EP4676934A1 - Porphyrins and their pharmaceutical uses - Google Patents

Abstract

The present invention relates to a method of preparing a porphyrin compound of formula (I) or metallo derivatives, salts and solvates thereof, in particular, to a tin (IV) phosphate derivative thereof; wherein each of R₁, R₂, R₃, R₄, R₅, and R₆, are independently selected from H, methyl, ethyl, halogen, methoxy, ethoxy, NR'R'', C_1-2 haloalkyl, C_1-2 alkanamine, and C(O)CH₃. The present invention also relates to such porphyrins for use as a medicament, in particular for the treatment of proliferative and/or malignant diseases (such as cancer) and for use for treating or inhibiting metastasis and to pharmaceutical compositions containing such porphyrins.

Description

PORPHYRINS AND THEIR PHARMACEUTICAL USES

FIELD OF THE INVENTION

The present invention relates to a method of preparing porphyrins, or metallo derivatives, salts and solvates thereof, especially tin mesoporphyrins, and to compounds prepared by such methods. The present invention also relates to such porphyrins or metallo derivatives, salts and solvates thereof, for use as a medicament, in particular for the treatment of proliferative and/or malignant diseases (such as cancer) and to pharmaceutical compositions containing such porphyrins.

BACKGROUND TO THE INVENTION

Cancer is a leading cause of death worldwide. There remains an urgent need for new cancer treatments to tackle the disease.

One promising treatment involves immunotherapies that target particular proteins. Preclinical data demonstrates that heme oxygenase-1 (HO-1) represents an important immunotherapeutic target in cancer (5).

The HO family of enzymes are responsible for the breakdown of haem to the biologically active products biliverdin, ferrous iron (Fe²⁺) and carbon monoxide (CO) (6). CO has been demonstrated to be immune suppressive (7-9), suggesting that it may play a role in blocking the immune response against tumours. Using an aggressive spontaneous model of breast cancer (MMTV-PyMT), the present inventors have demonstrated that a clinically relevant small molecule inhibitor of HO-1, tin mesoporphyrin (SnMP), in combination with standard of care chemotherapies, alleviates immune suppression and permitted CD8+ T-cells (the immune cell which can specifically target cancer) to control tumour growth (5, 10).

SnMP is the tin adduct of mesoporphyrin IX (3,3'-(7,i2-diethyl-3,8,i3,i7- tetramethylporphyrin-2,i8-diyl)dipropionic acid), as shown in Scheme 1 below. KC1- HO-ii is the tin phosphate salt of mesoporphyrin IX, its synthesis is shown in Scheme 2 below, and KCl-HO-ii is an HO inhibitor as shown in the examples below. SnMP and KCl-HO-ii cross react HO-1/2. The conventional synthesis of SnMP uses hemin (usually bovine derived) which raises issues with certification in certain markets and may be problematic due to ethical, material sourcing and health concerns.

SnMP, as a chloride salt, has been described in the literature as a HO-i and HO-2 inhibitor. SnMP has shown tumour suppression in combination with existing chemotherapeutic agents or immunotherapeutic agents. Studies by the present inventors suggest that SnMP in combination with standard care chemotherapies enables NK cells to play a role in controlling tumour growth.

Infant hyperbilirubinemia (also known as infant jaundice or neonatal hyperbilirubinemia) occurs in a new-born when the liver is unable to conjugate bilirubin so it can be excreted at a rate commensurate with bilirubin formation. Bilirubin comes from the release of haem as part of the physiological conversion from foetal to adult haemoglobin at birth. Stannsoporfin (tin (IV) mesoporphyrin IX dichloride) has been demonstrated to be of therapeutic value in treating hyperbilirubinemia.

Scheme 1: Mesoporphyrin IX.

The synthesis and use of SnMP has been described.

WO-A-2013/083659 discloses cancer therapies involving combinations of HO-i inhibitors, such as SnMP, and immunotherapeutic agents.

JP-A-2013/232129 discloses synthesizing stanzaporphin (tin (IV) mesoporphyrin IX dichloride) in high purity and in large quantities using hemin as a starting material, and to the resulting composition. US-B-io, 533,024 discloses methods for synthesizing metal mesoporphyrins by hemin transmetallation and subsequent hydrogenation of the tin protoporphyrin IX to form a metal mesoporphyrin.

US-B-8,530,458 discloses methods for synthesizing stannsoporfin (tin (IV) mesoporphyrin IX dichloride) in large quantities at high purity, and the compositions so produced and their use for treating infant hyperbilirubinemia.

There is a need, however, for improved methods of producing mesoporphyrin and dervatives thereof, especially for methods that are synthetic and do not rely on animal product (such as hemin). There is also a need for improved therapeutic forms of mesoporphyrin and derivatives thereof including new salt forms.

The present inventors have found that certain tin salts of mesophorphyrin dervatives can provide improved treatments of proliferative and/or malignant diseases; and also in inhibiting or reducing metastasis.

The present invention seeks to address these needs and to overcome problems associated with the prior art.

SUMMARY OF THE INVENTION

The present invention accordingly provides in a first aspect a method of preparing a compound of formula (I): or metallo derivatives, salts and solvates thereof, wherein the method comprises the step of adding a dicarboxylic acid compound of the following formula: or salts and solvates thereof, to a dialdehyde compound of the following formula: or salts and solvates thereof, and reacting to form a porphyrin diester compound of the following formula: or metallo derivatives, salts and solvates thereof; wherein each of Ri, R₂, R₃, R4, R₅, and Re, are independently selected from H, methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, C1-2 haloalkyl, Ci-₂ alkanamine, and C(O)CH₃; and R₇ and Rs are independently selected from C1-6 alkyl; and each R’ and R” are independently selected from H and C1-6 alkyl.

In a further aspect, there is provided a compound of formula (I) or metallo derivatives, salts and solvates thereof, wherein the compound is:

or salts and solvates thereof.

More suitably, there is provided a compound of formula (II) or (III) of the following formulas: or salts and solvates thereof.

More suitably for the compound of formula (II) or (III), Ri, R₃, R₅, and Re are methyl; and R₂, and R₄ are ethyl, resulting in a compound of formula (VI) or (VII): or salts and solvates thereof.

In a further aspect, there is provided a compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof, for use as a medicament.

In a further aspect, there is provided a compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof, for use in the treatment of a proliferative and/or malignant disease.

In a further aspect, there is provided a compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof, for use in a method of therapy.

In a further aspect, there is provided a pharmaceutical composition comprising a compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof, and a pharmaceutically acceptable excipient, carrier or diluent. In a further aspect, there is provided a compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof, for use in the manufacture of a medicament for treating a proliferative and/or malignant disease.

In a further aspect, there is provided a method of treatment of a patient suffering from a proliferative and/or malignant disease, comprising administering to said patient a therapeutically effective amount of a compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof .

In a further aspect, there is provided a compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof, wherein the compound is administered on its own or either simultaneously or sequentially with one or more immunotherapeutic agent or other anti-cancer agent for use in the treatment of a proliferative and/or malignant disease.

In a further aspect, there is provided a method of preparing a compound of formula (I): or metallo derivatives, salts and solvates thereof, wherein each of Ri, R₂, R₃, R4, R₅, and Re, are independently selected from H, methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, C1-2 haloalkyl, C1-2 alkanamine, and C(O)CH₃; and each R’ and R” are independently selected from H and C1-6 alkyl and the method comprises the steps of

(a) reacting a pyrrole A: salts and solvates thereof, with pyrrole B: salts and solvates thereof, wherein R_g and R_i0 are independently selected from benzyl, para-methoxybenzyl, and 2,4-dimethoxybenzyl; to form a diester compound of the following formula: solvates thereof;

(b) subjecting the diester compound or salts and solvates thereof, to a deprotection reaction to form a dicarboxylic acid compound of the following formula: solvates thereof,

(c) adding the dicarboxylic acid compound or salts and solvates thereof, to a dialdehyde compound of the following formula: salts and solvates thereof, and reacting to form a porphyrin diester compound of the following formula:

or salts and solvates thereof, wherein R₇ and Rs are each independently selected from C1-6 alkyl;

(d) hydrolysis of the porphyrin diester compound or salts and solvates thereof, to provide a porphyrin dicarboxylic acid of formula (I):

(e) converting the porphyrin dicarboxylic acid of formula (I) or salts and solvates thereof, to a metallo derivative of the porphyrin dicarboxylic acid of formula (I) or salts and solvates thereof; suitably, converting the porphyrin dicarboxylic acid of formula (I) or salts and solvates thereof, to a tin compound of the porphyrin dicarboxylic acid of formula (I) or salts and solvates thereof.

In a further aspect, the compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof or mixtures thereof, may be administered alone or in combination with other treatments, either simultaneously or sequentially depending upon the condition to be treated. In a further aspect, there is provided a compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof for use in treating or inhibiting metastasis.

DEFINITIONS

The following abbreviations are used throughout the specification Ac acetyl -C(O)CH₃; Et ethyl; E₃N triethyl amine; 5-FU Fluorouracil; . KCl-HO-ii tin phosphate salt of mesoporphyrin IX; Me methyl; MeOH methanol; Ph (phenyl); p-TSA/PTSA p- Toluenesulfonic acid; TBAC tetrabutylammonium chloride; TEOF triethyl orthoformate; TFA trifluoroacetic acid; THF tetrahydrofuran; and Ts (tosylate) p- toluene sulfonyl group [-SO₂-C6H₄-CH₃]; i.p. intraperitoneal; SD Standard deviation; and SEM Standard Error of the Mean.

Adoptive cell therapy is an immunotherapy using a subject’s own immune cells (or a donor’s immune cells) to treat diseases such as, for example, cancer or a viral infection. In adoptive cell therapy, T cells are isolated based upon their ability to expand in response to tumour or are genetically modified to target certain molecules on cells, such as antigens on cancer cells. The tumour reactive T cells are then expanded and infused back into the subject.

“TIL therapy” is a form of adoptive cell therapy that involves tumour infiltrating lymphocytes. In TIL therapy, tumour infiltrating lymphocytes that have penetrated a tumour are collected from a tumour biopsy taken from a subject and expanded in vitro for re-infusion into the patient. The tumour infiltrating lymphocytes are actively engaged in tumour destruction. In one method, following excision of the biopsy, DNA isolated from the tumour is sequenced to identify mutations found in the cancer that are recognized as neoantigens. In this method, mutated neoantigens are inserted into autologous dendritic cells, which are co-cultured with the tumour infiltrating lymphocytes. Tumour infiltrating lymphocytes are then assayed for neoantigen recognition. Those tumour infiltrating lymphocytes that recognize the neoantigen are then selected, expanded, and transfused back into the subject. In other methods, the T cells are expanded in number due to their capacity to recognize the tumour biopsy from which they were isolated and are infused back into the patient.

Another approach to adoptive cell therapy is called TCR therapy which involves engineering a subject’s or donor’s T cells to express a specific T-cell receptor (“TCR”). The T cell receptor is a heterodimer consisting of two subunits, TCRa and TCRp. Each subunit contains a constant region that anchors the receptor to the cell membrane and a hypervariable region that functions in antigen recognition. TCRs can recognize tumour specific proteins/peptides presented on the outside of cells. In TCR therapy, T cells are harvested from a subject’s or donor’s blood. The T cells are genetically modified in the laboratory to express a newT cell receptor. The T cells are expanded in number and infused back into the subject. The T cells with the new T cell receptor may target a patient’s cancer.

A further adoptive cell therapy is chimeric antigen receptor (“CAR”) T cell therapy (“CAR-T therapy”). In CAR-T therapy, one or more parts of a T cell receptor is changed into an antigen binding moiety, such as an antibody, antibody fragment or receptor ligand. A cancer associated antigen (tumour association antigen or “TAA”) is often expressed by tumours. The antibody or antibody fragment is targeted to the TAA. T cells targeted to a TAA may directly attack cancer cells. In CAR-T therapy, T cells are harvested from a subject’s or donor’s blood and genetically modified to express a CAR. T cells are expanded in number and infused back into the subject. CAR-T modifications target T cells specifically to the subject’s cancer.

“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.

“Independently” or “Independently selected” is used in the context of statement that, for example, “each R₁₅ R₂, is independently H, methyl, ethyl,...” and means that each instance of the functional group, e.g. Ri, is selected from the listed options independently of any other instance of Ri or R₂ in the compound. Hence, for example, H may be selected for the first instance of Ri in the compound; methyl may be selected for the next instance of Ri in the compound; and ethyl may be selected for the first instance of R₂ in the compound.

Ci-6 alkyl: refers to straight chain and branched saturated hydrocarbon groups, generally having from i to 6 carbon atoms; suitably a Ci-₅ alkyl; more suitably a Ci-₄ alkyl; more suitably a Ci-₃ alkyl; more suitably methyl or ethyl. Examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, pent- 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, n-octyl and the like. Ci-2 alkanamine refers to a methyl or ethyl group substituted with an amine NR’R” group wherein each R’ an R” is independently selected from H and Ci-6 alkyl. Hence, the Ci- 2 alkanamine is a CH₂NR’R”, CHNR”R”-CH₃, or CH₂-CH₂NR’R”.

Ci-₂ haloalkyl, refers to a methyl or ethyl group substituted with one or more halogen groups. Thus, the Ci-₂ haloalkyl is CH₂X, CHX₂, CX₃, CH₂CH₂X, CH₂CHX₂, CH₂CX₃, CHXCH₃, CHXCH₂X, CHXCHX₂, CHXCX₃, CX₂CH₃, CX₂CH₂X, CX₂CHX₂, or CX₂CX₃, wherein X is a halogen. More suitably, the Ci-₂ haloalkyl is CH₂X, CX₃, CH₂CH₂X, CHXCH₃, or CX₂CX₃. Each halogen may be selected independently. More suitably, each halogen in a Ci-₂ haloalkyl is the same.

“Halo,” “halogen” and “halogeno” maybe used interchangeably and each halogen is independently selected from fluoro, chloro, bromo, and iodo. Suitably each halogen is independently fluoro or chloro.

The term “or metallo derivatives, salts and solvates thereof’ means the porphyrin compound may alternatively comprise none of, or one or more of a metallo group, salts and solvates. Hence, the compound may be a metallo derivative, a salt, or a solvate or any combination of these options. Metallo derivatives are compounds where a metal is hosted in the center of the porphyrin ring. The metallo group of most interest is tin. For example, a compound of formula (II) or (III) as described herein is a tin metallo derivative of the compound of formula (I), which also comprises a phosphate salt.

The term “oncolytic virus” is used in accordance with its plain ordinary meaning and refers to a virus that preferentially infects and kills cancer cells. As the infected cancer cells are destroyed by oncolysis (directly lysing cells), they release new infectious virus particles or virions to help destroy the remaining tumour. Oncolytic viruses are thought not only to cause direct destruction of the tumour cells, but also to stimulate host antitumour immune system responses. Suitable oncolytic viruses are adenovirus, reovirus, measles, herpes simplex, Newcastle disease virus, vaccinia, or senecavirus.

The term “or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof’ means that pharmaceutically acceptable salt, solvate, tautomeric, stereoisomeric forms of the shown structure are also included. Mixtures thereof means that mixture of these forms maybe present, for example, the compounds of the invention may include both a tautomeric form and a pharmaceutically acceptable salt. “Pharmaceutically acceptable” substances refer to those substances which are within the scope of sound medical judgment suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit-to-risk ratio, and effective for their intended use.

“Pharmaceutical composition” refers to the combination of one or more drug substances and one or more excipients.

The term “or salts and solvates thereof’ means that the compound may alternatively comprise none of, or one or more of salts and solvates. Thus, the compound may be a salt, the compound may be a solvate, or the compound may be both a salt and a solvate. Suitably, the broader term “or metallo derivatives, salts and solvates thereof’ may be restricted to “or salts and solvates thereof’. Where the compound comprises salts, there maybe one or more salts, for example, there could be situations where only one of the carboxylic acid groups is a salt, or other situations where both carboxylic acid groups are salts. Suitably, any salt is a pharmaceutically acceptable salt.

As used herein, “solvate” refers to a complex of variable stoichiometry formed by a solute and a solvent. Pharmaceutically acceptable solvates may be formed for crystalline compounds wherein solvent molecules are incorporated into the crystalline lattice during crystallization. The incorporated solvent molecules can be water molecules or non-aqueous molecules, such as but not limited to, ethanol, isopropanol, dimethyl sulfoxide, acetic acid, ethanolamine, and ethyl acetate molecules.

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

The term “subject” as used herein refers to a human or non-human mammal. Examples of non-human mammals include livestock animals such as sheep, horses, cows, pigs, goats, rabbits and deer; and companion animals such as cats, dogs, rodents, and horses. Suitably the subject is a human.

“Therapeutically effective amount” of a drug refers to the quantity of the drug or composition that is effective in treating a subject and thus producing the desired therapeutic, ameliorative, inhibitory, or preventative effect. The therapeutically effective amount may depend on the weight and age of the subject and the route of administration, among other things.

“Treating” refers to reversing, alleviating, inhibiting the progress of, or preventing a disorder, disease or condition to which such term applies, or to reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of such disorder, disease or condition. “Treatment” refers to the act of “treating”, as defined immediately above.

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

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

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

Deprotection Reaction

Suitably the method further comprises a step of subjecting a diester compound of the following formula: or salts and solvates thereof, to a deprotection reaction to form a dicarboxylic acid compound of the following formula: or salts and solvates thereof.

More suitably, a step of subjecting a diester compound of the following formula: thereof, to a deprotection reaction to form a dicarboxylic acid compound of the following formula: salts and solvates thereof.

Suitably the diester compound or salts and solvates thereof, is subjected to the deprotection reaction to form a dicarboxylic acid compound using (i) tin and concentrated HC1; (ii) iron and ammonium formate; (iii) CrO₃ and acetic acid; (iv) N- bromo succinimide (NBS) and ozone; or (v) a hydrogenation reaction.

Suitably the deprotection reaction is carried out by refluxing the diester compound or salts and solvates thereof, with (i) tin and concentrated HC1.

Suitably the deprotection reaction is carried out using (ii) iron and ammonium formate in ethanol and water.

Suitably the deprotection reaction is carried out using(iii) CrO₃ and acetic acid.

Suitably the deprotection reaction is carried out using (iv) N-bromo succinimide (NBS) and ozone.

Suitably the deprotection reaction is (v) a hydrogenation reaction. Suitably the hydrogenation reaction is carried out using a Pd/ C catalyst.

Suitably the hydrogenation reaction is carried out in the presence of an organic base.

Suitably the hydrogenation is carried out in the presence of triethylamine. Diester Formation

Suitably the method comprises a step of reacting pyrrole A: with pyrrole B: solvates thereof, to form a diester compound of the following formula: salts and solvates thereof.

Suitably pyrrole A is: More suitably Ri is methyl and the method comprises a step of reacting pyrrole A: , to form a diester compound of the following formula:

Suitably pyrrole A is reacted with pyrrole B in the presence of a protic acid or montmorillonite clay. Suitably pyrrole A is reacted with pyrrole B in the presence of a protic acid. Suitably pyrrole A is reacted with pyrrole B in the presence of montmorillonite clay. Suitably the protic acid is a mineral acid, an organic sulfonic acid or trifluoroacetic acid. Suitably the mineral acid is boric acid, hydrobromic acid, hydrochloric acid, hydrofluoric acid, hydroiodic acid, nitric acid, perchloric acid, phosphoric acid, or sulfuric acid. Suitably, the organic sulfonic acid is benzene sulfonic acid, camphor sulfonic acid, ethane sulfonic acid, methane sulfonic acid, or para- toluene-sulfonic acid. More suitably, the protic acid is para-toluenesulfonic acid. Suitably the montmorillonite clay is K10 montmorillonite clay.

Suitably pyrrole A is reacted with pyrrole B in methanol.

Porphyrin Diester Formation

Suitably the method comprises the step of adding a di carboxylic acid compound of the following formula: thereof, to a dialdehyde compound of the following formula: salts and solvates thereof, and reacting to form a porphyrin diester compound of the following formula: ,or metallo derivatives, salts and solvates thereof.

More suitably a di carboxylic acid compound of the following formula: thereof, to a dialdehyde compound of the following formula: porphyrin diester compound of the following formula: metallo derivatives, salts and solvates thereof. Suitably (i) the dicarboxylic acid compound or salts and solvates thereof, is added to the dialdehyde compound or salts and solvates thereof, in the presence of a protic acid or montmorillonite clay. Suitably this addition is in the presence of a protic acid. Suitably this addition is in the presence of montmorillonite clay. Suitably the protic acid is a mineral acid, an organic sulfonic acid or trifluoroacetic acid. Suitably the mineral acid is boric acid, hydrobromic acid, hydrochloric acid, hydrofluoric acid, hydroiodic acid, nitric acid, perchloric acid, phosphoric acid, or sulfuric acid. Suitably, the organic sulfonic acid is benzene sulfonic acid, camphor sulfonic acid, ethane sulfonic acid, methane sulfonic acid, or para-toluene-sulfonic acid. More suitably, the protic acid is para-toluene sulfonic acid. Suitably the montmorillonite clay is Kio montmorillonite clay.

Suitably (i) the dicarboxylic acid compound or salts and solvates thereof, is added to the dialdehyde compound or salts and solvates thereof, using a trichloromethane and methanol solvent system. Suitably (i) trichloromethane is the major component (that is greater than 50% by volume) of the trichloromethane and methanol solvent system. Suitably (i) trichloromethane is present in a ratio of trichloromethane : methanol of at least 2:1; suitably in a ratio of at least 3:1; suitably in a ratio of at least 4:1. Suitably (i) trichloro methane : methanol is present in a ratio of about 5:1.

Suitably, subsequent to the addition of the dicarboxylic acid compound or salts and solvates thereof, with the dialdehyde compound or salts and solvates thereof, (ii) the intermediate is treated with zinc acetate and oxygen; or with zinc hexafluoroacetylacetonate dihydrate. More suitably, (ii) the intermediate is treated with zinc acetate and oxygen.

Suitably, subsequent to the treatment with zinc acetate and oxygen, or with zinc hexafluoroacetylacetonate dihydrate (iii) the further intermediate is treated with trifluoroacetic acid.

Hydrolysis of the Porphyrin Diester

Suitably the method further comprises the step of hydrolysis of the porphyrin diester compound of the following formula:

or metallo derivatives, salts and solvates thereof, to provide a dicarboxylic acid of formula (I): metallo derivatives, salts and solvates thereof. More suitably, the step of hydrolysis of the porphyrin diester compound of the following formula: provide a di carboxylic acid of formula (I): metallo derivatives, salts and solvates thereof.

Suitably the hydrolysis step is a base hydrolysis step. Suitably the base hydrolysis step is carried out using sodium hydroxide or lithium hydroxide.

Suitably the hydrolysis step is carried out in the presence of tributyl ammonium chloride (TBAC).

Suitably the hydrolysis step is carried out in di chloromethane and methanol solvent system.

Suitably the hydrolysis step is carried out in methanol.

In some aspects, the hydrolysis is carried out using lithium hydroxide in ethanol.

Converting to a Metallo Derivative

Suitably the method further comprises the step of converting the compound of formula (I) to a metallo derivative of the porphyrin dicarboxylic acid of formula (I) or salts and solvates thereof.

Suitably the metallo derivative is a tin compound of the porphyrin dicarboxylic acid of formula (I) or salts and solvates thereof. More suitably the metallo derivative is a tin mesylate compound, a tin oxalate compound, a tin phosphate compound or a tin tartrate compound of the porphyrin dicarboxylic acid of formula (I) or salts and solvates thereof.

Suitably the method further comprises the step of converting the compound of formula (I) or metallo derivatives, salts and solvates thereof, to a tin (IV) compound or salts and solvates thereof. Suitably the compound of formula (I) or salts and solvates thereof, is contacted with a source of tin to produce a tin compound of the porphyrin dicarboxylic acid of formula (I) or salts and solvates thereof. Suitably the method further comprises the step of converting the compound of formula (I) or metallo derivatives, salts and solvates thereof, to a metallo derivative that is a tin compound, wherein the tin compound is a tin acetate compound, a tin mesylate compound, a tin oxalate compound, a tin phosphate compound or a tin tartrate compound of the porphyrin dicarboxylic acid of formula (I) or salts and solvates thereof.

Suitably the method further comprises the step of converting the compound of formula (I) or metallo derivatives, salts and solvates thereof, to a metallo derivative that is a tin compound, wherein the tin compound is a tin mesylate compound, a tin oxalate compound, a tin phosphate compound or a tin tartrate compound of the porphyrin dicarboxylic acid of formula (I) or salts and solvates thereof.

More suitably the method further comprises the step of converting the compound of formula (I) or metallo derivatives, salts and solvates thereof, to a tin (IV) compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof. More suitably the method further comprises the step of converting the compound of formula (I) or metallo derivatives, salts and solvates thereof, to a metallo derivative that is a tin compound, wherein the tin compound is:

Suitably the compound of formula (I) or metallo derivatives, salts and solvates thereof, is converted to a tin (IV) compound using SnO or tin(II) chloride. Suitably the reaction with SnO is carried out in the presence of HC0₂H. Suitably the reaction with SnO is carried out in the presence of CH₃CO₂H.

Suitably the reaction with tin(II) chloride is carried out in the presence of acetic acid. Suitably the compound of formula (I) or salts and solvates thereof is refluxed with tin(II) chloride in acetic acid. Suitably subsequent to the reaction with SnO, the intermediate is treated with an acid.

Suitably subsequent to the reaction with SnO, the intermediate is treated with hydrochloric acid.

In other aspects, more suitably, the method further comprises the step of converting the compound of formula (I) or metallo derivatives, salts and solvates thereof, to a tin phosphate of formula (II) or (III): or salts and solvates thereof. contacted with a source of tin and a source of phosphate to produce More suitably, the compound of formula (I) or metallo derivatives, salts and solvates thereof, is converted to a tin (IV) compound using tin(II) pyrophosphate. More suitably the reaction with tin(II) pyrophosphate is carried out in the presence of acetic acid. More suitably the compound of formula (I) or metallo derivatives, salts and solvates thereof is refluxed with tin(II) pyrophosphate in acetic acid.

More suitably the method further comprises the step of converting the compound of formula (I) or metallo derivatives salts and solvates thereof, to a tin (IV) phosphate salt of formula (VII) or (VIII): or salts and solvates thereof.

Compounds

Suitably the compound of formula (I) or metallo derivatives, salts and solvates thereof is a tin salt of a compound of formula (I) wherein the tin salt is a tin acetate salt, a tin mesylate salt, a tin oxalate salt, a tin phosphate salt or a tin tartrate salt.

Suitably, the tin salt of a compound of formula (I) is a tin mesylate salt, a tin oxalate salt, a tin phosphate salt or a tin tartrate salt. More suitably, the tin salt is a tin mesylate salt, a tin oxalate salt, or a tin phosphate salt; or alternatively the tin salt is a tin oxalate salt, a tin phosphate salt or a tin tartrate salt; or alternatively the tin salt is a tin mesylate salt, a tin phosphate salt or a tin tartrate salt.

More suitably, the tin salt is a tin mesylate salt or a tin phosphate salt; or alternatively the tin salt is a tin oxalate salt or a tin phosphate salt or a tin tartrate salt; A tin salt of a compound of formula (I) wherein the tin salt is a tin acetate salt, a tin mesylate salt, a tin oxalate salt or a tin phosphate salt

More suitably, for any of the above tin salts of a compound of formula (I) the substituents Ri, R₃, R₅, and Re are methyl; and R₂, and R4 are ethyl. Suitably the compound of formula (I) or metallo derivatives, salts and solvates thereof, is a compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof.

More suitably the compound of formula (I) or metallo derivatives, salts and solvates thereof, is a compound of compound of formula (II), or (III), or (IV), or (V), or (VI), wherein the substituents Ri, R₃, R₅, and Re are methyl; and R₂, and R4 are ethyl. Such compounds have the formula: or (XI), or salts and solvates thereof.

Synthesis of Pyrrole A

Suitably, the method further comprises (i) a step of reacting a compound of the following formula: hereof, to produce an oxime of the following formula: salts and solvates thereof.

More suitably, step (i) reacts a compound of the following formula: hereof, to produce an oxime of the following formula: salts and solvates thereof.

Suitably, the step (i) uses sodium nitrite and acetic acid to produce the oxime.

Suitably, the method further comprises a step (ii) of a cyclisation reaction of the oxime or salts and solvates thereof with R₂-CH₂C(0)CH₃ to produce a pyrrole of the following formula:

20 salts and solvates thereof.

Suitably R₂ is acetyl such that R₂-CH₂C(0)CH₃ is acetyl acetone and the cyclisation reaction step (ii) of the oxime or salts and solvates thereof with acetyl acetone produces a pyrrole of the following formula: salts and solvates thereof.

Suitably the cyclisation reaction of step (ii) is carried out in the presence of one or more of sodium acetate, acetic acid and zinc.

Suitably the method further comprises (iii) a step of a selective reduction of: hereof, to produce a pyrrole compound of the following formula: or salts and solvates thereof.

Suitably, the selective reduction is a Wolf-Kishner reduction, a Clemmenson reduction, a catalytic hydrogenation or using TsNHNH₂, then a NaBH₄CN reduction, such selective reductions are well-known in the literature (11). Suitably, the Wolf-Kishner reduction is carried out using NH₂NH₂ and KOH. Suitably, the Clemmensen reduction is carried out using Zn-Hg and HC1. Suitably, the catalytic hydrogenation is carried out using Pt-C, H₂ under pressure. Suitably, the method further comprises (iv) a step or reacting a pyrrole compound of the following formula: or salts and solvates thereof, to add an acyloxy group to form Pyrrole A: or salts and solvates thereof.

Suitably, step (iv) is catalysed with lead tetraacetate.

More suitably, Ri is methyl, and step (iv) forms Pyrrole A: Synthesis of Pyrrole B

Suitably the method further comprises (a) a step of converting an amine of the following formula: or salts and solvates thereof, to an isocyanide of the following formula: or salts and solvates thereof. Suitably step (a) is carried out using KOH and Benzyltrimethylammonium bromide.

Suitably the method further comprises (b) a step of a cyclisation reaction between an isocyanide of the following formula: thereof, and an alkyne of the following formula: or salts and solvates thereof, to form pyrrole compound of the following formula: salts and solvates thereof. 3, and the alkyne has the following formula: ts and solvates thereof.

Cyclisation reactions of this type have been discussed in the literature (12). Suitably the cyclisation step (b) is carried out using a Cu catalyst. Suitably the cyclisation step (b) is carried out using a.Cu catalyst in the presence of triphenylphospine (PPh₃). Suitably the Cu catalyst is Cu₂0. Suitably the Cu catalyst is present in from 1 to 10 mol%; suitably from 4 to 6 mol %; more suitably in 5 mol%. Suitably the triphenylphosphine is present in from 10 to 30 mol%; suitably in from 15 to 25 mol%; more suitably in 20 mol%. Suitably R4 is C(O)CH₃, and the method further comprises (c) a step of a selective reduction of the pyrrole compound: ereof, to form Pyrrole B: salts and solvates thereof.

More suitably, R₃ is methyl, and step (c) forms Pyrrole B: salts and solvates thereof.

Suitably, the selective reduction is a Wolf-Kishner reduction; a Clemmenson reduction; a catalytic hydrogenation; or using TsNHNH₂, then a NaBH₄CN reduction; such selective reductions are well-known in the literature (11). Suitably, the Wolf-Kishner reduction is carried out using NH₂NH₂ and KOH. Suitably, the Clemmensen reduction is carried out using Zn-Hg and HC1. Suitably, the catalytic hydrogenation is carried out using Pt-C, H₂ under pressure.

Synthesis of Dialdehyde

Suitably, the method further comprises (i) a step of reacting a compound of the following formula: or salts and solvates thereof, to produce an oxime of the following formula: solvates thereof.

Suitably, the step (i) uses sodium nitrite and acetic acid to produce the oxime. Suitably, the method further comprises a step (ii) of a cyclisation reaction of the oxime or salts and solvates thereof, with a compound of the following formula: Suitably, the method further comprises (ia) a step of reacting a compound of the following formula: or salts and solvates thereof, to produce an oxime of the following formula: solvates thereof.

Suitably, the method further comprises a step (iia) of a cyclisation reaction of the oxime or salts and solvates thereof, with a compound of the following formula: Suitably the cyclisation reaction of step (iia) is carried out in the presence of one or more of sodium acetate, acetic acid and zinc.

Suitably the method further comprises a step (iii) where a pyrrole compound of the following formula: salts and solvates thereof; is coupled with a pyrrole compound of the following formula: salts and solvates thereof; to produce a pyrrole diester compound of the following formula: salts and solvates thereof.

Suitably, the method further comprises (i) a step of reacting a compound of the following formula:

O O

Suitably, the method further comprises a step (ii) of a cyclisation reaction of the oxime or salts and solvates thereof, with a compound of the following formula: produce a pyrrole of the following formula: salts and solvates thereof.

Suitably, the step (i) or (ia) uses sodium nitrite and acetic acid to produce the oxime.

Suitably the cyclisation reaction of step(ii) or (iia) is carried out in the presence of one or more of sodium acetate, acetic acid and zinc. Suitably the cyclisation reaction of step(ii) or (iia) is carried out in the presence of sodium acetate, acetic acid and zinc.

Suitably, step (iii) uses Br₂ as a reagent to produce the pyrrole diester compound.

Suitably step (iii) is carried out in methanol.

Suitably step (iii) couples two identical pyrrole compounds, i.e. R₅ = Re and R₇ = Rs.

Suitably the method further comprises a step (iv) where the pyrrole diester compound of the following formula: or salts and solvates thereof; is subjected to selective hydrolysis to produce the pyrrole diacid compound of the following formula: salts and solvates thereof. Suitably, the selective hydrolysis of step (iv) is carried out under hydrogenation conditions. Suitably, the selective hydrolysis of step (iv) is carried out using a Pd/ C catalyst.

Suitably the method further comprises a step (v) a selective reduction of the pyrrole diacid compound of the following formula: or salts and solvates thereof; to a dialdehyde compound of the following formula: or salts and solvates thereof.

Ri

Ri is selected from H, methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl, Ci- 2 alkanamine, and C(O)CH₃. Suitably Ri is selected from H, methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl and Ci-₂ alkanamine.

Suitably, in some aspects, Ri is selected methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-2 haloalkyl, Ci-₂ alkanamine, and C(O)CH₃.

In an alternative aspect, Ri is H. More suitably, Ri is selected from H, methyl, ethyl, halogen, methoxy, ethoxy and NR’R”. More suitably, Ri is selected from methyl, ethyl, methoxy and ethoxy.

More suitably, Ri is methyl or ethyl. Most suitably Ri is methyl.

R₂ R₂ is selected from H, methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl, Ci- ₂ alkanamine, and C(O)CH₃. Suitably R₂ is selected from H, methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl and Ci-₂ alkanamine.

Suitably, in some aspects, R₂ is selected methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl, Ci-₂ alkanamine, and C(O)CH₃.

In an alternative aspect, R₂ is H.

More suitably, R₂ is selected from H, methyl, ethyl, halogen, methoxy, ethoxy and NR’R” and C(O)CH₃. More suitably, R₂ is selected from methyl, ethyl, methoxy, ethoxy and C(O)CH₃.

More suitably, R₂ is methyl or ethyl. Most suitably R₂ is ethyl.

R3

R₃ is selected from H, methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, C_x-2 haloalkyl, Ci- ₂ alkanamine, and C(O)CH₃. Suitably R₃ is selected from H, methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl and Ci-₂ alkanamine.

Suitably, in some aspects, R₃ is selected methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-2 haloalkyl, Ci-₂ alkanamine, and C(O)CH₃.

In an alternative aspect, R₃ is H.

More suitably, R₃ is selected from H, methyl, ethyl, halogen, methoxy, ethoxy and NR’R”. More suitably, R₃ is selected from methyl, ethyl, methoxy and ethoxy.

More suitably, R₃ is methyl or ethyl. Most suitably R₃ is methyl.

R4 is selected from H, methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl, Ci- ₂ alkanamine, and C(O)CH₃. Suitably R4 is selected from H, methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl and Ci-₂ alkanamine.

Suitably, in some aspects, R4 is selected methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl, Ci-₂ alkanamine, and C(O)CH₃.

In an alternative aspect, R4 is H.

More suitably, R4 is selected from H, methyl, ethyl, halogen, methoxy, ethoxy and NR’R” and C(O)CH₃. More suitably, R4 is selected from methyl, ethyl, methoxy, ethoxy and C(O)CH₃. More suitably, R4 is methyl or ethyl. Most suitably R₄ is ethyl.

R5

R₅ is selected from H, methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl, Ci- 2 alkanamine, and C(O)CH₃. Suitably R₅ is selected from H, methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl and Ci-₂ alkanamine.

Suitably, in some aspects, R₅ is selected methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, C1-2 haloalkyl, Ci-₂ alkanamine, and C(O)CH₃.

In an alternative aspect, R₅ is H.

More suitably, R₅ is selected from H, methyl, ethyl, halogen, methoxy, ethoxy and NR’R”. More suitably, R₅ is selected from methyl, ethyl, methoxy and ethoxy.

More suitably, R₅ is methyl or ethyl. Most suitably R₅ is methyl.

£6

Re is selected from H, methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl, Ci- ₂ alkanamine, and C(O)CH₃. Suitably Re is selected from H, methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl and Ci-₂ alkanamine.

Suitably, in some aspects, Re is selected methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl, Ci-₂ alkanamine, and C(O)CH₃.

In an alternative aspect, Re is H.

More suitably, Re is selected from H, methyl, ethyl, halogen, methoxy, ethoxy and NR’R”. More suitably, Re is selected from methyl, ethyl, methoxy and ethoxy.

More suitably, Re is methyl or ethyl. Most suitably Re is methyl.

R1, R₂, R₂, Rd, R- and Re

Suitably, one, two or three of Ri, R₂, R₃, R4, R₅ and Re, are independently selected from H, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl, Ci-₂ alkanamine, and C(O)CH₃; and the remaining of Ri, R₂, R₃, R4, R₅ and Re are independently selected from methyl and ethyl.

Suitably, one, two or three of Ri, R₃, R₅ and Re are independently selected from H, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl, Ci-₂ alkanamine, and C(O)CH₃; and the remaining of Ri, R₃, R₅ and Re are methyl; and R₂ and R4 is ethyl. More suitable, one of Ri, R₃, R₅ and Re is independently selected from H, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl, Ci-₂ alkanamine, and C(O)CH₃; and the remaining of Ri, R₃, R₅ and Re are methyl; and R₂ and R4 is ethyl

Additionally suitably, optionally one or two of Ri, R₃, R₅ and Re are independently selected from H, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl, Ci-₂ alkanamine, and C(O)CH₃; and the remaining of Ri, R₃, R₅ and Re are methyl; and one of R₂ and R4 is selected from H, methyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl, Ci-₂ alkanamine, and C(O)CH₃; and the other of R₂ and R₄ is ethyl. Hence, when the option is not taken then R_x, R₃, R₅ and Re are methyl.

Additionally suitably, optionally one of Ri, R₃, R₅ and Re are independently selected from H, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl, Ci-₂ alkanamine, and C(O)CH₃; and the remaining of Ri, R₃, R₅ and Re are methyl; and R₂ and R4 are independently selected from H, methyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl, Ci-₂ alkanamine, and C(O)CH₃. Hence, when the option is not taken then Ri, R₃, R₅ and Re are methyl.

For any of the above four paragraphs, suitably the relevant number of Ri, R₃, R₅ and Re are independently selected from H, ethyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl, and Ci-₂ alkanamine. More suitably, the relevant number of Ri, R₃, R₅ and Re are independently selected from H, ethyl, halogen, methoxy, ethoxy and NR’R”. More suitably the relevant number of Ri, R₃, R₅ and Re are independently selected from ethyl, methoxy and ethoxy.

For either of the preceding paragraphs starting additionally suitably, then more suitably the relevant number of R₂ and R4 are independently selected from H, methyl, halogen, methoxy, ethoxy, NR’R”, Ci-₂ haloalkyl and Ci-₂ alkanamine. More suitably the relevant number of R₂ and R₄ are independently selected from H, methyl, halogen, methoxy, ethoxy and NR’R”. More suitably the relevant number of R₂ and R₄ are independently selected from methyl, methoxy and ethoxy.

More suitably, Ri, R₃, R₅, and Re are methyl; and R₂, and R₄ are ethyl.

R₇ is C1-6 alkyl. Suitably R₇ is selected from Ci-₅ alkyl. More suitably, R₇ is selected from Ci-₄ alkyl.

Suitably, R₇ is selected from methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, and t-butyl. Suitably, R₇ is selected from methyl, ethyl, n-propyl and i-propyl. More suitably, R₇ is selected from methyl and ethyl. Most suitably, R₇ is methyl. Rs

Rs is Ci-6 alkyl. Suitably Rs is selected from Ci-₅ alkyl. More suitably, Rs is selected from Ci-4 alkyl.

Suitably, Rs is selected from methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, and t-butyl. Suitably, Rs is selected from methyl, ethyl, n-propyl and i-propyl. More suitably, Rs is selected from methyl and ethyl. Most suitably, Rs is methyl.

Suitably R₇ and Rs are the same.

Ba

R₉ is selected from benzyl, para-methoxybenzyl, and 2,4-dimethoxybenzyl. Suitably, R₉ is selected from benzyl and para-methoxybenzyl. More suitably, R₉ is benzyl.

Rio

Rio is selected from benzyl, para-methoxybenzyl, and 2,4-dimethoxybenzyl. Suitably, Rio is selected from benzyl and para-methoxybenzyl. More suitably, R_i0 is benzyl.

Suitably R_g and R_i0 are the same.

NR’R”

For NR’R” each R’ and R” are independently selected from H and C1-6 alkyl. Thus, NR’R” is NH₂, NHCI-6 alkyl or N(CI-6 alkyl )₂. Suitably, in some aspects, NR’R” is NH₂.

Suitably in other aspects, NR’R” is NHC1-6 alkyl or N(CI-6 alkyl)₂. More suitably, NR’R” is NHCI-6 alkyl. More suitably, NR’R” is NHCH₃ or NHCH₂CH₃. More suitably, NR’R” is NHCH₃.

Proliferative and/or Malignant Disease

There is described a compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof for use in the treatment of a proliferative and/ or malignant disease.

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

For example, a proliferative and/or malignant disease may comprise a solid tumour, for example, a carcinoma or a sarcoma. Carcinomas include malignant neoplasms derived from epithelial cells which infiltrate, for example, invade, surrounding tissues and give rise to metastases. Adenocarcinomas are carcinomas derived from glandular tissue, or from tissues that form recognizable glandular structures.

The invention finds application in the treatment of proliferative and/ or malignant diseases.

In certain aspects a method of treating a proliferative and/or malignant disease is provided, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the disclosure or salts and solvates thereof or a composition comprising a compound of the disclosure or salts and solvates thereof.

Suitably, the proliferative and/or malignant disease is selected from breast, lung, brain and central nervous system cancers, carcinomas, gastrointestinal cancers, hormonal cancers, leukemias, liver cancer, lymphomas, sarcomas and fibrosarcomas, skin cancers and melanomas, urinary tract and reproductive cancers, and miscellaneous other cancers.

Suitably, the proliferative and/ or malignant disease is selected from brain and central nervous system cancers, carcinomas, gastrointestinal cancers, hormonal cancers, leukemias, liver cancer, lymphomas, sarcomas and fibrosarcomas, skin cancers and melanomas, urinary tract and reproductive cancers, and miscellaneous other cancers.

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

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

Gastrointestinal cancers that may be treated include extrahepatic bile duct cancer, bowel cancer, colon cancer, colon and rectum cancer, colorectal cancer, gallbladder cancer, gastric ( stomach) cancer, gastrointestinal carcinoid tumour, gastrointestinal carcinoid tumours, gastrointestinal stromal tumours, bladder cancers, islet cell carcinoma (endocrine pancreas), pancreatic cancer, islet cell pancreatic cancer, prostate cancer, rectal cancer, salivary gland cancer, small intestine cancer, colon cancer, and polyps associated with colorectal neoplasia. Lung and respiratory cancers that maybe treated include bronchial adenomas/carcinoids, esophagus cancer esophageal cancer, esophageal cancer, hypopharyngeal cancer, laryngeal cancer, hypopharyngeal cancer, lung carcinoid tumour, non-small cell lung cancer, small cell lung cancer , small cell carcinoma of the lungs , mesothelioma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nasopharyngeal cancer, oral cancer, oral cavity and lip cancer, oropharyngeal cancer; paranasal sinus and nasal cavity cancer, and/ or pleuropulmonary blastoma.

Hormonal cancers that may be treated include: parathyroid cancer, pineal and supratentorial primitive neuroectodermal tumours, pituitary tumour, thymoma and thymic carcinoma, thymoma, thymus cancer, thyroid cancer, cancer of the adrenal cortex, and/or ACTH-producing tumours.

Leukemias that may be targeted (may also be considered to include other blood cell malignancies) include acute lymphoblastic, acute myeloid, acute lymphocytic, acute myelogenous leukemia, chronic myelogenous, hairy cell, erythroleukemia, lymphoblastic, myeloid, lymphocytic, myelogenous, leukemia, hairy cell, T-cell, monocytic, myeloblastic, granulocytic, gross, hand mirror-cell, basophilic, hemoblastic, histiocytic, leukopenic, lymphatic, Schilling’s, stem cell, myelomonocytic, monocytic, prolymphocytic, promyelocytic, micromyeloblastic, megakaryoblastic, megakaryocytic, rieder cell, bovine, aleukemic, mast cell, myelocytic, plamsa cell, subleukemic, multiple myeloma, nonlymphocytic, chronic myelogenous leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin’s disease, non-Hodgkin’s lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease , myelodysplastic syndrome, and myelodysplasia and/or chronic myelocytic leukemias.

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

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

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

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

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

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

More suitably, the proliferative and/or malignant disease is bladder cancer, brain cancer, breast cancer, hepatocellular carcinoma, acute myeloid leukaemia, lung cancer, non-small cell lung cancer, non-Hodgkin's lymphoma, glioma, glioblastoma, melanoma, metastatic melanoma, ovarian cancer or prostate cancer.

Suitably, the compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof, is administered either on its own or simultaneously or sequentially with one or more further therapeutic agents.

Suitably, the one or more further therapeutic agents are selected from one or more immunotherapeutic agent or other anti-cancer agent.

Suitably the immunotherapeutic agent may include one or more cancer vaccine and/ or therapeutic antibody and/or small molecule, and/or RNA/DNA-based agent, and/or viral agent (such as oncolytic viruses or carriers) and/ or cellular therapy or genetically engineered cellular therapy. A cancer vaccine is an agent, a cell -based agent, molecule, or immunogen which stimulates, harnesses or elicits an endogenous immune response in an individual or subject against one or more tumour antigens.

Cellular therapy or genetically engineered cellular therapy comprises adoptive cell therapies such as TIL therapy, CAR-T therapy and other immune effector cells. In some embodiments, the immune effector cells comprise or consist of NK cells, T cells, B-cells, dendritic cells, macrophages, peripheral blood mononuclear cells (PBMCs), ab T cells, gd T cells, regulatory T cells, NK T cells, or mesenchymal cells or a combination thereof.

As used herein, a tumour antigen is broadly defined as an antigen specifically expressed within or by tumour or cancer cells. The antigen may be expressed at the cell surface where it is recognized by components of the humoral immune system such as B lymphocytes (B cells). Intracellular tumour antigens (including those associated with secreted, cytoplasmic and nuclear proteins) are processed into shorter peptide fragments which form complexes with major histocompatibility complex (MHC) molecules I and II, also referred to as human leukocyte antigen (HLA) (and are presented on the cell surface of cancer cells, where they are recognized by the T cell receptors (TCF s) of T lymphocytes (T cells). Antigens can also include foreign and selflipids (such as those presented on CDi molecules) or aberrant glycosylation or post translational modifications. Preferably, the tumour antigen is one which is not expressed by normal cells, or at least not expressed to the same level as in tumour cells. Currently produced cancer vaccines being developed for the treatment of humans activate the humoral immune system (i.e., the antibody dependent immune response) or the cell-mediated immune system including T lymphocytes (T cells) which are capable of specifically recognizing and killing tumour cells.

A cancer vaccine may enhance the presentation of one or more tumour antigens to both antigen presenting cells (e.g., macrophages and dendritic cells) and/or to other immune cells such as T cells, B cells, and NK cells. In some examples, preparations and/or formulations of cancer vaccines may be used together with one or more adjuvants that are well known in the art, to induce an immune response or to increase an immune response.

An adjuvant is a substance incorporated into or administered with antigen which potentiates the immune response. Adjuvants may enhance the immunological response by providing a reservoir of antigen (extracellularly or within macrophages/DCs), activating antigen presenting cells to stimulate specific sets of lymphocytes. Adjuvants of many kinds are well known in the art. Specific examples of adjuvants include monophosphoryl lipid A (MPL, SmithKline Beecham) , a congener obtained after purification and acid hydrolysis of Salmonella Minnesota Re 595 lipopolysaccharide; saponins , including QS21 ( SmithKlineBeecham) a pure QA-21 saponin purified from Quillj a saponaria extract ; DQS21 , described in PCT application WO96/33739 ( SmithKline Beecham) ; QS-7, QS-17, QS-18, and QS-L1 (So et al., Mol Cells (1997) 7 : 178-186) ; ISCOMATRIX adjuvant, a cage-like structure composed of saponin, phospholipid, and cholesterol ( see, e.g., Maraskovsky et al. , Clin. Cancer Res. (2004) 10:2879-2890); incomplete Freund’s adjuvant; complete Freund’s adjuvant; montanide ; alum; CpG oligonucleotides (see e.g. Kreig et al., Nature 374:546-9, 1995) and other immunostimulatory oligonucleotides including poly-IC and poly-ICLC (Hiltonol©); and various water-in-oil emulsions prepared from biodegradable oils such as squalene and/or tocopherol.

Suitably, cancer cells in an individual express a tumour antigen which is immunologically cross reactive with the cancer vaccine. Suitably the tumour antigen is expressed in the cancer cells but not normal somatic cells of the individual. Suitable tumour antigens for use in an immunotherapeutic agent such as a cancer vaccine include: : P1A, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-I, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-I, RAGE-1, LB33/MUM-1, FRAME, NAG, MAGE-Xp2 (MAGE-B2) , MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), tyrosinase, brain glycogen phosphorylase, Melan-A, MAGE- C1/CT7, MAGE-C2, NY-ESO-I, LAGE-I, SSX-I, SSX- 2 (HOM-MEL-40) , SSX-3, SSX-4, SSX-5, SCP-I and XAGE and immunogenic fragments thereof. Other types of tumour antigens include overexpressed or mutated proteins and differentiation antigens particularly melanocyte differentiation antigens such as p53, ras, CEA, MUC1, PMSA, PSA, tyrosinase, Melan-A, MART-1, gpioo, gp75, alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-i, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA- A11, hsp7O-2, KIAAO2O5, Mart2, Mum-2, and 3 , neo-PAP, myosin class I, OS-9, pml- RAR.alpha, fusion protein, PTPRK, K-ras, N-ras, Triosephosphate isomeras, GnTV, Herv-K-mel, NA-88, SP17, TRP2-Int2, (MART-I), E2A-PRL, H4-RET, IGH-IGK, MYL- RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, pi8serbB2, pi8oerbB-3, c-met, nm- 23H1, PSA, TAG- 72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, alpha-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\17OK, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, and TPS and tyrosinase related proteins such as TRP-1, TRP-2. Other suitable antigens include cancer antigens in the following classes: cancer testis antigens (e.g., HOM-MEL-40), differentiation antigens (e.g., HOM-MEL-55), overexpressed gene products (HOM- MD-21), mutated gene products (NY-COL-2), splice variants (HOM-MD-397), splice peptides (13, 14, 15), gene amplification products (HOM-NSCLC-11) and cancer related autoantigens (HOM- MEL-2.4) as reviewed in Cancer Vaccines and Immunotherapy (2000) Eds Stern, Beverley and Carroll, Cambridge University Press, Cambridge. In some exemplary embodiments, the antigen is a tumour antigen selected from the group consisting of MUC1, MAGE, BAGE, RAGE, CAGE, SSX-2, NY-ESO-1, FRAME, PSMA, tyrosinase, melan-A, and mixtures thereof. In some variations, the cancer antigen is a mammalian protein. In some variations, the cancer antigen is a human protein. In some variations, the full-length protein may be employed as the antigen. In some variations, peptides comprising an antigenic fragment of these proteins may be used as the tumour antigen.

Other suitable tumour antigens are well known in the art (see for example WO 00/20581 ) The sequences of these tumour antigens are readily available from public databases but are also found in WO 1992/020356, WO 1994/005304, WO 1994/023031, WO 1995/020974, WO 1995/023874, and WO 1996/026214.

In some embodiments, a method of treating a subject using a compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof, and an immunotherapeutic agent as disclosed herein, may further comprise administering one or more tumour therapies to treat the tumour. Such therapies include, for example, tumour medicaments, radiation and surgical procedures.

As used herein, a “tumour medicament” refers to an agent which is administered to a subject for the purpose of treating a cancer. Various types of medicaments for the treatment of tumours are described herein.

Tumour medicaments function in a variety of ways. Some cancer medicaments work by targeting physiological mechanisms that are specific to tumour cells. Examples include the targeting of specific genes and their gene products (i.e. proteins primarily) which are mutated in tumours. Such genes include but are not limited to oncogenes (e.g., Ras, Her2, bcl-2 ) , tumour suppressor genes (e.g., EGF, P53 , Rb) , and cell cycle targets (e.g. CDK4 , p2i , telomerase). Tumour medicaments can alternately target signal transduction pathways and other molecular mechanisms which are altered in tumour cells. Immunotherapeutic agents for use in combination with compounds, as described herein, may include biological response modifiers. Biological response modifiers for use in combination therapies as disclosed herein may include interferon-alpha, IL-2, G- CSF and GM-CSF; miscellaneous agents including platinum coordination complexes such cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (o,p’-DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethyl stilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; nonsteroidal antiandrogens such as flutamide; kinase inhibitors, histone deacetylase inhibitors, methylation inhibitors, proteasome inhibitors, oxidants, anti-oxidants, telomerase inhibitors, BH3 mimetics, ubiquitin ligase inhibitors, stat inhibitors; receptor tyrosine kinase inhibitors such as Imatinib (Gleevec or Glivec), and Erlotinib (an EGF receptor inhibitor) now marketed as Tarceva.

Additional biological response modifiers may include inhibitors of the immunomodulatory enzymes indoleamine 2,3-dioxygenase (IDO or (IDO or INDO EC 1.13.11.52) tryptophan 2,3-dioxygenase (TDO, (EC 1.13.11.11).

Tryptophan 2, 3-dioxygenase (TDO) is a homotetrameric heme- containing cytosolic enzyme encoded by gene TD02 and expressed at high levels in the liver. It catalyses the first and rate- limiting step of tryptophan degradation along the kynurenine pathway and thereby regulates systemic tryptophan levels. The role of TDO in cancer was shown in studies by Pilotte et al. (Proc Natl Acad Sci U S A. 2012 Feb 14; 109(7): 2497-502. Epub 2012 Jan 30). These studies detected TDO expression in a significant proportion of human tumours. In a preclinical model, TDO expression by tumours prevented their rejection by immunized mice. The studies used a TDO inhibitor, which, upon systemic treatment, restored the ability of mice to reject TDO-expressing tumours thus providing evidence that TDO inhibitors can be effective in cancer therapy. TDO inhibitors include LM10.

Indoleamine 2,3 dioxygenase (IDO) is an enzyme that in humans is encoded by the ID01 gene. This enzyme also catalyzes the first and rate-limiting step in the degradation of the essential amino acid L-tryptophan to N-formylkynurenine, and is normally expressed in tumour cells and in activated immune cells. IDO dampens the immune response by degrading the indole moiety of tryptophan, locally depleting tryptophan levels, and increasing proapoptotic kynurenines. Consequently, IDO blocks the proliferation and activation of T-cells, which are extremely sensitive to Trp shortage. This creates an environment in which tumour-specific cytotoxic T lymphocytes are rendered functionally inactive or are no longer able to attack a patient’s cancer cells. The observation that many human tumours constitutively express IDO introduced the hypothesis that its inhibition could enhance the effectiveness of cancer immunotherapy. Results from in vitro and in vivo studies show that the efficacy of therapeutic vaccination of cancer patients may indeed be improved by concomitant administration of an IDO inhibitor.

Small molecule inhibitors of IDO are available in the art to treat IDO-related diseases such as cancer. For example, W099/ 29310 reports methods for altering T-cell mediated immunity comprising altering local extracellular concentrations of tryptophan and tryptophan metabolites among others. Additional compounds having IDO inhibitory activity are reported in W02004/094409, US8088803 (which reports the INCB024360 compound), US20110165188 and US20110159017. Immunotherapeutic agents for use in combination with compounds, as described herein, may include cytokines.

Cytokines that are effective in inhibiting tumour growth/metastasis may be used in combination with compounds described herein. Such cytokines, lymphokines, or other hematopoietic factors include, but are not limited to, M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL- 11, IL- 12, IL- 13, IL- 14, IL- 15, IL-16, IL- 17, IL-18, IFN (I and II types), TNFct, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin.

As used herein, the term “immunotherapy”, “immunotherapeutic agent” or “immunotherapeutic” generally refers to any therapeutic approach aimed at mobilizing or manipulating a patient’s immune system to treat or cure disease, particularly diseases such as cancer. The term “immunotherapy”, “immunotherapeutic agent” or “immunotherapeutic” includes the targeting of tumour cells via the recognition of immunogenic proteins or antigens expressed by said tumour cells, accomplished by utilizing either passively transferred immune molecules such as antibodies, or cancer vaccine preparations designed to induce antibodies or T lymphocytes (T cells) recognizing a localized region of an antigen or epitope specific to the tumour cell. Immunotherapeutic agents for use in combination with the compounds, as described herein, may be antibodies.

Suitably the therapeutic antibody comprises one or more anti-Her2/neu receptor antibody for example trastuzumab (marketed as Herceptin); Alemtuzumab, a CD52 antibody marketed as Campath, MabCampath or Campath-iH currently under further development as Lemtrada; Gemtuzumab, an anti-CD33 monoclonal antibody linked to a calicheamicin marketed by Wyeth as Mylotarg; an anti-CD20 antibody, such as Rituximab (marketed as Rituxan and MabThera) or Ibritumomab tiuxetan sold under the trade name Zevalin; anti-TNF-alpha antibodies such as Infliximab (marketed as Remicade), or Adalimumab (marketed as Humira), or a soluble TNFR2 molecule such as etanercept (also known as Enbrel); an antibody to the CD25 chain of the IL-2 receptor such as basiliximab (trade name Simulect); an anti CD40/CD40L antibody such as a humanized IgGl anti-human CD40 antibody (SGN-40); an anti-CTLA-4 blocking antibody, such as Ipilimumab (also known as MDX-101 or MDX-010, and marketed as Yervoy) or tremelimumab; anti-PD-i antibody (programmed cell death protein 1, also designated as CD279); an anti-PDL-i (programmed cell death ligand); an antibody against glucocorticoid-induced TNFR family-relate gene, or anti-GITR antibody; or an anti-OX-40 (CD 134) antibody.

Other suitable immunotherapeutic agents may include soluble Lymphocyte-activation gene 3 (also known as LAG3 or CD223)-based immune modulators such as LAG3-Ig (IMP321); Toll-like receptor agonists like MPL, CpG, single-stranded R A, nucleotides, nucleotide analogue, CL087 (a TLR7-specific ligand), loxoribine, polyinosine- polycytidylic acid, flagellin, resiquimod, immiquimod, gardiquimod NOD ligands like muramyl dipeptide, murabutide, peptidoglycan, muramyldipeptide and anti-virals such as oseltamivir phosphate, Amphotericin B, and palivizumab.

In some embodiments, a therapy as disclosed herein may comprise administration of a compound as described herein, such as a compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof and one or more antibodies selected from the group consisting of an anti-PDi antibody, an anti-PDL-i antibody, an anti-CTLA-4 antibody an anti-GITR antibody and an anti-OX4O antibody.

An anti-PDi antibody maybe a monoclonal antibody directed against the negative immunoregulatory human cell surface receptor PD-1 with immunopotentration activity. An exemplary anti-PDi antibody is human monoclonal antibody MDX-1106 which binds and blocks the activation of PD-i by its ligands PD-Li and PD-L2, resulting in the activation of T-cells and cell-mediated immune responses against tumour cells. In some embodiments, the anti-PD-i antibody is a monoclonal antibody directed against the protein ligand PD-L1 with immunomodulating and antineoplastic activities. An exemplary anti-PD-Li antibody is human monoclonal antibody MDX-1105 which binds PD-Li and blocks its binding to and activation of its receptor PD-1, which may enhance the T-cell-mediated immune response to neoplasms and reverse T-cell inactivation in chronic infections disease. PD- Li is expressed broadly on hematopoietic and parenchymal tissues.

An anti-CTLA-4 antibody may be a monoclonal antibody directed against the T-cell receptor protein cytotoxic T-lymphocyte associated protein 4 (CTLA-4). An exemplary anti-CTLA-4 antibody is human IgG2 monoclonal antibody tremelimumab which binds to CTLA4 and blocks binding of the antigen presenting cell ligands B7-1 and B7-2 to CTLA-4, resulting in inhibition of B7- CTLA4-mediated down-regulation of T-cell activation. Another exemplary anti-CTLA-4 antibody is human IgGi monoclonal antibody ipilimumab which binds to CTLA4 and blocks binding of the antigen presenting cell ligands B7-1 and B7-2 to CTLA-4, resulting in inhibition of B7-CTLA4- mediated down-regulation of T-cell activation. Ipilimumab is undergoing clinical trials for the treatment of non-small cell lung carcinoma, small cell lung cancer and metastatic hormone-refractory prostate cancer.

An anti-GITR antibody may be a monoclonal antibody directed against glucocorticoid- induced tumour necrosis factor receptor (GITR) which blocks the interaction of GITR with its ligand, enhances cytotoxicity of natural human killer cells and/or down- modulates GITR expression on peripheral blood lymphocytes.

An anti-OX4O antibody may be an agonistic monoclonal antibody that mimicks the natural OX4O ligand and selectively binds to and activates the OX4O receptor. Receptor activation induces proliferation of memory and effector T cells.

Suitably, the anti-cancer agents may comprise any known agent with desirable anticancer properties. Suitably the anti-cancer agents are one or more of taxoids such as Taxol®, Taxotere©, Abraxane or other chemotherapeutics, such as cis-platin (and other platin intercalating compounds), etoposide and etoposide phosphate, bleomycin, mitomycin C, CCNU, doxorubicin, daunorubicin, idarubicin, ifosfamide, and the like. Other anticancer agents may be contemplated for use in combination therapies as disclosed herein include aspirin, sulindac, curcumin, alkylating agents including: nitrogen mustards , such as mechlor-ethamine , cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); ethyienimines/methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5- azacytidine, 2,2’-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6- thioguanine, azathioprine, 2’-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2- chlorodeoxyadenosine (cladribine, 2-CdA); natural products including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate; epipodophylotoxins such as etoposide and tenyposide; antibiotics such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin), mitomycinC , and actinomycin; and enzymes such as L-asparaginase. The anticancer agent may also be a biological agent such as a protein that inhibits tumour growth, such as interferon (IFN)-gamma, tumour necrosis factor (TNF) -alpha, TNF-beta, GM-CSF, and similar cytokines, or an anti-angiogenic factor such as angiostatin and endostatin or inhibitors of FGF or VEGF such as soluble forms of receptors for angiogenic factors, including soluble VGF/VEGF receptor. Further anticancer agents can be platinum coordination complexes such cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (0, p’-DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethyl stilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropinreleasing hormone analogs and leuprolide; non-steroidal antiandrogens such as flutamide; kinase inhibitors, histone deacetylase inhibitors, methylation inhibitors, proteasome inhibitors, monoclonal antibodies, oxidants, anti-oxidants, telomerase inhibitors, BH3 mimetics , ubiquitin ligase inhibitors, stat inhibitors and receptor tyrosin kinase inhibitors such as imatinib mesylate (marketed as Gleevac or Glivac ) and erlotinib (an EGF receptor inhibitor) now marketed as Tarveca; and anti-virals such as oseltamivir phosphate, Amphotericin B, and palivizumab. Also including STING agonists such as DMXAA.

Suitably, the anti-cancer agents comprise one or more pyrimidine analogs and antimitotic drugs. More suitably, the anti-cancer agents comprise one or more of 5- fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2’-difluorodeoxycytidine, paclitaxel, vinblastine (VLB), vincristine, vinorelbine, taxotere, estramustine, and estramustine phosphate

Suitably the anti-cancer agents may further comprise an antibody linked, directly or indirectly to one or more agents with desirable anti-cancer properties to form an antibody-drug conjugate. A suitably antibody may be chosen that directs the conjugate to the cells of interest, e.g. tumour cells. Such antibody-drug conjugate are well-known in the art (16).

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 and/or malignant condition for any particular cell type.

Suitably subjects are human, livestock animals and companion animals. More suitably, the subjects are human.

Antimetastatic

Compound of formula (I) or metallo derivatives, salts and solvates thereof, wherein the compound is a compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof, such as compounds of formula (VII), or (VIII), or (IX), or (X), or (XI), or salts and solvates thereof have been found to have antimetastatic activity.

Hence, compounds of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof, may find use in the manufacture of a medicament for treating or inhibiting metastasis.

Suitably, the compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof for use in treating or inhibiting metastasis is a compound of formula (VII), or (VIII), or (IX), or (X), or (XI), or salts and solvates thereof. More suitably, the compound is a compound of formula (II) or (III), or salts and solvates thereof. More suitably, the compound is a compound of formula (VII) or (VIII), or salts and solvates thereof.

Treating metastases, comprising the administration of a therapeutically effective amount of a compound as described herein, or a pharmaceutical composition as described herein, to a subject in need thereof.

Suitably, the metastasis is derived from bladder cancer, breast cancer, colorectal cancer, esophageal cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, or stomach cancer or any combination thereof.

The spread of cancer cells from a primary tumour site to distant organs is known as metastasis. Cancer tumour metastasis is responsible for many therapeutic failures when the disease is treated, as patients succumb to the multiple tumour growth. The extent to which metastasis occurs varies with the individual type of tumour. Melanoma, breast cancer, lung cancer and prostate cancer are particularly prone to metastasize.

When metastasis takes place, the secondary tumours can form at a variety of sites in the body, with one of the more common sites for metastasis being the lung.

Thus, inhibition of tumour metastasis to any extent would be beneficial, regardless of whether the agent involved in the inhibition had any effect on the primary tumour. However, if the agent also inhibited the primary tumour, this would be an additional advantage for the agent.

Inhibition of metastasis disclosed herein may be used alone or in combination as part of a treatment regimen for an animal or human patient having a cancer that is prone to metastasis, particularly, melanoma, breast cancer, lung cancer and prostate cancer. Treatment to inhibit the formation of metastases is best administered as soon after the detection of the cancer as possible. By utilizing the treatment regimen in patients at an early stage, a treating physician maximizes the chances that significant metastasis has not yet occurred. This maximizes chances for successful treatment. In such a regimen, the antimetastatic factor or its salts may, and generally will, be administered in combination with another form of therapy which controls the primary tumour itself.

Suitably the compound of formula (I) or metallo derivatives, salts and solvates thereof, for use in treating or inhibiting metastasis, is administered either simultaneously or sequentially with one or more immunotherapeutic agent or other anti-cancer agent. The treatment described herein may also be used conjointly with (i.e., either preceding or subsequent to) a surgical procedure to remove the primary tumorous material from the body. Frequently, surgical procedures to remove tumorous material from the body are avoided because of the fear that metastasis of tumour cells will occur as a result of the physical manipulation involved. However, if an antimetastatic as described herein is administered to the patient prior to the surgical procedure, then the risk of metastasis which may result from surgery can be reduced and surgery would be a more attractive treatment option.

Within the scope of sound medical judgement, the dosage of antimetastatic as described herein and the method of administration will vary with the severity and nature of the particular condition being treated, the duration of treatment, the adjunct therapy used, the age and physical condition of the patient, and like factors within the specific knowledge and expertise of the attending physician. However, single dosages can typically range from o.oi to 2000 milligrams per kilogram of body weight, preferably 1 to 200 milligrams per kilogram (unless otherwise specified, the unit designated “mg/kg”, as used herein, refers to milligrams per kilogram of body weight). Up to four doses per day can be used routinely, but this can be varied according to the needs of the patient, consistent with a sound benefit/ risk ratio. Variation in patient response may be expected but the higher dosages within the ranges indicated are usually required in the case of oral administration while the lower dosages indicated would apply for intravenous administration.

Pharmaceutical Composition

Suitably the pharmaceutical composition further comprises one or more further therapeutic agents.

Suitably, the pharmaceutical composition further comprises one or more further therapeutic agents selected from one or more immunotherapeutic agent or other anticancer agent.

Suitably, the pharmaceutical composition further comprises one or more other anticancer agent.

Suitably the pharmaceutical composition further comprises one or more cancer vaccine or therapeutic antibody. Suitably the pharmaceutical composition further comprises an adjuvant. Suitably the pharmaceutical composition comprises a compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof, a cancer vaccine or therapeutic antibody, an adjuvant and a pharmaceutically acceptable excipient, carrier or diluent.

Suitably the pharmaceutical composition is formulated as a nanoparticle carrier vehicles or as a viral vector.

Suitably the pharmaceutical composition is formulated as a nanoparticle carrier vehicles. Suitably the nanoparticle carrier vehicle is one of silica nanoparticles, liposomes, micelles, nanogel or polymeric nanoparticles. Suitably the nanoparticle carrier vehicle is a liposome.

Suitably the pharmaceutical composition is formulated as a viral vector. Suitably the viral vector is an adenovirus, adeno-associated virus, retrovirus, lentivirus, or herpes viral vectors. The use of viral vectors is a well-known delivery method (17, 18).

Administration & Dose

Compounds of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof, 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 (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof.

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

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

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.

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

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 deliveiy applications. The compounds of the invention may also be administered via fast dispersing or fast dissolving dosages forms.

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

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

Modified release and pulsatile release dosage forms may contain excipients such as those detailed for immediate release dosage forms together with additional excipients that act as release rate modifiers, these being coated on and/or included in the body of the device. Release rate modifiers include, but are not exclusively limited to, hydroxypropylmethyl cellulose, methyl cellulose, sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate, polyethylene oxide, Xanthan gum, Carbomer, ammonio methacrylate copolymer, hydrogenated castor oil, carnauba wax, paraffin wax, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, methacrylic acid copolymer and mixtures thereof. Modified release and pulsatile release dosage forms may contain one or a combination of release rate modifying excipients. Release rate modifying excipients maybe present both within the dosage form i.e. within the matrix, and/or on the dosage form i.e. upon the surface or coating.

Fast dispersing or dissolving dosage formulations (FDDFs) may contain the following ingredients: aspartame, acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, diascorbic acid, ethyl aciylate, ethyl cellulose, gelatin, hydroxypropylmethyl cellulose, magnesium stearate, mannitol, methyl methacrylate, mint flavouring, polyethylene glycol, fumed silica, silicon dioxide, sodium starch glycolate, sodium stearyl fumarate, sorbitol, xylitol.

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

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

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

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

By “sequential” it is meant that the different agents are administered at different points in time, provided that the activity of the first administered agent is present and ongoing in the subject at the time the second agent is administered. For example, an immunotherapeutic agent may be administered first, such that an immune response against a tumour antigen is generated, followed by administration of a compound of formula (II), or (III), or (IV), or (V), or (VI), or derivative compound thereof, or salts and solvates thereof, such that immunosuppression at the site of the tumour is reduced. This sequential administration may occur by the same route or by different routes of administration. Preferably, a sequential dose will occur such that the second of the two agents is administered within 48 hours, preferably within 24 hours, such as within 12, 6, 4, 2 or 1 hour(s) of the first agent. Preferably, one agent may be administered daily and a second agent may be administered every two, or every three, or eveiy four, or eveiy five or every six, or every seven days.

Multiple doses of the compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof, or may be administered, for example 2, 3, 4, 5 or more than 5 doses may be administered after administration of the one or more immunotherapeutic agent or other anticancer agent. The administration of the compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof, may continue for sustained periods of time after administration of the immunotherapeutic agent or other anticancer agent. For example, treatment with the compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof, maybe continued for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month or at least 2 months. Treatment with a compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof, may be in cycles with a break and then resumption of treatment. Treatment with the compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof, may be continued for as long as is necessary to achieve complete tumour rejection.

Multiple doses of the one or more immunotherapeutic agent or other anticancer agent may be administered, for example 2, 3, 4, 5 or more than 5 doses may be administered after administration of the compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof. The administration of the immunotherapeutic agent or other anti cancer agent may continue for sustained periods of time after administration of the compound of formula (II), or (III), or (IV), or (V), or (VI), or salts and solvates thereof. For example, treatment with the immunotherapeutic agent or other anticancer agent maybe continued for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month or at least 2 months. Treatment with the immunotherapeutic agent or other anticancer agent may be continued for as long as is necessary to achieve complete tumour rejection.

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 too 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, tautomeric and protected forms of these substituents. For example, a reference to carboxylic acid (-RCOOH) also includes the anionic (carboxylate) form (-RCOO ), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-RN+H R‘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.

Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, mesomeric stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and 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. Ci-₇ alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not apply to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol, imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

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

Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof.

Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

Unless otherwise specified, a reference to a particular compound also includes ionic, salt, solvate, and protected forms of thereof, for example, as discussed below. In some embodiments, the compound of the disclosure and salts and solvates thereof, comprises pharmaceutically acceptable salts of the compounds of the disclosure.

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

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

For example, if the compound is anionic, or has a functional group which maybe anionic (e.g. -RCOOH may be -RCOO ), then a base salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, metal cations, such as an alkali or alkaline earth metal cation, ammonium and substituted ammonium cations, as well as amines. Examples of suitable metal cations include sodium (Na⁺) potassium (K⁺), magnesium (Mg²⁺), calcium (Ca²⁺), zinc (Zn²⁺), and aluminum (A13+). 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 ethylenediamine, 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 Pharm aceutical Salts: Properties, Selection, and Use (2011)

Pharmaceutically acceptable salts may be prepared using various methods. For example, one may react a compound of the disclosure with an appropriate acid or base to give the desired salt. One may also react a precursor of the compound of the disclosure with an acid or base to remove an acid- or base-labile protecting group or to open a lactone or lactam group of the precursor. Additionally, one may convert a salt of the compound of the disclosure to another salt through treatment with an appropriate acid or base or through contact with an ion exchange resin. Following reaction, one may then isolate the salt by filtration if it precipitates from solution, or by evaporation to recover the salt. The degree of ionization of the salt may vary from completely ionized to almost non-ionized.

It maybe 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 may be isotopically substituted (e.g., D₂0, acetone-d6, DMS0-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 lie 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, nonstoichiometry will typically be observed.

Synthetic Strategies

The method of the invention may include one or more of the techniques described below. Some of the schemes and examples may omit details of common reactions, including oxidations, reductions, and so on, separation techniques (extraction, evaporation, precipitation, chromatography, filtration, trituration, crystallization, and the like), and analytical procedures, which are known to persons of ordinary skill in the art of organic chemistry. The details of such reactions and techniques can be found in a number of treatises, including Richard Larock, Comprehensive Organic Transform ations, A Guide to Functional Group Preparations, 2nd Ed (2010), and the multi-volume series edited by Michael B. Smith and others, Compendium of Organic Synthetic Methods (1974 et seq.). Starting materials and reagents may be obtained from commercial sources or may be prepared using literature methods. Some of the reaction schemes may omit minor products resulting from chemical transformations (e.g., an alcohol from the hydrolysis of an ester, C0₂ from the decarboxylation of a diacid, etc.). In addition, in some instances, reaction intermediates maybe used in subsequent steps without isolation or purification (i.e., in situ .

In some of the reaction schemes and examples below, certain compounds can be prepared using protecting groups, which prevent undesirable chemical reaction at otherwise reactive sites. Protecting groups may also be used to enhance solubility or otherwise modify physical properties of a compound. For a discussion of protecting group strategies, a description of materials and methods for installing and removing protecting groups, and a compilation of useful protecting groups for common functional groups, including amines, carboxylic acids, alcohols, ketones, aldehydes, and so on, see T. W. Greene and P. G. Wuts, Protecting Groups in Organic Chem istry , 4th Edition, (2006) and P. Kocienski, Protective Groups, 3rd Edition (2005).

Generally, the chemical transformations described throughout the specification may be carried out using substantially stoichiometric amounts of reactants, though certain reactions may benefit from using an excess of one or more of the reactants. Additionally, many of the reactions disclosed throughout the specification may be carried out at about room temperature (RT) and ambient pressure, but depending on reaction kinetics, yields, and so on, some reactions may be run at elevated pressures or employ higher temperatures (e.g., reflux conditions) or lower temperatures (e.g., -78°C. to o°C.). Any reference in the disclosure to a stoichiometric range, a temperature range, a pH range, etc., whether or not expressly using the word "range," also includes the indicated endpoints.

Many of the chemical transformations may also employ one or more compatible solvents, which may influence the reaction rate and yield. Depending on the nature of the reactants, the one or more solvents may be polar protic solvents (including water), polar aprotic solvents, non-polar solvents, or some combination. Representative solvents include saturated aliphatic hydrocarbons (e.g., n-pentane, n-hexane, n- heptane, n-octane); aromatic hydrocarbons (e.g., benzene, toluene, xylenes); halogenated hydrocarbons (e.g., methylene chloride, chloroform, carbon tetrachloride); aliphatic alcohols (e.g., methanol, ethanol, propan-i-ol, propan-2-ol, butan-i-ol, 2- methyl-propan-i-ol, butan-2-ol, 2-methyl-propan-2-ol, pentan-i-ol, 3-methyl-butan-i- ol, hexan-i-ol, 2-methoxy-ethanol, 2-ethoxy-ethanol, 2-butoxy-ethanol, 2-(2-methoxy- ethoxy)-ethanol, 2-(2-ethoxy-ethoxy)-ethanol, 2-(2-butoxy-ethoxy)-ethanol); ethers (e.g., diethyl ether, di-isopropyl ether, dibutyl ether, 1,2-dimethoxy-ethane, 1,2- diethoxy-ethane, i-methoxy-2-(2-methoxy-ethoxy)-ethane, i-ethoxy-2-(2-ethoxy- ethoxy)-ethane, tetrahydrofuran, 1,4-dioxane); ketones (e.g., acetone, methyl ethyl ketone); esters (methyl acetate, ethyl acetate); nitrogen-containing solvents (e.g., formamide, N,N-dimethylformamide, acetonitrile, N-methyl-pyrrolidone, pyridine, quinoline, nitrobenzene); sulfur-containing solvents (e.g., carbon disulfide, dimethyl sulfoxide, tetrahydro-thiophene-1,1, -dioxide); and phosphorus-containing solvents (e.g.,HMPA, hexamethylphosphoramide).

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.

BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the present invention will now be described further, with reference to the accompanying drawings, in which:

Figure 1 is a graph of the serum concentration (ng/mL) of drug in mouse plasma samples after SnMP (38 mg/kg, PO) administration analyzed through LC-MS/MS. The mean ± SD values vs. time were plotted in line chart (semi-log).

Figure 2 is a graph of the serum concentration (ng/mL) of drug in mouse plasma samples after KCL-HO-ii (NS) (44 mg/kg, PO) administration analyzed through LC- MS/MS. The mean ± SD values vs. time were plotted in line chart (semi-log).

Figure 3 is a combined graph of the data in Figures 1 and 2.

Figure 4A & 4B show schematic representations of intraperitoneal (i.p.) dosing strategies for KCL-HO-ii or SnMP and/or Fluorouracil (5-FU) or Gemcitabine or vehicle in MMTV-PyMT mice bearing established tumors.

Figure 4C-4G shows growth curves of established spontaneous tumours in MMTV- PyMT mice that had received single treatments of vehicle, SnMP, KCL-HO-ii, 5-FU or Gemcitabine as indicated according to the dosing strategies shown in Figure 41 & 4B. Each line represents an individual tumour and mouse.

Figure 4H-4J shows growth curves of established spontaneous tumours in MMTV- PyMT mice that had received combined treatments of SnMP/5-FU, KCL-HO-ii/5-FU and KCL-HO-ii/gemcitabine as indicated according to the dosing strategies shown in Figure 4A & 4B. Each line represents an individual tumour and mouse.

Figure 4K shows averages of the mouse growth curves of established spontaneous tumours that were given vehicle, KCL-HO-ii, 5-FU or KCL-HO-ii and 5-FU combination as indicated.

Figure 4L shows averages of the mouse growth curves of established spontaneous tumours that were given vehicle, KCL-HO-ii, Gemcitabine or KCL-HO-ii and Gemcitabine combination as indicated.

Figure 5 shows tumour sizes at day 8 post initiation of treatment in MMTV-PyMT mice that had received SnMP, KCL-HO-ii alongside Fluorouracil (5-FU). Each dot represents an individual mouse. * P<O.O5.

Figure 6 shows growth curves of established spontaneous tumours in MMTV-PyMT mice that had received commercial SnMP, SnMP synthesized according to the invention (Vegan SnMP), and/or Fluorouracil (5-FU) as indicated. Each line represents an individual mouse.

Figure 7A shows a schematic of the experiment and representation of the strategy used to determine the plasma concentration.

Figure 7B shows a graph of the plasma concentration over time for male mice that received either intraperitoneal administration (left panel) or oral administration (right panel) of commercial SnMP, SnMP synthesized according to the invention (veSnMP) or KCL-HOli.

Figure 7C shows a schematic representation of the HO-i^luc/eGFP reporter mouse model used (top) and experimental outline (below).

Figure 7D shows representative bioluminescence images for Vehicle, commercial SnMP, SnMP synthesized according to the invention (VeSnMP) or KCL-HOli at To (upper panel) and after 24 hours of treatment (bottom panel) of the whole body in the HO-i^luc/eGFP reporter mouse (C).

Figure 7E shows relative quantification of the luciferase expression (photons/sec) for the whole body images shown in (D) in the HO-i^luc/eGFP reporter mouse (C).

Figure 7F shows the change HO-1 expression of each individual mouse tissue (from those shown in D) normalised to the Vehicle treated group in the HO-i^luc/^eGFP reporter mouse (C).

Figure 7G shows an evaluation of the inhibition of HO-1 activity in rat microsomes using a range of doses of KCL-HO-ii and SnMP.

Figure 8 A shows a schematic representation of the dosing strategy for the KCL-HO-ii and gemcitabine in MMTV-PyMT mice.

Figure 8 B shows how the tumour volumes (mm³) for each mouse changed over time (days). KCL-HO-ii was delivered via oral delivery and gemcitabine was delivered I.P. The dashed lined represents the start of treatment.

Figure 9 shows a bar graph of the CD8 T-cell infiltration into the tumour of MMTV- PyMT mice that were treated with vehicle, commercial SnMP(cSnMP) or KCL-HO-ii Figure 10 A shows a schematic representing the i.p. dosing strategy for non-immune IgG and immune-depleting anti-CD8a antibodies that were also given alongside KCL- HO-ii and gemcitabine and in MMTV-PyMT mice.

Figure 10 B shows growth curves of each individual mouse treated with non-immune IgG.

Figure 10 C shows growth curves of each individual mouse treated with immune- depleting anti-CD8a antibodies.

Figure 10 D shows growth curves of each individual mouse treated with immune- depleting anti-CD8a antibodies, KCL-HO-ii and gemcitabine.

Figure 10 E shows growth curves as a line chart where each line displays the mean for each treatment.

Figure 11A shows a schematic representation of the i.p. dosing strategy for KCL-HO-ii and/or 5-FU or gemcitabine or vehicle in MMTV-PyMT mice bearing established tumours. Tumours were analysed by flow cytometry 36 h post initiation of treatment.

Figure 11B shows the change in tumour growth over the 36 h treatment period.

Figure 11C shows the stromal compositions for the enzyme-dispersed tumours for each of the different treatments.

Figure 11D shows a bar chart representing CD8⁺ T-cells infiltration in the tumour microenvironment for each of the different treatments.

Figure HE shows a bar chart representing CD8⁺ T-cell effector function as assessed by their expression of IFN-gamma for each of the different treatments.

Figure HF shows a bar chart representing CD8⁺ T-cells infiltration in the tumour microenvironment for vehicle and SnMP.

Figure HG shows a representative dot plot of FACS gated live (7AAD ), CD45⁺CD3⁺CD8⁺ T-cells showing the expression of CD44 and CD62L in vehicle treated mouse.

Figure 11H shows histograms representing the CD8⁺ T-cells sub populations (as shown in Fig. 11G) across the different treatment groups. Figure 12 A shows a schematic representing the dosing strategy for mice bearing established MMTV-PyMT tumours which were treated with KCL-HO-ii and/ or 5-FU or gemcitabine or vehicle and tumour tissue was analyzed at 36 hours by bulk RNAseq analysis.

Figure 12B shows a Venn diagram of all DEGs for the respective treatments against vehicle and their intercepts between groups.

Figure 12 C shows a Venn diagram showing all upregulated DEGs for KCL-HO-ii based treatments against vehicle and their intercepts between groups

Figure 12D shows a heatmap of hierarchical clustered common upregulated DEGs between treatment groups (419 genes) that are secreted genes (91 genes) across treatments.

Figure 12E shows a Venn diagram of all upregulated DEGs for treatments including chemotherapy and their intercepts between groups.

Figure 12F shows a Venn diagram of chemokine and upregulated DEGs associated dual therapy treatments and their intercepts between groups.

Figure 12 G shows a heatmap of hierarchical clustered chemokine and cytokine upregulated DEGs associated with dual treatment groups.

Figure 13 A shows a schematic representing the dosing strategy for KCL-HO-ii and/or gemcitabine or vehicle in MMTV-PyMT mice bearing established tumours

Figure 13 B shows tumour growth curves for the respective treatments represented in Figure 13A.

Figure 13 C is a graph showing the weights of the mice during the respective treatments.

Figure 13 D shows the ratio of serum liver aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the blood for the respective treatments.

Figure 13 E shows blood immune cell compositions the respective treatments.

Figure 13 F shows a heatmap of the animal’s physical characteristics as accessed at day 21 of treatment.

Figure 14 A shows a schematic representing the oral delivery dosing strategy for KCL- HO-ii and/or i.p. delivered gemcitabine or vehicle in C57BI/6 mice bearing established sub cut. sarcoma tumours (MN-MCA-1 cell line) Figure 14 B shows tumour growth curves for the respective treatments represented in Figure 14A.

Figure 15 A shows the tumour volume for mice that at day 12 post tumour injection received either KCL-HO-ii (blue curve) or Vehicle (black curve) daily for 14 days.

Figure 15B shows a histogram that represents the metastatic nodules that were counted from lungs that were harvested at day 26 post tumour injection.

EXAMPLES

Surprisingly, the inventors have discovered that the phosphate salt, KCL-HO-ii, showed unexpected in vivo anti-cancer activity and demonstrated superiority over commercial SnMP dichloride in in vivo studies (see section 2). It also appears that KCL-HO-ii has a superior solubility profile compared to commercially available SnMP.

A synthesis of KCL-HO-ii from SnMP is set out below. Generally, SnMP maybe contacted with a source of phosphate at a predetermined temperature and for a predetermined time or MP may be contacted with a source of tin and a source of phosphate at a predetermined temperature and for a predetermined time.

1) Synthesis of KCL-HO-li

Scheme 2. synthesis of KCL-HO-li using tin pyrophosphate.

2) KCL-HO-li as an HO inhibitor with anti-tum our efficacy. We have explored KCL-HO-ii preclinically and demonstrated that it is superior to dichloride SnMP (as shown in Figures 3, 4, 5, 7 and 9). Administration of KCL-HO-ii to MMTV-PyMT bearing tumours mice has demonstrated that KCL-HO-ii displays superior anti-tumour efficacy in combination with chemotherapy 5-FU (Fig. 4) where at day 8 post-initiation of treatment KCL-HO-ii had surprisingly eradicated all tumour burden where SnMP treated animals still had residual disease (Fig. 5). Thus, KCL-HO- ii provides a measurable improvement in the anti-tumour immune response when combined with 5-FU compared to standard commercial SnMP (Fig. 5).

Currently, MP is synthesised using animal product starting materials, specifically hemin.

There is a need for a synthetic route to improve efficiency and scale of manufacture, and address ethical and religious concerns, and which is vegan-friendly as it is free from animal products. Surprisingly, the present inventors provide a method to synthesise MP without the use of animal products using a method that would allow for large scale manufacture of MP.

3) Large-scale manufacture of synthetic KCL-HO-li

The synthetic route illustrated in Scheme 3, below, is a possible route to KCL-HO-ii that does not require animal starting materials.

SnMP produced using the synthetic route according to the method of the invention is active in delivering anti-tumour efficacy in combination with the chemotherapy 5-FU in the MMTV-PyMT murine model of cancer (see Figure 6). 90%

Pyrrole A Pyrrole B Stage 1

Scheme 3. Optimized synthesis for the large-scale production of synthetic synthesis of tin mesoporphyrin phosphate salt (KCL-HO-ii) according to the invention. KCL-HO-ii maybe synthesised from the MP produced in the large-scale synthesis at stage 4, or the SnMP produced at stage 5 by contacting the MP or SnMP with a source of tin and a source of phosphate (e.g. tin pyrophosphate as set out in 1, above).

Stage 1

Pyrrole A, (1), (200 g), Pyrrole B, (2), (309 g) and methanol (3.99 L) were charged to the reactor under nitrogen and the contents heated to reflux. A solution of para- toluenesulphonic acid monohydrate (1.21 g) in methanol (10 mL) was added over 3 minutes. The reaction mass was stirred at reflux for 32 minutes, then cooled on a controlled ramp to o °C, over 2:30 hours. Spontaneous crystallisation occurred between 38 and 36 °C. Once at 2 °C, the slurry was discharged to a filter, deliquored and the vessel washed to the filter with methanol (2 x 500 mL) cooled to <5 °C. The cake was fully deliquored and dug off to a drying tray and dried under vacuum at 40 °C for 8 hours to give the product (3) (286.8g, (90.7% yield).

Stage 2

Compound (3), (210.0 g), tetrahydrofuran (1050 mL) and triethylamine (147 mL) were charged to the flask and the contents stirred until a solution formed. 5% Pallidum on alumina (42.0 g) was charged to the flask and hydrogen sparged through the stirring mixture at 500 mLmin-i for 52 minutes, during which time the contents of the flask increased in temperature from 17 to 37 °C. After this time, the reaction temperature began to decrease and the flow of gas venting from the batch increased markedly by visual observation. After a reaction time of 60 minutes, the mixture was passed through a 25 mm deep celite bed into a 3 L pear shaped flask. The filter bed was washed with tetrahydrofuran (4 x too mL). The filtrate and washings were combined and concentrated under reduced pressure to give a damp off-white solid, which was dried under vacuum at 40 °C for 20 hours to give the dry intermediate (174.2 g, 98.6% yield) which was used without purification (identity confirmed by ^XH NMR).

Stage 3 para-Toluenesulphonic acid monohydrate (372.4 g), chloroform (10.61 L) and methanol (2.12 L) were charged to the flask and placed under nitrogen atmosphere. A solution of crude compound (26 ), TEA salt (174.1 g) from stage 2, and the dialdehyde (157.6 g) in 5:1 chloroform/methanol (1.50 L) was charged over 16:30 hours via HPLC pump. 5:1 chloroform/methanol (3 x 40 mL) was charged as vessel and line rinses. The mixture was stirred for an hour. A solution of zinc acetate dihydrate (172.4 g) and methanol (2.50 L) was added. Air was sparged through the mixture for 6 hours. The mixture was stirred open to atmosphere for approximately 10 hours. The mixture was washed with deionised water (2 x 2.50 L) followed by 20%w/w aqueous potassium carbonate (2.10 L, 2.5 kg), and further water (2.50 L). The chlorinated solution was concentrated under reduced pressure to give dark brown/purple oily mass, which was dried under vacuum 40 °C for 20 hours to give the dry intermediate, (21), (255.6 g, 99% yield) which was used without further purification, or analysis. The crude zinc porphyrin complex, (21), (511.7 g) and a solution of 5% v/v sulphuric acid in methanol (2.50 L) were charged to the flask and stirred until solids dissolved. Stirring was continued for 4 hours before the mixture was transferred to a 20 litre vessel and diluted with dichloromethane (5.00 L). The mixture was washed with deionised water (2.50 L). The aqueous layer was back extracted with dichloromethane (2 x 0.5 L). The chlorinated phases were combined and washed with deionised water (2.50 L), followed by 20%w/w aqueous potassium carbonate (2.10 L, 2.5 kg), and further water (2.50 L). The chlorinated solution was concentrated under reduced pressure to give darkbrown/black oily mass, which was dried under vacuum 40 °C for 20 hours to give the dry crude Stage 3 product, (6), (490.1 g).

Crude Stage 3 product (480 g) was dissolved in dichloromethane (~3-5 L) and loaded onto a 5kg HP-Sphere silica cartridge in a Biotage Flash 150L system that had been equilibrated with dichloromethane. The cartridge was eluted at ~8oo mLmin-i with dichloromethane (40 L), followed by 0.5% methanol in dichloromethane (130 L) taking ~io L fractions. Elution was continued with 0.5% methanol in dichloromethane (30 L) taking a further 6 x ~5 L fractions. The desired fractions were combined and concentrated under reduced pressure to give the Stage 3 product as a brown/purple solid (214.3 g, 44-3% yield).

Stage 4 6

Mesoporphyrin IX

Compound (6), (85.0 g) from Stage 3, tetrabutylammonium chloride (850 mg), dichloromethane (1.70 L), methanol (1.70 L) and 2M aqueous sodium hydroxide (268 mL) were charged to the flask and the contents heated to reflux (45 °C). The mixture was stirred at reflux for 4 hours after which time the reaction had reached completion, as determined by HPLC. The mixture was allowed to cool to room temperature. Concentrated hydrochloric acid (56.2 g) was added dropwise over 10 minutes. The mixture was stirred for an hour before being discharged to a filter, deliquored and the vessel washed to the filter with a 1:1 mixture of dichloromethane and methanol (2 x 150 mL) followed by water (2 x 100 mL). The filter cake was fully deliquored and dug off.

The damp product cake, deionised water (300 mL) and methanol (300 mL) were charged to the flask and the contents heated to 70 °C and stirred for 30 minutes before cooling to room temperature. The slurry was discharged to a filter, deliquored and the vessel washed to the filter with a 1:1 mixture of methanol and deionised water (2 x 150 mL) followed by methanol (100 mL). The filter cake was fully deliquored and dug off to a drying tray and dried under vacuum at 45 °C for 20 hours to give the Stage 4 product as a brown/purple powder (71.6 g). The liquors from both filtrations were concentrated under reduced pressure to give a slurry with a volume of ~6oo mL which was discharged to a filter and deliquored. The filter cake was washed with deionised water (2 x 100 mL). The filter cake was fully deliquored and dug off to a drying tray and dried under vacuum at 45 °C for 20 hours to give a second crop of Stage 4, (7), product as a brown/purple powder (7.2 g). The Stage 4 product (7), was combined to give a brown/purple powder (78.8 g, 97.3% yield).

Stage 5

200mg of Mesoporphyrin IX (0.35 mmol, teq.) and 576 mg of tin (II) pyrophosphate (1.4 mmol, 4 eq.) were added to a 50 ml round-bottom flask covered with tin foil. 5 ml of glacial acetic acid was added and the flask was flushed with nitrogen gas. The mixture then left to stir under reflux at 115 C. Once the temperature was stabilised, the reaction was allowed to open to air to introduce oxygen to the system and the reaction was then left for 24 hours. The mixture then cooled down to room temperature, quenched with 4 ml of HPLC grade water, filtered, and left to air dry for 10 minutes. The filtered solid was placed into another 50ml flask with 3 ml of HPLC grade water and 0.48 ml of concentrated HC1 and then left to stir for 30 minutes at 90C. The mixture cooled down to room temperature and then filtered and washed with cold water. After air-drying for 30 minutes, I25mg of the product (KCL-HO-ii) was obtained as a red powder (40% isolated yield). 1H NMR (400 MHz, DMS0-d6) 8 10.36 (s, 1H), 10.13 (s, 1H), 10.09 (d, J=4 Hz, 2H), 7.11 (s, 4H), 3.82 - 3.96 (m, 5H), 3.62 (q, , J=14 Hz, 4H), 3.17 (d, J=12 Hz, 12H), 2.64 (t, J=6 Hz, 5H), 1.82 (t, J=2 Hz, 16H), 1.25 (t, J=6 Hz, 8H).

Alternative Stage 5

This general reaction scheme can also be applied to preparing SnMP by using the following alternative stage 5.

Mesoporphyrin IX Th Mesoporphyrin

Tin(II) oxide (101.7 g) and acetic acid (2.50 L) were charged to the flask and the contents heated to 65 °C. A solution of Stage 4, (7), (115 g) in a mixture formic acid (590 mL) and concentrated hydrochloric acid (43.2 g) was added dropwise over 3:40 hours maintaining the temperature of the mixture in the flask between 60 and 70 °C. Formic acid (3 x 20 mL) was charged as line rinses; the formation of a crimson precipitate became evident after ~2:45 hours (—75% through the addition). The mixture was stirred between 63 and 68 °C for 17:25 hours. Deionised water (1.05 L) was added over 20 minutes. The mixture was cooled to 20 °C, over 3 hours and stirred for a further 2 hours. The slurry was discharged to a filter, deliquored and the vessel washed to the filter with deionized water (2 x 100 mL). The filter cake was deliquored, dug off and recharged to the flask. 1M aqueous hydrochloric acid (1.75 L) was charged to the flask and the contents heated to 85 °C. The mixture was stirred between 85 and 90 °C for 80 minutes before the slurry was discharged to a filter, deliquored and the vessel washed to the filter with water (2 x 250 mL). The filter cake was fully deliquored and dug off to a drying tray and dried under vacuum at 40 °C for 40 hours to give Tin Mesoporphyrin as a crimson powder (135.7 g, 88.7% yield).

An inductively coupled plasma mass spectrometry (ICP-MS) comparison was carried out between SnMP (the tin dichloride salt of mesoporphyrin IX) and two samples of KCL-HO-ii (the tin phosphate salt of mesoporphyrin IX) to provide an elemental analysis of the tin content of these compounds. Hence, this elemental analysis data shows that KCL-HOii has on average 3 times more Sn compared to SnMP.

This general reaction scheme can also be applied to the synthesis of tartrate salt of tin mesophorphorin IX (TA-191-149) by using the following alternative stage 5.

200mg of Mesoporphyrin IX (0.35 mmol, leq.) and 372.5 mg of tin (II) tartrate (1.4 mmol, 4 eq.) were added to a 50 ml round-bottom flask covered with tin foil. 5 ml of glacial acetic acid was added and the flask was flushed with nitrogen gas twice. The mixture then left to stir under reflux at 120 C. Once the temperature was stabilised, the reaction was allowed to open to air to introduce oxygen to the system and the reaction was then left for 24 hours. The mixture then cooled down to room temperature, quenched with 4 ml of HPLC grade water, filtered, and left to air dry for 10 minutes. The filtered solid was placed into another 50ml flask with 3.5 ml of 1M HC1 and then left to stir for 30 minutes at 90C. The mixture cooled down to room temperature and then filtered and washed with cold water. After air-drying for 30 minutes, 125 mg of the product was obtained as a brick-red powder (32% isolated yield). M.P: 287.2 °C; ^XH NMR (400 MHz, methanol-d₄) d 11.21 (br. s., 1H), 11.01 (br. s., 1H), 10.97 (br. s., 2H), 4.45 (d, J = 7.06 Hz, 5H), 3.95 (s, 7H), 3.98 (s, 8H), 3.47 (br. s., 5H), 2.65 (br. s., 2H), 1.88 - 2.16 (m, 8H).

This general reaction scheme can also be applied to the synthesis of oxalate salt of tin mesophorphorin IX (TA-191-151) by using the following alternative stage 5.

200mg of Mesoporphyrin IX (0.35 mmol, leq.) and 290 mg of tin (II) oxalate (1.4 mmol, 4 eq.) were added to a 50 ml round-bottom flask covered with tin foil. 5 ml of glacial acetic acid was added and the flask was flushed with nitrogen gas twice. The mixture then left to stir under reflux at 120 C. Once the temperature was stabilised, the reaction was allowed to open to air to introduce oxygen to the system and the reaction was then left for 24 hours. The mixture then cooled down to room temperature, quenched with 4 ml of HPLC grade water, filtered, and left to air dry for 10 minutes. The filtered solid was placed into another 50ml flask with 3.5 ml of 1M HC1 and then left to stir for 30 minutes at 90C. The mixture cooled down to room temperature and then filtered and washed with cold water. After air-drying for 30 minutes, 130 mg of the product was obtained as a brick-red powder (38% isolated yield). M.P: 263.2 °C; ^XH NMR (400 MHz, methanol-d₄) d 11.08 (d, J = 8.34 Hz, 1H), 10.89 (d, J = 6.69 Hz, 1H), 10.85 Cd, J = 5.41 Hz, 2H), 4.19 - 4.39 (m, 5H), 3.67 - 3.93 (m, 14H), 3.27 - 3.47 (m, 5H), 3.23 (s, 1H), 1.91 (q, J = 7.40 Hz, 7H)

This general reaction scheme can also be applied to the synthesis of mesylate salt of tin mesop horphorin IX (TA-i9i-i53)by using the following alternative stage 5. mesoporphyrin

200mg of Mesoporphyrin IX (0.35 mmol, leq.) and 290 mg of tin (II) methanesulfornate (1.4 mmol, 4 eq.) were added to a 50 ml round-bottom flask covered with tin foil. 5 ml of glacial acetic acid was added and the flask was flushed with nitrogen gas twice. The mixture then left to stir under reflux at 120 C. Once the temperature was stabilised, the reaction was allowed to open to air to introduce oxygen to the system and the reaction was then left for 24 hours. The mixture then cooled down to room temperature, quenched with 4 ml of HPLC grade water, filtered, and left to air dry for 10 minutes. The filtered solid was placed into another 50ml flask with 3.5 ml of 1M HC1 and then left to stir for 30 minutes at 90C. The mixture cooled down to room temperature and then filtered and washed with cold water. After air-drying for 30 minutes, 72 mg of the product was obtained as a red powder (24% isolated yield). M.P: 257.8 °C; ^XH NMR (400 MHz, methanol-d₄) d 11.25 (d, J = 16.05 Hz, 1H), 10.94 - H-14 (m, 3H), 4.71 - 4.80 (m, 4H), 4.39 - 4.53 (m, 4H), 3.91 - 4.04 (m, 12H), 3.47 (t, J = 7.24 Hz, 4H), 2.70 (s, 6H), 2.03 (t, J = 7.57 Hz, 6H)

4) Synthesis of Pyrrole A

The selective reduction step (iii) may be achieved with (a) a Wolff- Kishner reduction using N₂H₂, KOH; (b) a Clemmensen Reduction using Zn-Hg, HC1; (c) a catalytic hydrogenation using Pt-C, H₂ under pressure; or (d) using TsNHNH₂, then NaBH₃CN.

4) Synthesis of Pyrrole B

Pyrrole B

(a) KOH, Benzyltrimethylammonium bromide, CHC1₃; (b) pen-3-yn-2-one, 5 mol% Cu catalyst (as Cu₂0), 20 mol% PPh₃, dioxane, 100 C for 6 to 8 hour, see (19); (c) a selective reduction using either (i) a Wolff-Kishner reduction using N₂H₂, KOH; (ii) a Clemmensen Reduction using Zn-Hg, HC1; (iii) a catalytic hydrogenation using Pt-C, H₂ under pressure; or (iv) using TsNHNH₂, then NaBH₃CN.

5) Synthesis of Dialdehyde

Dialdehyde

For more details regarding step (v) see (20).

Pharmacokinetic (PK) Study

A pharmacokinetic (PK) study was performed in male ICR following oral (PO) administration of SnMP (CR) and KCL-HO-ii (NS) at 38 and 44 mg/kg, respectively. The plasma samples were collected at predetermined times after PO administration. Two formulations containing SnMP (CR) and KCL-HO-ii 7 (NS)] were prepared as set out in Table 1, below. The dosing volume was 9.5 mL/kg for SnMP (CR) and 11 mL/kg for KCL-HO-ii (NS).

SnMP (CR) preparation (4 mg/mL). 23.38 mg of SnMP (CR) powder in 1.17 mL of 0.1M sodium hydroxide (NaOH); once fully dissolved, then added 4.68 mL of 0.5M sodium bicarbonate (NaHCO₃) at pH 7 to achieve the final concentration at 4 mg/mL.

KCL-HO-ii (NS) preparation (4 mg/mL). 26.68 mg of KCL-HO-ii (NS) powder in 1.334 mL of 0.1M NaOH; once fully dissolved, then added 5.336 mL of 0.5M NaHCO₃ at pH 7 to achieve the final concentration at 4 mg/ mL.

Male ICR mice, at 25 ± 5 g, were acquired by BioLasco Taiwan (under Charles River Laboratories Licensee). Animals were acclimated for 3 days prior to use and were confirmed with good health. All animals were maintained in a hygienic environment with controlled temperature (20 - 24°C), humidity (30% - 70%) and 12 hours light/dark cycles. Free access to sterilized standard lab diet [MFG (Oriental Yeast Co., Ltd., Japan)] and autoclaved tap water were granted. All aspects of this work, including housing, experimentation, and disposal of animals were performed in general accordance with the Guide for the Care and Use of Laboratory Animals: Eighth Edition (National Academy Press, Washington, D. C., 2011) in our AAALAC-accredited laboratory animal facility. The animal care and use protocol was reviewed and approved by the IACUC at Pharmacology Discovery Services Taiwan, Ltd.

Animal dosing design is set out in Table 2, below.

Plasma Sample Collection from Mice (Parallel)

Blood aliquots were collected via cardiac puncture (~3OO pL) from euthanatized mice in tubes coated with lithium heparin, mixed gently, and centrifuged at 2,500 xg for 15 minutes at 4°C, within 1 hour of collection. The plasma samples were then harvested and kept frozen at -7O°C until further processing.

Quantitative Bioanalysis (Plasma Samples) & Pharmacokinetics

The plasma samples were processed using methanol precipitation and analyzed by LC- MS/MS. The exposure levels (ng/mL) of the formulations [SnMP or KCL-HO-ii] in plasma samples were then determined by LC-MS/MS. The exposure levels (ng/mL) SnMP in mouse plasma samples after SnMP (38 mg/kg, PO) administration are shown in Figure 1, for KCL-HO-ii in Figure 2 and the combined plots are shown in Figure 3.

(a) This is based upon visual observation: S: soluble; SS: slight soluble I: insoluble (suspension or precipitation)

(b) Y: formula is kept in brown tube or vial, or covered with aluminium foil.

(c) RT: prepared fresh and stored between 2O-25°C. 4°C: prepared fresh and stored in the refrigerator or kept on ice.

Table 1: Formulations

Table 2: Animal Dosing Design In vivo Anti -tumoral Effect of Various Single and Combination Treatments

Mice bearing established tumours (MMTV-PyM) an aggressive model of breast cancer underwent an intraperitoneal (i.p.) dosing strategy providing individual treatment of KCL-HO-ii (25pMol/kg/day) or SnMP (25pMol/kg/day) or 5-FU (40 mg/kg/4 days) or gemcitabine (64mg/kg/7days) or vehicle injection, or a combined dual treatment of (i) KCL-HO-ii or SnMP with (ii) 5-FU or gemcitabine (see Fig. 4A & 4B). Treatment was initiated at day o which is marked by the vertical dashed line in each of Fig. 4C-4L. Tumour growth was monitored, and tumour volumes were calculated for each individual mouse tumour and the results are shown as growth curves for the different single (see Fig. 4C-4G) and combined treatments (see Fig. 4H-4J). KCL-HO-ii in combination with chemotherapy (with either 5-FU or Gemictabine) provides durable anti-tumour efficacy as illustrated in Fig. 4K & 4L where each line displays the mean (n=5 for each group of treatment) (** P<o.oi). Surprisingly, treatment of KCL-HO-ii and 5-FU had eradicated all tumour burden by day 8 post-initiation of treatment which provides a measurable improvement in the anti-tumour immune response as compared to treatment of standard commercial SnMP and 5-FU where animals still had residual disease (see Fig. 5).

Bioavailability

Plasma concentration was measured in a three stage process comprising (1) injection (ip) or oral gavage of SnMP (25pMol/kg) or KCL-HOli (25pMol/kg); (2) blood collections; and (3) LC/MS/MS measurements. Figure 7(A) provides a schematic representation of this process. Male mice received either intraperitoneal administration, see Figure 7(B) (left panel), or oral administration, see Figure 7(B) (right panel), of commercial SnMP or KCL-HOli. Blood aliquots were collected via cardiac puncture in tubes coated with lithium heparin at 0.167, 0.5, 1, 2, 4, 6, 8 and 24 hours after the oral administration of SnMP. After centrifugation, the plasma samples were harvested and kept in - 7O°C until processing. The exposure levels (ng/mL) of the SnMP (blue curves), veSnMP and KCL-HOli (red curves) are shown in Figure 7(B) (left panel) for intraperitoneal administration, and in Figure 7(B) (right panel) for oral administration, in plasma samples. These exposure levels were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Figure 7A). The plasma concentration of SnMP and KCL-HOli versus time in mice plasma are depicted as the mean ± SEM (Standard Error of Mean). Bar charts represent the mean and the dots show individual data points from individual mouse. A bioavailability study was carried out testing KCL-HOli against commercial SnMP using bioluminescence. A schematic representation of the HO-i^luc/^eGFP mouse model used and the strategy followed for bioluminescence is shown in Figure 7(C). The HO- iiuc/eGFP _{mjce were} injected with D-luciferin and imaged at To and 24 hours after being treated. The organs have been collected and imaged. Representative bioluminescence images comparing the relative luciferase expression between the HO- i^luc/^eGFP mouse treated with Vehicle, commercial SnMP or KCL-HOli at To (upper panel) and after 24 hours of treatment (bottom panel) of the whole body are shown in Figure 7(D). A relative quantification of the luciferase expression (photons/sec) for the whole body and across the tissues is shown in Figure 7(E). A representation of the total HO-1 and the change HO-i expression of each individual tissue and mouse was obtained by normalised to the Vehicle group. Unexpectedly KCL-HO-ii does not induce HO-1 expression that is seen as an undesirable side effect of commercial SnMP (Figure 7E, F). Figure 7(G) shows an evaluation of the inhibition of HO-1 activity in rat microsomes using a range of doses of KCL-HO-ii and SnMP (n=3 biological repeats) with the concentration (pM) of the KCL-HO-ii and SnMP displayed on a logarithmic scale.

(* P<O.O5, " P<0.01, ****P<0.001).

These results demonstrate that KCL-HOli shows a better availability than commercial SnMP in vivo.

In vivo Anti-tumoral Effect of Oral KCl-HO-ii in Combination with Gemcitabine

Mice bearing spontaneous tumours (MMTV-PyM) an aggressive model of breast cancer were given, gemcitabine (given intraperitoneally) in combination with KCL-HO-ii (given orally), as indicated in Figure 8A. Tumour measurement was started at day zero (cohorts of n=2 mice). Tumour growth was monitored, and tumour volumes were calculated for each mouse (as shown in Figure 8B where each line represents an individual mouse and tumour). KCL-HO-ii in combination with gemcitabine resulted in a control of tumour growth.

Study of CD8 T-cell infiltration into the tumour of MMTV-PyMT mice.

MMTV-PyMT spontaneous tumour mice with tumours that had reached 5OO-75Omm3 were used. Mice were given equivalent doses of commercial SnMP (cSnMP) or KCL- HO-ii on day o and 1, then the tumours were harvested on day 2. (thirty-six hours after the first injection). Tumours were enzyme-digested to release single cells and the proportion of CD8+CD3+CD45+ T-cells were assessed by flow cytometry. The CD8 T- cell infiltration into the tumour of MMTV-PyMT mice that were treated with vehicle, commercial SnMP(cSnMP) or KCL-HO-ii is shown as a bar graph in Figure 9 where the dots represent individual tumours and mice. ”* P< 0.001.

Effect of Immune-Depleting Anti-CD8a antibodies On In vivo Anti-tumoral Effect of KCl-HO-ii in Combination with Gemcitabine

Mice bearing spontaneous tumours (MMTV-PyM) were treated with non-immune IgG or with immune-depleting anti-CD8a antibodies. The mice were given the respective antibody treatment at day -2 as a loading dose and subsequent doses every 4 days. Some of these mice were also given KCL-HO-ii (25pMol/kg/day) and gemcitabine (64mg/kg/7days) starting at day o. Tumour growth was monitored, and tumour volumes were calculated for each mouse and the tumour growth curves are shown in Fig. 10B-10E. For Fig. 10B, 10C & 10D dots represent individual tumours and mice. Fig. 10E is a line chart where each line displays the mean for each treatment and the bars. In Fig. 10 a dashed black line marks the start of treatment (day o), a red line marks 250mm³, and ** P<o.oi. These results, in particular for KCL-HO- ii/gemcitabine/anti-CD8a antibodies as compared to KCL-HO-ii/gemcitabine (see Fig. 10E) indicate that the anti-tumour effect of KCL-HO-ii and gemcitabine is immunotherapeutic.

Investigation of T-cell Infiltration to the tumour and T-cell effector function.

Mice bearing established tumours (MMTV-PyM) were treated with KCL-HO-ii (25pMol/kg/day) and/or 5-FU (40 mg/kg/4 days) or gemcitabine (64mg/kg/7days) or vehicle. Tumours were analysed before treatment started and again at 36 h post initiation of treatment. The tumour growth over the 36 h treatment period are shown in the line chart Fig. 11B for each treatment. Tumours from treated MMTV-PyMT were removed, collected and investigated by flow cytometry to evaluate the tumour microenvironment populations by analysing the tumour for their stromal composition. There were no significant changes in the stromal populations of most cells (see Fig.nC). From an examination of CD8⁺ T-cells it was found that KCL-HO-ii caused an influx of CD8⁺ T-cells into the tumor microenvironment which was boosted by combining KCL-HOli with chemotherapy (see bar chart Fig. 11D). KCL-HO-ii treatment improved CD8⁺ T-cell effector function as assessed by their expression of IFN-gamma and this improvement was increased by combining KCL-HOli with chemotherapy (see bar chart Fig. 11E). There is evidence of synergy between KCL-HOli and the chemotherapeutic agents tested. In contrast to KCL-HOli, it was found that SnMP does not elicit CD8⁺ T-cell infiltration into the tumor microenvironment in MMTV-PyMT mice using an equivalent dose and regimen to that of KCL-HO-ii (see bar chart Fig. nF). The flow cytometry included using fluorescence-activated cell sorting (FACS) with gating to investigate and quantify populations of interest. A representative dot plot of FACS gated live (7AAD ), CD45⁺CD3⁺CD8⁺ T-cells showing the expression of CD44 and CD62L in vehicle treated mouse is shown in Fig. 11G and histograms represent the CD8⁺ T-cells sub populations (that are shown in Fig. 11G) across the different treatment groups are shown in Fig. 11H.

Bar charts show the mean and the dots show individual data points from individual tumors and mice. Line charts display the mean and SEM. * P<o.O5, ** P<o.oi.

Investigation by bulk RNAseq analysis

Mice bearing established MMTV-PyMT tumors were intraperitoneally dosed with KCL- HO-ii (25pMol/kg/day) and/or 5-FU (40 mg/kg) or gemcitabine (64mg/kg) or vehicle and tumor tissue was removed, collected and analyzed at 36 hours by bulk RNAseq analysis (cohorts of n=5 mice and tumors) as shown schematically in Fig. 12A. From this analysis Venn diagrams showing all upregulated DEGs (see Fig. 12B), and showing KCL-HO-ii treatment associated upregulated DEGs (see Fig. 12C) for the respective treatments against vehicle and their intercepts between groups were plotted. A heatmap was also prepared of hierarchical clustered common upregulated DEGs between treatment groups (419 genes) that are secreted genes (91 genes) across treatments where the scale is from low to high of the greatest change from vehicle treated tumours (see Fig 12D). Also from this analysis, a Venn diagram showing all of the upregulated DEGs for treatments that include chemotherapy (5-FU or Gemcitabine either alone or with KCL-HO-ii) against vehicle were plotted (see Fig 12E). A Venn diagram showing the chemokine and upregulated DEGs associated dual therapy treatments (KCL-HO-ii/5-FU or KCL-HO-ii/Gemcitabine) against vehicle and their intercepts between groups was plotted (see Fig. 12F). A heatmap was also prepared of hierarchical clustered chemokine and cytokine upregulated DEGs associated with dual treatment groups across treatments where the scale is from low to high of the greatest change from vehicle treated tumours (see Fig. 12G). In vivo Treatment Oral KCl-HO-ii in Combination with Gemcitabine with Analysis of Animals Physical characteristics and Blood

Mice bearing established MMTV-PyMT tumors were oral dosed with KCL-HO-ii (25|uMol/kg/day) and/or intraperitoneally delivered gemcitabine (64mg/kg/7days) or vehicle (see Fig. 13A). Line charts of the tumour growth where each line displays the mean for the mice undergoing each treatment are shown in Fig 13B from which it can be seen that KCL-HO-ii in combination with gemcitabine resulted in a control of tumour growth. The body weights of the mice were measured during treatment and were found to stay close to the starting body weight (see Fig. 13C). At 21 days post treatment blood samples were taken and the plasma was separated from the blood by centrifugation. Serum liver aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzymes were evaluated by enzyme-linked immunosorbent assay (ELISA) and the AST/ALT ratio were accessed and depicted (see Fig. 13D) in the histogram (n=5 for each group of treatment). From this it was found that there were no overt changes in any of the treatment groups as compared to treatment with vehicle (see Fig. 13D). The blood immune cell composition was measured (see Fig. 13E) and the animals physical characteristics were examined by their behavioural responses at day 21 for any signs of distress and these were scored on a scale of o to 5 (see heatmap shown in Fig. 13F). No signs of distress were found in the animals and these results along with the body weight measurements and the blood analysis all indicate that these treatments were non-toxic at the doses tested.

Bar charts show the mean and the dots show individual data points from individual tumors and mice. Line charts display the mean and SEM. * P<o.O5, ** P<o.oi.

Pathohistological evaluation of organs from mice treated with KCL-HO-ii and/or Gemcitabine.

Mice were dosed with KCL-HO-ii (25pMol/kg/day using a regimen of 5 days on and 2 days off treatment) using oral gavage delivery and/or gemcitabine (64mg/kg/7days) delivered i.p. or vehicle in MMTV-PyMT mice bearing established tumors (n=5 for each group of treatment). At 21 days post initiation of treatment the indicated tissues were harvested for histological examination and tissue sections from each mouse and tissue were stained with hematoxylin and eosin stain (H&E) and scored by a pathologist. These scores used a non-linear, semi-quantitative grading system from o to 5, where o = no significant change and 5 = whole organ or tissue affected for each observation. From the histological examination of tissues from the brain, heart, kidney and liver little or no significant change was observed for all of the treatments. However, the pathologist scored the mice up to a score of 3 for the KCL-HO-ii and gemcitabine dual treatment when scoring the mononuclear inflammatory cell infiltrate, multifocal (perivascular) pathology in the histological examination of tissues from the lungs. Also, for the lungs the pathologist scored the mice up to a score of 2 for the KCL-HO-ii and gemcitabine dual treatment when scoring the mixed inflammatory cell infiltrate, focal (alveolar). However, scores of o were recorded for the lungs when scoring metastases and when scoring foamy macrophages (alveolar) pathologies in the histological examination of tissues from the lungs.

A further patho histological evaluation of lungs from mice was carried out on nontumor bearing C57BI/6 mice that were dosed per os with a dual treatment of KCL-HO- ii (25pMol/kg/day) and gemcitabine (64mg/kg/7days) or vehicle for 21 days. At the end of treatment, lungs were then excised from one cohort of mice and a second left 30 days without treatment prior to sacrifice. Lung sections were H&E stained and assessed by a pathologist and scored on the same non-linear, semi-quantitative grading system. For the dual treatment at the end of treatment scores of up to 3 were again observed for the mononuclear inflammatory cell infiltrate, multifocal (perivascular) pathology and scores up to 2 for the mixed inflammatory cell infiltrate, focal (alveolar) in the histological examination of tissues from the lungs. However, 30 days post end of treatment lower scores were observed for these pathologies and for some mice a score of o was recorded in the histological examination of tissues from the lungs.

These pathohistological evaluations indicate that -HO-ii and gemcitabine dual treatment shows some lung toxicity but that these issues start to resolve after treatment stops. The absence of metastases following the dual treatment indicates that KCL-HO- ii may have an antimetastatic effect.

Further In vivo Anti-tumoral Effect of Oral KCl-HO-ii in Combination with Gemcitabine

Mice (C57BI/6) bearing established sub cut. sarcoma tumours (MN-MCA-i cell line) were given KCL-HO-ii (25pMol/kg/day, administered orally) and/or intraperitoneally administered gemcitabine (64mg/kg/7days) or vehicle, as indicated schematically in Figure 14A. Tumour measurement was started at day zero. Tumour growth was monitored, and tumour volumes were calculated for each mouse. Tumour growth curves for the respective treatments are shown in Figure 14B where the lines display the mean of the mice undergoing the treatment and the bars show the SEM. KCL-HO-ii in combination with gemcitabine resulted in a control of tumour growth for this sub cut. sarcoma tumors. ** P<o.oi.

Anti-Metastatic Effects.

4T1 mammary adenocarcinoma cells were orthotopically implanted in Balb/c mice (BioLasco Taiwan under Charles River Laboratories Licensee) for tumours. A total of 2.5 x to⁵ cells in toopl Roswell Park Memorial Institute medium (RPMI 1640 medium) were injected subcutaneously into the mammary fat pad of syngeneic female mice, then the tumour growth monitored and this is depicted in Figure 15A. At day 12 post tumour injection, the mice received intraperitoneally either KCL-HO-ii (25 pMol/kg) or vehicle daily for 14 days. At day 26 post tumour injection, the lungs were harvested and the metastatic nodules were counted for both the KCL-HO-ii and the vehicle treated group and this is represented in the histogram depicted in Figure 15B. KCL-HO-ii exhibited an anti-metastatic effect in 4T1 mice compared to the vehicle treated group (see Figure 15B) where the dots represent individual mice, **P<o.oi). This model shows that the primary tumour growth is not affected, so the lack of lung metastasis observed for the KCL-HO-ii vehicle treated group is not simply a reflection of the lower tumour burden.

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All publications mentioned in the above specification are herein incorporated by reference. Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiment and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.

Claims

1. A method of preparing a compound of formula (I): or metallo derivatives, salts and solvates thereof, wherein the method comprises the step of adding a dicarboxylic acid compound of the following formula: or salts and solvates thereof, to a dialdehyde compound of the following formula: or salts and solvates thereof, and reacting to form a porphyrin diester compound of the following formula:

or metallo derivatives, salts and solvates thereof; wherein each of Ri, R2, R3, R4, Rs, and Re, are independently selected from H, methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, C1-2 haloalkyl, C1-2 alkanamine, and C(0)CH3; and R7 and Rs are independently selected from C1-6 alkyl; and each R’ and R” are independently selected from H and C1-6 alkyl.

2. A method according to claim 1, wherein the method further comprises the step of hydrolysis of the porphyrin diester compound or metallo derivatives, salts and solvates thereof; to provide a dicarboxylic acid of formula (I) or metallo derivatives, salts and solvates thereof.

3. A method according to claim 1 or 2, wherein the preparation of the dicarboxylic acid compound further comprises a step of reacting pyrrole A: or salts and solvates thereof with pyrrole B: or salts and solvates thereof; to form a diester compound of the following formula: or salts and solvates thereof, wherein R9 and Rio are independently selected from benzyl, para-methoxybenzyl, and 2,4-dimethoxybenzyl.

4. A method according to claim 3, wherein the method further comprises a step of subjecting a diester compound of the following formula: or salts and solvates thereof, to a deprotection reaction to form a dicarboxylic acid compound of the following formula: or salts and solvates thereof.

5. A method according to any of the preceding claims, wherein the method further comprises the step of converting the compound of formula (I) or metallo derivatives, salts and solvates thereof, to a metallo derivative that is a tin compound, wherein the tin compound is a tin mesylate compound, a tin oxalate compound, a tin phosphate compound or a tin tartrate compound of the porphyrin dicarboxylic acid of formula (I) or salts and solvates thereof.

6. A method according to any of the preceding claims, wherein the method further comprises the step of converting the compound of formula (I) or metallo derivatives, salts and solvates thereof, to a metallo derivative that is a tin compound, wherein the tin compound is:

7. A method according to any of the preceding claims wherein the method further comprises the step of converting the compound of formula (I) or metallo derivatives salts and solvates thereof, to a tin (IV) phosphate salt of formula (II) or (III):

or salts and solvates thereof.

8. A compound of formula (I) or metallo derivatives salts and solvates thereof, wherein the compound is:

or salts and solvates thereof, wherein each of Ri, R2, R3, R4, Rs, and Re, are independently selected from H, methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, C1-2 haloalkyl, C1-2 alkanamine, and C(0)CH3; and each R’ and R” are independently selected from H and C1-6 alkyl. or salts and solvates thereof, wherein each of Ri, R2, R3, R4, Rs, and Re, are independently selected from H, methyl, ethyl, halogen, methoxy, ethoxy, NR’R”, C1-2 haloalkyl, C1-2 alkanamine, and C(0)CH3; and each R’ and R” are independently selected from H and C1-6 alkyl.

10. A compound of formula (II) or (III) according to claim 9, wherein Ri, R3, Rs, and Re are methyl; and R2, and R4 are ethyl, which may be represented by formula (VII) or (VIII):

or salts and solvates thereof.

11. A compound of formula (I) or salts and solvates thereof according to claim 8, 9, or 10, for use in the treatment of a proliferative and/or malignant disease.

12. A compound of formula (I) or salts and solvates thereof for use in the treatment of a proliferative and/or malignant disease according to claim 11, wherein the proliferative and/or malignant disease is selected from breast, lung, brain and central nervous system cancers, carcinomas, gastrointestinal cancers, hormonal cancers, leukemias, liver cancer, lymphomas, sarcomas and fibrosarcomas, skin cancers and melanomas, urinary tract and reproductive cancers, and miscellaneous other cancers.

13. A compound of formula (I) or salts and solvates thereof for use in the treatment of a proliferative and/or malignant disease, according to claim 11 or 12, wherein the compound is administered either simultaneously or sequentially with one or more immunotherapeutic agent or other anti-cancer agent.

14. A compound of formula (I) or salts and solvates thereof according to claim 8, 9, or 10, for use in treating or inhibiting metastasis.

15. A pharmaceutical composition comprising a compound of formula (I) or salts and solvates thereof according to claim 8, 9, or 10, and a pharmaceutically acceptable excipient, carrier or diluent.