WO2025224196A1 - Compounds - Google Patents

Abstract

The invention relates to pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) compounds comprising a halogen or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, and pharmaceutical compositions thereof which are useful as medicaments, as drug payloads in targeting moiety-drug conjugates and, in particular, in treating proliferative and/or malignant diseases. These PDBs are a compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof.

Description

COMPOUNDS

FIELD OF THE INVENTION

The invention relates to pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) compounds comprising a halogen or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, and pharmaceutical compositions thereof which are useful as medicaments, as drug payloads, for example, in antibody-drug conjugates and, in treating proliferative and/or malignant diseases.

BACKGROUND

Pyrrolobenzodiazepines (PBDs) are tricyclic compounds, originally discovered in Streptomyces species [1-5], comprising fused 6-7-5-membered rings with an anthranilate (A ring), a diazepine (B ring) and a pyrrolidine (C ring) [3], They have an electrophilic N 10=011 imine group (as shown below along with the position numbers) or the hydrated equivalent, a carbinolamine [NH-CH(OH)], or a carbinolamine alkyl ether ([NH-CH(OR, where R = alkyl)] which can form a covalent bond to a C2-amino group of guanine in DNA to form a DNA adduct [6],

Carbinolamine Imine Carbinolamine alkyl ether

PBD natural products interact in the minor groove of the DNA helix with excellent fit (i.e. , good “isohelicity”) due to a right-handed longitudinal twist induced by a chiral C11a-position which has the (S)-configuration [6], The DNA adduct has been reported to inhibit a number of biological processes including the binding of transcription factors [7-9] and the function of enzymes such as endonucleases [10, 11] and RNA polymerase [12],

PBDs are thought to interact with DNA by first locating at a low-energy binding sequence (i.e., a 5’-Pu-G-Pu-3’ triplet) through Van der Waals, hydrogen bonding and electrostatic interactions [7], Then, once in place, a nucleophilic attack by the exocyclic C2-amino group of the central guanine occurs to form the covalent adduct [7], Once bound, the PBD remains anchored in the DNA minor groove, avoiding DNA repair by causing negligible distortion of the DNA helix [3], The ability of PBDs to form an adduct in the minor groove and crosslink DNA enables them to interfere with DNA processing and, hence, their potential for use as antiproliferative agents.

A range of monomeric PBD structures have been isolated from Streptomyces species, including anthramycin [14], tomamycin [15], and usabamycin [16], This has led to the development of a large range of synthetic analogues which have been reviewed [1, 17], Many PBDs have potent cytotoxicity and several PBD dimer compounds have been developed as drug payloads for targeting moiety-drug conjugates, in particular, for antibody-drug conjugates (ADCs). Such PBD compounds are typically attached to a linker and through this linker to turn or- targeting antibodies to create ADCs. However, some PBD dimer-containing ADCs have had toxicity issues, and the clinical experience of such ADCs has been discussed in reviews [18, 19], Several monomeric PBD structures that are linked through their C8 position to pyrroles and imidazoles have also been reported WO 2004/043963, WO 2007/039752, WO 2013/164593 [20-24], An example [25] of such a monomeric PBD structures with pyrroles attach via the C8 position is GWL-78

A subsequent study [26] showed that some PBD compounds with a shortened C8 side chains such as a derivative with a p-tolyl end group (designated below as MH01): showed activity of the same order of magnitude as GWL-78 when tested against a two cell lines. This study also tested two compounds containing a pyridyl end group, including the compound 27 shown below: and reported that this structural change of replacing the end tolyl group for a pyridyl group eliminated the compound activity at the dose range tested against the cell lines. Thus, there remains a need to develop compounds that have high potency but with less toxicity to normal cells and which provide a better therapeutic window.

The present inventors have surprisingly found that certain PBDs with shortened C8 side chains that comprise a halogen (i.e. halogenated PBDs) are significantly more effective against a panel of solid tumours than the prior art compound MH01. These halogenated PBDs include an example with a halogenated pyridyl end groups which was surprisingly potent against the panel of solid tumours given the lack of activity demonstrated by compound 27 in the earlier study [26], In addition, some of these halogenated PBD compounds have demonstrated selective toxicity towards cancer cells, with no toxicity at 50 mg/Kg dose in Galleria mellonella. In contrast, in a comparable test GWL-78 showed marked toxicity in Galleria mellonella. Thus, these halogenated PBD compounds provide a better therapeutic window. These halogenated PBD compounds also showed further advantages such having high aqueous solubility, high metabolic stability, low intrinsic clearance, high membrane permeability and as being less susceptible to efflux. Many of these halogenated PBD compounds provided greater synthetic flexibility including, for example, the option to bind to an antibody via suitable groups on the C8 side chain. These halogenated PBD compounds also preferentially bind to GC sequence which is significant for inhibiting transcription factors.

SUMMARY

In a first aspect, the present invention provides a compound of formula (I): or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, wherein: the dotted lines indicates the optional presence of a double bond between C1 and 02, or 02 and 03;

Xi is O, S, NH, 0(0), C(O)NH or C(0)-0;

L is C1-12 alkylene;

Ri is H, OH, halogen, ON, =CH2, =CH(CI-6 alkyl), C1-6 alkyl, or OC1-6 alkyl;

R2 is H, OH, halogen, ON, =CH2, =CH(CI-6 alkyl), C1-6 alkyl, or OC1-6 alkyl;

R3 is H, OH, halogen, ON, =CH2, =CH(CI-6 alkyl), C1-6 alkyl, or OC1-6 alkyl;

R4 is halogen, OH, OC1-6 alkyl, or OCH2Ph; Rs is:

X₂ is N or CH; X₃ is N or CH;

Yi is C-R11 or N;

Y₂ is S or O;

Y₃ is S, N or O; X₄ is N or CH;

Y₄ is S or O; either (a) Rs is R’, Ci-₃ alkyl, OCi-₃ alkyl, or NH₂; R9 is R’; and R10 is H;

(b) Rs and R_w are independently R’; and R9 is Ci-₃ alkyl;

(c) Rs is R’; R9 is OC_2.3 alkyl; and R_w is H; or

(d) one of Rs and R9 is R’ and the other is H; and R_w is H; one of R14 and R15 is independently R’ and the other of R14 and R15 is H, independently R’, Ci-₃ alkyl, OCi-₃ alkyl, or NH₂; each R11 , RI₃, R16, and Ris is, H, independently R’, Ci-₃ alkyl, OCi-₃ alkyl, or NH₂; each RI₂, and R17 is independently R’; either (1a) R19 is H; R₂o is H, R’, Ci-₃ alkyl, OCi-₃ alkyl, or NH₂; R₂I is R’; and R₂₂ is H, R’, Ci-₃ alkyl, OCi-₃ alkyl, or NH₂; or

(1b) R19 is H, R’, Ci-₃ alkyl, OCi-₃ alkyl, or NH₂; R₂o is R’; R₂I is H, R’, Ci-₃ alkyl, OCi-₃ alkyl, or NH₂; and R₂₂ is H; one of R₂₃ and R₂4 is H and the other is H; and R₂s is independently R’; each R’ is independently a halogen; z is 0 or 1 ; and either:

(i) Rs and R7 together form a double bond; or

(ii) Rs is H, SO₃H, or nitrogen protecting groups; and R7 is H, OH or OC1-6 alkyl. In a further aspect, there is provided a pharmaceutical composition comprising a compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, as described herein, and one or more of a pharmaceutically acceptable carrier, diluent, excipient, or lipid nanopaticle.

In a further aspect, there is provided a compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, as described herein, or a pharmaceutical composition, as described herein, for use as a medicament.

In a further aspect, there is provided a compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, as described herein, or a pharmaceutical composition, as described herein, for use in the treatment of a proliferative and/or malignant disease.

In a further aspect, there is provided a compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, or a pharmaceutical composition, for use in the treatment of a proliferative and/or malignant disease, as described herein, wherein the compound is administered either simultaneously or sequentially with one or more other therapeutic agent.

In a further aspect, there is described a method of treatment of a patient suffering from a proliferative and/or malignant disease, comprising administering to said patient a therapeutically effective amount of a compound or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, as described herein, or a pharmaceutical composition as described herein.

In a further aspect, there is described a use of a compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, as described herein, in the manufacture of a medicament for treating a proliferative and/or malignant disease.

In a further aspect, there is described a compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, as described herein, for use as a drug in a targeting moiety-drug conjugate, suitably an antibody-drug conjugate.

Further particular and preferred aspects are set out in the accompanying independent and dependent claims. Features of the dependent claims may be combined with features of the independent claims as appropriate, and in combinations other than those explicitly set out in the claims.

ABBREVIATIONS

ADC Antibody-Drug Conjugate; Alloc allyloxycarbonyl; BAIB Bis(acetoxy)iodobenzene; BTFFH Fluoro-dipyrrolidinocarbenium hexafluorophosphate; DCM Dichloromethane; DHP 3,4-Dihydropyran; DI PEA N,N-Diisopropylethylamine; DMAP 4- Dimethylaminopyridine; DMF N,N-Dimethylformamide; DMSO Dimethylsulfoxide; DNA Deoxyribonucleic Acid; DOX Doxorubicin; dppf 1,1'-Bis(diphenylphosphino)ferrocene; EDC 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide; EDTA Ethylenediaminetetraacetic acid; FRET Forster resonance energy transfer; HATLI 1- [Bis(dimethylamino)methylene]-1/7-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate, /V-[(Dimethylamino)-1/7-1,2,3-triazolo-[4,5-b]pyridin-1- ylmethylene]-/V-methylmethanaminium hexafluorophosphate /V-oxide; HER2 Human epidermal growth factor receptor 2; hERG the human Ether-a-go-go-Related Gene; HRMS High-resolution mass spectrometry; LC-MS Liquid Chromatography-Mass Spectrometry; MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NFKB Nuclear factor kappa-light-chain-enhancer of activated B cells;NMR Nuclear Magnetic Resonance Spectroscopy; PBDs pyrrolo[2,1-c][1,4]benzodiazepines; PBS Phosphate- buffered saline; P-gp P-glycoprotein; PNP p-nitrophenol; PTFE Polytetrafluoroethylene; PTSA p-Toluenesulfonic acid; TEMPO 2,2,6,6-Tetramethylpiperidinyloxy; TFA Trifluoroacetic acid; and TLC Thin-layer chromatography.

C1-6 alkyl: refers to straight chain and branched saturated hydrocarbon groups, generally having from 1 to 6 carbon atoms; suitably a C1-5 alkyl; more suitably a C1-4 alkyl; more suitably a C1-3 alkyl; more suitably methyl or ethyl. Examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, pent-1- yl, pent-2-yl, pent-3-yl, 3-methylbut-1-yl, 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2,2- trimethyleth-1 -yl, n-hexyl, n-heptyl, or n-octyl and the like.

“Aryl”: refers to fully unsaturated monocyclic, bicyclic and polycyclic aromatic hydrocarbons having at least one aromatic ring and having a specified number of carbon atoms that comprise their ring members (e.g., Ce-14 aryl refers to an aryl group having 6 to 14 carbon atoms as ring members). The aryl group may be attached to a parent group or to a substrate at any ring atom and may include one or more nonhydrogen substituents unless such attachment or substitution would violate valence requirements. Examples of aryl groups include phenyl. “Substituted”, when used in connection with a chemical substituent or moiety (e.g., an alkyl group), means that one or more hydrogen atoms of the substituent or moiety have been replaced with one or more non-hydrogen atoms or groups, provided that valence requirements are met and that a chemically stable compound results from the substitution.

“Halogen”, “Halo,” and “halogeno” may be used interchangeably and suitably each halogen is independently selected. Suitably a halogen is F, Cl, Br or I. Suitably a halogen is F, Cl, or Br. More suitably, a halogen is F or Cl. Most suitably, a halogen is F.

“Independently” is used in the context of “each R’ is independently a halogen” or “... independently R’...” mean that each instance of the functional group e.g. R’ is selected from the listed options independently of any other instance of R’ in the compound. Hence, for example, the first instance of R’ may be F and the next instance of R’ may be Br.

“Pharmaceutically acceptable” substances refer to those substances which are within the scope of sound medical judgment suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit-to-risk ratio, and effective for their intended use.

“Tautomer” refers to two or more isomers of a compound which exist together in equilibrium, and are readily interchanged by migration of an atom, group, or double bond within the molecule.

The term “or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof” means that the compound may comprise one or more tautomers, one or more salts, or one or more salts and one or more tautomers. Suitably, any salt is a pharmaceutically acceptable salt.

The term “subject” as used herein refers to a human or non-human mammal. Examples of non-human mammals include livestock animals such as sheep, horses, cows, pigs, goats, rabbits and deer; and companion animals such as cats, dogs, rodents, and horses. Suitably the subject is a human. “Therapeutically effective amount” of a drug refers to the quantity of the drug or composition that is effective in treating a subject and thus producing the desired therapeutic, ameliorative, inhibitory, or preventative effect. The therapeutically effective amount may depend on the weight and age of the subject and the route of administration, among other things.

“Treating” refers to reversing, alleviating, inhibiting the progress of, or preventing a disorder, disease or condition to which such term applies, or to reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of such disorder, disease or condition.

“Treatment” refers to the act of “treating”, as defined immediately above.

As used herein the term “comprising” means “including at least in part of” and is meant to be inclusive or open ended. When interpreting each statement in this specification that includes the term “comprising”, features, elements and/or steps other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner.

The term “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. When the phrase “consisting essentially of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause.

The term “consisting of” excludes any element, step, or ingredient not specified in the claim; “consisting of” defined as “closing the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase “consists of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole. It should be understood that while various embodiments in the specification are presented using “comprising” language, under various circumstances, a related embodiment is also described using “consisting essentially of” or “consisting of” language.

Dotted Lines The compound of formula (I) is drawn with dotted lines that indicates the optional presence of a double bond between C1 and C2, or C2 and C3. Hence, suitably, the compound formula (I) may be a compound of formula (II) or (III): or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof.

More suitably, there is no optional double bond between C1 and C2, or C2 and C3, and the dotted lines represent single bonds and the of formula (I) is a compound of formula (IV): or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof.

Xi

Xi links a group L to the C8 position of the PBD ring system (which can be represented as -L-Xi-PBD). Suitably, where Xi is C(O)NH or C(0)-0 this amide or ester group may link the PBD to L in either direction. Thus, the amide may be -L-C(O)-NH-PBD or -L- NH-C(O)-PBD and the ester may be -L-C(O)-O-PBD or -L-O-C(O)-PBD.

Suitably, Xi is O, S, NH, C(O), C(O)NH or C(O)-O;

Most suitably Xi is O.

X2

Suitably, X2 is CH.

Alternatively, suitably, X2 is N.

Suitably, X3 is CH.

Alternatively, suitably, X3 is N. x₂ &x₃

Suitably, X₂ and X₃ are CH.

Suitably, one of X₂ and X₃ is CH and the other is N.

Alternatively, suitably, at least one of X₂ and X₃ are N.

Suitably, X₂ and X₃ are N.

X4

Suitably, X4 is CH.

Alternatively, more suitably, X4 is N.

Yi

Suitably, Y1 is N.

Alternatively, more suitably, Y1 is C-R11.

Y2

Suitably, Y₂ is O.

Alternatively, more suitably, Y₂ is S.

Y3

Suitably, Y₃ is N or O.

Suitably, Y₃ is S or N.

More suitably, Y₃ is S or O.

Suitably, Y₃ is N.

Suitably, Y₃ is O

More suitably, Y₃ is S.

Y₃ & X₄

In an aspect, suitably, Y₃ is S ,N or O; and X4 is CH.

In another aspect, more suitably, Y₃ is S, N or O; and X4 is N.

More suitably, Y₃ is S or O; and X4 is N.

Most suitably, Y₃ is S; and X4 is N.

Y4

Suitably, Y₄ is O.

Alternatively, more suitably, Y₄ is S.

L Suitably L is an alkylene chain containing from 1 to 11 carbon atoms, from 1 to 10 carbon atoms, from 1 to 9 carbon atoms, from 1 to 8 carbon atoms, from 1 to 7 carbon atoms, from 1 to 6 carbon atoms, from 1 to 5 carbon atoms.

More suitably, L is CH₂, CH₂CH₂, CH₂CH₂CH₂, CH₂CH₂CH₂CH₂ or CH₂CH₂CH₂CH₂CH₂.

Most suitably, L is CH₂CH₂CH₂.

Ri

Suitably in one aspect, Ri is OH, halogen, CN, =CH₂, =CH(CI-6 alkyl), C1-6 alkyl, or OCi-6 alkyl.

Suitably, Ri is H, OH, halogen, CN, C1-6 alkyl, or OC1-6 alkyl.

Suitably, Ri is H, or C1-6 alkyl.

Alternatively, suitably Ri is H, C1-3 alkyl, or OC1-3 alkyl.

More suitably, Ri is H, methyl or ethyl.

Most suitably, Ri is H.

Rz

Suitably in one aspect, R₂ is OH, halogen, CN, =CH₂, =CH(CI-6 alkyl), C1-6 alkyl, or OC1-6 alkyl.

Suitably, R₂ is H, OH, halogen, CN, C1-6 alkyl, or OC1-6 alkyl.

Suitably, R₂ is H, or C1-6 alkyl.

Alternatively, suitably R₂ is H, C1-3 alkyl, or OC1-3 alkyl.

More suitably, R₂ is H, methyl or ethyl.

Most suitably, R₂ is H.

R3

Suitably in one aspect, R3 is OH, halogen, CN, =CH₂, =CH(CI-6 alkyl), C1-6 alkyl, or OC1-6 alkyl.

Suitably, R₂ is H, OH, halogen, CN, C1-6 alkyl, or OC1-6 alkyl.

Suitably, R3 is H, or C1-6 alkyl.

Alternatively, suitably R3 is H, C1-3 alkyl, or OC1-3 alkyl.

More suitably, R3 is H, methyl or ethyl.

Most suitably, R3 is H.

Ri, R₂ & R3

Suitably, at least one of Ri, R₂ & R3 is H. Suitably, at least two of Ri, R2 & R3 is H.

More suitably, R1 is H, R2 is H and R3 are is H.

R4

Suitably, R4 is F, Cl, Br, I, OH, OC1-6 alkyl, or OCH2Ph.

More suitably, R4 is F, Cl, Br, I, or OC1-6 alkyl.

More suitably, R4 is F, Cl, Br, OCH3, OCH2CH3 or OCH2CH2CH3. More suitably, R4 is F, Cl, Br, OCH₃, or OCH₂CH₃.

In some alternatives, more suitably, R4 is F, Cl or Br.

In other alternatives, more suitably, R4 is OCH3, OCH2CH3 or OCH2CH2CH3.

More suitably, R4 is F, or OCH3.

More suitably, R4 is F.

Most suitably, R4 is OCH3.



Benzofused aromatic groups attached to the PBD through the 5-membered ring (as shown above) have the advantage of providing greater synthetic flexibility than such groups attached to the PBD through the 6-membered ring. For example, one of the R19 to R22 groups may be used to attach a linker group and bind to a suitable targeting moiety, such as an antibody. Suitably, Rs is

Alternatively, more suitably, Rs is: More suitably, Rs is:

In one aspect, suitably, Rs is wherein each R’ is independently a halogen. Suitably, Rs does not include the two options comprising an H2N- substituent in the above benzothiazole groups.

Alternatively, more suitably, Rs is

10

In another aspect, suitably Rs is wherein each R’ is independently a halogen. More suitably, in this aspect, Rs does not include the benzothiazole group attached to the rest of the molecule through the 6- membered benzene ring.

More suitably, Rs is in this aspect, Rs does not include the benzothiazole group attached to the rest of the molecule through the 6-membered benzene ring.

In some aspects, more suitably Rs is In this aspect, more suitably Rs is H, C1-3 alkyl or NH2 and R9 is R’.

More suitably, Rs is

Re and R₇

Suitably, either (i) Rs and R₇ together form a double bond; or

(ii) R₆ is H; and R₇ is H, OH, OCH₃, OCH2CH3, or OCH₂CH₂CH₃; or

(iii) Rs is SO3H, or nitrogen protecting groups; and R₇ is H.

Suitably, either (i) Re and R₇ together form a double bond; or

(ii) R₆ is H and R₇ is OH, OCH₃, OCH2CH3, or OCH₂CH₂CH₃.

More suitably Re and R₇ together form a double bond.

Nitrogen protecting groups

Nitrogen protecting groups are well known in the art and are groups that block or protect the nitrogen groups from further reaction. Nitrogen protecting groups are exemplified by carbamates, such as methyl or ethyl carbamate, 9-fluorenylmethyloxy- carbonyl (Fmoc), substituted ethyl carbamates, carbamates cleaved by 1 ,6-beta- elimination, ureas, amides, peptides, alkyl and aryl derivatives. Carbamate protecting groups have the general formula:

In this specification a zig-zag line indicates the point of attachment of the shown group (e.g. the protecting group above) to the rest of the compound. A large number of possible carbamate nitrogen protecting groups are listed on pages 706 to 771 of Wuts, P.G.M. and Greene, T.W., Protective Groups in Organic Synthesis, 4^th Edition, Wiley- Interscience, 2007, and in P. Kocienski, Protective Groups, 3rd Edition (2005) which are incorporated herein by reference.

Particularly preferred protecting groups include acetyl, allyloxycarbonyl (Alloc), trifluoroacetyl, 2,2,2-Trichloroethyl carbonate (Troc), 2-(Trimethylsilyl)ethoxycarbonyl (Teoc), tert-butyloxycarbonyl(BOC), 2,4-dimethylpent-3-yloxycarbonyl (Doc), cyclohexyloxy-carbonyl (Hoc), 2,2,2-trichloro-tert-butyloxycarbonyl (TcBOC), benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), 1- Adamantyloxycarbonyl and 2-adamantyloxycarbonyl (2-Adoc).

Rs, R9. and R10

In some aspects, suitably either (a) Rs is R’, C1-3 alkyl, OC1-3 alkyl, or NH2; R9 is R’; and R10 is H; (b) Rs is R’; R9 is C1-3 alkyl; and R10 is R’; or (c) Rs is R’; R9 is OC2-3 alkyl; and R10 is H.

In other aspects, suitably either (a) Rs is R’, C1-3 alkyl, OC1-3 alkyl, or NH2; R9 is R’; and R10 is H; (b) Rs is R’; R9 is C1-3 alkyl; or (d) one of Rs and R9 is R’ and the other is H; and R10 is H.

In other aspects, suitably either (a) Rs is R’, C1-3 alkyl, OC1-3 alkyl, or NH2; R9 is R’; and R10 is H; and R_w is R’; or (c) Rs is R’; R9 is OC2-3 alkyl; and R10 is H; or (d) one of Rs and R9 is R’ and the other is H; and R10 is H.

In other aspects, suitably either (a) Rs is R’, C1-3 alkyl, OC1-3 alkyl, or NH2; R9 is R’; and R10 is H; or (b) Rs is R’; R9 is C1-3 alkyl; and R10 is R’.

In other aspects, suitably either (a) Rs is R’, C1-3 alkyl, OC1-3 alkyl, or NH2; R9 is R’; and R10 is H; and R_w is R’; or (c) Rs is R’; R9 is OC2-3 alkyl; and R10 is H.

In other aspects, suitably either (a) Rs is R’, C1-3 alkyl, OC1-3 alkyl, or NH2; R9 is R’; and R10 is H; or (d) one of Rs and R9 is R’ and the other is H; and R10 is H.

In an aspect, suitably (a) Rs is R’, C1-3 alkyl, OC1-3 alkyl, or NH2; R9 is R’; and R_w is H.

In some aspects, suitably for (a) Rs is R’, or NH2; R9 is R’; and R_w is H.

In other aspects, suitably for (a) Rs is R’, C1-3 alkyl, OC1-3 alkyl; R9 is R’; and R_w is H. In other aspects, suitably for (a) Rs is C1-3 alkyl, OC1-3 alkyl, or NH2; R9 is R’; and R_w is H;

Suitably for (a) Rs is CH3, CH2CH3, OCH3, OCH2CH3, or NH2; R9 is R’; and R_w is H. Suitably for (a) Rs is CH3, CH2CH3, OCH3, OCH2CH3, or NH2; R9 is R’; and R_w is H. More suitably, for (a) Rs is CH3, CH2CH3, or NH2; R9 is R’; and R_w is H. More suitably, for (a) Rs is NH2; 9 is R’; and R_w is H. Most suitably, for (a) Rs is NH2; R9 is F; and R10 is H.

In an aspect, suitably (b) Rs and R10 are independently R’; and R9 is C1-3 alkyl;

Suitably for (b) Rs and R_w are independently R’; and R9 is CH3, or CH2CH3.

Suitably, for (b) Rs is F, Cl or Br; R9 is CH3, or CH2CH3; and R10 is F, Cl or Br.

Suitably for (b) Rs and R_w are the same halogen.

More suitably for b) Rs is F; R9 is CH3, or CH2CH3; and R_w is F.

In an aspect, suitably (c) Rs is R’; R9 is OC2-3 alkyl; and R10 is H.

In some aspects, suitably for (c) Rs is F, Cl or Br; R9 is OCH2CH3, or OCH2CH2CH3; and R10 is H.

In other aspects, suitably for (c) Rs is R’; R9 is OCH2CH3; and R_w is H.

Suitably for (c) R9 is OCH3.

Most suitably for (c) Rs is F; R9 is OCH2CH3; and R_w is H.

In an aspect, suitably (d) one of Rs and R9 is R’ and the other is H; and R_w is H.

In some aspects, suitably for (d) Rs is R’; R9 is H; and R10 is H.

In other aspects, suitably for (d) Rs is H; R9 is R’; and R_w is H.

R11, R12, R13

In some aspect, suitably Rn is independently R’, C1-3 alkyl, OC1-3 alkyl, or NH2.

In other aspects, suitably Rn is H, C1-3 alkyl, OC1-3 alkyl, or NH2.

In other aspects, suitably Rn is H, independently R’, C1-3 alkyl or OC1-3 alkyl.

In some aspect, suitably Rn is H, independently R’, C1-3 alkyl, or NH2.

In some aspects, suitably, Rn is NH2.

In other aspects, suitably Rn is H, or C1-3 alkyl.

More suitably, Rn is H.

Suitably, R12 is F, Cl or Br.

In some aspects, suitably R12 is Br.

In other aspects, suitably R12 is Cl.

In other aspects, more suitably R12 is F. In some aspect, suitably R13 is independently R’, C1-3 alkyl, OC1-3 alkyl, or NH2.

In other aspects, suitably R13 is H, C1-3 alkyl, OC1-3 alkyl, or NH2.

In other aspects, suitably R13 is H, independently R’, C1-3 alkyl or OC1-3 alkyl.

In some aspect, suitably R13 is H, independently R’, C1-3 alkyl, or NH2.

In some aspects, suitably, R13 is NH2.

In other aspects, suitably R13 is H, or C1-3 alkyl.

More suitably, R13 is H.

Suitably, one of Rn, and R13 is H.

Suitably, one of Rn, and R13 is H, and the other of Rn, and R13 is NH2.

Alternatively, more suitably, both of Rn and R13 are H.

More suitably, Rn is H, R12 is F, and R13 is H.

In some aspects, Y1 is N, and R13 is NH2.

In other aspects, Y1 is N, and R13 is H.

R14, and R15

In some aspect, suitably, one of R14 and R15 is independently R’ and the other of R14 and R15 is independently R’, C1-3 alkyl, OC1-3 alkyl, or NH2;

In other aspects, suitably one of R14 and R15 is independently R’ and the other of R14 and R15 is H, C1-3 alkyl, OC1-3 alkyl, or NH2.

In other aspects, suitably one of R14 and R15 is independently R’ and the other of R14 and R15 is H, independently R’, C1-3 alkyl or OC1-3 alkyl.

In some aspect, suitably one of R14 and R15 is independently R’ and the other of R14 and R15 is H, independently R’, C1-3 alkyl, or NH2.

In some aspects, suitably one of R14 and R15 is independently R’ and the other of R14 and R15 is NH2.

Suitably, R14 is F, Cl or Br.

In some aspects, suitably R14 is Br.

In other aspects, suitably R14 is Cl.

In other aspects, more suitably R14 is F.

In some aspect, suitably R15 is independently R’, C1-3 alkyl, OC1-3 alkyl, or NH2.

In other aspects, suitably R15 is H, C1-3 alkyl, OC1-3 alkyl, or NH2.

In other aspects, suitably R15 is H, independently R’, C1-3 alkyl or OC1-3 alkyl.

In some aspect, suitably R15 is H, independently R’, C1-3 alkyl, or NH2. In some aspects, suitably, R15 is NH2.

In other aspects, suitably R15 is H, or C1-3 alkyl.

More suitably, R15 is H.

More suitably, R14 is F, and R15 is H.

R16, R17, and RI₈

In some aspect, suitably R16 is independently R’, C1-3 alkyl, OC1-3 alkyl, or NH2.

In other aspects, suitably R16 is H, C1-3 alkyl, OC1-3 alkyl, or NH2.

In other aspects, suitably R16 is H, independently R’, C1-3 alkyl or OC1-3 alkyl.

In some aspect, suitably R16 is H, independently R’, C1-3 alkyl, or NH2.

In some aspects, suitably, R16 is NH2.

In other aspects, suitably R16 is H, or C1-3 alkyl.

More suitably, R16 is H.

Suitably, R17 is F, Cl or Br.

In some aspects, suitably R17 is Br.

In other aspects, suitably R17 is Cl.

In other aspects, more suitably R17 is F.

In some aspect, suitably Ris is independently R’, C1-3 alkyl, OC1-3 alkyl, or NH2.

In other aspects, suitably Ris is H, C1-3 alkyl, OC1-3 alkyl, or NH2.

In other aspects, suitably Ris is H, independently R’, C1-3 alkyl or OC1-3 alkyl.

In some aspect, suitably Ris is H, independently R’, C1-3 alkyl, or NH2.

In some aspects, suitably, Ris is NH2.

In other aspects, suitably Ris is H, or C1-3 alkyl.

More suitably, Ris is H.

Suitably, one of R16 and Ris is H.

More suitably, both of R16 and Ris are H.

More suitably, R16 is H, R17 is F, and Ris is H.

R19; R20, R21; and R22

In some aspects, suitably (1a) R19 is H; R20 is H, R’, C1-3 alkyl, OC1-3 alkyl, or NH2; R21 is R’; and R22 is H, R’, C1-3 alkyl, OC1-3 alkyl, or NH2.

In some aspect, suitably for (1a) R20 is independently R’, C1-3 alkyl, OC1-3 alkyl, or NH2. In other aspects, suitably for (1a) R20 is H, C1-3 alkyl, OC1-3 alkyl, or NH2.

In other aspects, suitably for (1a) R20 is H, independently R’, C1-3 alkyl or OC1-3 alkyl.

In some aspect, suitably for (1a) R20 is H, independently R’, C1-3 alkyl, or NH2.

In some aspects, suitably for (1a) R20 is NH2.

In other aspects, suitably for (1a) R20 is H, or C1-3 alkyl.

More suitably for (1a), R20 is H.

Suitably for (1a), R21 is F, Cl or Br.

In some aspects, suitably for (1a) R21 is Br.

In other aspects, suitably for (1a) R21 is Cl.

In other aspects, more suitably for (1a) R21 is F.

In some aspect, suitably for (1a) R22 is independently R’, C1-3 alkyl, OC1-3 alkyl, or NH2.

In other aspects, suitably for (1a) R22 is H, C1-3 alkyl, OC1-3 alkyl, or NH2.

In other aspects, suitably for (1a) R22 is H, independently R’, C1-3 alkyl or OC1-3 alkyl.

In some aspect, suitably for (1a) R22 is H, independently R’, C1-3 alkyl, or NH2.

In some aspects, suitably for (1a), R22 is NH2.

In other aspects, suitably for (1a) R22 is H, or C1-3 alkyl.

More suitably for (1a), R22 is H.

Suitably for (1a), one of R20 and R22 is H.

Suitably, in some aspects for (1a), R19 is H; R20 is H, R’, C1-3 alkyl, OC1-3 alkyl, or NH2; R21 is R’; and R22 is H.

Alternatively, more suitably, for (1a), R19 is H; R20 is R’, or NH2; R21 is R’; and R22 is H.

Alternatively, more suitably, for (1a), R19 is H; R20 is NH2; R21 is R’; and R22 is H.

Alternatively, more suitably, for (1a), R19 is H; R20 is NH2; R21 is F; and R22 is H.

Suitably, in other aspects for (1a), R19 is H; R20 is H; R21 is R’; and R22 is H, R’, C1-3 alkyl, OC1-3 alkyl, or NH2.

Alternatively, more suitably, for (1a), R19 is H; R20 is H; R21 is R’; and R22 is R’, or NH2.

Alternatively, more suitably, for (1a), R19 is H; R20 is H; R21 is R’; and R22 is NH2.

Alternatively, more suitably, for (1a), R19 is H; R20 is H; R21 is F; and R22 is NH2.

More suitably for (1a), both of R20 and R22 are H.

More suitably for (1a), R19 is H; R20 is H; R21 is R’; and R22 is H.

More suitably for (1a), R19 is H; R20 is H; R21 is F; and R22 is H.

In other aspects, suitably (1b) R19 is H, R’, C1-3 alkyl, OC1-3 alkyl, or NH2; R20 is R’; R21 is H, R’, C1-3 alkyl, OC1-3 alkyl, or NH2; and R22 is H. In some aspect, suitably for (1b) R19 is independently R’, C1-3 alkyl, OC1-3 alkyl, or NH2.

In other aspects, suitably for (1b) R19 is H, C1-3 alkyl, OC1-3 alkyl, or NH2.

In other aspects, suitably for (1b) R19 is H, independently R’, C1-3 alkyl or OC1-3 alkyl.

In some aspect, suitably for (1b) R19 is H, independently R’, C1-3 alkyl, or NH2.

In some aspects, suitably for (1b) R19 is NH2.

In other aspects, suitably for (1b) R19 is H, or C1-3 alkyl.

More suitably for (1b), R19 is H.

Suitably for (1b), R20 is F, Cl or Br.

In some aspects, suitably for (1b) R20 is Br.

In other aspects, suitably for (1b) R20 is Cl.

In other aspects, more suitably for (1b) R20 is F.

In some aspect, suitably for (1b) R21 is independently R’, C1-3 alkyl, OC1-3 alkyl, or NH2.

In other aspects, suitably for (1b) R21 is H, C1-3 alkyl, OC1-3 alkyl, or NH2.

In other aspects, suitably for (1b) R21 is H, independently R’, C1-3 alkyl or OC1-3 alkyl.

In some aspect, suitably for (1b) R21 is H, independently R’, C1-3 alkyl, or NH2.

In some aspects, suitably for (1b), R21 is NH2.

In other aspects, suitably for (1a) R21 is H, or C1-3 alkyl.

More suitably for (1b), R21 is H.

Suitably for (1a), one of R19 and R21 is H.

Suitably, in some aspects for (1b), R19 is H, R’, C1-3 alkyl, OC1-3 alkyl, or NH2; R20 is R’; R21 is H; and R22 is H.

Alternatively, more suitably, for (1b), R19 is R’, or NH2; R20 is R’; R21 is H; and R22 is H. Alternatively, more suitably, for (1b), R19 is NH2; R20 is F; R21 is H; and R22 is H. Suitably, in other aspects for (1b), R19 is H; R20 is R’; R21 is H, R’, C1-3 alkyl, OC1-3 alkyl, or NH2; and R22 is H.

Alternatively, more suitably, for (1b), R19 is H; R20 is R’; R21 is R’, or NH2; and R22 is H.

Alternatively, more suitably, for (1b), R19 is H; R20 is R’; R21 is NH2; and R22 is H.

Alternatively, more suitably, for (1b), R19 is H; R20 is F; R21 is NH2; and R22 is H.

More suitably for (1a), both of R19 and R21 are H.

More suitably for (1a), R19 is H, R20 is F, R21 is H, and R22 is H.

R23 & R24 — N'^

In some aspects, suitably, R23 is H and R24 is H. In these aspects, Rs may be represented as:

— N'^ . More suitably, when R23 is H and R24 is H, then R4 is halogen.

— N'^

In other aspects, suitably, R23 is H and R24 is H . In these aspects, Rs may be represented as:

— N'^

. More suitably, when R23 is H and R24 is H , then R4 is

R25

Suitably, each R25 is independently F, Cl, Br or I. More suitably, each R25 is independently F, Cl or Br. More suitably, each R25 is independently F or Cl. Most suitably, R25 is F.

Suitably, each R’ is independently F, Cl, Br or I.

More suitably, each R’ is independently F, Cl or Br.

More suitably, each R’ is independently F or Cl.

Most suitably, R’ is F. z

Suitably, z is 0.

Alternatively, suitably, z is 1.

Structures

Suitably, the compound of formula (I) is: or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof.

Suitably, the compound of formula (I) is: or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof.

More suitably, the compound of formula (I) is: or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof. More suitably, the compound of formula (I) is:

or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof. or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof.

In some aspects, more suitably, the compound of formula (I) is:

MH02 MH04 MH64 10 or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof.

More suitably, the compound of formula (I) is MH02, MH03, MH04, MH05, MH07, MH10, MH13, MH17, MH19, MH22, MH32, MH33, MH34, MH44, MH63, or MH64, 10, 11, 12, 13, 14, 16, 19 or SL-226- 19 or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof.

More suitably, the compound of formula (I) is MH02, MH03, MH04, MH10, MH13, MH17, MH22, MH33, MH34, MH63, or MH64, 10, 11, 12, 13, 14, 16, 19 or SL-226-19 or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof.

More suitably, the compound of formula (I) is MH22, 16, 19 or SL-226-19 or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof. Alternatively, more suitably, the compound of formula (I) is MH02, MH03, MH04,

MH05, MH07, MH10, MH13, MH17, MH19, MH22, MH32, MH33, MH34, MH44, MH54,

MH63, or MH64, or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof.

In some aspects, more suitably, the compound of formula (I) is: or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof; wherein R16, R20 and R21 are independently H, R’, C1-3 alkyl, OC1-3 alkyl, or NH2; and each R’ is independently a halogen.

Targeting moiety-drug conjugates and linker groups

In one aspect, a compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, is linked directly, or indirectly via a linker group, to a targeting moiety to form a targeting moiety-drug conjugate.

Suitably, the targeting moiety is an aptamer, an antibody, an antibody fragment or a lipid nanoparticle.

Suitably, where the targeting moiety is a lipid nanoparticle the lipid nanoparticle encapsulates the compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof.

More suitably, the targeting moiety is an antibody or an antibody fragment.

More suitably, the targeting moiety-drug conjugate is an antibody-drug conjugate.

Antibody therapy has been established for the targeted treatment of patients with cancer, immunological and angiogenic disorders [27]. The use of antibody-drug conjugates (ADCs), i.e. immunoconjugates, for the local delivery of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumour cells in the treatment of cancer, targets delivery of the drug moiety to tumours and intracellular accumulation therein. In contrast, systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells [28],

Thus, ADCs seek to provide maximal efficacy with minimal toxicity. Efforts to design and refine ADCs have focused on the selectivity of monoclonal antibodies (mAbs) as well as drug mechanism of action, drug-linking, drug/antibody ratio (loading), and drugreleasing properties [29], also see US 7521541; US 7723485; W02009/052249. Numerous ADCs containing PBDs have also been reported [18, 19],

Suitably, there is described a compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, for use as a drug in an antibody-drug conjugate. Suitably, a compound of formula (I), or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, for use as a drug in an antibody-drug conjugate is prepared by attaching a compound of formula (I), or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, to an antibody or antibody fragment, either directly or via an optional linker group. Suitably, the compound of formula (I), (or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, is attached to an antibody or antibody fragment via a linker group. Suitably, the compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, is linked directly, or indirectly via a linker group, to an antibody, or antibody fragment, to form an antibody-drug conjugate.

In some aspects, the present invention relates to a compound of formula (I), or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, for use as a drug in a targeting moiety-drug conjugate, suitably an antibody-drug conjugate. Suitably, a compound of formula (I), or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, may be used directly to prepare a targeting moiety-drug conjugate, suitably an antibody-drug conjugate, when a compound of formula (I), or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, comprises one or more functional groups (such as amine, hydroxyl or carboxylic acid groups) for attaching the drug to the targeting moiety, suitably an antibody, either directly or via a linker group. Suitably, a compound of formula (I), or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, may be used in preparing a targeting moiety-drug conjugate, suitably an antibody-drug conjugate, by being modified to comprise one or more functional groups (such as amine, hydroxyl or carboxylic acid groups) for attaching the drug to the targeting moiety, suitably an antibody, either directly or via a linker group. Suitably, a compound of formula (I), or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, may be used in preparing a targeting moiety-drug conjugate, suitably an antibody-drug conjugate, by being modified to contain one or more linker groups, wherein the targeting moiety, suitably an antibody, is attached to the drug through the one or more linker groups. Therefore, the present disclosure provides for a compound of formula (I), or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, further comprising one or more linker groups. Suitably, a compound of formula (I), or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, may further comprise 1 , 2, or 3 linker groups. Suitably, a compound of formula (I), or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, may contain 1 or 2 linker groups. Suitably, a compound of the formula (I), or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, may contain 1 linker group. In some aspects, one or more atoms or groups of the compound of formula (I), or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, may be eliminated during the attachment of the drug to the targeting moiety, suitably an antibody, or the attachment of the linker to the drug or the modification of the drug to contain one or more functional groups (such as amine, hydroxyl or carboxylic acid groups) for attaching the drug to the targeting moiety, suitably an antibody, either directly or via a linker group.

Thus, suitably the compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, as described herein, may contain a linker group, wherein the targeting moiety, suitably an antibody, or antibody fragment, is attached to the compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, through the linker group.

Hence, there is described a targeting moiety-drug conjugate, suitably an antibody-drug conjugate, wherein one or more compounds of formula (I) a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, are linked, directly or indirectly, to the targeting moiety, such as an antibody. Therefore, the compound compounds of formula (I) a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, as described herein, may be used as a payload on a targeting moiety-drug conjugate, suitably an antibodydrug conjugate.

More suitably, the compound of formula (I) a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, as described herein, is linked via a linker group, to a targeting moiety, such as an antibody, or antibody fragment, to form a targeting moiety- drug conjugate, suitably an antibody-drug conjugate.

Antibody-drug conjugates as described herein may be formed in stages. Hence, the compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, as described herein, may be linked to a linker group.

A variety of suitable linker groups are known in the art and may be used as described herein. For example, the maleimide methodology is routinely used as a method to attach targeting moieties, such as antibodies, to drug compounds by providing a linker attached to the drug with a terminal maleimide group. In addition, methodologies using diarylcyclooctyne moeities (such as, but not limited to, dibenzylcyclooctyne (DBCO), or dibenzoazacyclooctyne (DIBAC)) are also alternatives used in the art.

Diarylcyclooctynes react with azides to provide attachment via the formation of stable triazoles. Diarylcyclooctynes are thermostable with very narrow and specific reactivity toward azides, resulting in almost quantitative yields of stable triazoles. Furthermore, the reaction does not require a cytotoxic Cu(l) catalyst (that is toxic to most organisms) and thus, prevents its use in many biological systems. Still further, alkoxyamine methodologies are also alternatives used in the art. For site-specific conjugation of the drug to an antibody, the antibodies may comprise a “tag” (which may be proprietary) that will react with a dairylcyclooctyne (for example, DBCO), an alkyloxyamine and/or maleimide group to attach the antibody to the drug. The tag in some instances may be a mutated amino acid. Suitable linker groups incorporating the various groups described above are available in the art.

Suitably, the compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, as described herein, may be linked through the N10 of the PBD by replacement of the Re group with a linker group, for example, to produce the compound, in a reaction scheme as shown below.

Alternatively, the linkage may be through a suitable substituent group on the C8 side chain such as by substitution of a hydrogen of an amine with the linker group, for example, in a reaction as shown below.

Such linker groups for use with targeting moiety-drug conjugates, such as ADCs, are well-known in the art and have been reviewed [30, 31, 32], These linker groups comprise a group for attachment to the targeting moiety, such as an antibody or antibody fragment. Suitably, the linker group comprises an alkoxyamine, an amine, an amino acid, a diarylcyclooctyne, a maleimide, a triazole, or a thiol group.

Suitably, the diarylcyclooctyne is dibenzylcyclooctyne (DBCO), or dibenzoazacyclooctyne (DIBAC).

Suitably, the linker group comprises a peptide group comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid units. More suitably, the linker group comprises a peptide group comprising 2, 3, or 4 amino acid units. More suitably, the linker group comprises a dipeptide.

Suitably, the linker group comprises a peptide group comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid units and a group selected from an alkoxyamine, an amine, an amino acid, a diarylcyclooctyne, a maleimide, a triazole, or a thiol group. More suitably, the linker group comprises a peptide group comprising 2, 3, or 4 amino acid units and a group selected from an alkoxyamine, an amine, an amino acid, a di benzylcyclooctyne (DBCO), a dibenzoazacyclooctyne (DIBAC), a maleimide, a triazole, or a thiol group. More suitably, the peptide group is a dipeptide. More suitably, the dipeptide is valinecitrulline (Val-Cit), valine-alanine (Val-Ala) or phenylalanine-lysine (Phe-Lys). More suitably, the peptide group is valine-alanine (Val-Ala).

In some aspects, the present invention relates to the use of a compound of formula (I), a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, as a drug in an antibody-drug conjugate. Suitably, the use of a compound of formula (I), a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, as a drug in an antibody-drug conjugate is accomplished by attaching a compound of formula (I) a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, to an antibody, either directly or via an optional linker group. Suitably, the compound of formula (I) a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, is attached to an antibody via a linker group. Suitably, the antibody-drug conjugate is for use in for treatment of a disease, more specifically of a proliferative and/or malignant disease. Suitably, the drug may be attached by any suitable functional group that it contains to the antibody either directly or via a linker group. Typically, the drug contains, or can be modified to contain, one or more functional groups such as amine, hydroxyl or carboxylic acid groups for attaching the drug to the antibody either directly or via a linker group. In some aspects, the antibody of the antibody drug conjugate is an antibody fragment, such as, but not limited to a single chain antibody. In some aspects, one or more atoms or groups of the compound of formula (I), a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, may be eliminated during the attachment of the drug to the antibody. In some aspects, the antibody binds to a cell surface receptor or a tumour-associated antigen.

Aptamers

Aptamers are oligonucleotides that are capable of specifically binding to selected targets. They are created by an in vitro selection from a library of random sequence oligonucleotides through a combinatorial process named SELEX (“Systematic Evolution of Ligands by Exponential enrichment”). A typical aptamer is 10-15 kDa in size (30-45 nucleotides), binds its target with sub-nanomolar affinity, and discriminates against closely related targets (e.g., aptamers will typically not bind other proteins from the same gene family). A series of structural studies have shown that aptamers are capable of using the same types of binding interactions (e.g., hydrogen bonding, electrostatic complementarity, hydrophobic contacts, steric exclusion) that drive affinity and specificity in antibody-antigen complexes. Target specific aptamers can be readily generated using techniques generally known in the art (see, e.g., Gold et al., Annu. Rev. Biochem. 64:763, 1995; Brody and Gold, J. Biotechnol. 74:5, 2000; Sun, Curr. Opin. Mol. Ther. 2:100, 2000; Kusser, J. Biotechnol. 74:27, 2000; Hermann and Patel, Science 287:820, 2000; and Jayasena, Clinical Chem. 45:1628, 1999).

Antibody and antibody fragments

The term “antibody” specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), intact antibodies and antibody fragments, so long as they exhibit the desired biological activity, for example, the ability to bind a desired antigen on a target cell or tissue. Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen [33], A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on the antibody. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e. , a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin can be of any type (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass, or allotype (e.g. human G1 ml , G1 m2, G1 m3, non-G1 ml [that, is any allotype other than G1 ml], G1 m17, G2m23, G3m21 , G3m28, G3m1 1 , G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1 , A2m2, Km1 , Km2 and Km3) of immunoglobulin molecule. The immunoglobulins can be derived from any species, including human, murine, or rabbit origin.

As used herein, “binds an epitope” is used to mean the antibody binds an epitope with a higher affinity than a non-specific partner such as Bovine Serum Albumin (BSA, Genbank accession no. CAA76847, version no. CAA76847.1 Gl:3336842, record update date: Jan 7, 201 1 02:30 PM). In some embodiments the antibody binds an epitope with an association constant (Ka) at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10⁴, 10⁵ or 10⁶-fold higher than the antibody's association constant for BSA, when measured at physiological conditions. The term “antibody fragment” refers to a portion of a full length antibody, for example, the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and scFv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-ld) antibodies, CDR (complementary determining region), single-chain antibody molecules; and multispecific antibodies formed from antibody fragments and epitope-binding fragments of any of the above which immunospecifically bind to target antigens, such as, for example, cancer cell antigens, viral antigens or microbial antigens. The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant or epitope on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described in 1975 [34], or may be made by recombinant DNA methods (see, US 4816567). The monoclonal antibodies may also be isolated from phage antibody libraries using known techniques [35] or from transgenic mice carrying a fully human immunoglobulin system [36],

The antibodies, including monoclonal antibodies, herein specifically include “chimeric” antibodies in which a portion of the antibody structure, for example the heavy and/or light chain, is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity [see US 4816567; and [37], Chimeric antibodies include “primatized” antibodies comprising variable domain antigen-binding sequences derived from a non- human primate (e.g. Old World Monkey or Ape) and human constant region sequences. An “intact antibody” herein is one comprising VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof. The intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1 q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors such as B cell receptor and BCR.

The antibodies disclosed herein may be modified. For example, to make them less immunogenic to a human subject. This may be achieved using any of a number of techniques familiar to the person skilled in the art, such as humanisation.

Proliferative and/or Malignant Disease

There is described a compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, or a pharmaceutical composition, as described herein, for use in the treatment of a proliferative and/or malignant disease.

Suitably, the proliferative and/or malignant disease may be a metastatic or non- metastatic cancer. The cancer may be familial or sporadic. Suitably, the proliferative and/or malignant disease that can be treated may comprise, for example, benign or in- situ lesions and malignant solid tumours and benign and malignant non-solid tumours.

A method of treating a proliferative and/or malignant disease is described, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the disclosure, or a tautomer thereof, or salts thereof, or a salt of a tautomer thereof, or a composition comprising a compound of the disclosure or a tautomer thereof, or salts thereof, or a salt of a tautomer thereof.

Suitably, the proliferative and/or malignant disease is selected from breast cancers, brain cancers, central nervous system cancers, carcinomas, gastrointestinal cancers, hormonal cancers, leukemias, liver cancers, lung cancers, respiratory cancers, lymphomas, sarcomas, fibrosarcomas, skin cancers, melanomas, urinary tract cancers, reproductive cancers, or miscellaneous other cancers. Suitably, breast cancers that may be treated is invasive breast cancer of no special type, ductal carcinoma in situ (DCIS), ductal invasive breast cancer, inflammatory breast cancer, invasive lobular breast cancer, Paget’s disease of the breast, or triple negative breast cancer.

Suitably, brain cancers, or central nervous system (CNS) cancers and tumours that may be treated include astrocytomas (including cerebellar and cerebral), brain stem glioma, brain tumours, malignant gliomas, ependymoma, glioblastoma, medulloblastoma, supratentorial primitive neuroectodermal tumours, visual pathway and hypothalamic gliomas, primary central nervous system lymphoma, ependymoma, brain stem glioma, visual pathway and hypothalamic glioma, extracranial germ cell tumour, medulloblastoma, myelodysplastic syndromes, oligodendroglioma, myelodysplastic/myeloproliferative diseases, myelogenous leukemia, myeloid leukemia, multiple myeloma, myeloproliferative disorders, neuroblastoma, plasma cell neoplasm/multiple myeloma, central nervous system lymphoma, intrinsic brain tumours, astrocytic brain tumours, gliomas, and/or metastatic tumour cell invasion in the central nervous system.

Carcinomas that may be treated include acinar, acinic cell, acinous, adenocystic, adenoid cystic, adenoid squamous cell, cancer adenomatosum, adenosquamous, adnexel, cancer of adrenal cortex, adrenocortical, aldosterone-producing, aldosterone- secreting, alveolar, alveolar cell, ameloblastic, ampullary, anaplastic cancer of thyroid gland, apocrine, basal cell, alveolar, comedo basal cell, cystic basal cell, morphea-like basal cell, multicentric basal cell, nodulo-ulcerative basal cell, pigmented basal cell, sclerosing basal cell, superficial basal cell, basaloid, basosquamous cell, bile duct, extrahepatic bile duct, intrahepatic bile duct, bronchioalveolar, bronchiolar, bronchioloalveolar, bronchoalveolar, bronchoalveolar cell, bronchogenic, cerebriform, cholangiocellular, chorionic, choroids plexus, clear cell, cloacogenic anal, colloid, comedo, corpus, cancer of corpus uteri, Cortisol-producing, cribriform, cylindrical, cylindrical cell, duct, ductal carcinoma, ductal cancer of the prostate, ductal cancer in situ (DCIS), eccrine, embryonal, cancer en cuirasse, endometrial, cancer of endometrium, endometroid, epidermoid, cancer ex mixed tumour, cancer ex pleomorphic adenoma, exophytic, fibrolamellar, cancer fibro’ sum, follicular cancer of thyroid gland, gastric, gelatinform, gelatinous, giant cell, giant cell cancer of thyroid gland, cancer gigantocellular, glandular, granulose cell, hepatocellular, Hurthle cell, hypernephroid, infantile embryonal, islet cell carcinoma, breast cancer, inflammatory cancer of the breast, cancer in situ, intraductal, intraepidermal, intraepithelial, juvenile embryonal, Kulchitsky-cell, large cell, leptomeningeal, lobular, infiltrating lobular, invasive lobular, lobular cancer in situ (LCIS), lymphoepithelial, cancer medullare, medullary, medullary cancer of thyroid gland, medullary thyroid, melanotic, meningeal, Merkel cell cancer, metatypical cell, micropapillary, mucinous, cancer muciparum, cancer mucocellulare, mucoepidermoid, cancer mucosum, mucous, nasopharyngeal, neuroendocrine cancer of the skin, noninfiltrating, non-small cell, non-small cell lung cancer (NSCLC), oat cell, cancer ossificans, osteoid, Paget’s, papillary, papillary cancer of thyroid gland, periampullary, preinvasive, prickle cell, primary intraosseous, renal cell carcinoma, scar, schistosomal bladder, Schneiderian, scirrhous, sebaceous, signet-ring cell, cancer simplex, small cell, small cell lung cancer (SCLC), spindle cell, cancer spongiosum, squamous, squamous cell carcinoma, terminal duct, anaplastic thyroid, follicular thyroid, medullary thyroid, papillary thyroid, trabecular cancer of the skin, transitional cell , tubular, undifferentiated cancer of thyroid gland, uterine corpus, verrucous, villous, cancer villosum, yolk sac, squamous cell particularly of the head and neck, esophageal squamous cell, and/or oral cancers and carcinomas.

Gastrointestinal cancers that may be treated include extrahepatic bile duct cancer, bowel cancer, colon cancer, colon and rectum cancer, colorectal cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumour, gastrointestinal carcinoid tumours, gastrointestinal stromal tumours, bladder cancers, islet cell carcinoma (endocrine pancreas), pancreatic cancer, islet cell pancreatic cancer, prostate cancer, rectal cancer, salivary gland cancer, small intestine cancer, colon cancer, and polyps associated with colorectal neoplasia.

Lung cancers, or respiratory cancers that may be treated include bronchial adenomas/carcinoids, esophagus cancer, esophageal cancer, laryngeal cancer, hypopharyngeal cancer, lung carcinoid tumour, non-small cell lung cancer, small cell lung cancer, small cell carcinoma of the lungs, mesothelioma, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, nasopharyngeal cancer, oral cancer, oral cavity cancer, lip cancer, oropharyngeal cancer, paranasal sinus and nasal cavity cancer, and/or pleuropulmonary blastoma.

Hormonal cancers that may be treated include: parathyroid cancer, pineal and supratentorial primitive neuroectodermal tumours, pituitary tumour, thymoma and thymic carcinoma, thymoma, thymus cancer, thyroid cancer, cancer of the adrenal cortex, and/or ACTH-producing tumours. Leukemias that may be targeted (may also be considered to include other blood cell malignancies) include acute lymphoblastic, acute myeloid, acute lymphocytic, acute myelogenous leukemia, chronic myelogenous, hairy cell, erythroleukemia, lymphoblastic, myeloid, lymphocytic, myelogenous, leukemia, hairy cell, T-cell, monocytic, myeloblastic, granulocytic, gross, hand mirror-cell, basophilic, hemoblastic, histiocytic, leukopenic, lymphatic, Schilling’s, stem cell, myelomonocytic, monocytic, prolymphocytic, promyelocytic, micromyeloblastic, megakaryoblastic, megakaryocytic, heder cell, bovine, aleukemic, mast cell, myelocytic, plamsa cell, subleukemic, multiple myeloma, nonlymphocytic, chronic myelogenous leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin’s disease, non-Hodgkin’s lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease , myelodysplastic syndrome, and myelodysplasia and/or chronic myelocytic leukemias.

Liver cancers that may be targeted include extrahepatic bile duct cancer, and/or hepatocellular cancers.

Lymphomas that may be treated include AIDS-related, non- Hodgkin’s, Hodgkin’s, T- cell, T-cell leukemia/lymphoma, African, B-cell, B-cell monocytoid, bovine malignant, Burkitt’s, centrocytic, lymphoma cutis, diffuse; diffuse, large cell; diffuse, mixed small and large cell; diffuse, small cleaved cell; follicular, follicular center cell, follicular, mixed small cleaved and large cell, follicular, predominantly large cell, follicular, predominantly small cleaved cell, giant follicle, giant follicular, granulomatous, histiocytic, large cell, immunoblastic, large cleaved cell, large nocleaved cell, Lennert’s, lymphoblastic, lymphocytic, intermediate; lymphocytic, intermediately differentiated, plasmacytoid; poorly differentiated lymphocytic, small lymphocytic, well differentiated lymphocytic, lymphoma of cattle; MALT, mantle cell, mantle zone, marginal zone, Mediterranean lymphoma mixed lymphocytic-histiocytic, nodular, plasmacytoid, pleomorphic, primary central nervous system, primary effusion, small B-cell, small cleaved cell, small concleaved cell, T-cell lymphomas; convoluted T-cell, cutaneous t- cell, small lymphocytic T-cell, undefined lymphoma, u-cell, undifferentiated, aids- related, central nervous system, cutaneous T-cell, effusion (body cavity based), thymic lymphoma, and/or cutaneous T-cell lymphomas.

Suitable proliferative and/or malignant diseases include sarcomas, or fibrosarcomas, which are tumours whose cells are embedded in a fibrillar or homogeneous substance, such as embryonic connective tissue. Sarcomas that may be targeted include adipose, alveolar soft part, ameloblastic, avian, botryoid, sarcoma botryoides, chicken, chloromatous, chondroblastic, clear cell sarcoma of tendon sheaths, clear cell sarcoma of kidney, embryonal, endometrial stromal, epithelioid, Ewing’s, fascial, fibroblastic, fowl, giant cell, granulocytic, hemangioendothelial, Hodgkin’s, idiopathic multiple pigmented hemorrhagic, immunoblastic sarcoma of B cells, immunoblastic sarcoma of T cells, Jensen’s, Kaposi’s, kupffer cell, leukocytic, lymphatic, melanotic, mixed cell, multiple, lymphangio, idiopathic hemorrhagic, multipotential primary sarcoma of bone, osteoblastic, osteogenic, parosteal, polymorphous, pseudo-kaposi, reticulum cell , reticulum cell sarcoma of the brain, rhabdomyosarcoma, rous, soft tissue, spindle cell, synovial sarcoma, telangiectatic, sarcoma (osteosarcoma)/malignant fibrous histiocytoma of bone, and/or soft tissue sarcomas.

Skin cancers, or melanomas that may be treated include cutaneous T-cell lymphoma, intraocular melanoma, metastatic melanoma, non-melanoma skin cancer, tumour progression of human skin keratinocytes, basal cell carcinoma, and squamous cell cancer. Eye cancers that may be targeted include intraocular melanoma, retinoblastoma, and/or intraocular melanoma.

Urinary tract and reproductive cancers that may be treated include cervical cancer, endometrial cancer, ovarian epithelial cancer, extragonadal germ cell tumour, extracranial germ cell tumour, extragonadal germ cell tumour, ovarian germ cell tumour, gestational trophoblastic tumour, spleen, kidney cancer, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumour, ovarian low malignant potential tumour, penile cancer, renal cancer, retin renal cell cancer (including carcinomas), renal cell cancer, transitional cell cancer of the renal pelvis and ureter, gestational trophoblastic tumour, testicular cancer, ureter and renal pelvis, transitional cell cancer, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, ovarian carcinoma, primary peritoneal epithelial neoplasms, cervical carcinoma, urinary system cancer, uterine cancer and solid tumours in the ovarian follicle, superficial bladder tumours, invasive transitional cell carcinoma of the bladder, and/or muscle-invasive bladder cancer.

Miscellaneous other cancers that may be targeted include advanced cancers, AIDS- related, anal cancer adrenal cortical, aplastic anemia, aniline, betel, bone cancer, buyo cheek, carcinoid (gastrointestinal and bronchal) Castleman’s disease, chronic myeloproliferative disorders, cerebriform, chimney-sweeps, clay pipe, colloid, contact, cystic, dendritic, cancer a deux, duct, dye workers, encephaloid, cancer en cuirasse, endometrial, endothelial, epithelial, Ewing’s family of tumours, glandular, head and neck cancer, hemangiopericytoma, cancer in situ, kang, kangri, latent, lip and oral cavity cancer, medullary, melanotic, metastatic squamous neck cancer with occult primary, multiple endocrine neoplasia syndrome, multiple myeloma/plasma cell neoplasm, mule-spinners’, mycosis fungoides, occult cancer, paraffin, peritoneal effusion, malignant pleural effusion, pheochromocytoma, pitch workers’, scar, schistosomal bladder, scirrhous, sezary syndrome, lymph node, soft, soot, spindle cell, supratentorial primitive neuroectodermal tumours, swamp, tar, tubular cancers, trophoblastic neo-plasms, unknown primary site, and Wilms’ tumour.

Suitably the proliferative and/or malignant disease is a solid cancer.

As used herein, a “solid cancer” refers to one or more cells which are growing or have grown in an uncontrolled manner to form cancer tissue. As used herein, the term “solid cancer” includes, but is not limited to “carcinomas”, “adenocarcinomas” and “sarcomas”. “Sarcomas” are cancers of the connective tissue, cartilage, bone, muscle, and so on. “Carcinomas” are cancers of epithelial (lining) cells. “Adenocarcinoma” refers to carcinoma derived from cells of glandular origin. The terms “cancer” and “tumour” are used interchangeably throughout the subject specification.

Solid cancers may arise in nearly any tissue of the body and the treatment of any solid cancer is contemplated by the present invention. Exemplary “solid cancers” which may be treated as described herein are AIDS related cancer, adenocystic carcinoma, adrenocortical carcinoma, alveolar soft-part sarcoma, anal cancer, angiosarcoma, aplastic anaemia, astrocytoma, basal cell carcinoma (bcc), bladder cancer, bone cancers, bowel cancer, brain stem glioma, brain cancer, breast cancer, central nervous system (CNS) cancers, carcinoid tumours, cervical cancer, colorectal cancers, cutaneous T-Cell lymphoma, ductal carcinoma, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extra hepatic bile duct cancer, eye cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancers, gastrointestinal carcinoid tumours, germ cell cancers, gestational trophoblastic tumours, glioma, head and neck cancer, hepatocellular cancer, Hodgkin's disease, hypopharynx cancer, intraocular melanoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip cancer, liver cancer, lung cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, medulloblastoma, melanoma, merkel cell cancer, mesothelioma, metastatic cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndromes, multiple myeloma, myeloproliferative disorders, nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-melanoma skin cancer, non-small cell lung cancer (nsclc), oral cavity cancer, osteosarcoma, pancreatic cancer, paranasal cancer, parathyroid cancer, penile cancer, pituitary tumours, polycythemia vera, prostate cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, sezary syndrome, skin cancer, small cell lung cancer (scic), soft tissue sarcoma, squamous cell carcinoma (see), stomach cancer, synovial sarcoma, testicular cancer, thymus cancer, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, urinary system cancer, uterine sarcoma, uterine cancer, vaginal Cancer, vulvar cancer, Waldenstrom's macroglobulinemia or Wilms' tumours.

More suitably, the proliferative disease is adenocystic carcinoma, basal cell carcinoma, bladder cancer, bone cancer, bowel cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon carcinoma, colorectal cancer, ductal carcinoma, eye: melanoma, head and neck cancer, intra-ocular melanoma, liver cancer, lung cancer, lymphoma, melanoma, oesophageal cancer, oral cavity cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, retinoblastoma, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine sarcoma or uterus cancer.

Any type of cell may be treated, including but not limited to, bone, eye, head and neck, lung, gastrointestinal (including, e.g. mouth, oesophagus, bowel, colon), breast (mammary), cervix, ovarian, uterus, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain, or skin.

Other Therapeutic Agents

A compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, or a pharmaceutical composition, may be used in the treatment of a proliferative and/or malignant disease, wherein the compound is administered either simultaneously or sequentially with one or more other therapeutic agent. Suitably, the one or more other therapeutic agents may comprise one or more cytotoxic agents, and/or one or more chemotherapeutic agents and/or one or more checkpoint inhibitor agents and/or one or more other therapeutic antibodies or agents.

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. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, ^Re188’ Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclosed below.

A “chemotherapeutic agent” refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9- tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetyl camptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammal l and calicheamicin omegall (see, e.g., Nicolaou et al., Angew. Chem Inti. Ed. Engl., 33 : 183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCI liposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®), peglylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-Fll); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2- ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2’-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C"); thiotepa; taxoid, e.g., paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANETM), and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vincas, which prevent tubulin polymerization from forming microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP- 16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1 ,3- dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC- alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF- R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU- 11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitor (e.g., celecoxib or etoricoxib), proteosome inhibitor (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors; serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®); farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASARTM); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATINTM) combined with 5-FU and leucovorin.

Chemotherapeutic agents as defined herein include “anti-hormonal agents” or “endocrine therapeutics” which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer. They may be hormones themselves, including, but not limited to: anti-estrogens with mixed agonist/antagonist profile, including, tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs) such as SERM3; pure anti-estrogens without agonist properties, such as fulvestrant (FASLODEX®), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors such as anastrazole (A RF Ml DEX®), letrozole (FEMARA®) and aminoglutethimide, and other aromatase inhibitors include vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and 4(5)-imidazoles; lutenizing hormone- releaseing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; sex steroids, including progestines such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, and androgens/retinoids such as fluoxymesterone, all transretionic acid and fenretinide; onapristone; anti-progesterones; estrogen receptor down- regulators (ERDs); anti-androgens such as flutamide, nilutamide and bicalutamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above.

The term “checkpoint inhibitor agents” refers to a substance that affect the way the immune system functions. For example, checkpoint inhibitor agents may target lymphocyte receptors or their ligands in order to enhance the endogenous antitumor activity of the immune system. Immune checkpoint agents can be stimulatory or inhibitory. Tumours can use these checkpoints to protect themselves from immune system attacks. Checkpoint inhibitor agents can block inhibitory checkpoints, restoring immune system function. Checkpoint proteins include programmed cell death 1 protein (PDCD1, PD-1; also known as CD279) and its ligand, PD-1 ligand 1 (PD-L1, CD274), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), A2AR (Adenosine A2A receptor), B7-H3 (or CD276), B7-H4 (or VTCN1), BTLA (B and T Lymphocyte Attenuator, or CD272), GITR (glucocorticoid-induced tumour necrosis factor receptor), IDO (Indoleamine 2,3-dioxygenase), KIR (Killer-cell Immunoglobulin-like Receptor), LAG3 (Lymphocyte Activation Gene-3), 0X40 (Receptor activation induces proliferation of memory and effector T cells), TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3), TIGIT (T-cell immunoglobulin and ITIM domain receptor) and VISTA (V- domain Ig suppressor of T cell activation).

Checkpoint inhibitor agents may comprise one or more antibodies that are an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-A2AR antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-BTLA antibody, an anti-GITR antibody, an anti-IDO antibody, an anti-KIR antibody, an anti-LAG3 antibody, an anti- 0X40 antibody an anti-TIM-3 antibody, an anti-TIGIT antibody and an anti-VISTA antibody.

Because such antibodies act primarily by regulating the immune response to diseased cells, tissues or pathogens, but do not target tumour cells directly, they may ideally be used in combination with other therapeutic groups, such as antibody-drug conjugates (ADCs), and/or cytotoxic agents, and/or chemotherapeutic agents, to enhance the antitumour effect of these other therapeutic groups.

Exemplary anti-PD1 antibodies include pembrolizumab (MK-3475, MERCK), nivolumab (BMS-936558, Bristol-Myers Squibb), and pidilizumab (CT-011 , Curetech Ltd.). Anti- PD1 antibodies are commercially available, for example from Abeam (AB137132), Biolegend (EH12.2H7, RMP1-14) and Affymetric Ebioxcience (J105, J116, MIH4). Exemplary anti-PDL1 antibodies include MDX-1105 (Medarex), durvalumab (MEDI4736, Medimmune) atezolizumab (TECENTRIQ®, MPDL3280A, Genetech) and BMS-936559 (Bristol-Myers Squibb). Anti-PDL1 antibodies are also commercially available, for example from Affymetric Ebioxcience (MIH1).

Exemplary anti-CTLA4 antibodies include ipilimumab (Bristol-Myers Squibb) and tremelimumab (Pfizer). Anti-PD1 antibodies are commercially available, for example from Abacam (AB134090), Sino Biological Inc. (11159-H03H, 11159-H08H), and ThermoFisher Scientific (PA5-29572, PA5-23967, PA5-26465, MA1-12205, MAI- 35914).

The one or more other therapeutic antibodies or agents includes therapeutic antibody comprises one or more anti-Her2/neu receptor antibody for example trastuzumab (marketed as Herceptin); Alemtuzumab, a CD52 antibody marketed as Campath, MabCampath or Campath-1 H currently under further development as Lemtrada; Gemtuzumab, an anti-CD33 monoclonal antibody linked to a calicheamicin marketed by Wyeth as Mylotarg; an anti-CD20 antibody, such as Rituximab (marketed as Rituxan and MabThera) or Ibritumomab tiuxetan sold under the trade name Zevalin; anti-TNF- alpha antibodies such as Infliximab (marketed as Remicade), or Adalimumab (marketed as Humira), or a soluble TNFR2 molecule such as etanercept (also known as Enbrel); an antibody to the CD25 chain of the IL-2 receptor such as basiliximab (trade name Simulect); an anti CD40/CD40L antibody such as a humanized IgGI antihuman CD40 antibody (SGN-40); and/or one or more other The term “other therapeutic agents that may include soluble Lymphocyte-activation gene 3 (also known as LAG3 or CD223)-based immune modulators such as LAG3-lg (IMP321); Toll-like receptor agonists like MPL, CpG, single-stranded R A, nucleotides, nucleotide analogue, CL087 (a TLR7-specific ligand), loxoribine, polyinosine-polycytidylic acid, flagellin, resiquimod, immiquimod, gardiquimod NOD ligands like muramyl dipeptide, murabutide, peptidoglycan, muramyldipeptide and/or anti-virals such as oseltamivir phosphate, Amphotericin B, and palivizumab.

Pharmaceutical Composition

Suitably the pharmaceutical composition further comprises one or more other therapeutic agents. Suitably, the pharmaceutical composition further comprises one or more cytotoxic agents. Suitably, the pharmaceutical composition further comprises one or more chemotherapeutic agents. Suitably, the pharmaceutical composition further comprises one or more checkpoint inhibitor agents. Suitably, the pharmaceutical composition further comprises one or more other therapeutic antibodies or agents

Suitably the pharmaceutical composition comprises (i) a compound of formula or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof; (ii) one or more other therapeutic agents; and (iii) one or more of a pharmaceutically acceptable carrier, diluent, excipient or lipid nanoparticle.

Suitably, the pharmaceutical composition comprises a lipid nanoparticle. Suitably the lipid nanoparticle encapsulates the compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof.

Lipid Nanoparticles

As used herein, the term “lipid nanoparticle” refers to a particle comprising one or more lipids. Suitably, the lipid nanoparticle comprises one or more cationic lipids, anionic lipids, neutral lipids, amphipathic lipids and/or structural lipids.

Suitably the cationic lipids are one or more of 3p-[N — (N',N'-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride (DC-Chol); 1,2-dioleoyl-3-trimethylammonium- propane (DOTAP); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); dimethyldioctadecylammonium bromide salt (DDAB); 1,2-dilauroyl-sn-glycero-3- ethylphosphocholine chloride (DL-EPC); N-[1-(2, 3-dioleyloyx) propyl]-N — N — N- trimethyl ammonium chloride (DOTMA); N-[1-(2, 3-dioleyloyx) propyl]-N — N — N- dimethyl ammonium chloride (DODMA); N,N-dioctadecyl-N,N-dimethylammonium chloride (DODAC); N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N- dimethylammonium trifluoracetate (DOSPA); 1,2-dimyristyloxypropyl-3- dimethylhydroxyethyl ammonium bromide (DMRIE); and/or dioctadecylamidoglycylspermine (DOGS).

Suitable anionic lipids are one or more of oleic acids, linoleic acids, and linolenic acids; cholesteryl hemisuccinate; 1 ,2-di-O-tetradecyl-sn-glycero-3-phospho-(1 -rac-glycerol) (Diether PG); 1,2-dimyristoyl-sn-glycero-3-phospho-(T-rac-glycerol) (sodium salt); 1,2- dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1-hexadecanoyl,2-(9Z,12Z)- octadecadienoyl-sn-glycero-3-phosphate; 1 ,2-dioleoyl-sn-glycero-3-[phosphor-rac-(1 - glycerol)] (DOPG); dioleoylphosphatidic acid (DOPA); and/or 1 ,2-dioleoyl-sn-glycero-3- phospho-L-serine (DOPS).

Suitable neutral lipids are one or more of phosphatidylcholine (PC), phosphatidylethanolamine, ceramide, cerebrosides, sphingomyelin, cephalin, cholesterol, diacylglycerols, glycosylated diacylglycerols, prenols, lysosomal PLA2 substrates, and/or N-acylglycines.

Suitable amphiphatic lipids are one or more saturated or (poly)unsaturated phospholipids.

Suitable structural lipids are one or more of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, and/or steroids.

Administration & Dose

A compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, may be administered alone or in combination with one or more other therapeutic agents, which are pharmacologically active compounds which are different from the compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof.

Compounds of the invention may suitably be combined with various components to produce compositions of the invention. Suitably the compositions are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition (which may be for human or animal use). Suitable carriers and diluents include isotonic saline solutions (for example phosphate-buffered saline), water, ethanol, propylene glycol, glycerin, and combinations thereof. Useful pharmaceutical compositions and methods for their preparation may be found in standard pharmaceutical texts. See, for example, Handbook for Pharmaceutical Additives, 3rd Edition (eds. M. Ash and I. Ash), 2007 (Synapse Information Resources, Inc., Endicott, New York, USA) and Remington: The Science and Practice of Pharmacy, 21st Edition (ed. D. B. Troy) 2006 (Lippincott, Williams and Wilkins, Philadelphia, USA) which are incorporated herein by reference.

The compounds of the invention may be administered by any suitable route. Suitably the compounds of the invention will normally be administered orally or by any parenteral route, in the form of pharmaceutical preparations comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Suitably the parenteral route is selected from intramuscular, subcutaneous, intravenous and intradermal administration. More suitably, the parenteral route selected from intramuscular and intravenous administration.

A compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

Suitable pharmaceutically acceptable excipient includes without limitation any adjuvant, disintegrants, excipient, glidant, granulation binders, lubricating agents, sweetening agent, preservative, dye/colorant, flavour enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

An adjuvant is a substance incorporated into or administered with antigen which potentiates the immune response. Adjuvants may enhance the immunological response by providing a reservoir of antigen (extracellularly or within macrophages/DCs), activating antigen presenting cells to stimulate specific sets of lymphocytes. Adjuvants of many kinds are well known in the art. Specific examples of adjuvants include monophosphoryl lipid A (MPL, SmithKline Beecham) , a congener obtained after purification and acid hydrolysis of Salmonella Minnesota Re 595 lipopolysaccharide; saponins , including QS21 ( SmithKlineBeecham) a pure QA-21 saponin purified from Quillj a saponaria extract ; DQS21 , described in PCT application WO96/33739 ( SmithKline Beecham) ; QS-7, QS-17, QS-18, and QS-L1 (So et al., Mol Cells (1997) 7 : 178-186) ; ISCOMATRIX adjuvant, a cage-like structure composed of saponin, phospholipid, and cholesterol ( see, e.g., Maraskovsky et al. , Clin. Cancer Res. (2004) 10:2879-2890); incomplete Freund’s adjuvant; complete Freund’s adjuvant; montanide ; alum; CpG oligonucleotides (see e.g. Kreig et al., Nature 374:546-9, 1995) and other immunostimulatory oligonucleotides including poly-IC and poly-ICLC (Hiltonol©); and various water-in-oil emulsions prepared from biodegradable oils such as squalene and/or tocopherol. For example, the compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, can be administered orally, buccally or sublingually in the form of tablets, capsules (including soft gel capsules), ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, controlled-release or pulsatile delivery applications. The compounds of the invention may also be administered via fast dispersing or fast dissolving dosages forms.

Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethyl cellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

Modified release and pulsatile release dosage forms may contain excipients such as those detailed for immediate release dosage forms together with additional excipients that act as release rate modifiers, these being coated on and/or included in the body of the device. Release rate modifiers include, but are not exclusively limited to, hydroxypropylmethyl cellulose, methyl cellulose, sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate, polyethylene oxide, Xanthan gum, Carbomer, ammonio methacrylate copolymer, hydrogenated castor oil, carnauba wax, paraffin wax, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, methacrylic acid copolymer and mixtures thereof. Modified release and pulsatile release dosage forms may contain one or a combination of release rate modifying excipients. Release rate modifying excipients maybe present both within the dosage form i.e. within the matrix, and/or on the dosage form i.e. upon the surface or coating. Fast dispersing or dissolving dosage formulations (FDDFs) may contain the following ingredients: aspartame, acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, diascorbic acid, ethyl acrylate, ethyl cellulose, gelatin, hydroxypropylmethyl cellulose, magnesium stearate, mannitol, methyl methacrylate, mint flavouring, polyethylene glycol, fumed silica, silicon dioxide, sodium starch glycolate, sodium stearyl fumarate, sorbitol, xylitol.

The compounds of the invention can also be administered parenterally, for example, intravenously, intra-arterially, or they may be administered by infusion techniques. For such parenteral administration they are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

Suitably formulation of the invention is optimised for the route of administration e.g. oral, intravenously, etc.

Administration may be in one dose, continuously or intermittently (e.g. in divided doses at appropriate intervals) during the course of treatment. Methods of determining the most effective means and dosage are well known to a skilled person and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and the dose regimen being selected by the treating physician, veterinarian, or clinician.

Where more than one agent is to be administered, these agents may be provided by simultaneous, or sequential administration. By “simultaneous” administration, it is meant that the different agents are administered to the individual at the same time. This may be achieved as a single dose by the same route of administration or by different routes of administration which occur at the same time. This may occur for example where one agent is administered by infusion or parenterally and the other is given orally during the course of the infusion or parenteral administration.

By “sequential” it is meant that the different agents are administered at different points in time, provided that the activity of the first administered agent is present and ongoing in the subject at the time the second agent is administered. For example, an chemotherapeutic agent may be administered first, followed by administration of a compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, such that both agent and compound may act on the tumour. This sequential administration may occur by the same route or by different routes of administration. Preferably, a sequential dose will occur such that the second of the two agents is administered within 48 hours, preferably within 24 hours, such as within 12, 6, 4, 2 or 1 hour(s) of the first agent. Preferably, one agent may be administered daily and a second agent may be administered every two, or every three, or every four, or every five or every six, or every seven days.

Multiple doses of the compound of compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, or may be administered, for example 2, 3, 4, 5 or more than 5 doses may be administered after administration of the one or more other therapeutic agent. The administration of the compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, may continue for sustained periods of time after administration of the other therapeutic agent. For example, treatment with the compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, may be continued for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month or at least 2 months. Treatment with a compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, may be in cycles with a break and then resumption of treatment. Administration with the compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, may be continued for as long as is necessary to inhibit growth of, or reduce, or to achieve complete treatment of the tumour.

Multiple doses of the one or more or other therapeutic agent may be administered, for example 2, 3, 4, 5 or more than 5 doses may be administered after administration of the compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof. The administration of the other therapeutic agent may continue for sustained periods of time after administration of the compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof. For example, treatment with the other therapeutic agent may be continued for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month or at least 2 months. Administration with the other therapeutic agent may be continued for as long as is necessary to to inhibit growth of, or reduce or to achieve complete treatment of the tumour. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses. For example, a typical dosage for an adult human may be 100 ng to 25 mg (suitably about 1 micro g to about 10 mg) per kg body weight of the subject per day.

Suitably guidance may be taken from studies in test animals when estimating an initial dose for human subjects. For example, when a particular dose is identified for mice, suitably an initial test dose for humans may be approx. 0.5x to 2x the mg/Kg value given to mice.

Other Forms

Unless otherwise specified, included in the above are the well-known ionic, salt, solvate, tautomeric and protected forms of these substituents. For example, a reference to carboxylic acid (-RCOOH) also includes the anionic (carboxylate) form (- RCOO'), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-RN⁺HR¹R²), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (-O'), a salt or solvate thereof, as well as conventional protected forms.

Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, mesomeric stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and I- forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; alpha- and beta-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”).

Note that, except as discussed below for tautomeric forms, specifically excluded from the term “isomers”, as used herein, are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, -OCH3, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, - CH2OH. A reference to a class of structures may well include structurally isomeric forms falling within that class (e.g. C1-7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl). The above exclusion does not apply to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol, imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

Note that specifically included in the term “isomer” are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including ¹H, ²H (D), and ³H (T); C may be in any isotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopic form, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

Unless otherwise specified, a reference to a particular compound also includes ionic, salt, solvate, and protected forms of thereof, for example, as discussed below.

In some embodiments, the compound of the disclosure and salts and solvates thereof, comprises pharmaceutically acceptable salts of the compounds of the disclosure. Compounds of the disclosure, which include compounds specifically named above, may form pharmaceutically acceptable complexes, salts, solvates and hydrates. These salts include nontoxic acid addition salts (including di-acids) and base salts.

If the compound is cationic or has a functional group which may be cationic (e.g. -NH2 may be -NH₃ ⁺), then an acid addition salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids hydrochloric acid, nitric acid, nitrous acid, phosphoric acid, sulfuric acid, sulphurous acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid, phosphoric acid and phosphorous acids. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2- acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose. Such salts include acetate, adipate, aspartate, benzoate, besylate, bicarbonate, carbonate, bisulfate, sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfonate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate, hydrogen phosphate, dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts.

For example, if the compound is anionic, or has a functional group which may be anionic (e.g. -RCOOH may be -RCOO'), then a base salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, metal cations, such as an alkali or alkaline earth metal cation, ammonium and substituted ammonium cations, as well as amines. Examples of suitable metal cations include sodium (Na⁺) potassium (K⁺), magnesium (Mg²⁺), calcium (Ca²⁺), zinc (Zn²⁺), and aluminum (Al³⁺). Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH4⁺) and substituted ammonium ions (e.g. NHsR⁺, NH2R2⁺, NHRa⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4⁺. Examples of suitable amines include arginine, N,N'-dibenzylethylene- diamine, chloroprocaine, choline, diethylamine, diethanolamine, dicyclohexylamine, ethylenediamine, glycine, lysine, N-methylglucamine, olamine, 2-amino-2- hydroxymethyl-propane-1 ,3-diol, and procaine. For a discussion of useful acid addition and base salts, see S. M. Berge et al., J. Pharm. Sci. (1977) 66:1-19; see also Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (2011) Pharmaceutically acceptable salts may be prepared using various methods. For example, one may react a compound of the disclosure with an appropriate acid or base to give the desired salt. One may also react a precursor of the compound of the disclosure with an acid or base to remove an acid- or base-labile protecting group or to open a lactone or lactam group of the precursor. Additionally, one may convert a salt of the compound of the disclosure to another salt through treatment with an appropriate acid or base or through contact with an ion exchange resin. Following reaction, one may then isolate the salt by filtration if it precipitates from solution, or by evaporation to recover the salt. The degree of ionization of the salt may vary from completely ionized to almost non-ionized.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described further, with reference to the accompanying drawings, in which:

Figure 1 shows a PBD core structure of the tested compounds whose Rs groups are shown in Figures 2-6.

Figure 2 shows the anticancer activity against a panel three solid cancers of four halogenated PBD and two reference PBD compounds and one non-PBD reference compound Doxorubicin (Dox).

Figure 3 shows the activity of a further halogenated PBD compound and one reference PBD compound against the cancer panel.

Figure 4 shows the activity of three further halogenated PBD compounds and one reference PBD compound that comprise thiazole groups against the cancer panel. Figure 5A-5C shows the inhibition of canonical NF-KB subunits for p50 & p65 subunits for one reference compound GWL-78 and five halogenated PBD compounds.

Figure 6A-6C shows the inhibition of canonical NF-KB subunits for p52 subunits for for one reference compound GWL-78 and five halogenated PBD compounds.

Figure 7 shows the cytotoxicity profile of selected halogenated PBD compounds, MH10 and MH22, and a reference PBD compound, GWL-78.

Figure 8 shows the cytotoxicity profile of MH64.

Figure 9 shows the aqueous solubility (PBS, pH 7.4) of selected PBD compounds and six reference FDA-approved drugs.

Figure 10 shows the in vitro absorption of halogenated PBD compounds, MH10 and MH22, and reference compounds measured in the Caco-2 permeability model.

Figure 11 shows the protein binding of halogenated PBD compounds, MH10 and MH22, and reference compounds. Figure 12 shows the intrinsic clearance for two halogenated PBD compounds, MH10 and MH22.

Figure 13 shows the half-life of two halogenated PBD compounds, MH10 and MH22, and four refence drugs.

Figure 14 shows a typical plate design used for anticancer screening against different cell lines for two compounds, columns 1-6 and 7-11 , respectively.

Figure 15 shows an overview of TransAM NFKB family ELISA assay.

Figure 16 shows a bovine serum albumin (BSA) standard curve to interpolate the concentrations of nuclear extracts.

Figure 17 shows the hERG channel inhibition by verapamil.

Figure 18 shows the principle and workflow of FRET DNA melting study where F indicates FAM and Q indicates TAMRA.

Figure 19 shows DNA minor groove fitting and interactions of superimposed structures of reference PBD monomer MH01 (light black) and its fluorinated counterpart MH02 (yellow) are shown in A and their docking results (B-C).

Figure 20 shows the superimposed DNA minor groove fitting between MH02 (brown) and MH05 (light black) is shown in A. DNA minor groove fitting (3D image; left) and interactions (2D image; right) of reference PBD monomer MH05 (B) and its fluorinated counterpart H06 (C).

Figure 21 shows the superimposed structure of MH10 (light black) and MH22 (yellow) is shown in A. DNA minor groove fitting (3D image; left) and interactions (2D image; right) of mono-alkyl and fluorine-containing short-PBD analogue MH10 (B) and its 08-linkable counterpart H22 (C).

Figure 22 shows the relative position of fluorine of MH10 and MH22 inside the DNA minor groove.

Figure 23 The close view of the interaction of fluorine of MH32 within the DNA minor groove, while the non-fluorinated MH31 compound did not form any interactions.

Figure 24 shows a close view within the DNA minor groove of the superimposed structure of H61 and H64, along with drawings of these structures.

Figure 25 shows A) the interactions of MH10 in a P-gp efflux pump inhibitor binding pocket; B) shows the % cell viability of MH10 & MH10 + verapamil at a range of MH10 concentrations 72 h after treatment of a MDA-MB-231 cell line; and C) shows the structure of MH10.

Figure 26 shows A) the interactions of MH13 in a P-gp efflux pump inhibitor binding pocket; B) shows the % cell viability of MH13 & MH13 + verapamil at a range of MH13 concentrations 72 h after treatment of a MDA-MB-231 cell line; and C) shows the structure of MH13. Figure 27 shows A) the interactions of MH37 in a P-gp efflux pump inhibitor binding pocket; B) shows the % cell viability of MH37 & MH37 + verapamil at a range of MH37 concentrations 72 h after treatment of a MDA-MB-231 cell line; and C) shows the structure of MH37.

Figure 28 shows the chromatogram for the monoclonal antibody trastuzumab with absorbance under 280 nm as the y-axis and the eluting volume (ml) as the x-axis. Figure 29 shows the chromatogram of the antibody drug conjugate of trastuzumab with SL-226-26 with absorbance under 280 nm as the y-axis and the eluting volume (ml) as the x-axis.

EXPERIMENTAL

Synthetic building blocks and chemicals were sourced from several suppliers, including Sigma-Aldrich (Merck KGaA, USA), Thermo Fisher Scientific (UK, including Acros Organics, Maybridge and Alfa Aesar), Fluorochem (UK), Activate Scientific (UK), VWR International (USA), and Apollo Scientific (UK). Solvents were purchased from Sigma- Aldrich and Thermo Fisher Scientific. Silica gel and thin-layer chromatography plates were purchased from Sigma-Aldrich. LC-MS vials were purchased from Agilent (USA). NMR tubes were purchased from Fluorochem (UK). Thin-layer chromatography (TLC) was used to separate the components of a crude mixture. The TLC plates were coated with silica gel (Merck silica gel 60 F254 plates). The separated components were visualized using ultraviolet (UV) light at a wavelength of 254 nm or by staining with potassium permanganate solution followed by gentle heating. Flash column chromatography used columns purchased from Dixon Science UK with a PTFE stopcock. Manual flash chromatography used silica gel (Millipore 109385, 230-400 mesh ASTM, 0.040-0.063 mm) as a stationary phase. The solvent system, mobile phase, was determined using a TLC system from diethyl ether, hexane, DCM and ethyl acetate. Flash column chromatography used. Stuart™ SMP30 (Merck Z675407) melting point apparatus was used to measure the melting points (MP) of the final PBD monomers. The solid materials were taken into a closed 10 mm tube (Jaytec Glass™ CAP-MPG-100) and the tube was filled up to 2 mm. The machine was set at 60 °C in a plateau state and then ramped at 8 °C per minute until the sample started melting. The data was collected at the start and the end of the melting.

Analytical Liquid Chromatography-Mass Spectrometry (LC-MS)

Analytical LCMS was utilised for reaction monitoring, compound identification, and purity analysis of synthesised compounds. Method A (5 min): Flow rate 1 .0 mL/min, 100 pL was split via a zero dead volume T piece, which passed into the mass spectrometer. The wavelength range of the UV detector was 220-500 nm. i) from 95% A/5% B to 10% A/90% B over three minutes; ii) from 10% A/90% B to 5% A/95% B over 30 seconds; iii) held constant at 5% A/95% B for a further minute; iv) from 5% A/95% B to 95% A/5% B over 30 seconds.

Method B (10 min): Flow rate 0.5 mL/min, 200 pL was split via a zero dead volume T piece, which passed into the mass spectrometer. The wavelength range of the UV detector was 220-400 nm i) .from 95% A/5% B to 50% A/50% B over three minutes; ii) from 50% A/50% B to 20% A/80% B over two minutes; iii) from 20% A/80% B to 5% A/95% B over 1.5 minutes; iv) held constant at 5% A/95% B for a further 1.5 minutes; v) from 5% A/95% B to 95% A/5% B over 0.2 minutes; vi) held constant at 95% A/5% B for a further 1.8 minutes.

Analytical liquid chromatography used the following parameters: injection volume 10 pL; draw speed 100 pL/min; ejection speed 400 pL/min; wait time after drawing 1.2 s. Mass spectrometry data (both ESI+ and ESI- modes) were collected using the following parameters: capillary voltage 4 kV (ESI+), 3.5 kV (ESI-); drying gas flow 13.0 L/min; nebuliser pressure 50 psig (method A), 30 psig (method B), 60 psig (maximum); drying gas temperature 350°C; mass range 150 - 1 ,200 Da; fragmentor 70; gain 1.00; stepsize 0.10; speed 2,600 u/sec.

High-Resolution Mass Spectrometry (HRMS)

High-resolution mass spectrometry (HRMS) data of the final PBD monomers was obtained using an electrospray ionisation technique. Agilent InfinityLab LC/MSD System consisting of an Agilent 1290 Infinity II Analytical-Scale LC Purification System coupled to a 6120 Quadrupole mass spectrometer

Nuclear Magnetic Resonance Spectroscopy (NMR)

All NMR spectra were obtained at room temperature using a Bruker Ascend 400 MHz NMR spectrometer. The proton NMR was run at 400Hz, while the carbon samples were run at 100Hz. The temperature for all samples was 300 K. All the raw data were processed and analyzed using ACD Labs/NMR Processor Academic Edition software. Chemical shifts (5 H) are expressed in parts per million (ppm) relative to the residual signal of deuterated chloroform (CDCh, 5 H = 7.26 ppm). The other reference compounds used are deuterated acetonitrile (CD3CN, 5 H = 1.94 ppm), deuterated dimethyl sulfoxide ((CDs^SO, 5 H = 2.50 ppm), and deuterated methanol (CD3OD, 5 H = 3.31 ppm). Coupling constants are expressed in hertz. Multiplicities in 1 H NMR spectra are indicated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets, ddd = doublet of doublet of doublets, dt = doublet of triplets, td = triplet of doublets, spt = septet, and br = broad. We used Wilmad economy 5mm NMR tubes for all NMR analysis, purchased from Fluorochem Ltd. (UK).

General Amide Coupling Method I (EDC/DMAP in DMF)

The acid (1 eq) was dissolved in DCM (5 mL for 100 mg of starting material) in a round- bottomed flask fitted with a magnetic stirrer. Then EDC (2 eq) and DMAP (2 eq) were added to the reaction mixture and stirred for at least 30 minutes. After that, the amine (1.1 eq) was added to the reaction mixture. The reaction was stirred until the TLC or LC-MS showed the consumption of the acid. The reaction usually finishes within 3 hours but the reaction could be stirred overnight depending on the reactivity of the amine moiety. Once finished, the reaction was quenched using ice/water 30 mL, followed by extraction with ethyl acetate. The organic layer was washed with 1M citric acid, saturated NaHCOs, water and brine (30 mL of each). The organic layer was then dried over MgSO4, and the solvent was removed using a rotary evaporator. Flash chromatography could be used if the crude has shown more than one spot in TLC using the abovementioned general procedures.

General Amide Coupling Method II (BTFFH/DIPEA in DCM)

The acid (1 eq) was dissolved in DMF (4 mL for 75 mg of starting material) in a microwave reaction vial (maximum capacity 10 mL) fitted with a magnetic stirrer. Once dissolved, the coupling reagent BTFFH was added to the reaction mixture (1.5 eq). The base DI PEA was then added drop-by-drop to the mixture 4.5 eq) and stirred for 30 minutes. Finally, the amine (1.5 eq) was added to the reaction mixture. The vial was then sealed and heated to 80 °C while stirring. The reaction was then allowed to be stirred until the TLC/LC-MS confirmed the complete consumption of the acid. Once finished, the reaction mixture was dried over a rotary evaporator, and the desired compound was purified using flash chromatography. Synthesis of PBD Core

Methyl 4-(4-formyl-2-methoxyphenoxy)butanoate (1.2) A mixture of vanillin (18.5 g, 0.122 mol, 1.0 eq), methyl 4-bromobutanoate (23.13 g, 16.2 mL, 0.128 mol, 1.05 eq) and potassium carbonate (25.21 g, 0.183 mol, 1.5 eq) was dissolved in DMF (75 mL). The reaction mixture was stirred at room temperature overnight. Thin-layer chromatography (TLC) was used to monitor the reaction. The reaction was completed after 13 hours, and the reaction mixture was diluted with water (800 mL) and cooled down in an ice bath for 30 minutes. A white precipitate was formed, filtered, washed with cold water and dried initially on the filter under vacuum and later in a vacuum oven overnight at 40°C to yield 28.3 g of product 1.2 (90%).

Methyl 4-(4-formyl-2-methoxy-5-nitrophenoxy)butanoate (1.3)

Previously synthesized 1.2 (28g, 0.111 mol, 1 eq) was added into a 250 mL round- bottomed flask containing TFA (30 mL) and allowed to dissolve completely by stirring.

Potassium nitrate (14 g, 1.2 eq) was added to stirring TFA (30 mL) in a round-bottomed flask fitted in an ice bath. After waiting a while, the mixture was cooled down and dissolved 1.2 in TFA was added dropwise and kept, stirring the reaction mixture at room temperature overnight. Once TLC showed the completion of the reaction, the reaction mixture was dried in a rotary evaporator and obtained a brownish oily residue. The residue was dissolved in 100 mL of ethyl acetate (EtOAc) and washed with brine (100 mL x3). The organic phase was kept and dried over MgSO4, followed by a rotary evaporator. The residue was dried in a vacuum oven overnight at 40°C to yield 26 g of product 1.3 (82%).

5-methoxy-4-(4-methoxy-4-oxobutoxy)-2-nitrobenzoic acid (1.4)

A solution of nitro-aldehyde 1.3 (26 g, 0.087 mol, 1 eq) in acetone (600 mL) in a 2L flask fitted with a hotplate and a condenser kept stirring at 70°C. A hot aqueous solution (600 mL) of 10% (w/v) potassium permanganate was quickly added (within 5 to 10 minutes) to the nitroaldehyde solution. The reaction mixture was refluxed vigorously after adding the initial 150-200 mL potassium permanganate solution. Refluxing was maintained until the end of the reaction, which TLC confirmed. The reaction mixture was slowly cooled to room temperature and filtered through celite packed in a sintered funnel. The brown residue on the celite bed was washed with 600 mL of hot water. The filtrate was transferred to a larger beaker, and a sodium bisulphite solution (96 g in 600 mL 1 N HCI) was added. The pH of the final solution was adjusted to 1-2 using concentrated HCI. The mixture was then extracted with DCM (500 mL x3) and combined with organic phases. The organic phase was then dried in a rotary evaporator and a high vacuum for extended periods. Product 1.4 was found to be pure by TLC. The yield was 22 g (80%).

Methyl (S)-4-(4-(2-(hydroxymethyl)pyrrolidine-1-carbonyl)-2-methoxy-5- nitrophenoxy)butanoate (1.5)

The acid 1.4 (5 g, 15.96 mmol) was dissolved in oxalyl chloride (2M solution in DCM) in a round-bottomed flask fitted with a balloon and magnetic stirrer. Within a few minutes, the bubble formation was observed in the balloon. After 20 minutes, the bubbling ceased. The reaction mixture was then dried over a rotary evaporator. The remaining residues were then dissolved in toluene (15 mL) and dried again in the rotary evaporator to confirm the complete removal of oxalyl chloride. The residue was then dissolved in 10 mL of DCM. Place the reaction mixture in a cooling bath (ice bath). Once the reaction mixture turned cooled, the (S)-(+)-2-Pyrrolidinemethanol (1.7 g, 1.65 mL, 16.76 mmol, 1.05 eq) was added drop by drop, followed by triethylamine (1.5 eq, 3.34 mL) separately dissolved in 20 mL dry DCM. The reaction mixture was then allowed to stir at room temperature and kept stirring overnight.

After 12 hours, the LC-MS confirmed that the reaction was finished. The reaction mix was washed with hydrochloric acid (1M, 10 ml) and brine (20 ml) and dried over magnesium sulphate. The solution was concentrated using a rotary evaporator, and the crude product was purified by flash column chromatography. The final compound was eluted at 5% methanol in ethyl acetate to afford 1.5 3.75 g yield (59%). This reaction was repeated a few times to get a good amount (about 12 g) to move to the next steps.

Methyl (S)-4-(5-amino-4-(2-(hydroxymethyl)pyrrolidine-1-carbonyl)-2- methoxyphenoxy)butanoate(1.6)

The pyrrole containing nitro compound 1.5 (3.8 g, 9.59 mmol) was dissolved in 10 mL of ethyl acetate into a reaction bottle (cat no. 66CA3, Parr Instrument Company) and then added 380 mg of Pd/C (10% w/w) into the reaction bottle. Finally, the ammonium formate (2 eq, 1.21 g, 19.17 mmol) was added and settled the bottle to the Perr Shaking Hydrogenator (3911 series). The H₂ pressure was kept at 45 psi. Within one hour, the H₂ pressure stabilized. The LC-MS and TLC confirmed the reaction completion after 3 hours of reaction. The reaction mixture was then filtered through celite. The celite bed was washed with 400 mL of ethyl acetate. The filtrate was then dried over a rotary evaporator to afford a yellow oily pure 1.6 3.236 g (92%). This step was repeated a few times as the maximum solvent we could use in 500 mL of the reaction bottle was about 10-12 mL to avoid spillage.

1.6 1 HNMR (400 MHz, CDCI3): 5 7.69(s, 1 H), 6.80(s, 1 H),

Dark 4.38(d, J=5.8 Hz, 1 H), 4.14(t, J=6.2 Hz, 2H), 3.95(s, 3H),

¹H-NMR brown 3.85(m, 2H), 3.70(s, 3H), 3.16(t, J=6.6 Hz, 2H), 2.55(t, paste J=7.2 Hz, 2H), 2.19(p, J=6.6 Hz, 3H), 1.83(m, 3H)

Methyl (S)-4-(5-(((allyloxy)carbonyl)amino)-4-(2-(hydroxymethyl)pyrrolidine-1-carbonyl)- 2-methoxyphenoxy)butanoate (1.7) The amine 1.6 (17.87 mmol; 6.55 g) was dissolved in 100 mL of dry DCM. Dry pyridine (2.5 eq, 3.54 g; 44.69 mmol) was then added (N2 flushed) and placed the reaction mixture in a cooling bath (Ice: Acetone, 1 :1). Once the reaction mixture was cooled, the allyl chloroformate was added to the mixture and kept stirring. The LC-MS showed that the reaction was finished after 2 hours. The reaction mixture was washed with 100 mL of CuSO4 (aqueous), followed by 100 mL of NaHCCh and brine and collected from the organic layer. The organic layer was then dried over MgSO4, and the solvent was evaporated in a rotary evaporator. The crude product was then purified using flash chromatography. The compound 1.7 eluted at 35% ethyl acetate in DCM. The final yield of 1.7 was 5.3 g (65%).

Allyl (11aS)-11-hydroxy-7-methoxy-8-(4-methoxy-4-oxobutoxy)-5-oxo-2,3,11 ,11a- tetrahydro-1 H-benzo[e]pyrrolo[1 ,2-a][1 ,4]diazepine-10(5H)-carboxylate (1.8)

1.8 (65%) The alloc-protected compound 1.7 (2.7 g; 5.99 mmol) was dissolved in DCM (120 mL) in a 250 mL round-bottomed flask. The solution turned pinkish. BAI B was added immediately (1.5 eq, 2.90 g; 8.99 mmol), and the colour changed to yellow. Finally, a catalytic amount of TEMPO (0.1 eq, 93.65 mg; 0.599 mmol) and the colour of the reaction turned reddish-yellow. We kept stirring overnight, and the reaction was finished, as the LCMS confirmed. The reaction mixture was then washed with sodium bisulfite (100 mL) to remove the trace amount of TEMPO/BAIB, followed by extraction sequentially with 100 mL of NaHCCh and brine and collected the organic layer. The organic layer was then dried over MgSCL, and the solvent was evaporated in a rotary evaporator. The crude product was then purified using flash chromatography. The compound 1.8 eluted at 25% ethyl acetate in DCM. The final yield of 1.8 was 63% (1.7 g). The reaction was done twice as it required a large amount of solvents to afford 3.45

Allyl (11aS)-7-methoxy-8-(4-methoxy-4-oxobutoxy)-5-oxo-11-((tetrahydro-2H-pyran-2- yl)oxy)-2,3, 11 ,11 a-tetrahydro-1 H-benzo[e]pyrrolo[1 , 2-a][ 1 ,4]diazepine-10(5H)- carboxylate (1.9)

The alcohol of 1.8 was protected using DHP/PTSA. Compound 1.8 (3.37 g, 7.52 mmol) was transferred to a clean 250 mL round bottomed flask and dissolved the compound in 40 mL of ethyl acetate. While stirring, the 3,4-Dihydropyran (10 eq, 6.32 g, 75.17 mmol) drop by drop to the reaction mixture, followed by the addition of a catalytic amount of p-Toluenesulfonic acid (0.05 eq, 64.72 mg) and kept stirring. The reaction was finished after 3.5 hours, as per the LC-MS confirmed. The reaction mixture was washed in NaHCCh (50 mL x 2), followed by 50 mL of water and brine. The organic fraction was then dried over MgSCL, and the solvent was evaporated in a rotary evaporator. The crude product was then purified using flash chromatography. The compound 1.9 eluted at 10% acetone in DCM. The final yield of 1.9 was 3.55 g (88%).

4-(((11aS)-10-((allyloxy)carbonyl)-7-methoxy- 5-oxo- 11-((tetrahydro-2H-pyran-2-yl)oxy)-

2,3,5, 10, 11 , 11 a-hexahydro-1 H-benzo[e]pyrrolo[1 ,2-a][ 1 ,4]diazepin-8-yl)oxy)butanoic

The alloc-THP-protected PBD ester 1.9 (3.5 g, 6.58 mmol) was transferred to a clean round- bottomed flask. About 70 mL of dioxane was added to the same flask. In another beaker, 2.63 g (10 eq, 65.81 mmol) of sodium hydroxide was dissolved in 30 ml dd- water. The dissolved sodium hydroxide solution was then added to the reaction mixture and stirred for 2 hours at room temperature until the LC-MS confirmed the completion of the reaction. The dioxane was removed from the reaction mixture using a rotary evaporator, and the remaining residue was diluted with water (25 ml). The solution was acidified using citric acid (1M; 20 mL) to convert the free acid to its associated protonated form. This is important because only the protonated form of an organic acid is soluble in an organic solvent. The acid was extracted using ethyl acetate (2X 50ml), and the combined organic fractions were then washed with brine (50ml). The resulting solution was dried over magnesium sulphate and concentrated over a rotary evaporator. The resulting white solid 1.10 was resolved in 87% yield (3.01 g). 1.10

Cream solid 87%

Synthesis of PBD monomers

The synthesis of PBD monomers consists of single phenyl derivatives as sidechain was synthesised using two methods, Amide coupling I or II, depending on the electrondeficient nature and yield of the reactions as described in Chapter 02. The compounds are synthesised using the amide coupling method listed in Table 1.

Amide coupling method I Amide coupling method II

Compound MH01 , MH02, MH04, MH05, MH03, MH06, MH07, MH17

ID MH10, MH13, MH19

Table 1 Synthesis of MH01

The PBD core 1.10 (1 eq, 50 mg, 0.09642 mmol) was dissolved in 2.5 mL of DMF in a round- bottomed flask fitted with stirring apparatus. Once dissolved, the EDC (2 eq, 36.97 mg) and DMAP (2 eq, 23.56 mg) were added to the reaction mixture. Kept stirring for 30 minutes and then added The p-toluidine (1.1 eq, 11.36 mg) was added to the reaction mixture and kept stirring overnight. The LC-MS showed the completion of the reaction after 13 hours from the addition of amine. Once finished, quench the reaction using ice/water 30 mL, followed by extraction with ethyl acetate. The organic layer was washed with 1M citric acid, Saturated NaHCCh, water and brine (30 mL of each). The organic layer was then dried over MgSO₄, and the solvent was removed using a rotary evaporator. The crude was directly used for deprotection without further characterization and purification.

The crude was then dissolved into 5 mL of DCM. After dissolving, the pyrrolidine (1.2 eq, 9 pL) was added to the reaction, followed by triphenylphosphine (0.25 eq, 5.93 mg). Finally, a catalytic amount of tetrakis(triphenylphosphine)palladium(0) (0.05 eq, 5.23 mg) was added to the reaction and kept stirring. The LC-MS showed that the reaction was finished after 30 minutes. The reaction mixture was then dried over a rotary evaporator and dissolved the crude into 2 mL of DCM. The crude was then purified using flash chromatography, and the compound MH01 was eluted at 5% methanol in ethyl acetate and afforded 30 mg (78%) of opaque crystals. The NMR data of the compound was found to be consistent with the literature [26],

Synthesis of MH02: Amine used: 4-fluoroaniline; Protocol: see synthesis of MH01

(S)-N-(4-fluorophenyl)-4-((7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1 H- benzo[e]pyrrolo[1 , 2-a][1 ,4]diazepin-8-yl)oxy)butanamide Synthesis of MH03

The acid 1.10 (1 eq, 75 mg, 0.144 mmol) was dissolved in 4 mL of DCM in a 10 mL microwave reaction vial fitted with a magnetic stirrer. Once dissolved, the coupling reagent BTFFH was added to the reaction mixture (1.5 eq, 68.6 mg, 0.216 mmol). The base DI PEA was then added drop-by-drop to the mixture (4.5 eq, 84.12 mg, 113.34 pL) and stirred for 30 minutes. Finally, the 4-bromoaniline (1.5 eq, 37.32 mg) was added to the reaction mixture. The vial was then sealed and heated to 80 °C while stirring. The TLC/LC-MS confirms the completion of the reaction after 14 hours. Once finished, the reaction mixture was dried over a rotary evaporator. The crude was then dissolved into 5 mL of DCM. After dissolving, the pyrrolidine (1.5 eq, 17.77 pL) was added to the reaction, followed by triphenylphosphine (0.25 eq, 9.46 mg). Finally, a catalytic amount of tetrakis(triphenylphosphine)palladium(0) (0.05 eq, 8.33 mg) was added to the reaction and kept stirring. The LC-MS showed that the reaction was finished after 30 minutes. The reaction mixture was then dried over a rotary evaporator and redissolved the crude into 2 mL of DCM. The crude was then purified using flash chromatography, and the compound MH03 was eluted at 3% methanol in ethyl acetate and afforded 62.23 mg (88.3%) of yellow solid.

Synthesis of MH04: Amine used: 4-chloroaniline; Protocol: see synthesis of MH01

(S)-N-(4-chlorophenyl)-4-((7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1 H- Synthesis of MH05: Amine used: 3-fluoroaniline; Protocol: see synthesis of MH01

(S)-N-(3-fluorophenyl)-4-((7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1 H- benzo[e]pyrrolo[1 , 2-a][1 ,4]diazepin-8-yl)oxy)butanamide

Synthesis of MH06: Amine used: 2-fluoroaniline; Protocol: see synthesis of MH03

(S)-N-(2-fluorophenyl)-4-((7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1 H- benzo[e]pyrrolo[1 , 2-a][1 ,4]diazepin-8-yl)oxy)butanamide 26.1820 ([M+H]⁺), theoretical 426.18236 tes (Method B); purity 99%

Synthesis of MH07: Amine used: 3,4-difluoroaniline; Protocol: see synthesis of MH03

(S)-N-(3,4-difluorophenyl)-4-((7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1 H- benzo[e]pyrrolo[1 , 2-a][1 ,4]diazepin-8-yl)oxy)butanamide Synthesis of MH10: Amine used: 4-fluoro-3-methylaniline; Protocol: see synthesis of MH01

Synthesis of MH13: Amine used: 3,5-difluoro-4-methylaniline; Protocol: see synthesis of MH01 Synthesis of MH17: Amine used: 4-fluoro-3-methoxyaniline; Protocol: see synthesis of

MH03

(S)-N-(4-fluoro-3-methoxyphenyl)-4-((7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1 H- benzo[e]pyrrolo[1 , 2-a][1 ,4]diazepin-8-yl)oxy)butanamide Synthesis of MH19: Amine used: 4-ethoxy-3-fluoroaniline; Protocol: see synthesis of MH01

Synthesis of MH21 a) EDC/DMAP, DMF; b) DCM/TFA, rt, 20 mins; c) Pd(PPh₃)₄/PPh₃, Pyrrolidine; DCM

The PBD core 1.10 (1 eq, 200 mg, 0.385 mmol) was dissolved in 4 mL of DMF in a round- bottomed flask fitted with stirring apparatus. Once dissolved, the EDC (2.5 eq, 154.03 mg) and DMAP (2 eq, 78.5 mg) were added to the reaction mixture. Kept stirring for 30 minutes and then added the tert-butyl (4-aminophenyl)carbamate (2 eq, 133.87 mg) was added to the reaction mixture and kept stirring overnight. The LC-MS showed the completion of the reaction after 14 hours from the addition of amine. Once finished, quench the reaction using ice/water 30 mL, followed by extraction with ethyl acetate. The organic layer was washed with 1 M citric acid, Saturated NaHCO₃, water and brine (30 mL of each). The organic layer was then dried over MgSO4, and the solvent was removed using a rotary evaporator. The crude was directly used for Boc- deprotection without further characterization and purification.

The crude was dissolved in DCM 3 mL and stirred in a magnetic stirrer. Then, 1 mL of TFA was added to the reaction mixture. The LC-MS showed that the reaction was completed in 30 minutes. The reaction mixture was then dried over a rotary evaporator. The dry residue was then dissolved in 5 mL of 1M NaOH and 10 mL of ethyl acetate and sonicated to dissolve completely. The crude was then extracted with ethyl acetate (15 mL x 03). The organic layer was then collected and dried over MgSC>4, and the solvent was removed using rotary evaporation. The crude was about 125 mg.

The 125 mg crude was then dissolved into 5 mL of DCM. After dissolving, the pyrrolidine (1.2 eq, 24 pL) was added to the reaction, followed by triphenylphosphine (0.25 eq, 15.63 mg). Finally, a catalytic amount of tetrakis

(triphenylphosphine)palladium(O) (0.05 eq, 13.77 mg) was added to the reaction and kept stirring. The LC-MS showed that the reaction was finished after 30 minutes. The reaction mixture was then dried over a rotary evaporator and dissolved the crude into 2 mL of DCM. The crude was then purified using flash chromatography, and the compound MH21 was eluted at 3% methanol in ethyl acetate and afforded 73 mg (44.8%) of opaque crystals.

(S)-N-(4-aminophenyl)-4-((7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1 H- benzo[e]pyrrolo[1 , 2-a][1 ,4]diazepin-8-yl)oxy)butanamide

Synthesis of MH22: Amine used: 4-fluorobenzene-1 ,3-diamine; Protocol: see synthesis of MH21

(S)-N-(3-amino-4-fluorophenyl)-4-((7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1 H- benzo[e]pyrrolo[1 , 2-a][1 ,4]diazepin-8-yl)oxy)butanamide

Synthesis of MH31 : Amine used: thiazol-2-amine; Protocol: see synthesis of MH03

(S)-4-((7-methoxy-5-oxo-2,3,5, 11 a- tetrahydro- 1 H-benzo[e]pyrrolo[1 ,2-

Synthesis of MH34: Amine used: 5-chlorothiazol-2-amine; Protocol: see synthesis of

MH03 Synthesis of MH37: Amine used: 5-methylthiazol-2-amine; Protocol: see synthesis of

MH03

Synthesis of MH54: Amine used: 2-chlorobenzo[d]thiazol-5-amine; Protocol: see synthesis of MH03.

(S)-N-(2-chlorobenzo[d]thiazol-5-yl)-4-((7-methoxy-5-oxo-2,3,5, 11a-tetrahydro-1 H- .643minutes (Method B); purity 98%

Synthesis of MH63: Amine used: 6-fluorobenzo[d]thiazol-2-amine; Protocol: see synthesis of MH03.

Synthesis C7 fluorine PBD payloads and linker payloads.

General Reaction Scheme for Synthesis of 4C-Alloc-THP-Protected 07 fluorine PBD unit

8 9

Synthesis of Methyl 5-fluoro-4-hydroxy-2-nitrobenzoate (1)

Methyl 4-bromo-5-fluoro-2-nitrobenzoate (5.0 g, 1 equiv.) was dissolved in dioxane (40 mL) and sequentially added of bis(pinacolato)diboron (5.04 g, 1.1 equiv.), Pd(dppf)Ch ■CH2CI2 (0.44 g, 0.03 equiv.) and potassium acetate (5.2 g, 3 equiv.). The reaction mixture was left heating at reflux, under magnetic stirrer and N2 atmosphere overnight until TLC showed the total consumption of the starting material. The reaction mixture was then filtered on Celite path eluting with DCM, the solvent evaporated under reduced pressure and the crude purified by filtration on silica gel eluting with DCM. The collected organic solvent was filtrated under reduced pressure giving a black oil. The crude of reaction was dissolved in THF (100 mL) and added of acetic acid (8 mL). The solution was kept at 0°C and H2O2 35% (15 mL) added dropwise. The reaction mixture was then left under magnetic stirrer at r.t. for 1 hour until TLC showed total consumption of the starting material. Iced water (50 mL) was then added to the reaction mixture along with sodium metabisulphite (10 g). The reaction mixture was washed with ethyl acetate (3 x 50mL) and the collected organic phases dried over dried over MgSC>4 and concentrated by rotary evaporator to give a yellow oil. The crude was purified by column chromatography (mobile phase: 100% DCM) affording pure 1 as an orange solid (2.3 g, 59%). ¹H NMR (400 MHz, METHANOL-d₄) 5: 7.56 (d, J = 10.58 Hz, 1 H), 7.39 (d, J = 7.30 Hz, 1 H), 3.85 (s, 3H). ¹³C NMR (101 MHz, METHANOL-d₄) 5: 164.2, 153.5, 151.1 , 148.6, 117.5, 117.2, 113.0, 51.9. m/z (+EI) calc, for C₈H₆FNO₅ (M)+ 215.0 found 214.0 ([M]-H)’; LCMS (5 min method): Rt 3.05 min.

Synthesis of 5-fluoro-4-hydroxy-2-nitrobenzoic acid (2)

NaOH 1 M aqueous solution in excess was added to a solution of 1 (2.1 g, 9.7 mmol) in dioxane (60 mL). The reaction mixture was left at r.t. under magnetic stirrer overnight until TLC showed completion of the reaction. Dioxane was evaporated under reduced pressure and water (40 mL) was added to the crude. Citric acid 1 M aqueous solution was added until acid pH is reached. The aqueous layer was extracted with ethyl acetate (2 x 70 mL). The combined organic layers were dried over MgSO₄ and concentrated under reduced pressure, giving pure 2 (1.9 g, >95%) as a light yellow solid. ¹H NMR (400 MHz, METHANOL-d₄) 5 7.30 (d, J = 10.83 Hz, 1 H), 7.03 (d, J = 7.30 Hz, 1 H). ¹³C NMR (101 MHz, METHANOL-d₄) 5 175.1 , 171.8, 164.7, 153.0, 117.2, 112.6, 72.4. m/z (+EI) calc, for C₇H₄FNO₅ (M)+ 201.0 found 200.0 ([M]-H)’; LCMS (5 min method): Rt 2.09 min.

Synthesis of (S)-(5-fluoro-4-hydroxy-2-nitrophenyl)(2-(hydroxymethyl)pyrrolidin-1- yl)methanone (3)

HATU (4.5 g, 1.2 equiv.) and DI PEA (3.6 mL, 2 equiv.) were sequentially added to a solution of 2 (2.0 g, 1 equiv.) in DMF (30 mL) and the reaction mixture was left under magnetic stirrer for 30 minutes. (S)-pyrrolidinemethanol (0.97 mL, 1 equiv.) was then added to the solution and the reaction mixture was left under magnetic stirrer overnight until no changes by TLC were observed. The reaction did not go to completion. The reaction mixture was added of ethyl acetate (40 mL) and extracted with citric acid 0.1 M aqueous solution (3x50mL). The organic phase was then dried over MgSC>4 and concentrated under reduced pressure. The crude was further purified by column chromatography (mobile phase: 100% ethyl acetate) affording pure 3 (2.0 g, 71 %) as a yellow oil. ¹H NMR (400 MHz, METHANOL-d₄) 5 7.77 (d, J = 7.55 Hz, 1 H), 7.38 (d, J = 10.32 Hz, 1 H), 4.23 - 4.31 (m, 1 H), 3.73 - 3.88 (m, 2H), 3.28 (t, J = 6.80 Hz, 2H), 2.04 - 2.15 (m, 2H), 1.96 - 2.02 (m, 1 H), 1.81 - 1.91 (m, 1 H). ¹³C NMR (101 MHz, DMSO) 5 164.4, 145.9, 141.1 , 125.7, 116.0, 115.8, 113.7, 60.6, 58.6, 48.6, 27.0, 23.6. m/z (+EI) calc, for C12H13FN2O5 (M)+ 284.0 found 285.0 ([M]+H)⁺; LCMS (5 min method): Rt 2.31 min.

Synthesis of (methyl (S)-4-(2-fluoro-4-(2-(hydroxymethyl)pyrrolidine-1-carbonyl)-5- nitrophenoxy)butanoate (4)

Methyl 4-bromobutanoate (0.81 mL, 1.1 equiv.) and potassium carbonate (1.2 g, 3 equiv.) were added to a solution of 3 (1.7 g, 5.9 mmol) in DMF (20 mL). The suspension was stirred at room temperature overnight, until TLC showed completion. Iced water (60 mL) was added to the reaction mixture that was subsequently extracted with ethyl acetate (3X50 mL). The collected organic phases were dried over MgSO₄ and concentrated under reduced pressure. The crude was then purified by column chromatography (mobile phase: ethyl acetate/MeOH, 99/1, v/v) affording pure 4 (1.7 g, 74%) as a yellow oil. ¹H NMR (400 MHz, CHLOROFORM-d) 5 7.70 (d, J = 7.30 Hz, 1H), 7.11 (d, J = 9.82 Hz, 1 H), 4.36 (dd, J = 4.28, 7.05 Hz, 1 H), 4.16 - 4.25 (m, 1 H), 4.10 (t, J = 6.40 Hz, 2H), 3.65 - 3.71 (m, 1 H), 3.59 (s, 3H), 3.10 (t, J = 6.29 Hz, 2H), 2.46 (t, J = 7.05 Hz, 2H), 1.98 - 2.13 (m, 3H), 1.78 - 1.88 (m, 1 H), 1.65 - 1.77 (m, 2H). ¹³C NMR (101 MHz, CHLOROFORM-d) 5 173.2, 166.5, 156.8, 154.2, 147.5, 140.5, 127.2, 115.5, 110.6, 68.7, 64.7, 51.7, 49.5, 38.5, 30.0, 28.0, 24.0. m/z (+EI) calc, for C17H21 FN2O7 (M)+ 384.1 found 385.0 ([M]+H)⁺; LCMS (5 min method): Rt 3.01 min.

Synthesis of methyl (S)-4-(5-amino-2-fluoro-4-(2-(hydroxymethyl)pyrrolidine-1- carbonyl)phenoxy)butanoate (5)

A solution of the starting nitro derivative was prepared dissolving 4 (1.6 g, 4.1 mmol) in EtOH (30 mL). A catalytic amount of Pd/C (10% w/w) was added to the solution that was hydrogenated in a Parr hydrogenator at 60 psi until TLC showed the completion of the reaction. The reaction mixture was then filtered on celite path eluting with DCM. The organic phase was evaporated under reduced pressure to give pure 5 (1.4 g, >95%) as a transparent oil. ¹H NMR (400 MHz, CHLOROFORM-d) 5 6.94 (d, J = 11.58 Hz, 1 H), 6.26 (d, J = 7.30 Hz, 1 H), 4.65 (br. s., 2H), 4.28 - 4.43 (m, 1 H), 4.02 (t, J = 6.04 Hz, 2H), 3.73 - 3.82 (m, 1 H), 3.68 (s, 3H), 3.55 - 3.66 (m, 2H), 3.44 - 3.53 (m, 1 H), 2.53 (t, J = 7.18 Hz, 2H), 2.05 - 2.21 (m, 3H), 1.81 - 1.94 (m, 1 H), 1.56 - 1.81 (m, 2H). ¹³C NMR (101 MHz, CHLOROFORM-d) 5 173.5, 171.1 , 149.0, 145.6, 143.9, 143.2, 115.4, 110.7, 102.4, 67.8, 61.0, 51.7, 38.6, 30.3, 28.4, 25.0, 24.3. m/z (+EI) calc, for C17H23FN2O5 (M)+ 354.1 found 355.2 ([M]+H)⁺; LCMS (5 min method): Rt 2.77 min.

Synthesis of methyl (S)-4-(5-(((allyloxy)carbonyl)amino)-2-fluoro-4-(2-(hydroxymethyl) pyrrolidine-1-carbonyl)phenoxy)butanoate (6)

Compound 5 (0.9 g, 2.5 mmol) was dissolved in dry DCM (50 mL) and the solution was kept at -10°C under N2 atmosphere. Dry pyridine (2.0 mL) and a solution of allyl chloroformate (0.3 mL, 0.95 equiv.) in anhydrous DCM (5 mL) were sequentially added to the obtained solution. The reaction mixture was left under magnetic stirrer at room temperature for 2 hours, until TLC showed completion of reaction. The reaction mixture was then sequentially washed with saturated CuSCL solution (70 mL), saturated aqueous NaHCCh (100 mL) and brine (100 mL). The organic phase was dried over MgSCL and concentrated under reduced pressure using a rotary evaporator. The crude of reaction was subsequently purified by column chromatography (mobile phase: Ethyl acetate/MeOH, 99/1 , v/v) giving pure 6 (0.86 g, 78%) as a transparent oil. ¹H NMR (400 MHz, CHLOROFORM-d) 5 9.02 (br. s., 1 H), 7.81 (d, J = 7.81 Hz, 1 H), 7.06 (d, J = 11.33 Hz, 1 H), 5.80 - 6.03 (m, 1 H), 5.32 (qd, J = 1.51 , 17.37 Hz, 1 H), 5.22 (qd, J = 1.30, 10.45 Hz, 1 H), 4.60 (qd, J = 1.48, 5.63 Hz, 2H), 4.34 (br. s., 1 H), 4.24 (br. s., 1 H), 4.09 (t, J = 6.80 Hz, 2H), 3.81 (br. s., 1 H), 3.58 - 3.70 (m, 4H), 3.42 - 3.57 (m, 2H), 2.51 (t, J = 7.30 Hz, 2H), 2.09-2.16 (m, 3H), 1.83 - 1.92 (m, 1 H), 1.66 - 1.77 (m, 2H). ¹³C NMR (101 MHz, CHLOROFORM-d) 5 173.4, 171.2, 153.5, 148.7, 148.6, 147.9, 145.5, 136.6, 132.3, 118.2, 115.2, 106.4, 67.9, 65.8, 65.6, 60.9, 51.7, 30.4, 28.0, 25.1 , 24.3. m/z (+EI) calc, for C2iH₂7FN₂O7 (M)+ 438.1 found 439.1 ([M]+H)⁺; LCMS (5 min method): Rt 3.31 min.

Synthesis of allyl (11aS)-7-fluoro-11-hydroxy-8-(4-methoxy-4-oxobutoxy)-5-oxo- 2,3,11 ,11 a-tetrahydro-1/7-benzo[e]pyrrolo[1 ,2-a][1 ,4]diazepine-10(5/-/)-carboxylate (7)

Compound 6 (0.8 g, 1.8 mmol) was dissolved in DCM (25 mL) and sequentially added of BAIB (0.67 g, 1.2 equiv.) and a catalytic amount of TEMPO (0.03 g, 0.1 equiv.). The reaction mixture was left overnight under magnetic stirrer until TLC showed total disappearance of the starting material. The reaction mixture was the sequentially washed by saturated sodium metabisulphite aqueous solution (20 mL), saturated aqueous NaHCOs solution (20 mL) and brine (20 mL). The organic phase was dried over MgSO4 and concentrated under reduced pressure using a rotary evaporation. The crude of reaction was further purified by column chromatography (mobile phase: from ethyl acetate 100% to ethyl acetate/MeOH, 99/1 , v/v) affording pure 7 (0.65 g, 81%) as a transparent oil. ¹H NMR (400 MHz, CHLOROFORM-d) 5 7.44 (d, J = 11.08 Hz, 1 H), 6.79 (d, J = 6.29 Hz, 1 H), 5.69 - 5.91 (m, 1 H), 5.52 - 5.69 (m, 1 H), 5.14 (br. s., 2H), 4.50 - 4.70 (m, 2H), 4.32 - 4.49 (m, 1 H), 3.99 - 4.09 (m, 2H), 3.61 - 3.70 (m, 4H), 3.35 - 3.57 (m, 2H), 2.52 (m, 2H), 2.04 - 2.19 (m, 4H), 1.91 - 2.01 (m, 2H). ¹³C NMR (101 MHz, CHLOROFORM-d) 5 173.4, 165.9, 155.6, 152.6, 150.1, 148.8, 131.7, 126.4, 118.1 , 116.1 , 115.8, 85.8, 68.2, 66.8, 60.4, 57.7, 46.4, 30.2, 28.7, 24.2, 23.0. m/z (+EI) calc, for C₂IH₂₅FN₂O₇ (M)+ 436.1 found 437.1 ([M]+H)⁺; LCMS (5 min method): Rt 2.99 min.

Synthesis of allyl (11aS)-7-fluoro-8-(4-methoxy-4-oxobutoxy)-5-oxo-11-((tetrahydro-2/7- pyran-2-yl)oxy)-2,3, 11 ,11 a- tetrahydro- 1 /7-benzo[e]pyrrolo[1 ,2-a][1 ,4]diazepine-10(5/7)- carboxylate (8)

DHP (1.1 mL, 10 equiv.) along with a catalytic amount of PTSA (10 mg) was added to a solution of 7 (0.55 g, 1.2 mmol) in ethyl acetate (20 mL). The reaction mixture was left under magnetic stirrer for 4 hours until TLC showed completion of reaction and then washed with saturated aqueous NaHCCh (2X20 mL) and brine (20 mL). The organic phase was dried over MgSCL, and evaporated using a rotary evaporator under reduced pressure. The crude of reaction was purified by column chromatography (mobile phase: ethyl acetate 100%) affording pure 8 (0.57 g, 91%) as a transparent oil. ¹H NMR (400 MHz, CHLOROFORM-d, mix of isomers) 5 7.32 - 7.44 (m, 2H),6.91 (d, J = 6.80 Hz, 1 H), 6.63 (d, J = 6.80 Hz, 1 H), 5.58 - 5.87 (m, 4H), 4.92 - 5.09 (m, 5H), 4.80 (br. s., 1 H), 4.34 - 4.64 (m, 2H), 3.90-3.96 (m, 1 H), 3.83-3.87 (m, 2H), 3.62 (s, 6H), 3.54 - 3.60 (m, 3H), 3.37 - 3.50 (m, 6H), 2.49 (t, J = 7.05 Hz, 4H), 2.03 - 2.13 (m, 7H), 1.91 - 1.96 (m, 4H), 1.64 - 1.74 (m, 5H), 1.38 - 1.55 (m, 13H). ¹³C NMR (101 MHz, CHLOROFORM-d, mix of isomers ) 5 173.3, 166.0, 155.4, 152.9, 150.5, 148.9, 148.6, 131.8, 127.2, 117.4, 117.2, 116.6, 116.0, 115.5, 100.3, 96.0, 94.3, 91.6, 88.2, 68.2, 67.9, 66.6, 66.4, 64.2, 63.8, 63.6, 59.9, 59.8, 51.6, 32.0, 31.1 , 30.8, 30.2, 30.0, 29.0, 28.7, 25.1 , 24.2, 24.1 , 23.2, 20.4, 19.9. m/z (+EI) calc, for C26H33FN2O8 (M)+ 520.2 found 521.2 ([M]+H)⁺; LCMS (5 min method): Rt 3.74 min.

Synthesis of 4-(((11aS)-10-((allyloxy)carbonyl)-7-fluoro-5-oxo-11-((tetrahydro-2/7- pyran-2-yl)oxy)-2,3,5, 10, 11 , 11 a-hexahydro-1 /7-benzo[e]pyrrolo[1 ,2-a][1 ,4]diazepin-8- yl)oxy) butanoic acid (9)

NaOH 1 M aqueous solution in excess was added to a solution of 8 (0.6 g, 1.1 mmol) in dioxane (20mL). The reaction mixture was left at r.t. under magnetic stirrer overnight until TLC showed completion of the reaction. Dioxane was evaporated under reduced pressure and water (20 mL) was added to the crude. Citric acid 1 M aqueous solution was added until acid pH is reached. The aqueous layer was extracted with ethyl acetate (2 x 35 mL). The combined organic layers were dried over MgSC>4 and concentrated under reduced pressure, giving pure 9 (0.56 g, >95%) as a yellowish solid. ¹H NMR (400 MHz, CHLOROFORM-d, mix of isomers) 5 7.40 - 7.51 (m, 2H), 6.98 (d, J = 7.30 Hz, 1 H), 6.62 - 6.70 (m, 1 H), 5.89 (d, J = 9.06 Hz, 1 H), 5.75 (d, J = 9.32 Hz, 2H), 4.99 - 5.16 (m, 4H), 4.88 - 4.99 (m, 1 H), 4.52 - 4.71 (m, 2H), 4.36 - 4.52 (m, 1 H), 4.00 - 4.10 (m, 3H), 3.86 - 3.97 (m, 2H), 3.59 - 3.73 (m, 4H), 3.42 - 3.59 (m, 6H), 2.55 - 2.64 (m, 4H), 2.09 - 2.20 (m, 7H), 1.96 - 2.04 (m, 5H), 1.71 - 1.83 (m, 5H), 1.42 - 1.62 (m, 11 H). ¹³C NMR (100 MHz, CHLOROFORM-d) 5: 177.4, 177.3, 166.4, 166.2, 155.5, 153.1 , 150.6, 132.0, 131.8, 127.3, 117.3, 116.8, 116.2, 115.9, 100.5,

96.1 , 94.5, 91.6, 88.3, 68.2, 67.9, 66.5, 64.4, 63.8, 63.6, 60.4, 46.5, 31.8, 31.1 , 30.9,

30.2, 30.1 , 29.0, 28.8, 25.2, 25.1 , 24.1 , 24.0, 23.2, 23.1 , 20.2, 19.9. m/z (+EI) calc, for C25H31FN2O8 (M)+ 506.2 found 507.2 ([M]+H)⁺; LCMS (5 min method): Rt 3.36 min.

Synthesis of (S)-4-((7-fluoro-5-oxo-2,3,5,11a-tetrahydro-1/7-benzo[e]pyrrolo[1 ,2- a][1 ,4]diazepin-8-yl)oxy)-/V-(5-fluorobenzo[d]thiazol-2-yl)butanamide (10, PA-208-169)

Compound 9 (200 mg, 0.4 mmol), was dissolved in DMF (2 mL) and /\/-(3- dimethylaminopropyl)-/V'-ethylcarbodiimide hydrochloride (96 mg, 2.5 equiv.) and 4- (dimethylamino)pyridine (86 mg, 3.5 equiv.) were added, followed by 5- fluorobenzo[c(]thiazol-2-amine (75 mg, 1.1 equiv.). The mixture was stirred at 65°C for 8 hours before being diluted with EtOAc (50 mL) and washed with a saturated aqueous solution of NaHCC>3 (50 mL), a 10% aqueous solution of acetic acid (50 mL) and brine (50 mL). The organic layer was then concentrated in vacuo and the resulting crude purified by silica gel column chromatography (mobile phase: DCM/MeOH, 90/10, v/v). The pure product was then redissolved in DCM (2 mL) and tetrakis(triphenylphosphine) palladium(O) (10 mol%) and pyrrolidine (26 pL, 1.05 equiv.) were sequentially added. The reaction was stirred at room temperature for 30 min and then concentrated under vacuum. The crude was finally purified by silica gel column chromatography (mobile phase: from DCM to DCM/MeOH, 90/10, v/v) to obtain 30 mg (16%) of 10 (PA-208- 169) as a clear viscous oil. ¹H NMR (400 MHz, DMSO) <5 12.45 (s, 1 H), 7.98 (dd, J = 8.7, 5.5 Hz, 1 H), 7.86 (d, J = 4.5 Hz, 1 H), 7.59 - 7.50 (m, 2H), 7.17 (td, J = 9.1 , 2.5 Hz, 1 H), 7.03 (d, J = 8.0 Hz, 1 H), 4.25 (dt, J = 9.8, 6.2 Hz, 1 H), 4.16 (dt, J = 9.8, 6.3 Hz, 1 H), 3.72 (ddd, J = 7.8, 4.5, 3.2 Hz, 1 H), 3.59 (dt, J = 11.5, 5.7 Hz, 1 H), 3.41 - 3.35 (m, 1 H), 2.70 (t, J = 7.2 Hz, 2H), 2.28 (ddd, J = 13.1, 9.9, 6.5 Hz, 1 H), 2.18 - 2.04 (m, 3H), 1.98 - 1.90 (m, 2H). ¹³C NMR (101 MHz, DMSO) 5 171.9, 166.0, 162.4, 160.1 , 148.6, 148.5, 148.0, 143.5, 127.2, 122.9, 122.8, 120.1 , 116.1 , 111.9, 106.7, 106.5, 68.2, 54.9, 53.4, 46.4, 31.7, 28.7, 23.6. m/z (+EI) calc, for C23H20F2N4O3S (M)+ 470.12 found 471.1 ([M]+H)⁺; LCMS: (5 min method) Rt 3.22 min, (10 min method) Rt 7.10 min;

HRMS (+EI) calc, for C23H20F2N4O3S (M)+ 470.122 found 471.130 ([M]+H)⁺.

Synthesis of (S)-4-((7-fluoro-5-oxo-2,3,5,11a-tetrahydro-1/7-benzo[e]pyrrolo[1 ,2- a][1 ,4]diazepin-8-yl)oxy)-/V-(4-fluoropyridin-2-yl)butanamide (11, PA234-4)

Compound 9 (100 mg, 0.2 mmol), was dissolved in DMF (1 mL) and HATLI (90 mg, 1.2 equiv.) and triethyl amine (68 pL, 2.5 equiv.) were added, followed by 4-fluoropyridin- 2-amine (33 mg, 1.5 equiv.). The mixture was stirred at 50°C for 8 hours before being diluted with EtOAc (50 mL) and washed with a saturated aqueous solution of NaHCCh (2 x 50 mL). The organic layer was then concentrated in vacuo and the resulting crude purified by silica gel column chromatography (mobile phase: from DCM to DCM/MeOH, 90/10, v/v). The pure product was then redissolved in DCM (1.5 mL) and tetrakis (triphenylphosphine)palladium(O) (5 mol%) and pyrrolidine (5 pL, 0.32 equiv.) were sequentially added. The reaction was stirred at room temperature for 1 hour and then concentrated under vacuum. The crude was finally purified by silica gel column chromatography (mobile phase: DCM/MeOH, 90/10, v/v) to obtain 5 mg (5%) of 11 (PA234-4) as a clear viscous oil. ¹H NMR (400 MHz, DMSO) 5 10.80 - 10.69 (m, 1 H), 8.26 (ddd, J = 9.3, 5.7, 1.6 Hz, 1 H), 7.85 (dd, J = 12.2, 2.4 Hz, 1 H), 7.80 (d, J = 4.5 Hz, 1 H), 7.49 (d, J = 12.1 Hz, 1 H), 7.02 - 6.89 (m, 2H), 4.18 - 3.95 (m, 2H), 3.69 - 3.59 (m, 1 H), 3.52 (tt, J = 10.2, 5.2 Hz, 1 H), 3.43 - 3.30 (m, 1 H), 2.53 (t, J = 7.2 Hz, 2H), 2.00 - 1.93 (m, 2H), 1.91 - 1.80 (m, 3H), 1.61 (dtd, J = 13.5, 8.7, 4.8 Hz, 1 H). ¹³C NMR (101 MHz, DMSO) 5 172.0, 166.0, 162.4,0 150.6, 150.5, 148.5, 143.4, 116.1 , 115.9, 111.9, 107.3, 107.1 , 100.6, 100.4, 68.3, 53.4, 46.4, 32.4, 30.7, 28.7, 23.6. m/z (+EI) calc, for C21H20F2N4O3 (M)+ 414.15 found 415.1 ([M]+H)⁺; LCMS: (5 min method) Rt 2.76 min, (10 min method) Rt 6.04 min; (+EI) calc, for C21H20F2N4O3 (M)+ 414.150 found 415.157 ([M]+H)⁺.

Synthesis of (S)-4-((7-fluoro-5-oxo-2,3,5,11a-tetrahydro-1 H-benzo[e]pyrrolo[1 ,2- a][1 ,4]diazepin-8-yl)oxy)-N-(6-fluorobenzo[d]thiazol-2-yl)butanamide (12, PA-234-11)

Compound 9 (130 mg, 0.26 mmol), was dissolved in DMF (3 mL) and /V-(3-dimethyl- aminopropyl)-/V -ethylcarbodiimide hydrochloride (128 mg, 2.5 equiv.) and 4-(dimethyl- amino)pyridine (94 mg, 3.5 equiv.) were added, followed by 6-fluorobenzo[c(]thiazol-2- amine (52 mg, 1.2 equiv.). The mixture was stirred at 80°C for 5 hours before being diluted with EtOAc (100 mL) and washed with a saturated aqueous solution of NaHCCh (100 mL), a 10% aqueous solution of acetic acid (100 mL) and brine (50 mL). The organic layer was then concentrated in vacuo and the resulting crude purified by silica gel column chromatography (mobile phase: DCM/MeOH, 90/10, v/v). The pure product was then redissolved in DCM (2 mL) and tetrakis(triphenylphosphine)palladium(0) (5 mol%) and pyrrolidine (19 pL, 0.85 equiv.) were sequentially added. The reaction was stirred at room temperature for 2 hours and then concentrated under vacuum. The crude was finally purified by silica gel column chromatography (mobile phase: from DCM to DCM/MeOH, 90/10, v/v) to obtain 25 mg (21%) of 12 (PA-234-11) as a white solid. ¹H NMR (400 MHz, DMSO) 5 12.38 (s, 1 H), 7.91 - 7.84 (m, 2H), 7.73 (dd, J = 8.9, 4.8 Hz, 1 H), 7.56 (d, J = 12.2 Hz, 1 H), 7.27 (td, J = 9.1 , 2.7 Hz, 1 H), 7.04 (d, J = 8.0 Hz, 1 H), 4.25 (dt, J = 9.7, 6.2 Hz, 1 H), 4.16 (dt, J = 9.8, 6.3 Hz, 1 H), 3.72 (ddd, J = 7.8, 4.5, 3.2 Hz, 1 H), 3.59 (dt, J = 11.4, 5.6 Hz, 1H), 3.41 - 3.35 (m, 1 H), 2.69 (t, J = 7.3 Hz, 2H), 2.33 - 2.25 (m, 1 H), 2.22 - 2.06 (m, 3H), 1.98 - 1.90 (m, 2H). ¹³C NMR (101 MHz, DMSO) 5 171.8, 166.0, 159.8, 148.6, 145.2, 143.5, 132.6, 121.6, 120.2, 116.1 , 115.9, 114.2, 111.9, 108.2, 108.0, 68.2, 54.9, 53.4, 46.4, 31.6, 28.7, 23.6. m/z (+EI) calc, for C23H20F2N4O3S (M)+ 470.12 found 471 .0 ([M]+H)⁺ ; LCMS (5 min method) Rt 3.23 min, (10 min method) Rt 7.10 min; HRMS (+EI) calc, for C23H20F2N4O3S (M)+ 470.122 found 471.130 ([M]+H)⁺.

Synthesis of (S)-4-((7-fluoro-5-oxo-2,3,5,11a-tetrahydro-1 H-benzo[e]pyrrolo[1 ,2- a][1 ,4]diazepin-8-yl)oxy)-N-(5-fluorothiazol-2-yl)butanamide (13, PA234-12)

Compound 9 (130 mg, 0.26 mmol), was dissolved in DMF (3 mL) and /V-(3-dimethyl- aminopropyl)-/V -ethylcarbodiimide hydrochloride (128 mg, 2.5 equiv.) and 4-(dimethyl- amino)pyridine (94 mg, 3.5 equiv.) were added, followed by 6-fluorobenzo[c(]thiazol-2- amine (52 mg, 1.2 equiv.). The mixture was stirred at 70°C for 5 hours before being diluted with EtOAc (100 mL) and washed with a saturated aqueous solution of NaHCCh (100 mL), a 10% aqueous solution of acetic acid (100 mL) and brine (100 mL). The organic layer was then concentrated in vacuo and the resulting crude purified by silica gel column chromatography (mobile phase: from DCM to DCM/MeOH, 90/10, v/v). The pure product was then redissolved in DCM (2 mL) and tetrakis(triphenylphosphine) palladium(O) (5 mol%) and pyrrolidine (8.3 pL, 0.4 equiv.) were sequentially added. The reaction was stirred at room temperature for 2 hours and then concentrated under vacuum. The crude was finally purified by silica gel column chromatography (mobile phase: from DCM to DCM/MeOH, 90/10, v/v) to obtain 5.2 mg (5%) of 13 (PA234-12) as a clear viscous oil. ¹H NMR (400 MHz, DMSO) 5 12.16 (s, 1 H), 7.87 (d, J = 4.5 Hz, 1 H), 7.56 (d, J = 12.1 Hz, 1 H), 7.26 (d, J = 2.5 Hz, 1 H), 7.02 (d, J = 8.0 Hz, 1 H), 4.22 (dt, J = 9.8, 6.2 Hz, 1 H), 4.13 (dt, J = 9.8, 6.3 Hz, 1 H), 3.72 (ddd, J = 7.9, 4.5, 3.2 Hz, 1 H), 3.60 (dt, J = 11.4, 5.7 Hz, 1 H), 3.40 - 3.35 (m, 1 H), 2.60 (t, J = 7.3 Hz, 2H), 2.34 - 2.19 (m, 2H), 2.08 (q, J = 6.8 Hz, 2H), 1.99 - 1.91 (m, 2H). ¹³C NMR (101 MHz, DMSO) 5 170.9, 166.1 , 162.4, 155.7, 147.3, 143.5, 118.1 , 118.0, 116.1 , 115.9, 111.9, 68.1 , 54.9, 53.4, 46.4, 31.1 , 28.7, 23.6. m/z (+EI) calc, for C19H18F2N4O3S (M)+ 420.11 found 421.0 ([M]+H)⁺; LCMS (5 min method) Rt 2.86 min, (10 min method) Rt 6.35 min; HRMS (+EI) calc, for C19H18F2N4O3S (M)+ 420.107 found 421.114 ([M]+H)⁺.

Synthesis of (S)-/V-(4-fluoro-3-methylphenyl)-4-((7-fluoro-5-oxo-2,3,5,11a-tetrahydro- 1/7-benzo[e]pyrrolo[1 ,2-a][1,4]diazepin-8-yl)oxy)butanamide (14, PA234-14)

Compound 9 (100 mg, 0.2 mmol), was dissolved in DMF (2 mL) and /V-(3-dimethyl- aminopropyl)-/V'-ethylcarbodiimide hydrochloride (95 mg, 2.5 equiv.) and 4-(dimethyl- amino)pyridine (72 mg, 3.5 equiv.) were added, followed by 4-fluoro-3-methylaniline (37 mg, 1.5 equiv.). The mixture was stirred at 60°C for 3 hours before being diluted with EtOAc (100 mL) and washed with a saturated aqueous solution of NaHCOs (100 mL), a 10% aqueous solution of acetic acid (100 mL) and brine (100 mL). The organic layer was then concentrated in vacuo and the resulting crude purified by silica gel column chromatography (mobile phase: DCM/Acetone, 50/50, v/v). The pure product was then redissolved in DCM (2 mL) and tetrakis(triphenylphosphine)palladium(0) (5 mol%) and pyrrolidine (22 pL, 1.4 equiv.) were sequentially added. The reaction was stirred at room temperature for 2 hours and then concentrated under vacuum. The crude was finally purified by silica gel column chromatography (mobile phase: from DCM to DCM/MeOH, 90/10, v/v) to obtain 35 mg (34%) of 14 (PA234-14) as a clear viscous oil. ¹H NMR (400 MHz, DMSO) 5 9.93 (s, 1 H), 7.87 (d, J = 4.5 Hz, 1 H), 7.58 (d, J = 12.1 Hz, 1 H), 7.53 - 7.48 (m, 1 H), 7.44 - 7.36 (m, 2H), 7.04 (t, J = 9.1 Hz, 1 H), 4.24 (dt, J = 9.7, 6.3 Hz, 1 H), 4.15 (dt, J = 9.8, 6.3 Hz, 1 H), 3.72 (ddd, J = 7.8, 4.5, 3.2 Hz, 1 H), 3.64 - 3.60 (m, 1 H), 3.45 - 3.38 (m, 1 H), 2.48 (t, J = 3.6 Hz, 2H), 2.19 (s, 3H), 2.17 - 2.10 (m, 1 H), 2.05 (q, J = 5.8 Hz, 3H), 1.97 - 1.91 (m, 2H). ¹³C NMR (101 MHz, DMSO) 5 170.3, 166.1 , 164.4, 157.6, 155.2, 143.5, 135.3, 123.9, 122.0, 118.3, 114.9, 111.9, 106.2, 102.2, 67.7, 58.8, 53.4, 46.4, 32.4, 30.7, 22.4, 14.4. m/z (+EI) calc, for C23H23F2N3O3 (M)+ 427.17 found 428.1 ([M]+H)⁺; LCMS: (5 min method) Rt 3.16 min, (10 min method) Rt 6.98 min; (+EI) calc, for C23H23F2N3O3 (M)+ 427.171 found 428.180 ([M]+H)⁺.

Synthesis of allyl terf-butyl (4-fluoro-1,3-phenylene)dicarbamate (15) terf-Butyl N-(3-amino-4-fluorophenyl)carbamate (500 mg, 2.21 mmol, 1 equiv.) was dissolved in DCM (15 mL) and pyridine (445 pL, 2.5 equiv.) was added. To this solution allyl chloroformate (235 pL, 1 equiv.) was also added dropwise. The reaction mixture was then stirred at room temperature for 3 hours before been diluted with EtOAc (100mL). This was washed with a saturated aqueous solution of CuSO4 and concentrated under vacuum. The resulting crude was purified by silica gel column chromatography (mobile phase: from DCM to DCM/Acetone, 70/30, v/v) to obtain 464 mg of pure product (68%) of 15 as a pink solid. ¹H NMR (400 MHz, DMSO) 5 9.36 (s, 1 H), 9.31 (s, 1 H), 7.80 (d, J = 6.9 Hz, 1 H), 7.18 - 7.05 (m, 2H), 5.96 (ddt, J = 17.2, 10.6, 5.4 Hz, 1 H), 5.35 (dq, J = 17.2, 1.7 Hz, 1 H), 5.22 (dq, J = 10.5, 1.5 Hz, 1 H), 4.59 (dt, J = 5.3, 1.5 Hz, 2H), 1.46 (s, 9H). ¹³C NMR (101 MHz, DMSO) 5 153.6, 152.8, 135.7, 133.2, 125.8, 125.6, 117.5, 115.4, 115.2, 79.1 , 64.91 , 40.20, 28.10. m/z (+EI) calc, for C15H19FN2O4 (M)+ 310.13 found 333.1([M]+Na)⁺; LCMS (5 min method): Rt 3.79 min.

Synthesis of (S)-/V-(3-amino-4-fluorophenyl)-4-((7-fluoro-5-oxo-2,3,5,11 a-tetrahydro- 1/7-benzo[e]pyrrolo[1 ,2-a][1,4]diazepin-8-yl)oxy)butanamide (16, PA234-24)

Compound 15 (95 mg, 0.31 mmol, 1.2 equiv.) was boc-deprotected in MeOH (2 ml) and HCI 4M in dioxane (3 mL). The solution was left under magnetic stirrer for 3 hours until TLC showed total cleavage of protecting group. The reaction mixture was subsequently evaporated using a rotary evaporator, obtaining a solid. PBD fluorine unit 9 (130 mg, 0.257 mmol, 1 equiv.) was dissolved in DMF (1 mL) and /V-(3-dimethyl- aminopropyl)-/V'-ethylcarbodiimide hydrochloride (2 equiv.) and 4-(dimethylamino) pyridine (3 equiv.) were added. The deprotected amine was dissolved in DMF (1 mL) and added to the reaction mixture, which was then stirred at 60 °C for 6 hours. The reaction was quenched by addition of water (0 mL) that was then extracted with ethyl acetate (3 X 30 mL). The organic phase was then sequentially washed with brine (50 mL), NaHCOs saturated aqueous solution (50 mL) and a 10% v/v acetic acid aqueous solution (50 mL). The collected organic phase was concentrated using a rotary evaporator. The resulting crude was purified by column chromatography (mobile phase: from DCM /acetone, 90/10, v/v to DCM /acetone, 60/40/, v/v). The protected intermediate was redissolved in DCM (3 mL) and added of tetrakis(triphenylphosphine) palladium(O) (5 mol%) and pyrrolidine (22 pL, 1.02 equiv.). The reaction mixture was kept under magnetic stirrer for 2 hours when TLC showed completion of reaction. At was purified by column chromatography (mobile phase: from DCM to DCM /MeOH, 90/10/, v/v) affording 35 mg (32%) of pure 16 (PA234-24) as a clear viscous oil. ¹H NMR (400 MHz, DMSO) 5 9.70 (d, J = 10.2 Hz, 1 H), 7.25 (d, J = 12.0 Hz, 1 H), 7.12 (dd, J = 8.5, 2.5 Hz, 1 H), 6.85 (ddd, J = 11.4, 8.8, 4.8 Hz, 1 H), 6.72 (dd, J = 7.8, 2.3 Hz, 1 H), 6.67 (dp, J = 6.5, 2.6 Hz, 1 H), 6.33 (s, 1 H), 5.10 (s, 2H), 4.14 - 3.89 (m, 3H), 3.50 - 3.39 (m, 2H), 2.48 - 2.40 (m, 2H), 2.07 - 1.97 (m, 3H), 1.91 - 1.79 (m, 3H). ¹³C NMR (101 MHz, DMSO) 5 170.2, 160.8, 148.8, 146.5, 140.8, 136.1 , 135.7, 129.5, 121.1 , 114.9, 112.4, 106.7, 102.0, 101.3, 68.5, 67.8, 55.8, 32.1, 29.6, 24.4, 22.5. m/z (+EI) calc, for C22H22F2N4O3 (M)+ 428.17 found 429.1 ([M]+H)⁺; LCMS: (5 min method) Rt 2.63 min, (10 min method) Rt 5.74 min; HRMS: (+EI) calc, for C22H22F2N4O3 (M)+ 428.166 found 429.174 ([M]+H)⁺.

Synthesis of 5-fluoro-6-nitrobenzo[d]thiazol-2-amine (17)

Method A

To a solution of 5-fluorobenzo[d]thiazol-2-amine (1.5 g, 9.13 mmol, 1 equiv.) in trifluoroacetic acid (7 mL) a solution of KNOa (1.02 g, 1.1 equiv.) in trifluoroacetic acid (8 mL) was slowly added at 0°C. The mixture was then allowed to reach room temperature and kept under magnetic stirring for 16 hours. The reaction was then quenched by slowly adding a saturated aqueous solution of K2CC>3 (100 mL). Product was then extracted in EtOAc (3 x 50 mL) and the combined organic fractions were concentrated under reduced pressure. The resulting crude was purified by silica gel column chromatography (mobile phase: from DCM to DCM /MeOH, 90/10/, v/v) affording 745 mg (39%) of pure 17 as a yellow powder. ¹H NMR (400 MHz, DMSO) 5 8.62 (d, J = 7.8 Hz, 1 H), 8.42 (s, 2H), 7.36 (d, J = 13.2 Hz, 1 H). ¹³C NMR (101 MHz, DMSO) 5 173.4, 159.2, 156.3, 153.8, 127.0, 119.3, 105.2. m/z (+EI) calc, for C7H4FN3O2S (M)+ 213.00 found 213.9 ([M]+H)⁺ ; LCMS (5 min method): Rt 2.85 min.

Method B 5-fluorobenzo[d]thiazol-2-amine (146 mg, 0.868 mmol, 1 ,0 eq) was added into a round- bottomed flask containing trifluoroacetic acid (2 mL). The product was allowed to dissolve completely by stirring. Potassium nitrate (105.31 mg, 1.042 mmol, 1.2 eq) was added into another round-bottom flask containing trifluoroacetic acid (2 mL). The product was stirred in an ice bath. After waiting a while for the mixture to cool down the dissolved amine was added dropwise and kept stirring at 55 °C overnight. The reaction was monitored by LC-MS and it showed finished after 15 hours. The mixture was then dried in a rotary evaporator and a brownish oily residue was obtained. The residue was dissolved in 20 mL of ethyl acetate and washed with brine (20 mL x 3). The organic phase was kept and dried over MgSCL. The solvent was removed via rotary evaporator, followed by a high vacuum overnight to provide 110.4 mg of crude 17. Further purification was needed if there were two peaks on the LC-MS profile otherwise the crude 17 was used directly in the synthesis of SL-226-19.

Synthesis of allyl (11aS)-7-fluoro-8-(4-((5-fluoro-6-nitrobenzo[c(]thiazol-2-yl)amino)-4- oxobutoxy)-5-oxo-11-((tetrahydro-2/7-pyran-2-yl)oxy)-2,3, 11 ,11 a- tetrahydro- 1 H- benzo[e]pyrrolo[1 ,2-a][1 ,4]diazepine-10(5/7)-carboxylate (18)

Compound 9 (100 mg, 0.2 mmol), was dissolved in DMF (0.5 mL) and HATU (82 mg,

1.1 equiv.) and triethyl amine (68 pL, 2.5 equiv.) were added, followed by intermediate 18 (56 mg, 1.3 equiv.). The mixture was stirred at 60°C for 8 hours before being diluted with EtOAc (50 mL) and washed with a saturated aqueous solution of NaHCC>3 (50 mL), a 10% v/v solution of acetic acid in water (50 mL) and brine (50 mL). The organic layer was then concentrated in vacuo and the resulting crude purified by silica gel column chromatography (mobile phase: from DCM to DCM/Acetone, 40/60, v/v) to obtain 39 mg (28%) of 18 as a clear viscous oil. ¹H NMR (400 MHz, CDCh) 6 8.38 (dd, J = 7.1 , 1.1 Hz, 1 H), 7.37 (d, J = 11.6 Hz, 1 H), 7.30 (d, J = 11.0 Hz, 1 H), 6.53 (d, J =

7.2 Hz, 1 H), 5.83 - 5.47 (m, 2H), 4.97 - 4.80 (m, 2H), 4.49 - 4.18 (m, 2H), 3.97 (dtt, J = 9.7, 6.6, 3.8 Hz, 2H), 3.73 (ddd, J = 11.0, 6.4, 3.0 Hz, 1 H), 3.53 - 3.27 (m, 4H), 2.63 (t, J = 7.2 Hz, 2H), 2.14 - 2.08 (m, 2H), 1.98 - 1.82 (m, 3H), 1.66 - 1.49 (m, 2H), 1.47 - 1.23 (m, 5H). ¹³C NMR (101 MHz, CDCh) 6 171.9, 166.3, 162.5, 156.4, 153.8, 153.1 , 150.7, 131.8, 127.7, 119.9, 116.4, 109.3, 109.1 , 96.3, 68.2, 63.8, 60.2, 46.6, 36.2, 32.2, 31.2, 31.0, 28.9, 25.3, 23.3, 20.0. m/z (+EI) calc, for C32H33N5O9S (M)+ 701.2 found 702.1 ([M]+H)⁺ ; LCMS (5 min method): Rt 3.90 min.

Synthesis of (S)-/V-(6-amino-5-fluorobenzo[d]thiazol-2-yl)-4-((7-fluoro-5-oxo-2,3,5,11a- tetrahydro-1/7-benzo[e]pyrrolo[1 ,2-a][1,4]diazepin-8-yl)oxy)butanamide (19, PA234-47)

18 19

To a solution of compound 18 (36 mg, 0.051 mmol, 1 equiv.) in THF (5 mL) zinc powder was added (100 mg, 30 equiv.) followed by an aqueous solution (5 mL) of ammonium chloride (163 mg, 60 equiv.). The mixture was stirred at room temperature for 3 hours before being filtered through celite pad, which was washed with MeOH (100 mL). The filtrate was then concentrated in vacuo and the resulting crude purified by silica gel column chromatography (mobile phase: DCM/MeOH, 90/10, v/v). The pure product was then redissolved in DCM (2 mL) and tetrakis(triphenylphosphine) palladium(O) (5 mol%) and pyrrolidine (4 pL, 1 equiv.) were sequentially added. The reaction was stirred at room temperature for 30 min and then concentrated under vacuum. The crude was finally purified by silica gel column chromatography (mobile phase: from DCM to DCM/MeOH, 85/15, v/v) to obtain 8.5 mg (34%) of 19 (PA234-47) as a clear viscous oil. ¹H NMR (400 MHz, DMSO) 5 11.97 (s, 1 H), 7.48 (d, J = 5.6 Hz, 1 H), 7.43 (d, J = 13.4 Hz, 1 H), 7.39 (d, J = 11.9 Hz, 1 H), 7.20 (d, J = 8.7 Hz, 1 H), 6.47 (d, J = 7.6 Hz, 1 H), 5.18 (s, 2H), 4.09 - 3.96 (m, 2H), 3.69 (dd, J = 8.2, 5.7 Hz, 1 H), 3.61 - 3.44 (m, 2H), 2.64 (t, J = 7.2 Hz, 2H), 2.10 - 2.04 (m, 2H), 2.00 - 1.81 (m, 3H), 1.73 - 1.64 (m, 1 H). ¹³C NMR (101 MHz, DMSO) 5 170.99, 163.78, 141.34, 134.07, 127.86, 117.64, 113.96, 109.64, 106.64, 105.96, 103.59, 102.14, 68.49, 58.13, 53.89, 32.07, 29.58, 23.92, 22.31. m/z (+EI) calc, for C24H24N5O3S (M)+ 485.13 found 486.1 ([M]+H)⁺; LCMS: (5 min method) Rt 2.72 min; (10 min method) Rt 5.94 min; HRMS: (+EI) calc, for C32H33N5O9S (M)+ 701.2 found 702.1 ([M]+H)⁺; HRMS: (+EI) calc, for C24H24N5O3S (M)+ 485.133 found 486.140 ([M]+H)⁺.

Synthesis of methyl 4-(5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-2-fluoro-4-((S)-2- (hydroxymethyl)pyrrolidine-1-carbonyl)phenoxy)butanoate (20)

5 20

Compound 5 (242 mg, 0.68 mmol, 1 equiv.) was added to a solution of Alloc-Val-Ala- PAB-PNP (CAS No. 1884578-27-1) (369 mg, 1 equiv.) in DMF (3 mL) followed by the sequential addition of /V,/V-diisopropylethylamine (295 pL, 2.5 equiv.) and 1- hydroxybenzotriazole hydrate (183 mg, 2 equiv.). The reaction mixture was stirred at 70°C for 6 hours and then diluted with EtOAc (50 mL). The organic mixture was washed with brine (2 x 50 mL) and then concentrated under reduced pressure. The obtained crude was purified by reverse phase chromatography (mobile phase: water/ acetonitrile, from 95/5 to 25/75 v/v) obtaining 350 mg (68%) of pure 20 as a cream viscos oil. ¹H NMR (400 MHz, DMSO) 5 9.97 (d, J = 32.9 Hz, 1 H), 8.16 (d, J = 8.4 Hz, 1 H), 7.63 - 7.53 (m, 2H), 7.41 - 7.23 (m, 4H), 7.09 (dd, J = 12.1 , 2.1 Hz, 1 H), 5.91 (s, 1 H), 5.34 - 5.17 (m, 2H), 5.07 (s, 1 H), 4.74 (s, 1 H), 4.56 - 4.38 (m, 4H), 4.26 (s, 1 H), 4.08 (d, J = 8.1 Hz, 3H), 3.98 (d, J = 6.9 Hz, 1 H), 3.90 (s, 1 H), 3.62 - 3.60 (m, 3H), 3.18 (s, 3H), 2.44 (t, J = Q.7 Hz, 2H), 2.04 - 1.87 (m, 6H), 1.31 (d, J = 6.5 Hz, 3H), 0.91 - 0.84 (m, 6H). ¹³C NMR (101 MHz, DMSO) 5 172.9, 161.2, 146.1 , 139.3, 135.9, 133.6, 132.8, 131.7, 128.8, 127.7, 126.5, 124.9, 119.0, 116.9, 115.3, 83.3, 67.8, 61.3, 56.7, 51.3, 49.3, 48.6, 46.4, 41.0, 30.3, 29.8, 29.8, 28.9, 27.1 , 24.0, 19.1 , 18.1. m/z (+EI) calc, for C37H48FN6O11 (M)+ 757.33 found 758.3 ([M]+H)⁺; LCMS (5 min method): Rt 3.20 min.

Synthesis of 4-((10-(((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutan- amido)propanamido)benzyl)oxy)carbonyl)-7-fluoro-11-hydroxy- 5-0X0-2,3,5,10,11 , 11a- hexahydro-1 H-benzo[e]pyrrolo[1 ,2-a][1 ,4]diazepin-8-yl)oxy)butanoic acid (21)

20 21

Compound 20 (350 mg, 0.46 mmol, 1 equiv.) was dissolved in dioxane (3 mL) and a 0.2M aqueous solution of NaOH (2 mL) was added. The reaction mixture was stirred at room temperature for 3 hours and then quenched with the addition of a 10% v/v aqueous solution of acetic acid (100 mL). Product was extracted from the aqueous mixture in EtOAc (3 x 50 mL) and the combined organic fractions were concentrated in vacuo. The resulting crude was purified by silica gel column chromatography (mobile phase: from DCM to DCM/MeOH, 90/10, v/v) to obtain 94 mg (0.126 mmol, 1 equiv.) of the pure acid intermediate. This was then redissolved in DCM (1 mL) and Dess Martin periodane (107 mg, 2 equiv.) was added to the solution. The reaction mixture was then stirred at room temperature for 3 hours. The reaction was then quenched with a saturated solution of sodium metabisulfite (50 mL) and acidified to pH approximatively of 4 with acetic acid. Product was them extracted in EtOAc (2 x 50 mL) and the combined organic fractions were concentrated under vacuum. The resulting rude was purified by silica gel column chromatography (mobile phase: from DCM to DCM/MeOH, 90/10, v/v) to obtain 55 mg (16%) of pure 21 as a brown viscous oil. ¹H NMR (400 MHz, CDCI₃) 6 9.03 (s, 1 H), 7.47 - 7.40 (m, 2H), 7.10 (d, J = 8.2 Hz, 2H), 6.58 (d, J = 7.1 Hz, 1 H), 5.98 - 5.72 (m, 2H), 5.62 (d, J = 9.8 Hz, 1 H), 5.39 (d, J = 11.4 Hz, 1 H), 5.26 - 5.10 (m, 2H), 4.76 - 4.45 (m, 5H), 4.16 - 3.95 (m, 1 H), 3.83 (s, 1 H), 3.72 - 3.60 (m, 2H), 3.56 - 3.49 (m, 1 H), 3.45 - 3.39 (m, 1 H), 2.56 - 2.35 (m, 2H), 2.16 - 2.07 (m, 3H), 2.03 - 1.93 (m, 3H), 1.45 - 1.39 (m, 3H), 0.96 - 0.88 (m, 6H). ¹³C NMR (101 MHz, CDCh) 6 179.1 , 170.8, 166.2, 156.8, 155.9, 150.2, 149.0, 138.0, 132.5, 131.7, 129.1 , 126.3, 120.6, 120.4, 118.2, 100.1 , 95.4, 86.1 , 68.3, 67.6, 66.3, 60.2, 50.9, 49.9, 46.6, 31.1 , 29.8, 28.9, 24.3, 23.2, 19.33, 18.0. m/z (+EI) calc, for C37H45FN4O11 (M)+ 741.31 found 742.3 ([M]+H)⁺and 764.2 ([M]+Na)⁺; LCMS (5 min method): Rt 3.08 min.

Synthesis of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)- propanamido)benzyl 8-fluoro-7-(4-((4-fluoro-3-methylphenyl)amino)-4-oxobutoxy)-4- hydroxy- 10-oxo-2,3,3a,4, 10,10a-hexahydrobenzo[b]cyclopenta[e]azepine-5(1 /-/)- carboxylate (22)

Compound 21 (48 mg, 0.065 mmol, 1 equiv.), was dissolved in DMF (1 mL) and /\/-(3- Dimethylaminopropyl)-/V'-ethylcarbodiimide hydrochloride (31 mg, 2.5 equiv.) and 4- (dimethylamino)pyridine (24 mg, 3 equiv.) were added, followed by 4-fluoro-3-methyl- aniline (12 mg, 1.5 equiv.). The mixture was stirred at 50°C for 8 hours before being diluted with EtOAc (50 mL) and washed with a saturated aqueous solution of NaHCOs (30 mL), a 10% aqueous solution of acetic acid (30 mL) and brine (50 mL). The organic layer was then concentrated in vacuo and the resulting crude purified by silica gel column chromatography (mobile phase: from DCM to DCM/MeOH, 90/10, v/v) to obtain 50 mg (92%) of 22 as a clear viscous oil. ¹H NMR (400 MHz, CDCI₃) 6 7.54 - 7.43 (m, 1 H), 7.35 - 7.16 (m, 3H), 7.08 - 7.04 (m, 1 H), 6.82 (t, J = 9.0 Hz, 1 H), 6.70 (t, J = 9.0 Hz, 1 H), 6.41 (ddd, J = 20.9, 7.6, 3.6 Hz, 2H), 5.88 - 5.70 (m, 1 H), 5.66 - 5.31 (m, 2H), 5.27 - 4.98 (m, 2H), 4.66 - 4.34 (m, 3H), 4.00 - 3.53 (m, 4H), 3.53 - 3.08 (m, 4H), 2.48 - 2.19 (m, 2H), 2.14 (d, J = 2.0 Hz, 2H), 2.10 (d, J = 2.0 Hz, 3H), 2.03 (q, J = 5.5 Hz, 2H), 1.91 (s, 3H), 1.33 (t, J = 7.0 Hz, 3H), 1.02 - 0.68 (m, 6H). ¹³C NMR (101 MHz, CDCh) 6 172.0, 171.0, 166.0, 154.2, 148.9, 146.4, 141.7, 138.3, 132.4, 129.3, 125.5, 125.2, 123.5, 120.1 , 118.1 , 115.8, 115.5, 115.3, 113.7, 86.1 , 68.5, 66.3, 49.8, 46.5, 33.4, 31.6, 29.8, 28.9, 24.9, 23.1 , 19.4, 19.3, 17.8, 14.7. m/z (+EI) calc, for C44H51 F2N5O10 (M)+ 848.36 found 871.3 ([M]+Na)⁺; LCMS (5 min method): Rt 3.56 min.

Synthesis of 4-((S)-2-((S)-2-(5-(2,5-dioxo-2,5-dihydro-1/7-pyrrol-1-yl)pentanamido)-3- methylbutanamido)propanamido)benzyl 7-fluoro-8-(4-((4-fluoro-3-methylphenyl)amino)- 4-oxobutoxy)-11-hydroxy-5-oxo-2,3,11 , 11a- tetrahydro- 1 H-benzo[e]pyrrolo[1 ,2- a][1 ,4]diazepine-10(5/-/)-carboxylate (23, PA248-12)

Compound 22 (50 mg, 0.064 mmol, 1 equiv.) was dissolved in DCM (0.5 mL) and tetrakis(triphenylphosphine)palladium(0) (10 mol%) and pyrrolidine (14 pL, 2.5 equiv.) were sequentially added. The reaction was stirred at room temperature for 6 hours and then concentrated under vacuum. The crude was purified by silica gel column chromatography (mobile phase: from DCM to DCM/MeOH, 85/15, v/v). The purified intermediate was then redissolved in DCM (0.5 mL) and 6-(2,5-dioxo-2,5-dihydro-1/7- pyrrol-1-yl)hexanoic acid (20 mg, 1.5 equiv.) and /\/-(3-dimethylaminopropyl)-/\/'-ethyl- carbodiimide hydrochloride (30, 2.5 equiv.) were sequentially added. The mixture was then stirred at room temperature for 4 hours, until TCL showed total consumption of the starting material. The mixture was then concentrated in vacuo and purified by silica gel column chromatography (mobile phase: from DCM to DCM/MeOH, 90/10, v/v) to obtain 23 mg (38%)of pure product (23) (PA248-12) as a viscous clear oil. ¹H NMR (400 MHz, DMSO) 59.94 (d, J= 9.4 Hz, 2H), 8.16 (d, J= 6.8 Hz, 1H), 7.79 (d, J= 8.6 Hz, 1H), 7.54 (ddd, J= 13.5, 9.4, 5.5 Hz, 3H), 7.41 -7.31 (m, 2H), 7.18 (s, 1H), 7.08-6.96 (m, 4H), 6.59 (s, 1H), 5.48 (d, J= 7.7 Hz, 1H), 5.13 (d, J= 12.1 Hz, 1H), 4.85 (d, J= 10.9 Hz, 1H), 4.38 (dt, J= 13.6, 6.6 Hz, 1H), 4.18-4.06 (m, 5H), 3.94 (s, 1H), 3.52-3.45 (m, 1H), 3.37 (d, J=6.9 Hz, 2H), 2.18 (d, J= 1.5 Hz, 3H), 2.17-2.09 (m, 2H), 2.03 (s, 3H), 1.96-1.81 (m, 4H), 1.51 - 1.43 (m, 4H), 1.31 - 1.13 (m, 7H), 0.89-0.79 (m, 6H). ¹³C NMR (101 MHz, DMSO) 5173.2, 172.7, 171.5, 170.7, 168.5, 165.3, 156.3, 149.6, 135.8, 134.9, 131.3, 129.0, 124.8, 122.5, 119.3, 118.8, 115.6, 115.4, 115.1, 113.4, 80.4, 57.9, 49.5, 49.1, 43.5, 37.5, 35.4, 32.9, 30.7, 28.7, 28.2, 26.3, 25.4, 24.8, 23.2, 19.7, 18.6, 18.4, 16.5, 14.8. m/z (+EI) calc, for C49H57F2N7O11 (M)+ 957.41 found 980.3 ([M]+Na)⁺; LCMS (5 min method) Rt 3.76 min, (10 min method) Rt 7.61; HRMS: (+EI) calc, for C49H57F2N7O11 (M)+ 957.408 found 958.414 ([M]+H)⁺.

Synthesis of allyl ((S)-1-(((S)-1-((5-((terf-butoxycarbonyl)amino)-2-fluorophenyl)amino)- 1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (24)

24

To a solution of Alloc-Val-Ala-OH (500 mg, 1.84 mmol, 1 equiv.) HATLI (838 mg, 1.2 equiv.) and triethylamine (642 pL, 2.5 equiv.) were added, followed by tert-butyl (3- amino-4-fluorophenyl)carbamate (416 mg, 1 equiv.). The reaction mixture was then stirred at room temperature for 16 hours. The reaction was then diluted with EtOAc (100 mL) and washed with a saturated aqueous solution of NaHCC>3(100 mL), a 10% aqueous solution of acetic acid (100 mL) and brine (100 mL). The organic layer was then concentrated in vacuo and the resulting crude purified by silica gel column chromatography (mobile phase: from DCM to DCM/acetone, 60/40, v/v) to obtain 478 mg (54%) of 24 as a cream viscous oil. ¹H NMR (400 MHz, DMSO) 59.58 (d, J = 23.0 Hz, 1H), 9.37 (s, 1H), 8.35-8.15 (m, 1H), 8.08-7.94 (m, 1H), 7.25 (t, J= 8.9 Hz, 1H), 7.16-7.07 (m, 2H), 5.94-5.90 (m, 1H), 5.31 -5.23 (m, 2H), 4.53 (dd, J= 9.1, 5.0 Hz, 1H), 4.46-4.43 (m, 2H), 3.92-3.83 (m, 1H), 1.95- 1.90 (m, 1H), 1.45 (d, J= 2.9 Hz, 9H), 1.30 (dd, J = 7.0, 2.8 Hz, 3H), 0.84 - 0.77 (m, 6H). ¹³C NMR (101 MHz, DMSO) 5 171.3, 171.2, 171.2, 155.8, 152.8, 135.6, 133.6, 117.0, 116.7, 115.2, 114.3, 113.9, 79.0, 64.4, 60.2, 54.9, 48.6, 40.2, 39.9, 39.7, 39.5, 39.3, 39.1 , 38.9, 28.1, 19.2, 14.6. m/z (+EI) calc, for C23H33FN4O6 (M)+ 480.24 found 503.2([M]+Na)⁺; LCMS (5 min method): Rt 3.64 min.

Synthesis of allyl (11aS)-8-(4-((3-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methyl- butanamido)propanamido)-4-fluorophenyl)amino)-4-oxobutoxy)-7-fluoro-5-oxo-11- ((tetrahydro-2/7-pyran-2-yl)oxy)-2,3, 11 ,11 a-tetrahydro-1 /7-benzo[e]pyrrolo[1 ,2-a][1 ,4] diazepi ne-10(5/7)-carboxylate (25)

24 25

Compound 24 (100 mg, 0.21 mmol, 1 equiv.) was boc-deprotected in MeOH (2 ml) and HCI 4M in dioxane (3 mL). The solution was left under magnetic stirrer for 3 hours until TLC showed total cleavage of protecting group. The reaction mixture was subsequently evaporated using a rotary evaporator, obtaining a brown viscus oil. Compound 9 (108 mg, 0.21 mmol, 1 equiv.) was dissolved in DMF (1 mL) and /\/-(3-dimethylaminopropyl)- /V'-ethylcarbodiimide hydrochloride (2 equiv.) and 4-(dimethylamino)pyridine (3 equiv.) were added. The deprotected amine was dissolved in DMF (1 mL) and added to the reaction mixture, which was then stirred at 60 °C for 6 hours. The reaction was quenched by addition of water (50 mL) that was then extracted with ethyl acetate (3 X 30 mL). The organic phase was then sequentially washed with brine (50 mL), NaHCCh saturated aqueous solution (50 mL) and a 10% v/v acetic acid aqueous solution (50 mL). The collected organic phase was concentrated using a rotary evaporator. The resulting crude was purified by column chromatography (mobile phase: from DCM to DCM /acetone, 50/50/, v/v) affording 36 mg (20%) of pure 25 as a light brown viscous oil. ¹H NMR (400 MHz, CDCI3) 5 8.11 - 8.02 (m, 1 H), 7.53 (d, J = 10.7 Hz, 1 H), 7.48 - 7.40 (m, 1 H), 6.98 (t, J = 9.6 Hz, 1 H), 6.72 (s, 1 H), 5.96 - 5.81 (m, 2H), 5.78 - 5.54 (m, 2H), 5.36 - 5.02 (m, 4H), 4.76 - 4.53 (m, 4H), 4.37 (dd, J = 27.6, 14.2 Hz, 1 H), 4.09 (dd, J = 11 .2, 5.9 Hz, 3H), 3.91 (d, J = 7.5 Hz, 1 H), 3.74 - 3.62 (m, 2H), 3.60 - 3.49 (m, 2H), 2.61 - 2.51 (m, 2H), 2.22 - 1.97 (m, 8H), 1.79 - 1.67 (m, 2H), 1.52 (d, J = 5.1 Hz, 3H), 1 .42 (t, J = 7.2 Hz, 3H), 1 .02 - 0.78 (m, 6H). ¹³C NMR (101 MHz, CDCI3) 6 176.0, 172.6, 170.8, 166.3, 162.4, 155.6, 150.7, 149.2, 144.2, 132.6, 132.1 , 131.9, 127.5, 118.0, 110.1 , 100.6, 91.8, 68.7, 66.1 , 60.2, 50.9, 49.7, 46.6, 38.7, 31.5, 31.0, 29.8, 29.4, 29.0, 25.4, 24.7, 23.4, 20.1 , 19.2, 17.9. m/z (+EI) calc, for C43H54F2N6O11 (M)+ 868.38 found 869.4 ([M]+H)⁺and 891.3 ([M]+Na)⁺; LCMS (5 min method): Rt 3.71 min.

Synthesis of 6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((2-fluoro-5-(4- (((S)-7-fluoro-5-oxo-2,3,5, 11 a- tetrahydro- 1 H-benzo[e]pyrrolo[1 , 2-a] [ 1 ,4]diazepin-8-yl)- oxy)butanamido)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)- hexanamide (26, PA248-13)

25 26

Compound 25 (25 mg, 0.029 mmol, 1 equiv.) was dissolved in DCM (0.5 mL) and tetrakis(triphenylphosphine)palladium(0) (10 mol%) and pyrrolidine (83 pL, 3.5 equiv.) were sequentially added. The reaction was stirred at room temperature for 6 hours and then concentrated under vacuum. The crude was purified by silica gel column chromatography (mobile phase: from DCM to DCM/MeOH, 85/15, v/v). The purified intermediate was then redissolved in DCM/MeOH (90/10 v/v, 0.5 mL) and 6-(2,5-dioxo- 2,5-dihydro-1/7-pyrrol-1-yl)hexanoic acid (9 mg, 1.5 equiv.) and 2-ethoxy-1- ethoxycarbonyl-1 ,2-dihydroquinoline (11 mg, 1.5 equiv.) were sequentially added. The mixture was then stirred at room temperature for 4 hours, until TCL showed total consumption of the starting material. The mixture was then concentrated in vacuo and purified by silica gel column chromatography (mobile phase: from DCM to DCM/MeOH, 90/10, v/v) to obtain 13 mg (57%) of pure product (26) (PA248-13) as a viscous clear oil. ¹H NMR (400 MHz, DMSO) 5 10.03 (d, J = 2.7 Hz, 2H), 9.62 (s, 1 H), 8.23 (d, J = 6.8 Hz, 1 H), 8.15 - 8.09 (m, 1 H), 7.86 (d, J = 4.5 Hz, 1 H), 7.81 (d, J = 8.9 Hz, 1 H), 7.57 (d, J = 12.1 Hz, 1 H), 7.15 (t, J = 9.8 Hz, 1 H), 7.00 (s, 2H), 6.47 (d, J = 7.6 Hz, 1 H), 4.53 - 4.48 (m, 1 H), 4.26 - 4.10 (m, 2H), 4.01 (ddd, J = 14.5, 10.0, 6.7 Hz, 2H), 3.71 (ddd, J = 13.9, 8.0, 4.6 Hz, 1 H), 3.62 - 3.55 (m, 1 H), 3.45 (dt, J = 8.7, 6.0 Hz, 1 H), 3.36 (t, J = 7.1 Hz, 2H), 2.28 - 2.10 (m, 3H), 2.05 (d, J = 6.5 Hz, 2H), 1.94 (dd, J = 11.2, 4.5 Hz, 2H), 1.68 (ddd, J = 15.4, 7.6, 4.1 Hz, 1 H), 1.47 (dt, J = 14.2, 6.3 Hz, 4H), 1.30 (d, J = 7.1 Hz, 3H), 1.23 - 1.09 (m, 4H), 0.88 - 0.81 (m, 6H);.¹³C NMR (101 MHz, DMSO) 5 172.1 , 171.4, 171.2, 171.1 , 170.4, 166.1 , 163.8, 162.4, 141.4, 135.4, 134.4, 117.9, 117.6, 115.6, 115.0, 114.4, 111.9, 109.6, 102.1 , 87.1 , 67.7, 58.1 , 57.3, 53.9, 48.6, 46.4, 37.0, 34.9, 32.4, 30.7, 27.8, 25.8, 24.9, 22.3, 19.2, 18.2, 17.7. m/z (+EI) calc, for C40H47F2N7O8 (M)+ 791.35 found 792.3 ([M]+H)⁺; LCMS (5 min method): Rt 3.00 min.

Synthesis of (S)-N-(6-amino-5-fluorobenzo[d]thiazol-2-yl)-4-((7-methoxy-5-oxo-

2,3,5, 11a-tetrahydro-1 H-benzo[e]pyrrolo[1 ,2-a][1 ,4]diazepin-8-yl)oxy)butanamide (SL-

226-19)

SL-226-19 a) BTFFH, DI PEA, DCM, 80 °C; b) Fe, NH₄CI, EtOH, H₂O, 80 °C; c) PPH₃, Pd(PPh₃)₄, pyrrolidine, DCM Step a)

To a solution of the PBD core 1.10 (178.9 mg, 0.345 mmol, 1.0 eq) in 4 mL of DCM in a 10 mL microwave vial was added fluoro-/V,/V,/V /V'-bis(tetramethylene)formamidinium hexafluorophosphate (163.79 mg, 0.518 mmol, 1.5 eq). N,N-Diisopropylethylamine (200.85 mg, 266 pL, 1.554 mmol, 4.5 eq) was then added drop by drop to the mixture and kept stirring under room temperature for 30 minutes to activate the PBD core. Crude 17 (110.4 mg, 0.518 mmol, 1.5 eq) was then added to the reaction mixture. The vial was then sealed and heated to 80°C by using the microwave and kept stirring overnight. The reaction completed after 15 hours as confirmed by LC-MS. The mixture was dried over a rotary evaporator and was redissolved in 2 mL of DCM for purification. The purification was done by a flash column chromatography and the product was eluted with 70% ethyl acetate/ DCM to afford 102.8 mg of the intermediate.

Step b)

A solution of the intermediate (102.8 mg, 0.145 mmol, 1.0 eq) in 4 mL of ethanol was kept stirring in a round- bottomed flask. A solution of NH4CI (69.8 mg, 1.305 mmol, 9.0 eq) in water was added dropwise to the previous solution. The reaction mixture was stirred at 50°C for 15 minutes, followed by adding iron (48.59 mg, 0.87 mmol, 6.0 eq) to the flask. After that, the temperature was increased to 80°C for another 15 minutes until the LC-MS confirmed the completion of the reaction. The mixture was cooled down to room temperature and the iron was removed through celite. The celite pad was washed with 200 mL of ethanol and the solvent was removed by a rotary evaporator. The residue was redissolved in 60 mL of water and extracted with ethyl acetate (60 mL x 3), followed by drying over MgSC>4 and a rotary evaporator to give 96 mg of the crude. The crude was directly used for the next step.

Step c)

The crude (96 mg, 0.141 mmol, 1.0 eq) was dissolved in 5 mL of DCM. Once dissolved, the pyrrolidine (12.02 mg, 14 pL, 0.169 mmol, 1.20 eq) was added to the reaction, followed by triphenylphosphine (9.18 mg, 0.035 mmol, 0.25 eq). Finally, a catalytic amount of tetrakis(triphenylphosphine)palladium(0) (8.09 mg, 0.007 mmol, 0.05 eq) was added to the reaction and kept stirring. The LC-MS confirmed the completion of the reaction after 30 minutes. The reaction mixture was then dried over a rotary evaporator and purified by a flash column chromatography. The crude was eluted in 5% methanol/ ethyl acetate to provide 34.1 mg (49%) of SL-226-19 as a yellow solid. ¹H NMR (400 MHz, CDCI₃) 6 7.65 (d, J = 4.4 Hz, 1 H), 7.51 (s, 1 H), 7.38 (d, J = 11.4 Hz, 1 H), 7.12 (dd, J = 8.4, 4.8 Hz, 1 H), 6.79 (s, 1 H), 4.17 - 4.07 (m, 2H), 3.88 - 3.68 (m, 8H), 3.49 (s, 2H), 3.32 (s, 1 H), 2.71 (q, J = 6.8 Hz, 2H), 2.35 - 2.24 (m, 3H). ¹³C NMR (100 MHz, CDCI₃) 6 171.32, 164.61 , 162.52, 156.48, 150.32, 147.80, 140.57, 132.64, 120.65, 111.65, 110.73, 107.08, 67.66, 60.41 , 56.05, 53.73, 50.85, 46.72, 33.01 , 29.71 , 24.31 , 24.20, 14.21 , 13.15. LCMS Rt 2.551 minutes (5 min method), Rt 5.596 (10 min method); purity: 98.44%; HRMS: (ESI⁺) found: 498.1606 [M+H]⁺, theoretical: 498.1533 [M+H]⁺.

General Method Of Synthesis of Linker-Payload From Amine Containing Payloads

To a solution of SL-217-19 (1.7 eq) in 10% methanol/DCM mixture (5 mL), ethyl 2- ethoxyquinoline-1 (2H)-carboxylate (EEDQ) (2.0 eq) was added and allowed to stir in an ice bath for 40 minutes to activate the linker. The amine containing payload (1.0 eq) was added to the mixture and it was stirred at room temperature. The LC-MS showed the end of the reaction after 18 hours. The reaction mixture was dried over a rotary evaporator and redissolved in 2 mL DCM for purification. The purification was done by using flash chromatography and the final compound was eluted at 4% methanol in DCM. 6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((5-fluoro-2-(4-(((S)-7- methoxy-5-oxo-2,3,5,11a-tetrahydro-1 H-benzo[e]pyrrolo[1 ,2-a][1 ,4]diazepin-8- yl)oxy)butanamido)benzo[d]thiazol-6-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-

SL-226-25 llsing the amine containing payload SL-226-19 in the general method above, linker-payload compound SL-226-25 was synthesised as a brown solid (20%). ¹H NMR (400 MHz, DMSO) 5 12.42 (s, 1 H), 9.74 (d, J = 16.7 Hz, 1 H), 8.40 (d, J = 7.4 Hz, 1 H), 8.22 (dd, J = 11.0, 6.9 Hz, 2H), 7.80 - 7.72 (m, 2H), 7.63 (dd, J = 11.3, 5.1 Hz, 1 H), 7.00 (d, J = 2.6 Hz, 3H), 4.20 - 4.17 (m, 2H), 4.04 (q, J = 7.1 Hz, 2H), 3.75 (s, 3H), 3.38 (dd, J = 7.0, 3.1 Hz, 4H), 2.16 - 2.06 (m, 6H), 1.99 (s, 3H), 1.95 - 1.89 (m, 2H), 1.49 (d, J = 8.9 Hz, 9H), 1.35 (d, J = 6.1 Hz, 7H), 1.25 - 1.23 (m, 10H). ¹³C NMR (100 MHz, MeOD) 5 174.83, 174.56, 173.08, 171.16, 169.15, 161.58, 159.37, 154.55, 150.43, 149.12, 146.40, 139.58, 133.93, 133.22, 123.27, 122.49, 114.68, 114.64, 107.19, 106.42, 106.15, 104.83, 102.09, 73.76, 67.92, 61.80, 58.83, 58.48, 54.46, 37.00, 35.16, 30.60, 30.53, 30.42, 27.88, 25.95, 25.06, 18.35, 18.23, 17.39, 16.56, 15.92. LCMS Rt 2.942 minutes (5 min method), Rt 6.584 minutes (10 min method); purity: 87.92%; HRMS: (ESI⁺) found: 861.3402 [M+H]⁺, theoretical: 861.3327 [M+H]⁺.

6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((2-fluoro-5-(4-(((S)-7- methoxy- 5-0X0-2,3,5,11 a-tetrahydro-1 H-benzo[e]pyrrolo[1 , 2-a][1 , 4]diazepin-8- yl)oxy)butanamido)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2- yl)hexanamide (SL-226-26)

Using the amine containing payload MH22 in the general method above, linker-payload compound SL-226-25 was synthesised as a yellow solid (23%). ¹H NMR (400 MHz, MeOD) 5 8.01 (dd, J = 7.0, 2.7 Hz, 1 H), 7.32 (ddt, J = 9.1 , 4.5, 2.4 Hz, 1 H), 7.24 (s, 1 H), 7.06 (s, 1 H), 6.98 (dd, J = 10.4, 8.9 Hz, 1 H), 6.67 (d, J = 9.6 Hz, 3H), 6.49 (s, 1 H), 6.23 (s, 1 H), 4.52 - 4.40 (m, 3H), 4.09 (d, J = 7.4 Hz, 1 H), 3.71 - 3.63 (m, 6H), 3.37 (t, J = 7.1 Hz, 3H), 3.25 (s, 6H), 3.20 - 3.08 (m, 1 H), 2.47 (dd, J = 8.4, 6.4 Hz, 3H), 1.37 - 1.17 (m, 15H). ¹³C NMR (100 MHz, MeOD) 5 174.80, 172.40, 171.17, 167.18, 152.64, 144.73, 141.57, 140.02, 138.46, 133.93, 119.92, 115.70, 114.74, 112.17, 109.19, 107.24, 101.50, 95.60, 88.75, 67.79, 67.44, 59.46, 59.06, 58.76, 55.63, 55.44, 54.46, 37.02, 35.14, 32.80, 30.44, 30.19, 28.87, 27.87, 25.97, 25.03, 24.76, 22.43, 18.36, 17.39, 16.50. LCMS Rt 2.871 minutes (5 min method Rt 6.429 (10 min method); purity: 88.77%; HRMS: (ESI⁺) found: 804.3745 [M+H]⁺, theoretical: 804.3654 [M+H]⁺.

ADC Conjugation

100 pL sample of trastuzumab (10 mg/mL) in PBS 2 mM ethylenediaminetetraacetic acid [EDTA] was reacted with 10,0 eq. of tris(2-carboxyethyl)phosphine hydrochloride [TCEP] (10 mM, 10.3 uL) and left to mix in the thermoshaker (VWR) set to 20°C for 1.5 hours. Then, 16 eq. of linker-payload (10 mM in DMSO, 11 uL) was added and left to react in thermoshaker set to 20°C for 1 hour. After that, 30 eq. of N-acetylcysteine (10 mM, 20.6 pL) was added and left to mix at 20°C for 15 minutes to quench the conjugation. The reaction mixture was then buffer exchanged three times to water via Zeba™ Spin Desalting Columns (7K MWCO).

The Zeba™ Spin Desalting Column was placed in a 2 mL centrifuge tube with the bottom closure removed and cap loosened and centrifuged at 1500 x g for 1 minute to remove storage solution. The slanted upward resin in the column was marked and was placed in the centrifuge with the mark facing outward in all subsequent centrifugation steps. 300 pL of water was added on top of the resin bed and was centrifuged at 1500 x g for 1 minute to remove buffer. This step was repeated three additional times, followed by discarding buffer from the collection tube. A new collection tube was replaced for the ADC collection. 30 - 130 pL of ADC sample was then added on top of the resin. The sample was then centrifuged at 1500 x g for 2 minutes and collected. Final concentration of the sample was determined using BioDrop-pLITE UV/Vis spectrophotometer.

ADC Characterisation

ADC characterisation was performed via the AKTA system. HiTrap Butyl HP 1 mL column (Cytiva, CN#1775490) was used for ADC characterisation. ADC mixtures were prepared in 1.5 mL of buffer A containing 20 mM sodium phosphate by adding 50mM sodium phosphate and 0.9 M ammonium sulfate by adding 3.96 M ammonium sulfate solution for sample loading. Before loading, the sample was spun at 25000 rpm for 10 minutes and the supernatant was taken for injection if there was any precipitate. Buffer A was 0.9 M ammonium sulfate and 20 mM sodium phosphate, pH 7, and buffer B was 20 mM sodium phosphate and 25% isopropanol, pH 7. The sample was loaded using AKTA injection pump. The flow rate of HiTrap Butyl HP 1 mL column was 5 mL min^-1. The separation program was set up as column volume (CV). The linear gradient conditions were from 0% B to 50% B for 40 CV, or 0% B to 100% B for 40 CV and then 100% B wash the column for 10 CV. The separations were monitored at UV 280 nm. The chromatogram for the monoclonal antibody trastuzumab, using a gradient of 0% B to 50% B for 40 CV, shows a peak at 21.30 ml (see Figure 28). Trastuzumab was reacted with the linker-payload SL-226-26 using the ADC conjugation method. Following the ADC conjugation reaction the chromatogram, under the same linear gradient conditions, shows that the peak at 21.30 ml for free trastuzumab has disappeared and the formation of other peaks at a higher eluting volume for the antibody drug conjugate of trastuzumab with SL-226-26.

ADC Stability Test

An ADC stability test was carried out using a 1 mL sample of trastuzumab (10 mg/mL) that was prepared for the conjugation. The ADC was divided into 5 groups with 300 pL for each group. The ADC was stored at 4°C for 0 week, 2 weeks, 5 weeks, 8 weeks and -80 °C for 8 weeks. At each time point, the ADC was prepared following the same procedure as described above for the sample for characterisation and injected into the AKTA system. The result was compared with 0 week.

Biological Assays

Anticancer Screening Anticancer screening of synthesised drugs was conducted using three different cancer cell lines (CT26, B16-F10 and MDA-MD-231) to evaluate their anticancer potential (see Table 2)

Table 2 Different cell lines used in study

For culturing cell lines, a combination of media and supplements is used for different cell lines (Table 3). The cell lines were obtained from the in-house cell bank. All cells were grown at 37 °C in a humidified condition, maintaining 5% CO2. For sub-culturing, the standard protocol was followed [38, 39], In brief, when cells were confluent enough (60-80%), the old media was removed and washed twice with PBS (Gibco™ 70011044). The attached cells were then treated with Trypsin-EDTA (0.05%) solution (Gibco™ 25300054), incubated for 3-5 minutes, and resuspended into respective media for the cell line. They then diluted 1:6 with new media and set it for the intended use

Supplements

Cell lines Media

Serum L-glutamine Antibiotics

B16-F10; RPMI 1640

Penicillin-

(Gibco™ Newborn Calf

GlutaMAX™ Streptomycin

CT-26 31870025) Serum 10%

1% v/v (10 000 U/mL)

Advanced v/v (Gibco™

(Gibco™ 1% v/v

RPMI 1640 16010159)

MDA-MB-231 35050061) (Gibco™

(Gibco™ 15140122) 12633012)

Table 3 Media supplements used for culturing different cell lines

MTT Assay

The anticancer screening was done on a 96-well flat-bottom plate. A confluent T75 flask of a cancer cell line was treated with trypsin for five minutes at 37°C and gently tapped on the plates for complete cell detachment. Detached cells were diluted and counted on the automated cell counter Countess™ (Catalog number: AMQAX2000; Life Technologies). We have seeded 5,000 live cells per well. The seeded cells were allowed to settle into the plates overnight at 37 °C and 5% CO2. The next day, the media was discarded and refilled with fresh media for the respective cell lines containing different drug concentrations, see Figure 14. For each drug concentration, we treated five wells as technical repeats. The treated plates were incubated for 72 hours at 37 °C and 5% CO2. After incubation, the drug concentrations were discarded, and cells in the wells were treated with 0.1 mg MTT (3- (4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) per well in 100 pL of respective media for the cell line. The plates were incubated for 4 hours at 37 °C and 5% CO2. The media containing MTT was removed carefully. Formazan, the reduced form of MTT converted by cellular mitochondrial reductase [39], was dissolved in 200 pL of DMSO and shaken for 5 minutes in a rotary shaker to dissolve completely. Finally, the absorbance of the plates was measured in a plate reader at 570 nm.

Calculation of inhibitory concentrations (IC50)

The raw absorbance data of the MTT assay obtained from the plate reader was processed in the Excel desktop app of the Microsoft 365 package. Firstly, the mean value of absorbance data for the media blank repeats was determined and considered 100% cell viability. Based on this value, we calculated the mean % of cell viability for each concentration of the tested drug and the standard deviation (SD). The computed mean and SD values were then plotted in GraphPad Prism 9.5.1. The IC50 value for each tested compound was calculated by transforming the data into log concentrations, followed by the nonlinear regression model: log(inhibitor) vs. response (three parameters)

Calculation of the mean IC50

The IC50 values calculated for each sample from at least two independent experiments involving 05 repeats were plotted in GraphPad Prism to generate a bar chart showing each compound's comparative anticancer activity.

Summary of the mean IC50

The anticancer activity observed for a range of PDB compounds with the PBD Core Structure (shown below) against a panel of three solid tumour cell lines (CT26, B16- F10 and MDA-MD-231) is summarized in Table 4. These results include for comparison the prior art compound MH01 [26], and reference compounds MH21 and MH31. PBD Core Structure

Table 4 provides a summary of the anticancer activity observed for a range of compounds with the PBD Core Structure against a panel of three solid tumour cell lines. The anticancer activity of four PBDs (MH02, MH03, MH04 & MH05) comprising an end phenyl ring with a halogen substituent are shown graphically in Figure 2. The activity of MH01 & GWL-78 that are both prior art [26] PBD compounds and also of a non-PBD reference compound Doxorubicin (Dox) are also shown in Figure 2. As shown in Figure 3, the fluorinated PBD compound MH22 demonstrates good anticancer activity across the tested panel of cell lines whereas the related nonhalogenated PBD MH21 is significantly less active. The strong anticancer activity of three PBD compounds comprising a halogenated thiazole group are shown in Figure 3 which contrast strongly to the much poorer activity of the related PBD comprising a non-halogenated thiazole. A series of compounds, fluorinated at the 7-position of the PBD core, were also prepared and tested against the solid tumour cell lines (MDA-MD-231) and these results are summarized in Table 5. core structure fluorinated at the 7-position Table 5 summarizes the anticancer activity of a range of compounds, fluorinated at the

7-position of the PBD, against the MDA-MB-231 solid tumour cell line.

The tested compounds, fluorinated at the 7-position of the PBD, all demonstrated good anticancer activity across against the MDA-MB-231 solid tumour cell line. The anticancer activity with selected compounds were carried out against the triple negative breast cancer cell line MDA-MB-231 and against the breast adenocarcinoma cell line M DA- MB- 361 (ATCC HTB-27™; ECACC 92029423). These selected componds included two of the payload-linker compounds were also tested against these cell lines (see Table 6).

Table 6 summarizes the anticancer activity of selected compounds against the MDA- MB-231 & MDA-MB-361 cell lines.

Transcription Factor NF-KB Family Assay

To understand the mechanism of action of the synthesised compounds at the molecular level, we have conducted the transcription factor NFKB family assay using the TransAM ELISA kit developed by Active Motif, Inc. (Catalog No. 43296). The kit is very convenient for monitoring the activation or deactivation of NFKB in cells, tissues or animal samples, see US Patent No. 6,150,090, [40],

A summary of workflows for the TransAM ELISA assay is outlined below (see Figure 15). The procedure is divided into two parts: /) getting nuclear extracts of treated and untreated cells and //) developing the NFKB family assay using the kit purchased from Active Motif, Inc.

Nuclear extraction protocol

MDA-MB-231 cells were detached from the T75 flask by treating 1 mL of Trypsin-EDTA (0.05%; Gibco™ 25300054) for 5 minutes at 37 °C. The detached cells were diluted in fresh media mentioned above for the cell line and counted using the automated cell counter Countess® II. The suspended cells were seeded in a 6-well plate with 2x10^A6 cells per well in 2 mL media. Furthermore, it incubated at 37 °C and 5% CC>2for 24 h. After 24 hours, the cell confluency was checked, and the next steps would be if it reached between 80% and 90%. The media was discarded and washed with 2 mL of 1xPBS thrice. The wells were finally treated with the desired concentrations of drugs (10 nM and 100 nM) and vehicle (0.01% DMSO). All drug concentrations were made using a serial dilution of the 10 mM drug dissolved in DMSO using a vehicle. All treated plates were then incubated for 4 hours using the same conditions.

Nuclear extracts were prepared using the Nuclear Extract Kit from Signosis Inc., USA (Cat no. SK-0001). The working solutions of Buffer I and Buffer II were freshly prepared following the manual from Signosis Inc. After incubation, the media from the wells of 6 well-plates was discarded and washed with 1xPBS thrice and 250 pL of Buffer I of the nuclear extraction kit. The plate was kept on ice and shaken on a rocking platform for 10 minutes. Then, the cells from each were released from the wells using Fisherbrand™ Cell Scrapers and transferred into labelled centrifuge tubes. The tubes were centrifuged at 12000 rpm for 05 minutes at 04 °C using Eppendorf™ Centrifuge 5804. The supernatants were discarded entirely, and 50 pL Buffer II working was added to each tube. Tubes were then tapped gently so that the pellets were floating in Buffer II. All tubes were kept on ice and rocked for 2 hours in the shaking platform — finally, centrifuge at 12000 rpm for 05 minutes at 04 °C. The supernatant was collected into new microcentrifuge tubes as 10 pL aliquotes, which were used for total protein quantification and NF-KB family ELISA assay.

Quantification of nuclear extracts

The total protein in obtained nuclear extracts was determined using a colourimetric protein assay kit purchased from Bio-Rad (Cat no. 5000002). We have used the microtiter plate protocol for determining total protein as it is suitable for determining the concentrations between 1 to 20 pg of protein from 10 pL of samples in an assay done in a 96-well plate.

The dye reagent with the kit was diluted by adding 4 parts of distilled de-ionised (DDI) water. The diluted dye reagent was then filtered through the Whatman #1 filter. Six bovine serum albumin (BSA) standard dilutions were prepared using the following Table 7.

Dil. Desired cone. Stock (pL) DDI Water (pL)

1 0.8 mg/mL 53.3 of 1.5 mg/mL 46.7

2 0.4 mg/mL 50 pL of dil l 50

3 0.2 mg/mL 50 pL of dil 2 50 4 0.1 mg/mL 50 pL of dil 3 50

5 0.05 mg/mL 50 pL of dil 4 50

6 0.025 mg/mL 50 pL of dil 5 50

Table 7 - Concentrations used to prepare BSA standard dilutions.

Once the dye working solution and BSA were ready, we pipet 10 pL of each standard dilution into three different wells in a 96-well plate. Sample solutions of nuclear extract were pipetted to the same plate into designated wells as per the pre-designed layout. Then, the 200 pL dye solution was added to each well using a multichannel pipette, mixed well by pipetting and incubated for 05 minutes at room temperature. The absorbances were measured at 595 nm using a microplate reader. The sample protein concentrations were interpolated by plotting the absorbance data of the BSA standards into GraphPad Prism 9.5.1 to get the standard curve (Figure 16); the nonlinear regression model was used.

NF-KB family ELISA assay

An ELISA assay developed by Active Motif Inc. was utilized. TransAM NF-KB Kits have a plate with 96 wells, and each well has a small piece of DNA attached to it. This DNA piece has the same sequence (5'-GGGACTTTCC-3') as the part of the gene that NFKB can bind to. The active form of NF-KB subunits in the nuclear extract specifically binds to this immobilised oligonucleotide. Once binding is done, the primary antibodies were used to identify the epitope on the p65, p52 and p50 subunits of the NF-KB family, accessible only when the subunit is bound to oligonucleotide and activated. Finally, an HRP-conjugated secondary antibody can bind to the primary antibody and provide a sensitive colourimetric signal in spectrophotometry. The step-by-step procedure is provided below.

Preparing the nuclear extracts

Previously quantified nuclear extracts were diluted using the “Complete Lysis Buffer” of the kit. In this study, we have used 02 pg of nuclear extracts for individual samples per well of the assay plate coated with the oligonucleotide specific to NF-KB. The experiment was carried out in triplicates. In the case of the positive control, we used Raji nuclear extract, which was included in the kit. “Complete Lysis Buffer” was used as negative control or blanks.

NF-KB binding to the consensus DNA sequence Once we had prepared the nuclear extracts for each sample, positive and negative controls, the oligo-containing wells were filled with 30 pL of “Complete Binding Buffer” and followed by each sample, 20 pL, in “Complete Lysis Buffer” (Table 8). The wells were sealed with the adhesive cover and incubated for one hour at room temperature with mild agitation at 100 rpm on a rocking platform (IKA™ MTS 2/4). After the incubation period, the wells were washed with 200 pL 1X wash buffer included with the kit. For each wash, the plate was flicked over a sink to empty the wells, followed by tapping the inverted plates on absorbent paper towels. The process was repeated thrice.

Table 8 - A layout of plate design to carry out the nuclear binding assay of NF-KB subunits.

Binding primary antibody

The working solution of the primary antibody for the tested NF-KB subunit was added, 100 pL, to each well, including blank wells. Covered the plate with the adhesive cover and incubated for 1 hour at room temperature without further shaking. After the incubation period, the wells were washed thrice, as described in the previous step.

Binding the secondary antibody

The working solution of 100 pL of the HRP-conjugated secondary antibody was added to each well, including blank wells. Covered the plate with the adhesive cover and incubated for 1 hour at room temperature without further shaking. After the incubation period, the wells were washed four times, as described in the previous step.

Colourimetric reaction and absorbance measurements

Finally, we added 100 pL of developing solution to each well. After 5 minutes of incubation, we checked the colour change of the positive control, and if it reached blue, then added 100 pL stop solution to each well. Due to the acidic nature of the solution, it turned the bluish colour of the well into yellow. For p65, we incubated for 10 minutes before adding the stop solution; otherwise, the rest of the NF-KB subunit's 5 minutes of developing time was okay. Once the stop solution was added to the assay plate, we measured the absorbance in a 96-well plate reader at 450 and 655 nm (used as a reference wavelength).

Data analysis and statistics

The raw data from the plate reader was analysed primarily in the Microsoft Excel desktop application. We subtracted the “blank well” absorbance to get the actual absorbance for each sample well. These values were then converted to percentages by taking the absorbance of the untreated well as 100%. Finally, the percentage data were utilised in GraphPad Prism to visualise the relative reduction of each subunit at different treatment concentrations — the 2-way ANOVA test for the multiple comparisons to assess the level of significance, if any.

Summary of NF-KB family assay

In summary, all three tested compounds, including the classical PBD monomer GWL- 78, have shown that they can similarly inhibit the canonical (p50 and p65) (see Figure 5A) and non-canonical (p52) subunits (see Figure 6A) of the NF-KB transcription factor family. A higher degree of nuclear inhibition of NF-KB subunits was observed with the treatment of high concentrations for all compounds. But out of these three, MH22, the 08-linkable PBD monomer, exhibited superior inhibition in all three NF-KB subunits, especially against p50, even with the lowest concentration (69.85% inhibition at 10 nM). This superior inhibition indicated that the amine group in the MH22 contributed to the efficient binding with the highly conserved NF-KB recognising DNA sequences.

For both single heterocycles containing PBDmonomers, 5-membered thiazol-containing short-PBD MH32 and 6-membered pyridine-containing short-PBD MH44, we found that they can inhibit the canonical (p50 and p65) (see Figure 5B) and non-canonical (p52) (see Figure 6B) subunits of NF-KB signalling pathways. Both compounds showed statistically significant inhibition for p50 subunits for canonical pathways (87.67% and 90.33%, respectively) at 100 nM concentration, which aligned with the reference PBD monomer GWL-78 (73.77%). Moreover, both compounds can inhibit the p65 subunit of NF-KB, but we did not find them statistically significant due to high variability in repeated experiments. But both the compounds did well in inhibiting the non-canonical subunit p52; even at 10 nM concentration, they can significantly inhibit the DNA binding of p52 (44%, 33.29% and 30.28% for MH32, MH44 and GWL-78, respectively).

In summary, the fluorinated benzothiazole-containing PBD monomer MH64 can significantly reduce the nuclear binding of non-canonical NF-KB subunit p52 (see Figure 60), even at a lower concentration of 10 nM (48.9%, p=0.003). But it cannot do the same for p65 (see Figure 50). In the case of p50 (see Figure 50), it can only inhibit its binding significantly at the higher concentration of 100 nM (91.67%, p=0.0028).

Comparatively, MH64 was found to inhibit slightly differently than the classic PBD monomer GWL-78 — further comprehensive transcription factor screening assay with this compound is required to reach any conclusion.

Toxicity in Galleria mellonella model

To test the toxicity of synthesised compounds, we used the Galleria mellonella model in this study. The Galleria mellonella was obtained from the UK Health and Security Agency and followed the protocol described below.

Materials

The following materials were used in this analysis.

• Multi-channel pipette 300 pL

• Pipettes and tips

• Falcon tubes (15 mL and 50 mL)

• Filter sterilised PBS/Water

• Hamilton syringe (25 pL)

• One-time use, sterile Petri dishes (90 mm)

• Galleria mellonella

• Drug concentrations

Experimental procedure

Handling of the Galleria mellonella

The G. mellonella boxes were checked that they were filled with sufficient sawdust. Each lot was used within two weeks upon receipt. The plastic boxes of G. mellonella were kept at 04 °C. Before starting the toxicity test, the boxes were removed from the refrigerator and were incubated at room temperature for at least one hour before the experiments commenced. Within hours, G. mellonella started moving freely. Darkened and non-moving G. mellonella were not considered for the studies. Those unsuitable for studies and the dead G. mellonella were removed from the box and disposed of. For disposal of dead G. mellonella, whether during the research or regular checking, the dead G. mellonella were sealed into a clean petri dish. Afterwards, incubate the Petri dish in the -20 °C freezer overnight. Finally, the plates were discarded in clinical west bins.

Preparation of test drug compounds

Before each experiment, ten healthy (motile and not darkened) G. mellonella were weighed in a clean petri dish to find the average weight. The average weight observed during this study for different drug experiments ranged between 260 mg and 310 mg. Based on the weight measurement of G. mellonella, we have determined the quantity required to prepare 1 mL of the desired dose of the highest concentration (50 mg/kg), weighed and dissolved it in the vehicle solution (10% DMSO in PBS). Finally, the remaining concentrations (1 mg/kg to 25 mg/kg) were made using the vehicle solution and vortexed to create a homogenous solution/suspension.

Injection protocol

An injection station was prepared for G. mellonella by securely taping a piece of a white roll to the bench. Securely tape a 1000 pL pipette tip in the centre of the white roll. The infection stage was equipped with pipettes, tips, falcon tubes containing 70% ethanol, selected healthy G. mellonella in Petri dishes, a Hamilton syringe with a needle, and boxes containing drug concentrations. Before injection, the syringe was washed with the 70% ethanol solution three times, followed by rinsing with PBS 3 times to remove the trace amount of ethanol. 10 pL of the desired drug or vehicle solution was withdrawn with the syringe and parked in the needle holder.

Once the solution was injected, one G. mellonella was removed from a previously prepared Petri dish containing a set of 10. The G. mellonella was bent over the fixed pipette tip on the stage so that its prolegs were facing upwards. We used tweezers to hold the G. mellonella firmly in place. Finally, the Hamilton syringe injected 10 pL of the desired drug into one of the opened prolegs. Once the needle was inserted, the G. mellonella was released whilst the solution was slowly expelled from the syringe into the G. mellonella. This minimises the percentage of solution lost by blebbing.

Afterwards, the plunger was gradually depressed to prevent blebbing and discomfort to the G. mellonella. The inoculated G. mellonella was placed into a clean, appropriately labelled petri dish with sufficient sawdust. The same protocol was repeated until the inoculation of 10 was finished.

The inoculated G. mellonella were observed 24, 48, 72, 96 and 120 hours postinjection to record whether they were alive or dead. The state of G. mellonella was tested by a light touch with a pipette tip. If the G. mellonella responded and moved in any way, it was recorded as alive, but if not, it was recorded as dead. The data recorded for individual G. mellonella was used to prepare the survival curve using GraphPad Prism 9.0.

Cytotoxicity results in Galleria mellonella

In this study, five different drug doses (1 mg/kg — 50 mg/kg) were injected and the vehicle (10% DMSO in PBS). The observational data of how many injected larvae survived was taken at one-day intervals, as described in the procedure detailed above.

The cytotoxicity profile of our synthesised drugs was compared with the reference PBD monomer GWL-78, which contains a bis-pyrrole sidechain connected through the 08 position to the PBD core. The data of cytotoxicity study of the reference compound showed that the reference compound GWL-78 at concentrations above 1 mg/kg dose was responsible for killing most of the larvae. For 50 mg/kg and 25 mg/kg, we have seen 60% and 70% death of the G. mellonella larvae after day 02, respectively. 100% of larvae with these two concentrations were found dead on day 03. At the same time, we saw 80% deaths for the 5 mg/kg and 10 mg/kg on day 03, which ultimately ended up at 90% after day 05 (see Figure 7).

Compared with GWL-78, both MH10 and MH22 look to have a safe drug profile (Figure 7).

Analogous experiments were carried out on MH64 a PBD which comprises a fluorinated benzothiazole side chain. Again, compared with GWL-78, MH64 look to have a safe drug profile (Figure 8).

Solution Properties

The aqueous solubility of two selected compounds, MH10 and MH22, was investigated at a pH of 7.4 to simulate the in vivo environment. Phosphate-buffered saline (PBS) was used as the aqueous solvent to achieve this target pH. The aqueous solubility of each test compound was determined by comparing the peak area of the principal peak in a calibration standard (200 pM) containing methanol/water (60/40, v/v) to the peak area of the corresponding peak in a buffer sample.

The solubility data shows compounds MH10 and MH22 were soluble in PBS at pH 7.4 at 184.41 pM and 193.15 pM, respectively suggesting very high solubility which is unusual for PBD type compounds (see Figure 9). While the solubility of the reference FDA-approved drugs ranged between 39.01 pM and 177.83 pM. The solubility at higher concentrations indicated that both MH 10 and MH22 are suitable for oral drug absorption, dissolution, and systemic delivery, and can be easily conjugated with an antibody as aggregation, which limits conjugation, is unlikely [41 , 42],

In vitro absorption

The human colon adenocarcinoma cell line (Caco-2) is a well-established intestinal epithelial cell model for drug permeability assays. Candidate compounds' in vitro absorption performance was evaluated using the Caco-2 permeability model at pH 6.5 and 7.4. The incubation time for A-B and B-A permeability tests was 0 and 60 minutes and 0 and 40 minutes at 37 °C, respectively. Test concentrations for all cased was 10 pM. Concentrations were measured as the peak area of the LCMS spectra. The reference compounds, colchicine, labetalol, propranolol, and ranitidine, were used as positive controls, as they are all orally available and have a bioavailability of over 25%.

The data on permeability showed that our compounds' permeability was similar (4.63 and 7.78 x 10^A-6 cm/s for MH 10 and MH22, respectively) to the reference drug labetalol (5.44 x 10^A-6 cm/s) and about half of the propranolol (12.67 x 10^A-6 cm/s). Labetalol is considered a highly permeable drug according to the Eurofins model. However, our test compounds had superior permeability than that we found for colchicine (0.423 x 10^A-6 cm/s) and ranitidine (0.33 x 10^A-6 cm/s) (Figure 10). The oral bioavailability of the three drugs, propranolol, colchicine and ranitidine, to which our testing compounds are superior or similar, was found in the literature at 50%, 45% and 50%, respectively [43,44, 45], Hence, both MH10 and MH22 can be considered to have a high permeability level and are expected to have good in-vivo absorption.

The comparison of the apparent permeability (Papp) between A-B and B-A showed a pattern in the case of the reference drugs. The Papp was about twice as high in B-A for all the reference drugs compared to A-B, but the opposite was the case for MH22, and the B-A and A-B permeability was very similar for MH10 (Figure 10). This data suggests that the compounds are not substrates of P-gp efflux pumps. Substrates of P- gp efflux pumps show a high B-A permeability due to the active efflux of compounds from the cell. We assume the presence of primary amine made the MH22 unsuitable to pass through membrane protein hydrophobic channels.

Distribution - protein binding Plasma protein binding assays were conducted to assess the binding characteristics of the two short-PBD analogues (MH10 and MH22) and four reference compounds, namely acebutolol, quinidine, sertraline and warfarin. Acebutolol represented a cardio- selective beta-1 blocker, quinidine was a class I antiarrhythmic agent, sertraline was a selective serotonin reuptake inhibitor, and warfarin was an anticoagulant for comparison — the methodology employed equilibrium dialysis. The assay involved partitioning the test compounds between a protein-containing compartment (plasma compartment) and a protein-free compartment (buffer compartment) through a semi- permeable membrane that allowed only small molecules to permeate. Subsequently, the concentration of the test compound was determined by measuring peak areas in HPLC spectra at equilibrium, enabling the calculation of the percentage binding using the following formulas.

Protein binding (%) = • 100 Where, Ap= Peak area of analvte in the protein

Recovery (%)=^±^ < 100 matrix

Ab= Peak area of analvte in buffer

Ac= Peak area of analvte in the control sample

The data we received for the protein binding assay demonstrated that our reference drugs belonged to low to high plasma protein binders, with acebutolol having the lowest binding of 23.1% and sertraline as the highest protein binding drug with 98.6%. Interestingly, our C8-linkable short-PBD monomer, MH22, belongs to the intermediate plasma protein binders with a binding percentage 48.60. In comparison, its counterpart (MH10) fell into the high plasma protein binder with a binding percentage 90.50 (Figure 11). A high fraction bound value (>90%) indicates the extensive binding to plasma proteins. The extent of binding will impact the distribution of the compound. Higher binding will retain more compounds in the plasma, leading to a small distribution volume. As a result, a reduced amount of compound will be left to act on the target protein, and hence, the compound's therapeutic effects will also be reduced. In addition, the highly binding compound could also cause limited metabolism and, as a result, lead to a reduced compound clearance and prolonged half-life.

In vitro metabolism - intrinsic clearance

The intrinsic clearance of two PBD compounds, MH10 and MH22, was determined in human liver microsomes (0.1 mg/mL) at 37 °C. The test compounds were incubated individually with the liver microsomes, and sample aliquots were removed at different time points ( 0, 15, 20, 45 and 60 minutes). The remaining compounds were detected and quantified via HPLC-MS/MS.

The half-life (T 1/2) was estimated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) vs. time, assuming the first-order kinetics. The apparent intrinsic clearance (CLint, in pL/min/mg) was calculated according to the following formula:

,-,_T 0.693

The data of in vitro metabolism in human liver microsomes at different time points for two PBD compounds showed that both drugs are metabolically stable. After an hour, MH10 remained at more than 80%, while MH22 remained at more than 93% (Figure 12). The data indicated that these compounds are quite resistant to liver metabolism even after an hour of exposure. This can be considered a significant advantage for an ADC payload as ADCs typically have a long half-life.

As it was observed that more than 80% of MH10 and MH22 were recovered after one hour of exposure to the liver microsomes, a high half-life was expected. The data showed that the half-life of the two drugs was determined to be 266.35 minutes and 686 minutes, respectively (Figure 13). The half-life of propranolol, 233.75 minutes, was about one-third of MH22. But the value was similar to that of MH10. The other three reference drugs were found to have comparatively shorter half-lives than their PBD counterparts (Figure 13).

Compound Test concentration CLint (pL/min/mg)

MH10 100 pM <57.8 MH22 100 pM <57.8 Imipramine 100 pM <57.8 Propranolol 100 pM <57.8 Terfenadine 100 pM 570.80

Verapamil 100 pM 216.80

Table 9 Intrinsic clearance of two PBD compounds and four reference compounds

The longer the half-life, the slower the clarity of the drug. A similar correlation was observed when calculating the intrinsic clearance of the two testing and four reference drugs. Two of our PBD and two reference drugs with long half-lives had an intrinsic clearance of less than 57.8 pL/min/mg, and this means that for every minute, a volume of 57.8 pL of the drug is cleared from 1 mg of liver microsome tissue, see Table 9. At the same time, terfenadine and verapamil showed a higher rate of intrinsic clearance, 570.80 and 216.80 pL/min/mg. In summary, our testing compounds are less likely to degrade by the liver enzymes and, therefore, can remain inside for an extended period to exert their biological activity. This can reduce the dose frequency and the expected pharmacokinetic properties of anticancer drugs.

In vitro toxicity - hERG interactions

The blockage of hERG (human Ether-a-go-go-Related Gene) potassium channels is a frequent off-target biological activity that can lead to undesirable results in drug development.

To measure hERG current amplitude, a whole-cell patch clamp assay was employed. This assay measures the activity of hERG channels in individual cells. The protocol involves first holding the cell at a negative potential to close the hERG channels. A 500-ms pulse to -40 mV is then delivered to measure the leak current, subtracted from the hERG current online. The cell is then depolarized to +40 mV for 500 ms, followed by a 100-ms ramp to -80 mV. This protocol is repeated every 8 s to monitor the current amplitude.

The hERG inhibition data of two tested compounds were satisfactory compared to the reference compound, verapamil. At the test concentration, the mean inhibition of the channel when treated with MH10 was found to be 18.28%, while for MH22, it was 19.28%. In contrast, the vehicle, 0.33% DMSO, had an average inhibition of 15.72%, indicating our drug components' good cardiac safety profile, see Table 10.

Compound Concentration % inhibition ID (pM) n1 n2 mean

MH10 10 20.55 15.99 18.27

MH22 10 19.25 19.32 19.28

Vehicle - 15.66 15.77 15.72

(0.33%

DMSO)

Table 10 - The hERG channel inhibition of selected PBD compounds

It is worth mentioning that the test was carried out at a concentration of 10 pM, while the mean IC50 of the two compounds was 1.6 nM (>6000 times less) and 15.66 nM (>600 times less) for MH10 and MH22, respectively. In contrast, the reference hERG antagonist verapamil, a non-dihydropyridine calcium channel blocker that can block hERG channels at high concentrations, showed that it can block the hERG channel by 90% at 03 pM. The IC50 of verapamil against the hERG channel was determined to be 0.326 pM, see Figure 17. This result suggests that the tested compounds are not hERG inhibitors.

DNA melting assay

A chromophore-based FRET (Fluorescence Resonance Energy) DNA melting assay was utilised to screen PBD compounds with better anticancer activity. FRET is an energy transfer mechanism between two light-sensitive molecules (chromophores). The assay utilised the change in fluorescence as DNA molecules denature over time with increasing temperature from 30 °C to 94 °C on a thermal cycler.

The principle of the assay is that a donor chromophore (FAM) and an acceptor chromophore (TAMRA) are attached to a hairpin DNA strand. When the strand is cooled, the DNA gets hybridised; therefore, the chromophores are close enough to undergo FRET and emit a specific fluorescence signal. When the DNA samples are submitted to the thermal cycler and temperature increases at a rate, the chromophores are separated when the DNA starts to denature by heating. FRET is disrupted, resulting in a different fluorescence signal. By measuring the changes in fluorescence as a function of temperature, we determined the melting temperature of the DNA (Tm), which reflects its stability and specificity (Figure 18). In a subsequent experiment, we incubated the same DNA with PBD compounds and did the same to see the changes in the Tm to obtain the ATm. (46).

Annealing of DNA

Two customed DNA hairpin oligonucleotides were designed and used in this study. One is AT-rich (FAM-TAT-AAG-ATA-TAT-ATA-TTT-TTT-TAT-ATA-TAT-CTT-ATA- TAMRA) and the other one is GC rich (FAM-GCT-AGC-TAG-CTA-TTT-TTT-TAG-CTA- GCT-AGC-TAMRA). All the oligos with chromophores were purchased from Kaneka Eurogentec S.A., Belgium. A stock solution 20 pM was prepared from the received oligos by diluting with nuclease-free water (Cat no. PD092; Omega Bio-tek, Inc). The stock solution was diluted further with FRET buffer (50 mM potassium cacodylate, pH 7.4) to get a working solution of 400 nM. Once prepared, the required quantity was taken for each experimental set-up into microcentrifuge tubes and incubated them into a benchtop heat block (Grant-bio; cat no. PCH-2) for 5 minutes at 85 °C. The DNA samples were then cooled at room temperature for 3 hours to allow complete annealing and formation of hairpins before incubation with synthesised drugs. Drug sample preparation

Each synthesised PBD compound was dissolved in DMSO to prepare a 10 mM stock solution. From the stock, we have prepared dilutions of 20 pM, 10 pM, 05 pM and 2.5 pM sample solutions using FRET buffer before incubating with the annealed DNA.

Incubation of DNA with drugs

The annealed DNA and the diluted drug, 25 pL each, were taken into a 96-Well PCR plate (Multiplate™, low profile, unskirted, clear; cat no. MLL9601; Bio-Rad Laboratories, Inc.) (Table 11). The final volume of the reaction mixture in each well was 50 25 pL, and the concentration of DNA and drug were 200 nM and 1.25 - 10 pM, respectively. The 96-well plate was then incubated at room temperature for 24 hours before being in a thermal cycler machine for the melting experiment.

A B C D E F G H

Drug 1 Drug 2 Drug 3 Drug 4 Drug 5 Drug 6 Drug 7 Table 11 - A typical 96-well plate design used in this study. The first six wells of column A were used as a reference for calculating the Tm of each compound.

DNA melting

After incubation, the 96-well plate was subjected to the thermal cycler system (Opticon 2; Bio-Rad Laboratories, Inc.). The program was customised to take fluorescence readings at intervals of 0.5 °C over the range between 30 and 94 °C. Moreover, each temperature point was maintained for 30 seconds before each reading. The incident radiation was set to 450-495 nm with 515-545 nm detection wavelengths.

Data analysis

The fluorescence data was visualised using the default software for the PCR system, Opticon Monitor. The raw data were exported as comma-separated values (.csv) and analysed in Origin Pro by using a custom script written to find out the melting temperature, Tm, for each well by determining the maximum rate of changing the fluorescence with an increase of the same unit of temperature (ARFU/AT) (46, 47). Once the raw data was fed into the script, we got the Tm for each well, including the reference DNA wells (Table 11). The Tm values were then used to calculate the mean and standard deviation to determine the ATm (see Table 12 for the values for various compounds). The raw melting curves were generated using the data exported from the thermal cycler machine using GraphPad software.

Table 12 - The ATm of a range of PBDs after 24 h of incubation with AT- and GC-rich DNA hairpin structure ^a Incubation time with annealed DNA was 24 h. ^b The mean anticancer activity of each compound against three cell lines (CT26, B16- F10 and MDA-MB-231) ^c Reference compound

Generally, a trend of having relatively higher ATm for GC-rich DNAs over AT-rich counterparts was observed.

The halogen containing thiazole derivatives (MH32, MH33, MH34), halogen containing pyridine derivative (MH44) and halogen containing benzothiazole derivatives (MH63, MH64) were found to bind more strongly to DNA, in particular, to GC-rich DNA, than the phenyl-containing compounds (MH02 to MH22). Whilst the heteroatoms play a role in DNA binding, the halogen containing thiazole derivatives also bound more strongly to DNA than an unsubstituted thiazole derivative (MH31).

In summary, these compounds have relatively low DNA binding which makes them less toxic against healthy cells which is further supported by the Galleria tox data. The relatively low DNA binding for these short PBDs as compared to PBDs with longer side chains at the 8-position is considered to be due to their smaller size, as this means these short PBDs have a limited number of interacting heteroatoms. However, they are highly toxic against cancer cells as the relatively low DNA binding gives them sufficient occupancy to inhibit transcription factors which is supported by their NFkB inhibition data. The presence of halogens as substituents of the phenyl or the heteroaryl ring also make them more metabolically stable.

Molecular Docking

The procedure employed for the molecular docking of short PBDs with reference DNA is described in the following section.

Preparation of the macromolecule (DNA)

The 3D structures of the desired B-form DNA sequence (5'- TATAGGGACAGCGCTATATATAGCGCTGTCCCTATA-3') (48) were generated using PyMOL 2.5 structure Builder (The PyMOL Molecular Graphics System, Schrodinger, LLC.). The DNA structures were processed (energy minimization and addition of polar hydrogens) using MGLTools v1.5.7 (https://autodock.scripps.edu/). The grid box was configured for each DNA macromolecule to cover the whole length of the structure so that the ligand could find the best possible binding sites along with the DNA structures, including both the major and minor grooves.

Preparation of ligands

3D structures were generated using the Chem3D 20.0 program for short-PBDs used in this study. The process of generating is to upload the ChemDraw file to the Chem3D program which will convert it to the 3D afterwords by default. The 3D models of each short-PBD were then subjected to energy minimization using the built-in command “MM2 minimize” and the minimized conformation was used for the docking experiments.

Molecular docking

The previously prepared ligands are further processed with the MGLTools v1.5.7 to generate the input files (pdbqt) for the open-source Docking software, AutoDock Vina v.1.2.0 (https://vina.scripps.edu/) (47). The default AutoDock docking parameters were kept for docking where the ligands were kept as flexible and the DNA as rigid. The parameters we used for our docking are tabulated below. After doing a few repeats we have chosen the number of output poses to default (a total of 9 poses with the best conformation and binding energy). x=0.070

Centre of the Grid Box y=0.306 z=28.478 x=36

Size of the Grid Box y=38 z=114

Exhaustiveness 100

Table 13 - Docking parameters used

Post-processing and analysis

The post-processing of the output files was curated using PyMOL 2.5. For the superposition of two different docked PBD structures, we have chosen the poses according to the following criteria which are in line with the previous report(i) poses fit within the same core region of the DNA minor groove (ii) the difference in binding energy < 5% (iii) if the binding energy difference was not fit with the ccriterion then an alternative position was chosen. Finally, the selected poses and their molecular interactions were visualized using the Discovery Studio Visualizer (BIOVIA, Dassault Systemes, Discovery Studio, v20.1, San Diego: Dassault Systemes, 2022).

DNA minor groove fitting of MH compounds

Molecular docking analysis was used to demonstrate the role of the C8 substituents on the DNA minor groove fitting and how that possibly influences their anticancer activities. A comparison of MH01 with MH02 (the role of fluorine in activity); MH02, MH05 with MH06 (positional role of fluorine); and MH10 with MH22 (methyl vs amine while fluorine kept at the para position).

Role of halogen in DNA binding

To see whether halogen helps PBDs fit better within the DNA minor groove, MH01 and MH02 (fluorine-containing analogue) were docked against the same piece of DNA molecule used in the previous study (48) on single unit sidechain containing PBD monomers in 2019. From the docking experiment, it was found that fluorine at the para position of MH02 can contribute to additional van der Waals interaction with the cytosine (C32) while MH01 cannot. (Figure 19). The additional means of interaction of MH02 through the fluorine at the para position might help the compound to fit better within the DNA minor groove and might contribute to its anticancer activity compared to MH01 (IC₅₀ of 0.60 nM vs 298.45 nM).

Positional role of fluorine in DNA binding

Biological and biophysical data and docking experiments showed that the fluorine at position four or para position, as in MH02, of the phenyl side chain of the PBD monomer plays a crucial role in the activity and DNA binding. Further docking data for two other analogues, one having fluorine at the meta position (MH05) and the other with the ortho position (MH06), have been used to elucidate the fit of these compounds into the DNA minor groove. The molecular docking of MH06 showed that the fluorine at the ortho position protrudes outward of the groove and does not contribute to any interaction, which means it is not helping the PBD to fit well inside the DNA minor groove (Figure 20C). The docking results of short-PBD containing fluorine at the meta position, MH05, showed that this compound could fit well within the groove but cannot create any additional halogen-mediated interaction with the Cytosine residue of DNA, as we have seen for the para position in MH02 (Figure 20A and B). These could be linked with the mean IC50 values (0.60 nM, 7.88 nM, 1190.38 nM against three cell lines for MH02, MH05, MH06, respectively). Hence, this suggests that substitution at the ortho position is unhelpful.

Influence of methyl vs. amine at the meta position with para-fluorine

The mean anticancer activity of primary amine-containing analogue, MH22, was similar to MH10 (1.60 nM vs 15.66 nM against three cell lines). To explore the comparative DNA minor groove fit of MH10 and MH22, the molecular docking of these two compounds was examined. The superimposed structure of these showed that the amine-containing analogue MH22 could fit more towards the centre of the axis of the DNA minor groove than its counterpart MH10 (Figure 21). Moreover, the fluorine at the para position of MH22 can form an additional van der Waals interaction with cytosine (C32) like MH02 (sole fluorine-containing analogue), while its counterpart MH10 cannot (Figure 21).

Keeping the chemical environment in mind, looking into the zoomed superimposed structures of sidechains of MH10 and MH22 in Figure 22, both having fluorine at the para position and its position within the hydrophobic region is crucial for DNA fitting and binding. As the MH10 contains a methyl group positioned towards the centre of the minor groove is a hydrophobic sidechain, accommodating both the methyl and fluorine within the hydrophobic region is less likely. Incorporating two hydrophobic or less polar moieties ultimately pushed the fluorine outside, and consequently, it failed to form the additional bond needed for more efficient DNA binding (Figure 22). In contrast, MH22 contains an amine group at the same meta position as MH10. However, as it is polar, it can penetrate deep inside the groove towards the polar centre and allows the fluorine at the para position to stick in the optimal hydrophobic region within the DNA minor groove, leading to forming the required additional bond with the Cytosine residue (Figure 22). The relative central position of F within the minor groove helps the moiety to create an additional bond with DNA (MH02; mean IC50 0.6 nM) or better fitting (MH10; mean IC50 1.6 nM). However, the relative central position of F within the minor groove is influenced by the adjacent substitutions.

We have observed that the addition of F can not only help to create an additional bond with the DNA base for the phenyl compounds but also heteroaromatic compounds (MH31 vs MH32; Figure 23) and benzo fused compounds (MH61 vs MH64; Figure 24). The additional halogen bonding with DNA bases for MH32 and MH64 may help these compounds to fit better with the DNA minor groove as compared to the nonhalogenated compounds (MH31 and MH61).

Efflux Resistance

Molecular Modelling

Molecular modelling was used to investigate the fit in the P-glycoprotein (P-gp) efflux pump inhibitor binding pocket compounds MH10, MH13 and for reference compound MH 37 and to determine residues within the hydrophobic binding pocket that interact with each compound.

In Vitro Experiment

MDA-MB-231 breast cancer cells were seeded onto 96-well plates at a concentration of 0.5 million cells/well in a final volume of 200 pL. After 30 mins of incubation with the P- gp pump inhibitor verapamil at a concentration of 10 pM, compounds were added at different concentrations ranging from 1nM to 10 pM for PBD derivatives with a serial dilution of 10-fold with a 2-fold serial dilution across the plate. After a 72-hour incubation period with the test compounds, both with and without the inhibitor, cell viabilities were assessed with MTT assay. These tests were carried out for compounds MH13 and MH10 and also for reference compound MH37. A further test was carried out for MH10, SL-226-19, and for reference compound DC-1-170. DC-1-170 Results

For both MH10 and MH13 the molecular modelling (see Figures 25A & 26A) determined that efficient interaction within the p-Gp efflux pump inhibitor binding pocket makes it less susceptible to efflux (key residues within the binding pocket are PHE336, ILE 340, ILE 306 and PHE 343 that form the hydrophobic pocket). For reference compounds MH37 the molecular modelling (see Figure 27A) determined that MH37 does not bind to the same pocket and cannot interact with the key amino acid residues and is therefore expected to be effluxed by p-GP.

For both compounds MH10 and MH13 the % cell viability measurements showed little change between the values observed with and without the P-gp pump inhibitor verapamil (see Figures 25B and 26B). For MH10 and MH 13 the experimental results with p-Gp inhibitor verapamil (10 pM) shows that these compounds do not get effluxed with almost identical IC50 observed in the presence and absence of verapamil.

In contrast, when the reference compound MH37 was tested the experimental data shows that the IC50 significantly changes in the presence of P-gp inhibitor verapamil as the % cell viability curve shifts markedly (see Figure 27B). This marked difference indicates that MH37 is normally subject to efflux and that addition of P-gp pump inhibitor verapamil suppresses this efflux and shows that more of the compound stays within the cell and as a result greater cytotoxicity is observed in the presence of verapamil.

Hence, these results show that the novel compounds comprising a halogen (MH13 and MH 10) disclosed herein are less susceptible to efflux than non-halogenated derivatives. These results validate the molecular modelling evidence that the novel compounds are less susceptible to efflux and also support the in vitro absorption results using the Caco-2 model for drug permeability assays.

In the further test comparing MH10, SL-226-19, and for reference compound DC-1-170 the observed cytotoxicity for the compounds alone and in combination with verapamil are provided in Table 14.

Table 14 cytotoxicity of compounds alone and in combination with Verapamil to assess efflux liability

A higher than 1 efflux ratio indicates the compounds are getting effluxed by P-gp as the addition of verapamil reduces efflux and improves activity (i.e., lower IC50). The new generation PBD payloads MH10 and SL-226-19, have 5-to- 10-fold better activity than comparable literature compound DC-1-170, and are efflux resistant. DC-1-170 has an efflux ratio of 3.03 which suggests it gets effluxed by P-gp. All publications mentioned in this specification are herein incorporated by reference. Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiment and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.

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Claims

1. A compound of formula (I): or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, wherein: the dotted lines indicates the optional presence of a double bond between C1 and C2, or C2 and C3;

Xi is O, S, NH, C(O), C(O)NH or C(O)-O;

L is C1-12 alkylene;

R1 is H, OH, halogen, CN, =CH2, =CH(CI-6 alkyl), C1-6 alkyl, or OC1-6 alkyl;

R2 is H, OH, halogen, CN, =CH2, =CH(CI-6 alkyl), C1-6 alkyl, or OC1-6 alkyl;

R3 is H, OH, halogen, CN, =CH2, =CH(CI-6 alkyl), C1-6 alkyl, or OC1-6 alkyl;

R4 is halogen, OH, OC1-6 alkyl, or OCH2Ph;

Rs is:

X₂ is N or CH;

X3 is N or CH;

Y1 is C-R11 or N;

Y2 is S or O;

Y₃ is S, N or O; X₄ is N or CH;

Y₄ is S or O; either (a) Rs is R’, C1-3 alkyl, OC1-3 alkyl, or NH2; R9 is R’; and R10 is H;

(b) Rs and R_w are independently R’; and R9 is C1-3 alkyl;

(c) Rs is R’; R9 is OC2-3 alkyl; and R_w is H; or

(d) one of Rs and R9 is R’ and the other is H; and R_w is H; one of R14 and R15 is independently R’ and the other of R14 and R15 is H, independently R’, C1-3 alkyl, OC1-3 alkyl, or NH2; each R11 , R13, R16, and Ris is, H, independently R’, C1-3 alkyl, OC1-3 alkyl, or NH2; each R12, and R17 is independently R’; either (1a) R19 is H; R20 is H, R’, C1-3 alkyl, OC1-3 alkyl, or NH2; R21 is R’; and R22 is H, R’, C1-3 alkyl, OC1-3 alkyl, or NH2; or

(1b) R19 is H, R’, C1-3 alkyl, OC1-3 alkyl, or NH2; R20 is R’; R21 is H, R’, C1-3 alkyl, OC1-3 alkyl, or NH2; and R22 is H; one of R23 and R24 is H and the other is H; and R25 is independently R’; each R’ is independently a halogen; z is 0 or 1 ; and either:

(i) Re and R7 together form a double bond; or

(ii) Re is H, SO3H, or nitrogen protecting groups; and R7 is H, OH or OC1-6 alkyl.

2. A compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, according to claim 1 , wherein Rs is:

3. A compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, according to claim 1 or 2, wherein Rs is:

4. A compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, according to claim 1 or 2, wherein Rs is

5. A compound of formula (I) according to one of the preceding claims, wherein the compound is: or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof.

6. A compound of formula (I) according to any one of the preceding claims, wherein the compound is: or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof.

7. A compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, according to any one of the preceding claims, wherein R4 is F, Cl, Br, OCH₃, or OCH2CH3.

8. A compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, according to any one of the preceding claims, wherein Rs is: Rs is H, C1-6 alkyl or NH2 and R9 is halogen.

9. A compound of formula (I) according to any one of the preceding claims, wherein the compound is:

io or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof.

10. A compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, according to any one of the preceding claims, wherein it is linked to a linker group.

11. A compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, according to any one of the preceding claims, wherein it is linked directly, or indirectly via a linker group, to a targeting moiety to form a targeting moiety- drug conjugate.

12. A pharmaceutical composition comprising a compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, according to any one of claims 1 to 11 , and one or more of a pharmaceutically acceptable carrier, diluent, excipient, or lipid nanoparticle.

13. A compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, of any one of claims 1 to 11, or a pharmaceutical composition according to claim 12, for use in the treatment of a proliferative and/or malignant disease.

14. A compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, or a pharmaceutical composition, for use in the treatment of a proliferative and/or malignant disease, according to claim 13, wherein the proliferative and/or malignant disease is selected from breast cancers, brain cancers, central nervous system cancers, carcinomas, gastrointestinal cancers, hormonal cancers, leukemias, liver cancers, lung cancers, respiratory cancers, lymphomas, sarcomas, fibrosarcomas, skin cancers, melanomas, urinary tract cancers, reproductive cancers, or miscellaneous other cancers.

15. A compound of formula (I) or a tautomer thereof, or a salt thereof, or a salt of a tautomer thereof, or a pharmaceutical composition, for use in the treatment of a proliferative and/or malignant disease, according to claim 13 or 14, wherein the compound is administered either simultaneously or sequentially with one or more other therapeutic agent.