# Patent

## Title
Redox-active therapeutics for treatment of mitochondrial diseases and other conditions and modulation of energy biomarkers

## Abstract

Methods of treating or suppressing mitochondrial diseases, such as Friedreich's ataxia (FRDA), Leber's Hereditary Optic Neuropathy (LHON), mitochondrial myopathy, encephalopathy, lactacidosis, stroke (MELAS), or Kearns-Sayre Syndrome (KSS) are disclosed, as well as compounds useful in the methods of the invention, such as alpha-tocopherol quinone. Methods and compounds useful in treating other disorders are also disclosed. Energy biomarkers useful in assessing the metabolic state of a subject and the efficacy of treatment are also disclosed. Methods of modulating, normalizing, or enhancing energy biomarkers, as well as compounds useful for such methods, are also disclosed.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the priority benefit of U.S. Provisional Application No. 60/686,826, filed on Jun. 1, 2005, U.S. Provisional Application No. 60/701,815, filed Jul. 21, 2005, and U.S. Provisional Application No. 60/776,028, filed Feb. 22, 2006. The entire contents of those applications are hereby incorporated by reference herein.


TECHNICAL FIELD
The application discloses compositions and methods useful for treatment or suppression of diseases due to mitochondrial disorders, such as Friedreich's ataxia, Leber's Hereditary Optic Neuropathy, Kearns-Sayre Syndrome, and mitochondrial myopathy, encephalopathy, lactacidosis, stroke, and for modulating energy biomarkers in a subject.
BACKGROUND
Mitochondria are organelles in eukaryotic cells, popularly referred to as the “powerhouse” of the cell. The molecule adenosine triphosphate (ATP) functions as an energy “currency” or energy carrier in the cell, and eukaryotic cells derive the majority of their ATP from biochemical processes carried out by mitochondria. These biochemical processes include the citric acid cycle (the tricarboxylic acid cycle, or Kreb's cycle), which generates reduced nicotinamide adenine dinucleotide (NADH+H+) from oxidized nicotinamide adenine dinucleotide (NAD+), and oxidative phosphorylation, during which NADH+H+ is oxidized back to NAD+. (The citric acid cycle also reduces flavin adenine dinucleotide, or FAD, to FADH2; FADH2 also participates in oxidative phosphorylation.)
The electrons released by oxidation of NADH+H+ are shuttled down a series of protein complexes (Complex I, Complex II, Complex III, and Complex IV) known as the respiratory chain. These complexes are embedded in the inner membrane of the mitochondrion. Complex IV, at the end of the chain, transfers the electrons to oxygen, which is reduced to water. The energy released as these electrons traverse the complexes is used to generate a proton gradient across the inner membrane of the mitochondrion, which creates an electrochemical potential across the inner membrane. Another protein complex, Complex V (which is not directly associated with Complexes I, II, III and IV) uses the energy stored by the electrochemical gradient to convert ADP into ATP.
The citric acid cycle and oxidative phosphorylation are preceded by glycolysis, in which a molecule of glucose is broken down into two molecules of pyruvate, with net generation of two molecules of ATP per molecule of glucose. The pyruvate molecules then enter the mitochondria, where they are completely oxidized to CO2 and H2O via oxidative phosphorylation (the overall process is known as aerobic respiration). The complete oxidation of the two pyruvate molecules to carbon dioxide and water yields about at least 28-29 molecules of ATP, in addition to the 2 molecules of ATP generated by transforming glucose into two pyruvate molecules. If oxygen is not available, the pyruvate molecule does not enter the mitochondria, but rather is converted to lactate, in the process of anaerobic respiration.
The overall net yield per molecule of glucose is thus approximately at least 30-31 ATP molecules. ATP is used to power, directly or indirectly, almost every other biochemical reaction in the cell. Thus, the extra (approximately) at least 28 or 29 molecules of ATP contributed by oxidative phosphorylation during aerobic respiration are critical to the proper functioning of the cell. Lack of oxygen prevents aerobic respiration and will result in eventual death of almost all aerobic organisms; a few organisms, such as yeast, are able to survive using either aerobic or anaerobic respiration.
When cells in an organism are temporarily deprived of oxygen, anaerobic respiration is utilized until oxygen again becomes available or the cell dies. The pyruvate generated during glycolysis is converted to lactate during anaerobic respiration. The buildup of lactic acid is believed to be responsible for muscle fatigue during intense periods of activity, when oxygen cannot be supplied to the muscle cells. When oxygen again becomes available, the lactate is converted back into pyruvate for use in oxidative phosphorylation.
Genetic defects in the proteins making up the respiratory chain lead to severe disease states. One such disease is Friedreich's ataxia (FRDA or FA). Friedreich's ataxia is an autosomal recessive neurodegenerative and cardiodegenerative disorder caused by decreased levels of the protein frataxin. Frataxin is important for the assembly, of iron-sulfur clusters in mitochondrial respiratory-chain complexes. Estimates of the prevalence of FRDA in the United States range from 1 in every 22,000-29,000 people (see World-Wide-Web address .nlm.nih.gov/medlineplus/ency/article/001411.htm) to 1 in 50,000 people (World-Wide-Web address .umc-cares.org/health_info/ADAM/Articles/001411.asp). The disease causes the progressive loss of voluntary motor coordination (ataxia) and cardiac complications. Symptoms typically begin in childhood, and the disease progressively worsens as the patient grows older; patients eventually become wheelchair-bound due to motor disabilities.
Another disease linked to mitochondrial dysfunction is Leber's Hereditary Optic Neuropathy (LHON). The disease is characterized by blindness which occurs on average between 27 and 34 years of age (World-Wide-Web address .ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=535000); blindness can develop in both eyes simultaneously, or sequentially (one eye will develop blindness, followed by the other eye two months later on average). Other symptoms may also occur, such as cardiac abnormalities and neurological complications.
Yet another devastating syndrome resulting from mitochondrial defects is mitochondrial myopathy, encephalopathy, lactacidosis, and stroke (MELAS). The disease can manifest itself in infants, children, or young adults. Strokes, accompanied by vomiting and seizures, are one of the most serious symptoms; it is postulated that the metabolic impairment of mitochondria in certain areas of the brain is responsible for cell death and neurological lesions, rather than the impairment of blood flow as occurs in ischemic stroke. Other severe complications, including neurological symptoms, are often present, and elevated levels of lactic acid in the blood occur.
Another mitochondrial disease is Keams-Sayre Syndrome (KSS). KSS is characterized by a triad of features including: (1) typical onset in persons younger than age 20 years; (2) chronic, progressive, external ophthalmoplegia; and (3) pigmentary degeneration of the retina. In addition, KSS may include cardiac conduction defects, cerebellar ataxia, and raised cerebrospinal fluid (CSF) protein levels (e.g., >100 mg/dL). Additional features associated with KSS may include myopathy, dystonia, endocrine abnormalities (e.g., diabetes, growth retardation or short stature, and hypoparathyroidism), bilateral sensorineural deafness, dementia, cataracts, and proximal renal tubular acidosis. Thus, KSS may affect many organ systems.
The four diseases above appear to be caused by defects in complex I of the respiratory chain. Electron transfer from complex I to the remainder of the respiratory chain is mediated by the compound coenzyme Q (also known as ubiquinone). Oxidized coenzyme Q (CoQox or ubiquinone) is reduced by complex I to reduced coenzyme Q (CoQred or ubiquinol). The reduced coenzyme Q then transfers its electrons to complex III of the respiratory chain (skipping over complex II), where it is re-oxidized to CoQox (ubiquinone). CoQox can then participate in further iterations of electron transfer.
Very few treatments are available for patients suffering from these diseases. Recently, the compound idebenone has been proposed for treatment of Friedreich's ataxia. While the clinical effects of idebenone have been relatively modest, the complications of mitochondrial diseases can be so severe that even marginally useful therapies are preferable to the untreated course of the disease. Another compound, MitoQ, has been proposed for treating mitochondrial disorders (see U.S. Patent Application Publication No. 2005/0043553); clinical results for MitoQ have not yet been reported. For KSS, administration of coenzyme Q10 (CoQ10) and vitamin supplements have shown only transient beneficial effects in individual cases.
Accordingly, there is a serious and unmet need for effective treatments of mitochondrial disorders, such as Friedreich's ataxia, Leber's hereditary optic neuropathy, MELAS, and Keams-Sayre Syndrome.
The ability to adjust biological production of energy has applications beyond the diseases described above. Various other disorders can result in suboptimal levels of energy biomarkers (sometimes also referred to as indicators of energetic function), such as ATP levels. Treatments for these disorders are also needed, in order to modulate one or more energy biomarkers to improve the health of the patient. In other applications, it can be desirable to modulate certain energy biomarkers away from their normal values in an individual that is not suffering from disease. For example, if an individual is undergoing an extremely strenuous undertaking, it can be desirable to raise the level of ATP in that individual.
DISCLOSURE OF THE INVENTION
In one embodiment, the invention embraces compounds of formula I/III:




where Rh is selected from the group consisting of:





where the * indicates the point of attachment of Rh to the remainder of the molecule;

where R1, R2, and R3 are independently selected from —C1-C4 alkyl, —C1-C4 haloalkyl, —CN, —F, —Cl, —Br, and —I, with the proviso that at least one of R1, R2, and R3 is not methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.

In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula I/III as described above.
In another embodiment, the invention embraces compounds of formula I:




where R1, R2, and R3 are independently selected from —C1-C4 alkyl, —C1-C4 haloalkyl, —CN, —F, —Cl, —Br, and —I, with the proviso that at least one of R1, R2, and R3 is not methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.

In another embodiment, the invention embraces compounds of formula Ia:




where R1, R2, and R3 are independently selected from —C1-C4 alkyl, with the proviso that at least one of R1, R2, and R3 is not methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.

In another embodiment, the invention embraces compounds of formula Ia, where R1 is independently selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R1 to the remainder of the molecule can be at any location on the alkyl fragment; where R2 is independently selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R2 to the remainder of the molecule can be at any location on the alkyl fragment; and where R3 is independently selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R3 to the remainder of the molecule can be at any location on the alkyl fragment; with the proviso that at least one of R1, R2, and R3 is not methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula Ia where R1, R2, and R3 are independently selected from methyl, ethyl, n-propyl, and n-butyl, with the proviso that at least one of R1, R2, and R3 is not methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula Ia where R1, R2, and R3 are independently selected from C2-C4 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula Ia where R1, R2, and R3 are independently selected from C2-C4 n-alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula Ia, where R1 is independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R1 to the remainder of the molecule can be at any location on the alkyl fragment; where R2 is independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R2 to the remainder of the molecule can be at any location on the alkyl fragment; and where R3 is independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R3 to the remainder of the molecule can be at any location on the alkyl fragment; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula Ia wherein any one of R1, R2, and R3 is methyl and the remaining groups are independently selected from C2-C4 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. The C2-C4 alkyl groups are independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of the C2-C4 alkyl group to the remainder of the molecule can be at any location on the alkyl fragments.
In another embodiment, the invention embraces compounds of formula Ia wherein any two of R1, R2, and R3 are methyl and the remaining group is independently selected from C2-C4 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. The C2-C4 alkyl group is independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of the C2-C4 alkyl group to the remainder of the molecule can be at any location on the alkyl fragment.
In another embodiment, the invention embraces compounds of the formula Ib:




where R1, R2, and R3 are as defined above for formula I, formula Ia, and all embodiments of formula Ia.

In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula I, formula Ia, or formula Ib, or of the embodiments of formula I, formula Ia, or formula Ib, as described above.
In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula II:




where R11, R12, and R13 are independently selected from H, —C1-C4 alkyl, —C1-C4 haloalkyl, —CN, —F, —Cl, —Br, and —I, with the proviso that if any of R11, R12, or R13 is H, then at least one of the other two substituents is neither H nor methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. In one embodiment, R11, R12, and R13 are all methyl. In another embodiment, at least one of R11, R12, and R13 is not methyl.

In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of alpha-tocopherol quinone.
In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula IIa:




where R11 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R11 to the remainder of the molecule can be at any location on the alkyl fragment; where R12 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R12 to the remainder of the molecule can be at any location on the alkyl fragment; and where R13 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R13 to the remainder of the molecule can be at any location on the alkyl fragment; with the proviso that if any of R11, R12, or R13 is H, then at least one of the other two substituents is neither H nor methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. In one embodiment, R11, R12, and R13 are all methyl. In another embodiment, at least one of R11, R12, and R13 is not methyl.

In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula IIa where R11, R12, and R13 are independently selected from H, methyl, ethyl, n-propyl, and n-butyl; with the proviso that if any of R11, R12, or R13 is H, then at least one of the other two substituents is neither H nor methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula IIa where R11, R12, and R13 are independently selected from —C1-C4 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula IIa where R11, R12, and R13 are independently selected from —C1-C4 n-alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula IIb:




where R11, R12, and R13 are as described above for formula II or formula IIa. In one embodiment, R11, R12, and R13 are all methyl. In another embodiment, at least one of R11, R12, and R13 is not methyl.

In another embodiment, the invention embraces compounds of formula III:




where R1, R2, and R3 are independently selected from —C1-C4 alkyl, —C1-C4 haloalkyl, —CN, —F, —Cl, —Br, and —I, with the proviso that at least one of R1, R2, and R3 is not methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.

In another embodiment, the invention embraces compounds of formula IIIa:




where R1, R2, and R3 are independently selected from —C1-C4 alkyl, with the proviso that at least one of R1, R2, and R3 is not methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.

In another embodiment, the invention embraces compounds of formula IIIa, where R1 is independently selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R1 to the remainder of the molecule can be at any location on the alkyl fragment; where R2 is independently selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R2 to the remainder of the molecule can be at any location on the alkyl fragment; and where R3 is independently selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R3 to the remainder of the molecule can be at any location on the alkyl fragment; with the proviso that at least one of R1, R2, and R3 is not methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula IIIa where R1, R2, and R3 are independently selected from methyl, ethyl, n-propyl, and n-butyl, with the proviso that at least one of R1, R2, and R3 is not methyl; and all salts, stereo isomers, mixtures of stereoisomers, pro drugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula IIIa where R1, R2, and R3 are independently selected from C2-C4 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula IIIa where R1, R2, and R3 are independently selected from C2-C4 n-alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula IIIa, where R1 is independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R1 to the remainder of the molecule can be at any location on the alkyl fragment; where R2 is independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R2 to the remainder of the molecule can be at any location on the alkyl fragment; and where R3 is independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R3 to the remainder of the molecule can be at any location on the alkyl fragment; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula IIIa wherein any one of R1, R2, and R3 is methyl and the remaining groups are independently selected from C2-C4 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. The C2-C4 alkyl groups are independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of the C2-C4 alkyl group to the remainder of the molecule can be at any location on the alkyl fragments.
In another embodiment, the invention embraces compounds of formula IIIa wherein any two of R1, R2, and R3 are methyl and the remaining group is independently selected from C2-C4 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. The C2-C4 alkyl group is independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of the C2-C4 alkyl group to the remainder of the molecule can be at any location on the alkyl fragment.
In another embodiment, the invention embraces compounds of the formula IIIb:




where R1, R2, and R3 are as defined above for formula III, formula IIIa, and all embodiments of formula IIIa.

In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula III, formula IIIa, or formula IIIb, or of the embodiments of formula III, formula IIIa, or formula IIIb, as described above.
In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula IV:




where R11, R12, and R13 are independently selected from H, —C1-C4 alkyl, —C1-C4 haloalkyl, —CN, —F, —Cl, —Br, and —I, with the proviso that if any of R11, R12, or R13 is H, then at least one of the other two substituents is neither H nor methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. In one embodiment, R11, R12, and R13 are all methyl. In another embodiment, at least one of R11, R12, and R13 is not methyl.

In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula IVa:




where R11 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R11 to the remainder of the molecule can be at any location on the alkyl fragment; where R12 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R12 to the remainder of the molecule can be at any location on the alkyl fragment; and where R13 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R13 to the remainder of the molecule can be at any location on the alkyl fragment; with the proviso that if any of R11, R12, or R13 is H, then at least one of the other two substituents is neither H nor methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. In one embodiment, R11, R12, and R13 are all methyl. In another embodiment, at least one of R11, R12, and R13 is not methyl.

In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula IVa where R11, R12, and R13 are independently selected from H, methyl, ethyl, n-propyl, and n-butyl; with the proviso that if any of R11, R12, or R13 is H, then at least one of the other two substituents is neither H nor methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula IVa where R11, R12, and R13 are independently selected from —C1-C4 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula IVa where R11, R12, and R13 are independently selected from —C1-C4 n-alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula IVb:




where R11, R12, and R13 are as described above for formula IV or formula IVa. In one embodiment, R11, R12, and R13 are all methyl. In another embodiment, at least one of R11, R12, and R13 is not methyl.

In another embodiment, the invention embraces compounds of the formula V:




where R1, R2, and R3 are independently selected from —C1-C4 alkyl, —C1-C4 haloalkyl, —CN, —F, —Cl, —Br, and —I, with the proviso that at least one of R1, R2, and R3 is not methyl; R8 is independently selected from H and —C1-C6 alkyl optionally substituted with —OR9 or —N(R9)2, where each R9 is independently selected from H and —C1-C6 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. In another embodiment, R8 is independently selected from H and —C1-C6 alkyl optionally substituted with —OR9.

In another embodiment, the invention embraces compounds of formula Va:




where R1, R2, and R3 are independently selected from —C1-C4 alkyl, with the proviso that at least one of R1, R2, and R3 is not methyl; R8 is independently selected from H and —C1-C6 alkyl optionally substituted with —OR9 or —N(R9)2, where each R9 is independently selected from H and —C1-C6 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. In another embodiment, R8 is independently selected from H and —C1-C6 alkyl optionally substituted with —OR9.

In another embodiment, the invention embraces compounds of formula Va, where R1 is independently selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R1 to the remainder of the molecule can be at any location on the alkyl fragment; where R2 is independently selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R2 to the remainder of the molecule can be at any location on the alkyl fragment; and where R3 is independently selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R3 to the remainder of the molecule can be at any location on the alkyl fragment; with the proviso that at least one of R1, R2, and R3 is not methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula Va where R1, R2, and R3 are independently selected from methyl, ethyl, n-propyl, and n-butyl, with the proviso that at least one of R1, R2, and R3 is not methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula Va where R1, R2, and R3 are independently selected from methyl, ethyl, n-propyl, and n-butyl, with the proviso that when R1, R2, and R3 are all methyl, then R8 is C5 alkyl or C6 alkyl optionally substituted with —OR9 or —N(R9)2; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. In another embodiment, R8 is independently selected from C5 alkyl or C6 alkyl optionally substituted with —OR9.
In another embodiment, the invention embraces compounds of formula Va where R1, R2, and R3 are independently selected from C2-C4 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula Va where R1, R2, and R3 are independently selected from C2-C4 n-alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula Va, where R1 is independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R1 to the remainder of the molecule can be at any location on the alkyl fragment; where R2 is independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R2 to the remainder of the molecule can be at any location on the alkyl fragment; and where R3 is independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R3 to the remainder of the molecule can be at any location on the alkyl fragment; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula Va wherein any one of R1, R2, and R3 is methyl and the remaining groups are independently selected from C2-C4 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. The C2-C4 alkyl groups are independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of the C2-C4 alkyl group to the remainder of the molecule can be at any location on the alkyl fragments.
In another embodiment, the invention embraces compounds of formula Va wherein any two of R1, R2, and R3 are methyl and the remaining group is independently selected from C2-C4 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. The C2-C4 alkyl group is independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of the C2-C4 alkyl group to the remainder of the molecule can be at any location on the alkyl fragment.
In another embodiment, the invention embraces compounds of the formula Vb:




where R1, R2, R3, and R8 are as defined above for formula V, formula Va, and all embodiments of formula Va.

In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula V, formula Va, or formula Vb, or of any of the embodiments of formula V, formula Va, or formula Vb, as described above.
In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula VI:




where R11, R12, and R13 are independently selected from H, —C1-C4 alkyl, —C1-C4 haloalkyl, —CN, —F, —Cl, —Br, and —I; R8 is independently selected from H and —C1-C6 alkyl optionally substituted with —OR9 or —N(R9)2, where each R9 is independently selected from H and —C1-C6 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. In one embodiment, R11, R12, and R13 are all methyl. In another embodiment, at least one of R11, R12, and R13 is not methyl. In another embodiment, R8 is independently selected from H and —C1-C6 alkyl optionally substituted with —OR9. In another embodiment, the proviso is added that if any of R11, R12, or R13 is H, then at least one of the other two substituents is neither H nor methyl.

In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula VIa:




where R11 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R11 to the remainder of the molecule can be at any location on the alkyl fragment; where R12 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R12 to the remainder of the molecule can be at any location on the alkyl fragment; and where R13 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R13 to the remainder of the molecule can be at any location on the alkyl fragment; R8 is independently selected from H and —C1-C6 alkyl optionally substituted with —OR9 or —N(R9)2, where each R9 is independently selected from H and —C1-C6 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. In one embodiment, R11, R12, and R13 are all methyl. In another embodiment, at least one of R11, R12, and R13 is not methyl. In another embodiment, R8 is independently selected from H and —C1-C6 alkyl optionally substituted with —OR9. In another embodiment, the proviso is added that if any of R11, R12, or R13 is H, then at least one of the other two substituents is neither H nor methyl.

In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula VIa where R11, R12, and R13 are independently selected from H, methyl, ethyl, n-propyl, and n-butyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula VIa where R11, R12, and R13 are independently selected from —C1-C4 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula VIa where R11, R12, and R13 are independently selected from —C1-C4 n-alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula VIb:




where R11, R12, and R13 are as described above for formula VI or formula VIa. In one embodiment, R11, R12, and R13 are all methyl. In another embodiment, at least one of R11, R12, and R13 is not methyl.

In another embodiment, the invention embraces compounds of formula VII-O, formula VII-R, formula VIII-O, formula VIII-R, formula IX-O, or formula IX-R:




where R1, R2, and R3 are independently selected from —C1-C4 alkyl, with the proviso that at least one of R1, R2, and R3 is not methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.

In another embodiment, the invention embraces compounds of formula VII-O, formula VII-R, formula VIII-O, formula VIII-R, formula IX-O, or formula IX-R, where R1 is independently selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R1 to the remainder of the molecule can be at any location on the alkyl fragment; where R2 is independently selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R2 to the remainder of the molecule can be at any location on the alkyl fragment; and where R3 is independently selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R3 to the remainder of the molecule can be at any location on the alkyl fragment; with the proviso that at least one of R1, R2, and R3 is not methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula VII-O, formula VII-R, formula VIII-O, formula VIII-R, formula IX-O, or formula IX-R where R1, R2, and R3 are independently selected from methyl, ethyl, n-propyl, and n-butyl, with the proviso that at least one of R1, R2, and R3 is not methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula VII-O, formula VII-R, formula VIII-O, formula VIII-R, formula IX-O, or formula IX-R where R1, R2, and R3 are independently selected from C2-C4 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula VII-O, formula VII-R, formula VIII-O, formula VIII-R, formula IX-O, or formula IX-R where R1, R2, and R3 are independently selected from C2-C4 n-alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula VII-O, formula VII-R, formula VIII-O, formula VIII-R, formula IX-O, or formula IX-R, where R1 is independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R1 to the remainder of the molecule can be at any location on the alkyl fragment; where R2 is independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R2 to the remainder of the molecule can be at any location on the alkyl fragment; and where R3 is independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R3 to the remainder of the molecule can be at any location on the alkyl fragment; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces compounds of formula VII-O, formula VII-R, formula VIII-O, formula VIII-R, formula IX-O, or formula IX-R wherein any one of R1, R2, and R3 is methyl and the remaining groups are independently selected from C2-C4 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. The C2-C4 alkyl groups are independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of the C2-C4 alkyl group to the remainder of the molecule can be at any location on the alkyl fragments.
In another embodiment, the invention embraces compounds of formula VII-O, formula VII-R, formula VIII-O, formula VIII-R, formula IX-O, or formula IX-R wherein any two of R1, R2, and R3 are methyl and the remaining group is independently selected from C2-C4 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. The C2-C4 alkyl group is independently selected from ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of the C2-C4 alkyl group to the remainder of the molecule can be at any location on the alkyl fragment.
In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula X-O, formula X-R, formula XI-O, formula XI-R, formula XII-O, or formula XII-R:




where R11, R12, and R13 are independently selected from H, —C1-C4 alkyl, —C1-C4 haloalkyl, —CN, —F, —Cl, —Br, and —I, with the proviso that if any of R11, R12, or R13 is H, then at least one of the other two substituents is neither H nor methyl; and all salts, stereo isomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. In one embodiment, R11, R12, and R13 are all methyl. In another embodiment, at least one of R11, R12, and R13 is not methyl.

In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula X-O, formula X-R, formula XI-O, formula XI-R, formula XII-O, or formula XII-R:




where R11 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R11 to the remainder of the molecule can be at any location on the alkyl fragment; where R12 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R12 to the remainder of the molecule can be at any location on the alkyl fragment; and where R13 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, cyclopropyl-methyl, and methyl-cyclopropane, where the point of attachment of R13 to the remainder of the molecule can be at any location on the alkyl fragment; with the proviso that if any of R11, R12, or R13 is H, then at least one of the other two substituents is neither H nor methyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. In one embodiment, R11, R12, and R13 are all methyl. In another embodiment, at least one of R11, R12, and R13 is not methyl.

In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula X-O, formula X-R, formula XI-O, formula XI-R, formula XII-O, or formula XII-R where R11, R12, and R13 are independently selected from methyl, ethyl, n-propyl, and n-butyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula X-O, formula X-R formula XI-O, formula XI-R, formula XII-O, or formula XII-R where R11, R12, and R13 are independently selected from —C1-C4 alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formula X-O, formula X-R, formula XI-O, formula XI-R, formula XII-O, or formula XII-R where R11, R12, and R13 are independently selected from —C1-C4 n-alkyl; and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof.
In another embodiment, the invention embraces a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers, by administering a therapeutically effective amount or effective amount of one or more compounds of formulas VII-i, VIII-i, or IX-i, and all salts, stereoisomers, mixtures of stereoisomers, prodrugs, metabolites, solvates, and hydrates thereof. Compound VII-i is:




which is α-tocotrienolquinone (alternatively named as 2-(3-hydroxy-3,7,11,15-tetramethyl-6,10,14-hexadecatrienyl)-3,5,6-trimethyl-2,5-cyclohexadiene-1,4-dione or 2-(3-hydroxy-3,7,11,15-tetramethyl-6,10,14-hexadecatrienyl)-3,5,6-trimethyl-p-benzoquinone, CAS Registry number 14101-66-7). Compound VIII-i is:





which is 2,3,5-trimethyl-6-(3,7,11,15-tetramethylhexadecyl)-2,5-cyclohexadiene-1,4-dione (alternatively named 6-(3,7,11,15-tetramethylcetyl)-p-pseudocumoquinone, CAS Registry number 75917-94-1). Compound IX-i is:





which is 2,3,5-trimethyl-6-(3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl)-2,5-cyclohexadiene-1,4-dione (alternatively named trimethyl(3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl)-p-benzoquinone, CAS Registry Number 65647-38-3).

In other embodiments, including any of the foregoing embodiments, the mitochondrial disorder is selected from the group consisting of inherited mitochondrial diseases; Myoclonic Epilepsy with Ragged Red Fibers (MERRF); Mitochondrial Myopathy, Encephalopathy, Lactacidosis, Stroke (MELAS); Leber's Hereditary Optic Neuropathy (LHON); Leigh Disease; Keams-Sayre Syndrome (KSS); Friedreich's Ataxia (FA); other myopathies; cardiomyopathy; encephalomyopathy; renal tubular acidosis; neurodegenerative diseases; Parkinson's disease; Alzheimer's disease; amyotrophic lateral sclerosis (ALS); motor neuron diseases; other neurological diseases; epilepsy; genetic diseases; Huntington's Disease; mood disorders; schizophrenia; bipolar disorder; age-associated diseases; macular degeneration; diabetes; and cancer.
In another embodiment, including any of the foregoing embodiments, the mitochondrial disorder is selected from the group consisting of inherited mitochondrial diseases; Myoclonic Epilepsy with Ragged Red Fibers (MERRF); Mitochondrial Myopathy, Encephalopathy, Lactacidosis, Stroke (MELAS); Leber's Hereditary Optic Neuropathy (LHON); Leigh Disease; Keams-Sayre Syndrome (KSS); and Friedreich's Ataxia (FA).
In another embodiment of the invention, including any of the foregoing embodiments, the mitochondrial disorder is Friedreich's ataxia (FRDA). In another embodiment of the invention, the mitochondrial disorder is Leber's Hereditary Optic Neuropathy (LHON). In another embodiment of the invention, the mitochondrial disorder is mitochondrial myopathy, encephalopathy, lactacidosis, stroke (MELAS). In another embodiment of the invention, the mitochondrial disorder is Kearns-Sayre Syndrome (KSS). In another embodiment of the invention, the mitochondrial disorder is Myoclonic Epilepsy with Ragged Red Fibers (MERRF). In another embodiment of the invention, the mitochondrial disorder is Parkinson's disease.
In another embodiment of the invention, including any of the foregoing embodiments, the compounds described herein are administered to subjects suffering from a mitochondrial disorder to modulate one or more of various energy biomarkers, including, but not limited to, lactic acid (lactate) levels, either in whole blood, plasma, cerebrospinal fluid, or cerebral ventricular fluid; pyruvic acid (pyruvate) levels, either in whole blood, plasma, cerebrospinal fluid, or cerebral ventricular fluid; lactate/pyruvate ratios, either in whole blood, plasma, cerebrospinal fluid, or cerebral ventricular fluid; phosphocreatine levels, NADH (NADH+H+) or NADPH (NADPH+H+) levels; NAD or NADP levels; ATP levels; reduced coenzyme Q (CoQred) levels; oxidized coenzyme Q (CoQox) levels; total coenzyme Q (CoQtot) levels; oxidized cytochrome C levels; reduced cytochrome C levels; oxidized cytochrome C/reduced cytochrome C ratio; acetoacetate levels; beta-hydroxy butyrate levels; acetoacetate/beta-hydroxy butyrate ratio; 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels; levels of reactive oxygen species; oxygen consumption (VO2), carbon dioxide output (VCO2), respiratory quotient (VCO2/VO2), and to modulate exercise intolerance (or conversely, modulate exercise tolerance) and to modulate anaerobic threshold. Energy biomarkers can be measured in whole blood, plasma, cerebrospinal fluid, cerebroventricular fluid, arterial blood, venous blood, or any other body fluid, body gas, or other biological sample useful for such measurement. In one embodiment, the levels are modulated to a value within about 2 standard deviations of the value in a healthy subject. In another embodiment, the levels are modulated to a value within about 1 standard deviation of the value in a healthy subject. In another embodiment, the levels in a subject are changed by at least about 10% above or below the level in the subject prior to modulation. In another embodiment, the levels are changed by at least about 20% above or below the level in the subject prior to modulation. In another embodiment, the levels are changed by at least about 30% above or below the level in the subject prior to modulation. In another embodiment, the levels are changed by at least about 40% above or below the level in the subject prior to modulation. In another embodiment, the levels are changed by at least about 50% above or below the level in the subject prior to modulation. In another embodiment, the levels are changed by at least about 75% above or below the level in the subject prior to modulation. In another embodiment, the levels are changed by at least about 100% above or at least about 90% below the level in the subject prior to modulation.
In another embodiment, including any of the foregoing embodiments, the subject or subjects in which a method of treating or suppressing a mitochondrial disorder, modulating one or more energy biomarkers, normalizing one or more energy biomarkers, or enhancing one or more energy biomarkers is performed is/are selected from the group consisting of subjects undergoing strenuous or prolonged physical activity; subjects with chronic energy problems; subjects with chronic respiratory problems; pregnant females; pregnant females in labor; neonates; premature neonates; subjects exposed to extreme environments; subjects exposed to hot environments; subjects exposed to cold environments; subjects exposed to environments with lower-than-average oxygen content; subjects exposed to environments with higher-than-average carbon dioxide content; subjects exposed to environments with higher-than-average levels of air pollution; airline travelers; flight attendants; subjects at elevated altitudes; subjects living in cities with lower-than-average air quality; subjects working in enclosed environments where air quality is degraded; subjects with lung diseases; subjects with lower-than-average lung capacity; tubercular patients; lung cancer patients; emphysema patients; cystic fibrosis patients; subjects recovering from surgery; subjects recovering from illness; elderly subjects; elderly subjects experiencing decreased energy; subjects suffering from chronic fatigue; subjects suffering from chronic fatigue syndrome; subjects undergoing acute trauma; subjects in shock; subjects requiring acute oxygen administration; subjects requiring chronic oxygen administration; or other subjects with acute, chronic, or ongoing energy demands who can benefit from enhancement of energy biomarkers.
In another embodiment, the invention embraces one or more compounds of formula I, Ia, Ib, II, IIa, IIb, III, IIIa, IIIb, IV, IVa, IVb, V, Va, Vb, VI, VIa, VIb, VII-O, VII-R, VIII-O, VIII-R, IX-O, IX-R, X-O, X-R, XI-O, XI-R, XII-O, and/or XII-R, in combination with a pharmaceutically acceptable excipient, carrier, or vehicle.
In another embodiment, the invention embraces the use of one or more compounds of formula I, Ia, Ib, II, IIa, IIb, III, IIIa, IIIb, IV, IVa, IVb, V, Va, Vb, VI, VIa, VIb, VII-O, VII-R, VIII-O, VIII-R, IX-O, IX-R, X-O, X-R, XI-O, XI-R, XII-O, and/or XII-R in therapy. In another embodiment, the invention embraces the use of one or more compounds of formula I, Ia, Ib, II, IIa, IIb, III, IIIa, IIIb, IV, IVa, IVb, V, Va, Vb, VI, VIa, VIb, VII-O, VII-R, VIII-O, VIII-R, IX-O, IX-R, X-O, X-R, XI-O, XI-R, XII-O, and/or XII-R in the therapy of mitochondrial disease. In another embodiment, the invention embraces the use of one or more compounds of formula I, Ia, Ib, II, IIa, IIb, III, IIIa, IIIb, IV, IVa, IVb, V, Va, Vb, VI, VIa, VIb, VII-O, VII-R, VIII-O, VIII-R, IX-O, IX-R, X-O, X-R, XI-O, XI-R, XII-O, and/or XII-R in the manufacture of a medicament for use in therapy of mitochondrial disease.
For all of the compounds and methods described above, the quinone form can also be used in its reduced (hydroquinone) form when desired. Likewise, the hydroquinone form can also be used in its oxidized (quinone) form when desired.
MODES FOR CARRYING OUT THE INVENTION
The invention embraces compounds useful in treating or suppressing mitochondrial disorders, and methods of using such compounds for modulation of energy biomarkers. The redox active therapeutics for treatment or suppression of mitochondrial diseases and associated aspects of the invention are described in more detail herein.
By “subject,” “individual,” or “patient” is meant an individual organism, preferably a vertebrate, more preferably a mammal, most preferably a human.
“Treating” a disease with the compounds and methods discussed herein is defined as administering one or more of the compounds discussed herein, with or without additional therapeutic agents, in order to reduce or eliminate either the disease or one or more symptoms of the disease, or to retard the progression of the disease or of one or more symptoms of the disease, or to reduce the severity of the disease or of one or more symptoms of the disease. “Suppression” of a disease with the compounds and methods discussed herein is defined as administering one or more of the compounds discussed herein, with or without additional therapeutic agents, in order to suppress the clinical manifestation of the disease, or to suppress the manifestation of adverse symptoms of the disease. The distinction between treatment and suppression is that treatment occurs after adverse symptoms of the disease are manifest in a subject, while suppression occurs before adverse symptoms of the disease are manifest in a subject. Suppression may be partial, substantially total, or total. Because many of the mitochondrial disorders are inherited, genetic screening can be used to identify patients at risk of the disease. The compounds and methods of the invention can then be administered to asymptomatic patients at risk of developing the clinical symptoms of the disease, in order to suppress the appearance of any adverse symptoms. “Therapeutic use” of the compounds discussed herein is defined as using one or more of the compounds discussed herein to treat or suppress a disease, as defined above. An “effective amount” of a compound is an amount of the compound sufficient to modulate, normalize, or enhance one or more energy biomarkers (where modulation, normalization, and enhancement are defined below). A “therapeutically effective amount” of a compound is an amount of the compound, which, when administered to a subject, is sufficient to reduce or eliminate either a disease or one or more symptoms of a disease, or to retard the progression of a disease or of one or more symptoms of a disease, or to reduce the severity of a disease or of one or more symptoms of a disease, or to suppress the clinical manifestation of a disease, or to suppress the manifestation of adverse symptoms of a disease. A therapeutically effective amount can be given in one or more administrations. An “effective amount” of a compound embraces both a therapeutically effective amount, as well as an amount effective to modulate, normalize, or enhance one or more energy biomarkers in a subject.
“Modulation” of, or to “modulate,” an energy biomarker means to change the level of the energy biomarker towards a desired value, or to change the level of the energy biomarker in a desired direction (e.g., increase or decrease). Modulation can include, but is not limited to, normalization and enhancement as defined below.
“Normalization” of, or to “normalize,” an energy biomarker is defined as changing the level of the energy biomarker from a pathological value towards a normal value, where the normal value of the energy biomarker can be 1) the level of the energy biomarker in a healthy person or subject, or 2) a level of the energy biomarker that alleviates one or more undesirable symptoms in the person or subject. That is, to normalize an energy biomarker which is depressed in a disease state means to increase the level of the energy biomarker towards the normal (healthy) value or towards a value which alleviates an undesirable symptom; to normalize an energy biomarker which is elevated in a disease state means to decrease the level of the energy biomarker towards the normal (healthy) value or towards a value which alleviates an undesirable symptom.
“Enhancement” of, or to “enhance,” energy biomarkers means to intentionally change the level of one or more energy biomarkers away from either the normal value, or the value before enhancement, in order to achieve a beneficial or desired effect. For example, in a situation where significant energy demands are placed on a subject, it may be desirable to increase the level of ATP in that subject to a level above the normal level of ATP in that subject. Enhancement can also be of beneficial effect in a subject suffering from a disease or pathology such as a mitochondrial disease, in that normalizing an energy biomarker may not achieve the optimum outcome for the subject; in such cases, enhancement of one or more energy biomarkers can be beneficial, for example, higher-than-normal levels of ATP, or lower-than-normal levels of lactic acid (lactate) can be beneficial to such a subject.
By modulating, normalizing, or enhancing the energy biomarker Coenzyme Q is meant modulating, normalizing, or enhancing the variant or variants of Coenzyme Q which is predominant in the species of interest. For example, the variant of Coenzyme Q which predominates in humans is Coenzyme Q10. If a species or subject has more than one variant of Coenzyme Q present in significant amounts (i.e., present in amounts which, when modulated, normalized, or enhanced, can have a beneficial effect on the species or subject), modulating, normalizing, or enhancing Coenzyme Q can refer to modulating, normalizing or enhancing any or all variants of Coenzyme Q present in the species or subject.
While the compounds described herein can occur and can be used as the neutral (non-salt) compound, the description is intended to embrace all salts of the compounds described herein, as well as methods of using such salts of the compounds. In one embodiment, the salts of the compounds comprise pharmaceutically acceptable salts. Pharmaceutically acceptable salts are those salts which can be administered as drugs or pharmaceuticals to humans and/or animals and which, upon administration, retain at least some of the biological activity of the free compound (neutral compound or non-salt compound). The desired salt of a basic compound may be prepared by methods known to those of skill in the art by treating the compound with an acid. Examples of inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Examples of organic acids include, but are not limited to, formic acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, sulfonic acids, and salicylic acid. Salts of basic compounds with amino acids, such as aspartate salts and glutamate salts, can also be prepared. The desired salt of an acidic compound can be prepared by methods known to those of skill in the art by treating the compound with a base. Examples of inorganic salts of acid compounds include, but are not limited to, alkali metal and alkaline earth salts, such as sodium salts, potassium salts, magnesium salts, and calcium salts; ammonium salts; and aluminum salts. Examples of organic salts of acid compounds include, but are not limited to, procaine, dibenzylamine, N-ethylpiperidine, N,N′-dibenzylethylenediamine, and triethylamine salts. Salts of acidic compounds with amino acids, such as lysine salts, can also be prepared.
The invention also includes all stereoisomers of the compounds, including diastereomers and enantiomers. The invention also includes mixtures of stereoisomers in any ratio, including, but not limited to, racemic mixtures. Unless stereochemistry is explicitly indicated in a structure, the structure is intended to embrace all possible stereoisomers of the compound depicted. If stereochemistry is explicitly indicated for one portion or portions of a molecule, but not for another portion or portions of a molecule, the structure is intended to embrace all possible stereoisomers for the portion or portions where stereochemistry is not explicitly indicated.
The compounds can be administered in prodrug form. Prodrugs are derivatives of the compounds which are themselves relatively inactive, but which convert into the active compound when introduced into the subject in which they are used, by a chemical or biological process in vivo, such as an enzymatic conversion. Suitable prodrug formulations include, but are not limited to, peptide conjugates of the compounds of the invention and esters of compounds of the inventions. Further discussion of suitable prodrugs is provided in H. Bundgaard, Design of Prodrugs, New York: Elsevier, 1985; in R. Silverman, The Organic Chemistry of Drug Design and Drug Action, Boston: Elsevier, 2004; in R. L. Juliano (ed.), Biological Approaches to the Controlled Delivery of Drugs (Annals of the New York Academy of Sciences, v. 507), New York: New York Academy of Sciences, 1987; and in E. B. Roche (ed.), Design of Biopharmaceutical Properties Through Prodrugs and Analogs (Symposium sponsored by Medicinal Chemistry Section, APhA Academy of Pharmaceutical Sciences, November 1976 national meeting, Orlando, Fla.), Washington: The Academy, 1977.
The various compounds of the invention, particularly compounds V, Va, Vb, VI, VIa, and VIb, as well as their various embodiments, can be administered either as therapeutic agents in and of themselves, or as prodrugs which will convert to other therapeutically effective or effective substances in the body.
Metabolites of the compounds are also embraced by the invention. However, metabolites of substances which occur naturally in subjects, such as metabolites of alpha-tocopherol quinone, are excluded from the claimed compounds of the invention.
“C1-C4 alkyl” is intended to embrace methyl (Me), ethyl (Et), propyl (Pr), n-propyl (nPr), isopropyl (iPr), butyl (Bu), n-butyl (nBu), isobutyl (iBu), sec-butyl (sBu), t-butyl (tBu), cyclopropyl (cyclPr), cyclobutyl (cyclBu), cyclopropyl-methyl (cyclPr-Me) and methyl-cyclopropane (Me-cyclPr), where the C1-C4 alkyl groups can be attached via any valence on the C1-C4 alkyl groups.
“Halogen” or “halo” substituents designates fluoro (—F), chloro (—Cl), bromo (—Br), and iodo (—I).
“C1-C4 haloalkyl” is intended to embrace any C1-C4 alkyl substituent having at least one halogen substituent; the halogen can be attached via any valence on the C1-C4 alkyl group. One subset of C1-C4 haloalkyl is —CF3, —CCl3, —CBr3, and —CI3. Another subset of C1-C4 haloalkyl is the subset with exactly one halogen substituent. Another subset of C1-C4 haloalkyl is the subset of C1-C4 perhaloalkyl; that is, C1-C4 alkyl with all available valences replaced by halogens. Another subset of C1-C4 haloalkyl is the subset of C1-C4 perfluoroalkyl; that is, C1-C4 alkyl with all available valences replaced by fluorines. Another subset of C1-C4 haloalkyl is the subset of C1-C4 perchloroalkyl; that is, C1-C4 alkyl with all available valences replaced by chlorines.
One compound of interest, which can be used in any of the methods of the invention, is α-tocopherol quinone. The structure of alpha-tocopherol quinone (D-α-tocopherol quinone; alpha-tocopherylquinone; 2-[(3R,7R,11R)-3-hydroxy-3,7,11,15-tetramethylhexadecyl]-3,5,6-trimethyl-2,5-cyclohexadiene-1,4-dione, CAS Registry number 7559-04-8) is:




An alternate name for alpha-tocopherol quinone is alpha-tocopherylquinone. This compound corresponds to the compound of formula IIb where R11, R12, and R13 are all methyl. In human cell culture models of FRDA, alpha-tocopherol quinone possesses an EC50 105-fold lower (i.e., 100,000 times more potent) than idebenone, the current therapeutic agent of choice for FRDA patients; see Example 2. In this same cell culture model, alpha-tocopherol quinone has an EC50 104-fold lower (i.e., 10,000 times more potent) than alpha-D-tocopherol, (2R)-3,4-dihydro-2,5,7,8-tetramethyl-2-[(4R,8R)-4,8,12-trimethyltridecyl]-2H-1-benzopyran-6-ol, a common form of vitamin E.

Another group of compounds of interest are represented by formula IIm:




where the lack of indication of stereochemistry indicates that this structure is intended to represent all eight possible stereoisomers, as there are 2 different orientations possible at the 3, 7, and 11 positions as indicated in the drawing of formula IIm. Formula IIm corresponds to formula II where R11, R12, and R13 are all methyl. The eight stereoisomers embraced by this structure drawing include: [(3R,7R,11R)-3-hydroxy-3,7,11,15-tetramethylhexadecyl]-3,5,6-trimethyl-2,5-cyclohexadiene-1,4-dione); the 3R, 7R, 11S-compound; the 3R, 7S, 11R-compound; the 3S, 7R, 11R-compound; the 3R, 7S, 11S-compound; the 3S, 7R, 11S-compound; the 3S, 7S, 11R-compound; and the 3S, 7S, 11S-compound.
Synthesis of Compounds of Formula I and Formula II
The compounds of formula I and formula II can be readily synthesized by a variety of methods. The synthesis of alpha-tocopherol quinone is detailed in several references, e.g., U.S. Pat. No. 3,406,188 (GB 1,059,155) and U.S. Pat. No. 4,310,465. The synthesis of benzoquinone-type compounds is disclosed in U.S. Pat. Nos. 5,229,385 and 4,393,075.
In several of the following methods, an oxidizing agent is used. Suitable oxidizing agents which can be used in the synthetic methods include, but are not limited to, ceric ammonium nitrate (CAN), FeCl3, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), or atmospheric oxygen (i.e., air oxidation).
Several methods can be used to make the compounds of Formulas I, Ia, Ib, II, IIa, IIb, III, IIIa, IIIb, IV, IVa, and IVb. One such method utilizes an oxysulfonium rearrangement as follows:




The starting material 4-hydroxy-2,3,6-trimethyl phenyl acetate, 51, is prepared as described in Weichet et al., Coll. Czech. Chem. Comm. 31:4598 (1966). Compound 52 is prepared and reacted with compound 51 and sulfuryl chloride as in Inoue et al., J. Org. Chem. Soc. 52:5495 (1987) to yield compound 53. Removal of the isopropyl-sulfur moiety with Raney nickel, followed by removal of the acetyl groups with lithium aluminum hydride, yields the hydroquinone compound 54 corresponding to Formula IVb where R11, R12, and R13 are methyl. Oxidation of the hydroquinone compound 54 with iron (III) chloride (Shiraishi et al., J. Med. Chem. 32:2214-2221 (1989)) yields the quinone compound 55 corresponding to Formula IIb where R11, R12, and R13 are methyl.
By using starting material of the form:




in place of 51, the entire range of compounds of Formulas I, Ia, Ib, II, IIa, IIb, III, IIIa, IIIb, IV, IVa, and IVb can be synthesized. (For the compounds of Formulas III, IIIa, IIIb, IV, IVa, and IVb, the synthesis would be completed after the Raney nickel and lithium aluminum hydride treatment, before the FeCl3 oxidation.) The dihydroquinone starting material can be prepared by a variety of processes, e.g., those described in U.S. Pat. Nos. 3,909,376, 5,132,468, and 6,303,801, and in German Patent No. DE 3818696; the acetylated dihydroquinone precursor for the synthesis above can then be prepared as in Weichet et al., Coll. Czech. Chem. Comm. 31:4598 (1966).

An alternative method for synthesizing compounds of Formulas I, Ia, Ib, II, IIa, and IIb utilizes an epoxide opening as described in Hübscher et al., Helvetica Chimica Acta 73:1068-1083 (1990).




2,5-dimethoxy-3,4,6-trimethyl benzyllithium 61 is reacted with the epoxide compound 62 to yield 1,4-dimethoxybenzene derivative 63. Subsequent oxidation with ceric ammonium nitrate (CAN) yields the compound 64 corresponding to Formula IIb where R11, R12, and R13 are methyl.
An additional method of synthesizing compounds of Formulas I, Ia, Ib, II, IIa, and IIb makes use of a Claisen rearrangement as follows, in a procedure adapted from Green et al., J. Chem. Soc. (C) 1422 (1966) and Zheng et al., J. Org. Chem. 64:156 (1999).




An allyl group is introduced onto 71 to yield 72, and then subjected to conditions that cause rearrangement to 73 (e.g., in the manner as described in Scheme 3 of Zheng et al., J. Org. Chem. 64:156 (1999)). Hydrogenation of 73 (e.g., with palladium on charcoal) then yields the propyl-substituted hydroquinone (not depicted) (e.g., for compounds of formulas III, IIIa, IIIb, IV, IVa, and IVb); subsequent oxidation with FeCl3 yields the propyl-substituted quinone 74 (e.g., for compounds of formulas I, Ia, Ib, II, IIa, and IIb).

Another method of synthesizing the compounds of Formulas I, Ia, Ib, II, IIa, and IIb, as well as synthesizing compounds of Formulas V, Va, Vb, VI, VIa, and VIb utilizes an aldehyde condensation (Adelwöhrer et al., Tetrahedron 61:9070 (2005)) as follows.




Starting with a γ-tocopherol compound 81, a fused dioxane-chroman compound 82 is formed. Hydrogenation with palladium on charcoal yields the chroman compound 83 (e.g., compounds of formulas V, Va, Vb, VI, VIa, and VIb), while ceric ammonium nitrate (CAN) oxidation then yields the quinone compounds (e.g., compounds of formulas I, Ia, Ib, II, IIa, and IIb).

A method of synthesizing compounds of the formula:




where the chemistry for conversion of duroquinone 91 into 3,6-dimethoxy-1,2,4,5-tetramethyl-1,4-cyclohexadiene 92 is described in Thomas et al., Journal of Organic Chemistry 51(22):4160 (1986); the chemistry for conversion of 3,6-dimethoxy-1,2,4,5-tetramethyl-1,4-cyclohexadiene 92 into the 3,6-dimethoxy-1-methylene lithium-2,4,5-trimethyl-1,4-cyclohexadiene 93 intermediate is described in Hübscher et al., Helvetica Chimica Acta 73(4):1068 (1990); and the chemistry for conversion of the 3,6-dimethoxy-1-alkyl-2,4,5-trimethyl-1,4-cyclohexadiene 94 into the 2-alkyl-3,5,6-trimethyl-1,4-benzoquinone 95 is described in Shiraishi et al., Journal of Medicinal Chemistry 32(9):2214 (1989). This synthesis can be easily modified to produce compounds of formula IIm:





by using the following intermediate:





where PG indicates a protecting group, such as a methyl methoxymethyl (MOM) or methoxy ethoxymethyl (MEM) group. Other suitable protecting groups, for this and other reactions described herein, are detailed in the text by Theodora W. Greene and Peter G. M. Wuts, Protective Groups in Organic Synthesis, 3rd edition, Hoboken, N.J.: Wiley-Interscience, 1999.

Another method of making compounds of the formula 95 is as follows:




where the chemistry of converting 1,4-hydroxy-2,3,5-trimethylbenzene 100 into 2,3,5-trimethyl-1,4-benzoquinone 101 is described in Pelter et al., J. Chem. Soc., Perkin Trans. 1, (16), 1891 (1993), the chemistry of converting the benzoquinone compound 102 into the 2-alkyl-3,5,6-trimethyl-1,4-benzoquinone 95 is described in Fieser et al., Journal of the American Chemical Society 64(9):2060 (1942), and the chemistry of converting the alkanoyl chloride 103 into the dialkanoyl peroxide 104 is described in Silbert et al., Journal of the American Chemical Society 81(10):2364 (1959). The alkanoyl chloride 106





can be used to prepare compounds of the formula IIm via this route. Compounds of formula I and formula II can be prepared via this route by starting with the appropriate 1,4-dihydroxy-2,3,5-substituted-1,4-benzoquinone and using the intermediate 106.

Methods suitable for making compounds of the inventions with halogen substituents on the quinone ring are depicted as follows. (See Fujishima et al. Arch. Pharm. Pharm. Med. Chem. 329:27-34 (1996) for additional information.)




2,6-dimethylquinone 111 is reduced with sodium dithionite to the hydroquinone 112, which is then reacted with 3,7,11,15-tetramethyl-3-hydroxy-1-hexadecene 113 and ZnCl2 to form the 6-chromanol 114. Conversion to the protected intermediate 115 is followed by bromination with Br2 and silver trifluoroacetate to form the bromide 116. Finally, 116 can be deprotected and oxidized with ceric ammonium nitrate (CAN) to yield 117.

Iodine can be introduced onto the quinone ring using a procedure as outlined in the following scheme (see Kumadaki, I. et al. Synthetic Communications 19:173-177 (1989) for more information).




The protected chromanol 120 is treated with I2 and silver trifluoroacetate to yield the iodinated derivative 121, followed by deprotection/oxidation with ceric ammonium nitrate to give 122.

A method suitable for synthesizing nitrile-containing compounds of formula I, formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, formula X, formula XI, or formula XII (including all variations on the formulas) is depicted in the following scheme. The scheme illustrates the synthesis starting from α-tocopherol and ending in the cyano-substituted quinone, but can be readily generalized to the other compounds of the invention by using appropriate groups in place of the 2-methyl and 3-methyl groups and the appropriate tail group.




The synthesis from conversion of α-tocopherol 131 to the 5-bromomethyl derivative with acetate-protected 6-hydroxy group 132, followed by oxidation to the aldehyde intermediate 133 with anhydrous N-methylmorpholine-N-oxide (NMMO), is described in Mazzini et al., Tetrahedron 813-817 (2005). Hydroxylamine is then used to form an oxime 134, followed by dehydration of the oxime with acetic anhydride (see, e.g., the procedure described in Organic Syntheses, Coll. Vol. 3, p. 690 (1955); Vol. 20, p. 74 (1940)) to give 135. Removal of the acetate protection groups, and oxidation with ceric ammonium nitrate (CAN) yields 136.

Another method suitable for synthesizing nitrile-containing compounds of formula I, formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, formula X, formula XI, or formula XII (including all variations on the formulas) is depicted in the following scheme, starting from intermediate 114 from one of the syntheses depicted above for making compounds of the inventions with halogen substituents on the quinone ring.




Compound 114 is treated with trimethylsilyl cyanide and trifluoromethane sulfonic acid in trifluoroacetic acid to introduce the formyl group, resulting in compound 142. The phenolic group is protected, and hydroxylamine is used to convert the aldehyde compound 142 into the oxime compound 143. Dehydration of the oxime to give the nitrile 144 can be followed by deprotection and oxidation to form 145; alternatively, 144 can be deprotected to give the 6-chromanol-type compounds. (See Fujishima et al. Arch. Pharm. Pharm. Med. Chem. 329:27-34 (1996) for additional information.)

The regioisomer 157




can be prepared by synthesizing the regioisomer 154





in a manner analogous to the synthesis of compound 9 described in Dean et al., Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry, (5) 1437-42 (1981), as outlined in the following scheme.









Synthesis of Compounds of Formula III and Formula IV

The compounds of formula III and of formula IV are similar to the compounds of formula I and formula II, except that the “head group” is a benzene-1,4-diol moiety instead of a 1,4-benzoquinone. That is, the head group of formulas III and IV is the reduced form of the head group of formulas I and II. Thus, compounds of formulas III and IV can be readily prepared by simple reduction of the compounds of formulas I and II. This reduction can be done chemically (e.g., with Na2S2O4) or electrochemically, as is well known in the art.
Synthesis of Compounds of Formula V and Formula VI
The compounds of Formula V and Formula VI can be synthesized by the following procedure, as discussed in Omura, J. Org. Chem. 54:1987 (1989).




By using alcohols of the form R8OH, other alkoxy groups can be introduced, in a manner analogous to the methoxy group introduction as illustrated. An alternative synthesis is described in Goodhue et al., Biochemistry 4:854 (1965).

The corresponding hydroxy compound (R8=H for the compounds of general formula V and general formula VI) can be synthesized by the procedure described in Dürckheimer et al., J. Am. Chem. Soc. 86:4388 (1964), involving oxidation of alpha-tocopherol with tetrachloro-o-quinone in an acetonitrile/water mixture:




The compounds of formula V and formula VI can also be synthesized by treating the compounds of formula I or formula II with p-toluenesulfonic acid in benzene in the presence of R8OH (R8OH can be, e.g., methanol), as follows (adapted from Cohen et al., J. Org. Chem. 46:2445 (1981)).




Compounds of Formula VII-i, Formula VIII-i, and Formula IX-i

Information pertaining to the compound of formula VII-i can be found in the following publications: US 2004/0116715; Storozhok et al., Biomeditsinskaya Khimiya (2003), 49(1), 96-104; Bertalan et al., Olaj, Szappan, Kozmetika (2000), 49 (Kulonszam), 40-45; Dompert et al., Fette, Seifen, Anstrichmittel (1976), 78(3), 108-11; Berndorfer-Kraszner et al., Elelmezesi Ipar (1971), 25(11), 339-45; and Whittle et al., Biochemical Journal (1967), 103(3), 21C-22C.
Information pertaining to the compound of formula VIII-i can be found in the following publications: JP 58-193689; Mahmood et al., Phytochemistry (Elsevier) (1984), 23(8), 1725-7; Hughes et al., Journal of Biological Chemistry (1980), 255(24), 11802-6; Deuel et al., Journal of Biological Chemistry (1941), 139, 479-80; and Tishler et al., Journal of Biological Chemistry (1941), 139, 241-5. See Example 1 (Example 1A) below for a synthetic route to a mixture of stereoisomers of this compound.
Information pertaining to the compound of formula IX-i can be found in the following publications: JP 2003-137716 and JP 52-111576. See Example 1 (Example 1B) below for a synthetic route to a mixture of stereoisomers of this compound.
Interconvertibility of Quinone, Dihydroquinone Forms
The quinone and dihydroquinone forms of the compounds disclosed herein are readily interconverted with appropriate reagents. For example, the quinone form of a compound can be reduced to the dihydroquinone form with reducing agents such as sodium dithionite. The hydroquinone form can be oxidized to the quinone form with oxidizing agents such as ceric ammonium nitrate or ferric chloride. The quinone and hydroquinone forms are also readily converted electrochemically, as is well known in the art. See, e.g., Section 33.4 of Streitweiser & Heathcock, Introduction to Organic Chemistry, New York: Macmillan, 1976.
When the compounds of the invention are drawn as the quinone or hydroquinone form, that specific form is intended. However, when the quinone form is drawn and followed by the phrase “reduced counterpart thereof” or “reduced form” or the like, the structure and the subsequent phrase are intended to embrace both the quinone and hydroquinone. Similarly, when the hydroquinone form is drawn and followed by the phrase “oxidized counterpart thereof” or “oxidized form thereof” or the like, the structure and the subsequent phrase are intended to embrace both the hydroquinone and quinone.
Diseases Amenable to Treatment or Suppression with Compounds and Methods of the Invention
A variety of diseases are believed to be caused or aggravated by mitochondrial disorders and impaired energy processing, and can be treated or suppressed using the compounds and methods of the invention. Such diseases include, but are not limited to, inherited mitochondrial diseases, such as Myoclonic Epilepsy with Ragged Red Fibers (MERRF), Mitochondrial Myopathy, Encephalopathy, Lactacidosis, Stroke (MELAS), Leber's Hereditary Optic Neuropathy (LHON, also referred to as Leber's Disease, Leber's Optic Atrophy (LOA), or Leber's Optic Neuropathy (LON)), Leigh Disease or Leigh Syndrome, Keams-Sayre Syndrome (KSS), Friedreich's Ataxia (FA), other myopathies (including cardiomyopathy and encephalomyopathy), and renal tubular acidosis; neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease), motor neuron diseases; other neurological diseases such as epilepsy; genetic diseases such as Huntington's Disease (which is also a neurological disease); mood disorders such as schizophrenia and bipolar disorder; and certain age-associated diseases, particularly diseases for which CoQ10 has been proposed for treatment, such as macular degeneration, diabetes, and cancer.
Clinical Assessment of Mitochondrial Dysfunction and Efficacy of Therapy
Several readily measurable clinical markers are used to assess the metabolic state of patients with mitochondrial disorders. These markers can also be used as indicators of the efficacy of a given therapy, as the level of a marker is moved from the pathological value to the healthy value. These clinical markers include, but are not limited to, one or more of the previously discussed energy biomarkers, such as lactic acid (lactate) levels, either in whole blood, plasma, cerebrospinal fluid, or cerebral ventricular fluid; pyruvic acid (pyruvate) levels, either in whole blood, plasma, cerebrospinal fluid, or cerebral ventricular fluid; lactate/pyruvate ratios, either in whole blood, plasma, cerebrospinal fluid, or cerebral ventricular fluid; phosphocreatine levels, NADH (NADH+H+) or NADPH (NADPH+H+) levels; NAD or NADP levels; ATP levels; anaerobic threshold; reduced coenzyme Q (CoQred) levels; oxidized coenzyme Q (CoQox) levels; total coenzyme Q (CoQtot) levels; oxidized cytochrome C levels; reduced cytochrome C levels; oxidized cytochrome C/reduced cytochrome C ratio; acetoacetate levels, β-hydroxy butyrate levels, acetoacetate/β-hydroxy butyrate ratio, 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels; levels of reactive oxygen species; and levels of oxygen consumption (VO2), levels of carbon dioxide output (VCO2), and respiratory quotient (VCO2/VO2). Several of these clinical markers are measured routinely in exercise physiology laboratories, and provide convenient assessments of the metabolic state of a subject. In one embodiment of the invention, the level of one or more energy biomarkers in a patient suffering from a mitochondrial disease, such as Friedreich's ataxia, Leber's hereditary optic neuropathy, MELAS, or KSS, is improved to within two standard deviations of the average level in a healthy subject. In another embodiment of the invention, the level of one or more of these energy biomarkers in a patient suffering from a mitochondrial disease, such as Friedreich's ataxia, Leber's hereditary optic neuropathy, MELAS, or KSS is improved to within one standard deviation of the average level in a healthy subject. Exercise intolerance can also be used as an indicator of the efficacy of a given therapy, where an improvement in exercise tolerance (i.e., a decrease in exercise intolerance) indicates efficacy of a given therapy.
Several metabolic biomarkers have already been used to evaluate efficacy of CoQ10, and these metabolic biomarkers can be monitored as energy biomarkers for use in the methods of the current invention. Pyruvate, a product of the anaerobic metabolism of glucose, is removed by reduction to lactic acid in an anaerobic setting or by oxidative metabolism, which is dependent on a functional mitochondrial respiratory chain. Dysfunction of the respiratory chain may lead to inadequate removal of lactate and pyruvate from the circulation and elevated lactate/pyruvate ratios are observed in mitochondrial cytopathies (see Scriver C R, The metabolic and molecular bases of inherited disease, 7th ed., New York: McGraw-Hill, Health Professions Division, 1995; and Munnich et al., J. Inherit. Metab. Dis. 15(4):448-55 (1992)). Blood lactate/pyruvate ratio (Chariot et al., Arch. Pathol. Lab. Med. 118(7):695-7 (1994)) is, therefore, widely used as a noninvasive test for detection of mitochondrial cytopathies (see again Scriver C R, The metabolic and molecular bases of inherited disease, 7th ed., New York: McGraw-Hill, Health Professions Division, 1995; and Munnich et al., J. Inherit. Metab. Dis. 15(4):448-55 (1992)) and toxic mitochondrial myopathies (Chariot et al., Arthritis Rheum. 37(4):583-6 (1994)). Changes in the redox state of liver mitochondria can be investigated by measuring the arterial ketone body ratio (acetoacetate/3-hydroxybutyrate: AKBR) (Ueda et al., J. Cardiol. 29(2):95-102 (1997)). Urinary excretion of 8-hydroxy-2′-deoxyguanosine (8-OHdG) often has been used as a biomarker to assess the extent of repair of ROS-induced DNA damage in both clinical and occupational settings (Erhola et al., FEBS Lett. 409(2):287-91 (1997); Honda et al., Leuk. Res. 24(6):461-8 (2000); Pilger et al., Free Radic. Res. 35(3):273-80 (2001); Kim et al. Environ Health Perspect 112(6):666-71 (2004)).
Magnetic resonance spectroscopy (MRS) has been useful in the diagnoses of mitochondrial cytopathy by demonstrating elevations in cerebrospinal fluid (CSF) and cortical white matter lactate using proton MRS (1H-MRS) (Kaufmann et al., Neurology 62(8):1297-302 (2004)). Phosphorous MRS (31P-MRS) has been used to demonstrate low levels of cortical phosphocreatine (PCr) (Matthews et al., Ann. Neurol. 29(4):435-8 (1991)), and a delay in PCr recovery kinetics following exercise in skeletal muscle (Matthews et al., Ann. Neurol. 29(4):435-8 (1991); Barbiroli et al., J. Neurol. 242(7):472-7 (1995); Fabrizi et al., J. Neurol. Sci. 137(1):20-7 (1996)). A low skeletal muscle PCr has also been confirmed in patients with mitochondrial cytopathy by direct biochemical measurements.
Exercise testing is particularly helpful as an evaluation and screening tool in mitochondrial myopathies. One of the hallmark characteristics of mitochondrial myopathies is a reduction in maximal whole body oxygen consumption (VO2max) (Taivassalo et al., Brain 126(Pt 2):413-23 (2003)). Given that VO2max is determined by cardiac output (Qc) and peripheral oxygen extraction (arterial-venous total oxygen content) difference, some mitochondrial cytopathies affect cardiac function where delivery can be altered; however, most mitochondrial myopathies show a characteristic deficit in peripheral oxygen extraction (A-VO2 difference) and an enhanced oxygen delivery (hyperkinetic circulation) (Taivassalo et al., Brain 126(Pt 2):413-23 (2003)). This can be demonstrated by a lack of exercise induced deoxygenation of venous blood with direct AV balance measurements (Taivassalo et al., Ann. Neurol. 51(1):38-44 (2002)) and non-invasively by near infrared spectroscopy (Lynch et al., Muscle Nerve 25(5):664-73 (2002); van Beekvelt et al., Ann. Neurol. 46(4):667-70 (1999)).
Several of these energy biomarkers are discussed in more detail as follows. It should be emphasized that, while certain energy biomarkers are discussed and enumerated herein, the invention is not limited to modulation, normalization or enhancement of only these enumerated energy biomarkers.
Lactic acid (lactate) levels: Mitochondrial dysfunction typically results in abnormal levels of lactic acid, as pyruvate levels increase and pyruvate is converted to lactate to maintain capacity for glycolysis. Mitochondrial dysfunction can also result in abnormal levels of NADH+H+, NADPH+H+, NAD, or NADP, as the reduced nicotinamide adenine dinucleotides are not efficiently processed by the respiratory chain. Lactate levels can be measured by taking samples of appropriate bodily fluids such as whole blood, plasma, or cerebrospinal fluid. Using magnetic resonance, lactate levels can be measured in virtually any volume of the body desired, such as the brain.
Measurement of cerebral lactic acidosis using magnetic resonance in MELAS patients is described in Kaufmann et al., Neurology 62(8): 1297 (2004). Values of the levels of lactic acid in the lateral ventricles of the brain are presented for two mutations resulting in MELAS, A3243G and A8344G. Whole blood, plasma, and cerebrospinal fluid lactate levels can be measured by commercially available equipment such as the YSI 2300 STAT Plus Glucose & Lactate Analyzer (YSI Life Sciences, Ohio).
NAD, NADP, NADH and NADPH Levels:
Measurement of NAD, NADP, NADH (NADH+H+) or NADPH (NADPH+H+) can be measured by a variety of fluorescent, enzymatic, or electrochemical techniques, e.g., the electrochemical assay described in US 2005/0067303.
Oxygen Consumption (vO2 or VO2), Carbon Dioxide Output (vCO2 or VCO2), and Respiratory Quotient (VCO2/VO2):
vO2 is usually measured either while resting (resting vO2) or at maximal exercise intensity (vO2 max). Optimally, both values will be measured. However, for severely disabled patients, measurement of vO2 max may be impractical. Measurement of both forms of vO2 is readily accomplished using standard equipment from a variety of vendors, e.g. Korr Medical Technologies, Inc. (Salt Lake City, Utah). VCO2 can also be readily measured, and the ratio of VCO2 to VO2 under the same conditions (VCO2/VO2, either resting or at maximal exercise intensity) provides the respiratory quotient (RQ).
Oxidized Cytochrome C, Reduced Cytochrome C, and Ratio of Oxidized Cytochrome C to Reduced Cytochrome C:
Cytochrome C parameters, such as oxidized cytochrome C levels (Cyt Cox), reduced cytochrome C levels (Cyt Cred), and the ratio of oxidized cytochrome C/reduced cytochrome C ratio (Cyt Cox)/(Cyt Cred), can be measured by in vivo near infrared spectroscopy. See, e.g., Rolfe, P., “In vivo near-infrared spectroscopy,” Annu. Rev. Biomed. Eng. 2:715-54 (2000) and Strangman et al., “Non-invasive neuroimaging using near-infrared light” Biol. Psychiatry 52:679-93 (2002).
Exercise Tolerance/Exercise Intolerance:
Exercise intolerance is defined as “the reduced ability to perform activities that involve dynamic movement of large skeletal muscles because of symptoms of dyspnea or fatigue” (Piña et al., Circulation 107:1210 (2003)). Exercise intolerance is often accompanied by myoglobinuria, due to breakdown of muscle tissue and subsequent excretion of muscle myoglobin in the urine. Various measures of exercise intolerance can be used, such as time spent walking or running on a treadmill before exhaustion, time spent on an exercise bicycle (stationary bicycle) before exhaustion, and the like. Treatment with the compounds or methods of the invention can result in about a 10% or greater improvement in exercise tolerance (for example, about a 10% or greater increase in time to exhaustion, e.g. from 10 minutes to 11 minutes), about a 20% or greater improvement in exercise tolerance, about a 30% or greater improvement in exercise tolerance, about a 40% or greater improvement in exercise tolerance, about a 50% or greater improvement in exercise tolerance, about a 75% or greater improvement in exercise tolerance, or about a 100% or greater improvement in exercise tolerance. While exercise tolerance is not, strictly speaking, an energy biomarker, for the purposes of the invention, modulation, normalization, or enhancement of energy biomarkers includes modulation, normalization, or enhancement of exercise tolerance.
Similarly, tests for normal and abnormal values of pyruvic acid (pyruvate) levels, lactate/pyruvate ratio, ATP levels, anaerobic threshold, reduced coenzyme Q (CoQred) levels, oxidized coenzyme Q (CoQox) levels, total coenzyme Q (CoQtot) levels, oxidized cytochrome C levels, reduced cytochrome C levels, oxidized cytochrome C/reduced cytochrome C ratio, acetoacetate levels, β-hydroxy butyrate levels, acetoacetate/β-hydroxy butyrate ratio, 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels, and levels of reactive oxygen species are known in the art and can be used to evaluate efficacy of the compounds and methods of the invention. (For the purposes of the invention, modulation, normalization, or enhancement of energy biomarkers includes modulation, normalization, or enhancement of anaerobic threshold.)
Table 1, following, illustrates the effect that various dysfunctions can have on biochemistry and energy biomarkers. It also indicates the physical effect (such as a disease symptom or other effect of the dysfunction) typically associated with a given dysfunction. It should be noted that any of the energy biomarkers listed in the table, in addition to energy biomarkers enumerated elsewhere, can also be modulated, enhanced, or normalized by the compounds and methods of the invention. RQ=respiratory quotient; BMR=basal metabolic rate; HR (CO)=heart rate (cardiac output); T=body temperature (preferably measured as core temperature); AT=anaerobic threshold; pH=blood pH (venous and/or arterial).










TABLE 1





Site of

Measurable Energy



Dysfunction
Biochemical Event
Biomarker
Physical Effect







Respiratory
↑ NADH
Δ lactate,
Metabolic


Chain

Δ lactate:pyruvate ratio;
dyscrasia &




and
fatigue




Δ acetoacetate:β-hydroxy




butyrate ratio


Respiratory
↓ H+ gradient
Δ ATP
Organ dependent


Chain


dysfunction


Respiratory
↓ Electron flux
Δ VO2, RQ, BMR, ΔT,
Metabolic


Chain

AT, pH
dyscrasia &





fatigue


Mitochondria &
↓ ATP, ↓ VO2
Δ Work, ΔHR (CO)
Exercise


cytosol


intolerance


Mitochondria &
↓ ATP
Δ PCr
Exercise


cytosol


intolerance


Respiratory
↓ Cyt COx/Red
Δλ ~700–900 nM (Near
Exercise


Chain

Infrared Spectroscopy)
intolerance


Intermediary
↓ Catabolism
Δ C14-Labeled substrates
Metabolic


metabolism


dyscrasia &





fatigue


Respiratory
↓ Electron flux
Δ Mixed Venous VO2
Metabolic


Chain


dyscrasia &





fatigue


Mitochondria &
↑ Oxidative stress
Δ Tocopherol &
Uncertain


cytosol

Tocotrienols, CoQ10,




docosahexanoic acid


Mitochondria &
↑ Oxidative stress
Δ Glutathionered
Uncertain


cytosol


Mitochondria &
Nucleic acid
Δ8-hydroxy 2-deoxy
Uncertain


cytosol
oxidation
guanosine


Mitochondria &
Lipid oxidation
ΔIsoprostane(s),
Uncertain


cytosol

eicasanoids


Cell membranes
Lipid oxidation
ΔEthane (breath)
Uncertain


Cell membranes
Lipid oxidation
ΔMalondialdehyde
Uncertain









Treatment of a subject afflicted by a mitochondrial disease in accordance with the methods of the invention may result in the inducement of a reduction or alleviation of symptoms in the subject, e.g., to halt the further progression of the disorder.
Partial or complete suppression of the mitochondrial disease can result in a lessening of the severity of one or more of the symptoms that the subject would otherwise experience. For example, partial suppression of MELAS could result in reduction in the number of stroke-like or seizure episodes suffered.
Any one, or any combination of, the energy biomarkers described herein provide conveniently measurable benchmarks by which to gauge the effectiveness of treatment or suppressive therapy. Additionally, other energy biomarkers are known to those skilled in the art and can be monitored to evaluate the efficacy of treatment or suppressive therapy.
Use of Compounds for Modulation of Energy Biomarkers
In addition to monitoring energy biomarkers to assess the status of treatment or suppression of mitochondrial diseases, the compounds of the invention can be used in subjects or patients to modulate one or more energy biomarkers. Modulation of energy biomarkers can be done to normalize energy biomarkers in a subject, or to enhance energy biomarkers in a subject.
Normalization of one or more energy biomarkers is defined as either restoring the level of one or more such energy biomarkers to normal or near-normal levels in a subject whose levels of one or more energy biomarkers show pathological differences from normal levels (i.e., levels in a healthy subject), or to change the levels of one or more energy biomarkers to alleviate pathological symptoms in a subject. Depending on the nature of the energy biomarker, such levels may show measured values either above or below a normal value. For example, a pathological lactate level is typically higher than the lactate level in a normal (i.e., healthy) person, and a decrease in the level may be desirable. A pathological ATP level is typically lower than the ATP level in a normal (i.e., healthy) person, and an increase in the level of ATP may be desirable. Accordingly, normalization of energy biomarkers can involve restoring the level of energy biomarkers to within about at least two standard deviations of normal in a subject, more preferably to within about at least one standard deviation of normal in a subject, to within about at least one-half standard deviation of normal, or to within about at least one-quarter standard deviation of normal.
When an increase in an energy biomarker level is desired to normalize the one or more such energy biomarker, the level of the energy biomarker can be increased to within about at least two standard deviations of normal in a subject, more preferably increased to within about at least one standard deviation of normal in a subject, increased to within about at least one-half standard deviation of normal, or increased to within about at least one-quarter standard deviation of normal, by administration of one or more compounds according to the invention. Alternatively, the level of one or more of the energy biomarkers can be increased by about at least 10% above the subject's level of the respective one or more energy biomarkers before administration, by about at least 20% above the subject's level of the respective one or more energy biomarkers before administration, by about at least 30% above the subject's level of the respective one or more energy biomarkers before administration, by about at least 40% above the subject's level of the respective one or more energy biomarkers before administration, by about at least 50% above the subject's level of the respective one or more energy biomarkers before administration, by about at least 75% above the subject's level of the respective one or more energy biomarkers before administration, or by about at least 100% above the subject's level of the respective one or more energy biomarkers before administration.
When a decrease in a level of one or more energy biomarkers is desired to normalize the one or more energy biomarkers, the level of the one or more energy biomarkers can be decreased to a level within about at least two standard deviations of normal in a subject, more preferably decreased to within about at least one standard deviation of normal in a subject, decreased to within about at least one-half standard deviation of normal, or decreased to within about at least one-quarter standard deviation of normal, by administration of one or more compounds according to the invention. Alternatively, the level of the one or more energy biomarkers can be decreased by about, at least 10% below the subject's level of the respective one or more energy biomarkers before administration, by about at least 20% below the subject's level of the respective one or more energy biomarkers before administration, by about at least 30% below the subject's level of the respective one or more energy biomarkers before administration, by about at least 40% below the subject's level of the respective one or more energy biomarkers before administration, by about at least 50% below the subject's level of the respective one or more energy biomarkers before administration, by about at least 75% below the subject's level of the respective one or more energy biomarkers before administration, or by about at least 90% below the subject's level of the respective one or more energy biomarkers before administration.
Enhancement of the level of one or more energy biomarkers is defined as changing the extant levels of one or more energy biomarkers in a subject to a level which provides beneficial or desired effects for the subject. For example, a person undergoing strenuous effort or prolonged vigorous physical activity, such as mountain climbing, could benefit from increased ATP levels or decreased lactate levels. As described above, normalization of energy biomarkers may not achieve the optimum state for a subject with a mitochondrial disease, and such subjects can also benefit from enhancement of energy biomarkers. Examples of subjects who could benefit from enhanced levels of one or more energy biomarkers include, but are not limited to, subjects undergoing strenuous or prolonged physical activity, subjects with chronic energy problems, or subjects with chronic respiratory problems. Such subjects include, but are not limited to, pregnant females, particularly pregnant females in labor; neonates, particularly premature neonates; subjects exposed to extreme environments, such as hot environments (temperatures routinely exceeding about 85-86 degrees Fahrenheit or about 30 degrees Celsius for about 4 hours daily or more), cold environments (temperatures routinely below about 32 degrees Fahrenheit or about 0 degrees Celsius for about 4 hours daily or more), or environments with lower-than-average oxygen content, higher-than-average carbon dioxide content, or higher-than-average levels of air pollution (airline travelers, flight attendants, subjects at elevated altitudes, subjects living in cities with lower-than-average air quality, subjects working in enclosed environments where air quality is degraded); subjects with lung diseases or lower-than-average lung capacity, such as tubercular patients, lung cancer patients, emphysema patients, and cystic fibrosis patients; subjects recovering from surgery or illness; elderly subjects, including elderly subjects experiencing decreased energy; subjects suffering from chronic fatigue, including chronic fatigue syndrome; subjects undergoing acute trauma; subjects in shock; subjects requiring acute oxygen administration; subjects requiring chronic oxygen administration; or other subjects with acute, chronic, or ongoing energy demands who can benefit from enhancement of energy biomarkers.
Accordingly, when an increase in a level of one or more energy biomarkers is beneficial to a subject, enhancement of the one or more energy biomarkers can involve increasing the level of the respective energy biomarker or energy biomarkers to about at least one-quarter standard deviation above normal, about at least one-half standard deviation above normal, about at least one standard deviation above normal, or about at least two standard deviations above normal. Alternatively, the level of the one or more energy biomarkers can be increased by about at least 10% above the subject's level of the respective one or more energy biomarkers before enhancement, by about at least 20% above the subject's level of the respective one or more energy biomarkers before enhancement, by about at least 30% above the subject's level of the respective one or more energy biomarkers before enhancement, by about at least 40% above the subject's level of the respective one or more energy biomarkers before enhancement, by about at least 50% above the subject's level of the respective one or more energy biomarkers before enhancement, by about at least 75% above the subject's level of the respective one or more energy biomarkers before enhancement, or by about at least 100% above the subject's level of the respective one or more energy biomarkers before enhancement.
When a decrease in a level of one or more energy biomarkers is desired to enhance one or more energy biomarkers, the level of the one or more energy biomarkers can be decreased by an amount of about at least one-quarter standard deviation of normal in a subject, decreased by about at least one-half standard deviation of normal in a subject, decreased by about at least one standard deviation of normal in a subject, or decreased by about at least two standard deviations of normal in a subject. Alternatively, the level of the one or more energy biomarkers can be decreased by about at least 10% below the subject's level of the respective one or more energy biomarkers before enhancement, by about at least 20% below the subject's level of the respective one or more energy biomarkers before enhancement, by about at least 30% below the subject's level of the respective one or more energy biomarkers before enhancement, by about at least 40% below the subject's level of the respective one or more energy biomarkers before enhancement, by about at least 50% below the subject's level of the respective one or more energy biomarkers before enhancement, by about at least 75% below the subject's level of the respective one or more energy biomarkers before enhancement, or by about at least 90% below the subject's level of the respective one or more energy biomarkers before enhancement.
Use of Compounds in Research Applications, Experimental Systems, and Assays
The compounds of the invention can also be used in research applications. For example, alpha-tocopherol quinone can be used in vitro, in vivo, or ex vivo experiments to modulate one or more energy biomarkers in an experimental system. Such experimental systems can be cell samples, tissue samples, cell components or mixtures of cell components, partial organs, whole organs, or organisms. Any one or more of the compounds of formula I, Ia, Ib, II, IIa, IIb, III, IIIa, IIIb, IV, IVa, IVb, V, Va, Vb, VI, VIa, VIb, VII-O, VII-R, VIII-O, VIII-R, IX-O, IX-R, X-O, X-R, XI-O, XI-R, XII-O, and/or XII-R can be used in experimental systems or research applications. Such research applications can include, but are not limited to, use as assay reagents, elucidation of biochemical pathways, or evaluation of the effects of other agents on the metabolic state of the experimental system in the presence/absence of one or more compounds of the invention.
Additionally, the compounds of the invention can be used in biochemical tests or assays. Such tests can include incubation of one or more compounds of the invention with a tissue or cell sample from a subject to evaluate a subject's potential response (or the response of a specific subset of subjects) to administration of said one or more compounds, or to determine which compound of the invention produces the optimum effect in a specific subject or subset of subjects. One such test or assay would involve 1) obtaining a cell sample or tissue sample from a subject in which modulation of one or more energy biomarkers can be assayed; 2) administering one or more compounds of the invention to the cell sample or tissue sample; and 3) determining the amount of modulation of the one or more energy biomarkers after administration of the one or more compounds, compared to the status of the energy biomarker prior to administration of the one or more compounds. Another such test or assay would involve 1) obtaining a cell sample or tissue sample from a subject in which modulation of one or more energy biomarkers can be assayed; 2) administering at least two compounds of the invention to the cell sample or tissue sample; 3) determining the amount of modulation of the one or more energy biomarkers after administration of the at least two compounds, compared to the status of the energy biomarker prior to administration of the at least compounds, and 4) selecting a compound for use in treatment, suppression, or modulation based on the amount of modulation determined in step 3).
Pharmaceutical Formulations
The compounds described herein can be formulated as pharmaceutical compositions by formulation with additives such as pharmaceutically acceptable excipients, pharmaceutically acceptable carriers, and pharmaceutically acceptable vehicles. Suitable pharmaceutically acceptable excipients, carriers and vehicles include processing agents and drug delivery modifiers and enhancers, such as, for example, calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidinone, low melting waxes, ion exchange resins, and the like, as well as combinations of any two or more thereof. Other suitable pharmaceutically acceptable excipients are described in “Remington's Pharmaceutical Sciences,” Mack Pub. Co., New Jersey (1991), and “Remington: The Science and Practice of Pharmacy,” Lippincott Williams & Wilkins, Philadelphia, 20th edition (2003) and 21st edition (2005), incorporated herein by reference.
A pharmaceutical composition can comprise a unit dose formulation, where the unit dose is a dose sufficient to have a therapeutic or suppressive effect or an amount effective to modulate, normalize, or enhance an energy biomarker. The unit dose may be sufficient as a single dose to have a therapeutic or suppressive effect or an amount effective to modulate, normalize, or enhance an energy biomarker. Alternatively, the unit dose may be a dose administered periodically in a course of treatment or suppression of a disorder, or to modulate, normalize, or enhance an energy biomarker.
Pharmaceutical compositions containing the compounds of the invention may be in any form suitable for the intended method of administration, including, for example, a solution, a suspension, or an emulsion. Liquid carriers are typically used in preparing solutions, suspensions, and emulsions. Liquid carriers contemplated for use in the practice of the present invention include, for example, water, saline, pharmaceutically acceptable organic solvent(s), pharmaceutically acceptable oils or fats, and the like, as well as mixtures of two or more thereof. The liquid carrier may contain other suitable pharmaceutically acceptable additives such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickening agents, viscosity regulators, stabilizers, and the like. Suitable organic solvents include, for example, monohydric alcohols, such as ethanol, and polyhydric alcohols, such as glycols. Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, and the like. For parenteral administration, the carrier can also be an oily ester such as ethyl oleate, isopropyl myristate, and the like. Compositions of the present invention may also be in the form of microparticles, microcapsules, liposomal encapsulates, and the like, as well as combinations of any two or more thereof.
Time-release or controlled release delivery systems may be used, such as a diffusion controlled matrix system or an erodible system, as described for example in: Lee, “Diffusion-Controlled Matrix Systems”, pp. 155-198 and Ron and Langer, “Erodible Systems”, pp. 199-224, in “Treatise on Controlled Drug Delivery”, A. Kydonieus Ed., Marcel Dekker, Inc., New York 1992. The matrix may be, for example, a biodegradable material that can degrade spontaneously in situ and in vivo for, example, by hydrolysis or enzymatic cleavage, e.g., by proteases. The delivery system may be, for example, a naturally occurring or synthetic polymer or copolymer, for example in the form of a hydrogel. Exemplary polymers with cleavable linkages include polyesters, polyorthoesters, polyanhydrides, polysaccharides, poly(phosphoesters), polyamides, polyurethanes, poly(imidocarbonates) and poly(phosphazenes).
The compounds of the invention may be administered enterally, orally, parenterally, sublingually, by inhalation (e.g. as mists or sprays), rectally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. For example, suitable modes of administration include oral, subcutaneous, transdermal, transmucosal, iontophoretic, intravenous, intraarterial, intramuscular, intraperitoneal, intranasal (e.g. via nasal mucosa), subdural, rectal, gastrointestinal, and the like, and directly to a specific or affected organ or tissue. For delivery to the central nervous system, spinal and epidural administration, or administration to cerebral ventricles, can be used. Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques. The compounds are mixed with pharmaceutically acceptable carriers, adjuvants, and vehicles appropriate for the desired route of administration. Oral administration is a preferred route of administration, and formulations suitable for oral administration are preferred formulations. The compounds described for use herein can be administered in solid form, in liquid form, in aerosol form, or in the form of tablets, pills, powder mixtures, capsules, granules, injectables, creams, solutions, suppositories, enemas, colonic irrigations, emulsions, dispersions, food premixes, and in other suitable forms. The compounds can also be administered in liposome formulations. The compounds can also be administered as prodrugs, where the prodrug undergoes transformation in the treated subject to a form which is therapeutically effective. Additional methods of administration are known in the art.
Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in propylene glycol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable nonirritating excipient such as cocoa butter and polyethylene glycols that are solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.
Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, cyclodextrins, and sweetening, flavoring, and perfuming agents.
The compounds of the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.W., p. 33 et seq (1976).
The invention also provides articles of manufacture and kits containing materials useful for treating or suppressing mitochondrial diseases. The article of manufacture comprises a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition having an active agent which is effective for treating or suppressing mitochondrial diseases. The active agent in the composition is one or more of the compounds of formulas I, Ia, Ib, II, IIa, IIb, III, IIIa, IIIb, IV, IVa, IVb, V, Va, Vb, VI, VIa, VIb, VII-O, VII-R, VIII-O, VIII-R, IX-O, IX-R, X-O, X-R, XI-O, XI-R, XII-O, and/or XII-R. The label on the container indicates that the composition is used for treating or suppressing mitochondrial diseases, and may also indicate directions for either in vivo or in vitro use, such as those described above.
The invention also provides kits comprising any one or more of the compounds of formulas I, Ia, Ib, II, IIa, IIb, III, IIIa, IIIb, IV, IVa, IVb, V, Va, Vb, VI, VIa, VIb, VII-O, VII-R, VIII-O, VIII-R, IX-O, IX-R, X-O, X-R, XI-O, XI-R, XII-O, and/or XII-R. In some embodiments, the kit of the invention comprises the container described above. In other embodiments, the kit of the invention comprises the container described above and a second container comprising a buffer. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any methods described herein.
In other aspects, the kits may be used for any of the methods described herein, including, for example, to treat an individual with a mitochondrial disorder, or to suppress a mitochondrial disorder in an individual.
The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host to which the active ingredient is administered and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, body area, body mass index (BMI), general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the type, progression, and severity of the particular disease undergoing therapy. The pharmaceutical unit dosage chosen is usually fabricated and administered to provide a defined final concentration of drug in the blood, tissues, organs, or other targeted region of the body. The therapeutically effective amount or effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician.
Examples of dosages which can be used are an effective amount within the dosage range of about 0.1 μg/kg to about 300 mg/kg, or within about 1.0 μg/kg to about 40 mg/kg body weight, or within about 1.0 μg/kg to about 20 mg/kg body weight, or within about 1.0 μg/kg to about 10 mg/kg body weight, or within about 10.0 μg/kg to about 10 mg/kg body weight, or within about 100 μg/kg to about 10 mg/kg body weight, or within about 1.0 mg/kg to about 10 mg/kg body weight, or within about 10 mg/kg to about 100 mg/kg body weight, or within about 50 mg/kg to about 150 mg/kg body weight, or within about 100 mg/kg to about 200 mg/kg body weight, or within about 150 mg/kg to about 250 mg/kg body weight, or within about 200 mg/kg to about 300 mg/kg body weight, or within about 250 mg/kg to about 300 mg/kg body weight. Other dosages which can be used are about 0.01 mg/kg body weight, about 0.1 mg/kg body weight, about 1 mg/kg body weight, about 10 mg/kg body weight, about 20 mg/kg body weight, about 30 mg/kg body weight, about 40 mg/kg body weight, about 50 mg/kg body weight, about 75 mg/kg body weight, about 100 mg/kg body weight, about 125 mg/kg body weight, about 150 mg/kg body weight, about 175 mg/kg body weight, about 200 mg/kg body weight, about 225 mg/kg body weight, about 250 mg/kg body weight, about 275 mg/kg body weight, or about 300 mg/kg body weight. Compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided dosage of two, three or four times daily.
α-tocopherol quinone is a naturally-occurring substance, which is normally found in serum (Pollok et al., J. Chromatogr. A. 1056:257 (2004)) and mitochondrial membranes (Gregor et al., Biochem Pharmacol. 71:1589 (2006)). Accordingly, when α-tocopherol quinone is administered to treat or suppress mitochondrial diseases or to modulate energy biomarkers, it can be administered in an amount sufficient to raise serum levels, intracellular levels, or mitochondrial membrane levels of α-tocopherol quinone by at least about 10%, by at least about 25%, by at least about 50%, by at least about 75%, by at least about 100%, by at least about 150%, or by at least about 200% as compared to the level of α-tocopherol quinone prior to α-tocopherol quinone administration. Reduced α-tocopherol quinone also occurs naturally. Accordingly, when α-tocopherol quinone is administered to treat or suppress mitochondrial diseases or to modulate energy biomarkers, it can be administered in an amount sufficient to raise serum levels, intracellular levels, or mitochondrial membrane levels of its reduced counterpart, reduced α-tocopherol quinone, by at least about 10%, by at least about 25%, by at least about 50%, by at least about 75%, by at least about 100%, by at least about 150%, or by at least about 200% as compared to the level of reduced α-tocopherol quinone prior to α-tocopherol quinone administration. Alternatively, reduced α-tocopherol quinone can be administered instead of α-tocopherol quinone in order to treat or suppress mitochondrial diseases or to modulate energy biomarkers, and can be administered in an amount sufficient to raise serum levels, intracellular levels, or mitochondrial membrane levels of reduced α-tocopherol quinone by at least about 10%, by at least about 25%, by at least about 50%, by at least about 75%, by at least about 100%, by at least about 150%, or by at least about 200% as compared to the level of reduced α-tocopherol quinone prior to reduced α-tocopherol quinone administration
While the compounds of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other agents used in the treatment or suppression of disorders. Representative agents useful in combination with the compounds of the invention for the treatment or suppression of mitochondrial diseases include, but are not limited to, Coenzyme Q, vitamin E, idebenone, MitoQ, vitamins, and antioxidant compounds.
When additional active agents are used in combination with the compounds of the present invention, the additional active agents may generally be employed in therapeutic amounts as indicated in the Physicians' Desk Reference (PDR) 53rd Edition (1999), which is incorporated herein by reference, or such therapeutically useful amounts as would be known to one of ordinary skill in the art.
The compounds of the invention and the other therapeutically active agents can be administered at the recommended maximum clinical dosage or at lower doses. Dosage levels of the active compounds in the compositions of the invention may be varied so as to obtain a desired therapeutic response depending on the route of administration, severity of the disease and the response of the patient. When administered in combination with other therapeutic agents, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.
The invention will be further understood by the following nonlimiting examples.


EXAMPLES
Example 1
Synthesis of Compounds
Example 1A




Step 1: A 50 mL RBF was charged with (R,R,R)-2-(3-hydroxy-3,7,11,15-tetramethyl-hexadecyl)-3,5,6-trimethyl-[1,4]benzoquinone (Ex-1A-1) (2.0 g, 4.40 mmol) and pyridine (10 mL) and the reaction was cooled to 0 C. Neat POCl3 (520 L, 5.60 mml) was added. The reaction was allowed to warm to RT and stirred for 16 h. The reaction was monitored by TLC (3:1 Heptane.:ETOAc). The reaction was diluted with saturated NH4Cl (10 mL) and MTBE (10 mL) and then extracted with MTBE (3×10 mL). The combined MTBE layers were passed through a silica plug and then washed with 0.1 HCl (3×10 mL). The MTBE layer was then concentrated by rotary evaporation to yield a yellow oil (1.95 g, 100%). The crude material, which was a mixture of alkene regioisomers and geometric isomers, was taken to the next step without further purification.
Step 2: A crude mixture of alkene regioisomers and geometric isomers (13.3 g, 31.0 mmol, prepared as described in step 1) was dissolved in EtOAc (100 mL) and hydrogenated using PtO2 (250 mg) at 50 psi H2. After 6 h,  ̃30% unsaturated material remained (1H NMR). Additional PtO2 (250 mg) was added and hydrogenation was continued for 16 h. The reaction mixture was filtered through celite, which was then rinsed with EtOAc (50 mL). The filtrate was concentrated by rotary evaporation to yield (R/S,R,R)-2,3,5-trimethyl-6-(3,7,11,15-tetramethyl-hexadecyl)-benzene-1,4-diol (Ex-1A-2) as a waxy white solid (12.7 g, 95%). 1H NMR (400 MHz, CDCl3) δ (ppm): 4.36 (broad-s, 1H), 4.33 (broad-s, 1H), 2.70-2.54 (m, 2H), 2.20 (s, 3H), 2.18 (s, 6H), 1.57-1.04 (m, 24H), 1.00 (d, J=6.4 Hz, 3H), 0.89-0.86 (m, 12H).
Step 3: A solution of (R/S,R,R)-2,3,5-trimethyl-6-(3,7,11,15-tetramethyl-hexadecyl)-benzene-1,4-diol (Ex-12A-2) (10.2 g, 0.24 g) in DCM (100 mL) was allowed to stir in the presence of SiO2 (500 mg) for 4 days exposed to air. The reaction mixture was then filtered and concentrated by rotary evaporation to yield an orange oil (10.0 g, 98%). A portion of the crude product (5.0 g) was purified using a Biotage automated chromatography instrument (eluted with DCM: Hept gradient) to yield pure (R/S,R,R)-2,3,5-trimethyl-6-(3,7,11,15-tetramethyl-hexadecyl)-[1,4]benzoquinone (Ex-1A-3, mixture of stereoisomers of compound VIII-i) (1.98 g, 40%). 1H NMR (400 MHz, CDCl3) δ (ppm): 2.54-2.40 (m, 2H), 2.03 (s, 3H), 2.03 (s, 6H), 1.56-1.02 (m, 24H), 0.96 (d, J=6.5 Hz, 3H), 0.89-0.85 (m, 12H).
Example 1B




Step 1: A 2 L 3-N flask was charged with 2,3,5-trimethyl-benzene-1,4-diol (Ex-1B-1) (50 g, 0.33 mol) and MEK (750 mL) to yield an amber solution. Potassium carbonate (210 g, 1.64 mol) was charged to the solution. After 30 min at RT, MeI (81.2 mL, 1.31 mol) was added to the brown suspension. The reaction mixture was heated to 65° C. for 72 h. After cooling to RT, the reaction mixture was concentrated to dryness by rotary evaporation to give a white paste. The paste was washed with EtOAc (3×300 mL). The EtOAc extracts were combined and concentrated by rotary evaporation. The resulting yellow-brown oil was chromatographed (80:20/Heptanes:EtOAc) to yield 1,4-dimethoxy-2,3,5-trimethyl-benzene (Ex-1B-2) (47.2 g, 80%). 1H NMR (400 MHz, CDCl3) δ (ppm): 6.55 (s, 1H), 3.80 (s, 3H), 3.68 (s, 3H), 2.30 (s, 3H), 2.22 (s, 3H), 2.14 (s, 3H).
Step 2: A flask was charged with 1,4-dimethoxy-2,3,5-trimethyl-benzene (Ex-1B-2) (47.2 g, 0.26 mol), glacial acetic acid (250 mL), and paraformaldehyde (39.3 g, 1.31 mol) to yield a yellow suspension. Anhydrous HCl gas was then slowly bubbled through the reaction mixture for 1.5 h producing a clear amber solution. The reaction mixture was then diluted with water (300 mL) and extracted with MTBE (3×300 mL). The combined MTBE layers were dried over Na2SO4, filtered and concentrated by rotary evaporation. Purification of the crude product by column chromatography (95:5/Heptanes: EtOAc) yielded 48.7 g of 1-chloromethyl-2,5-dimethoxy-3,4,6-trimethyl-benzene (Ex-1B-3) (81%). 1H NMR (400 MHz, CDCl3) δ (ppm): 4.76 (s, 2H), 3.81 (s, 3H), 3.68 (s, 3H), 2.36 (s, 3H), 2.23 (s, 3H), 2.21 (s, 3H).
Step 3: A flask was charged with 1-chloromethyl-2,5-dimethoxy-3,4,6-trimethyl-benzene (Ex-1B-3) (6.37 g, 27.9 mmol) and ACN (10 mL) then cooled to 0° C. A solution of CAN (31.3 g, 57.1 mmol) in water (10 mL) was added to the flask. After 1 h the reaction mixture was extracted with MTBE (3×50 mL). The combined MTBE layers were then washed with water (50 mL), dried over MgSO4, filtered and concentrated by rotary evaporation. Trituration of the crude product with MeOH yielded 4.49 g of 2-chloromethyl-3,5,6-trimethyl-[1,4]benzoquinone (Ex-1B-4) (81%) as a bright orange-yellow solid. 1H NMR (400 MHz, CDCl3) δ (ppm): 4.77 (s, 2H), 2.17 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H).
Step 4: A 3-N 100 mL flask was charged with PCl3 (2.8 mL, 31.6 mmol) and dry DMF (32 mL) then stirred at RT for 1 h. In a separate 50 mL flask, farnesol (Ex-1B-5) (10.0 g, 45.2 mmol) and DMF (10 mL) was charged. The PCl3/DMF solution was then transferred to the farnesol, solution and the resulting dark orange solution was stirred for 1 h. The reaction was quenched by addition of solid NaHCO3 (2.5 g, 63.2 mmol). The solvent was removed by high vacuum rotary evaporation to yield an oily orange residue. To the residue was added MTBE (40 mL) and water (40 mL). The aqueous phase was washed with MTBE (3×20 mL). The MTBE layers were combined, washed with brine (2×20 mL), dried over MgSO4, filtered and finally concentrated by rotary evaporation to yield 1-chloro-3,7,11-trimethyl-dodeca-2,6,10-triene (Ex-1B-6) as a yellow oil (9.89 g, 92%). 1H NMR (400 MHz, CDCl3) δ (ppm): 5.47 (broad-t, J=8.3 Hz, 1H), 5.15-5.07 (m, 2H), 4.12 (d, J=8.1 Hz, 2H), 2.18-1.95 (m, 8H), 1.75 (s, 3H), 1.70 (s, 3H), 1.62 (s, 6H).
Step 5: A 3-N 250 mL flask was inerted and charged with TMS-propyne (6.90 mL, 46.2 mmol) and THF (90 mL). The reaction was cooled to −40° C. after which time BuLi (18.5 mL, 46.2 mmol) was added. After 45 min, the reaction was cooled further (−70° C.) and a precooled (−70° C.) solution of 1-chloro-3,7,11-trimethyl-dodeca-2,6,10-triene (Ex-1B-6) (8.9 g, 37.0 mmol) in THF (50 mL) was added over 10 min. After 1 h, the reaction was warmed to RT and quenched by addition of saturated NH4Cl (20 mL) and MTBE (25 mL). The aqueous layer was separated and washed with MTBE (25 mL). The combined organic layers were then washed with brine, dried over MgSO4, filtered and concentrated to yield a yellow liquid (10.3 g). The crude oil was further purified by column chromatography (99:1/Heptanes:MTBE) to provide trimethyl-(6,10,14-trimethyl-pentadeca-5,9,13-trien-1-ynyl)-silane (Ex-1B-7). 1H NMR (400 MHz, CDCl3) δ (ppm): 5.22-5.16 (m, 1H), 5.16-5.08 (m, 2H), 2.27-2.22 (m, 4H), 2.15-1.94 (m, 8H), 1.70 (s, 3H), 1.65 (s, 3H), 1.62 (s, 6H), 0.17 (s, 9H).
Step 6: A 3-N 250 mL flask was charged with trimethyl-(6,10,14-trimethyl-pentadeca-5,9,13-trien-1-ynyl)-silane (Ex-1B-7) (19.38 g, 64.1 mmol) and NaOEt (42 mL of a 21% w/w solution, 112 mmol). The reaction mixture was stirred at 60° C. for 4 h. After cooling to RT, the reaction mixture was diluted with MTBE (100 mL) and water (100 mL) and then filtered to remove solid present at the phase interface. The aqueous layer was extracted with MTBE (3×100 mL). The combined MTBE layers were washed with brine (100 mL), dried over MgSO4, filtered, and concentrated by rotary evaporation to yield 12.43 g of 6,10,14-trimethyl-pentadeca-5,9,13-trien-1-yne (Ex-1B-8) as a dark orange oil (96%). 1H NMR (400 MHz, CDCl3) δ (ppm): 5.23-5.17 (m, 1H), 5.17-5.07 (m, 2H), 2.29-1.95 (m, 13H), 1.70 (s, 3H), 1.65 (s, 3H), 1.62 (s, 6H).
Step 7: A 3-N 250 mL flask equipped with a thermometer, a stirbar, and stopcock fitted vacuum adapter was evacuated, flame-dried, and flushed with N2 (3×) via a single manifold Schlenk line. To the flask was then charged bis(cyclopentadienyl)zirconium dichloride (Cp2ZrCl2) (2.16 g, 7.4 mmol) and dry DCE (40 mL). The reaction mixture was cooled to −20° C. AlMe3 (36.8 mL, 73.6 mmol) was added dropwise over 5 min to generate a yellow slurry. After 15 min. at −20° C., water (220 μL, 12.3 mmol) was added dropwise over 5 min. to yield a greenish-yellow solution. After stirring for 30 min. at −20° C., a solution of 6,10,14-trimethyl-pentadeca-5,9,13-trien-1-yne (Ex-1B-8) (6.0 g, 24.6 mmol) in dry DCE (20 mL) was added dropwise over 5 min. The reaction mixture became dark brown then amber in color. The reaction was allowed to warm to RT over 2 h. 1H NMR analysis of a DCl quenched aliquot revealed 95% deuterium incorporation. The solvent was removed in vacuo at RT. The resulting residue was washed with heptanes (2×40 mL) through a sintered glass frit into an inerted 250 mL 3-N flask equipped with a stirbar and stopcock fitted vacuum adapter. The reaction was allowed to stir overnight.
The solvent was removed in vacuo and replaced by addition of dry degassed THF (40 mL). A quenched aliquot of the reaction mixture revealed >92% deuterium incorpration by 1H NMR spectroscopy. A solution of 2-chloromethyl-3,5,6-trimethyl-[1,4]benzoquinone (Ex-1B-4) (3.0 g, 15.0 mmol) in dry degassed THF (20 mL) was added to the flask which was then cooled to 0° C. In a separate inerted 50 mL flask, (PPh3)2NiCl2 (750 mg, 1.3 mmol) and dry degassed THF (20 mL) was charged. BuLi (1.4 mL, 2.6 mmol) was added to the brown Ni(II) suspension to generate a blood red solution. The solution was stirred for 5 min. then added to the vinylalane/quinone (Ex-1B-9/Ex-1B-4) solution. The amber solution became blue gray in color. After 5 min, the reaction was complete by 1H NMR analysis of a quenched aliquot.
The reaction was quenched by very slow addition of 1 M HCl (great caution must be used in this procedure, as it is extremely exothermic) such that the temperature did not exceed 15° C. The reaction mixture was diluted with MTBE (20 mL). The resulting suspension was filtered. The aqueous layer of the filtrate was washed with MTBE (3×25 mL). The combined MTBE layers were dried over MgSO4, filtered, and concentrated by rotary evaporation to yield an amber oil (12 g). Purification of the crude oil by column chromatography (heptane to 1:2/heptane:DCM) yielded pure 2,3,5-trimethyl-6-(3,7,11,15-tetramethyl-hexadeca-2,6,10,14-tetraenyl)-[1,4]benzoquinone (Ex-1B-11, or Compound IX-i) (5.25 g, 83%, >96% a/a by HPLC). 1H NMR (400 MHz, CDCl3) δ (ppm): 5.11-5.05 (m, 3H), 4.98-4.95 (m, 1H), 3.21 (d, J=6.9 Hz, 2H), 2.10-1.94 (m, 21H), 1.76 (s, 3H), 1.69 (s, 3H), 1.61 (s, 3H), 1.60 (s, 3H), 1.59 (s, 3H).
Example 1C




Step 1: A 25 mL RBF was charged with butyraldehyde (155 mg, 2.16 mmol), AcOH (2 mL) and H2SO4 (1 drop). To the flask was then added a solution of (+)-γ-tocopherol (Ex-1C-1) (300 mg, 0.72 mmol) in AcOH (3 mL) dropwise over 2 h via syringe pump. The reaction was then stirred for 16 h and monitored by TLC (9:1 Hept:EtOAc). The reaction was then diluted with water (15 mL) and extracted with DCM (3×20 mL). The combined organic layers were washed with water (3×15 mL), dried over Na2SO4, filtered and concentrated by rotary evaporation to yield 7,9,10-trimethyl-2,4-dipropyl-7-(4,8,12-trimethyl-tridecyl)-4,5,6,7-tetrahydro-1,3,8-trioxa-phenanthrene (Ex-1C-2) as a brownish oil (425 mg, >100%), which was used without further purification.
Step 2: A solution of 7,9,10-trimethyl-2,4-dipropyl-7-(4,8,12-trimethyl-tridecyl)-4,5,6,7-tetrahydro-1,3,8-trioxa-phenanthrene (Ex-1C-2) (180 mg of crude material form above) in AcOH (10 mL) and conc. H2SO4 (10 drops) was hydrogenated (H2, 50 psi, RT) with 5% Pd/C (20 mg of 50% w/w wet) at RT for 16 h. The reaction mixture was then filtered through celite. The celite was rinsed with DCM (2×2 mL). The DCM layer was concentrated by rotary evaporation to yield a light brown oil. The oil was dissolved in DCM (15 mL) and passed through a silica plug. The DCM was concentrated by rotary evaporation to yield (R,R,R)-5-butyl-2,7,8-trimethyl-2-(4,8,12-trimethyl-tridecyl)-chroman-6-ol (Ex-1C-3) as a cloudy yellow oil (165 mg, >100%), which was used directly without further purification.
Step 3: A 50 mL RBF flask was charged with (R,R,R)-5-butyl-2,7,8-trimethyl-2-(4,8,12-trimethyl-tridecyl)-chroman-6-ol (Ex-1C-3) (120 mg, 0.25 mmol) and ACN (25 mL), then cooled to 0° C. A solution of CAN (268 mg, 0.49 mmol) in water (1 mL) was added dropwise over 1 min to the reaction resulting in a bright orange solution. After 10 min, the reaction was deemed complete (TLC—9:1 hept:EtOAc). The reaction was diluted with DCM (10 mL) and water (10 mL). The aqueous layer was washed with DCM (10 mL). The DCM layers were washed with brine (5 mL), passed through a silica plug and concentrated by rotary evaporation to yield (R,R,R)-2-butyl-3-(3-hydroxy-3,7,11,15-tetramethyl-hexadecyl)-5,6-dimethyl-[1,4]benzoquinone (Ex-1C-4) as an orange oil (105 mg, 85%). 1H NMR (400 MHz, CDCl3) δ (ppm): 2.56-2.52 (m, 2H), 2.47 (broad-t, J=6.9 Hz, 2H), 2.02 (s, 6H), 1.55-1.02 (m, 28H), 0.95 (broad-t, J=5.6 Hz, 3H), 0.89-0.85 (m, 15H).
Example 1D




Step 1: (+)-γ-tocopherol (Ex-1D-1) (300 mg, 0.72 mmol), K2CO3 (199 mg, 1.44 mmol), allyl bromide (182 μL, 1.44 mmol) and acetone (8 mL) were charged to a 50 mL RBF. The reaction was heated to reflux for 20 h after which time it was deemed complete by TLC (1:5 EtOAc:Hept). The reaction was diluted with water (10 mL). The aqueous layer was separated and washed with DCM (3×10 mL). The combined DCM layers were dried over Na2SO4, filtered and concentrated by rotary evaporation to yield a pale yellow oil. The oil was flashed through a silica plug (1:1: DCM:Heptane). After concentration of the eluent, (R,R,R)-6-allyloxy-2,7,8-trimethyl-2-(4,8,12-trimethyl-tridecyl)-chroman (Ex-1D-2) was obtained as a clear, colorless oil (334 mg, >100%), which was used without further purification.
Step 2: (R,R,R)-6-allyloxy-2,7,8-trimethyl-2-(4,8,12-trimethyl-tridecyl)-chroman (Ex-1D-2) (0.33 g, 0.72 mmol) was heated to 200° C. for 1 h after which time the reaction was deemed complete (TLC). The reaction was then cooled to RT and purified by flash chromatography (1:1 DCM:Heptane) to yield rearranged product (R,R,R)-5-allyl-2,7,8-trimethyl-2-(4,8,12-trimethyl-tridecyl)-chroman-6-ol (Ex-1D-3) (112 mg, 34%), which was used without further purification.
Step 3: A 50 mL RBF flask was charged with (R,R,R)-5-allyl-2,7,8-trimethyl-2-(4,8,12-trimethyl-tridecyl)-chroman-6-ol (Ex-1D-3) (120 mg, 0.26 mmol) and ACN (20 mL), then cooled to 0° C. A solution of CAN (285 mg, 0.52 mmol) in water (1 mL) was added dropwise over 1 min to the reaction resulting in a bright orange solution. After 15 min, the reaction was deemed complete (TLC—9:1 hept:EtOAc). The reaction was diluted with MTBE (10 mL) and water (10 mL). The aqueous layer was washed with MTBE (3×10 mL). The combined, MTBE layers were washed with brine (5 mL), dried over MgSO4, filtered and concentrated by rotary evaporation to yield an orange oil. The oil was dissolved in DCM (10 mL) and passed through a silica plug. The DCM eluent was concentrated by rotary evaporation to yield (R,R,R)-2-allyl-3-(3-hydroxy-3,7,11,15-tetramethyl-hexadecyl)-5,6-dimethyl-[1,4]benzoquinone (Ex-1D-4) as an orange oil (100 mg, 80%). 1H NMR (400 MHz, CDCl3) δ (ppm): 5.83 (ddt, 1H), 5.10-5.05 (m, 2H), 3.29 (d, J=6.2 Hz, 2H), 2.59-2.45 (m, 2H), 2.04 (s, 6H), 1.56-1.00 (m, 24H), 1.25 (s, 3H), 0.89-0.85 (m, 12H).
Step 4: (R,R,R)-2-allyl-3-(3-hydroxy-3,7,11,15-tetramethyl-hexadecyl)-5,6-dimethyl-[1,4]benzoquinone (Ex-1D-4) (50 mg, 0.1 mmol) was hydrogenated using PtO2 (5 mg) at 50 psi for 2 h in a solution of EtOAc (5 mL). The suspension was filtered through celite, which was rinsed with DCM (2×2 mL). The pale yellow solution was concentrated by rotary evaporation to yield a pale yellow oil (Ex-1D-5). The oil was dissolved in DCM (5 mL) and stirred with silica ( ̃20 mg) for 5 days. The bright yellow suspension was filtered through a cotton plug and concentrated by rotary evaporation to yield (R,R,R)-2-(3-hydroxy-3,7,11,15-tetramethyl-hexadecyl)-5,6-dimethyl-3-propyl-[1,4]benzoquinone (Ex-1D-6) as a bright yellow oil (38 mg, 76%). 1H NMR (400 MHz, CDCl3) δ (ppm): 2.57-2.52 (m, 2H), 2.48-2.44 (m, 2H), 2.02 (s, 6H), 1.57-1.04 (m, 26H), 0.99 (t, J=7.4 Hz, 3H), 0.89-0.85 (m, 15H).
Example 1E




Step 1: (+)-δ-tocopherol (Ex-1E-1) (1.04 g, 2.58 mmol), K2CO3 (715 mg, 5.17 mmol), allyl bromide (450 μL, 5.17 mmol) and acetone (10 mL) were charged to a 50 mL RBF. The reaction was heated to reflux for 16 h after which time it was deemed complete by TLC (1:5 EtOAc:Hept). The reaction was diluted with water (10 mL) and DCM (10 mL). The aqueous layer was separated and washed with DCM (3×10 mL). The combined DCM layers were dried over MgSO4, filtered and concentrated by rotary evaporation to yield a pale yellow liquid (1.09 g). The liquid wash flashed through a silica plug (1:1: DCM:hept). After concentration of the eluent, (R,R,R)-6-allyloxy-2,8-dimethyl-2-(4,8,12-trimethyl-tridecyl)-chroman (Ex-1E-2) was obtained as a clear, colorless oil (0.97 g, 85%).
Step 2: (R,R,R)-6-allyloxy-2,8-dimethyl-2-(4,8,12-trimethyl-tridecyl)-chroman (Ex-1E-2) (0.97 g, 2.19 mmol) was heated to 200° C. for 3 h after which time the reaction was deemed complete (1H NMR —4:1 mixture of isomers). The reaction was then cooled to RT to yield (R,R,R)-5-allyl-2,8-dimethyl-2-(4,8,12-trimethyl-tridecyl)-chroman-6-ol (Ex-1E-3) as a brown oil (0.97 g, 100%), which was carried on to the next step without further purification.
Step 3: A 50 mL RBF flask was charged with (R,R,R)-5-allyl-2,8-dimethyl-2-(4,8,12-trimethyl-tridecyl)-chroman-6-ol (Ex-1E-3) (280 mg, 0.63 mmol) and ACN (14 mL), then cooled to 0° C. A solution of CAN (710 mg, 1.30 mml) in water (2 mL) was added dropwise over 1 min to the reaction resulting in a bright orange solution. After 15 min, the reaction was deemed complete (TLC—5:1 hept:EtOAc). The reaction was extracted with MTBE (3×15 mL). The combined MTBE layers were dried over Na2SO4, filtered and concentrated by rotary evaporation to yield (R,R,R)-2-allyl-3-(3-hydroxy-3,7,11,15-tetramethyl-hexadecyl)-5-methyl-[1,4]benzoquinone (Ex-1E-4) as an orange oil (270 mg, 96%). 1H NMR (400 MHz, CDCl3) δ (ppm): 6.59 (d, J=1.4 Hz, 1H), 5.82 (ddt, 1H), 5.10-5.06 (m, 2H), 3.27 (d, J=6.2 Hz, 2H), 2.60-2.56 (m, 2H), 2.06 (s, 6H), 1.59-1.04 (m, 21H), 0.89-0.85 (m, 15H).
Step 4: (R,R,R)-2-allyl-3-(3-hydroxy-3,7,11,15-tetramethyl-hexadecyl)-5-methyl-[1,4]benzoquinone (Ex-1E-4) (115 mg, 0.25 mmol) was hydrogenated using PtO2 (6 mg) at 50 psi for 3 h in a solution of EtOAc (7 mL). The suspension was filtered through silica, which was rinsed with EtOAc (40 mL). The solution was concentrated by rotary evaporation to yield (R,R,R)-3-(3-hydroxy-3,7,11,15-tetramethyl-hexadecyl)-5-methyl-2-propyl-benzene-1,4-diol (Ex-1E-5) as a clear, colorless oil (110 mg, 96%), which was carried on to the next step without further purification.
Step 5: A 50 mL RBF flask was charged with (R,R,R)-3-(3-hydroxy-3,7,11,15-tetramethyl-hexadecyl)-5-methyl-2-propyl-benzene-1,4-diol (Ex-1E-5) (110 mg, 0.24 mmol), ACN (15 mL) and DCM (2 mL), then cooled to 0° C. A solution of CAN (269 mg, 0.49 mmol) in water (1 mL) was added dropwise over 1 min to the reaction resulting in a bright orange solution. The reaction was stirred for 15 min then was diluted with water (5 mL). The aqueous layer was washed with DCM (3×30 mL). The combined DCM layers were dried over Na2SO4, filtered and concentrated by rotary evaporation to yield an orange oil. The oil was purified by column chromatography (gradient—hept to 20:1 hept.:EtOAc) to yield (R,R,R)-3-(3-hydroxy-3,7,11,15-tetramethyl-hexadecyl)-5-methyl-2-propyl-[1,4]benzoquinone (Ex-1E-6) as an orange oil (50 mg, 44%). 1H NMR (400 MHz, CDCl3) δ (ppm): 6.56 (s, 1H), 2.58-2.54 (m, 2H), 2.45 (t, J=7.9 Hz, 2H), 2.04 (s, 3H), 1.55-1.04 (m, 26H), 1.00 (t, J=7.4 Hz, 3H), 0.89-0.85 (m, 15H).
Example 1F




Step 1: (+)-γ-tocopherol (Ex-1F-1) (300 mg, 0.72 mmol), K2CO3 (199 mg, 1.44 mmol), 3-chloro-2-methyl propene (450 μL, 5.17 mmol), NaI ( ̃10 mg) and acetone (8 mL) were charged to a 50 mL RBF. The reaction was heated to reflux for 20 h after which time it was deemed complete by TLC (1:9 EtOAc:Hept). The reaction was diluted with water (15 mL) and DCM (10 mL). The aqueous layer was separated and washed with DCM (3×10 mL). The combined DCM layers were dried over Na2SO4, filtered and concentrated by rotary evaporation to yield (R,R,R)-2,7,8-trimethyl-6-(2-methyl-allyloxy)-2-(4,8,12-trimethyl-tridecyl)-chroman as a pale yellow liquid (Ex-1F-2) (324 mg, 95%). The isolated product was used without any further purification.
Step 2: (R,R,R)-2,7,8-trimethyl-6-(2-methyl-allyloxy)-2-(4,8,12-trimethyl-tridecyl)-chroman (Ex-1F-2) (325 mg, 0.691 mmol) was heated to 200° C. for 4.5 h after which time the reaction was deemed complete (TLC—10:1 Heptane:EtOAc). The reaction was then cooled to RT to yield (R,R,R,)-2,7,8-trimethyl-5-(2-methyl-allyl)-2-(4,8,12-trimethyl-tridecyl)-chroman-6-ol (Ex-1F-3) (302 mg, 93%), which was taken to the next step without further purification.
Step 3: A 50 mL RBF flask was charged with crude (R,R,R,)-2,7,8-trimethyl-5-(2-methyl-allyl)-2-(4,8,12-trimethyl-tridecyl)-chroman-6-ol (Ex-1F-3) (150 mg, 0.32 mmol) and ACN (20 mL), then cooled to 0° C. A solution of CAN (362 mg, 0.66 mmol) in water (1 mL) was added dropwise over 1 min to the reaction resulting in a bright orange solution. After 15 min, the reaction was deemed complete (TLC—9:1 hept:EtOAc). The reaction was diluted with DCM (10 mL) and water (5 mL). The aqueous layer was washed with DCM (10 mL). The DCM layers were washed with brine (5 mL), dried over MgSO4, filtered and concentrated by rotary evaporation to yield an orange oil. The oil was dissolved in DCM (10 mL) and passed through a silica plug. The DCM eluent was concentrated by rotary evaporation to yield (R,R,R,)-2-(3-hydroxy-3,7,11,15-tetramethyl-hexadecyl)-5,6-dimethyl-3-(2-methyl-allyl)-[1,4]benzoquinone (Ex-1F-4) as an orange oil (100 mg, 61%). 1H NMR δ (ppm): 4.78 (s, 1H), 4.54 (s, 1H), 3.22 (s, 2H), 2.55-2.51 (m, 2H), 2.04 (s, 6H), 1.55-1.04 (m, 30H), 0.89-0.85 (m, 12H).
Step 4: (R,R,R,)-2-(3-hydroxy-3,7,11,15-tetramethyl-hexadecyl)-5,6-dimethyl-3-(2-methyl-allyl)-[1,4]benzoquinone (Ex-1F-4) (50 mg, 0.10 mmol) was hydrogenated using PtO2 (5 mg) at 50 psi for 3 h in a solution of EtOAc (5 mL). The suspension was filtered through celite, which was rinsed with EtOAc (5 mL). The solution was concentrated by rotary evaporation to yield a clear, colorless oil (Ex-1F-5) (40 mg). The oil was dissolved in CDCl3 (1 mL) and stirred with silica ( ̃20 mg) for 5 days. The bright yellow suspension was filtered through a cotton plug and concentrated by rotary evaporation to yield (R,R,R)-2-(3-hydroxy-3,7,11,15-tetramethyl-hexadecyl)-3-isobutyl-5,6-dimethyl-[1,4]benzoquinone (Ex-1F-6) as a bright yellow oil (38 mg, 76%). 1H NMR δ (ppm): 2.58-2.53 (m, 2H), 2.40 (d, J=7.2 Hz, 2H), 2.02 (s, 6H), 1.84 (sept, J=6.9 Hz, 1H) 1.56-1.03 (m, 27H), 0.93 (d, J=6.6 Hz, 6H), 0.90-0.84 (m, 12H).
Example 2
Initial Screen for Effective Redox Compounds
An initial screen was performed to identify compounds effective for the amelioration of redox disorders. Test samples, 4 reference compounds (idebenone, decylubiquinone, Trolox and α-tocopherol acetate), and solvent controls were tested for their ability to rescue FRDA fibroblasts stressed by addition of L-buthionine-(S,R)-sulfoximine (BSO), as described in Jauslin et al., Hum. Mol. Genet. 11(24):3055 (2002), Jauslin et al., FASEB J. 17:1972-4 (2003), and International Patent Application WO 2004/003565. Human dermal fibroblasts from Friedreich's Ataxia patients have been shown to be hypersensitive to inhibition of the de novo synthesis of glutathione (GSH) with L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of GSH synthetase (Jauslin et al., Hum. Mol. Genet. 11(24):3055 (2002)). This specific BSO-mediated cell death can be prevented by administration of antioxidants or molecules involved in the antioxidant pathway, such as α-tocopherol, short chain quinones, selenium, or small molecule glutathione peroxidase mimetics. However, antioxidants differ in their potency, i.e. the concentration at which they are able to rescue BSO-stressed FRDA fibroblasts. With this assay EC50 concentrations of the test compounds were determined and compared to known reference antioxidants.
MEM (a medium enriched in amino acids and vitamins, catalog no. 1-31F24-I) and Medium 199 (M199, catalog no. 1-21F22-I) with Earle's Balanced Salts, without phenol red, were purchased from Bioconcept. Fetal Calf Serum was obtained from PAA Laboratories. Basic fibroblast growth factor and epidermal growth factor were purchased from PeproTech. Penicillin-streptomycin-glutamine mix, L-buthionine (S,R)-sulfoximine, (+)-α-tocopherol acetate, decylubiquinone, and insulin from bovine pancreas were purchased from Sigma. Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) was obtained from Fluka. Idebenone was obtained from Chemo Iberica. Calcein AM was purchased from Molecular Probes. Cell culture medium was made by combining 125 ml M199 EBS, 50 ml Fetal Calf Serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, 10 μg/ml insulin, 10 ng/ml EGF, and 10 ng/ml bFGF; MEM EBS was added to make the volume up to 500 ml. A 10 mM BSO solution was prepared by dissolving 444 mg BSO in 200 ml of medium with subsequent filter-sterilization. During the course of the experiments, this solution was stored at +4° C. The cells were obtained from the Coriell Cell Repositories (Camden, N.J.; repository number GM04078) and grown in 10 cm tissue culture plates. Every third day, they were split at a 1:3 ratio.
The test samples were supplied in 1.5 ml glass vials. The compounds were diluted with DMSO, ethanol or PBS to result in a 5 mM stock solution. Once dissolved, they were stored at −20° C. Reference antioxidants (idebenone, decylubiquinone, α-tocopherol acetate and trolox) were dissolved in DMSO.
Test samples were screened according to the following protocol: A culture with FRDA fibroblasts was started from a 1 ml vial with approximately 500,000 cells stored in liquid nitrogen. Cells were propagated in 10 cm cell culture dishes by splitting every third day in a ratio of 1:3 until nine plates were available. Once confluent, fibroblasts were harvested. For 54 micro titer plates (96 well-MTP) a total of 14.3 million cells (passage eight) were re-suspended in 480 ml medium, corresponding to 100 μl medium with 3,000 cells/well. The remaining cells were distributed in 10 cm cell culture plates (500,000 cells/plate) for propagation. The plates were incubated overnight at 37° C. in a atmosphere with 95% humidity and 5% CO2 to allow attachment of the cells to the culture plate.
MTP medium (243 μl) was added to a well of the microtiter plate. The test compounds were unfrozen, and 7.5 μl of a 5 mM stock solution was dissolved in the well containing 243 μl medium, resulting in a 150 μM master solution. Serial dilutions from the master solution were made. The period between the single dilution steps was kept as short as possible (generally less than 1 second).
Plates were kept overnight in the cell culture incubator. The next day, 10 μl of a 10 mM BSO solution were added to the wells, resulting in a 1 mM final BSO concentration. Forty-eight hours later, three plates were examined under a phase-contrast microscope to verify that the cells in the 0% control (wells E1-H1) were clearly dead. The medium from all plates was discarded, and the remaining liquid was removed by gently tapping the plate inversed onto a paper towel.
100 μl of PBS containing 1.2 μM Calcein AM were then added to each well. The plates were incubated for 50-70 minutes at room temperature. After that time the PBS was discarded, the plate gently tapped on a paper towel and fluorescence (excitation/emission wavelengths of 485 nm and 525 nm, respectively) was read on a Gemini fluorescence reader. Data was imported into Microsoft Excel (EXCEL is a registered trademark of Microsoft Corporation for a spreadsheet program) and used to calculate the EC50 concentration for each compound.
The compounds were tested three times, i.e., the experiment was performed three times, the passage number of the cells increasing by one with every repetition.
The solvents (DMSO, ethanol, PBS) neither had a detrimental effect on the viability of non-BSO treated cells nor did they have a beneficial influence on BSO-treated fibroblasts even at the highest concentration tested (1%). None of the compounds showed auto-fluorescence. The viability of non-BSO treated fibroblasts was set as 100%, and the viability of the BSO- and compound-treated cells was calculated as relative to this value.
The following table summarizes the EC50 for alpha-tocopherol quinone and the four control compounds.













EC50 [μM]












Compound
Value 1
Value 2
Value 3
Average
Stdev















α-tocopherol
0.000001
0.000003
2E−07
1.40E−06
1.44E−06


quinone


decylubi-
0.05
0.035
0.03
0.038
0.010


quinone


α-tocopherol
0.4
0.15
0.35
0.30
0.13


acetate


idebenone
1.5
1
1
1.2
0.3


Trolox
9
9
8
8.7
0.6









Example 3
Screening Compounds of the Invention
Compounds of the invention are tested using the screen as described in Example 2 for their ability to rescue human dermal fibroblasts from FRDA patients from oxidative stress. This data is used to estimate their potential as disease treatments.
Example 4
Administration of Compounds of the Invention
A compound of the invention, such as alpha-tocopherol quinone, is presented in a capsule containing 300 mg of compound in a pharmaceutically acceptable carrier. A capsule is taken orally, once a day, preferably during breakfast or lunch. In case of very young children, the capsule is broken and its contents mixed with food.
The disclosures of all publications, patents, patent applications and published patent applications referred to herein by an identifying citation are hereby incorporated herein by reference in their entirety.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is apparent to those skilled in the art that certain minor changes and modifications will be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention.

## Claims

1. A method of treating a mitochondrial disorder, comprising administering to a subject suffering from the mitochondrial disorder a composition comprising a therapeutically effective amount of one or more compounds of the formula:



where R11, R12, and R13 are independently selected from the group consisting of H, —C1-C4 alkyl, —C1-C4 haloalkyl, —CN, —F, —Cl, —Br, and —I, with the proviso that if any of R11, R12, or R13 is H, then at least one of the other two substituents is neither H nor methyl; and all stereoisomers, and mixtures of stereoisomers thereof;
wherein said one or more compounds are the sole active pharmaceutical agent or agents present in a therapeutically effective amount in the composition;
and wherein the mitochondrial disorder is selected from the group consisting of Leber's Hereditary Optic Neuropathy (LHON); Friedreich's Ataxia (FA); Parkinson's disease; amyotrophic lateral sclerosis (ALS); and Huntington's Disease.

2. The method of claim 1, wherein the one or more compounds are selected from compounds of the formula:



and all stereoisomers, and mixtures of stereoisomers thereof.

3. The method of claim 2, wherein the one or more compounds is the compound:

4. The method of claim 1, wherein none of R11, R12, and R13 is H and at least one of R11, R12, and R13 is not methyl, and all stereoisomers, and mixtures of stereoisomers thereof.

5. The method of claim 1, wherein the mitochondrial disorder is Friedreich's Ataxia (FA).

6. The method of claim 1, wherein the mitochondrial disorder is Leber's Hereditary Optic Neuropathy (LHON).

7. The method of claim 1, wherein the mitochondrial disorder is Parkinson's disease.

8. The method of claim 1, wherein the mitochondrial disorder is amyotrophic lateral sclerosis (ALS).

9. The method of claim 1, wherein the mitochondrial disorder is Huntington's Disease.

10. The method of claim 2, wherein the mitochondrial disorder is Friedreich's Ataxia (FA).

11. The method of claim 2, wherein the mitochondrial disorder is Leber's Hereditary Optic Neuropathy (LHON).

12. The method of claim 2, wherein the mitochondrial disorder is Parkinson's disease.

13. The method of claim 2, wherein the mitochondrial disorder is amyotrophic lateral sclerosis (ALS).

14. The method of claim 2, wherein the mitochondrial disorder is Huntington's Disease.

15. The method of claim 3, wherein the mitochondrial disorder is Friedreich's Ataxia (FA).

16. The method of claim 3, wherein the mitochondrial disorder is Leber's Hereditary Optic Neuropathy (LHON).

17. The method of claim 3, wherein the mitochondrial disorder is Parkinson's disease.

18. The method of claim 3, wherein the mitochondrial disorder is amyotrophic lateral sclerosis (ALS).

19. The method of claim 3, wherein the mitochondrial disorder is Huntington's Disease.

20. The method of claim 2, wherein the one or more compounds are the sole active pharmaceutical agent or agents in the composition.

21. The method of claim 3, wherein the compound is the sole active pharmaceutical agent in the composition.

22. The method of claim 12, wherein the one or more compounds are the sole active pharmaceutical agent or agents in the composition.

23. The method of claim 14, wherein the one or more compounds are the sole active pharmaceutical agent or agents in the composition.

24. The method of claim 17, wherein the compound is the sole active pharmaceutical agent in the composition.

25. The method of claim 19, wherein the compound is the sole active pharmaceutical agent in the composition.

26. The method of claim 2, wherein the method consists of administering the composition to the subject.

27. The method of claim 3, wherein the method consists of administering the composition to the subject.

28. The method of claim 12, wherein the method consists of administering the composition to the subject.

29. The method of claim 14, wherein the method consists of administering the composition to the subject.

30. The method of claim 17, wherein the method consists of administering the composition to the subject.

31. The method of claim 19, wherein the method consists of administering the composition to the subject.

32. A method of treating a mitochondrial disorder, comprising administering to a subject suffering from the mitochondrial disorder a composition comprising a therapeutically effective amount of one or more compounds of the formula:



where R11, R12, and R13 are independently selected from the group consisting of H, —C1-C4 alkyl, —C1-C4 haloalkyl, —CN, —F, —Cl, —Br, and —I, with the proviso that if any of R11, R12, and R13 is H, then at least one of the other two substituents is neither H nor methyl; and all stereoisomers, and mixtures of stereoisomers thereof;
wherein the one or more compounds are the sole active pharmaceutical agent or agents used to treat the mitochondrial disorder;
and wherein the mitochondrial disorder is selected from the group consisting of Leber's Hereditary Optic Neuropathy (LHON); Friedreich's Ataxia (FA); Parkinson's disease; amyotrophic lateral sclerosis (ALS); and Huntington's Disease.

33. The method of claim 32, wherein the one or more compounds are selected from compounds of the formula:



and all stereoisomers, and mixtures of stereoisomers thereof.

34. The method of claim 32, wherein the one or more compounds is the compound:

35. The method of claim 33, wherein the mitochondrial disorder is Friedreich's Ataxia (FA).

36. The method of claim 33, wherein the mitochondrial disorder is Leber's Hereditary Optic Neuropathy (LHON).

37. The method of claim 33, wherein the mitochondrial disorder is Parkinson's disease.

38. The method of claim 33, wherein the mitochondrial disorder is amyotrophic lateral sclerosis (ALS).

39. The method of claim 33, wherein the mitochondrial disorder is Huntington's Disease.

40. The method of claim 34, wherein the mitochondrial disorder is Friedreich's Ataxia (FA).

41. The method of claim 34, wherein the mitochondrial disorder is Leber's Hereditary Optic Neuropathy (LHON).

42. The method of claim 34, wherein the mitochondrial disorder is Parkinson's disease.

43. The method of claim 34, wherein the mitochondrial disorder is amyotrophic lateral sclerosis (ALS).

44. The method of claim 34, wherein the mitochondrial disorder is Huntington's Disease.

45. A method of treating a mitochondrial disorder, comprising administering to a subject suffering from the mitochondrial disorder a composition comprising a therapeutically effective amount of one or more compounds of the formula:



where R11, R12, and R13 are independently selected from the group consisting of H, —C1-C4 alkyl, —C1-C4 haloalkyl, —CN, —F, —Cl, —Br, and —I, with the proviso that if any of R11, R12, or R13 is H, then at least one of the other two substituents is neither H nor methyl; and all stereoisomers, and mixtures of stereoisomers thereof;
and wherein the mitochondrial disorder is selected from the group consisting of Leber's Hereditary Optic Neuropathy (LHON); Friedreich's Ataxia (FA); and amyotrophic lateral sclerosis (ALS).

46. The method of claim 45, wherein the one or more compounds are selected from compounds of the formula:



and all stereoisomers, and mixtures of stereoisomers thereof.

47. The method of claim 45, wherein the one or more compounds is the compound:

48. The method of claim 46, wherein the mitochondrial disorder is Friedreich's Ataxia (FA).

49. The method of claim 46, wherein the mitochondrial disorder is Leber's Hereditary Optic Neuropathy (LHON).

50. The method of claim 46, wherein the mitochondrial disorder is amyotrophic lateral sclerosis (ALS).

51. The method of claim 47, wherein the mitochondrial disorder is Friedreich's Ataxia (FA).

52. The method of claim 47, wherein the mitochondrial disorder is Leber's Hereditary Optic Neuropathy (LHON).

53. The method of claim 47, wherein the mitochondrial disorder is amyotrophic lateral sclerosis (ALS).