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WO2025059726A1 - Nouvelle thérapie - Google Patents

Nouvelle thérapie Download PDF

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Publication number
WO2025059726A1
WO2025059726A1 PCT/AU2024/051009 AU2024051009W WO2025059726A1 WO 2025059726 A1 WO2025059726 A1 WO 2025059726A1 AU 2024051009 W AU2024051009 W AU 2024051009W WO 2025059726 A1 WO2025059726 A1 WO 2025059726A1
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Prior art keywords
hydrogen
compound
subject
methyl
salt
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Inventor
Jack Gordon Parsons
Silas Bond
Penelope Jane Huggins
Daniel Jacob KOSMAN
Danielle Kristine BAILEY
Rhudwan NIHLAWI
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Alterity Therapeutics Ltd
Research Foundation of the State University of New York
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Alterity Therapeutics Ltd
Research Foundation of the State University of New York
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Publication of WO2025059726A1 publication Critical patent/WO2025059726A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants

Definitions

  • 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.
  • ATH434 is a small molecule with moderate ferric and ferrous iron affinities (Kd 10 -10, Kd 10 -5 respectively, Finkelstein et al, Acta Neuropathologica Communications, 2017, 5:53; the entire contents of which are incorporated herein by this reference).
  • ATH434 It reduces excess brain iron, reduces aggregated ⁇ -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.
  • 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.
  • R 1 and R 2 are each independently halogen or hydrogen
  • R 3 is 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
  • R 4 is hydrogen, -CH2OH or -CH2NR 6 R 7 , wherein R 6 and R 7 are each independently hydrogen or C 1-4 alkyl
  • 2 R 5 is 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).
  • ROS reactive oxygen species
  • 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.
  • 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 R 1 and R 2 are each independently halogen or hydrogen; R 3 is 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; R 4 is hydrogen, -CH2OH or -CH2NR 6 R 7 , wherein R 6 and R 7 are each independently hydrogen or C1-4 alkyl; and R 5 is hydrogen or methyl; or a salt thereof, or of a pharmaceutical composition
  • R 1 and R 2 are each independently
  • 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.
  • R 1 and R 2 are each independently halogen or hydrogen
  • R 3 is hydrogen, C 1-4 alkyl, -CH 2 -C 3-7 cycloalkyl or -CH 2 Het, 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
  • R 4 is hydrogen, -CH 2 OH or -CH 2 NR 6 R 7 , wherein R 6 and R 7 are each independently hydrogen or C1-4 alkyl
  • R 5 is 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.
  • 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 R 1 and R 2 are each independently halogen or hydrogen; R 3 is hydrogen, C 1-4 alkyl, -CH 2 -C 3-7 cycloalkyl or -CH 2 Het, 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 R 4 is hydrogen, -CH2OH or -CH2NR 6 R 7 , wherein R 6 and R 7 are each independently hydrogen or C 1-4 alkyl; and R 5 is hydrogen or methyl; or a salt thereof, or of a pharmaceutical composition comprising the compound or salt and a pharmaceutical excipient, to the subject.
  • R 1 and R 2 are each independently halogen or hydrogen
  • R 3 is hydrogen, C 1-4 alkyl,
  • 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.
  • ROS reactive oxygen species
  • 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 R 1 and R 2 are each independently halogen or hydrogen; R 3 is hydrogen, C 1-4 alkyl, -CH 2 -C 3-7 cycloalkyl or -CH 2 Het, 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; R 4 is hydrogen, -CH 2 OH or -CH 2 NR 6 R 7 , wherein R 6 and R 7 are each independently hydrogen or C 1-4 alkyl; and R 5 is hydrogen or methyl; or a salt thereof,
  • ROS reactive oxygen species
  • R 1 and R 2 are each independently halogen or hydrogen
  • R 3 is hydrogen, C 1-4 alkyl, -CH 2 -C 3-7 cycloalkyl or -CH 2 Het, 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
  • R 4 is hydrogen, -CH 2 OH or -CH 2 NR 6 R 7 , wherein R 6 and R 7 are each independently hydrogen or C1-4 alkyl
  • R 5 is 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.
  • ROS reactive oxygen species
  • the subject is human.
  • the disease or disorder is a neurodegenerative disease or disorder.
  • 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.
  • 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 R 1 and R 2 are each independently halogen or hydrogen; R 3 is 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; R 4 is hydrogen, -CH2OH or -CH2NR 6 R 7 , wherein R 6 and R 7 are each independently hydrogen or C1-4 alkyl; and R 5 is hydrogen or methyl; or a salt thereof, or
  • ROS reactive oxygen species
  • R 1 and R 2 are each independently halogen or hydrogen
  • R 3 is hydrogen, C 1-4 alkyl, -CH 2 -C 3-7 cycloalkyl or -CH 2 Het, 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
  • R 4 is hydrogen, -CH2OH or -CH2NR 6 R 7 , wherein R 6 and R 7 are each independently hydrogen or C 1-4 alkyl
  • R 5 is 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.
  • ROS reactive oxygen species
  • the subject is human.
  • 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 R 1 and R 2 are each independently halogen or hydrogen; R 3 is hydrogen, C 1-4 alkyl, -CH 2 -C 3-7 cycloalkyl or -CH 2 Het, 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; R 4 is hydrogen, -CH 2 OH or -CH 2 NR 6 R 7 , wherein R 6 and R 7 are each independently hydrogen or C1-4 alkyl; and R 5 is hydrogen or methyl; or a salt thereof, or of a pharmaceutical composition comprising the compound or salt and
  • R 1 and R 2 are each independently halogen or hydrogen
  • R 3 is hydrogen, C 1-4 alkyl, -CH 2 -C 3-7 cycloalkyl or -CH 2 Het, 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
  • R 4 is hydrogen, -CH 2 OH or -CH 2 NR 6 R 7 , wherein R 6 and R 7 are each independently hydrogen or C1-4 alkyl
  • R 5 is 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.
  • ROS reactive oxygen species
  • 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.
  • R 1 and R 2 are each chlorine.
  • R 3 is methyl. In some embodiments, R 3 is C 1-4 alkyl. In some embodiments, R 4 is -CH2NHEt. In some embodiments, R 5 is H.
  • the compound of formula (I) is 9 (ATH434) or a salt thereof. In some embodiments, a mesylate salt of the compound is used.
  • 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 20 ⁇ M 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 20 ⁇ M 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 0.2 ⁇ M, 2 ⁇ M, 20 ⁇ M and 200 ⁇ M concentrations, for ATH434, ATH434-met, Dfp, and Dfx respectively.
  • ORAC Oxygen Radical Ab
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • “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.
  • “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.
  • “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.
  • 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.
  • R 1 and R 2 are each independently halogen or hydrogen
  • R 3 is hydrogen, C 1-4 alkyl, -CH 2 -C 3-7 cycloalkyl or -CH 2 Het, 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
  • R 4 is hydrogen, -CH 2 OH or -CH 2 NR 6 R 7 , wherein R 6 and R 7 are each independently hydrogen or C1-4 alkyl
  • R 5 is hydrogen or methyl.
  • C a to C b or “C a-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.
  • a “C1 to C4 alkyl”, or “C1-4-alkyl” group includes alkyl groups having from 1 to 4 carbons, e.g. CH 3 -, CH 3 CH 2 -, CH 3 CH 2 CH 2 -, (CH 3 ) 2 CH-, CH 3 CH 2 CH 2 CH 2 -, CH 3 CH 2 CH(CH 3 )- and (CH 3 ) 3 C-.
  • groupAgroupB groups of groups that are intended to refer to a groupA when linked by a divalent form of groupB.
  • 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.
  • 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.
  • 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 “C 1-4 alkyl” or similar designations.
  • C 1-4 alkyl 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.
  • cycloalkyl means a monocyclic or polycyclic saturated carbon ring. Examples of cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptanyl.
  • a C 3-7 cycloalkyl is a cycloalkyl group containing between 3 and 7 carbon atoms.
  • 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.
  • 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.
  • 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, adenin
  • heteroaryl 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.
  • 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.
  • halo or “halogen,” mean, in the context of the compounds defined herein, a fluorine, chlorine, bromine, or iodine atom
  • R 1 and R 2 are each halogen.
  • R 1 and R 2 are each independently selected from the group consisting of fluorine, chlorine and iodine.
  • R 1 and R 2 are each independently selected from the group consisting of chlorine and iodine.
  • R 1 and R 2 are each chlorine.
  • R 3 is C 1-4 alkyl. In some embodiments, R 3 is methyl, ethyl, n-propyl, or isopropyl.
  • R 3 is methyl. In some embodiments, R 3 is hydrogen. 14 In some embodiments, R 3 is -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, R 3 is -CH 2 Het, 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.
  • 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.
  • Het is a 5 to 10-membered heterocycle which is substituted with 1 methyl group.
  • R 3 is selected from the group consisting of
  • R 4 is -CH 2 NR 6 R 7 , wherein R 6 and R 7 are each independently a C 1-4 alkyl.
  • R 4 is -CH2NR 6 R 7 , wherein R 6 and R 7 are each independently methyl or ethyl.
  • R 4 is -CH 2 NR 6 R 7 , wherein R 6 and R 7 are each hydrogen.
  • R 4 is -CH2NR 6 R 7 , wherein R 6 is hydrogen and R 7 is C1-4alkyl.
  • R 4 is -CH2NR 6 R 7 , wherein R 6 is hydrogen and R 7 is ethyl. 15 In some embodiments, R 4 is hydrogen. In some embodiments, R 4 is -CH 2 OH. In some embodiments, R 5 is H. In other embodiments, R 5 is 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 . In some embodiments, the compound of formula (I) is .
  • 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).
  • the compound is present in the form of a mesylate salt.
  • a salt of ATH434 is used.
  • a mesylate salt of ATH434 is used.
  • a fumarate salt of ATH434 is used.
  • 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.
  • an acid e.g. methanesulfonic acid
  • 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.
  • the present disclosure provides a compound of formula (IA) 18 wherein R 1 and R 2 are each independently halogen or hydrogen; R 3 is hydrogen, C 1-4 alkyl, R 3 is -CH 2 -C 3-7 cycloalkyl or -CH 2 Het, 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; R 4 is -CH 2 OH; and R 5 is hydrogen or methyl; or a salt thereof; or a pharmaceutical composition comprising the compound or salt and a pharmaceutically acceptable excipient.
  • R 1 and R 2 are each independently halogen or hydrogen
  • R 3 is hydrogen, C 1-4 alkyl
  • R 3 is -CH 2 -C 3-7 cycloalkyl or -CH 2 Het, wherein Het is a 5 to 10- membered heterocycle, said heterocycle being unsubstituted or substituted with 1 or
  • the present disclosure provides a compound of formula (IB) wherein R 1 and R 2 are each independently halogen or hydrogen; R 3 is selected from the group consisting of R 4 is hydrogen, -CH2OH or -CH2NR 6 R 7 , wherein R 6 and R 7 are each independently hydrogen or C 1-4 alkyl; and R 5 is hydrogen or methyl; 19 or a salt thereof; or a pharmaceutical composition comprising the compound or salt and a pharmaceutically acceptable excipient.
  • R 1 and R 2 are each independently halogen or hydrogen
  • R 3 is selected from the group consisting of R 4 is hydrogen, -CH2OH or -CH2NR 6 R 7 , wherein R 6 and R 7 are each independently hydrogen or C 1-4 alkyl
  • R 5 is hydrogen or methyl
  • 19 or a salt thereof or a pharmaceutical composition comprising the compound or salt and a pharmaceutically acceptable excipient.
  • the present disclosure provides a compound of formula (IB) wherein R 1 and R 2 are each independently halogen or hydrogen; R 3 is -CH 2 -C 3-7 cycloalkyl; R 4 is hydrogen, -CH2OH or -CH2NR 6 R 7 , wherein R 6 and R 7 are each independently hydrogen or C1-4 alkyl; and R 5 is hydrogen or methyl; or a salt thereof; or a pharmaceutical composition comprising the compound or salt and a pharmaceutically acceptable excipient.
  • 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.
  • ORAC oxygen radical absorbance capacity
  • FRAP ferric reducing antioxidant power
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • inert diluents such as calcium carbonate, lactose, calcium phosphate or sodium phosphate
  • granulating and disintegrating agents such as corn starch or alginic acid
  • binding agents such as starch, gelatin or acacia
  • 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.
  • a time delay material such as gly
  • 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.
  • the agents may be added or dissolved in an appropriate biologically acceptable buffer and added to a cell or tissue.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • 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.
  • compositions 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.
  • different daily doses can be used depending on activity of the compound, manner of administration, nature and severity of the disorder, age and body weight of the subject. 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.
  • 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.
  • animal models may be used to determine effective dosages for treatment of the cytotoxic side effects.
  • 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.
  • 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
  • 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.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • 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.
  • 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.
  • the compound of formula (I) interacts directly with one or more types of ROS.
  • 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.
  • 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.
  • 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.
  • the compound of formula (I) is used for direct scavenging and/or direct reduction of ROS in a subject.
  • 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.
  • treating and the like are used herein to mean affecting a subject, tissue or cell to obtain a desired pharmacologic and/or physiologic effect.
  • 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.
  • therapeutically effective amount is meant an amount of a compound of formula (I) effective to yield a desired therapeutic response.
  • the subject has an elevated concentration of one or more ROS.
  • 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.
  • 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.
  • the subject has normal concentrations of total iron.
  • 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).
  • ROS reactive oxygen species
  • 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.
  • the disease or disorder is a neurodegenerative disease or disorder.
  • 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.
  • 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).
  • ROS reactive oxygen species
  • 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.
  • 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.
  • ROS mitochondrial reactive oxygen species
  • 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.
  • signs of aging include skin aging, muscle weakness and loss of muscle tone.
  • subject 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.
  • 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.
  • the subject is human.
  • the subject is an adult human.
  • the subject is at least 40, or at least 50, or at least 60, or at least 70 years old.
  • the subject is male.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 65 o C; ii) MeI, K2CO3, acetone reflux; iii) 2N NaOH, MeOH 60 o C; iv) 1.SOCl2 reflux, 2.8.0M MeNH2, CH2Cl2 rt.; v) Fe powder, AcOH 80 o C; vi) ClCH2COCl, AcOH 90 o C; vii) EtNH 2 , THF rt.; viii) 48% HBr 120 o C; ix) NaHCO 3 , Methanesulfonic acid.
  • 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.
  • ABTS radical stock was prepared by mixing 25 ⁇ L potassium persulfate (70mM) with 700 ⁇ L 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, 312.6 ⁇ L 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.
  • Compound EC 50 range ( ⁇ M) (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.
  • Deferiprone 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.
  • ORAC Oxygen Radical Absorbance Capacity
  • 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 4 ⁇ M 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 150 ⁇ L fluorescein working solution and 25 ⁇ L test compound at various concentrations (0.2-200 ⁇ M) and incubated at 37°C for 10min, then the reaction was initiated with 25 ⁇ L 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 20 ⁇ M 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 ⁇ M are shown in Figure 2a and Trolox equivalent response ranges are shown in the below table. 35 At 20 ⁇ M, ATH434 HAT activity was 3.5-fold higher than Trolox. ATH434-met had minimal HAT activity at 20 ⁇ M. Dfx and Dfp did have detectable HAT activity of 2-fold and 7-fold increase compared to Trolox at the same concentration.
  • 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 280 ⁇ L of FRAP assay mix and 20 ⁇ L of sample.
  • 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).
  • TMRM tetramethylrhodamine methyl ester
  • MMP mitochondrial membrane potential
  • the TMRM assay gives a readout of mitochondrial health and function.
  • ROS reactive 37 oxygen species
  • 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 ⁇ M menadione in DM for 20 hours at 37C. In some cases, cells were pretreated with 20 ⁇ M ATH434 or other potential antioxidants.
  • DM differentiation media
  • Thermo Fisher Thermo Fisher
  • 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.
  • 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.

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Abstract

Sont proposés dans les présentes des procédés et des utilisations de composés pour le piégeage direct et/ou la réduction directe de la concentration d'un ou de plusieurs dérivés réactifs de l'oxygène (ROS), et pour la thérapie de maladies et de troubles associés. Sont également proposés dans les présentes des procédés et des utilisations des composés pour prévenir la perte, le maintien ou la restauration de la fonction mitochondriale, et pour la thérapie de maladies et de troubles associés. Sont également proposés dans les présentes des procédés et des utilisations des composés pour traiter ou prévenir des signes de vieillissement.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005095360A1 (fr) * 2004-04-02 2005-10-13 Prana Biotechnology Limited Composes neurologiquement actifs
WO2008074068A1 (fr) * 2006-12-20 2008-06-26 Prana Biotechnology Limited Dérivés de quinoline substitués utilisés comme agents non-amyloïdogéniques
WO2010071944A1 (fr) * 2008-12-24 2010-07-01 Prana Biotechnology Limited Composés de quinazolinone

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005095360A1 (fr) * 2004-04-02 2005-10-13 Prana Biotechnology Limited Composes neurologiquement actifs
WO2008074068A1 (fr) * 2006-12-20 2008-06-26 Prana Biotechnology Limited Dérivés de quinoline substitués utilisés comme agents non-amyloïdogéniques
WO2010071944A1 (fr) * 2008-12-24 2010-07-01 Prana Biotechnology Limited Composés de quinazolinone

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Title
BAILEY DANIELLE K., CLARK WHITNEY, KOSMAN DANIEL J.: "The iron chelator, PBT434, modulates transcellular iron trafficking in brain microvascular endothelial cells", PLOS ONE, PUBLIC LIBRARY OF SCIENCE, US, vol. 16, no. 7, 1 July 2021 (2021-07-01), US , pages e0254794, XP093297333, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0254794 *
HERAS‐GARVIN ANTONIO, REFOLO VIOLETTA, SCHMIDT CLAUDIO, MALFERTHEINER KATJA, WENNING GREGOR K., BRADBURY MARGARET, STAMLER DAVID, : "ATH434 Reduces α‐Synuclein‐Related Neurodegeneration in a Murine Model of Multiple System Atrophy", MOVEMENT DISORDERS, RAVEN PRESS, NEW YORK, NY, US, vol. 36, no. 11, 1 November 2021 (2021-11-01), US , pages 2605 - 2614, XP093297338, ISSN: 0885-3185, DOI: 10.1002/mds.28714 *
SIWECKA NATALIA, SARAMOWICZ KAMIL, GALITA GRZEGORZ, ROZPĘDEK-KAMIŃSKA WIOLETTA, MAJSTEREK IRENEUSZ: "Inhibition of Protein Aggregation and Endoplasmic Reticulum Stress as a Targeted Therapy for α-Synucleinopathy", PHARMACEUTICS, MDPI AG, SWITZERLAND, vol. 15, no. 8, 1 July 2023 (2023-07-01), Switzerland, pages 2051, XP093297328, ISSN: 1999-4923, DOI: 10.3390/pharmaceutics15082051 *

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