Field of the invention

The present invention relates to a novel fluorescent probe for detecting the components of Fenton chemistry in biological samples and systems, and methods comprising use of same.

Background of the invention

Fenton chemistry (the reaction of iron and hydrogen peroxide in the cell to produce damaging superoxide radicals) has long been known as a mechanism of oxidative stress and cellular death.

However, more recently there has been increasing interest in the role of Fenton chemistry in the genesis of many diseases, and a concomitant focus on mitigating Fenton reactions in order to treat or prevent disease. At present, there are no methods for simultaneously measuring both of the reactants of Fenton chemistry, nor are there high through-put methods to assess the effect of drug candidates on Fenton reactions in disease models.

There is a need for reagents and methods for studying Fenton chemistry in samples and in living systems cells, including for identifying and/or quantifying one or both of the Fenton reactants in biological samples.

There is a need for improvements in reagents and methods for assessing assess the effect of therapeutic candidates on Fenton reactions.

Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

Summary of the invention

In a first aspect, the present invention provides a compound of formula I:

wherein R ¹ is S or O;

X is N or O; Y is O, Si, Se or Ge; and

R ² -R ⁷ is any substituent or absent; or any salt or solvate thereof.

Preferably, R ¹ and Y are O, X is N, R ² and R ³ are absent, and R ⁴ -R ⁷ is a C2 alkyl such that in a further, aspect, the present invention provides a compound of formula II: or a salt or solvate thereof. In a further aspect, the present invention provides a composition comprising the compound of formula I, or II or a salt or solvate thereof.

In any aspect, the invention provides a compound of formula I or II when conjugated to iron, or a composition comprising the same.

Still further, the present invention provides a method for determining the concentration of ferric ions (Fe2+, herein Fe(ll)) in a test sample, the method comprising:

- contacting the test sample with the compound of Formula I or II, or a salt or solvate thereof;

- optionally contacting the test sample with an exogenous source of hydrogen peroxide;

- exposing the test sample to a source of light sufficient to excite the compound;

- detecting fluorescence from the compound, wherein fluorescence of the compound is indicative of the presence of Fe(ll) in the test sample, thereby determining the concentration of Fe( 11) in the test sample.

In a further aspect, the present invention provides a method for determining the concentration of hydrogen peroxide in a test sample, the method comprising:

- contacting the test sample with an amount of the compound of Formula I or II, or a salt or solvate thereof;

- optionally contacting the test sample with an exogenous source of Fe(ll);

- exposing the test sample to a source of light sufficient to excite the compound,

- detecting fluorescence from the compound, thereby determining the concentration of hydrogen peroxide in the sample.

In a further aspect, the present invention provides a method for determining the concentration of hydrogen peroxide in a test sample, the method comprising: - contacting the test sample with an amount of the compound of Formula I or II, or a salt or solvate thereof, wherein the compound is conjugated to Fe(ll);

- exposing the test sample to a source of light sufficient to excite the compound;

- detecting fluorescence from the compound, thereby determining the concentration of hydrogen peroxide in the sample.

In a further aspect, the present invention provides a method for determining the presence or absence of ferric ions (Fe2+, herein Fe(ll)) and hydrogen peroxide in a test sample, the method comprising:

- contacting a test sample with an amount of the compound of Formula I or II , or a salt or solvate thereof;

- exposing the test sample to a source of light sufficient to excite the compound;

- detecting fluorescence from the compound, wherein the fluorescence of the compound indicates the presence of both Fe(ll) and hydrogen peroxide in the test sample, thereby determining the presence or absence of Fe(ll) and hydrogen peroxide in the test sample.

In any embodiment of the above aspects of the invention, the sample in which the concentration of Fe(ll) and/or hydrogen peroxide is to be determined, may be selected from the group consisting of: a sample comprising a population of live cells (wherein the cells may be obtained from a tissue biopsy or comprise a cell-line), a tissue sample, a sample comprising cell lysates, a sample of biological fluid or an environmental sample.

The sample of live cells may comprise a population of cells that has been contacted with an agent that has or is suspected of having one of more of the following effects on the cells: modifying the concentration of Fe(ll) in the cells, modifying the redox state of the cells, increasing or reducing the presence of Fe(ll) and/or FteC in the cell, inducing ferroptosis in the cells, or combinations thereof. The cell lysate may be derived or obtained from one or more cells that have been contacted with an agent that has or is suspected of having one of more of the following effects on the cells: modifying the concentration of Fe(ll) in the cells, modifying the redox state of the cells, increasing or reducing the presence of Fe( 11) and/or H2O2 in the cell, inducing ferroptosis in the cells, or combinations thereof.

The sample of live cells or cell lysate may be derived from a biopsy obtained from a subject for whom a diagnosis is required for a disease or condition characterised by one or more of: altered Fe(ll) haemostasis, altered Redox state, oxidative stress, increased presence of Fenton reactants and ferroptosis. Non-limiting examples of such diseases or conditions include: neurodegenerative disease, haemochromatosis, erythroid pathologies, iron-related disorders, diabetes.

Accordingly, the present invention provides a method for determining the likelihood that a subject is suffering from a disease or condition associated with ferroptosis, the method comprising:

- providing a test biological sample from a subject suspected of suffering from a disease or condition associated with ferroptosis;

- contacting the test biological sample with a compound of Formula I or II, or a salt thereof;

- exposing the test sample to a source of light sufficient to excite the compound;

- detecting fluorescence from the compound; wherein the presence of fluorescence indicates that the subject is suffering from a disease or condition associated with ferroptosis. The biological sample may be selected from: a bodily fluid obtained from the subject (optionally blood, saliva, lacrimal fluid), or a tissue sample.

The method may further comprise obtaining the biological sample from the subject.

In embodiments where the sample comprises a population of live cells, the methods of the present invention may also be utilised to determine subcellular localisation of Fe (II) and/or hydrogen peroxide. Further still, the present invention provides a method for screening for an agent capable of inducing ferroptosis in a cell, the method comprising:

- providing a candidate agent suspected of being capable of inducing ferroptosis in a cell;

- contacting a cell or population of cells with the candidate agent;

- contacting the cells with a compound of Formula I or II, or a salt thereof;

- exposing the cells to a source of light sufficient to excite the compound;

- detecting fluorescence from the compound;

- wherein the presence of fluorescence indicates that the agent induces ferroptosis in the cell.

In any aspect of the invention, the source of light comprises a wavelength of between about 500 nm to about 600 nm, preferably between about 520 nm to about 580 nm, more preferably between about 550 nm to 560 nm.

In any aspect of the invention, detecting the fluorescence of the compound comprises measuring fluorescence emission in the range of between about 550 nm to about 670 nm, preferably between about 570 nm to about 600 nm.

The present invention also provides a kit comprising a compound of Formula I or Formula II, or a salt or solvate thereof, and/or instructions for carrying out a method of detecting the presence or absence of hydrogen peroxide and/or Fe(ll) therewith, optionally in a common package or container.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the drawings

Figure 1. Scheme 1 - synthesis of compound of Formula II. Figure 2. Proposed mechanism by which the compound of Formula II (RTFtl ) can detect the two substrates of the Fenton reaction, Fe(ll) and H2O2.

Figure 3. Fluorescence excitation (dotted line) and emission (solid line) spectra of RTFT1 (10pM, 10% DMSO in MllliQ water), in the absence and presence of Fe(ll).

Figure 4. Fluorescence of RTFT1 (50pM) in presence of Fe(ll) (100 pM) and other biological metals (20pM) in a solution of MilliQ H2O/DMSO/H2O2 (89/10/1 , v/v/v): 1 ) control; 2) Fe(ll); 2) Fe(lll); 4) Cu ²⁺ ; 5) Zn ²⁺ ;6) Ca ²⁺ ; 7) Na ⁺ ; 8) K ⁺ ; 9) Mg ²⁺ _; 10) Cu ⁺ . A _ex /em=556/580 nm. Error bars denote S. E. M.

Figure 5. Fluorescence emission of probes in presence of Fe(ll). (1 ) RTFtl . (2) late stage addition of 2,2'-bipyridyl of condition (1 ). (3) RTFt-dimer. (4) RTFt-O. (10 pM probes, 20 pM Fe(ll) and H2O2, 100 pM 2,2'-bipyridyl. Aex/em = 556/580 nm).

Figure 6. Confocal fluorescence microscope images of DLD-1 cells treatment with RTFT1 (20 pM) in HBSS buffer, 1 hr. (a) Control cells, with no staining; (b) pretreatment with BPD (2,2’-bipyridyl, 1 mM in HBSS buffer, 1 h); (c) RTFT1 alone; and (d) treatment with Fe(ll) citrate (100pM) in HBSS buffer, 0.5 hr after RTFT1. (e) - (h) Rhodamine channel (Aex/em=561/570-670 nm) images of the same fields of view of (a)- (d) respectively, (i) Mean of quantified fluorescence intensity. Error bars denote SEM, n= approximately 30 cells from 3 fields of view, N=3 individual experiments. Statistical significant was assessed by an unpaired /-test. ****p < 0.0001. Scale bars indicate 10 pm.

Figure 7. Confocal fluorescence microscope images of DLD-1 cells treated with RTFT1 (20 pM) in HBSS buffer, 1 hr. (a) Control cells, with no staining; (b) pre-treatment with catalase (2000-5000 units/mL in adv. DMED media, 4hr; (c) RTFT1 alone; and (d) treatment with H2O2 (100pM) in HBSS buffer, 1 hr after RTFT1. (e) - (h) Rhodamine channel (Aex/em=561/570-670 nm) images of the same fields of view of (a)-(d) respectively, (i) Mean of quantified fluorescence intensity. Error bars denote SEM, n= approximately 30 cells from 3 fields of view, N=3 individual experiments. Statistical significant was assessed by an unpaired /-test. ****p<0.0001 . Scale bars indicate 10 pm. Figure 8. Confocal fluorescence microscope images of DLD-1 cells treated with RTFT1 (20 pM in HBSS buffer), 1 hr. (a) Control cells, with no staining; (b) RTFT1 alone; (c) treatment with Fe(ll) citrate (100pM in HBSS buffer), 0.5hr after RTFT1 ; (d) treatment with H2O2 (100 pM in HBSS buffer), 1 hr after RTFT1 and (e) treatment with Fe(ll) citrate (100 pM in HBSS buffer) 0.5hr, H2O2 (100 pM in HBSS buffer), 1 hr after RTFT1. (f) - (j) rhodamine channel (Aex/em=561/570-670 nm) images of the same fields of view of (a)-(e) respectively, (k) Mean of quantified fluorescence intensity. Error bars denote SEM, n= approximately 30 cells from 3 fields of view, N=3 individual experiments. Statistical significant was assessed by an unpaired t-test. *** £<0.0002 ****p<0.0001. Scale bars indicate 10 pm.

Figure 9. Confocal fluorescence microscope images of DLD-1 cells treated with RTFT1 (20 pM) in HBSS buffer, 1 hr. (a) Control cells, with no staining; (b) pre-treatment with catalase (2000-5000 units/mL) in adv. DMED media, 4h and BPD (2,2’-bipyridyl), 1 mM in HBSS buffer, 1 hr; (c) pre-treatment with BPD (2,2’-bipyridyl) 1 mM, in HBSS buffer, 1 hr; (d) pre-treatment with catalase (2000-5000 units/mL in adv. DMED media, 4hr; (e) RTFT1 alone, (f) - (j) Rhodamine channel (Aex/em=561/570-670 nm) images of the same fields of view of (a)-(e) respectively, (k) Mean of quantified fluorescence intensity. Error bars denote SEM, n= approximately 30 cells from 3 fields of view, N=3 individual experiments. Statistical significant was assessed by an unpaired t-test. *** £<0.0002 ****p<0.0001. Scale bars indicate 10 pm.

Figure 10. Confocal fluorescence microscope images of living DLD-1 cells loaded with erastin (20 pM in adv. DMED media) and RTFtl (20 pM in HBSS buffer, 1 h). (a) RTFtl alone; (b) pre-treatment with erastin (2 h) before RTFtl ; (c) pre-treatment with erastin (4 h) before RTFtl ; (d) pre-treatment with erastin (6 h) before RTFtl ; (e) pretreatment with erastin (8 h) before RTFtl ; (f) pre-treatment with erastin (10 h) before RTFtl . (g) Means of quantified fluorescence intensity of (a)-(f). Aex/em = 561/570-670 nm. Error bars denote standard error of the mean, n = approximate 50 cells from 3 fields of view, N = 3 individual experiments. Statistical significance was assessed by unpaired t- test. **£ < 0.01 , ****£ < 0.0001 . Scale bars indicate 50 pm.

Figure 11. Confocal fluorescence microscope images of living HT-1080 cells loaded with erastin (20 pM in adv. DMED media) and RTFtl (20 pM in HBSS buffer, 1 h). (a) RTFtl alone; (b) pre-treatment with erastin (15 min) before RTFtl ; (c) pretreatment with erastin (30 min) before RTFtl ; (d) pre-treatment with erastin (1 h) before RTFtl ; (e) pre-treatment with erastin (2 h) before RTFtl . (f)-(j) Rhodamine channel ( ex/em = 561/570-670 nm) images of the same view fields of (a)-(e) respectively, (k) Means of quantified fluorescence intensity of (f)-(j). n = approximate 50 cells from 3 fields of view, N = 3 individual experiments. Scale bars indicate 10 m.

Figure 12. Confocal fluorescence microscope images of living HT-1080 cells loaded with erastin (20 pM in adv. DMED media) and rhodamine B methyl ester (1 pM in HBSS buffer, 1 h). (a) rhodamine B methyl ester alone; (b) pre-treatment with erastin (30 min) before rhodamine B methyl ester; (c) pre-treatment with erastin (1 h) before rhodamine B methyl ester; (d) pre-treatment with erastin (2 h) before rhodamine B methyl ester; (e) pre-treatment with erastin (3 h) before rhodamine B methyl ester, (f) pre-treatment with erastin (4 h) before rhodamine B methyl ester, (g)-(l) Rhodamine channel (Xex/em = 561/570-670 nm) images of the same view fields of (a)-(f) respectively, (k) Means of quantified fluorescence intensity of (g)-(l). n = approximate 60 cells from 3 fields of view, N = 3 individual experiments. Scale bars indicate 50 pm.

Figure 13. Confocal fluorescence microscope images of DLD-1 cells loaded with RTFtl (20 pM, 1 h) and cisplatin (20 pM). (a)-(e) cisplatin stained for 15 min, 30 min, 60 min, 120 min and 180 min respectively after RTFtl stain, (f) Cells incubated in HBSS buffer for 180 min after RTFtl stain, (g) Means of quantified fluorescence intensity of (a)-(f). Aex/em = 561/570-670 nm. Error bars denote standard error of the mean, n = approximate 50 cells from 3 fields of view, N = 3 individual experiments. Statistical significance was assessed by an unpaired t-test. ****p < 0.0001 . Scale bars indicate 50 pm.

Figure 14. Standard Alamar blue assay test for DLD-1 cells viability and IC50 measurements of cisplatin alone and cisplatin together with 30 pM 2,2'-bi pyridyl.

Figure 15. Confocal fluorescence microscope images of DLD-1 cells treated with RTFtl (20 pM,1 h) cisplatin (20 pM), and/or tubastatin A (10 pM). (a) Cells incubated for 7 h after RTFtl . (b) Cells stained with cisplatin for 2 h, and incubated for 4 h after RTFtl ; (c) Cells incubated for 2 h, then stained with tubastatin A for 4 h after RTFtl ; (d) Cells stained with cisplatin for 2 h, then tubastatin A for 4 h after RTFtl ; (e) Means of quantified fluorescence intensity of (a)-(d). Aex/em = 561/570-670 nm. Error bars denote SEM, N = 3 individual experiments with at least 50 cells from 3 fields of view, Statistical significance assessed by an unpaired /-test. *p < 0.05; ***p < 0.001 ; ****p < 0.0001. Scale bars indicate 50 |im.

Figure 16. Standard Alamar blue assay test for DLD-1 cells viability and IC50 measurements of cisplatin alone and cisplatin together with 10 jiM tubastatin A.

Figure 17. Standard Alamar blue assay test for DLD-1 cell viability, (a) DLD-1 cells viability of vehicle control (0.5% DMSO) and 10 jiM tubastatin A. (b) Normalized DLD-1 cells viability of 20 jiM cisplatin and 20 jiM cisplatin together with 10 jiM tubastatin A.

Detailed description of the embodiments

Fluorescent probes offer many advantages for monitoring cellular processes in real time and several have been developed for detecting the presence of metal ions such as Ca(ll), Zn(ll), Cu(ll), Hg(ll) ions. Existing probes for detection of iron include the commercially available calcein and PhenGreen, as well as those in development like CP655 and RDA. All of these iron probes work by a fluorescence “switch-off” mechanism resulting from iron-induced fluorescence quenching. In order to measure labile iron in cells by using these probes, a high-affinity chelator is used to compete iron away from the probe and restore its fluorescence. This change upon dequenching provides the concentration of iron that was accessible to the probe chelator. A concern with any of these probes is that their mere presence shifts the equilibrium of iron and other metals in the cell by acting as a “vacuum cleaner” that labilizes iron from stores and redistributes it.

While various probes for iron detection are known, they are typically not very selective for iron.

In contrast to the approaches of the prior art, the inventors have developed a probe for detecting the reactants of Fenton chemistry (iron and hydrogen peroxide) using a “switch-on” approach. The probe of the invention is understood to provide an advantage over the prior art because of its selectivity towards Fe(ll) and hydrogen peroxide, requiring both reactants to activate the switch-on of fluorescence. This advantageously provides the ability to differentiate Fenton Chemistry from other conditions in the cell that induce changes in either Fe(ll) or H2O2. The probe is also selective towards Fe(ll) in the presence of other metal ions, such as Fe(lll) and Cu(ll), which confers a further advantage over other prior fluorescence probes for detecting Fe(ll).

The inventors believe that a considerable advantage of this probe is that it is biocompatible and can therefore be used in living cells to detect Fenton reaction. The simultaneous detection of both of the Fenton reactants represents its particular advantage over current probes. Furthermore, the sensing process can be divided into two steps: chelation and reaction.

The inventors have demonstrated the utility of the probe of the invention to detect levels of Fe(ll) in live cells, to monitor Fenton chemistry in live cells; and to detect oxidative stress/ferroptosis caused by drugs (such as cisplatin and erastin) in cells.

Thus, the inventors anticipate that the probe of the invention will likely be useful for:

• screening for new drugs that induce Fenton chemistry;

• in the clinic, to identify evidence of Fenton chemistry in tissues/bodily fluids which may assist in diagnosis of diseases where Fenton chemistry is known to contribute to underlying pathologies; and

• to investigate oxidative stress in living cells (including to further understand the underlying biochemistry in different disease pathologies).

Accordingly, the present invention provides a compound of Formula I:

wherein R ¹ is S or O

X is N or O

Y is O, Si, Se or Ge, and R ² -R ⁷ is any substituent or absent; or any salt or solvate thereof.

A "substituent" as used herein, refers to a molecular moiety that is covalently bonded to an atom within a molecule of interest. For example, a "substituent" may be a moiety such as a halogen, alkyl group, or other substituent described herein that is covalently bonded to an atom, preferably a carbon or nitrogen atom. The term "substituted," as used herein, means that any one or more hydrogens on the designated atom is replaced with a selection from the indicated substituents, provided that the designated atom's normal valence is not exceeded, and that the substitution results in a stable compound, i.e., a compound that can be isolated, characterised and tested for biological activity.

Examples of substituents include but are not limited to:

Ci-C ₆ alkyl, Ci-Ce haloalkyl, Ci-Ce haloalkoxy, Ci-Ce hydroxyalkyl, C3-C7 heterocyclyl, C3-C7 cycloalkyl, Ci-Ce alkoxy, Ci-Ce alkylsulfanyl, Ci-Ce alkylsulfenyl, Ci- Ce alkylsulfonyl, Ci-Ce alkylsulfonylamino, arylsulfonoamino, alkylcarboxy, alkylcarboxyamide, oxo, hydroxy, mercapto, amino, acyl, carboxy, carbamoyl, aminosulfonyl, acyloxy, alkoxycarbonyl, nitro, cyano or halo.

As used herein the term "alkyl" refers to a straight or branched chain hydrocarbon radical having from one to ten carbon atoms, or any range between, i.e. it contains 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The alkyl group is optionally substituted with substituents, multiple degrees of substitution being allowed. Examples of "alkyl" as used herein include, but are not limited to, methyl, ethyl (Et), n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, and the like.

As used herein, the term "C1-6 alkyl" refers to an alkyl group, as defined above, containing at least 1 , and at most 6 carbon atoms respectively, or any range in between (e.g. alkyl groups containing 2-5 carbon atoms are also within the range of Ci-e). Unless the context requires otherwise, the term “Ci -ealkyl” also encompasses alkyl groups containing one less hydrogen atom such that the group is attached via two positions i.e. divalent. “Ci-4alkyl” and “Ci-3alkyl” including methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl are preferred with methyl being particularly preferred.

Preferably the alkyl groups contain from 1 to 6 carbons and more preferably are methyl, ethyl (Et) or propyl.

The term “heterocyclyl” refers to a moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound which moiety has from 3 to 10 ring atoms (unless otherwise specified), of which 1 , 2, 3 or 4 are ring heteroatoms each heteroatom being independently selected from O, S and N. Heterocyclyl groups include monocyclic and polycyclic (such as bicyclic) ring systems, such as fused, bridged and spirocyclic systems, provided at least one of the rings of the ring systm contains at least one heteroatom.

In this context, the prefixs 3-, 4-, 5-, 6-, 7-, 8-, 9- and 10- membered denote the number of ring atoms, or range of ring atoms, whether carbon atoms or heteroatoms. For example, the term “3-10 membered heterocylyl”, as used herein, pertains to a heterocyclyl group having 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms. Examples of heterocylyl groups include 5-6-membered monocyclic heterocyclyls and 9-10 membered fused bicyclic heterocyclyls. Heterocyclyls encompass aromatic heterocyclyls and non-aromatic heterocyclyls. Such groups may be substituted or unsubstituted.

As used herein, the term "aryl" refers to an optionally substituted benzene ring. The aryl group is optionally substituted with substituents, multiple degrees of substitution being allowed.

As used herein, the term "heteroaryl" refers to a monocyclic five, six or seven membered aromatic ring containing one or more nitrogen, sulfur, and/or oxygen heteroatoms, where N-oxides and sulfur oxides and dioxides are permissible heteroatom substitutions and may be optionally substituted with up to three members. Examples of "heteroaryl" groups used herein include furanyl, thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, oxopyridyl, thiadiazolyl, isothiazolyl, pyridyl, pyridazyl, pyrazinyl, pyrimidyl and substituted versions thereof.

The term “halo” refers to fluoro, chloro, bromo or iodo.

The term “thiol” refers to the group -SH.

The terms “thioxo” refer to the group =S.

Unless otherwise defined, the term “optionally substituted” or “optional substituent” as used herein refers to a group which may or may not be further substituted with 1 , 2, 3, 4 or more groups, preferably 1 , 2 or 3, more preferably 1 or 2 groups selected from the group consisting of Ci ealkyl, C2-ealkenyl, C2-ealkynyl, C3- scycloalkyl, hydroxyl, oxo, Ci ealkoxy, aryloxy, Ci ealkoxyaryl, halo, Ci ealkylhalo (such as CF3), Ci ealkoxyhalo (such as OCF3), carboxyl, esters, cyano, nitro, amino, substituted amino, disubstituted amino, acyl, ketones, substituted ketones, amides, aminoacyl, substituted amides, disubstituted amides, thiol, alkylthio, thioxo, sulfates, sulfonates, sulfinyl, substituted sulfinyl, sulfonyl, substituted sulfonyl, sulfonylamides, substituted sulfonamides, disubstituted sulfonamides, aryl, arylCi ealkyl, heterocyclyl and heteroaryl wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heterocyclyl and groups containing them may be further optionally substituted. Optional substituents in the case of heterocycles containing N may also include but are not limited to Ci ealkyl i.e. N-Ci-3alkyl. Preferably, R ¹ and Y are O, X is N, R ² and R ³ are absent, and R ⁴ -R ⁷ is C2 alkyl.

Accordingly, the present invention provides a compound of Formula II: or a salt or solvate thereof.

Suitable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, malic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.

Base salts include, but are not limited to: those formed with pharmaceutically acceptable cations, such as: sodium, potassium, lithium, calcium, magnesium, zinc, ammonium and alkylammonium; salts formed from triethylamine; alkoxyammonium salts such as those formed with ethanolamine; and salts formed from ethylenediamine, choline or amino acids such as arginine, lysine or histidine.

Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.

Salts, or other derivatives of the compound of Formula I or Formula II may be provided in the form of solvates. Solvates contain either stoichiometric or non- stoichiometric amounts of a solvent, and may be formed during the process of crystallization with pharmaceutically acceptable solvents such as water, alcohols such as methanol, ethanol or isopropyl alcohol, DMSO, acetonitrile, dimethyl formamide (DMF) and the like with the solvate forming part of the crystal lattice by either non- covalent binding or by occupying a hole in the crystal lattice. Hydrates are formed when the solvent is water, alcoholates are formed when the solvent is alcohol. Solvates of the compounds of the present invention can be conveniently prepared or formed during the processes described herein. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the compounds and methods provided herein.

As used herein the term fluorophore refers to a chemical moiety that emits light following excitation. Any suitable fluorophore may be used. Suitable fluorophores include, but are not limited to rhodamine and derivatives thereof, such as rhodamine B, rhodamine 6G, rhodamine 123, tetramethylrhodamine, silicon rhodamine, fluorescein and derivatives thereof.

The probe of the present invention is prepared from 3 synthetic steps from a commercially available fluorophore, for example rhodamine. While the probe of the invention is weakly fluorescent, in the presence of Fe(ll) and hydrogen peroxide, the highly fluorescent rhodamine B is released. The response relies on two separate steps:

Chelation step: the probe firstly chelates Fe(ll) which can be provided from an exogenous or an endogenous source, resulting in an open ring conformation. Chelation of Fe(ll) results in quenching of the fluorescence and at this stage, the probe remains weakly fluorescence .

Reaction step: there are two reactions in this step, Fenton reaction and subsequent hydrolysis. Chelated Fe(ll) further reacts with endogenous or exogenous hydrogen peroxide to generate hydroxyl anion and hydroxyl radical. The products of this reaction initiate a second reaction: hydrolysis, which results in cleavage between rhodamine and the thio-phenol group. As Fe(ll) is no longer chelated to the rhodamine fluorophore (and should be oxidised for Fe(lll)), the fluorescent rhodamine B is released. It will be appreciated that the compound of the present invention may also be provided conjugated to iron for the purposes of detecting hydrogen peroxide in a sample.

It will be appreciated that the compound of the invention has utility in detecting the Fenton reactants (alone or in combination) in a variety of different settings. For example, the sample in which the concentration of Fe(ll) and/or hydrogen peroxide is to be determined, may be a sample comprising a population of live cells, a sample comprising cell lysates, a sample of biological fluid (e.g., blood, urine, tears, saliva), a tissue sample, or an environmental sample.

In embodiments where the sample comprises a population of live cells, the methods of the present invention may also be utilised to determine subcellular localisation of Fe (II) and/or hydrogen peroxide. This may be by simple visualisation of the location of fluorescence within cells, for example using standard confocal microscopic techniques.

The cells may include plant, animal, and microbial cells. Cells may be individual cells in vitro or in a cell culture, or cells residing in a tissue, which tissue is ex vivo or in vitro in a tissue culture.

Plant cells may be from or of any suitable plant, including angiosperms and gymnosperms, and including monocots and dicots. Examples include but are not limited to wheat, soy, corn, potato, tomato, orange, lemon, pine, oak, etc.

Animal cells may be from or of any animal, including but not limited to avian, reptile, amphibian, and mammalian species, such as mouse, rat, cat, dog, horse, cow, sheep, rabbit, and primate such as human.

Microbial cells may be from any microorganism, including yeast, fungi, protozoa, and bacteria, including gram negative and gram positive bacteria.

In certain embodiments, the invention provides for methods for screening an agent or panel of agents, for their ability to induce one or more of the following changes in a cell, population of cells or biological system: modifying the concentration of Fe(ll) in the cells, modifying the redox state of the cells, increasing or reducing the presence of Fe(ll) and/or H2O2 in the cell, inducing ferroptosis in the cells, or combinations thereof.

As used herein, ferroptosis refers to a type of programmed cell death dependent on iron and characterized by the accumulation of lipid peroxides, and is genetically and biochemically distinct from other forms of regulated cell death such as apoptosis. Ferroptosis may be initiated by the failure of the glutathione-dependent antioxidant defences, resulting in unchecked lipid peroxidation and eventual cell death. The hallmark feature of ferroptosis is the iron-dependent accumulation of oxidatively damaged phospholipids (i.e. lipid peroxides). This typically occurs when free radical molecules take electrons from a lipid molecule, promoting their oxidation by oxygen. The primary cellular mechanism of protection against ferroptosis is mediated by GPX4, a glutathione-dependent peroxidase that converts lipid peroxides into non-toxic lipid alcohols. A second parallel protective pathway has also been identified and which involves the oxidoreductase FSP1/AIFM2. It is thought that FSP1/AIFM2 enzymatically reduces non-mitochondrial Coenzyme Q10, thereby generating a potent lipophilic antioxidant to suppresses the propagation of lipid peroxides.

As used herein, altered redox state will be understood to refer to any alteration in the redox environment of a cell or system that results in an increase in oxidative stress or free radicals. The alteration in redox state may be direct or indirect. For example, in the context of a direct alteration in redox state, this may arise from changes or failure of the glutathione-dependent antioxidant defences of a cell, reduction or alteration in the amount, activity or expression of intracellular antioxidants,

The methods of the invention may find particular applicability in studies for identifying therapeutic agents for use in treating diseases or conditions where ferroptosis or Fenton Chemistry is desirable. Conversely, the methods of the invention may also be useful for screening a therapeutic agent or panel of agents for potentially deleterious effects on a cell, population of cells or biological system. In other words, the methods may be useful for excluding an agent from therapeutic use if it is identified that the agent induces undesirable effects such as modifying the concentration of Fe(ll) in the cells, modifying the redox state of the cells, increasing or reducing the presence of Fe(ll) and/or F C in the cell, inducing ferroptosis in the cells, or combinations thereof. The skilled person will appreciate that a common approach for treating cancer is to induce ferroptosis in cancer cells. Accordingly, the present invention finds application in methods for determining the efficacy of an agent or a treatment for inducing ferroptosis in a cancer cell. Such methods may be applied during ongoing treatment, such that measurement of ferroptosis in cells can be used as a means to determine the efficacy of ongoing cancer treatment. Alternatively, the methods may comprise methods for screening one or more agents (such as a panel of agents) which are candidates for use in inducing ferroptosis and for treatment of cancer.

In such methods, the compound of the invention may be contacted with a population of cells that has been contacted with an agent that has or is suspected of having one of more of the following effects on the cells: modifying the concentration of Fe(ll) in the cells, modifying the redox state of the cells, increasing or reducing the presence of Fe(ll) and/or H2O2 in the cell, inducing ferroptosis in the cells, or combinations thereof. Alternatively, the compound of the invention may be contacted with a cell lysate derived from a population of cells that has been contacted with such an agent. Still further, the methods of the invention find application in identifying agents that are suspected of having the capacity to reduce conditions leading to ferroptosis, or for suppressing Fenton chemistry in cells.

Cells used in screening assays may comprise a cell-line or may comprise primary cells in culture.

The compound of the invention may be used to detect Fenton reactants in a test biological sample obtained from a patient in need of diagnosis or for determining the success of treatment with an agent that induces Fenton chemistry. The test sample may comprise a bodily fluid obtained from a patient (such as saliva, tears, urine or blood), or may comprise cells or tissue sample obtained from a biopsy, or derivatives thereof.

The patient may require diagnosis with a disease or condition characterised by one or more of: altered Fe(ll) haemostasis, altered Redox state, oxidative stress, increased presence of Fenton reactants and ferroptosis.

Ferroptosis is associated with various diseases including acute kidney injury, cancer, and cardiovascular, pulmonary, neurodegenerative, and hepatic diseases. Accordingly, the methods of the present invention also find application in diagnosing or determining the likelihood that a subject has a disease or condition characterised by ferroptosis. Thus, the methods of the present invention may be used as an adjunct to diagnostic methods for determining whether a subject has acute kidney injury, cancer, and cardiovascular, pulmonary, neurodegenerative, and hepatic diseases associated with ferroptosis.

It will also be appreciated that the methods and compounds of the present invention can be used to characterise a disease or pathology for the presence of ferroptosis.

As used herein, a disease or condition that may be diagnosed using the methods of the present invention includes any disease or condition in which there is an alteration of Fe(ll) and/or hydrogen peroxide levels. Accordingly, a disease or condition that may be diagnosed using the methods of the present invention may include: neurodegenerative diseases, haemochromatosis, erythroid pathologies, iron-related disorders, and diabetes.

Compounds of the invention may be contacted with cells or compositions as described herein by any suitable technique, such as simply combining with the mixture, adding to a cell or tissue culture, injection into a subject, etc., and fluorescence measured therefrom in accordance with known techniques or variations thereof which will be apparent to those skilled in the art.

The methods for measuring (or detecting) Fe(ll) ions and/or hydrogen peroxide may comprise mixing a test sample with a compound of the present invention in a suitable buffer solution, incubating the mixture, and irradiating the incubated mixture with excitation light to measure the fluorescence.

In the context of in vitro or ex vivo studies, the compound of the invention may be provided dissolved in a solutions of buffer. Buffers for use in accordance with the methods of the invention are not particularly limited, and examples include known buffer solutions, such as HEPES buffer solution (pH of 7.4), Hank's Balanced Salt Solution (HBSS), and Dulbecco’s Modified Eagle Medium (DMEM). The concentration of the compound represented by formula (I) in a buffer solution is not particularly limited, and is typically about 0.1 pM to 1 mM, preferably about 1 pM to 0.1 mM, and more preferably about 5 pM to 20 pM.

The temperature and the time period for incubation are not particularly limited. For example, the incubation can be performed at about 0 to 40° C for about 10 minutes to 2 hours. When cells or tissues serve as a specimen, the temperature suitable for the culturing is preferably applied (e.g., 37° C. for human-derived cells or tissues).

The term “measurement” used in the specification should be construed in its broadest sense, including quantitative and qualitative measurement.

Detecting Fe(ll), hydrogen peroxide, or both, in a test sample can be performed in any setting which permits exciting the compound with the required wavelength of light. The fluorescence can be measured by using a commercially available fluorometer. Accordingly, in certain embodiments, the reactants can be measured in a cuvette using a fluorimeter or plate-reader. The dynamics of Fenton chemistry in live cells can be investigated by observation using any known technique, such as a fluorescence microscope and a confocal laser scanning fluorescence microscope. Such methods will be familiar to the skilled person. Methods of utilising high content screening instrumentation will also be familiar to the skilled person.

Kits useful for carrying out the present invention may comprise one or more compounds as described herein, and/or instructions (e.g., printed instructions) for carrying out a method as described herein, optionally packaged together in a common package or container.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention. Examples

The invention will be further described by way of non-limiting example(s). It will be understood to persons skilled in the art of the invention that many modifications may be made without departing from the spirit and scope of the invention.

The practice of the present invention employs, unless otherwise indicated, conventional organic synthesis, analysis and molecular characterisation techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature.

Example 1 . Synthesis of RTFtl , RTFtl dimer and RTFt-O.

Rhodamine thiophenol (RTFtl ) and RTFtl dimer were synthesised according to scheme 1 (see Figure 1).

Reagents and conditions: (i) hydrazine hydrate, MeOH, reflux, overnight; (ii) B0C2O, DMAP, THF, r.t., 1 h; (iii) NBS, benzoyl peroxide, CCk, 60 °C, overnight; (iv) 2, K2CO3, MeCN, 50 °C, 20 h; (v) TFA, DCM, r.t., 4 h; (vi) l ₂ , MeOH, r.t., 1 h.

2-Methylbenzenethiol (5 g) was added to a 100 ml round bottom flask (RBF) containing THF (40 mL), equipped with magnetic stirrer. Di-Tert-Butyl Dicarbonate (8.8 g) and 4-Dimethylaminopyridine (0.2 g) were added and the reaction mixture was stirred for 1 h at room temperature. Reaction mixture was dried by vacuo and further purified by silica-gel column chromatography (EA/hexane, 1/30, v/v) affording compound 3 as colorless oil (8.9 g, 98 %). ¹ H NMR (300 MHz, CDCh) 5 (ppm) :7.58 (d, 1 H), 7.38-7.21 (m, 3 H), 2.50 (s, 3 H), 1 .56 (s, 9 H).

Boc-protected 2-Methylbenzenethiol (3, 8 g) was added to a 100 ml RBF containing carbon tetrachloride (40 mL), equipped with magnetic stirrer and water cooled-condenser. NBS (6.4 g) and BPO (410 mg) are added and the reaction mixture was heated at 60 °C for 12 h. The reaction was quenched by sat. Na2S20s solution and extracted by DCM (100 mL, 3 times). The combined organic layer was concentrated by vacuo to generate crude product. Further purification was applied by silica-gel column chromatography (EA/hexane, 1 :30, v/v) affording compound 2 as colorless solid (7.5 g, 69 %). ¹ H NMR (300 MHz, CNCD3) 5 (ppm) :7.36 (d, 1 H), 7.33 (d, 1 H), 7.23 (t, 1 H), 7.16 (t, 1 H), 4.48 (s, 2 H), 1.26 (s, 9 H). ¹³ C NMR (75 MHz, CNCD ₃ ) 6 (ppm): 166.94, 142.30, 138.64, 131.77, 131.63, 130.34, 129.30, 118.08, 86.85, 32.90, 28.31. ESI-MS calcd. for Ci2HisNaBrO2S ⁺ [M + Na] ⁺ 324.99, found 325.07.

Rhodamine B (4.2 g) was added to a 100 mL RBF, equipped with magnetic stirrer and water cooled-condenser. MeOH (40 mL) and hydrazine hydrate (4 mL) were added to the flask and the mixture was refluxed overnight. Reaction mixture was poured into H2O (100 mL), and extracted by DCM (100 mL) for 3 times. The combined organic layer was dried by anhydrous sodium sulfate and vacuo, further purified by silica-gel column chromatography (MeOH/DCM, 1/20, v/v) affording compound 1 as gray powder (3.8 g, 93 %). ¹ H NMR (200 MHz, CDCh) 5 (ppm): 7.93 (dd, 1 H), 7.45 (t, 1 H), 7.40 (t, 1 H), 7.08 (dd, 1 H), 6.44 (d, 2 H), 6.42 (s, 2 H), 6.25 (dd, 1 H), 3.61 (s, 2 H), 3.33 (q, 8 H), 1.15 (t, 12 H). ESI-MS calcd. for C28H ₃ 2N ₄ NaO2 ⁺ [M + Na] ⁺ 479.24, found 479.29.

Compound 1 (1 g) and 2 (750 mg) were added to a 100 ml RBF containing acetonitrile (40 mL), equipped with magnetic stirrer and water cooled-condenser. Then potassium carbonate (1 g) was added and the reaction mixture was heated at 50 °C overnight. Reaction mixture was poured into H2O (50 mL), and extracted by DCM (30 mL) for 3 times. The combined organic layer was dried by anhydrous sodium sulfate and vacuo, further purified by silica-gel column chromatography (EA/Hexane, 1/3, v/v) affording compound 4 as white powder (870 mg, 58 %). ¹ H NMR (300 MHz, CDCh) 6 (ppm): 7.97 (dd, 1 H), 7.51 (t, 2 H), 7.43 (d, 1 H), 7.29 (d, 1 H), 7.23-7.15 (m, 2 H), 6.47- 6.44 (m, 4H), 6.27 (dd, 2 H), 4.64 (t, 1 H), 4.02 (d, 2 H), 3.45 (q, 8 H), 1 .52 (s, 9 H), 1 .22 (t, 12 H). ¹³ C NMR (75 MHz, CDCh) 5 (ppm): 167.77, 166.47, 153.97, 151.37, 148.67, 141.71 , 136.28, 132.52, 130.77, 130.63, 129.58, 128.53, 128.10, 127.53, 124.08, 122.66, 107.69, 106.07, 97.93, 85.13, 65.58, 53.06, 44.32, 28.15, 12.68. ESI-MS calcd. for C4OH47N ₄ 04S ⁺ [M + H] ⁺ 679.33, found 679.43

Compound 4 (RTFtl) (500 mg) was added to a 50 ml RBF containing magnetic stirrer and DCM/TFA (20 mL/ 4mL), and stirred for 4 h at room temperature. The reaction mixture was neutralized by sat. NaHCCh solution and extracted by DCM (30 mL, 3 times). The combined organic layer was dried by anhydrous sodium sulfate and vacuo, purified by silica-gel column chromatography (EA/Hexane, 1/3, v/v). Product generated from column was concentrated by vacuo and dissolved in diethyl ether (10 mL). RTFtl was precipitated out after hexane (30 mL) was added into the solution as white powder (210 mg, 49 %). ¹ H NMR (300 MHz, DMSO) 5 (ppm): 7.83 (dd, 1 H), 7.53 (t, 3 H), 7.16 (d, 1 H), 7.08-6.97 (m, 4 H), 6.40-6.30 (m, 6 H), 5.25 (t, 3 H), 4.93 (s, 1 H), 3.85 (d, 2 H), 3.30 (q, 8 H), 1.07(t, 12 H). ¹³ C NMR (75 MHz, DMSO) 5 165.65, 153.11 , 151.66, 148.22, 134.83, 132.85, 132.53, 130.58, 129.98, 129.71 , 128.38, 127.89, 125.12, 123.76, 122.17, 107.86, 105.23, 97.37, 64.65, 52.90, 43.68, 39.52, 27.64, 12.38. HRMS calcd. for C ₃₅ H39N4O2S ⁺ [M + H] ⁺ 579.2788, found 579.2788.

RTFtl (200 mg) was added to a 25 ml RBF containing magnetic stirrer together with I2 (50 mg) and MeOH (10 mL), stirred for 1 h at room temperature. The reaction mixture was quenched by sat. Na2S20a solution and extracted by DCM (20 mL, 3 times). The combined organic layer was dried by anhydrous sodium sulfate and vacuo, purified by silica-gel column chromatography (EA/Hexane, 1/5, v/v), afforded RTFtl - dimmer as white powder (17 5 g, 88 %). ¹ H NMR (300 MHz, CDCh) 5 (ppm): 7.98 (dd,

1 H), 7.51 (t, 2 H), 7.31 (dd, 1 H) 7.22-6.96 (m, 4 H), 6.51 (d, 2 H), 6.47(d, 2 H), 6.27(dd,

2 H), 4.29 (t, 1 H), 4.05 (d 2 H), 3.29 (q, 8 H), 1 .13 (t, 12 H). ¹³ C NMR (75 MHz, CDCh) 5 (ppm): 166.88, 154.11 , 152.02, 148.82, 137.35, 135.45, 132.70, 130.61 , 130.06, 128.63, 128.24, 128.10, 127.45, 126.08, 124.21 , 122.72, 107.67, 105.88, 98.22, 65.71 , 52.85, 44.35, 12.72. HRMS calcd. for C oHysNsC^ [M + H] ⁺ 1154.5274, found 1154.5274.

RTFt-0 (structure shown below) was synthesized from a similar way as RTFtl with 17% yield from rhodamine B. ¹ H NMR (300 MHz, CDCh) 5 (ppm): 8.81 (s, 1 H), 7.97 (dd, 1 H), 7.51 (t, 2 H), 7.16-7.07(m, 2 H), 6.84 (td, 1 H), 6.68 (td, 1 H), 6.47(dd, 4 H), 6.26(dd, 2 H) 4.23 (t, 1 H), 3.87 (d 2 H) 3.35 (q, 8 H), 1.18 (t, 12 H). ESI-MS calcd. for C35H39N4O3 ⁺ [M + H] ⁺ 563.30, found 563.41 .

Rhodamine B (500 mg) was added to a 50 ml RBF containing magnetic stirrer and DCM (15 mL), thionyl chloride (0.5 mL) was added to the reaction mixture and stirred for 4 h at room temperature. MeOH (2 mL) was then slowly added to the reaction and stirred for 10 min before quenching by H2O (20 mL). The reaction mixture was extracted by DCM (20 mL, 3 times) and further dried by anhydrous sodium sulfate and vacuo, purified by silica-gel column chromatography (MeOH/DCM, 1/20, v/v), afforded RTFt1-0 as dark solid (457 mg, 89 %). ¹ H NMR (300 MHz, CDCh) 5 (ppm): 8.81 (s, 1 H), 7.97 (dd, 1 H), 7.51 (t, 2 H), 7.16-7.07(m, 2 H), 6.84 (td, 1 H), 6.68 (td, 1 H), 6.47(dd, 4 H), 6.26(dd, 2 H) 4.23 (t, 1 H), 3.87 (d 2 H) 3.35 (q, 8 H), 1.18 (t, 12 H). ESI-MS calcd. for C35H39N ₄ O3 ⁺ [M + H] ⁺ 457.25, found 457.25.

Example 2: General Methods

Fluorescence measurements using platereader: All experiments were carried out in 96-wells plates in an aqueous solution (milliQ H2O/DMSO = 90/10, v/v). Aex/em= 556/576 nm. Ferrous ammonium sulfate was used as Fe(ll) source, 2.2’-bipyridyl was used as Fe(ll) chelator. 20 pM Fe(ll) and H2O2, (1% volume) was used to simulate Fenton conditions.

Cell culture

All cell lines (Human DLD-1 colorectal adenocarcinoma (DLD-1 ), HT-1080 firbosarcoma cells (HT-1080), and (human prostate cancer cells (PC-3)) were cultured in Advanced Dulbecco’s Modified Medium (DMEM) supplemented with 2% Fetal Bovine serum, and2mM glutamine and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. After growing to 80% confluence, the cells were treated with trypsin, and then seeded on glass bottom dishes for 24 h for further experiments.

Confocal microscopy imaging

Approximately 2 x 10 ⁵ cells were seeded onto MatTek glass bottom dishes (precoated with L-proline) for imaging the next day. Cells were stained with 20 pM RTFtl and incubated at 37°C for 1 h. Cells were imaged using an Olympus FV3000 confocal microscope equipped with a SAPO 60 x objective and a 561 nm LED laser. Z-stacked images with a step-size of 1.0 micron were acquired. Fluorescence images presented were as maximum projections of z-stacked micrographs. A 561 nm laser was used to excite the fluorescent product, 570-670 nm was collected as emission. Before imaging, manually lock cells’ top and bottom, between which a serial of stacked images was taken.

Cytotoxicity assay

Cell viability (as a measure of cytotoxity) was performed using Alamar Blue or MTT assays, according to standard protocols. Approximately 5 x 10 ³ cells were seeded to determine cytotoxicity of compounds over 24 h.

Viable cells were determined using Alamar Blue assay or MTT assay. Approximately 5 x 10 ³ cells were seeded to determine cytotoxicity of compounds over 24 h.

Monitoring positive/negative Fenton conditions

Simulation of positive Fenton condition: Citrate Fe(ll) and H2O2 were used to simulate positive Fenton conditions. Cells were stained with RTFT1 (10pM in HBSS buffer, 0.5% DMSO) for 1 h in an incubator, then washed by HBSS buffer 3 times, followed by treatment with citrate Fe(ll) (50 pM in HBSS buffer) for another 1 h in the incubator. The cells were finally incubated with 100 pM H2O2 in HBSS for 1 h, and washed 3 times. The cells were then imaged by confocal microscopy.

Simulation of negative Fenton conditions: Catalase, an H2O scavenger, and 2,2’- bipyridyl (BPD), a Fe(ll) chelator, were used to simulate negative Fenton conditions. Cells were treated with catalase (2000 - 5000 units/mL in DMEM media for 4h), then washed in HBSS buffer for 3 times before incubating cells with 2,2’-bipyridyl (500 pM in HBSS buffer, 0.5% DMSO). Cells were finally incubated with 10pM RTFT1 in HBSS for 1 h, and washed 3 times, then imaged by confocal microscopy.

Monitoring Fenton conditions initiated by cisplatin: Cells were contacted with RTFT1 (10 pM in HBSS buffer, 0.5% DMSO) and cisplatin (20 pM in HBSS buffer 0.2% DMSO) for 2 hours in an incubator then washed with HBSS 3 times prior to imaging.

Monitoring Fenton conditions during erastin-initiated ferroptosis: Cells were contacted with RTFT1 (10 pM in HBSS buffer, 0.5% DMSO) for 1 hour in uncubaotr, then washed in HBSS buffer (3x) prior to treatment with erastin (30 pM in HBSs buffer, 0.5% DMSO) for 30 minutes. Cells were washed in HBSS (3x) prior to confocal microscopy imaging.

Example 3. Response of RPT to Fenton conditions.

Figure 2 shows the proposed mechanism by which RTFtl can detect the two Fenton substrates Fe(ll) and F C .Figure 3 shows fluorescence excitation (dotted) and emission (solid) spectra of RTFT1 in the presence or absence of Fe(ll) (10 pM) and H2O2 (1% v/v). RTFtl alone exhibited extremely weak absorption and emission. Under Fenton conditions, both absorption and emission increased significantly, with maxima at 556 nm and 580 nm respectively.

Example 4. Selectivity of RTFT1 selectivity towards biological metals in Fenton condition.

The selectivity of RTFT1 towards different biological metals in the presence of H2O2was measured.

The fluorescence response of RTFT1 (50pM) toward Fe(ll) (100 pM) and other biological metals (100pM) in a solution of milliQ H2O/DMSO/H2O2 (89/10/1 , v/v/v) was measured in Wellplate experiments as described in example 2, using Aex/em=556/580 nm. The results are shown in Figure 4. Metals tested were: : 1 ) control; 2) Fe(ll); 2) Fe(lll); 4) Cu ²⁺ ; 5) Zn ²⁺ ;6) Ca ²⁺ ; 7) Na ⁺ ; 8) K ⁺ ; 9) Mg ²⁺ _; 10) Cu ⁺ .

Example 5. Role of the -SH group in the chelation of Fed I)

To further confirm the role of Fe(ll) chelation, optical spectra of RTFtl in the absence or presence of Fe(ll) and H2O2 were compared. Although the probe was slightly turned on by Fe(ll) alone, there was a shift in the peak maximum after subsequent H2O2 addition (576 nm to 580 nm), consistent with emission from different fluorophores. It was hypothesized that the fluorescence from RTFtl with Fe(ll) comes the chelated spirolactam ring-open form. As expected for chelation, this first step is reversible process, as the fluorescence change could be reversed by the addition of 2,2'-bipyridyl, a selective Fe(ll) chelator (Figure 5). The binding mode was further investigated by preparing analogues of RTFtl with a disrupted binding site. Since it was proposed that the thiol group is essential for Fe(ll) binding, RTFtl was converted to the oxidized RTFt-dimer (Scheme S1 ), and the sulfur changed for oxygen to give RTFt-0 (Figure 5). Neither of these analogues showed significant emission changes when subjected to Fe(ll) (Figure 5), confirming the importance of the thiol for Fe(ll) recognition.

Example 6. Sensitivity of the RTFtl probe towards Fe(ll) alone

The biocompatibility of RTF1t in DLD-1 colorectal carcinoma cells was confirmed by standard MTT assay (data not shown). Viability was 92% when cells were incubated with 50 pM RTFtl for 24 hours.

Figure 6 shows confocal microscopy images and mean quantified fluorescence intensity of DLD-1 cells treated with RTFtl in the presence or absence of the Fe(ll) chelator BPD or with the addition of Fe( 11) citrate.

Addition of Fe(ll) (as Fe(ll) citrate), leads to an increase in fluorescence; starvation of Fe(ll) using the iron chelator BPD leads to a decrease in fluorescence compared to cells treated with RTFtl alone. These results show that intracellular H2O2 levels are sufficient to turn on the probe in the presence of Fe(ll), but in the presence of a chelator there is less observable Fenton chemistry.

Example 7: Sensitivity of the RTFT1 probe towards H2O2 alone

Figure 7 shows confocal microscopy images and mean quantified fluorescence intensity of DLD-1 cells treated with RTFT1 in the presence of absence of the hydrogen peroxide scavenger catalase, or with the addition of H2O2.

Addition of H2O2 leads to an increase in fluorescence; sequestration of H2O2 using scavenger catalase leads to a decrease in fluorescence compared to cells treated with RTFtl alone. These results show that intracellular Fe(ll) levels are sufficient to turn on the probe in the presence of H2O2, but in the presence of a scavenger there is less observable Fenton chemistry.

Example 8. Fenton condition sensing by RTFT1 probe in live cells

Figure 8 shows confocal microscopy images and mean quantified fluorescence intensity for DLD-1 cells treated with RTFT1 alone (b), Fe(ll) citrate following treatment with RTFT1 (c), hydrogen peroxide following treatment with RTFT1 (d), and Fe(ll) citrate and hydrogen peroxide following treatment with RTFT1 (e).

As for previous results, adding both reagents leads to even greater turn-on of probe (corresponding to greater Fenton chemistry).

In all cases the greatest fluorescence signals were observed in positive Fenton conditions. RTFtl has next to no fluorescence on its own, and in the presence of only one of Fe(ll) or H2O2 showed significantly less fluorescence compared to the combination of all three. This is shown in two ways, in Figure 8 the reactants are added singly and in combination. Figure 9 shows the reverse process where scavengers are added to starve the cells of the reagents (singly and combined).

Example 9. Monitoring Fenton conditions during Erastin-initated ferroptosis.

Ferroptosis is a regulated cell death pathway involving thiols, lipid peroxidation and iron. Although a chronological signaling pathway that initiates ferroptosis is yet to be well-defined, recent research has established the central role of Fenton chemistry as an up-steam regulator of ferroptosis. Since the frequency of the intracellular Fenton reaction is a metric of ferroptosis, it was proposed that RTFtl would be able to sense ferroptosis. Ferroptosis-susceptible DLD-1 cells and HT-1080 fibrosarcoma cells were selected for imaging studies.

Before RTFtl staining, cells were pre-treated with erastin, a reported inducer of ferroptosis. A fluorescence decrease was first observed 2 h after erastin treatment compared to the vehicle control, and the signal further in-creased over time (Figure 10). This fluorescence decrease was also observed at shorter timepoints (Figure 1 1 ). It was confirmed that erastin did not have an effect on uptake of the non-responsive rhodamine B methyl ester (Figure 12), suggesting that there is a decrease in Fenton reactants during the first 2 h after erastin treatment, with an increase at subsequent timepoints. At longer timepoints, changes in the subcellular localization of fluorescence were observed, with evident cytoplasmic localization (white arrows) compared to the perinuclear staining at shorter time points. It was hypothesized that this difference arises from changed H2O2 distribution after erastin treatment. These results confirm that RTFtl can be used to report on ferroptosis. Example 10. Monitoring Fenton conditions initiated by cisplatin.

Platinum-based drugs are used in up 50% of all chemo-therapeutic regimens. While they are known to cross-link nuclear DNA thus inducing apoptosis, some studies have suggested that cisplatin induces production of ROS. Since H2O2 is one the Fenton reactants, it was proposed that the Fenton reaction, triggered by increased intracellular H2O2 can be another source of cisplatin’s cytotoxicity in addition to its DNA effects.

DLD-1 cells were pre-stained with RTFtl for 1 h before treatment with cisplatin. Fluorescence from vehicle control cells did not change over-time, but clear fluorescence changes were observed in cells 2 h after cisplatin treatment, with subsequent increases over time, indicating increased Fenton biochemistry in cisplatin-loaded cells (Figure 13). To provide further evidence of the role of Fenton biochemistry in cisplatin cytotoxicity, the intracellular Fenton reaction was suppressed by addition of 2 ,2’— bipyridyl .

Cell viability upon cisplatin treatment was assessed by a standard Alamar Blue assay. The 24 h IC50 induced by cisplatin (20 pM) was tripled in the presence of 30 pM 2,2’— bipyridyl (60 pM) providing further indication that Fenton biochemistry could contribute to the cytotoxicity of cisplatin (Figure 14).

Example 1 1 . Monitoring Fenton conditions initiated by cisplatin in combination with tubastatin A

Inhibition of histone deacetylase (HDAC) was first proposed to be an effective method of increasing the cytotoxicity of anticancer drugs in 2003, and since that time a series of HDAC inhibitors have been shown to enhance the efficacy of existing drugs. HDAC inhibitors have been previously related to Fenton Chemistry. Therefore the effect of combining platinum drugs and HDAC inhibitors was explored.

The IC50 of DLD-1 cells treated with cisplatin was compared to DLD-1 cells treated with cisplatin in the presence of 10 pM tubastatin A, an HDAC6 inhibitor (Figure 15). The cytotoxicity of tubastatin A in presence or absence of cisplatin was also determined (Figure 16). These results showed that tubastatin A enhanced the cytotoxicity of cisplatin. The Fenton biochemistry in DLD-1 cells in the presence of cisplatin and/or tubastatin A using RTFtl was explored. Cells treated with tubastatin A alone showed a slight fluorescence decrease, matching previously findings of its role in suppressing oxidative stress (Figure 17). Interestingly, fluorescence from cells co-treated with cisplatin and tubastatin A is much higher than cells treated with cisplatin alone, indicating that the enhancement of cisplatin-induced cytotoxicity by tubastatin A may be due to the elevation of intracellular Fenton reaction (Figure 17).

These results show the potential of the probes of this invention to shed light on the role of Fenton chemistry for treatments involving combinations of platinum drugs and HDAC inhibitors.