WO2020152462A1 - Pyrrolobenzodiazepine derivatives as inhibitors of nf-kappa b for the treatment of proliferative diseases - Google Patents

Abstract

The invention relates to a compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof, wherein the dotted lines indicate the optional presence of a double bond between C1 and C2 or C2 and C3; and R1 and R2 are either (i) R1 and R2 together form a double bond; (ii) R1 is H and R2 is OH; or (iii) R1 is H and R2 is OC1-6 alkyl; Y is N-RB, S or O; Y1 is N or C-R8; q is o or 1; Het is where the carbonyl of the Het group is attached to the Y- & Y1-containing heterocyclic ring; Y2 is N-RB, S or O; Y3 is N or C-R10; Y4 is N-RB, S or O; Y5 is N or C-R10; Rx is H, RB, (CH2)m-ORB, halo, (CH2)m-NHRB and CO2RB; and each RB is independently selected from H, C1-6 alkyl and C1-6 haloalkyl. The invention also describes pharmaceutical compositions, and kits comprising compounds of formula (I) and their use for treating proliferative diseases such as multiple myeloma or chronic lymphocytic leukaemia and as NF-κΒ inhibitors.

Description

PYRROLOBENZODIAZEPINE DERIVATIVES AS INHIBITORS OF NF-KAPPA B FOR THE TREATMENT OF PROLIFERATIVE DISEASES

FIELD OF THE INVENTION

The invention relates to novel pyrrolo[2,i-c][i,4]benzodiazepines (PBDs) compounds and the use of such compounds to treat proliferative diseases, such as, multiple myeloma or chronic lymphocytic leukaemia and as NF kappa B (NF-KB) inhibitors. The invention also relates to pharmaceutical compositions, and kits comprising the compounds.

BACKGROUND

Traditional alkylating agents are still widely used for the treatment of a number of human cancers, however they are associated with severe side effects that include hair loss, gut toxicity and bone marrow suppression, which are caused by collateral damage to non- malignant cells (1). Although there is growing emphasis on the use of molecular targeted agents, DNA still remains a critical target in anti-cancer treatment, so a focus on increased selectivity towards malignant cells, in order to reduce side effects, is of particular interest and importance (2).

Pyrrolo[2,i-c][i,4]benzodiazepines (PBDs) are naturally occurring molecules produced by Streptomyces bacteria whose family members include anthramycin (the methyl ester is shown below) and tomaymycin (3, 4).

PBDs are a class of sequence-specific DNA minor groove binding agents that are selective for GC-rich sequences, which have been evaluated as potential chemotherapeutic agents in recent years (5). PBDs fit perfectly in the minor groove of DNA due to their chiral Ciia(S)- position which provides a right-handed longitudinal twist isohelical with double-stranded DNA. They are tricyclic in nature, and are comprised of fused 6-7-5-membered rings and can be identified as an anthranilate (A ring), a diazepine (B ring) and a pyrrolidine (C ring). They also possess an electrophilic N10-C11 imine moiety (or the carbinolamine or carbinolamine methyl ether equivalent, as shown below) that can form a covalent aminal linkage between their Cn-position and the nucleophilic C2-NH2 group of a guanine base. Carbinolamine Imine Carbinolamine alkyl ether

Interconvertible forms of the PBD Basher et ah, Biophysical Chemistry, Vol. 230. 2017, pages 53-61 discloses sequence selective binding of C8 conjugated PBDs. WO-A-2017/039752 discloses PBD monomers and methods of synthesising PBD monomers.

The most effective synthetic modifications to PBD cores have involved the conjugation of two DNA-interactive moieties via their C8-position to create PBD dimers capable of cross-linking duplex DNA which improves cytotoxicity. Similarly, other groups have focussed on adding large substituents to the C8-position of the PBD core to improve DNA-interaction and cytotoxicity. PBD based compounds are in clinical development as standalone agents (e.g., SJG-136, shown below). More recently, members of the PBD family have been developed as cytotoxic payloads for attachment to antibodies to form Antibody-Drug Conjugates (ADCs) (e.g., SGN-CD33A), and a number of these are currently undergoing clinical evaluation (6).

SJG-136 Previous research has shown that PBD monomers such as GWL-78 (shown below) inhibit the transcription factor NF-Y (7), whilst PBD monomers such as the DC-81-indole hybrid (8) and KMR-28-39 have been implicated in NF-KB inhibition (9).

- 1- n o e y r

IN4CPBD (when n =4) and IN6CPBD (when n = 6) Nuclear factor kappa B (NF-KB) denotes a family of homo- and heterodimeric transcription factors composed of five subunits: p05 (Rel A), p50, Rel B, P52 and c-Rel (to). These subunits exert their effects via the canonical or non-canonical signalling pathways (11). NF- KB is maintained in an inactive state in the cytoplasm but following IKB kinase (IKK) activation NF-KB is shuttled into the nucleus where it exerts its transcriptional effects (12). NF-KB regulates the transcription of genes that are essential for cell survival, proliferation, inflammation and invasion/metastasis. These processes are de-regulated in numerous cancers, including haematological malignancies, and are associated with the constitutive activation of NF-KB (11, 13). Indeed, NF-KB has been shown to play a role in disease progression and drug resistance in multiple myeloma and chronic lymphocytic leukaemia [CLL] (14, 15). Whilst treatment with currently established therapies, such as the proteasome inhibitor bortezomib, are initially effective in a significant proportion of patients (16), there is evidence to suggest that treatment with bortezomib causes an increase in canonical NF-KB activation which has been linked to drug resistance (17). Therefore, direct inhibition of NF- KB could potentially resensitise tumour cells, thus highlighting this transcription factor as a potential therapeutic target (18-20).

The present inventors identified a group of novel compounds (designated DC-1-192, DC-1-92 and DC-1-170 and shown below) from a libraiy screen of 87 novel synthetic C8-linked benzofused PBD monomeric hybrids based on their in vitro cytotoxicity.

in malignant and non-malignant cells and their mechanisms of action. In this regard, PBD monomers can recognise and bind to specific sequences of DNA and therefore have the potential to act as competitive inhibitors of transcription factors.

The lead novel C8-linked benzofused PBD monomeric hybrids and showed a degree of selectivity for NF-KB DNA consensus sequences. When tested in multiple myeloma cell lines and primary chronic lymphocytic leukaemia cells, these PBD compounds showed low nanomolar LD₅₀ values, with normal age-matched B- and T-cells 2.4-fold and 4.6-fold less susceptible to their cytotoxic effects respectively. In the multiple myeloma JJN3 cell line, the lead compound, DC-1-192, showed significant inhibition of p65, p50 and Rel B NF-KB DNA binding after just 4h exposure, demonstrating potent dual inhibitory properties on both the canonical and non-canonical NF-KB pathways. RNA-sequencing confirmed gene set enrichment for NF-KB pathway genes although as expected, other canonical pathways were also affected. In addition, DC-1-192 showed synergism when combined with bortezomib or ibrutinib in JJN3 cells and primary CLL cells respectively. Further, in vivo studies showed that such combinations were well tolerated in test subjects. Finally, in vivo efficacy studies in NOD/SCID mice, using a systemic RPMI 8226 human multiple myeloma xenograft model, showed that DC-1-192 significantly prolonged survival (median survival 68 days) and this was as effective as bortezomib (median survival 63 days). Taken together these data provide a strong rationale for the use of DC-1-192 and analogs in the treatment of NF-KB-driven haematological cancers both alone and in combination with existing drugs.

SUMMARY

In a first aspect, there is provided a compound of formula (I):

or salts, solvates, stereoisomers, tautomers or combinations thereof,

wherein the dotted lines indicate the optional presence of a double bond between Cl and C2 or C2 and C3;

R and R₂ are either

(i) R and R₂ together form a double bond;

(ii) R is H and R₂ is OH; or

(iii) R is H and R₂ is OC -₆ alkyl;

R₃, R₄ and R₅ are independently R_A, OR_A, halo, =0, =CH-R_A, (CH₂)_m-0R_A, (CH₂)_m-NHR , (CH₂)_m-C0₂R_A, (CH₂)_m-C(0)R_A, (CH₂)_m-S0₂R_A, 0-S0₂R or CN;

each m is independently o, 1, 2, 3, 4 or 5; where R_A is H; a C -₁₂ alkyl group; a C_6-12 aryl group; a C_5-i0 heteroaryl group; a C_7-is aralkyl group; or a C_6-16 heteroaralkyl group; whereof the alkyl, aralkyl, or heteroaralkyl group optionally contains one or more carbon-carbon double or triple bonds, which may form part of a conjugated system; and the alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group is optionally substituted by one or more independently selected halo, hydroxy, amino, or nitro groups;

R_¾ is H, C -₆ alkyl or CH₂Ph;

p is 1, 2 or 3;

Ry is RB, ORB or halo;

Y is N-R_B, S or O;

Y¹ is N or C-Rs;

Rs is RB, ORB or halo;

q is o or 1;

Het is

where the carbonyl of the Het group is attached to the Y- & Y’-containing heterocyclic ring; Rg is R_B, OR_B or halogen;

Y² is N-RB, S or O;

Y³ is N or C-R o;

Y4 is N-R_B, S or O;

Y⁵ is N or C-R o;

R o is RB, ORB or halogen;

R^x is H, R_b, (CH₂)_m-0R_B, halo, (CH₂)_m-NHR_B and C0₂R_B; and

each R is independently selected from H, C -₆ alkyl and C -₆ haloalkyl.

In a further aspect, there is provide a pharmaceutical composition comprising a compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof as described herein and a pharmaceutically acceptable carrier or diluent.

In a further aspect, there is provide a pharmaceutical composition comprising a compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof as described herein and a pharmaceutically acceptable carrier or diluent, and further comprising a therapeutic agent for treating a proliferative disease. In a further aspect, there is provide a kit comprising :

(a) a compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof as described herein; and

(b) a therapeutic agent for treating a proliferative disease.

In a further aspect, there is provided a compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof, or a pharmaceutical composition, or a kit as described herein, for use as a medicament.

In a further aspect, there is provided a compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof, or a pharmaceutical composition, or a kit as described herein, for use in the treatment of a proliferative disease wherein the proliferative disease is a haematological malignancy or a solid tumour.

In a further aspect, there is provided a use of a compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof, or a pharmaceutical composition, or a kit as described herein, for the manufacture of a medicament for the treatment of a proliferative disease.

In a further aspect, there is provided a method of treating a proliferative disease in a patient comprising administering a therapeutically effective amount of a compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof, or a pharmaceutical composition, or a kit as described herein,

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.

Where an apparatus feature is described as being operable to provide a function, it will be appreciated that this includes an apparatus feature which provides that function or which is adapted or configured to provide that function.

DEFINTIONS AND ABBREVIATIONS

ANOVA - Analysis of variance; Bid - Twice a day; BIW - Twice a week; BW - Body weight; BWL - Body weight loss; Cone. - Concentration; CR - Complete regression, which is defined as tumours that are reduced to below the limit of palpation (62.5 mm3) at the end of the treatment period; FFPE - Formalin Fixed Paraffin Embedded; ILS - Increase in life-span; i.p. - Intraperitoneal (ly); i.m. - Intramuscular(ly); i.v. - Intravenous(ly); MTD - Maximum tolerated dose; MTV - Mean tumour volume; N/A - Not available or not applicable; p.o. - Oral(ly); PR - Partial regression, which is defined as tumours that are reduced from initial tumour volume but still palpable (>62.5 mm3) at the end of the treatment period; Qd (or QD) - Once a day; Q2d (or Q2D) - Every other day; (¾d (or Q3D) - Every tree days (one day dosing and 2 days off; Q4d (or Q4D) - Every four days (one day dosing and 3 days off); QW (or q7d) - Once a week; RA - Reference article; RT - Room temperature; RTV - Relative tumour volume; s.c. - Subcutaneous(ly); SD - Standard deviation; SEM - Standard error of mean; SOC - Standard of care used in clinic setting for a specific disease; SPSS - Statistical Product and Service Solutions; TA - Test article; TBD - To be determined; TGD - Tumour growth delay; TGI - Tumour growth inhibition; Tid - Thrice a day; TV - Tumour volume; and TW - Tumour weight.

“C -₁₂ alkyl”: refers to straight chain and branched saturated hydrocarbon groups, generally having from 1 to 12 carbon atoms; more suitably C -₇ alkyl; more suitably C -₆ alkyl; more suitably C -₃ alkyl. Examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n- butyl, s-butyl, i-butyl, t-butyl, pent-i-yl, pent-2-yl, pent-3-yl, 3-methylbut-i-yl, 3-methylbut- 2-yl, 2-methylbut-2-yl, 2,2,2-trimethyleth-i-yl, n-hexyl, n-heptyl, and the like.

“Aryl”: refers to fully unsaturated monocyclic, bicyclic and polycyclic aromatic hydrocarbons having at least one aromatic ring. Suitably, an aryl group is a C_6-12 aryl and having a specified number of carbon atoms that comprise their ring members (e.g., 6 to 12 carbon atoms as their ring members). The aryl group may comprise fused rings, at least one of which is a fully unsaturated ring, for example indanyl and 5,6,7,8-tetrahydronaphthalenyl. The aiyl group may be attached to a parent group or to a substrate at any ring atom and may include one or more non-hydrogen substituents unless such attachment or substitution would violate valence requirements. Examples of aryl groups include phenyl, biphenyl, indanyl, indenyl, naphthalenyl, 5,6-dihydronaphthalenyl, 7,8-dihydronaphthalenyl and 5,6,7,8- tetrahydronaphthalenyl.

“C_7-I8 aralkyl and C_6-16 heteroaralkyl,” represent alkyl substitutents that are substituted with the named ring structure. For example, an aralkyl group comprises an alkyl group

substituted with an aryl group. Suitably, C_7-is aralkyl and C_6-16 heteroaralkyl groups comprise a C -₆ alkyl group substituted with a C_6-12 aryl, or a C_5-i0 heteroaryl group respectively.

Suitably, the alkyl group in C_7-i5 aralkyl or C_6-13 heteroaralkyl comprising a C -₃ alkyl; suitably, comprising a C or C₂ alkyl group; more suitably, comprising a C alkyl group. 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 be described using“consisting of’ or“consisting essentially of’ language.

“Drug”,“drug substance”,“active pharmaceutical ingredient”, and the like, refer to a compound (e.g., compounds of Formula (I) and compounds specifically named above) that maybe used for treating a subject in need of treatment.

“Excipient” refers to any substance that may influence the bioavailability of a drug, but is otherwise pharmacologically inactive.

“Halogen” or“halo” refers to a halogen selected from fluoro, chloro, bromo, and iodo.

Suitably the halogen may be selected from fluoro, chloro and iodo.

“C -₆ haloalkyl” refers to a C -₆ alkyl group where at least one of the H atoms have been replaced with halo group. Suitably, all of the H atoms of the alkyl group have been replaced with halo groups. Examples of C -₆ alkyl groups maybe selected from CH₂F, CHF₂, CF₃, CH_aCl, CHC1₂, CC1₃, CH₂Br, CHBr₂, CBr₃, CHJ, CHI₂ and CI₃.

“C_5-io heteroaryl”: refers to unsaturated monocyclic or bicyclic aromatic groups comprising from 5 to 10 ring atoms, whether carbon or heteroatoms, of which from 1 to 5 are ring heteroatoms. Suitably, the heteroaryl group is a 5- to 10-membered ring heteroaryl comprising 5 to 10 ring atoms, whether carbon or heteroatoms, of which from 1 to 5 are ring heteroatoms. Suitably, any monocyclic heteroaryl ring has from 5 to 6 ring atoms including from 1 to 3 ring heteroatoms. Suitably each ring heteroatom is independently selected from nitrogen, oxygen, and sulfur. The bicyclic rings include fused ring systems and, in particular, include bicyclic groups in which a monocyclic heterocycle comprising 5 ring atoms is fused to a benzene ring. The heteroaryl group may be attached to a parent group or to a substrate at any ring atom and may include one or more non-hydrogen substituents unless such attachment or substitution would violate valence requirements or result in a chemically unstable compound.

Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from:

Nu pyrrole, pyridine;

(X: furan;

Su thiophene isoxazole, isoxazine;

N Ou oxazole, isoxazole;

N₂0_I: oxadiazole (e.g. i-oxa-2,3-diazolyl, i-oxa-2,4-diazolyl, i-oxa-2,5-diazolyl, i-oxa-3,4- diazolyl);

N₃0_I: oxatriazole;

N Su thiazole, isothiazole;

N₂S_I: thiadiazole (e.g. 1,3,4-thiadiazole);

N₂: imidazole, pyrazole, pyridazine, pyrimidine, pyrazine;

N₃: triazole, triazine; and,

N₄: tetrazole.

Examples of heteroaryl which comprise fused rings, include, but are not limited to, those derived from:

(X: benzofuran, isobenzofuran;

Nu indole, isoindole, indolizine, isoindoline;

Su benzothiofuran; N O : benzoxazole, benzisoxazole;

N S : benzothiazole;

N₂: benzimidazole, indazole;

0₂: benzodioxole;

N₂0_I: benzofurazan;

N₂S_I: benzothiadiazole;

N₃: benzotriazole; and

N₄: purine (e.g., adenine, guanine).

“Independently selected” is used in the context of statement that, for example,“each R is independently selected from H, C -₆ alkyl,...” and means that each instance of the functional group, e.g. R_B, is selected from the listed options independently of any other instance of R_B in the compound. Hence, for example, H may be selected for the first instance of R_B in the compound; and methyl may be selected for the next instance of R_B in the compound.

“Pharmaceutically acceptable” substances refers 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.

“Optionally substituted” refers to a parent group which may be unsubstituted or which may be substituted with one or more substituents. Suitably, unless otherwise specified, when optional substituents are present the optional substituted parent group comprises from one to three optional substituents. Where a group may be“optionally substituted with up to three groups”, this means that the group may be substituted with o, l, 2 or 3 of the optional substituents. Where a group may be“optionally substituted with one or two optional substituents”, this means that the group may be substituted with o, 1 or 2 of the optional substituents. Suitably groups maybe optionally substituted with o or 1 optional

substituents.

Optional substituents may be selected from C -_i2 alkyl, C_2-12 alkenyl, C_2-12 alkynyl, C_5-i2 aryl, C_3-10 cycloalkyl, C_3-i0 cycloalkenyl, C_3-i0 cycloalkynyl, C_3-20 heterocyclyl, C_3-i0 heteroaryl, acetal, acyl, acylamido, acyloxy, amidino, amido, amino, aminocarbonyloxy, azido, carboxy, cyano, ether, formyl, guanidino, halo, hemiacetal, hemiketal, hydroxamic acid, hydroxyl, imidic acid, imino, ketal, nitro, nitroso, oxo, oxycarbonyl, oxycarboyloxy, sulfamino, sulfamyl, sulfate, sulfhydryl, sulfinamino, sulfinate, sulfino, sulfinyl, sulfinyloxy, sulfo, sulfonamido, sulfonamino, sulfonate, sulfonyl, sulfonyloxy, uredio groups. “Pharmaceutical composition” refers to the combination of one or more drug substances and one or more excipients.

“Salts, solvates, stereoisomers, tautomers or combinations thereof’ means that the compound may be a combination of these options such as being both a tautomer and a pharmaceutically acceptable salt.

“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.

The term“subject” or“patient” 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.

“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. The“effective amount” includes an amount of the compound of formula (I) that will elicit a biological or medical response of a subject, for example, the reduction or inhibition of enzyme or protein activity related to a bacterial infection, amelioration of symptoms of a bacterial infection, or the slowing or delaying of progression of a bacterial infection. In some embodiments, the language“effective amount” includes the amount of a compound of formula (I), that when administered to a subject, is effective to at least partially alleviate, inhibit, and/or ameliorate a bacterial infection and/or reduce or inhibit the bacterial growth, replication or bacterial load of a bacteria in a subject.

“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. Suitable Structures

Suitably, the compound of formula (I), is a compound wherein the structure is:

or salts, solvates, stereoisomers, tautomers or combinations thereof.

Suitably, the compound of formula (I), is a compound wherein the structure is:

or salts, solvates, stereoisomers, tautomers or combinations thereof.

Suitably, the compound of formula (I), is a compound wherein the structure is:

or salts, solvates, stereoisomers, tautomers or combinations thereof.

Suitably, the compound of formula (I), is a compound wherein the structure is:

or salts, solvates, stereoisomers, tautomers or combinations thereof.

Suitably, the compound of formula (I), is a compound wherein the structure is:

or salts, solvates, stereoisomers, tautomers or combinations thereof.

or salts, solvates, stereoisomers, tautomers or combinations thereof.

Suitably, the compound of formula (I) is:

Suitably, the compound of formula (I) is (i), (ii), (iii), (iv), (v), (vi), (vii), (viii), (ix) or salts, solvates, stereoisomers, tautomers or combinations thereof.

Suitably, the compound of formula (I) is (i), (ii), (iii), (iv), (v), (vii), (viii), (ix) or salts, solvates, stereoisomers, tautomers or combinations thereof.

Suitably, the compound of formula (I) is (i), (ii), (iii), (iv), (v), (vi) or salts, solvates, stereoisomers, tautomers or combinations thereof.

Suitably, the compound of formula (I) is (i), (ii), (iii), (iv) or salts, solvates, stereoisomers, tautomers or combinations thereof.

Suitably, the compound of formula (I) is (iv), (v), (vi) or salts, solvates, stereoisomers, tautomers or combinations thereof.

Optional Double Bond

In some aspects, suitably, the PBD moiety comprises a double bond between Cl and C2. Thus, suitably, the PBD moiety comprises a double bond between Cl and C2, to provide a structure:

or salts, solvates, stereoisomers, tautomers or combinations thereof.

In some aspects, suitably, the PBD moiety comprises a double bond between C2 and C3. Hence, suitably, the PBD moiety comprises a double bond between C2 and C3, to provide a structure:

or salts, solvates, stereoisomers, tautomers or combinations thereof.

In some aspects, more suitably, the optional double bond is absent. Therefore, more suitably, the optional double bond is absent and the compound of formula (I) is:

or salts, solvates, stereoisomers, tautomers or combinations thereof.

R and R

Suitably, (ii)R is H and R₂ is OH.

Suitably, (iii)R is H and R₂ is OCi-6 alkyl.

More suitably, (i) R and R₂ together form a double bond.

R₂. R_| and R_^

Suitably, R₃, R4 and R₅ are independently RA, ORA, halo, =0, =CH-R_A, (CH₂)_m-0R_A, (CH₂)_m- NHRB, (CH₂)_m-C0₂R_A, (CH₂)_m-C(0)R_A, (CH₂)_m-S0₂R_A, 0-S0₂R or CN. Suitably, R₃, R₄ and R₅ are independently H, C -₁₂ alkyl, =CH₂, =CH-(C_I-I2 alkyl group), group), (CH₂)_m-NH₂, (CH₂)_m-C0₂H, (CH₂)_m-C0₂CH₃,

, 0-S0₂CH₃ or CN.

Suitably, R₃, R₄ and R₅ are independently H, CH₃, CH₂CH₃, OH, OCH₃, OCH₂CH₃, NH₂, C0₂H, C0₂CH₃, C(0)CH₃, S0₂CH₃, 0-S0₂CH₃ or CN.

Suitably, R₃, R₄ and R₅ are independently H, CH₃, CH₂CH₃, OH, OCH₃ or OCH₂CH₃.

Suitably, one of R₃, R₄ and R₅ is H.

Suitably, two of R₃, R₄ and R₅ are H.

More suitably, in some aspects, each of R₃, R₄ and R₅ are H. in

Suitably, each m is independently o, 1, 2 or 3. Suitably, each m is o or 1.

More suitably, each m is o.

RA

Suitably, in some aspects, each R_A is H.

Suitably, in some aspects, each R_A is an optionally substituted C - ₂ alkyl.

Suitably, in some aspects, each R_A is an optionally substituted C_6-12 aryl.

Suitably, in some aspects, each R_A is an optionally substituted C_5-i0 heteroaryl.

Suitably, in some aspects, each R_A is an optionally substituted C_7-is aralkyl.

Suitably, in some aspects, each R_A is an optionally substituted C_6-16 heteroaralkyl.

Suitably, the optionally substituted alkyl, aralkyl, or heteroaralkyl group is optionally substituted with 1, 2 or 3 carbon-carbon double or triple bonds. Suitably, the optionally substituted alkyl, aralkyl, or heteroaralkyl group is optionally substituted with 1 carbon- carbon double or triple bond.

Suitably, the optionally substituted alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group is optionally substituted by 1, 2 or 3 independently selected halo, hydroxy, amino, or nitro groups. More suitably, each R_A is H or an optionally substituted C -_i2 alkyl. More suitably, each R_A is H or C -₆ alkyl. More suitably, each R_A is H, CH₃ or CH₂CH₃.

Rs

Suitably, R₆ is H or C -₆ alkyl.

Suitably, R₆ is C -₆ alkyl or CH₂Ph.

More suitably, R₆ is CH₃ or CH₂CH₃. More suitably, R₆ is CH₃.

R

Suitably, p is l or 3. More suitably, p is 1.

R

Suitably, R₇ is RB or ORB. More suitably, R₇ is RB.

Suitably, in some aspects, R₇ is H, CH₃, CH₂CH₃, CF₃, OH, OCH₃, OCH₂CH₃, OCF₃, F, Cl or Br. More suitably, R₇ is H, CH₃, CH₂CH₃, OH, OCH₃ or OCH₂CH₃. More suitably, R₇ is H.

Y

Suitably Y is N-R . Suitably, Y is N-H or N-(C_I-6 alkyl). Suitably, Y is N-CH₃.

Suitably, Y is S or O. Suitably, Y is O. More suitably, Y is S.

Yl

Suitably, Y¹ is N.

More suitably, Y¹ is C-Rs.

Rs

Suitably, Rs is RB or ORB. More suitably, Rs is RB.

Suitably, in some aspects, Rs is H, CH₃, CH₂CH₃, CF₃, OH, OCH₃, OCH₂CH₃, OCF₃, F, Cl or Br. More suitably, Rs is H, CH₃, CH₂CH₃, OH, OCH₃ or OCH₂CH₃. More suitably, Rs is H. a

q maybe o. Where q is o in formula (I), suitably, Y is N-R or O. More suitably the compound may be:

More suitably, q is 1. Het

Suitably Het is

The zig-zag or wavy line indicates where the Het group is attached to the rest of the compound of Formula (I).

Suitably,

Suitably,

Suitably Het is

More suitably, Het is

More suitably,

More suitably,

More suitably,

R₉

Suitably, R_g is RB or ORB. More suitably, R_g is RB.

Suitably, in some aspects, R_g is H, CH₃, CH CH₃, CF₃, OH, OCH₃, OCH CH₃, OCF₃, F, Cl or Br. More suitably, R_g is H, CH₃, CH CH₃, OH, OCH or OCH CH₃. More suitably, R_g is H.

Y2

Suitably Y is N-R . Suitably, Y is N-H or N-(C_I alkyl). Suitably, Y is N-CH₃. Suitably, Y is S or O. Suitably, Y is O. More suitably, Y is S. Y³

Suitably, Y³ is N.

More suitably, Y³ is C-R ₀. More suitably, Y³ is C-H.

Y!

Suitably, Y⁴ is S.

Suitably, Y⁴ is O.

More suitably, Y⁴ is N-R . More suitably, Y⁴ is N-H or N-(C_I-6 alkyl). More suitably, Y⁴ is N- CH₃.

Y5

Suitably, Y⁵ is N.

More suitably, Y⁵ is C-R ₀. More suitably, Y⁵ is C-H.

R o

Suitably, R₁₀ is RB or ORB. More suitably, R₁₀ is RB.

Suitably, in some aspects, R_i0 is H, CH₃, CH₂CH₃, CF₃, OH, OCH₃, OCH₂CH₃, OCF₃, F, Cl or Br. More suitably, R_i0 is H, CH₃, CH₂CH₃, OH, OCH₃ or OCH₂CH₃. More suitably, R_i0 is H.

Rx

Suitably, R^x is H, CH₃, CH₂CH₃, CF₃, (CH₂)_m-0H, (CH₂)_m-OCH₃, (CH₂)_m-0CH₂CH₃, (CH₂)_m- OCF₃, F, Cl, Br, I, (CH₂)_m-NH₂, (CH₂)_m-NHCH₃, C0₂H, C0₂CH₃ or C0₂CH₂CH₃.

More suitably, R^x is H, CH₃, CH₂CH₃, (CH₂)_m-0H, (CH₂)_m-OCH₃, (CH₂)_m-0CH₂CH₃, (CH₂)_m- NH₂, (CH₂)_m-NHCH₃, C0₂H, C0₂CH₃ or C0₂CH₂CH₃.

More suitably, R^x is H, CH₃, CH₂CH₃, OH, OCH₃, OCH₂CH₃, C0₂CH₃ or C0₂CH₂CH₃. More suitably, R^x is H, C0₂CH₃ or C0₂CH₂CH₃.

RB Suitably, each R is independently selected from H, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, CH₂F, CHF₂, CF₃, CH₂C1, CHC1₂, CC1₃, CH₂Br, CHBr₂, CBr₃, CH₂I, CHI₂ and CI₃.

More suitably, each R is independently H, CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂, CH₂F, CHF₂ or CF₃.

More suitably, each R_B is independently H, CH₃ or CH₂CH₃,

Pharmaceutical Composition

Suitably, the pharmaceutical composition comprising a compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof describe herein, and a

pharmaceutically acceptable carrier or diluent.

Suitably, the pharmaceutical composition, further comprises a therapeutic agent for treating a proliferative disease.

Suitably, the pharmaceutical composition, further comprises a therapeutic agent for treating a proliferative disease, wherein the therapeutic agent is selected from 5-fluorouracil, afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, carfilzomib, ixazomib, marizomib, oprozomib, delanzomib, bosutinib, bryostatin-i, busulfan, calicheamycin, camptothecin, carboplatin, 10- hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans,

cyclophosphamide, crizotinib, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano- morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptor binding agents, etoposide (VP16), etoposide glucuronide, etoposide phosphate, exemestane, fmgolimod, flavopiridol, floxuridine (FUdR), 3',5'-0-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitors, fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib, acalabrutinib (ACP-196), ONO/GS-4059, BGB-3111, idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase, lapatinib,

lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine, melphalan,

mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine, sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (an aqueous form of DTIC), transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine, vincristine, vinca alkaloids, ZD1839, ipilimumab, tremelimumab, nivolumab, cemiplimab, pembrolizumab, avelumab, durvalumab, atezolizumab and combinations thereof.

Suitably, the pharmaceutical composition further comprises a therapeutic agent for treating a proliferative disease, wherein the therapeutic agent is a proteasome inhibitor, a Bruton’s tyrosine kinase inhibitor, a checkpoint inhibitor or combinations thereof.

Suitable proteasome inhibitors comprise, but are not limited to, bortezomib, carfilzomib, ixazomib, marizomib, oprozomib and delanzomib.

Suitable Bruton’s tyrosine kinase inhibitors comprise, but are not limited to, ibrutinib, acalabrutinib (ACP-196), ONO/GS-4059 and BGB-3111.

Suitable checkpoint inhibitors comprise, but are not limited to, ipilimumab, tremelimumab, nivolumab, cemiplimab, pembrolizumab, avelumab, durvalumab and atezolizumab. Suitably, the pharmaceutical composition further comprises a therapeutic agent for treating a proliferative disease, wherein the therapeutic agent is a proteasome inhibitor, a Bruton’s tyrosine kinase inhibitor or combinations thereof.

More suitably, the pharmaceutical composition, further comprises a therapeutic agent for treating a proliferative disease, wherein the therapeutic agent is bortezomib, ibrutinib or a combination thereof In one aspect, suitably the therapeutic agent is bortezomib. In another aspect, suitably the therapeutic agent is ibrutinib.

Applications

The compound of formula (I) or salts, solvates, isomers or tautomers thereof, or a pharmaceutical compositions comprising such compounds of formula (I) find application as a medicament.

The invention finds application in the treatment of a proliferative disease.

In certain aspects a method of treating a proliferative disease is provided comprising administering to a subject a therapeutically effective amount of a compound of the formula (I) or salts, solvates, isomers or tautomers thereof or a composition comprising a compound of formula (I) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or combinations thereof. In certain aspects a method of treating a proliferative disease is provided comprising administering to a subject a therapeutically effective amount of a targeted conjugate comprising a compound of the formula (I) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or combinations thereof. In certain aspects a method of treating a proliferative disease is provided, the method comprising administering to a subject a therapeutically effective amount of an antibody-drug conjugate comprising a compound of the formula (I) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or combinations thereof. The term“proliferative disease” refers to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo. Examples of proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g. histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, bone cancer, bowel cancer, brain cancer, colon cancer, head and neck cancer, hepatoma, breast cancer, glioblastoma, laryngeal cancer, cervical cancer, ovarian cancer, oesophageal [or esophageal] cancer, oral cancer, oral carcinoma, prostate cancer, testicular cancer, liver cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, uterine cancer, salivary gland carcinoma, kidney or renal cancer, parathyroid cancer, prostate cancer, vulval cancer, skin cancer, testicular cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, head and neck cancer, bladder cancer, pancreas cancer, brain cancer, sarcoma, osteosarcoma, Kaposi’s sarcoma, melanoma), leukaemia (e.g. adult T-cell leukaemia (HTLV-i), acute lymphocyctic leukaemia, acute myeloid leukaemia, chronic lymphocytic leukaemia or chronic myeloid leukaemia), lymphoma (e.g. mantle cell lymphoma, mucosa-associated lymphoid tissue lymphoma or MALT lymphoma, diffuse large B-cell lymphoma, Hodgkin’s lymphoma), melanoma, multiple myeloma, myelodysplasic syndrome, psoriasis, retinoblastoma, squamous cell carcinoma (skin), squamous cell carcinoma (head and neck), bone diseases, cylindroma, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis.

Suitably the proliferative disease is a haematological malignancy or a solid tumour. Suitably, the haematological malignancy is multiple myeloma, mantle cell lymphoma, mucosa-associated lymphoid tissue lymphoma or MALT lymphoma, diffuse large B-cell lymphoma, Hodgkin’s lymphoma, myelodysplastic syndrome, adult T-cell leukaemia (HTLV- 1), acute lymphocyctic leukaemia, acute myeloid leukaemia, chronic lymphocytic leukaemia or chronic myeloid leukaemia.

More suitably, the solid tumour is astrocyoma, glioblastoma, breast cancer, bladder cancer, cervical cancer, colon cancer, cylindroma, endometrial carcinoma, esophageal [or oesophageal] cancer, gastric or stomach cancer, laryngeal cancer, liver cancer, lung cancer, melanoma, oral carcinoma, ovarian cancer, pancreatic cancer, parathyroid cancer, prostate cancer, renal cancer, retinoblastoma, squamous cell carcinoma (skin), squamous cell carcinoma (head and neck) or thyroid cancer.

Most suitably, the proliferative disease is multiple myeloma or chronic lymphocytic leukaemia.

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, and skin.

A skilled person is readily able to determine whether or not a candidate compound treats a proliferative condition for any particular cell type.

Suitably subjects are human, livestock animals and companion animals.

In a further aspect, the compound of formula (I) or salts, solvates, tautomers, stereoisomers or combinations thereof, may be linked, either directly or indirectly, to a targeting agent (e.g., a protein, a portion of a protein, a polypeptide, a nucleic acid, a hormone, an antibody or an antibody fragment, etc.) to provide a targeted conjugate. The target conjugates of the present disclosure may contain one or multiple compounds of formula (I) (or

pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or combinations thereof). A variety of target conjugates are known in the art and may be used with a compound of formula (I) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or combinations thereof. For example, in a particular aspect the target conjugate is an antibody-drug conjugate, wherein one or more compounds of formula (I) are linked, directly or indirectly, to the antibody. Therefore, the compound of formula (I) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or combinations thereof, may be used as a payload on a targeted conjugate.

There is disclosed a compound of formula (I) or salts, solvates, tautomers, stereoisomers or combinations thereof, for use as a drug in a targeted conjugate. Such a targeting conjugate may be prepared by attaching a compound of formula (I) or salts, solvates, isomers or tautomers thereof to a targeting agent, either directly or via an optional linker group.

Suitably, the compound of formula (I) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or combinations thereof, is attached to a targeting agent via a linker group. Suitably, the targeted conjugate is for use in the treatment of a disease, more specifically of a proliferative disease. Suitably, the drug may be attached by any suitable functional group that it contains to the targeting agent 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 targeting agent either directly or via a linker group. In some aspects, one or more atoms or groups of the compound of formula (I) maybe eliminated during the attachment of the drug to the antibody. In some aspects, the targeting agent binds to a cell surface receptor or a tumour- associated antigen. In some aspects, the targeting agent is an antibody. In some aspects, the targeting agent is an antibody fragment. In some aspects, the targeting agent is a hormone. In some aspects, the targeting agent is a protein. In some aspects, the targeting agent is a polypeptide. In some aspects, the targeting agent is a small molecule (for example, folic acid). Suitably, the targeting agent is selected from a protein, a portion of a protein, a polypeptide, a nucleic acid, an antibody or an antibody fragment. More suitably, the targeting agent is an antibody or an antibody fragment. More suitably, the targeting agent is an antibody.

Suitably, the present invention relates to a compound of formula (I) or salts, solvates, tautomers, stereoisomers or combinations thereof, for use in preparing a targeting conjugate (e.g. an antibody-drug conjugate). Suitably, a compound of formula (I) or salts, solvates, tautomers, stereoisomers or combinations thereof, may be used directly to prepare a targeting conjugate when a compound of formula (I) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or combinations thereof, contains one or more functional groups such as amine, hydroxyl or carboxylic acid groups for attaching the drug to the targeting agent either directly or via a linker group. Suitably, a compound of formula (I) or salts, solvates, tautomers, stereoisomers or combinations thereof, may be used in preparing a targeting conjugate by being modified to contain one or more functional groups such as amine, hydroxyl or carboxylic acid groups for attaching the drug to the targeting agent either directly or via a linker group. Suitably, a compound of formula (I) or salts, solvates, tautomers, stereoisomers or combinations thereof, may be used in preparing a targeting conjugate by being modified to contain one or more linker groups, wherein the targeting agent (such as an antibody) is attached to the drug through one or more linker groups.

Therefore, the present invention provides for a compound of formula (I) further comprising one or more linker groups or salts, solvates, tautomers, stereoisomers or combinations thereof. Suitably, a compound of formula (I) further comprises 1, 2 or 3 linker groups.

Suitably, a compound of formula (I) further comprises 1 or 2 linker groups. Suitably, a compound of formula (I) further comprises 1 linker group. In some aspects, one or more atoms or groups (such as H atoms or hydroxyl groups) of the compound of formula (I) may be eliminated during the attachment of the drug to the targeting agent (such as 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 antibody either directly or via a linker group. In some aspects, where the compound of formula (I) further comprises a linker group that is attached to the rest of the compound of formula (I) by eliminating one or more atoms or groups (such as H atom or atoms or hydroxyl groups) from an R_A group or by eliminating the R₇ group from a N-R₇ group.

Suitably such linker groups may comprise from 1-200 non-hydrogen atoms selected from C, N, O, S or halogen and may be branched, cyclic and/ or unsaturated and, optionally, such linker groups may incorporate ether, oxo, carboxamidyl, urethanyl, heterocyclyl, aryl, heteroaryl, azide, alkyne, bisulfone, carbohydrazide, hydrazine, hydroxylamine,

iodoacetamide, isothiocyanate, maleimide, phosphine, pyrridopyridazine, R_A,

semihydrazide, succinimidyl ester, sulfodichlorophenol ester, sulfonyl halide,

sulfosuccinimidyl ester, 4-sulfotetrafluorophenyl ester, tetrafluorophenyl ester and thiazole moieties.

The compounds of formula (I) find application as payloads for antibodies or antibody fragments. The compounds of formula (I) readily allow conjugation to antibodies or antibody fragments.

Administration & Dose

Compounds of formula I may be administered alone or in combination with one or another or with one or more pharmacologically active compounds which are different from the compounds of formula I. 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. 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, NewYork, 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.

The compounds of the invention, their pharmaceutically acceptable salts, and

pharmaceutically acceptable solvates of either entity 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.

For example, the compounds of the invention or salts or solvates 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 methaciylate copolymer,

hydrogenated castor oil, carnauba wax, paraffin wax, cellulose acetate phthalate,

hydroxypropylmethyl cellulose phthalate, methacrylic acid copolymer and combinations 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 maybe 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.

Depending upon the disorder and patient to be treated, as well as the route of

administration, the compositions maybe 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 maybe 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, and protected forms of these substituents. For example, a reference to carboxylic acid (-COOH) also includes the anionic (carboxylate) form (-COO ), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-N⁺HR’R²), 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 (-0 ), a salt or solvate thereof, as well as conventional protected forms.

Isomers. Salts and Solvates

Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, 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 1- 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, -CH₂0H.

A reference to a class of structures may well include structurally isomeric forms falling within that class (e.g. C -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/hydroxyazo, 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 Ή, ²H (D), and ³H (T); C maybe in any isotopic form, including ¹²C, ¹³C, and ¹⁴C; O maybe in any isotopic form, including ^l60 and ^l80; and the like.

Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other combinations 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 formula (I) and salts and solvates thereof, comprises pharmaceutically acceptable salts of the compounds of formula (I).

Compounds of formula (I), which include compounds specifically named above, may form salts, solvates (such as hydrates), isomers or tautomers. Suitably, these are pharmaceutically acceptable salts, solvates, isomers or tautomers. 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 maybe anionic (e.g. -COOH may be -COO ), 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 aluminium (Al3⁺). Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH4⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, 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'-dibenzyl ethylene-diamine, chloroprocaine, choline, diethylamine, diethanolamine, dicyclohexylamine, ethylenediamine, glycine, lysine, N-methylglucamine, olamine, 2-amino-2-hydroxymethyl-propane-i,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 formula (I) with an appropriate acid or base to give the desired salt. One may also react a precursor of the compound of formula (I) 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 formula (I) 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.

It may be convenient or desirable to prepare, purify, and/ or handle a corresponding solvate of the active compound. The term“solvate” describes a molecular complex comprising the compound and one or more pharmaceutically acceptable solvent molecules (e.g., EtOH).

The term“hydrate” is a solvate in which the solvent is water. Pharmaceutically acceptable solvates include those in which the solvent maybe isotopically substituted (e.g., D₂0, acetone-d6, DMSO-d6).

A currently accepted classification system for solvates and hydrates of organic compounds is one that distinguishes between isolated site, channel, and metal-ion coordinated solvates and hydrates. See, e.g., K. R. Morris (H. G. Brittain ed.) Polymorphism in Pharmaceutical Solids (1995). Isolated site solvates and hydrates are ones in which the solvent (e.g., water) molecules are isolated from direct contact with each other by intervening molecules of the organic compound. In channel solvates, the solvent molecules he in lattice channels where they are next to other solvent molecules. In metal-ion coordinated solvates, the solvent molecules are bonded to the metal ion.

When the solvent or water is tightly bound, the complex will have a well-defined

stoichiometry independent of humidity. When, however, the solvent or water is weakly bound, as in channel solvates and in hygroscopic compounds, the water or solvent content will depend on humidity and drying conditions. In such cases, non-stoichiometry will typically be observed.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Figure l: shows that PBDs induced apoptosis in multiple myeloma cell lines in a dose- dependent manner. (A) An example of Annexin V and propidium iodide bivariate plots obtained from JJN3 cells treated with increasing concentrations of DC-1-92. A dose- dependent increase in the proportion of Annexin V⁺/PE and Annexin V⁺/PI⁺ was observed. (B) Sigmoidal dose-response curves illustrating the comparative effects of each compound on the H929, JJN3 and MMi.S multiple myeloma cell lines. (C) Comparative analysis of the three PBD lead compounds in the three multiple myeloma cell lines revealed significant differential sensitivity of the PBDs within each cell line. (D) DC-1-192 was the most potent PBD in all three of the multiple myeloma cell lines. All experiments were performed in triplicate. P-values: *<0.05, **<0.01, ***<0.001 and ****<0.0001.

Figure 2: PBDs showed preferential cytotoxicity in primary CLL cells compared with healthy non-malignant B- and T-lymphocytes. (A) Non-malignant B- and T-lymphocytes were identified using the gating strategy shown, which allowed the enumeration of the percentage of apoptotic cells in each lymphocyte subset. (B) Apoptotic response graphs illustrating the comparative effects of DC-1-92, DC-1-170 and DC-1-192 on primary CLL cells and non- malignant B- and T-lymphocytes. (C) Comparison of the mean LD₅₀ values showed that all three PBDs were significantly more potent in primary CLL cells when compared with age- matched normal B- and T-lymphocytes. (D) DC-1-192 showed the greatest positive therapeutic index when comparing CLL cells with normal B-lymphocytes (2.4 fold) and CLL cells with normal T-lymphocytes (4.6 fold). P-values: *<0.05, **<0.01, ***<0.001 and ****<0.0001.

Figure 3: PBDs show marked inhibitory effects on both canonical and non-canonical NF-KB subunits. (A) JJN3 cells were treated with DC-1-92, DC-1-170 and DC-1-192 for 4 hours, nuclear extracts were then generated from these samples and the amount of (A) p6s, (B) P50, (C) p52 and (D) Rel B was quantified and expressed relative fold change as a function of the untreated controls. The p6s, p50 and Rel B NF-KB subunits all showed significant reductions in nuclear expression following exposure to DC-1-92, DC-1-170 and DC-1-192. All experiments were performed in triplicate. P-values: *<0.05, **<0.01, ***<0.001 and

****<0.0001. ns denotes that the changes observed were not significant.

Figure 4: PBDs demonstrated cytotoxic synergy with bortezomib and ibrutinib. Synergy between DC-1-92 (1:8), DC-1-170 (1:8) and DC-1-192 (1:15) with bortezomib and DC-1-92 (1:2000), DC-1-170 (1:1250) and DC-1-192 (1:3000) with ibrutinib was investigated in JJN3 and primary CLL cells respectively. The fixed molar ratios for each combination were derived from the individual LD₅₀ values of the PBDs and the clinically achievable doses of bortezomib and ibrutinib. Apoptosis was determined using the Annexin V/PI assay. (A) The fraction affected plots for the individual PBDs, bortezomib and their respective combinations in JJN3 cells. (B) The fraction affected plots for the individual PBDs, ibrutinib and their respective combinations in primary cells. (C) and (D) Isobologram plots indicating the synergistic effects at the level of LD₅₀, LD₇₅ and LD_go for each respective combination. (E) The combination indices for the combination of DC-1-92, DC-1-170 and DC-1-192 with bortezomib at the level of LD₅₀, LD₇₅ and LD_go in JJN3 cells (F) The combination indices for the combination of DC-1-92, DC-1-170 and DC-1-192 with Ibrutinib at the level of LD₅₀, LD₇₅ and LD_go in primary CLL cells. All experiments were performed in triplicate.

Figure 5: PBDs induced similar transcriptomic changes in JJN3 cells including a marked inhibition of NF-KB. (A) Unsupervised hierarchical clustering revealed a strong drug- associated transcriptional signature for both DC-1-170 and DC-1-192. (B) The majority of the significantly altered transcripts were down-regulated in response to drug 4418/5077 (87%). Strikingly, 4040/5077 (80%) of the changes were common to both DC-1-170 and DC-1-192. Both (C) DC-1-170 and (D) DC-1-192 showed a significant enrichment for the inhibition of genes involved in NF-KB signalling. (E) Gene set enrichment analysis identified numerous canonical pathways that were over represented in the significantly altered gene lists for each PBD analysed; several these involved NF-KB signalling pathways.

Figure 6: DC-1-192 significantly prolonged survival in a murine xenograft model of myeloma. (A) In order to investigate the in vivo anti-tumour effects of DC-1-192, NOD/SCID mice were systemically inoculated with the human RPMI 8226 myeloma cell line. DC-1-192 (lmg/kg) significantly prolonged the survival of the mice when compared to untreated control mice. (B) Bortezomib (o.6mg/kg) also prolonged survival in this model although this did not reach statistical significance. (C) Comparison of the Kaplan-Meier curves for DC-1- 192 and bortezomib showed that the ability of the two agents to extend survival were not significantly different. Figure 7: shows bar graphs of the DNA binding of GWL-78 and DC-1-192 when compared to untreated control for NF-KB subunits p65, p50, Rel B and P52.

Figure 8: shows the body weight changes of mice in the different groups treated with DC-1- 192 (1 mg/kg) or with 0.5 mg/kg of an in vivo test of maximum tolerated dose.

Figure 9: shows the DC-1-192 in vivo efficacy in myeloma model including in combination with protease inhibitor.

Figure 10: shows the body weight changes of RPMI-8226 bearing mice in different treatment groups.

Figure 11: shows survival curves of RPMI-8226 bearing mice in different treatment groups. Figure 12: shows individual survival curves of RPMI-8226 bearing mice in different treatment groups at day 78.

DESCRIPTION OF THE EMBODIMENTS

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.

CHEMICAL EXPERIMENTAL

General material and methods

All solvents and reagents for the synthesis were obtained from commercial available sources including among others Sigma-Aldrich, Fisher Scientific, Fluorochem and Alfa Aesar. Thin- layer-chromatography (TLC) analysis was performed on silica gel plates (E. Merck silica gel 60 F254 plates) and visualized by ultra-violet (UV) radiation at 254 nm. Flash

chromatography for the purification of compound was performed with silica gel as a stationary phase (Merck 60, 230-400 mesh). Ή and ¹³C nuclear magnetic resonance (NMR) analyses were performed on a Bruker Spectrospin 400Hz spectrometer. IR spectra were collected with an FT/IR IRAffinity-iS IR spectrophotometer (Shimadzu). HRMS was performed on a Thermo Scientific-Exactive HCD Orbitrap Mass Spectrometer. LC-MS analyses were performed on a Waters Alliance 2695 system

LCMS gradient conditions are described as follows.

Method A (10 min): from 95% A/5% B to 50% B over 3 min. Then from 50% B to 80% B over 2 min. Then from 80% B to 95% B over 1.5 min and held constant for 1.5 min. This was then reduced to 5% B over 0.2 min and maintained to 5% B for 1.8 min. The flow rate was 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. Method B (5 min): from 95% A/5% B to 90% B over 3 min. Then from 90% B to 95% B over 0.5 min and held constant for 1 min. This was then reduced to 5% B over 0.5 min. The flow rate was 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.

PBD Core Synthesis

Synthesis of methyl 4-(4-formyl-2-methoxyphenoxy)butanoate (2.4)

A suspension of 2.3 (20 g, 131 mmol), methyl 4-bromobutanoate (24.986 g, 1.05 equiv.) and potassium carbonate (27.25 g, 1.5 equiv.) was prepared in DMF (too ml) and stirred at room temperature for six hours. After completion of reaction was confirmed by LC-MS analysis, the reaction mixture was diluted with water (500 ml). A white precipitate formed. This precipitate was filtered and washed with cold water to yield a white solid product which was dried in a vacuum oven at 40 °C to yield 28.1 g of 2.4 (85% yield).

Synthesis of methyl 4-(4-formyl-2-methoxy-5-nitrophenoxy)butanoate (2.5)

Acetic anhydride (20 ml) was added drop wise to a stirred solution of nitric acid (70%, 90 ml) at -10 °C (caution, rapid exotherm possible). The solution was stirred at this temperature for 30 minutes. 2.4 (5 g, 0.0198 mol) was dissolved in acetic anhydride (25 ml). This solution was then added drop wise to the stirred acetic anhydride/nitric acid solution. A colour change from colourless to pale yellow occurred upon addition. After the solution was added, the temperature was allowed to rise to 5 °C and maintained until LC-MS showed completion of reaction after 3 hours. The reaction mixture was then poured into a solution of ice water (500 ml) to quench the reaction. The product was extracted with dichloromethane (400 ml) and concentrated using a rotary evaporator. The crude product was purified by flash chromatography using a 0.15:0.85 gradient of ethyl acetate/n-hexane to yield 3.62 g of

2.5 as a bright yellow solid (56% yield).

Method 2

2.4 (5g, 19.8 mmol) was dissolved in trifluoroacetic acid (12 ml). This solution was added drop wise at 0-5 °C to a stirred solution of potassium nitrate (2.5 g, 1.25 equiv.) in TFA (12 ml). A colour change from colourless to pale yellow occurred on addition. The reaction mix was stirred at room temperature for 20 mins at which point TLC ( 15% ethyl acetate/n- hexane) showed completion of reaction. The product was concentrated using a rotary evaporator and the residue dissolved in ethyl acetate (200 ml). The solution was washed with brine (3 x 50ml) and dried over magnesium sulphate. The material was concentrated using a rotary evaporator and dried in a vacuum oven at 40 °C to yield 5.81 g of 2.5, a bright yellow solid (95% yield). The product was taken to the next step without further purification.

Synthesis of 5-methoxy-4-(4-methoxy-4-oxobutoxy)-2-nitrobenzoic acid (2.6)

2.5 (5g, 16.83 mmol) was dissolved in acetone (160 ml) and placed in a three necked round bottomed flask with fitted with a reflux condenser. A solution of potassium permanganate (10% w/v, 100 ml) was prepared and heated to 70 °C. This solution was quickly added to the stirred solution of 2.5 and the reaction mix was heated at 70 °C. The reaction mix was left for three hours and completion of reaction observed by TLC (15% ethyl acetate/ hexane) and colour change from purple to brown. The reaction vessel was allowed to cool and the reaction mix passed filtered through celite packed into a sintered funnel. The brown residue on the celite was washed with hot water (300 ml). A solution of sodium bisulphite (10 g) in hydrochloric acid (lM, 160 ml) was then added to the filtrate. The pH of the product was then adjusted to 1 through the addition of lM hydrochloric acid. This caused partial precipitation of 2.6. The product was extracted from the filtrate with dichloromethane (400 ml) and dried using magnesium sulphate. The DCM extract was concentrated using a rotaiy evaporator and dried at 40 °C in a vacuum oven to yield 4.155 g of 2.6 as a pale yellow solid (79% yield).

Synthesis of (-S) -methyl 4-(4-(2-(hydroxymethyl)pyrrolidine-i-carbonyl)-2- methoxy-5-nitrophenoxy)butanoate (2.7)

2.6 (5 g, 15.9 mmol, 1.05 equiv.) was dissolved in anhydrous dichloromethane under nitrogen. Oxalyl chloride (1.926 g, 0.0151 mol) was added to the flask along with two drops of anhydrous dimethylformamide as a catalyst. Vigorous bubbling was observed. The reaction was left at room temperature for three hours to ensure complete formation of the acid chloride. A solution of triethylamine (2.2 equiv.) and + (S)-pyrrolidinemethanol (1.1 equiv.) was prepared in dry DCM (40 ml) under nitrogen and cooled to -30 °C. The acid chloride solution was added dropwise to the pyrrolidine solution. A mild exotherm was observed upon addition of the acid chloride. After three hours, TLC (2% methanol/ethyl acetate) showed completion of reaction and the reaction mixture was washed with 1 M hydrochloric acid (2 x 40 ml), water (2 x 30 ml) and brine (25 ml). The solution was dried over

magnesium sulphate and concentrated using a rotary evaporator to yield yellow oil 2.7. The crude product was purified by flash column chromatography 0.02:0.98 methanol/ethyl acetate to yield a yellow solid in 74% yield.

Method 2

2.6 (5 g, 15.9 mmol) was placed in a round bottomed flask to which a gas bubbler was attached. Oxalyl chloride (4.4 g) was added to the flask as a solvent and the formation of HC1 gas was observed through the bubbler. After 20 minutes, the bubbling ceased and the reaction mix was concentrated in a rotary evaporator. The resulting residue was dissolved in toluene (to ml) and the solution concentrated once more to remove any excess oxalyl chloride. The resulting residue was dissolved in dichloromethane (to ml) and added drop wise to a solution of + GS)-pyrrolidinemethanol (1.05 equiv.) and triethylamine (1.5 equiv.) in dichloromethane (20 ml) at o °C. The reaction allowed to warm to room temperature and left until TLC/LC-MS (2% methanol/ethyl acetate) showed completion of the reaction at about three hours. The reaction mix was washed with hydrochloric acid (lM, 10 ml) and brine (10 ml) and dried over magnesium sulphate. The solution was concentrated using a rotary evaporator and the crude product purified by flash column chromatography 0.02:0.98 methanol/ ethyl acetate to yield 2.7 in similar yields to method 1.

Synthesis of (-S) -methyl 4-(5-amino-4-(2-(hydroxymethyl)pyrrolidine-i- carbonyl)-2-methoxyphenoxy)butanoate (2.8)

Method 1

2.7 (7.5 g, 18.9 mmol) was dissolved in an 80:20 mix of ethanol/ethyl acetate (too ml). A catalytic amount of palladium over carbon (10% wt. loading) was carefully added to the flask and the resulting suspension was hydrogenated at 45 psi until the uptake of H₂ by the reaction vessel ceased. After three hours, TLC (2% methanol/ethyl acetate) and LC-MS showed completion of the reaction. A layer of Celite was packed into a sintered funnel and the reaction mixture was filtered. The palladium/ carbon residue on the Celite was washed with ethyl acetate and the filtrate was concentrated using a rotary evaporator to yield 2.8 as a red oil, which on exposure to hard vacuum formed a foam in 92% yield. Method 2

2.7 (2 g, 5.4 mmol) was dissolved in methanol. Ammonium formate (5 equiv.) was then added to the flask, together with a catalytic amount of palladium on carbon (10% wt. loading). The solution was refluxed at 60 °C for 30 minutes, after which LC-MS/TLC (2% methanol/ethyl acetate) showed completion of the reaction. The solution was then filtered through Celite, with the Pd/C residue remaining on the Celite washed with methanol. The filtrate was concentrated using a rotary evaporator to yield 2.8 as a red oil in similar yields to those obtained for method 1.

Synthesis of (S') -methyl 4-(5-(((allyloxy)carbonyl)amino)-4-(2- (hydroxymethyl)pyrrolidine-i-carbonyl)-2-methoxyphenoxy)butanoate (2.9)

2.8 (5 g, 13.7 mmol) was dissolved in anhydrous dichloromethane (too ml) and anhydrous pyridine (2.54 ml, 2.3 equiv.) under nitrogen and the resulting solution cooled to -10 °C. A solution of allyl chloroformate (1.73 g, 1.05 equiv.) was dissolved in anhydrous

dichloromethane (too ml) and added drop wise to the solution of 2.8. The reaction mix was then allowed to reach room temperature and reaction progression monitored using TLC (5% acetone/DCM). After about two hours, the reaction was complete. The reaction mix was sequentially washed with saturated copper sulphate solution (too ml), water (too ml) and sodium bicarbonate solution (too ml). The reaction mix was dried over magnesium sulphate and concentrated in a rotary evaporator to yield 2.9 in 90% yield as a red oil.

Synthesis of allyl (iiaS^,)-ii-hydroxy-7-methoxy-8-(4-methoxy-4-oxobutoxy)-5- 0x0-2, 3,11, iia-tetrahydro-iii-benzo[e]pyrrolo[i,2-a][i,4]diazepine-io(5H)- carboxylate (2.10)

2.9 (3-5 g, 7.8 mmol) was dissolved in dichloromethane (175 ml). To this solution, (2, 2,6,6- tetramethylpiperidine-i-yl)oxyl (TEMPO) (0.122 g, 0.1 equiv.) and bis(acetoxy)iodobenzene (BAIB) (3.01 g, 1.2 equiv.) were added and the reaction mix monitored for reaction progression via TLC (5% acetone/DCM) and LC-MS. After six hours the reaction went to completion. The reaction mixture was washed sequentially with saturated sodium metabisulphite (75 ml), saturated sodium bicarbonate solution (75 ml), water (75 ml) and brine (75 ml). The solution was concentrated using a rotary evaporator and the crude 2.10 product was purified using flash column chromatography using a 50:50 Ethyl acetate/n- hexane gradient as a yellow oil. The product was recrystallized using diethyl ether overnight to form a white powder with a yield of 75%.

Synthesis of allyl (iiaS')-7-methoxy-8-(4-methoxy-4-oxobutoxy)-5-oxo-ii- ((tetrahydro-2JT-pyran-2-yl)oxy)-2,3,ii,iia-tetrahydro-iiT-benzo[e]pyrrolo[i,2- a][i,4]diazepine-io(5ii)-carboxylate (2.11)

2.10 (3.5 g, 7.8 mmol) was dissolved in ethyl acetate (50 ml). Dihydropyran (6.571 g, 10 equiv.) and a catalytic amount of PTSA (35 mg) were added to the reaction mix. The progression of the reaction was monitored using TLC (5% acetone/DCM) and LC-MS and after two hours the reaction was complete. The reaction mix was diluted with a further amount of ethyl acetate (50 ml) and washed sequentially with saturated sodium bicarbonate solution (50 ml) and brine (75 ml). The ethyl acetate layer was dried using magnesium sulphate and concentrated using a rotary evaporator to produce 2.11 in 90% yield.

Synthesis of 4-(((naS')-io-((allyloxy)carbonyl)-7-methoxy-5-oxo-ii- ((lelrahydro-2//-pyran-2-yl)oxy)-2,3,5,io,ii,iia-hexahydro-i//- benzo[e]pyrrolo[i,2-a][i,4]diazepin-8-yl)oxy)butanoic acid (2.12)

2.11 (3.5 g, 6.571 mmol) was dissolved in dioxane (70 ml), and a solution of sodium hydroxide (0.5 M) was added in excess to the solution. The reaction was monitored using LC-MS and went to completion after 5 hours. The dioxane was removed from the reaction mix using a rotary evaporator, and the remaining residue was diluted with water (25 ml). The solution was acidified using citric acid solution (lM, 25 ml). The acid was extracted using ethyl acetate (2 x 50 ml) and the combined organic fractions then washed with brine (50 ml). The resulting solution was dried over magnesium sulphate and concentrated over a rotary evaporator. The resulting white solid 2.12 was resolved in 95% yield.

Synthesis of Double Benzofused Side Chains

Each of the four double benzofused second generation side chains were synthesised from combinations of 2.i3a/b and 2.i7a/b type building blocks (described in detail in scheme 4.2). General method below was used to couple the building blocks with the synthesis of

4.12a below as an example.

General Method

The hoc protected compound 2.i7a/b (1.2 eq) was dissolved in DMF (5 mL) to which 2.0 eq of EDCI and 2.5 eq of DMAP were added. The mixture was allowed to stir for 30 minutes after which 2.i3a/b (1.0 eq) was added. The reaction mixture was allowed to stir for a further 6 hour until TLC showed completion of reaction. The reaction was quenched by pouring it onto a mixture of ice/ water mixture and the resulting mixture was extracted with ethyl acetate (3 x 150 mL). The combined extracts were sequentially washed saturated aqueous NaHC0₃ (50 mL), water (50 mL), brine (50 mL) and finally dried over MgS04. Excess ethyl acetate was evaporated by rotary evaporator under reduced pressure and the crude product which was purified by flash chromatography (n-hexane/ethyl acetate).

Four protected side chains were synthesised via this method (4.12a, 4.12b, 4.12c and 4.i2d), with yields listed in the characterisation tables below.

The side chains were then deprotected via standard deprotection procedures known in the art, e.g. see T. W. Greene and P. G. Wuts, Protecting Groups in Organic Chemistry, 4th Edition, (2006) and P. Kocienski, Protective Groups, 3rd Edition (2005). Hence, the Boc protecting groups (4.12a, 4.12c) were removed by dissolving in MeOH and slowly adding 4M HC1 in dioxane with stirring for 6 h until TLC showed completion of reaction. Solvent was the evaporated and the product purified with flash chromatography (n-hexane/ethyl acetate). For the alloc protected compounds (4.12b, 4.121I) (1 equiv) was dissolved in DCM (4 mL) and added of palladium tetrakis(triphenylphosphine) (0.05 equiv.), triphenyl- phospine (0.25 equiv.) and pyrrolidine (1.2 equiv). The reaction mixture was kept under magnetic stirrer for 2 hours until TLC showed completion of reaction Solvent was the evaporated and the product purified by column chromatography (n-hexane/ethyl acetate. Both deprotection reactions proceeded efficiently to produce the deprotected side chains 4.13a, 4.13b, 4.13c and 4.13d, with yields listed in the characterisation tables below.

Synthesis of DC-1-192 and PBD compounds with double benzofused rings

General Method .

A solution of Alloc-THP protected PBD acid 2.12 (1.2 equivalent) was dissolved in DMF EDCI (2 0 eq) and DMAP (2.5 eq) were added to the stirred solution of 2.12 at room temperature and the mixture was allowed to stir for 30 minutes after which the side chain 4.133-4, 13d (1.0 eq) was added. The reaction mixture was allowed to stir for a further 2 hour at which point TLC showed completion of reaction. The reaction was quenched by pouring it onto a mixture of ice/water mixture and the resulting mixture was extracted with ethyl acetate (3 x 150 ml). The combined extracts were sequentially washed with citric acid (200 ml), saturated aqueous NaHCo 3(250 ml), water (250 ml), brine (250 ml) and finally dried over MgSo 4. Excess ethyl acetate was evaporated by rotary evaporator under reduced pressure and the crude product of each reaction was purified by column chromatography (mobile phase: from DCM /acetone, 90/10, v/vto DCM /acetone, 60/40/, v/v depending on the substrate). The protected PBD-conjugates (1 equiv) was dissolved in DCM (4 mL) and added of Tetrakis Pd (0.05 equiv.), triphenylphospine (0.25 equiv.) and pyrrolidine (1.2 equiv). The reaction mixture was kept under magnetic stirrer for 20 minutes when TLC showed completion of reaction. At that point the solvent was evaporated using a rotary evaporator and the crude of reaction was purified by column chromatography (mobile phase: from DCM /acetone, 90/10, v/vto DCM /acetone, 40/60/, v/v, depending on the substrate) affording pure final compounds.

BIOLOGICAL EXAMPLES

These examples determined the biological properties of selected novel C8-linked benzofused PBD compounds by investigating their cytotoxic profiles in multiple myeloma cell lines, primary CLL cells and age-matched normal B- and T-lymphocytes. Their ability to inhibit NF-KB and whether they could potentiate the effects of the targeted agents bortezomib and ibrutinib currently used in the treatment of myeloma and CLL respectively was also investigated.

Example l

Materials and Methods

Culture conditions for cell lines, primary CLL cells and normal lymphocytes

Primary chronic lymphocytic leukaemia (CLL) cells were obtained from patients attending outpatients’ clinics at the University Hospital of Wales with informed consent in accordance with the ethical approval granted by South East Wales Research Ethics Committee

(02/4806). Age-matched normal B- and T-lymphocytes were obtained from age-matched healthy volunteers again with informed consent. Three multiple myeloma cell lines, JJN3, MMi.S and H929, were maintained in liquid culture at densities ranging between 0.5-2X10⁶ cells/ml. JJN3 cells were maintained in DMEM media containing 20% foetal bovine serum (FBS), 1% sodium pyruvate and 1% penicillin and streptomycin. Both H929 and MMi.S cells were maintained in RPMI media containing 10% FBS, 1% L-glutamate and 1% penicillin and streptomycin. All cell lines were purchased from DSMZ and were used for these experiments within 6 months of purchase. In each case, the provenance of the cell lines was verified by multiplex PCR of minisatellite markers, and all were certified mycoplasma-free. Primary CLL and normal lymphocytes were isolated by density gradient centrifugation using Histopaque (Sigma-Aldrich) and were then maintained in RPMI media containing 10% FBS, 5ng/ml IL- 4, 1% L-glutamine and 1% penicillin and streptomycin. All cells were cultured at 37°C in 5% C0₂ atmospheric conditions. Cell counts and viability were determined using the Vi-Cell XR cell counter (Beckman Coulter).

Measurement of in vitro apoptosis

Aliquots of each cell type (lxio⁶ cells) were cultured for 48h, harvested by centrifugation (300XC7 for 5 mins) and then resuspended in i95pl of a calcium-rich buffer. Subsequently, 5m1 of Annexin V (eBiosciences) was added to the cell suspension, and cells were incubated in the dark for 10 mins prior to washing. Cells were finally resuspended in I90m1 of calcium-rich buffer together with iomΐ of propidium iodide. Apoptosis was assessed by dual-colour immunofluorescent flow cytometry using an Accuri C6 flow cytometer, and data were analysed using CFlow software (BD Biosciences).

Measurement of apoptosis in normal B- and T-lymphocytes

Peripheral blood mononuclear cells from age-matched healthy donors (lxio⁶ cells) were treated with concentrations of DC-1-92, DC-1-170 and DC-1-192 between inM-ioonM for 48 hours. Cells were then harvested and stained with APC-conjugated CD19, PE-conjugated CD3 and FITC-conjugated Annexin V. Using an Accuri C6 flow cytometer, a gating strategy (shown in Figure 1) was employed to quantify apoptosis in CDi9⁺ B-lymphocytes and CD3⁺ T-lymphocytes, with appropriate compensation applied.

Enzyme Linked Immuno-sorbent Assay (ELISA) for NF-KB subunits

JJN3 cells were treated for 4 hours with DC-1-92, DC-1-170 (ionM-30nM) and DC-1-192

(2.5nM-ionM). Pellets containing 5x1o⁶ cells were then harvested, and subsequently, nuclear extracts were produced prepared using a nuclear extraction kit (Active Motif). Total protein was determined by DC protein assay (Biorad) in each nuclear extract using a standard curve of known concentrations of BSA. Nuclear extracts containing lpg of total protein from each treatment were then added to an NF-KB family kit (Active Motif) in accordance with the manufacturer’s instructions. Levels of p05, p50, P52 and Rel B DNA binding were then assessed to determine relative levels of each subunit in the nucleus. The absorbance measurements (450nm) were subsequently converted into ng/ pg of nuclear extract for each sample.

Synergy with bortezomib and ibrutinib The synergy between the three PBD compounds in combination with either bortezomib or ibrutinib was determined in the JJN3 cells and primary CLL cells respectively. Different treatment ratios were experimentally determined using the previously established LD₅₀ values for each compound and the clinically achievable concentrations of bortezomib and ibrutinib. The fixed molar ratio of DC-1-92: Bortezomib and DC-i-i70:bortezomib was 1:8 and for DC-i-i92:bortezomib was 1:15. The fixed molar ratio of DC-i-92:ibrutinib was 1:2000, DC-i-i70:ibrutinib was 1:1250 and DC-i-i92:ibrutinib was 1:3000. Cells were treated with each drug individually and in combination at the defined molar ratio. Treated cells were incubated alongside untreated controls for 48 hours, before being labelled with Annexin V-FITC/PI and then analysed on an Accuri C6 flow cytometer. CalcuSyn software was used to establish whether synergy was evident between the PBD compounds and bortezomib or ibrutinib and expressed as a combination index (Cl); Cl values <1 are considered to demonstrate synergy. RNA Isolation

JJN3 cells were treated with either DC-1-170 or DC- 1-192 at sonM in triplicate alongside untreated controls for 4 hours. From each sample, 5x1o⁶ cells were then harvested, washed in ice cold PBS and re-suspended in iml of Trizol reagent (Thermo Fisher). RNA was extracted following the addition of chloroform and 70% ethanol, and an RNeasy mini-kit (Qiagen) was then used in accordance with the manufacturer’s instructions to isolate RNA to be used in RNA sequencing (RNA-seq) analysis.

RNA Sample Preparation and Sequencing

Total RNA quality and quantity was assessed using an Agilent 2100 Bioanalyser and an RNA Nano 6000 kit (Agilent Technologies) ioo- oong of Total RNA with an RNA integrity number (RIN) >8 was depleted of ribosomal RNA, and the sequencing libraries were prepared using the Alumina® TruSeq® Stranded Total RNA with Ribo-Zero Gold™ kit (Alumina Inc.). The steps included rRNA depletion and cleanup, RNA fragmentation, 1^st strand cDNA synthesis, 2^nd strand cDNA synthesis, adenylation of 3’-ends, adapter ligation, PCR amplification (12-cycles) and validation. The manufacturer’s instructions were followed except for the cleanup after the Ribo-Zero depletion step where Ampure®XP beads

(Beckman Coulter) and 80% Ethanol were used. The libraries were validated using the Agilent 2100 Bioanalyser and a high-sensitivity kit (Agilent Technologies) to ascertain the insert size, and the Qubit® (Life Technologies) was used to perform the fluorometric quantitation. Following validation, the libraries were normalised to 4nM, pooled together and clustered on the cBot™2 following the manufacturer’s recommendations. The pool was then sequenced using a 75-base paired-end (2x75bp PE) dual index read format on the Illumina® HiSeq2500 in high-output mode according to the manufacturer’s instructions. Subsequently, analysis was performed after trimming to remove adaptor sequences and low- quality base calls. Trimmed reads were then mapped to the standard reference 'hgi9' using the alignment software package 'bwa-mem'. Downstream analysis of the data was performed using GenView2 software (in-house analysis tool developed by Peter Giles) and Ingenuity Pathway Analysis (Qiagen).

In vivo systemic xenograft: model of myeloma inNOD/SCID mice

Female NOD/SCID mice were sourced from Beijing AK Bio-Technology Co. Ltd. (Beijing, China). The care and use of animals was conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Mice were sub-1 ethally irradiated with 200 cGy with a ^6oCo source one day before inoculation with human myeloma cells, then each mouse was inoculated intravenously into the tail vein with RPMI8226 tumour cells (1 x 10⁷) in 0.1 ml of PBS to initiate tumour development. The date of tumour cell inoculation was denoted as Day o; treatment with vehicle only; 0.05% DMSO in saline (n = 7), lmg/kg DC-1-192 (n = 7) or o.6mg/kg bortezomib (n = 7) was started at Day 5. Survival was evaluated from the first day of treatment until death.

Statistical Analysis

All statistical analysis was performed using Graphpad Prism 6.0 software. All toxicity data from drug treatment were used to produce sigmoidal dose-response curves from which LD₅₀ values were calculated. Toxicity data from synergy experiments were processed using CalcuSyn software using the median effect method to subsequently calculate the

combination index for each pair of agents (21).

Results

Cytotoxic screening ofPBD compounds identified three lead compounds

Initial screening of a library of 87 novel synthetic C8-linked benzofused PBD monomeric hybrids identified three lead compounds based on their cytotoxicity in the multiple myeloma cell line, JJN3. The chemical structures of all three compounds, together with that of Anthramycin on which they are based, are shown in Figure 1.

Cytotoxicity of the lead PBD compounds in multiple myeloma cell lines

After the intial screening, the relative cytotoxicity of the three lead compounds was assessed in three different multiple myeloma cell lines, H929, JJN3 and MMi.S. The cells were cultured for 48I1 in increasing concentrations (inM-ioonM) of DC-1-92, DC-1-170 and DC-1- 192 and were compared with untreated controls. Each compound showed a dose-dependent increase in apoptosis; a representative example of the data generated is shown in Figure lA. The dose-response curves for each compound were compared in each cell line using overlaid sigmoidal plots (Figure lB). Mean LD₅₀ values were then calculated for each treatment and plotted in the scatter plot shown in Figure lC. Although each cell line showed different sensitivity to the three DC compounds, in every case DC-1-192 was the most cytotoxic compound and DC-1-170 was the least cytotoxic (Figure lD).

Toxicity in primary CLL and normal B- and T-lymphocytes

Primary CLL cells and age-matched normal B- and T-lymphocytes obtained from healthy donors were treated with increasing concentrations of DC-1-92, DC-1-170 and DC-1-192.

Apoptosis was measured using CDi9/CD3/Annexin V labelling to determine the percentage of apoptosis induced by the PBDs in CDi9⁺ B-cells and CD3⁺ T-cells as shown in Figure 2A. Figure 2B shows the comparative dose-responses in each of the cell types indicating that normal lymphocytes were less susceptible to the effects of the DC compounds. As was the case with the three multiple myeloma cell lines, DC-1-192 was the most potent cytotoxic agent in primary CLL cells. Figure 2C shows the mean LD₅₀ values for each compound in each cell type and confirms that CLL cells were significantly more sensitive to the effects of the DC compounds when compared with age-matched normal B- and T-lymphocytes (DC-1- 92: P = 0.01 and P = 0.001, DC-1-170: P = 0.002 and P = 0.002, DC-1-192: P = 0.02 and P = 0.02 respectively). The therapeutic indices for each agent, calculated from the comparative

LD_5O values in primary CLL cells versus normal B- and T-lymphocytes, are shown in Figure 2D. DC-1-192 showed the highest differential toxicity in both lymphocyte subsets.

Nuclear localisation ofNF-kB subunits following treatment with PBD

compounds

We have previously shown that PBD monomers, such as KMR-28-39, have NF-KB inhibitory effects (9). We, therefore, determined the NF-KB inhibitory properties of this new series of compounds. In order to explore this, we used JJN3 cells as they have been previously shown to overexpress both the canonical and non-canonical NF-KB subunits (22). JJN3 cells were treated for 4h with up to 20nM of each agent and the relative change in nuclear p65, p50, P52 and Rel B DNA binding was determined as a function of the untreated control (Figure 3). All compounds showed significant inhibition of the p05 and p50 canonical subunits as well as the non-canonical Rel B subunit. In contrast, no significant changes in P52 were observed under any of the conditions tested.

Synergy between PBD compounds with bortezomib and ibrutinib Over expression of NF-KB is associated with chemotherapeutic drug resistance in both CLL and multiple myeloma (23, 24). Having established that the PBD compounds inhibited NF- KB subunits, we set out to determine whether these inhibitory properties could enhance the killing effect of both bortezomib and ibrutinib in the JJN3 myeloma cell line and primary CLL cells, respectively. To investigate synergy, JJN3 and primary CLL cells were treated with increasing concentrations of each drug individually and in combination with bortezomib in JJN3 cells and ibrutinib in CLL samples. The fixed molar ratios employed in the combination studies were determined experimentally using the LD₅₀ values calculated from previous toxicity data. The fraction affected plots for JJN3 cells (Figure 4A), and primary CLL cells (Figure 4B) show that the cytotoxic effects all three DC compounds are potentiated by the addition of bortezomib and ibrutinib, respectively. The isobologram plots in Figures 4C and 4D show that all three DC compounds were synergistic with bortezomib and ibrutinib respectively. The combination of the DC compounds with bortezomib (Figure 4E) and ibrutinib (Figure 4F) showed synergy (Cl values <1) at the level of LD₅₀, LD₇₅ and LD_go with an incremental increase in synergy from LD₅₀ to LD_go in every case. The degree of synergy observed with DC-1-192 was less striking than for DC-1-92 and DC-1-170. Given that DC-1- 192 was the most potent DC compound, these data suggest that the experimentally- determined fixed molar ratios for DC-1-192 may require further optimisation. RNA-seq analysis of the global effects of DC-1-170 and DC-1-192 on JJN3 cells

As predicted, RNA-seq analysis of DC-1-170 and DC-1-192 revealed a dominant inhibitory effect on gene transcription with a smaller subset of genes showing increased transcription following exposure to the drug (Figure 5A). Strikingly, 4040/5077 (80%) of the genes altered by exposure to the drugs were common to both compounds (Figure 5B) confirming that their structural similarity resulted in the inhibition of a conserved set of genes. Furthermore, gene ontology analysis using Ingenuity Pathway Assist (Qiagen) confirmed that both compounds significantly inhibited the NF-KB signalling pathway (Figures 5C and 5D) although it should be noted that other canonical pathways were also shown to be over represented in the significantly altered gene list (Figure 6E). These data suggest that inhibition of NF-KB target genes may contribute to the cytotoxic synergy observed with bortezomib and ibrutinib.

DC-1-192 shows in vivo efficacy in a systemic xenograft murine model

In order to investigate the anti-tumour effects of DC-1-192 in a more clinically relevant model, we systemically inoculated NOD/SCID mice with the human RPMI 8226 myeloma cell line. DC-1-192 (lmg/kg) significantly prolonged the survival of the mice (P = 0.017, HR = 2.98; Figure 6A). These observed anti-tumour effects were similar to those observed in mice treated with bortezomib (o.6mg/kg) in the same xenograft model (Figure 6B and 6C). The median survival in the DC-1-192 cohort was 68 days versus 63 days in the bortezomib- treated mice (P = 0.93, HR = 1.05).

Discussion

NF-KB is a master regulator of vital cellular processes that contribute to cancer progression including cell survival and proliferation. It is also often implicated in drug resistance, highlighting its potential as a therapeutic target (19, 20). The interest in small molecular DNA-binding agents such as the PBD monomers has increased in recent years due to their ability to selectively bind to specific sequences within the minor groove of DNA; a characteristic that separates them from traditional alkylating agents and raises the possibility that they can selectively inhibit transcription factors (5). This study set out to determine the in vitro and in vivo biological effects of a series of novel C8-linked PBD- benzofused hybrids. Initially, three different multiple myeloma cell lines, H929, MMi.S and JJN3, were studied to reflect the clinical heterogeneity of multiple myeloma patients. Each compound showed high potency with LD₅₀ values in the low nanomolar range for each cell line. These cytotoxic effects were produced in a dose-dependent manner and were mediated via the induction of apoptosis. Subsequently, the PBDs showed similar high potency in primary leukaemia cells derived from CLL patients (mean LD₅₀ for DC-1-92 = 5.2nM, DC-1-170 = 7.8nM and DC-1- 192 = 2.65nM). Furthermore, the compounds all demonstrated preferential cell killing effects in malignant cells when compared with normal age-matched B- and T-lymphocytes; DC-1-192 showed a 2.4-fold and 4.6-fold positive therapeutic index respectively. Given that our previous research showed that PBDs can inhibit the transcription factor NF- KB (9), we next explored this as a potential mechanism of action in the JJN3 MM cell line. We used this cell line as our model because it showed constitutive expression of both canonical and non-canonical NF-KB subunits (22). All three compounds showed significant inhibition of the p65 and p50 canonical subunits as well as the non-canonical subunit Rel B after 4h exposure. In contrast, no significant change in P52 was observed following treatment with the PBDs. The rapid reduction in nuclear p65, p50 and Rel B NF-KB subunit expression indicates that NF-KB inhibition precedes apoptosis in these cells and is likely to contribute to the efficacy of the PBDs. It is well established that MM cells are heavily dependent on over-activity of the NF-KB pathway to sustain cellular growth, proliferation and survival (25). Activating mutations in both the canonical and non-canonical pathways have been described so there is probably a need to target both pathways to completely block NF-KB activity (26). However, the frequency of mutations found in the non-canonical pathway in multiple myeloma point to its importance in the progression of this malignancy. One such mutation found in JJN3 cells involves NIK (NF-KB Inducing Kinase) which is the key kinase involved in the cleavage of pioo to p52 (22, 27, 28). The presence of this mutation may explain the lack of inhibitory effect the PBDs had on the P52 subunit in these experiments.

Activation of NF-KB has also been implicated in the development of chemotherapeutic drug resistance (29). Several alkylating agents, including melphalan, have been shown to induce the activity of NF-KB, thereby contributing to cellular resistance to the cytotoxic effects of these treatments (30). Bortezomib is a proteasome inhibitor that has shown clinical success following administration in relapsed MM patients (31) and has been found to re-sensitise malignant cells to the effects of chemotherapy (32). However, the emergence of a

bortezomib-resistant sub-clones ultimately leads to further relapse in patients (33). One putative mechanism of bortezomib resistance is the constitutive expression of NF-KB.

Although bortezomib can prevent de novo activation of the canonical pathway, it has no significant effect on constitutive NF-KB activity (24). In this study, we showed that direct competitive inhibition of NF-KB at the site of transcription led to the re-sensitisation of multiple myeloma cells to the effects of bortezomib. This synergistic effect is likely to be multi-factorial, but indicates that bortezomib and the PBDs have different molecular targets.

Chronic lymphocytic leukaemia (CLL) is another haematological malignancy that is frequently associated with constitutive NF-KB activation expression and over-activity of signalling pathways that promote survival and proliferation (34). The B-cell receptor (BCR) signalling pathway has been strongly implicated in CLL progression and is a key mediator of NF-KB signalling (35). Bruton’s tyrosine kinase (BTK) is a critical downstream mediator of BCR signalling in CLL and is constitutively activated in CLL patients. The targeting of this kinase with the BTK inhibitor, ibrutinib has shown notable effects in patients with relapsed CLL (36, 37) and this is mediated, at least in part, by the distal inhibition of NF-KB (38). In this study, we show the effects of PBDs on primary CLL cells in combination with ibrutinib. This combination produces cytotoxic synergy suggesting that the PBDs and ibrutinib target NF-KB through different mechanisms and/or that they have other, non-overlapping, molecular targets. Finally, we set out to determine whether the in vitro efficacy observed with DC-1-192 could be recapitulated in a system in vivo model of myeloma. Using an RPMI 8226 xenograft model we were able to show that DC-1-192 significantly prolonged the survival of the mice and this was at least as effective as bortezomib in the same model. The next step will be to evaluate the potential for synergistic interactions with DC-1-192 in vivo to determine how best to deploy this promising class of drugs for the treatment of NF-KB-driven tumours. In summary, the PBD monomer hybrids evaluated in this study showed low nanomolar toxicity in both primary CLL cells and myeloma cell lines. In addition, primary CLL cells were shown to be preferentially sensitive to the cytotoxic effects of the PBDs when compared with normal age-matched B- and T-lymphocytes. The PBDs demonstrated promising dual inhibitory properties on both the canonical and non-canonical NF-KB pathways; a characteristic that has been previously linked to significant anti-tumour effects in multiple myeloma (39). Furthermore, the PBDs evaluated here showed in vitro synergism with bortezomib and ibrutinib in MM and CLL respectively, providing a strong rationale for the use of these agents in the treatment of these B-cell neoplasms. Finally, the lead compound, DC-1-192, prolonged survival in a murine xenograft model of myeloma and was as effective as bortezomib at the concentrations used. Taken together, these data provide a strong rationale for the further development of these novel PBDs as anti-cancer therapeutics.

Example 2

Comparison of the NF-KB inhibitory effects of GWL-78 and DC-1-192

Enzyme Linked Immunosorbent Assay (ELISA) for NF-KB subunits

The human myeloma cell line JJN3 was treated for 4 hours with DC-1-192 (2.5nM and 5nM) or GWL-78 (2.5nM and 5nM). Cells treated with vehicle alone (0.05% DMSO in PBS) were used as a control. Pellets containing 5x1o⁶ cells were then harvested, and subsequently, nuclear extracts were produced prepared using a nuclear extraction kit (Active Motif). Total protein was determined by DC protein assay (Biorad) in each nuclear extract using a standard curve of known concentrations of BSA. Nuclear extracts containing lpg of total protein from each treatment were then added to an NF-KB family kit (Active Motif) in accordance with the manufacturer’s instructions. Levels of p05, p50, P52 and Rel B DNA binding were then assessed to determine relative levels of each subunit in the nucleus. The absorbance measurements (450nm) were subsequently converted into ng/ pg of nuclear extract for each sample. Finally, the data were expressed as a percentage of the untreated control and the effects of DC-1-192 and GWL-78 were compared using a paired t-test. Results

Both GWL-78 and DC-1-192 significantly inhibited the canonical NF-kB subunits, p05 and P50 when compared to untreated control cells. However, only DC-1-192 significantly inhibited the non-canonical subunit, P52. Furthermore, mole for mole DC-1-192 was a significantly more potent inhibitor of all four subunits when compared to GWL-78 (see Figure 8).

Example 3

DC-1-192: In Vivo Efficacy in Myeloma Model including Synergy with

Proteasome Inhibitor

Dosing: QD x 5 per week for three weeks from days 5-23.

Inoculation with Myeloma Cells: Day = o

Survival curves are shown in Figure 9.

Example 4

In vivo Test of Maximum Tolerated Dose (MTD) of DC-1-192 in NOD/SCID Female Mice

Study Objective - The objective of the study was to evaluate the maximum tolerated dose (MTD) of DC-1-192 in NOD/SCID female mice.

Experimental Design - The administration of the test articles and the animal numbers in each study group are shown in the following experimental design table.

Table 1. Study Design

Group N Treatment Dose Dosing Schedule

(mg/kg) Route

1 3 DC-1-192 1 i.v. Qd x 5 per week for 2 weeks

2 3 DC-1-192 0.5 i.v. Qd x 5 per week for 2 weeks

Note:

1. N: animal number;

2. Dosing volume: loul/g;

Study endpoints: The major endpoint is to evaluate the body weight loss and animal survival. The body weights will be recorded daily or every other day during dosing. The animal death is checked daily for survival. The tolerated dose is defined as the dose that results in less than 10% mean body-weight loss, and no treatment related death during the study. Materials

Animal Housing

Animals

Species: Mus Musculus Strain: NOD/SCID

Age: 6-8 weeks (age at inoculation) Sex: female

BW: 19.5-20.9 g Total number: 6 mice

Animal supplier: Beijing AK Bio-Technology Co. Ltd. (Beijing, China)

Animal Certificate No.:ii4024000i2536

Housing condition

The mice were kept in Individually Ventilated Cage (IVC) systems at constant temperature and humidity with 3 animals in each cage.

- Temperature: 20.5-24.3^

- Humidity: 45-57 %

Cages: Made of polycarbonate. The size is 325 mm x 210 mm x 180 mm. The bedding material was corn cob.

Diet: Mouse diet, Co⁶⁰ irradiation sterilized dry granule food. Animals had free access during the entire study period.

Water: Reverse osmosis RO water, autoclaved before using. Animals had free access to sterile drinking water.

Cage identification: the identification labels for each cage contained the following information: number of animals, sex, strain, receiving date, treatment, study number, group number, and the starting date of the treatment.

Animal identification: Animals were marked by ear coding (notch).

Test Articles

Testing article

Product identification: DC-1-192 Manufacturer: Transcriptogen Limited

Physical description: White Powder

Package and Storage condition: 9.i9mg/bottle, stored at -20°C

Experimental Methods and Procedures

Group assignment

Before commencement of treatment, all animals’ weights were measured. Since body weight can affect the effectiveness of any given treatment, mice were assigned to groups using a randomized block design based on their body weight. This ensures that both groups have comparable baselines.

The randomized block design was used to assign experimental animals to groups. First, the experimental animals were divided into homogeneous blocks according to their initial body weight. Secondly, within each block, randomization of experimental animals to treatments were conducted. By using a randomized block design to assign experimental animals, we ensured that each animal had the same probability of being assigned to a given treatment and therefore systematic error was reduced

Formulation - The dosing solutions were prepared in a sterile biosafety cabinet. The dosing solutions were freshly made before dosing.

Volume type: Adjust dosing volume for body weight (Dosing volume = io pl/g) Detailed formulation Instructions:

Table 2. Test Article preparation

Compounds Preparation Cone Dose Physical Storage

(mg/ (mg/ description

ml) kg)

Vehicle 5% DMSO, 95% saline Solution freshly made

Suspension before dosing

DC-1-192 9.19 mg DC-1-192 were dissolved 1 Solution -20°C (

Stock in 9.19ml DMSO, vortex to mix Suspension aliquots)

Solution well.

DC-1-192 0.14ml of lmg/ ml stock solution 0.1 1 Solution freshly made were diluted in 1.26ml saline, Suspension before dosing mix well to prepare 1.4ml of

o.img/ml dosing solution

DC-1-192 0.45ml of o.img/ ml solution 0.05 0.5 Solution freshly made were diluted in 0.45ml saline, Suspension before dosing mix well to prepare 0.9ml of

0.05mg/ml dosing solution

Ensure that formulation is mixed immediately before use by gently turning the tube up and down.

Test article administration

The treatment was initiated immediately post grouping per study design (Table 1). Observation and data collection

The animals were checked daily for morbidity and mortality. At the time of routine monitoring, the animals were checked for treatments on normal behaviour such as mobility, visual estimation of food and water consumption, body weight gain/loss, eye/hair matting and any other abnormal effect. Death and observed clinical signs were recorded in detail in the comment section of the datasheet for each animal. The body weight was recorded daily. The entire procedures of dosing and body weight measurement was conducted in a Laminar Flow Cabinet.

Animals showing obvious signs of severe distress were euthanized. Gross necropsy was performed on euthanized animals and observations on the major organs (heart, lung, liver, kidney, spleen, and intestine) were reported.

Termination - The study was termination 12 days post the first-dose.

Sampling - The liver, kidney, lungs and bone marrow of the 3 mice in group- 1 were collected and stored at -80°C according to the client’s request.

Results

Body Weights

The results of the body weight changes in the mice are shown in Figure 8.

Result Summary

In this study, the maximum tolerated dose of DC-1-192 was tested in non-tumour bearing NOD/SCID female mice. The body weight changes were shown in Figure 8. No animals in either study group were found dead or exhibited significant body weight loss during the whole study. On PG-D12, Administration of DC-1-192 0.5 and l.omg/kg once daily for five days in each of two weeks resulted in mean body weight increases of 4.5% 1.8% respectively. As a single agent, body weights of mice were maintained during the study.

Compliance

The protocol and any amendment(s) or procedures involving the care and use of animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee (LACUC) prior to conduct. During the study, the care and use of animals was conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratoiy Animal Care (AAALAC). Body Weights

Body Weights of the mice are provided in Table 3

able 3 - Body Weights of the Mice

65

Example 5

In Vivo Efficacy Study of Test Compound in the Systemic RPMI8226

Human Multiple Myeloma Xenograft Model in NOD/SCID Mice

Study Objective

The objective of the study was to investigate the in vivo efficacy of test compound DC-1- 192 alone and in combination with bortezomib in the treatment of the systemic RPMI8226 human multiple myeloma xenograft model in NOD/SCID Mice.

Experimental Design Abbreviations

The treatments were started at Day 5 (D5) following inoculation of the study animals with RPMI8226 myeloma tumour cells. The test article regimen and animal numbers in each group are shown in the following experimental design table.

Table 4. Study Design

Group N Treatment Dose Dosing Schedule

(mg/kg) Route

1 7 Vehicle i.v. Qd x 5 per week for 3 weeks

2 7 DC-1-192 1 i.v. Qd x 5 per week for 3 weeks

3 7 DC-1-192 1-3 i.v. Qd x 5 per week for 3 weeks

4 7 Bortezomib 0.6 i.v. BIW for 3 weeks

5 7 DC-1-192 1 i.v. Qd x 5 per week for 3 weeks

Study endpoints: The major endpoints of the study included the following:

1. Animal survival time: The major endpoint was animal survival. Animals were checked daily and any animals that were deteriorating clinically or moribund would be humanely euthanized with C0₂. Median Survival Times (MST) were calculated for each group. The increase in life-span (ILS) was calculated as follows: ILS (%) = Median Survival Time of drug treated group/ Median Survival Time of vehicle group - 1

2. Body Weight: Animal body weights were measured every day.

3. Histological evaluation: Gross autopsies were performed on the euthanized mice. Materials

Animal Housing

Animals

Species: Mus Musculus Strain: NOD SCID

Age: 8-9 weeks (age at inoculation) Sex: Female

BW:i5-9-23.6 g Total number: 42 (35 plus spare) mice Animal supplier: Beijing Anikeeper Biotech Co., Ltd (Beijing, China)

Animal Certificate No.: 11402400012541

Housing condition

The mice were kept in individually ventilated cage (IVC) systems at constant temperature and humidity with up to 5 animals in each cage.

- Temperature: 22.i-25.o°C.

- Humidity: 32-66 %

Cages: Made of polycarbonate. The size is 325 mm x 210 mm x 180 mm. The bedding material was corn cob.

Diet: Mouse diet, Co⁶⁰ irradiation sterilized dry granule food. Animals had free access during the entire study period.

Water: Reverse osmosis (RO) water, autoclaved before using. Animals had free access to sterile drinking water.

Cage identification: the identification labels for each cage contained the following information: number of animals, sex, strain, receiving date, treatment, study number, group number, and the starting date of the treatment.

Animal identification: Animals were marked by ear coding (notch). Test Articles

Test article

Product identification: DC-1-192 Manufacturer: Transcriptogen Limited

Physical description: powder

Package and Storage condition: 9.i9mg/bottle, stored at -20°C

Active Reference article

Product identification: Bortezomib Manufacturer: Meilun.

Physical description: powder

Package and Storage condition: lOOmg/vial, stored at -20°C

Experimental Methods and Procedures

Cell Culture

The RPMI8226 tumour cells were maintained in vitro in RPMI1640 medium supplemented with 10% heat inactivated fetal bovine serum at 37°C in an atmosphere of 5% C0₂ in air. The tumour cells were routinely sub-cultured twice per week by trypsin-EDTA treatment. The cells growing in an exponential growth phase were harvested and counted for tumour inoculation. Tumour Inoculation

One day before tumour inoculation, each mouse was irradiated with Co⁶⁰ (150 rad). On Study Day o, each mouse was inoculated intravenously into the tail vein with

RPMI8226 tumour cells (1 x ioe7) in 0.1 ml of PBS for tumour development. Study treatments were started at Day 5. The test article regimen for each study group is given in the experiment design table above.

Group assignment

Before grouping and treatment, all animals were weighed. Mice were assigned into groups using a randomized block design as follows:

First, the experimental animals were divided into homogeneous blocks based on their body weight. Secondly, within each block, randomization of experimental animals to different groups was undertaken. Using randomized block design to assign

experimental animals ensured that each animal had the same probability of being assigned to any given treatment groups, minimizing any systematic error.

Formulation - The dosing solutions were prepared in a sterile biosafety cabinet.

Table 5 The test article formulation preparation

Compounds Preparation Cone. Storage

(mg/ml)

Vehicle for 1.9 ml saline was added to 0.1ml DMSO, and Freshly

DC-1-192 vortexed to mix well to 2 ml Vehicle. made

DC-1-192 9-i9mg DC-1-192 was dissolved in 9.19ml 1 -20°C

Stock 100% DMSO, and vortexed to mix well.

Solution

DC-1-192 0.234ml of lmg/ml stock solution was diluted 0.13 Freshly in 1.566ml saline, and mixed well to prepare made 1.8 ml of o.i3mg/ml dosing solution

DC-1-192 0.42ml of lmg/ml stock solution was diluted in 0.1 Freshly

3.78ml saline, and mixed well to prepare 4.2 made ml of o.img/ml dosing solution

Vehicle for 20 mg mannitol was added to 50 ml saline, 4°C Bortezomid stirred and vortexed until clear. The solution weekly were filtered with a 0.2-micron filter to

produce the vehicle

Bortezomib 3 mg Bortezomib was added to 5 ml vehicle 0.6 Freshly stock (0.04% mannitol) and sonicated to produce made solution 5ml of 0.6 mg/ml homogeneous dosing

solution for single use.

Bortezomib 0.5 mL of 0.6 mg/ mL Bortezomib stock 0.06 Freshly solution was added to 4.5 mL vehicle (0.04% made mannitol) to make 5 mL of 0.06 mg/mL dosing

solution.

Ensure that formulation was mixed immediately before use by gently turning the tube up and down.

Observations and Data Collection

After inoculation, the animals were checked daily for morbidity and mortality. At the time of routine monitoring, the animals were checked for any effects of tumour growth and treatments on normal behaviour such as mobility, visual estimation of food and water consumption, body weight gain/loss (body weights were measured twice weekly or every day), eye/hair matting and any other abnormal effect. Death and observed clinical signs were recorded on the basis of the numbers of animals within each subset. The entire procedures of dosing as well as body weight measurement were conducted in a Laminar Flow Cabinet.

Termination

The whole study was terminated at Day 83. During the study, mice that reached the ethical endpoint (in a continuing deteriorating condition, showing obvious signs of severe distress and/or pain, having other severe clinical signs, i.e., prolonged diarrhoea, persistent anorexia, lethargy or failure to respond to gentle stimuli, laboured respiration, or inability to get to adequate food or water, etc.) were euthanized and necropsy performed. At termination, all the remaining mice were euthanized and necropsy performed. No samples were collected for the study.

Statistical Analysis

A Kaplan-Meier survival analysis was performed with the event of interest being animal death. The survival time was defined as the time from the day of tumour cell inoculation to either one day before the animal died or the day it was euthanized. For each group, the median survival time (MST) and the increased in life-span (ILS) were calculated. Kaplan-Meier plots were constructed for each group and the log-rank test was used to compare survival curves between groups. All data were analyzed using SPSS 18.0, with p < 0.05 considered to be statistically significant.

Results

Body Weights - The results of the body weight changes in the tumour bearing mice are shown in Figure 10. Body Weight (g)

Days after Tumour Inoculation

The survival times of different groups are shown in Table 6.

Table 6 Effects of DC-1-192 and Bortezomib on the Survival of Tumour Bearing Mice Groups Groups Treatment MST (days) ILS(%) P

Valueb

1 Vehicle 56 (40-6s)a

2 DC-1-192 (lmg/kg) 68 (58-8o)a 21.4 0.017

3 DC-1-192 (i.3mg/kg) 63 (52-69)a 12.5 0.152

4 Bortezomib (o.6mg/kg) 63 (50-84) a 12.5 0.091

5 DC-1-192+ Bortezomib (img/kg+o.6mg/kg) 63 (50-78) a 12.5 0.111

Note: a: The range of survival times. b:,vs. vehicle control

Survival Curves

The Kaplan-Meier curves of different treatment groups are shown in Figure 11. Individual survival curves of RPMI-8226 bearing mice in different treatment groups at Day 78 are shown in Figure 12.

Results: Summary and Discussion

In this study, the safety and therapeutic efficacy of DC-1-192 (lmg/kg), DC-1-192 (i.3mg/kg), Bortezomib (o.6mg/kg) and DC-i-192+Bortezomib (img/kg+o.6mg/kg) in the treatment of systemic RPMI8226 human multiple myeloma xenograft model were evaluated.

The life span of the vehicle group (Group-i) ranged from 40 to 65 days with an MST of 56 days. A significantly prolonged life span was observed in the DC-1-192 treated group

(lmg/kg , Group- 2) with an MST of 68 days and an ILS of 21.4% ip=o.ov ) when compared with the vehicle group . Treatment with DC-1-192 (i.3mg/kg, Group-3), Bortezomib (0.6 mg/kg, Group-4) and DC-i- 192+Bortezomib (1 mg/kg+0.6 mg/kg, Group-5) all produced a small anti-tumour effects with MSTs of 63 days, 63 days and 63 days, respectively. Calculated ILS were 12.5% (p= 0.152), 12.5% (p=o.09i), 12.5% (p=o.m) respectively, compared with vehicle group. No statistically significant difference in survival time was found between vehicle group and Group-3, 4 and 5.

Mice in Groups 2, 3, 4 and 5 lost body weight initially after the first dosing, which may have been related to treatment. However, they recovered by day 12. For the euthanized mice during the study, a variety of symptoms were observed, including hind limb paralysis, and tumour like growths on neck, axilla and lumbar vertebra.

In summary, the test compound DC-1-192 (lmg/kg) as a single agent showed statistically significant antitumour efficacy in a systemic RPMI8226 human multiple myeloma xenograft model in this study, and the treatment was well tolerated.

Compliance

The protocol and any amendment(s) or procedures involving the care and use of animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee (LACUC) prior to conduct. During the study, the care and use of animals was conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Conclusions

Thus, the examples demonstrate that compounds of formula (I) are cytotoxic in multiple myeloma cell lines and chronic lymphocytic leukaemia cells. In addition from the in vitro studies, compounds of formula (I) show synergism when combined with a proteasome inhibitor or a Bruton’s tyrosine kinase inhibitor in multiple myeloma cell lines and chronic lymphocytic leukaemia cells. In addition, in vivo studies showed that such combinations were well tolerated in test subjects.

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Claims

1. A compound of formula (I):

or salts, solvates, stereoisomers, tautomers or combinations thereof,

wherein the dotted lines indicate the optional presence of a double bond between Cl and C2 or C2 and C3;

R and R₂ are either

(i) R and R₂ together form a double bond;

(ii) R is H and R₂ is OH; or

(iii) R is H and R₂ is OC -₆ alkyl;

R₃, R₄ and R₅ are independently R_A, OR_A, halo, =0, =CH-R_A, (CH₂)_m-0R_A, (CH₂)_m- NHRB, (CH₂)_m-C0₂R_A, (CH₂)_m-C(0)R_A, (CH₂)_m-S0₂R_A, 0-S0₂R or CN;

each m is independently o, 1, 2, 3, 4 or 5;

where R_A is H; a C -₁₂ alkyl group; a C_6-12 aryl group; a C_5-10 heteroaryl group; a C_7-is aralkyl group; or a C_6-16 heteroaralkyl group; whereof the alkyl, aralkyl, or heteroaralkyl group optionally contains one or more carbon-carbon double or triple bonds, which may form part of a conjugated system; and the alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group is optionally substituted by one or more independently selected halo, hydroxy, amino, or nitro groups;

R_¾ is H, C -₆ alkyl or CH₂Ph;

p is 1, 2 or 3;

R₇ is R_B, OR_B or halo;

Y is N-R_B, S or O;

Y¹ is N or C-Rs;

Rs is RB, ORB or halo;

q is o or 1;

Het is

where the carbonyl of the Het group is attached to the Y- & Y’-containing heterocyclic ring;

Rg is R_B, OR_B or halogen;

Y² is N-RB, S or O;

Y3 is N or C-R_i0;

Y4 is N-R_B, S or O;

Y⁵ is N or C-R o;

R o is RB, ORB or halogen;

R^x is H, R_b, (CH₂)_m-0R_B, halo, (CH₂)_m-NHR_B and C0₂R_B; and

each R is independently selected from H, C -₆ alkyl and C -₆ haloalkyl.

2. A compound of formula (I) according to claim l, wherein the structure is:

or salts, solvates, stereoisomers, tautomers or combinations thereof.

3. A compound of formula (I) according to claim l or 2, wherein the compound is

or salts, solvates, stereoisomers, tautomers or combinations thereof.

4. A compound of formula (I) according to any of the preceding claims, wherein

R₃, R₄ and R₅ are independently H, C -_i2 alkyl, =CH₂, =CH-(C -_i2 alkyl group), (CH₂)_m- OH, (CH₂)_m-0-(C - ₂ alkyl group), (CH₂)_m-NH₂, (CH₂)_m-C0₂H, (CH₂)_m-C0₂CH₃, (CH₂)_m- C(0)CH₃, (CH₂)_m-S0₂CH₃, 0-S0₂CH₃ or CN.

5· A compound of formula (I) according to claim 1 or 2, wherein the compound is:

or salts, solvates, stereoisomers, tautomers or combinations thereof.

6. A compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof according to any of the preceding claims, wherein R^x is H,

C0₂CH₃, or C0₂CH₂CH₃.

7. A compound of formula (I) according to claim l, wherein the compound is:

or salts, solvates, stereoisomers, tautomers or combinations thereof.

8. A pharmaceutical composition comprising a compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier or diluent.

9. A pharmaceutical composition comprising a compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof according to claim 8, further comprising a therapeutic agent for treating a proliferative disease.

10. A pharmaceutical composition comprising a compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof according to claim 9, wherein the therapeutic agent for treating a proliferative disease is selected from 5- fluorouracil, afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, carfilzomib, ixazomib, marizomib, oprozomib, delanzomib, bosutinib, bryostatin-i, busulfan, calicheamycin,

camptothecin, carboplatin, io-hydroxycamptothecin, carmustine, celebrex,

chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans, cyclophosphamide, crizotinib, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptor binding agents, etoposide (VP16), etoposide glucuronide, etoposide phosphate, exemestane, fmgolimod, flavopiridol, floxuridine (FUdR), 3',5'-0-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitors, fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib, acalabrutinib (ACP-196), ONO/GS-4059, BGB- 3111, idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase, lapatinib,

lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine, sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (an aqueous form of DTIC), transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine, vincristine, vinca alkaloids, ZD1839, ipilimumab, tremelimumab, nivolumab, cemiplimab,

pembrolizumab, avelumab, durvalumab, atezolizumab and combinations thereof.

11. A pharmaceutical composition comprising a compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof, further comprising a therapeutic agent for treating a proliferative disease according to claim 9, wherein the therapeutic agent is a proteasome inhibitor, a Bruton’s tyrosine kinase inhibitor or a combination thereof.

12. A pharmaceutical composition comprising a compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof, further comprising a therapeutic agent for treating a proliferative disease according to claim 9, 10 or 11, wherein the therapeutic agent is bortezomib, ibrutinib or a combination thereof.

13. A kit comprising :

(a) a compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof according to any one of claims 1 to 7; and

(b) a therapeutic agent for treating a proliferative disease.

14. A compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof according to any one of claims 1 to 7, or a pharmaceutical composition according to any one of claims 8 to 12, or a kit according to claim 13, for use as a medicament.

15. A compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof according to any one of claims 1 to 7, or a pharmaceutical composition according to any one of claims 8 to 12, or a kit according to claim 13, for use in the treatment of a proliferative disease wherein the proliferative disease is a haematologic malignancy or a solid tumour.

16. A compound of formula (I) or salts, solvates, stereoisomers, tautomers or combinations thereof, or pharmaceutical composition, or kit, for use in the treatment of a proliferative disease according to claim 15, wherein the proliferative disease is multiple myeloma or chronic lymphocytic leukaemia.