WO2019125184A1

WO2019125184A1 - Use of biomarker in cancer therapy

Info

Publication number: WO2019125184A1
Application number: PCT/NZ2018/050180
Authority: WO
Inventors: Christopher Paul Guise; Adam Vorn Patterson; Cristin Gregor Print; Shevan SILVA; Jeffrey Bruce Smaill
Original assignee: Auckland Uniservices Ltd
Current assignee: Auckland Uniservices Ltd
Priority date: 2017-12-19
Filing date: 2018-12-18
Publication date: 2019-06-27
Anticipated expiration: 2020-06-19

Abstract

In certain aspects, provided herein are methods of using Six-Transmembrane Epithelial Antigen of Prostate 4 (STEAP4) in the metabolism of certain hypoxia-activated prodrugs (HAPs), and the application of such in cancer therapies, including methods of predicting the responsiveness of an individual with cancer to treatment with a HAP based on levels of STEAP4 expression.

Description

USE OF BIOMARKER IN CANCER THERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/683,157, filed June 11, 2018, U.S. Provisional Application No. 62/608,486, filed December 20, 2017, and U.S. Provisional Application No. 62/607,451, filed December 19, 2017, each of which is incorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application incorporates by reference a Computer Readable Form (CRF) of a Sequence Listing in ASCII text format submitted with this application, entitled "14506-008- 228_ST25.txt", was created on December 11, 2018, and is 30 kilobytes in size.

TECHNICAL FIELD

The present disclosure relates to the role played by Six-Transmembrane Epithelial Antigen of Prostate 4 (STEAP4) in the metabolism of certain hypoxia-activated prodrugs (HAPs), and the application of such in cancer therapies.

BACKGROUND

Cancer continues to be a significant unmet clinical indication. Expression of the enzyme STEAP4 has been shown to be associated with certain cancers (Gomes et al, 2012; Xue et al, 2017). STEAP4 has also been shown to be highly induced by hypoxia, i.e., low oxygen at the tissue level. Moreover, hypoxia within tumours is associated with poor prognosis of cancer patients and with treatment failure (Hunter et al., 2016), such as resistance to radiotherapy and traditional chemotherapy. Accordingly, there is a need in the art to identify cancer therapies that can be effectively used to treat or prevent cancers associated with high expression of STEAP4, and to identify patient populations with STEAP4 expressing cancers which are likely to be responsive to hypoxia-activated prodrugs (HAPs). It is therefore an object of the present disclosure to provide identification, predictive and treatment methods as provided herein based on this work, and/or at least to provide the public with a useful choice.

SUMMARY

The aspects and embodiments described and claimed herein have many attributes and embodiments including, but not limited to, those set forth or described or referenced in this Summary. It is not intended to be all-inclusive and the aspects and embodiments described and claimed herein are not limited to or by the features or examples identified in this Summary, which is included for purposes of illustration only and not restriction.

High levels of STEAP4 expression are associated with certain cancers. In certain embodiments, the inventors surprisingly found that when STEAP4 is highly expressed in cancers, STEAP4 can catalyse a reaction in HAPs leading to release of the drug payload and/or modification of the HAP to a form that is able to penetrate the cell membrane and cause cell death. Thus, the inventors surprising found that cancers with elevated STEAP4 expression levels respond to treatment by HAPs. Accordingly, the inventors have surprisingly found an effective treatment for STEAP4-associated cancers. Accordingly, in one aspect of the present disclosure there is provided a method of treating cancer in an individual in need thereof, the method comprising :

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells;

c) predicting the individual as being likely to be responsive to treatment by a HAP if the tumour cells exhibit an elevated level of STEAP4 expression; and

d) administering a therapeutically effective amount of a HAP to the individual.

In another aspect, the present disclosure provides a method of treating cancer in an individual in need thereof, where tumour cells of the individual exhibit an elevated level of STEAP4 expression, the method comprising :

a) administering a therapeutically effective amount of a HAP to the individual.

In yet another aspect, the present disclosure provides a method of predicting the responsiveness of an individual with cancer to treatment with a HAP, the method comprising :

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells;

c) predicting the individual as being likely to be responsive to a treatment with the HAP if the tumour cells exhibit an elevated level of STEAP4 expression.

a) determining the level of STEAP4 expression in a sample from the individual, wherein the sample comprises tumour cells;

b) predicting the individual as being likely to be responsive to a treatment with the HAP if the tumour cells exhibit an elevated level of STEAP4 expression.

The present disclosure also provides kits for performing the disclosed methods.

In some embodiments, provided herein are certain HAPs, for example, a HAP having the structures of Compound A, Compound C, and Compound E.

.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the amino acid sequence SEQ ID NO: l, which is isoform 1 of the

STEAP4 protein (NCBI Acc No. NM_024636.3), and comprises 459 amino acids.

Figure 2 shows the nucleotide sequence SEQ ID NO:2, which is variant 1 (and the predominant variant) encoding isoform 1 of the STEAP4 protein, and comprises 4488 base pairs.

Figure 3 shows the nucleotide sequence SEQ ID NO:3, which is variant 2 (and an alternative to variant 1) encoding isoform 1 of the STEAP4 protein, and comprises 4587 base pairs.

Figure 4 shows the amino acid sequence SEQ ID NO:4, which is isoform 2 of the STEAP4 protein (NCBI Acc No. NM_001205315.1), and comprises 283 amino acids. Figure 5 shows the nucleotide sequence SEQ ID NO: 5, which is variant 3 encoding isoform 2 of the STEAP4 protein, and comprises 3960 base pairs.

Figure 6 shows the effect of siRNA knockdown in HCC1954 and SiHa cells of five reductase genes ( STEAP4 , SDHA, DCXR, COQ6 and FOXRED1 ) on Compound A metabolism, as described in Example 3.

Figure 7 shows the enforced overexpression expression of STEAP4 in H1299 and C33A cells by western blot and the effect of STEAP4 overexpression in H1299 and C33A cells on Compound A metabolism under aerobic (oxic) and anoxic conditions, as described in Example 4.

Figure 8 shows the structures for the prodrugs TH-302 (also known as evofosfamide), tirapazamine (TPZ or SR4233), and Compound C (also known as PR-104A or SN27858), as referred to in Example 5.

Figure 9 is a graphic showing the layout of the 96-well plate, as described in Example 5.

Figure 10 shows the IC50 values for TH-302, TPZ, and Compound C in wild-type (WT) and STEAP4-expressing cells, where the concentration of prodrug required to inhibit cell growth by 50% (IC50) is shown, as described in Example 5.

Figure 11 shows the metabolism of the prodrugs Compound A and Compound C in wild-type A431 cells (A431^WT) and in cells over-expressing cytochrome P450 oxidoreductase (A431^por) under aerobic (oxic) and anoxic conditions, and metabolism of the prodrugs Compound A by cell-free recombinant POR under anoxia in the presence of NADPH, as described in Example 6.

Figure 12 shows the nucleotide sequence SEQ ID NOs: 6-l l, which may be used as probes to detect STEAP4 mRNA sequences, including as PCR primers, as herein described.

Figure 13 shows the results of the RT-PCR study demonstrating the feasibility of using selected primers for qPCR detection of STEAP4, as described further in Example 7.

Figure 14 shows the results of the qPCR where STEAP4 expression was normalised in reference to GAPDH expression, across a panel of human cancer cell lines, as described in Example 7.

Figure 15 shows the results of the Western blot for STEAP4 in H 1299^WT cells (lane 2) and H 1299^STEAP4 cells (lane 3), as described in Example 7.

Figure 16 shows the results of the functional assay for STEAP4 based on the ferrireductase activity of the STEAP4 enzyme in parental C33A^WT and C33A^STEAP4 cells, as described in Example 7.

Figure 17 shows the rate of Compound B production under aerobic and anoxic conditions in C33A^WT, C33A^STEAP4, H 1299^WT, and H 1299^STEAP4 cells following exposure to 10pM Compound A for 90 minutes, as described in Example 7. Figure 18 shows the expression of STEAP4 relative to GAPDH and HPRT expression in SiHa clones derived following transfection with the STEAP4 gRNA containing px458 plasmid, as described in Example 8.

Figure 19 shows the measured Compound A metabolism under anoxic conditions in parental SiHa^WT cells and SiHa clone #50 cells as measured by the rate of Compound B formation in each cell line, as described in Example 8.

Figure 20 shows the fold-change in STEAP4 expression in each isogenic cell line pairs relative to each parental (WT) cell line, as described in Example 9.

Figure 21 shows the Compound A metabolism under anoxic conditions in parental (WT) and STEAP4-overexpressing C33A and PC9 isogenic cell line pairs, as described in Example 9.

Figure 22 shows the change (increase) in Compound A metabolism (as determined by tumour Compound B concentration) in STEAP4-overexpressing PC9 and C33A xenografts relative to parental (WT) xenografts. Mean concentrations were obtained from n = 5 PC9^WT, n = 5 PC9^STEAP4, n = 3 C33A^WT and n = 4 C33A^STEAP4 tumours, as described in Example 9.

Figure 23 shows the intratumoural Compound B concentrations in individual parental and STEAP4 k/o SiHa tumours (n = 6 per tumour type), as described in Example 10.

Figure 24 shows the mean ± SEM Compound B concentrations in the same parental and STEAP4 k/o SiHa tumours as shown in Figure 23, and as described in Example 10.

Figure 25 shows the intratumour ratios of Compound B: Compound A in parental and STEAP4 k/o SiHa tumours, as described in Example 10.

Figure 26 shows STEAP4 expression by qPCR in 5/6 parental SiHa and 5/6 STEAP4 k/o SiHa tumours, as described in Example 10.

Figure 27 shows the products from the qPCR reaction run on a 2% agarose gel, as described in Example 10.

Figure 28 shows STEAP4 expression by immunohistochemical detection in parental C33A^WT tumours and C33A^STEAP4 tumour xenograft tissue sections engineered to overexpress STEAP4, as described in Example 7.

Figure 29 shows STEAP4 expression by immunohistochemical detection in parental SiHa^WT tumours and SiHa^{STEAP4 nu}" tumour xenograft tissue sections where the STEAP4 gene has been genomically disrupted and results in the absence of protein expression, overexpress STEAP4, as described in Example 10.

Figure 30 shows a schematic depicting an exemplary STEAP4-mediated activation of Compound A.

Figure 31 shows anti-tumour activity of tarloxotinib when administered as a single dose (48 mg/kg) in C33A^WT (WT) and STEAP4 overexpressing C33A^STEAP4 (STEAP4) tumours. The top three (3) lines correspond to WT #1, WT #2, and WT #3. The bottom three (3) lines correspond to STEAP4 #1, STEAP4 #2, and STEAP4 #3.

DETAILED DESCRIPTION

Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in organic chemistry, biochemistry, and medicine).

It is intended that reference to a range of numbers disclosed herein (e.g., 1 to 10) also incorporates reference to all related numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

The terms "alkyl", "alkenyl", "alkynyl" and "alkoxy" include both straight chain and branched chain groups, and unsubstituted and substituted groups. The optional substituents may include, without limitation, halogen, C1-C6 alkoxy, CN, OH, NH₂, N0₂, NH(CI-C₆ alkyl), N(CI-C₆ alkyl)₂, CONH₂, CO(Ci-C₆ alkyl), S0₂NH₂ and S0₂(Ci-C₆ alkyl).

The term "quaternisable nitrogen" means a fully substituted nitrogen of sufficient basicity (or nucleophilicity) to react with an electrophilic group such as an a-methyl halide/mesylate/tosylate or triflate to provide a quaternary ammonium salt of the nitrogen.

The term "aromatic nitroheterocycle" means an aromatic heterocyclic moiety substituted at any ring position by one or more nitro (N0₂) groups. The aromatic heterocyclic moiety may be a monocyclic or bicyclic ring containing 4 to 12 atoms of which at least one atom is chosen from nitrogen, sulphur or oxygen. The aromatic heterocyclic moiety may be carbon or nitrogen linked. The aromatic heterocyclic moiety may additionally be substituted by one or more additional substituents at any available ring carbon or heteroatom. The substituents may include, but are not limited to the groups as defined for R₂₆ in Formula V.

The term "aromatic nitrocarbocycle" means a benzene moiety substituted at any position by one or more nitro (N0₂) groups. In addition, two adjacent ring carbon atoms may optionally be linked to form a fused carbocyclic or heterocyclic ring. The benzene moiety (and optional fused ring) may additionally be substituted by one or more additional substituents at any available carbon or heteroatom. The substituents may include, but are not limited to, the groups as defined for R26 in Formula V.

The term "pharmaceutically acceptable" means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary as well as human pharmaceutical use.

The term "pharmaceutically acceptable salts" of a compound means salts that are pharmaceutically acceptable, as defined herein, and that possess the desired pharmacological activity of the parent compound. Such salts include: acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with organic acids such as acetic acid, methanesulfonic acid, maleic acid, tartaric acid, citric acid and the like; and salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminium ion; or coordinates with an organic or inorganic base. Acceptable organic bases include ethanolamine, diethanolamine, N-methylglucamine, triethanolamine and the like. Acceptable inorganic bases include aluminium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.

The term "prodrug" refers to a compound that, after administration, is metabolised or otherwise converted to a biologically active or more active compound (or drug) with respect to at least one property. A prodrug, relative to the drug, is modified chemically in a manner that renders it, relative to the drug, less active or inactive, but the chemical modification is such that the corresponding drug is generated by metabolic or other biological processes after the prodrug is administered. A prodrug may have, relative to the active drug, altered metabolic stability or transport characteristics, fewer side effects or lower toxicity, or improved flavour (for example, see the reference Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392, incorporated herein by reference). A prodrug may be synthesized using reactants other than the corresponding drug.

The term "hypoxic" refers to a concentration of oxygen in tissue that is significantly lower the normal physiological concentration of oxygen in healthy well perfused tissue, in particular oxygen tensions below approximately 1% (10,000 parts per million oxygen; 7.6 mmHg). The term "anoxia" or "anoxic conditions" (and similar) refers to an absence (or near absence) of oxygen in tissue, and in particular oxygen tensions below approximately 1 parts per million oxygen.

The terms "treat", "treating", and "treatment" as used herein refer to an action that occurs while an individual is suffering from the specified cancer, which reduces the severity of the cancer or the symptoms of the cancer, or retards or slows the progression of the cancer. For instance, in some embodiments, "treatment" may refer to a 5%, 10%, 25%, 50%, or 100% decrease in the rate of progress of a tumour. In some other embodiments, "treatment" may refer to a 5%, 10%, 25%, 50%, or 100% decrease in determined tumour burden (/.e., the number of cancerous cells present in the individual, or the size of the tumour). In some other embodiments, "treatment" may refer to a 5%, 10%, 25%, 50%, or 100% decrease in the physical symptoms of a cancer. In some other embodiments, "treatment" may refer to a 5%, 10%, 25%, 50%, or 100% increase in the general health of the individual, as determined by any suitable means, such as cell counts, assay results, or other suitable means.

The cancer may be any suitable cancer, including those as further defined herein.

The term "individual" or "subject" as used interchangeably herein refers to any mammalian animal including a human being. Suitably, the individual is a human being.

The phrase "providing tumour cells" may refer to the step of obtaining cells of the individual (e.g., by way of biopsy or otherwise), or may refer to the step of receiving a sample of tumour cells which has previously been obtained from the individual.

The tumour cells may comprise a sample. In some embodiments, the sample comprises a biological sample and can be, for instance, a cell, a cell culture, a tissue, or a biological fluid. Suitably, the biological sample may comprise a tumour cell biopsy, a plurality of samples from a clinical trial, or the like. The sample can be a crude sample, or can be purified to various degrees prior to storage, processing, or measurement.

The term "determining" as used herein generally refers to any form of measurement, and includes determining if the Six-Transmembrane Epithelial Antigen of Prostate 4 (STEAP4) protein or mRNA is present or not. The term "determining" includes both quantitative and/or qualitative determination. The STEAP4 expression levels may be determined by any suitable method known to those skilled in the art, including those as further defined herein. The expression "elevated level of STEAP4 expression" is further defined herein. The terms "determining", "measuring", "evaluating", "assessing" and "assaying" are used interchangeably herein.

The term "predict" generally means to determine or tell in advance. When used to "predict" the responsiveness to a treatment for example, the term "predict" can mean that the likelihood of the outcome of the cancer treatment can be determined at the outset, before the treatment has begun, or before the treatment period has progressed substantially. A predictive method may also be described as a prognostic method.

As used herein, "likelihood", "likely to", and similar generally refers to an increase in the probability of an event. Thus "likelihood", "likely to", and similar when used in reference to responsiveness to cancer therapy generally contemplates an increased probability that the individual will exhibit a reduction in the severity of cancer or the symptoms of cancer or the retardation or slowing of the cancer progression. The term "likelihood", "likely to", and similar when used in reference to responsiveness to cancer therapy can also generally mean the increase of indicators, such as mRNA or protein expression, that may evidence an increase in cancer treatment.

The hypoxia-activated prodrug and HAP refer to any suitable HAP, including those as further defined herein. Suitable administration methods for HAPs are further described herein.

The term "therapeutically effective amount" of a HAP refers to an amount of the HAP, alone or in combination with other therapies, which is sufficient to treat a cancer. A therapeutically effective amount of a compound refers to the amount of the compound that, when administered, is sufficient to prevent the development of, or alleviate to some extent, one or more of the symptoms of the cancer. The term also refers to the amount of the compound that is sufficient to elicit a biological or medical response of a biological molecule (e.g., a protein, enzyme, RNA, or DNA), cell, tissue, system, animal, or human, which is being sought by a researcher, veterinarian, medical doctor, or clinician. Furthermore, a therapeutically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment or management of the cancer. The term encompasses an amount of the compound that improves overall therapy, reduces, or avoids symptoms or causes of the cancer, or enhances the therapeutic efficacy of another therapeutic agent.

The term "level" refers to the amount, accumulation, or rate of a biomarker molecule. A level can be represented, for example, by the amount or the rate of synthesis of a messenger RNA (mRNA) encoded by a gene, the amount or the rate of synthesis of a polypeptide or protein encoded by a gene, the amount or the rate of synthesis of a biological molecule accumulated in a cell or biological fluid, or the biological activity of a biological molecule in a cell or biological fluid. The term "level" refers to an absolute amount of a molecule in a sample or to a relative amount of the molecule, determined under steady-state or non-steady-state conditions.

The term "responsiveness" or "responsive" when used in reference to a treatment refers to the degree of effectiveness of the treatment in lessening or decreasing the symptoms of a disease, disorder, or condition being treated. For example, the term "increased responsiveness" when used in reference to a treatment of a cell or a subject refers to an increase in the effectiveness in lessening or decreasing the symptoms of the disease when measured using any methods known in the art. In certain embodiments, the increase in the effectiveness is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%.

STEAP4 protein

The Six-Transmembrane Epithelial Antigen of Prostate (STEAP) protein family contains four members (STEAP1-4) though only STEAP2-4 have oxidoreductase activity.

The STEAP4 protein, also known as STAMP2 or TIARP, is a metalloreductase that reduces iron and copper ions. STEAP4 has equivalent activity under either physiological or acidic pH (pH5.5 - 7.5).

The full length nucleotide sequence encoding the STEAP4 protein (/.e., the STEAP4 gene) and the full length amino acid sequence of the STEAP4 protein are known in the art (see, e.g., NCBI Gene ID: 79689, NCBI Acc. No. NM_024636, NCBI Acc. No. NM_001205315.1, NCBI Acc. No. NM_001205316.1, and UniProt Acc. No. Q687X5). At least two isoforms of the STEAP4 protein exist. Isoform 1 is 459 amino acids in length and is shown in SEQ ID NO: l (also in Figure 1). This isoform 1 is encoded by two variants: variant 1 and variant 2. Variant 1 is the predominant nucleotide sequence encoding isoform 1 of the STEAP4 protein, and is shown in SEQ ID NO:2 (also in Figure 2). Variant 2 is an alternative nucleotide sequence encoding isoform 1 of the STEAP4 protein, and is shown in SEQ ID NO:3 (also in Figure 3). Isoform 2 of the STEAP4 protein is 283 amino acids long, and is shown in SEQ ID NO:4 (also in Figure 4). Variant 3 is a nucleotide sequence encoding this shorter isoform of STEAP4 protein (isoform 2) and is shown in SEQ ID NO: 5 (also in Figure 5).

As used herein, reference to the "STEAP4", "STEAP4 protein", or similar, refers to isoform 1 (SEQ ID NO: l), isoform 2 (SEQ ID NO:4), a protein encoded by variant 1 (SEQ ID NO:2), a protein encoded by variant 2 (SEQ ID NO:3), or a protein encoded by variant 3 (SEQ ID NO: 5), and/or to any other variant thereof. Variants contemplated within the scope of the present disclosure include protein variants which are substantially homologous to a native STEAP4 protein. The term "substantially homologous" as used herein refers to a protein having one or more naturally or non-naturally occurring amino acid deletions, insertions, or substitutions (e.g., derivatives, homologs, and fragments), as compared to the amino acid sequence of a native STEAP4 protein. The amino acid sequence of a STEAP4 variant may be at least about 40% identical, at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to a native STEAP4 protein. As used herein, a "native STEAP4 protein" refers to the STEAP4 proteins which are found in nature and are not manipulated by man, and includes isoform 1 (SEQ ID NO: l), isoform 2 (SEQ ID NO:4), a protein encoded by variant 1 (SEQ ID NO:2), a protein encoded by variant 2 (SEQ ID NO:3), and a protein encoded by variant 3 (SEQ ID NO: 5). Variants contemplated within the scope of the present disclosure also include proteins encoded by polynucleotide variants which have substantial sequence similarity or sequence identity to a native STEAP4 gene. The polynucleotide sequence of a STEAP4 variant may have at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% sequence similarity or sequence identity with a native STEAP4 gene. As used herein, a "native STEAP4 gene" refers to genes: (a) which are found in nature and are not manipulated by man, and encode STEAP4 proteins; and (b) which encode STEAP4 proteins that are found in nature and are not manipulated by man; and includes the gene having the polynucleotide sequence encoding isoform 1 (SEQ ID NO: l), the gene having the polynucleotide sequence encoding isoform 2 (SEQ ID NO:4), the gene with the polynucleotide sequence of variant 1 (SEQ ID NO:2), the gene with the polynucleotide sequence of variant 2 (SEQ ID NO:3), and the gene with the polynucleotide sequence of variant 3 (SEQ ID NO:5).

High levels of STEAP4 expression are associated with certain cancers. In certain embodiments, when STEAP4 is highly expressed in a cancer, the inventors have surprisingly found that STEAP4 can catalyse conversion of an administered drug into its active form, such as a reaction leading to release of a drug payload. Accordingly, in some embodiments, high STEAP4 expression leads to release of the drug payload in HAPs. In certain embodiments, the high STEAP4 expression is associated with hypoxic metabolism. In certain embodiments, the high STEAP4 expression is associated with a hypoxic environment. In certain embodiments, the high STEAP4 expression is associated with a hypoxic tumour. In certain embodiments, the high STEAP4 expression is not associated with low oxygen levels.

In certain embodiments, when STEAP4 is present in hypoxic tumour environments, STEAP4 catalyses one-electron reduction of cell-excluded quaternary ammonium salt HAPs, leading to their fragmentation selectively in pathophysiologically hypoxic tumour tissues, releasing the active drug which can then cross the cell wall and kill the cancer cell. Without wishing to be bound by theory, it is thought that STEAP4 is located on the plasma membrane. As such, in some embodiments, STEAP4 reduces the HAP extracellularly, forming a molecule that is capable of diffusing into the cell (e.g., a reduced form of the molecule). In some embodiments, STEAP4 is on the plasma membrane and reduces the HAP extracellularly, at which point the charged molecule undergoes fragmentation and diffuses into the cell to inhibit EGFR. In certain embodiments, the charged HAP molecule undergoes fragmentation under hypoxic conditions. In certain embodiments the charged HAP molecule undergoes fragmentation in the presence of elevated levels of STEAP4. In certain embodiments, the HAP is Compound A. In certain embodiments, the HAP is Compound C. In certain embodiments, the HAP is Compound E. In certain embodiments, the HAP is selected from the group consisting of Compound A, Compound C, and Compound E.

While NMQ prodrugs used in the disclosed methods function by releasing an active molecule having undergone reduction in the extracellular medium, other HAPs for use in the disclosed methods do not fragment and release an active molecule, but instead are effective because the reduced form of the molecule is able to penetrate the cell membrane and then cause cell death. In certain embodiments, the HAPs may have a limiting or low rate of membrane penetration such that extracelluclar metabolism by STEAP4 will contribute a significant proportion of total cellular metabolism and such that intracellular reductases will contribute a less significant proportion of total cellular metabolism. One such class of HAPs is the nitrophenyl mustards. For example, in some embodiments, HAPs with a net neutral charge, e.g., nitrophenyl mustards, may be hydrophilic in nature, e.g., may have a low partition coefficient, which can result in a limiting rate of cell membrane penetration. In certain embodiments when HAPs have a limiting rate of cell membrane penetration, extracellular metabolism by STEAP4 contributes a significant proportion of total cellular metabolism, and the remainder of HAP cellular metabolism is due to intracellular reductases.

In certain embodiments, HAPs with a low pKa may be protonated at physiologically relevant pH range and thus carry a net positive charge, resulting in a low partition coefficient, which can result in a limiting rate of cell membrane penetration. In certain embodiments when HAPs have a limiting rate of cell membrane penetration, extracellular metabolism by STEAP4 contributes a significant proportion of total cellular metabolism, and the remainder of HAP cellular metabolism is due to intracellular reductases.

The inventors surprisingly discovered, in part, a previously unidentified role played by the enzyme STEAP4 in hypoxic tumour environments in metabolising HAPs, including cell-excluded HAPs, at the cell surface, having the effect of releasing the active drug from the prodrug. This can enable the active drug to cross the cell membrane and deliver its payload to the intracellular targets and thus kill the cancer cell. The inventors have also shown, for example, that this activity appears to be unique to the STEAP4 reductase enzyme, and is not shared with the other STEAP proteins or with other reductases. These findings led the inventors to the discovery, in part, that individuals suffering from cancer can be stratified based on their STEAP4 expression levels, with those exhibiting elevated expression being identified as those likely to respond to HAP treatment. Contrary to the prevailing understanding of STEAP4 that has proposed the inhibition of STEAP4 in order to treat cancer, the inventors propose instead the exploitation of these elevated levels of STEAP4 in order to achieve better treatment outcome for patients.

Treatment methods and uses

The present application thus contemplates methods of treating or preventing cancer with a HAP in an individual in need thereof, where the individual exhibits an elevated level of STEAP4 expression, and related methods and uses.

Accordingly, the present disclosure provides a method of treating or preventing cancer in an individual in need thereof, the method comprising :

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells;

d) administering a therapeutically effective amount of a HAP to the individual.

In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4- expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E.

In another aspect, the present disclosure provides a method of treating or preventing cancer in an individual in need thereof, the method comprising :

a) determining the level of STEAP4 expression in tumour cells of the individual;

b) predicting the individual as being likely to be responsive to treatment by a HAP if the tumour cells exhibit an elevated level of STEAP4 expression; and

c) administering a therapeutically effective amount of a HAP to the individual.

a) determining the level of STEAP4 expression in a sample of tumour cells from the individual;

c) administering a therapeutically effective amount of a HAP to the individual.

In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4- expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E. .

In another aspect, the present disclosure provides a method of treating or preventing cancer in an individual in need thereof, where tumour cells of the individual exhibit an elevated level of STEAP4 expression; the method comprising administering a therapeutically effective amount of a HAP to the individual.

In another aspect, the present disclosure provides a method of treating or preventing cancer in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of a hypoxia -activated prodrug (HAP), wherein the cancer exhibits an elevated level of STEAP4 expression. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4- expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E.

In certain embodiments of the various methods provided herein, the prediction of whether an individual is likely to be responsive to treatment by a HAP may be made by comparing the determined STEAP4 expression levels in the tumour cells to a reference level of STEAP4 expression.

Accordingly, in another aspect, the present disclosure provides a method of treating or preventing cancer in an individual in need thereof, the method comprising :

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells;

c) predicting the individual as having a treatment-responsive cancer if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to a reference level of STEAP4 expression expression; and

d) administering a therapeutically effective amount of a HAP to the individual predicted as having the treatment-responsive cancer.

b) predicting the individual as having a treatment-responsive cancer if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to a reference level of STEAP4 expression; and

c) administering a therapeutically effective amount of a HAP to the individual predicted as having the treatment-responsive cancer. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4- expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E.

c) administering a therapeutically effective amount of a HAP to the individual predicted as having the treatment-responsive cancer.

In certain embodiments of the various methods provided herein, the prediction of whether an individual is likely to be responsive to treatment by a HAP may also be made by comparing the determined STEAP4 expression levels in the tumour cells to a level of STEAP4 expression in a control sample.

a) providing tumour cells of the individual;

b) providing a control sample;

c) determining the level of STEAP4 expression in the tumour cells;

d) determining the level of STEAP4 expression in the control sample; e) predicting the individual as having a treatment-responsive cancer if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to the level of STEAP4 expression in the control sample; and

f) administering a therapeutically effective amount of a HAP to the individual predicted as having the treatment-responsive cancer.

b) determining the level of STEAP4 expression in a control sample;

c) predicting the individual as having a treatment-responsive cancer if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to the level of STEAP4 expression in the control sample; and

b) predicting the individual as having a treatment-responsive cancer if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to the level of STEAP4 expression in the control sample; and c) administering a therapeutically effective amount of a HAP to the individual predicted as having the treatment-responsive cancer.

In another aspect, the present disclosure provides the use of a HAP in the manufacture of a medicament for the treatment or prevention of cancer in an individual, wherein the cancer exhibits an elevated level of STEAP4 expression.

In yet another aspect, the present disclosure provides the use of a HAP in the manufacture of a composition for the treatment or prevention of cancer in an individual in need thereof, where tumour cells of the individual exhibit an elevated level of STEAP4 expression.

In yet another aspect, the present disclosure provides the use of a HAP in the manufacture of a composition for the treatment or prevention of cancer in an individual in need thereof, where a sample of tumour cells from the individual exhibit an elevated level of STEAP4 expression. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4- expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E.

In another aspect, the present disclosure provides the use of a HAP for the treatment or prevention of cancer in an individual, wherein the cancer exhibits an elevated level of STEAP4 expression.

In yet another aspect, the present disclosure provides the use of a HAP in the treatment or prevention of cancer in an individual in need thereof, where tumour cells of the individual exhibit an elevated level of STEAP4 expression.

In yet another aspect, the present disclosure provides the use of a HAP in the treatment or prevention of cancer in an individual in need thereof, where a sample of tumour cells of the individual exhibit an elevated level of STEAP4 expression.

In another aspect, the present disclosure provides a compound which is a HAP, for use in the treatment or prevention of a cancer exhibiting an elevated level of STEAP4 expression, wherein the treatment comprises administering the HAP to an individual in need thereof.

In another aspect, the present disclosure provides a compound which is a HAP for use in the treatment or prevention of cancer in an individual in need thereof, where tumour cells of the individual exhibit an elevated level of STEAP4 expression .

In another aspect, the present disclosure provides a compound which is a HAP for use in the treatment or prevention of cancer in an individual in need thereof, where a sample of tumour cells of the individual exhibit an elevated level of STEAP4 expression .

In yet a further aspect, the present disclosure provides a HAP for use in the treatment or prevention of cancer in an individual in need thereof, where the cancer exhibits an elevated level of STEAP4 expression.

In yet a further aspect, the present disclosure provides a HAP for use in the treatment or prevention of cancer in an individual in need thereof, where tumour cells of the individual exhibit an elevated level of STEAP4 expression.

In yet a further aspect, the present disclosure provides a HAP for use in the treatment or prevention of cancer in an individual in need thereof, where a sample of tumour cells of the individual exhibit an elevated level of STEAP4 expression.

In yet a further aspect, the present disclosure provides a HAP for use in the treatment or prevention of cancer in an individual in need thereof, comprising : a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells; and

c) administering a therapeutically effective amount of a HAP to the individual if the tumour cells exhibit an elevated level of STEAP4 expression.

In yet a further aspect, the present disclosure provides a HAP for use in the treatment or prevention of cancer in an individual in need thereof, comprising :

a) determining the level of STEAP4 expression in tumour cells of the individual; and b) administering a therapeutically effective amount of a HAP to the individual if the tumour cells exhibit an elevated level of STEAP4 expression.

a) determining the level of STEAP4 expression in a sample of tumour cells from the individual; and

b) administering a therapeutically effective amount of a HAP to the individual if the tumour cells exhibit an elevated level of STEAP4 expression.

Predictive / Identification methods

The present application also contemplates methods of predicting the responsiveness of an individual with cancer to treatment with a HAP, methods of predicting whether an individual with cancer is likely to be responsive to treatment with a HAP, and methods of identifying an individual with cancer who is likely to be responsive to treatment with a HAP.

Accordingly, in yet another aspect, the present disclosure provides a method of predicting the responsiveness of an individual with cancer to treatment with a HAP, the method comprising :

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells;

In one embodiment, the method further comprises administration of the HAP to the individual. In another embodiment, the method further comprises administration of the HAP to the individual if the individual is predicted to be likely to be responsive to the treatment. In certain embodiments, the HAP is administered in a therapeutically effective amount. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4-expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E.

The prediction of an individual that is likely to be responsive to treatment by a HAP may be made by comparing the determined STEAP4 expression levels in the tumour cells to a reference level of STEAP4 expression.

Accordingly, in another aspect, the present disclosure provides a method of predicting the responsiveness of an individual with cancer to treatment with a HAP, the method comprising :

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells; and

c) predicting the individual as being likely to be responsive to treatment with a HAP if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to a reference level of STEAP4 expression.

In another aspect, the present disclosure provides a method of predicting the responsiveness of an individual with cancer to treatment with a HAP, the method comprising :

a) determining the level of STEAP4 expression in tumour cells of the individual; and b) predicting the individual as being likely to be responsive to treatment with a HAP if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to a reference level of STEAP4 expression.

b) predicting the individual as being likely to be responsive to treatment with a HAP if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to a reference level of STEAP4 expression.

In one embodiment, the method further comprises administration of the HAP to the individual. In another embodiment, the method further comprises administration of the HAP to the individual if the individual is predicted to be likely to be responsive to the treatment. In certain embodiments, the HAP is administered in a therapeutically effective amount. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4-expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E. In yet another aspect, the present disclosure provides a method of predicting the responsiveness of an individual with cancer to treatment with a HAP, the method comprising :

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells; and

c) comparing the level of STEAP4 expression in the tumour cells to a reference level of STEAP4 expression;

wherein the individual is likely to be responsive to the treatment with a HAP if the level of STEAP4 expression in the tumour cells is elevated relative to the reference level of STEAP4 expression.

a) determining the level of STEAP4 expression in tumour cells of the individual; and b) comparing the level of STEAP4 expression in the tumour cells to a reference level of STEAP4 expression;

b) comparing the level of STEAP4 expression in the tumour cells to a reference level of STEAP4 expression;

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells; and

c) comparing the level of STEAP4 expression in the tumour cells to a reference level of STEAP4 expression; wherein an elevated level of STEAP4 expression in the tumour cells relative to the reference level of STEAP4 expression correlates with an increased likelihood of responsiveness or an increased responsiveness of the individual to the treatment.

wherein an elevated level of STEAP4 expression in the tumour cells relative to the reference level of STEAP4 expression correlates with an increased likelihood of responsiveness or an increased responsiveness of the individual to the treatment.

In some embodiments of the various methods provided herein, the prediction of an individual that is likely to be responsive to treatment by a HAP is made by comparing the determined STEAP4 expression levels in the tumour cells to the level of STEAP4 expression in a control sample.

a) providing tumour cells of the individual;

b) providing a control sample;

c) determining the level of STEAP4 expression in the tumour cells;

d) determining the level of STEAP4 expression in the control sample; and

e) predicting and/or identifying the individual as having a treatment-responsive cancer if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to the level of STEAP4 expression in the control sample.

In another aspect, the present disclosure provides a method predicting the responsiveness of an individual with cancer to treatment with a HAP, the method comprising :

b) determining the level of STEAP4 expression in a control sample; and

c) predicting the individual as having a treatment-responsive cancer if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to the level of STEAP4 expression in the control sample.

b) predicting the individual as having a treatment-responsive cancer if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to the level of STEAP4 expression in the control sample.

a) providing tumour cells of the individual;

b) providing a control sample;

c) determining the level of STEAP4 expression in the tumour cells;

d) determining the level of STEAP4 expression in the control sample;

e) comparing the level of STEAP4 expression in the tumour cells with the level of STEAP4 expression in the control sample; and f) predicting the individual as having a treatment-responsive cancer if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to the level of STEAP4 expression in the control sample.

b) determining the level of STEAP4 expression in a control sample;

c) comparing the level of STEAP4 expression in the tumour cells with the level of STEAP4 expression in the control sample; and

d) predicting the individual as having a treatment-responsive cancer if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to the level of STEAP4 expression in the control sample.

In one embodiment, the method further comprises administration of the HAP to the individual. In another embodiment, the method further comprises administration of the HAP to the individual if the individual is predicted to be likely to be responsive to the treatment. In certain embodiments, the HAP is administered in a therapeutically effective amount. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4-expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E. In another aspect, the present disclosure provides a method of predicting the responsiveness of an individual with cancer to treatment with a HAP, the method comprising :

b) comparing the level of STEAP4 expression in the tumour cells with the level of STEAP4 expression in the control sample; and

In one embodiment, the method further comprises administration of the HAP to the individual. In another embodiment, the method further comprises administration of the HAP to the individual if the individual is predicted to be likely to be responsive to the treatment. In certain embodiments, the HAP is administered in a therapeutically effective amount. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4-expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E..

a) providing tumour cells of the individual;

b) providing a control sample;

c) determining the level of STEAP4 expression in the tumour cells;

d) determining the level of STEAP4 expression in the control sample; and

e) comparing the level of STEAP4 expression in the tumour cells with the level of STEAP4 expression in the control sample;

a) determining the level of STEAP4 expression in tumour cells of the individual; b) determining the level of STEAP4 expression in a control sample; and

c) comparing the level of STEAP4 expression in the tumour cells with the level of STEAP4 expression in the control sample;

a) determining the level of STEAP4 expression in a sample of tumour cells from the individual; and b) comparing the level of STEAP4 expression in the tumour cells with the level of STEAP4 expression in the control sample;

The present disclosure also provides a method of predicting the responsiveness of an individual with cancer to treatment with a HAP, the method comprising :

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells;

c) predicting the individual as being unlikely to be responsive to a treatment with the HAP if the tumour cells do not exhibit an elevated level of STEAP4 expression.

In yet another aspect, provided herein is a method of identifying an individual with cancer who is likely to be responsive to treatment with a HAP, the method comprising : a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells;

c) identifying the individual as being likely to be responsive to a treatment with the HAP if the tumour cells exhibit an elevated level of STEAP4 expression.

In one embodiment, the method further comprises administration of the HAP to the individual. In another embodiment, the method further comprises administration of the HAP to the individual if the individual is identified to be likely to be responsive to the treatment. In certain embodiments, the HAP is administered in a therapeutically effective amount. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4-expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E.

Accordingly, in yet another aspect, the present disclosure provides a method of identifying an individual with cancer who is likely to be responsive to treatment with a HAP, the method comprising :

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells;

In one embodiment, the method further comprises administration of the HAP to the individual. In another embodiment, the method further comprises administration of the HAP to the individual if the individual is identified as being likely to be responsive to the treatment. In certain embodiments, the HAP is administered in a therapeutically effective amount. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4- expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E. In yet another aspect, the present disclosure provides a method of identifying an individual with cancer who is likely to be responsive to treatment with a HAP, the method comprising :

b) identifying the individual as being likely to be responsive to a treatment with the HAP if the tumour cells exhibit an elevated level of STEAP4 expression.

In one embodiment, the method further comprises administration of the HAP to the individual. In another embodiment, the method further comprises administration of the HAP to the individual if the individual is identified as being likely to be responsive to the treatment. In certain embodiments, the HAP is administered in a therapeutically effective amount. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4- expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E.

The identification of an individual that is likely to be responsive to treatment by a HAP may be made by comparing the determined STEAP4 expression levels in the tumour cells to a reference level of STEAP4 expression.

Accordingly, in another aspect, the present disclosure provides a method of identifying an individual with cancer who is likely to be responsive to treatment with a HAP, the method comprising :

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells; and

c) identifying the individual as being likely to be responsive to treatment with a HAP if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to a reference level of STEAP4 expression.

In another aspect, the present disclosure provides a method of identifying an individual with cancer who is likely to be responsive to treatment with a HAP, the method comprising :

a) determining the level of STEAP4 expression in tumour cells of the individual; and b) identifying the individual as being likely to be responsive to treatment with a HAP if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to a reference level of STEAP4 expression.

b) identifying the individual as being likely to be responsive to treatment with a HAP if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to a reference level of STEAP4 expression.

In yet another aspect, the present disclosure provides a method of identifying an individual with cancer who is likely to be responsive to treatment with a HAP, the method comprising :

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells; and

a) determining the level of STEAP4 expression in tumour cells of the individual; and b) comparing the level of STEAP4 expression in the tumour cells to a reference level of STEAP4 expression; wherein the individual is likely to be responsive to the treatment with a HAP if the level of STEAP4 expression in the tumour cells is elevated relative to the reference level of STEAP4 expression.

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells; and

wherein an elevated level of STEAP4 expression in the tumour cells relative to the reference level of STEAP4 expression correlates with an increased likelihood of responsiveness or an increased responsiveness of the individual to the treatment. In one embodiment, the method further comprises administration of the HAP to the individual. In another embodiment, the method further comprises administration of the HAP to the individual if the individual is identified as being likely to be responsive to the treatment. In certain embodiments, the HAP is administered in a therapeutically effective amount. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4-expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E.

In some embodiments various methods provided herein, the identification of an individual that is likely to be responsive to treatment by a HAP is made by comparing the determined STEAP4 expression levels in the tumour cells to the level of STEAP4 expression in a control sample. In one embodiment, the method further comprises administration of the HAP to the individual. In another embodiment, the method further comprises administration of the HAP to the individual if the individual is identified as being likely to be responsive to the treatment. In certain embodiments, the HAP is administered in a therapeutically effective amount. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4-expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E.

a) providing tumour cells of the individual;

b) providing a control sample;

c) determining the level of STEAP4 expression in the tumour cells;

d) determining the level of STEAP4 expression in the control sample; and

e) identifying the individual as having a treatment-responsive cancer if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to the level of STEAP4 expression in the control sample.

In another aspect, the present disclosure provides a method identifying an individual with cancer who is likely to be responsive to treatment with a HAP, the method comprising : a) determining the level of STEAP4 expression in tumour cells of the individual;

b) determining the level of STEAP4 expression in a control sample; and

c) identifying the individual as having a treatment-responsive cancer if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to the level of STEAP4 expression in the control sample.

b) identifying the individual as having a treatment-responsive cancer if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to the level of STEAP4 expression in the control sample.

a) providing tumour cells of the individual;

b) providing a control sample;

c) determining the level of STEAP4 expression in the tumour cells;

d) determining the level of STEAP4 expression in the control sample; e) comparing the level of STEAP4 expression in the tumour cells with the level of STEAP4 expression in the control sample; and

f) identifying the individual as having a treatment-responsive cancer if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to the level of STEAP4 expression in the control sample.

b) determining the level of STEAP4 expression in a control sample;

d) identifying the individual as having a treatment-responsive cancer if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to the level of STEAP4 expression in the control sample.

a) providing tumour cells of the individual;

b) providing a control sample;

c) determining the level of STEAP4 expression in the tumour cells;

d) determining the level of STEAP4 expression in the control sample; and

wherein an elevated level of STEAP4 expression in the tumour cells relative to the reference level of STEAP4 expression correlates with an increased likelihood of responsiveness or an increased responsiveness of the individual to the treatment. In one embodiment, the method further comprises administration of the HAP to the individual. In another embodiment, the method further comprises administration of the HAP to the individual if the individual is identified as being likely to be responsive to the treatment. In certain embodiments, the HAP is administered in a therapeutically effective amount. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4- expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E.

b) determining the level of STEAP4 expression in a control sample; and

In another aspect, the present disclosure provides a method of identifying an individual with cancer who is likely to be responsive to treatment with a HAP, the method comprising : a) determining the level of STEAP4 expression in a sample of tumour cells from the individual; and

b) comparing the level of STEAP4 expression in the tumour cells with the level of STEAP4 expression in the control sample;

The present disclosure also provides a method of identifying an individual with cancer who is likely to be responsive to treatment with a HAP, the method comprising :

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells;

c) identifying the individual as being unlikely to be responsive to a treatment with the HAP if the tumour cells do not exhibit an elevated level of STEAP4 expression.

In one embodiment, the method further comprises administration of the HAP to the individual. In another embodiment, the method further comprises administration of the HAP to the individual if the individual is identified as being likely to be responsive to the treatment. In certain embodiments, the HAP is administered in a therapeutically effective amount. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4- expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E. In yet another aspect, provided herein is a method for determining whether an individual with cancer is sensitive to a treatment with a HAP, the method comprising :

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells;

c) diagnosing the individual as being sensitive to the treatment with the HAP if the tumour cells exhibit an elevated level of STEAP4 expression.

Accordingly, in yet another aspect, the present disclosure provides a method of determining whether an individual with cancer is sensitive to treatment with a HAP, the method comprising :

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells;

c) diagnosing the individual as being sensitive to treatment to the treatment with the HAP if the tumour cells exhibit an elevated level of STEAP4 expression.

In one embodiment, the method further comprises administration of the HAP to the individual. In another embodiment, the method further comprises administration of the HAP to the individual if the individual is predicted to be likely to be responsive to the treatment. In certain embodiments, the HAP is administered in a therapeutically effective amount. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4-expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E. In yet another aspect, the present disclosure provides a method of determining whether an individual with cancer is sensitive to treatment with a HAP, the method comprising :

b) diagnosing the individual as being sensitive to treatment to the treatment with the HAP if the tumour cells exhibit an elevated level of STEAP4 expression.

The determination of whether an individual is likely to be responsive to treatment by a HAP may be made by comparing the determined STEAP4 expression levels in the tumour cells to a reference level of STEAP4 expression.

Accordingly, in another aspect, the present disclosure provides a method of determining whether an individual with cancer is sensitive to treatment with a HAP, the method comprising :

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells; and

c) diagnosing the individual as being sensitive to treatment to the treatment with a HAP if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to a reference level of STEAP4 expression.

In another aspect, the present disclosure provides a method of determining whether an individual with cancer is sensitive to treatment with a HAP, the method comprising :

a) determining the level of STEAP4 expression in tumour cells of the individual; and b) diagnosing the individual as being sensitive to treatment to the treatment with a HAP if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to a reference level of STEAP4 expression.

b) diagnosing the individual as being sensitive to treatment to the treatment with a HAP if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to a reference level of STEAP4 expression.

In yet another aspect, the present disclosure provides a method of determining whether an individual with cancer is sensitive to treatment with a HAP, the method comprising :

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells; and

wherein the individual is likely to be sensitive to treatment to the treatment with a HAP if the level of STEAP4 expression in the tumour cells is elevated relative to the reference level of STEAP4 expression.

wherein the individual is likely to be sensitive to treatment to the treatment with a HAP if the level of STEAP4 expression in the tumour cells is elevated relative to the reference level of STEAP4 expression. In one embodiment, the method further comprises administration of the HAP to the individual. In another embodiment, the method further comprises administration of the HAP to the individual if the individual is predicted to be likely to be responsive to the treatment. In certain embodiments, the HAP is administered in a therapeutically effective amount. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4-expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E.

In yet another aspect, the present disclosure provides a method of determining whether an individual with cancer is sensitive to treatment with a HAP, the method comprising : a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells; and

In some embodiments various methods provided herein, the determination of whether an individual is likely to be responsive to treatment by a HAP is made by comparing the determined STEAP4 expression levels in the tumour cells to the level of STEAP4 expression in a control sample. In one embodiment, the method further comprises administration of the HAP to the individual. In another embodiment, the method further comprises administration of the HAP to the individual if the individual is predicted to be likely to be responsive to the treatment. In certain embodiments, the HAP is administered in a therapeutically effective amount. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4-expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E.

a) providing tumour cells of the individual;

b) providing a control sample;

c) determining the level of STEAP4 expression in the tumour cells;

d) determining the level of STEAP4 expression in the control sample; and

In another aspect, the present disclosure provides a method determining whether an individual with cancer is sensitive to treatment with a HAP, the method comprising :

b) determining the level of STEAP4 expression in a control sample; and

a) providing tumour cells of the individual;

b) providing a control sample;

c) determining the level of STEAP4 expression in the tumour cells;

d) determining the level of STEAP4 expression in the control sample;

e) comparing the level of STEAP4 expression in the tumour cells with the level of STEAP4 expression in the control sample; and f) identifying the individual as having a treatment-responsive cancer if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to the level of STEAP4 expression in the control sample.

In one embodiment, the method further comprises administration of the HAP to the individual. In another embodiment, the method further comprises administration of the HAP to the individual if the individual is predicted to be likely to be responsive to the treatment. In certain embodiments, the HAP is administered in a therapeutically effective amount. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4-expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E. In another aspect, the present disclosure provides a method determining whether an individual with cancer is sensitive to treatment with a HAP, the method comprising :

b) determining the level of STEAP4 expression in a control sample;

In one embodiment, the method further comprises administration of the HAP to the individual. In another embodiment, the method further comprises administration of the HAP to the individual if the individual is predicted to be likely to be responsive to the treatment. In certain embodiments, the HAP is administered in a therapeutically effective amount. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4-expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E. In another aspect, the present disclosure provides a method of determining whether an individual with cancer is sensitive to treatment with a HAP, the method comprising :

• determining the level of STEAP4 expression in a sample of tumour cells from the individual;

• comparing the level of STEAP4 expression in the tumour cells with the level of STEAP4 expression in the control sample; and

• identifying the individual as having a treatment-responsive cancer if the level of STEAP4 expression in the tumour cells was determined to be an elevated level relative to the level of STEAP4 expression in the control sample.

a) providing tumour cells of the individual;

b) providing a control sample;

c) determining the level of STEAP4 expression in the tumour cells;

d) determining the level of STEAP4 expression in the control sample; and

wherein an elevated level of STEAP4 expression in the tumour cells relative to the reference level of STEAP4 expression correlates with an increased likelihood of sensitivity of the individual to the treatment.

b) comparing the level of STEAP4 expression in the tumour cells with the level of STEAP4 expression in the control sample; wherein an elevated level of STEAP4 expression in the tumour cells relative to the reference level of STEAP4 expression correlates with an increased likelihood of sensitivity of the individual to the treatment.

Also provided herein is a method of determining whether an individual with cancer is sensitive to treatment with a HAP, the method comprising :

a) providing tumour cells of the individual;

b) determining the level of STEAP4 expression in the tumour cells;

c) identifying the individual as being unlikely to be sensitive to a treatment with the HAP if the tumour cells do not exhibit an elevated level of STEAP4 expression.

In one embodiment, the method is a method of identifying whether the individual with cancer is likely to be sensitive to treatment to the treatment. In one embodiment, the method further comprises administration of the HAP to the individual. In another embodiment, the method further comprises administration of the HAP to the individual if the individual is predicted to be likely to be responsive to the treatment. In certain embodiments, the HAP is administered in a therapeutically effective amount. In one embodiment, the level of STEAP4 expression is expression of a STEAP4 gene. In another embodiment, the level of STEAP4 expression is expression of a STEAP4-expressing target nucleic acid. In one embodiment, the target nucleic acid is DNA. In another embodiment, the target nucleic acid is mRNA. In another embodiment, the level of STEAP4 expression is expression of a STEAP4 protein. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E.

In some embodiments of these methods, the method further comprises administering a therapeutically effective amount of a HAP to the individual. In some embodiments, the method further comprises administering a therapeutically effective amount of a HAP to the individual who is likely to be responsive to the treatment. In certain embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In certain embodiments, the HAP is Compound A. In other embodiments, the HAP is Compound C. In other embodiments, the HAP is Compound E.

Cancer

The term "cancer" and "cancerous" as used herein may refer to any cancer or cancerous condition and refers to the physiological condition in mammals characterised by unregulated cell growth or a malignant tumour.

Suitable cancers include, but are not limited to, prostate cancer (including but not limited to neuroendocrine prostate cancer/NEPC), lung cancer (including but not limited to lung adenocarcinoma, lung squamous cell cancer, small cell lung cancer, non-small cell lung cancer/NSCLC, and mesothelioma), breast cancer, esophageal cancer, head and neck cancer, upper aerodigestive tract cancer, neuroblastoma, cancer of the brain (including but not limited to glioma), cancer of the kidney, leukemia, chronic lymphocytic leukemia (CLL), monoclonal B cell lymphocytosis (MBL), childhood B-cell acute lymphobastic leukemia (B- ALL), chronic myeloid leukemia (CML), T-cell acute lymphobastic leukemia (T-ALL), sarcoma (including but not limited to Ewings sarcoma), pancreatic cancer, gastric cancer (also known as stomach cancer), desmoplastic small-round-cell tumour (DESM), uterine cancer, uterine carcinosarcoma (UCS), acute myeloid leukemia (AML), B-cell acute lymphobastic leukemia (B-ALL), liver cancer, bladder cancer, cancer of the urinary tract, childhood acute myeloid leukemia (AML), renal cancer, colorectal cancer, cervical cancer, multiple myeloma, endometrial cancer, ovarian cancer, lymphoma (including but not limited to diffuse large B- cell lymphoma/DLBCL, Hodgkin's lymphma, and non-Hodgkin's lymphoma including but not limited to Burkitt lymphoma), glioblastoma, medulloblastoma, melanoma, cutaneous T-cell lymphoma (CTCL), astrocytoma, bile duct cancer (including but not limited to cholangiocarcinoma), osteosarcoma, meningioma, thyroid cancer, soft tissue sarcoma, and chondrosarcoma.

In one embodiment, the cancer is a prostate cancer. In one embodiment, the cancer is a neuroendocrine prostate cancer (NEPC). In another embodiment, the cancer is a lung cancer. In one embodiment, the cancer is a lung adenocarcinoma. In another embodiment, the cancer is a lung squamous cell cancer. In another embodiment, the cancer is a small cell lung cancer. In another embodiment, the cancer is a non-small cell lung cancer (NSCLC). In another embodiment, the cancer is a mesothelioma. In another embodiment, the cancer is a breast cancer. In another embodiment, the cancer is a esophageal cancer. In another embodiment, the cancer is a head and neck cancer. In another embodiment, the cancer is a upper aerodigestive tract cancer. In another embodiment, the cancer is a neuroblastoma. In another embodiment, the cancer is a cancer of the brain. In one embodiment, the cancer is a glioma). In another embodiment, the cancer is a cancer of the kidney. In another embodiment, the cancer is a leukemia. In another embodiment, the cancer is a chronic lymphocytic leukemia (CLL). In another embodiment, the cancer is a monoclonal B cell lymphocytosis (MBL) . In another embodiment, the cancer is a childhood B-cell acute lymphobastic leukemia (B-ALL). In another embodiment, the cancer is a chronic myeloid leukemia (CML). In another embodiment, the cancer is a T-cell acute lymphobastic leukemia (T-ALL). In another embodiment, the cancer is a sarcoma. In one embodiment, the cancer is a Ewings sarcoma. In another embodiment, the cancer is a pancreatic cancer. In another embodiment, the cancer is a gastric cancer (also known as stomach cancer) . In another embodiment, the cancer is a desmoplastic small-round-cell tumour (DESM). In another embodiment, the cancer is a uterine cancer. In another embodiment, the cancer is a uterine carcinosarcoma (UCS). In another embodiment, the cancer is a acute myeloid leukemia (AML). In another embodiment, the cancer is a B-cell acute lymphobastic leukemia (B-ALL). In another embodiment, the cancer is a liver cancer. In another embodiment, the cancer is a bladder cancer. In another embodiment, the cancer is a cancer of the urinary tract. In another embodiment, the cancer is a childhood acute myeloid leukemia (AML). In another embodiment, the cancer is a renal cancer. In another embodiment, the cancer is a colorectal cancer. In another embodiment, the cancer is a cervical cancer. In another embodiment, the cancer is a multiple myeloma. In another embodiment, the cancer is a endometrial cancer. In another embodiment, the cancer is a ovarian cancer. In another embodiment, the cancer is a lymphoma. In one embodiment, the cancer is a diffuse large B- cell lymphoma (DLBCL). In another embodiment, the cancer is a Hodgkin's lymphoma. In another embodiment, the cancer is a non-Hodgkin's lymphoma. In one embodiment, the cancer is a Burkitt lymphoma. In another embodiment, the cancer is a glioblastoma. In another embodiment, the cancer is a medulloblastoma. In another embodiment, the cancer is a melanoma. In another embodiment, the cancer is a cutaneous T-cell lymphoma (CTCL). In another embodiment, the cancer is a astrocytoma. In another embodiment, the cancer is a bile duct cancer. In one embodiment, the cancer is a cholangiocarcinoma. In another embodiment, the cancer is a osteosarcoma. In another embodiment, the cancer is a meningioma. In another embodiment, the cancer is a thyroid cancer. In another embodiment, the cancer is a soft tissue sarcoma. In another embodiment, the cancer is a and chondrosarcoma.

In specific embodiments, the cancer is selected from the group consisting of prostate cancer, lung cancer (including but not limited to lung adenocarcinoma, lung squamous cell cancer, and mesothelioma), breast cancer, esophageal cancer, gastric cancer, cervical cancer, squamous cell carcinoma of the cervix, squamous cell carcinoma of the head and neck, liver cancer, neuroblastoma, chronic myeloid leukemia, and sarcoma. In one embodiment, the cancer is a prostate cancer. In another embodiment, the cancer is a lung cancer. In one embodiment, the cancer is a lung adenocarcinoma. In another embodiment, the cancer is a lung squamous cell cancer. In another embodiment, the cancer is a mesothelioma. In an embodiment, the cancer is a breast cancer. In another embodiment, the cancer is a esophageal cancer. In another embodiment, the cancer is a gastric cancer. In an embodiment, the cancer is a cervical cancer. In one embodiment, the cancer is a squamous cell carcinoma of the cervix. In another embodiment, the cancer is a squamous cell carcinoma of the head and neck. In another embodiment, the cancer is a liver cancer. In another embodiment, the cancer is a neuroblastoma. In another embodiment, the cancer is a chronic myeloid leukemia. In one embodiment, the cancer is a sarcoma.

In certain embodiments, the combinations disclosed herein are administered to treat cancer. In certain embodiments, the cancer to be treated comprises lung cancer. In other embodiments, the lung cancer comprises non-small cell lung cancer. In yet other embodiments, the cancer comprises gastric cancer. In yet other embodiments, the cancer comprises breast cancer. In yet other embodiments, the cancer comprises head and neck squamous cell carcinoma (HNSCC). In yet other embodiments, the cancer comprises gastric/gastroesophageal (GE) junction cancer. In yet other embodiments, the cancer comprises oesophageal cancer. In yet other embodiments, the cancer comprises salivary cancer. In yet other embodiments, the cancer comprises ovarian cancer. In yet other embodiments, the cancer comprises endometrial cancer. In yet other embodiments, the cancer comprises uterine cancer. In yet other embodiments, the cancer comprises pancreatic cancer. In certain embodiments, the cancer comprises biliary tract cancer. In certain embodiments, the cancer comprises bladder cancer. In certain embodiments, the cancer comprises colorectal cancer. In certain embodiments, the cancer comprises renal cancer. In certain embodiments, the cancer comprises brain and/or spinal cord cancer (glioblastoma). In some embodiments, the cancer comprises lymphoma, e.g., primary central nervous system lymphoma. In some embodiments, the cancer comprises leukaemia, e.g., acute lymphoblastic leukaemia.

In certain embodiments, the cancer is selected from the group of lung cancer, gastric cancer, breast cancer, HNSCC, GE junction cancer, oesophageal cancer, salivary cancer, ovarian cancer, endometrial cancer, uterine cancer, prostate cancer, pancreatic cancer, colon cancer, biliary tract cancer, bladder cancer, colorectal, renal, glioblastoma, mesothelioma, adenocarcinoma, lymphoma, and leukaemia.

In certain embodiments, the cancer is selected from bone cancer, lung cancer, breast cancer, cancer of the head and neck, prostate cancer, pancreatic cancer, skin cancer, uterine cancer, ovarian cancer, cancer of the urethra, cancer of the adrenal gland, cancer of the small intestine, cancer of the kidney, cancer of the bladder, cancers of the brain, colorectal cancer, oesophageal cancer, gastric cancer, anal cancer, liver cancer, thyroid cancer, ocular cancer, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland and testicular cancer.

In certain embodiments, the cancer is selected from chronic or acute leukaemia, acute myeloid leukaemia, chronic myeloid leukaemia, acute lymphoblastic leukaemia, chronic lymphocytic leukaemia, carcinoma of the cervix, carcinoma of the vulva, carcinoma of the vagina, Hodgkin's Disease, brain stem glioma, melanoma, Merkel cell carcinoma, Urothelial carcinoma, lymphomas, gliomas, meningiomas, pituitary adenomas, nerve sheath tumours, and retinoblastoma, and non-small cell lung cancer.

In certain embodiments, the cancer is non-small cell lung cancer.

In certain embodiments, the cancer is prostate cancer.

In certain embodiments, the cancer is cervical carcinoma.

In certain embodiments, the cancer is breast cancer.

In certain embodiments, the cancer is brain cancer.

In certain embodiments, the cancer is glioblastoma.

In certain embodiments, the cancer is spinal cord cancer.

In certain embodiments, the cancer is a solid tumour. Solid tumours are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumours can be benign or malignant. Different types of solid tumours are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). The solid tumour can be sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumour, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, oesophageal adenocarcinoma, oesophageal squamous cell carcinoma, squamous cell carcinoma of the head and neck (HNSCC), oral carcinoma, gastric carcinoma, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumour, cervical cancer, testicular tumour, seminoma, bladder carcinoma, melanoma, and CNS tumours (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pineaioma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases).

In some embodiments, the solid tumour is malignant melanoma, adrenal carcinoma, breast carcinoma, renal cell cancer, carcinoma of the pancreas, non-small-cell lung carcinoma (NSCLC) or carcinoma of unknown primary.

In some embodiments, the solid tumour is breast cancer, ovarian cancer, brain cancer, gastric cancer, oesophageal cancer prostate cancer, lung cancer, colon cancer, skin cancer, liver cancer, pancreatic cancer, and thyroid cancer. In some embodiments, the solid tumour is selected from the groups consisting of carcinoma, melanoma, sarcoma, or chronic granulomatous disease.

Hypoxia-Activated Prodrug (HAP)

The hypoxia-activated prodrug (HAP) may be any suitable HAP that is capable of being converted to an active drug molecule in a hypoxic tumour environment where the active molecule is sufficiently long-lived and able to penetrate the cell membrane in order to kill the cell. Many such HAPs are known. All are potential candidates for use in any of the disclosed methods. In certain embodiments, the HAPs are nitromethylaryl quaternary ammonium salts (often referred to as NMQ prodrugs) or nitrophenyl mustards.

Certain NMQ prodrugs are broadly described in PCT publications WO 2010/104406 and WO 2011/028135. However, it should be appreciated that the NMQ prodrugs for use in the disclosed methods are not limited to the compounds described in WO 2010/104406 and WO 2011/028135. NMQ prodrugs are quaternary ammonium salts where at least one group bound to the ammonium nitrogen atom comprises a moiety capable of being reduced so that following reduction the fragmenting and release of an active molecule is triggered and the active molecule is able to penetrate the cell membrane and then cause cell death.

Provided herein are certain HAPs, for example, a HAP having the structures of Compound A.

Compound A, RN-4000, TRLX, or

tarloxotinib

In certain embodiments, STEAP4 increases and/or causes the metabolism of Compound A to its active metabolite, Compound B:

Compound B, or RN-4000E, or TRLX-TKI. Also provided herein are certain HAPs, for example, HAPs having the structure of Compound C and Compound E.

.

Provided herein are NMQ prodrugs of quaternary nitrogen salts of Formula I :

Formula I

where:

X is any negatively charged counterion;

Ri is a group of the formula -(CH2)_nTr, where Tr is an aromatic nitroheterocycle or aromatic nitrocarbocycle and -(CH2)_nTr acts as a reductively-activated fragmenting trigger; and n is an integer from 0 to 6; R₂, R₃ and R₄ may each independently be selected from aliphatic or aromatic groups of a tertiary amine kinase inhibitor (R₂)(R₃)(R₄)N, or two of R₂, R₃, and R₄ may form an aliphatic or aromatic heterocyclic amine ring of a kinase inhibitor, or one of R₂, R₃ and R₄ may be absent and two of R₂, R₃ and R₄ form an aromatic heterocyclic amine ring of a kinase inhibitor.

In certain embodiments, the NMQ prodrugs are quaternary ammonium salts of Formula II:

Formula II

where:

X is any negatively charged counterion;

Y is N or C-R7, where R₇ is selected from the group consisting of H, C1-C6 alkyl, C1-C6 alkoxy and groups of Formula III:

Formula III where * is the point of attachment, and where

T is selected from O, NH, N(C_I-C₆ alkyl) and a direct link;

m is selected from integers from 0 to 6;

U is selected from ORio, CF3, OCF3, CN, NR11R12, pyrrolidinyl, piperidinyl, piperazinyl, Nl-methylpiperazinyl, morpholinyl, CON(Ri3)(Ri₄), S02N(Ris)(Ri₆), N(RI₇)CORI_S, N(Rig)S02R2o, COR21, SOR22, SO2R23 and COOR24; and Re, R9, Rio, Rll, R12, R13, R14 R15, Rl6, Rl7, Rl8, Rl9, R20, R2I, R22, R23, R24 Q GQ independently selected from H and C₁-C₆ alkyl;

Z is N or C-CN;

n is an integer from 0 to 6;

Ri is a group of the formula (CH₂)_nTr where Tr is an aromatic nitroheterocycle or aromatic nitrocarbocycle and -(CH₂)_nTr acts as a reductively-activated fragmenting trigger; and n is an integer from 0 to 6;

R₂ and R₃ are independently selected from C₁-C₆ alkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, CH₂CH₂OH, CH₂CH₂0(C_I-C₆ alkyl), or R₂ and R₃ may together form a non-aromatic carbocyclic ring or non-aromatic heterocyclic ring containing at least one heteroatom;

R₅ is selected from anilines, indoles, indolines, amines, aminoindoles and aminoindazoles, each of which may be optionally substituted with one or more substituents selected from H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxy, F, Cl, Br, I, CN, CH2F, CHF2, CF₃, OH, NH₂, N0₂, NH(CI-C₆ alkyl), N(CI-C₆ alkyl)₂, CONH₂, CO(Ci-C₆ alkyl), SO2NH2 and S0₂(Ci-C₆ alkyl); and

R₆ is selected from H, C₁-C₆ alkyl, C₁-C₆ alkoxy, NH(CI-C₆ alkyl), N(CI-C₆ alkyl)2 and groups of Formula IV:

Formula IV where

* is the point of attachment;

V is selected from (CH₂)_k where k is an integer from 0 to 6, O, NH and N(C_I-C₆ alkyl); and

R2₅ is selected from H and C₁-C₆ alkyl.

In certain embodiments, X may be selected from halide (fluoride, chloride, bromide, iodide), methanesulfonate, trifluoromethanesulfonate, acetate, trifluoroacetate, tosylate, lactate, citrate and formate.

In certain embodiments, Ri may be selected from groups of Formula V:

Formula V

where

* is the point of attachment to the quaternary nitrogen of a compound of Formula II;

R2₆ is selected from H, C₁-C₆ alkyl, C₁-C₆ alkoxy, C2-C₆ alkenyl, C2-C₆ alkynyl, CF₃, OCF₃, F, Cl, Br, I, NO2, CN, COOH, COO(Ci-C₆ alkyl), CONH₂, CONH(CI-C₆ alkyl), CON(CI-C₆ alkyl)₂, CO(Ci-C₆ alkyl), SO2NH2, S0₂NH(Ci-C₆ alkyl), S0₂N(Ci-C₆ alkyl)₂, S0₂(Ci-C₆ alkyl) and groups of Formula Ilia as defined above but where * is the point of attachment to a group of Formula V;

R₂₇ is selected from the group consisting of H, C₁-C₆ alkyl and groups of Formula Ilia as defined above but where * is the point of attachment to a group of Formula V; and

R2₈ is selected from H and C₁-C₆ alkyl.

In certain embodiments, Ri is a group of one of the following Formulas Vr-Vae:

y z aa ab ac ad ae

In certain embodiments, Ri may be selected from groups of Formula Vc, where R₂₆ is H and R27 is CH₃. In certain embodiments, Ri may also be selected from groups of Formula Vd, where

R2₆ is selected from H, C₁-C₆ alkyl (such as methyl), C₁-C₆ alkoxy (such as OCH₃), C2-C₆ alkynyl (such as ethynyl), CONH₂, CONHMe, CF₃, OCF₃, Br, NO₂ and CN, and R₂₇ is selected from CH₃, CH2CH2CONH2 and CH2CH2CN.

In certain embodiments, Ri may also be selected from groups of Formula Vd :

Formula Vd where

* is a point of attachment, R₂₆ is selected from H and C₁-C₃ alkyl, and R₂₇ is selected from H and C1-C6 alkyl .

In certain embodiments, R₂₆ is H and R₂₇ is C₁-C₃ alkyl e.g., methyl .

In certain embodiments, Ri is selected from groups of Formula Vd, where R26 is 1- propynyl and R27 IS CH₃.

In certain embodiments, Ri is selected from groups of Formula Vq, where R26 is selected from H, C₁-C₆ alkyl (such as methyl or ethyl) and C₁-C₆ alkoxy (such as OCH₃), and R27 is CH₃.

In certain embodiments, Ri is a group of Formula Vq, where R26 is selected from H,

C₁-C₆ alkyl (e.g., methyl or ethyl) and C₁-C₆ alkoxy (e.g., OCH₃), and R27 is CH₃.

In certain embodiments, Ri is a group of any one of Formulas Vd⁽¹⁾-Vd⁽⁷⁾ :

Vd⁽¹⁾ Vd⁽²⁾ Vd⁽³⁾ Vd⁽⁴⁾ Vd⁽⁵⁾ Vd⁽⁶⁾ Vd⁽⁷⁾

In certain embodiments, R₂₇ is selected from methyl, ethyl and propyl . In certain embodiments R₂₇ is methyl . R₂ and R₃ may form a ring selected from pyrrolidinium, piperidinium, piperazinium, N l -methylpiperazinium and morpholinium.

R5 may be selected from groups of Formula VI :

Formula VI

where

* is the point of attachment;

R29, R36, R37, R39, R44, R49 and R54, are independently selected from H and C1-C6 alkyl;

R30_/ R31_/ R32, R33_/ R34, R35_/ R38_/ R40_/ R41, R42, R43_/ R45_/ R46_/ R47, R48_/ R50_/ Rsi_/ R52, R53_/ R₅₅, R₅₆, R₅₇ and R₅₈ are independently selected from H, C₁-C₆ alkyl, C2-C₆ alkenyl, C2-C₆ alkynyl, Ci-C₆ alkoxy, F, Cl, Br, I, CN, CH₂F, CHF₂, CF₃, OH, NH₂, N0₂, NH(CI-C₆ alkyl), N(CI-C₆ alkyl)₂, CONH₂, CO(Ci-C₆ alkyl), SO2NH2 and S0₂(Ci-C₆ alkyl); and

W is N or C-H.

In certain embodiments, Y is N, Z is N or C-CN;

Ri is selected from the following :

(a) a group of Formula Vc, where R₂₆ is H and R₂₇ is CH₃;

(b) groups of Formula Vd, where (i) R₂₆ is selected from H, C₁-C₆ alkyl (such as methyl), C₁-C₆ alkoxy (such as OCH₃), C₂-C₆ alkynyl (such as ethynyl), CF₃, OCF₃, Br, NO₂ and CN, and R₂₇ is selected from CH₃, CH₂CH₂CONH₂ and CH₂CH₂CN; or (ii) R₂₆ is 1-propynyl and R₂₇ is CH₃;

(c) groups of Formula Vf, where R₂₆ is H and R₂₇ is CH₃; and

(d) groups of Formula Vq, where R₂₆ is selected from H, C₁-C₆ alkyl (such as methyl or ethyl) and C₁-C₆ alkoxy (such as OCH₃), and R₂₇ is CH₃;

R₂ and R₃ are independently selected from C₁-C₆ alkyl, or together form a ring selected from pyrrolidinium, piperidinium, piperazinium, Nl-methylpiperazinium and morpholinium; R.₅ is selected from the following :

(a) a group of Formula Via, where * is the point of attachment, R₂₉ is H, and R₃₀, R₃₁, R₃₂ are independently selected from H, C₁-C₆ alkyl, C2-C₆ alkenyl, C2-C₆ alkynyl, C₁-C₆ alkoxy, F, Cl, Br, I, CN, CH₂F, CHF₂, CF₃, OH, NH₂, N0₂, NH(CI-C₆ alkyl), N(CI-C₆ alkyl)₂;

(b) a group of Formula VId, where * is the point of attachment, R₃₉ is H, and R₄o and R_4I are independently selected from H, C₁-C₆ alkyl, C2-C₆ alkenyl, C2-C₆ alkynyl, C₁-C₆ alkoxy, F, Cl, Br, I, CN, CH₂F, CHF₂, CF₃, OH, NH₂, N0₂, NH(CI-C₆ alkyl), N(CI-C₆ alkyl)₂; R₄₂ and R₄₃ are independently selected from H, C₁-C₆ alkyl, C2-C₆ alkenyl, C2-C₆ alkynyl, C₁-C₆ alkoxy, F, Cl, Br, I, CN, CH₂F, CHF₂, CF₃, OH, NH₂, N0₂, NH(CI-C₆ alkyl), N(CI-C₆ alkyl)₂, W is N or C-H; and

(c) a group of Formula Vlf, where * is the point of attachment, R₄₉ is H, and R₅₀ and R₅₁ are independently selected from H or F; R₅₂ and R₅₃ are independently selected from H, C₁-C₆ alkyl, F, Cl, Br, I, CH₂F, CHF₂, CF₃; W is N or C-H; R₆ is H; X is any negatively charged counterion; and n = l or 2.

In certain embodiments, Y is C-H or C-(Ci-C₆ alkoxy), Z is N or C-CN;

Ri is selected from the following :

(a) a group of Formula Vc, where R2₆ is H; and R27 is CH₃;

(b) groups of Formula Vd, where R2₆ is selected from H, C₁-C₆ alkyl, C₁-C₆ alkoxy, C2-C₆ alkynyl, CF₃, OCF₃, Br, NO2 and CN, and R27 is selected from CH₃, CH2CH2CONH2 and CH₂CH₂CN; or R_{26 IS} 1-propynyl and R₂₇ is CH₃;

(c) groups of Formula Vf, where R2₆ is H and R27 is CH₃; and

(d) groups of Formula Vq, where R₂₆ is selected from H, C₁-C₆ alkyl (such as methyl or ethyl) and C₁-C₆ alkoxy (such as OCH₃), and R27 is CH₃;

R₂ and R₃ are independently selected from C₁-C₆ alkyl, or together form a ring selected from pyrrolidinium, piperidinium, piperazinium, Nl-methylpiperazinium and morpholinium;

R₅ is selected from the following :

(a) a group of Formula Via, where * is the point of attachment; R₂₉ is H; and R₃₀, R₃₁, R₃₂ are independently selected from H, C₁-C₆ alkyl, C2-C₆ alkenyl, C2-C₆ alkynyl, C₁-C₆ alkoxy, F, Cl, Br, I, CN, CH₂F, CHF₂, CF₃, OH, NH₂, N0₂, NH(CI-C₆ alkyl), N(CI-C₆ alkyl)₂;

(b) a group of Formula VId, where * is the point of attachment; R₃₉ is H; and R₄₀ and R₄₁ are independently selected from H, C₁-C₆ alkyl, C2-C₆ alkenyl, C2-C₆ alkynyl, C₁-C₆ alkoxy, F, Cl, Br, I, CN, CH₂F, CHF₂, CF₃, OH, NH₂, N0₂, NH(CI-C₆ alkyl), N(CI-C₆ alkyl)₂; R₄₂ and R₄₃ are independently selected from H, C₁-C₆ alkyl, C2-C₆ alkenyl, C2-C₆ alkynyl, C₁-C₆ alkoxy, F, Cl, Br, I, CN, CH₂F, CHF₂, CF₃, OH, NH₂, N0₂, NH(CI-C₆ alkyl), N(CI-C₆ alkyl)₂; and W is N or C-H; and (c) a group of Formula Vlf, where * is the point of attachment; R₄₉ is H; and R₅₀ and R₅₁ are independently selected from H or F; R₅₂ and R₅₃ are independently selected from H, Ci-Ce alkyl, F, Cl, Br, I, CH₂F, CHF₂, CF₃; and W is N or C-H;

R₆ is H;

X is any negatively charged counterion; and

n = l or 2.

In certain embodiments, Y is C-R7, where R7 is a group of Formula Illb; Z is N or C-

CN;

Ri is selected from the following :

(a) a group of Formula Vc, where R₂₆ is H; and R₂₇ is CH₃;

(b) groups of Formula Vd, where R₂₆ is selected from H, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₂-C₆ alkynyl, CF₃, OCF₃, Br, N0₂ and CN, and R₂₇ is selected from CH₃, CH₂CH₂CONH₂ and CH₂CH₂CN; or R₂₆ is 1-propynyl; and R₂₇ is CH₃;

(c) groups of Formula Vf, where R₂₆ is H and R₂₇ is CH₃; and

(d) groups of Formula Vq, where R₂₆ is selected from H, C₁-C₆ alkyl (such as methyl or ethyl) and C₁-C₆ alkoxy (such as OCH₃); and R₂₇ is CH₃;

R₅ is selected from the following :

(a) a group of Formula Via, where * is the point of attachment to a compound of Formula II; R_2g is H; and R₃₀, R₃₁, R₃₂ are independently selected from H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, Ci-C₆ alkoxy, F, Cl, Br, I, CN, CH₂F, CHF₂, CF₃, OH, NH₂, N0₂, NH(Ci- C₆ alkyl), N(CI-C₆ alkyl)₂;

(b) a group of Formula VId, where * is the point of attachment to a compound of Formula II; R₃₉ is H; and R_4o and R_4I are independently selected from H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, Ci-C₆ alkoxy, F, Cl, Br, I, CN, CH₂F, CHF₂, CF₃, OH, NH₂, N0₂, NH(Ci- C₆ alkyl), N(CI-C₆ alkyl)₂; R₄₂ and R₄₃ are independently selected from H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, Ci-C₆ alkoxy, F, Cl, Br, I, CN, CH₂F, CHF₂, CF₃, OH, NH₂, N0₂, NH(Ci- C₆ alkyl), N(C_I-C₆ alkyl)₂; and W is N or C-H; and

(c) a group of Formula Vlf, where * is the point of attachment to a compound of Formula II; R_4g is H; and R₅₀ and R₅₁ are independently selected from H or F; R52 and R₅₃ are independently selected from H, C₁-C₆ alkyl, F, Cl, Br, I, CH₂F, CHF₂, CF₃; and W is N or C-H;

R₆ is H;

X is any negatively charged counterion; and

n = l or 2. Also provided herein are compounds of Formula VII as kinase inhibitors released from an NMQ prodrug :

Formula VII wherein either:

(a) R59 is H, and

(i) R.60 is (3-chlorobenzyl)oxy- and R_6I is chloro;

(ii) R_6O and R_6i, together with the carbon atoms to which they are attached, form l-(3-fluorobenzyl)-lH-pyrazole;

(iii) R_6O is 2-pyridinylmethoxy and R_6I is chloro;

(iv) R_6O and R_6I are both chloro;

(v) R_6O is chloro and R_6I is bromo;

(vi) R_6O and R_6I are both bromo;

(vii) R_6O is fluoro and R_6I is ethynyl;

(viii) R_6O is chloro and R_6I is ethynyl;

(ix) R_6O is bromo and R_6I is ethynyl;

(x) other than when R₆o is in the 3-position in combination with R_6I in the 4- position, R₆o is bromo and R_6I is fluoro;

(xi) R_6O is 2-pyridinylmethoxy and R_6I is fluoro; or

(xii) R_6O is 2-pyridinylmethoxy and R_6I is bromo;

or

(b) at least one of R59, R60 and R_6I is selected from benzyloxy, 3-chlorobenzyloxy and 2- pyridinylmethoxy and when at least one of R59, R60 and R_6I is not benzyloxy, 3- chlorobenzyloxy or 2-pyridinylmethoxy, each of the others is independently selected from H, halogen, and C2-C4 alkynyl, with the proviso that when one of R59, R60 and R₆I is benzyloxy or 2-pyridinylmethoxy, the other two of R59, R60 and R_6I are not H;

or

two of R59, Reo and R_6i, together with the carbon atoms to which they are attached, form 1- (3-fluorobenzyl)-l/-/-pyrazole, and the other is selected from H, halogen and C2-C4 alkynyl. In certain embodiments, the compound of Formula VII is a compound according to Formula VIII:

Formula VIII

wherein R₆2 is H, and either

(a) R.63 is (3-chlorobenzyl)oxy- and R₆4 is chloro;

(b) R₆3 and R₆4, together with the carbon atoms to which they are attached, form l-(3-fluorobenzyl)-lH-pyrazole;

(c) R₆3 is 2-pyridinylmethoxy and R₆4 is chloro;

(d) R₆3 and R₆4 are both chloro;

(e) R₆3 is chloro and R₆4 is bromo;

(f) R₆3 is bromo and R₆4 is chloro

(g) R₆3 and R₆4 are both bromo;

(h) R₆3 is fluoro and R₆4 is ethynyl;

(i) R₆3 is chloro and R₆4 is ethynyl;

(j) R₆3 is bromo and R₆4 is ethynyl;

(k) R₆3 is bromo and R₆4 is fluoro;

(L) R₆3 is 2-pyridinylmethoxy and R₆4 is fluoro; or

(m) R₆3 is 2-pyridinylmethoxy and R₆4 is bromo.

In certain embodiments, the compound of Formula VII is selected from the group consisting of:

(2E)-/V-(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-c/]pyrimidin-6-yl)-4- (dimethylamino)-2-butenamide (1),

(2E)-4-(dimethylamino)-/V-(4-{[l-(3-fluorobenzyl)-l/-/-indazol-5- yl]amino}pyrido[3,4-c/]pyrimidin-6-yl)-2-butenamide (2),

(2E)-/V-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6-yl}-4- (dimethylamino)-2-butenamide (3),

(2E)-/V-[4-(3,4-dichloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]-4-(dimethylamino)-2- butenamide (4), (2E)-/V-[4-(3-bromo-4-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]-4- (dimethylamino)-2-butenamide (5),

(2E)-/V-[4-(4-bromo-3-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]-4- (dimethylamino)-2-butenamide (6),

(2E)-/V-[4-(3,4-dibromoanilino)pyrido[3,4-c/]pyrimidin-6-yl]-4-(dimethylamino)-2- butenamide (7),

(2E)-4-(dimethylamino)-/V-[4-(3-ethynyl-4-fluoroanilino)pyrido[3,4-c/]pyrimidin-6- yl]-2-butenamide (8),

(2E)-/V-[4-(4-chloro-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]-4- (dimethylamino)-2-butenamide (9),

(2E)-/V-[4-(4-bromo-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]-4- (dimethylamino)-2-butenamide (10),

(2E)-/V-[4-(4-bromo-3-fluoroanilino)pyrido[3,4-c/]pyrimidin-6-yl]-4-(dimethylamino)- 2-butenamide (11),

(2E)-4-(dimethylamino)-/V-{4-[3-fluoro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4- c/]pyrimidin-6-yl}-2-butenamide (89) and

(2E)-/V-{4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6-yl}-4- (dimethylamino)-2-butenamide (90).

The structures of the compounds in the list above are below:

In certain embodiments, the NMQ prodrugs are quaternary ammonium salts of Formula X:

Formula X wherein X is any negatively charged counterion, R59, R60 and R₆I are as defined for Formula VII, R₆5 is selected from H, methyl, ethyl, trifluoromethyl, -CN, -CONH2 and propyn-l-yl, and R₆₆ is C1-C6 alkyl. In certain embodiments, the NMQ prodrugs are quaternary ammonium salts of

Formula XI :

Formula XI wherein X is any negatively charged counterion, R59, R60 and R₆I are as defined for Formula VII and R₆7 is selected from H, methyl, ethyl, trifluoromethyl, -CN, -CONH2 and propyn-l-yl.

In certain embodiments, the NMQ prodrugs are quaternary ammonium salts of

Formula XII :

Formula XII wherein X is any negatively charged counterion, R₆2, R63 and R₆4 are as defined for Formula VIII and R₆₈ is selected from H, methyl, ethyl, trifluoromethyl, -CN, -CONH2 and propyn-l-yl.

In certain embodiments, X is selected from halide (fluoride, chloride, bromide, iodide), methanesulfonate, trifluoromethanesulfonate, acetate, trifluoroacetate, tosylate, lactate, citrate and formate. In certain embodiments, the NMQ prodrugs are quaternary ammonium salts selected from the group consisting of:

(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-c/]pyrimidin-6- yl)amino]-/V,/V-dimethyl-/V-[(l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl]-4-oxo-2-buten-l- ammonium bromide (12),

(2E)-4-[(4-{[l-(3-fluorobenzyl)-lH-indazol-5-yl]amino}pyrido[3,4-c/]pyrimidin-6- yl)amino]-/V,/V-dimethyl-/V-[(l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl]-4-oxo-2-buten-l- ammonium bromide ( 13),

(2E)-/V-[(l,2-dimethyl-4-nitro-l/-/-imidazol-5-yl)methyl]-4-[(4-{[l-(3-fluorobenzyl)- l/-/-indazol-5-yl]amino}pyrido[3,4-c/]pyrimidin-6-yl)amino]-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide ( 14),

(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V,/V-dimethyl-/V-[(l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl]-4-oxo-2-buten-l- ammonium bromide ( 15),

(2E)-4-{[4-(3,4-dichloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-/V- [(l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide (16),

(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-[(l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide (17),

(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide (18),

(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-/V- [(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide (19),

(2E)-4-{[4-(3-ethynyl-4-fluoroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide (20),

(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide (21),

(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-c/]pyrimidin-6- yl)amino]-/V-[(l,2-dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten- 1-ammonium bromide (22),

(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-c/]pyrimidin-6- yl)amino]-/V-[(2-ethyl-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2- buten-l-ammonium bromide (23), (2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-c/]pyrimidin-6- yl)amino]-/V,/V-dimethyl-/V-{[l-methyl-4-nitro-2-(tnfluoromethyl)-l/-/-imidazol-5- yl]methyl}-4-oxo-2-buten-l-ammonium bromide (24),

(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-c/]pyrimidin-6- yl)amino]-/V-[(2-cyano-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2- buten-l-ammonium bromide (25),

(2E)-/V-{[2-(aminocarbonyl)-l-methyl-4-nitro-l/-/-imidazol-5-yl]methyl}-4-[(4-{3- chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-c/]pyrimidin-6-yl)amino]-/V,/V-dimethyl-4- oxo-2-buten-l-ammonium bromide (26),

(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-c/]pyrimidin-6- yl)amino]-/V,/V-dimethyl-/V-{[l-methyl-4-nitro-2-(l-propynyl)-l/-/-imidazol-5-yl]methyl}-4- oxo-2-buten-l-ammonium bromide (27),

(2E)-/V-[(2-ethyl-l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl]-4-[(4-{[l-(3- fluorobenzyl)-l/-/-indazol-5-yl]amino}pyrido[3,4-c/]pyrimidin-6-yl)amino]-/V,/V-dimethyl-4- oxo-2-buten-l-ammonium bromide (28),

(2E)-4-[(4-{[l-(3-fluorobenzyl)-lH-indazol-5-yl]amino}pyrido[3,4-c/]pyrimidin-6- yl)amino]-/V,/V-dimethyl-/V-{[l-methyl-4-nitro-2-(trifluoromethyl)-l/-/-imidazol-5- yl]methyl}-4-oxo-2-buten-l-ammonium bromide (29),

(2E)-/V-[(2-cyano-l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl]-4-[(4-{[l-(3- fluorobenzyl)-l/-/-indazol-5-yl]amino}pyrido[3,4-c/]pyrimidin-6-yl)amino]-/V,/V-dimethyl-4- oxo-2-buten-l-ammonium bromide (30),

(2E)-/V-{[2-(aminocarbonyl)-l-methyl-4-nitro-l/-/-imidazol-5-yl]methyl}-4-[(4-{[l- (3-fluorobenzyl)-l/-/-indazol-5-yl]amino}pyrido[3,4-c/]pyrimidin-6-yl)amino]-/V,/V-dimethyl- 4-oxo-2-buten-l-ammonium bromide (31),

(2E)-4-[(4-{[l-(3-fluorobenzyl)-lH-indazol-5-yl]amino}pyrido[3,4-c/]pyrimidin-6- yl)amino]-/V,/V-dimethyl-/V-{[l-methyl-4-nitro-2-(l-propynyl)-l/-/-imidazol-5-yl]methyl}-4- oxo-2-buten-l-ammonium bromide (32),

(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V-[(l,2-dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten- 1-ammonium bromide (33),

(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V-[(2-ethyl-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2- buten-l-ammonium bromide (34),

(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V,/V-dimethyl-/V-{[l-methyl-4-nitro-2-(trifluoromethyl)-l/-/-imidazol-5- yl]methyl}-4-oxo-2-buten-l-ammonium bromide (35), (2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V-[(2-cyano-l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2- buten-l-ammonium bromide (36),

(2E)-/V-{[2-(aminocarbonyl)-l-methyl-4-nitro-l/-/-imidazol-5-yl]methyl}-4-({4-[3- chloro-4-(2-pyndinylmethoxy)anilino]pyrido[3,4-c/]pynmidin-6-yl}amino)-/V,/V-dimethyl-4- oxo-2-buten-l-ammonium bromide (37),

(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V,/V-dimethyl-/V-{[l-methyl-4-nitro-2-(l-propynyl)-l/-/-imidazol-5-yl]methyl}-4- oxo-2-buten-l-ammonium bromide (38),

(2E)-4-{[4-(3,4-dichloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(l,2- dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (39),

(2E)-4-{[4-(3,4-dichloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(2-ethyl-l- methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (40),

(2E)-4-{[4-(3,4-dichloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-/V- {[l-methyl-4-nitro-2-(trifluoromethyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2-buten-l- ammonium bromide (41),

(2E)-/V-[(2-cyano-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-{[4-(3,4- dichloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (42),

(2E)-/V-{[2-(aminocarbonyl)-l-methyl-4-nitro-lH-imidazol-5-yl]methyl}-4-{[4-(3,4- dichloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (43),

(2E)-4-{[4-(3,4-dichloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-/V- {[l-methyl-4-nitro-2-(l-propynyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2-buten-l-ammonium bromide (44),

(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(l,2- dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (45),

(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(2- ethyl-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (46),

(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-{[l-methyl-4-nitro-2-(trifluoromethyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2- buten-l-ammonium bromide (47), (2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(2- cyano-l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (48),

(2E)-/V-{[2-(aminocarbonyl)-l-methyl-4-nitro-l/-/-imidazol-5-yl]methyl}-4-{[4-(3- bromo-4-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (49),

(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-{[l-methyl-4-nitro-2-(l-propynyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2-buten-l- ammonium bromide (50),

(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(l,2- dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (51),

(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(2- ethyl-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (52),

(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-{[l-methyl-4-nitro-2-(trifluoromethyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2- buten-l-ammonium bromide (53),

(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(2- cyano-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (54),

(2E)-/V-{[2-(aminocarbonyl)-l-methyl-4-nitro-lH-imidazol-5-yl]methyl}-4-{[4-(4- bromo-3-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (55),

(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-{[l-methyl-4-nitro-2-(l-propynyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2-buten-l- ammonium bromide (56),

(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(l,2- dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (57),

(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(2-ethyl-l- methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (58),

(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-/V- {[l-methyl-4-nitro-2-(trifluoromethyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2-buten-l- ammonium bromide (59), (2E)-/V-[(2-cyano-l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl]-4-{[4-(3,4- dibromoanilino)pyndo[3,4-c/]pynmidin-6-yl]amino}-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (60),

(2E)-/V-{[2-(aminocarbonyl)-l-methyl-4-nitro-l/-/-imidazol-5-yl]methyl}-4-{[4-(3,4- dibromoanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (61),

(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-/V- {[l-methyl-4-nitro-2-(l-propynyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2-buten-l-ammonium bromide (62),

(2E)-/V-[(l,2-dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-4-{[4-(3-ethynyl-4- fluoroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (63),

(2E)-/V-[(2-ethyl-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-{[4-(3-ethynyl-4- fluoroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (64),

(2E)-4-{[4-(3-ethynyl-4-fluoroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-{[l-methyl-4-nitro-2-(trifluoromethyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2- buten-l-ammonium bromide (65),

(2E)-/V-[(2-cyano-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-{[4-(3-ethynyl-4- fluoroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (66),

(2E)-/V-{[2-(aminocarbonyl)-l-methyl-4-nitro-lH-imidazol-5-yl]methyl}-4-{[4-(3- ethynyl-4-fluoroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (67),

(2E)-4-{[4-(3-ethynyl-4-fluoroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-{[l-methyl-4-nitro-2-(l-propynyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2-buten-l- ammonium bromide (68),

(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(l,2- dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (69),

(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(2- ethyl-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (70),

(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-{[l-methyl-4-nitro-2-(trifluoromethyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2- buten-l-ammonium bromide (71), (2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(2- cyano-l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (72),

(2E)-/V-{[2-(aminocarbonyl)-l-methyl-4-nitro-l/-/-imidazol-5-yl]methyl}-4-{[4-(4- chloro-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (73),

(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-{[l-methyl-4-nitro-2-(l-propynyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2-buten-l- ammonium bromide (74),

(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide (75),

(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(l,2- dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (76),

(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(2- ethyl-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (77),

(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-{[l-methyl-4-nitro-2-(trifluoromethyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2- buten-l-ammonium bromide (78),

(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(2- cyano-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (79),

(2E)-/V-{[2-(aminocarbonyl)-l-methyl-4-nitro-lH-imidazol-5-yl]methyl}-4-{[4-(4- bromo-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (80),

(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-{[l-methyl-4-nitro-2-(l-propynyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2-buten-l- ammonium bromide (81),

(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide (82),

(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(l,2- dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (83), (2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(2- ethyl-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (84),

(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-{[l-methyl-4-nitro-2-(trifluoromethyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2- buten-l-ammonium bromide (85),

(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(2- cyano-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (86),

(2E)-/V-{[2-(aminocarbonyl)-l-methyl-4-nitro-l/-/-imidazol-5-yl]methyl}-4-{[4-(4- bromo-3-fluoroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (87),

(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-{[l-methyl-4-nitro-2-(l-propynyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2-buten-l- ammonium bromide (88),

(2E)-4-({4-[3-fluoro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V,/V-dimethyl-/V-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l- ammonium bromide (91),

(2E)-/V-[(l,2-dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-4-({4-[3-fluoro-4-(2- pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6-yl}amino)-/V,/V-dimethyl-4-oxo-2-buten- 1-ammonium bromide (92),

(2E)-/V-[(2-ethyl-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-({4-[3-fluoro-4-(2- pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6-yl}amino)-/V,/V-dimethyl-4-oxo-2-buten- 1-ammonium bromide (93),

(2E)-4-({4-[3-fluoro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V,/V-dimethyl-/V-{[l-methyl-4-nitro-2-(trifluoromethyl)-l/-/-imidazol-5- yl]methyl}-4-oxo-2-buten-l-ammonium bromide (94),

(2E)-/V-[(2-cyano-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-({4-[3-fluoro-4-(2- pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6-yl}amino)-/V,/V-dimethyl-4-oxo-2-buten- 1-ammonium bromide (95),

(2E)-/V-{[2-(aminocarbonyl)-l-methyl-4-nitro-lH-imidazol-5-yl]methyl}-4-({4-[3- fluoro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6-yl}amino)-/V,/V-dimethyl-4- oxo-2-buten-l-ammonium bromide (96),

(2E)-4-({4-[3-fluoro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V,/V-dimethyl-/V-{[l-methyl-4-nitro-2-(l-propynyl)-l/-/-imidazol-5-yl]methyl}-4- oxo-2-buten-l-ammonium bromide (97), (2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V,/V-dimethyl-/V-[(l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl]-4-oxo-2-buten-l- ammonium bromide (98),

(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V-[(l,2-dimethyl-4-nitro-l/-/-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten- 1-ammonium bromide (99),

(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V-[(2-ethyl-l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2- buten-l-ammonium bromide (100),

(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V,/V-dimethyl-/V-{[l-methyl-4-nitro-2-(trifluoromethyl)-l/-/-imidazol-5- yl]methyl}-4-oxo-2-buten-l-ammonium bromide (101),

(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V-[(2-cyano-l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2- buten-l-ammonium bromide (102),

(2E)-/V-{[2-(aminocarbonyl)-l-methyl-4-nitro-lH-imidazol-5-yl]methyl}-4-({4-[3- bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6-yl}amino)-/V,/V-dimethyl-4- oxo-2-buten-l-ammonium bromide (103) and

(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V,/V-dimethyl-/V-{[l-methyl-4-nitro-2-(l-propynyl)-l/-/-imidazol-5-yl]methyl}-4- oxo-2-buten-l-ammonium bromide (104).

The structures of the prodrug compounds in the list above are below:

O₂

² 44

JOC

ϊ¾ϊ¾¾

O,N,^K NI

50

5

In certain embodiments, the compounds are selected from the group consisting of:

(2E)-4-{[4-(3-bromoanilino)-6-quinazolinyl]amino}-N,N-dimethyl-N-(4-nitro- benzyl)-4-oxo-2-buten-l-ammonium bromide (105);

(2E)-4-{[4-(3-bromoanilino)-6-quinazolinyl]amino}-N,N-dimethyl-N-(2-nitro- benzyl)-4-oxo-2-buten-l-ammonium bromide (106);

(2E)-4-{[4-(3-bromoanilino)-6-quinazolinyl]amino}-N,N-dimethyl-N-[(l-methyl-5- nitro-lH-pyrrol-2-yl)methyl]-4-oxo-2-buten-l-ammonium bromide(107);

(2E)-4-{[4-(3-bromoanilino)-6-quinazolinyl]amino}-N,N-dimethyl-N-[(l-methyl-4- nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten- 1-ammonium bromide ( 108);

(2E)-4-{[4-(3-bromoanilino)-6-quinazolinyl]amino}-N,N-dimethyl-N-[(l-methyl-4- nitro-lH-imidazol-2-yl)methyl]-4-oxo-2-buten- 1-ammonium bromide ( 109);

(2E)-4-{[4-(3-bromoanilino)-6-quinazolinyl]amino}-N,N-dimethyl-N-[(l-methyl-4- nitro-lH-pyrazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide ( 110);

(2E)-4-{[4-(3-bromoanilino)-6-quinazolinyl]amino}-N,N-dimethyl-N-[(3- nitroimidazo[l,2-a]pyridin-2-yl)methyl]-4-oxo-2-buten-l-ammonium bromide (111); l-((2E)-4-{[4-(3-bromoanilino)-6-quinazolinyl]amino}-4-oxo-2-butenyl)-l-[-(l- methyl-4-nitro-lH-imidazol-5-yl)methyl]piperidinium bromide (112);

4-((2E)-4-{[4-(3-bromoanilino)-6-quinazolinyl]amino}-4-oxo-2-butenyl)-4-[-(l- methyl-4-nitro-lH-imidazol-5-yl)methyl]morpholin-4-ium formate (113); (2E)-4-{[4-(3-chloro-4-fluoroanilino)-7-methoxy-6-quinazolinyl]amino}-N,N- dimethyl-N-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide (114);

(2E)-4-{[4-(3-bromo-4-fluoroanilino)-6-quinazolinyl]amino}-N,N-dimethyl-N-[(l- methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide (115);

(2E)-4-{[4-(4-fluoro-3-methoxyanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N- dimethyl-N-[(l-methyl-4-nitro-lH-imidazol-5-yl) methyl]-4-oxo-2-buten-l-ammonium bromide (116);

(2E)-4-{[4-(3-bromo-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-N- dimethyl-N-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide (117);

(2E)-4-{[4-(3-bromo-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-[(l,2- dimethyl-4-nitro-lH-imidazol-5-yl)methyl]N,N-dimethyl-4-oxo-2-buten-l-ammonium bromide (118);

(2E)-4-{[4-(3-bromo-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-[(2- methoxy-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-l- ammonium bromide ( 119);

(2E)-4-{[4-(3-bromo-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-[(2- ethynyl-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-l- ammonium bromide (120);

(2E)-4-{[4-(3-bromo-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-N- dimethyl-N-{[l-methyl-4-nitro-2-(trifluoromethyl)-lH-imidazol-5-yl]methy-l}-4-oxo-2- buten-l-ammonium bromide (121);

(2E)-N-{[l-(3-amino-3-oxopropyl)-4-nitro-lH-imidazol-5-yl]methyl}-4-{[4-(-3- bromo-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-4-oxo-2-buten-l- ammonium bromide (122);

(2E)-4-{[4-(3-bromo-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-[(2- cyano-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-l- ammonium bromide ( 123);

(2E)-4-{[4-(3-bromo-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-[(2- cyano-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-l- ammonium trifluoroacetate (124);

(2E)-4-{[4-(3-bromo-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-{[l-(2- cyanoethyl)-4-nitro-lH-imidazol-5-yl]methyl}-N,N-dimethyl-4-oxo-2-buten-l-ammonium bromide (125); (2E)-4-({4-[4-fluoro-3-(trifluoromethyl)anilino]pyrido[3,4-d]pyrimidin-6-yl}amino)- N,N-dimethyl-N-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide (126);

(2E)-N-[(l,2-dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-4-({4-[4-fluoro-3- (tnfluoromethyl)anilino]pyndo[3,4-d]pyrimidin-6-yl}amino)-N,N-dimethyl-4-oxo-2-buten-l- ammonium bromide (127);

(2E)-4-({4-[4-fluoro-3-(trifluoromethyl)anilino]pyrido[3,4-d]pyrimidin-6-yl}amino)- N-[(2-methoxy-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-l- ammonium bromide (128);

(2E)-N-[(2-ethynyl-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-({4-[4-fluoro-3- (trifluoromethyl)anilino]pyrido[3,4-d]pyrimidin-6-yl}amino)-N,N-dimethyl-4-oxo-2-buten-l- ammonium bromide (129);

(2E)-4-({4-[4-fluoro-3-(trifluoromethyl)anilino]pyrido[3,4-d]pyrimidin-6-yl}amino)- N,N-dimethyl-N-{[l-methyl-4-nitro-2-(trifluoromethyl)-lH-imidazo- l-5-yl]methyl}-4-oxo- 2-buten-l-ammonium bromide (130);

(2E)-N-{[l-(3-amino-3-oxopropyl)-4-nitro-lH-imidazol-5-yl]methyl}-4-({4-[-4- fluoro-3-(trifluoromethyl)anilino]pyrido[3,4-d]pyrimidin-6-yl}amino)-N,N-dimethyl-4-oxo-2- buten-l-ammonium bromide(1310);

(2E)-N-[(2-cyano-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-({4-[4-fluoro-3- (trifluoromethyl)anilino]pyrido[3,4-d]pyrimidin-6-yl}amino)-N,N-dimethyl-4-oxo-2-buten-l- ammonium bromide(132);

(2E)-N-{[l-(2-cyanoethyl)-4-nitro-lH-imidazol-5-yl]methyl}-4-({4-[4-fluoro-3- (trifluoromethyl)anilino]pyrido[3,4-d]pyrimidin-6-yl}amino)-N,N-dimethyl-4-oxo-2-buten-l- ammonium bromide(133);

(2E)-4-{[4-(3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimet- hyl-N- [(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide(134);

(2E)-N-[(l,2-dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-4-{[4-(3-ethynylan- ilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-4-oxo-2-buten-l-ammonium bromide(135);

(2E)-4-{[4-(3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-[(2-methoxy-l- methyl-4-nitro-lH-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-l-ammonium bromide(136);

(2E)-4-{[4-(3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-[(2-ethynyl-l- methyl-4-nitro-lH-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-l-ammonium bromide(137); (2E)-4-{[4-(3-ethynylanilino)pyndo[3,4-d]pynmidin-6-yl]amino}-N,N-dimethyl-N- {[l-methyl-4-nitro-2-(tnfluoromethyl)-lH-imidazol-5-yl]methyl}-4-oxo-2-buten-l- ammonium bromide(138);

(2E)-N-{[l-(3-amino-3-oxopropyl)-4-nitro-lH-imidazol-5-yl]methyl}-4-{[4-(-3- ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-4-oxo-2-buten-l- ammonium bromide(139);

(2E)-N-[(2-cyano-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-{[4-(3- ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-4-oxo-2-buten-l- ammonium bromide (140);

(2E)-N-{[l-(2-cyanoethyl)-4-nitro-lH-imidazol-5-yl]methyl}-4-{[4-(3-ethyn- ylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-4-oxo-2-buten-l-ammonium bromide(141);

(2E)-4-({4-(3-chloro-4-fluoroanilino)-7-[(3S)-tetrahydro-3-furanyloxy]-6- quinazolinyl}amino)-N,N-dimethyl-N-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2- buten-l-ammonium trifluoroacetate (142);

(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6- quinolinyl}amino)-N,N-dimethyl-N-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2- buten-l-ammonium trifluoroacetate (143);

(2E)-4-{[4-(3-chloro-4-fluoroanilino)-3-cyano-7-ethoxy-6-quinolinyl]amino-}-N,N- dimethyl-N-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten- -1-ammonium bromide (144);

2-(4-{[6-(2,6-dichlorophenyl)-8-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2- yl]amino}phenoxy)-N,N-diethyl-N-[(l-methyl-4-nitro-lH-imidazol-5- yl)methyl]ethanammonium bromide (145);

2-(4-{[6-(2,6-dichlorophenyl)-8-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2- yl]amino}phenoxy)-N-[(l,2-dimethyl-4-nitro-lH-imidazol-5-yl)methyl-]N,N- diethylethanammonium bromide(146);

4-{[6-(2,6-dichlorophenyl)-8-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2- yl]amino}-l-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]pyridinium bromide (147);

l-[2-(4-{[6-(2,6-dichlorophenyl)-8-methyl-7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2-yl]amino}phenoxy)ethyl]-l-[(l-methyl-4-nitro-lH-imidazol-5-yl)

methyl]piperidinium bromide (148);

N,N-diethyl-2-[({5-[(Z)-(5-fluoro-2-oxo-l,2-dihydro-3H-indol-3-ylidene)methyl]- 2,4-dimethyl-lH-pyrrol-3-yl}carbonyl)amino]-N-[(l-methyl-4-nitro-lH-imidazol-5- yl)methyl]ethanammonium trifluoroacetate (149); N-[(l,2-dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-N,N-diethyl-2-[({5-[(Z)-(5- fluoro-2-oxo-l,2-dihydro-3H-indol-3-ylidene)methyl]2, 4-dimethyl- lH-pyrrol-3- yl}carbonyl)amino]ethanammonium bromide (150);

4-({[4-(4-bromo-2-fluoroanilino)-6-methoxy-7-quinazolinyl]oxy}methyl)-l-m- ethyl-l-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]piperidinium trifluoroacetate;

(2E)-4-{[4-(3-bromo-4-fluoroanilino)pyndo[3,4-d]pyrimidin-6-yl]amino}-N-[(2- ethyl-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-l-ammonium bromide (151);

(2E)-4-{[4-(3-bromo-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-N- dimethyl-N-{[l-methyl-4-nitro-2-(l-propynyl)-lH-imidazol-5-yl]methyl}-4-oxo-2-buten-l- ammonium bromide ( 152);

(2E)-4-{[4-(3-bromo-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-N- dimethyl-N-[(l-methyl-2-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide (153);

(2E)-4-{[4-(3-bromo-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-[(4- ethyl-l-methyl-2-nitro-lH-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-l-ammonium bromide (154); and

(2E)-N-[(2-ethyl-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-{[4-(3-ethyn- ylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-4-oxo-2-buten-l ammonium bromide (155).

In certain embodiments, the HAP is a nitrophenyl mustard compound. Certain nitrophenyl mustards are broadly described in PCT publications WO 2005/042471 and WO 2014/031012. These compounds include compounds of Formula XIII:

Formula XIII

wherein

Xi represents at any available ring position -CONH-, -SO2NH-, -0-, -CH2-, -NHCO- or -NHSO2-;

R.69 represents a lower Ci-₆ alkyl optionally substituted with one or more groups including hydroxy, amino and N-oxides therefrom or dialkylamino and N-oxides therefrom;

Yi represents at any available ring position -N-aziridinyl, -N(CH2CH2W_I)2 or

-N(CH2CHMeWi)2, where each Wi is independently selected from halogen or -OSC^Me; Zi represents at any available ring position -NO₂, -halogen, -CN, -CF₃ or -S0₂Me.

In certain embodiments, the compound of Formula (XIII) is selected from a compound represented by Formula XIV, Formula XV or Formula XVI:

Formula XIV Formula XV Formula XVI wherein Y₂ may represent

and wherein

n represents 1 to 6

Z₂ represents -NO₂, -halogen, -CN, -CF₃ or -S0₂Me; and

where each W2 is independently selected from halogen or -OSC^Me.

In certain embodiments, the phosphate compound of Formula XIII is selected from the group consisting of:

2-[[2-[Bis(2-bromoethyl)amino]-3,5-dinitrobenzoyl]amino]ethyl dihydrogen phosphate (156);

3-[[5-[Bis(2-chloroethyl)amino]-2,4-dinitrobenzoyl]amino]propyl dihydrogen phosphate (157);

3-[[5-[Bis(2-bromoethyl)amino]-2,4-dinitrobenzoyl]amino]propyl dihydrogen phosphate (158);

2-[[2-[Bis(2-chloroethyl)amino]-3,5-dinitrobenzoyl]amino]ethyl dihydrogen phosphate (159);

2-[(2-Chloroethyl)-2,4-dinitro-6-[[[2-(phosphonooxy)ethyl]amino]-carbonyl]- anilino]ethyl methanesulfonate (160);

2-({2-[Bis(2-bromopropyl)amino]-3,5-dinitrobenzoyl}amino)ethyl dihydrogen phosphate (161);

2-[(2-Bromoethyl)-2,4-dinitro-6-[[[2-(phosphonooxy)ethyl]amino]-carbonyl]- anilino]ethyl methanesulfonate (162);

2-[[2-[Bis(2-iodoethyl)amino]-3,5-dinitrobenzoyl]amino]ethyl dihydrogen phosphate

(163); 2-[(2-Iodoethyl)-2,4-dinitro-6-({[2-(phosphonooxy)ethyl]amino}ca rbonyl)-anilino]- ethyl methanesulfonate; (164);

2-[(2-Chloroethyl)-2,4-dinitro-3-[[[3-(phosphonooxy)propyl]amino]-carbonyl]- anilino]ethyl methanesulfonate (165);

3-({3-[Bis(2-bromoethyl)amino]-2,6-dinitrobenzoyl}amino)propyl dihydrogen phosphate (166);

2-[(2-Bromoethyl)-2,4-dinitro-3-[[[2-(phosphonooxy)ethyl]amino]-carbonyl]- anilino]ethyl methanesulfonate (167);

2-[(2-Bromoethyl)-2,4-dinitro-3-[[[3-(phosphonooxy)propyl]amino]-carbonyl]- anilino]ethyl methanesulfonate (168); and

2-[(2-Iodoethyl)-2,4-dinitro-3-[[[3-(phosphonooxy)propyl]amino]-carbonyl]- anilino]ethyl methanesulfonate (169).

The nitrophenyl mustards also include compounds of Formula XVII:

Formula XVII

wherein

X₃ represents at any available ring position -CONH-, -SO2NH-, -0-, -CH2-, -NHCO- or -NHSO2-;

Y₃ represents at any available ring position -N-aziridinyl, -N(CH₂CH₂W₃)₂, or

-N(CH₂CH MeW₃)₂ where each W₃ is independently selected from halogen or - 0S0₂Me;

Z₃ represents at any available ring position -NO₂, -halogen, -CN, -CF₃ or -S0₂Me;

R₇₀ represents a lower Ci-₆ alkyl optionally substituted with one or more groups including hydroxy, amino and N-oxides therefrom or dialkylamino and N-oxides therefrom.

In certain embodiments, the alcohol compound of Formula XVII is selected from a compound represented by Formulae XVIII, Formula XIX or Formula XX:

Formula XVIII Formula XIX Formula XX

I

wherein Y₄ may represent or N

LA

and wherein

n represents 1 to 6

Z₄ represents -NO₂, -halogen, -CN, -CF₃ or -S0₂Me; and

where each W₄ is independently selected from halogen or -OSC^Me.

In certain embodiments, the compound of Formula XVII is selected from the group consisting of:

N-(3-Hydroxypropyl)-5-[bis(2-chloroethyl)amino]-2,4-dinitrobenzamide (170);

N-(3-Hydroxypropyl)-5-[bis(2-bromoethyl)amino]-2,4-dinitrobenzamide (171); N-(2-Hydroxyethyl)-5-[bis(2-bromoethyl)amino]-2,4-dinitrobenzamide (172);

N-(4-Hydroxybutyl)-5-[bis(2-bromoethyl)amino]-2,4-dinitrobenzamide (173);

N-(5-Hydroxypentyl)-5-[bis(2-bromoethyl)amino]-2,4-dinitrobenzamide (174); N-(6-Hydroxyhexyl)-5-[bis(2-bromoethyl)amino]-2,4-dinitrobenzamide (175);

5-[Bis(2-bromoethyl)amino]-N-(2-hydroxyethyl)-4-(methylsulfonyl)-2- nitrobenzamide (176);

2[(2-Bromoethyl)-5-[[(3-hydroxypropyl)amino]carbonyl]-2,4-dinitroanilino]ethyl methanesulfonate (177);

5-[Bis(2-iodoethyl)amino]-N-(2-hydroxyethyl)-2, 4-dinitrobenzamide (178);

2-[Bis(2-Chloroethyl)amino]-N-(2-hydroxyethyl)-3,5-dinitrobenzamide (179);

2-[Bis(2-bromoethyl)amino]-N-(2-hydroxyethyl)-3,5-dinitrobenzamide (180);

2-[Bis(2-chloroethyl)amino]-N-(3-hydroxypropyl)-3,5-dinitrobenzamide (181);

2-[Bis(2-bromoethyl)amino]-N-(3-hydroxypropyl)-3,5-dinitrobenzamide (182); 2-[Bis(2-chloroethyl)amino]-N-(4-hydroxybutyl)-3,5-dinitrobenzamide (183);

2-[Bis(2-bromoethyl)amino]-N-(4-hydroxybutyl)-3,5-dinitrobenzamide (184);

2-[Bis(2-chloroethyl)amino]-N-(5-hydroxypentyl)-3,5-dinitrobenzamide (185);

2-[Bis(2-bromoethyl)amino]-N-(5-hydroxypentyl)-3,5-dinitrobenzamide (186); 2-[Bis(2-chloroethyl)amino]-N-(6-hydroxyhexyl)-3,5-dinitrobenzamide (187);

2-[Bis(2-bromoethyl)amino]-N-(6-hydroxyhexyl)-3,5-dinitrobenzamide (188);

2-[Bis(2-bromopropyl)amino]-N-(2-hydroxyethyl)-3,5-dinitrobenzamide (189); 2-((2-Bromoethyl)-2-{[(2-hydroxypropyl)amino]carbonyl}-4,6-dinitroanilino)ethyl methanesulfonate (190);

2-((2-Bromoethyl)-2-{[(2-hydroxyethyl)amino]carbonyl}-4,6-dinitroanilino)ethyl methanesulfonate (191);

2-((2-Chloroethyl)-2-{[(2-hydroxyethyl)amino]carbonyl}-4,6-dinitroanilino)ethyl methanesulfonate (192);

2-[Bis(2-iodoethyl)amino]-N-(2-hydroxyethyl)-3,5-dinitrobenzamide (193);

2-((2-Iodoethyl)-2-{[(2-hydroxyethyl)amino]carbonyl}-4,6-dinitroanilino)ethyl methanesulfonate (194);

3-[Bis(2-bromoethyl)amino]-N-(2-hydroxyethyl)-2,6-dinitrobenzamide (195);

2-((2-Bromoethyl)-3-{[(2-hydroxyethyl)amino]carbonyl}-2,4-dinitroanilino)ethyl methanesulfonate (196);

3-[Bis(2-bromoethyl)amino]-N-(3-hydroxypropyl)-2,6-dinitrobenzamide (197);

2-((2-bromoethyl)-3-{[(3-hydroxypropyl)amino]carbonyl}-2,4-dinitroanilino)ethyl methanesulfonate (198);

3-[Bis(2-bromoethyl)amino]-N-(4-hydroxybutyl)-2,6-dinitrobenzamide (199);

2-((2-Bromoethyl)-3-{[(4-hydroxybutyl)amino]carbonyl}-2,4-dinitroanilino)ethyl methanesulfonate (200);

2-((2-Chloroethyl)-3-{[(3-hydroxypropyl)amino]carbonyl}-2,4-dinitroanilino)ethyl methanesulfonate (201); and

2-((2-Iodoethyl)-3-{[(3-hydroxypropyl)amino]carbonyl}-2,4-dinitroanilino)ethyl methanesulfonate (202).

In certain embodiments, the nitrophenyl mustards are compounds of the Formula

XXI:

Formula XXI wherein W₅ represents Cl, Br, I, OSO₂R₇₁,

X₅ represents Cl, Br, I, OSO2R7₁,

Y₅ represents H, CN, SO2R7₁,

each R71 independently represents a C1-6 alkyl group,

Z₅ is selected from any of the radicals of Formula XXII:

Formula XXII

wherein

R72 represents H, or a Ci-₆ alkyl group;

R7₃ and R7₄ may independently represent H, or a C₁-C₆ alkyl group, or R₇₃ and R₇₄ together may be linked to form a substituted or unsubstituted heterocyclic ring comprising 5 or 6 members;

n represents 2 to 6;

* represents a point of attachment to Formula XXI.

Nitrophenyl mustards of WO 2014/031012 include compounds of Formula XXIII:

Formula XXIII wherein

Y₆ represents H, CN, SO2R75,

R₇₅ represents a methyl or ethyl group,

Z₆ is selected from any of the radicals of Formula XXIV:

Formula XXIV

wherein

R76 represents H, or a C1-C6 alkyl group;

R77 and R78 may independently represent H, or a C1-C6 alkyl group, or

R77 and R78 together may be linked to form a substituted or unsubstituted heterocyclic ring comprising 5 or 6 members;

n represents 2 to 6; and

* represents a point of attachment to Formula XXIII.

Nitrophenyl mustards of WO 2014/031012 also include compounds of Formula XXV:

Formula XXV wherein

n represents 2 to 6,

W₇ represents Cl, Br, I, OSO2R79,

X7 represents Cl, Br, I, OSO2R79,

each R79 independently represents a C1-C6 alkyl group, and

Rso represents H, or a C1-C6 alkyl group.

W₇ may be bromine or iodine.

X7 may be bromine or OS0₂Me.

R₇₉ may be methyl or ethyl. Rso may be hydrogen, methyl or ethyl

n may represent 2 or 3.

Nitrophenyl mustards of WO 2014/031012 also include compounds of Formula XXVI:

Formula XXVI wherein n represents 2 to 6, and f represents methyl or ethyl.

Some specific nitrophenyl mustards of WO 2014/031012 include:

2-(5-(bis(2-bromoethyl)amino)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamido)ethyl dihydrogen phosphate (203),

2-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)ethyl dihydrogen phosphate (204),

3-(5-(bis(2-bromoethyl)amino)-N-methyl-4-(methylsulfonyl)-2- nitrobenzamido)propyl dihydrogen phosphate (205),

3-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)propyl dihydrogen phosphate (206),

2-(5-(bis(2-bromoethyl)amino)-4-cyano-N-methyl-2-nitrobenzamido)ethyl dihydrogen phosphate (207),

3-(5-(bis(2-bromoethyl)amino)-4-cyano-N-methyl-2-nitrobenzamido)propyl dihydrogen phosphate(208),

2-(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrobenzamido)ethyl dihydrogen phosphate(209),

3-(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrobenzamido)propyl dihydrogen phosphate (210),

3-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzamido)propyl dihydrogen phosphate (211),

2-((2-bromoethyl)(2-cyano-5-(methyl(2-(phosphonooxy)ethyl)ca rbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (212), and 2-((2-bromoethyl)(2-cyano-5-(methyl(3-(phosphonooxy)propyl)carbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (213).

Nitrophenyl mustards of WO 2014/031012 also include compounds of Formula XXVII:

Formula XXVII wherein n represents 2 to 6,

Ws represents Cl, Br, I, OSO2R82,

Xs represents Cl, Br, I, OSO2R82,

each R_S2 independently represents a C1-C6 alkyl group, and

R_S3 represents H, or a C1-C6 alkyl group.

Ws may be bromine or iodine.

Xs may be bromine or OS0₂Me.

R82 may be methyl or ethyl.

R_S3 may be hydrogen, methyl or ethyl,

n may represent 2 or 3.

Nitrophenyl mustards of WO 2014/031012 also include compounds of Formula

XXVIII:

Formula XXVIII wherein n represents 2 to 6, and Rs₄ represents methyl or ethyl.

Some specific nitrophenyl mustards of WO 2014/031012 include:

5-(bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-N-methyl-4-(methylsulfonyl)-2- nitrobenzamide (214),

5-(bis(2-bromoethyl)amino)-N-(3-hydroxypropyl)-N-methyl-4-(methylsulfonyl)-2- nitrobenzamide (215),

5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-(2-hydroxyethyl)-N-methyl-2- nitrobenzamide (216),

5-(bis(2-bromoethyl)amino)-4-cyano-N-(2-hydroxyethyl)-N-methyl-2- nitrobenzamide (217),

5-(bis(2-bromoethyl)amino)-4-cyano-N-(3-hydroxypropyl)-N-methyl-2- nitrobenzamide (218),

5-(bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-4-(methylsulfonyl)-2- nitrobenzamide (219),

5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-(3-hydroxypropyl)-N-methyl-2- nitrobenzamide (220),

5-(bis(2-bromoethyl)amino)-N-(3-hydroxypropyl)-4-(methylsulfonyl)-2- nitrobenzamide (221),

5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-(3-hydroxypropyl)-2- nitrobenzamide (222),

2-((2-bromoethyl)(2-cyano-5-((2-hydroxyethyl)(methyl)carbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (223), and

2-((2-bromoethyl)(2-cyano-5-((3-hydroxypropyl)(methyl)carbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (224).

Nitrophenyl mustards of WO 2014/031012 also include compounds of Formula XXIX:

Formula XXIX wherein W represents Cl, Br, I, OSO2R85,

X9 represents Cl, Br, I, OSO2R85,

each R_S5 independently represents a C1-C6 alkyl group,

R_S6 represents H, or a C1-C6 alkyl group.

W9 May be bromine or iodine.

X9 may be bromine or OSC>2Me.

R_S5 may be methyl or ethyl.

R₈₆ may be methyl, ethyl, propyl or iso-propyl.

Further specific nitrophenyl mustards of WO 2014/031012 include:

2-((2-bromoethyl)(5-(4-methylpiperazine-l-carbonyl)-2-(methylsulfonyl)-4- nitrophenyl)amino)ethyl methanesulfonate (225),

2-((2-bromoethyl)(5-(4-ethylpiperazine-l-carbonyl)-2-(methylsulfonyl)-4- nitrophenyl)amino)ethyl methanesulfonate (226),

2-((2-bromoethyl)(5-(4-ethylpiperazine-l-carbonyl)-2-(ethylsulfonyl)-4- nitrophenyl)amino)ethyl methanesulfonate (227),

4-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-(methylsulfonyl)-2- nitrobenzoyl)-l-methylpiperazin-l-ium methanesulfonate (228),

4-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-(methylsulfonyl)-2- nitrobenzoyl)-l-ethylpiperazin-l-ium methanesulfonate (229), and

4-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-(ethylsulfonyl)-2- nitrobenzoyl)-l-ethylpiperazin-l-ium methanesulfonate (230).

Nitrophenyl mustards of WO 2014/031012 also include compounds of Formula XXX:

Formula XXX wherein Rs7 represents a C1-C6 alkyl group, and Rss represents H, or a C1-C6 alkyl group. R_S7 may be methyl or ethyl.

R.₈₈ may be methyl or ethyl.

Further specific nitrophenyl mustards of WO 2014/031012 include:

(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-methylpiperazin- l-yl)methanone (231),

(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-ethylpiperazin-l- yl)methanone (232),

(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4- isopropylpiperazin-l-yl)methanone (233),

(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrophenyl)(4-methylpiperazin-l- yl)methanone (234),

(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrophenyl)(4-ethylpiperazin-l- yl)methanone (235),

(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrophenyl)(4-isopropylpiperazin-

1-yl)methanone (236),

5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-N-(2-morpholinoethyl)-2- nitrobenzamide (237),

5-(bis(2-bromoethyl)amino)-N-methyl-4-(methylsulfonyl)-N-(2-morpholinoethyl)-2- nitrobenzamide (238),

5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-N-(3-morpholinopropyl)-2- nitrobenzamide (239),

5-(bis(2-bromoethyl)amino)-N-methyl-4-(methylsulfonyl)-N-(3-morpholinopropyl)-2- nitrobenzamide (240),

5-(bis(2-bromoethyl)amino)-N-(2-(4-methylpiperazin-l-yl)ethyl)-4-(methylsulfonyl)-

2-nitrobenzamide (241),

5-(bis(2-bromoethyl)amino)-N-methyl-N-(2-(4-methylpiperazin-l-yl)ethyl)-4- (methylsulfonyl)-2-nitrobenzamide (242),

5-(bis(2-bromoethyl)amino)-N-(3-(4-methylpiperazin-l-yl)propyl)-4- (methylsulfonyl)-2-nitrobenzamide (243),

5-(bis(2-bromoethyl)amino)-N-methyl-N-(3-(4-methylpiperazin-l-yl)propyl)-4- (methylsulfonyl)-2-nitrobenzamide (244),

5-(bis(2-bromoethyl)amino)-N-(2-(dimethylamino)ethyl)-4-(methylsulfonyl)-2- nitrobenzamide (245),

5-(bis(2-bromoethyl)amino)-N-(2-(dimethylamino)ethyl)-N-methyl-4- (methylsulfonyl)-2-nitrobenzamide (246), 5-(bis(2-bromoethyl)amino)-N-(3-(dimethylamino)propyl)-4-(methylsulfonyl)-2- nitrobenzamide (247),

5-(bis(2-bromoethyl)amino)-N-(3-(dimethylamino)propyl)-N-methyl-4- (methylsulfonyl)-2-nitrobenzamide (248),

5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-(2-morpholinoethyl)-2- nitrobenzamide (249),

5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-N-(2-morpholinoethyl)-2- nitrobenzamide (250),

5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-(3-morpholinopropyl)-2- nitrobenzamide (251),

5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-N-(3-morpholinopropyl)-2- nitrobenzamide (252),

5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-(2-(4-methylpiperazin-l-yl)ethyl)- 2-nitrobenzamide (253),

5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-N-(2-(4-methylpiperazin-l- yl)ethyl)-2-nitrobenzamide (254),

5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-(3-(4-methylpiperazin-l-yl)propyl)- 2-nitrobenzamide (255),

5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-N-(3-(4-methylpiperazin-l- yl)propyl)-2-nitrobenzamide) (256),

5-(bis(2-bromoethyl)amino)-N-(2-(dimethylamino)ethyl)-4-(ethylsulfonyl)-2- nitrobenzamide (257),

5-(bis(2-bromoethyl)amino)-N-(2-(dimethylamino)ethyl)-4-(ethylsulfonyl)-N-methyl- 2-nitrobenzamide (258),

5-(bis(2-bromoethyl)amino)-N-(3-(dimethylamino)propyl)-4-(ethylsulfonyl)-2- nitrobenzamide (259),

5-(bis(2-bromoethyl)amino)-N-(3-(dimethylamino)propyl)-4-(ethylsulfonyl)-N- methyl-2-nitrobenzamide (260),

3-(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrobenzamido)propanoic acid

(261),

4-(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrobenzamido)butanoic acid

(262),

3-(5-(bis(2-bromoethyl)amino)-N-methyl-4-(methylsulfonyl)-2- nitrobenzamido)propanoic acid (263),

4-(5-(bis(2-bromoethyl)amino)-N-methyl-4-(methylsulfonyl)-2- nitrobenzamido)butanoic acid (264), 3-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzamido)propanoic acid

(265),

4-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzamido)butanoic acid

(266),,

3-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2- nitrobenzamido)propanoic acid (267),

4-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2- nitrobenzamido)butanoic acid (268),

2-(bis(2-bromoethyl)amino)-4-(4-methylpiperazine-l-carbonyl)-5-nitrobenzonitrile

(269),

2-(bis(2-bromoethyl)amino)-4-(4-ethylpiperazine-l-carbonyl)-5-nitrobenzonitrile

(270),

2-(bis(2-bromoethyl)amino)-4-(4-isopropylpiperazine-l-carbonyl)-5-nitrobenzonitrile

(271),

3-(5-(bis(2-bromoethyl)amino)-4-cyano-2-nitrobenzamido)propanoic acid (272),

4-(5-(bis(2-bromoethyl)amino)-4-cyano-2-nitrobenzamido)butanoic acid (273),

3-(5-(bis(2-bromoethyl)amino)-4-cyano-N-methyl-2-nitrobenzamido)propanoic acid

(274),

4-(5-(bis(2-bromoethyl)amino)-4-cyano-N-methyl-2-nitrobenzamido)butanoic acid

(275),

5-(bis(2-bromoethyl)amino)-4-cyano-N-(2-morpholinoethyl)-2-nitrobenzamide

(276),

5-(bis(2-bromoethyl)amino)-4-cyano-N-methyl-N-(2-morpholinoethyl)-2- nitrobenzamide (277),

5-(bis(2-bromoethyl)amino)-4-cyano-N-(3-morpholinopropyl)-2-nitrobenzamide

(278),

5-(bis(2-bromoethyl)amino)-4-cyano-N-methyl-N-(3-morpholinopropyl)-2- nitrobenzamide (279),

5-(bis(2-bromoethyl)amino)-4-cyano-N-(2-(4-methylpiperazin-l-yl)ethyl)-2- nitrobenzamide (280),

5-(bis(2-bromoethyl)amino)-4-cyano-N-methyl-N-(2-(4-methylpiperazin-l-yl)ethyl)- 2-nitrobenzamide (281),

5-(bis(2-bromoethyl)amino)-4-cyano-N-(3-(4-methylpiperazin-l-yl)propyl)-2- nitrobenzamide (282),

5-(bis(2-bromoethyl)amino)-4-cyano-N-methyl-N-(3-(4-methylpiperazin-l- yl)propyl)-2-nitrobenzamide (283), 5-(bis(2-bromoethyl)amino)-4-cyano-N-(2-(dimethylamino)ethyl)-2-nitrobenzamide

(284),,

5-(bis(2-bromoethyl)amino)-4-cyano-N-(2-(dimethylamino)ethyl)-N-methyl-2- nitrobenzamide (285),

5-(bis(2-bromoethyl)amino)-4-cyano-N-(3-(dimethylamino)propyl)-2- nitrobenzamide (286),

5-(bis(2-bromoethyl)amino)-4-cyano-N-(3-(dimethylamino)propyl)-N-methyl-2- nitrobenzamide (287),

2-((2-bromoethyl)(5-(4-methylpiperazine-l-carbonyl)-2-(methylsulfonyl)-4- nitrophenyl)amino)ethyl methanesulfonate (288),

2-((2-bromoethyl)(5-(4-ethylpiperazine-l-carbonyl)-2-(methylsulfonyl)-4- nitrophenyl)amino)ethyl methanesulfonate (289),

2-((2-bromoethyl)(5-(4-isopropylpiperazine-l-carbonyl)-2-(methylsulfonyl)-4- nitrophenyl)amino)ethyl methanesulfonate (290),

2-((2-bromoethyl)(2-(ethylsulfonyl)-5-(4-methylpiperazine-l-carbonyl)-4- nitrophenyl)amino)ethyl methanesulfonate (291),

2-((2-bromoethyl)(5-(4-ethylpiperazine-l-carbonyl)-2-(ethylsulfonyl)-4- nitrophenyl)amino)ethyl methanesulfonate (292),

2-((2-bromoethyl)(2-(ethylsulfonyl)-5-(4-isopropylpiperazine-l-carbonyl)-4- nitrophenyl)amino)ethyl methanesulfonate (293),

2-((2-bromoethyl)(2-(methylsulfonyl)-5-((2-morpholinoethyl)carbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate, (294),

2-((2-bromoethyl)(5-(methyl(2-morpholinoethyl)carbamoyl)-2-(methylsulfonyl)-4- nitrophenyl)amino)ethyl methanesulfonate (295),

2-((2-bromoethyl)(2-(methylsulfonyl)-5-((3-morpholinopropyl)carbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (296),

2-((2-bromoethyl)(5-(methyl(3-morpholinopropyl)carbamoyl)-2-(methylsulfonyl)-4- nitrophenyl)amino)ethyl methanesulfonate (297),

2-((2-bromoethyl)(5-((2-(4-methylpiperazin-l-yl)ethyl)carbamoyl)-2- (methylsulfonyl)-4-nitrophenyl)amino)ethyl methanesulfonate (298),

2-((2-bromoethyl)(5-(methyl(2-(4-methylpiperazin-l-yl)ethyl)carbamoyl)-2- (methylsulfonyl)-4-nitrophenyl)amino)ethyl methanesulfonate (299),

2-((2-bromoethyl)(5-((3-(4-methylpiperazin-l-yl)propyl)carbamoyl)-2- (methylsulfonyl)-4-nitrophenyl)amino)ethyl methanesulfonate (300),

2-((2-bromoethyl)(5-(methyl(3-(4-methylpiperazin-l-yl)propyl)carbamoyl)-2- (methylsulfonyl)-4-nitrophenyl)amino)ethyl methanesulfonate (301), 2-((2-bromoethyl)(5-((2-(dimethylamino)ethyl)carbamoyl)-2-(methylsulfonyl)-4- nitrophenyl)amino)ethyl methanesulfonate (302),

2-((2-bromoethyl)(5-((2-(dimethylamino)ethyl)(methyl)carbamoyl)-2- (methylsulfonyl)-4-nitrophenyl)amino)ethyl methanesulfonate (303),

2-((2-bromoethyl)(5-((3-(dimethylamino)propyl)carbamoyl)-2-(methylsulfonyl)-4- nitrophenyl)amino)ethyl methanesulfonate (304),

2-((2-bromoethyl)(5-((3-(dimethylamino)propyl)(methyl)carbamoyl)-2- (methylsulfonyl)-4-nitrophenyl)amino)ethyl methanesulfonate (305),

2-((2-bromoethyl)(2-(ethylsulfonyl)-5-((2-morpholinoethyl)carbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (306),

2-((2-bromoethyl)(2-(ethylsulfonyl)-5-(methyl(2-morpholinoethyl)carbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (307),

2-((2-bromoethyl)(2-(ethylsulfonyl)-5-((3-morpholinopropyl)carbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (308),

2-((2-bromoethyl)(2-(ethylsulfonyl)-5-(methyl(3-morpholinopropyl)carbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (309),

2-((2-bromoethyl)(2-(ethylsulfonyl)-5-((2-(4-methylpiperazin-l-yl)ethyl)carbamoyl)- 4-nitrophenyl)amino)ethyl methanesulfonate (310),

2-((2-bromoethyl)(2-(ethylsulfonyl)-5-(methyl(2-(4-methylpiperazin-l- yl)ethyl)carbamoyl)-4-nitrophenyl)amino)ethyl methanesulfonate (311),

2-((2-bromoethyl)(2-(ethylsulfonyl)-5-((3-(4-methylpiperazin-l- yl)propyl)carbamoyl)-4-nitrophenyl)amino)ethyl methanesulfonate (312),

2-((2-bromoethyl)(2-(ethylsulfonyl)-5-(methyl(3-(4-methylpiperazin-l- yl)propyl)carbamoyl)-4-nitrophenyl)amino)ethyl methanesulfonate (313),

2-((2-bromoethyl)(5-((2-(dimethylamino)ethyl)carbamoyl)-2-(ethylsulfonyl)-4- nitrophenyl)amino)ethyl methanesulfonate (314),

2-((2-bromoethyl)(5-((2-(dimethylamino)ethyl)(methyl)carbamoyl)-2- (ethylsulfonyl)-4-nitrophenyl)amino)ethyl methanesulfonate (315),

2-((2-bromoethyl)(5-((3-(dimethylamino)propyl)carbamoyl)-2-(ethylsulfonyl)-4- nitrophenyl)amino)ethyl methanesulfonate (316),

2-((2-bromoethyl)(5-((3-(dimethylamino)propyl)(methyl)carbamoyl)-2- (ethylsulfonyl)-4-nitrophenyl)amino)ethyl methanesulfonate (317),

3-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-(methylsulfonyl)-2- nitrobenzamido)propanoic acid (318),

4-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-(methylsulfonyl)-2- nitrobenzamido)butanoic acid (319), 3-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-N-methyl-4- (methylsulfonyl)-2-nitrobenzamido)propanoic acid (320),

4-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-N-methyl-4- (methylsulfonyl)-2-nitrobenzamido)butanoic acid (321),

3-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-(ethylsulfonyl)-2- nitrobenzamido)propanoic acid (322),

4-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-(ethylsulfonyl)-2- nitrobenzamido)butanoic acid (323),

3-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-(ethylsulfonyl)-N- methyl-2-nitrobenzamido)propanoic acid (324),

4-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-(ethylsulfonyl)-N- methyl-2-nitrobenzamido)butanoic acid (325),

2-((2-bromoethyl)(2-cyano-5-(4-methylpiperazine-l-carbonyl)-4- nitrophenyl)amino)ethyl methanesulfonate (326),

2-((2-bromoethyl)(2-cyano-5-(4-ethylpiperazine-l-carbonyl)-4- nitrophenyl)amino)ethyl methanesulfonate (326),

2-((2-bromoethyl)(2-cyano-5-(4-isopropylpiperazine-l-carbonyl)-4- nitrophenyl)amino)ethyl methanesulfonate, (327),

3-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-cyano-2- nitrobenzamido)propanoic acid (328),

4-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-cyano-2- nitrobenzamido)butanoic acid (329),

3-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-cyano-N-methyl-2- nitrobenzamido)propanoic acid (330),

4-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-cyano-N-methyl-2- nitrobenzamido)butanoic acid (331),

2-((2-bromoethyl)(2-cyano-5-((2-morpholinoethyl)carbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (332),

2-((2-bromoethyl)(2-cyano-5-(methyl(2-morpholinoethyl)carbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (333),

2-((2-bromoethyl)(2-cyano-5-((3-morpholinopropyl)carbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (334),

2-((2-bromoethyl)(2-cyano-5-(methyl(3-morpholinopropyl)carbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (335),

2-((2-bromoethyl)(2-cyano-5-((2-(4-methylpiperazin-l-yl)ethyl)carbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (336), 2-((2-bromoethyl)(2-cyano-5-(methyl(2-(4-methylpiperazin-l-yl)ethyl)carbamoyl)- 4-nitrophenyl)amino)ethyl methanesulfonate (337),

2-((2-bromoethyl)(2-cyano-5-((3-(4-methylpiperazin-l-yl)propyl)carbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (338),

2-((2-bromoethyl)(2-cyano-5-(methyl(3-(4-methylpiperazin-l-yl)propyl)carbamoyl)- 4-nitrophenyl)amino)ethyl methanesulfonate (339),

2-((2-bromoethyl)(2-cyano-5-((2-(dimethylamino)ethyl)carbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (340),

2-((2-bromoethyl)(2-cyano-5-((2-(dimethylamino)ethyl)(methyl)carbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (341),

2-((2-bromoethyl)(2-cyano-5-((3-(dimethylamino)propyl)carbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (342), and

2-((2-bromoethyl)(2-cyano-5-((3-(dimethylamino)propyl)(methyl)ca rbamoyl)-4- nitrophenyl)amino)ethyl methanesulfonate (343),

In certain embodiments, the HAP is selected from an aminodihydropyrimidopyrimidinone, an aminopyridopyrimidinone, an aminopteridinone, an aminodihydropyridopyrimidinone, an aminonaphthyridinone, and an aminopyridopyrazinone. For example, also provided herein are HAPs of a compound of Formula XXXI:

XXXI

wherein :

Wio is N or CH;

Yio is N, CH or CH₂;

ZIO is N or C;

Ar is an aryl group optionally substituted with halogen, alkyl or alkoxy; denotes either a single bond or a double bond;

R_S9 is hydrogen, or is selected from the group comprising C1-C6 alkyl, C3-C6 cycloalkyl, heterocyclyl, aryl, and heteroaryl each of which is optionally substituted with hydroxy, alkyl, alkenyl, alkynyl, alkoxy, acetyl, thiol, alkylthio, arylthio, aralkylthio, halogen, carboxylic acid, carboxylate alkyl ester, carboxamide, alkylca rboxamide, dialkylcarboxamide, alkylsulfonyl, alkylsulfoxide, haloalkyl, haloalkoxy, amino, al kylamino, dialkylamino, morpholinyl, thiomorpholinyl, piperazinyl, N-alkylpiperazinyl, N-acetylpiperazinyl, N-alkylsulfonylpiperazinyl, pyrrolidinyl, piperidinyl, imidazolyl, or nitro;

L is a radical of selected from the group comprising Formulae (A)-(J) :

wherein :

* is a point of attachment to the heterocyclic nitrogen atom of Formula

(i) ;

** is a point of attachment to R₉₀; and

R91, R92, R93 and R94 are independently selected from hydrogen and Ci- C₆ alkyl ;

R90 is a radical of Formula (K), (L) or (L-i) :

m m **--■)

wherein :

R95 and R96 are independently C1-C6 alkyl or may together form a heterocyclic ring ; and

** is a point of attachment to L;

or a pharmaceutically acceptable salt thereof, or a solvate thereof.

In some embodiments of the disclosure, Rs9 is C1-C6 alkyl or is a radica l selected from the group comprising Formulae (M)-(FF) :

(U) (W) (Y) (Z) (AA) (BB) (CC) (DD) (EE) (FF)

In some embodiments of the disclosure, Ar is a radical selected from the group comprising Formulae (GG)-(LLL) :

In some embodiments of the disclosure, the compounds have a formula selected from the group comprising Formulae (XXXII) to (XXXVII) :

wherein Ar, R_8g and Rg_o are as defined above.

Certain embodiments of the disclosure include those where R_8g is alkylamino. Preferably, the alkylamino is -(CH2)_nNRg₈Rg9 where n is an integer from 1-6, Rg₈ is H or alkyl, and R99 is H or alkyl, or Rgs and Rgg taken together form a non-aromatic heterocyclic ring.

In some embodiments of the disclosure, L is a radical of Formula (B), (C) or (H) as defined above. Preferably, L is a radical of Formula (B) as defined above.

In some embodiments of the disclosure, Rgi, R92, R93 and Rg₄ are each independently hydrogen or methyl. For example, Rgi, R92, R93 and Rg₄ may all be hydrogen.

In some embodiments of the disclosure, Rgo is a radical of Formula (K). Preferably, R95 and Rg₆ together form a heterocyclic ring, or R95 and Rg₆ may both be methyl. The heterocyclic ring may preferably be a pyrrolidinyl, piperidinyl, piperazinyl, /V-alkylpiperazinyl or morpholinyl ring.

The prodrug is preferably a compound where Rgo is a radical of Formula (MMM) or Formula (MMM-i) :

wherein R₉₅ and R₉₆ are as defined above, and R₉₇ is hydrogen or C₁-C₆ alkyl.

In some embodiments of the disclosure, X is fluoride, chloride, bromide, iodide, acetate, trifluoroacetate, methanesulfonate or tosylate. Preferably, X is bromide.

Additional compounds of Formula XXXI include:

(S,E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-3-yl)-7-(methylamino)-3,4-dihydropyrimido[4,5-c/]pyrimidin-2(l/-/)-one

(344),

(345),

(R,f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pyrrolidin-3-yl)-7-(methylamino)-3,4-dihydropyrimido[4,5-c/]pyrimidin-2(l/-/)-one

(346),

(S, f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pyrrolidin-3-yl)-7-(methylamino)-3,4-dihydropyrimido[4,5-c/]pyrimidin-2(l/-/)-one

(347),

(S,E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pyrrolidin-3-yl)-7-(isopropylamino)-3,4-dihydropyrimido[4,5-c/]pyrimidin-2(lH)- one (348),

(S,E)-7-(cyclohexylamino)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4- (dimethylamino)but-2-enoyl)pyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-c/]pyrimidin- 2(lH)-one (349),

(S,E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pyrrolidin-3-yl)-7-(phenylamino)-3,4-dihydropyrimido[4,5-c/]pyrimidin-2(l/-/)-one

(350),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(methylamino)-3,4-dihydropyrimido[4,5-c/]pyrimidin-2(l/-/)-one

(351), (f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(isopropylamino)-3,4-dihydropynmido[4,5-c/]pynmidin-2(lH)- one (352),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-((tetrahydrofuran-3-yl)amino)-3,4-dihydropynmido[4,5- c/]pyrimidin-2(lH)-one (353),

(f)-7-(cyclohexylamino)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4- (dimethylamino)but -2-enoyl)piperidin-4-yl)-3,4-dihydropyrimido[4,5-c/]pyrimidin- 2(lH)-one (354),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2-enoyl)pipendin -4 -yl)-7-((tetrahydro-2H-pyran-4-yl)amino)-3,4-dihydropyrimido[4,5-c/]pynmidin- 2(lH)-one (355),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(phenylamino)-3,4-dihydropynmido[4,5-c/]pynmidin-2(l/-/)-one

(356),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-((3-methoxyphenyl)amino)-3,4-dihydropynmido[4,5- c/]pyrimidin-2(lH)-one (357),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-(m-tolylamino)-3,4-dihydropynmido[4,5-c/]pynmidin-2(l/-/)-one

(358),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-((3-fluorophenyl)amino)-3,4-dihydropynmido[4,5-c/]pynmidin- 2(lH)-one (359),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-((3,5-difluorophenyl)amino)-l-(l-(4- (dimethylamino)but-2-enoyl)pipendin-4-yl)-3,4-dihydropynmido[4,5-c/]pynmidin-2(l/-/)- one (360),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-((3,4-difluorophenyl)amino)-l-(l-(4- (dimethylamino)but-2-enoyl)pipendin-4-yl)-3,4-dihydropynmido[4,5-c/]pynmidin-2(l/-/)- one (361),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl) -7-((4-fluoro-3-methoxyphenyl)amino)-3,4-dihydropyrimido[4,5-c/] pyrimidin-2(lH)-one (362),

(E)-7-((3-chloro-4-fluorophenyl)amino)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4- (dimethylamino)but-2-enoyl)pipendin-4-yl)-3,4-dihydropynmido[4,5-c/]pynmidin-2(l/-/)- one (363), (E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-((4-fluorophenyl)amino)-3,4-dihydropynmido[4,5-c/]pynmidin- 2(lH)-one (364),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-((2-methoxyphenyl)amino)-3,4-dihydropynmido[4,5- c/]pyrimidin-2(lH)-one (365),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-((4-methoxyphenyl)amino)-3,4-dihydropynmido[4,5- c/]pyrimidin-2(lH)-one (366),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(methylamino)-l-(l-(4-morpholinobut-2- enoyl)piperidin-4-yl)-3,4-dihydropynmido[4,5-c/]pynmidin-2(lH)-one (367),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(methylamino)-l-(l-(4-(piperidin-l-yl)but- 2-enoyl)piperidin-4-yl)-3,4-dihydropynmido[4,5-c/]pynmidin-2(lH)-one (368),

(E)-N-(3-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(methylamino)-2-oxo-3,4- dihydropyrimido[4,5-c/]pynmidin-l(2/-/)-yl)propyl)-4-(dimethylamino)but-2-enamide

(369),

(E)-/V-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(methylamino)-2-oxo-3,4- dihydropyrimido[4,5-c/]pynmidin-l(2/-/)-yl)cydohexyl)-4-(dimethylamino)but-2-enamide

(370),

(E)-/V-(3-((3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(methylamino)-2-oxo-3,4- dihydropyrimido[4,5-c/]pynmidin-l(2/-/)-yl)methyl)phenyl)-4-(dimethylamino)but-2- enamide (371),

(E)-3-(2-chloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2-enoyl)piperidin-4- yl)-7-(phenylamino)-3,4-dihydropyrimido[4,5-c/]pynmidin-2(lH)-one (372),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-((4-morpholinophenyl)amino)-3,4-dihydropynmido[4,5- c/]pyrimidin-2(lH)-one (373),

(E)-3-(2-chloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2-enoyl) piperidin-4- yl)-7-((4-morpholinophenyl)amino)-3,4-dihydropyrimido[4,5-c/]pyrimidin-2(l/-/)-one

(374),

(E)-3-(3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2-enoyl)piperidin-4-yl)-7-((4- morpholinophenyl)amino)-3,4-dihydropyrimido[4,5-c/]pynmidin-2(l/-/)-one (375),

(E)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-(methylamino)pyndo[2,3-c/]pynmidin-7(8H)-one (376),

(E)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-(isopropylamino)pyndo[2,3-c/]pynmidin-7(8H)-one (377), (E)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-((tetrahydrofuran-3-yl)amino)pyndo[2,3-c/]pynmidin-7(8H)-one

(378),

(E)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-((tetrahydro-2H-pyran-4-yl)amino)pyndo[2,3-c/]pynmidin- 7(8H)-one (379),

(E)-2-(cyclohexylamino)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4- (dimethylamino)but-2-enoyl)piperidin-4-yl)pyndo[2,3-c/]pynmidin-7(8H)-one (380), (E)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-(((tetrahydro-2H-pyran-4-l)methyl)amino)pyndo[2,3- c/]pyrimidin-7(8H)-one (381),

(E)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-(phenylamino)pyndo[2,3-c/]pynmidin-7(8H)-one (382),

(E)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-((4-fluorophenyl)amino)pyndo[2,3-c/]pynmidin-7(8H)-one

(383),

(E)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-((3-methoxyphenyl)amino)pyndo[2,3-c/]pynmidin-7(8H)-one

(384),

(E)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-((4-fluoro-3-methoxyphenyl)amino)pyndo[2,3-c/]pynmidin- 7(8H)-one (385),

(E)-2-((3-chloro-4-fluorophenyl)amino)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4- (dimethylamino)but-2-enoyl)piperidin-4-yl)pyndo[2,3-c/]pynmidin-7(8H)-one (386), (E)-/V-(3-((6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)-7-oxopyrido[2,3- c/]pynmidin-8(7/-/)-yl)methyl)phenyl)-4-(dimethylamino)but-2-enamide (387),

(E)-/V-(3-((6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(ethylamino)-7-oxopyrido[2,3- c/]pynmidin-8(7H)-yl)methyl)phenyl)-4-(dimethylamino)but-2-enamide (388),

(E)-/V-(3-((6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(isopropylamino)-7-oxopyrido[2,3- c/]pynmidin-8(7H)-yl)methyl)phenyl)-4-(dimethylamino)but-2-enamide (389),

(E)-/V-(3-(l-(6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)-7-oxopyrido[2,3- c/]pynmidin-8(7H)-yl)ethyl)phenyl)-4-(dimethylamino)but-2-enamide (390),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(isopropylamino)pyndo[3,4-b]pyrazin-2(lH)-one (391),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(methylamino)-l,6-naphthyndin-2(lH)-one (392), (f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(isopropylamino)-l,6-naphthyndin-2(lH)-one (393),

(S,E)-3-(3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2-enoyl)pyrrolidin-3-yl)-7- (methylamino)-3,4-dihydropyrimido[4,5-c/]pynmidin-2(lH)-one (394),

(E)-3-(2,6-dibromo-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(isopropylamino)-3,4-dihydropyrimido[4,5-c/]pynmidin-2(lH)- one (395),

(f)-6-(2,6-dibromo-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-(phenylamino)pyndo[2,3-c/]pynmidin-7(8H)-one (396),

(f)-6-(2,6-dibromo-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-(isopropylamino)pyndo[2,3-c/]pynmidin-7(8H)-one (397),

(f)-3-(2,6-dibromo-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-(isopropylamino)pyndo[3,4-b]pyrazin-2(lH)-one (398),

(E)-3-(2,6-dibromo-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-(isopropylamino)-l,6-naphthyndin-2(lH)-one (399),

l-(l-acryloylpipendin-4-yl)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(methylamino)-3,4- dihydropyrimido[4,5-c/]pynmidin-2(lH)-one (400),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-(pyndin-2-ylamino)-3,4-dihydropynmido[4,5-c/]pynmidin-2(l/-/)- one (401),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-(pyndin-3-ylamino)-3,4-dihydropynmido[4,5-c/]pynmidin-2(l/-/)- one (402),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(pyndin-4-ylamino)-3,4-dihydropynmido[4,5-c/]pynmidin-2(l/-/)- one (403),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-((l-methyl-lH-pyrazol-3-yl)amino)-3,4-dihydropynmido[4,5- c/]pyrimidin-2(lH)-one (404),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-((5-morpholinopyndin-2-yl)amino)-3,4-dihydropynmido[4,5- c/]pyrimidin-2(lH)-one (405),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-(pynmidin-4-ylamino)-3,4-dihydropynmido[4,5-c/]pynmidin- 2(lH)-one (406), (S,f)-4-(3-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(methylamino)-2-oxo-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)pyrrolidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl- 4-nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (407),

(S,f)-4-(3-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(isopropylamino)-2-oxo-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)pyrrolidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl- 4-nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (408),

(E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(isopropylamino)-2-oxo-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (409),

(E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-2-oxo-7-((tetrahydro-2H-pyran-4- yl)amino)-3,4-dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl- /V-((l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium 2,2,2- trifluoroacetate (410),

(E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-2-oxo-7-(phenylamino)-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-lH-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (411),

(E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-((4-fluorophenyl)amino)-2-oxo-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium 2,2,2-trifluoroacetate (412),

(E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-((4-methoxyphenyl)amino)-2-oxo-

3,4-dihydropyrimido[4,5-d]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l- methyl-4-nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (413),

(E)-4-(4-(3-(2,6-dibromo-3,5-dimethoxyphenyl)-7-(isopropylamino)-2-oxo-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (414),

(E)-4-(4-(6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)-7-oxopyrido[2,3-c/] pyrimidin-8(7/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4-nitro-l/-/-imidazol-5- yl)methyl)-4-oxobut-2-en-l-aminium bromide (415),

(E)-4-(4-(6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(isopropylamino)-7-oxopyrido[2,3- c/]pyrimidin-8(7H)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4-nitro-l/-/-imidazol-5- yl)methyl)-4-oxobut-2-en-l-aminium bromide (416),

(E)-4-(4-(6-(2,6-dibromo-3,5-dimethoxyphenyl)-2-(isopropylamino)-7-oxopyrido[2,3- c/]pyrimidin-8(7/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-N-((l-methyl-4-nitro-lH-imidazol-5- yl)methyl)-4-oxobut-2-en-l-aminium bromide (417),

(E)-4-(4-(6-(2,6-dichloro-3,5-dimethoxyphenyl)-7-oxo-2-(phenylamino)pyrido[2,3-c/] pyrimidin-8(7/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4-nitro-l/-/-imidazol-5- yl)methyl)-4-oxobut-2-en-l-aminium bromide (418), (f)-4-(4-(6-(2,6-dibromo-3,5-dimethoxyphenyl)-7-oxo-2-(phenylamino)pyrido[2,3-c/] pyrimidin-8(7/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4-nitro-l/-/-imidazol-5- yl)methyl)-4-oxobut-2-en-l-aminium bromide (419),

(f)-4-((3-((6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(isopropylamino)-7-oxopyrido[2,3- c/]pyrimidin-8(7/-/)-yl)methyl)phenyl)amino)-/V,/V-dimethyl-/V-((l-methyl-4-nitro-l/-/- imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (420),

(E)-4-((4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-((4-methoxyphenyl)amino)-2-oxo-

3.4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)cyclohexyl)amino)-/V,/V-dimethyl-/V-((l- methyl-4-nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (421), (E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(isopropylamino)-2-oxo-l,6- naphthyridin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4-nitro-lH-imidazol-5- yl)methyl)-4-oxobut-2-en-l-aminium bromide (422),

(E)-4-(4-(3-(2,6-dibromo-3,5-dimethoxyphenyl)-7-(isopropylamino)-2-oxo-l,6- naphthyridin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4-nitro-l/-/-imidazol-5- yl)methyl)-4-oxobut-2-en-l-aminium bromide (423),

(E)-4-(4-(3-(2-chloro-3,5-dimethoxyphenyl)-2-oxo-7-(phenylamino)-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (424),

(E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-((4-morpholinophenyl)amino)-2-oxo-

3.4-dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l- methyl-4-nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (425), (E)-4-(4-(3-(2-chloro-3,5-dimethoxyphenyl)-7-((4-morpholinophenyl)amino)-2-oxo-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (426),

(E)-4-(4-(3-(3,5-dimethoxyphenyl)-7-((4-morpholinophenyl)amino)-2-oxo-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (427),

(E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-2-oxo-7-(pyridin-2-ylamino)-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (428),

(E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-2-oxo-7-(pyridin-3-ylamino)-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium 2,2,2-trifluoroacetate (429), (f)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-2-oxo-7-(pyridin-4-ylamino)-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium 2,2,2-trifluoroacetate (430), (E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-((l-methyl-lH-pyrazol-3-yl)amino)- 2-oxo-3,4-dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V- ((l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (431), (E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-((5-morpholinopyridin-2-yl)amino)-2- oxo-3,4-dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l- methyl-4-nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (432), (E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-2-oxo-7-(pyrimidin-4-ylamino)-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (433), and

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(phenylamino)-l,6-naphthyridin-2(l/-/)-one (434).

Examples of NMQ prodrugs used in the disclosed methods include, but are not limited to, the following compounds:

In certain embodiments, the HAP is Compound A (tarloxotinib bromide; ((2E)-4-{[4- (3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-N-[(l-methyl- 4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide, having the chemical formula :

In certain embodiments, the nitrophenyl mustard compound is:

Other examples of nitrophenyl mustard compounds include, but are not limited to, the following compounds:

In certain embodiments, the compound of Formula XXXI includes, but is not limited to Compound E:

Compound E.

The compounds (e.g., the HAPs) described herein may form salts with acids, and such salts are included in the present application. In one embodiment, the salts are pharmaceutically acceptable salts. The term "salts" embraces addition salts of free acids that are useful within the methods disclosed herein. The term "pharmaceutically acceptable salt" refers to salts that possess toxicity profiles within a range that affords utility in pharmaceutical applications. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present application, such as for example utility in process of synthesis, purification or formulation of compounds useful within the methods disclosed herein.

Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include sulphate, hydrogen sulphate, hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulphuric, and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate). Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4- hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2- hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, b-hydroxybutyric, salicylic, galactaric, galacturonic acid, glycerophosphonic acids and saccharin (e.g., saccharinate, saccharate). Salts may be comprised of a fraction of one, one or more than one molar equivalent of acid or base with respect to any compound contemplated herein.

Suitable pharmaceutically acceptable base addition salts of compounds contemplated herein include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.

Administration of a Hypoxia-Activated Prodrug (HAP)

Any route of administration of a HAP may be used. For example, an HAP can be administered by oral, parenteral, intravenous, transdermal, intramuscular, rectal, sublingual, mucosal, nasal, or other means. Suitably, the HAP is administered intravenously administration or intraperitoneally administration or is formulated for intravenous administration or for intraperitoneal administration.

The dosage amount of HAP will depend on the specific HAP used, the type of cancer being treated, and any optional additional agents concurrently administered to the patient. Typical dosage amounts comprise an amount of from about 0.001 to about 2500 mg/m². Accordingly, suitable dosage amounts comprises from about 0.001, 0.01, 0.1, 1, 2, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 50, 100, 150, 200, 250, or 300 to about 10, 12.5, 15, 17.5, 20, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2250, or 2500 mg. In a suitable embodiment, the dosage amount comprises Compound A in an amount of about 10 mg/m² to about 270 mg/m², suitably about 110 mg/m² to about 170 mg/m², more suitably about 140 mg/m² to about 160 mg/m², most suitably about 150 mg/m².

In certain embodiments, the dosage amount of HAP is from about 0.1 mg/kg of body weight of a subject to about 200 mg/kg of body weight of a subject. In certain embodiments, the dosage amount of HAP is from about 0.1 mg/kg of body weight of a subject to about 100 mg/kg of body weight of a subject, from about 0.1 mg/kg of body weight of a subject to about 50 mg/kg of body weight of a subject, from about 0.1 mg/kg of body weight of a subject to about 25 mg/kg of body weight of a subject, from about 0.1 mg/kg of body weight of a subject to about 20 mg/kg of body weight of a subject, from about 0.1 mg/kg of body weight of a subject to about 15 mg/kg of body weight of a subject, from about 0.1 mg/kg of body weight of a subject to about 10 mg/kg of body weight of a subject, from about 0.1 mg/kg of body weight of a subject to about 5 mg/kg of body weight of a subject, or from about 0.1 mg/kg of body weight of a subject to about 1 mg/kg of body weight of a subject. In certain embodiments, the dosage amount of HAP is from about 1 mg/kg of body weight of a subject to about 100 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 50 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 25 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 20 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 15 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 10 mg/kg of body weight of a subject, or from about 1 mg/kg of body weight of a subject to about 5 mg/kg of body weight of a subject. In certain embodiments, the dosage amount of HAP is from about 10 mg/kg of body weight of a subject to about 100 mg/kg of body weight of a subject, from about 10 mg/kg of body weight of a subject to about 50 mg/kg of body weight of a subject, from about 10 mg/kg of body weight of a subject to about 25 mg/kg of body weight of a subject, from about 10 mg/kg of body weight of a subject to about 20 mg/kg of body weight of a subject, or from about 10 mg/kg of body weight of a subject to about 15 mg/kg of body weight of a subject. In certain embodiments, the dosage amount of HAP is from about 20 mg/kg of body weight of a subject to about 100 mg/kg of body weight of a subject, from about 20 mg/kg of body weight of a subject to about 50 mg/kg of body weight of a subject, or from about 20 mg/kg of body weight of a subject to about 25 mg/kg of body weight of a subject. In certain embodiments, the HAP is Compound A. In certain embodiments, the HAP is Compound C. In certain embodiments, the HAP is Compound E.

In certain embodiments, the dosage amount of HAP is from about 0.1 mg/kg of body weight of a subject to about 300 mg/m² of body weight of a subject. In certain embodiments, the dosage amount of HAP is from about 0.1 mg/m² of body weight of a subject to about 200 mg/m² of body weight of a subject, from about 0.1 mg/m² of body weight of a subject to about 150 mg/m² of body weight of a subject, from about 0.1 mg/m² of body weight of a subject to about 100 mg/m² of body weight of a subject, from about 0.1 mg/m² of body weight of a subject to about 50 mg/m² of body weight of a subject, from about 0.1 mg/m² of body weight of a subject to about 25 mg/m² of body weight of a subject, from about 0.1 mg/m² of body weight of a subject to about 20 mg/m² of body weight of a subject, from about 0.1 mg/m² of body weight of a subject to about 15 mg/m² of body weight of a subject, from about 0.1 mg/m² of body weight of a subject to about 10 mg/m² of body weight of a subject, from about 0.1 mg/m² of body weight of a subject to about 5 mg/m² of body weight of a subject, or from about 0.1 mg/m² of body weight of a subject to about 1 mg/m² of body weight of a subject. In certain embodiments, the dosage amount of HAP is from about 1 mg/m² of body weight of a subject to about 150 mg/m² of body weight of a subject, about 1 mg/m² of body weight of a subject to about 100 mg/m² of body weight of a subject, from about 1 mg/m² of body weight of a subject to about 50 mg/m² of body weight of a subject, from about 1 mg/m² of body weight of a subject to about 25 mg/m² of body weight of a subject, from about 1 mg/m² of body weight of a subject to about 20 mg/m² of body weight of a subject, from about 1 mg/m² of body weight of a subject to about 15 mg/m² of body weight of a subject, from about 1 mg/m² of body weight of a subject to about 10 mg/m² of body weight of a subject, or from about mg/m² of body weight of a subject to about 5 mg/m² of body weight of a subject. In certain embodiments, the dosage amount of HAP is from about 10 mg/m² of body weight of a subject to about 150 mg/m² of body weight of a subject, from about 10 mg/m² of body weight of a subject to about 100 mg/m² of body weight of a subject, from about 10 mg/m² of body weight of a subject to about 50 mg/m² of body weight of a subject, from about 10 mg/m² of body weight of a subject to about 25 mg/m² of body weight of a subject, from about 10 mg/m² of body weight of a subject to about 20 mg/m² of body weight of a subject, or from about 10 mg/kg of body weight of a subject to about 15 mg/m² of body weight of a subject. In certain embodiments, the dosage amount of HAP is from about 20 mg/m² of body weight of a subject to about 150 mg/m² of body weight of a subject, from about 20 mg/m² of body weight of a subject to about 100 mg/m² of body weight of a subject, from about 20 mg/m² of body weight of a subject to about 50 mg/m² of body weight of a subject, or from about 20 mg/m² of body weight of a subject to about 25 mg/m² of body weight of a subject. In certain embodiments, the dosage amount of HAP is from about 50 mg/m² of body weight of a subject to about 150 mg/m² of body weight of a subject, from about 50 mg/m² of body weight of a subject to about 100 mg/m² of body weight of a subject, from about 50 mg/m² of body weight of a subject to about 80 mg/m² of body weight of a subject, or from about 50 mg/m² of body weight of a subject to about 75 mg/m² of body weight of a subject. In certain embodiments, the dosage amount of HAP is from about 75 mg/m² of body weight of a subject to about 150 mg/m² of body weight of a subject, or from about 75 mg/m² of body weight of a subject to about 100 mg/m² of body weight of a subject. In certain embodiments, the dosage amount of HAP is from about 80 mg/m² of body weight of a subject to about 150 mg/m² of body weight of a subject, or from about 80 mg/m² of body weight of a subject to about 100 mg/m² of body weight of a subject. In certain embodiments, the dosage amount of HAP is from about 0.1 mg/m², about 1 mg/m², about 5 mg/m², about 10 mg/m², about 15 mg/m², about 20 mg/m², about 25 mg/m², about 30 mg/m², about 40 mg/m², about 50 mg/m², about 60 mg/m², about 70 mg/m², about 75 mg/m², about 80 mg/m², about 100 mg/m², about 150 mg/m², about 200 mg/m², or about

300 mg/m². In certain embodiments, the HAP is Compound A. In certain embodiments, the HAP is Compound C. In certain embodiments, the HAP is Compound E.

In some embodiments, the HAP is administered, or is formulated for administration, by intravenous or intraperitoneal administration. In some embodiments, the HAP is administered, or is formulated for administration by at least one route selected from the group consisting of inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal, intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, epidural, intrapleural, intraperitoneal, intratracheal, optic, intraocular, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical. In certain embodiments, the HAP is administered orally, parenterally, rectally, topically, intravenously, intramuscularly, subcutaneously, or intraperitoneally. In certain embodiments the HAP is administered intravenously.

In some embodiments, the HAP is administered at a frequency of at least once per day, at least once per week or at least once per month. Suitably, the HAP may be administered for a period of 1-3 weeks or up to at least 30 weeks or up, at least 1 day or up to at least 150 days. In some embodiments, longer periods of administration are employed.

In one embodiment, the dosage amount comprises Compound A in an amount of about 140 mg/m² to about 160 mg/m², more suitably about 150 mg/m², where the dosage is administered once per week. Suitably, the dosage is administered intravenously over a period of approximately one hour. In other embodiments, the dosage is administered intravenously over a period of two to four hours. In one embodiment, the HAPs is administered to the patient in dosages that range from one to five times per day or more. In another embodiment, the HAP is administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the HAPs contemplated herein varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the present disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.

It is understood that the amount of HAP dosed per day may be administered, in non- limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.

In one embodiment, the HAP is administered once, twice, three times, four times, five times, or six times a day. In another embodiment, the HAP is administered once, twice, three times, four times, five times, or six times a week. In yet another embodiment, the HAP is administered every week, every two weeks, every three weeks, every four weeks, every five weeks, or every six weeks.

In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the HAPs contemplated herein is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (/.e., a "drug holiday"). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the disease or disorder, to a level at which the improved disease is retained. In one embodiment, patients require intermittent treatment on a long- term basis upon any recurrence of symptoms and/or infection Pharmaceutical compositions provided herein can also contain one of more pharmaceutically acceptable excipients. See, e.g., Rowe et al, Handbook of Pharmaceutical Excipients, 4th Ed. (2003), entirety of which is incorporated herein by reference. In one embodiment, the HAPs contemplated herein are formulated in a pharmaceutical composition using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical compositions contemplated herein comprise a therapeutically effective amount of a combination contemplated herein and a pharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.

Suitable administration methods for a HAP compounds, as well as suitable dosage forms and pharmaceutical compositions, can be found in PCT/NZ2010/000040, PCT/NZ2010/000174, and PCT/US2015/063806, each of which is incorporated by reference herein in its entirety. Additional dosage forms include dosage forms as described in U.S. Patents Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Further dosage forms include dosage forms as described in PCT Applications Nos. WO 03/35041; WO 03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO 02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO 98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.

In some embodiments, the HAP may be administered in combination with other agents, including but not limited to a further HAP, a chemotherapeutic agent, a radiotherapeutic agent, and/or an immunotherapeutic agent. In one suitable embodiment, the HAP may comprise Compound A and may be administered in combination with another tyrosine kinase inhibitor selected from the group consisting of crizotinib, alectinib, ceritinib, erlotinib, dacomitinib, osimertinib, afatinib, gefitinib, rociletinib, cetuximab, icotinib, sapitinib, lapatinib, neratinib, brigatinib, poziotinib, naquotinib, TAS-121, panitumumab, nimotuzumab, catumaxomab, duligotuzumab patritumumab, abemaciclib, acalabrutinib, axitinib, baricitinib, binimetinib, bosutinib, brigatinib, cabozantinib, cobimetinib, dabrafenib, dasatinib, encorafenib, everolimus, fostamatinib, gilteritinib, ibrutinib, matinib, lapatinib, larotrectinib, lenvatinib, lorlatinib, midostaurin, neratinib, nilotinib, nintedanib, palbociclib, pazopanib, ponatinib, regorafenib, ribociclib, ruxolitinib, sirolimus, sorafenib, sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib, and vemurafenib.

In a non-limiting example, the HAPs contemplated herein, or a salt or solvate thereof, can be used concurrently or in combination with one or more agents known to be useful in treating or preventing cancer, such as the cancers described herein. Non-limiting examples of additional anti-proliferative agents contemplated include, but are not limited to, compounds listed on the cancer chemotherapy drug regimens in the 14^th Edition of the Merck Index (2006), which is hereby incorporated by reference, such as asparaginase, bleomycin, carboplatin, carmustine, chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, doxorubicin (adriamycine), epirubicin, etoposide, 5-fluorouracil, hexamethylmelamine, hydroxyurea, ifosfamide, irinotecan, leucovorin, lomustine, mechlorethamine, 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitoxantrone, prednisolone, prednisone, procarbazine, raloxifen, streptozocin, tamoxifen, thioguanine, topotecan, vinblastine, vincristine, and vindesine. Additional anti- proliferative agents include other molecular targeted agents that modulate parallel pathways such as MEK 1/2 inhibitors, AKT inhibitors and mTOR inhibitors, monoclonal antibodies (such as Cetuximab), oxaliplatin, gemcitabine, gefinitib, taxotere, ara A, ara C, herceptin, BCNU, CCNU, DTIC, and actinomycin D. Still further anti-proliferative agents include but are not limited to those compounds acknowledged to be used in the treatment of neoplastic diseases in Goodman and Gilman's The Pharmacological Basis of Therapeutics (Eleventh Edition), editor Molinoff et al., publ. by McGraw-Hill, pages 1225-1287 (2006), which is hereby incorporated by reference, such as aminoglutethimide, L-asparaginase, azathioprine, 5-azacytidine cladribine, busulfan, diethylstilbestrol, 2 ,2 - difluorodeoxycytidine, docetaxel, erythrohydroxynonyladenine, ethinyl estradiol, 5- fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, fludarabine phosphate, fluoxymesterone, flutamide, hydroxyprogesterone caproate, idarubicin, interferon, medroxyprogesterone acetate, megestrol acetate, melphalan, mitotane, paclitaxel, pentostatin, N-phosphonoacetyl-L-aspartate (PALA), plicamycin, semustine, tenipdside, testosterone propionate, thiotepa, trimethylmelamine, uridine, vinorelbine, taxanes, paclitaxel (Taxol), docetaxel, cabazitaxel, and enzalutamide. .

In certain embodiments, a HAP is disclosed herein is co-administered with at least one additional agent. In certain embodiments, a HAP is disclosed herein is is co-formulated with at least one additional agent.

In one embodiment, the formulations of the present application may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations. The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.

For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material that provides sustained release properties to the compounds. As such, the compounds for use in the methods disclosed herein may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.

In one embodiment, the compounds contemplated herein are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that may, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.

The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.

The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.

As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.

As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.

As discussed earlier, the disclosed methods are based at least in part on the surprising discovery by the inventors that in certain embodiments, the cell surface reductase STEAP4 plays a role in metabolising HAPs and therefore that individuals with elevated STEAP4 expression may be identified as those likely to response to treatment by a HAP. It is believed that this subgroup of individuals (/.e., individuals with elevated levels of STEAP4 expression as a subgroup of all individuals) may be more responsive to treatment by a HAP due to their elevated levels of STEAP4 expression. Accordingly, in some embodiments, the identification of individuals with elevated STEAP4 expression and subsequent treatment of those individuals may permit the HAP to be administered at a low dosage.

As used herein a "low dosage" may refer to a dosage that is lower than the dosage conventionally employed. Suitably, the HAP may be administered at a dosage that is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 35%, 50%, 60%, or more lower than the conventionally employed HAP dosage. A conventionally employed HAP dosage may be the dosage approved for administration by medical regulatory authorities in a particular jurisdiction, or may be the dosage employed in previous clinical studies, or elsewhere.

It is also believed that the identification of individuals with elevated STEAP4 expression levels will enable treatment by a HAP to be targeted to cancer cells and thus avoid or minimise delivery of the HAP at other sites in the body where the cytotoxic metabolite of the HAP may have undesirable effects. Accordingly, in some embodiments, the identification of individuals with elevated STEAP4 expression and subsequent treatment of those individuals may permit the HAP to be administered at a high dosage.

As used herein a "high dosage" may refer to a dosage that is higher than the dosage conventionally employed. Suitably, the HAP may be administered at a dosage that is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 35%, 50%, 60%, or more higher than the conventionally employed HAP dosage. A conventionally employed HAP dosage may be the dosage approved for administration by medical regulatory authorities in a particular jurisdiction, or may be the dosage employed in previous clinical studies, or elsewhere.

It is also believed that the identification of individuals with elevated STEAP4 expression levels will enable treatment of those individuals with fewer or reduced side effects. As used herein, "fewer or reduced side effects" (or similar) refers to a smaller number or a smaller magnitude of side effects compared to those experienced in a group of individuals receiving HAP treatment who have not been stratified to identify those with elevated levels of STEAP4 expression (e.g., a group comprising individuals with elevated levels of STEAP4 expression and individuals without elevated levels of STEAP4 expression), and/or compared to one or more individuals who does not exhibit elevated levels of STEAP4 expression. Suitably, the side effects that may be reduced in number or in magnitude include, but are not limited to, hearing loss, muscle cramping, diarrhoea, skin rash, nausea, and vomiting. Elevated STEAP4 expression levels

As used herein, the phrase "elevated STEAP4 expression levels" and similar may refer generally to an absolute level of STEAP4 expression which is considered to be elevated, or a level of STEAP4 expression which is elevated relative to (e.g., higher or greater than) a comparative level (e.g., a reference level or a level in a control sample). The term "expression" and similar terms as used herein refers to a transcription from a gene to give an RNA nucleic acid molecule at least complementary in part to a region of one or more of the two nucleic acid strands of the gene. The term "expressed" or "expression" as used herein also refers to the translation from the RNA molecules to give a protein, a polypeptide, or a portion thereof.

Suitably, the phrase "elevated STEAP4 expression levels" refers to an increase of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 100%, 150%, 200%, 300%, 500%, or more STEAP4 mRNA level relative to a comparative level (e.g., reference level or level in a control sample) of STEAP4 mRNA level or an increase of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 100%, 150%, 200%, 300%, 500%, or more STEAP4 protein level relative to a comparative level (e.g., reference level or level in a control sample) of STEAP4 protein.

In certain embodiments, the reference level of STEAP4 expression and/or the level of STEAP4 expression in a control sample is one that a treatment decision is made based on whether an individual has an expression level of STEAP4 (e.g., in their tumour cells) that is elevated as compared to the reference level of STEAP4 expression or as compared to the level of STEAP4 expression in a control sample. An individual who has a level of STEAP4 expression that is elevated as compared to the reference level of STEAP4 expression or as compared to a level of STEAP4 expression in a control sample has a different probability of responsiveness to the treatment than an individual who has a level of STEAP4 expression the same as or lower than the reference level of STEAP4 expression and/or the level of STEAP4 expression in a control sample.

In certain embodiments, the reference level of STEAP4 expression and/or the level of STEAP4 expression in a control sample is determined simultaneously with the determination of the level of STEAP4 expression in the tumour cells of the individual or in the sample of tumour cells from the individual. In certain embodiments, the reference level of STEAP4 expression level and/or the level of STEAP4 expression in a control sample is determined independently from the level of STEAP4 expression in the tumour cells of the individual or in the sample of tumour cells from the individual.

The reference level of STEAP4 expression may suitably be determined in a sample of non-tumour cells from the same individual, and/or in a sample of non-tumour cells from a different individual (including from a different individual who does not have cancer), and/or in a sample of non-tumour cells from a group of individuals (including from one or more individuals who do not have cancer).

In some embodiments, the control sample may suitably comprise a sample of non- tumour cells from the same individual, and/or a sample of non-tumour cells from a different individual (including from a different individual who does not have cancer), and/or a sample of non-tumour cells from a group of individuals (including from one or more individuals who do not have cancer).

In certain embodiments, the reference level of STEAP4 expression is predetermined. In certain embodiments, the level of STEAP4 expression in the control sample is predetermined.

Levels of STEAP4 expression are reported (see, for example, oncomine.orq) at a macrolevel, for instance across disease (cancer) types. See also, the database of The Cancer Genome Atlas (cancerqenome.nih.gov, and specifically at firebrowse.Org/viewGene.html7qene^~STEAP4# , last accessed October 2017). It is believed that the cause of elevated levels of STEAP4 expression may be due to amplification of the STEAP4 gene or region where the STEAP4 gene is located, mutation of the STEAP4 gene or region where the STEAP4 gene is located or elsewhere, deletion of a genetic region, or multiple genetic alterations. Amplification of the STEAP4 gene or region where the STEAP4 gene is located appears to be the most common reason for elevated levels of STEAP4 expression.

In some embodiments, the phrase "elevated STEAP4 expression levels" refers to a STEAP4 expression level which is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more, higher relative to the expession of a reference gene, such as a house-keeping gene. Suitably, the reference gene may comprise glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) and/or hypoxanthine phosphoribosyl transferase ( HPRT ).

In some embodiments, the phrase "elevated STEAP4 expression levels" refers to a rate of reduction of Fe³⁺ to Fe²⁺ that is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 100%, 150%, 200%, or more relative to a comparative level (e.g., reference level or level in a control sample). Suitably, the rate may be measured by measuring formation of the Fe²⁺-ferrozine complex.

In some embodiments, the phrase "elevated STEAP4 expression levels" refers to a rate of production of Compound B above 100 picomole/hr/10⁶ cells under anoxic conditions (< 1 ppm oxygen). More suitably, the phrase "elevated STEAP4 expression levels" refers to a rate of production of Compound B above 200 picomole/hr/10⁶ cells under anoxic conditions (<1 ppm oxygen). Measuring STEAP4 expression levels

As mentioned above, levels of STEAP4 expression are reported in the art at a macrolevel, and thus there exist methods known in the art for measuring STEAP4 expression levels. Suitable methods include, for example, those described in published PCT applications WO 01/40276 and WO 2017/055322, the contents of each of which is incorporated herein by reference.

STEAP4 expression levels may be determined by assessing the level of STEAP4 gene expression, by assessing the level of STEAP4 protein itself, or by assessing a biomarker in order to determine STEAP4 expression levels. In the methods of the present disclosure, any suitable method of detecting the level of STEAP4 expression levels can be used.

Assessing STEAP4 gene expression

STEAP4 expression levels may be determined by assessing the level of STEAP4 gene expression, which may include measuring STEAP4 mRNA levels.

Several methods of quantitating mRNA levels are known in the art. Exemplary methods include but are not limited to cDNA hybridisation, northern blots, ribonuclease protection assays, PCR-based methods, and the like. When the molecule to be detected is an mRNA molecule, the mRNA sequence of STEAP4, or a fragment thereof, can be used to prepare a probe that is at least partially complementary. The probe can then be used to detect the mRNA sequence in a sample, using any suitable assay, such as PCR-based methods, Northern blotting, a dipstick assay, and the like. The term "probe" as used herein, refers to a capture agent that is directed to a specific target mRNA biomarker sequence. Accordingly, each probe of a probe set has a respective target mRNA biomarker. A probe/target mRNA duplex is a structure formed by hybridising a probe to its target mRNA biomarker.

Suitable probes of the present disclosure, include the following sequences (see also Figure 12) :

GCAAAGCATCCAGTGGTCAA (SEQ ID NO: 6)

ATGACAGCAAAGCCAAGCAA (SEQ ID NO: 7)

GACTGGCTTGACCACTGGAT (SEQ ID NO: 8)

TTGCCTGGGTAACGGTTAAG (SEQ ID NO: 9)

GGCACAAAATACCGTCGATT (SEQ ID NO: 10)

TTTGGTTTACCTCCCTGGTG (SEQ ID NO: 11)

In other embodiments, a nucleic acid assay for testing for immunomodulatory activity in a biological sample can be prepared. An assay typically contains a solid support and at least one nucleic acid contacting the support, where the nucleic acid corresponds to at least a portion of the STEAP4 mRNA. The assay can also have a means for detecting the altered expression of the mRNA in the sample. The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to describe a polymer of any length comprised of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, or compounds produced synthetically, which can hybridise with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids. The term "nucleotide" (and also the term "nucleoside") is intended to include those moieties that contain not only the known purine and pyrimidine bases, but also other heterocylic bases that have been modified. Such modifications including methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles. In addition, the term "nucleotide" includes those moieties that contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalised as ethers, amines, or the like. The term "nucleotide" also intends to include analogues thereof, including molecules having structural features that are recognised in the literature as being mimetics, derivatives, having analogous structures, or other like terms, and include, for example, polynucleotides incorporating non-natural nucleotides, nucleotide mimetics such as 2'-modified nucleosides, peptide nucleic acids, oligomeric nucleoside phosphonates, and any polynucleotide that has added substituent groups, such as protecting groups or linking moieties.

The assay method can be varied depending on the type of mRNA information desired. Exemplary methods include but are not limited to Northern blots and PCR-based methods (e.g., qRT-PCR). Methods such as qRT-PCR can also accurately quantitate the amount of the mRNA in a sample.

Any suitable assay platform can be used to determine the presence of the mRNA in a sample. For example, an assay may be in the form of a dipstick, a membrane, a chip, a disk, a test strip, a filter, a microsphere, a slide, a multiwell plate, or an optical fiber. An assay system may have a solid support on which a nucleic acid corresponding to the mRNA is attached. The solid support may comprise, for example, a plastic, silicon, a metal, a resin, glass, a membrane, a particle, a precipitate, a gel, a polymer, a sheet, a sphere, a polysaccharide, a capillary, a film a plate, or a slide. The assay components can be prepared and packaged together as a kit for detecting an mRNA.

The nucleic acid can be labeled, if desired, to make a population of labelled mRNAs. In general, a sample can be labeled using methods that are well known in the art (e.g., using DNA ligase, terminal transferase, or by labeling the RNA backbone, etc.; see, e.g., Ausubel, et al., 1995 and Sambrook et al., 2001). In some embodiments, the sample is labeled with fluorescent label. Exemplary fluorescent dyes include but are not limited to xanthene dyes, fluorescein dyes, rhodamine dyes, fluorescein isothiocyanate (FITC), 6 carboxyfluorescein (FAM), 6 carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 6 carboxy 4’, 5’ dichloro 2’, T dimethoxyfluorescein (JOE or J), N.N.N’.Nl tetramethyl 6 carboxyrhodamine (TAMRA or T), 6 carboxy X rhodamine (ROX or R), 5 carboxyrhodamine 6G (R6G5 or G5), 6 carboxyrhodamine 6G (R6G6 or G6), and rhodamine 110; cyanine dyes, e.g., Cy3, Cy5 and Cy7 dyes; Alexa dyes, e.g., Alexa-fluor-555; coumarin, Diethylaminocoumarin, umbelliferone; benzimide dyes, e.g., Hoechst 33258; phenanthridine dyes, e.g., Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, BODIPY dyes, quinoline dyes, Pyrene, Fluorescein Chlorotriazinyl, Rl 10, Eosin, JOE, R6G, Tetramethylrhodamine, Lissamine, ROX, Napthofluorescein, and the like.

In some embodiments, the mRNA sequences comprise at least one mRNA selected from the group consisting of STEAP4 mRNA, or a fragment thereof (including, but not limited to, any of the STEAP4 mRNA probes described above and in any one or more of SEQ ID NOs:6-l l). The nucleic acids may be present in specific, addressable locations on a solid support.

A typical mRNA assay method can contain the steps of 1) obtaining surface-bound subject probes; 2) hybridisation of a population of mRNAs to the surface-bound probes under conditions sufficient to provide for specific binding (3) post-hybridisation washes to remove nucleic acids not bound in the hybridisation; and (4) detection of the hybridised mRNAs. The reagents used in each of these steps and their conditions for use may vary depending on the particular application.

Hybridisation can be carried out under suitable hybridisation conditions, which may vary in stringency as desired. Typical conditions are sufficient to produce probe/target complexes on a solid surface between complementary binding members, i.e., between surface-bound subject probes and complementary mRNAs in a sample. The term "complementary" as used herein refers to specific binding between polynucleotides based on the sequences of the polynucleotides. Portions of polynucleotides are complementary to each other if they follow conventional base-pairing rules, e.g., A pairs with T (or U) and G pairs with C, although small regions (e.g., less than about 3 bases) of mismatch, insertion, or deleted sequence may be present. In certain embodiments, stringent hybridisation conditions may be employed, and thus a first polynucleotide and a second polynucleotide may be considered complementary in some embodiments if they bind to each other in a hybridisation assay under stringent conditions, e.g., if they produce a given or detectable level of signal in a hybridisation assay. The term "stringent assay conditions" refers to conditions that are compatible to produce binding pairs of nucleic acids, e.g., probes and target mRNAs, of sufficient complementarity to provide for the desired level of specificity in the assay while being generally incompatible to the formation of binding pairs between binding members of insufficient complementarity to provide for the desired specificity. The term stringent assay conditions generally refers to the combination of hybridisation and wash conditions.

Hybridisation is typically performed under stringent hybridisation conditions. Standard hybridisation techniques (e.g., under conditions sufficient to provide for specific binding of target mRNAs in the sample to the probes) are described in Kallioniemi et al, (1992) and published PCT application WO 93/18186. Several guides to general techniques are available, e.g., Tijssen (1993). For descriptions of techniques suitable for in situ hybridisations, see Gall et al. (1971) and Angerer et al. (1985). Selection of appropriate conditions, including temperature, salt concentration, polynucleotide concentration, hybridisation time, stringency of washing conditions, and the like will depend on experimental design, including source of sample, identity of capture agents, degree of complementarity expected, etc., and may be determined as a matter of routine experimentation for those of ordinary skill in the art. The term "capture agent", as used herein, refers to an agent that binds an mRNA or protein through an interaction that is sufficient to permit the agent to bind and concentrate the mRNA or protein from a homogeneous mixture.

Those of ordinary skill will readily recognise that alternative but comparable hybridisation and wash conditions can be utilised to provide conditions of similar stringency.

After the mRNA hybridisation procedure, the surface bound polynucleotides are typically washed to remove unbound nucleic acids. Washing may be performed using any convenient washing protocol, where the washing conditions are typically stringent, as described above. The hybridisation of the target mRNAs to the probes is then detected using standard techniques.

An exemplary method of determining STEAP4 mRNA abundance is by using Affymetrix GeneChip PrimeView arrays.

Other methods, such as PCR-based methods, can also be used to detect STEAP4 expression levels. Examples of PCR methods can be found in the literature. Examples of PCR assays can be found in US patent 6,927,024, which is incorporated by reference herein in its entirety. Examples of RT-PCR methods can be found in US patent 7,122,799, which is incorporated by reference herein in its entirety. A method of fluorescent in situ PCR is described in US patent 7,186,507, which is incorporated by reference herein in its entirety.

In some embodiments, Real-Time Reverse Transcription-PCR (qRT-PCR) can be used for both the detection and quantification of RNA targets (Bustin, et al, 2005, Clin. ScL, 109:365-379). Quantitative results obtained by qRT-PCR are generally more informative than qualitative data. Thus, in some embodiments, qRT-PCR-based assays can be useful to measure mRNA levels during cell-based assays. The qRT-PCR method is also useful to monitor patient therapy. Examples of qRT-PCR-based methods can be found, for example, in US patent 7,101,663, which is incorporated by reference herein in its entirety.

In contrast to regular reverse transcriptase-PCR and analysis by agarose gels, real- time PCR gives quantitative results. An additional advantage of real-time PCR is the relative ease and convenience of use. Instruments for real-time PCR, such as the Applied Biosystems 7500, are available commercially, as are the reagents, such as TaqMan Sequence Detection chemistry. For example, TaqMan® Gene Expression Assays can be used, following the manufacturer's instructions. These kits are p re -formulated gene expression assays for rapid, reliable detection and quantification of human, mouse and rat mRNA transcripts. An exemplary PCR program, for example, is 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, then 60°C for 1 minute.

To determine the cycle number at which the fluorescence signal associated with a particular amplicon accumulation crosses the threshold (referred to as the CT), the data can be analysed, for example, using a 7500 Real-Time PCR System Sequence Detection software vl .3 using the comparative CT relative quantification calculation method. Using this method, the output is expressed as a fold-change of expression levels. In some embodiments, the threshold level can be selected to be automatically determined by the software. In some embodiments, the threshold level is set to be above the baseline but sufficiently low to be within the exponential growth region of an amplification curve.

In some embodiments, mRNA may be detected by real-time quantitative PCR (qPCR) using suitable primers and methods of detection relative to house keeping genes. Analysis of the crossing threshold point (CT) for the amplification curves for each sample is determined and absolute quantitation is performed with an internal standard curve with results expressed as the ratio of STEAP4/reference gene copies, following normalisation against calibrator RNA.

In some embodiments where a PCR detection method is used, a pair of the STEAP4 mRNA probes described above may comprise the PCR primers (in 5' - 3' direction), for instance: SEQ ID NO:6 may comprise the forward primer and SEQ ID NO:7 may comprise the reverse primer; SEQ ID NO:8 may comprise the forward primer and SEQ ID NO:9 may comprise the reverse primer; SEQ ID NO: 10 may comprise the forward primer and SEQ ID NO:9 may comprise the reverse primer; or SEQ ID NO: 11 may comprise the forward primer and SEQ ID NO:9 may comprise the reverse primer.

In some embodiments, single cell capture technologies may be employed and for instance may be followed by droplet PCR. In some embodiments, single cell qPCR (for instance to measure STEAP4 levels in circulating tumour cells), may be employed. Assessing STEAP4 protein levels

STEAP4 expression levels may be determined by assessing the level of STEAP4 protein itself. Where the level of STEAP4 itself is measured, the protein level can be measured using any method of protein quantification that is well-known in the art, including but not limited to an antibody-based method. Exemplary methods that can be used include but are not limited to immunoblotting (western blot), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), flow cytometry, cytometric bead array, mass spectroscopy, including mass spectrometry detection of proteotypic peptide fragments from enzymatic digests of STEAP4 (Picotti et al), and the like. Several types of ELISA are commonly used, including direct ELISA, indirect ELISA, and sandwich ELISA.

Assessing biomarkers to determine STEAP4 expression levels

STEAP4 expression levels may be determined by assessing a biomarker in order to determine STEAP4 expression levels.

One suitable biomarker is a metabolite of a compound that is metabolised by STEAP4, for instance a metabolite of a HAP. Such a biomarker can be used to inform on STEAP4 expression levels. Suitably, the method may comprise measuring the rate of metabolism of Compound A to Compound B under anoxic conditions. The rate of production of Compound B can be used to inform of STEAP4 expression levels. Suitably, a rate of anoxic Compound B production of greater than 100, 200, 500, 1000, 1200, 1400, or 1800 pmol/hr/10⁶ cells indicates that the cells have elevated STEAP4 expression levels.

Kits

The present disclosure also provides kits for performing the disclosed methods.

Accordingly, in one embodiment the present disclosure provides a kit useful for predicting whether an individual with cancer is likely to be responsive to treatment with a HAP.

In some embodiments, a kit for detecting the STEAP4 mRNA biomarkers can be prepared. The kits can include, for example, a probe or probe set comprising oligonucleotides that can bind to the mRNA biomarker(s) of interest for a given disease, compound, or other parameter. Suitably, the probe may be complementary to at least 20, 50, 100, 200, 350, or more basis of STEAP4 mRNA. In some embodiments, the probe may comprise one or more nucleotide sequences selected from the group consisting of SEQ ID NOs:6-l l. Suitably, the probe may comprise a pair of probes for use as PCR primers, selected from the group consisting of (in 5' - 3' direction) : SEQ ID NO:6 as the forward primer and SEQ ID NO:7 as the reverse primer; SEQ ID NO:8 as the forward primer and SEQ ID NO:9 as the reverse primer; SEQ ID NO: 10 as the forward primer and SEQ ID NO:9 as the reverse primer; and SEQ ID NO: 11 as the forward primer and SEQ ID NO:9 as the reverse primer. Washing solutions, reagents for performing a hybridisation assay, mRNA isolation or purification means, detection means, as well as positive and negative controls can also be included. The kit can also include instructions for using the components of the kit. The kit can be tailored for in-home use, clinical use, or research use.

In some embodiments, a kit for detecting STEAP4 protein levels can be prepared. The kits can include, for example, a dipstick coated with an antibody that recognises the protein, washing solutions, reagents for performing the assay, protein isolation or purification means, detection means, as well as positive and negative controls. The kit can also include instructions for using the components of the kit. The kit can be tailored for in- home use, clinical use, or research use.

In some other embodiments, the kit comprises a solid support, and a means for detecting the protein expression of STEAP4 mRNA in a sample (e.g., a biological sample). Such a kit can employ, for instance, a dipstick a membrane, a chip, a disk, a test strip, a filter, a microsphere, a slide, a multiwell plate, or an optical filter.

The kits described here may be employed in the methods described herein.

The kits described here may be employed in the methods of the present disclosure. These Examples are provided for the purpose of illustration only and the disclosure should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

EXAMPLES

Example 1: Synthesis of NMQ prodrugs

The synthesis of NMQ prodrugs is described in PCT publications WO 2010/104406 and WO 2011/028135.

Example 2: Synthesis of nitrophenyl mustards

The synthesis of nitrophenyl mustards is described in PCT publications WO 2005/042471 and WO 2014/031012. Example 3: Effect of siRNA knockdown of five candidate Compound A reductases: STEAP4, SDHA, DCXR, COQ6 and FOXRED1 on anoxic Compound A metabolism in HCC1954 and SiHa cells

Method

Cells were cultured in sterile tissue culture flasks prior to the assay being conducted. SiHa cells were maintained in a-MEM + 5% FCS; HCC1954 cells were maintained in DMEM + 10% FCS.

The five siRNA's against STEAP4, SDHA, DCXR, COQ6 and FOXRED1 were purchased from Dharmacon (ON-TARGETplus siRNA SMARTpools). Triplicate wells of a 6-well plate were assigned for each siRNA complex. The siGLO Green Transfection Indicator was used as a positive control that enabled visualisation of delivery of a non-toxic siRNA complex to cells. Each siRNA complex was prepared in 250 pL of Opti-MEM medium containing 9 pl_ of Lipofectamine® RNAiMAX Transfection reagent per well. The amount of each siRNA added to the medium containing the transfection reagent was sufficient to achieve a final concentration of siRNA in each well of 100 nM. The siRNA and transfection reagent were incubated together at room temperature for 5 min. The transfection was performed as a reverse transfection where the siRNA complexes were added first to the wells followed by addition of the cells. Cells suspensions were prepared to obtain a density of 5 x 10⁵ cells per well in 2 mL of medium (a-MEM + 5% FCS for SiHa, DMEM + 10% FCS for HCC1954) in each well. The medium was renewed and replaced with 2 mL of the respective medium in each well 24 h following the transfection. Compound Ametabolism was assessed 24 h following medium renewal (48 h after siRNA transfection).

For the Compound A metabolism assay, cell numbers were calculated to ensure a seeding density of 5x10^s cells per well of a 24-well plate (385 mI per well, with all conditions done in triplicate). Cells were seeded under anoxic conditions using a 5% H2/palladium catalyst scrubbed Bactron anaerobic chamber (Sheldon Manufacturing, Cornelius, Oregon) to achieve severe anoxia (< 10 ppm 0₂ gas phase) during prodrug exposure. After 1 h incubation to enable cell attachment to the plates and equilibrate to anoxia, 15 mI of media containing Compound A was added to each well to give a final prodrug concentration of 10 mM. Cells were incubated in Compound A for 90 minutes, after which plates were removed from the anoxic chamber. Plates were placed on ice and crashed with 800 pL ice cold acetonitrile spiked with 0.5 mM D6 internal standard for Compound B. A standard curve of Compound B ranging from 0.003 mM - 10 mM was also prepared in media and spiked with the same acetonitrile/internal standard mix as the samples. All samples/standards were stored at -80 °C until analysis by LC/MS/MS. Results and Discussion

The five candidate reductase genes whose transcript abundance had the highest correlation to Compound A metabolism were STEAP4, SDHA, DCXR, COQ6 and FOXRED1. siRNA SMARTpools targeting these genes were purchased for transfection into cells which had both high transcript abundance and high rates of Compound A metabolism. The siGLO Green Transfection Indicator (Dharmacon Inc.) was used as a positive control for cell uptake of siRNA complexes which results in the nuclear localisation of a green fluorescent oligonucleotide duplex. HCC1954 and SiHa cells were selected for transfection of siRNA SMARTpools against the five candidate reductases as both cell lines showed efficient uptake of the siGLO transfection indicator 24 h following transfection. HCC1954 and SiHa cells transfected with the five indicated siRNA SMARTpools and siGLO were assessed for anoxic Compound A metabolism using non-transfected cells as a negative control.

The results are shown in Figure 6. In HCC1954 cells, transfection with the siRNA SMARTpool against STEAP4 (siSTEAP4) resulted in an approximately 70% decrease in rate of Compound B Compound Bformation relative to non-transfected cells (P < 0.001) while transfection of SiHa cells with siSTEAP4 resulted in an approximately 73% decrease in rate of Compound B Compound Bformation relative to non-transfected cells (P < 0.001). Transfection with siRNA SMARTpools against either SDHA, DCXR, COQ6 or FOXRED1 did not lead to a change in anoxic Compound A metabolism.

These findings suggest that STEAP4 is a one-electron reductase of TH-4000.

Example 4: Effect of STEAP4 over-expression on Compound A metabolism in C33A and HI 299 cells

Method

The open reading frame (ORF) for human STEAP4 transcript variant 1 (NM_024636.2) was cloned into the F279-V5 expression vector. This provides transcription of a bicistronic mRNA from the human immediate-early cytomegalovirus (CMV) promoter, which encodes the open reading frames for the gene of interest and the pac (puromycin resistance) gene.

Human cervical carcinoma (C33A) and non-small cell lung cancer (H1299) cells were transfected using Lipofectamine 3000 reagent complexed with F279-STEAP4-V5 plasmid DNA. The transfection mix was added drop wise and after 48 hours the culture media was replaced with fresh a-MEM (with 5% FBS) medium containing 1 mM puromycin, escalating to 3 mM puromycin over time. Cells were grown to confluence in the wells before being pooled together and transferred to a cell culture flask of desired volume. Cells were maintained in a-MEM supplemented with 3 pM puromycin. Successful plasmid-based expression of STEAP4 was confirmed by preparing cell lysates that were run on western blot with detection of STEAP4 protein by rabbit anti-human STEAP4 antibody (Proteintech; Cat No. 11944-1-AP). Cell line pairs were designated : C33A^WT/C33A^STEAP4 and ^1299^/^1299^ .

On the day of the experiment, cells were harvested with Trypsin/EDTA and counted. A seeding density of 5xl0⁵ cells per well of a 24-well plate (385 pi per well, with all conditions done in triplicate). Cells were seeded under aerobic (oxic) or anoxic conditions, the latter using a 5% H2/palladium catalyst scrubbed Bactron anaerobic chamber (Sheldon Manufacturing, Cornelius, Oregon) to achieve severe anoxia (< 1 ppm 0₂ gas phase) during prodrug exposure. After 1 h incubation to enable cell attachment to the plates (and equilibrate to anoxia in the case of the anoxic samples), 15 pi of media containing Compound A was added to each well to give a final concentration of 10 mM. Cells were incubated in the prodrug for 90 minutes, after which plates were removed from the anoxic chamber. Plates were placed on ice and crashed with 800 mI_ ice cold acetonitrile spiked with 0.5 mM D6 internal standard for Compound B. A standard curve of Compound B ranging from 0.003 mM - 10 mM was also prepared in media and spiked with the same acetonitrile/internal standard mix as the samples. All samples/standards were stored at -80 °C until analysis by LC/MS/MS.

Results and Discussion

The open reading frame for STEAP4 (NCBI Reference Sequence: NM_024636.2) was introduced to the F279-V5 vector and the resultant expression plasmid was used for transfection of H1299 and C33A cells. Expression of V5 tagged STEAP4 protein in suppressor supernatant-induced H 1299^STEAP4 and C33A^STEAP4 cells was detected by Western blot using an anti-V5 tag antibody, whilst V5 tagged protein was not detected in the non- transfected parental cell lines. The expression of the V5 tag in the STEAP4-transfected cell lines strongly supports the conclusion that complete in-frame translation of the STEAP4 mRNA to the corresponding full-length protein has resulted in a predictable manner.

When screened for Compound A metabolism in both isogenic pairs of cell lines, STEAP4-transfected cells showed a significant increase in rates of anoxic Compound A metabolism relative to parental cells. The results are shown in Figure 7. C33A^STEAP4 cells had a 52-fold increase in rate of anoxic Compound B Compound Bformation relative to C33A^WT cells (P < 0.001) while H1299^STEAP4 cells had an 18-fold increase in rate of anoxic Compound B Compound Bformation relative to H1299^WT cells (P < 0.001). Direct confirmation of STEAP4 expression notwithstanding, the increase in anoxic Compound A metabolism in the STEAP4-transfected cell lines is wholly consistent with the conclusion that STEAP4 functions as a bona fide one-electron reductase of Compound A. As expected, no activity was detected under oxygen-rich conditions. Example 5: Evaluating the cytotoxicity of three HAPs in parental (WT) neoplastic cell lines and paired lines engineered to overexpress human STEAP4 cDNA.

The following HAPs evaluated in this example: TH-302, Tirapazamine (TPZ) and Compound C, the structure for each prodrug is shown in Figure 8.

Method:

Cloning of STEAP4

The open reading frame (ORF) for human STEAP4 transcript variant 1 (NM_024636.2) was cloned into the F279-V5 expression vector. This provides transcription of a bicistronic mRNA from the human immediate-early cytomegalovirus (CMV) promoter, which encodes the open reading frames for the gene of interest and the pac (puromycin resistance) gene; the former harbouring an occult C-terminal V5-Tag inducible with TAG- On-Demand™ technology.

Human cervical carcinoma (C33A) and non-small cell lung cancer (H1299) cells were transfected using Lipofectamine 3000 reagent complexed with F279-STEAP4-V5 plasmid DNA. The transfection mix was added drop wise and after 48 hours the culture media was replaced with fresh a-MEM (with 5% FBS) medium containing 1 mM puromycin, escalating to 3 mM puromycin over time. Cells were grown to confluence in the wells before being pooled together and transferred to a cell culture flask of desired volume. Cells were maintained in a-MEM supplemented with 3 pM puromycin. Cell line pairs were designated: C33A^WT/C33A^STEAP4 and H1299^WT/H1299^STEAP4.

In vitro cytotoxicity assays to determine the sensitivity of WT and STEAP4 expressing cells to HAPs

Cell lines were maintained in a-MEM 5% FCS in sterile tissue culture flasks prior to the experiment. Cells engineered to express STEAP4 were routinely cultured in media supplemented with 1 pM puromycin to maintain a selection pressure on the transfected cells, although puromycin was omitted from the media during experiments. On the day of the experiment, cells were harvested with Trypsin/EDTA and counted. The required number of cells was transferred to a 50 ml falcon tube and centrifuged to pellet cells (1000 rpm, 5 min). Excess media was removed by aspiration leaving cell pellets sitting in 50-100 pi media. The cell number was calculated to ensure a seeding density of 400 cells per well of a 96-well plate, with cells resuspended in a-MEM + 10% FCS, -P/S + lOmM D-Glucose + 0.2mM 2'-deoxycytidine. Cells were seeded under aerobic or anoxic conditions, the latter using a 5% H2/palladium catalyst scrubbed Bactron anaerobic chamber (Sheldon Manufacturing, Cornelius, Oregon) to achieve severe anoxia (<10 ppm 0₂ gas phase) during prodrug exposure. The layout of the plate is described in Figure 9, where column 1 (the left-most column) consists of 8 media only control wells. Column 2 (second from the left) consists of 8 cells only (no prodrug) control wells. The remaining columns (3-12) consist of cells exposed to a range of prodrug concentrations. A high concentration of prodrug was added to column 12 (the right-most column) in each row, and 3-fold dilutions were performed across the plate using a multi-channel pipette. Thus, a total of four prodrugs could be evaluated on a single plate, with duplicate wells per prodrug. In this example, the three prodrugs were evaluated with the range of concentrations shown in the following table:

Table 1 : Concentration ranges for prodrugs in the 96-well plate

After 2 hours incubation to enable cell attachment to the plates (and equilibrate to anoxia in the case of the anoxic samples), cells were exposed to a range of prodrug concentrations for 4 hours under aerobic or anoxic conditions. After prodrug exposure, plates were transferred to aerobic conditions. A vacuum immunowash was used to aspirate the media and wells were washed three times with 150 pi of prodrug-free aMEM 5% FCS + P/S. The plates were then incubated under aerobic conditions for 5 days for cell growth. Cells were fixed by adding 50mI of cold 40% trichloroacetic acid (TCA) to each well by layering on top without mixing, this gave a final concentration of 10% TCA. Plates were incubated in a fridge for 1 hour before the TCA was washed off and the plates rinsed in running tap water. 50mI of 0.4% SRB in 1% acetic acid was added to each well and plates were incubated for 30 minutes in the dark. Excess stain was flicked off and plates were washed four times in 2L of tap water containing 1% acetic acid. Excess fluid was flicked from the plates and the remaining SRB stain solubilized by adding 100mI of lOmM unbuffered Tris to each well. Plates were then left on a shaker for at least 2 hours in the dark before analysis using a plate reader (set at an optical density of 490 nM for measurement filter and 450nM for reference filter). The IC50 value is the concentration of prodrug required to inhibit cell growth by 50%.

The results from the IC50 assay showed that overexpression of STEAP4 increased the sensitivity of C33A and H1299 cells to Compound C, but not to TH-302 or TPZ. This is predicted to result from the extra-cellular site of metabolism (see, e.g., Figure 30). The active (cytotoxic) metabolite of Compound C is able to cross the cell membrane and exert its biological effect (DNA cross-linking). In contrast, evofosfamide (TH-302) releases a negatively charged Br-IPM active metabolite that is unable to traverse the cell membrane and exert its biological effect (DNA cross-linking). Further, the active metabolite of tirapazamine (TPZ) is a short lived free-radical species that is unable to penetrate the cell.

The results are shown graphically in Figure 10. The hypoxic cytoxicity ratio (aerobic IC50 value divided by anoxic IC50 value), is indicated above the anoxic bar for each cell group.

Consequently STEAP4 metabolism only results in PR-104A sensitivity. The prodrugs TH-302 and TPZ are consumed BUT this does not result in cell kill. Therefore STEAP4 functions as a detoxifying pathway due to futile, unproductive metabolism.

Example 5A: Evaluating the cytotoxicity of three HAPs in parental (WT) neoplastic cell lines and paired lines engineered to overexpress human STEAP4 cDNA

The following HAPs evaluated in this example: Compound A (Tarloxotinib), Compound C (PR-104A), Compound G (SN29176), Compound H (SN27686), the structure for each prodrug is shown in Figure 8.

Method:

Cloning of STEAP4

Human cervical carcinoma (C33A) cells were transfected using Lipofectamine 3000 reagent complexed with F279-STEAP4-V5 plasmid DNA. The transfection mix was added drop wise and after 48 hours the culture media was replaced with fresh a-MEM (with 5% FBS) medium containing 1 mM puromycin, escalating to 3 mM puromycin over time. Cells were grown to confluence in the wells before being pooled together and transferred to a cell culture flask of desired volume. Cells were maintained in a-MEM supplemented with 3 pM puromycin. Cell lines were designated C33A^WT/C33A^STEAP4. In vitro cytotoxicity assays to determine the sensitivity of WT and STEAP4 expressing cells to HAPs

Cell lines were maintained in a-MEM 5% FCS in sterile tissue culture flasks prior to the experiment. Cells engineered to express STEAP4 were routinely cultured in media supplemented with 1 mM puromycin to maintain a selection pressure on the transfected cells, although puromycin was omitted from the media during experiments. On the day of the experiment, cells were harvested with Trypsin/EDTA and counted. The required number of cells was transferred to a 50 ml falcon tube and centrifuged to pellet cells (1000 rpm, 5 min). Excess media was removed by aspiration leaving cell pellets sitting in 50-100 pi media. The cell number was calculated to ensure a seeding density of 400 cells per well of a 96-well plate, with cells resuspended in a-MEM + 10% FCS, -P/S + lOmM D-Glucose + 0.2mM 2'-deoxycytidine. Cells were seeded under aerobic or anoxic conditions, the latter using a 5% H2/palladium catalyst scrubbed Bactron anaerobic chamber (Sheldon Manufacturing, Cornelius, Oregon) to achieve severe anoxia (<10 ppm 0₂ gas phase) during prodrug exposure.

The layout of the plate is described in Figure 9, where column 1 (the left-most column) consists of 8 media only control wells. Column 2 (second from the left) consists of 8 cells only (no prodrug) control wells. The remaining columns (3-12) consist of cells exposed to a range of prodrug concentrations. A high concentration of prodrug was added to column 12 (the right-most column) in each row, and 3-fold dilutions were performed across the plate using a multi-channel pipette. Thus, a total of four prodrugs could be evaluated on a single plate, with duplicate wells per prodrug. In this example, the three prodrugs were evaluated with the range of concentrations shown in the following table:

Table 1A: Concentration ranges for prodrugs in the 96-well plate

After 2 hours incubation to enable cell attachment to the plates (and equilibrate to anoxia in the case of the anoxic samples), cells were exposed to a range of prodrug concentrations for 4 hours under aerobic or anoxic conditions. After prodrug exposure, plates were transferred to aerobic conditions. A vacuum immunowash was used to aspirate the media and wells were washed three times with 150 mI of prodrug-free aMEM 5% FCS + P/S. The plates were then incubated under aerobic conditions for 5 days for cell growth. Cells were fixed by adding 50mI of cold 40% trichloroacetic acid (TCA) to each well by layering on top without mixing, this gave a final concentration of 10% TCA. Plates were incubated in a fridge for 1 hour before the TCA was washed off and the plates rinsed in running tap water.

50mI of 0.4% SRB in 1% acetic acid was added to each well and plates were incubated for 30 minutes in the dark. Excess stain was flicked off and plates were washed four times in 2L of tap water containing 1% acetic acid. Excess fluid was flicked from the plates and the remaining SRB stain solubilized by adding 100mI of lOmM unbuffered Tris to each well. Plates were then left on a shaker for at least 2 hours in the dark before analysis using a plate reader (set at an optical density of 490 nM for measurement filter and 450nM for reference filter). The IC50 value is the concentration of prodrug required to inhibit cell growth by 50%.

The results from the IC50 assay showed that overexpression of STEAP4 increased the sensitivity of C33A cells to Compound A, Compound C, Compound G, and Compound H. The results are shown in Table 2A.

Table 2A: IC50 values for Compound A, Compound C, Compound G (SN29176) and Compound H (SN27686) in wild-type (WT) and STEAP4-expressing C33A cells, where the concentration of prodrug required to inhibit cell growth by 50% (IC50) is shown, as described in Example 5.

Example 6: Effect of Cytochrome P450 oxidoreductase (POR) over-expression on metabolism of Compound A and Compound C

Method:

The open reading frame (ORF) for POR was purchased from Invitrogen's Ultimate ORFTM collection (Clone ID IOH21456) and cloned into the F527-V5 expression vector. Human cutaneous squamous cell carcinoma cell line (A431) was transfected using Lipofectamine 3000 reagent complexed with F279-POR-V5 plasmid DNA to generate the stable transfected A431^POR cell line. The A431^POR cell line was maintained under puromycin selection using a-MEM + 5% FCS supplemented with 3 mM puromycin.

A431^WT and A431^POR cells were cultured in sterile tissue culture flasks using a-MEM + 5% FCS.

On the day of the experiment, cells were harvested with Trypsin/EDTA and counted. A seeding density of 5x10^s cells per well of a 24-well plate (385 pi per well, with all conditions done in triplicate). Cells were seeded under aerobic or anoxic conditions, the latter using a 5% H2/palladium catalyst scrubbed Bactron anaerobic chamber (Sheldon Manufacturing, Cornelius, Oregon) to achieve severe anoxia (<1 ppm 0₂ gas phase) during prodrug exposure. After 1 h incubation to enable cell attachment to the plates (and equilibrate to anoxia in the case of the anoxic samples), 15 mI of media containing either Compound A (SN33999) or Compound C (SN27858) was added to each well to give a final prodrug concentration of 10 pmol/L. Cells were incubated in prodrug for 90 minutes, after which plates were removed from the anoxic chamber. Plates were placed on ice and crashed with 800 mI_ ice cold acetonitrile spiked with either 0.5 mM D6 internal standard for Compound B or 0.5 mM D4 internal standard for Compound D (Compound D is also known as PR-104H or PR104-H and is the active metabolite (hydroxylamine) of Compound C). Standard curves of Compound B and Compound D ranging from 0.003 - 10 pmol/L were also prepared in media and spiked with the respective acetonitrile/internal standard solutions as per the samples. All samples/standards were stored at -80 °C until analysis by LC/MS/MS.

Results:

The results are shown graphically in Figure 11.

A431 cells were engineered to overexpress human cytochrome P450 reductase (POR) as detected by western blotting. When anoxic metabolism of Compound C was assessed in parental A431^WT cells and A431^POR cells, there was a significant difference in rates of Compound C formed between the two cell lines (t-test, P < 0.001). Comparison of Compound C metabolism in the two cell lines showed a 460% increase (P < 0.001) in the rate of anoxic Compound C metabolite (Compound D) formation in A431^POR cells relative to A431^WT cells. This demonstrates intracellular POR is active in the A431^POR cells. No significant aerobic conversion of Compound C to Compound D was detected for either cell line. These results indicate that Compound C is able to freely diffuse into A431 cells and be converted to Compound D by POR. The oxygen-sensitive nature of the metabolism is consistent with one-electron reduction. This confirms the A431^POR cells have elevated intracellular one-electron reductase activity relative to parental A431^WT cells and POR is able to bio-activate Compound C.

In contrast, when anoxic Compound A metabolism was assessed in parental A431^WT cells and A431^POR cells, there was no significant difference in rates of Compound B formed between the two cell lines (t-test, P > 0.1). Similarly, no effect was seen under aerobic conditions. This indicates Compound A is unable to be activated by POR, an enzyme well known to activate a wide variety of HAPs. Therefore, unexpectedly, POR was found not to be a predominant one-electron reductase involved in catalysing the reduction (bioactivation) of TH-4000.

Notably, recombinant POR is readily able to convert Compound A to Compound B in cell-free systems indicating the presence of a plasma membrane barrier prevents Compound A from interacting with intracellular POR expressed in the A431^POR cell line. Compound A metabolism by recombinant cytochrome P450 reductase (POR; 0.1 mg/ml_) was measured at 1 hour using 10 mM Compound A in the presence of NAPDH cofactor (100 pmol/L). Heat-inactivation was achieved by incubating POR at 70°C for 10 minutes and demonstrates active POR enzyme is required. Thus, the oxidoreductase POR serves as an example (sentinel) for all intracellular reductase activity and indicates Compound A is metabolised by enzymes other than those inside of cells. This observation is consistent with the requirement for the cell surface reductase STEAP4 to perform the activity of one- electron addition to Compound A in the extracellular space.

Example 7: Measuring STEAP4 expression levels / function

1. SYBR Green-based qPCR for STEAP4 gene expression

a. Validation of STEAP4 primers by RT-PCR

The following primers were selected:

STEAP4 forward primer sequence (5' - 3') : GACTGGCTTGACCACTGGAT

STEAP4 reverse primer sequence (5' - 3') : TTGCCTGGGTAACGGTTAAG cDNA was prepared with 1 pg of RNA using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer's recommended protocol. The resulting cDNA was diluted 1 :20 in ultrapure water prior to RT-PCR or qPCR.

RT-PCR was then performed on the prepared cDNA of representative human cancer cell lines (PC9 parental, 5 PC9 clones, and H2126), and the expected product size of 148 bp was detected by electrophoresis on a 2% agarose gel.

These results are shown in Figure 13, where lane 1 is the lkb+ ladder, lane 2 shows the results of PC9 (parental), lanes 3-7 show the results of the PC9 clones, and lane 8 shows the results of the H2126 cell line. This confirmed that the primers were suitable for use as STEAP4 primers in PCR methods. b. qPCR detection of STEAP4

qPCR reactions were then carried out using SYBR Green-based detection to determine both STEAP4 expression and expression of the reference (house-keeping) gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

The primers for STEAP4 were as above in a.

The primers for GAPDH were as follows (5' - 3'):

Forward - ACGGGAAGCTTGTCATCAAT (SEQ ID NO: 12)

Reverse - TGGACTCCACGACGTACTCA (SEQ ID NO: 13)

Each reaction consisted of the following components with triplicate wells for each sample/primer combination :

(i) 5 pL of SYBR® Green PCR Master Mix (Applied Biosystems)

(ii) 0.25 pl_ of 8 mM forward primer (STEAP4 or GAPDH)

(iii) 0.25 mI_ of 8 mM reverse primer (STEAP4 or GAPDH)

(iv) 4.5 mI_ of cDNA (diluted 1 :20 as described above)

The results are shown in Figure 14 where the STEAP4 expression was normalised to expression of the reference gene GAPDH, across the panel of human cancer cell lines.

2. Western blotting for STEAP4 expression

H1299 parental cells (H1299^WT) and STEAP4-overexpression H1299 cells (H1299^STEAP4) were prepared as described earlier.

To prepare the cells for Western blotting, the cells were lysed in a buffer consisting of 10% Triton X-100, 1% SDS, 20% glycerol 8i 60 mM Tris. The whole cell lysate was homogenised by probe sonication. A rabbit polyclonal STEAP4 antibody (Proteintech, catalogue number 11944-1-AP) was then used at a dilution of 1 : 1000.

The results are shown in Figure 15 where lane 1 shows the marker (with 50kDa marker indicated), lane 2 shows the results of the H1299^WT cells, and lane 3 shows the results of the H1299^STEAP4 cells following SDS-PAGE separation of equal amount of protein from each cell line (20 pg).

3. STEAP4 ferricreductase activity (functional assay)

This assay was based on the ferrireductase activity of STEAP4 as described by Ohgami et al.

C33A parental cells (C33A^WT) and STEAP4-overexpression C33A cells (C33A^STEAP4) were prepared as described earlier. Cells were seeded at 10⁵/well in a 96-well plate in the presence of 100 pl_ of iron uptake buffer supplemented with 50 mM ferric (Fe³⁺) nitrilotriacetate and 200 mM ferrozine. The iron uptake buffer consisted of 25 mM MES, 25 mM MOPS, 140 mM NaCI, 5 mM glucose, 5.4 mM KCI, 1.8 mM CaCh and 800 mM MgC - Absorbance was monitored at 562 nm.

The results are shown in in Figure 16, where the ferrireductase activity of STEAP4 in parental C33A^WT and C33A^STEAP4 cells as measured by absorbance at 562 nm over a 3 hour time period is shown. Reduction of Fe³⁺ to Fe²⁺ leads to formation of the Fe²⁺-ferrozine complex which leads to an increase in absorbance at 562 nm.

4. Compound A metabolism

The assay that was performed here relied on the STEAP4-mediated anoxic metabolism of the prodrug Compound A to the effector tyrosine kinase inhibitor Compound B.

C33A parental cells (C33A^WT), STEAP4-overexpression C33A cells (C33A^STEAP4), H1299 parental cells (H1299^WT), and STEAP4-overexpression H1299 cells (H1299^STEAP4) were prepared as described earlier.

5 X 10⁵ cells/well were seeded in a 24-well plate following which Compound A was added to a final concentration of 10 mM for 90 minutes. Cells and medium from each well were crashed with 2 equivalents of acetonitrile containing 0.5 mM of the internal standards for Compound A and Compound B. Rate of Compound B production was determined by LC- MS/MS mass spectrometry. The results are shown in Figure 17, where the rate of Compound B production under oxic (aerobic) or anoxic conditions in C33A^WT, C33A^STEAP4, H 1299^WT, and H1299^STEAP4 cells following exposure to IOmM Compound A for 90 minutes is shown.

5. Immunohistochemical detection of STEAP4 expression

C33A parental xenografts (C33A^WT) and STEAP4-overexpression C33A tumour xenografts cells (C33A^STEAP4) were established in nude mice. Briefly, cells were harvested and resuspended in MEMa media (without FBS) to achieve an inoculum of 5 x 10⁶ cells in a volume of 0.1 mL per mouse. The inoculum was injected subcutaneously on the right flank of each mouse. Tumour xenografts of C33A^WT and C33A^STEAP4 cells Parental and STEAP4 overexpressing tumours grown in nude mice were harvested and fixed in formalin for 48 hours prior to being embedded in paraffin. Sections were cut to 5 pm thickness on to Superfrost™ Plus microscope slides (Thermo Scientific). Slides were dried at 60°C for 20 minutes, deparaffinised twice in xylene (15 minutes each) and rehydrated successively in 100%, 95% and 80% ethanol (5 minutes each), followed by 60% and 30% ethanol (3 minutes each). Slides were briefly soaked in ultrapure water for 1 minute, blocked in Dual Endogenous Enzyme Block (Dako) for 30 minutes at room temperature and rinsed in Tris- buffered saline (TBS pH 7.4). Antigen retrieval was performed by brief, high temperature heat denaturation (pressure cooker) with the slides immersed in 10 mM sodium citrate buffer (pH 6.0). The slides were the cooled to room temperature for 30 minutes, rinsed in TBS-T (TBS with 0.01% Tween-20) for 5 minutes and blocked with 1% bovine serum albumin (BSA) in TBS-T for 1 hour at room temperature. The primary antibody (rabbit polyclonal anti-STEAP4, Proteintech Group, catalogue number 11944-1-AP) was applied to each slide at a dilution of 1 : 50 for 30 minutes at room temperature and rinsed in TBS-T for 15 minutes with gentle agitation. The HRP-conjugated secondary antibody (as part of the Dako REAL™ Envision™ Detection System, Peroxidase/DAB+, Rabbit/Mouse kit) was applied directly to each slide for 30 minutes at room temperature and rinsed three times in TBS-T for 5 minutes each . DAB staining was performed with the Dako REAL™ EnVision™ Detection System, Peroxidase/DAB+, Rabbit/Mouse according to the manufacturer's recommended protocol for approximately 5 minutes. The reaction was halted by rinsing the slides in TBS-T and water for 5 minutes each. Nuclear counterstaining was performed with Mayer's Hematoxylin Solution (Sigma Aldrich) for 10 minutes, rinsed in water and dipped ten times in Scott's tap water. Dehydration of slides was carried out by immersing slides in 70%, 95% and 100% ethanol for 3 minutes each, followed by xylene for 3 minutes. Slides were air dried briefly and mounted using DPX Mountant for histology (Sigma Aldrich). Images were acquired on a MetaSystems VSlide slide scanner and are shown in Figure 28. Example 8: Functional characterisation of a STEAP4 knock-out SiHa clone

The expression of STEAP4 in SiHa clones that were derived following transfection with a gRNA sequence against STEAP4 was evaluated by qPCR, and metabolism of the prodrug Compound A in the SiHa clones with low or no relative STEAP4 expression was then assessed.

Method:

Fifteen clones were derived from SiHa cells and sorted based on GFP-positivity following transfection with the px458 vector carrying a gRNA against STEAP4 (gRNA target sequence = AATACCGTCGATTCCCAGAC) (SEQ ID NO: 14).

cDNA synthesis was then performed using 1 pg of RNA from each clonal cell line. qPCR was then performed to evaluate STEAP4 expression, and using both GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and HPRT (hypoxanthine phosphoribosyl transferase) as reference genes, using the primers shown in Table 2.

Table 2: Primers used for qPCR analysis

The reaction was carried out in a 384-well plate with 3 independent wells per clone / gene of interest. The reaction in each well consisted of:

1. 5 pL of SYBR Green PCR Master Mix;

2. 0.5 pL of mixed (forward and reverse primer made to an 8 pM stock); and

3. 4.5 pL cDNA diluted in water to 1 :20.

Compound A metabolism was carried out inside a H2/palladium-catalyst scrubbed anaerobic chamber (Bactron, Shellab) using plasticware and medium that was equilibrated to anoxic conditions inside the chamber at least 3 days prior to the experiment. Cells were exposed to 10 pM of Compound A for 90 minutes. Metabolism was suppressed by addition of two volumes of ice-cold acetonitrile containing the deuterated internal standards (D6) for Compound A and Compound B. Rates of Compound B formation were measured by mass spectrometry with reference to the D6 internal standard. Results:

qPCR results of the SiHa clones revealed all clones apart from clone #50 had STEAP4 expression levels that were comparable to parental SiHa^WT cells, as shown in Figure 18. STEAP4 mRNA was however not detected in SiHa #50 relative to either reference genes GAPDH or HPRT.

Anoxic Compound A metabolism in the SiHa #50 cell line was approximately 92% lower relative to the parental SiHa^WT cells, as shown in Figure 19.

Conclusion:

A SiHa clone (#50) that was characterised as having no STEAP4 expression by qPCR following delivery of a gRNA against STEAP4 was shown to have significantly lower (92%) rate of Compound A metabolism relative to parental SiHa^WT cells.

Example 9: STEAP4 overexpression leads to increased Compound A metabolism in vitro and in vivo

Method:

Parental C33A^WT and PC9^WT cells were transfected with the F279-V5 expression plasmid encoding the sequence verified open reading frame of STEAP4. Stably transfected cell lines expressing the plasmids were selected following puromycin selection.

qPCR was performed to determine relative expression of STEAP4 in C33A^STEAP4 and PC9^STEAP4 using GAPDH as a reference control. The primers used for this SYBR-Green based qPCR are shown in Table 3.

Table 3: Primers used for qPCR analysis

The metabolism experiment was carried out inside a H2/palladium-catalyst scrubbed anaerobic chamber (Bactron, Shellab) using plasticware and medium that was equilibrated to anoxic conditions inside the chamber at least 3 days prior to the experiment. Cells were exposed to 10 mM of Compound A for 90 minutes. Metabolism was suppressed by addition of two volumes of ice-cold acetonitrile containing the deuterated internal standards (D6) for Compound A and Compound B. Rates of Compound B formation were measured by mass spectrometry with reference to D6 internal standard. For in vivo evaluation in NIH-III female mice, each cell line was administered subcutaneously on the right flank of each mouse as a 100 mI_ inoculum containing 5 x 10⁶ cells. Six mice were assigned to each xenograft type. However, only n = 5 PCS ¹, n = 5 PC9^STEAP4, n = 3 C33A^WT and n = 4 C33A^STEAP4 tumours reached the desired tumour volume within a 3-month period.

When tumours reached desired volumes (~400 mm³), mice were administered a dose of 48 mg/kg of Compound A. Mice were sacrificed 3 hours later and tumours snap frozen in liquid nitrogen. Tumour samples were minced to a fine powder using a bio- pulverizer. Four volumes of ice-cold acetonitrile containing D6 internal standards for Compound A and Compound B was added followed by vigorous vortexing for 2 minutes to enable analyte extraction. Tumour Compound B concentrations were measured by mass spectrometry with reference to D6 internal standard.

Results:

C33A^STEAP4 tumour cells had a >70, 000-fold increase in STEAP4 expression relative to parental C33A^WT tumour cells, as shown in Figure 20. For three C33A^WT tumours and three C33A^STEAP4 tumours, the individual qPCR reaction results were run on a gel and are illustrated in Figure 20. pc9^STEAP4 tumour cells had a 123-fold increase in STEAP4 expression relative to parental PC9^WT tumour cells, also as shown in Figure 20.

C33A^STEAP4 cells had a 37-fold increase in Compound A metabolism relative to parental C33A^WT cells, while PC9^STEAP4 cells had a 9-fold increase in Compound A metabolism relative to parental PC9^WT cells, also as shown in Figure 21.

Both STEAP4-overexpressing PC9 and C33A xenografts showed significantly increased concentrations of Compound B generated following Compound A administration relative to the respective parental xenografts. With regards to the PC9 pair of xenografts, STEAP4-overexpressing tumours showed a 194% increase in concentration of Compound B in comparison to parental (WT) PC9 tumours, as shown in Figure 22. C33A^STEAP4 tumours showed a 367% increase in tumour Compound B concentration relative to parental C33A^WT tumours, also as shown in Figure 22.

Conclusions:

C33A^STEAP4 and PC9 ^STEAP4 cells had increased expression of STEAP4 relative to the corresponding parental cell lines, as determined by qPCR.

Both STEAP4-overexpressing cell lines showed increased Compound A metabolism in vitro and in vivo xenograft models relative to parental cell lines. Example 10: SiHa STEAP4 knock-out (clone #50) pilot tumour growth

Method:

Parental SiHa^WT and SiHa STEAP4 knock-out (k/o) clone #50 (from Example 8) were inoculated into NIH-III female mice by subcutaneous injection of ~7 x 10⁶ cells per inoculum. On the day the tumours were harvested, the mice were dosed with EF5 (SN31691) (60 mg/kg, i.p.) and then with Compound A (60 mg/kg, i.p.) 3 hours after EF5 administration. 3 hours after EF5 adminsitration, the mice were sacrificed and tumours collected.

Each tumour was split for various assays, where a sample of each tumour was fixed in 10 mL of 10% neutral buffered formalin immediately after excision, and where additional tumour samples for Compound A metabolism and Western blotting/q PCR were snap frozen in liquid nitrogen and stored at -80 °C (untill required for assay).

Compound A and Compound B concentrations were quantified in tumours as follows. Frozen tumour samples were cryofractured to fine particulates using a pre-cooled (-80 °C) BioPulverizer system (BioSpec Products Inc., USA) and transferred to a pre-weighed 1.5 mL microcentrifuge tube. The weights of the cryofractured tumour samples were calculated and supplemented with 4x (w/v) of ice-cold acetonitrile spiked with 0.5 mM D6 internal standards for Compound A and Compound B. The samples were vortexed vigorously for 5 minutes, placed on ice and centrifuged at 13,000 rpm for 5 minutes. 40 pL of the supernatant was diluted 1 :2 in 0.1% formic acid/water prior to injection on the LC-MS/MS system. Analyte concentrations (Compound A and Compound B) were determined by LC- MS/MS.

For qPCR, ~0.2 g of cryofractured tumour sample was dissolved in 1 mL of TRIzol. RNA was extracted from each sample based on standard guanidinium thiocyanate-phenol- chloroform extraction. cDNA was prepared from 1 pg of RNA of each sample using the QuantiTect Reverse Transcription Kit (Qiagen). Diluted cDNA (1 :20 in RNAse free water) was used for SYBR Green-based qPCR using the primers shown in Table 4

Table 4: Primers used for qPCR analysis

SiHa parental xenografts (SiHa^WT) and STEAP4-deleted SiHa tumour xenografts cells (SiHa^{STEAP4 nul1}) were established in nude mice. Briefly, cells were harvested and resuspended in MEMa media (without FBS) to achieve an inoculum of 5 x 10⁶ cells in a volume of 0.1 mL per mouse. The inoculum was injected subcutaneously on the right flank of each mouse. Tumour xenografts of SiHa^WT and SiHa^{STEAP4 nu}" cells grown in nude mice were harvested and fixed in formalin for 48 hours prior to being embedded in paraffin. Sections were cut to 5 pm thickness on to Superfrost™ Plus microscope slides (Thermo Scientific). Slides were dried at 60°C for 20 minutes, deparaffinised twice in xylene (15 minutes each) and rehydrated successively in 100%, 95% and 80% ethanol (5 minutes each), followed by 60% and 30% ethanol (3 minutes each). Slides were briefly soaked in ultrapure water for 1 minute, blocked in Dual Endogenous Enzyme Block (Dako) for 30 minutes at room temperature and rinsed in Tris-buffered saline (TBS pH 7.4). Antigen retrieval was performed by brief, high temperature heat denaturation (pressure cooker) with the slides immersed in 10 mM sodium citrate buffer (pH 6.0). The slides were the cooled to room temperature for 30 minutes, rinsed in TBS-T (TBS with 0.01% Tween-20) for 5 minutes and blocked with 1% bovine serum albumin (BSA) in TBS-T for 1 hour at room temperature. The primary antibody (rabbit polyclonal anti-STEAP4, Proteintech Group, catalogue number 11944-1-AP) was applied to each slide at a dilution of 1 : 50 for 30 minutes at room temperature and rinsed in TBS-T for 15 minutes with gentle agitation. The HRP-conjugated secondary antibody (as part of the Dako REAL™ EnVision™ Detection System, Peroxidase/DAB+, Rabbit/Mouse kit) was applied directly to each slide for 30 minutes at room temperature and rinsed three times in TBS-T for 5 minutes each. DAB staining was performed with the Dako REAL™ EnVision™ Detection System, Peroxidase/DAB+, Rabbit/Mouse according to the manufacturer's recommended protocol for approximately 5 minutes. The reaction was halted by rinsing the slides in TBS-T and water for 5 minutes each. Nuclear counterstaining was performed with Mayer's Hematoxylin Solution (Sigma Aldrich) for 10 minutes, rinsed in water and dipped ten times in Scott's tap water. Dehydration of slides was carried out by immersing slides in 70%, 95% and 100% ethanol for 3 minutes each, followed by xylene for 3 minutes. Slides were air dried briefly and mounted using DPX Mountant for histology (Sigma Aldrich). Images were acquired on a MetaSystems VSlide slide scanner.

Results:

Intratumour Compound B concentrations

5 of the 6 parental SiHa tumours ranged in volume from 776 - 911 mm³ while the smallest tumour was 312 mm³ (mean ± SEM of all 6 tumours was 766 ± 93 mm³)

5 of the 6 SiHa STEAP4 k/o tumours ranged from 687 - 918 mm³ while one tumour was 139 mm³ (mean ± SEM of all 6 tumours was 682 ± 113 mm³).

Table 5 provides further details. Table 5: Details of weight of each mouse, tumour volume and volumes of EF5 and Compound A administered.

* Injection volume of 0.02 ml/g of 9.93 mM stock of EF5 (SN31691) with saline as the vehicle.

**Injection volume of 0.01 ml/g of 8.8 mM stock of Compound A with 20% cyclodextrin/water as the vehicle

The smallest tumours from each tumour type (#3 for WT and #6 for STEAP4 k/o) had enough material only for Compound A metabolism and formalin fixation (i.e. not enough for qPCR/Western blotting).

STEAP4 k/o SiHa tumours had a 76% decrease in intratumoural Compound B concentrations relative to parental SiHa tumours, as shown in Figures 23 and 24. This difference in mean Compound B concentrations was statistically significant (P = 0.011). The Compound B concentrations amongst the parental SiHa tumours had a higher standard deviation than the STEAP4 k/o SiHa tumours (Table 6). Table 6: Mean, SEM and SD in Compound B concentrations from n = 6 parental SiHa tumours and n = 6 STEAP4 k/o SiHa tumours

Compound B concentrations were normalised relative to the Compound A prodrug concentrations in each tumour. These ratios are shown in Figure 25. STEAP4 k/o SiHa tumours had a 89% decrease in Compound B: Compound A ratio in comparison to parental SiHa tumours (P < 0.001).

STEAP4 expression in parental and STEAP4 k/o tumours by gPCR

Relative to parental (WT) SiHa tumours, STEAP4 k/o tumours had no STEAP4 expression when STEAP4 expression in each sample was normalised to the reference gene HPRT (see Figure 26). cDNA samples from parental (WT) SiHa cells and STEAP4 k/o #50 cells grown in vitro were used as reference samples for this assay.

The qPCR products were electrophoresed on a 2% agarose gel which recapitulated the lack of a ~148 bp product corresponding to STEAP4 in each of the STEAP4 k/o tumours (Figure 27).

IHC results of the SiHa clones grown as subcutaneous tumour xenografts revealed detection of plasma membrane localised STEAP4 expression in SiHa^WT tissues but no signal inand SiHa^{STEAP4 nu}" tumours, as shown in Figure 29.

Conclusion

5 of the 6 STEAP4 k/o tumours characterised for STEAP4 expression by qPCR showed a lack of STEAP4 while all 6 tumours analysed for tumour Compound A metabolism showed significantly lower Compound B concentrations relative to parental SiHa tumours. These results suggest a lack of functional STEAP4 leads to a significant suppression in Compound A metabolism in vivo. Similarly, these results also suggest that STEAP4 is the predominant one-electron donor which leads to one-electron reduction of Compound A. EXAMPLE 11: STEAP4 knockout by CRISPR/Cas9 leads to Compound A resistance in EGFR exon 20 A767_V769dupASV mutant CUTO-14 NSCLC cells

Method:

Generation of STEAP4 knockout clones

CUTO-14 cells ( EGFR exon 20 A767_V769dupASV mutant) were seeded in 6-well plates at a density of 2.5 x 10⁵ cells/well. The following day the two plasmids (purchased from Genscript USA Inc.) from the pSpCas9(BB)-2A-GFP backbone encoding the guide RNA constructs against STEAP4 and Cas9 nuclease were used to transfect CUTO-14 cells using Lipofectamine 3000 (Life Technologies™). The genomic regions targeted by the two gRNAs are as follows:

• AATACCGTCGATTCCCAGAC (gRNA 1) (SEQ ID NO: 14)

• TGCAGAGTACCTTGCTCATT (gRNA 6) (SEQ ID NO: 17)

Cells were grown in the presence of the transfection mix for 24 hours prior to sorting. Simultaneously, 24 hours prior to cell sorting a 5 mL suspension of CUTO-14 cells (1 x 10⁶ cells/mL) was places in a 75mm³ tissue culture flask and irradiated to 100 Gy. The irradiated cell suspension was plated out into 96-well plates at a density of 1 x 10⁵ cells/well. GFP-positive cells were sorted on a BD FACSAria II system (BD Biosciences, USA) as single cells into 96-well plates containing 100 pL of medium per well along with the irradiated CUTO-14 cells. Individual clones were monitored and cultured to increasing cell densities in successively larger culture vessels until a sufficient quantity of cells was obtained.

Assessment of Compound A metabolism

For the Compound A (tarloxotinib) metabolism experiment, cells were seeded in 96-well plates at a density of 1 x 10⁵ cells/well in 90 pL of MEMa (with 10% FBS) and allowed to attach for 1 hour inside A Fh/palladium-catalyst anaerobic chamber (Bactron, Shellab) using plasticware and medium that was equilibrated to anoxic conditions inside the chamber at least 3 days prior to the experiment. A stock solution of Compound A was prepared inside the anaerobic chamber to supply a final concentration in each well of 10 pM. Exposure to Compound A was for 90 minutes at which time metabolism was suppressed by addition of two volumes of ice-cold acetonitrile containing the deuterated internal standards (D6) for Compound A and Compound B (tarloxotinib-E, the Compound A metabolite) (both D6 standards present in same solution to final concentrations of 0.5 pM). Compound A metabolism is significantly suppressed in CUTO-14 STEAP4 knockout clones relative to WT CUTO-14 cells (Table 7). Table 7: Percentage residual Compound B produced in clones transfected with the STEAP4 gRNA relative to WT CUTO-14 cells.

In vitro cytotoxicity assays to determine the sensitivity of STEAP4 knockout clones to Compound A and Compound C

Parental CUTO-14 cells and clones were maintained in RPMI 10% FCS in sterile tissue culture flasks prior to the experiment. On the day of the experiment, cells were harvested with Trypsin/EDTA and counted. The required number of cells was transferred to a 50 ml falcon tube and centrifuged to pellet cells (1000 rpm, 5 min). Excess media was removed by aspiration leaving cell pellets sitting in 50-100 pi media. The cell number was calculated to ensure a seeding density of 6000 cells per well of a 96-well plate, with cells resuspended in RPMI + 10% FCS, -P/S + lOmM D-Glucose + 0.2mM 2'-deoxycytidine. Cells were seeded under anoxic conditions using a 5% H2/palladium catalyst scrubbed Bactron anaerobic chamber (Sheldon Manufacturing, Cornelius, Oregon) to achieve severe anoxia (< 10 ppm 0₂ gas phase) during prodrug exposure.

The layout of the plate is described in Figure 9, where column 1 (the left-most column) consists of 8 media only control wells. Column 2 (second from the left) consists of 8 cells only (no prodrug) control wells. The remaining columns (3-12) consist of cells exposed to a range of prodrug concentrations. A high concentration of prodrug was added to column 12 (the right-most column) in each row, and 3-fold dilutions were performed across the plate using a multi-channel pipette. Thus, a total of four prodrugs could be evaluated on a single plate, with duplicate wells per prodrug.

After 2 hours incubation to enable cell attachment to the plates (and equilibrate to anoxia in the case of the anoxic samples), cells were exposed to a range of prodrug concentrations for 4 hours under aerobic or anoxic conditions. After prodrug exposure, plates were transferred to aerobic conditions for 20 hours. A vacuum immunowash was then used to aspirate the media and wells were washed three times with 150 pi of prodrug-free RPMI 10% FCS + P/S. The plates were then incubated under aerobic conditions for 4 days for cell growth. Cells were fixed by adding 50mI of cold 40% trichloroacetic acid (TCA) to each well by layering on top without mixing, this gave a final concentration of 10% TCA. Plates were incubated in a fridge for 1 hour before the TCA was washed off and the plates rinsed in running tap water. 50mI of 0.4% SRB in 1% acetic acid was added to each well and plates were incubated for 30 minutes in the dark. Excess stain was flicked off and plates were washed four times in 2L of tap water containing 1% acetic acid. Excess fluid was flicked from the plates and the remaining SRB stain solubilized by adding 100mI of lOmM unbuffered Tris to each well. Plates were then left on a shaker for at least 2 hours in the dark before analysis using a plate reader (set at an optical density of 490 nM for

measurement filter and 450nM for reference filter). The IC50 value is the concentration of prodrug required to inhibit cell growth by 50%.

For Compound A, the three clones were resistant to exposure, with IC50 values above 1.0 uM (Table 8). In contrast the WT CUTO-14 cells were sensitive (Table 8) while the same clones screened against Compound C showed increases in IC50 values relative to WT cells (Table 9).

Table 8 : Anti-proliferative activity (IC50) of Compound A in CUTO-14 WT and STEAP4 gRNA transfected clones under anoxic conditions

Table 9 : Anti-proliferative activity (IC50) of Compound C (PR-104A) in CUTO-14 WT and STEAP4 gRNA transfected clones under anoxic conditions

Example 12: Evaluating the in vivo efficacy of Compound A in a parental (WT) neoplastic cell line and paired line engineered to overexpress human STEAP4 cDNA

Method:

Cloning of STEAP4 utilising the same method as described in Example 5

The open reading frame (ORF) for human STEAP4 transcript variant 1

(NM_024636.2) was cloned into the F279-V5 expression vector. This provides transcription of a bicistronic mRNA from the human immediate-early cytomegalovirus (CMV) promoter, which encodes the open reading frames for the gene of interest and the pac (puromycin resistance) gene; the former harbouring an occult C-terminal V5-Tag inducible with TAG- On-Demand™ technology.

Human cervical carcinoma (C33A) cells were transfected using Lipofectamine 3000 reagent complexed with F279-STEAP4-V5 plasmid DNA. The transfection mix was added drop wise and after 48 hours the culture media was replaced with fresh o-MEM (with 5% FBS) medium containing 1 mM puromycin, escalating to 3 mM puromycin over time. Cells were grown to confluence in the wells before being pooled together and transferred to a cell culture flask of desired volume. Cells were maintained in o-MEM supplemented with 3 mM puromycin. Cell lines were designated C33A^WT/C33A^STEAP4.

Mouse strain and housing

Specific pathogen-free female NIH-III mice (NIH-Lyst^{b9 J}Foxnl^nuBtk^xid) were obtained from the Vernon Jansen Unit (VJU), The University of Auckland. To minimize exogenous infection, mice were maintained in individually ventilated cages at a maximum of 6 mice per cage (Green Line IVC Sealsafe PLUS, Mouse, Tecniplast, USA). The temperature of the vivarium was maintained at 21 - 22 °C and mice provided with an ad libitum diet of Harlan™ Teklad Global Diets with 18% protein content.

Subcutaneous implantation of tumours

Cells were harvested and resuspended in a 1 : 1 mixture of MEMo (without FBS) and BD Matrigel™ Basement Membrane Matrix to achieve an inoculum of 5 x 10⁶ cells in a volume of 0.1 mL per mouse. The inoculum was injected subcutaneously on the right flank of each mouse. A single dose of Compound A at 48 mg/kg was administered by

intraperitoneal injection. Tumour volumes were calculated assuming a prolate ellipsoid shape: n/6 x (length x width x width).

As shown in Figure 31, STEAP4 overexpressing C33A tumours showed a greater degree of tumour regression relative to parental C33A^WT tumours indicating an improved therapeutic outcome for Compound A in tumours with high expression of STEAP4. EXEMPLARY EMBODIMENTS

1. In one embodiment, disclosed herein is a hypoxia-activated prodrug (HAP) for use in the treatment or prevention of cancer in an individual in need thereof, where tumour cells of the individual exhibit an elevated level of STEAP4 expression.

2. In one embodiment, disclosed herein is a HAP for use in the treatment or prevention of cancer in an individual in need thereof, comprising :

3. In one embodiment, disclosed herein is a HAP for use in the treatment or prevention of cancer in an individual in need thereof, comprising :

a) providing tumour cells of the individual;

b) determining a level of STEAP4 expression in the tumour cells;

d) administering a therapeutically effective amount of a HAP to the individual.

4. In one embodiment, disclosed herein is a method of treating or preventing cancer in an individual in need thereof, where tumour cells of the individual exhibit an elevated level of STEAP4 expression, the method comprising administering to the individual a therapeutically effective amount of a hypoxia-activated prodrug (HAP).

5. In one embodiment, disclosed herein is a method of treating or preventing cancer in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of a hypoxia-activated prodrug (HAP), wherein the cancer exhibits an elevated level of STEAP4 expression.

6. In one embodiment, disclosed herein is a method of treating or preventing cancer in an individual in need thereof, the method comprising :

a) determining a level of STEAP4 expression in a sample from the individual, wherein the sample comprises tumour cells; b) predicting the individual as being likely to be responsive to treatment by a HAP if the tumour cells exhibit an elevated level of STEAP4 expression; and

c) administering a therapeutically effective amount of a HAP to the individual.

7. In one embodiment, disclosed herein is a method of treating or preventing cancer in an individual in need thereof, the method comprising :

a) providing tumour cells of the individual;

b) determining a level of STEAP4 expression in the tumour cells;

d) administering a therapeutically effective amount of a HAP to the individual.

8. In one embodiment, disclosed herein is a method of treating or preventing cancer in an individual, where an elevated level of STEAP4 expression is detected in tumour cells of the individual, wherein the method comprises administering a therapeutically effective amount of a HAP to the individual.

9. In one embodiment, disclosed herein is a method of predicting whether an individual with cancer is likely to be responsive to treatment with a HAP, the method comprising : a) determining the level of STEAP4 expression in a sample from the individual, wherein the sample comprises tumour cells;

10. In one embodiment, disclosed herein is a method of predicting whether an individual with cancer is likely to be responsive to treatment with a HAP, the method comprising : a) providing tumour cells of the individual;

b) determining a level of STEAP4 expression in the tumour cells; and

11. In one embodiment, disclosed herein is a method of predicting whether an individual with cancer is likely to be responsive to treatment with a HAP, wherein the method comprises detecting the level of STEAP4 expression in a sample of tumour cells from the individual; wherein the individual is likely to be responsive to the treatment with a HAP if the tumour cells exhibit an elevated level of STEAP4 expression.

12. In one embodiment, disclosed herein is a method of identifying an individual with cancer who is likely to be responsive to treatment with a HAP, wherein the method comprises:

a) providing tumour cells of the individual;

b) determining a level of STEAP4 expression in the tumour cells;

13. In one embodiment, disclosed herein is a method of increasing metabolism of a HAP in an individual suffering from cancer comprising administering to the individual a therapeutically effective amount of the HAP or a pharmaceutically acceptable salt thereof;

wherein the cancer exhibits an elevated level of STEAP4 expression; and wherein the elevated level of STEAP4 expression results in an increase in the HAP metabolism as compared to a normal level of STEAP4 expression.

14. In certain embodiments, the increase in HAP metabolism comprises about a 9-fold increase in HAP metabolism as compared to the HAP metabolism in the presence of a normal level of STEAP4 expression.

15. In certain embodiments, the increase in HAP metabolism comprises up to about a 9-fold increase in HAP metabolism as compared to the HAP metabolism in the presence of a normal level of STEAP4 expression.

16. In one embodiment, disclosed herein is a compound which is a HAP, for use in the treatment or prevention of a cancer exhibiting an elevated level of STEAP4 expression, wherein the treatment comprises administering the HAP to an individual in need thereof.

17. In one embodiment, disclosed herein is a compound which is a HAP, for use in the treatment or prevention of a cancer exhibiting an elevated level of STEAP4 expression, wherein the treatment comprises administering the HAP to an individual in need thereof.

18. In one embodiment, disclosed herein is use of a HAP for the treatment of cancer in an individual, wherein the cancer exhibits an elevated level of STEAP4 expression. 19. In one embodiment, disclosed herein is use of a HAP for the treatment of cancer in an individual, wherein tumour cells of the individual exhibit an elevated level of STEAP4 expression.

20. In one embodiment, disclosed herein is use of a HAP in the manufacture of a medicament for the treatment of cancer in an individual, wherein the cancer exhibits an elevated level of STEAP4 expression.

21. In one embodiment, disclosed herein is use of a HAP in the manufacture of a medicament for the treatment of cancer in an individual, wherein tumour cells of the individual exhibit an elevated level of STEAP4 expression.

22. In one embodiment, disclosed herein is a composition for use in treating or preventing cancer, wherein the cancer exhibits an elevated level of STEAP4 expression, the composition comprising a therapeutically effective amount of a HAP.

23. In one embodiment, disclosed herein is a composition for treating or preventing cancer, wherein the cancer exhibits an elevated level of STEAP4 expression, the composition comprising a therapeutically effective amount of a HAP and a pharmaceutically acceptable excipient.

24. In certain embodiments or combinations of the above-mentioned embodiments, the level of STEAP4 expression is expression of a STEAP4 gene.

25. In certain embodiments or combinations of the above-mentioned embodiments, the level of STEAP4 expression is expression of a STEAP4-expressing target nucleic acid.

26. In certain embodiments or combinations of the above-mentioned embodiments, the nucleic acid is DNA.

27. In certain embodiments or combinations of the above-mentioned embodiments, the nucleic acid is mRNA.

28. In certain embodiments or combinations of the above-mentioned embodiments, the level of STEAP4 expression is expression of a STEAP4 protein. 29. 1n certain embodiments or combinations of the above-mentioned embodiments, the level of STEAP4 expression is associated with a low oxygen level.

30. In certain embodiments or combinations of the above-mentioned embodiments, the cancer is selected from the group consisting of bone cancer, lung cancer, breast cancer, cancer of the head and neck, prostate cancer, pancreatic cancer, skin cancer, uterine cancer, ovarian cancer, cancer of the urethra, cancer of the adrenal gland, cancer of the small intestine, cancer of the kidney, cancer of the bladder, cancers of the brain, colorectal cancer, oesophageal cancer, gastric cancer, anal cancer, liver cancer, thyroid cancer, ocular cancer, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland and testicular cancer.

31. In certain embodiments or combinations of the above-mentioned embodiments, the cancer is prostate cancer.

32. In certain embodiments or combinations of the above-mentioned embodiments, the cancer is chronic or acute leukaemia, acute myeloid leukaemia, chronic myeloid leukaemia, acute lymphoblastic leukaemia, chronic lymphocytic leukaemia, carcinoma of the cervix, carcinoma of the vulva, carcinoma of the vagina, Hodgkin's Disease, brain stem glioma, melanoma, Merkel cell carcinoma, cervical carcinoma, Urothelial carcinoma, lymphomas, gliomas, meningiomas, pituitary adenomas, nerve sheath tumours, retinoblastoma, or non-small cell lung cancer

33. In certain embodiments or combinations of the above-mentioned embodiments, the cancer is cervical carcinoma.

34. In certain embodiments or combinations of the above-mentioned embodiments, the cancer is non-small cell lung cancer (NSCLC).

35. In certain embodiments or combinations of the above-mentioned embodiments, the cancer is glioblastoma.

36. In certain embodiments or combinations of the above-mentioned embodiments, a dosage of the HAP is from about 0.1 mg/m² of body weight of an individual to about 300 mg/m² of body weight of an individual. 37. 1n certain embodiments or combinations of the above-mentioned embodiments, a dosage of the HAP is from about 0.1 mg/m² of body weight of an individual to about 150 mg/m² of body weight of an individual.

38. In certain embodiments or combinations of the above-mentioned embodiments, a dosage of the HAP is about 150 mg/m² of body weight of an individual.

39. In certain embodiments or combinations of the above-mentioned embodiments, the HAP is administered orally, parenterally, rectally, topically, intravenously, intramuscularly, subcutaneously, or intraperitoneally.

40. Certain embodiments or combinations of the above-mentioned embodiments, further comprising administering an additional agent.

41. In certain embodiments or combinations of the above-mentioned embodiments, the additional agent is an anti-cancer agent selected from the group consisting of asparaginase, bleomycin, carboplatin, carmustine, chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, doxorubicin (adriamycine), epirubicin, etoposide, 5-fluorouracil, hexamethylmelamine, hydroxyurea, ifosfamide, irinotecan, leucovorin, lomustine, mechlorethamine, 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitoxantrone, prednisolone, prednisone, procarbazine, raloxifen, streptozocin, tamoxifen, thioguanine, topotecan, vinblastine, vincristine, vindesine, a MEK 1/2 inhibitor, an AKT inhibitor, a mTOR inhibitor, a monoclonal antibody, oxaliplatin, gemcitabine, gefinitib, taxotere, ara A, ara C, herceptin, BCNU, CCNU, DTIC, actinomycin D, aminoglutethimide, L-asparaginase, azathioprine, 5-azacytidine cladribine, busulfan, diethylstilbestrol, 2', 2'- difluorodeoxycytidine, docetaxel, erythrohydroxynonyladenine, ethinyl estradiol, 5- fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, fludarabine phosphate, fluoxymesterone, flutamide, hydroxyprogesterone caproate, idarubicin, interferon, medroxyprogesterone acetate, megestrol acetate, melphalan, mitotane, paclitaxel, pentostatin, N-phosphonoacetyl-L-aspartate (PALA), plicamycin, semustine, tenipdside, testosterone propionate, thiotepa, trimethylmelamine, uridine, and vinorelbine.

42. In certain embodiments, provided herein is a pharmaceutical composition comprising a HAP of any combinations of the above-mentioned embodiments, and a pharmaceutically acceptable excipient. In certain embodiments or combinations of the above-mentioned embodiments, the

HAP is a nitromethylaryl quaternary ammonium salt, a nitrophenyl mustard, an aminodihydropyrimidopyrimidinone, an aminopyridopyrimidinone, an aminopteridinone, an aminodihydropyridopyrimidinone, an aminonaphthyridinone, or an aminopyridopyrazinone. 1n certain embodiments or combinations of the above-mentioned embodiments, the nitromethylaryl quaternary ammonium salt is a compound of Formula I:

Formula I

where:

X is any negatively charged counterion;

Ri is a group of the formula -(CH₂)_nTr, where Tr is an aromatic nitroheterocycle or aromatic nitrocarbocycle and -(CH₂)_nTr acts as a reductively- activated fragmenting trigger; and n is an integer from 0 to 6;

R₂, R₃ and R₄ may each independently be selected from aliphatic or aromatic groups of a tertiary amine kinase inhibitor (R₂)(R₃)(R₄)N, or two of R₂, R₃, and R₄ may form an aliphatic or aromatic heterocyclic amine ring of a kinase inhibitor, or one of R₂, R₃ and R₄ may be absent and two of R₂, R₃ and R₄ form an aromatic heterocyclic amine ring of a kinase inhibitor. In certain embodiments or combinations of the above-mentioned embodiments, the nitromethylaryl quaternary ammonium salt is a compound of Formula II:

Formula II

where:

X is any negatively charged counterion;

Y is N or C-R.₇, where R₇ is selected from the group consisting of H, C₁-C₆ alkyl, C₁-C₆ alkoxy and groups of Formula III:

a b c

Formula III where * is the point of attachment, and where

T is selected from O, NH, N(C_I-C₆ alkyl) and a direct link;

m is selected from integers from 0 to 6;

U is selected from ORio, CF₃, OCF₃, CN, NR₁₁R₁₂, pyrrolidinyl, piperidinyl, piperazinyl, Nl-methylpiperazinyl, morpholinyl, CON(Ri₃)(Ri₄), S02N(Ris)(Ri₆), N(RI₇)CORI8, N(Rig)S02R2o, COR21, SOR22, SO2R23 and COOR24; and

Re, R9, Rio, Rll, R12, R13, R14 R15, Rl6, Rl7, Rl8, Rl9, R20, R2I, R22, R23, R24 QGQ independently selected from H and C₁-C₆ alkyl;

Z is N or C-CN;

n is an integer from 0 to 6;

Ri is a group of the formula (CH₂)_nTr where Tr is an aromatic nitroheterocycle or aromatic nitrocarbocycle and -(CH₂)_nTr acts as a reductively- activated fragmenting trigger; and n is an integer from 0 to 6;

R₅ is selected from anilines, indoles, indolines, amines, aminoindoles and aminoindazoles, each of which may be optionally substituted with one or more substituents selected from H, C₁-C₆ alkyl, C2-C₆ alkenyl, C2-C₆ alkynyl, C₁-C₆ alkoxy, F, Cl, Br, I, CN, CH₂F, CHF₂, CF₃, OH, NH₂, N0₂, NH(CI-C₆ alkyl), N(CI-C₆ alkyl)₂, CONH2, CO(Ci-C₆ alkyl), SO2NH2 and S0₂(Ci-C₆ alkyl); and

R₆ is selected from H, C₁-C₆ alkyl, C₁-C₆ alkoxy, NH(CI-C₆ alkyl), N(CI-C₆ alkyl)₂ and groups of Formula IV:

Formula IV where

* is the point of attachment;

V is selected from (CH2)_k where k is an integer from 0 to 6, O, NH and N(Ci-

C₆ alkyl); and

R2₅ is selected from H and C₁-C₆ alkyl.

46. In certain embodiments or combinations of the above-mentioned embodiments, the HAP is selected from the group comprising :

(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-c/]pyrimidin-6- yl)amino]-/V,/V-dimethyl-/V-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l- ammonium bromide (12),

(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide (17),

(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide (21), (2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-c/]pyrimidin-6- yl)amino]-/V-[(l,2-dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten- 1-ammonium bromide (22),

(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-c/]pyrimidin-6- yl)amino]-/V-[(2-ethyl-l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2- buten-l-ammonium bromide (23),

(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-c/]pyrimidin-6- yl)amino]-/V,/V-dimethyl-/V-{[l-methyl-4-nitro-2-(trifluoromethyl)-l/-/-imidazol-5- yl]methyl}-4-oxo-2-buten-l-ammonium bromide (24),

(2E)-/V-{[2-(aminocarbonyl)-l-methyl-4-nitro-lH-imidazol-5-yl]methyl}-4-[(4-{3- chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-c/]pyrimidin-6-yl)amino]-/V,/V-dimethyl-4- oxo-2-buten-l-ammonium bromide (26),

(2E)-/V-[(2-ethyl-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-[(4-{[l-(3- fluorobenzyl)-l/-/-indazol-5-yl]amino}pyrido[3,4-c/]pyrimidin-6-yl)amino]-/V,/V-dimethyl-4- oxo-2-buten-l-ammonium bromide (28),

(2E)-/V-[(2-cyano-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-[(4-{[l-(3- fluorobenzyl)-l/-/-indazol-5-yl]amino}pyrido[3,4-c/]pyrimidin-6-yl)amino]-/V,/V-dimethyl-4- oxo-2-buten-l-ammonium bromide (30),

(2E)-/V-{[2-(aminocarbonyl)-l-methyl-4-nitro-lH-imidazol-5-yl]methyl}-4-[(4-{[l- (3-fluorobenzyl)-l/-/-indazol-5-yl]amino}pyrido[3,4-c/]pyrimidin-6-yl)amino]-/V,/V-dimethyl- 4-oxo-2-buten-l-ammonium bromide (31),

(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V-[(l,2-dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten- 1-ammonium bromide (33), (2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V-[(2-ethyl-l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2- buten-l-ammonium bromide (34),

(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V,/V-dimethyl-/V-{[l-methyl-4-nitro-2-(trifluoromethyl)-l/-/-imidazol-5- yl]methyl}-4-oxo-2-buten-l-ammonium bromide (35),

(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V-[(2-cyano-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2- buten-l-ammonium bromide (36),

(2E)-/V-{[2-(aminocarbonyl)-l-methyl-4-nitro-l/-/-imidazol-5-yl]methyl}-4-({4-[3- chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6-yl}amino)-/V,/V-dimethyl-4- oxo-2-buten-l-ammonium bromide (37),

(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(l,2- dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (45), (2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(2- ethyl-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (46),

(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-{[l-methyl-4-nitro-2-(trifluoromethyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2- buten-l-ammonium bromide (47),

(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(2- cyano-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (48),

(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(l,2- dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (57), (2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(2-ethyl-l- methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (58),

(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-c/]pynmidin-6-yl]amino}-/V,/V-dimethyl-/V- {[l-methyl-4-nitro-2-(tnfluoromethyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2-buten-l- ammonium bromide (59),

(2E)-/V-[(2-cyano-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-{[4-(3,4- dibromoanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (60),

(2E)-/V-{[2-(aminocarbonyl)-l-methyl-4-nitro-lH-imidazol-5-yl]methyl}-4-{[4-(3,4- dibromoanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (61),

(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(l,2- dimethyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (69), (2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(2- ethyl-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (70),

(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-{[l-methyl-4-nitro-2-(trifluoromethyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2- buten-l-ammonium bromide (71),

(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(2- cyano-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l- ammonium bromide (72),

(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-{[l-methyl-4-nitro-2-(l-propynyl)-l/-/-imidazol-5-yl]methyl}-4-oxo-2-buten-l- ammonium bromide (81), (2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V,/V- dimethyl-/V-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide (82),

(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(l,2- dimethyl-4-nitro-l/-/-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (83),

(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-c/]pyrimidin-6-yl]amino}-/V-[(2- ethyl-l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2-buten-l-ammonium bromide (84),

(2E)-/V-[(2-cyano-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-({4-[3-fluoro-4-(2- pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6-yl}amino)-/V,/V-dimethyl-4-oxo-2-buten- 1-ammonium bromide (95), (2E)-/V-{[2-(aminocarbonyl)-l-methyl-4-nitro-l/-/-imidazol-5-yl]methyl}-4-({4-[3- fluoro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6-yl}amino)-/V,/V-dimethyl-4- oxo-2-buten-l-ammonium bromide (96),

(2E)-4-({4-[3-fluoro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V,/V-dimethyl-/V-{[l-methyl-4-nitro-2-(l-propynyl)-l/-/-imidazol-5-yl]methyl}-4- oxo-2-buten-l-ammonium bromide (97),

(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V,/V-dimethyl-/V-[(l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl]-4-oxo-2-buten-l- ammonium bromide (98),

47. In certain embodiments or combinations of the above-mentioned embodiments, the HAP is Compound A (tarloxotinib bromide; ((2E)-4-{[4-(3-bromo-4- chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-N-[(l-methyl-4-nitro- lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide, having the chemical formula :

48. In certain embodiments or combinations of the above-mentioned embodiments, the nitrophenyl mustard is a compound of Formula XIII:

Formula XIII where

Xi represents at any available ring position -CONH-, -SO2NH-, -0-, -CH2-, - NHCO- or -NHSO2-;

R₆9 represents a lower Ci-₆ alkyl optionally substituted with one or more groups including hydroxy, amino and N-oxides therefrom or dialkylamino and N- oxides therefrom;

Yi represents at any available ring position -N-aziridinyl, -N(CH2CH2W_I)2 or

-N(CH2CHMeWi)2, where each Wi is independently selected from halogen or -0SC>₂Me; and

Zi represents at any available ring position -NO₂, -halogen, -CN, -CF₃ or -SC^Me.

49. In certain embodiments or combinations of the above-mentioned embodiments, the

HAP is:

(Compound C). In certain embodiments or combinations of the above-mentioned embodiments, the HAP is a compound of Formula XXXI:

XXXI. In certain embodiments or combinations of the above-mentioned embodiments, the HAP is selected from the group consisting of:

(344),

(345),

(346),

(S, f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pyrrolidin-3-yl)-7-(methylamino)-3,4-dihydropyrimido[4,5-c/]pyrimidin-2(l/-/)-one (347),

(S,f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pyrrolidin-3-yl)-7-(isopropylamino)-3,4-dihydropyrimido[4,5-c/]pyrimidin-2(lH)- one (348),

(S,f)-7-(cyclohexylamino)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4- (dimethylamino)but-2-enoyl)pyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-c/]pyrimidin-

2(lH)-one (349),

(S,f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pyrrolidin-3-yl)-7-(phenylamino)-3,4-dihydropyrimido[4,5-c/]pyrimidin-2(l/-/)-one

(350), (f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(methylamino)-3,4-dihydropynmido[4,5-c/]pynmidin-2(l/-/)-one

(351),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(isopropylamino)-3,4-dihydropynmido[4,5-c/]pynmidin-2(lH)- one (352),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-((tetrahydrofuran-3-yl)amino)-3,4-dihydropynmido[4,5- c/]pyrimidin-2(lH)-one (353),

(f)-7-(cyclohexylamino)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4- (dimethylamino)but -2-enoyl)piperidin-4-yl)-3,4-dihydropyrimido[4,5-cf]pyrinnidin- 2(lH)-one (354),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2-enoyl)pipendin -4 -yl)-7-((tetrahydro-2H-pyran-4-yl)amino)-3,4-dihydropyrimido[4,5-c/]pyrimidin- 2(lH)-one (355),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-(phenylamino)-3,4-dihydropynmido[4,5-c/]pynmidin-2(l/-/)-one

(356),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-((3-methoxyphenyl)amino)-3,4-dihydropynmido[4,5- c/]pyrimidin-2(lH)-one (357),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(m-tolylamino)-3,4-dihydropynmido[4,5-c/]pynmidin-2(l/-/)-one

(358),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-((3-fluorophenyl)amino)-3,4-dihydropynmido[4,5-c/]pynmidin- 2(lH)-one (359),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl) -7-((4-fluoro-3-methoxyphenyl)amino)-3,4-dihydropyrimido[4,5-c/] pyrimidin-2(lH)-one (362), (f)-7-((3-chloro-4-fluorophenyl)amino)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4- (dimethylamino)but-2-enoyl)pipendin-4-yl)-3,4-dihydropyrimido[4,5-c/]pynmidin-2(l/-/)- one (363),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-((4-fluorophenyl)amino)-3,4-dihydropynmido[4,5-c/]pynmidin- 2(lH)-one (364),

(369),

(370),

(374),

(E)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-(methylamino)pyndo[2,3-c/]pynmidin-7(8H)-one (376), (E)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-(isopropylamino)pyndo[2,3-c/]pynmidin-7(8H)-one (377), (E)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-((tetrahydrofuran-3-yl)amino)pyndo[2,3-c/]pynmidin-7(8H)-one

(378),

(383),

(384),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(isopropylamino)pyndo[3,4-b]pyrazin-2(lH)-one (391), (f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-(methylamino)-l,6-naphthyridin-2(lH)-one (392),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(isopropylamino)-l,6-naphthyndin-2(lH)-one (393),

(S,E)-3-(3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2-enoyl)pyrrolidin-3-yl)-7- (methylamino)-3,4-dihydropyrimido[4,5-c/]pyrimidin-2(lH)-one (394),

(f)-3-(2,6-dibromo-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(isopropylamino)-3,4-dihydropyrimido[4,5-c/]pynmidin-2(lH)- one (395),

(E)-3-(2,6-dibromo-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-(isopropylamino)pyndo[3,4-b]pyrazin-2(lH)-one (398),

(f)-3-(2,6-dibromo-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-(isopropylamino)-l,6-naphthyndin-2(lH)-one (399),

l-(l-acryloylpiperidin-4-yl)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(methylamino)-3,4- dihydropyrimido[4,5-c/]pynmidin-2(lH)-one (400),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(pyndin-2-ylamino)-3,4-dihydropynmido[4,5-c/]pynmidin-2(l/-/)- one (401),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-(pyndin-3-ylamino)-3,4-dihydropynmido[4,5-c/]pynmidin-2(l/-/)- one (402),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-(pyndin-4-ylamino)-3,4-dihydropynmido[4,5-c/]pynmidin-2(l/-/)- one (403),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-((l-methyl-lH-pyrazol-3-yl)amino)-3,4-dihydropynmido[4,5- c/]pyrimidin-2(lH)-one (404),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-((5-morpholinopyndin-2-yl)amino)-3,4-dihydropynmido[4,5- c/]pyrimidin-2(lH)-one (405),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-(pynmidin-4-ylamino)-3,4-dihydropynmido[4,5-c/]pynmidin- 2(lH)-one (406), (S,f)-4-(3-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(methylamino)-2-oxo-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)pyrrolidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl- 4-nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (407),

(E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-2-oxo-7-(pyridin-3-ylamino)-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium 2,2,2-trifluoroacetate (429), (f)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-2-oxo-7-(pyridin-4-ylamino)-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium 2,2,2-trifluoroacetate (430), (E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-((l-methyl-lH-pyrazol-3-yl)amino)- 2-oxo-3,4-dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V- ((l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (431), (E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-((5-morpholinopyridin-2-yl)amino)-2- oxo-3, 4-dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l- methyl-4-nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (432), (E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-2-oxo-7-(pyrimidin-4-ylamino)-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (433), and

* * *

All patents, publications, scientific articles, web sites, and other documents and materials referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the disclosure pertains, and each such referenced document and material is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such patents, publications, scientific articles, web sites, electronically available information, and other referenced materials or documents.

Although the disclosure has been described by way of example, it should be appreciated that variations and modifications may be made without departing from the scope of the disclosure as defined in the claims. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred in this specification. The specific compositions and methods described herein are representative of preferred examples and are exemplary and not intended as limitations on the scope of the disclosure. Other aspects and examples will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the disclosure as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the disclosure disclosed herein without departing from the scope and spirit of the disclosure. The disclosure illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed as essential. Thus, for example, in each instance described or used herein, in embodiments or examples of the present disclosure, any of the terms "comprising", "consisting essentially of", and "consisting of" may be replaced with either of the other two terms in the specification. Also, the terms "comprising", "including", containing", etc. are to be read expansively and without limitation. The methods illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims. Further, as used or described herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein.

The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognised that various modifications are possible within the scope of the disclosure as claimed. Thus, it will be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts disclosed herein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as described herein, and as defined by the appended claims.

The disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the disclosure with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims. In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognise that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

REFERENCES

1. Angerer et at. in Genetic Engineering : Principles and Methods (Setlow and Hollaender, Eds.) Vol 7, pgs 43-65 (Plenum Press, New York 1985).

2. Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons 1995

3. Gall JG, Pardue ML, "Nucleic acid hybridization in cytological preparations" Methods in Enzymology (1971) : 21; 470-480.

4. Gomes IM, Maia CJ, Santos CR, "STEAP Proteins: From Structure to Applications in Cancer Therapy" Mol Cancer Res (May 2012) : 10(5); 573-587.

5. Hunter FW, Wouters BG, and Wilson WR, "Hypoxia-activated prodrugs: paths forward in the era of personalised medicine" British Journal of Cancer (2016) : 114(10); 1071-1077, doi : 10.1038/bjc.2016.79.

6. Jin Y, Wang L, Qu S, Sheng X, Kristian A, Maelandsmo GM, Pallmann N, Yuca E, Tekedereli I, Gorgulu K, Alpay N, Sood A, Lopez-Berestein G, Fazli L, Rennie P, Risberg B, Waehre H, Danielsen HE, Ozpolat B, Saatcioglu F, "STAMP2 increased oxidative stress and is critical for prostate cancer" EMBO Molecular Medicine (2015): 7(3); 315-331.

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8. Korkmaz CG, Korkmas KS, Kurys P, Elbi C, Wang L, Klokk TI, et al., "Molecular cloning and characterization of STAMP2, and androgen-regulated six transmembrane protein that is overexpressed in prostate cancer" Oncogene (2005) : 24; 4934-4945.

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l l. Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, 2001 Cold Spring Harbor, N.Y.

12. Tijssen, Hybridisation with Nucleic Acid Probes, Parts I and II (Elsevier, Amsterdam 1993).

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Claims

1. A hypoxia-activated prodrug (HAP) for use in the treatment or prevention of cancer in an individual in need thereof, where tumour cells of the individual exhibit an elevated level of STEAP4 expression.

2. A HAP for use in the treatment or prevention of cancer in an individual in need thereof, comprising :

a. determining the level of STEAP4 expression in tumour cells of the individual; b. predicting the individual as being likely to be responsive to treatment by a HAP if the tumour cells exhibit an elevated level of STEAP4 expression; and c. administering a therapeutically effective amount of a HAP to the individual if the tumour cells exhibit an elevated level of STEAP4 expression.

3. A HAP for use in the treatment or prevention of cancer in an individual in need thereof, comprising :

a. providing tumour cells of the individual;

b. determining a level of STEAP4 expression in the tumour cells;

c. predicting the individual as being likely to be responsive to treatment by a HAP if the tumour cells exhibit an elevated level of STEAP4 expression; and d. administering a therapeutically effective amount of a HAP to the individual.

4. The HAP as claimed in any one of claims 1 to 3, wherein the level of STEAP4 expression is expression of a STEAP4 gene.

5. The HAP as claimed in any one of claims 1 to 3, wherein the level of STEAP4 expression is expression of a STEAP4-expressing target nucleic acid.

6. The HAP as claimed in claim 5, wherein the nucleic acid is DNA.

7. The HAP as claimed in claim 5, wherein the nucleic acid is mRNA.

8. The HAP as claimed in any one of claims 1 to 3, wherein the level of STEAP4 expression is expression of a STEAP4 protein.

9. The HAP as claimed in any one of claims 1 to 8, wherein the level of STEAP4 expression is associated with a low oxygen level.

10. The HAP as claimed in any one of claims 1 to 9, wherein the cancer is selected from the group consisting of bone cancer, lung cancer, breast cancer, cancer of the head and neck, prostate cancer, pancreatic cancer, skin cancer, uterine cancer, ovarian cancer, cancer of the urethra, cancer of the adrenal gland, cancer of the small intestine, cancer of the kidney, cancer of the bladder, cancers of the brain, colorectal cancer, oesophageal cancer, gastric cancer, anal cancer, liver cancer, biliary tract cancer, thyroid cancer, ocular cancer, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland and testicular cancer.

11. The HAP as claimed in claim 10, wherein the cancer is prostate cancer.

12. The HAP as claimed in any one of claims 1 to 9, wherein the cancer is chronic or acute leukaemia, acute myeloid leukaemia, chronic myeloid leukaemia, acute lymphoblastic leukaemia, chronic lymphocytic leukaemia, carcinoma of the cervix, carcinoma of the vulva, carcinoma of the vagina, Hodgkin's Disease, brain stem glioma, melanoma, Merkel cell carcinoma, cervical carcinoma, Urothelial carcinoma, lymphomas, gliomas, meningiomas, pituitary adenomas, nerve sheath tumours, retinoblastoma, or non-small cell lung cancer

13. The HAP as claimed in claim 12, wherein the cancer is cervical carcinoma.

14. The HAP as claimed in claim 12, wherein the cancer is non-small cell lung cancer (NSCLC).

15. The HAP as claimed in claim 12, wherein the cancer is glioblastoma.

16. The HAP as claimed in claim 12, wherein the cancer is breast cancer

17. The HAP as claimed in any one of claims 1 to 16, wherein a dosage of the HAP is from about 0.1 mg/m² of body weight of an individual to about 300 mg/m² of body weight of an individual.

18. The HAP as claimed in any one of claims 1 to 17, wherein a dosage of the HAP is from about 0.1 mg/m² of body weight of an individual to about 150 mg/m² of body weight of an individual.

19. The HAP as claimed in any one of claims 1 to 18, wherein a dosage of the HAP is about 150 mg/m² of body weight of an individual.

20. The HAP as claimed in any one of claims 1 to 10, wherein the HAP is administered orally, parenterally, rectally, topically, intravenously, intramuscularly, subcutaneously, or intraperitoneally.

21. The HAP as claimed in any one of claims 1 to 20, further comprising administering an additional agent.

22. The HAP as claimed in claim 21, wherein the additional agent is an anti-cancer agent selected from the group consisting of asparaginase, bleomycin, carboplatin, carmustine, chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, doxorubicin (adriamycine), epirubicin, etoposide, 5- fluorouracil, hexamethylmelamine, hydroxyurea, ifosfamide, irinotecan, leucovorin, lomustine, mechlorethamine, 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitoxantrone, prednisolone, prednisone, procarbazine, raloxifen, streptozocin, tamoxifen, thioguanine, topotecan, vinblastine, vincristine, vindesine, a MEK 1/2 inhibitor, an AKT inhibitor, a mTOR inhibitor, a monoclonal antibody, oxaliplatin, gemcitabine, gefinitib, taxotere, ara A, ara C, herceptin, BCNU, CCNU, DTIC, actinomycin D, aminoglutethimide, L-asparaginase, azathioprine, 5-azacytidine cladribine, busulfan, diethylstilbestrol, 2',2'-difluorodeoxycytidine, docetaxel, erythrohydroxynonyladenine, ethinyl estradiol, 5-fluorodeoxyuridine, 5- fluorodeoxyuridine monophosphate, fludarabine phosphate, fluoxymesterone, flutamide, hydroxyprogesterone caproate, idarubicin, interferon, medroxyprogesterone acetate, megestrol acetate, melphalan, mitotane, paclitaxel, pentostatin, N-phosphonoacetyl-L- aspartate (PALA), plicamycin, semustine, tenipdside, testosterone propionate, thiotepa, trimethylmelamine, uridine, vinorelbine, taxanes, paclitaxel, docetaxel, cabazitaxel, and enzalutamide.

23. The HAP as claimed in any one of claims 1 to 22, wherein the HAP is a nitromethylaryl quaternary ammonium salt, a nitrophenyl mustard, an aminodihydropyrimidopyrimidinone, an aminopyridopyrimidinone, an aminopteridinone, an aminodihydropyridopyrimidinone, an aminonaphthyridinone, or an aminopyridopyrazinone.

24. The HAP as claimed in any one of claims 1 to 23, wherein the nitromethylaryl quaternary ammonium salt is a compound of Formula I:

Formula I

where:

X is any negatively charged counterion;

Ri is a group of the formula -(CH2)_nTr, where Tr is an aromatic nitroheterocycle or aromatic nitrocarbocycle and -(CH2)_nTr acts as a reductively- activated fragmenting trigger; and n is an integer from 0 to 6;

R₂, R₃ and R₄ may each independently be selected from aliphatic or aromatic groups of a tertiary amine kinase inhibitor (R₂)(R₃)(R₄)N, or two of R₂, R₃, and R₄ may form an aliphatic or aromatic heterocyclic amine ring of a kinase inhibitor, or one of R₂, R₃ and R₄ may be absent and two of R₂, R₃ and R₄ form an aromatic heterocyclic amine ring of a kinase inhibitor. 25. The HAP as claimed in claim 24, wherein the nitromethylaryl quaternary ammonium salt is a compound of Formula II:

Formula II

where:

X is any negatively charged counterion;

Y is N or C-R.7, where R₇ is selected from the group consisting of H, C1-C6 alkyl, C1-C6 alkoxy and groups of Formula III:

a b e

Formula III where * is the point of attachment, and where

T is selected from O, NH, N(C_I-C₆ alkyl) and a direct link;

m is selected from integers from 0 to 6;

Z is N or C-CN;

n is an integer from 0 to 6;

Formula IV where

* is the point of attachment;

V is selected from (CH2)_k where k is an integer from 0 to 6, O, NH and N(Ci-

C₆ alkyl); and

R2₅ is selected from H and C₁-C₆ alkyl.

26. The HAP as claimed in claim 25, wherein the HAP is selected from the group comprising :

(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-c/]pyrimidin-6- yl}amino)-/V-[(2-cyano-l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-/V,/V-dimethyl-4-oxo-2- buten-l-ammonium bromide (102),

27. The HAP as claimed in claim 25, wherein the HAP is Compound A (tarloxotinib bromide;

((2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N- dimethyl-N-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l-ammonium bromide, having the chemical formula :

28. The HAP as claimed in any one of claims 1 to 23, wherein the nitrophenyl mustard is a compound of Formula XIII:

Formula XIII where

Yi represents at any available ring position -N-aziridinyl, -N(CH2CH2W_I)2 or -N(CH2CHMeWi)2, where each Wi is independently selected from halogen or -OSC>2Me; and

29. The HAP as claimed in claim 28, wherein the HAP is:

(Compound C).

30. The HAP as claimed in any one of claims 1 to 23, wherein the HAP is a compound of Formula XXXI:

XXXI.

31. The HAP as claimed in claim 30, wherein the HAP is selected from the group consisting of:

(344),

(345),

(346),

(347),

(351),

(356),

(358),

(369),

(370),

(374),

(E)-3-(3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2-enoyl)piperidin-4-yl)-7-((4- morpholinophenyl)amino)-3,4-dihydropyrimido[4,5-c/]pynmidin-2(l/-/)-one (375), (E)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-(methylamino)pyndo[2,3-c/]pynmidin-7(8H)-one (376),

(E)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-(isopropylamino)pyndo[2,3-c/]pynmidin-7(8H)-one (377),

(E)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-((tetrahydrofuran-3-yl)amino)pyndo[2,3-c/]pynmidin-7(8H)-one

(378),

(E)-2-(cyclohexylamino)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4- (dimethylamino)but-2-enoyl)pipendin-4-yl)pyrido[2,3-c/]pynmidin-7(8H)-one (380), (E)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-(((tetrahydro-2H-pyran-4-l)methyl)amino)pyndo[2,3- c/]pyrimidin-7(8H)-one (381),

(383),

(384),

(E)-2-((3-chloro-4-fluorophenyl)amino)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-8-(l-(4- (dimethylamino)but-2-enoyl)pipendin-4-yl)pyrido[2,3-c/]pynmidin-7(8H)-one (386), (E)-/V-(3-((6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)-7-oxopyrido[2,3- c/]pynmidin-8(7/-/)-yl)methyl)phenyl)-4-(dimethylamino)but-2-enamide (387),

(E)-/V-(3-(l-(6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)-7-oxopyrido[2,3- c/]pynmidin-8(7H)-yl)ethyl)phenyl)-4-(dimethylamino)but-2-enamide (390), (f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(isopropylamino)pyndo[3,4-b]pyrazin-2(lH)-one (391),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(methylamino)-l,6-naphthyndin-2(lH)-one (392),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(isopropylamino)-l,6-naphthyridin-2(lH)-one (393),

(E)-6-(2,6-dibromo-3,5-dimethoxyphenyl)-8-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-2-(isopropylamino)pyndo[2,3-c/]pynmidin-7(8H)-one (397),

(f)-3-(2,6-dibromo-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(isopropylamino)pyndo[3,4-b]pyrazin-2(lH)-one (398),

(E)-3-(2,6-dibromo-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(isopropylamino)-l,6-naphthyndin-2(lH)-one (399),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(pyndin-3-ylamino)-3,4-dihydropynmido[4,5-c/]pynmidin-2(l/-/)- one (402),

(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(pyndin-4-ylamino)-3,4-dihydropynmido[4,5-c/]pynmidin-2(l/-/)- one (403),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-((l-methyl-lH-pyrazol-3-yl)amino)-3,4-dihydropyrimido[4,5- c/]pyrimidin-2(lH)-one (404),

(f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)pipendin-4-yl)-7-((5-morpholinopyndin-2-yl)amino)-3,4-dihydropynmido[4,5- c/]pyrimidin-2(lH)-one (405), (f)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(l-(4-(dimethylamino)but-2- enoyl)piperidin-4-yl)-7-(pyrimidin-4-ylamino)-3,4-dihydropyrimido[4,5-c/]pyrimidin- 2(lH)-one (406),

(S,f)-4-(3-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(methylamino)-2-oxo-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)pyrrolidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl- 4-nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (407),

(f)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(isopropylamino)-2-oxo-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (409),

(f)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-2-oxo-7-((tetrahydro-2H-pyran-4- yl)amino)-3,4-dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl- /V-((l-methyl-4-nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium 2,2,2- trifluoroacetate (410),

(E)-4-(4-(6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(isopropylamino)-7-oxopyrido[2,3- c/]pyrimidin-8(7H)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4-nitro-l/-/-imidazol-5- yl)methyl)-4-oxobut-2-en-l-aminium bromide (416), (f)-4-(4-(6-(2,6-dibromo-3,5-dimethoxyphenyl)-2-(isopropylamino)-7-oxopyrido[2,3- c/]pyrimidin-8(7/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-N-((l-methyl-4-nitro-lH-imidazol-5- yl)methyl)-4-oxobut-2-en-l-aminium bromide (417),

(f)-4-(4-(6-(2,6-dichloro-3,5-dimethoxyphenyl)-7-oxo-2-(phenylamino)pyrido[2,3-c/] pyrimidin-8(7/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4-nitro-l/-/-imidazol-5- yl)methyl)-4-oxobut-2-en-l-aminium bromide (418),

(E)-4-(4-(6-(2,6-dibromo-3,5-dimethoxyphenyl)-7-oxo-2-(phenylamino)pyrido[2,3-c/] pyrimidin-8(7/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4-nitro-l/-/-imidazol-5- yl)methyl)-4-oxobut-2-en-l-aminium bromide (419),

(E)-4-((3-((6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(isopropylamino)-7-oxopyrido[2,3- c/]pyrimidin-8(7/-/)-yl)methyl)phenyl)amino)-/V,/V-dimethyl-/V-((l-methyl-4-nitro-l/-/- imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (420),

(E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-2-oxo-7-(pyridin-2-ylamino)-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (428), (f)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-2-oxo-7-(pyridin-3-ylamino)-3,4- dihydropyrimido[4,5-c/]pynmidin-l(2/-/)-yl)pipendin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-lH-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium 2,2,2-trifluoroacetate (429), (f)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-2-oxo-7-(pyridin-4-ylamino)-3,4- dihydropyrimido[4,5-c/]pynmidin-l(2/-/)-yl)pipendin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-lH-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium 2,2,2-trifluoroacetate (430), (E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-((l-methyl-lH-pyrazol-3-yl)amino)- 2-oxo-3,4-dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V- ((l-methyl-4-nitro-lH-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (431), (E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-((5-morpholinopyridin-2-yl)amino)-2- oxo-3, 4-dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l- methyl-4-nitro-l/-/-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (432), (E)-4-(4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-2-oxo-7-(pyrimidin-4-ylamino)-3,4- dihydropyrimido[4,5-c/]pyrimidin-l(2/-/)-yl)piperidin-l-yl)-/V,/V-dimethyl-/V-((l-methyl-4- nitro-lH-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium bromide (433), and

32. The HAP as claimed in claim 31, wherein the HAP is:

Compound E.

33. A pharmaceutical composition comprising a HAP as claimed in any one of the preceeding claims, and a pharmaceutically acceptable excipient.