Novel therapy

Abstract

Provided herein are methods and uses of compounds for direct scavenging of and / or direct reduction of the concentration of one or more reactive oxygen species (ROS), and for therapy of associated diseases and disorders. Also provided herein are methods and uses of the compounds for preventing loss of, maintaining, or restoring mitochondrial function, and for therapy of associated diseases and disorders. Also provided herein are methods and uses of the compounds for treating or preventing signs of aging.

Description

[0001]  Novel Therapy Field This disclosure relates to methods and uses of compounds for direct scavenging of and / or direct reduction of the concentration of one or more reactive oxygen species (ROS), and for therapy of associated diseases. It also relates to methods and uses of the compounds for preventing loss of, maintaining, or restoring mitochondrial function, and for therapy of related diseases and disorders. The disclosure also related to uses of the compounds for treating and / or preventing one or more signs of aging. Background In all cells, there is a balance between cellular oxidation and cellular reduction reactions which defines the redox state. Redox reactions are intrinsic to energy metabolism, including mitochondrial respiration. During the transitions in redox reactions such as those during aerobic metabolism, free radicals form including reactive oxygen species (ROS), a set of distinct molecular oxygen derivative with uneven electrons (i.e. superoxide anion, peroxide, hydroxyl radical, hydroxyl ion). Hydrogen peroxide is an additional non-radical ROS that can oxidize proteins and that can form hydroxyl radical in the presence of ferrous iron. The cellular redox state is maintained through a network of enzymatic reactions and factors that generate ROS for subsequent reactions and that eliminate them through reduction reactions. When endogenous antioxidant systems are overwhelmed, the ROS are able to interact with DNA, lipids and proteins in a manner that causes cellular injury due to the uneven number of electrons in free radicals and the oxidizing capacity of hydrogen peroxide. Exposure of cells to maladaptive levels of ROS due to an imbalance in endogenous antioxidant control mechanisms is thought to contribute to the tissue damage in several diseases as well as in aging. ATH434 is a small molecule with moderate ferric and ferrous iron affinities (Kd 10-10,Kd 10-5respectively, Finkelstein et al, Acta Neuropathologica Communications, 2017, 5:53; the entire contents of which are incorporated herein by this reference).   1     (ATH434) It reduces excess brain iron, reduces aggregated α-synuclein and improves neuronal survival. There is a need for further agents which can directly reduce levels of ROS to limit tissue damage and / or protect mitochondrial function from the effects of ROS. There is also a need for further therapies of diseases and disorders associated with elevated levels of ROS and / or or with mitochondrial dysfunction. Summary The efficacy of ATH434 as a potential antioxidant and mitochondrial protectant has been investigated, using a menadione-induced model of oxidative stress in the glutamatergic neuronal HT22 cell line. ATH434 rescued the reduction in the mitochondrial membrane potential associated with menadione treatment, demonstrating an ability to preserve mitochondrial function in stressed neurons. Consistent with the cellular effects, ATH434 possesses in-solution antioxidant capacity in an ABTS radical scavenging assay. The chemical antioxidant effect and cellular effects with an ATH434 analog possessing reduced iron binding affinity indicates a direct interaction between ATH434 and free radicals. Together, these results indicate that ATH434 has important hitherto undiscovered properties as a direct antioxidant, and that it also has mitochondria-protective activities. Accordingly, in a first aspect there is provided use of a compound of formula (I) wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10-membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4alkyl; and 2     R5is hydrogen or methyl; or a salt thereof; or a pharmaceutical composition comprising the compound or salt and a pharmaceutically acceptable excipient; for direct scavenging and / or direct reduction of the concentration of one or more reactive oxygen species (ROS). In some embodiments, the compound or salt is used for direct scavenging of and / or direct reduction of the concentration of one or more ROS in a subject. In another aspect, there is provided a method of directly scavenging and / or directly reducing the concentration of one or more reactive oxygen species (ROS) in a subject, comprising administering an effective amount of a compound of formula (I) wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10-membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4 alkyl; and R5is hydrogen or methyl; or a salt thereof, or of a pharmaceutical composition comprising the compound or salt and a pharmaceutical excipient, to the subject. In some embodiments, the subject is human. In some embodiments, the subject has an elevated concentration of one or more ROS. In some embodiments, the subject has a disease or disorder in which elevated concentrations of total iron, labile iron, and / or in which improperly stored iron, is not the determinant of the disease or disorder. In another aspect, there is provided use of a compound of formula (I) 3     wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10-membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4 alkyl; and R5is hydrogen or methyl; or a salt thereof; or a pharmaceutical composition comprising the compound or salt and a pharmaceutically acceptable excipient; for maintaining, preventing loss of, or restoring mitochondria and / or mitochondrial function in a subject. In another aspect, there is provided a method of maintaining, preventing loss of, or restoring mitochondria and / or mitochondrial function in a subject, comprising administering an effective amount of a compound of formula (I) wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10-membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; 4     R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4alkyl; and R5is hydrogen or methyl; or a salt thereof, or of a pharmaceutical composition comprising the compound or salt and a pharmaceutical excipient, to the subject. In some embodiments, the subject is human. In some embodiments, the subject has an elevated concentration of one or more reactive oxygen species (ROS). In some embodiments, the subject has a disease or disorder in which elevated concentrations of total iron, labile iron, and / or in which improperly stored iron, is not the determinant of the disease or disorder. In another aspect, there is provided a method of treating or preventing a disease or disorder which is associated with elevated levels of one or more reactive oxygen species (ROS), and in which elevated concentrations of total iron, labile iron, and / or in which improperly stored iron, is not the determinant of the disease or disorder, in a subject, comprising administering an effective amount of a compound of formula (I) wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10-membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4alkyl; and R5is hydrogen or methyl; or a salt thereof, or of a pharmaceutical composition comprising the compound or salt and a pharmaceutical excipient, to the subject. In another aspect, there is provided use of a compound of formula (I), 5     wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10-membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4 alkyl; and R5is hydrogen or methyl; or a salt thereof, for the manufacture of a medicament for treating or preventing a disease or disorder which is associated with elevated levels of one or more reactive oxygen species (ROS), and in which elevated concentrations of total iron, labile iron, and / or in which improperly stored iron, is not the determinant of the disease or disorder, in a subject. In some embodiments, the subject is human. In some embodiments, the disease or disorder is a neurodegenerative disease or disorder. In some embodiments the disease or disorder is selected from the group consisting of Pelizaeus-Merzbacher disease, diabetes mellitus, renal cancer, cardiovascular disease and partial bladder outlet obstruction. In another aspect, there is provided a method of treating and / or preventing one or more signs of aging which is associated with elevated levels of one or more reactive oxygen species (ROS), and in which elevated concentrations of total iron, labile iron, and / or in which improperly stored iron, is not the determinant of the sign of aging, in a subject, comprising administering an effective amount of a compound of formula (I) 6     wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10-membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4 alkyl; and R5is hydrogen or methyl; or a salt thereof, or of a pharmaceutical composition comprising the compound or salt and a pharmaceutical excipient, to the subject. In another aspect, there is provided use of a compound of formula (I), wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10-membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4alkyl; and R5is hydrogen or methyl; 7     or a salt thereof, for the manufacture of a medicament for treating or preventing one or more signs of aging which is associated with elevated levels of one or more reactive oxygen species (ROS), and in which elevated concentrations of total iron, labile iron, and / or in which improperly stored iron, is not the determinant of the sign of aging, in a subject. In some embodiments, the subject is human. In another aspect, there is provided a method of treating or preventing a disease of disorder associated with loss of mitochondrial function and / or loss of mitochondria, and which is associated with elevated levels of one or more reactive oxygen species (ROS), in a subject, comprising administering an effective amount of a compound of formula (I) wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10-membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4 alkyl; and R5is hydrogen or methyl; or a salt thereof, or of a pharmaceutical composition comprising the compound or salt and a pharmaceutical excipient, to the subject. In another aspect, there is provided use of a compound of formula (I) 8     wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10-membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4 alkyl; and R5is hydrogen or methyl; or a salt thereof; for the manufacture of a medicament for treating or preventing a disease or disorder associated with loss of mitochondrial function and / or loss of mitochondria, and which is associated with elevated levels of one or more reactive oxygen species (ROS), in a subject. In some embodiments, the subject is human. In some embodiments, the subject has a disease or disorder in which elevated concentrations of total iron, labile iron, and / or in which improperly stored iron, is not the determinant of the disease or disorder. In some embodiments, R1and R2are each chlorine. In some embodiments, R3is methyl. In some embodiments, R3is C1-4alkyl. In some embodiments, R4is -CH2NHEt. In some embodiments, R5is H. In some embodiments, the compound of formula (I) is 9       (ATH434) or a salt thereof. In some embodiments, a mesylate salt of the compound is used. Brief Description of the Drawings Figure 1 shows a chart showing the antioxidant capacity of example compounds of the disclosure versus compound concentration in an ABTS antioxidant potential assay, together with results for comparator compounds. Figure 2 presents: a) a chart showing the hydrogen atom transfer activity of example compounds of the disclosure at 20μM concentration in an Oxygen Radical Absorbance Capacity (ORAC) assay compared to the compound Trolox, together with results for comparator compounds; b) a chart showing remaining fluorescein (RFU) normalized to unquenched control against time in the ORAC assay for Trolox, compounds of the disclosure, comparator compounds, and DMSO, at 20μM concentration; and c) - f) charts showing remaining fluorescein (RFU) normalized to unquenched control against time in the ORAC assay for DMSO and test compound at 0.2μM, 2μM, 20μM and 200μM concentrations, for ATH434, ATH434-met, Dfp, and Dfx respectively. Figure 3 presents a chart showing the electron transfer activity of example compounds of the disclosure in a Ferric Reducing Antioxidant Power (FRAP) assay, together with results for comparator compounds. Figure 4 presents charts showing the reduction potential (mV) of ATH434 and ATH434- met, when analysed using cyclic voltammetry, which measures the oxidation and reduction cycling of a solution of compound. Figure 5 presents a chart showing TMRM dye fluorescence and that allows monitoring of mitochondrial function for cells pre-treated with compounds of the disclosure and challenged with a pro-oxidant, menadione. 10     Figure 6 presents a chart showing results of a lipid peroxidation assay experiment conducted on ATH434 in glutamatergic neuronal HT22 cells, showing the ratio of oxidized (510nm) to reduced (590 nm) BODIPY, which is a measure of upstream ROS generation. Figure 7 presents a chart showing results of a Seahorse metabolic assay experiment showing a) oxygen consumption rate (OCR) against time, and b) extracellular acidification rate (ECAR) against time for ATH434, an example compound of the present disclosure. Figure 8 presents charts showing results from the Seahorse metabolic assay experiment showing effects of different concentrations of ATH434 on basal respiration rate, maximal respiration rate and non-mitochondrial respiration. Figure 9 presents charts showing results from the Seahorse metabolic assay experiment showing effects of different concentrations of ATH434 on ATP-linked respiration, proton leak and coupling efficiency (ATP production / basal respiration). Figure 10 presents charts showing results from the Seahorse metabolic assay experiments showing effects of different concentrations of ATH434 on total ATP production rate, non- mitochondrial ATP production rate (glycolysis) and mitochondrial ATP production rate. Detailed Description Definitions Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art. It is to be understood that if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art. As used herein, the term “and / or”, e.g., “X and / or Y” shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning. As used herein, the term about, unless stated to the contrary, refers to + / - 10%, of the designated value. Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context clearly 11     dictates otherwise. For example, reference to "a" includes a single as well as two or more; reference to "an" includes a single as well as two or more; reference to "the" includes a single as well as two or more and so forth. Unless otherwise indicated, terms such as "first," "second," etc. are used herein merely as labels, and are not intended to impose ordinal, positional, or hierarchical requirements on the items to which these terms refer. Moreover, reference to a “second” item does not require or preclude the existence of lower-numbered item (e.g., a “first” item) and / or a higher-numbered item (e.g., a “third” item). As used herein, the phrase “at least one of”, when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed. The item may be a particular object, thing, or category. In other words, “at least one of” means any combination of items or number of items may be used from the list, but not all of the items in the list may be required. For example, “at least one of item A, item B, and item C” may mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C. In some cases, “at least one of item A, item B, and item C” may mean, for example and without limitation, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination. As used herein, the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps. Each embodiment of the present disclosure described herein is to be applied mutatis mutandis to each and every other embodiment unless specifically stated otherwise or required otherwise by context. Compounds The present disclosure relates to uses and methods involving a compound of formula (I), or a salt thereof. Compounds of formula (I) have the following formula:     wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10-membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4 alkyl; and R5is hydrogen or methyl. As used herein, “Cato Cb” or “Ca-b” in which “a” and “b” are integers refer to the number of carbon atoms in the specified group. That is, the group can contain from “a” to “b”, inclusive, carbon atoms. Thus, for example, a “C1 to C4 alkyl”, or “C1-4-alkyl” group includes alkyl groups having from 1 to 4 carbons, e.g. CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)- and (CH3)3C-. Terms written as "groupAgroupB" are intended to refer to a groupA when linked by a divalent form of groupB. For example, "hydroxyalkyl" is a hydroxy group when linked by an alkylene group, "haloalkyl" is a halo group when linked by an alkylene group, "alkoxyalkyl" is an alkoxy group when linked by an alkylene group, etc. Similarly, "alkoxy" denotes alkyl, as herein defined, when linked by an oxygen atom, "aryloxy" denotes aryl, as herein defined, when linked by an oxygen atom, etc. As used herein, the term “alkyl” refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). The alkyl group of the compounds may be designated as “C1-4alkyl” or similar designations. By way of example only, “C1-4alkyl” indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t- butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like. As used herein, the term “cycloalkyl” means a monocyclic or polycyclic saturated carbon ring. Examples of cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptanyl. A C3-7cycloalkyl is a cycloalkyl group containing between 3 and 7 carbon atoms. As used herein, the term “heterocyclic” means an aromatic or non-aromatic ring system that contain(s) one or more heteroatoms, that is, an element other than carbon, such as N, O and / or S, in the ring backbone. The remaining ring atoms are typically carbon atoms. In some embodiments, a heterocyclic group contains 1, 2, or 3 heteroatoms. A heterocyclic ring can for example be a 13     heterocycloalkyl ring or can for example be a heterocyclic aromatic (also known as heteroaryl) group, or if polycyclic, any combination thereof. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. A heterocyclic ring can also include one or more double bonds, e.g. it may be a heterocycloalkenyl group. The phrase “heterocyclic group” includes fused ring species including those comprising fused aromatic and non-aromatic groups. Heterocyclic groups include, but are not limited to, pyrrolidinyl, piperidinyl, homopiperidinyl, piperazinyl, homo piperazinyl, morpholinyl, homomorpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. As used herein, the term “heteroaryl”, “heteroaromatic” or “aromatic heterocycle” means an aromatic ring system that contain(s) one or more heteroatoms, that is, an element other than carbon, such as N, O and / or S, in the ring backbone. The remaining ring atoms are typically carbon atoms. In some embodiments, the heteroaromatic group has from 5 to 9 ring atoms. Examples of heteroaromatic rings include, but are not limited to, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, thiophenyl, benzothiophenyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothienyl. As used herein, the terms “halo” or “halogen,” mean, in the context of the compounds defined herein, a fluorine, chlorine, bromine, or iodine atom In some embodiments, R1and R2are each halogen. In some embodiments, R1and R2are each independently selected from the group consisting of fluorine, chlorine and iodine. In some embodiments, R1and R2are each independently selected from the group consisting of chlorine and iodine. In some embodiments, R1and R2are each chlorine. In some embodiments, R3is C1-4alkyl. In some embodiments, R3is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R3is methyl. In some embodiments, R3is hydrogen. 14     In some embodiments, R3is -CH2-C3-7cycloalkyl, i.e. it contains a methylene group connected to a 3-7 membered cycloalkyl ring. For example, it may be ,  In some embodiments, R3is -CH2Het, wherein Het is a 5 to 10-membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen.  In some embodiments the heterocycle is an aromatic heterocycle. In other embodiments, the heterocycle is a non-aromatic heterocycle. In some embodiments, the heterocycle is a monocyclic heterocycle. In some other embodiments, the heterocycle is a bicyclic heterocycle. In some embodiments, the heterocycle is a thiazolyl, an imidazolyl, a pyridyl or a benzimidazolyl group. In some embodiments, Het is an unsubstituted 5-10-membered heterocycle. In some embodiments, Het is a 5 to 10-membered heterocycle which is substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen. In some embodiments, Het is a 5 to 10-membered heterocycle which is substituted with 1 or 2 methyl groups. In some embodiments, Het is a 5 to 10-membered heterocycle which is substituted with 1 methyl group. In some embodiments, R3is selected from the group consisting of In some embodiments, R4is -CH2NR6R7,wherein R6and R7are each independently a C1-4alkyl. In some embodiments, R4is -CH2NR6R7, wherein R6and R7are each independently methyl or ethyl. In some embodiments, R4is -CH2NR6R7,wherein R6and R7are each hydrogen. In some embodiments, R4is -CH2NR6R7, wherein R6is hydrogen and R7is C1-4alkyl. In some embodiments R4is -CH2NR6R7, wherein R6is hydrogen and R7is ethyl. 15   In some embodiments, R4is hydrogen. In some embodiments, R4is -CH2OH. In some embodiments, R5is H. In other embodiments, R5is methyl. In some embodiments, the compound of formula (I) is ATH434 (ATH434). In some embodiments, the compound of formula (I) is In some embodiments, the compound of formula (I) is . In some embodiments, the compound of formula (I) is In some embodiments, the compound of formula (I) is   .  In some embodiments, the compound of formula (I) is .  In some embodiments, the compound of formula (I) is . In some embodiments, the compound of formula (I) is . In some embodiments, the compound of formula (I) is . In some embodiments, the compound of formula (I) is . Compounds disclosed herein may optionally be present in neutral form, e.g. as the ‘free base’, or alternatively may be present in the form of a salt, e.g. a pharmaceutically acceptable salt. All such forms of the compounds and salts are encompassed within the scope of the present disclosure. Examples of salts include chloride, bromide, sulfate, fumarate, methanesulfonate 17     (mesylate), benzenesulfonate (besylate), and toluenesulfonate (tosylate). In some embodiments, the compound is present in the form of a mesylate salt. In some embodiments, a salt of ATH434 is used. In some embodiments, a mesylate salt of ATH434 is used. In some embodiments, a fumarate salt of ATH434 is used. Those skilled in the art will appreciate that many organic compounds can form complexes in solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as “solvates”. For example, a complex with water is known as a “hydrate”. Solvates, such as hydrates, exist when the compound incorporates solvent. It will be understood that the compounds of the present disclosure, as well as salts thereof, may be present in the form of solvates. Solvates of the compounds which are suitable are those where the associated solvent is pharmaceutically acceptable. Suitable solvates are pharmaceutically acceptable solvates including hydrates. It will be understood that the present disclosure encompasses unsolvated forms of the compounds and salts thereof, as well as solvated forms, such as hydrates. Compounds or salts disclosed herein may exist in one or more crystalline or amorphous forms. It will be understood that all such forms of the compounds and salts are within the scope of the present disclosure. Compounds of formula (I) may be prepared by any suitable method known to a person of skill in the art. For example, compounds according to the present disclosure may be prepared, as described in US 8,084,459. Salt forms of compounds of formula (I) may be produced by any suitable method. For example, an acid (e.g. methanesulfonic acid) in a suitable solvent may be added gradually to a solution of the neutral form of the compound in a suitable solvent, leading to formation of the appropriate salt, which may be recovered by precipitation and / or crystallisation of the salt from the mixture, of by removal of the solvent (e.g. by evaporation, for example under reduced pressure). The present disclosure also provides novel compounds per se and salts thereof, which find use in directly scavenging and / or directly reducing ROS, and / or which find use in maintaining, preventing loss of, or restoring mitochondria and / or mitochondrial function. Accordingly, in another aspect the present disclosure provides a compound of formula (IA) 18     wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, R3is -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10- membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is -CH2OH; and R5is hydrogen or methyl; or a salt thereof; or a pharmaceutical composition comprising the compound or salt and a pharmaceutically acceptable excipient. In another aspect, the present disclosure provides a compound of formula (IB) wherein R1and R2are each independently halogen or hydrogen; R3is selected from the group consisting of R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4alkyl; and R5is hydrogen or methyl; 19     or a salt thereof; or a pharmaceutical composition comprising the compound or salt and a pharmaceutically acceptable excipient. In another aspect, the present disclosure provides a compound of formula (IB) wherein R1and R2are each independently halogen or hydrogen; R3is -CH2-C3-7cycloalkyl; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4 alkyl; and R5is hydrogen or methyl; or a salt thereof; or a pharmaceutical composition comprising the compound or salt and a pharmaceutically acceptable excipient. In another aspect there is provided a compound selected from the group consisting of 20     or a salt thereof; or a pharmaceutical composition comprising the compound or salt and a pharmaceutically acceptable excipient. In another aspect, there is provided a mesylate salt of ATH434. In another aspect, there is provided a fumarate salt of ATH434. In some embodiments, the compound or salt has one or more of the following: a hydrogen transfer activity as determined by oxygen radical absorbance capacity (ORAC) assay which is within 5-fold that of Trolox; an electron transfer activity as determined by ferric reducing antioxidant power (FRAP) assay which is within 5-fold that of Trolox. Pharmaceutical Compositions The methods and uses of the present disclosure may involve administration of a pharmaceutical composition comprising the compound of formula (I) or salt thereof and a pharmaceutically acceptable excipient (such as a diluent, carrier, or adjuvant). The excipient(s) (e.g. carrier, diluent, adjuvant) must be pharmaceutically “acceptable” in the sense of being compatible with the other ingredients of the composition and not injurious to the subject. Compositions include those suitable for oral and parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the excipient or excipients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid or solid carriers, diluents, adjuvants and / or other excipients. Methods for preparation and pharmaceutical carriers for preparation of pharmaceutical compositions are well known in the art, as set out in textbooks such as Remington's Pharmaceutical Sciences, 20th Edition, Williams & Wilkins, Pennsylvania, USA. As used herein, a “pharmaceutical carrier” includes a pharmaceutically acceptable solvent, suspending agent or vehicle for delivering the compound of formula I to the subject. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Each carrier must be pharmaceutically “acceptable” in the sense of being compatible with other ingredients of the composition and non-injurious to the subject. The compound of formula I may for example be administered orally or parenterally in a formulation containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, 21     and / or vehicles. The term parenteral as used herein includes subcutaneous injections, aerosol for administration to lungs or nasal cavity, intravenous, intramuscular, intrathecal, intracranial, injection or infusion techniques. The present disclosure also provides suitable topical, oral, and parenteral pharmaceutical formulations for use in the novel methods of treatment. The compounds of the present disclosure may for example be administered orally as tablets, aqueous or oily suspensions, lozenges, troches, powders, granules, emulsions, capsules, syrups or elixirs. The composition for oral use may contain one or more agents selected from the group of sweetening agents, flavouring agents, colouring agents and preserving agents in order to produce pharmaceutically elegant and palatable preparations. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc: Suitable time delay agents include glyceryl monostearate or glyceryl distearate. Where for example the composition is a tablet, the tablet may contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, (1) inert diluents, such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; (2) granulating and disintegrating agents, such as corn starch or alginic acid; (3) binding agents, such as starch, gelatin or acacia; and (4) lubricating agents, such as magnesium stearate, stearic acid or talc. These tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Coating may also be performed using techniques described in the U.S. Pat. Nos.4,256,108; 4,160,452; and 4,265,874 to form osmotic therapeutic tablets for control release. The compound of formula (I) may for example be administered, for in vivo application, parenterally by injection or by gradual perfusion over time independently or together. Administration may be intravenously, intraarterial, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally or by infusion using, for example, an osmotic pump. For in vitro studies the agents may be added or dissolved in an appropriate biologically acceptable buffer and added to a cell or tissue. 22     Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, anti-microbials, growth factors and inert gases and the like. Frequently used carriers or auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyhydric alcohols. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial, anti-oxidants, chelating agents and inert gases. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in Remington's Pharmaceutical Sciences, 20th ed. Williams and Wilkins (2000) and The British National Formulary 43rd ed. (British Medical Association and Royal Pharmaceutical Society of Great Britain, 2002; http: / / bnf.rhn.net), the contents of which are hereby incorporated by reference. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See Goodman and Gilman's The Pharmacological Basis for Therapeutics (7th ed., 1985). The pharmaceutical composition is preferably prepared and administered in dose units. Solid dose units may be tablets, capsules and suppositories. For treatment of a subject, depending on activity of the compound, manner of administration, nature and severity of the disorder, age and body weight of the subject, different daily doses can be used. Under certain circumstances, however, higher or lower daily doses may be appropriate. The administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals. The pharmaceutical compositions according to the disclosure may be administered locally or systemically in a therapeutically effective dose. Amounts effective for this use will, of course, depend on the severity of the disease and the weight and general state of the subject. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration 23     of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of the cytotoxic side effects. Various considerations are described, e.g., in Langer, Science, 249: 1527, (1990). Formulations for oral use may be in the form of hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin. They may also be in the form of soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil. Aqueous suspensions typically contain the compound of formula (I) in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients may be (1) suspending agent such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; (2) dispersing or wetting agents which may be (a) naturally occurring phosphatide such as lecithin; (b) a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate; (c) a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadecaethylenoxycetanol; (d) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexitol such as polyoxyethylene sorbitol monooleate, or (e) a condensation product of ethylene oxide with a partial ester derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate. The pharmaceutical compositions may for example be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Compounds of formula I may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines. 24     Direct Antioxidant Properties and Mitochondrial Protection As described herein, the compounds of formula (I) have unexpectedly been found to have beneficial properties as direct antioxidants and direct scavengers of reactive oxygen species (ROS). Accordingly, the compounds of formula (I), salts thereof, and pharmaceutical compositions comprising the compound or salt, find use in direct scavenging and / or direct reduction of the concentration of one or more reactive oxygen species (ROS). Examples of ROS include superoxide anion, peroxide, hydroxyl radical, hydroxyl ion, and hydrogen peroxide. The compound of formula (I) is used for direct scavenging and / or direct reduction of ROS. Without being bound by any particular theory, it is considered that the compound of formula (I) interacts directly with one or more types of ROS. As used herein, the terms ‘direct scavenging’ and ‘direct reduction’ refer to scavenging and / or reduction of ROS via a process which does not require binding or chelation of the compound to a metal ion such as iron. In some embodiments, the compound of formula (I) is used for direct scavenging and / or direct reduction of one or more ROS in a system having normal physiological levels of metal ions, e.g. iron. The reduction of the level of ROS may depend on factors such as the concentration of ROS in the system, and the amount of compound used. In some embodiments, the level of ROS is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%. The term ‘scavenging’ as used herein refers to reduction of the amount of ROS present in, and / or the removal of one or more ROS from, a system. The compound of formula (I) may be used to reduce levels of ROS either in vivo or in vitro. In some embodiments, the compound of formula (I) is used for direct scavenging and / or direct reduction of ROS in a subject. As used herein, the terms “treatment” or “treating” include curing a disease or disorder, as well as alleviation of or reduction of symptoms associated with a disease or disorder or condition. The term treating also includes slowing the progression of a disease or disorder. The terms “treating”, “treatment” and the like are used herein to mean affecting a subject, tissue or cell to obtain a desired pharmacologic and / or physiologic effect. It may include one or more of a) inhibiting the disease, i.e., arresting its development; or (b) relieving or ameliorating the effects of the disease, i.e., cause regression of the effects of the disease. 25     As used herein, the terms “prevention” or “preventing” include prophylaxis, and includes reducing the likelihood of contracting a disease or disorder or a symptom thereof. The effect may be prophylactic in terms of completely or partially preventing a disease or disorder or sign or symptom thereof. As used herein, the term “therapeutically effective amount” is meant an amount of a compound of formula (I) effective to yield a desired therapeutic response. The specific “therapeutically effective amount” will, obviously, vary with such factors as the particular condition being treated, the physical condition of the subject, the type of subject being treated, the duration of the treatment, and the nature of concurrent therapy (if any). In some embodiments, the subject has an elevated concentration of one or more ROS. For example, the subject may have a concentration of one or more ROS (e.g. superoxide anion, peroxide, hydroxyl radical, hydroxyl ion, or hydrogen peroxide) which is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15% or at least 20% higher than normal concentrations. ROS levels may be determined using conventional techniques. In some embodiments, the subject has a disease or disorder in which elevated concentrations of total iron, labile iron, and / or in which improperly stored iron, is not the determinant of the disease or disorder. In some embodiments, the subject has normal concentrations of total iron. In some embodiments, the subject has normal concentrations of labile iron. The compound of formula (I), salts thereof, and pharmaceutical compositions comprising the compound or salt accordingly find use in treating or preventing a disease or disorder which is associated with elevated levels of one or more reactive oxygen species (ROS). For example, those diseases and disorders may be characterised such that elevated concentrations of total iron, labile iron, and / or in which improperly stored iron, are not the determinant of the disease or disorder, in a subject. In some embodiments, the disease or disorder is a neurodegenerative disease or disorder. In some embodiments the disease or disorder is selected from the group consisting of Pelizaeus-Merzbacher disease, diabetes mellitus, renal cancer, cardiovascular disease and partial bladder outlet obstruction. It has also been found that compounds of formula (I) can exert mitochondrial protective effects, and thus find use in maintaining, preventing loss of, or restoring mitochondria and / or mitochondrial function in a subject. The compounds, salts and pharmaceutical compositions also find use in treating or preventing diseases or disorders associated with loss of mitochondrial 26     function and / or loss of mitochondria, and associated with elevated levels of one or more reactive oxygen species (ROS). For example, the compounds salts and pharmaceutical compositions may find use in treating or preventing diabetes mellitus and / or insulin resistance, or one or more signs or symptoms thereof. In the case of diabetes mellitus, hyperglycemia glycolysis leads to ROS which impact enzymes that ultimately lead to increased levels of glyceraldehyde phosphate, which is a pro-oxidant. Mitochondrial impairment can contribute to the development of age-dependent insulin resistance, which can be exacerbate normal sequelae of aging and increase diabetes mellitus risk. Intracellular hyperglycemia can promote mitochondrial reactive oxygen species (ROS), increased formation of intracellular advanced glycation end-products, activation of protein kinase C, and / or increased polyol pathway flux. Reduced mitochondrial function and / or presence of elevated levels of ROS are often associated with signs of aging. Accordingly, the compounds, salts and pharmaceutical compositions also find use in treating or preventing one or more signs of aging, for example one or more signs of aging associated with elevated levels of one or more reactive oxygen species (ROS), and in which elevated concentrations of total iron, labile iron, and / or in which improperly stored iron, is not the determinant of the sign of aging. Examples of signs of aging include skin aging, muscle weakness and loss of muscle tone. The term “subject” as used herein refers to any animal having a disease or condition which requires treatment with a pharmaceutically-active agent. The subject may be a mammal, preferably a human, or may be a domestic or companion animal. While it is particularly contemplated that the compound of formula (I) is for use in medical treatment of humans, it is also applicable to veterinary treatment, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, ponies, donkeys, mules, llama, alpaca, pigs, cattle and sheep, or zoo animals such as primates, felids, canids, bovids, and ungulates. In some embodiments, the subject is human. In some embodiments the subject is an adult human. In some embodiments, the subject is at least 40, or at least 50, or at least 60, or at least 70 years old. In some embodiments, the subject is male. In some embodiments, the subject is female. The compound of formula (I) may additionally be combined with other medicaments to provide an operative combination. It is intended to include any chemically compatible combination of pharmaceutically-active agents, as long as the combination does not eliminate the activity of the compound of formula I. It will be appreciated that the compound of formula (I) and the other medicament may be administered separately, sequentially or simultaneously. 27     A suitable dosage amount of the compound of formula (I) or salt thereof may be administered. For example, it may be in the range of from about 0.25 mg to about 20 mg per kilogram body weight per day, or from about 0.5 mg to about 20 mg per kilogram body weight per day, or from about 0.5 mg to about 15 mg per kilogram body weight per day, or from about 0.5 mg to about 10 mg per kilogram body weight per day, or from about 0.5 mg to about 5 mg per kilogram body weight per day, or from about 500 mg to about 3000 mg per patient per day, or from about 25 mg to about 500 mg per patient per day, or from about 25 mg to about 250 mg per patient per day. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for oral administration to humans may contain about 5 mg to 1 g of an active compound with an appropriate and convenient amount of carrier material which may vary from about 5 to 95 percent of the total composition. Dosage unit forms will generally contain between from about 5 mg to 500 mg of active ingredient. Optionally the compound of formula (I) or salt may be administered in a divided dose schedule, for example the compound or salt may be administered twice per day, three times per day or four times per day. If desired, and if appropriate for the specific condition, the compound of formula (I) may also be administered less frequently, for example once every other day, or once a week. It will be understood, however, that the specific dose level for any particular patient may depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy. Examples The disclosure is further illustrated by way of reference only to the following non- limiting examples. Example 1: Synthesis of ATH434 from nitro-benzoic acid 1   28       Scheme 1: Synthesis of ATH434 from nitro-benzoic acid 1 i) MeI, K2CO3, DMF 65oC; ii) MeI, K2CO3, acetone reflux; iii) 2N NaOH, MeOH 60oC; iv) 1.SOCl2 reflux, 2.8.0M MeNH2, CH2Cl2 rt.; v) Fe powder, AcOH 80oC; vi) ClCH2COCl, AcOH 90oC; vii) EtNH2, THF rt.; viii) 48% HBr 120oC; ix) NaHCO3, Methanesulfonic acid. Methyl 4,6-dichloro-3-hydroxy-2-nitrobenzoate (2) Benzoic acid 1 (25.0 g, 99.2 mmol, US 8,084,459) was dissolved in anhydrous DMF (250 mL), to which K2CO3(20.6 g, 149.0 mmol) and MeI (35.2 g, 248.0 mmol) were added, and the resulting mixture heated at 65oC with stirring under an argon atmosphere for 16 hours. Upon cooling the reaction was concentrated and then acidified to pH 0-2 with conc. HCl. The reaction was then diluted with H2O (250 mL) and extracted with EtOAc (4 x 150 mL). The combined organic extracts were washed with brine (2 x 200 mL), dried (Na2SO4), filtered and concentrated 29     to afford the title compound 2 as a reddish-brown gummy solid (28.56g, crude yield – DMF present).1H NMR (500 MHz, d6-DMSO): ^ ^3.82 (s, 3H), 8.05 (s, 1H). Methyl 4,6-dichloro-3-methoxy-2-nitrobenzoate (3) The methyl ester 2 (28.5g crude, 107.1 mmol) was dissolved in acetone (250 mL) to which K2CO3 (22.2 g, 160.7 mmol) and MeI (45.6 g, 321.3 mmol) were added, and the resulting mixture stirred at reflux under an argon atmosphere for 24 hours. Upon cooling the reaction was concentrated and taken up with H2O (200 mL), then extracted with CH2Cl2(3 x 200 mL). The combined organic extracts were washed with brine (2 x 200 mL), dried (Na2SO4), filtered and concentrated to yield the dimethylated compound 3 as a clear brown oil (26.92 g, 97% - some DMF still present).1H NMR (500 MHz, d6-DMSO): ^ ^3.86 (s, 3H), 3.95 (s, 3H), 8.25 (s, 1H). 4,6-Dichloro-3-methoxy-2-nitrobenzoic acid (4) The dimethylated compound 3 (26.8 g, 95.7 mmol) was heated at 60oC in 2N NaOH (150 mL) and MeOH (200 mL) for 3 hours. Upon cooling the MeOH was removed in vacuo leaving an aqueous solution which was extracted with EtOAc (200 mL) to remove organic impurities. The aqueous layer was then acidified with conc. HCl and extracted with EtOAc (3 x 200 mL). The organic extracts were washed with brine (200 mL), dried (Na2SO4), filtered and concentrated to afford the benzoic acid 4 as a light yellow solid (23.3 g, 91%).1H NMR (500 MHz, d6-DMSO): ^ ^3.94 (s, 3H), 8.15 (s, 1H). 4,6-Dichloro-3-methoxy-N-methyl-2-nitrobenzamide (5) Compound 4 (23.2 g, 87.2 mmol) was heated at reflux in neat SOCl2 (150 mL) for 1 hour. The excess SOCl2 was then distilled off under reduced pressure. Residual SOCl2 was further removed by azeotropic distillation with toluene leaving a red oil which readily crystallised on standing. The acid chloride was dissolved in anhydrous CH2Cl2 (150 mL) and cooled to 0oC, upon which an 8.0M solution of MeNH2 in EtOH (50 mL 400 mmol) was added dropwise, and the reaction stirred under an argon atmosphere for 20 hours at ambient temperature. The reaction mixture was then concentrated, partitioned between H2O (200 mL) and EtOAc (200 mL), and then further extracted with EtOAc (2 x 200 mL). The organic extracts were washed with brine (200 mL), dried (Na2SO4), filtered and concentrated to afford the amide as a highly crystalline brown solid (17.3 g, 71%).1H NMR (500 MHz, d6-DMSO): ^ ^ ^ ^ ^ ^ ^(d, J = 4.5 Hz, 3H) 3.94 (s, 3H), 8.17 (s, 1H), 8.83 (d, J = 4.5 Hz, 1H). ^ 30     2-Amino-4,6-dichloro-3-methoxy-N-methylbenzamide (6) To a suspension of the nitro compound 5 (17.2 g, 61.6 mmol) in glacial AcOH (200 mL) was added Fe powder (15 g, 269 mmol), and the resulting mixture was heated at 80oC for 1.5 hours. The reaction was then filtered hot through a small pad of celite, which was washed through with EtOAc and a further small amount of AcOH. The filtrate was concentrated and the brown residue taken up in EtOAc (250 mL) and sat. NaHCO3 soln. (250 mL) resulting in a significant emulsion. Upon settling, the aqueous layer was extracted with again with EtOAc (3 x 200 mL). The organic extracts were washed with brine (300 mL), dried (Na2SO4), filtered and concentrated to afford the yield the aniline 6 as a tan coloured solid (14.4 g, 94%).1H NMR (500 MHz, d6-DMSO): ^ ^ ^ ^ ^ ^ ^ ^d, J = 4.5 Hz, 3H) 3.67 (s, 3H), 5.32 (s, 2H), 6.72 (s, 1H), 8.34 (d, J = 4.5 Hz, 1H). 5,7-Dichloro-2-(chloromethyl)-8-methoxy-3-methylquinazolin-4(3H)-one (7) The aniline derivative 6 (14.4 g, 57.8 mmol) was dissolved in glacial AcOH (150 mL), to which chloroacetyl chloride (18.4 mL, 231 mmol) was added. The resulting solution was added stirred at 90oC for 2.5 hours. After cooling, the volume of solvent was reduced, and then 2N NaOH was added until the pH was ~ 6. The mixture was extracted with CH2Cl2(3 x 200 mL), and then the extracts were washed with brine (200 mL), dried (Na2SO4), filtered and concentrated. The solid residue was then purified by flash chromatography eluting with 20-40% EtOAc / hexanes to afford the desired cyclised material 7 as a light orange solid (8.31 g, 47%).1H NMR (500 MHz, d6- DMSO): ^ 3.54 (s, 3H), 4.00 (s, 3H), 4.91 (s, 2H), 7.69 (s, 1H). 5,7-Dichloro-2-((ethylamino)methyl)-8-methoxy-3-methylquinazolin-4(3H)-one (8) A solution of the cyclised material 7 (8.2 g, 26.7 mmol) in anhydrous THF (100 mL) was cooled to 0oC and stirred under an atmosphere of argon. At this point a 2.0M solution of EtNH2 in THF (53 mL, 106 mmol) was added dropwise, and then the resulting mixture was allowed to warm to room temperature, followed by further stirring for 1 hour. The reaction was concentrated and taken up into CH2Cl2(200 mL) and H2O (200 mL), and the aqueous layer extracted with CH2Cl2(2 x 200 mL). The organic extracts were washed with brine (200 mL), dried (Na2SO4), filtered and then concentrated to yield the free amine 8 as an orange solid (8.40 g, 99%).1H NMR (500 MHz, d6-DMSO): ^ ^ ^ ^ ^ ^ ^ ^t, J = 7.5 Hz, 3H), 2.40 (br s, 1H), 2.65 (q, J = 7.5 Hz, 2H), 3.49 (s, 3H), 3.86 (s, 2H), 3.98 (s, 3H), 7.60 (s, 1H). 31     5,7-Dichloro-2-((ethylamino)methyl)-8-hydroxy-3-methylquinazolin-4(3H)-one hydrobromide (9) The methyl ether 8 (8.3 g, 26.3 mmol) was heated at 120oC in 48% aqueous HBr (120 mL) for 3 hours. The HBr was then distilled off under reduced pressure, and then a small amount of MeOH (20 mL) was added to the residue and concentrated in vacuo. A further amount of MeOH (40 mL) was added to the residue resulting in a brown suspension which was sonicated and left to stand for 15 minutes. The solid material was then filtered and carefully washed with a minimum amount of MeOH (x 4), leaving behind phenol 9 as a fluffy light brown powder (8.5 g, 85%).1H NMR (500 MHz, d6-DMSO): ^ ^ ^ ^ ^ ^ ^ ^t, J = 7.5 Hz, 3H), 3.16 (br s, 2H), 3.44 (s, 3H), 4.57 (s, 2H), 7.62 (s, 1H), 9.06 (br s, 2H), 10.24 (s, 1H);    13C NMR (125 MHz,d6-DMSO):    ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ 8.53 min (> 98%). 5,7-dichloro-2-((ethylamino)methyl)-8-hydroxy-3-methylquinazolin-4(3H)-one mesylate (ATH434) 5,7-dichloro-2-((ethylamino)methyl)-8-hydroxy-3-methylquinazolin-4(3H)-one hydrobromide 9 (8 g, 20.9 mmoL) was suspended in saturated aqueous NaHCO3(100 mL) solution. The resulting gluggy suspension was washed with H2O and Methanol, then dried under high vacuum. The resulting free base was then dissolved in Methanol (150 mL), then Methanesulfonic acid (3mL) was added dropwise to the solution. Solvent was removed to 1 / 3 volume. The solution was allowed to cool, and ATH434 was collected by filtration, as a white solid (5.8 g, 70% for two steps).1H NMR (500 MHz, d6-DMSO): ^ ^ ^ ^ ^ ^ ^ ^ ^t, J = 7.5 Hz, 3H), 2.33 (s, 3H), 3.15 (br s, 2H), 3.43 (s, 3H), 4.52 (s, 2H), 7.63 (s, 1H), 8.80 (br s, 2H), 10.36 (s, 1H); m / z 302 [M+H]+.Example 2: Determination of compound antioxidant properties using ABTS assay The ABTS assay was used to assess the reducing potential of compounds, i.e. their ability to reduce the ABTS+radical produced via oxidation of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline- 6-sulfonic acid) with potassium persulfate. The assay uses Trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid), a derivative of the known antioxidant Vitamin E, as a positive control. To characterize the antioxidant potential of unknown compounds, compounds were first run in the assay using a wide, log-scale concentration range and, if positive for activity, further characterized using a narrow concentration range to identify the EC50. 32     Methods: An ABTS radical stock was prepared by mixing 25µL potassium persulfate (70mM) with 700µL ABTS (7mM). This was incubated at room temperature in the dark for 16 hours. To create a ABTS working solution for every 96 well plate, 312.6µL of ABTS radical stock was combined with 10mL 95% ethanol. Replicate wells were set up with sample and blanks according to the table shown below. The reaction was quantified after 90min incubation at room temperature in the dark using a plate reader. Absorbance was read for the experiment at 734nm. Blank 1 Blank 2 Sample Drug / antioxidant - - 20μL DMSO 20μL 20μL - Ethanol - 100μL - 0.1M Tris Buffer 80μL 80μL 80μL ABTS working 100μL - 100μL solution Final Volume 200μL 200μL 200μL Results: The results are shown in Figure 1, and EC50 ranges are shown in the below table. ATH434 was found to have an EC50of ~28µM. As the activity was similar to that of the positive control Trolox, these data confirm that ATH434 possesses strong antioxidant activity. ATH434’s reducing capacity exceeded that of ascorbic acid and 2-methoxyhydroxyquinone. Compound EC50range (μM) (actual) ATH434 20-30 (28.7) ATH434-met 130-170 (160) ATH434-NDE 20-30 (27.3) Deferasirox (Dfx) 180-220 (197.9) 33     Deferiprone (Dfp) Not calculable due to lack of reducing capacity at tested concentrations (up to 1mM) Ascorbic acid 380-470 (425.9) 2-methoxyhydroquinone 360-440 (387.3) Trolox 20-30 (23.1) Dunnett’s T3 multiple comparisons test Summary Adjusted P Value 434 vs.434-Met **** <0.0001 434 vs. Dfp ns >0.9999 434 vs. Dfx *** 0.0004 434 vs 2-methoxyhydroquinone **** <0.0001 434 vs.434-NDE ns >0.9999 434 vs. Trolox * 0.0189 434 vs. M12 Ns >0.9999 434 vs.8-quinolinol **** <0.0001 434 vs. ascorbic acid **** <0.0001 ATH434-met has the following structure: . ATH434-NDE has the following structure: . Deferiprone (DFP) is an approved high ferric iron affinity drug (Kd 10-21, Hider 2014) designed to clear iron from cellular storage in systemic iron-overload disorders, 34     Deferasirox (DFX) is an approved high affinity oral iron chelating agent used to treat chronic iron overload. Example 3: ORAC (Hydrogen Atom Transfer) The Oxygen Radical Absorbance Capacity (ORAC) assay was used to quantify the hydrogen atom transfer activity of the test compounds. ORAC activity is measured by the quenching of a fluorescein probe by the radical generator 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), which can be inhibited by addition of an antioxidant. Methods: A 75mM AAPH solution was freshly prepared in phosphate buffer (pH 7.4). A fluorescein stock solution was made using the 75mM phosphate buffer at 4µM final concentration. Before use, the fluorescein stock solution was diluted in 75mM phosphate buffer to reach an 80nM working solution concentration. A 96 well plate was loaded with 150µL fluorescein working solution and 25µL test compound at various concentrations (0.2-200 µM) and incubated at 37°C for 10min, then the reaction was initiated with 25µL AAPH solution. Fluorescence measurements were taken every 5 mins for 1200 mins total (20hrs) total at ex / em 485 / 528nm. The AUC was calculated for each test compound and blank corrected to the DMSO vehicle control to determine the net AUC. The AUC for each compound at 20µM was expressed as percent of Trolox AUC as the Trolox equivalent, reported in the table as mean, SD, and SEM. Statistical significance was determined using one-way ANOVA compared to Trolox, with p-values noted in the table. Results: The responses at 20 µM are shown in Figure 2a and Trolox equivalent response ranges are shown in the below table. 35     At 20μM, ATH434 HAT activity was 3.5-fold higher than Trolox. ATH434-met had minimal HAT activity at 20μM. Dfx and Dfp did have detectable HAT activity of 2-fold and 7-fold increase compared to Trolox at the same concentration. Table: Trolox-equivalent values for compounds at 20μM Trolox ATH434 ATH434-met Dfp Dfx Mean 100.0 354.1 19.39 206.1 693.6 Std. Deviation 16.48 35.34 6.12 32.78 30.70 Std. Error of 8.24 17.67 3.53 16.39 15.35 the Mean P-value - <0.0001 0.0065 0.0003 <0.0001 compared to Trolox The full concentrations responses for the test compounds are presented in Figures 2b-2f. Example 4: FRAP & Cyclic Voltammetry (Electron Transfer) The Ferric Reducing Antioxidant Power (FRAP) assay was used to quantify the electron transfer activity of test compounds. FRAP antioxidant activity was quantified by measuring the change in absorbance at 593nm of an Fe(III)-TPTZ complex upon reduction by antioxidants. This electron transfer antioxidant activity was confirmed by cyclic voltammetry, which measures the oxidation and reduction cycling of a solution of compound. Methods (FRAP): A FRAP assay mix was made fresh with 10 parts volume of 300mM acetate buffer (pH 3.6), 1 part TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine; Sigma #93285), and 1 part 20mM ferrous chloride. A 96-well plate was loaded with 280µL of FRAP assay mix and 20μL of sample. The plate was gently shaken to mix and incubated at 37 ^C in the dark for 30 minutes. Absorbance was read at 593nm. Various concentrations of compounds (1µM-1000µM) were assayed to generate dose- response curves, data were transformed using linear regression to calculate the slopes and expressed as Trolox equivalent (TE). Methods (Cyclic Voltammetry): 36     Cyclic voltammetry was performed on ATH434 using a WaveNowXVpotentiostat and analyzed using Aftermath software (Pine Research Instrumentation, Inc, Durham NC). Compounds were prepared as 5mM solutions in 0.1M KCl, 10% DMSO, pH 7.4, then bubbled with nitrogen for 5-10min prior to measurement. Currents for each sample were recorded at a glassy carbon electrode with 100mV / s scan rate from -500mV to 1200mV under ambient atmosphere at 25 ^C. Samples were measured against an Ag / AgCl reference electrode, 6 segments per scan, n = 3 scans per compound. Data was plotted as current ( ^A) versus potential (mV), and midpoint potentials, E1 / 2, were calculated for the oxidation and reduction peaks. Results: The FRAP assay results are shown in Figure 3 and the table below. Compound Trolox equivalent response range (μM) (actual) Trolox 0.80-1.2 (1.0) ATH434 0.50-0.8 (0.67) ATH434-met 0.01-0.05 (0.03) Dfp 0.50-0.80 (0.61) Dfx 0.20-0.80 (0.53) The Cyclic Voltammetry assay results are shown in Figure 4a and 4b. ATH434 was found to have electron transfer activity but not ATH434-met, as determined by the FRAP assay and confirmed by cyclic voltammetry (ATH434 mean reduction potential was 348.8 mv (SD=37.16, SEM=18.58, n=4)). Example 5: Effect of compounds on mitochondrial health status in menadione injury model Mitochondrial status can be monitored in cells using the dye tetramethylrhodamine methyl ester (TMRM). The dye fluoresces when localized into mitochondria that maintain a functional mitochondrial membrane potential (MMP). The TMRM assay gives a readout of mitochondrial health and function. To assess the potential protective function of ATH434, cells were pretreated with ATH434 and subsequently challenged with known a pro-oxidant, menadione. Menadione generates reactive 37     oxygen species (ROS) and mitochondrial oxidative stress. The mitochondrial health status of treated cells was examined using TMRM. Methods: HT22 cells were plated in 24-well plates coated with poly-D-lysine, at 20K cells / well. The cells were grown in DMEM with 10% Fetal Bovine Serum, 1X penicillin-streptomycin, and 2mM L-Glutamine. Plate progress was monitored by checking plate confluency. When 60-70% confluent, the cells were differentiated in differentiation media (DM; Neurobasal media containing 1X N2 supplement (Thermo Fisher), 2mM L-Glutamine) along with specified treatments. Cells were challenged with 6 µM menadione in DM for 20 hours at 37C. In some cases, cells were pretreated with 20μM ATH434 or other potential antioxidants. In others, ATH434 or other potential antioxidants were present in the menadione-containing DM. A staining mix containing TMRM (10µg / mL), Hoechst (0.7µg / mL), MitoTracker Green (100nM) in HEPES buffered saline solution (HBSS) was made up, comprised of components: ^ 140mM NaCl ^ 2.5mM KCl ^ 1.8mM CaCl2^ 1.0mM MgCl2 ^ 20mM HEPES ^ pH = 7.4 After 20 hours treatment incubation, media was aspirated and 500µL of staining mix was applied to each well for 30min at 37oC. Cells were washed twice in HBSS. Fluorescence was quantified using an BMG Labtech FLUOstar Omega plate reader, using 3x3 matrix well scanning. Fluorescence wavelengths for TMRM, Hoechst and MitoTracker Green were as follows: ^ TMRM: ex / em 544 / 620nm ^ Hoechst: ex / em 355 / 460nm ^ MitoTracker Green: ex / em 480 / 520nm Numerical values for TMRM were normalized to Hoechst for cell count and MitoTracker Green for mitochondrial signal (i.e. TMRM / Hoechst / MitoTracker Green). Data were expressed relative to untreated control, mean + / - SEM. For analysis, data was split into groups by + / - 38     menadione, then compared for rescue of menadione effect by the compounds using two-way ANOVA. Results: The results are shown in Figure 5. The results showed that menadione “control” produces a ~50% decrease in mitochondrial staining, understood to be associated with loss of mitochondrial function. The levels of staining were rescued to control levels following ATH434 pre-treatment. Rescue is also present for the ATH434-met compound which has been found not to bind iron. By contrast, iron chelators deferasirox and deferiprone did not increase the TMRM signal and are those considered inactive in rescuing the mitochondrial function. Example 6: Lipid Peroxidation Assay Lipid peroxidation was used as a measure of upstream ROS generation, specifically that of the hydroxyl radical which is highly reactive and likely to non-specifically react with many subcellular compartments. Cellular lipid peroxidation was measured using the Image-iT Peroxidation kit (Thermo Fisher), which uses BODIPY 581 / 591 as a reporter. This is a ratiometric reporter that shifts from the reduced, red (590nm) form to the oxidized, green (510nm) form upon oxidation in cells. Therefore, lipid peroxidation is detectable by an increase in the green:red ratio (decreased red, increased green fluorescence). Menadione was used in this assay and showed the expected increase in the green:red ratio using fluorescence microscopy. Methods: HT22 cells were plated in 24-well plates coated with poly-D-lysine, at 20K cells / well. The cells were grown in DMEM with 10% Fetal Bovine Serum, 1X penicillin-streptomycin, and 2mM L-Glutamine. When cells on the plates were 60-70% confluent, cells were differentiated in differentiation media (DM; Neurobasal media containing 1X N2 supplement (Thermo Fisher), 2mM L-Glutamine) along with specified treatments. For pre-treatments with ATH434, DM containing 20µM ATH434 was added for treatment for 2 hours prior to beginning the assay. After pre-treatment, DM containing 6µM menadione was added in the presence or absence of ATH434 and cells were incubated for an additional 20 hours at 37oC. Staining mix containing Image-iT lipid peroxidation sensor (10µM) and Hoechst (0.7µg / mL) in DM was made. After a 20-hour treatment incubation, media was aspirated and 39     500µL of staining mix was applied to each well for 30min at 37oC. Cells were washed twice in HBSS. Fluorescence was quantified using a BioTek Cytation 5 plate reader, using 3x3 matrix well scanning. Fluorescence wavelengths for both Image-iT sensor oxidized and reduced spectra and Hoechst were as follows: ^ Image-iT sensor, reduced: ex / em 596 / 615nm ^ Image-iT sensor, oxidized: ex / em 490 / 525nm ^ Hoechst: ex / em 355 / 460nm The ratio of oxidized (510nm) to reduced (590 nm) BODIPY was calculated, and Hoechst was used to validate cell count. Data are expressed as percent of treatment control, mean + / - SEM. For analysis, data was split into groups by + / - menadione, then compared for rescue of menadione effect by the compounds using two-way ANOVA. Results: The results are shown in Figure 6 and the table below. Compound Response to menadione: (Menadione response as % Control) - Control response, defined as 100%) Vehicle 100-125% ATH434 10%-20% ^-tocopherol10%-20%Trolox 10%-20% The results showed that menadione induced an approximate 2-fold increase (i.e. 100% increase over control) in lipid peroxidation in HT22 cells. This increase in lipid peroxidation was rescued by pre-treatment with ATH434 as well as by known antioxidants Trolox and ^-tocopherol. Example 7: Seahorse Metabolic Assay The metabolic status of cells is reflective of their overall health and mitochondrial activity. The Seahorse assay is used to assess the contributions to ATP production from both glycolysis and mitochondrial oxidative phosphorylation, as well as extrapolating information on specific 40     metabolic parameters such as coupling efficiency and proton leak. In response to prooxidants or oxidative stress, cells will typically have decreased mitochondrial ATP production, either to limit the production of ROS or because their mitochondrial electron transport chain no longer functions properly to link electron transfer and the generated proton gradient to ATP production. This is often accompanied by decreases in ATP-linked respiration and coupling efficiency, and in some cases a shift towards non-mitochondrial ATP production (glycolysis) or overall decreased total ATP production from both metabolic processes. With the Seahorse assay, the baseline metabolic activity in HT22 cells treated in the presence or absence of different concentrations of ATH434 (0.2, 2, 20µM) was investigated. Methods: HT22 cells were plated on a poly-D-lysine coated 96-well Seahorse cell culture plate (Agilent) at 7.5K cells / well in DMEM with 10% Fetal Bovine Serum, 1X penicillin-streptomycin, and 2mM L-Glutamine. Cells were left at room temperature for 1 hour to adhere, then placed in a 37oC incubator overnight. The next day, cells were switched to differentiation media (DM; Neurobasal media containing 1X N2 supplement (Thermo Fisher), 2mM L-Glutamine) in the presence or absence of ATH434 (0.2, 2, 20µM) and allowed to incubate at 37oC overnight. The day prior to the assay, the Seahorse cartridge was incubated with milliQ H2O in a non- CO2 incubator at 37oC overnight to equilibrate the sensors. The next morning, the Seahorse cartridge was switched to pre-warmed calibrant solution and equilibrated in a 37oC, non-CO2incubator for 1 hour. After equilibration, the Seahorse assay compounds were loaded into the corresponding ports (A: oligomycin, 2.5µM; B: Carbonyl cyanide-p- trifluoromethoxyphenylhydrazone (FCCP), 1µM; C: rotenone & antimycin A, 0.5µM each; final concentration in well) and the cartridge was calibrated in the Seahorse XFe96 Analyzer Instrument (Agilent). While the sensors were calibrating, the cell culture plate was switched to Seahorse XF DMEM pH 7.4 media containing 10mM glucose, 1mM pyruvate, and 2mM L-glutamine added fresh and the cells were allowed to equilibrate in a 37oC, non-CO2incubator for 1 hour. After calibration of the cartridge, the cell plates were loaded into the Seahorse instrument and the assay was run. After the run, the cells were lysed and protein content was measured with a bicinchoninic acid assay (Thermo). The total mg of protein per well was used to normalize the 41     Seahorse data using the Agilent Seahorse Analytics software. Data was exported to Excel and plotted using Graphpad Prism. Results: The results are shown in Figures 7a and 7b. The OCR (oxygen consumption rate, Figure 3a) and ECAR (extracellular acidification rate, Figure 3b) traces for HT22 cells treated with ATH434 show that low concentrations of ATH434 (0.2, 2µM) increased overall metabolic activity compared to control cells, while 20µM ATH434 decreased overall oxygen consumption compared to control cells. When specific parameters are extrapolated from the OCR and ECAR data (Figures 8 and 9), these data show that 0.2µM and 2µM ATH434 increased cellular respiration, specifically basal respiration, maximal respiration, non-mitochondrial respiration (glycolysis), and ATP-linked respiration. On the other hand, 20µM ATH434 decreased respiration compared to control cells but also decreased proton leak, an effect that is likely mitoprotective as increased proton leak is typically associated with increased ROS. From the Seahorse data the relative contributions of glycolysis and mitochondrial metabolism to total ATP production can also be extrapolated (see Figure 10). Both 0.2µM and 2µM ATH434 increased total ATP production which is comprised of both increased glycoATP and increased mitoATP production. Treatment with 20µM ATH434 increased glycoATP production commensurate with the lower concentrations of ATH434, but decreased mitoATP production, suggesting that treatment with higher concentrations of ATH434 will trigger mitoprotective mechanisms to shift away from the ROS-generating mitochondrial metabolism. It will be apparent to the person skilled in the art that while this disclosure has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification. 42

Claims

Claims 1. Use of a compound of formula (I)wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10- membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4 alkyl; and R5is hydrogen or methyl; or a salt thereof; or a pharmaceutical composition comprising the compound or salt and a pharmaceutically acceptable excipient; for direct scavenging and / or direct reduction of the concentration of one or more reactive oxygen species (ROS).

2. Use as claimed in claim 1, wherein the compound or salt is used for direct scavenging of and / or direct reduction of the concentration of one or more ROS in a subject.

3. A method of directly scavenging and / or directly reducing the concentration of one or more reactive oxygen species (ROS) in a subject, comprising administering an effective amount of a compound of formula (I) 43   wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10- membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4 alkyl; and R5is hydrogen or methyl; or a salt thereof, or of a pharmaceutical composition comprising the compound or salt and a pharmaceutical excipient, to the subject.

4. Use as claimed in claim 2, or a method as claimed in claim 3, wherein the subject is human.

5. Use or method as claimed in any of claims 2 to 4, wherein the subject has an elevated concentration of one or more ROS.

6. Use or method as claimed in any of claims 2 to 5, wherein the subject has a disease or disorder in which elevated concentrations of total iron, labile iron, and / or in which improperly stored iron, is not the determinant of the disease or disorder.

7. Use of a compound of formula (I) 44   wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10- membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4 alkyl; and R5is hydrogen or methyl; or a salt thereof; or a pharmaceutical composition comprising the compound or salt and a pharmaceutically acceptable excipient; for maintaining, preventing loss of, or restoring mitochondria and / or mitochondrial function in a subject.

8. A method of maintaining, preventing loss of, or restoring mitochondria and / or mitochondrial function in a subject, comprising administering an effective amount of a compound of formula (I)wherein R1and R2are each independently halogen or hydrogen; 45    R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10- membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4alkyl; and R5is hydrogen or methyl; or a salt thereof, or of a pharmaceutical composition comprising the compound or salt and a pharmaceutical excipient, to the subject.

9. Use as claimed in claim 7, or method as claimed in claim 8, wherein the subject is human.

10. Use or method as claimed in any of claims 7 to 9, wherein the subject has an elevated concentration of one or more reactive oxygen species (ROS).

11. Use or method as claimed in any of claims 7 to 10, wherein the subject has a disease or disorder in which elevated concentrations of total iron, labile iron, and / or in which improperly stored iron, is not the determinant of the disease or disorder.

12. A method of treating or preventing a disease or disorder which is associated with elevated levels of one or more reactive oxygen species (ROS), and in which elevated concentrations of total iron, labile iron, and / or in which improperly stored iron, is not the determinant of the disease or disorder, in a subject, comprising administering an effective amount of a compound of formula (I)wherein R1and R2are each independently halogen or hydrogen; 46    R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10- membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4alkyl; and R5is hydrogen or methyl; or a salt thereof, or of a pharmaceutical composition comprising the compound or salt and a pharmaceutical excipient, to the subject.

13. Use of a compound of formula (I),wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10- membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4 alkyl; and R5is hydrogen or methyl; or a salt thereof, for the manufacture of a medicament for treating or preventing a disease or disorder which is associated with elevated levels of one or more reactive oxygen species (ROS), and in which elevated concentrations of total iron, labile iron, and / or in which improperly stored iron, is not the determinant of the disease or disorder, in a subject.

14. A method as claimed in claim 12, or use as claimed in claim 13, wherein the subject is human. 47    15. A method or use as claimed in any of claims 12 to 14, wherein the disease or disorder is a neurodegenerative disease or disorder.

16. A method or use as claimed in any of claims 12 to 14, wherein the disease or disorder is selected from the group consisting of Pelizaeus-Merzbacher disease, diabetes mellitus, renal cancer, cardiovascular disease and partial bladder outlet obstruction.

17. A method of treating and / or preventing one or more signs of aging which is associated with elevated levels of one or more reactive oxygen species (ROS), and in which elevated concentrations of total iron, labile iron, and / or in which improperly stored iron, is not the determinant of the sign of aging, in a subject, comprising administering an effective amount of a compound of formula (I)wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10- membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4 alkyl; and R5is hydrogen or methyl; or a salt thereof, or of a pharmaceutical composition comprising the compound or salt and a pharmaceutical excipient, to the subject.

18. Use of a compound of formula (I), 48   wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10- membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4 alkyl; and R5is hydrogen or methyl; or a salt thereof, for the manufacture of a medicament for treating or preventing one or more signs of aging which is associated with elevated levels of one or more reactive oxygen species (ROS), and in which elevated concentrations of total iron, labile iron, and / or in which improperly stored iron, is not the determinant of the sign of aging, in a subject.

19. A method as claimed in claim 17, or use as claimed in claim 18, wherein the subject is human.

20. A method of treating or preventing a disease of disorder associated with loss of mitochondrial function and / or loss of mitochondria, and which is associated with elevated levels of one or more reactive oxygen species (ROS), in a subject, comprising administering an effective amount of a compound of formula (I) 49   wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10- membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4 alkyl; and R5is hydrogen or methyl; or a salt thereof, or of a pharmaceutical composition comprising the compound or salt and a pharmaceutical excipient, to the subject.

21. Use of a compound of formula (I)wherein R1and R2are each independently halogen or hydrogen; R3is hydrogen, C1-4alkyl, -CH2-C3-7cycloalkyl or -CH2Het, wherein Het is a 5 to 10- membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or 2 substituents each independently selected from the group consisting of methyl and halogen; R4is hydrogen, -CH2OH or -CH2NR6R7, wherein R6and R7are each independently hydrogen or C1-4 alkyl; and 50    R5is hydrogen or methyl; or a salt thereof; for the manufacture of a medicament for treating or preventing a disease or disorder associated with loss of mitochondrial function and / or loss of mitochondria, and which is associated with elevated levels of one or more reactive oxygen species (ROS), in a subject.

22. A method as claimed in claim 20, or use as claimed in claim 21, wherein the subject is human.

23. A method or use as claimed in any of claims 20 to 22, wherein the subject has a disease or disorder in which elevated concentrations of total iron, labile iron, and / or in which improperly stored iron, is not the determinant of the disease or disorder.

24. Use or method as claimed in any of claims 1 to 23, wherein R1and R2are each chlorine.

25. Use or method as claimed in any of claims 1 to 24, wherein R3is C1-4 alkyl.

26. Use or method as claimed in claim 25, wherein R3is methyl.

27. Use or method as claimed in any of claims 1 to 26, wherein R4is CH2NHEt.

28. Use or method as claimed in any of claims 1 to 27, wherein R5is H.

29. Use or method as claimed in any of claims 1 to 28, wherein the compound of formula (I) is  (ATH434) or a salt thereof. 51    30. Use or method as claimed in any of claims 1 to 29, wherein a mesylate salt of the compound is used.

31. Use or method as claimed in any of claims 1 to 30, wherein the compound or salt has one or more of the following: a hydrogen transfer activity as determined by oxygen radical absorbance capacity (ORAC) assay which is within 5-fold that of Trolox; an electron transfer activity as determined by ferric reducing antioxidant power (FRAP) assay which is within 5-fold that of Trolox. 52

Images