Compounds for treating mitochondrial disease

Abstract

The invention relates to novel compounds that are useful for modulating cellular ROS. The compounds are amide-derivatives of 2-hydroxy-2-methyl-4-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-butanoic acid. The compounds of the invention are formulated into pharmaceutical or cosmetic compositions. The invention further relates to methods wherein the compounds of the invention are used for treating or preventing diseases associated with increased ROS levels mitochondrial disorders and/or conditions associated with mitochondrial dysfunction, including adverse drug effects. The invention also relates to cosmetic methods for treating or delaying further aging of the skin and veterinary applications.

Claims

1. A compound of general structure (I): ##STR00048## wherein wherein L is selected from —CH.sub.2—CH.sub.2—NH—C(O)—CH.sub.2—, —CH.sub.2—CH.sub.2—NH—C(NH.sub.2)═, —CH.sub.2—CH.sub.2—NH—C(O)—CH.sub.2—NH—C(NH.sub.2)═, —CH.sub.2—CH.sub.2—CH.sub.2—NH—C(NH.sub.2)═, —CH.sub.2—CH.sub.2—NH—C(Me)═, —CH.sub.2—CH.sub.2—NH—C(O)—CH.sub.2—NH—C(Me)═, —CH.sub.2—CH.sub.2—CH.sub.2—NH—C(Me)═, —CH.sub.2—CH.sub.2—NR.sup.1′—C(NH.sub.2)═, —C(CO.sub.2H)—CH.sub.2—CH.sub.2—CH.sub.2—, —C(CO.sub.2H)—CH.sub.2—CH.sub.2—CH.sub.2—NH—C(NH.sub.2)—, —C(CO.sub.2H)—CH.sub.2—, —C(CO.sub.2H)—CH.sub.2—CH.sub.2—, —C(CO.sub.2H)—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—, —CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—, —CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—, —CHR.sup.2′—C(O)—, —CHR.sup.2′—CH.sub.2—, —CHR.sup.5—CH.sub.2—NR.sup.5′—C(Me)═, —CHR.sup.2′—CH.sub.2—CH.sub.2—, —CH.sub.2—CH.sub.2—CHR.sup.1′—, —CH.sub.2—CH.sub.2—CHR.sup.1′—NH—C(O)—C(Me)-, —CH.sub.2—CHR.sup.1′—, —CH.sub.2—CHR.sup.1′—NH—C(Me)═, or —CHR.sup.5—CH.sub.2—CH.sub.2—CHR.sup.5′—; R.sup.1 and R.sup.2 are each independently selected from H, C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkenyl, or R.sup.1 is joined with R.sup.1′ in a cyclic structure and/or R.sup.2 is joined with R.sup.2′ in a cyclic structure, or R.sup.1 and R.sup.2 are joined together and thus form a second linker between the amide nitrogen atom and the distal nitrogen atom (N*) wherein L is a linker comprising 1 to 10 optionally substituted backbone atoms selected from carbon, nitrogen and oxygen; R.sup.3 is selected from H, C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkenyl, wherein the alkyl or alkenyl moiety may be substituted with one or more halogen atoms or (halo)alkoxy moieties, or R.sup.3 is absent when the distal nitrogen atom is part of an imine moiety; and R.sup.4 is absent; and R.sup.5 is joined with R.sup.5′ in a cyclic structure, and X is absent.

2. The compound according to claim 1, wherein R.sup.2 is joined with a backbone atom of the linker L in a saturated cyclic structure.

3. The compound according to claim 2, wherein R.sup.2 is joined with a backbone atom of the linker L in a piperidine ring.

4. The compound according to claim 1, wherein L=—C(CO.sub.2H)(CH.sub.2).sub.3—, R.sup.1═R.sup.2═R.sup.3═H; L=—(CH.sub.2).sub.4—, R.sup.1═H, R.sup.2═R.sup.3=Me; L=—CHR.sup.2′CH.sub.2—R.sup.1═R.sup.3═H, R.sup.2-R.sup.2′═—(CH.sub.2).sub.3—; L=—CHR.sup.5(CH.sub.2).sub.2CHR.sup.5′—, R.sup.1═R.sup.2═R.sup.3═H, R.sup.5-R.sup.5′═—(CH.sub.2).sub.2—; or L=—CHR.sup.5(CH.sub.2).sub.2CHR.sup.5′—, R.sup.1═R.sup.2═R.sup.3═H, and R.sup.5-R.sup.5′═—(CH.sub.2).sub.2—, which is in the S,R-configuration.

5. A pharmaceutical or cosmetic composition comprising a compound according to claim 1 and a physiologically acceptable carrier.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1. Effect of L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of glutathione synthesis, on the viability of primary human fibroblasts derived from healthy individuals or derived from Complex-I deficient patients. Two days after treatment of 48 h with 200 μM BSO, cells were washed and stained with Calcein-AM. Cell viability was determined as a function of fluorescence intensity.

(2) FIG. 2A-FIG. 2H Effect of the compounds on oxidative stress-induced cell death. Primary human fibroblasts derived from Complex-I deficient patients were treated with increasing concentrations of the compounds in combination with 200 μM BSO. After 2 days the cells were washed, stained with Calcein-AM and the fluorescence was measured. Cell viability is depicted as normalized against untreated cells. Each graph depicts the potency of the closed form and corresponding open form of the compound to prevent cell death at selected concentrations. FIG. 2A S,R—X.sup.II (R.sup.4═H, X═Cl) and S,R—X (R.sup.4═H, X=formate) in addition to the known compounds EPI743 and Idebenone, FIG. 2B Trolox and open Trolox, FIG. 2C R-T.sup.II (R.sup.4═H, X═Cl) and R-T (R.sup.4═H, X=formate), FIG. 2D R,R—N.sup.II (R.sup.4═H, X═Cl) and R,R—N(R.sup.4═H, X=formate), FIG. 2E S,R—N.sup.II (R.sup.4═H, X═Cl) and S,R—N(R.sup.4═H, X=formate), FIG. 2F R,R—X.sup.II (R.sup.4═H, X═Cl) and R,R—X (R.sup.4═H, X=formate), FIG. 2G R,S—X.sup.II (R.sup.4═H, X═Cl) and R,S—X (R.sup.4═H, X=formate) and FIG. 2H R,trans-AE.sup.II (R.sup.4═H, X═Cl) and R,trans-AE (R.sup.4═H, X=formate).

(3) FIG. 3. The effect of the compounds on cellular ROS levels. Primary human fibroblasts derived from Complex-I deficient patients were incubated with 5 μM CM-H.sub.2DCFDA for 20 minutes, followed by the addition of the compound EPI743, Idebenone, S,R—X.sup.II (R.sup.4═H, X═Cl) or S,R—X (R.sup.4═H, X=formate), all with a final concentration 10 μM. Untreated cells (vehicle) served as controls. Approximately 8 min. after the addition of the compounds, H.sub.2O.sub.2 was added to a final concentration of 100 μM and CM-DCF fluorescence was measured for 30 minutes.

(4) FIG. 4A-FIG. 4E Effect of the compounds on cellular superoxide production. Primary human fibroblasts derived from Complex-I patients were incubated with increasing concentrations of the compounds. The following days cells were stained with Hydroethidine (Het) (10 μM) and fluorescence was measured. Results are depicted as percentage of vehicle-treated cells. FIG. 4A S,R—X.sup.II (R.sup.4═H, X═Cl) and S,R—X (R.sup.4═H, X=formate), in addition to the known compounds EPI743 and Idebenone, FIG. 4B R-T (R.sup.4═H, X═Cl) and R-T (R.sup.4═H, X=formate), FIG. 4C R,R—X.sup.II (R.sup.4═H, X═Cl) and R,R—X (R.sup.4═H, X=formate), FIG. 4D R,S—X.sup.II (R.sup.4═H, X═Cl) and R,S—X (R.sup.4═H, X=formate) and FIG. 4E R,trans-AE.sup.II (R.sup.4═H, X═Cl) and R,trans-AE (R.sup.4═H, X=formate).

EXAMPLES

Example 1. Syntheses of the Compounds

(5) Synthesis of the compounds according to the invention was performed by first preparing the closed chroman derivative of general structure (II). These closed-form derivatives of the compounds according to the invention are designated with an superscript II. For example, the closed form of compound T as defined above is referred to as compound T.sup.II. Compounds of general structure (II) are prepared according to WO 2014/011047.

(6) ##STR00031##

(7) Unless noted otherwise, materials were purchased from commercial suppliers and used as received. CH.sub.2Cl.sub.2 (DCM) was freshly distilled from calcium hydride. All air and moisture sensitive reactions were carried out under an inert atmosphere of dry nitrogen. Column chromatography was performed using Acros silica gel (0.035-0.070 mm, 6 nm).

(8) GENERAL PROCEDURE A for the EDCI/HOAt coupling of amines to Trolox™: To a mixture of Trolox™ (1 eq) and amine (1 eq) in DMF (dry, ˜0.2M) under nitrogen atmosphere were added EDCI.HCl (1.1 eq) and HOAt (0.1 eq). The mixture was stirred at room temperature until complete conversion (LCMS). The mixture was diluted with H.sub.2O (20 mL) and extracted with EtOAc (3×20 mL). The combined organic phases were successively washed with 0.5M KHSO.sub.4 (20 mL), sat. aq. NaHCO.sub.3 (20 mL) and brine (3×20 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo, to obtain intermediate A.

(9) GENERAL PROCEDURE B for BOC-deprotection: To a solution of intermediate A (1 eq) in DCM (˜0.03M) was added 4N HCl in dioxane (36 eq). The mixture was stirred at room temperature until complete conversion (LCMS), concentrated, coevaporated with DCM (2×), purified by reversed phase column chromatography (H.sub.2O+0.01% (w/w) formic acid/MeCN) and freeze-dried.

(10) GENERAL PROCEDURE C for the ring opening of the chroman derivatives: The compound of formula (II) was oxidized with cerium ammonium nitrate (CAN) or with Iron(III) chloride hexahydrate in the presence of water and acetonitrile as solvent. The compound of formula (II) (0.2 mmol; 1 eq) was dissolved in MeCN (4 ml). Cerium(IV) diammonium nitrate (2.1 eq) was dissolved in H.sub.2O (800 μl) to form an orange solution, and added to the reaction mixture. The mixture was stirred for 30 min at room temperature (colour changed from orange to dark yellow). Then a sample was taken and analysed by LCMS: in all instances complete and clean conversion of starting material into one peak with mass of the desired compound was observed. The reaction mixture was quenched by addition of solid NaHCO.sub.3 (3 eq.). The mixture (colour changed from dark yellow to yellow) was stirred for 45 min at room temperature and then the MeCN was removed under reduced pressure. 3 ml H.sub.2O was added and the solids were filtered off and washed with water. The filtrate was partially concentrated and directly purified by reversed phase column chromatography (12 g C18 material, eluens MeCN and H.sub.2O+0.01% (w/w) formic acid, collected at 225 nm and 265 nm): (1) 3 min 0% MeCN; (2) 13 min 0% to 100% MeCN; and (3) 3 min 100% MeCN. The pure fractions were combined and freeze-dried overnight to obtain the product in 40-80% yield as a fluffy solid.

(11) All compounds of general structure (II) and of general structure (I) are obtained as formate salts, in other words R.sup.4═H, X=formate. These formate salts are used in the examples here below. Thus, even if not indicated, R.sup.4═H and X=formate. Compound S,R—X was also prepared as HCl salt, by using 0.01% (w/w) HCl solution during reversed phase column chromatography. In examples 2-4, the HCl salt of compound S,R—X was also tested, and no difference in activity compared to the formate salt of compound S,R—X, of which the results are present below, was observed.

Example 2. Effect of the Compounds on Oxidative Stress-Induced Cell Death

(12) Methods: To assess the ability of the compounds to protect patient cells against oxidative stress-induced cell death an assay was established using stressed primary human fibroblasts derived from a Complex-I deficient patient. Utilizing the inherent oxidative stress of fibroblasts from patients with mitochondrial disease, their oxidative burden was further increased by depleting cellular glutathione with an inhibitor of glutathione synthesis, L-buthionine-(S,R)-sulfoximine (BSO). As a result, while fibroblasts from healthy individuals retained full viability, patient fibroblasts exhibited complete cell death within 48 h of the BSO insult (200 μM) (FIG. 1).

(13) Cells were seeded at a density of 3000 cells/well in a 96-well format plate and incubated with increasing concentrations of compounds in combination with BSO (200 μM, Sigma-Aldrich). Two days after treatment, the cells were washed twice and stained with a solution of 5 μM Calcein-AM (Life technologies C3100MP) during 25 min in 199 medium without phenol red (Life technologies, 11043-023) at 37° C., 5% CO.sub.2. After 2 washes with PBS the plate was read on a fluorescence plate reader (Fluostar Omega, BMG labtech) and the percentage of cell viability was determined as a function of fluorescence intensity (FIG. 2).

(14) Results: The tested compounds are shown in the table below. Except for Trolox and open Trolox, all compounds are in salt form, i.e. R.sup.4═H and X=formate.

(15) TABLE-US-00003 Closed compounds Open compounds 0

(16) For each experiment, the closed and corresponding open compound was tested for the ability to protect cells against oxidative stress-induced cell death. As shown in FIG. 2A-2H, the open compound outperformed the corresponding closed compound in each case. Hence, the open compounds are more potent in protecting cells against oxidative stress-induced cell death as compared to the corresponding closed compound.

(17) In addition as shown in FIG. 2A, the open-form compound S,R—X is also more potent than the known compounds EPI743 from Edison pharmaceutical and Idebenone from Santhera in protecting the cells against oxidative stress-induced cell death. In particular, the EC.sub.50 for S,R—X was 16.71 (+/−5.19) nM, while the EC.sub.50 for EPI743 and Idebenone was respectively 35.32 (+/−4.12) nM and 1469.70 (+/−97.10) nM.

Example 3. Effect of the Compounds on Cellular ROS Levels

(18) Methods: CM-H.sub.2DCFDA is a cell-permeable reporter molecule for reactive oxygen species (ROS) that is converted into non-fluorescent and membrane-impermeable CM-H.sub.2DCF following removal of its acetate groups by intracellular esterases. Upon oxidation by ROS, CM-H.sub.2DCF is converted into fluorescent CM-DCF. It is widely accepted that a wide variety of ROS can be responsible for the CM-H.sub.2DCF oxidation, making it a suitable reporter of cellular oxidant levels. The average cellular CM-DCF fluorescence intensity is considered an indirect measure of cellular ROS levels.

(19) The effect of the compounds on the intracellular ROS levels was measured in response to an induction of ROS by hydrogen peroxide. Primary human fibroblasts derived from a patient with mitochondrial disease were seeded at a density of 2500 cells/well in a 96-well format plate. The following day, the culture medium was replaced with 100 μl M199 medium without FBS and phenol red and containing CM-H.sub.2DCFDA at a final concentration of 5 μM (Life Technologies). The cell culture plate containing CM-H.sub.2DCFDA was placed for 20 minutes at 37° C., 5% CO.sub.2. Next, the cells were washed twice with PBS, and 100 μl M199 medium without FBS and phenol red and containing the compounds (final conc. 10 μM) was added to each well. Wells without cells were used to correct for the background fluorescence. The plate was read on a fluorescence plate reader (Fluostar Omega, BMG labtech) in a kinetic mode with a 2 min interval cycle. After 4 cycles, H.sub.2O.sub.2 (final concentration 100 μM) was added using onboard injectors and the fluorescence measurement was resumed for 1 hour. After background correction the fluorescence intensities were plotted as a function of time.

(20) Results: As depicted in FIG. 3, the addition of H.sub.2O.sub.2 led to a significant increase in cellular CM-DCF fluorescence. CM-DCF fluorescence intensity is a measure of cellular ROS levels, indicating that H.sub.2O.sub.2 increased intracellular ROS levels. This H.sub.2O.sub.2-mediated induction of cellular ROS levels was slightly diminished after the addition of the commercially available compound Idebenone from Santhera or after the addition of EPI743 from Edison pharmaceutical. The addition of S,R—X.sup.II further damped the H.sub.2O.sub.2-mediated induction of ROS levels. Strikingly the compound S,R—X, which is the corresponding open form of S,R—X.sup.II, was significantly more potent than S,R—X.sup.II (and EPI743 and Idebenone) in limiting H.sub.2O.sub.2-mediated induction of ROS levels (FIG. 3).

Example 4. Effect of the Compounds on Intracellular Superoxide Production

(21) Methods: HEt (Hydroethidine) is a non-fluorescent compound, which can enter the cells freely. There it is oxidized by superoxide to its fluorescent products E.sup.+ and 2OHE.sup.+, which accumulate in negatively charged cellular compartments (i.e. nucleus and mitochondria). E.sup.++2OHE.sup.+ fluorescence is thus considered a measure of superoxide production within the cell.

(22) The effect of the compounds on the intracellular superoxide levels was measured. Primary human fibroblasts obtained from a patient with mitochondrial disease were seeded at a density of 3000 cells/well in a 96-well format. The following day, the culture medium was replaced with 100 μl medium containing the compounds at different concentrations. After approximately 24 hours the cells were incubated with 100 μL medium without FBS and phenol red and containing HEt at a final concentration 10 μM (Life Technologies). The cell culture plate containing HEt was placed for 10 minutes at 37° C., 5% CO.sub.2. Next, the cells were washed twice with medium, placed in culture medium without FBS and phenol red and visualized by fluorescence microscopy (BD Pathway 855, BDBiosciences). From the obtained images superoxide levels were analysed using using Image Pro plus (Media Cybernetics) software.

(23) Results: Selected compounds were tested for their superoxide scavenging capabilities. As indicated in FIG. 4A, the compounds S,R—X.sup.II, EPI743 (Edison pharmaceutical) and Idebenone (Santhera) demonstrated only a limited ability to scavenge superoxide. In contrast the compound S,R—X, which is the corresponding open-form of S,R—X.sup.II, significantly lowered superoxide levels (FIG. 4A). In fact as indicated in FIG. 4A-E, each of the tested open-form compounds was more potent than the corresponding closed-form compound in scavenging superoxide, especially at higher concentrations. Hence, the open-form compounds are more potent than their closed counterparts in scavenging intracellular superoxide.