WO2010110684A1 - Donneurs d'oxyde nitrique - Google Patents

Donneurs d'oxyde nitrique Download PDF

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Publication number
WO2010110684A1
WO2010110684A1 PCT/NZ2010/000059 NZ2010000059W WO2010110684A1 WO 2010110684 A1 WO2010110684 A1 WO 2010110684A1 NZ 2010000059 W NZ2010000059 W NZ 2010000059W WO 2010110684 A1 WO2010110684 A1 WO 2010110684A1
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alkylene
compound
group
mitosnap
aryl
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PCT/NZ2010/000059
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English (en)
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Robin Andrew James Smith
Michael Patrick Murphy
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Robin Andrew James Smith
Michael Patrick Murphy
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/54—Quaternary phosphonium compounds
    • C07F9/5407—Acyclic saturated phosphonium compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the invention relates to nitric oxide donor compounds and uses thereof, in particular nitric oxide donor compounds comprising a thionitrite conjugated to triphenylphosphonium cation.
  • NO nitric oxide
  • All nitrogen-oxygen bonded compounds have the potential to decompose, be oxidized, or be reduced to produce reactive nitrogen species. Accordingly, a diverse range of NO donors has been developed including organic nitrates and nitrites, metal-NO complexes, N- nitrosamines, thionitrites, furoxans and benzofuroxans, oximes and N-hydroxyguanidines.
  • NO donors developed to date are poorly taken up by cells and are not targeted to particular compartments of the cell. Therefore, the presently known NO donors mainly produce NO in the circulation and thus expose a range of NO receptors to NO.
  • S-Nitrosylation (also referred to as S-nitrosation) is the post-translational addition of a nitrosyl group to a protein.
  • NO donors can also transfer a nitrosonium group to a protein thiol to form an S-nitrosylated product.
  • the modulation of S-nitrosylation may be involved in regulating a number of pathways important in cell metabolism ⁇ Nature Reviews .Molecular Cell Biology, 2005, 6:2, pp 150-166).
  • mitochondrial ROS reactive oxygen species
  • Transfer of the nitrosonium group to protein thiols may also be involved in regulating mitochondrial function, for example at complex I (Dahm, C. C, Moore, K., Murphy, M. P., J. Biol. Chem. 2006, 281, pp 10056-10065).
  • S-Nitrosylation of particular mitochondrial thiol proteins such as complex I may play a protective role in conditions such as ischaemia- reperfusion injury ⁇ Journal of Molecular and Cellular Cardiology, 2007, 42:4, pp 812-825).
  • a NO donor compound that selectively released NO in the mitochondria.
  • a number of medical conditions are influenced by the selective, reversible inhibition of mitochondria. Therefore a mitochondrially targeted NO donor would have potential as a therapeutic agent.
  • the invention provides a compound comprising a lipophilic cation linked by a linker group to a thionitrite moiety
  • lipophilic cation is capable of mitochondrially targeting the thionitrite moiety.
  • the lipophilic cation is a substituted or unsubstituted triphenylphosphonium cation.
  • a substituted triphenylphosphonium cation is substituted with one or more alkyl groups, preferably methyl or ethyl groups.
  • the linker group is selected from the group comprising
  • alkylene group is optionally substituted with one or more functional groups independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, haloalkyl, aryl, aminoalkyl, hydroxyalkyl, alkoxyalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, carboxyalkyl, cyano, oxy, amino, alkylamino, aminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl, aralkylaminocarbonyl, alkylcarbony
  • ⁇ L ⁇ is a linker group and X is an optional anion where the linker group is selected from the group comprising (a) (C 1 -C 3 O) alkylene,
  • alkylene group is optionally substituted with one or more functional groups independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, haloalkyl, aryl, aminoalkyl, hydroxyalkyl, alkoxyalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, carboxyalkyl, cyano, oxy, amino, alkylamino, aminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl, aralkylaminocarbonyl, alkylcarbonylamino, ary
  • n is from 0 to 27
  • X is an optional anion and R 1 and R 2 are independently selected from the group comprising hydrogen, alkyl and aryl.
  • n is from 0 to 27
  • X is an optional anion and R]
  • R 2 , R 3 and R 4 are independently selected from the group comprising hydrogen, alkyl and aryl.
  • the invention provides a compound of formula (IV)
  • n is from 0 to 27
  • X is an optional anion and Rj, R 2 and R 3 are independently selected from the group comprising hydrogen, alkyl and aryl.
  • n is from 0 to 24
  • X is an optional anion and R 1 , R 2 , R 3 , R 4 and R 5 are independently selected from the group comprising hydrogen, alkyl and aryl.
  • the invention provides a compound of formula (VI)
  • n is from 0 to 24
  • X is an optional anion
  • Ri, R 2 , R 3 , and R 4 are independently selected from the group comprising hydrogen, alkyl and aryl.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound of the invention in combination with one or more pharmaceutically acceptable excipients, carriers or diluents.
  • the invention provides a method of treating Alzheimer's disease in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of the invention or a pharmaceutical composition comprising same.
  • the invention provides a method of treating Parkinson's disease in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of the invention or a pharmaceutical composition comprising same.
  • the invention provides a method of treating a disease or disorder selected from the group comprising cancer, neoplasms, tumor growth, metastatsis, angina, stroke, myocardial infarction and ischaemia-reperfusion injury in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of the invention or a pharmaceutical composition comprising same.
  • the invention provides a method of inhibiting angiogenesis in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of the invention or a pharmaceutical composition comprising same
  • the invention provides a method for generating NO in the mitochondria of a subject comprising administering to the subject, a therapeutically effective amount of a compound of the invention or a pharmaceutical composition comprising same
  • the invention provides a method for inhibiting cytochrome oxidase in the mitochondria of a subject comprising administering to the subject, a therapeutically effective amount of a compound of the invention or a pharmaceutical composition comprising same.
  • the invention provides a method for S-nitrosylating proteins in the mitochondria of a subject comprising " administering to the subject, a therapeutically effective amount of a compound of the invention or a pharmaceutical composition comprising same.
  • FIG. 1 is a schematic diagram showing the effects of a compound of the invention (MitoSNO) when delivered to a cell.
  • MitoSNO accumulates within the mitochondria and releases NO, inhibiting cytochrome oxidase by competing with oxygen.
  • Figure 2 is a trace showing NO release from a compound of the invention (MitoSNO) measured using a NO electrode in the presence and absence of glutathione (GSH). The trace shown is typical of two experiments.
  • Figure 3 is a trace showing MitoSNO uptake into energized respiring mitochondria in a ⁇ - dependent manner. Oxygen (black trace) and TPP cation (dashed trace) concentrations were measured simultaneously. The trace shown is typical of two experiments.
  • FIG 4 is a trace showing MitoSNO uptake into respiring mitochondria in a ⁇ -dependent manner.
  • Oxygen (black trace) and TPP (triphenylphosphonium) cation (dashed trace) concentrations were measured.
  • the trace shown is typical of two experiments.
  • MitoSNO uptake leads to production of NO inside the mitochondria which gradually inhibits respiration, as indicated by the shape of the oxygen electrode trace. This also decreases the membrane potential, as shown by the decreased uptake of MitoSNO.
  • Figure 5 is a graph showing the concentration of nitrate formed over time from a compound of the invention (MitoSNAP) compared to SNAP.
  • the NO released from MitoSNAP reacts with oxygen to form nitrite.
  • Figure 6 is a trace showing release of NO from MitoSNAP, as assessed by measuring the formation of NO with an NO electrode then degrading the NO to nitrate by addition of oxyhemoglobin (Oxy Hb).
  • Figure 7 is a trace showing the interaction of isolated mitochondria with MitoSNAP.
  • the trace shows the results from an NO-electrode (measuring NO) and an oxygen electrode (measuring respiration of the mitochondria).
  • NO-electrode measuring NO
  • oxygen electrode measuring respiration of the mitochondria.
  • addition of succinate to energise the mitochondria leads to a large increase in NO formation due to accumulation of MitoSNAP into the mitochondria and subsequent NO release.
  • the released NO then interacts with cytochrome oxidase to decrease respiration, as indicated by the curving off of the oxygen electrode trace.
  • FCCP abolishes the membrane potential preventing uptake of MitoSNAP into the mitochondria.
  • Figure 8 is a trace showing the uptake of MitoSNAP and MitoNAP by energized mitochondria measured using an ion-selective electrode.
  • mitochondria (2 mg protein/ml) were incubated in KCl buffer supplemented with rotenone in a stirred 3 ml chamber thermostatted at 37° C and shielded from ambient light.
  • An electrode selective for the TPP cation was inserted and calibrated by five sequential 1 ⁇ M additions of MitoNAP (a) or MitoSNAP (b). Succinate (10 mM) was then added to energize the mitochondria, followed by the uncoupler FCCP (0.5 ⁇ M).
  • Figure 9 is a series of traces and graphs showing membrane potential-dependent MitoSNAP mitochondrial uptake and NO * generation,
  • (a-d) Liver mitochondria were incubated in KCl medium supplemented with rotenone in a stirred 3 ml chamber and electrodes were used to measure the concentrations of O 2 and NO " simultaneously.
  • succinate (10 mM) OxyHb (5 ⁇ M) or FCCP (500 nM) were added.
  • succinate (10 mM
  • OxyHb 5 ⁇ M
  • FCCP 500 nM
  • Mitochondria (1 mg protein) were incubated in 1 ml KCl medium without neocuproine or DTPA, supplemented with rotenone and succinate in the absence (e) or presence (f) of FCCP (500 nM). Mitochondria were then pelleted by centrifugation (10,000 x g) then aliquots of the supernatant (700 ⁇ l) were mixed with 700 ⁇ l 0.1% TFA and analyzed by RP-HPLC. The amount of MitoSNAP and MitoNAP were calculated as the percentage of their peak areas relative to the sum of both peak areas.
  • Figure 10 is a series of traces and graphs showing the effect of MitoSNAP on respiration in cells, (a-d) Jurkat cells (55 x 10 6 cells) were incubated in PBS supplemented with 25 mM
  • FIG 11 is a series of graphs showing S-nitrosation of mitochondrial thiols by MitoSNAP.
  • NEM JV-ethylmaleimide
  • C2C12 cells were preincubated for 5 min in 2 ml cell culture medium containing either 20 ⁇ M FCCP or DMSO carrier, then 5 ⁇ M MitoSNAP or SNAP was added After further incubation for 5 min at 37°C in the dark, the medium was removed and the content of ⁇ S-nitrosated proteins was measured. Data are means ⁇ range of the duplicate determinations. Control incubations with 5 ⁇ M MitoNAP generated no RSNO. (d) iS-nitrosation of a mitochondria-enriched cell subfraction.
  • C2C12 cells were preincubated for 5 min in cell culture medium containing either 20 ⁇ M FCCP or dimethylsulfoxide (DMSO) carrier, then 5 ⁇ M MitoSNAP was added After 5 min at 37°C in the dark, the medium was removed and the cells were processed to isolate a mitochondria-enriched fraction and S- nitrosated protein was then determined. Data are mean ⁇ sem of triplicate determinations (+MitoSNOl), or mean ⁇ range of duplicate determinations (+FCCP). (e & f) Visualisation of iS-nitrosated mitochondrial proteins by copper/ascorbate-dependent labelling of thiols with a Cy3 fluorophore.
  • Liver (e) or heart (f) mitochondria (1 mg protein) were incubated in 1 ml KCl buffer supplemented with rotenone and succinate in the presence of no additions, 10 ⁇ M MitoSNAP or 500 ⁇ M diamide for 5 min.
  • S-nitrosated thiols were then selectively tagged with Cy3 maleimide and protein (10 ⁇ g) was then separated on a 12.5% SDS PAGE gel which was stained with coomassie blue and scanned for Cy3 fluorescence.
  • the Cy3 fluorescence (SNO) and coomassie staining (coomassie) of each gel is shown. The experiment shows a typical result repeated twice.
  • Figure 12 is a graph showing the effect of OxyHb on S-nitrosation of mitochondrial thiols by MitoSNAP.
  • Time course of 5-nitrosation of mitochondrial proteins by MitoSNAP Aliquots of liver mitochondria (1 mg protein/ml) were incubated at 37°C in 1 ml KCl buffer supplemented with rotenone and succinate in the presence of 5 ⁇ M MitoSNAP and 5 ⁇ M OxyHb and at various times mitochondria were treated with 10 mM NEM and isolated by centrifugation and the S'-nitrosothiol content assessed. Data are of a typical experiment showing means ⁇ range of duplicate samples for each time point, each measured in duplicate. The experiment was repeated twice with the same result.
  • Figure 13 is a trace and graph showing limited S-nitrosation of mitochondria by DetaNONOate.
  • Rat liver mitochondria (1 mg protein/ml) were incubated in KCl medium supplemented with rotenone and succinate at 37°C in the 3 ml stirred chamber of an NO ' electrode. Where indicated 500 ⁇ M DetaNONOate was added and the concentration of NO * was measured over time, before 5 ⁇ M OxyHb was added.
  • B Rat liver mitochondria (1 mg protein/ml) were incubated in 1 ml KCl medium supplemented with rotenone and succinate at 37°C with 500 ⁇ M DetaNONOate for 5 min.
  • Figure 14 is a graph showing the time course of S-nitrosation of cell proteins by MitoSNAP.
  • Six 25 cm 2 flasks were seeded with C2C12 cells in 2 ml cell culture medium and incubated overnight. Then 5 ⁇ M MitoSNAP was added to each flask and they were incubated for the indicated times at 37°C in the dark. The cells were then processed to measure S-nitrosated protein thiols. The experiment was repeated twice with similar results.
  • FIG. 15 is a series of graphs and pictures showing inhibition and S-nitrosation of complex I by MitoSNAP.
  • Heart mitochondria (1 mg protein/ml) were incubated in 1 ml KCl medium supplemented with 1 mM phosphate in an oxygen electrode with 5 mM glutamate and 5 mM malate, or succinate, and incubated with 10 ⁇ M MitoSNAP, 10 ⁇ M MitoNAP or carrier for 2 min (succinate) or 3 min (glutamate/malate), then 250 ⁇ M adenosine diphosphate (ADP) was added and the rate of respiration was measured.
  • ADP adenosine diphosphate
  • Bovine heart mitochondrial membranes (0.25 mg protein/ml) were incubated in 1 ml KCl medium in an O 2 electrode at 37°C, and incubated with 75 ⁇ M MitoSNAP or MitoNAP, or ethanol carrier for 5 min in the presence or absence of rotenone, then 1 mM NADH or 10 mM succinate was added and the rate of respiration was measured. Data are expressed as respiration in the presence of MitoSNAP or MitoNAP as a percentage of the appropriate controls and are means ⁇ range of two separate experiments, each determined in triplicate.
  • Figure 16 is a series of graphs showing the vasodilatory and tissue protective effects of
  • MitoSNAP protects against I/R injury in Langendorff- perfused mouse hearts. Hearts from C57BL6 mice were Langendorff perfused and subjected to 25 min global normothermic ischemia followed by 1 h of reperfusion. MitoSNAP or MitoNAP (100 nM final), or vehicle carrier, was added via an infusion port above the aortic cannula during reperfusion. Hearts were then stained with 2,3,5-triphenyltetrazolium chloride (TTC) to visualize infarct, (c) Protection of heart function by MitoSNAP during I/R injury. Left ventricular contractile function was recorded throughout the I/R protocol.
  • TTC 2,3,5-triphenyltetrazolium chloride
  • RRP Rate Pressure Product
  • systolic minus diastolic The arrow indicates where infusion with MitoSNAP or MitoNAP was initiated.
  • (d) Decreased cardiac infarct size following infusion with MitoSNAP. Upper panel shows typical infarct staining in vehicle control, MitoNAP and MitoSNAP treated hearts. Pale white staining is necrotic infarct, while live tissue stains deep red. Lower panel shows quantitation of infarct size versus area at risk.
  • Figure 17 is a graph showing vasorelaxation of endothelium-denuded rat small mesenteric artery preparations by MitoSNAP and SNAP.
  • To measure vessel relaxation in endothelium- denuded rat mesenteric artery male Wistar rats (250-350 g) were anesthetized with sodium pentobarbitone (60 mg/kg ip Sagatal, Rhone Merieux, Harlow, Essex UK). The mesentery was removed and placed in ice-cold, gassed (95% O 2 /5% CO 2 ) Krebs-Henseleit buffer.
  • Segments (2 mm in length, 250 - 350 ⁇ m in diameter) of third order branches of the superior mesenteric artery were removed, endothelium was removed by rubbing the intima with a human forearm hair and mounted in a Mulvany-Halpern myograph (Model 500A, JP trading, Aarhus, Denmark) as described 2 .
  • Vessels were maintained at 37°C in Krebs-Henseleit solution containing indomethacin (10 ⁇ M) and bubbled with 95%O 2 /5 % CO 2 and were allowed to equilibrate under zero tension for 60 min. After equilibration vessels were normalized to a tension equivalent that generated at 90% of the diameter of the vessel at 100 mm Hg 3 .
  • FIG. 18 is a graph showing the effect of pre-ischemic administration of MitoSNAP or MitoNAP on recovery of heart function (rate pressure product) from IR injury. Experiments were carried out exactly as described in Figs. 16c & 16d, except that MitoSNOl or MitoNAP were infused into the heart for 20 min, followed by a 2 min wash-out period, prior to the onset of ischemia.
  • White symbols vehicle control
  • black symbols MitoSNOl
  • gray symbols MitoNAP.
  • Data are means ⁇ sem, n > 6.
  • Figure 19 is a pictorial representation of the area at risk (AR) and infarct zone, stained with Evans' blue and TTC respectively, then scanned.
  • the infarct/AR ratios were determined with NIH ImageJ software.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e. C 1 -C 1O means one to ten carbons).
  • saturated hydrocarbon radicals include groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)ethyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n- octyl, and the like.
  • An unsaturated alkyl group is one having one or more double bonds or triple bonds.
  • unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2- isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
  • alkyl unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below as “cycloalkyl” and “alkylene.”
  • alkylene by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified by -CH 2 CH 2 CH 2 CH 2 -.
  • an alkyl group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention.
  • a “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
  • cycloalkyl by itself or in combination with other terms, represent, unless otherwise stated, a cyclic versions of “alkyl”.
  • examples of cycloalkyl include cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.
  • alkenyl means a hydrocarbon radical having at least one double bond including, but not limited to, ethenyl, propenyl, 1-butenyl, 2-butenyl and the like.
  • alkynyl means a hydrocarbon radical having at least one triple bond including, but not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl and the like.
  • halo or halogen
  • substituents mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • fluoroalkyl are meant to include monofluoroalkyl and polyfluoroalkyl.
  • aryl employed alone or in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) means, unless otherwise stated, an aromatic substituent which can be a single ring or multiple rings (up to three rings) which are fused together or linked covalently.
  • the rings may each contain from zero to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized.
  • the aryl groups that contain heteroatoms may be referred to as "heteroaryl" and can be attached to the remainder of the molecule through a heteroatom.
  • Non-limiting examples of aryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2- pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4- oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2- thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4- pyridyl, 2-pyrimidyl 4-pyrimidyl, 2-benzothiazolyl, 5-benzothiazolyl, 2-benzoxazolyl, 5- benzoxazolyl, purin
  • alkoxy means an O-alkyl group wherein “alkyl” is defined above.
  • sulfonyl refers to a radical -S(O) 2 R where R is an alkyl, substituted alkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, or substituted heteroaryl group as defined herein. Representative examples include, but are not limited to methylsulfonyl, ethylsulfonyl, propylsulfonyl, butylsulfonyl, and the like.
  • sulf ⁇ nyl refers to a radical -S(O)R where R is an alkyl, substituted alkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, or substituted heteroaryl group as defined herein.
  • Representative examples include, but are not limited to, methylsulfinyl, ethylsulfinyl, propylsulfinyl, butylsulfinyl, and the like.
  • aralkyl or "arylalkyl” means an aryl-alkyl- group in which the aryl and alkyl are as previously described. Preferred aralkyls comprise a lower alkyl group attached to the aryl group. Non-limiting examples of suitable aralkyl groups include phenymethylene, 2-phenethyl and naphthalenylmethyl. The bond to the parent moiety is through the alkyl.
  • pharmaceutically acceptable refers to compounds, ingredients, materials, compositions, dosage forms and the like, which are within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. human) without excessive toxicity, ⁇ irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Each carrier, diluent, exipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • subject refers to a human or non-human mammal.
  • non-human mammals include livestock animals such as sheep, horses, cows, pigs, goats, rabbits, deer, ostriches and emus; and companion animals such as cats, dogs, rodents, and horses.
  • treatment as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of human or animal, in which some desired therapeutic effect is achieved, for example, the inhibition of progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, and cure of the condition.
  • Treatment as a prophylactic measure i.e., prophylaxis
  • Treatment also includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously.
  • a therapeutically effective amount' of a compound of the invention could be combined with or used in conjunction with radiation therapy or chemotherapy in the treatment of cancer.
  • terapéuticaally-effective amount refers to that amount of an active compound, or a material, composition or dosage form comprising an active compound, which is effective for producing some desired therapeutic or prophylactic effect, commensurate with a reasonable benefit/risk ratio.
  • R , R and R are said to be independently selected from a group of substituents, mean that R 1 ' R 2 and R 3 are independently selected, but also that where an R, R 1 , R 2 and R 3 variable occurs more than once in a molecule, each occurrence is independently selected (e.g., if R is -OR 6 wherein R 6 is hydrogen, R 2 can be -OR 6 wherein R 6 is lower alkyl).
  • R is -OR 6 wherein R 6 is hydrogen
  • R 2 can be -OR 6 wherein R 6 is lower alkyl
  • Some compounds of the invention have at least one asymmetrical carbon atom and therefore all isomers, including enantiomers, stereoisomers, rotamers, tautomers and racemates of the compounds are contemplated as being part of this invention.
  • the invention includes d and 1 isomers in both pure form and in admixture, including racemic mixtures.
  • Isomers can be prepared using conventional techniques, either by reacting optically pure or optically enriched starting materials or by separating isomers of a compound of the invention. Isomers may also include geometric isomers, e.g., when a double bond is present.
  • RSNO Thionitrites
  • Intracellular production of NO may have applications for regulating signaling pathways dependent on NO such as vasodilation, regulating activity of intracellular signaling pathways that impact on cell function and on the regulation of mitochondrial biogenesis.
  • NO itself may act as an antioxidant, therefore the intracellular production of NO may to protect against oxidative damage in pathologies (Current Medicinal Chemistry: Anti-inflammatory and Anti-Allergy agents, 2004, 3:3, pp 181-188).
  • the compounds of the invention not only release free NO but can also transfer a nitrosonium group to a protein thiol, thereby forming a S-nitrosylated (S-nitrosated) product. Therefore, the compounds of the invention allow for manipulation of both free NO and the S-nitrosylation (S-nitrosation) status of cells, in order to manipulate and modify the cell's metabolism and function.
  • the compounds of the invention can selectively -release NO in the mitochondria and/or can S-nitrosylate proteins in the mitochondria.
  • Mitochondria have a substantial membrane potential of up to 180 mV across their inner membrane (negative inside). Because of the potential, membrane permeant lipophilic cations such as the triphenylphosphonium cation accumulate several-hundred fold within the mitochondrial matix. This is illustrated in Figure 1 which shows the effect of a compound of the invention (MitoSNO) when delivered to a whole cell.
  • MitoSNO a compound of the invention
  • MitoSNO is taken up by cells, courtesy of the plasma membrane potential ( ⁇ p), and further accumulates in the mitochondrial matrix, driven by the mitochondrial membrane potential ( ⁇ m). Once inside mitochondria, MitoSNO releases NO, which results in direct inhibition of respiration at complex (IV) of the respiratory chain (Brown, G. C. and Cooper, C. E. FEBS Lett. 1994, 356, 295 - 298; Cleeter, M. J. W., Cooper, J. M., Darley-Usmer, V. M., Moncada, S. and Schapira, A. H. V. FEBS Lett. 1994, 345, 50-54; Schweizer, M. and Richter, C. Biochem Biophys. Res. Commum. 1994, 204, 169-175).
  • the invention provides a compound comprising a lipophilic cation linked by a linker group to a thionitrite moiety; and a pharmaceutically acceptable anion wherein the lipophilic cation is capable of mitochondrially targeting the thionitrite moiety.
  • Lipophilic cations may be targeted to the mitochondrial matrix because of their positive charge. Such ions are accumulated provided they are sufficiently lipophilic to screen the positive charge or delocalize it over a large surface area, also provided that there is no active efflux pathway and the cation is not metabolized or immediately toxic to a cell.
  • the lipophilic cation is a substituted or unsubstituted triphenylphosphonium cation.
  • Other lipophilic cations that may be linked to the thionitrite moiety include tribenzyl ammonium and phosphonium cations, arsonium cations or other aromatic and delocalized systems that can act as lipophilic cations and be accumulated inside mitochondria in response to the membrane potential.
  • ⁇ L ⁇ is a linker group and X is an optional anion.
  • the linker group ( ⁇ L ⁇ ) linking the lipophilic cation to the thionitrite moiety may be any chemically non-active distance-making group (spacer) which joins the triphenylphosphonium cation moiety to the thionitrite moiety, and enables the two moieties to remain bonded together when crossing the plasma and mitochondrial membranes.
  • ⁇ L ⁇ is stable under physiological conditions.
  • the linker group will be an alkylene group.
  • alkylene as used herein, means a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 1 to 30 carbon atoms, preferably 2 to 20, more preferably 2 to 10, even more preferably 3 to 5 and most preferably 4, which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated.
  • alkylene includes the sub-classes alkenylene, alkynylene, and cycloalkylene.
  • the linker group is (C]-C 3 o) alkylene or substituted (C 1 -C 3O ) alkylene.
  • the linker group is (C 2 -C 20 ) alkylene or substituted (C 2 -C 2 o) alkylene.
  • the linker group is (C 2 -Cio) alkylene or substituted (C 2 -Ci O ) alkylene.
  • the linker group is (C 3 -C5) alkylene or substituted (C3-C5) alkylene.
  • the linker group may also be substituted by one or more substituent groups that increases the solubility of the molecule, increases the uptake of the molecule across the plasma and/or mitochondrial membranes, or decreases the rate of degradation of the molecule in vivo.
  • the linker group is substituted with one or more functional groups independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, haloalkyl, aryl, aminoalkyl, hydroxyalkyl, alkoxyalkyl, alkylthio, alkylsulfmyl, alkylsulfonyl, carboxyalkyl, cyano, oxy, amino, alkylamino, aminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl, aralkylaminocarbonyl, alkylcarbonylamino, arylcarbonylamino, aralkylcarbonylamino, alkylcarbonyl, heterocyclocarbonyl, aminosulfonyl, alkylaminosulfonyl, alkylsulfonyl, and heterocyclosulfonyl,
  • the linker group may be substituted by alkyl, hydroxyl, thio, amino, carboxy, amido groups or groups derived from sugars or sugar derivatives.
  • the linker group is alkyl or aryl substituted.
  • the linker group is substituted with one or more substituents selected from the group comprising hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl and aryl.
  • the linker group is substituted at the carbon ⁇ to the S atom.
  • the linker group is disubstituted with alkyl or aryl at the carbon ⁇ to the S atom. More preferably, the linker group is disubstituted with methyl groups at the carbon ⁇ to the S atom.
  • the linker group is substituted at the carbon ⁇ to the S atom.
  • the linker group is substituted with a group selected from alkylcarbonylamino, arylcarbonylamino, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl and aryloxycarbonyl. More preferably, the linker group is substituted with alkylcarbonylamino at the carbon ⁇ to the S atom.
  • the linker group is alkyl-substituted (C 3 -C 5 ) alkylene, preferably alkyl substituted butylene, more preferably, dimethyl substituted butylene.
  • the linker group is (C1-C3 0 ) alkylene substituted at the carbon ⁇ to the S atom.
  • the linker group is (C 2 -C 2 o) alkylene substituted at the carbon ⁇ to the S atom.
  • the linker group is (C 2 -C 10) alkylene substituted at the carbon ⁇ to the S atom.
  • the linker group is (C 3 -Cs) alkylene substituted at the carbon ⁇ to the S atom.
  • the linker group is dimethyl substituted at the carbon ⁇ to the S atom.
  • the linker group may also include within its structure, one or more aryl groups.
  • the aryl group(s) may be positioned anywhere in the linker.
  • the linker group is an aryl-containing alkylene chain.
  • the linker group is (C 2 -C]s) alkylene-aryl-(C 2 -C 1 s) alkylene or substituted (C 2 -C ⁇ s) alkylene-aryl-substituted (C 2 -Cis) alkylene.
  • the linker group is (C 2 -C 10) alkylene-aryl-(C 2 -C 1 o) alkylene or substituted (C 2 -Ci 0 ) alkylene-aryl-substituted (C 2 -C 1 O) alkylene.
  • the linker group is (C 2 -C 5 ) alkylene-aryl-(C 2 -C 5 ) alkylene or substituted (C 2 -C 5 ) alkylene-aryl- substituted (C 2 -C 5 ) alkylene.
  • the aryl group of the linker is substituted.
  • the aryl group is substituted with one or more functional groups independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, haloalkyl, aryl, aminoalkyl, hydroxyalkyl, alkoxyalkyl, alkylthio, alkyls ⁇ lf ⁇ nyl, alkylsulfonyl, carboxyalkyl, cyano, amino, alkylamino, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, aralkylaminocarbonyl, alkylcarbonylamino, arylcarbonylamino, aralkylcarbonylamino, alkylcarbonyl, heterocyclocarbonyl, aminosulfonyl, alkylaminosulfonyl, alkylsulfonyl, and heterocyclosulfon
  • the linker group may also include within its structure, one or more heteroatoms such as N, O and S.
  • the heteroatom may be positioned anywhere in the linker.
  • the linker group is (C 2 -Ci 5 ) alkylene-NR-(C 2 -Ci 5 ) alkylene or substituted (C 2 -Ci 5 ) alkylene-NR-substituted (C 2 -Ci 5 ) alkylene.
  • the linker group is (C 2 -Cio) alkylene-NR-(C2-Cio) alkylene or substituted (C 2 -Cio) alkylene-NR- substituted (C 2 -Cio) alkylene.
  • the linker group is (C 2 -C 5 ) alkylene-NR-(Cr- C 5 ) alkylene or substituted (C 2 -C 5 ) alkylene-NR-substituted (C 2 -C 5 ) alkylene.
  • R is hydrogen.
  • the linker group is substituted with a group selected from alkylcarbonylamino, arylcarbonylamino, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl and aryloxycarbonyl. More preferably, the linker group is substituted with alkylcarbonylamino at the carbon ⁇ to the S atom. Preferably, the linker group is substituted with an alkyl or aryl group at the carbon ⁇ to the S atom.
  • R is hydrogen.
  • either end of the linker may be attached to the triphenylphosphonium ion.
  • R is hydrogen.
  • the linker group is substituted with a group selected from alkylcarbonylamino, arylcarbonylamino, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl and aryloxycarbonyl. More preferably, the linker group is substituted with alkylcarbonylamino at the carbon ⁇ to the S atom. Preferably, the linker group is substituted with an alkyl or aryl group at the carbon ⁇ to the S atom.
  • the linker group is (C 2 -C] 5 ) alkylene-O-(C 2 -Ci 5 ) alkylene or substituted (C 2 -Ci S ) alkylene-O- substituted (C 2 -Ci 5 ) alkylene.
  • the linker group is (C 2 -Cio) alkylene-O-(C 2 - Cio) alkylene or substituted (C 2 -Cio) alkylene-O-substituted (C 2 -CiO) alkylene.
  • the linker group is (C 2 -C 5 ) alkylene-O- ⁇ r-Cs) alkylene or substituted (C 2 -C 5 ) alkylene-O-substituted (C 2 -Cs) alkylene.
  • the linker group is substituted with a group selected from alkylcarbonylamino, arylcarbonylamino, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl and aryloxycarbonyl. More preferably, the linker group is substituted with alkylcarbonylamino at the carbon ⁇ to the S atom. Preferably, the linker group is substituted with an alkyl or aryl group at the carbon ⁇ to the S atom.
  • the linker group is (C 2 -C 15 ) alkylene-O-C(O)-(C 2 -Ci 5 ) alkylene or substituted (C 2 - C 15 ) alkylene-O-C(O)-substituted (C 2 -Ci 5 ) alkylene.
  • the linker group is (C 2 - Cio) alkylene-O-C(O)-(C 2 -C 10 ) alkylene or substituted (C 2 -Ci 0 ) alkylene-O-C(O)-substituted (C 2 -Ci 0 ) alkylene.
  • the linker group is (C 2 -C 5 ) alkylene-O-C(O)-(C 2 -C 5 ) alkylene or substituted (C 2 -Cs) alkylene-O-C(O)-substituted (C 2 -Cs) alkylene.
  • the linker group is substituted with a group selected from alkylcarbonylamino, arylcarbonylamino, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl and aryloxycarbonyl. More preferably, the linker group is substituted with alkylcarbonylamino at the carbon ⁇ to the S atom. Preferably, the linker group is substituted with an alkyl or aryl group at the carbon ⁇ to the S atom.
  • the linker group is (C 2 -Ci S ) alkylene-S-(C 2 -Cis) alkylene or substituted (C 2 -Ci 5 ) alkylene-S-substituted (C 2 -Ci 5 ) alkylene.
  • the linker group is (C 2 -Ci 0 ) alkylene-S-(C 2 -Ci 0 ) alkylene or substituted (C 2 -Ci 0 ) alkylene-S-substituted (C 2 -Ci 0 ) alkylene.
  • the linker group is (C 2 -Cs) alkylene-S-(C 2 -C 5 ) alkylene or substituted (C 2 -Cs) alkylene-S-substituted (C 2 -C 5 ) alkylene.
  • the linker group is substituted with a group selected from alkylcarbonylamino, arylcarbonylamino, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl and aryloxycarbonyl. More preferably, the linker group is substituted with alkylcarbonylamino at the carbon ⁇ to the S atom. Preferably, the linker group is substituted with an alkyl or aryl group at the carbon ⁇ to the S atom.
  • Linker groups containing aryl and/or heteroatoms may also be substituted at other positions within the linker.
  • the linker is optionally substituted with one or more functional groups independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, haloalkyl, aryl, aminoalkyl, hydroxyalkyl, alkoxyalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, carboxyalkyl, cyano, oxy, amino, alkylamino, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, aralkylaminocarbonyl, alkylcarbonylamino, arylcarbonylamino, aralkylcarbonylamino, alkylcarbonyl, heterocyclocarbonyl, aminosulfonyl, alkylaminosulfonyl, alkylsulfon
  • the linker group is optionally substituted with one or more functional groups independently selected from the group consisting of alkyl, aryl, alkoxycarbonyl, aryloxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonylamino and arylcarbonylamino.
  • one or more functional groups independently selected from the group consisting of alkyl, aryl, alkoxycarbonyl, aryloxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonylamino and arylcarbonylamino.
  • the anion comprises a suitable inorganic or organic anion known in the art and is present when required for overall electrical neutrality.
  • suitable inorganic anions include, but are not limited to, those derived from hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric or phosphorous acid or from an alkylsulfonic or an arylsulfonic acid.
  • Suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyuvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. All are generally recognized as pharmaceutically acceptable salts.
  • the anion is an anion derived from hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric or phosphorous acid, or from an alkylsulfonic or an arylsulfonic acid.
  • the anion is chloride, bromide, iodide, most preferably bromide.
  • the anion is methanesulfonate.
  • the invention provides a compound of formula (II)
  • n is from 0 to 27
  • X is an optional anion and R 1 and R 2 are independently selected from the group comprising hydrogen, alkyl and aryl.
  • n is from 0 to 20. Preferably n is from 0 to 10. More preferably n is from 0 to 2. Most preferably, n is 1.
  • R 1 and R 2 are independently selected from the group comprising hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl and aryl.
  • Ri and R 2 are methyl.
  • the anion is an inorganic anion derived from hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric or phosphorous acid, or from an alkylsulfonic or an arylsulfonic acid.
  • the anion is chloride, bromide, iodide or methanesulfonate.
  • the anion is bromide or methanesulfonate.
  • the linker group comprises (C 3 -C 3 o) alkylene optionally substituted with alkyl or aryl at the carbon ⁇ to the sulfur atom.
  • the invention provides a compound of formula (III)
  • n is from 0 to 27
  • X is an optional anion and R 1 , R 2 , R 3 and R 4 are independently selected from the group comprising hydrogen, alkyl and aryl.
  • n is from 0 to 20.
  • n is from 0 to 10. More preferably n is from 1 to 5. Most preferably, n is 3.
  • Ri R 2 , R 3 and R 4 are independently selected from the group comprising hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl and aryl.
  • R] R 2 and R 3 are methyl, and R 4 is hydrogen.
  • the anion is an inorganic anion derived from hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric or phosphorous acid, or from an alkylsulfonic or an arylsulfonic acid.
  • the anion is chloride, bromide, iodide or methanesulfonate.
  • the anion is bromide or methanesulfonate.
  • the invention provides a compound of formula (IV)
  • n is from 0 to 27
  • X is an optional anion
  • Ri, R 2 and R 3 are independently selected from the group comprising hydrogen, alkyl and aryl.
  • n is from 0 to 20.
  • n is from 0 to 10. More preferably n is from 1 to 5. Most preferably, n is 3.
  • Ri R 2 and R 3 are independently selected from the group comprising hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl and aryl. Preferably, Ri R 2 and R 3 are methyl.
  • the anion is an inorganic anion derived from hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric or phosphorous acid, or from an alkylsulfonic or an arylsulfonic acid.
  • the anion is chloride, bromide, iodide or methanesulfonate.
  • the anion is bromide or methanesulfonate.
  • the invention provides a compound of formula (V)
  • n is from 0 to 27
  • X is an optional anion and R 1 , R 2 , R 3 , R 4 and R 5 are independently selected from the group comprising hydrogen, alkyl and aryl.
  • n is from 0 to 20.
  • n is from 0 to 10. More preferably n is from 1 to 5. Most preferably, n is 3.
  • Ri R 2 , R 3 , R 4 and R 5 are independently selected from the group comprising hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl and aryl.
  • Ri R 2 , R 3 , R 4 and R 5 are methyl.
  • the anion is an inorganic anion derived from hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric or phosphorous acid, or from an alkylsulfonic or an arylsulfonic acid.
  • the anion is chloride, bromide, iodide or methanesulfonate.
  • the anion is bromide or methanesulfonate.
  • the invention provides a compound of formula (VI)
  • n is from 0 to 27
  • X is an optional anion
  • Ri, R 2 , R 3 and R 4 are independently selected from the group comprising hydrogen, alkyl and aryl.
  • n is from 0 to 20.
  • n is from 0 to 10. More preferably n is from 1 to 5. Most preferably, n is 3.
  • Ri R 2 , R 3 and R 4 are independently selected from the group comprising hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl and aryl.
  • Ri R 2 , R 3 and R 4 are methyl.
  • the anion is an inorganic anion derived from hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric or phosphorous acid, or from an alkylsulfonic or an arylsulfonic acid.
  • the anion is chloride, bromide, iodide or methanesulfonate.
  • the anion is bromide or methanesulfonate.
  • the compounds of the invention can be made using any convenient synthetic process.
  • the compounds of the invention can be synthesised by reacting the convenient intermediate thiol of formula (VII) with nitrous acid under an inert atmosphere as shown in Scheme 1 below.
  • the intermediate thiol (VII) can be prepared in a number of ways depending on the final compound required.
  • the intermediate thiol (VII) can be prepared using Scheme 2 as outlined below (Burns, R. J., Smith, R. A. J. and Murphy, M. P. Archives of Biochemistry and Biophysics, 1995, 322, 60-68).
  • a linker group including a bromine group at one end and an alkylene group at another end are reacted with thioacetic acid using the radical generator 2,2'- azobisisobutyronitrile (AIBN).
  • AIBN 2,2'- azobisisobutyronitrile
  • step 2 The product of step 1 is then fused together with triphenylphosphine (PPh 3 ).
  • the intermediate thiol (VII) is generated by base hydrolysis with NaOH and anion exchange.
  • Scheme 3 outlines the synthesis of a protected thiol intermediate compound incorporating an ether functionality in the linker.
  • Base hydrolysis provides a compound of the invention wherein the linker group is C 4 alkylene-O-C 4 alkylene.
  • Scheme 4 outlines the synthesis of a protected thiol intermediate compound incorporating a benzene functionality in the linker.
  • THP is tetrahydrophyran.
  • Base hydrolysis provides a compound of the invention wherein the linker group is an aryl-containing alkylene chain, C 6 alkylene-aryl-C 4 alkylene.
  • Scheme 5 outlines the synthesis of a protected thiol intermediate compound which is dimethyl substituted at the carbon ⁇ to the S atom.
  • Thietanes are sulfur- containing 4-membered ring compounds.
  • Scheme 6 shows the synthesis of compounds of intermediate thiols that include these functionalities in the chain of the linker and are substituted with alkylcarboxylamine groups. Reaction of these intermediates with nitrous acid provides the equivalent thionitrites of the invention
  • Compounds of formula (VIII) are prepared using a triphenylphosphonium cation attached to a linker group that includes an amine group at the opposite end.
  • Compounds of formula (IX) are prepared using a triphenylphosphonium cation with a hydroxy attached to the end of the linker group.
  • the remainder of the linker group is an alkylene chain but the linker group of the thiol intermediate can be altered further by using triphenylphosphonium cations linked to the amine or hydroxy groups via other chains, for example, substituted alkylene chains.
  • the amine or alcohol group at the end of the linker group opens the thiotane ring to provide the thiol intermediate.
  • the amine group is a secondary amine (R 4 is alkyl or aryl)
  • the resulting compound of formula (VIII) is N-alkyl or aryl substituted.
  • Reaction with the thietane shown above provides a thiol intermediate that is substituted with an amide group at the position ⁇ to the sulfur atom and also substituted at the carbon ⁇ to the sulfur atom.
  • the nature of the substitution at R 1 , R 2 and R 3 depends on the thietane used.
  • Many thietanes are commercially available, for example, N-(2-ethyl-2-methyl-4-oxo-3- thietanyl) acetamide.
  • Other alkyl and aryl groups can be introduced using thiotanes with different Ri , R 2 and R 3 substituents. Such thiotanes can easily be prepared by a person skilled in the art using known techniques.
  • thietanes for use in synthesising compounds of the invention can also be prepared by reaction of modified cysteine, as shown in scheme 7 below.
  • R t , R 2 and R 3 will be alkyl or aryl.
  • the functional groups of intermediate compounds may need to be protected by protecting groups.
  • Functional groups which it is desirable to protect include, but are not limited to hydroxyl, amino and carboxylic acid.
  • Protecting groups may be added and removed in accordance with techniques that are well known to those skilled in the art. The use of protecting groups is fully described in "Protective Groups in Organic Chemistry", edited by J.W.F McOmie, Plenum Press (1973), and “Protective Groups in Organic Synthesis", 2 nd Ed, T. W. Greene and P.G.M Wutz, Wiley-Interscience (1991).
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound of the invention in combination with one or more pharmaceutically acceptable excipients, carriers or diluents.
  • Suitable excipients, carriers and diluents can be found in standard pharmaceutical texts. See, for example, Handbook for Pharmaceutical Additives, 2 nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA) and Remington's Pharmaceutical Science, (ed. A. L. Gennaro) 2000 (Lippincott, Williams and Wilkins, Philadelphia, USA) which are incorporated herein by reference.
  • Excipients for use in the compositions of the invention include, but are not limited to microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine may be employed along with various disintegrants such as starch (and preferably corn, potato or tapioca starch), alginic acid and certain complex silicates, together with granulation binders like polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes.
  • compositions of a similar type may also be employed as fillers in gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols.
  • preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols.
  • the active ingredient may be combined with various sweetening or flavouring agents, colouring matter or dyes, and, if so desired, emulsifying and/or suspending agents as well, together with such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.
  • Pharmaceutical carriers include solid diluents or fillers, sterile aqueous media and various non-toxic organic solvents, and the like.
  • NO has been shown to be a reversible inhibitor of the mitochondrial respiratory chain by competing with oxygen at cytochrome oxidase.
  • Selective delivery of NO to mitochondria by the compounds of the invention enables the respiration rate to be reversibly modulated within patients. Consequently, the compounds of the invention may be useful in the treatment of conditions that are affected by the inhibition of cytochrome oxidase.
  • the compounds of the invention do not act like other cytochrome oxidase inhibitors such as cyanide, because they compete with oxygen and thereby only affect respiration when the oxygen concentration is low. This effect is reversed once the oxygen concentration increases. Accordingly, the compounds of the invention can be considered modulators of the effective K m of cytochrome oxidase.
  • Ischaemia is the condition suffered by tissues and organs when deprived of blood flow. It is mostly the result of inadequate nutrient and oxygen supply.
  • Reperfusion injury refers to the tissue damage inflicted when blood flow is restored after an ischemic period of more than about ten minutes. Ischaemia and reperfusion can cause serious brain damage in stroke or cardiac arrest.
  • Example 14 shows that compounds of the invention protected against cardiac I/R injury when the compound was administered during the reperfusion phase in an ex vivo Langendorff heart I/R injury model.
  • Example 15 demonstrates the efficacy of the compounds in a well established murine model of in vivo I/R injury by subjecting mice to occlusion of the left anterior descending coronary artery (LAD) for 30 min followed by reperfusion and recovery over 24 h.
  • LAD left anterior descending coronary artery
  • the compounds of the invention may be used pre-surgically to minimise damage to organs and tissues caused by reperfusion, for example, during heart surgery.
  • Compounds of the invention may also be useful for preserving organs during organ transplant procedures.
  • the compounds of the invention have activity as angiogenesis inhibitors to prevent tumour growth and metastasis. Tumors above a certain size have to attract blood vessels to them in order to grow and blocking angiogenesis has proven to be a successful approach to targeting cancers.
  • the signaling pathway for angiogenesis involves sensing that oxygen concentration is lowered due to poor blood supply. This is done by the oxygen dependent degradation of hypoxia inducible factor 1- ⁇ (HIF- l ⁇ ). Under hypoxia, HIF- l ⁇ is no longer degraded and acts as a transcription factor inducing a range of processes including angiogenesis. It has been shown that exposure to NO can facilitate HIF- l ⁇ degradation by partially inhibiting respiration and thereby increasing the local oxygen concentration (Hagen, T., Taylor, C. T., Lam, F., Moncada, S. Science, 2003, 302, 1975-1978).
  • the compounds of the invention act as angiogenesis inhibitors by inhibiting cytochrome oxidase thereby increasing the local oxygen concentration in hypoxic tumours. This destabilises the transcription factor hypoxia inducible factor 1- ⁇ (HIF- l ⁇ ) thereby blocking angiogenesis to the tumour.
  • the selective production of NO within mitochondria by the compounds of the invention may act to switch off angiogenesis in tumors without affecting respiration in fully aerobic tissues. This will result in a slowing of tumour development.
  • the compounds of the invention have also shown to relax the smooth muscle in blood vessel walls thereby acting as vasodilators (see Example 13). Consequently, they may have application in the treatment of angina and high blood pressure.
  • the compounds of the invention can also be used in conjunction with other therapies such as anti cancer agents and other pharmaceuticals.
  • the conjugates or pharmaceutical compositions of the invention can be administered via oral, parenteral (such as subcutaneous, intravenous, intramuscular, intracisternal and infusion techniques), rectal, intranasal or topical routes.
  • parenteral such as subcutaneous, intravenous, intramuscular, intracisternal and infusion techniques
  • rectal intranasal or topical routes.
  • these compounds are administered in doses ranging from about 0.5 to about 500 mg per day, in single or divided doses (such as from 1 to 4 doses per day).
  • appropriate dosages of the compounds, and compositions comprising the compounds can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects.
  • the selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition and the general health and prior medical history of the patient.
  • the active compounds of this invention can be administered alone or in combination with pharmaceutically acceptable excipients, carriers or diluents by any of the routes previously indicated, and such administration may be carried out in single or multiple doses.
  • novel therapeutic agents of this invention can be administered in a wide variety of different dosage forms, they may be combined with various pharmaceutically acceptable inert carriers in the form of tablets, capsules, lozenges, troches, hard candies, powders, sprays, creams, salves, suppositories, jellies, gels, pastes, lotions, ointments, aqueous suspensions injectable solutions, elixirs, syrups, and the like.
  • Such carriers include solid diluents or fillers, sterile aqueous media and various non-toxic organic solvents, and the like. Injectable forms are preferred.
  • oral pharmaceutical compositions can be suitably sweetened and/or flavoured.
  • the conjugates of the invention are present in such dosage forms at concentration levels ranging from about 1.0% to about 70% by weight, preferably about 1.0% to about 70% by weight.
  • the conjugates can be administered, for example, in the form of tablets or capsules, or as an aqueous solution or suspension. Tablets may contain various excipients such as described above.
  • solutions of a compound of the present invention in oils such as sesame or peanut oil, or an aqueous propylene glycol may be employed.
  • the aqueous solutions should be suitably buffered (preferably pH less than 8) if necessary and the liquid diluent first rendered isotonic.
  • These aqueous solutions are suitable for intravenous injection purposes.
  • the oily solutions are suitable for intra-muscular and subcutaneous injection purposes. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
  • sterile solutions of the active ingredient can be prepared, and the pH of the solutions should be suitably adjusted and buffered.
  • the total concentration of solutes should be controlled to render the preparation isotonic.
  • Nitrite was analyzed by the Griess assay (Molecular Probes). Nitric oxide was measured using an NO ' electrode (World Precision Instruments Ltd, UK) connected to an Apollo 4000 Free Radical Analyser. The electrode was inserted into a stirred, sealed 3 ml chamber with a Clark type O 2 electrode (Rank Brothers, UK) built into its base and was thermostatted at 37 0 C. The NO ' electrode was calibrated by adding SNAP to argon-purged, saturated CuCl. OxyHb was prepared by reduction of bovine methemoglobin with sodium dithionite.
  • Protein S-nitrosothiols were measured using a chemiluminescence assay in an EcoMedics CLD 88 Exhalyzer (Annex, Herts, UK) (Feelisch, M. et al. Concomitant S-, N-, and heme-nitros(yl)ation in biological tissues and fluids: implications for the fate of NO in vivo. FASEB. J. 16, 1775-85 (2002)).
  • fetal calf serum FCS
  • penicillin 100 U.mT 1
  • streptomycin 100 ⁇ g.ml "1
  • Jurkat cells were cultured in Roswell park memorial institute (RPMI) 1640 medium supplemented with 2 mM Glutamax and 25 mM Hepes.
  • RPMI Roswell park memorial institute
  • HeLa cells were grown in minimum essential eagle medium (MEM) containing 2 mM glutamine and non-essential amino acids and were grown to 100% confluence prior to use.
  • MEM minimum essential eagle medium
  • C2C12 cells were grown in Dulbecco's modified Eagle's medium (DMEM) and were seeded at -25,000-30,000 cells/cm 2 and grown overnight prior to experiments.
  • DMEM Dulbecco's modified Eagle's medium
  • a mitochondria-enriched fraction from C2C12 cells was prepared from C2C12 cells, as described below.
  • Rat liver mitochondria were prepared in 250 mM sucrose, 5 mM Tris-HCl, 1 mM ethylene glycol-bis( ⁇ -aminoethyl ether)-N,N,N',N'- tetraacetic acid (EGTA) 5 pH 7.4.
  • Rat heart mitochondria were prepared in 250 mM sucrose, 5 mM Tris-HCl, 1 mM EGTA, 0.1% bovine serum albumin (BSA), pH 7.4.
  • BSA bovine serum albumin
  • Glutaredoxin 2 Catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: implications for mitochondrial redox regulation and antioxidant defense. J Biol. Chem. 279, 47939-51 (2004)). Protein concentration was determined by the biuret assay using BSA as a standard or by the bicinchoninic acid assay.
  • Mitochondrial incubations were usually in KCl buffer (120 mM KCl, 10 mM HEPES, 1 mM EGTA, 100 ⁇ M N r /V-bis(2-bis[carboxymethyl]aminoethyl) glycine (DTPA) and 10 ⁇ M neocuproine, pH 7.2) with 10 mM succinate and 4 ⁇ g/ml rotenone in the dark at 37"C, unless stated otherwise.
  • An electrode selective for the TPP moiety of MitoS ⁇ AP and Mito ⁇ AP was made and used as described previously (Asin-Cayuela, J., Manas, A.R., James, A.M., Smith, R. A.
  • Sprague-Dawley rats male 250 - 350 g were anesthetized by intraperitoneal injection of ketamine and xylazine (100 and 16 mg/kg, respectively) and thoracic aortas were excized and placed in Krebs-Henseleit buffer (pH 7.3) at 37°C.
  • Aortic segments were cut into seven to eight ⁇ 3 mm-long rings, mounted in baths containing 15 ml Krebs-Henseleit buffer equilibrated with 21% O 2 , 5% CO 2 at 37°C and isometric vessel tension was established using a vessel bioassay system (Radnoti, Monrovia CA).
  • mice Male C57BL6 mice (30 - 35 g) were obtained from Harlan (Indianapolis, IN) and were maintained with food and water available ad libitum. All procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication #85-23, 1996). Isolated mouse hearts were retrograde reperfused in Langendorff mode under constant flow (4 ml/min), essentially as described for rat hearts (Nadtochiy, S.M., Tompkins, AJ. & Brookes, P. S. Different mechanisms of mitochondrial proton leak in ischaemia/reperfusion injury and preconditioning: implications for pathology and cardioprotection. Biochem. J. 395, 611-8 (2006)).
  • mice Male C57BL/6 mice ( ⁇ 25g) were purchased from the Jackson Laboratory and handled in accordance with the procedures of the University of Rochester Committee on Animal Research. All surgical procedures were sterile and performed as described previously. Briefly, analgesics (acetaminophen 0.75 mg/ml in drinking water) were administered 24 hr before, and during the perioperative period. Acetaminophen does not impact on I/R injury.
  • the mitochondrial, or mitochondrial membrane, suspensions were supplemented with 10 mM NEM and 1 min later was pelleted by centrifugation (10,000 x g for 5 min) and resuspended 1 ml KCl buffer supplemented with 10 mM NEM and pelleted once more. The pellet was then resuspended in 1 ml 25 mM HEPES pH 7.2, 100 ⁇ M DTPA, 10 ⁇ M neocuproine and 10 mM NEM, snap frozen on dry ice/ ethanol, freeze/thawed (x3) and stored at -2O 0 C until analysis.
  • a control experiment was carried out in which mitochondria were incubated with 5 ⁇ M MitoSNAP for 5 min when the mitochondrial suspension was supplemented with 10 mM NEM and 1 min later was pelleted by centrifugation (10,000 x g for 5 min) as above.
  • the pellet was then resuspended in 1 ml 5% sulfosalicylic acid to fully lyse mitochondria and precipitate protein which was pelleted and washed again in 1 ml 5% sulfosalicylic acid, when the protein pellet was resuspended in 1 ml 25 mM HEPES pH 7.2, 100 ⁇ M DTPA, 10 ⁇ M neocuproine and 10 mM NEM KCl buffer supplemented with 10 mM NEM and assessed for SNOs as above.
  • mitochondria (lmg protein/ml) were incubated in 1 ml KCl Buffer for 5 min, pelleted by centrifugation and the pellet resuspended in 75 ⁇ l 250 mM sucrose, 5 mM HEPES, 1 mM EGTA, pH 7.4 supplemented with 0.1 % dodecylmaltoside.
  • C2C12 cells were seeded ( ⁇ 2 x 10 6 cells per 14 cm 2 dish) and grown with 25 ml medium/dish for 46 h giving ⁇ 7 xlO 6 cells per 14 cm 2 dish. The medium was removed and replaced with 10 ml medium. To this DMSO or 20 ⁇ M FCCP was added and incubated for 5 min at 37°C in the dark. After this 5 ⁇ M MitoSNAP or SNAP or ethanol was added and incubated for a further 5 min in the dark.
  • the supernatant was aspirated and the cell sheet was washed first in PBS/10 mM NEM then in buffer A (100 mM sucrose, 1 mM EGTA, 20 mm MOPS, pH 7.4, 10 mM NEM, 100 ⁇ M DTPA, 10 ⁇ M neocuproine) while on ice. Cells were scraped into 1 ml SEM buffer and the plate washed in 4 ml of the same buffer.
  • buffer A 100 mM sucrose, 1 mM EGTA, 20 mm MOPS, pH 7.4, 10 mM NEM, 100 ⁇ M DTPA, 10 ⁇ M neocuproine
  • buffer B buffer A supplemented with 0.1 mg/ml digitonin and 10 mM triethanolamine
  • the supernatant (cytosol fraction) was snap frozen and the pellets combined in 500 ⁇ l 25 mM HEPES pH 7.2, 100 ⁇ M DTPA, 10 ⁇ M neocuproine and 10 mM NEM and then snap frozen. Samples were freeze thawed 3 x and aliquots prepared for S-nitrosothiol analysis.
  • Isolated rat liver or rat heart mitochondria (4 mg protein/ml) were suspended in KCl buffer supplemented with 10 mM succinate and 8 ⁇ g/ml rotenone and were incubated with no additions, 10 ⁇ M MitoSNAP or 500 ⁇ M diamide at 37 0 C for 5 min with occasional mixing. Mitochondria were then pelleted by centrifugation and resuspended in a blocking buffer containing 250 mM HEPES, pH 7.7, 1 mM EDTA, 1 mM DTPA, 10 ⁇ M neocuproine, 1% SDS, and 50 mM NEM.
  • This blocking reaction was carried out for 5 min at 4O 0 C.
  • Cy3 maleimide 200 ⁇ M; Amersham product number PA 13131), 1 mM ascorbate, and 10 ⁇ M CuSO 4 were added and the mixture gently vortexed and then incubated for 30 min at 37 0 C.
  • the protein (10 ⁇ g) was then separated on a 12.5 % SDS PAGE gel. After electrophoresis, the gel image was acquired with a Typhoon 9410 variable mode imager.
  • bovine heart mitochondrial membranes 250 ⁇ g/ml were incubated in KCl buffer ⁇ 75 ⁇ M MitoSNAP at 37°C for 5 min with occasional mixing. Then 10 mM NEM was added and incubated for 5 min at 40 0 C. The membranes were then pelleted by centrifugation and washed three times in 1 ml PBS buffer. The pellet was then resuspended in 100 ⁇ l PBS supplemented with 200 ⁇ M Cy3 maleimide, 1 mM ascorbate, and 10 ⁇ M CuSO 4 and incubated for 30 min at 37°C.
  • MitoSNAP, MitoNAP and their derivatives were separated by reverse phase HPLC (RP- HPLC) on a Cl 8 column (Jupiter 300 A, Phenomenex) with a Widepore C18 guard column (Phenomenex), using a Gilson 321 pump.
  • RP- HPLC reverse phase HPLC
  • HeLa cells were seeded and grown to 100% confluence with 15 ml medium/dish on 165 cm 2 dishes. The medium was aspirated and replaced with 15 ml fresh medium. The dishes were then placed in a humidified hypoxia workstation (Coy Laboratories) at 1% O 2 concentration with 5% CO 2 and the balance N 2 for 60 min. The indicated concentration of MitoNAP, MitoSNAP and myxothiazol was added to individual dishes which were incubated in the dark for a further 30 min. Extracellular p ⁇ 2 measurements were taken by fluorescence quenching oximetry (Oxylite-2000; Oxford Optronix). Statistical analysis was performed using SPSS 12.0.1.
  • a compound of formula (X) was prepared as described in Schemes 1 and 2 above.
  • Rat liver mitochondria (1 mg protein/ml) in KCl buffer was incubated at 37 0 C with 5 ⁇ M MitoSNO. 1 ⁇ M additions of MitoSNO were made sequentially before addition of 10 mM succinate. 200 nM FCCP was then added to uncouple the mitochondria. The concentrations of O 2 and MitoSNO in solution were measured simultaneously using a Clark-type O 2 electrode in combination with an ion-selective electrode for triphenylphosphonium cation (TPP + ). MitoSNO was found to be taken up by isolated mitochondria and inhibited respiration by NO release. This can be seen in Figure 3.
  • the concentration of MitoSNO within mitochondria is ⁇ 5 mM. This represents a 2000 fold concentration relative to the 2.5 ⁇ M present in the external medium, and is consistent with a Nernstian uptake of MitoSNO.
  • MitoSNO released NO within energized mitochondria and inhibited mitochondrial respiration
  • 5 ⁇ M MitoSNO was incubated with a low concentration of rat liver mitochondria (0.5 mg protein/ml) in KCl buffer at 37°C, to enable a longer incubation period in order for sufficient NO release from MitoSNO to affect respiration.
  • 1 ⁇ M additions of MitoSNO were made sequentially before addition of 10 mM succinate. The mitochondria were then allowed to respire until oxygen consumption reached a plateau. At low oxygen concentrations, inhibition of respiration and a concomitant gradual redistribution of MitoSNO, or its derived TPP cations, to the extramitochondrial solution through decreased ⁇ were observed (Figure 4).
  • rat liver mitochondria (1 mg protein /ml) were incubated KCl buffer (as above) supplemented with rotenone (4 ⁇ g/ml) +/- FCCP (0.5 ⁇ M) at 37°C and the oxygen and NO concentrations were measured using appropriate electrodes.
  • MitoSNAP 50 ⁇ M was added followed by succinate (1OmM) which induced a mitochondrial membrane potential leading to the uptake of MitoSNAP inside mitochondria which led to its activation by thiols within mitochondria and to the release of large amounts of NO.
  • This NO led to the inhibition of respiration at cytochrome oxidase and this was reversed by the addition of oxyhemoglobin ( ⁇ 5 ⁇ M) to degrade all accumulated NO to nitrate.
  • MitoSNAP is rapidly accumulated by energized mitochondria, driven by the ⁇ , and once inside the matrix it interacts with GSH and other thiols to produce NO ' and MitoNAP.
  • the NO * thus generated competes with O 2 for the active site of cytochrome c oxidase, reversibly inhibiting respiration.
  • MitoSNAP is a ⁇ -dependent source of intramitochondrial NO ' that can reversibly modulate mitochondrial O 2 consumption, particularly at low O 2 concentrations.
  • the effective K n , of cytochrome c oxidase for O 2 is very low ( ⁇ 1 ⁇ M) (Wikstrom, M., Krab, K. & Saraste, M. Cytochrome oxidase - a synthesis London: Academic press (1981)), illustrated by the sharp transition from maximal O 2 consumption to zero respiration in Fig. 10b. This contrasts with the situation in the presence of MitoSNAP (Fig. 10a) where there is a gradual decline in O 2 consumption rate.
  • MitoSNAP leads to the extensive S-nitrosation of mitochondrial thiol proteins following its ⁇ -dependent uptake into the matrix and this S-nitrosation is predominantly due to the direct transfer OfNO + from MitoSNAP to thiolates within the matrix.
  • MitoSNAP The interaction of MitoSNAP with mitochondrial thiol proteins is likely to initiate a dynamic process that may lead to other protein thiol modifications, such as disulfide formation, glutathionylation, sulfenylation or sulfenylamide formation, in addition to persistent S- nitrosation.
  • other protein thiol modifications such as disulfide formation, glutathionylation, sulfenylation or sulfenylamide formation, in addition to persistent S- nitrosation.
  • the fluorescently-tagged proteins can then be sensitively detected by scanning the fluorescence of the gels.
  • This technique showed that exposure of liver (Fig. lie) or heart (Fig. llf) mitochondria to MitoSNAP led to the ⁇ S-nitrosation of a large number of different proteins.
  • the thiol oxidant diamide did not label proteins, indicating that this technique is selective for SNOs over other thiol oxidative modifications.
  • MitoSNAP S-nitrosates a range of mitochondrial proteins, although only a small proportion of mitochondrial protein thiols are modified. This 5-nitrosation persists in the presence of a reduced mitochondrial glutathione pool.
  • MitoSNAP the major entry point for electrons into the mitochondrial respiratory chain, can be S-nitrosated in vitro and in vivo, and this correlates with potentially important alterations to its activity.
  • MitoSNAP inhibited respiration by about 30% relative to MitoNAP on the complex I-linked substrates glutamate/malate, but had very little effect on respiration with the complex II substrate succinate (Fig. 15a). As this inhibition occurred in the presence of high O 2 concentrations, did not vary as the O 2 concentration decreased and did not affect respiration on succinate, it was not due to NO * inhibition at cytochrome c oxidase (cf. Fig.
  • MitoSNAP is either inhibiting NADH oxidation by complex I or affecting NADH supply.
  • respiration by fragments of heart mitochondrial membranes that directly oxidize both succinate and NADH (Fig. 15b)
  • Glutaredoxin 2 Catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: implications for mitochondrial redox regulation and antioxidant defense. J Biol. Chem. 279, 47939-51 (2004).
  • Results showed that MitoSNAP inhibited NADH respiration by about 35% while succinate was unaffected (Fig. 15b), strongly pointing to a specific effect of MitoSNAP on complex I.
  • MitoSNAP The uptake of MitoSNAP into mitochondria within vascular tissue and the subsequent NO " release may make MitoSNAP a potent vasodilator. This was evaluated by determining whether MitoSNAP could relax the smooth muscle in blood vessel walls. Sections of rat thoracic aorta were mounted in a myograph, pre-contracted and then cumulative amounts of MitoSNAP were added (Fig. 16a). This showed that MitoSNAP was a vasodilator, and that pretreating MitoSNAP to degrade its SNO moiety rendered it ineffective (Fig. 16a).
  • MitoSNAP was a more potent vasodilator than SNAP with an EC50 of 4.5 nM compared to 19.5 nM for SNAP (Fig. 16b). MitoSNAP was also an effective vasodilator in endothelium-denuded rat resistance mesenteric arteries (Fig. 17). These data demonstrate MitoSNAP is an effective endothelium-independent vasodilator acting via NO * release within tissues.
  • MitoSNAP protects against cardiac ischemia-reperfusion injury (ex vivo)
  • IPC ischemic preconditioning
  • MitoSNAP As MitoSNAP is rapidly taken up by mitochondria within cells where it S'-nitrosates mitochondrial thiol proteins, including complex I (Figs. 11 & 15), it may also protect heart mitochondria from I/R damage. Therefore we determined if MitoSNAP could decrease I/R injury in a mouse heart Langendorff model (Fig. 16c & d). When mouse hearts were Langendorff perfused and subjected to 25 min global normothermic ischemia followed by 1 h of normoxic reperfusion there was extensive heart damage, as indicated by the decrease in heart function (rate pressure product, RPP) (Fig. 16c) and by the large infarcted area (Fig. 16d).
  • RPP rate pressure product
  • the small protective effect of MitoNAP may be due to the antioxidant effect of accumulating the N-acetylpenicillamine group, or the TPP cation, in mitochondria.
  • a small degree of protection was also observed when MitoSNAP was delivered before the I/R injury, similar to that observed with the parent compound MitoNAP (Fig. 18).
  • the rapid metabolism of MitoSNAP in cells may be such that pre-treatment does not induce S-nitrosation at the critical time point of reperfusion required for maximal protection and the mild protection seen during pre-treatment is therefore likely to be due to residual antioxidant activity of the N- acetylpenicillamine function on MitoNAP.
  • MitoSNAP protects against cardiac ischemia-reperfusion injury (in vivo)
  • mice were intubated with a 20 gauge PE catheter which was connected to a rodent ventilator (Harvard MiniVent, 120 cycles/min., 0.3 ml tidal volume).
  • a thoracotomy was performed and the left anterior descending coronary artery (LAD) was then ligated and occluded with a 9-0 proline suture for 30 min.
  • MitoSNAP or MitoNAP were dissolved in 0.9% NaCl saline (final volume 50 ⁇ l) and then administered 5 min prior to reperfusion into left ventricle (LV) using 30G'/4 needle. Reperfusion was initiated by removing the suture.
  • the compounds of the invention provide a delivery system for NO directly into the mitochondria.
  • the compounds are selectively taken up by the mitochondria driven by the membrane potential. Selective delivery of NO in the mitochondria allows targeting of local NO-sensitive variables, such as the inhibition of respiration.
  • the compounds of the invention can also S-nitosylate proteins present in the mitochondria. Conventional NO donors produce NO throughout the cell and affect a great number of NO signaling pathways.
  • the compounds of the invention are of use in the treatment of conditions which are affected by mitochondrial respiration such as ischaemia-reperfusion injury. They are also able to inhibit angiogenesis and therefore are of use in the treatment and prevention of tumour growth and/or metastasis.

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Abstract

L'invention porte sur des composés thionitrites de triphénylphosphonium qui acheminent sélectivement de l'oxyde nitrique (NO) vers les mitochondries. Les composés de l'invention comprennent un cation triphénylphosphonium lipophile lié à une fraction thionitrite par l'intermédiaire d'un groupe de liaison et un anion pharmaceutiquement acceptable. Les composés sont utiles dans le traitement de maladies affectées par l'indication d'une activité de NO telles que des cancers, des affections cardiaques, des troubles cognitifs et un accident vasculaire cérébral.
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CZ307146B6 (cs) * 2015-03-31 2018-02-07 Kkcg Se Trifenylfosfoniové analogy biguanidu, způsob jejich přípravy a jejich použití jako léčiva
US9919049B2 (en) 2014-06-02 2018-03-20 University Of Exeter Combinations of a photosensitizer with a hydrogen sulfide donor, thioredoxin inhibitor or nitroxide for use in photodynamic therapy
US10058100B2 (en) 2011-09-30 2018-08-28 The University Of Exeter Hydrogen sulfide releasing compounds and their use

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10058100B2 (en) 2011-09-30 2018-08-28 The University Of Exeter Hydrogen sulfide releasing compounds and their use
US9919049B2 (en) 2014-06-02 2018-03-20 University Of Exeter Combinations of a photosensitizer with a hydrogen sulfide donor, thioredoxin inhibitor or nitroxide for use in photodynamic therapy
US10149907B2 (en) 2014-06-02 2018-12-11 University Of Exeter Combinations of a photosensitizer with a hydrogen sulfide donor, thioredoxin inhibitor or nitroxide for use in photodynamic therapy
CZ307146B6 (cs) * 2015-03-31 2018-02-07 Kkcg Se Trifenylfosfoniové analogy biguanidu, způsob jejich přípravy a jejich použití jako léčiva

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