NOVEL CELL-PERMEABLE SUCCINATE COMPOUNDS

Abstract

The present invention provides novel cell-permeable succinates and cell permeable precursors of succinate aimed at increasing ATP-production in mitochondria. The main part of ATP produced and utilized in the eukaryotic cell originates from mitochondrial oxidative phosphorylation, a process to which high-energy electrons are provided by the Kreb's cycle. Not all Kreb's cycle intermediates are readily permeable to the cellular membrane, one of them being succinate. The provision of the novel cell permeable succinates is envisaged to allow passage over the cellular membrane and thus the cell permeable succinates can be used to enhance mitochondrial ATP-output.

Claims

1. A method for treating or preventing metabolic diseases, treating diseases of mitochondrial dysfunction or disease related to mitochondrial dysfunction, treating or suppressing of mitochondrial disorders, stimulating mitochondrial energy production, treating cancer, hypoxia, ischemia, stroke, myocardial infarction, acute angina, an acute kidney injury, coronary occlusion or atrial fibrillation, or avoiding or counteracting reperfusion injuries, said method comprising administering to a subject a compound of Formula (I) ##STR00111## or a pharmaceutically acceptable salt thereof, wherein the dotted bond between A and B denotes an optional bond so as to form a ring closed structure, and wherein Z is selected from —CH.sub.2—CH.sub.2— or >CH(CH.sub.3), A is selected from —SR, —OR and NHR, and R is ##STR00112## B is selected from —O—R′, —NHR″, —SR′″ or —OH; and R′ is selected from the formulas below: ##STR00113## R′, R″ and R′″ are independently different or identical and is selected from the formulas below: ##STR00114## R.sub.1 and R3 are independently different or identical and are selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH.sub.2Xalkyl, CH.sub.2X-acyl, F, CH.sub.2COOH, CH.sub.2CO.sub.2alkyl, X is selected from O, NH, NR.sub.6, S, R.sub.2 is selected from Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, C(O)CH.sub.3, C(O)CH.sub.2C(O)CH.sub.3, C(O)CH.sub.2CH(OH)CH.sub.3, p is an integer and is 1 or 2 R.sub.6 is selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, or formula (II), or formula (VIII) X.sub.5 is selected from —H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, —COOH, —C(═O)XR.sub.6, CONR.sub.1R.sub.3 or is formula ##STR00115## X.sub.7 is selected from R.sub.1, —NR.sub.1R.sub.3, R.sub.9 is selected from H, Me, Et or O.sub.2CCH.sub.2CH.sub.2COXR.sub.8 R.sub.10 is selected from Oacyl, NHalkyl, NHacyl, or O.sub.2CCH.sub.2CH.sub.2COX.sub.6R.sub.8 X.sub.6 is selected from O, NR.sub.8, NR.sub.6R.sub.8, wherein R.sub.6 and R.sub.8 are independently different or identical and are is selected from H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, or formula (II), or formula (VIII), R.sub.11 and R.sub.12 are independently different or identical and are selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, propionyl, benzoyl, —CH.sub.2Xalkyl, —CH.sub.2Xacyl, where X is O, NR.sub.6 or S, R.sub.c and R.sub.d are independently different or identical and are selected from CH.sub.2Xalkyl, CH.sub.2Xacyl, where X=O, NR.sub.6 or S, R.sub.13, R.sub.14 and R.sub.15 are independently different or identical and are selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, —COOH, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH.sub.2Xalkyl; substituents on R13 and R14 or R13 and R15 may bridge to form a cyclic system, R.sub.f, R.sub.g and R.sub.h are independently different or identical and are selected from Xacyl, —CH.sub.2Xalkyl, —CH.sub.2X—acyl and R.sub.9, alkyl is selected from Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acyl is selected from formyl, acetyl, propionyl, isopropionyl, butyryl, tert-butyryl, pentanoyl, benzoyl, acyl and/or alkyl may be optionally substituted, and when the dotted bond between A and B is present, the compound according to formula (I) is ##STR00116## wherein X.sub.4 is selected from —COOH, —C(═O)XR.sub.6, ##STR00117##

2. The method according to claim 1, for preventing or treating drug-induced mitochondrial side-effects.

3. The method according to claim 2, wherein the drug-induced mitochondrial side-effects relates to drug interaction with Complex I.

4. The method according to claim 1, wherein diseases of mitochondrial dysfunction or disease related to mitochondrial dysfunction involve Complex I, II, III or IV deficiency or an enzyme deficiency.

5. The method according to claim 1, wherein the diseases of mitochondrial dysfunction or disease related to mitochondrial dysfunction are selected from the group consisting of Alpers Disease (Progressive Infantile Poliodystrophy), Amyotrophic lateral sclerosis (ALS), Autism, Barth syndrome (Lethal Infantile Cardiomyopathy), Beta-oxidation Defects, Bioenergetic metabolism deficiency, Carnitine-Acyl-Carnitine Deficiency, Carnitine Deficiency, Creatine Deficiency Syndromes, Cerebral Creatine Deficiency Syndromes (CCDS), Guanidinoaceteate Methyltransferase Deficiency (GAMT Deficiency), L-Arginine:Glycine Amidinotransferase Deficiency (AGAT Deficiency), and SLC6A8-Related Creatine Transporter Deficiency (SLC6A8 Deficiency), Co-Enzyme Q10 Deficiency Complex I Deficiency (NADH dehydrogenase (NADH-CoQ reductase deficiency), Complex II Deficiency (Succinate dehydrogenase deficiency), Complex III Deficiency (Ubiquinone-cytochrome c oxidoreductase deficiency), Complex IV Deficiency/COX Deficiency (Cytochrome c oxidase deficiency, Complex V Deficiency (ATP synthase deficiency), COX Deficiency, CPEO (Chronic Progressive External Ophthalmoplegia Syndrome), CPT I Deficiency, CPT II Deficiency, Friedreich's ataxia (FRDA or FA), Glutaric Aciduria Type II, KSS (Kearns-Sayre Syndrome), Lactic Acidosis, LCAD (Long-Chain Acyl-CoA Dehydrogenase Deficiency), LCHAD, Leigh Disease or Syndrome (Subacute Necrotizing Encephalomyelopathy), LHON (Leber's hereditary optic neuropathy), Luft Disease, MCAD (Medium-Chain Acyl-CoA Dehydrogenase Deficiency), MELAS (Mitochondrial Encephalomyopathy Lactic Acidosis and Strokelike Episodes), MERRF (Myoclonic Epilepsy and Ragged-Red Fiber Disease), MIRAS (Mitochondrial Recessive Ataxia Syndrome), Mitochondrial Cytopathy, Mitochondrial DNA Depletion, Mitochondrial Encephalopathy including: Encephalomyopathy and Encephalomyelopathy, Mitochondrial Myopathy, MNGIE (Myoneurogastointestinal Disorder and Encephalopathy, NARP (Neuropathy, Ataxia, and Retinitis Pigmentosa), Neurodegenerative disorders associated with Parkinson's, Alzheimer's or Huntington's disease, Pearson Syndrome, Pyruvate Carboxylase Deficiency, Pyruvate Dehydrogenase Deficiency, POLG Mutations, Respiratory Chain Deficiencies, SCAD (Short-Chain Acyl-CoA Dehydrogenase Deficiency), SCHAD, and VLCAD (Very Long-Chain Acyl-CoA Dehydrogenase Deficiency).

6. The method according to claim 5, wherein the mitochondrial dysfunction or disease related to mitochondrial dysfunction is a complex I dysfunction selected from the group consisting of Leigh Syndrome, Leber's hereditary optic neuropathy (LHON), MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) and MERRF (myoclonic epilepsy with ragged red fibers).

7. The method according to claim 1 having Formula (IA) ##STR00118## or a pharmaceutically acceptable salt thereof, wherein Z is —CH.sub.2—CH.sub.2—, A is selected from —SR, —OR and NHR, and R is ##STR00119## B is selected from —O—R′, —NHR″, —SR′″ or —OH; and R′, R″ and R′″ are independently different or identical and is selected from one or the formulas below: ##STR00120## R.sub.1 and R.sub.3 are independently different or identical and are selected from H, Me, Et, propyl, O-Me, O-Et, O-propyl, X is selected from O, NH, S, p is an integer and is 1, R.sub.6 is selected from H, Me, Et, X.sub.6 is selected from —H, Me, Et, —COOH, —C(═O)XR.sub.6, CONR.sub.1R.sub.3 X.sub.7 is selected from R.sub.1, —NR.sub.1R.sub.3, R.sub.13, R.sub.14 and R.sub.15 are independently different or identical and are selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, —COOH, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH.sub.2Xalkyl, wherein alkyl and acyl are as defined herein before.

8. The method according to claim 1, wherein R.sub.13, R.sub.14 and R.sub.15 are independently different or identical and are selected from H, Me, Et, —COOH.

9. The method according to claim 1, wherein Z is —CH.sub.2CH.sub.2— and A is —SR.

10. The method according to claim 1, wherein Z is —CH.sub.2CH.sub.2—, A is —SR, and B is —OR′, OH or S′″.

11. The method according to claim 1, wherein Z is —CH.sub.2CH.sub.2—, A is —SR, B is —OR′, OH or SR′″, where R′″ is ##STR00121##

12. The method according to claim 1, wherein B is —OR′ and R′ is ##STR00122##

13. The method according to claim 1, wherein Z is —CH.sub.2CH.sub.2—, A is —SR and R is ##STR00123## p is 1, and B is —OR′ and R′ is ##STR00124##

14. The method according to claim 1, wherein X.sub.5 is H and R.sub.13, R.sub.14 and R.sub.15 are H.

15. The method according to claim 1, wherein Z is —CH.sub.2CH.sub.2— and A is SR and B is OH.

16. The method according to claim 1, wherein Z is —CH.sub.2CH.sub.2—, A is NHR, B is OH and R is ##STR00125## and X is S.

17. The method according to claim 1, wherein R and/or R′″ is ##STR00126## and p=1 and X.sub.5 is —H.

18. The method according to claim 1, wherein R and/or R′″ is ##STR00127## and p=1 and X.sub.5 is COXR.sub.6.

19. The method according to claim 1, wherein R and/or R′″ is ##STR00128## and p=1 and X.sub.5 is CONR.sub.1R.sub.3.

20. The method according to claim 1, wherein the compound is selected from the group consisting of: ##STR00129## ##STR00130## ##STR00131## ##STR00132## ##STR00133## ##STR00134##

Description

[0380] The invention is illustrated in the following figures:

[0381] FIG. 1. Schematic figure of evaluation assay for enhancement of mitochondrial energy producing function in complex I inhibited cells. Protocol for evaluating the compounds according to the invention. In the assay, mitochondrial function in intact cells is repressed with the respiratory complex I inhibitor rotenone. Drug candidates are compared with endogenous (non cell-permeable) substrates before and after permeabilization of the plasma membrane to evaluate bioenergetic enhancement or inhibition.

[0382] FIG. 2. Schematic figure of assay for enhancement and inhibition of mitochondrial energy producing function in intact cells. Protocol for evaluating the potency of compounds according to the invention. In the assay, mitochondrial activity is stimulated by uncoupling the mitochondria with the protonophore FCCP. Drug candidates are titrated to obtain the level of maximum convergent (complex I- and complex II-derived) respiration. After rotenone addition, complex II-dependent stimulation is obtained. The complex III-inhibitor Antimycin is added to evaluate non mitochondrial oxygen consumption.

[0383] FIG. 3. Schematic figure of assay for prevention of lactate accumulation in cells exposed to a mitochondrial complex I inhibitor. Protocol for evaluating the potency of compounds according to the invention. In the assay, mitochondrial function in intact cells is repressed with the respiratory complex I inhibitor rotenone. As the cells shift to glycolysis lactate is accumulated in the medium. Drug candidates are compared with endogenous (non cell-permeable) substrates and decreased rate of lactate accumulation indicates restoration of mitochondrial ATP production.

[0384] FIG. 4. Figure of lactate accumulation in an acute metabolic crisis model in pig.

[0385] Lactate accumulation in an acute metabolic crisis model in pig. In the animal model, mitochondrial function is repressed by infusion of the respiratory complex I inhibitor rotenone. As the cells shift to glycolysis lactate is accumulated in the body. Mean arterial lactate concentrations are demonstrated for rotenone and vehicle treated animals at indicated infusion rates. Drug candidates are evaluated in rotenone treated animals and decreased rate of lactate accumulation indicates restoration of mitochondrial ATP production.

[0386] FIGS. 5A-5C Effect of metformin on mitochondrial respiration in permeabilized human peripheral blood mononuclear cells (PBMCs) and platelets. (FIG. 5A) Representative traces of simultaneously measured O.sub.2 consumption of metformin—(1 mM, black trace) or vehicle-treated (H.sub.2O, grey trace) permeabilized PBMCs assessed by applying sequential additions of indicated respiratory complex-specific substrates and inhibitors. The stabilization phase of the traces, disturbances due to reoxygenation of the chamber and complex IV substrate administration have been omitted (dashed lines). Boxes below traces state the respiratory complexes utilized for respiration during oxidation of the given substrates, complex I (CI), complex II (CII) or both (CI+II), as well as the respiratory states at the indicated parts of the protocol. Respiratory rates at three different respiratory states and substrate combinations are illustrated for PBMCs (FIG. 5B) and platelets (FIG. 5C) for control (H.sub.2O) and indicated concentrations of metformin: oxidative phosphorylation capacity supported by complex I substrates (OXPHOS.sub.CI), complex II-dependent maximal flux through the electron transport system (ETS.sub.CII) Following titration of the protonophore FCCP, and complex IV (CIV) capacity. Values are depicted as mean±SEM. *=P<0.05, **=P<0.01 and ***=P<0.001 using one-way ANOVA with Holm-Sidak's multiple comparison method, n=5. OXPHOS=oxidative phosphorylatation. ETS=electron transport system. ROX=residual oxygen concentration.

[0387] FIG. 6 Dose-response comparison of the toxicity displayed by metformin and phenformin on mitochondrial respiratory capacity during oxidative phosphorylation supported by complex I-linked substrates (OXPHOS.sub.CI) in permeabilized human platelets. Rates of respiration are presented as mean±SEM and standard non-linear curve fitting was applied to obtain half maximal inhibitory concentration (IC.sub.50) values for metformin and phenformin. *=P<0.05, **=P<0.01 and ***=P<0.001 compared to control using one-way ANOVA with Holm-Sidak's multiple comparison method, n=5.

[0388] FIGS. 7A-7B Time- and dose-dependent effects of metformin on mitochondrial respiration in intact human platelets. (FIG. 7A) Routine respiration of platelets, i.e. respiration of the cells with their endogenous substrate supply and ATP demand, was monitored during 60 min incubation of indicated concentrations of metformin or vehicle (H2O), which was followed by (FIG. 7B) maximal respiratory capacity induced by titration of the protonophore FCCP to determine maximal flux through the electron transport system (ETS) of the intact cells. Data are expressed as mean±SEM, n=5. *=P<0.05, **=P<0.01 and ***=P<0.001 using one-way ANOVA (b) and two-way ANOVA (a) with Holm-Sidak's post-hoc test.

[0389] FIG. 8 Effect of metformin and phenformin on lactate production and pH in suspensions of intact human platelets. Platelets were incubated in phosphate buffered saline containing glucose (10 mM) for 8 h with either metformin (10 mM, 1 mM), phenformin (0.5 mM), the complex I inhibitor rotenone (2 μM), or vehicle (DMSO, control). (Top) Lactate levels were determined every 2 h (n=5), and (bottom) pH was measured every 4 h (n=4). Data are expressed as mean±SEM. *=P<0.05, **=P<0.01 and ***=P<0.001 using two-way ANOVA with Holm-Sidak's post-hoc test.

[0390] FIG. 9 Human intact thrombocytes (200.Math.10.sup.6/ml) incubated in PBS containing 10 mM glucose. (Left) Cells incubated with 10 mM metformin were treated with either succinate or NV118 in consecutive additions of 250 μM each 30 minutes. Prior to addition of NV118 at time 0 h, cells have been incubated with just metformin or vehicle for 1 h to establish equal initial lactate levels (data not shown). Lactate concentrations were sampled each 30 minutes. (Center) Lactate production was calculated with a non-linear fit regression and 95% confidence intervals for the time lactate curves were calculated. Cells incubated with metformin had a significantly higher production of lactate than control, and succinate additions did not change this. Lactate production was significantly decreased when NV118 was added to the cells incubated with metformin. (Right) Lactate production induced by rotenone could similarly be attenuated by repeated additions of NV118.

[0391] FIG. 10 Human intact thrombocytes (200.Math.10.sup.6/ml) incubated in PBS containing 10 mM glucose. (Left) Cells incubated with 10 mM metformin were treated with either succinate or NV189 in consecutive additions of 250 μM each 30 minutes. Prior to addition of NV189 at time 0 h, cells have been incubated with just metformin or vehicle for 1 h to establish equal initial lactate levels (data not shown). Lactate concentrations were sampled each 30 minutes. (Center) Lactate production was calculated with a non-linear fit regression and 95% confidence intervals for the time lactate curves were calculated. Cells incubated with metformin had a significantly higher production of lactate than control, and succinate additions did not change this. Lactate production was significantly decreased when NV189 was added to the cells incubated with metformin. (Right) Lactate production induced by rotenone could similarly be attenuated by repeated additions of NV189. When antimycin also was added, the effect of NV189 on complex 2 was abolished by antimycin's inhibitory effect on complex III.

[0392] FIG. 11 Human intact thrombocytes (200.Math.10.sup.6/ml) incubated in PBS containing 10 mM glucose. (Left) Cells incubated with 10 mM metformin were treated with either succinate or NV241 in consecutive additions of 250 μM each 30 minutes. Prior to addition of NV241 at time 0 h, cells have been incubated with just metformin or vehicle for 1 h to establish equal initial lactate levels (data not shown). Lactate concentrations were sampled each 30 minutes. (Center) Lactate production was calculated with a non-linear fit regression and 95% confidence intervals for the time lactate curves were calculated. Cells incubated with metformin had a significantly higher production of lactate than control, and succinate additions did not change this. Lactate production was significantly decreased when NV241 was added to the cells incubated with metformin. (Right) Lactate production induced by rotenone could similarly be attenuated by repeated additions of NV241.

[0393] FIGS. 12A-12B Thrombocytes (200.Math.10.sup.6/ml) incubated in PBS containing 10 mM of glucose with sampling of lactate concentrations every 30 minutes. (FIG. 12A) During 3 hour incubation, cells treated with either rotenone (2 μM) or its vehicle is monitored for change in lactate concentration in media over time. Also, cells were incubated with rotenone together with NV189 and cells with rotenone, NV189 and the complex Ill inhibitor antimycin (1 μg/mL) are monitored. Prior to addition of NV189 at time 0 h, cells have been incubated with just rotenone or vehicle for 1 h to establish equal initial lactate levels (data not shown). Rotenone increase the lactate production of the cells, but this is brought back to normal (same curve slope) by co-incubation with NV189 (in consecutive additions of 250 μM each 30 minutes). When antimycin also is present, NV189 cannot function at complex II level, and lactate production is again increased to the same level as with only rotenone present. (FIG. 12B) A similar rate of lactate production as with rotenone can be induced by incubation with Metformin at 10 mM concentration.

EXPERIMENTAL

[0394] General Biology Methods

[0395] A person of skill in the art will be able to determine the pharmacokinetics and bioavailability of the compound of the invention using in vivo and in vitro methods known to a person of skill in the art, including but not limited to those described below and in Gallant-Haidner et al, 2000 and Trepanier et al, 1998 and references therein. The bioavailability of a compound is determined by a number of factors, (e.g. water solubility, cell membrane permeability, the extent of protein binding and metabolism and stability) each of which may be determined by in vitro tests as described in the examples herein, it will be appreciated by a person of skill in the art that an improvement in one or more of these factors will lead to an improvement in the bioavailability of a compound. Alternatively, the bioavailability of the compound of the invention may be measured using in vivo methods as described in more detail below, or in the examples herein.

[0396] In order to measure bioavailability in vivo, a compound may be administered to a test animal (e.g. mouse or rat) both intraperitoneally (i.p.) or intravenously (i.v.) and orally (p.o.) and blood samples are taken at regular intervals to examine how the plasma concentration of the drug varies over time. The time course of plasma concentration over time can be used to calculate the absolute bioavailability of the compound as a percentage using standard models. An example of a typical protocol is described below.

[0397] For example, mice or rats are dosed with 1 or 3 mg/kg of the compound of the invention i.v. or 1, 5 or 10 mg/kg of the compound of the invention p.o. Blood samples are taken at 5 min, 15 min, 1 h, 4 h and 24 h intervals, and the concentration of the compound of the invention in the sample is determined via LCMS-MS. The time-course of plasma or whole blood concentrations can then be used to derive key parameters such as the area under the plasma or blood concentration-time curve (AUC—which is directly proportional to the total amount of unchanged drug that reaches the systemic circulation), the maximum (peak) plasma or blood drug concentration, the time at which maximum plasma or blood drug concentration occurs (peak time), additional factors which are used in the accurate determination of bioavailability include: the compound's terminal half-life, total body clearance, steady-state volume of distribution and F %. These parameters are then analysed by non-compartmental or compartmental methods to give a calculated percentage bioavailability, for an example of this type of method see Gallant-Haidner et al, 2000 and Trepanier et al, 1998, and references therein.

[0398] The efficacy of the compound of the invention may be tested using one or more of the methods described below:

[0399] I. Assays for Evaluating Enhancement and Inhibition of Mitochondrial Energy Producing Function in Intact Cells

[0400] High Resolution Respirometry—A—General Method

[0401] Measurement of mitochondrial respiration is performed in a high-resolution oxygraph (Oxygraph-2k, Oroboros Instruments, Innsbruck, Austria) at a constant temperature of 37° C. Isolated human platelets, white blood cells, fibroblasts, human heart muscle fibers or other cell types containing live mitochondria are suspended in a 2 mL glass chamber at a concentration sufficient to yield oxygen consumption in the medium of ≥10 pmol O.sub.2 s.sup.−1 mL.sup.−1.

[0402] High-Resolution Respirometry—B (Used in Lactate Studies)

[0403] Real-time respirometric measurements were performed using high-resolution oxygraphs (Oxygraph-2k, Oroboros Instruments, Innsbruck, Austria). The experimental conditions during the measurements were the following: 37° C., 2 mL active chamber volume and 750 rpm stirrer speed. Chamber concentrations of O.sub.2 were kept between 200-50 μM with reoxygenation of the chamber during the experiments as appropriate (Sjövall et al., 2013a). For data recording, DatLab software version 4 and 5 were used (Oroboros Instruments, Innsbruck, Austria). Settings, daily calibration and instrumental background corrections were conducted according to the manufacturer's instructions. Respiratory measurements were performed in either a buffer containing 0.5 mM EGTA, 3 mM MgCl.sub.2, 60 mM K-lactobionate, 20 mM Taurine, 10 mM KH.sub.2PO.sub.4, 20 mM HEPES, 110 mM sucrose and 1 g/L bovine serum albumin (MiR05) or phosphate buffered saline (PBS) with glucose (5 mM) and EGTA (5 mM), as indicated in the corresponding sections. Respiratory values were corrected for the oxygen solubility factor both media (0.92) (Pesta and Gnaiger, 2012). Lactate production of intact human platelets was determined in PBS containing 10 mM glucose. All measurements were performed at a platelet concentration of 200×10.sup.6 cells per mL or a PBMC concentration of 5×10.sup.6 cells per mL.

[0404] Evaluation of Compounds

[0405] Four Typical Evaluation Protocols in Intact Cells are Utilized.

[0406] (1) Assay for Enhancement of Mitochondrial Energy Producing Function in Cells with Inhibited Respiratory Complex I

[0407] Cells are placed in a buffer containing 110 mM sucrose, HEPES 20 mM, taurine 20 mM, K-lactobionate 60 mM, MgCl.sub.2 3 mM, KH.sub.2PO.sub.4 10 mM, EGTA 0.5 mM, BSA 1 g/l, pH 7.1. After baseline respiration with endogenous substrates is established, complex I is inhibited with Rotenone 2 μM. Compounds dissolved in DMSO are titrated in a range of 10 μM to 10 mM final concentration. Subsequently, cell membranes are permeabilised with digitonin (1 mg/1*10.sup.6 plt) to allow entry of extracellularly released energy substrate or cell impermeable energy substrates. After stabilized respiration, Succinate 10 mM is added as a reference to enable respiration downstream of complex I. After the respiration stabilized the experiment is terminated by addition of Antimycin at final concentration 1 μg/mL and any residual non-mitochondrial oxygen consumption is measured. An increase in respiration rate in the described protocol is tightly coupled to ATP synthesis by oxidative phosphorylation unless cells are uncoupled (i.e. proton leak without production of ATP). Uncoupling is tested for by addition of the ATP synthase inhibitor oligomycin (1-2 μg mL.sup.−1) in a protocol 3 where the extent of uncoupling corresponds to the respiratory rate following oligomycin addition.

[0408] (2) Assay for Enhancement and Inhibition of Mitochondrial Energy Producing Function in Intact Cells

[0409] In the second protocol the same buffer is used as described above. After basal respiration is established, the mitochondrial uncoupler FCCP is added at a concentration of 2 nM to increase metabolic demand. Compounds dissolved in DMSO are titrated in several steps from 10 μM to 10 mM final concentration in order to evaluate concentration range of enhancement and/or inhibition of respiration. The experiment is terminated by addition of 2 μM Rotenone to inhibit complex I, revealing remaining substrate utilization downstream of this respiratory complex, and 1 μg/mL of the complex III inhibitor Antimycin to measure non-mitochondrial oxygen consumption.

[0410] (3) Assay to Assess Uncoupling in Intact Cells

[0411] In the third protocol, the same buffer as described above is used. After basal respiration is established, 1 mM of compound dissolved in DMSO is added. Subsequently, the ATP-synthase-inhibitor Oligomycin is added. A reduction in respiration is a measure of how much of the oxygen consumption that is coupled to ATP synthesis. No, or only a slight, reduction indicate that the compound is inducing a proton leak over the inner mitochondrial membrane. The uncoupler FCCP is then titrated to induce maximum uncoupled respiration. Rotenone (2 μM) is then added to inhibit complex I, revealing remaining substrate utilization downstream of this respiratory complex. The experiment is terminated by the addition of 1 μg/mL of the complex III inhibitor Antimycin to measure non-mitochondrial oxygen consumption.

[0412] (4) Assay for Enhancement of Mitochondrial Energy Producing Function in Cells with Inhibited Respiratory Complex I in Human Plasma

[0413] Intact human blood cells are incubated in plasma from the same donor. After baseline respiration with endogenous substrates is established, complex I is inhibited with Rotenone 2 μM. Compounds dissolved in DMSO are titrated in a range of 10 μM to 10 mM final concentration. The experiment is terminated by addition of Antimycin at final concentration 1 μg/mL and any residual non-mitochondrial oxygen consumption is measured.

[0414] Properties of Desired Compound in Respiration Assays

[0415] The ideal compound stimulates respiration in the described protocols in intact cells at low concentration without inhibitory effect on either succinate stimulated respiration after permeabilization in protocol 1 or the endogenous respiration in protocol 2. The concentration span between maximal stimulatory effect and inhibition should be as wide as possible. After inhibition of respiration with mitochondrial toxins at or downstream of complex III, respiration should be halted. Please refer to FIG. 1 and the listing below.

[0416] Desired properties of compounds: [0417] maximum value of a reached at low drug concentration. [0418] a substantially more than a″ [0419] a approaches b′ [0420] c approaches c′ [0421] d approaches d′

[0422] Compounds impermeable to the cellular membrane are identified in the assay as: [0423] a approaches a′

[0424] Non mitochondrial oxygen consumption induced by drug candidate is identified when [0425] d more than d′

[0426] II. Assay for Prevention of Lactate Accumulation in Cells Exposed to a Mitochondrial Complex I Inhibitor

[0427] Intact human platelets, white blood cells, fibroblasts, or other cell types containing live mitochondria are incubated in phosphate buffered saline containing 10 mM glucose for 8 h with either of the complex I inhibiting drugs metformin (10 mM), phenformin (0.5 mM) or rotenone (2 μM). The inhibition of mitochondrial ATP production through oxidative phosphorylation by these compounds increases lactate accumulation by glycolysis. Lactate levels are determined every 2 h (or more frequent eg every 30 min) using the Lactate Pro™ 2 blood lactate test meter (Arkray, Alere AB, Lidingö, Sweden) or similar types of measurements. Incubation is performed at 37° C. pH is measured at start, after 4 and after 8 h (or more frequently) of incubation using a Standard pH Meter, e.g. PHM210 (Radiometer, Copenhagen, Denmark). Drug candidates are added to the assay from start or following 30-60 min at concentrations within the range 10 μM-5 mM. The prevention of lactate accumulation is compared to parallel experiments with compound vehicle only, typically DMSO. In order to evaluate the specificity of the drug candidate, it is also tested in combination with a down-stream inhibitor of respiration such as the complex III inhibitor Antimycin at 1 μg/mL, which should abolish the effect of the drug candidate and restore the production of lactate. The use of antimycin is therefore also a control for undue effects of drug candidates on the lactate producing ability of the cells used in the assay. (See e.g. FIGS. 9, 10 and 11).

[0428] Data Analysis

[0429] Statistical analysis was performed using Graph Pad PRISM software (GraphPad Software version 6.03, La Jolla, Calif., USA). All respiratory, lactate and pH data are expressed as mean±SEM. Ratios are plotted as individuals and means. One-way ANOVA was used for one-factor comparison of three or more groups (concentration of drugs) and two-way mixed model ANOVA was used for two-factor comparison (time and concentration of drugs/treatment) of three or more groups. Post-hoc tests to compensate for multiple comparisons were done according to Holm-Sidak. Correlations were expressed as r.sup.2 and P-values. Standard non-linear curve fitting was applied to calculate half maximal inhibitory concentration (IC.sub.50) values. Results were considered statistically significant for P<0.05.

[0430] Properties of Desired Compound in Cellular Lactate Accumulation Assay [0431] (1) The ideal compound prevents the lactate accumulation induced by complex I inhibition, i.e. the lactate accumulation approaches a similar rate as that in non complex I-inhibited cells. (2) The prevention of lactate accumulation is abolished by a downstream respiratory inhibitor such as Antimycin.

[0432] III. Assay for Prevention of Lactate Accumulation and Energetic Inhibition in an Acute Metabolic Crisis Model in Pig

[0433] Lead drug candidates will be tested in a proof of concept in vivo model of metabolic crisis due to mitochondrial dysfunction at complex I. The model mimics severe conditions that can arise in children with genetic mutations in mitochondrial complex I or patients treated and overdosed with clinically used medications such as metformin, which inhibits complex I when accumulated in cells and tissues.

[0434] Female landrace pigs are used in the study. They are anaesthetized, taken to surgery in which catheters are placed for infusions and monitoring activities. A metabolic crisis is induced by infusion of the mitochondrial complex I inhibitor rotenone at a rate of 0.25 mg/kg/h during 3 h followed by 0.5 mg/kg/h infused during one hour (vehicle consisting of 25% NM P/4% polysorbate 80/71% water). Cardiovascular parameters such as arterial blood pressure is measured continuously through a catheter placed in the femoral artery. Cardiac output (CO) is measured and recorded every 15 minutes by thermo-dilution, and pulmonary artery pressure (PA, systolic and diastolic), central venous pressure (CVP), and SvO.sub.2 is recorded every 15 min and pulmonary wedge pressure (PCWP) every 30 min from a Swan-Ganz catheter. Indirect calorimetry is performed e.g. by means of a Quark RMR ICU option (Cosmed, Rome, Italy) equipment. Blood gases and electrolytes are determined in both arterial and venous blood collected from the femoral artery and Swan-Ganz catheters and analysed with use of an ABL725 blood gas analyser (Radiometer Medical Aps, Brønshøj, Denmark). Analyses include pH, BE, Hemoglobin, HCO.sub.3, pO.sub.2, pCO.sub.2, K.sup.+, Na.sup.+, Glucose and Lactate.

[0435] Properties of Desired Compound in a Proof of Concept In Vivo Model of Metabolic Crisis

[0436] The ideal compound should reduce the lactate accumulation and pH decrease in pigs with metabolic crisis induced by complex I inhibition. The energy expenditure decrease following complex I inhibition should be attenuated. The compound should not induce any overt negative effects as measured by blood and hemodynamic analyses.

[0437] Metabolomics Method

[0438] White blood cells or platelets are collected by standard methods and suspended in a MiR05, a buffer containing 110 mM sucrose, HEPES 20 mM, taurine 20 mM, K-lactobionate 60 mM, MgCl.sub.2 3 mM, KH.sub.2PO.sub.4 10 mM, EGTA 0.5 mM, BSA 1 g/I, with or without 5 mM glucose, pH 7.1. The sample is incubated with stirring in a high-resolution oxygraph (Oxygraph-2k, Oroboros Instruments, Innsbruck, Austria) at a constant temperature of 37° C.

[0439] After 10 minutes rotenone in DMSO is added (2 μM) and incubation continued. Following a further 5 minutes test compound in DMSO is added, optionally with further test compound after and a further period of incubation. During the incubation O.sub.2 consumption is measured in real-time.

[0440] At the end of the incubation the cells are collected by centrifugation and washed in 5% mannitol solution and extracted into methanol. An aqueous solution containing internal standard is added and the resultant solution treated by centrifugation in a suitable microfuge tube with a filter.

[0441] The resulting filtrate is dried under vacuum before CE-MS analysis to quantify various primary metabolites by the method of Ooga et al (2011) and Ohashi et al (2008).

[0442] In particular the levels of metabolite in the TCA cycle and glycolysis are assessed for the impact of compounds of the invention.

[0443] Ooga et al, Metabolomic anatomy of an animal model revealing homeostatic imbalances in dyslipidaemia, Molecular Biosystems, 2011, 7, 1217-1223 Ohashi et al, Molecular Biosystems, 2008, 4, 135-147

[0444] Materials

[0445] Unless otherwise indicated, all reagents used in the examples below are obtained from commercial sources.

EXAMPLES

Example 1

[0446] ##STR00038##

[0447] Succinyl chloride (0.1 mol) and triethylamine (0.4 mol) is dissolved in DCM and cysteine is added. The reaction is stirred at room temperature. The reaction is added to aqueous dilute hydrochloric acid and then is washed water and brine. The organic layers are dried over magnesium sulfate and reduced in vacuo. The target compound is the purified by silica gel chromatography.

Example 2—Synthesis of S,S-bis(2-propionamidoethyl) butanebis(thioate) (NV038, 01-038)

[0448] ##STR00039##

[0449] To a solution of cysteamine hydrochloride (5.0 g, 44 mmol) in CH.sub.3OH (50 mL) was added Et.sub.3N (4.4 g, 44 mmol), followed by (Boc).sub.2O (10.5 g, 48.4 mmol) and the mixture was stirred at room temperature for 1h. The reaction mixture was concentrated in vacuo. The obtained residue was dissolved in CH.sub.2Cl.sub.2, washed with 2M HCl aqueous solution and brine, dried over Na.sub.2SO.sub.4, filtered and evaporated to yield tert-butyl 2-mercaptoethylcarbamate as a colorless oil which was used in the next step without further purification.

##STR00040##

[0450] tert-Butyl 2-mercaptoethylcarbamate (9.8 g, 55.0 mmol) and Et.sub.3N (5.6 g, 55.0 mmol) were dissolved in CH.sub.2Cl.sub.2 (100 mL), the mixture cooled to 0° C., succinyl chloride (2.1 g, 13.8 mmol) was added with dropwise. Then the mixture was stirred at room temperature for 2h. The reaction mixture concentrated and the residue was purified by column chromatography (petrol ether/EtOAc=1/10 to 1/1). S,S-bis(2-(tert-butoxycarbonylamino)ethyl) butanebis(thioate) was obtained as a white solid.

##STR00041##

[0451] A mixture of S,S-bis(2-(tert-butoxycarbonylamino)ethyl) butanebis(thioate) (2.0 g, 4.58 mmol) and TFA (10 mL) in CH.sub.2Cl.sub.2 (10 mL) was stirred at room temperature for 4 hours. The reaction mixture was concentrated to yield S,S-bis(2-aminoethyl) butanebis(thioate) as a yellow oil which was used in the next step without further purification.

##STR00042##

[0452] S,S-bis(2-aminoethyl) butanebis(thioate) (1.1 g, 4.58 mmol) and Et.sub.3N (1.4 g, 13.74 mmol) were dissolved in CH.sub.2Cl.sub.2 (15 mL), the mixture cooled to 0° C., propionyl chloride (0.9 g, 10.07 mmol) was added with dropwise. Then the mixture was stirred at room temperature for 3 hours. The reaction mixture concentrated and the residue was purified by preparative TLC (CH.sub.2Cl.sub.2/MeOH=15/1). S,S-bis(2-Propionamidoethyl) butanebis(thioate) was obtained as a white solid.

Example 3—synthesis of (R)-4-(2-carboxy-2-propionamidoethylthio)-4-oxobutanoic acid (NV-041, 01-041)

[0453] ##STR00043##

[0454] To a mixture of L-cysteine (2.00 g, 16.5 mmol) in THF/H.sub.2O (8 mL/2 mL) was added NaOAc (2.70 g, 33.0 mmol). The mixture was stirred at room temperature for 20 min. The reaction was cooled to 5° C. before propionic anhydride (2.30 g, 17.6 mmol) was added dropwise. The reaction mixture was stirred at room temperature overnight and then heated to reflux for 4 hours. The reaction mixture was cooled and acidified to pH 5 by adding 4N HCl. The resulting solution was evaporated under reduced pressure to remove THF. The residue was purified by prep-HPLC (eluting with H.sub.2O (0.05% TFA) and CH.sub.3CN) to give 1.00 g of (R)-3-mercapto-2-propionamidopropanoic acid as colourless oil.

##STR00044##

[0455] A solution of (R)-3-mercapto-2-propionamidopropanoic acid (1.00 g, 5.65 mmol), succinic anhydride (565 mg, 5.65 mmol) and Et.sub.3N (572 mg, 5.65 mmol) in 10 mL of THF was heated under reflux overnight. The reaction mixture concentrated and the residue was purified by preparative-HPLC (eluting with H.sub.2O (0.05% TFA) and CH.sub.3CN) to yield (R)-4-(2-carboxy-2-propionamidoethylthio)-4-oxobutanoic acid as a colourless oil.

Example 4

[0456] ##STR00045##

[0457] Step 1

[0458] Triethylamine (0.24 mol) is added to a solution of N-acetylcysteamine (0.2 mol) in DCM. 4-Chloro-4-oxobutanoic acid (0.1 mol) is added dropwise, and the reaction mixture is stirred at room temperature. The mixture is added to aqueous dilute hydrochloric acid and is extracted with ethyl acetate, and then is washed water and brine. The organic layers are dried over magnesium sulfate and reduced in vacuo.

[0459] Step 2

[0460] The product of step 3 (0.1 mol), acetic acid 1-bromoethyl ester (0.1 mol) and caesium carbonate (0.12 mol) is suspended in DMF and stirred at 60° C. under an inert atmosphere. The suspension is allowed to cool to room temperature and ethyl acetate added and is washed successively with aqueous dilute hydrochloric acid and water. The organics are dried over magnesium sulfate and reduced in vacuo. The residue is purified by column chromatography.

Example 5

[0461] ##STR00046##

[0462] Step 1

[0463] Triethylamine (0.24 mol) is added to a solution of N-acetylcysteamine (0.2 mol) in DCM. 4-Chloro-4-oxobutanoic acid (0.1 mol) is added dropwise, and the reaction mixture is stirred at room temperature. The mixture is added to aqueous dilute hydrochloric acid and is extracted with ethyl acetate, and then is washed water and brine. The organic layers are dried over magnesium sulfate and reduced in vacuo.

[0464] Step 2

[0465] Dimethylamine (0.1 mol) and triethylamine (0.1 mol) are diluted in dichloromethane, the solution is cooled to 0° C. and 2-chloropropionyl chloride (0.1 mol) in DCM is added and the solution is allowed to warm to room temperature and is left to stir under an inert atmosphere. The solution is washed with water. The organics are combined and the volatiles are removed in vacuo. The residue is purified by silica gel chromatography.

[0466] Step 3

[0467] 2-Chloro-N,N-dimethyl-propionamide (0.1 mol), the product of step 1 (0.1 mol), caesium carbonate (0.1 mol), and sodium iodide (0.01 mol) is suspended in DMF and the suspension stirred at 80° C. under an inert atmosphere. The suspension is cooled to room temperature, is diluted with ethyl acetate and is washed with water. The organics are reduced in vacuo. The residue is purified by silica gel chromatography to yield the target compound.

Example 6—synthesis of 4-oxo-4-(2-propionamidoethylthio)butanoic acid (NV114, 01-114)

[0468] ##STR00047##

[0469] Propionic anhydride (11.7 g, 89.7 mmol) and aqueous KOH (8 M, to maintain pH=8) were added dropwise to a stirred solution of cysteamine hydrochloride (3.40 g, 30.0 mmol) in 24 mL of water. The mixture was neutralized by adding 2N HCl and stirred for 1 hour at room temperature. The solution was cooled with an ice bath and solid KOH (6.00 g, 105 mmol) was added slowly. The mixture was stirred for 50 minutes at room temperature. After saturated with NaCl and neutralized with 6N HCl, the mixture was extracted with CH.sub.2Cl.sub.2 (4×30 mL). The combined CH.sub.2Cl.sub.2 extracts were dried (Na.sub.2SO.sub.4) and concentrated in vacuo to give N-(2-mercaptoethyl)propionamide as colourless oil, which was used for next step without further purification.

##STR00048##

[0470] A solution of N-(2-mercaptoethyl)propionamide (2.00 g, 15.0 mmol), succinic anhydride (1.50 g, 15.0 mmol) and Et.sub.3N (1.50 g, 15.0 mmol) in 20 mL of THF was heated under reflux overnight. The reaction mixture was concentrated and the residue was purified by preparative-HPLC (eluting with H.sub.2O (0.05% TFA) and CH.sub.3CN) to yield 4-oxo-4-(2-propionamidoethylthio)butanoic acid as colourless oil.

Example 7—synthesis of 4-(2-acetamidoethylthio)-4-oxobutanoic acid (NV108, 01-108)

[0471] ##STR00049##

[0472] Acetic anhydride (8.48 mL, 90.0 mmol) and aqueous KOH (8 M, to maintain pH=8) were added dropwise to a stirred solution of cysteamine hydrochloride (3.40 g, 30.0 mmol) in 24 mL of water. The pH was then adjusted to 7 with adding 2N HCl. The mixture was stirred for 1 hour at room temperature, and then the solution was cooled with an ice bath. To the above solution, solid KOH (6.0 g, 105 mmol) was added slowly, and the resulting mixture was stirred for 50 minutes at room temperature. After saturated with NaCl and neutralized with 6N HCl, the mixture was extracted with CH.sub.2Cl.sub.2 (4×30 mL). The combined CH.sub.2Cl.sub.2 extracts were dried (Na.sub.2SO.sub.4) and concentrated in vacuo to give N-(2-mercaptoethyl)acetamide as colourless oil, which was used for next step without further purification.

##STR00050##

[0473] A solution of N-(2-mercaptoethyl)acetamide (1.50 g, 12.7 mmol), succinic anhydride (1.3 g, 12.7 mmol) and Et.sub.3N (1.3 g, 12.7 mmol) in 20 mL of THF was heated under reflux overnight. The reaction mixture was concentrated and the residue was purified by preparative HPLC (eluting with H.sub.2O (0.05% TFA) and CH.sub.3CN) to yield 4-(2-acetamidoethylthio)-4-oxobutanoic acid as colourless oil.

Example 8—The synthesis of (R)-3-(4-((R)-2-carboxy-2-propionamidoethylthio)-4-oxobutanoylthio)-2-propionamidopropanoic acid (NV099, 01-099)

[0474] ##STR00051##

[0475] To a mixture of N-hydroxysuccinimide (3.00 g, 26.1 mmol) and Et.sub.3N (3.20 g, 31.3 mmol) in CH.sub.2Cl.sub.2 (60 mL) was added dropwise succinyl chloride (2.00 g, 13.0 mmol). The mixture was stirred at room temperature for 3 hours before diluted with water (60 mL). The resulting suspension was filtered, washed with water and CH.sub.2Cl.sub.2. The cake was collected and dried to give bis(2,5-dioxopyrrolidin-1-yl) succinate as a grey solid.

##STR00052##

[0476] A mixture of N-(2-mercaptoethyl)propionamide (400 mg, 2.26 mmol), bis(2,5-dioxopyrrolidin-1-yl) succinate (353 mg, 1.13 mmol) and TEA (286 mg, 2.83 mmol) in 3.0 mL of CH.sub.3CN was stirred at room temperature for 2 hours. The clear reaction solution was purified by preparative-HPLC (eluting with H.sub.2O (0.05% TFA) and CH.sub.3CN) directly to yield (R)-3-(4-((R)-2-carboxy-2-propionamidoethylthio)-4-oxobutanoylthio)-2-propionamidopropanoic acid as colorless oil.

Example 9—Synthesis of (R)-4-(1-carboxy-2-(propionylthio)ethylamino)-4-oxobutanoic acid (NV122, 01-122)

[0477] ##STR00053##

[0478] To a mixture of (R)-3-mercapto-2-propionamidopropanoic acid (1.00 g, 8.25 mmol) and propionic acid (1.0 mL) in CHCl.sub.3 (10 mL) were added propionic anhydride (1.13 g, 8.67 mmol) dropwise. The reaction mixture was heated to reflux overnight. The reaction mixture was cooled and succinic anhydride (1.00 g, 9.99 mmol) was added. The mixture was refluxed overnight before concentrated under reduced pressure. The residue was purified by prep-HPLC (eluting with H.sub.2O (0.05% TFA) and CH.sub.3CN) to yield (R)-4-(1-carboxy-2-(propionylthio)ethylamino)-4-oxobutanoic acid as an off-white solid.

Example 10—The synthesis of 4-(1-acetamido-2-methylpropan-2-ylthio)-4-oxobutanoic acid (NV188, 01-188)

[0479] ##STR00054##

[0480] To a stirred solution of cysteamine hydrochloride (2.00 g, 14.1 mmol) in 15 mL of water was added acetic anhydride (4.30 g, 42.4 mmol) and aqueous KOH (8 M, to maintain pH=8) dropwise. The mixture was then neutralized by adding 2N HCl and stirred for 1 hour at room temperature. To the solution cooled with an ice bath was added slowly solid KOH (2.80 g, 49.4 mmol) and the mixture was stirred for 50 minutes at room temperature. After saturated with NaCl and neutralized with 6N HCl, the mixture was extracted with CH.sub.2Cl.sub.2 twice. The combined CH.sub.2Cl.sub.2 extracts were dried (Na.sub.2SO.sub.4) and concentrated in vacuo to yield N-(2-mercapto-2-methylpropyl)acetamide as a white solid which was used for next step without further purification.

##STR00055##

[0481] A solution of N-(2-mercapto-2-methylpropyl)acetamide (400 mg, 2.72 mmol), succinic anhydride (326 mg, 3.26 mmol) and Et.sub.3N (330 mg, 3.26 mmol) in 6 mL of THF was heated under overnight. The reaction mixture was concentrated and the residue was purified by preparative-HPLC (eluting with H.sub.2O (0.05% TFA) and CH.sub.3CN) to yield 4-(1-acetamido-2-methylpropan-2-ylthio)-4-oxobutanoic acid as yellow oil.

Example 11—The synthesis of S,S-bis((R)-3-(diethylamino)-3-oxo-2-propionamidopropyl) butanebis(thioate) (NV185, 01-185)

[0482] ##STR00056##

[0483] To a solution of (R)-3-mercapto-2-propionamidopropanoic acid (5.00 g, 28.0 mmol) in DMF (50 mL) was added triphenylmethyl chloride (8.70 g, 31.0 mmol) at 0° C. The mixture was stirred at 0° C. for 30 min and then warmed to room temperature overnight. The mixture was treated with water and extracted with EtOAc twice. The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH.sub.2Cl.sub.2/MeOH=80/1˜50/1) to yield (R)-2-propionamido-3-(tritylthio)propanoic acid as a white solid.

##STR00057##

[0484] To a stirred solution of (R)-2-propionamido-3-(tritylthio)propanoic acid (1.7 g, 4.0 mmol) in CH.sub.2Cl.sub.2 (50 mL) was added DCC (1.7 g, 8.0 mmol) and HOBT (0.50 g. 4.0 mmol) at room temperature. The mixture was stirred at room temperature for 1 h and then diethylamine (0.80 g, 8.0 mmol) was added. The mixture was stirred at room temperature overnight. The mixture was washed with water, dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to give the crude product which was purified by silica gel column chromatography (EtOAc/petrol ether=1/6˜1/1) to yield (R)-N,N-diethyl-3-mercapto-2-propionamidopropanamide as yellow oil.

##STR00058##

[0485] To a solution of (R)-N,N-diethyl-3-mercapto-2-propionamidopropanamide (400 mg, 0.800 mmol) in CH.sub.2Cl.sub.2 (10 mL) at 0° C. was added TFA (1 mL) and i-Pr3SiH (253 mg, 1.60 mmol). The mixture was warmed to room temperature and stirred for 2 hours. The solution was evaporated under reduced pressure. The residue was purified by preparative-HPLC (eluting with H.sub.2O (0.5% TFA) and CH.sub.3CN) to yield (R)-N,N-diethyl-3-mercapto-2-propionamidopropanamide as yellow oil.

##STR00059##

[0486] A mixture of (R)-N,N-diethyl-3-mercapto-2-propionamidopropanamide (150 mg, 0.600 mmol), Et.sub.3N (242 mg, 2.40 mmol) and bis(2,5-dioxopyrrolidin-1-yl) succinate (94 mg, 0.30 mmol) in CH.sub.3CN (100 mL) was stirred at room temperature overnight. The mixture was evaporated under reduced pressure. The residue was purified by preparative HPLC (eluting with H.sub.2O (0.5% TFA) and CH.sub.3CN) to yield S,S-bis((R)-3-(diethylamino)3-oxo-2-propionamidopropyl) butanebis(thioate) (36% yield) as a yellow solid.

Example 12—The synthesis of 4-(2-(2-(diethylamino)-2-oxoethoxy)ethylthio)-4-oxobutanoic acid (NV193, 01-193)

[0487] ##STR00060##

[0488] To a solution of 2-bromoacetyl bromide (4.00 g, 20.0 mmol) and DIPEA (2.60 g, 20 mmol) in CH.sub.2Cl.sub.2 (50 mL) was added dropwise diethylamine (1.60 g, 20.0 mmol) at 0° C. The mixture was stirred at 0° C. for 30 min. The solution was evaporated under reduced pressure to remove CH.sub.2Cl.sub.2. The residue was purified by silica gel column chromatography (EtOAc/petrol ether=1/5˜1/2) to yield 2-bromo-N,N-diethylacetamide as yellow oil.

##STR00061##

[0489] A solution of 2-mercaptoethanol (2.50 g, 32.0 mmol), triphenylmethyl chloride (10.7 g, 38.4 mmol) in 100 mL of THF was heated under reflux overnight. The reaction mixture was concentrated and the residue was purified by silica gel column chromatography (EtOAc/petrol ether=1/5˜1/1) to yield 2-(2,2,2-triphenylethylthio)ethanol as a white solid.

##STR00062##

[0490] To a solution of 2-(2,2,2-triphenylethylthio)ethanol (3.50 g, 10.9 mmol) in THF (30 mL) was added NaH (0.500 g, 13.0 mmol, 60% in oil) in portions at 0° C. The reaction mixture was stirred at 0° C. for 1 hour. Then a solution of 2-bromo-N,N-diethylacetamide (2.1 g, 10.9 mmol) in THF (5 mL) was added dropwise. The resulting mixture was warmed to room temperature over 2 hours. The mixture was quenched with water and extracted with EtOAc twice. The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (EtOAc/petrol ether=1/5˜1/2) to yield N,N-diethyl-2-(2-(tritylthio)ethoxy)acetamide as a white solid.

##STR00063##

[0491] To a solution of N,N-diethyl-2-(2-(tritylthio)ethoxy)acetamide (2.70 g, 6.30 mmol) in CH.sub.2Cl.sub.2 (20 mL) was added TFA (2 mL) and i-Pr.sub.3SiH (2.00 g, 12.6 mmol) at 0° C. The mixture was warmed to room temperature and stirred for 2 hours. The solution was evaporated under reduced pressure to remove CH.sub.2Cl.sub.2. The residue was purified by silica gel column chromatography (EtOAc/petrol ether=1/5˜1/1) to yield N,N-diethyl-2-(2-mercaptoethoxy)acetamide as colorless oil.

##STR00064##

[0492] A solution of N,N-diethyl-2-(2-mercaptoethoxy)acetamide (356 mg, 1.90 mmol), succinic anhydride (200 mg, 2.10 mmol) and Et.sub.3N (300 mg, 2.90 mmol) in 10 mL of THF was stirred at reflux overnight. The reaction mixture was concentrated in vacuo and the residue was purified by preparative HPLC (eluting with H.sub.2O (0.5% TFA) and CH.sub.3CN) to yield 4-(2-(2-(diethylamino)-2-oxoethoxy)ethylthio)-4-oxobutanoic acid as colorless oil.

Example 13—The synthesis of (R)-methyl 3-(4-((R)-3-methoxy-3-oxo-2-propionamidopropylthio)-4-oxobutanoylthio)-2-propionamidopropanoate (NV205, 01-205)

[0493] ##STR00065##

[0494] A mixture of (R)-3-(4-((R)-2-carboxy-2-propionamidoethylthio)-4-oxobutanoylthio)-2-propionamidopropanoic acid (300 mg, 0.69 mmol), CH.sub.3I (293 mg, 2.06 mmol) and K.sub.2CO.sub.3 (475 mg, 3.44 mmol) in 4.0 mL of DMF was stirred at room temperature overnight. The reaction mixture was filtered and the filtrate was purified by preparative-HPLC (eluting with H.sub.2O (0.05% TFA) and CH.sub.3CN) directly to yield (R)-methyl 3-(4-((R)3-methoxy-3-oxo-2-propionamidopropylthio)-4-oxobutanoylthio)-2-propionamidopropanoate as an off-white solid.

Example 14—Synthesis of NV189

[0495] ##STR00066##

[0496] A mixture of N-(2-mercapto-2-methylpropyl)acetamide (400 mg, 2.72 mmol), bis(2,5-dioxopyrrolidin-1-yl) succinate (339 mg, 1.09 mmol) and Et.sub.3N (550 mg, 5.44 mmol) in 6 mL of CH.sub.3CN was stirred at room temperature overnight. The reaction mixture was concentrated and the residue was purified by preparative HPLC (eluting with H.sub.2O (0.05% TFA) and CH.sub.3CN) to yield NV189 as an off-white solid.

Example 15—Synthesis of S,S-bis(2-(2-(diethylamino)-2-oxoethoxy)ethyl) butane-bis(thioate) (NV195, 01-195)

[0497] ##STR00067##

[0498] To a solution of N,N-diethyl-2-(2-mercaptoethoxy)acetamide (438 mg, 2.3 mmol) in CH.sub.3CN (10 mL) was added bis(2,5-dioxopyrrolidin-1-yl) succinate (374 mg, 1.2 mmol) and Et.sub.3N (232 mg, 2.3 mmol). The mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo and the residue was purified by preparative HPLC (eluting with H.sub.2O (0.5% TFA) and CH.sub.3CN) to yield S,S-bis(2-(2-(diethylamino)-2-oxoethoxy)ethyl) butanebis(thioate) as a colorless oil.

Example 16—Synthesis of NV206

[0499] ##STR00068##

[0500] A mixture of (R)-3-(4-((R)-2-carboxy-2-propionamidoethylthio)-4-oxobutanoylthio)-2-propionamidopropanoic acid (400 mg, 0.916 mmol), CH.sub.3I (156 mg, 1.1 mmol) and K.sub.2CO.sub.3 (190 mg, 1.37 mmol) in 4 mL of DMF was stirred at room temperature for 6 hours. The reaction mixture was filtered and the filtrate was purified by prep-HPLC (eluting with H.sub.2O (0.05% TFA) and CH.sub.3CN) directly to yield NV206 as a colorless gum.

Example 17

[0501] Results of Biological Experiments

[0502] The compounds given in the following table were subject to the assays (1)-(4) mentioned under the heading I. Assay for evaluating enhancement and inhibition of mitochondrial energy producing function in intact cells. In the following table the results are shown, which indicate that all compounds tested have suitable properties. Importantly, all compounds show specific effect on CII-linked respiration as seen from screening protocols 1 and 4, as well as a convergent effect, with Cl-substrates available, as seen in assay 2.

[0503] Results from Screening Protocols 1-4

[0504] Compound numbers as set out in Examples 1-16.

TABLE-US-00001 Convergent Convergent CII Comnpound NV (Routine) (FCCP) (plasma) CII Uncoupling Toxicity 01-193 (++) + (+) + + 5 mM 01-188 +++ +++ + + (+) 5 mM 01-185 (+) + + + (+) 2 mM 01-205 +++ ++ + ++ (+) 5 mM 01-114 +++ ++ + ++ (+) 10 mM 01-041 + +++ + ++ (+) 5 mM 01-108 ++ ++ (+) (++) + 10 mM

[0505] Legend: Convergent (Routine)—the increase in mitochondrial oxygen consumption induced by the compound under conditions described in screening assay 3; Convergent (FCCP)—the increase in mitochondrial oxygen consumption induced by the compound under conditions described in screening assay 2 (uncoupled conditions); Convergent (plasma)—the increase in mitochondrial oxygen consumption induced by the compound in cells with inhibited complex I incubated in human plasma, as described in screening assay 4; CII—the increase in mitochondrial oxygen consumption induced by the compound in cells with inhibited complex I as described in screening assay 1; Uncoupling—the level of oxygen consumption after addition of oligomycin as described in screening assay 3. The response in each parameter is graded either +, ++ or +++ in increasing order of potency. Brackets [( )] indicate an intermediate effect, i.e. (+++) is between ++ and +++. Toxicity—the lowest concentration during compound titration at which a decrease in oxygen consumption is seen as described in screening assay 2.

Examples 18-20

[0506] Metformin Studies

[0507] In the metformin study the following compounds were used (and which are referred to in the figures). The compounds are described in WO 2014/053857.

##STR00069##

[0508] Sample Acquisition and Preparation

[0509] The study was performed with approval of the regional ethical review board of Lund University, Sweden (ethical review board permit no. 2013/181). Venous blood from 18 healthy adults (11 males and 7 females) was drawn in K.sub.2EDTA tubes (BD Vacutainer® Brand Tube with dipotassium EDTA, BD, Plymouth, UK) according to clinical standard procedure after written informed consent was acquired. For platelet isolation the whole blood was centrifuged (Multifuge 1 S-R Heraeus, Thermo Fisher Scientifics, Waltham, USA) at 500 g at room temperature (RT) for 10 min. Platelet-rich plasma was collected to 15 mL falcon tubes and centrifuged at 4600 g at RT for 8 min. The resulting pellet was resuspended in 1-2 mL of the donor's own plasma. PBMCs were isolated using Ficol gradient centrifugation (Boyum, 1968). The blood remaining after isolation of platelets was washed with an equal volume of physiological saline and layered over 3 mL of Lymphoprep™. After centrifugation at 800 g at RT (room temperature) for 30 min the PBMC layer was collected and washed with physiological saline. Following a centrifugation at 250 g at RT for 10 min the pellet of PBMCs was resuspended in two parts of physiological saline and one part of the donor's own plasma. Cell count for both PBMCs and platelets were performed using an automated hemocytometer (Swelab Alfa, Boule Medical AB, Stockholm, Sweden).

[0510] Aim of Study Reported in Examples 18-19

[0511] Metformin induces lactate production in peripheral blood mononuclear cells and Platelets Through Specific Mitochondrial Complex I Inhibition

[0512] Metformin is a widely used anti-diabetic drug associated with the rare side-effect of lactic acidosis, which has been proposed to be linked to drug-induced mitochondrial dysfunction. Using respirometry, the aim of the study reported in Examples 1-2 below was to evaluate mitochondrial toxicity of metformin to human blood cells in relation to that of phenformin, a biguanide analog withdrawn in most countries due to a high incidence of lactic acidosis.

[0513] Aim of the Study Reported in Example 20

[0514] The aim is to investigate the ability of succinate prodrugs to alleviate or circumvent undesired effects of metformin and phenformin.

Example 18A

[0515] Effects of Metformin and Phenformin on Mitochondrial Respiration in Permeabilized Human Platelets

[0516] In order to investigate the specific target of biguanide toxicity, a protocol was applied using digitonin permeabilization of the blood cells and sequential additions of respiratory complex-specific substrates and inhibitors in MiR05 medium. After stabilization of routine respiration, i.e. respiration of the cells with their endogenous substrate supply and ATP demand, metformin, phenformin or their vehicle (double-deionized water) were added. A wide concentration range of the drugs was applied; 0.1, 0.5, 1, and 10 mM metformin and 25, 100 and 500 μM phenformin. After incubation with the drugs for 10 min at 37° C., the platelets were permeabilized with digitonin at a previously determined optimal digitonin concentration (1 μg 10.sup.−6 platelets) to induce maximal cell membrane permeabilization without disruption of the mitochondrial function and allowing measurements of maximal respiratory capacities (Sjövall et al. (2013a). For evaluation of complex I-dependent oxidative phosphorylation capacity (OXPHOS.sub.CI) first, the NADH-linked substrates pyruvate and malate (5 mM), then ADP (1 mM) and, at last, the additional complex I substrate glutamate (5 mM) were added sequentially. Subsequently the FADH.sub.2-linked substrate succinate (10 mM) was given to determine convergent complex I- and II-dependent OXPHOS capacity (OXPHOS.sub.CI+II). LEAKI+II state, a respiratory state where oxygen consumption is compensating for the back-flux of protons across the mitochondrial membrane (Gnaiger, 2008), was assessed by addition of the ATP-synthase inhibitor oligomycin (1 μg mL.sup.−1). Maximal uncoupled respiratory electron transport system capacity supported by convergent input through complex I and II (ETS.sub.CI+II) was evaluated by subsequent titration with the protonophore carbonyl-cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP). Addition of the complex I inhibitor rotenone (2 μM) revealed complex II-dependent maximal uncoupled respiration (ETS.sub.CII). The complex III inhibitor antimycin (1 μg mL.sup.−1) was then given to reveal residual oxygen consumption (ROX). Finally, the artificial complex IV substrate N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD, 0.5 mM) was added and the complex IV inhibitor sodium azide (10 mM) was given to measure complex IV activity and chemical background, respectively. Complex IV activity was calculated by subtracting the sodium azide value from the TMPD value. With exception of complex IV activity, all respiratory states were measured at steady-state and corrected for ROX. Complex IV activity was measured after ROX determination and not at steady-state. The integrity of the outer mitochondrial membrane was examined by adding cytochrome c (8 μM) during OXPHOS.sub.CI+II in presence of vehicle, 100 mM metformin or 500 μM phenformin.

Example 18B

[0517] Effect of Metformin on Mitochondrial Respiration in Permeabilized Human Peripheral Blood Mononuclear Cells and on Mitochondrial Respiration in Intact Human Platelets

[0518] For analysis of respiration of permeabilized PBMCs in response to metformin (0.1, 1 and 10 mM) the same protocol as for permeabilized platelets was used, except the digitonin concentration was adjusted to 6 μg 10.sup.−6 PBMCs (Sjövall et al., 2013b).

[0519] Results

[0520] Respiration using complex I substrates was dose-dependently inhibited by metformin in both permeabilized human PBMCs and platelets (FIG. 1). OXPHOS.sub.CI capacity decreased with increasing concentrations of metformin compared to controls with near complete inhibition at 10 mM (−81.47%, P<0.001 in PBMCs and −92.04%, P<0.001 in platelets), resulting in an IC.sub.50 of 0.45 mM for PBMCs and 1.2 mM for platelets. Respiratory capacities using both complex I- and complex II-linked substrates, OXPHOS.sub.CI+II and ETS.sub.CI+II, were decreased similarly to OXPHOS.sub.CI by metformin as illustrated by the representative traces of simultaneously measured O.sub.2 consumption of vehicle-treated and 1 mM metformin-treated permeabilized PBMCs (FIG. 5a). In contrast, ETS.sub.CII capacity and complex IV activity did not change significantly in presence of metformin compared to controls in either cell type (FIG. 5b, c) and neither did LEAK.sub.I+II respiration (the respiratory state where oxygen consumption is compensating for the back-flux of protons across the mitochondrial membrane, traditionally denoted state 4 in isolated mitochondria, data not shown). The mitochondrial inhibition of complex I induced by metformin did not seem to be reversible upon extra- and intracellular removal of the drug by washing and permeabilizing the cells, respectively. Although the severity of the insult of complex I inhibition was attenuated by removal (probably attributed to a shorter exposure time of the drug) platelets did not regain routine and maximal mitochondrial function comparable to control (data not shown). Phenformin likewise inhibited OXPHOS.sub.CI (FIG. 6), OXPHOS.sub.CI+II and ETS.sub.CI+II but not ETS.sub.CII or respiration specific to complex IV (data not shown). Phenformin demonstrated a 20-fold more potent inhibition of OXPHOS.sub.CI in permeabilized platelets than metformin (IC.sub.50 0.058 mM and 1.2 mM, respectively) (FIG. 2). Metformin and phenformin did not induce increased respiration following administration of cytochrome c and hence did not disrupt the integrity of the outer mitochondrial membrane.

[0521] After stabilization of routine respiration in MiR05 medium, either vehicle (double-deionized water) or 1, 10 and 100 mM metformin was added. Routine respiration was followed for 60 min at 37° C. before the ATP-synthase inhibitor oligomycin (1 μg mL.sup.−1) was added to assess LEAK respiration. Maximal uncoupled respiratory electron transport system capacity supported by endogenous substrates (ETS) was reached by titration of FCCP. Respiration was sequentially blocked by the complex I inhibitor rotenone (2 μM), the complex III inhibitor antimycin (1 μg mL.sup.−1) and the complex IV inhibitor sodium azide (10 mM) to assess ROX, which all respiration values were corrected for. In an additional experiment, whole blood was incubated in K.sub.2EDTA tubes with different metformin concentrations (0.1, 0.5 and 1 mM) over a period of 18 h prior to isolation of platelets and analyses of respiration.

[0522] Results

[0523] In intact human platelets, metformin decreased routine respiration in a dose- and time-dependent manner (FIG. 7a). When exposed to either metformin or vehicle the platelets showed a continuous decrease in routine respiration over time. After 60 min the routine respiration was reduced by −14.1% in control (P<0.05), by −17.27% at 1 mM (P<0.01), by −28.61% at 10 mM (P<0.001), and by −81.78% at 100 mM of metformin (P<0.001) compared to the first measurement after addition. Metformin at 100 mM decreased routine respiration significantly compared to control already after 15 min of exposure (−39.77%, P<0.01). The maximal uncoupled respiration of platelets (the protonophore-titrated ETS capacity) after 60 min incubation, was significantly inhibited by 10 mM (−23.86%, P<0.05) and 100 mM (−56.86%, P<0.001) metformin (FIG. 3b). LEAK respiration in intact cells was not significantly changed by metformin incubation (data not shown). When whole blood was incubated at a metformin concentrations of 1 mM over 18 h routine respiration of intact human platelets was reduced by 30.49% (P<0.05).

Example 19

[0524] Effect of Metformin and Phenformin on Lactate Production and pH of Intact Human Platelets

[0525] Platelets were incubated for 8 h with either metformin (1 mM, 10 mM), phenformin (0.5 mM), rotenone (2 μM), or the vehicle for rotenone (DMSO). Lactate levels were determined every 2 h (n=5) using the Lactate Pro™ 2 blood lactate test meter (Arkray, Alere AB, Lidingö, Sweden) (Tanner et al., 2010). Incubation was performed at 37° C. at a stirrer speed of 750 rpm, and pH was measured at start, after 4 and after 8 h of incubation (n=4) using a PHM210 Standard pH Meter (Radiometer, Copenhagen, Denmark).

[0526] Results

[0527] Lactate production increased in a time- and dose-dependent manner in response to incubation with metformin and phenformin in human platelets (FIG. 8). Compared to control, metformin—(1 and 10 mM), phenformin—(0.5 mM), and rotenone—(2 μM) treated platelets all produced significantly more lactate over 8 h of treatment. At 1 mM metformin, lactate increased from 0.30±0.1 to 3.34±0.2 over 8 h and at 10 mM metformin, lactate increased from 0.22±0.1 to 5.76±0.7 mM. The corresponding pH dropped from 7.4±0.01 in both groups to 7.16±0.03 and 7.00±0.04 for 1 mM and 10 mM metformin, respectively. Phenformin-treated platelets (0.5 mM) produced similar levels of lactate as 10 mM metformin-treated samples. The level of lactate increase correlated with the decrease in pH for all treatment groups. The increased lactate levels in metformin-treated intact platelets also correlated with decreased absolute OXPHOS.sub.CI respiratory values seen in metformin-treated permeabilized platelets (r.sup.2=0.60, P<0.001). A limited set of experiments further demonstrated that intact PBMCs also show increased lactate release upon exposure to 10 mM metformin (data not shown).

[0528] Discussion of the Results from Examples 18-19

[0529] This study demonstrates a non-reversible toxic effect of metformin on mitochondria specific for complex I in human platelets and PBMCs at concentrations relevant for the clinical condition of metformin intoxication. In platelets, we further have shown a correlation between decreased Complex I respiration and increased production of lactate. The mitochondrial toxicity we observed for metformin developed over time in intact cells. Phenformin, a structurally related compound now withdrawn in most countries due to a high incidence of LA, induced lactate release and pH decline in platelets through a complex I specific effect at substantially lower concentration.

[0530] In the present study, using a model applying high-resolution respirometry to assess integrated mitochondrial function of human platelets, we have demonstrated that the mitochondrial toxicity of both metformin and phenformin is specific to respiratory complex I and that a similar specific inhibition also is present in PBMCs. Complex I respiration of permeabilized PBMCs was 2.6-fold more sensitive to metformin than that of permeabilized platelets. However, due to the time-dependent toxicity of metformin (see below), the IC.sub.50 is possibly an underestimation and could be lower if determined after longer exposure time. These findings further strengthen that the mitochondrial toxicity of metformin is not limited to specific tissues, as shown previously by others, but rather a generalized effect on a subcellular level. The metformin-induced complex IV inhibition in platelets reported by (Protti et al., 2012a, Protti et al., 2012b) has not been confirmed in this study or in an earlier study by Dykens et al. (2008) using isolated bovine mitochondria. Further, metformin and phenformin did not induce respiratory inhibition through any unspecific permeability changes of the inner or outer mitochondrial membranes as there were no evidence of uncoupling or stimulatory response following cytochrome c addition in presence of the drugs. High-resolution respirometry is a method of high sensitivity and allows O.sub.2 measurements in the picomolar range. When applied to human blood cells ex vivo, it allows assessment of respiration in the fully-integrated state in intact cells, and permits exogenous supply and control of substrates to intact mitochondria in permeabilized cells. This is in contrast to enzymatic spectrophotometric assays which predominantly have been used in the research on mitochondrial toxicity of metformin, for instance by D kens et al. (2008) and Owen et al. 2000). These assays measure the independent, not-integrated function of the single complexes and hence, are less physiological, which may contribute to the differences in results between our studies.

[0531] The results of the study demonstrated significant respiratory inhibition, lactate increase and pH decrease in intact platelet suspensions caused by metformin at concentrations relevant for intoxication already after 8-18 h. The time-dependent inhibition of mitochondrial respiration in combination with the lack of reversal following exchange of the extracellular buffer and dilution of intracellular content of soluble metformin by permeabilization of the cell point towards intramitochondrial accumulation being a key factor in the development of drug-induced mitochondrial dysfunction-related LA, as has been proposed by others (Chan et al., 2005, Lalau, 2010).

[0532] Phenformin's mitochondrial toxicity has been shown previously, for instance on HepG2 cells, a liver carcinoma cell line, and isolated mitochondria of rat and cow. Here we have demonstrated specific mitochondrial toxicity also using human blood cells. Compared to metformin, phenformin had a stronger mitochondrial toxic potency on human platelets (IC.sub.50 1.2 mM and 0.058 mM, respectively). Phenformin and metformin show a 10 to 15-fold difference in clinical dosing and 3 to 10-fold difference in therapeutic plasma concentration. In this study we have observed a 20-fold difference between phenformin and metformin in the potential to inhibit complex I. If translated to patients this difference in mitochondrial toxicity in relation to clinical dosing could potentially explain phenformin's documented higher incidence of phenformin-associated LA.

[0533] Standard therapeutic plasma concentrations of metformin are in the range of 0.6 and 6.0 μM and toxic concentrations lie between 60 μM and 1 mM. In a case report of involuntary metformin intoxication, prior to hemodialysis, a serum level of metformin over 2 mM was reported (Al-Abri et al., 2013). Tissue distribution studies have further demonstrated that the metformin concentration under steady-state is lower in plasma/serum than in other organs. It has been shown to accumulate in 7 to 10-fold higher concentrations in the gastrointestinal tract, with lesser but still significantly higher amounts in the kidney, liver, salivary glands, lung, spleen and muscle as compared to plasma levels. Under circumstances where the clearance of metformin is impaired, such as predisposing conditions affecting the cardiovascular system, liver or kidneys, toxic levels can eventually be reached. The toxic concentration of metformin seen in the present study (1 mM) is thus comparable to what is found in the blood of metformin-intoxicated patients. Although metformin is toxic to blood cells, as shown in this study, it is unlikely that platelets and PBMCs are major contributors to the development of LA. As metformin is accumulated in other organs and additionally these organs are more metabolically active, increased lactate production is likely to be seen first in other tissues. Our results therefore strengthen what has been suggested by others (Brunmair et al., 2004, Protti et al., 2012b, Dykens et al., 2008), that systemic mitochondrial inhibition is the cause of metformin-induced LA.

[0534] Based on earlier studies and the present findings it is intriguing to speculate on the possibility that metformin's anti-diabetic effect may be related to inhibition of aerobic respiration. The decreased glucose levels in the liver and decreased uptake of glucose to the blood in the small intestine in metformin-treated diabetic patients might be due to partial complex I inhibition. Complex I inhibition causes reduced production of ATP, increased amounts of AMP, activation of the enzyme AMP-activated protein kinase (AMPK), and accelerated glucose turnover by increased glycolysis, trying to compensate for the reduced ATP production.

[0535] Until now, treatment measures for metformin-associated LA consist of haemodialysis and haemofiltration to remove the toxin, correct for the acidosis and increase renal blood flow.

Example 20

[0536] Intervention on Metformin-Induced Increase in Lactate Production with Cell-Permeable Succinate Prodrugs

[0537] Intervention of metformin-induced increase in lactate production in intact human platelets with newly developed and synthesized cell-permeable succinate prodrugs was done in PBS containing 10 mM glucose. The platelets were exposed to either rotenone alone (2 μM), rotenone (2 μM) and antimycin (1 μg/mL, only for cells treated with NV 189), or 10 mM metformin and after 60 min either vehicle (DMSO, control), either of the cell-permeable succinate prodrugs (NV118, NV189 and NV241), or succinate were added at a concentration of 250 μM each 30 minutes. Lactate levels were measured in intervals of 30 min with the onset of the experiment. Additionally, pH was measured prior to the first addition of vehicle (dmso, control), the different cell-permeable succinate prodrugs (NV118, NV189, NV241) or succinate and at the end of the experiment. The rate of lactate production was calculated with a nonlinear fit with a 95% Confidence interval (CI) of the lactate-time curve slope (FIGS. 9, 10, 11 and 12).

[0538] Results relating to Example 20 are based on the assays described herein.

[0539] Lactate Production Due to Rotenone and Metformin Incubation in Thrombocytes is Attenuated by the Addition of Cell-Permeable Succinate Prodrugs

[0540] The rate of lactate production in thrombocytes incubated with 2 μM Rotenone was 0.86 mmol lactate (200.Math.10.sup.6trc.Math.h).sup.−1 (95% Confidence Interval) [CI] 0.76-0.96) which was attenuated by NV118 (0.25 mmol [95% CI 0.18-0.33]), NV189 (0.42 mmol [95% CI 0.34-0.51]) and NV241 (0.34 mmol [95% CI 0.17-0.52]), which was not significantly different from cells not receiving rotenone (0.35 [95% CI 0.14-0.55]) (FIGS. 9,10 and 11). Cells incubated with antimycin in addition to rotenone and NV189 had a lactate production comparable to rotenone-treated cell (0.89 mmol [0.81-0.97]), demonstrating the specific mitochondrial effect of the cell-permeable succinate prodrugs (FIG. 10). Cells incubated with 10 mM Metformin produce lactate at a rate of 0.86 mmol lactate (200.Math.10.sup.9 trc.Math.h).sup.−1 (95% CI 0.69-1.04) compared 0.22 mmol (95% CI 0.14-0.30) in vehicle (water) treated cells (FIG. 12). Co-incubating with either of the three succinate prodrugs attenuate the metformin effect resulting in 0.43 mmol production (95% CI 0.33-0.54) for NV118 (FIG. 9), 0.55 mmol (95% CI 0.44-0.65) for NV189 (FIG. 10), and 0.43 mmol (95% CI 0.31-0-54) for NV241 (FIG. 11).

[0541] All references referred to in this application, including patent and patent applications, are incorporated herein by reference to the fullest extent possible.

[0542] Throughout the specification and the claims which follow, unless the context requires otherwise, the word ‘comprise’, and variations such as ‘comprises’ and ‘comprising’, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

[0543] The application of which this description and claims forms part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the following claims:

[0544] All references referred to in this application, including patent and patent applications, are incorporated herein by reference to the fullest extent possible.

[0545] Throughout the specification and the claims which follow, unless the context requires otherwise, the word ‘comprise’, and variations such as ‘comprises’ and ‘comprising’, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps. The word “comprise” includes “contain” and “consist of”.

[0546] General Description of the Class of Compounds to which the Compounds According to the Invention Belong and Specific Embodiments

[0547] The class of compounds may be defined by formula (IB) below,

##STR00070##

[0548] or a pharmaceutically acceptable salt thereof. Where the dotted bond between A and B denotes an optional bond so as to form a ring closed structure, wherein

[0549] Z is selected from —CH.sub.2—CH.sub.2— or >CH(CH.sub.3), —O, S,

[0550] A and B are independently different or identical and are selected from —O—R′, —NHR″, —SR′″ or —OH, with the proviso that both A and B cannot be H,

[0551] R′, R″ and R′″ are independently different or identical and selected from the formula (IIB) to (IXB) below:

##STR00071##

[0552] R.sub.1=H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH.sub.2Xalkyl, CH.sub.2X-acyl, F, CH.sub.2COOH, CH.sub.2CO.sub.2alkyl or any of the below formulas (a)-(f)

##STR00072##

[0553] In preferred structures, R.sub.1=H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH.sub.2Xalkyl, CH.sub.2X-acyl, F, CH.sub.2COOH.

[0554] X═O, NH, NR.sub.6, S

[0555] R.sub.2=Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, —C(O)CH.sub.3, —C(O)CH.sub.2C(O)CH.sub.3, —C(O)CH.sub.2CH(OH)CH.sub.3,

[0556] R.sub.3=R.sub.1, i.e. is the same or different groups as mentioned under R.sub.1 ═CR′.sub.3R′.sub.3, NR.sub.4

[0557] n=1-4,

[0558] p=1-2

[0559] X.sub.2=OR5, NR.sub.1R′.sub.2

[0560] R′.sub.3=H, Me, Et, F

[0561] R.sub.4=H, Me, Et, i-Pr

[0562] R.sub.5=acetyl, propionyl, benzoyl, benzylcarbonyl

[0563] R′.sub.2=H.HX.sub.3, acyl, acetyl, propionyl, benzoyl, benzylcarbonyl

[0564] X.sub.3=F, Cl, Br and I

[0565] R.sub.6=H, or alkyl such as e.g. Me, Et, n-propyl, i-propyl, butyl, iso-butyl, t-butyl, or acetyl, such as e.g. acyl, propionyl, benzoyl, or formula (IIB), formula (IIBI) or formula (VIIIB)

[0566] X.sub.5=—H, —COOH, —C(═O)XR.sub.6,

##STR00073##

[0567] X.sub.5 may also be CONR.sub.1R.sub.3.

[0568] R.sub.9=H, Me, Et or O.sub.2CCH.sub.2CH.sub.2COXR.sub.8

[0569] R.sub.10=Oacyl, NHalkyl, NHacyl, or O.sub.2CCH.sub.2CH.sub.2COX.sub.6R.sub.8

[0570] X.sub.6=O, NR.sub.8

[0571] R.sub.8=H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoylor formula (IIB),

[0572] R.sub.11 and R.sub.12 are independently H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, acyl, —CH.sub.2Xalkyl, —CH.sub.2Xacyl, where X=O, NR.sub.6 or S,

[0573] R.sub.c and R.sub.d are independently CH.sub.2Xalkyl, CH.sub.2Xacyl, where X=O, NR.sub.6 or S,

[0574] Rf, Rg and Rh are independently selected from Xacyl, —CH.sub.2Xalkyl, —CH.sub.2X—acyl and R.sub.9,

[0575] wherein alkyl is e.g. methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, neopentyl, isopentyl, hexyl, isohexyl, heptyl, octyl, nonyl or decyl and acyl is e.g. formyl, acetyl, propionyl, butyryl pentanoyl, benzoyl and the like, and wherein the acyls and alkyls may be optionally substituted,

[0576] the dotted bond between A and B denotes an optional bond to form a cyclic structure of formula (I) and with the proviso that when such a cyclic bond is present, the compound according to formula (I) is selected from

##STR00074##

[0577] wherein X.sub.4 is selected from —COOH, —C(═O)XR.sub.6,

##STR00075##

[0578] and wherein R.sub.x and R.sub.y are independently selected from R.sub.1, R.sub.2, R.sub.6 or R′, R″ or R′″ with the proviso that R.sub.x and R.sub.y cannot both be —H.

[0579] In preferred aspect, R′, R″ and R′″ are independently different or identical and selected from the formula (IIB), (VB), (VIIB) or VIIIB) below:

##STR00076##

[0580] Preferably, and with respect to formula (IIB), at least one of R.sub.1 and R.sub.3 is —H, such that formula II is:

##STR00077##

[0581] Preferably, and with respect to formula (VII), p is 1 or 2, preferably p is 1 and X.sub.5 is —H such that formula (VIIB) is

##STR00078##

[0582] Preferably, and with respect to formula (IXB), at least one of R.sub.f, R.sub.g, R.sub.h is —H or alkyl, with alkyl as defined herein. Moreover, it is also preferable with respect to Formula (IXB) that at least one of Rf, Rg, Rh is —CH.sub.2Xacyl, with acyl as defined herein.

[0583] An interesting subclass of the class mentioned above relates to the compounds of Formula (I)

##STR00079##

[0584] or a pharmaceutically acceptable salt thereof. The dotted bond between A and B denotes an optional bond so as to form a ring closed structure.

[0585] In formula (IC) Z is selected from —CH.sub.2—CH.sub.2— or >CH(CH.sub.3),

[0586] A is selected from —SR, —OR and NHR, and wherein R is

##STR00080##

[0587] B is selected from —O—R′, —NHR″, —SR′″ or —OH; R′ is selected from the formula (IIC) to (IXC) below:

##STR00081##

[0588] Preferably, R′ is selected from the formula (IIC), (VC), to (IXC) below:

##STR00082##

[0589] R′, R″ and R′″ are independently different or identical and is selected from formula (IVC-VIIIC) below:

##STR00083##

[0590] R.sub.1=H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH.sub.2Xalkyl, CH.sub.2X-acyl, F, CH.sub.2COOH, CH.sub.2CO.sub.2alkyl or any of formulae (a)-(f)

##STR00084##

[0591] Preferably, R.sub.1=H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, O-acyl, O-alkyl, Nacyl, N-alkyl, Xacyl, CH.sub.2Xalkyl, CH.sub.2X-acyl, F, CH.sub.2COOH, CH.sub.2CO.sub.2alkyl,

[0592] X=O, NH, NR.sub.6, S

[0593] R.sub.2=Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, C(O)CH.sub.3, C(O)CH.sub.2C(O)CH.sub.3, C(O)CH.sub.2CH(OH)CH.sub.3,

[0594] R.sub.3=R.sub.1, i.e. may be the same or a different group as defined under R.sub.1,

[0595] X.sub.1=CR′.sub.3R′.sub.3, NR.sub.4

[0596] n=1-4,

[0597] p=1-2

[0598] X.sub.2=OR.sub.5, NR.sub.1R′.sub.2

[0599] R′.sub.3=H, Me, Et, F

[0600] R.sub.4=H, Me, Et, i-Pr

[0601] R.sub.5=acetyl, propionyl, benzoyl, benzylcarbonyl

[0602] R′.sub.2=H.HX.sub.3, acyl, acetyl, propionyl, benzoyl, benzylcarbonyl

[0603] X.sub.3=F, Cl, Br and I

[0604] R.sub.6=H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, or formula (IIC), formula (IIIC) or formula (VIIIC)

##STR00085##

[0605] X.sub.5=—H, —COOH, —C(═O)XR.sub.6,

[0606] X.sub.5 may also be CONR.sub.1R.sub.3

[0607] R.sub.9=H, Me, Et or O.sub.2CCH.sub.2CH.sub.2COXR.sub.8

[0608] R.sub.10=Oacyl, NHalkyl, NHacyl, or O.sub.2CCH.sub.2CH.sub.2COX.sub.6R.sub.8

[0609] X.sub.6=O, NR.sub.8

[0610] R.sub.8=H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, or formula (IIC), formula (IIIC) or formula (VIIIC)

[0611] R.sub.11 and R.sub.12 are independently H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, acyl, —CH.sub.2Xalkyl, —CH.sub.2Xacyl, where X=O, NR.sub.6 or S

[0612] R.sub.c and R.sub.d are independently CH.sub.2Xalkyl, CH.sub.2Xacyl, where X=O, NR.sub.6 or S,

[0613] R.sub.f, R.sub.g and R.sub.h are independently selected from Xacyl, —CH.sub.2Xalkyl, —CH.sub.2X—acyl and R.sub.9

[0614] alkyl is e.g. Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl and acyl is e.g. formyl, acetyl, propionyl, isopropionyl, byturyl, tert-butyryl, pentanoyl, benzoyl and the likes and wherein the acyls and alkyls may be optionally substituted, and

[0615] when the dotted bond between A and B is present, the compound according to formula (I) is

##STR00086##

[0616] wherein X.sub.4 is selected from —COOH, —C(═O)XR.sub.6,

##STR00087##

[0617] Preferably, and with respect to formula (IIC), at least one of R.sub.1 and R.sub.3 is —H, such that formula II is:

##STR00088##

[0618] Preferably, and with respect to formula (VIIC), p is 1 or 2, preferably p is 1 and X.sub.5 is —H such that formula (VIIC) is

##STR00089##

[0619] Preferably, and with respect to formula (IXC), at least one of R.sub.f, R.sub.g, R.sub.h is —H or alkyl, with alkyl as defined herein. Moreover, it is also preferable with respect to Formula (IXC) that at least one of R.sub.f, R.sub.g, R.sub.h is —CH.sub.2Xacyl, with acyl as defined herein.

[0620] Interesting compounds according to formula (IC) are:

##STR00090## [0621] wherein X.sub.4 is selected from —COOH, —C(═O)XR.sub.6,

##STR00091## [0622] wherein R.sub.1 and X.sub.5 is as defined herein. Preferably X.sub.5 is —H.

##STR00092## [0623] wherein R.sub.6, X.sub.5 and R.sub.1 are as defined herein. Preferably X.sub.5 is —H.

##STR00093## [0624] wherein X.sub.5 and R.sub.1 are as defined herein.

[0625] Preferably X.sub.5 is —H.

Specific Embodiments

[0626] 1. A compound according to Formula (I)

##STR00094##

[0627] or a pharmaceutically acceptable salt thereof, wherein the dotted bond between A and B denotes an optional bond so as to form a ring closed structure, and wherein

[0628] Z is selected from —CH.sub.2—CH.sub.2— or >CH(CH.sub.3),

[0629] A is selected from —SR, —OR and NHR and R is

##STR00095##

[0630] B is selected from —O—R′, —NHR″, —SR′″ or —OH; and R′ is selected from the formula (II) to (IX) below:

##STR00096##

[0631] R′, R″ and R′″ are independently different or identical and is selected from formula (IV-VIII) below:

##STR00097##

[0632] R.sub.1=H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH.sub.2Xalkyl, CH.sub.2X-acyl, F, CH.sub.2COOH, CH.sub.2CO.sub.2alkyl or any of the below formulae (a)-(f)

##STR00098##

[0633] X=O, NH, NR.sub.6, S

[0634] R.sub.2=Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, C(O)CH.sub.3, C(O)CH.sub.2C(O)CH.sub.3, C(O)CH.sub.2CH(OH)CH.sub.3,

[0635] R.sub.3=R.sub.1, i.e. different or identical with the groups mentioned under R.sub.1,

[0636] X.sub.1=CR′.sub.3R′.sub.3, NR.sub.4

[0637] n=1-4,

[0638] p=1-2

[0639] X.sub.2=OR.sub.5, NR.sub.1R′.sub.2

[0640] R′.sub.3=H, Me, Et, F

[0641] R.sub.4=H, Me, Et, i-Pr

[0642] R.sub.5=acetyl, propionyl, benzoyl, benzylcarbonyl

[0643] R′.sub.2=H.HX.sub.3, acyl, acetyl, propionyl, benzoyl, benzylcarbonyl

[0644] X.sub.3=F, Cl, Br and I

[0645] R.sub.6=H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, or formula (II), formula (III) or formula (VIII)

[0646] X.sub.5=—H, —COOH, —C(═O)XR.sub.6,

##STR00099##

[0647] R.sub.9=H, Me, Et or O.sub.2CCH.sub.2CH.sub.2COXR.sub.8

[0648] R.sub.10=Oacyl, NHalkyl, NHacyl, or O.sub.2CCH.sub.2CH.sub.2CO X.sub.6R.sub.8

[0649] X.sub.6=O, NR.sub.8

[0650] R.sub.8=H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, or formula (II), formula (III) or formula (VIII)

[0651] R.sub.11 and R.sub.12 are independently H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, acyl, —CH.sub.2Xalkyl, —CH.sub.2Xacyl, where X=O, NR.sub.6 or S

[0652] R.sub.c and R.sub.d are independently CH.sub.2Xalkyl, CH.sub.2Xacyl, where X=O, NR.sub.6 or S,

[0653] R.sub.f, R.sub.g and R.sub.h are independently selected from Xacyl, —CH.sub.2Xalkyl, —CH.sub.2X—acyl and R.sub.9

[0654] alkyl is e.g. Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl and

[0655] acyl is e.g. formyl, acetyl, propionyl, isopropionyl, byturyl, tert-butyryl, pentanoyl, benzoyl and the likes and wherein the acyls or alkyls may be optionally substituted, and when the dotted bond between A and B is present, the compound according to formula (I) is

##STR00100##

[0656] wherein X.sub.4 is selected from —COOH, —C(═O)XR.sub.6,

##STR00101##

[0657] and with the further proviso that the compound is not any of the below compounds

##STR00102##

[0658] 2. A compound according to embodiment 1, wherein formula (II) is such that at least one of R1 and R.sub.3 is —H such that formula II is:

##STR00103##

[0659] 3. A compound according to embodiment 1, wherein formula (III) is such that R.sub.4 is —H and formula (III) is

##STR00104##

[0660] and X.sub.1 is NH

[0661] 4. A compound according to embodiment 1, wherein formula (VII) is such that, p=2 and X.sub.5 is —H and formula (VII) is

##STR00105##

[0662] 5. A compound according to embodiment 1, wherein formula (IX) is such that at least one of R.sub.f, R.sub.g, R.sub.h is —H or alkyl, with alkyl as defined herein.

[0663] 6. A compound according to embodiment 1 or 5, wherein formula (IX) is such that at least one of Rf, Rg, Rh is —CH.sub.2Xacyl, with acyl as defined herein.

[0664] 7. A compound according to any of embodiments 1-6, wherein Formula (I) is

##STR00106##

[0665] wherein X.sub.4 is selected from —COOH, —C(═O)XR.sub.6,

##STR00107##

[0666] 8. A compound according to any of embodiments 1-6, wherein Formula (I) is

##STR00108##

[0667] Wherein X.sub.5 and R.sub.1 is as defined in claim 1 and wherein X.sub.5 is preferably —H

[0668] 9. A compound according to any of embodiments 1-6, wherein Formula (I) is

##STR00109##

[0669] wherein X.sub.5 and R.sub.1 is as defined in embodiment 1 and wherein X.sub.5 is preferably —H

[0670] 10. A compound according to any of embodiments 1-6, wherein Formula (I) is

##STR00110##

[0671] Wherein X.sub.5, R.sub.1 and R.sub.6 is as defined in embodiment 1 and wherein X.sub.5 is preferably —H.

[0672] 11. A compound according to any of embodiments 1-10 for use in medicine

[0673] 12. A compound according to any of embodiments 1-10, for use in cosmetics

[0674] 13. A compound according to any of embodiments 1-10 for use in the treatment of or prevention of metabolic diseases, or in the treatment of diseases of mitochondrial dysfunction or disease related to mitochondrial dysfunction, treating or suppressing of mitochondrial disorders, stimulation of mitochondrial energy production, treatment of cancer and following hypoxia, ischemia, stroke, myocardial infarction, acute angina, an acute kidney injury, coronary occlusion and atrial fibrillation, or to avoid or counteract reperfusion injuries.

[0675] 14. A compound according for use according to embodiment 11, wherein the medical use is prevention or treatment of drug-induced mitochondrial side-effects.

[0676] 15. A compound for use according to embodiment 14, wherein the prevention or drug-induced mitochondrial side-effects relates to drug interaction with Complex I, such as e.g. metformin-Complex I interaction.

[0677] 16. A compound according to embodiment 13, wherein diseases of mitochondrial dysfunction involves e.g. mitochondrial deficiency such as a Complex I, II, III or IV deficiency or an enzyme deficiency like e.g. pyruvate dehydrogenase deficiency.

[0678] 17. A compound for use according to any of embodiments 13-16, wherein the diseases of mitochondrial dysfunction or disease related to mitochondrial dysfunction are selected from Alpers Disease (Progressive Infantile Poliodystrophy, Amyotrophic lateral sclerosis (ALS), Autism, Barth syndrome (Lethal Infantile Cardiomyopathy), Beta-oxidation Defects, Bioenergetic metabolism deficiency, Carnitine-Acyl-Carnitine Deficiency, Carnitine Deficiency, Creatine Deficiency Syndromes (Cerebral Creatine Deficiency Syndromes (CCDS) includes: Guanidinoaceteate Methyltransferase Deficiency (GAMT Deficiency), L-Arginine:Glycine Amidinotransferase Deficiency (AGAT Deficiency), and SLC6A8-Related Creatine Transporter Deficiency (SLC6A8 Deficiency), Co-Enzyme Q10 Deficiency, Complex I Deficiency (NADH dehydrogenase (NADHCoQ reductase deficiency), Complex II Deficiency (Succinate dehydrogenase deficiency), Complex III Deficiency (Ubiquinone-cytochrome c oxidoreductase deficiency), Complex IV Deficiency/COX Deficiency (Cytochrome c oxidase deficiency is caused by a defect in Complex IV of the respiratory chain), Complex V Deficiency (ATP synthase deficiency), COX Deficiency, CPEO (Chronic Progressive External Ophthalmoplegia Syndrome), CPT I Deficiency, CPT II Deficiency, Friedreich's ataxia (FRDA or FA), Glutaric Aciduria Type II, KSS (Kearns-Sayre Syndrome), Lactic Acidosis, LCAD (Long-Chain Acyl-CoA Dehydrogenase Deficiency), LCHAD, Leigh Disease or Syndrome (Subacute Necrotizing Encephalomyelopathy), LHON (Leber's hereditary optic neuropathy), Luft Disease, MCAD (Medium-Chain Acyl-CoA Dehydrogenase Deficiency), MELAS (Mitochondrial Encephalomyopathy Lactic Acidosis and Strokelike Episodes), MERRF (Myoclonic Epilepsy and Ragged-Red Fiber Disease), MIRAS (Mitochondrial Recessive Ataxia Syndrome), Mitochondrial Cytopathy, Mitochondrial DNA Depletion, Mitochondrial Encephalopathy including: Encephalomyopathy and Encephalomyelopathy, Mitochondrial Myopathy, MNGIE (Myoneurogastointestinal Disorder and Encephalopathy, NARP (Neuropathy, Ataxia, and Retinitis Pigmentosa), Neurodegenerative disorders associated with Parkinson's, Alzheimer's or Huntington's disease, Pearson Syndrome, Pyruvate Carboxylase Deficiency, Pyruvate Dehydrogenase Deficiency, POLG Mutations, Respiratory Chain Deficiencies, SCAD (Short-Chain Acyl-CoA Dehydrogenase Deficiency), SCHAD, VLCAD (Very Long-Chain Acyl-CoA Dehydrogenase Deficiency).

[0679] 18. A compound for use according to embodiment 17, wherein the mitochondrial dysfunction or disease related to mitochondrial dysfunction is attributed to complex I dysfunction and selected from Leigh Syndrome, Leber's hereditary optic neuropathy (LHON), MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) and MERRF (myoclonic epilepsy with ragged red fibers).

[0680] 19. A composition comprising a compound of Formula (I) as defined according any of embodiments 1-10 and one or more pharmaceutically or cosmetically acceptable excipients.

[0681] 20. A method of treating a subject suffering from diseases of mitochondrial dysfunction or disease related to mitochondrial dysfunction as defined in any of embodiments 16-18, the method comprising administering to the subject an efficient amount of a composition as defined in embodiment 19.

[0682] 21. A method according to embodiment 20, wherein the composition is administered parenterally, orally, topically (including buccal, sublingual or transdermal), via a medical device (e.g. a stent), by inhalation or via injection (subcutaneous or intramuscular) 22. A method according to any of embodiments 20-21, wherein the composition is administered as a single dose or a plurality of doses over a period of time, such as e.g. one daily, twice daily or 3-5 times daily as needed.

[0683] 23. A compound according to any of embodiments 1-10 for use in the treatment or prevention of lactic acidosis.

[0684] 24. A compound according to any of embodiments 1-10 for use in the treatment or prevention of a drug-induced side-effect selected from lactic acidosis and side-effects related to Complex I defect, inhibition or malfunction.

[0685] 25. A compound according to any of embodiments 1-10 for use in the treatment or prevention of a drug-induced side-effect selected from lactic acidosis and side-effects related to defect, inhibition or mal-function in aerobic metabolism upstream of complex I (indirect inhibition of Complex I, which would encompass any drug effect that limits the supply of NADH to Complex I, e.g. effects on Krebs cycle, glycolysis, beta-oxidation, pyruvate metabolism and drugs that affect the levels of glucose or other Complex I-related substrates).

[0686] 26. A combination of a drug substance and a compound according to any of embodiments 1-10 for use in the treatment and/or prevention of a drug-induced side-effect selected from i) lactic acidosis, ii) and side-effects related to a Complex I defect, inhibition or malfunction, and iii) side-effects related to defect, inhibition or malfunction in aerobic metabolism upstream of complex I (indirect inhibition of Complex I, which would encompass any drug effect that limits the supply of NADH to Complex I, e.g. effects on Krebs cycle, glycolysis, beta-oxidation, pyruvate metabolism and drugs that affect the levels of glucose or other Complex-I-related substrates), wherein

[0687] i) the drug substance is used for treatment of a disease for which the drug substance is indicated, and

[0688] ii) the succinate prodrug is used for prevention or alleviation of the side effects induced or inducible by the drug substance, wherein the side-effects are selected from lactic acidosis and side-effects related to a Complex I defect, inhibition or malfunction.

[0689] 27. A composition comprising a drug substance and a compound according to any of embodiments 1-10, wherein the drug substance has a potential drug-induced side-effect selected from i) lactic acidosis, ii) side-effects related to a Complex I defect, inhibition or malfunction, and iii) side-effects related to defect, inhibition or malfunction in aerobic metabolism upstream of complex I (indirect inhibition of Complex I, which would encompass any drug effect that limits the supply of NADH to Complex I, e.g. effects on Krebs cycle, glycolysis, beta-oxidation, pyruvate metabolism and even drugs that affect the levels of glucose or other Complex-I-related substrates).

[0690] 28. A kit comprising

[0691] i) a first container comprising a drug substance, which has a potential drug-induced side-effect selected i) from lactic acidosis, ii) and side-effects related to a Complex I defect, inhibition or malfunction, and iii) side-effects related to defect, inhibition or malfunction in aerobic metabolism upstream of complex I (indirect inhibition of Complex I, which would encompass any drug effect that limits the supply of NADH to Complex I, e.g. effects on Krebs cycle, glycolysis, beta-oxidation, pyruvate metabolism and even drugs that affect the levels of glucose or other substrates), and

[0692] ii) a second container comprising a compound according to any of embodiments 1-10, which has the potential for prevention or alleviation of the side effects induced or inducible by the drug substance, wherein the side-effects are selected from i) lactic acidosis, ii) side-effects related to a Complex I defect, inhibition or malfunction, and iii) side-effects related to defect, inhibition or malfunction in aerobic metabolism upstream of complex I (indirect inhibition of Complex I, which would encompass any drug effect that limits the supply of NADH to Complex I, e.g. effects on Krebs cycle, glycolysis, beta-oxidation, pyruvate metabolism and even drugs that affect the levels of glucose or other substrates).

[0693] 29. A method for treating a subject suffering from a drug-induced side-effect selected from i) lactic acidosis, ii) side-effect related to a Complex I defect, inhibition or malfunction, and iii) side-effects related to defect, inhibition or malfunction in aerobic metabolism upstream of complex I (indirect inhibition of Complex I, which would encompass any drug effect that limits the supply of NADH to Complex I, e.g. effects on Krebs cycle, glycolysis, beta-oxidation, pyruvate metabolism and even drugs that affect the levels of glucose or other substrates, the method comprises administering an effective amount of a compound according to any of embodiments 1-10 to the subject.

[0694] 30. A method for preventing or alleviating a drug-induced side-effect selected from i) lactic acidosis, ii) side-effect related to a Complex I defect, inhibition or malfunction, and iii) side-effects related to defect, inhibition or malfunction in aerobic metabolism upstream of complex I (indirect inhibition of Complex I, which would encompass any drug effect that limits the supply of NADH to Complex I, e.g. effects on Krebs cycle, glycolysis, beta-oxidation, pyruvate metabolism and even drugs that affect the levels of glucose or other substrates) in a subject, who is suffering from a disease that is treated with a drug substance, which potentially induce a side-effect selected from i) lactic acidosis, ii) side-effect related to a Complex I defect, inhibition or malfunction, and iii) side-effects related to defect, inhibition or malfunction in aerobic metabolism upstream of Complex I, such as in dehydrogenases of Kreb's cycle, pyruvate dehydrogenase and fatty acid metabolism, the method comprises administering an effective amount of a compound according to any of embodiments 1-10 to the subject.

[0695] 31. A method according to any one of embodiments 29-30, wherein the drug substance is an anti-diabetic substance.

[0696] 32. A method according to any one of embodiments 29-31, wherein the anti-diabetic substance is metformin.

[0697] 33. A compound according to any of embodiments 1-10, for use in the treatment of absolute or relative cellular energy deficiency.