combined organic layers were washed with water and brine, dried over Na2SO4 and concentrated. The crude was purified by preparative TLC (SiO2, petroleum ether / EtOAc, 10:1 , v / v) to afford 57 (56 mg, 34% yield) as a yellow oil. LCMS m / z = 174.1 [M+H]+.1H NMR (400 MHz, DMSO-d6) 5 8.98 (dd, = 4.4, 1.3 Hz, 1 H), 8.20 - 8.08 (m, 2H), 7.85 (ddd, J = 8.4, 6.9, 1 .4 Hz, 1 H), 7.72 (ddd, J = 8.4, 6.9, 1 .4 Hz, 1 H), 7.66 (dd, J = 4.4, 1.3 Hz, 1 H), 5.49 - 5.29 (m, 2H).

[0353] Synthesis of compound 58

[0354] DMF, 100 °C, 2 h

[0355] Compound 58

[0356] Scheme 24: Synthesis of compound 58.

[0357] To a solution of 4-bromoquinoline (100 mg, 0.48 mmol, 1.0 eq) in DMF (2 mL) was added Pd(PPhs)4 (56 mg, 0.05 mmol, 0.1 eq) and tributyl(1-methoxyvinyl)stannane (334 mg, 0.96 mmol, 2.0 eq) under nitrogen. The mixture was stirred at 100 °C under nitrogen for 2 hours. The mixture was diluted with water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic extracts were washed with brine, dried over Na2SC>4 and concentrated in vacuo. The crude residue was purified by preparative TLC (SiC>2, petroleum ether / EtOAc, 5:1 , v / v) to afford 58 (80 mg, 90% yield) as an oil. LCMS m / z = 186.2 [M+H]+.1H NMR (400 MHz, CDCh) 5 8.89 (d, J = 4.4 Hz, 1 H), 8.15 (dd, J = 17.2, 8.8 Hz, 2H), 7.71 (ddd, J = 8.4, 6.8, 1.4 Hz, 1 H), 7.56 (ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 7.42 (d, J= 4.4 Hz, 1H), 4.62 (d, J = 2.7 Hz, 1H), 4.53 (d, J = 2.8 Hz, 1 H), 3.84 (s, 3H).

[0358] Synthesis of compound 59 ci

[0359] (

[0360] ^LN^NH2Pd(dppf)CI2TEA, i-PrOH, 90°C, 3h

[0361] Compound 59

[0362] Scheme 25: Synthesis of compound 59.

[0363] 2-amino-4-chloropyridine (152.2mg, 1.18mmol, 1.0 eq) was dissolved in isopropyl alcohol (5.0mL) and stirred at room temperature. Once potassium vinyl trifluoroborate (172.8mg, 1.29mmol, 1.1 eq), Pd(dppf)Ch (25.8mg, 0.04mmol, 0.03 eq) and triethylamine (179 mg, 1.77mmol, 1.5 eq) were added, the reaction mixture was degassed with nitrogen / vacuum four times. The orange-brown solution was then heated under reflux to 90 °C, stirring under nitrogen, for 3 hours. The reaction mixture was filtered and concentrated in vacuo to yield crude product which was purified by column chromatography (SiO2, petroleum ether / EtOAc, 1 :1, v / v) to afford 59 (47.2mg, 33 % yield) as a white solid.1H NMR (300 MHz, MeOH-d6) 5 8.09 (d, J= 3.0Hz, 1H), 6.62 (d, J= 6.0Hz, 1 H), 6.49, (q, J= 9.0Hz, 1H), 6.22 (d, J= 3.0Hz, 1H), 5.52 (dd, J= 3.0, 9.0Hz, 1 H).

[0364] Synthesis of Compound 61

[0365] Scheme 26: Synthesis of Compound 61. To a solution of 4-chloroquinolin-8-amine (500 mg, 2.8 mmol, 1.0 eq) in HFIP (5 mL) was added MeOTf (920 mg, 5.6 mmol, 2.0 eq). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated in vacuo. The crude residue was purified by column chromatography (SiC>2, petroleum ether / EtOAc, 100:1 v / v) to afford 29A-1 (163 mg, 30% yield) as yellow oil. LCMS m / z = 193.10 [M+H]+.1H NMR (400 MHz, DMSO-cfe) 5 8.64 (d, J = 4.6 Hz, 1 H), 7.70 (d, J = 4.6 Hz, 1 H), 7.51 (t, J = 8.0 Hz, 1 H), 7.24 (d, J = 8.4 Hz, 1 H), 6.81 - 6.72 (m, 1 H), 6.68 (d, J = 7.8 Hz, 1 H), 2.91 (d, J = 5.0 Hz, 3H).

[0366] To a solution of 29A-1 (325 mg, 1.69 mmol, 1.0 eq) in DCE (5 mL) was added methyl 4-oxobutanoate (588 mg, 5.06 mmol, 3.0 eq) and AcOH (203 mg, 3.37 mmol, 2.0 eq). The reaction mixture was stirred at room temperature for 2h. NaBH(OAc)s (1.07 g, 5.06 mmol, 3.0 eq) was added and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with water (50 mL) and extracted with DCM (3 x 20 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue obtained was purified by prep-TLC (SiC>2, petroleum ether / EtOAc, 3:1 , v / v) to afford 29A-2 (300 mg, 61 % yield) as yellow oil. LCMS m / z = 293.10 [M+H]+.1H NMR (400 MHz, DMSO- d6) 5 8.70 (d, J = 4.6 Hz, 1 H), 7.70 (d, J = 4.6 Hz, 1 H), 7.65 (d, J = 8.2 Hz, 1 H), 7.57 (t, J = 8.0 Hz, 1 H), 7.17 (d, J = 7.6 Hz, 1 H), 3.60 - 3.54 (m, 2H), 3.53 (s, 3H), 2.97 (s, 3H), 2.35 (t, J = 7.2 Hz, 2H), 1.88 (q, J = 7.2 Hz, 2H).

[0367] To a solution of 29A-2 (300 mg, 1.02 mmol, 1.0 eq) in / -PrOH (5 mL) was added potassium vinyltrifluoroborate (275 mg, 2.0 mmol, 2.0 eq), TEA (2.07 g, 20.49 mmol, 20.0 eq) and Pd(dppf)Ch (75 mg, 0.1 mmol, 0.1 eq). The reaction mixture was stirred at 90 °C for 3 hours under N2 atmosphere. The reaction mixture was diluted water (50 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated in vacuo. The crude residue was purified by prep-TLC (SiO2, petroleum ether / EtOAc, 2:1 , v / v)to afford 29A- 3 (250 mg, 86% yield) as yellow oil. LCMS m / z = 285.20 [M+H]+.1H NMR (400 MHz, DMSO-cfe) 5 8.75 (d, J = 4.4 Hz, 1 H), 7.68 (d, J = 8.4 Hz, 1 H), 7.60 (d, J = 4.4 Hz, 1 H), 7.53 (dd, J = 17.2, 11.0 Hz, 1 H), 7.45 (t, J = 8.0 Hz, 1 H), 7.10 (d, J = 7.8 Hz, 1 H), 6.07 (dd, J = 17.2, 1.2 Hz, 1 H), 5.66 (dd, J = 11.0, 1.2 Hz, 1 H), 3.56 - 3.49 (m, 5H), 2.94 (s, 3H), 2.35 (t, J = 7.2 Hz, 2H), 1.86 (p, J = 7.2 Hz, 2H).

[0368] To a solution of 29A-3 (200 mg, 0.7 mmol, 1.0 eq) in MeCN (2 mL) at 0°C was added a solution of LiOH (84 mg, 3.52 mmol, 5.0 eq) in H2O (2 mL) drop-wise. The reaction mixture was stirred at room temperature overnight. The mixture was acidified to pH = 5~6 with 6M HCI (0.64 mL, 3.87 mmol, 5.5 eq), diluted with water (10 mL) and extracted with CH2CI2 (5 x 20 mL). The combined organic layers were dried over Na2SC>4, filtered and concentrated in vacuo to afford compound 61 (120 mg, 63% yield) as yellow oil. LCMS m / z = 271.20 [M+H]+.1H NMR (400 MHz, DMSO-cfe) 5 11.96 (s, 1 H), 8.77 (d, J = 4.4 Hz, 1 H), 7.67 (d, J = 8.4 Hz, 1 H), 7.60 (d, J = 4.4 Hz, 1 H), 7.52 (dd, J = 17.2, 11.0 Hz, 1 H), 7.45 (t, J = 8.0 Hz, 1 H), 7.10 (d, J = 7.6 Hz, 1 H), 6.07 (d, J = 17.2 Hz, 1 H), 5.66 (d, J = 11.0 Hz, 1 H), 3.51 (t, J = 7.4 Hz, 2H), 2.95 (s, 3H), 2.26 (t, J = 7.2 Hz, 2H), 1.84 (q, J = 7.2 Hz, 2H).

[0369] Synthesis of Compound 62

[0370] Compound 62 was synthesised from 1-chloroisoquinolin-5-amine according to the scheme below.

[0371] Scheme 27: Synthesis of Compound 64. Synthesis of Int 3

[0372] Scheme 28: Synthesis of intermediate Int 3 from compound 8.

[0373] To a solution of methyl 4-vinylquinoline-6-carboxylate (8) (141.2 mg, 0.66 mmol, 1.0 eq) in 1 ,4-dioxane / H2O (3 mL, 9:1 , v / v) was added sodium hydroxide (53 mg, 1.32 mmol, 2.0 eq). The resulting reaction mixture was stirred at room temperature overnight, then neutralised with 1.5M HCI solution and concentrated in vacuo to afford Int 1 (220 mg, quantitative) as a pale yellow solid. LCMS m / z = 200.1 [M+H]+.1H NMR (300 MHz, MeOD) 5 8.88 (d, J = 3.0 Hz, 1 H), 8.82 (d, J = 3.0 Hz, 1 H), 8.34 (m, 1 H), 8.0 (d, J = 9.0 Hz, 1 H), 7.69 (d, J = 3.0 Hz, 1 H), 7.63 (m, 1 H), 6.15 (d, J = 18.0 Hz, 1 H), 5.77 (d, J = 9.0 Hz ,1 H).

[0374] To a solution of Int 1 (2.37 g, 10.70 mmol, 1.2 eq) in DMF (30 mL) was added BocNH- PEG4-NH2 (3.0 g, 8.92 mmol, 1.0 eq) and DIPEA (3.5 g, 26.75, 3.0 eq) followed by addition of T3P (50% solution in EtOAc, 8.5 g, 13.38 mmol, 1.5 eq) at 0 °C. The resulting reaction mixture was stirred at room temperature overnight, diluted with water (100 mL) and extracted with EtOAc (2 x 100 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by RP column chromatography (C18 column, MeCN / FW, 1 :1 , v / v) to afford Int 2 (1.3 g, 23% yield) as a white oil. LCMS m / z = 518.4 [M+H]+.1HNMR (400 MHz, DMSO-ds) 6 8.94 (d, J = 4.6 Hz, 1 H), 8.91 - 8.84 (m, 1 H), 8.77 (d, J = 1 .8 Hz, 1 H), 8.20 (dd, J = 8.8, 1.9 Hz, 1 H), 8.08 (d, J = 8.8 Hz, 1 H), 7.76 (d, J = 4.6 Hz, 1 H), 7.73 - 7.64 (m, 1 H), 6.76 - 6.69 (m, 1 H), 6.22 (dd, J = 17.2, 1.2 Hz, 1 H), 5.80 (dd, J = 11.0, 1.1 Hz, 1 H), 3.61 - 3.53 (m, 6H), 3.51 - 3.49 (m, 3H), 3.48 - 3.43 (m, 7H), 3.36 - 3.33 (m, 2H), 3.07 - 3.01 (m, 2H), 1 .36 (s, 9H).

[0375] To a solution of Int 2 (1 g, 1.93 mmol, 1.0 eq) in CH2CI2 (10 mL) was added TFA (3 mL) and the resulting reaction mixture was stirred at 0 °C for 1 hour. The solvent was removed under reduced pressure to afford Int 3 (1.3 g, quantitative) as a yellow oil. LCMS m / z = 418.4 [M+H]+.1HNMR (400 MHz, DMSO-d6) 5 9.10 (d, J = 5.0 Hz, 1 H), 8.98 (s, 1 H), 8.88 (d, J = 1.8 Hz, 1 H), 8.33 (dd, J = 8.8, 1.8 Hz, 1 H), 8.19 (d, J = 8.8 Hz, 1 H), 8.01 (d, J = 5.0 Hz, 1 H), 7.81 - 7.73 (m, 4H), 6.40 (d, J = 17.2 Hz, 1 H), 5.96 (d, J = 11 .0 Hz, 1 H), 3.62 - 3.58 (m, 3H), 3.57 - 3.54 (m, 5H), 3.53 - 3.49 (m, 10H), 2.99 - 2.93 (m, 2H).

[0376] Synthesis of compound 13

[0377] Scheme 29: Synthesis of compound 13.

[0378] Dansyl chloride (151 mg, 0.56 mmol, 1.0 eq) was dissolved in CH2CI2 (9 mL), then tertbutyl methyl(2-(methylamino)ethyl)carbamate (110 pL, 0.56 mmol, 1.0 eq) and Et3N (78 pL, 0.56 mmol, 1.0 eq) was added and the mixture was stirred for 3.5 hours at room temperature. Upon reaction completion by LCMS, the mixture was evaporated to dryness to afford Int 4 (404 mg, quantitative) as an orange oil, which was used directly in the next step without further purification. LCMS m / z = 444.2 [M+Na]+.

[0379] Crude Int 4 (236 mg, 0.56 mmol, 1.0 eq) was dissolved with 4N HCI in 1 ,4-dioxane (2.8 mL, 11.20 mmol, 20 eq) and the mixture stirred at room temperature for 1 hour. The mixture was then evaporated to dryness to afford Int 5 (350 mg, quantitative) as a white solid, which was used directly in the next step without further purification. LCMS m / z = 322.2 [M+H]+.

[0380] To a solution of Boc-NH-PEGs-COOH (106 mg, 0.20 mmol, 1.0 eq) in DMF (0.5 mL) was added HATU (91 mg, 0.24 mmol, 1.2 eq) and DIPEA (105 pL, 0.60 mmol, 3.0 eq) at 0 °C. After 30 min, a solution of Int 5 (0.22 mmol, 1.0 eq) in DMF (0.5 mL) was added to the reaction mixture at 0 °C and stirred at room temperature for 2 hours. The mixture was then evaporated to dryness and purified by column chromatography (SiC>2, CH2Cl2 / MeOH, 100:0 to 4:1 , v / v) to afford Int 6 (160 mg, 95% yield) as a yellow oil. LCMS m / z = 845.5 [M+H]+

[0381] Int 6 (160 mg, 0.19 mmol, 1.0 eq) was dissolved with 4N HCI in 1 ,4-dioxane (950 pL, 3.78 mmol, 20 eq) and the mixture stirred at room temperature for 1 hour. The mixture was then evaporated to dryness to afford Int 7 (185 mg, quantitative) as crude orange oil, which was used directly in the next step without further purification. LCMS m / z = 745.4 [M+H]+.1H NMR (300 MHz, CDCh) 5 9.32 (d, J = 8.6 Hz, 1 H), 9.05 (d, J = 8.5 Hz, 1 H), 8.38 (d, J = 7.0 Hz, 1 H), 7.88 - 7.79 (m, 4H), 3.91 - 3.86 (m, 2H), 3.68 (s, 12H), 3.69 - 3.63 (m, 14H), 3.57 - 3.52 (m, 3H), 3.45 (s, 6H), 3.23 (dd, J = 9.7, 5.1 Hz, 3H), 3.15 - 3.03 (m, 7H), 1.39 (t, J = 7.3 Hz, 6H).

[0382] To a solution of Int 1 (16 mg, 0.072 mmol, 1 .0 eq) in DMF (0.5 mL) was added HATLI (33 mg, 0.086 mmol, 1.2 eq) at 0 °C. After 10 min, DIPEA (20 pL, 0.11 mmol, 1.5 eq) was added to the reaction mixture at 0 °C followed by a second addition of DI PEA (20 pL, 0.11 mmol, 1.5 eq) after an additional 10 min. The resulting solution was stirred at 0 °C for 30 min, after which a solution of Int 7 (59 mg, 0.08 mmol, 1.1 eq) in DMF (0.5 mL) was added to the reaction mixture and left to stir at room temperature for 6 hours. The mixture was then neutralized with HCI (0.5M, 2 mL) and extracted with CH2CI2 (4 x 2 mL). The combined organic layers were washed with a saturated aqueous NaHCCh solution (2 mL) and brine (2 mL), then dried over Na2SO4 and concentrated to dryness. The residue was purified by column chromatography (SiCh, C^Ch / MeOH, 100:0 to 1 :1 , v / v) to afford 13 (18 mg, 26% yield) as a yellow oil. LCMS m / z = 926.0 [M+H]+.1H NMR (300 MHz, CDCh) 5 8.92 (d, J = 4.6 Hz, 1 H), 8.71 (d, J = 1.5 Hz, 1 H), 8.53 (d, J = 8.5 Hz, 1 H), 8.27 (d, J = 8.7 Hz, 1 H), 8.22 - 8.09 (m, 4H), 7.60 - 7.46 (m, 4H), 7.17 (d, J = 7.2 Hz, 1 H), 6.05 (d, J = 17.3 Hz, 1 H), 5.74 (d, J = 11 .0 Hz, 1 H), 3.65 - 3.58 (m, 20H), 3.58 - 3.54 (m, 14H), 3.35 (t, J = 6.0 Hz, 3H), 2.91 (s, 3H), 2.90 (s, 3H), 2.87 (s, 6H), 2.45 (t, J = 6.9 Hz, 3H). Synthesis of compound 14

[0383] Scheme 30: Synthesis of compound 14.

[0384] Crude Int 5 (186 mg, 0.58 mmol, 1.0 eq) was dissolved in DMF (3 mL). HATLI (262 mg, 0.69 mmol, 1.2 eq), PL13-PEG8-COOH HCI (379 mg, 0.58 mmol, 1.0 eq) and DI PEA (1 mL, 5.79 mmol, 10 eq) were added and the solution stirred for 1 hour at room temperature. The mixture was then evaporated to dryness and purified by column chromatography (SiC>2, EtOAc / MeOH, 100:0 to 0:100, v / v) to afford 14 (175 mg, 33% yield) as an orange oil. LCMS m / z = 459.8 [M+2H]2+.1H NMR (300 MHz, CDCh) 6 8.52 (d, J = 8.4 Hz, 1 H), 8.25 (d, J = 8.7 Hz, 1 H), 8.08 (d, J = 6.3 Hz, 1 H), 7.54 - 7.45 (m, 2H), 7.17 (dd, J = 7.3, 2.8 Hz, 1H), 7.08 (d, J = 9.0 Hz, 2H), 6.66 - 6.53 (m, 1 H), 5.99 (dd, J = 17.6, 2.3 Hz, 1 H), 5.52 - 5.45 (m, 1 H), 3.75 - 3.47 (m, 43H), 3.40 (dd, J = 9.5, 5.6 Hz, 5H), 3.15 (dd, J = 15.2, 7.8 Hz, 7H), 3.08 (d, J = 7.2 Hz, 3H), 2.96 (s, 10H), 2.91 (dd, J = 4.6, 2.4 Hz, 7H), 2.87 (s, 6H), 2.76 (s, 7H), 2.54 (s, 3H), 1.45 - 1.36 (m, 50H).

[0385] Synthesis of Precursor 1

[0386] Scheme 31 : Synthesis of Precursor 1. To a solution of SM2 (2.0 g, 3.32 mmol, 1.0 eq) in DMF (12 mL) was added DIPEA (0.8 mL, 4.59 mmol, 1.4 eq) and bis(4-nitrophenyl) carbonate (2.02 g, 6.65 mmol, 2.0 eq). The reaction mixture was stirred at room temperature for 30 min. The mixture was diluted with water (100 mL) and extracted with EtOAc (2 x 100 mL). The combined organic extracts were washed with water (100 mL x 3), brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, CF^Ch / MeOH = 10:1) and recrystallised from MeOH to afford Int 8 (960 mg, 37 % yield) as a yellow solid. LCMS m / z = 767.45 [M+H]+.1H NMR (400 MHz, DMSO-ds) 5 10.13 (s, 1 H), 8.39 - 8.03 (m, 3H), 7.88 (d, J = 7.6 Hz, 2H), 7.77 - 6.85 (m, 13H), 6.03 - 5.92 (m, 1 H), 5.46 - 5.35 (m, 2H), 5.24 (s, 2H), 4.42 (q, J = 6.0, 4.4 Hz, 1 H), 4.34 - 4.21 (m, 3H), 3.96 - 3.90 (m, 1 H), 3.09 - 2.90 (m, 2H), 2.02 - 1 .95 (m, 1 H), 1.76 - 1.52 (m, 2H), 1.49 - 1.31 (m, 2H), 0.92 - 0.80 (m, 6H).

[0387] To a solution of Int 8 (830 mg, 1.08 mmol, 1.0 eq) in DMF (10 mL) was added DIPEA (279 mg, 2.16 mmol, 2.0 eq) and terf-butyl methyl(2-(methylamino)ethyl)carbamate (305 mg, 1.62 mmol, 1.5 eq). The reaction mixture was stirred at room temperature for 1 hour. The mixture was purified by column (C^Ch / MeOH = 10:1 to 2:1) to afford Int 9 (440 mg, 49 % yield) as a yellow solid. LCMS m / z = 816.60 [M+H]+.1H NMR (400 MHz, DMSO-ds) 5 10.05 (s, 1 H), 8.30 - 7.09 (m, 14H), 5.96 (t, J = 6.0 Hz, 1 H), 5.39 (s, 2H), 4.97 (s, 2H), 4.46 - 4.18 (m, 4H), 3.96 - 3.88 (m, 1 H), 3.15 - 2.61 (m, 11 H), 1 .99 (d, J = 1 .8 Hz, 2H), 1 .77 - 1 .30 (m, 13H), 0.89 - 0.83 (m, 6H).

[0388] To a solution of Int 9 (570 mg, 0.69 mmol) in CH2CI2 (12 mL) was added TFA (6 mL, 78.40 mmol). The reaction mixture was stirred at room temperature for 10 min. The mixture was filtered and concentrated to afford Int 10 (514 mg, quant.) which was used directly for the next step. LCMS m / z = 716.50 [M+H]+.

[0389] To a solution of SM1 (580 mg, 0.71 mmol, 1.0 eq.) and anhydrous HOBt (48.6 mg, 0.36 mmol, 0.5 eq.) in DMF (10 mL) was added DIPEA (185 mg, 1.44 mmol, 2.0 eq.) and a solution of Int 10 (as TFA salt) (514 mg, 0.71 mmol, 1.0 eq) in DMF (2.0 mL). The mixture was stirred at room temperature for 1 h. The mixture was diluted with water (20 mL) and extracted with EtOAc (2 x 20 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The crude was purified by preparative TLC (SiO2, C^Ch / MeOH = 15:1) (300 mg, -70% purity by LCMS) and RP column chromatography (C18 column, H2O / MeOH gradient = 5 - 100%,) to afford Precursor 1 (150 mg, 15% yield) as a red solid. LCMS m / z = 1383.80 [M+H]+.1H NMR (400 MHz, DMSO-d6) 514.03 (s, 1H), 13.24 (s, 1H), 10.04 (s, 1 H), 8.09 (s, 1 H), 7.88 (d, J = 23.6 Hz, 4H), 7.75 - 7.68 (m, 2H), 7.64 (s, 1 H), 7.55 (s, 2H), 7.44-7.35 (m, 3H), 7.30 (s, 4H), 5.96 (s, 1H), 5.49 (s, 1H), 5.39 (s, 2H), 5.19 (d, J = 23.2 Hz, 2H), 5.09-4.90 (m, 4H), 4.57 (s, 1H), 4.38 (d, J = 19.6 Hz, 1H), 4.22 (d, J = 5.8 Hz, 5H), 3.98 (s, 3H), 3.90 (d, J = 8.4 Hz, 2H), 3.48 - 3.38 (m, 7H), 3.30 (s, 4H), 2.82 (s, 13H), 1.99 (d, J = 11.2 Hz, 3H), 1.67 (s, 3H), 1.39 (d, J = 33.8 Hz, 2H), 0.88-0.83 (m, 6H).

[0390] Synthesis of PNU02

[0391] Scheme 32: Synthesis of PNU02.

[0392] To a solution of Int 14 (40 mg, 0.034 mmol, 1.0 eq), DIPEA (34.8 mg, 0.28 mmol, 8.2 eq) and PL13-PEG4-COOH HCI (18 mg, 0.038 mmol, 1.1 eq) in DMF (2 mL) was added HATLI (22.3 mg, 0.059 mmol, 1.5 eq) at 0°C. The reaction mixture was stirred at room temperature overnight, diluted with water and then extracted with EtOAc. The combined organic phase was washed with brine and dried over Na2SO4 and concentrated in vacuo. The residue was purified by preparative HPLC (C18 column, MeCN gradient in aqueous NH4HCO3 buffer) to afford PNU02 (4 mg, 7.3% yield) as a red solid. LCMS m / z = 791.4 [M+2H]2+.1H N MR (400 MHz, DMSO- cfe) 614.03 (s, 1H), 13.27 (s, 1H), 9.95 (s, 1H), 8.09 (d, J = 7.2 Hz, 1H), 7.95 - 7.89 (m, 3H), 7.86 (d, J = 8.4 Hz, 1H), 7.69 - 7.63 (m, 1H), 7.60 - 7.52 (m, 2H), 7.30 - 7.22 (m, 2H), 7.09 (d, J = 9.4 Hz, 2H), 6.64 (dd, J = 17.7, 10.9 Hz, 1H), 6.04 (d, J = 17.7 Hz, 1H), 6.00-5.94 (m, 1H), 5.54-5.48 (m, 1H), 5.45 (d, J = 10.9 Hz, 1H), 5.40 (s, 2H), 5.34-5.22 (m, 1H), 5.21 -5.14 (m, 1H), 5.04-4.88 (m, 3H), 4.57 (s, 1H), 4.41 -4.32 (m, 1H), 4.24 - 4.14 (m, 3H), 3.99 (s, 3H), 3.95 - 3.87 (m, 1 H), 3.70 - 3.62 (m, 2H), 3.61 - 3.55 (m, 3H), 3.48 - 3.46 (m, 10H), 3.30 (s, 3H), 3.21 - 3.15 (m, 3H), 3.07 - 2.57 (m, 16H), 2.47 - 2.44 (m, 3H), 2.40 (s, 3H), 2.38 - 2.30 (m, 3H), 2.13 - 2.04 (m, 1 H), 2.02 - 1.92 (m, 3H), 1.71 - 1.63 (m, 3H), 1.61 - 1.28 (m, 6H), 0.86 - 0.83 (m, 3H), 0.82 - 0.79 (m, 3H).

[0393] Synthesis of PNU04

[0394] Scheme 33: Synthesis of intermediate Int 13.

[0395] To a solution of SM3 (500 mg, 2.44 mmol, 1.0 eq) in H2O (5 mL) was added 1 M HCI (5 mL, 5 mmol, 2.0 eq) at 0 °C. The mixture was extracted with EtOAc (2 x 20 mL) and the combined organic extracts were concentrated in vacuo to afford Int 11 (330 mg, 68% yield) as a white solid, which was used directly without further purification.

[0396] To a solution of Int 11 (330 mg, 1.67 mmol, 1.0 eq) in DMF (5 mL) was added HOSu (286 mg, 2.49 mmol, 1.5 eq), DCC (513 mg, 2.49 mmol, 1.5 eq) and DMAP (31 mg, 0.25 mmol, 0.15 eq) at 0 °C. Then the mixture was stirred at room temperature for 3 hours. The mixture was diluted with water and extracted with EtOAc. The combined organic phases were concentrated under vacuum to afford Int 12 (250 mg, 51% yield) as a white solid, which was used directly for the next step.

[0397] LCMS: m / z = 297.1 [M+H]+

[0398] To a solution of Int 12 (250 mg, 0.84 mmol, 1.0 eq) in 1 ,4-dioxane (2 mL) and H2O (2 mL) was added H2N-PEG4-COOH (267 mg, 1.01 mmol, 1.2 eq) and NaHCO3(142 mg, 1 .69 mmol, 2.0 eq) at 0 °C. Then the mixture was stirred at room temperature for 3 hours. The reaction mixture was purified by RP column chromatography (C18 column, H2O / MeCN = 21%) to afford Int 13 (30 mg, 8% yield) as a white solid. LCMS: m / z = 445.2 [M-H]’.1H NMR (400 MHz, D2O) 5 8.80 (d, J = 4.8 Hz, 1 H), 8.55 (s, 1 H), 8.02 (s, 2H), 7.69 (d, J = 4.8 Hz, 1 H), 7.55 - 7.44 (m, 1 H), 6.13 (d, J = 17.4 Hz, 1H), 5.79 (d, J = 11.2 Hz, 1 H), 3.69 - 3.40 (m, 18H), 2.27 (t, J = 6.8 Hz, 2H). Scheme 34: Synthesis of PNU04.

[0399] To a solution of Precursor 1 (152 mg, 0.11 mmol, 1.0 eq) in THF (5 mL) and DMF (1 mL) was added DEA (252 mg, 3.45mmol, 31 eq) at 0 °C. Then the mixture was stirred at room temperature for 3 hours. The mixture was diluted with water extracted with Et2<D several times. The aqueous layer was lyophilised to afford Int 14 (117 mg, 92% yield) as a red solid. LCMS: m / z = 1161.6 [M+H]+

[0400] To a solution of Int 14 (27 mg, 0.02 mmol, 1.0 eq) in DMF (0.5 mL) was added Int 13 (28 mg, 0.06 mmol, 3.0 eq), T3P (50% solution in EtOAc, 45 mg, 0.07 mmol, 3.5 eq) and DI PEA (25 mg, 0.18 mmol) at 0 °C. The mixture was stirred at room temperature overnight. LCMS showed SM disappeared. The reaction mixture was diluted with water and extracted with EtOAc (3 x 20mL). The combined organic extacts were dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by prepartive HPLC (C18 column, MeCN / aqueous NH4HCO3 buffer) to give PNU04 (3.4 mg, 6% yield) as a red solid.

[0401] LCMS: m / z =795.7 [M+2H]2+.1H NMR (400 MHz, DMSO-d6) 59.97 (s, 1H), 8.93 (d, J = 4.6 Hz, 1H), 8.90-8.84 (m, 1H), 8.76 (s, 1H),8.20 (s, 1H), 8.17-8.09 (m, 1H), 8.05 (d, J = 8.7 Hz, 1H), 7.97-7.81 (m, 3H), 7.74 (d, J = 4.5 Hz, 1H), 7.72-7.47 (m, 4H), 7.32-7.20 (m, 2H), 6.21 (d, J = 17.2 Hz, 1H), 5.98 (s, 1H), 5.78 (d, J = 12.0 Hz, 1H), 5.49-5.35 (m, 3H), 5.27-5.12 (m, 2H), 5.04-4.91 (m, 4H), 4.58 (s, 1H), 4.39-4.33 (m, 1H), 4.26-4.15 (m, 3H), 3.98 (s, 3H), 3.93-3.88 (m, 1H), 3.68-3.63 (m, 1H), 3.54 (s, 23H), 3.04 - 2.73 (m, 12H), 2.72 - 2.58 (m, 3H), 2.45 - 2.26 (m, 5H), 2.14 - 1.92 (m, 4H), 1.73-1.51 (m, 5H), 1.46-1.31 (m, 3H), 0.86-0.79 (m, 6H).

[0402] Synthesis of MMAE29 and MMAE30

[0403] Int 15 (CAS # 229977-57-5, MedChemExpress) and BocNH-PEG4-CO2H (CAS #

[0404] 756525-91-4, BroadPharm) are commercially available.

[0405] Scheme 35: Synthesis of MMAE29 and MMAE30

Claims

CLAIMS1 . A linker-active agent conjugate comprising a linker and an active agent, wherein the linker is represented by Formula (la) or (lb):wherein:Ar is selected from the group consisting of phenyl, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrazole or imidazole; at least one of Ai and A2 is N and the other is selected from N or CR, with the proviso that when the linker is represented by Formula (lb) A1 is N and A2 is CR; n is an integer from 1 to 4; at least one R group is a connecting unit covalently linked to the active agent, wherein the active agent is a drug; and at each other occurrence R is independently selected from a hydrogen atom or an electron-withdrawing or electron-donating substituent.

2. The conjugate of claim 1 , wherein the connecting unit covalently linked to the active agent has the Formula:- W- Y -A wherein:W is a linking group;Y is an optional stretcher unit comprising one or more of a cleavable group, a hydrophilic spacer group, and a self-immolative group; andA is the active agent.

3. The conjugate of claim 2, wherein the hydrophilic spacer group is selected from a poly(alkylene glycol) or an amino acid residue.

4. The conjugate of claim 2 or 3, wherein the self-immolative group comprises paraaminobenzyl or para-aminobenzyloxycarbonyl.

5. The conjugate of any one of claims 2 to 4, wherein the cleavable group is a protease- cleavable peptide moiety, a glycosidase-cleavable sugar moiety, a pH sensitive moiety, or a hydrolysable moiety.

6. The conjugate of any one of claims 2 to 5, wherein the cleavable group is selected from a p-D-glucuronide linker, a p-D-galactoside linker, an O-glucosyl linker, an O-galactosyl linker or an O-mannosyl linker.

7. The conjugate of any one of claims 2 to 5, wherein the cleavable group is a dipeptide unit selected from -Valine-Citrulline- (-Val-Cit-), -Valine- Alanine-(-Val-Ala-), -Valine-Lysine-(- Val-Lys-), -Valine-Arginine-(-Val-Arg-), -Phenylalanine Acid-Citrulli ne-(-Phe-Cit-) , - Phenylalanine-Lysine-(-Phe-Lys-), -Phenylalanine-Arginine-(-Phe-Arg-), - Asparagine- Asparagine (-Asn-Asn-), -Asparagine-Phenylalanine Acid- (-Asn-Phe-), -Asparagine- Histidine- (-Asn-His-), -Asparagine-Alanine- (-Asn-Ala-), -Asparagine-Valine- (-Asn-Val-), - Asparagine-Lysine- (-Asn-Lys-), -Asparagine-Citrulline- (-Asn-Cit-), or -Asparagine- Tryptophan- (-Asn-Trp-).

8. The conjugate of any one of the preceding claims, wherein Ar is phenyl.

9. The conjugate of any one of the preceding claims, wherein in the drug is a cytotoxic drug.

10. The conjugate of claim 9, wherein the cytotoxic drug is selected from the group comprising dolastatins, auristatins, maytansinoids, tubulysins, calicheamicins, eribulin, hemiasterlin, thymidylate synthase inhibitors, duocarmycins, pyrrolobenzodiazepines, pyrridinobenzodiazepines, camptothecins, anthracyclines, cryptophycins, a-Amanitin and cyclopropabenzaindole analogues.

11. The conjugate of any one of the preceding claims, wherein the linker is represented by Formula (la) and wherein:(i) Ai is N and A2 is CH; or(ii) Ai is CH and A2 is N.

12. A conjugate comprising a protein or a peptide, a linker and an active agent, wherein the linker is represented by Formula (Ila) or (lib):wherein: the wavy line indicates the attachment site to the protein or peptide;Ar is selected from the group consisting of phenyl, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrazole or imidazole; at least one of A1 and A2 is N and the other is selected from N or CR, with the proviso that when the linker is represented by Formula (lib) A1 is N and A2 is CR; n is an integer from 1 to 4; at least one R group is a connecting unit covalently linked to the active agent, wherein the active agent is a drug; and at each other occurrence R is independently selected from a hydrogen atom or an electron-withdrawing or electron-donating substituent.

13. The conjugate of claim 12, wherein the connecting unit covalently linked to the active agent has the Formula:- W- Y -A wherein:W is a linking group;Y is an optional stretcher unit comprising one or more of a cleavable group, a hydrophilic spacer group, and a self-immolative group; andA is the active agent.

14. The conjugate of claim 12 or 13, wherein the hydrophilic spacer group is selected from a poly(alkylene glycol) or an amino acid residue.

15. The conjugate of any one of claims 12 to 14, wherein the self-immolative group comprises para-aminobenzyl or para-aminobenzyloxycarbonyl.

16. The conjugate of any one of claims 12 to 15, wherein the cleavable group is a protease- cleavable peptide moiety, a glycosidase-cleavable sugar moiety, a pH sensitive moiety, or a hydrolysable moiety17. The conjugate of any one of claims 12 to 16, wherein the cleavable group is selected from a p-D-glucuronide linker, a p-D-galactoside linker, an O-glucosyl linker, an O-galactosyl linker or an O-mannosyl linker.

18. The conjugate of any one of claims 12 to 16, wherein the cleavable group is a dipeptide unit selected from -Valine-Citrulline- (-Val-Cit-), -Valine- Alanine-(-Val-Ala-), - Valine-Lysine-(-Val-Lys-), -Valine-Arginine-(-Val-Arg-), -Phenylalanine Acid-Citrulline-(-Phe- Cit-), -Phenylalanine-Lysine-(-Phe-Lys-), -Phenylalanine-Arginine-(-Phe-Arg-), -Asparagine- Asparagine (-Asn-Asn-), -Asparagine-Phenylalanine Acid- (-Asn-Phe-), -Asparagine- Histidine- (-Asn-His-), -Asparagine-Alanine- (-Asn-Ala-), -Asparagine-Valine- (-Asn-Val-), - Asparagine-Lysine- (-Asn-Lys-), -Asparagine-Citrulline- (-Asn-Cit-), or -Asparagine- Tryptophan- (-Asn-Trp-).

19. The conjugate of any one of claims 12 to 18, wherein Ar is phenyl.

20. The conjugate of any one of claims 12 to 19, wherein in the drug, is a cytotoxic drug.

21. The conjugate of claim 20, wherein the cytotoxic drug is selected from the group comprising dolastatins, auristatins, maytansinoids, tubulysins, calicheamicins, eribulin, hemiasterlin, thymidylate synthase inhibitors, duocarmycins, pyrrolobenzodiazepines, pyrridinobenzodiazepines, camptothecins, anthracyclines, cryptophycins, a-Amanitin and cyclopropabenzaindole analogues.

22. The conjugate of any one of claims 12 to 21 , wherein the linker is represented by Formula (Ila) and wherein:(i) Ai is N and A2 is CH; or(ii) A1 is CH and A2 is N.

23. The conjugate of any one of claims 12 to 22, wherein the protein is an antibody or an antigen-binding fragment thereof, optionally wherein the antibody is a monoclonal antibody.

24. A linker compound represented by Formula (la) or (lb):wherein:Ar is selected from the group consisting of phenyl, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrazole or imidazole; at least one of A1 and A2 is N and the other is selected from N or CR4,with the proviso that when the linker is represented by Formula (lb) A1 is N and A2 is CR; n is an integer from 1 to 4; at least one R group is a connecting unit comprising a reactive group capable of binding to a drug; at each other occurrence R is independently selected from a hydrogen atom or an electron-withdrawing or electron-donating substituent; and wherein the connecting unit has the Formula:- W- Y - Z wherein:W is a linking group;Y is stretcher unit comprising one or more of a cleavable group, a hydrophilic spacer group, and a self-immolative group; andZ is the reactive group and is selected from an amine-reactive group, a hydroxyl-reactive group, a halide-reactive group, a carboxylic acid-reactive group, an ester-reactive group, an azide-reactive group, an alkyne-reactive group, a hydroxylamine-reactive group, an aldehyde-reactive group, or a ketone-reactive group.

25. The linker compound of claim 24, wherein the reactive group is selected from -C=CH, a dibenzocyclooctyne (DBCO) group, a bicyclononyne (BCN) group, a 4- dibenzocyclooctynol (DIBO) group, a Difluorooctyne (DIFO) group, -N3, -C(O)OH, -NH2, - NHRb; -NRb2, where Rbis C1-C5 alkyl group, -OH, Cl, Br, I, -C(O)X or -OX, where X is a leaving group.

26. The linker compound of claim 24 or 25, wherein the hydrophilic group, the self- immolative group and the cleavable group are as defined in any of claims 3 to 7.

27. A linker compound represented by Formula (la) or (lb):wherein:Ar is selected from the group consisting of phenyl, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrazole or imidazole; at least one of A1 and A2 is N and the other is selected from N or CR, with the proviso that when the linker is represented by Formula (lb) A1 is N and A2 is CR; n is an integer from 1 to 4; at least one R group is a connecting unit comprising a reactive group capable of binding to a drug, and is selected from:■ (CH2)e-Zi, or■ (CH2)f-Z2, or■ (CH2)e-Q-(CH2)f-Zi, or■ (CH2)e-Q-(CH2)f-Z2, or■ (CH2)e-Q-(CH2)f-Q-(CH2)f-Zi, or,■ (CH2)e-Q-(CH2)f-Q-(CH2)f-Z2, where:• Zi is independently selected from -C(O)OH, -OH, -C(O)X, -OX, or NHRb, where X is a leaving group and Rbis a C1-C5 alkyl group;• Z2is independently selected from -C=CH, a di benzocyclooctyne (DBCO) group, a bicyclononyne (BON) group, a 4-dibenzocyclooctynol (DI BO) group, a Difluorooctyne (DIFO) group, -N3, -NH2, -Cl, -Br, -I;• Q is independently selected from O, S, an amide group, NH, or NRb, where Rbis a C1-C5 alkyl group;• e is an integer from 0 to 10;• f is an integer from 1 to 10, and at each other occurrence R is independently selected from a hydrogen atom or an electron-withdrawing or electron-donating substituent.

28. The linker compound of any one of claims 24 to 27, wherein Ar is phenyl.

29. The conjugate or linker of any one of claims 24 to 28, wherein the linker is represented by Formula (la) and wherein:(i) A1 is N and A2is CH; or(ii) A1 is CH and A2is N.

30. A pharmaceutical composition comprising a conjugate according to any one of claims 12 to 23; and one or more pharmaceutically acceptable excipients, diluents, or carriers.

31. The pharmaceutical composition according to claim 30 for use as a medicament.

32. The pharmaceutical composition according to claim 31 , wherein the medicament is for use in the treatment of cancer and the active agent is a cytotoxin.

33. A method of treating cancer in a subject in need thereof, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition according to claim 32 to the subject.

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