Radiation-Activatable Compounds and Uses Thereof

FIELD

The present disclosure relates to a novel class of masking agents that can be activated under ionising radiation. In particular, the masking agents (which in some cases may be referred to as protecting groups) can be conjugated, linked or bonded to a parent compound to provide a masked compound. Ionising radiation activates the masking agent to release the parent compound. These masking agents or protecting groups may find application in a wide variety of fields, and may be particularly useful in therapy, e.g. in the formation of prodrugs and as linkers in antibody drug conjugates.

BACKGROUND

Cancer treatment often involves the use of cytotoxic agents, which results in significant side effects for a patient. Consequently, research efforts in this area have looked at ways to improve the therapeutic index of anticancer agents.

One approach is the use of prodrugs. A prodrug is an agent or compound that is converted into a pharmaceutically active form (often referred to as the parent compound) after administration into the body.

The major therapeutically relevant routes to prodrug activation are typically via enzymes e.g. Capecitabine, which is an orally available form of 5-fluorouracil undergoes three enzymatic reactions i.e. carboxylesterase, Cyd deaminase and dThdPase in liver or/and tumours, or physiological parameters such as pH e.g. Aldoxorubicin (INNO-206) which is an albumin-binding prodrug with release of doxorubicin under acidic conditions. Other, more academic prodrug stimuli have been explored in a variety of models, including electrochemical activation of metal-based prodrugs, ultrasound-induced release, or light mediated activation.

Concurrent chemotherapy and radiation has been shown to give significantly better overall survival for the treatment of rectal, lung and breast cancers than either alone. The combination of radiotherapy and hypoxia-activated prodrugs, such as evofosfamide and SN30000, (an analogue of tirapazamine) has also been reported. However, the 2 combined toxicities of the dual treatments are often prohibitive necessitating a reduction in the intensity of either or both treatment modalities and a reduction in efficacy.

Linear accelerator technology for the precise delivery of radiotherapy allows the treatment of a variety of tumour types, including breast, lung, head and neck, prostate, gastrointestinal and gynaecological cancers. Ionising radiation triggers a series of reactions in cells which generates a variety of reactive species (such as free electrons, and reactive oxygen species including 02·-, ·OH) in the tumour area. This gives rise to significant levels of DNA damage, including double-strand breaks, which in turn leads to reaction with intercellular oxygen, and ultimately cell death. This may explain why hypoxic tissues/cancers are radiation resistant, in the absence of oxygen, the DNA- derived free radicals are simply removed by glutathione.

US9408911 B (Korea Institute of Science and Technology) describes an anticancer prodrug consisting of peptide that is chemically linked to an anticancer drug. In this method, a patient is treated with radiation of radioactive ray or UV treatment to induce caspase activation in vivo. The anticancer prodrug is then cleaved by the activated caspase to release the anticancer drug.

W002/059122A1 (Auckland Uniservices Limited) describes 2, 3-dihydro-1 H-pyrrolo [3,2- f]quinoline complexes of cobalt and chromium and their use as prodrugs for the treatment of cancer. These complexes may be activated under hypoxic conditions by enzymes or by therapeutic ionising radiation. For example, the activation of metal complexes (via reduction - e.g. Co(lll) to Co(ll)) is described as triggering the release of a coordinated drug.

Tanabe et al (Bioorganic & medicinal chemistry letters 2012, 22(4): 1682-1685) describe a 5-fluorodeoxyuridine (5-FdUrd) derivative that contains an azide methyl group (N ₃ -CH2- FdUrd) and investigations into activation of this prodrug using X-ray radiation under hypoxic conditions.

Other examples of radiation- and photo-induced activation of related 5-fluorouracil prodrugs have been reported in Ito et al, Molecules, 2008, 13, 2370-2384; International Journal of Radiation Oncology*Biology*Physics, Volume 58, Issue 2, 2004, Pages 397- 402; Shibamoto et al, Jpn. J. Cancer Res. 91, 433-438, April 2000; and Mori et al, Org. 3

Chem. 2000 Jul 28;65(15):4641-7. However, there is a reported lack of in vivo effect (e.g. conversion to the active agent) when used at therapeutically relevant doses of radiation for all of the reported azide containing 5-fluorouracil-based prodrugs.

Thus, there is a need for new cancer therapies that address one or more of the above noted problems.

SUMMARY

The present disclosure is based on the finding that certain chemical moieties can be used as masking agents that can be activated, cleaved, removed and/or reacted upon application of ionising radiation. These masking agents may be conjugated, linked or bonded (e.g. covalently bonded) to a parent compound (such as an active agent, an imaging agent and/or probe compound) to provide a masked compound. In use, the application of ionising radiation may initiate, promote and/or facilitate the activation, cleavage, removal and/or reaction of the masking agent to yield the parent compound. In some instances, this approach can be used to promote and/or facilitate the unmasking of the masked compound at specific sites in a subject. In some examples, the masking agent may act as a linker and this approach can be used to promote and/or facilitate the cleavage of the linker at specific sites in a subject. This can provide a site-directed and/or targeted chemotherapy via the use of ionising radiation (e.g. radiotherapy) to unmask the masked compound at a target site.

In particular, the present disclosure relates to the finding that azide-containing moieties can be used as masking agents that may be conjugated, linked or bonded (e.g. covalently bonded) to a parent compound (such as an active agent, an imaging agent and/or probe compound) to provide a masked compound.

These azide-containing moieties may be activated, cleaved, removed and/or reacted upon application of ionising radiation. Indeed, it has been identified that certain azide- containing moieties may be activated, cleaved, removed and/or reacted upon application of therapeutic levels of ionising radiation. In other words, these types of azide-containing moieties are especially useful as masking agents as they can be unmasked (e.g. cleaved) to yield the parent compound at clinically relevant and/or therapeutically useful doses of ionising radiation. 4

Thus, according to a first aspect of this disclosure, there is provided a masked compound comprising a parent compound conjugated, linked or bonded to a masking agent. The masking agent may be or comprise an azide group. The masked compound may be unmasked to yield the parent compound upon application of or when subjected to a therapeutic level of ionising radiation.

In some examples, the masked compound may be a prodrug and the parent compound may be an active agent. In particular, the parent compound may be a cytotoxic agent. Accordingly, it is noted that any reference to a masked compound herein should also be considered to embrace the term “prodrug” and all the aspects, examples, and embodiments described in relation to a masked compound may be considered as equally applicable to the prodrug. It is additionally noted that any reference to a parent compound herein should also be considered to embrace the expressions “active agent”, “pharmaceutically active agent” and “cytotoxic agent”.

In other examples, the parent compound may be an imaging agent or probe compound (e.g. a fluorescent probe). In such examples, the masking group may be unmasked to yield the imaging agent or probe compound upon application of or when subjected to a therapeutic level of ionising radiation.

In some examples, the masking agent may act as a linker.

In some example, the masking agent may be used as a linker in an antibody drug conjugate (as is described in more detail below). In other examples, the masking agent may act as a chemical linker e.g. such as is found in solid phase synthesis and/or combinatorial chemistry. As used herein, solid phase synthesis may be a method in which molecules are covalently bound on a solid phase support material and synthesised step-by-step in a single reaction vessel using selective protecting group chemistry.

As such, the masking agent may act as a chemical linker attaching a chemical entity or chemical compound which is being synthesised to a solid phase support (e.g. a polymeric support material) or to another chemical entity. Further details of such synthetic strategies may be found in Linkers and Cleavage Strategies in Solid-Phase Organic Synthesis and Combinatorial Chemistry - Chem. Rev. 2000, 100, 2091-2157, 5 the contents of which are incorporated herein by reference. In use, the masking agent may be unmasked resulting in the cleavage of the chemical entity or compound upon application of or when subjected to ionising radiation (e.g. a therapeutic level of ionising radiation). Similar principles may apply when the masking agent is used as a linker in an antibody drug conjugate (which is explained in more detail below).

As used herein, ionising radiation may refer to subatomic particles or electromagnetic waves that have sufficient energy to ionize atoms or molecules by detaching electrons from them. Ionising radiation may be selected from gamma radiation, X-ray and higher ultraviolet radiation. As such, ionising radiation may mean radiation at the higher energy end of the electromagnetic spectrum, e.g. at energies greater than about 10eV, greater than about 25 eV, greater than about 50 eV or greater than about 100 eV. Ionising radiation may mean radiation at the shorter wavelength end of the electromagnetic spectrum (e.g. radiation having wavelengths less than about 10 nm).

Ionising radiation may be administered to a subject or patient by methods known in the art. Recent advances (e.g. linear accelerator technology such as the Cyberknife® and Gamma knife surgery which uses an array of intersecting beams of gamma radiation) have also facilitated a precise and accurate delivery of ionising radiation, meaning that radiotherapy may be tailored to conform to a target site in a patient (e.g. a tumour site). Accordingly, the approaches and methods described herein can be used to promote and/or facilitate the unmasking of the parent compound at specific sites in a subject

As used herein, a therapeutic level of ionising radiation may refer to a therapeutic dose of ionising radiation. For example, a therapeutic level of ionising radiation may be a dose between about 0.01 Gy and 100 Gy, between about 0.05 Gy and 75 Gy, between about 0.1 Gy and 50 Gy, between about 0.25 Gy and 25 Gy, between about 0.50 Gy and 10 Gy, or between about 1 and 7.5 Gy.

As noted above, the ionising radiation may initiate, promote and/or facilitate the activation, cleavage, removal and/or reaction of the masking agent to yield the parent compound. In some examples, the level of conversion and/or yield of the parent compound may depend upon the level or dose of radiation. The ionising radiation may be administered to the subject at a sufficient dosage and/or for a sufficient time period to provide and/or yield a desired and/or predetermined amount of the parent compound. 6

Additionally or alternatively, the masked compound may be administered to the subject at a sufficient dosage to provide and/or yield a predetermined amount of the parent compound. For example, in some cases, the ionising radiation and/or masked compound may be administered so as to provide and/or yield a therapeutically effective amount of the parent compound (e.g. an amount of parent compound that is sufficient to elicit a desired therapeutic effect).

In some cases, the application of ionising radiation may result in a partial or complete unmasking (e.g. conversion) of the masked compound to the parent compound. Following administration of a masked compound, the application of ionising radiation may provide a sufficient amount of parent compound to elicit a desired effect (e.g. a desired therapeutic effect). The degree of conversion of the masked compound to the parent compound is dependent upon the application of ionising radiation. The application of ionising radiation may cause the local activation of the masked compound (as is explained further in the later sections). As such, in some examples, the overall conversion of the masked compound to the parent compound may be low, however there may be a higher conversion of the masked compound to the parent compound at a site or location (e.g. in a subject) that is subjected to ionising radiation.

In other examples, at least about 1%, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the masked compound may be converted to the parent compound. In other examples, all (or substantially all) of the masked compound may be unmasked or converted to the parent compound.

The present inventors have surprisingly identified that aryl azides and sulfonyl azides may be unmasked or converted to aryl amines and sulfonamides respectively when subjected to therapeutic levels of ionising radiation. Thus, where a parent compound contains an aryl amine or sulfonamide moiety, the masking agent may be an azide group. In other words, an aryl amino group on the parent compound may be replaced with an aryl azide group to provide the masked compound. Similarly, a sulfonamide group on the parent compound may be replaced with a sulfonylazide group to provide the masked compound. 7

These findings have further led the present inventors to an azide-containing moiety (as shown and explained in more detail below) that can be activated, cleaved and/or removed to yield a parent compound. In particular, this azide-containing masking agent may be considered as a protecting group.

Accordingly, it is noted that any reference to a masking agent herein should also be considered to embrace the term “protecting group” and all the aspects, examples, and embodiments described in relation to the masking agent may be considered as equally applicable to the protecting group described below.

This azide-containing masking agent or protecting group may be conjugated, linked or bonded (e.g. covalently bonded) to a parent compound or compounds. Ionising radiation may initiate, promote and/or facilitate a self-immolative reaction of the azide-containing masking agent that results in the cleavage of the masking agent or protecting group to yield the parent compound (e.g. the self-immolative reaction of the azide-containing masking agent or protecting group may result in a cleavage of the bond between the protecting group and the parent compound). This masking agent or protecting group has the additional advantage of versatility in that it can be appended to a huge variety of parent compounds (and so is not limited to parent compounds having a specific chemistry, e.g. those containing an aryl amine or sulfonamide moiety). In addition, the masking agent or protecting group may be used as a linker.

As used herein, a self-immolative reaction may refer to a cascade of reactions that lead to the disassembly of a chemical moiety into a plurality of fragments. Within the context of the present disclosure, following activation by ionising radiation, an azide-containing masking agent or protecting group may be undergo a self-immolative reaction which results in its disassembly into a plurality of fragments and cleavage from the parent compound.

As used herein, the step of unmasking the masked compound to provide the parent compound may not be metal-activated and/or may not be mediated by an enzyme (such as an activated caspase enzyme).

In some cases, the masking agent or protecting group may be represented by formula

(I):

wherein:

A is selected from optionally substituted aryl and optionally substituted heteroaryl;

B is absent such that X is directly attached to an atom on the optionally substituted aryl or optionally substituted heteroaryl, or

B is -CR ¹ R ² 0(C=0)-, wherein R ¹ and R ² may each be independently selected from H and optionally substituted C1-C6 alkyl; and X represents the point of attachment of the parent compound.

On ring A, the azide (N ₃ ) and B groups may be held (e.g. covalently bonded) at any position on the optionally substituted aryl or heteroaryl (provided it has the correct valency and/or is chemically suitable). For example, the azide and B group may replace a hydrogen atom at any position on the aromatic or heteroaromatic group of ring A. In some cases, the azide and B group may be in a para or 1 ,4-substitution pattern on the aromatic ring. In some examples, the aryl or heteroaryl group of ring A may comprise one or more electron-withdrawing groups as substituents.

In some examples (e.g. where the masking agent or protecting agent is used as a linker in an antibody drug conjugate), the aryl or heteroaryl of ring A comprises at least one substituent that may serve as point of attachment for an antibody.

Where B is -CR ¹ R ² 0(C=0)-, this group may be attached to the ring A by way of the carbon of -CR ¹ CR ² - and to X via the carbon of the carbonyl group. As such, the masking agent may be represented by formula (la) 9

Wherein A, X, R ¹ and R ² are as defined above for formula (I).

As used herein, “C1-C6 alkyl” may be selected from straight or branched chain hydrocarbyl groups containing from 1 to 6 carbon atoms. Representative examples are methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, neohexyl, etc. When a C1-C6 alkyl group is substituted, any hydrogen atom(s), CH3,CH2 or CH group(s) may be replaced with the substituent(s), providing valencies are satisfied.

As used herein, the term "aryl" refers to a mono- or polycyclic aromatic hydrocarbon system having 6 to 14 carbon atoms. Representative examples of suitable "aryl" groups include, but are not limited to, phenyl, biphenyl, naphthyl, 1 -naphthyl, 2-naphthyl and anthracenyl. As used herein, “substituted aryl” refers to an aryl group as defined herein which comprises one or more substituents on the aromatic ring. When an aryl group is substituted, any hydrogen atom(s) may be replaced with the substituent(s), providing valencies are satisfied.

As used herein, “heteroaryl” may be a single or fused ring system having one or more aromatic rings containing 1 or more O, N and/or S heteroatoms. Representative examples of heteroaryl groups may include, but are not limited to, pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, indolyl, benzofuranyl, benzothiazolyl, benzimidazolyl, indazolyl, benzoxazolyl, benzisoxazolyl etc. As used herein, “substituted heteroaryl” refers to a heteroaryl group as defined herein which comprises one or more substituents on the 10 heteroaromatic ring. When a heteroaryl group is substituted, any hydrogen atom(s) may be replaced with the substituent(s), providing valencies are satisfied.

As used herein, the term “optionally substituted” means that the moiety may comprise one or more substituents.

As used herein, a “substituent” may include, but is not limited to, hydroxyl, thiol, carboxyl, cyano (CN), nitro (NO ₂ ), halo, haloalkyl (e.g. a Ci to C ₆ haloalkyl), an alkyl group (e.g. Ci to C ₁₀ or Ci to O _d ), aryl (e.g. phenyl and substituted phenyl for example benzyl or benzoyl), alkoxy group (e.g. Ci to C ₆ alkyl) or aryloxy (e.g. phenoxy and substituted phenoxy), thioether (e.g. Ci to C ₆ alkyl or aryl), keto (e.g. Ci to C ₆ keto), ester (e.g. Ci to O _d alkyl or aryl, which may be present as an oxyester or carbonylester on the substituted moiety), thioester (e.g. Ci to C ₆ alkyl or aryl), alkylene ester (such that attachment is on the alkylene group, rather than at the ester function which is optionally substituted with a Ci to O _d alkyl or aryl group), amine (including a five- or six-membered cyclic alkylene amine, further including a Ci to C ₆ alkyl amine or a Ci to C ₆ dialkyl amine which alkyl groups may be substituted with one or two hydroxyl groups), amido (e.g. which may be substituted with one or two Ci to C ₆ alkyl groups (including a carboxamide which is optionally substituted with one or two Ci to C ₆ alkyl groups), alkanol (e.g. Ci to C ₆ alkyl or aryl), or carboxylic acid (e.g. Ci to C ₆ alkyl or aryl), sulfoxide, sulfone, sulfonamide, and urethane (such as -0-C(0)-NR ₂ or-N(R)-C(0)-0-R, wherein each R in this context is independently selected from Ci to C ₆ alkyl or aryl).

As used herein, an electron withdrawing group may refer to any group which draws electron density away from neighbouring atoms and towards itself. Typically, the electron withdrawing group draws electron density away from neighbouring atoms and towards itself more strongly than a hydrogen substituent. Representative examples of suitable electron withdrawing groups include, but are not limited to, halo, Ci to C ₆ haloalkyl, -NO ₂ , -CONH ₂ , -CONH(Ci to C ₆ alkyl), -CON^ to C ₆ alkyl) ₂ , -SC^ to C ₆ alkyl), -CO ₂ H, - CC> ₂ (Ci to C ₆ alkyl), -CO(Ci to C ₆ alkyl) and -CN.

In some examples, and unless the context indicates otherwise, a “substituent” may include, but is not limited to, halo, Ci to C ₆ alkyl and C ₁ -C ₆ haloalkyl.

As used herein, a “halo” group may be F, Cl, Br, or I, typically F. 11

As used herein, “haloalkyl” may be an alkyl group in which one or more hydrogen atoms thereon have been replaced with a halogen atom. By way of a representative example, a C1-C ₆ haloalkyl may be a fluoroalkyl, such as trifluoromethyl (-CF ₃ ) or 1 , 1 -difluoroethyl (-CH2CHF2).

In some examples, the protecting group or masking agent may be represented by formula (lb): wherein X, R ¹ and R ² are as defined above for formula (I); n is in the range 0 to 4; and wherein each R ³ is independently selected from the group consisting of halo, C1-C6 alkyl and C1-C6 haloalkyl and/or wherein at least one R ³ may represent a point of attachment for an antibody.

In other words, when n is 0, the phenyl ring does not have any non-hydrogen substituents other than the azide group and the group linking to the parent compound X. Where n is 1 , 2, 3 or 4, each R ³ group replaces a hydrogen atom on the phenyl ring and is/are covalently bonded to a carbon atom(s) on the phenyl ring.

As shown in formula (lb), the substituent groups (e.g. N3, CR ¹ R ² 0(C=0)X and each R ³ (if present)) may be covalently bonded to the phenyl ring in any position and/or in any substitution pattern.

In some examples, the azide and CR ¹ R ² 0(C=0)X groups are covalently bonded to the phenyl ring in a 1,2-substitution or 1,4-substitution pattern. 12

Where the azide and CR ¹ R ² 0(C=0)X groups are bonded to the phenyl ring in a 1,4- substitution pattern, the azide-containing moiety may be represented by formula (lc): wherein X, R ¹ , R ² · R ³ and n are as defined above for formulae (I) to (lb).

In some examples of the disclosure, the masking agent according to any one of formulae (I) to (lc) comprises 1 or more halo substitutents (e.g. fluoro substituents) on the aryl or heteroaryl ring. By way of further example, one or more R ³ (as shown in formula (lb) or (lc)) may be halo, such as fluoro.

In some examples, the azide-containing moiety (e.g. the masking agent or protecting group) may comprise the following structure: 13

Wherein X is defined as above.

Without being bound by theory, under ionising radiation, the pendant azide group on the aryl azide group may be reduced to an amine and a subsequent 1,6 or 1,4 elimination reaction may cleave the masking agent or protecting group to yield the parent compound.

In the formulae shown above, X represents the point of attachment of the parent compound. The masking agent may be covalently bonded to the parent compound at any chemically suitable position e.g. by derivitisation of a chemically suitable position on the parent compound. By way of further example, the masking agent may be covalently bonded to the parent compound by way of an atom or moiety that is able to function as a leaving group. By way of example, the masking agent may be covalently bonded to the parent compound via a heteroatom on the parent compound (e.g. a nitrogen, oxygen or sulphur atom).

By way of example only, the parent compound may comprise an amino, hydroxyl or thiol moiety and the parent compound may be covalently linked to the masking agent or protecting group as described herein via this moiety.

In some cases, the masking agent may be covalently bonded to the parent compound by an -NR ⁴ - (wherein R ⁴ is optionally Ci to C ₆ alkyl, optionally substituted aryl, or optionally substituted heteroaryl), -O- or-S- linkage. 14

The parent compound may be covalently bonded to a masking agent as described herein via derivitisation of a pendant amino (-NH2), thiol (-SH) or hydroxyl (-OH) group that is present on the parent compound. In some examples, the parent compound is doxorubicin. The structure of doxorubicin is shown below: Doxorubicin is a chemotherapeutic agent that can be used to treat a variety of cancers (including, breast cancer, bladder cancer, Kaposi’s lymphoma and acute lymphocytic leukemia). However, serious side effects can accompany the use of this agent, e.g. it is known to cause heart damage. The present inventors have identified that the systemic toxicity of this compound can be shielded via the use of the masking agents described herein. In particular, a masked compound (or prodrug) comprising doxorubicin was found to significantly reduce and/or avoid the risk of heart damage in comparison to that observed when using doxorubicin.

In such examples, the masking agent or protecting group may be covalently bonded to doxorubicin via derivitisation of a pendant amino (-NH2) or hydroxyl (-OH) group.

In one example, the masked compound may comprise the following structure: 15

The masking agents or protecting groups as described herein (in particular with reference to formulae (I) to (lc)) may find further uses and application in antibody drug conjugates.

As used herein, an antibody drug conjugate comprises an antibody that is linked (e.g chemically and/or covalently linked) to an active agent (e.g. a pharmaceutically active agent, such as a cytotoxic agent). In general, in such conjugates, the antibody specifically binds an antigen, such as an antigen that is associated with and/or is more prevalent on diseased cells (e.g. tumour cells). This facilitates a targeted delivery of the active agent to the target cells and can limit the systemic toxicity of the active agent.

The masking agent or protecting group as described herein may be used as a linker between an antibody and an active agent. For example, the masking agent or protecting group may be conjugated, linked or bonded (e.g. covalently bonded) to the active agent in the manner described herein in relation to the parent compound. 16

The masking agent may be further conjugated, linked or bonded (e.g. covalently bonded) to the antibody at any chemically suitable position. By way of non-limiting example, the antibody may be conjugated, linked or bonded (e.g. covalently bonded) to the masking agent via a substituent on ring A of formulae (I) to (la). Additionally or alternatively, at least one R ³ on formula (lb) or (lc) may represent a point of attachment of an antibody to the masking agent.

An antibody may be conjugated, linked or bonded (e.g. covalently bonded) to the masking agent according to methods known in the art. For example, in some examples, the antibody may be covalently bonded to the masking agent via formation of an amide, carbamate, or disulfide linkage between a group on the masking agent and a group on the antibody (e.g. a reactive side chain of the antibody). By way of example only, a carboxylic acid group on ring A of formula (I) to (la) (or at an R ³ position in formula (lb) or (lc)) may react with an amino group on the antibody (e.g. as part of lysine residue) to covalently bond the masking agent to the antibody by way of an amide bond.

In use, ionising radiation (e.g. a therapeutic level of ionising radiation) may mediate, initiate, promote and/or facilitate the activation, cleavage, removal and/or reaction of the masking agent or protecting group from the masked compound to yield the parent compound as described previously. In other words, in such antibody drug conjugates, the masking agent or protecting group acts as a cleavable linker between the antibody and the active agent. Under ionising radiation, the masking agent or protecting group is activated and/or is cleaved to deliver the active agent (e.g. the “cargo”) to the target cell.

Such an approach may serve to further enhance the targeted and/or selective delivery of antibody drug conjugates in vivo and/or may allow the use of active agents that are too toxic to be used in isolation.

One example of such an active agent is monomethyl auristatin E. The structure of monomethyl auristatin E is illustrated below: 17

An exemplary antibody drug conjugate containing a masking agent as described herein may comprise a structure as illustrated schematically below:

In the example above, the masking agent comprises a 1,2 substitution pattern between the azide and carbamate group. It will be appreciated that those protecting groups with other substitution patterns may also be effective, e.g. a 1,4-substitution pattern. In particular, the linker in such antibody drug conjugates may be any of the masking agents or protecting groups described above (e.g. those described in formulae (I) to (lc)).

In other examples, where the parent compound comprises an optionally substituted aryl or optionally substituted heteroaryl amino moiety, the masking agent may be an azide group.

In other words, in these examples, the azide group replaces the pendant amino group on the aryl or heteroaryl ring of the parent compound. It has surprisingly been found that under therapeutic levels of ionising radiation, such aryl azides can be unmasked to provide the amino group of the parent compound. Under ionising radiation, this azide group may be cleaved and/or converted into the pendant amino group to provide the parent compound. As such, in some examples, the masked compound may be represented by formula (II): 18

Wherein C is selected from optionally substituted aryl and optionally substituted heteroaryl; and

X ¹ represents the remainder of the parent compound that is covalently bonded to ring C.

By way of non-limiting example only, the parent compound may be an amino-substituted coumarin. In such cases, the masked compound may have the following structure:

In yet other examples where the parent compound comprises a sulfonamide group, the masking agent may be a sulfonyl azide group. In other words, the sulfonyl azide group replaces the sulfonamide group of the parent compound. Again, it has unexpectedly been identified that under therapeutic levels of ionising radiation, such sulfonyl azides can be unmasked to provide the sulfonamide group of the parent compound.

As such, in some examples, the masked compound may be represented by formula (III):

X ² — SO ₂ N ₃ (HI) Wherein X ² represents the remainder of the parent compound. 19

By way of non-limiting example only, the parent compound may be pazopanib. Pazopanib comprises a sulfonamide group. As such, the masking agent may be a sulfonyl azide group. Accordingly, the masked compound may have the following structure:

According to a further aspect of the disclosure, there is provided a method of unmasking a masked compound. The method may comprise unmasking the masked compound using ionising radiation (e.g. a therapeutic level of ionising radiation).

Such methods have a wide range of applications, including for use in therapy, as probes, and as cleavable linkers (e.g. in solid phase synthesis and/or combinatorial chemistry).

The step of unmasking a masked compound may comprise using the therapeutic level of ionising radiation to initiate, promote and/or facilitate the activation, cleavage, removal and/or reaction of the masking agent to yield the parent compound.

The method of unmasking a masked compound may be carried out in vivo or in vitro.

When carried out in vitro, the masked compound may be contacted with a cell (or cells) in combination with ionising radiation.

When carried out in vivo, the masked compound may be administered to a subject in need thereof in combination with ionising radiation (e.g. a therapeutic level of ionising radiation). The application of the ionising radiation may be targeted or directed to a specific site in a subject such that the parent compound is unmasked at that site. 20

According to further aspects of the disclosure, there is provided a method of making a masked compound (e.g. a prodrug) as described herein.

The method may comprise covalently bonding a masking agent as described herein to a parent compound to provide the masked compound.

In some examples, the method may comprise the steps of:

(a) providing a parent compound;

(b) providing a masking agent as defined herein; and

(c) bonding (e.g. covalently bonding) the parent compound to the masking agent to provide the masked compound.

In some examples, the method may comprise converting, reacting and/or derivatizing an aryl amino on a parent compound to provide an aryl azide group.

In some examples, the method may comprise converting, reacting and/or derivatizing a sulfonamide on a parent compound to provide a sulfonyl azide group.

According to further aspects of the disclosure, there is provided a use of a masking agent as described herein (e.g. as shown in any of formulae (I) to (III) or otherwise exemplified above) to mask a parent compound and/or to provide a masked compound (e.g. a prodrug).

As described herein, the masked compound may be unmasked upon application of ionising radiation.

As noted above, in some instances the parent compound may be an active agent, such as a pharmaceutically or therapeutically active agent.

By way of representative example only, the parent compound may be an anticancer agent. In some examples, the parent compound may be selected from one of the following compounds: 21 idarubicin bleomycin; dactinomycin; etoposide; gemcitabine; toremifene; and vinorelbine.

In some examples, the parent compound may be one or more of the following compounds: amikacin, natamycin, amphotericin b, nystatin, vancomycin, sulphanilamide, quinethazone, hydrochlorothiazide, dichlofenamide, furosemide, hydroflumethiazide, chlorthalidone, metolazone, indapamide, celecoxib, cyclothiazide, bendroflumethiazide, benzthiazide, and polythiazide.

In those instances where the parent compound is an active agent, the masked compound may be considered as a prodrug. As used herein, a prodrug may refer to a compound that is converted into the pharmaceutically active agent following administration to a subject in need thereof. Within the context of the present disclosure, the prodrugs may 22 be converted into the pharmaceutically active agent upon application of or when subjected to ionising radiation (e.g. a therapeutic level of ionising radiation).

Accordingly, a further aspect of the disclosure provides a masked compound (or prodrug) as described herein for use in a method of therapy.

In particular, under ionising radiation (e.g. therapeutic levels of ionising radiation), it has been demonstrated that the masked compounds (or prodrugs) described herein can be activated to release and/or provide active agents that modulate (e.g. inhibit) cell growth and/or activity (e.g. a cytotoxic agent). As such, it will be appreciated that there are a number of medical and veterinary applications for these masked compounds (or prodrugs). For example, a masked compound or prodrug as described herein may be applied to the treatment and/or prevention of diseases in which aberrant cell growth, and/or aberrant cell activity is a factor.

Thus, there is provided:

(i) a prodrug as described herein for use in a method of treating and/or preventing a disease and/or condition caused, contributed to and/or characterised by aberrant cell growth and/or activity;

(ii) use of a prodrug in the manufacture of a medicament for use in a method of treating and/or preventing a disease and/or condition caused, contributed to and/or characterised by aberrant cell growth and/or activity;

(iii) a method of treating and/or preventing a disease and/or condition caused, contributed to and/or characterised by aberrant cell growth and/or activity, said method comprising the step of administering a therapeutically effective amount of a prodrug to a subject in need thereof.

The above uses and methods may comprise administering a prodrug as described herein to the subject in need thereof in combination with a therapeutic level of ionising radiation. The therapeutic level of ionising radiation may initiate, promote and/or facilitate the activation, cleavage, removal and/or reaction of the masking agent from the prodrug to yield an active agent that modulates (e.g. inhibits) cell growth or activity.

Diseases which are caused, contributed to or characterised by aberrant cell growth and/or activity may include, for example cell proliferation and/or differentiation disorders 23 including, those referred to or classified as benign or malignant conditions. For example, the term “cell proliferation and/or differentiation disorders” may include those diseases and/or conditions collectively referred to as “cancer”. The term “cancer” may include, but is not limited to, those cancers referred to as forms of breast cancer, bone cancer, brain cancer (gliomas), pancreatic cancer, lung cancer, prostate cancer, skin cancer, ovarian cancer, cervical cancer, head and neck cancers and bowel/colon cancer. The term “cancer” may also include those diseases and/or conditions collectively referred to as “leukaemias” (both chronic and acute) and any cancer affecting a mucosal/mucosal associated surface or tissue.

A subject in need thereof or indeed a subject to be administered a masked compound or prodrug as disclosed herein or a medicament comprising the same, may be any subject suffering (or suspected as suffering) from (i) a cell proliferation and/or differentiation disorder, (ii) cancer, (iii) any other disease and/or condition described herein; or (iv) a disease or condition caused, contributed to or characterised by aberrant cell growth and/or activity. Additionally, or alternatively, any subject may be a subject predisposed or susceptible to (i) a cell proliferation and/or differentiation disorder, (ii) cancer, (iii) any other disease and/or condition described herein; or (iv) a disease or condition caused, contributed to or characterised by aberrant cell growth and/or activity.

It should be understood that the treatment of a cell proliferation and/or differentiation disorder, a cancer or a disease or condition caused or contributed to (or characterised by) aberrant cell growth and/or activity, may involve the use of one or more masked compounds or prodrugs of this disclosure to treat, ameliorate or reduce, one or more symptoms of those diseases. By way of example, the symptoms of a disease such as cancer may include, for example, the presence of tumours and/or cell masses. As such, the prodrugs described herein may be used to modulate (for example stop, retard, inhibit or reduce) tumour formation and/or the metastasis thereof. The prodrugs of this disclosure may also be used to reduce the overall size of a tumour. Certain tumours, including those that are large and/or aggressive, are often easier to surgically remove if they have first been reduced in size. The methods described herein (which combine both chemotherapeutic and radiotherapeutic treatments) might be used to reduce the size of a tumour prior to a surgical procedure. Thus, the successful treatment of a tumour may therefore be characterised by a reduction in tumour size, a reduction in an observed or 24 detectable/detected level of tumour metastasis, angiogenesis within tumorigenic tissue and/or tissue invasion.

Thus, the prodrugs described herein may be of use in methods of modulating (for example inhibiting, restricting or reducing) tumour growth, development and/or metastasis in subjects in need thereof. The prodrugs described herein may be formulated as compositions for use in modulating tumour growth, development and/or metastasis or used in the manufacture of medicaments for achieving the same. This disclosure also provides a prodrug for use in treating a tumour. Further, described is the use of a prodrug for the manufacture of a medicament for treating a tumour. Also, the disclosure provides a method of treating a tumour, said method comprising administering a prodrug to a subject (or tumorigenic tissue) in need thereof in combination with therapeutic levels of ionising radiation.

Many anticancer agents are highly toxic and global systemic toxicity is a major drawback associated with many chemotherapies. However, the present inventors have recognised that the prodrugs described herein can mitigate and/or address some of these problems. In particular, the present inventors have identified that the prodrugs described herein can be locally activated to yield the active agent using radiotherapy (e.g. a therapeutic level of ionising radiation). As such, the compounds and methods disclosed herein can allow a “real time” unmasking (or decaging) of a prodrug at the target site, thus providing a targeted and/or directed delivery of the active agent.

As such, a prodrug described herein may find particular application in the treatment and/or prevention of cancer. Thus, there is provided:

(i) a prodrug for use in a method of treating and/or preventing cancer;

(ii) use of a prodrug in the manufacture of a medicament for use in a method of treating and/or preventing cancer; and/or

(iii) a method of treating and/or preventing cancer, said method comprising the step of administering a therapeutically effective amount of a prodrug to a subject in need thereof.

The above uses and methods may comprise administering a prodrug as described herein to the subject in need thereof in combination with a therapeutic level of ionising radiation. The therapeutic level of ionising radiation may initiate, promote and/or facilitate the 25 activation, cleavage, removal and/or reaction of the masking agent from the prodrug to yield an active agent that is effective in the treatment and/or prevention of cancer.

As used herein, the term “subject” or “patient” may refer to an animal, such as a mammal (e.g. a human), to whom treatment, including prophylactic treatment, with the compounds and/or compositions according to the present disclosure is provided. In some cases, “a subject in need thereof” may embrace any subject diagnosed or suspected to be suffering from cancer and/or any subject diagnosed or suspected as having a tumour and/or subjects that are identified as being predisposed and/or susceptible to tumours.

As explained previously, the prodrugs as described herein can be activated to yield the active agent using radiotherapy (e.g. a therapeutic level of ionising radiation). Thus, in some cases, the methods can be considered as a combination therapy wherein the prodrug is administered to a subject in need thereof in combination with the administration of ionising radiation (e.g. a therapeutic level or dose of ionising radiation.

As used herein, in the context of administering the prodrug in combination with the ionising radiation, the expression “in combination with” may refer to the delivery of the prodrug to a subject either concurrently or at a different time to the step of administering ionising radiation.

By way of example, the ionising radiation may be administered to the subject simultaneously with the administration of the prodrug.

In other examples, the prodrug may be administered to the subject in need thereof and, subsequently, ionising radiation may be administered to the subject. By way of example, the ionising radiation may be administered to the subject at a set time period following administration of the prodrug to the subject, e.g. at any time between about 30 seconds and 30 minutes, between about 1 minute and 60 minutes, between about 1 hour and 24 hours, or between about 1 day and 14 days after administration of the prodrug. In some examples, the subject may be administered ionising radiation between about 0.5 hour and 24 hours, between 1 hour and 10 hours, or between 2 hours and 6 hours, or about 4 hours after administration of the prodrug. 26

As explained previously, the use of radiotherapy (e.g. a therapeutic level of ionising radiation) to activate the prodrug to release the active agent (e.g. a cytotoxic agent) has the benefits of limiting the global systemic toxicity of many anticancer agents. In particular, the use of ionising radiation to activate the prodrug can allow a medical practitioner to target the timing and/or delivery of the active agent to specific locations in a subject, thus minimising the toxic effects of these agents in other areas of the body.

There is further provided a method (e.g. an in vitro method) of modulating the growth and/or activity of a cell, said method comprising contacting a cell (or cells) with a prodrug as described herein in combination with the administration of ionising radiation to the cell(s) (in particular a therapeutic level of ionising radiation). Such methods can be useful as assays to identify masked compounds (e.g. prodrugs) suitable for use in the methods described herein.

The method may further comprise detecting a change in the growth and/or activity of the cell(s) following administration of:

(i) the masked compound (or prodrug) in combination with the ionising radiation;

(ii) the masked compound (or prodrug) in the absence of ionising radiation; and/or

(iii) the parent compound.

The method may comprise comparing the change in the growth and/or activity in the cell(s) following administration of the masked compound (or prodrug) in combination with the ionising radiation to a change in the growth and/or activity of the cell(s) that is detected following administration of:

(i) the masked compound (or prodrug) in the absence of ionising radiation; and/or

(ii) the parent compound.

By way of example, a masked compound (or prodrug) suitable for the uses described herein e.g. as part of a combination treatment with ionising radiation, may show an increased level of inhibition and/or reduction in cell growth and/or activity when administered in combination with ionising radiation than is observed when the masked compound or prodrug is contacted with the cell(s) in the absence of ionising radiation.

Additionally or alternatively, a masked compound (or prodrug) suitable for the uses described herein e.g. as part of a combination treatment with ionising radiation, may 27 show a decreased level of inhibition and/or reduction in cell growth and/or activity than is observed when the parent compound (e.g. an active agent, such as a cytotoxic agent) is contacted with the cell(s).

A prodrug suitable for the uses described herein, in particular as part of combination treatment with ionising radiation, may show substantially the same level of modulation (e.g. inhibition) on the cell growth and/or activity as the parent compound (e.g. an active agent such as a cytotoxic compound). In some cases, the combination treatment may show an increased level of modulation (e.g. inhibition) on the cell growth and/or activity over that observed when the cell is contacted with the parent compound/active agent (e.g. cytotoxic agent).

In some cases, the combination treatment may show an increased level of modulation (e.g. inhibition) on the cell growth and/or activity over that shown by the parent compound. For example, in some cases, the combination of the prodrug and therapeutic level of ionising radiation has a synergistic therapeutic effect over the use of either the parent compound or the ionising radiation.

By way of an example, in those cases where the parent compound inhibits cell growth and/or activity, the prodrugs described herein (when administered in combination with ionising radiation) may show substantially the same level of inhibition on the cell growth and/or activity (e.g. about 100%, at least about 95%, at least about 90%, at least about 75% or at least about 50% of the inhibition shown by the parent compound). In some examples, the prodrugs described herein (when administered in combination with ionising radiation) may show an increased level of inhibition on the cell growth and/or inhibition in comparison to that observed when the cell is contacted with the parent compound or when the cell is administered the same level of ionising radiation in the absence of the drug.

One measure of the effect of the prodrugs on the growth and/or activity of a cell may be the cell viability. Therefore, the prodrugs described herein (when administered in combination with ionising radiation) may decrease and/or reduce cell viability. In some examples, the prodrugs may be administered in combination with ionising radiation (e.g. a therapeutic level of ionising radiation) to reduce cell viability by about 10%, about 20%, 28 about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, up to about 100%.

A prodrug suitable for the uses and methods described herein may show no effect (or substantially no effect) on the cell growth and/or activity in the absence of ionising radiation. For example, in the absence of ionising radiation, the prodrug may show no effect (or substantially no effect) on cell viability following contact with a cell (or cells). Alternatively, in the absence of ionising radiation, a prodrug suitable for the uses and methods described herein may show a decreased level of inhibition or reduction in cell growth and/or activity than is observed when the parent compound is contacted with the cell(s). For example, in the absence of ionising radiation, the prodrug may reduce cell viability to a lesser extent than the parent compound.

A prodrug may be formulated for use and as a therapeutic or pharmaceutical composition. The various compositions may comprise one or more of the prodrugs described herein and any given treatment may require the administration (together, concurrently or separately) of one or more of these compositions.

The various prodrugs described herein may be formulated for enteral (including oral), parenteral and/or topical administration and one of skill will appreciate that the precise formulation may vary depending on the route of administration. Pharmaceutical compositions according to the present invention may be prepared conventionally, comprising substances that are customarily used in pharmaceuticals and as described in, for example, Remington's The Sciences and Practice of Pharmacy, 22nd Edition (Pharmaceutical Press 2012) and/or Handbook of Pharmaceutical Excipients, 7th edition (compiled by Rowe et al, Pharmaceutical Press, 2012) - the entire content of all of these documents and references being incorporated by reference.

A therapeutic or pharmaceutical composition of this disclosure (that is a composition comprising a prodrug and for use in any of the medicaments or methods described herein - including the methods of or medicaments for, modulating cell growth and/or activity and/or treating cancer) may be formulated together with one or more pharmaceutically acceptable excipients, carriers, adjuvants and buffers. The compositions can be administered, e.g. orally (including mucosally), parentally, enterally, intramuscularly, subcutaneously, intravenously or via any other routes useful to achieve the desired effect 29

(in this case effects which include, modulation of cell growth/activity, treatment or prevention of diseases/conditions associated with the same and/or cancer and/or modulation of tumour growth). As stated, depending on the chosen route of administration, the exact composition of the formulation may vary.

A therapeutic or pharmaceutical formulation comprising a prodrug and for administration to a subject may be coated, encapsulated or enveloped in a material which protects the prodrug from the action of enzymes, acids and other natural compounds/conditions (including, for example, compounds (including antibodies), cells and processes of the immune system) which may inactivate the compound and/or cleave or remove the masking agent (e.g. prior to the application of ionising radiation).

Among the various standard and conventional excipients that may be available for use in compositions comprising the prodrugs described herein, are those pharmaceutically acceptable organic or inorganic carrier substances which are suitable for parenteral, enteral, oral (including mucosal) and other routes of administration that do not deleteriously react with the prodrugs described herein

Where the prodrugs as disclosed herein are to be formulated for parental administration, the compositions may be sterile.

The composition may comprise an oil-based or aqueous solution, a suspension and/or an emulsion.

In other embodiments, the composition may take the form of an implant, such as for example a (dissolvable or biodegradable) film, pessary or implant (including suppositories).

The pharmaceutical preparations comprising the prodrugs as described herein may be mixed with stabilizers, wetting agents, emulsifiers, salts (for use in influencing osmotic pressure), buffers and/or other substances that do not react deleteriously with the prodrugs. 30

One or more of the prodrugs described herein may be formulated for and administered, orally. As stated, oral administration would include mucosal administration which would itself would include administration intranasally and/or by inhalation.

Compositions for use may include solid dosage forms which are suitable for oral administration. These may include, for example capsules, tablets, pills, powders, and granules. In any given solid dosage form, a prodrug as described herein may be admixed with at least one inert pharmaceutically-acceptable excipient. Examples of suitable excipients will be known to one of skill in this field but may include, for example fillers or extenders, humectants, wetting agents, binders, disintegrating agents, solution retarders, absorption accelerators, adsorbents, lubricants or mixtures thereof. A tablet, pill or capsule may further comprise a buffering agent. Solid dosage forms such as tablets, dragees, capsules, pills and/or granules also can be prepared with coatings and shells, such as coatings which protect against the gastrointestinal environment and/or stomach acid.

A solid dosage form may contain opacifying agents and can also be formulated so as to ensure the delayed release of the prodrug in or to a specific part of the intestinal tract.

Solid compositions for oral administration can be formulated in a unit dosage form, each dosage containing an appropriate dose of the prodrug. The exact amount of prodrug contained within any given solid dosage form will vary depending on the intended use. A solid composition may contain a “unit dose” - a unit dose containing a quantity of the prodrug calculated to produce the desired effect (for example modulation of cell growth and/or activity) over the course of a treatment period.

Liquid dosage forms for oral administration may (as stated) include emulsions, solutions, suspensions, syrups, and elixirs. In addition to the compound or composition, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers.

The prodrug may be used in any suitable amount. As stated, the prodrugs may be formulated for oral, mucosal or parenteral administration and as such, the precise formulation may depend on the intended route of administration. The amount of prodrugs present in any given dose may be in the region of 0.1 pg -1000 pg. For example, amounts 31 of about 0.1 pg, 0.2 pg, 0.3 pg, 0.4 pg, 0.5 pg, 1 pg, 10 pg, 20 pg, 25 pg, 50 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg or about 900 pg. Higher amounts of any of the prodrugs described herein may also be used including amounts from about 1mg to about 1000mg. For example (and depending on the route of administration and/or the organism to which the prodrug is to be administered (for example a human)) about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 20 mg, 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg or about 900 mg of a prodrug may be administered. The selected amount of the prodrug may be formulated in a specific volume of a pharmaceutically acceptable excipient, diluent and/or buffer. The actual volume required may depend on the dose of prodrug to be administered, the route of administration and/or the duration of any treatment regime. The volume of excipient, diluent or buffer may be about 10 mI to 5 ml. For example, the required amount of prodrug may be combined (or formulated) with about 15 mI, 20 mI, 25 mI, 30 mI, 35 mI, 40 mI, 45 mI, 50 mI, 55 mI, 60 mI, 65 mI, 70 mI, 75 mI, 80 mI, 85 mI, 90 mI, 95 mI, 100 mI, 200 mI, 250 mI, 300 mI, 400 mI, 500 mI, 600 mI, 700 mI, 800 mI, 900 mI, 1 ml, 2 ml, 3 ml or 4ml. For example, an amount of 100 pg prodrug may be combined with about 250 pi of excipient to yield a final prodrug concentration of 400 pg/ml. Doses at concentrations of about 0.1 pg/ml-1 mg/ml may be used including, for example, doses at 5 pg/ml, 10 pg/ml, 20 pg/ml, 25 pg/ml, 50 pg/ml, 100 pg/ml, 200 pg/ml, 300 pg/ml, 500 pg/ml, 600 pg/ml, 700 pg/ml, 800 pg/ml or 900 pg/ml.

In use, a dose of a prodrug as described herein, administered as part of the treatment and/or prevention of a cell proliferation and/or differentiation disorder (for example cancer), may be administered multiple times over a number of days, weeks months or years. For example, after an initial (or first) administration, a dose of a prodrug may be administered again at about (+/- 1 or 2 days) 3, 4, 5, 6, 7, 17, 21, 28 and/or 35 days later. On any given day, a specific dose of a prodrug may be administered 1, 2, 3 or more times. Each time, the prodrug may be administered (by whatever route is considered best to affect a suitable treatment or to induce prophylaxis against the development of a cell proliferation and/or differentiation disorder.

According to a further aspect of the disclosure there is provided a kit or package comprising a masked compound as described herein (or a pharmaceutical composition thereof) and a set of instructions for use. 32

The set of instructions may instruct a user to administer the masked compound in combination with ionising radiation. The set of instructions may instruct the user to administer the masked compound to a subject in need thereof in combination with a therapeutic level of ionisation.

Throughout this specification, the terms “comprise”, “comprising” and/or “comprises” is/are used to denote that aspects and embodiments of this invention “comprise” a particular feature or features. It should be understood that this/these terms may also encompass aspects and/or embodiments which “consist essentially of” or “consist of” the relevant feature or features.

DETAILED DESCRIPTION

The present disclosure will now be described, by way of example only, with reference to the following Figures:

Figure 1 shows a schematic illustration of the strategy to activate a cancer prodrug by the application of clinically relevant doses of gamma/X-ray radiation. This may also be referred to as drug or probe decaging via X-ray irradiation. (Coumarin is given as an exemplary probe model, and pazopanib and doxorubicin were used as exemplary anticancer agents).

Figure 2 shows a model reaction of compounds 1 , 4 and 6 under X-ray irradiation (a) Model reaction of 4-acetamidobenzenesulfonyl azide (compound 1) under X-ray irradiation (b) HPLC traces of 1 and irradiated 1 (20 mM, 60 Gy, 6 Gy/min) that afforded products 2 (RT = 2.61 min) and 3 (RT = 2.83 min) with a full conversion of 1 (RT = 4.89 min) (c) Model reaction of 4-(hydroxymethyl)-2,3,5,6-tetrafluoroaryl azide (compound 4) under X-ray irradiation and resulted in 4-(hydroxymethyl)-2,3,5,6-tetrafluoroaniline (compound 5). (d) HPLC traces of 4 (RT = 4.31 min) and irradiated 4 (20 pM, 60 Gy, 6 Gy/min) that afforded products 5 (RT = 3.01 min) (e) Model reaction of 4-azidobenzoic acid (compound 6) under X-ray irradiation (f) HPLC traces of 6 (RT = 3.99 min) and irradiated 6 (20 pM, 60 Gy, 6 Gy/min) that afforded poor conversion to 4-aminobenzoic acid (compound 7) (RT = 1.79 min). HPLC chromatogram showing UV absorbance at 254 nm. 33

Figure 3 shows X-ray irradiation mediated activation of azido coumarin. (a) Irradiation of 7-azido-4-methylcoumarin 8 resulting in the generation of 7-amino-4-methylcoumarin 9 with fluorescence turn-on. (b) HPLC traces of the reaction mixture of 8 (RT = 4.57 min) after irradiation with 6 Gy to 60 Gy and afforded product 9 ( RT = 3.21 min) (c) Graph showing the yield of 9 generated with different irradiation doses from 6 Gy to 60 Gy. The data are presented as mean i s.d. (n = 3).(d) Fluorescence spectrum of 8 (100 mM in PBS, pH = 7.4, A _ex/em = 345/443) with irradiation with 6 Gy to 60 Gy with the fluorescence intensity increasing with irradiation dose (e) Flow cytometry analysis (A _ex /em = 360/450 nm) of cells incubated with 8 (100 pM) with 9 generated in cells following 0 Gy, 6 Gy, 36 Gy and 60 Gy irradiation.

Figure 4 shows the synthesis of the pazopanib prodrug 11 and development of the irradiation reaction (a) Synthesis of 11 from pazopanib (10, 83 mM) in H ₂ 0//-PrOH with imidazole-1 -sulfonyl azide (1.5 equiv.) and K2CO3 (4 equiv.), 18 h, yield 65%. (b) The reaction of prodrug 11 under irradiation afforded products, 10 (pazobanib) and 12. (c) HPLC traces of the reaction mixture of 11 (RT = 5.44 min) after irradiation with 6 Gy to 60 Gy and afforded product 10 (RT = 4.23 min) and 12 (RT = 4.83 min) with some remaining unreacted 11. (d) The yield of pazopanib 10 generated from 11 (5, 10 and 20 pM) under the irradiation from 6 Gy up to 60 Gy (6 Gy/min). The data are presented as mean i s.d. (n = 3). (e) HUVEC viability upon treatment of 11 (0, 5 and 10 pM) and X- ray irradiation (0, 6, 12 and 24 Gy). The data are presented as mean ± s.d. (n = 3) (f) HT- 29 tumour bearing BALB/c nude mice were treated with prodrug 11 by intratumoural injection. 4 h post injections, mice were treated with or without 6 Gy X-ray irradiation. Tumour burdens were measured every other day using a calliper. The data are presented as mean ± s.d. (n = 5) (g) Survival curves for HT29 tumour bearing mice. Mice were aged until moribund or the tumour volume reached 2000mm ³ . (n = 5)

Figure 5 shows DOX prodrug activation by X-ray irradiation (a) The reaction of the DOX prodrug 13 and the hypothetical mechanistic pathway for liberation of doxorubicin, 15. (b) HPLC traces of the reaction mixture of 13 after irradiation with 6 Gy to 60 Gy, showing unreacted prodrug 13 (RT = 5.40 min), 14 (RT = 4.36 min), DOX 15 (RT = 3.14 min) and 5 (RT = 3.03 min) (c) The yield of doxorubicin 15 generated from 13 (10 pM) under the irradiation from 6 Gy up to 60 Gy (1 to 10 min, 6 Gy/min, quantification were carried out 24 h after irradiation). The data are presented as mean ± s.d. (n = 3). (d) HeLa cells were incubated with prodrug 13 (0.5, 1 , 5 and 10 pM) for 4 h, followed by X-ray irradiation 34

(from 6 Gy to 60 Gy) and cell viability measured using an MTT assay after 24 h incubation at 37 °C. The data are presented as mean ± s.d. (n = 6). (e) HeLa tumour bearing BALB/c nude mice were treated with prodrug 13 by intratumoural injection. 4 h post injections, mice were treated with or without 6 Gy X-ray irradiation. Tumour burdens were measured every other day using a calliper. The data are presented as mean t s.d. (n = 5) (f) Survival curves for HT29 tumour bearing mice. Mice were aged until moribund or the tumour volume reached 2000mm ³ . (n = 5) (g) Biochemical markers CK, CK-MB and LDH levels in plasma 48 h after PBS, 13 and 15 treatments. The data are presented as mean t s.d. (n = 5). Statistical analysis was performed using one-way ANOVA with Dunnett post-test compared to PBS treated mice, ns (not significant).

Methods

Synthesis of compound 4 p-Toluenesuffonic acid

Compound 5 and 4 was synthesised following reported procedure (Matikonda, S. S.; Fairhall, J. M.; Fiedler, F.; Sanhajariya, S.; Tucker, R. A. J.; Hook, S.; Garden, A. L; Gamble, A. B., Mechanistic Evaluation of Bioorthogonal Decaging with trans- Cyclooctene: The Effect of Fluorine Substituents on Aryl Azide Reactivity and Decaging from the 1 ,2,3-Triazoline, Bioconjug. Chem. 2018, 29, 324-334).

Compound 5 was synthesised giving a brown solid in a 55% yield (950 mg). Compound 4 was synthesised giving a white solid in a 92% yield (99 mg).

Synthesis of prodrug 7 35

Pazopanib 10 (218 mg, 0.5 mmol) and K2CO3 (276 mg, 2 mmol) were dissolved in 6 ml_ of a 1 :1 mixture hhO/i-PrOH to which was then added imidazole-1 -sulfonylazide hydrochloride (157 mg, 0.75 mmol). After being stirred for 18 h at ambient temperature, the reaction mixture was diluted with saturated Na _H CC>3 (30 ml_) and extracted with EtOAc (2 x 60 ml_). The combined organic phases were washed twice with brine and dried over Na2SC>4 and the volatiles evaporated in vacuo. The residue was purified by silica gel chromatography (5% MeOH in DCM) to afford the prodrug 11 as a white solid (151 mg, 65%): ¹ H NMR (500 MHz, d6-DMSO) d (ppm) = 8.87 (d, J = 2.4 Hz 1 H), 7.88- 7.85 (m, 2H), 7.77 (d, J = 8.8 Hz, 1 H), 7.46 (s, 1H), 7.39 (d, J = 8.4 Hz, 1H), 6.90 (m, 1 H), 5.78 (d, J = 2.4 Hz 1H), 4.06 (s, 3H), 3.50 (s, 3H), 2.63 (s, 3H), 2.52 (s,3H); ¹³ C

NMR (125 MHz, d6-DMSO) d (ppm) = 162.40, 159.13, 155.76, 146.96, 141.74, 140.02, 135.66, 133.37, 132.21 , 128.47, 124.36, 121.93, 119.55, 117.94, 114.05, 97.14, 48.59, 37.97, 37.38, 18.98, 9.41.; IR (solid) cm- ¹ : 2934, 2848, 2123, 1734, 1614, 1573,, 1517,1423, 1402, 1361, 1234, 1163; HRMS (ESI) for C21H21N9O2S [M+H]+ : calcd.: 464.1612; found: 464.1604.

Synthesis of doxorubicin prodrug 13

Compound 13 was synthesised following a reported procedure giving a dark red solid in an 81% yield (39 mg) (see, for example, (Matikonda, S. S.; Fairhall, J. M.; Fiedler, F.; Sanhajariya, S.; Tucker, R. A. J.; Hook, S.; Garden, A. L; Gamble, A. B., Mechanistic Evaluation of Bioorthogonal Decaging with trans-Cyclooctene: The Effect of Fluorine 36

Substituents on Aryl Azide Reactivity and Decaging from the 1,2,3-Triazoline. Bioconjug. Chem. 2018, 29, 324-334).

X-ray source: A linear accelerator (Clinac iX from Varian Medical Systems) generated X-rays of nominal energy 6 MeV (with a Bremsstrahlung distribution of 2 - 6 MeV) were used, with a dose rate of 600 cGy per minute with samples treated at a depth of 1015 mm from the tungsten target with a build-up of 15 mm of solid water. The linear accelerator used was a Varian Medical Systems VitalBeam medical linear accelerator. The dosage rate of 6 Gy/min was used for all experiments.

Monitoring model reactions under X-ray irradiation

Selected model compounds (see chemical structures in Table 1 and Fig. S1) were dissolved in DMSO to give stock solutions of 100 mM and diluted in PBS (20 ml_) to give a final concentration of 10 mM. The solutions were degassed by bubbling Ar for 30 min, followed by X-ray irradiation (0 to 60 Gy, 0 to 10 min). The reaction mixtures after irradiation were analysed by HPLC.

Reaction of coumarin azide 8 under X-ray irradiation

A stock solution of coumarin azide 8 (1 M in DMSO) was diluted in PBS (20 ml_) to a final concentration of 100 pM. The solution was degassed by bubbling Ar for 30 min before X-Ray irradiation (0 to 60 Gy). The fluorescence intensity of the reaction mixtures was analysed. The product 9 was isolated by prep-HPLC and characterised by ¹ HNMR and HRMS.

Reaction of coumarin azide 8 in live cells under X-ray irradiation

Hela cells were seeded in 24-well plates at a density of 5 ^c 10 ⁴ cells per well and incubated overnight. The cells were then treated with coumarin azide 8 (100 pM) 1 h prior to irradiation (0, 6, 36 and 60 Gy) and analysed by flow cytometry using a DAPI filter (l _{c/e m} = 360/450 nm) and confocal microscopy.

Reaction of prodrug 11 under X-ray irradiation

A stock solution of coumarin azide 11 (100 mM in DMSO) was diluted in PBS (20 ml_) to a final concentration of 20 pM. The solution was degassed by bubbling Ar for 30 min before X-Ray irradiation (0 to 60 Gy). The reaction mixture was analysed by FTIR and 37 the products 10 and 12 were isolated by prep-HPLC and characterised ¹ H-NMR and HRMS.

Reaction of prodrug 13 under X-ray irradiation

A stock solution of Doxorubicin prodrug 13 (100 mM in DMSO) was diluted in PBS (20 ml_, with 0.1%, v/vTriton X100) to give a final concentration of 20 mM. The solution was degassed by bubbling Ar for 30 min before X-Ray irradiation (0 to 60 Gy). The reaction mixture was analysed by FTIR and the products 15 and 5 were isolated by prep-HPLC and characterised ¹ H-NMR and HRMS. (Note compound 15 and 5 co-eluted on the HPLC).

Evaluation of compound cytotoxicity

Compound cytotoxicities were evaluated using an MTT assay. Briefly, Hela and HEVEC cells were seeded in 96-well plates (1 ^c 10 ⁴ cells/well) and incubated overnight. The cells were treated with the desired compound at different concentrations in DMEM for 24 h. The media was removed and the cells were washed with PBS ( ^c 3) and incubated with 100 pL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (1 mg/mL) for 4 h at 37 °C. 100 pL of MTT solubilisation solution (10% Triton-X 100 in 0.1 N HCI in isopropanol) was added to each well and the plate was shaken horizontally for 60 min to dissolve the formazan crystals. The absorbances at 570 nm were measured on a multimode plate reader and cell viability was calculated compared to untreated cells.

Cell viability against prodrug 13 before and after X-ray irradiation

HeLa cell viability was evaluated using an MTT assay as described above. The cells were treated with prodrug 13 (0.5, 1, 5 and 10 pM) for 4 h, and irradiated with X-ray at 0 Gy, 6 Gy, 12 Gy, 24 Gy, 36 Gy, 48 Gy and 60 Gy. Cells treated with 50% DMSO in DMEM were used as a negative control.

Tumour model development

To establish the HT-29 tumour model, HT-29 cells suspended in Matrigel were subcutaneously injected into the right flank subcutaneous tissues of the 6-8 week aged female BALB/c nude mice (1 ^c 10 ⁶ cells/mouse). When the tumour size reached approximately 100 mm ³ , the mice were randomly divided into 6 groups (n = 5) for the treatment with PBS, pazopanib 10, prodrug 11 , PBS + X-ray, pazopanib 10 + X-ray and prodrug 11 + X-ray (doses for both pazopanib 10 and prodrug 11 : 100 mg/kg, 38 intratumoural injections). X-ray irradiation was conducted 4 h after injection (6 Gy, 6 Gy/min). Tumour sizes of each group were measured every other day using a calliper. Tumour volume = (length) ^c (width) ² /2. Relative tumour volume was calculated as V/Vo (Vo was the initial tumour volume). The body weights of the mice were measured every 2 days. The endpoint criteria of the study were a tumour size greater than 2,000 mm ³ and weight loss exceeding 15% of the starting weight. Tumours of mice in different treatment groups were sectioned for H&E staining and Ki67 immunostaining analysis.

Doxorubicin 15, prodrug 13, PBS + X-ray, doxorubicin 15 + X-ray and prodrug 13 + X- ray (doses for both 15 and 13: 10 mg/kg, intratumoural injections) were analysed in a similar manner. X-ray irradiation was conducted 4 h after injection (6 Gy, 6 Gy/min). T umour sizes and body weights of mice were analysis as conducted above.

Results and Discussion

Chemical reactions under X-ray radiation

A high-throughput screen was utilised to identify chemically relevant functional groups that were modified by gamma/X-ray irradiation - this included azides, disulfides, azobenzenes, triphenylphosphine, tetrazoles etc. with all reactions performed in an oxygen free environment in aqueous solution to mimic a hypoxic tumour environment (Table 1).

Table 1. Model reactions under X-ray Irradiation. ³  40 a: reactions were all carried out in PBS with concentration of 10 mM and under 60 Gy irradiation b: N = not reacted, Y = reacted; c: confirmed by HPLC chromatography (254 nm).

It was observed that molecules such as dipyridyl tetrazine and azobenzene did react under X-ray radiation (60 Gy), however, the resulting reaction mixtures were too complex to be useful. In contrast, it was also observed that a number of functional groups were converted cleanly and with high efficiency - thus 4-acetamidobenzenesulfonyl azide and some other aromatic azides reacted under irradiation to give simple reaction mixtures that could be fully characterised.

As seen in Figure 2, 4-acetamidobenzenesulfonyl azide 1 (10 mM in PBS) gave rise to 4-acetamidobenzenesulfonamide 2 in high conversion with traces of the sulfonic acid 3 (Figure 2a). A model reaction of 9-azidobenzoic acid 6 (10 mM in PBS) under X-ray irradiation afforded 4-aminobenzoic acid 7 although with a conversion <10% (Figure 2e) but 4-(hydroxymethyl)-2,3,5,6-tetrafluoroaryl azide 4 (10 pM in PBS) was cleanly reduced to 4-(hydroxymethyl)-2,3,5,6-tetrafluoroaniline 5 under X-ray radiation (60 Gy) (Figure 2c). It is reported that aromatic azides and sulfonyl azides can undergo decompositions in their exited states to generate a triplet-state nitrene by releasing one molecule of nitrogen. Without being bound by theory, the inventors hypothesise that the nitrene intermediates were involved in the X-ray mediated azide activation reactions, i.e. the azides were excited by the X-ray and release nitrogen to generate a nitrene, which further react with hydrogen radicals to give the amine products (as illustrated in Figure 2a).

Activation of coumarin fluorescence probe via X-ray irradiation

Following these observations an initial proof-of-concept involved X-ray irradiation unmasking of 7-azido-4-methylcoumarin 8 (Figure 3) to give 7-amino-4-methylcoumarin 9 allowing fluorescence assessment of activation (A _ex /em = 345/443 nm, Figure 3b). Conversion of the azide 8 (100 pM in PBS) by irradiation was > 90% with a linear relationship with irradiation dose (Figure 3b and 3c). When HeLa cells were incubated 41 with 7-azido-4-rnethylcoumarin 8 (100 mM) for 4 h and irradiation applied, flow cytometry (Figure 3e) also showed an increase in fluorescent intensity with increased irradiation dose.

Activation of a pazopanib prodrug - converting sulfonyl azides to sulfonamide with X-ray irradiation

Pazopanib (Votrien ^® ) is an orally available, small-molecule tyrosine kinase inhibitor of the vascular endothelial growth factor (VEGF) receptor 1, 2, and 3. Herein, pazopanib was used as one of the models for prodrug activation through X-ray irradiation. The sulfonyl azide 11 was generated from pazopanib 10 using imidazole-1 -sulfonyl azide hydrogen sulfate as the diazotransfer reagent (Figure 4) (Stevens et al, J. Org. Chem. 2014, 79, 4826-4831) and its reaction with X-ray irradiation examined. Reaction took place in a radiation dose dependent manner at ambient temperature yielding the drug pazopanib, 10 (and the by-product 12) (Figure 4). The reaction of 11 (5 mM, 10 mM and 20 mM in PBS) reached over 90% conversion following X-ray irradiation (60 Gy). Interestingly it was observed that the amount of pazopanib 10 increased when irradiation doses increased but reached its highest concentration at 36 Gy (Figure 4) where upon 11 started to decompose, generating the sulfonic acid by-product 12. Importantly, pazopanib, 10 (20 mM in PBS) remained unreactive under radiation (60 Gy). To evaluate the efficacy of the combination of radiotherapy and activation of the prodrug, HUVEC were incubated with 10 or 11 (between 5 mM to 20 mM) and irradiated with different doses (from 6 Gy to 60 Gy). It was observed that cell viability was both irradiation dose and drug concentration dependent. The prodrug 11 under irradiation generating active drug 10 (pazopanib) resulting in significant cytotoxicity in cells together with the effect of irradiation (Figure 4e).

The successful activation of the caged prodrug molecules was then confirmed in cellular level and then in an animal model. As expected, HUVEC cells treated with prodrug 11 and X-ray irradiation behaved similarly as the pazopanib 10 treated cells, i.e. showing less mobilities, metastasis abilities and tubule formation activities. The in vivo study showed that the tumour growths were more significantly inhibited by the combination (green line) of drug 10 treatment and X-ray irradiation than individual treatment. (Figure 4f). Mice treated with the combination of prodrug 11 and X-ray irradiation (purple line) showed slightly lower efficacy probably due the lower concentration of activated drugs, 42 although the survival period were prolonged compared to control groups treated with drug or irradiation individually (Figure 4g).

Activation of caged doxorubicin through X-ray radiation of a self-immolative linker

Based on the model reaction as shown in Figure 2c, where 4 was neatly reduced to a single product 5, (p-azido)-2,3,5,6-tetrafluorobenzyloxycarbonyl substituted doxorubicin 13 was synthesised as an additional prodrug model with another mode of activation, examining the reduction of azide to aniline and subsequent decaging of the drug doxorubicin (Figure 5). We observed that the prodrug 13 (10 mM) was also decaged in a radiation dose dependent manner, reaching 50% conversion with 60 Gy (see Figure 5c) with three products ( R _T = 4.36, 3.14 and 3.03 min) observed, consisting of doxorubicin 15 ( RT = 3.14 min), tetrafluoroaniline 5 (RT = 3.03 min) and the relatively instable “linker- DOX product” 14 (R _T = 4.36 min). ³⁶

The excellently shielded toxicity (in the absence of the radiation, the prodrug was non toxic when used up to 100 pM) revealed in the in vitro analysis (Figure 5d) drove the inventors to further investigate the anticancer efficacy of the prodrug system in animal level. In a HeLa tumour bearing mice model, tumour growth was significantly inhibited with overall survival prolonged by the combined treatment of X-ray and prodrug 13. Importantly, evaluations on body weight changes and key organ histological abnormalities showed that the prodrugs displayed no gross toxicities, indeed there was reduced heart toxicity compared to that usually associated with the use of doxorubicin.

Conclusion

A novel gamma/X-ray mediated strategy for the activation of prodrugs based on the reduction of sulfonyl azide and phenyl azide moieties has been devised. Without being bound by theory, it is hypothesised that the reactions are mediated via free radical chemistry and through the reductive loss of nitrogen. For the decaging of doxorubicin from the prodrug, it is hypothesised that DOX was decaged through a two-step reaction, reduction of phenyl azide to the amine and 1,6-self-immolation collapse of the linker.