EP2606022A2 - Novel benzoquinone derivatives and use thereof as modulators of mitochondrial function - Google Patents

Novel benzoquinone derivatives and use thereof as modulators of mitochondrial function

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Publication number
EP2606022A2
EP2606022A2 EP11746182.2A EP11746182A EP2606022A2 EP 2606022 A2 EP2606022 A2 EP 2606022A2 EP 11746182 A EP11746182 A EP 11746182A EP 2606022 A2 EP2606022 A2 EP 2606022A2
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EP
European Patent Office
Prior art keywords
group
linear
alkyl
independently selected
branched
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP11746182.2A
Other languages
German (de)
French (fr)
Inventor
Achim Feurer
Barbara Hoffmann-Enger
Holger Deppe
Michael Soeberdt
Nuri Gueven
Roman Haefeli
Fabrice Heitz
Michael Erb
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Santhera Pharmaceuticals Schweiz GmbH
Original Assignee
Santhera Pharmaceuticals Schweiz GmbH
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Filing date
Publication date
Application filed by Santhera Pharmaceuticals Schweiz GmbH filed Critical Santhera Pharmaceuticals Schweiz GmbH
Priority to EP11746182.2A priority Critical patent/EP2606022A2/en
Publication of EP2606022A2 publication Critical patent/EP2606022A2/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/08—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C225/00—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones
    • C07C225/24—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones the carbon skeleton containing carbon atoms of quinone rings
    • C07C225/26—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones the carbon skeleton containing carbon atoms of quinone rings having amino groups bound to carbon atoms of quinone rings or of condensed ring systems containing quinone rings
    • C07C225/28—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones the carbon skeleton containing carbon atoms of quinone rings having amino groups bound to carbon atoms of quinone rings or of condensed ring systems containing quinone rings of non-condensed quinone rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C07C233/24—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
    • C07C233/25—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
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    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/42—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/44—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring
    • C07C235/58—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring with carbon atoms of carboxamide groups and singly-bound oxygen atoms, bound in ortho-position to carbon atoms of the same non-condensed six-membered aromatic ring
    • C07C235/60—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring with carbon atoms of carboxamide groups and singly-bound oxygen atoms, bound in ortho-position to carbon atoms of the same non-condensed six-membered aromatic ring having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
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    • C07C235/70—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/72—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms
    • C07C235/76—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of an unsaturated carbon skeleton
    • C07C235/78—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of an unsaturated carbon skeleton the carbon skeleton containing rings
    • C—CHEMISTRY; METALLURGY
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C50/00—Quinones
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    • C07C50/30—Quinones containing groups having oxygen atoms singly bound to carbon atoms with polycyclic non-condensed quinoid structure
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    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D211/20—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by singly bound oxygen or sulphur atoms
    • C07D211/22—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by singly bound oxygen or sulphur atoms by oxygen atoms
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    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/60—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D211/62—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals attached in position 4
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/62—Oxygen or sulfur atoms
    • C07D213/63—One oxygen atom
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    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
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    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
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    • C07D233/56—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, attached to ring carbon atoms
    • C07D233/60—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, attached to ring carbon atoms with hydrocarbon radicals, substituted by oxygen or sulfur atoms, attached to ring nitrogen atoms
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    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
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    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/02—Systems containing only non-condensed rings with a three-membered ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/56—Ring systems containing bridged rings
    • C07C2603/58—Ring systems containing bridged rings containing three rings
    • C07C2603/70—Ring systems containing bridged rings containing three rings containing only six-membered rings
    • C07C2603/74—Adamantanes

Definitions

  • the present invention relates to novel benzoquinone derivatives.
  • the compounds are efficient as modulators of mitochondrial function and as such are useful for the treatment of pathological conditions where mitochondrial function is impaired.
  • Mitochondria sometimes described as "cellular power plants” because they generate most of the cell's supply of adenosine triphosphate (ATP), are essential to eukaryotic life.
  • mitochondria are also involved in a range of other processes, such as signalling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth.
  • Mitochondria have been implicated in several human diseases, including mitochondrial disorders and cardiac dysfunction and may play a role in the aging process.
  • Mitochondrial disorders are often present as neurological disorders, but can also occur as myopathy, diabetes, multiple endocrinopathy, or a variety of other systemic manifestations. These disorders can be caused by mutations of the mitochondrial DNA, by mutations of nuclear genes directly coding for oxidative phosphorylation enzymes and by defects in nuclear genes that are generally important for mitochondrial function. Environmental influences may also interact with hereditary predispositions and cause mitochondrial diseases. For example, there may be a link between pesticide exposure and the later onset of Parkinson's disease.
  • mitochondrial dysfunction Numerous other pathologies involving mitochondrial dysfunction include schizophrenia, bipolar disorder, autosomal dominant optic atrophy, epilepsy, stroke and autism (Jou et al. Chang Gung Med J. 2009; 32(4):370-9), dementia, Alzheimer's, Parkinson's and Huntington's disease (Yang et al. DNA Repair (Amst). 2008; 7(7): 1110-20), cardiovascular disease (Puddu et al. J Biomed Sci. 2009; 16:112), malignancies (Singh & Kulawiec; Methods Mol Biol. 2009; 471 :291-303), retinitis pigmentosa, metabolic syndrome, anorexia, obesity and diabetes mellitus (Burchell et al. Expert Opin Ther Targets 2010; 14(4):369-85; Pieczenik & Neustadt Exp Mol Pathol. 2007; 83(1):84-92).
  • Multiple sclerosis is the most common non-traumatic neurological disease in young adults, with a prevalence of 1:1000 in Northern Europe and North-America (Compston A Int MS J .2003. 10:29-31).
  • the disease course is generally episodic; exacerbations are followed by periods of remission which are characterized by focal infiltration of leukocytes and demyelination in the white matter.
  • the disease becomes more progressive, where demyelination of the grey matter and axonal degeneration in the white matter become more prominent (Hafler DA J. Clin. Invest. 2004; 113:788-794).
  • Available therapies are mainly immunomodulatory, which are effective in reducing the number of relapses. Disease progression, however, remains unchanged by these therapies (Filippini G et al. Lancet 2003; 361 :545-552.), demonstrating the need for novel therapeutic strategies oriented towards neuroprotection.
  • WO-A-2006130775 describes an attempt to treat or suppress mitochondrial diseases by modulating energy biomarkers such as lactic acid levels, levels of NAD, NADP, NADH and NADPH, and cytochrome C parameters.
  • energy biomarkers such as lactic acid levels, levels of NAD, NADP, NADH and NADPH, and cytochrome C parameters.
  • the compounds disclosed in the application have been tested for their ability to rescue human dermal fibroblasts from FRDA patients from oxidative stress. However, the data are not represented in the application.
  • the present invention relates to benzoquinone derivatives of structural formula (I)
  • R , R 2 , R 3 and R 4 are defined as described below.
  • the benzoquinone derivatives of structural formula (I) are effective as modulators of mitochondrial function and as such are useful for the treatment of pathological conditions where mitochondrial function is impaired. It has been found that the compounds of formula (I) are useful in the treatment of mitochondrial diseases such as Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy (DOA), macular degeneration, glaucoma, retinopathy, cataract, optic disc drusen (ODD), mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), myoclonic epilepsy with ragged red fibers (MERRF), myoneurogenic gastrointestinal encephalomyopathy (MNGIE), Kearns-Sayre syndrome, C0QIO deficiency, or mitochondrial complex deficiencies or neurodegenerative diseases such as Friedreich's ataxia (FRDA), amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, Strok
  • the present invention also relates to pharmaceutical compositions comprising the compounds of the present invention and a pharmaceutically acceptable carrier.
  • the present invention relates to benzoquinone derivatives which are capable to modulate mitochondrial function.
  • R 1 is a substituent represented by formula (II) Y 1 (X 1 ) a R 5
  • X 1 is -O- or -NR 6 -, wherein R 6 is H or linear or branched d. 6 alkyl;
  • R 5 is selected from the group consisting of
  • a linear or branched C 1-6 alkyl which is unsubstituted or substituted with 1 to 5 substituents independently selected from halogen, OH, NH 2 or CN, a 6-10 membered monocylic or bicyclic aryl group, optionally fused with a 5-6 membered heterocyclic or heteroaromatic group, the heterocyclic or heteroaryl group having 1 to 2 heteroatoms in the ring independently selected from N, O or S,
  • a 3-10 membered monocylic, bicylic or tricyclic cycloalkyl group wherein each aryl, heteroaryl, heterocyclyl and cycloalkyl is unsubstituted or substituted with 1 to 5 substituents independently from each other selected from the group consisting of halogen, hydroxy, linear or branched Ci-6 alkyl, linear or branched halo-Ci. 6 alkyl, Ci_ 6 alkoxy, -C(0)NH 2 , - NHC(0)C 1-6 alkyl, 5-6 membered heteroaryl having in the ring 1 to 3 heteroatoms independently selected from N, O or S,
  • X 1 and R 5 together form a 4-7 membered monocyclic heterocyclic group having 1 or 2 heteroatoms in the ring independently selected from N or S and wherein the heterocycle is unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of a halogen, a hydroxyl group and a Ci -6 alkoxy group;
  • Y 1 is a linear C 8 .i 2 alkylene group or a linear C 8 -12 alkenylene group
  • a is an integer of 0 or 1 ;
  • R 2 is a Ci-6 linear or branched alkyl group
  • R 3 is a Ci-e alkoxy group
  • R 4 is a C 1-6 alkoxy group
  • R 5 is not a 2H-pyrane group
  • R 1 is a substituent represented by formula (III)
  • R 7 and R 8 independently from each other are hydrogen, linear or branched d. 6 alkyl or halogen;
  • R 9 and R 10 independently from each other are hydrogen, linear or branched C 2 . 6 alkenyl or linear or branched C 2- 6 alkinyl;
  • R 9 and R 10 together are -(CH 2 ) X - wherein x is an integer of 2 to 6;
  • Y 2 is a linear C 5 . 9 alkylene group or a linear C 5 . 9 alkenylene group
  • R 2 is a linear or branched C 1-6 alkyl group
  • R 3 is a Ci- 6 alkoxy group
  • R 4 is a Ci -6 alkoxy group
  • R 1 is a substituent represented by formula (IV)
  • each alkyl, aryl, heteroaromatic, heterocyclic and cycloalkyi group is unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of halogen, hydroxy and Ci -6 alkoxy;
  • 4- 7 membered ring having in the ring 0 to 2 additional heteroatoms independently selected from N, O or S, wherein the ring is unsubstituted or substituted with 1 to 3 substituents selected from the group consisting of halogen, hydroxyl and C ⁇ alkoxy group;
  • Y 3 is a linear C 6 . 10 alkylene group or a linear C 6-10 alkenylene group
  • R 2 is a linear or branched 0 1-6 alkyl group
  • R 3 is a group
  • R 4 is a Ci. 6 alkoxy group
  • R 1 is hydrogen
  • R 2 is a substituent represented by X 2 -R 13
  • X 2 is selected from the group consisting of -O- and -NR 14 -, wherein R 14 is hydrogen or a d-e alkyl group, and
  • R 13 is a C4-12 alkyl group, C4- 12 alkenyl group, -(CH 2 )nCH 2 OH or -C 2 -1 0 alkenylene-CH 2 OH,
  • n is an integer of 1 to 10
  • X 2 -R 13 represents a piperidino group substituted in the 4-position with a C 2 -7 alkenylene-CH 2 OH group, a -(CH 2 ) m CH 2 OH group wherein m is an integer of 1 to 7, a -C(0)-NH-C 2 - 6 alkenylene-CH 2 OH group or a -C(0)-NH-(CH 2 ) p CH 2 OH group wherein p is an integer of 1 to 6;
  • R 3 is hydrogen; and R 4 is OR 15 ,
  • R 15 is a hydrogen atom or a d. 2 alkyl group
  • R 13 and R 15 independently represent a linear -C 2 - 7 alkenylene-CH 2 OH group or a -(CH 2 ) m CH 2 0H group wherein m is an integer of 1 to 7;
  • Y 4 is a linear C 8 -i 2 alkylene group or a linear C 8 -i 2 alkenylene group
  • R 2 is a linear or branched C 1 .6 alkyl group
  • R 3 is a Ci-e alkoxy group
  • R 4 is a Ci. 6 alkoxy group
  • Y 5 is a linear C 8 .i 2 alkylene group or a linear C 8 -i 2 alkenylene group;
  • R 2 is a linear or branched d-ealkyl group;
  • R 3 is a hydroxy group or a Ci. 6 alkoxy group
  • R 4 is a Ci-ealkoxy group
  • Y 6 is a linear C 6 -io alkylene group or a linear C 6 -io alkenylene group
  • R 2 is a linear or branched Ci. 6 alkyl group
  • R 3 is a d. 6 alkoxy group; and R 4 is a Ci. 6 alkoxy group.
  • R 1 has the formula (II);
  • a 1 ;
  • X 1 is an oxygen atom
  • R 5 is selected from the group consisting of a 6 membered heteroaryl group having 1 to 2 nitrogen atom(s) in the ring and a phenyl group wherein the heteroaryl and the phenyl group are unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of halogen, linear or branched C 1-6 alkyl, linear or branched halo On alkyl, C 1-6 alkoxy, -C(0)NH 2 , alkyl, 5-6 membered heteroaryl having in the ring 1 to 3 heteroatoms independently selected from N, O or S, and hydroxy.
  • R 1 has the formula (II);
  • a 1 ;
  • X 1 is a nitrogen atom (i.e., -NR 6 -);
  • R 5 is selected from the group consisting of
  • heteroaryl group, the phenyl group and the adamantly group are unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of halogen, linear or branched Ci -6 alkyl, linear or branched halo Ci -6 alkyl, d. 6 alkoxy, -C(0)NH 2 , -NHCiO ⁇ -e alkyl, 5-6 membered heteroaryl having in the ring 1 to 3 heteroatoms independently selected from N, O or S, and hydroxy or
  • X 1 and R 5 together form a 4-7 membered monocyclic heterocyclic group having 1 or 2 heteroatoms in the ring independently selected from N or S and whereby the heterocycle is unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of a halogen, a hydroxyl group and a Ci. 6 alkoxy group.
  • R 1 has the formula (II);
  • a 0;
  • R 5 is a 5-10 membered monocyclic or bicyclic heteroaryl group having 1 to 4 heteroatoms independently selected from N, O and S, wherein the heteroaryl group is unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of halogen, linear or branched 0 1-6 alkyl, linear or branched halo C 1-6 alkyl, d -6 alkoxy, - C(0)NH 2 , -NHC(0)Ci-6 alkyl, 5-6 membered heteroaryl having in the ring 1 to 3 heteroatoms independently selected from N, O or S, and hydroxy.
  • R 1 is hydrogen
  • R 2 is X 2 -R 13 wherein X 2 is -NR 14 wherein R 4 is hydrogen or a d-e alkyl group, and R 13 is a linear C 4-12 alkyl group, or -(CH 2 ) n CH 2 OH wherein n is an integer of 1 to 10, or
  • X 2 -R 13 represents a piperidino group substituted in the 4-position with a - (CH 2 ) m CH 2 OH group wherein m is an integer of 1 to 7 or a -C(0)-NH-(CH 2 ) p CH 2 OH group wherein p is an integer of 1 to 6;
  • R 3 is hydrogen
  • R 4 is -O-R 15 wherein R 15 is hydrogen or C -2 alkyl.
  • R 1 is represented by formula (II).
  • R has the formula (II);
  • X 1 is -O- or -NR 6 -, wherein R 6 is H or linear or branched Ci -6 alkyl;
  • R 5 is selected from the group consisting of
  • a linear or branched d -6 alkyl which is unsubstituted or substituted with 1 to 5 substituents independently selected from halogen, NH 2 or CN, a 6-10 membered monocylic or bicyclic aryl group, optionally fused with a 5-6 membered heterocyclic or heteroaromatic group, the heterocyclic or heteroaryl group having 1 to 2 heteroatoms in the ring independently selected from N, O or S,
  • X 1 and R 5 together form a 4-7 membered monocyclic heterocyclic group having 1 or 2 heteroatoms in the ring independently selected from N or S and wherein the heterocycle is unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of a halogen, a hydroxyl group and a Ci. 6 alkoxy group;
  • Y 1 is a linear C 8- 12 alkylene group or a linear C 8 .12 alkenylene group
  • a is an integer of 0 or 1 ;
  • R 2 is a C 1-6 linear or branched alkyl group;
  • R 3 is a d-ealkoxy group
  • R 4 is a group
  • R 5 is not a 2H-pyrane group.
  • R 1 has the formula (II);
  • R 5 is not a linear or branched Ci. 6 alkyl which is substituted with one substituent OH, or a phenyl or C 3 . 7 cylcoalkyl, wherein the phenyl or C 3 . 7 cylcoalkyl is substituted with one substituent OH or with one substituent C 1-4 alkoxy.
  • Y 1 is a linear C 8 -i 2 alkylene group or a linear C 8 -i 2 alkenylene group. In a preferred embodiment in combination with any of the above or below embodiments, Y 1 represents a linear C 8 . 12 alkylene group, more preferably a linear C 10 alkylene group.
  • index a represents an integer selected from the values 0 and 1.
  • the index a assumes the value 0. If a is 0, it is preferred that R 5 represents a 5-10 membered monocyclic or bicyclic heteroaryl group having 1 to 4 heteroatoms in the ring(s) independently selected from N, O or S, wherein the monocyclic heteroaryl group and one or both rings of the bicylic heteroaryl group is unsubstituted or substituted with 1 to 5 substituents independently from each other selected from the group consisting of halogen, linear or branched C 1 .6 alkyl, linear or branched halo-Ci -6 alkyl, C1.6 alkoxy, -C(0)NH 2 , -NHC(0)Ci.
  • R 5 is a 9-10 membered, more preferably a 9 membered bicyclic heteroaryi group having 1 to 4 heteroatoms in the ring(s) independently selected from N, O or S, wherein the heteroaryi group is unsubstituted or substituted with 1 to 3 Ci -6 alkyl groups.
  • the index a assumes the value 1.
  • X 1 represents -O- or -NR 6 - wherein R 6 is H or linear or branched C 1-6 alkyl, more preferably H or CH 3 .
  • X 1 is - 0-
  • R 5 is a 5-10 membered monocyclic or bicyclic heteroaryi group having 1 to 4 heteroatoms in the ring(s) independently selected from N, O or S, wherein the monocyclic heteroaryi group and one or both rings of the bicylic heteroaryi group is unsubstituted or substituted with 1 to 5 substituents independently from each other selected from the group consisting of halogen, hydroxy, linear or branched d-e alkyl, linear or branched halo-Ci. 6 alky Ci. 6 alkoxy, -C(0)NH 2 , - NHCiOJCi-e alkyl, 5-6 membered heteroaryi having in the ring 1 to 3 heteroatoms independently selected from N, O or S.
  • X 1 is -0-
  • R 5 is a 6-10 membered monocyclic or bicyclic aryl group which may be optionally fused with a 5-6 membered heterocylic or heteroaromatic group, wherein the heterocyclic or heteroaromatic group have 1 to 2 heteroatoms in the ring independently selected from N, O or S.
  • the aryl, the heterocyclic and the heteroaryi group are unsubstituted or substituted with 1 to 5 substituents independently from each other selected from the group consisting of halogen, hydroxy, linear or branched C1.6 alkyl, linear or branched halo-C 1-6 alkyl, C ⁇ alkoxy, -C(0)NH 2 , - ⁇ (0)0 1-6 alkyl, 5-6 membered heteroaryi having in the ring 1 to 3 heteroatoms independently selected from N, O or S. More preferably, R 5 is a phenyl group which is unsubstituted or substituted with 1 to 3 substituents independently selected from - C(0)NH 2 , halogen, -NHC(0)Ci.
  • 5-6 membered heteroaryi having in the ring 1 to 3 heteroatoms independently selected from N, O or S. More preferably said 5-6 membered heteroaryi group is a 5 membered heteroaryi group having in the ring 1 to 3, preferably 2 to 3 nitrogen atoms.
  • R 5 is preferably a 6-10 membered monocylic or bicyclic aryl group, optionally fused with a 5-6 membered heterocyclic or heteroaromatic group, the heterocyclic or heteroaromatic group having 1 to 2 heteroatoms in the ring independently selected from N, O or S; or a 5-10 membered monocyclic or bicyclic heteroaromatic group having 1 to 4 heteroatoms independently selected from N, O or; or a 5-7 membered monocyclic heterocyclic group having 1 to 4 heteroatoms in the ring independently selected from N, O or S, or a 3-10 membered monocylic, bicylic or tricyclic cycloalkyi group, wherein each aryl, heteroaryl, heterocyclyl and cycloalkyi is unsubstituted or substituted with 1 to 5 substituents independently from each other selected from the group consisting of hal
  • X 1 is -NR 6 -
  • R 5 is preferably a 5-10 membered monocyclic or bicyclic heteroaromatic group having 1 to 4 heteroatoms in the ring(s) independently selected from N, O or S, wherein the monocyclic heteroaryl group and one or both rings of the bicylic heteroaromatic group is unsubstituted or substituted with 1 to 5 substituents independently from each other selected from the group consisting of halogen, hydroxy, linear or branched C 1-6 alkyl, linear or branched halo-C -6 alkyl, Ci-6 alkoxy, -C(0)NH 2 , -NHCiOJC ⁇ alkyl, 5-6 membered heteroaryl having in the ring 1 to 3 heteroatoms independently selected from N, O or S.
  • R 1 is represented by formula (III).
  • R 1 has the formula (III);
  • R 2 when R 2 is methyl, R 3 is methoxy, R 4 is methoxy, Y 2 is a linear C 5 . 9 alkylene group, R 7 is hydrogen and R 8 is hydrogen,
  • Y 2 is a linear C 5-9 alkylene group or a linear C 5 . 9 alkenylene group.
  • Y 1 represents a linear C5-9 alkylene group, more preferably a linear C 7 alkylene group.
  • R 7 and R 8 both represent hydrogen
  • R 9 represents hydrogen
  • R 10 represents a C 2 -6 alkinyl group, more preferably a C 2 alkinyl group, or R 9 and R 10 together form a -CH 2 -CH 2 - bridge.
  • R 1 is represented by formula (IV).
  • Y 3 is a linear C 6- i 0 alkylene group or a linear C 6- io alkenylene group. In a preferred embodiment in combination with any of the above or below embodiments, Y 3 represents a linear C 6 -io alkylene group, more preferably a linear C 8 alkylene group.
  • R 1 and R 2 independently from each other represent hydrogen, linear or branched C1.6 alkyl, a 4-10 membered monocylic heterocyclic group having 1 to 4 heteroatoms in the ring independently selected from N, O or S, wherein each alkyl and heterocyclic group is unsubstituted or substituted with 1 to 5 substituents independently selected from halogen, hydroxy and C 1 -6 alkoxy, or R 1 and R 12 together with the nitrogen atom to which they are attached form a 4-7 membered ring having in the ring 0 to 2 additional heteroatoms independently selected from N, O or S, wherein the ring is unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of halogen, hydroxyl and C1.6 alkoxy group.
  • one of R 1 and R 1 represents a hydrogen and the other represents a linear or branched C 1 -6 alkyl, a 4-10 membered monocylic heterocyclic group having 1 to 4 heteroatoms in the ring independently selected from N, O or S, wherein each alkyl and heterocyclic group is unsubstituted or substituted with 1 to 5 substituents as defined above, or R 11 and R 12 together with the nitrogen atom to which they are attached form a 4-7 membered ring having in the ring 0 to 2 additional heteroatoms independently selected from N, O or S, wherein the ring is unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of halogen, hydroxyl and Ci.
  • R 1 and R 3 represent hydrogen
  • R 2 represents X -R 13 , wherein X 2 and R 13 are defined as above
  • R 4 represents OR 15 , wherein R 15 is defined as above.
  • X 2 represents -NR 14 - wherein R 14 is defined as above.
  • R 13 represents a C 4- i 2 alkyl group or -(CH 2 ) n CH 2 OH wherein n is an integer of 1 to 10.
  • X 2 and R 3 combine to represent a piperidino group substituted in the 4-position with a - (CH 2 ) m CH 2 OH group wherein m is an integer of 1 to 7 or a -C(0)-NH-(CH 2 ) p CH 2 OH group wherein p is an integer of 1 to 6;
  • Y 4 is preferably a C 8 alkylene group.
  • Alkyl is a straight chain or branched alkyl having preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl or 2-ethylbutyl.
  • Alkenyl is a straight chain or branched alkyl having preferably 2, 3, 4, 5, 6 carbon atoms and which contains at least one carbon-carbon double bond, preferably one or two double bonds, most preferably one double bound.
  • Preferred examples of a C 2 - 6 alkenyl group are ethenyl, prop-
  • More preferred examples of an alkenyl group are ethenyl and prop-1-enyl.
  • the alkenyl group is exclusively a linear alkenyl group.
  • preferred examples of an alkenyl group have 5 to 12 carbon atoms and at least one double bond up to three double bonds depending on the length of the carbon chain. Generally, the double bonds occur in an isolated or a conjugated order.
  • Alkinyl is a straight chain or branched alkyl having preferably 2, 3, 4, 5, or 6 carbon atoms and which contains at least one carbon-carbon triple bond, preferably one or two triple bonds, most preferably one triple bond.
  • Preferred examples of a C 2 -6 alkinyl group are ethinyl, prop-1-inyl, prop-2-inyl, n-but-1-inyl, n-but-2-inyl, n-but-3-inyl, n-pent-1-inyl, n-pent-2-inyl, n-pent-3-inyl, n- pent-4-inyl, n-pent-1 ,3-inyl, isopent-1-inyl, neopent-1-inyl, n-hex-1-inyl, n-hex-2-inyl, n-hex-3-inyl, n-hex-4-inyl, n-hex-5-
  • alkyl alkenyl and alkinyl equally apply to the groups alkylene, alkenylene and alkinylene used throughout the invention.
  • Cycloalkyl is one, two or three alkyl rings having preferably 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms at the most.
  • Preferred monocyclic examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl, more preferably cyclopropyl and cyclopentyl.
  • Preferred bicyclic examples include decalinyl.
  • Preferred tricyclic examples include adamantyl.
  • Heteroaryl is an aromatic moiety having in the ring at least one heteroatom independently selected from O, N or S.
  • the heteroaryl group may be monocylic or bicyclic.
  • Heteroaryl is preferably selected from thienyl, pyrrolyl, furanyl, imidazolyl, pyrazolyl, 1 ,2,3-triazolyl, 1 ,2,4- triazolyl, tetrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, thiazolyl, isothiazolyl, oxazolyl, isoxazyl, 1 ,2,3-oxadiazolyl, 1 ,2,4-oxadiazolyl 1 ,2,5-oxadiazolyl 1 ,3,4-oxadiazolyl indazolyl, quinolinyl, isquinolinyl, cinnolinyl, quinoxalin
  • Heterocyclyl is a saturated or partially unsaturated ring containing at least one heteroatom independently selected from O, N or S.
  • Preferred examples include tetrahydrofuranyl, azetidinyl, pyrrolidinyl, pyrrolinyl, piperidinyl, piperazinyl, pyranyl, morpholinyl, thiomorpholinyl, 1 ,4-dioxanyl, more preferred examples include piperidinyl, piperazinyl and pyrrolidinyl.
  • Aryl is either a phenyl or a naphthalin group.
  • Halogen is a halogen atom selected from F, CI, Br and I, preferably from F, CI and Br. Salts
  • salts refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids.
  • Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc and the like. Particularly preferred are the ammonium, calcium, lithium, magnesium, potassium and sodium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine, caffeine, choline, ⁇ , ⁇ '-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylamino-ethanol, ethanolamine, ethylenediamine, N- ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.
  • basic ion exchange resins such as
  • salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids.
  • acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, formic, furnaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, malonic, mucic, nitric, parnoic, pantothenic, phosphoric, propionic, succinic, sulfuric, tartaric, p-toluenesulfonic, trifluoroacetic acid and the like.
  • the compounds of formula (I) are preferably formulated into a dosage form prior to administration. Accordingly the present invention also includes a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof and a suitable pharmaceutical carrier.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art; e.g., in Remington's Pharmaceutical Sciences.
  • the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.
  • Any suitable route of administration may be employed for providing a mammal, especially a human with an effective dosage of a compound of the present invention.
  • Suitable administration routes include oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intraarticular, and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including dermal, buccal, sublingual and intraocular) administration although the most suitable route may depend upon for example the condition and disorder of the recipient.
  • the administration route is oral.
  • the administration route is topical ocular. In another preferred embodiment in combination with any of the above or below embodiments, the administration route is an intraocular injection.
  • the administration route is an intraocular depot implant.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association a compound of the present invention or a pharmaceutically acceptable salt or solvate thereof ("active ingredient") with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient.
  • the active ingredient may further be presented as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • compositions which can be used orally include tablets, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with binders, inert diluents, or lubricating, surface active or dispersing agents. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein. All formulations for oral administration should be in dosages suitable for such administration.
  • the push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • Dragee cores are provided with suitable coatings.
  • concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • the compounds of the present invention may be formulated for parenteral administration by injection, e.g. as intraocular, intraveneous, subcutaneous, intramuscular, or intraarterial injection.
  • the injection may be administered by bolus injection or continuous infusion.
  • the injection may be administered
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in powder form or in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or sterile pyrogen-free water, immediately prior to use.
  • sterile liquid carrier for example, saline or sterile pyrogen-free water
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Formulations for parenteral administration include aqueous and non-aqueous (oily) sterile injection solutions of the active compounds which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the compounds may also be formulated as a depot preparation.
  • Such long acting formulations may be administered by implantation (for example intraocular or subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • compositions may take the form of tablets, lozenges, pastilles, or gels formulated in conventional manner.
  • Such compositions may comprise the active ingredient in a flavoured basis such as sucrose and acacia or tragacanth.
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter, polyethylene glycol, or other glycerides.
  • Compounds of the present invention may be administered topically, that is by non-systemic administration. This includes the application of a compound of the present invention externally to the epidermis or the buccal cavity and the instillation of such a compound into the ear, eye and nose, such that the compound does not significantly enter the blood stream.
  • systemic administration refers to oral, intravenous, intraperitoneal and intramuscular administration.
  • Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of inflammation such as gels, liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.
  • the amount of compound of formula (I) will generally be in the range of 0.001 to 10% weight/volume (%w/v). Preferred concentrations range from 0.1 to 5 %w/v.
  • Topical administration to the eye is given one to six times per day.
  • a typical formulation contains 0.1 - 5 % of compound of formula (I), 0.5 %w/v hydroxypropylmethylcellulose (HMPC), 0.8 %w/v sodium chloride, 0.28 %w/v sodium phosphate, 0.01 %w/v edetate disodium, 0.01 %w/v benzalkonium chloride. The pH is adjusted to 7.2 - 7.4. Purified water is added q.s.
  • Gels for topical or transdermal administration of compounds of the subject invention may comprise a mixture of volatile solvents, nonvolatile solvents, and water.
  • the volatile solvent component of the buffered solvent system may preferably include lower (CVCe) alkyl alcohols, lower alkyl glycols and lower glycol polymers. More preferably, the volatile solvent is ethanol.
  • the volatile solvent component is thought to act as a penetration enhancer, while also producing a cooling effect on the skin as it evaporates.
  • the nonvolatile solvent portion of the buffered solvent system is selected from lower alkylene glycols and lower glycol polymers. Preferably, propylene glycol is used.
  • the nonvolatile solvent slows the evaporation of the volatile solvent and reduces the vapor pressure of the buffered solvent system.
  • the amount of this nonvolatile solvent component, as with the volatile solvent, is determined by the pharmaceutical compound or drug being used. When too little of the non-volatile solvent is in the system, the pharmaceutical compound may crystallize due to evaporation of volatile solvent, while an excess will result in a lack of bioavailability due to poor release of drug from solvent mixture.
  • the buffer component of the buffered solvent system may be selected from any buffer commonly used in the art; preferably, water is used.
  • Appropriate gelling agents can include, but are not limited to, semisynthetic cellulose derivatives (such as hydroxypropylmethylcellulose) and synthetic polymers, and cosmetic agents.
  • Lotions according to the present invention include those suitable for application to the skin or eye.
  • An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops.
  • Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.
  • Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or nonaqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy base.
  • the base may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap, a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil, wool fat or its derivatives or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or a macrogel.
  • the formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as a sorbitan ester or a polyoxyethylene derivative thereof.
  • Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.
  • Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions and may be prepared by dissolving the active ingredient in a suitable aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active agent.
  • Formulations for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatine and glycerin or sucrose and acacia.
  • the compounds according to the invention are conveniently delivered from an insufflator, nebulizer pressurized packs or other convenient means of delivering an aerosol spray.
  • Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetra-fluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the compounds according to the invention may take the form of a dry powder composition, for example a powder mix of the compound and a suitable powder such as lactose or starch.
  • the powder composition may be presented in unit form, in for example, capsules, cartridges, gelatine or blister packs from which the powder may be with the aid of an inhalator or insufflator.
  • Preferred unit dosage formulations are those containing an effective dose, as herein below recited or an appropriate fraction thereof, of the active ingredient.
  • the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
  • the compounds of the present invention may be administered via any route discussed above at a dose range for adult humans which is generally from 0.01 mg/kg/day to 60 mg/kg/day, more preferably from 0.01 mg/kg/day to 30 mg/kg day, most preferably from 0.01 mg/kg/day to 15 mg/kg/day.
  • 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 treated and the particular mode of administration.
  • the compounds of the subject invention can be administered in various modes, e.g. orally, topically, or by injection.
  • the precise amount of compound administered to a patient will be the responsibility of the attendant physician.
  • 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, general health, sex, diets, time of administration, route of administration, rate of excretion, drug combination, the precise disorder being treated, and the severity of the indication or condition being treated.
  • the route of administration may vary depending on the condition and its severity.
  • the compounds according to formula (I) are for use in the treatment of a mitochondrial disease, a neurodegenerative disease, a neuromuscular disease, psychiatric disorders, metabolic disorders, cancer, or immune dysfunction.
  • the compounds according to formula (I) are for use in the treatment of a mitochondrial disease.
  • the compounds of the present invention are modulators of mitochondrial function and as such are useful for the treatment of pathological conditions where mitochondrial function is impaired.
  • Mitochondria are e.g. critical for ocular function as they represent the major source of a cell's supply of energy and play an important role in cell differentiation and survival. Furthermore, the eye is one of the tissues with the highest energy consuption in the body (REF).
  • Ocular mitochondrial dysfunction can occur as a result of inherited mitochondrial syndromes and mutations, such as Leber's hereditary optic neuropathy (LHON), dominant optic atrophy (DOA, mutations in OPA1) dominant optic atrophy with cataracts (mutations in OPA3), vitelliform macular dystrophy (VD), Jensen syndrome, optic atrophy of Leigh and Leigh-like syndrome, maculopathy of neuropathy-ataxia-retinitis-pigmentosa (NARP), retinopathy of mitochondrial- encephalomyopathy-lactic-acidosis-stroke (MELAS), chronic progressive external ophthalmoplegia (CPEO) and optic atrophy caused by mutations in the ND3 subunit of complex 1 (MTND3), which are also associated with migraine and encephalopathy.
  • LHON Leber's hereditary optic neuropathy
  • DOA dominant optic atrophy
  • OPA1 dominant optic atrophy with cataracts
  • VD vitelliform macular dystrophy
  • VD
  • mitochondrial haplogroups Jones MM, Manwaring N, Wang JJ, Rochtchina E, Mitchell P, Sue CM. Arch Ophthalmol. 2007 Sep; 125(9): 1235-40
  • certain mitochondrial gene variants were implicated in an elevated risk of developing ophtalmological pathologies (Kanda A, Chen W, Othman M, Branham KE, Brooks M, Khanna R, He S, Lyons R, Abecasis GR, Swaroop A. Proc Natl Acad Sci U S A.
  • mitochondrial dysfunction whether inherited or aquired is considered one of the main molecular pathologies involved in the initiation of ocular disorders (such as macular degeneration, glaucoma, retinopathy and cataracts) (reviewed by Jarrett SG, Lewin AS, Boulton ME. Ophthalmic Res. 2010;44(3): 179-90; Brennan LA, Kantorow M. Exp Eye Res. 2009 Feb;88(2): 195-203).
  • AMD age-related macular degeneration
  • VD inherited macular degeneration
  • AMD a medical condition which results in a loss of vision in the center of the visual field (the macula) because of damage to the retina.
  • Glaucoma is a disease in which the optic nerve is damaged, leading to progressive, irreversible loss of vision, which is often, but not always, associated with increased pressure of the fluid in the eye.
  • the nerve damage involves loss of retinal ganglion cells in a characteristic pattern.
  • Raised intraocular pressure is a significant risk factor for developing glaucoma. Untreated glaucoma leads to permanent damage of the optic nerve and resultant visual field loss, which can progress to blindness.
  • Retinopathy is a general term that refers to some form of non-inflammatory damage to the retina of the eye.
  • spontaneous forms can be induced by drugs, toxins and radiation (ionizing and ultra violet).
  • retinopathies are ocular manifestations of systemic diseases such as diabetes, hypertension, sickle cell disease or ciliopathy such as Bardet-Biedl syndrome. Similar to macular degeneration and glaucoma, mitochondrial involvement in retinopathy has been well described (reviewed by Jarrett SG, Lewin AS, Boulton ME. Ophthalmic Res.
  • a cataract is a clouding that develops in the crystalline lens of the eye or in its envelope, varying in degree from slight to complete opacity and obstructing the passage of light.
  • the gradual yellowing and opacification of the lens may reduce the perception of blue colours.
  • Cataracts typically progress slowly to cause vision loss and are potentially blinding if untreated.
  • the condition usually affects both the eyes, but similar to the pathology of LHON and VD, one eye is almost always affected earlier than the other.
  • mitochondrial involvement in cataract formation is clearly described.
  • cataract formation is directly associated with some mitochondrial disorders such as dominant optic atrophy with cataracts, autosomal dominant progressive external ophthalmoplegia (PEOA3), PEOA2, mitochondrial myopathy caused by mutations of COX II or various others genes, Sengers syndrome, as well as mutations of mitochondrial genes such as mitochondrial tRNA-Ser (MTTS2), GFER, OPA3 but also large mitochondrial deletions.
  • mitochondrial disorders such as dominant optic atrophy with cataracts, autosomal dominant progressive external ophthalmoplegia (PEOA3), PEOA2, mitochondrial myopathy caused by mutations of COX II or various others genes, Sengers syndrome, as well as mutations of mitochondrial genes such as mitochondrial tRNA-Ser (MTTS2), GFER, OPA3 but also large mitochondrial deletions.
  • Optic disc drusen or optic nerve head drusen (ONHD) are globules of mucoproteins and mucopolysaccharides that progressively calcify in the optic disc. They are thought to be the remnants of the axonal transport system of degenerated retinal ganglion cells. ODD have also been referred to as congenitally elevated or anomalous discs, pseudopapilledema, pseudoneuritis, buried disc drusen, and disc hyaline bodies. They are associated with vision loss of varying degree occasionally resulting in blindness. Mitochondrial impairment due to Ca 2+ overload has been suggested in the process of drusen formation (Tso MO. Ophthalmology.
  • haplotype J mitochondrial haplotype J
  • haplotype H was associated with significantly lower risk
  • Chrysostomou et al. (Chrysostomou V, Trounce IA, Crowston JG. Ophthalmic Res. 2010;44(3): 173-8) suggested that mitochondrial dysfunction, inherited or as a cause or consequence of injury, renders ocular cells (in particular retinal ganglion cells) sensitive to degeneration.
  • Therapeutic approaches that target mitochondria should therefore provide a general means of protecting lens and retinal ganglion cells from degeneration, regardless of the etiology of the disease.
  • a further indication which can be allocated as mitochondrial disease is autosomal dominant optic atrophy (DOA).
  • DOA autosomal dominant optic atrophy
  • Pathogenic OPA1 mutations cause autosomal dominant optic atrophy, a condition characterized by the preferential loss of retinal ganglion cells and progressive optic nerve degeneration.
  • OPA1 was demonstrated to control both mitochondrial fusion and cristae morphology.
  • OPA1 loss-of-function studies have shown that OPA1 also regulates apoptosis induction (Liesa M et al. Physiol Rev. 2009; 89:799-845). Although it is expressed in all the tissues assayed, OPA1 shows a specific tissue expression pattern, with the highest expression in the retina, brain, testis, liver, heart, skeletal muscle, and pancreas.
  • Loss of OPA1 causes a marked reduction in mitochondrial membrane potential and a reduction in basal respiration and incapacity to enhance oxygen consumption in the presence of the uncoupler 2,4-dinitrophenol.
  • Human fibroblasts from patients with certain OPA1 mutations (that cause autosomal dominant optic atrophy or ADOA), in addition to decreased rates of mitochondrial fusion, also show impaired ATP synthesis driven by complex I substrates. Due to their ability to enhance ATP synthesis the compounds of the present invention are useful in the treatment of DOA.
  • the compounds of the present invention are suitable for use in the treatment of ophthalmological mitochondrial diseases such as Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy (DOA), and ophtalmological disorders displaying mitochondrial dysfunction such as macular degeneration, glaucoma, retinopathy, cataract, optic disc drusen (ODD).
  • LHON Leber's hereditary optic neuropathy
  • DOA autosomal dominant optic atrophy
  • ODD optic disc drusen
  • the compounds of the present invention are also suitable for use in the treatment of genetic neurodegenerative mitochondrial diseases with an ophthalmological component among the various symptoms such as MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms), MERFF (myoclonic epilepsy with ragged red fibers), MNGIE (myoneurogenic gastrointestinal encephalomyopathy), Kearns-Sayre syndrome, CoQ10 deficiency, or mitochondrial complex deficiencies (i.e. complex I, II, III, IV, V deficiency, and CPEO).
  • MELAS mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms
  • MERFF myoclonic epilepsy with ragged red fibers
  • MNGIE myoneurogenic gastrointestinal encephalomyopathy
  • Kearns-Sayre syndrome i.e. complex I, II, III, IV, V deficiency, and CPEO.
  • the following diseases are designated as a "mitochondrial disease”: Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy (DOA), macular degeneration, glaucoma, retinopathy, cataract, optic disc drusen (ODD), mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), myoclonic epilepsy with ragged red fibers (MERRF), myoneurogenic gastrointestinal encephalomyopathy (MNGIE), Kearns-Sayre syndrome, CoQ.10 deficiency, and mitochondrial complex deficiencies (1-5, CPEO).
  • LHON Leber's hereditary optic neuropathy
  • DOA autosomal dominant optic atrophy
  • macular degeneration glaucoma
  • ODD optic disc drusen
  • mitochondrial myopathy encephalomyopathy
  • lactic acidosis lactic acidosis
  • MELAS myoclonic epilepsy with ragged red fibers
  • the compounds according to formula (I) are modulators of mitochondrial disorders and are thus useful in the treatment of mitochondrial diseases such as Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy (DOA), macular degeneration, glaucoma, retinopathy, cataract, optic disc drusen (ODD), mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), myoclonic epilepsy with ragged red fibers (MERRF), myoneurogenic gastrointestinal encephalomyopathy (MNGIE), Kearns-Sayre syndrome, CoQ10 deficiency, or mitochondrial complex deficiencies.
  • mitochondrial diseases such as Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy (DOA), macular degeneration, glaucoma, retinopathy, cataract, optic disc drusen (ODD), mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (ME
  • the compounds according to formula (I) are for use in the treatment of a mitochondrial disease selected from Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy (DOA), macular degeneration, glaucoma, retinopathy, cataract, optic disc drusen (ODD), mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), myoclonic epilepsy with ragged red fibers (MERRF), myoneurogenic gastrointestinal encephalomyopathy (MNGIE), Kearns-Sayre syndrome, CoQ10 deficiency, or mitochondrial complex deficiencies (1-5, CPEO).
  • a mitochondrial disease selected from Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy (DOA), macular degeneration, glaucoma, retinopathy, cataract, optic disc drusen (ODD), mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms
  • the compounds according to formula (I) are for use in the treatment of a mitochondrial disease selected from Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy (DOA), macular degeneration, glaucoma, mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), or mitochondrial complex deficiencies (1-5, CPEO).
  • a mitochondrial disease selected from Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy (DOA), macular degeneration, glaucoma, mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), or mitochondrial complex deficiencies (1-5, CPEO).
  • the compounds according to formula (I) are for use in the treatment of a Leber's hereditary optic neuropathy (LHON).
  • LHON Leber's hereditary optic neuropathy
  • the compounds according to formula (I) are for use in the treatment of autosomal dominant optic atrophy (DOA). In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of macular degeneration.
  • DOA autosomal dominant optic atrophy
  • the compounds according to formula (I) are for use in the treatment of glaucoma.
  • the compounds according to formula (I) are for use in the treatment of retinopathy.
  • the compounds according to formula (I) are for use in the treatment of cataract.
  • the compounds according to formula (I) are for use in the treatment of optic disc drusen (ODD).
  • the compounds according to formula (I) are for use in the treatment of mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS).
  • the compounds according to formula (I) are for use in the treatment of myoclonic epilepsy with ragged red fibers (MERRF).
  • the compounds according to formula (I) are for use in the treatment of myoneurogenic gastrointestinal encephalomyopathy (MNGIE).
  • MNGIE myoneurogenic gastrointestinal encephalomyopathy
  • the compounds according to formula (I) are for use in the treatment of Keams-Sayre syndrome.
  • the compounds according to formula (I) are for use in the treatment of CoQ10 deficiency. In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of mitochondrial complex deficiencies (1 -5, CPEO).
  • the compounds according to formula (I) are for use in the treatment of a neurodegenerative disease.
  • the compounds of formula (I) can further be used to treat neurodegenerative diseases such as FRDA, ALS, Parkinson's disease, Alzheimer's disease, Huntington's disease, Stroke/Reperfusion Injury, or Dementia. Also associated with mitochondrial dysfunction are neuromuscular diseases such as DMD, BMD, LGMD, XLDCM, PKAN, SMA, Kugelberg- Welander disease, or Werdnig-Hoffmann disease which accordingly are also be treatable by using the compounds of formula (I).
  • neurodegenerative diseases such as FRDA, ALS, Parkinson's disease, Alzheimer's disease, Huntington's disease, Stroke/Reperfusion Injury, or Dementia.
  • neuromuscular diseases such as DMD, BMD, LGMD, XLDCM, PKAN, SMA, Kugelberg- Welander disease, or Werdnig-Hoffmann disease which accordingly are also be treatable by using the compounds of formula (I).
  • the compounds according to formula (I) are for use in the treatment of a neurodegenerative disease selected from Friedreich's ataxia (FRDA), amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, stroke/reperfusion injury, or dementia.
  • FRDA Friedreich's ataxia
  • ALS amyotrophic lateral sclerosis
  • Parkinson's disease Alzheimer's disease
  • Huntington's disease Huntington's disease
  • stroke/reperfusion injury or dementia.
  • the compounds according to formula (I) are for use in the treatment of Friedreich's ataxia (FRDA).
  • FRDA Friedreich's ataxia
  • the compounds according to formula (I) are for use in the treatment of amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • the compounds according to formula (I) are for use in the treatment of Parkinson's disease.
  • the compounds according to formula (I) are for use in the treatment of Alzheimer's disease.
  • the compounds according to formula (I) are for use in the treatment of Huntington's disease. In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of stroke/reperfusion injury.
  • the compounds according to formula (I) are for use in the treatment of dementia.
  • the compounds according to formula (I) are for use in the treatment of a neuromuscular disease.
  • the compounds according to formula (I) are for use in the treatment of a neuromuscular disease selected from Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Limb- Girdle muscular dystrophy (LGMD), X-linked dilated cardiomyopathy (XLDCM), Pantothenate kinase-associated neurodegeneration (PKAN), spinal muscular atrophy (SMA), multiple sclerosis and primary progressive multiple sclerosis (PP-MS), Kugelberg-Welander disease, and Werdnig- Hoffmann disease.
  • a neuromuscular disease selected from Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Limb- Girdle muscular dystrophy (LGMD), X-linked dilated cardiomyopathy (XLDCM), Pantothenate kinase-associated neurodegeneration (PKAN), spinal muscular atrophy (SMA), multiple sclerosis and primary progressive multiple sclerosis (PP-MS), Kugelberg-Welander disease, and Werdnig- Hoffmann disease
  • the compounds according to formula (I) are for use in the treatment of Duchenne muscular dystrophy (DMD).
  • DMD Duchenne muscular dystrophy
  • the compounds according to formula (I) are for use in the treatment of Becker muscular dystrophy (BMD).
  • the compounds according to formula (I) are for use in the treatment of Limb-Girdle muscular dystrophy (LGMD).
  • LGMD Limb-Girdle muscular dystrophy
  • the compounds according to formula (I) are for use in the treatment of X-linked dilated cardiomyopathy (XLDCM). In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of Pantothenate kinase- associated neurodegeneration (PKAN).
  • XLDCM X-linked dilated cardiomyopathy
  • PKAN Pantothenate kinase- associated neurodegeneration
  • the compounds according to formula (I) are for use in the treatment of spinal muscular atrophy (SMA).
  • the compounds according to formula (I) are for use in the treatment of multiple sclerosis.
  • the compounds according to formula (I) are for use in the treatment of primary progressive multiple sclerosis (PP-MS).
  • PP-MS primary progressive multiple sclerosis
  • the compounds according to formula (I) are for use in the treatment of Kugelberg-Welander disease.
  • the compounds according to formula (I) are for use in the treatment of Werdnig-Hoffmann disease.
  • RR-MS relapsing-remitting form of the disease
  • PP-MS primary progressive MS
  • PP-MS Primary progressive MS
  • RR-MS patients differ from RR-MS patients in several important characteristics: They tend to be older at the time of disease onset (mean 40 vs. 30 years); males and females tend to be affected equally; clinically there is a high prevalence of cortico-spinal dysfunction characterized by progressive weakness and spasticity; patients have more prominent involvement of the spinal cord and generally lower amount of distinct white matter lesions (i.e. plaques) in the brain and less evidence for brain inflammatory activity and, most importantly, PP-MS patients do not respond to immunomodulatory therapies with proven efficacy in RR-MS. Both new imaging modalities and pathological data suggest that in PP-MS, CNS pathology is more diffuse and occurs to some extent independently of focal lesions.
  • the compounds according to formula (I) are for use in the treatment of psychiatric disorders.
  • the compounds according to formula (I) are for use in the treatment of a psychiatric disorder selected from schizophrenia, major depressive disorder, bipolar disorder, or epilepsy.
  • the compounds according to formula (I) are for use in the treatment of schizophrenia. In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of major depressive disorder.
  • the compounds according to formula (I) are for use in the treatment of bipolar disorder.
  • the compounds according to formula (I) are for use in the treatment of epilepsy.
  • mitochondrial dysfunction has also been intimately linked to epilepsy [reviewed by Waldbaum S, Patel M Epilepsy Res. 2009 Oct 20 and Kudin AP, Zsurka G, Elger CE, Kunz WS Exp Neurol. 2009 Aug;218(2):326-32]. Therefore, the compounds of formula (I) are useful in the treatment of schizophrenia, major depressive disorder, bipolar disorder and epilepsy.
  • the compounds according to formula (I) are for use in the treatment of metabolic disorders.
  • the compounds according to formula (I) are for use in the treatment of a metabolic disorder selected from ageing-related physical decline, obesity, overweight, type II diabetes, or metabolic syndrome. In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of ageing-related physical decline.
  • the compounds according to formula (I) are for use in the treatment of obesity.
  • the compounds according to formula (I) are for use in the treatment of overweight.
  • the compounds according to formula (I) are for use in the treatment of type II diabetes.
  • the compounds according to formula (I) are for use in the treatment of metabolic syndrome.
  • the compounds of the present invention show a beneficial effect in diabetes type II, obesity and metabolic syndrome.
  • the compounds according to formula (I) are for use in the treatment of cancer.
  • White modulators of mitochondrial function have been suggested as anti-cancer agents mainly via an apoptosis-inducing function, recent data also links the metastatic potential of tumours directly to mitochondrial DNA and mitochondrial radical production [reviewed by Ishikawa K, Koshikawa N, Takenaga K, Nakada K, Hayashi J Mitochondrion. 2008; 8(4): 339-44]. Therefore, modulating mitochondria in this context could not only be employed as anti-cancer strategy but also to prevent metastasis of malignancies which makes the compounds according to formula (I) useful as cancer therapeutics.
  • the compounds according to formula (I) are for use in the treatment of immune dysfunction.
  • the compounds according to formula (I) are for use in the treatment of an immune dysfunction selected from arthritis, psoriasis or rheumatoid arthritis.
  • the compounds according to formula (I) are for use in the treatment of arthritis.
  • the compounds according to formula (I) are for use in the treatment of psoriasis.
  • the compounds according to formula (I) are for use in the treatment of rheumatoid arthritis.
  • Altered mitochondrial metabolism is also linked to multiple dysfunctions of the immune system.
  • reduced expression of mitochondrial respiratory chain genes was observed [Ishikawa S, Mima T, Aoki C, Yoshio- Hoshino N, Adachi Y, Imagawa T, Mori M, Tomiita M, Iwata N, Murata T, Miyoshi M, Takei S, Aihara Y, Yokota S, Matsubara K, Nishimoto N Ann Rheum Dis. 2009;68(2):264-72].
  • Oxidative stress and mitochondrial pathology has been reported to be associated with sepsis [Victor VM, Espulgues JV, Hernandez-Mijares A, Rocha M Infect Disord Drug Targets. 2009;9(4):376-89]. Furthermore, targeting mitochondrial function has been suggested as treatment strategy for several autoimmune disorders such as arthritis and psoriasis [J.J. Bednarski, R.E. Warner, T. Rao, F. Leonetti, R. Yung, B.C. Richardson, K.J. Johnson, J.A. Ellman, A.W. Opipari Jr., and G.D.
  • idebenone can be reacted with optionally substituted alcohols in the presence of two reagents such as PPh 3 and DEAD in an appropriate solvent such as THF to form the corresponding ether.
  • R in Reaction Scheme 1 is defined based on the corresponding moiety in the compounds of Examples 1 to 24.
  • idebenone can be reacted with a reagent such as Dess Martin reagent in an appropriate solvent such as DCM.
  • a reagent such as Dess Martin reagent in an appropriate solvent such as DCM.
  • the corresponding aldehyde can be reductively aminated.
  • Reaction with an optionally substituted amine in the presence of a reagent like sodium cyano borohydride in appropriate solvents such as MeOH and AcOH yields the target compounds.
  • R1 and R2 in Reaction Scheme 2 are defined like the corresponding moieties in the compounds of Examples 25 to 34.
  • a benzoquinone aldehyde can be reduced with a Grignard reagent in a solvent such as THF to give the corresponding substituted benzoquinone alcohol.
  • a hydroquinone methyl ester may be subjected to a Grignard reaction to yield the corresponding alcohol.
  • R in Reaction Scheme 3 is defined as in the corresponding moieties of Examples 35 and 36.
  • a benzoquinone methyl ester is subjected to a Kulinkovich reaction as shown in Reaction Scheme 4.
  • the benzoquinone methyl ester is reacted with Grignard reagent Et-MgBr in the presence of Ti(OiPr) 4 in a solvent such as THF to yield the desired compound.
  • a hydroquinone may be oxidised with a salt such as NO(S0 3 K) 2 in an aqueous solvent such as acetone and in the presence of a base such as KOH.
  • Reaction Scheme 5 is defined applicable to the preparation of all compounds wherein the oxidation of a hydroquinone is required.
  • R in Reaction Scheme 5 defines each possible moiety attached to the quinone as defined within the claims.
  • the compounds of the present invention were analyzed by analytical LC-MS. The conditions are summarized below.
  • Examples 25 to 33 which were synthesized according to general procedure (II) are: 2,3-Dimethoxy-5-methyl-6-[10-(2H-tetrazol-5-ylamino)-decyl]-[1 ,4]benzoquinone (25)
  • intermediate 35a (200 mg) was dissolved in dry THF (6 ml) and the solution was cooled to -10°C.
  • titanium (IV) isopropoxide (1.6 g) was added drop wise at -30°C to a 3M ethyl magnesium bromide solution in ether (4.6 ml) and THF (2 ml).
  • Intermediate 36b (0.84 g) dissolved in THF (2.5 ml) was added dropwise at -30°C and the reaction mixture was stirred for 15 min, and was monitored by LCMS.
  • Titanium (IV) isopropoxide (1.6 g), a 3M ethyl magnesium bromide solution in ether (4.6 ml) and THF (1 ml) were added and the reaction mixture was stirred for 15 min. at -30°C.
  • the solution was hydrolyzed at 0°C with a saturated ammonium chloride solution and extracted with ethyl acetate (20 ml). After phase separation, the aqueous layer was extracted again with ethyl acetate (2 x 20 ml). The combined organic layer was washed with brine (25 ml), dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure.
  • the crude product was purified by flash chromatography (AcOEt- cyclohexane).
  • the pH was adjusted to 8 by adding a 1% aqueous solution of potassium hydroxyde and acetone (1.5 ml) was added.
  • the reaction mixture was monitored by LCMS.
  • the reaction mixture was stirred for 14h at room temperature to obtain a complete conversion.
  • a 1 M aqueous hydrochloride solution (10 ml) was added and the aqueous layer was extracted with ethyl acetate (3x10ml).
  • the combined organic layer was dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure.
  • the crude product was purified with preparative HPLC-MS.
  • the reaction mixture was monitored by LCMS.
  • the reaction mixture was stirred for 14h at room temperature to obtain a complete conversion.
  • a 0.5M aqueous hydrochloride solution (300 ml) was added and the aqueous layer was extracted with ethyl acetate (4x200ml).
  • the combined organic layer was dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure.
  • the crude product was purified by flash-chromatography (AcOEt-cyclohexane).
  • intermediate 37a (6 g) was dissolved in dry THF (300 ml) and the solution was cooled to -60°C.
  • a 2M solution of LDA in THF (31 ml) and iodomethane (6 ml) were added and the reaction mixture was stirred for 1 h.
  • the reaction mixture was poured onto a saturated solution of ammonium chloride (300 ml) at 0°C. After phase separation, the aqueous layer was extracted with ethyl acetate (3x200 ml). The combined organic layer was washed with brine (200 ml), dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure.
  • the crude product was purified by flash-chromatography (AcOEt-cyclohexane).
  • intermediate 37b (201 mg) was dissolved in dry THF (5 ml) and lithium aluminium hydride (40 mg) was added at 0°C. The reaction mixture was stirred at this temperature for 15 min. and for 1 h at room temperature. The solution was diluted with ethyl acetate (30 ml) and washed with a saturated ammonium chloride solution (30 ml). The aqueous layer was extracted with ethyl acetate (25 ml). The combined organic layer was washed with brine (30 ml), dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure. The crude product was dissolved in toluene and silver carbonate (64 mg) was added. The reaction mixture was stirred for 2h and filtrated over Celite. The solvent was removed under reduced pressure and the crude product was purified with preparative HPLC-MS.
  • intermediate 37b (1 g) was dissolved in dry THF (60 ml) and the solution was cooled to -60°C.
  • the reaction mixture was poured onto a saturated ammonium chloride solution (50 ml) at 0°C. After phase separation, the aqueous layer was extracted with ethyl acetate (3x50 ml). The combined organic layer was washed with brine (100 ml), dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure.
  • the crude product was purified by flash-chromatography (AcOEt-cyclohexane).
  • intermediate 38a (103 mg) was dissolved in dry THF (4 ml) and lithium aluminium hydride (15 mg) was added at 0°C. The reaction mixture was stirred at this temperature for 15 min. The solution was diluted with ethyl acetate (15 ml) and washed with a saturated solution of ammonium chloride (15 ml). The aqueous layer was extracted with ethyl acetate (15 ml). The combined organic layer was washed with brine (15 ml), dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure. The crude product was dissolved in toluene (3 ml) and silver carbonate (36 mg) was added. The reaction mixture was stirred for 2h and filtrated over Celite. The solvent was removed under reduced pressure and the crude product was purified with preparative HPLC-MS.
  • intermediate 37a (600 mg) was dissolved in dry THF (10 ml) and the solution was cooled to -78°C.
  • a 2M solution of LDA in THF (3.3 ml) was added and the reaction mixture was stirred for 15 min. Then the solution was warmed up to -10°C and stirred for 30 min.
  • the reaction mixture was again cooled to -78°C and N-fluorodibenzenesulfonimide (2.0 g) was added.
  • the reaction mixture was stirred for 10 min. at -78°C for 14h at room temperature.
  • the solution was diluted with ethyl acetate (120 ml) and washed with a saturated solution of ammonium chloride (100 ml).
  • intermediate 39a (86 mg) was dissolved in dry THF (3.5 ml) and lithium aluminium hydride (25 mg) was added at 0°C. The reaction mixture was stirred at this temperature for 15 min. and 1 h at room temperature. The solution was diluted with ethyl acetate (15 ml) and washed with a saturated ammonium chloride solution (10 ml). The aqueous layer was extracted with ethyl acetate (15 ml). The combined organic layer was washed with brine (20 ml), dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure. The crude product was purified with preparative HPLC-MS.
  • Jones reagent was prepared by dissolving 670 mg of Cr0 3 in 1.25 ml dist. water. To this solution was added 0.58 ml of cone. H 2 S0 4 while cooling in an ice bath. After 5 min, the precipitating salts were brought into solution by dropwise addition of 0.15 ml of water.
  • Fmoc-Rink amide resin 200-400 mesh (PepChem, PC-01-0501) (273 mg, 0.21 mmol) was treated with a solution of 20% piperidine in DMF (3 ml) for 30 min. Excess of the reagent was removed by filtration. The resin was successively washed with DMF (3 x 3 ml), MeOH (3 x 3 ml) and DMF (3 x 3 ml) and treated with a solution of product from intermediate 59b (224 mg, 0.41 mmol) activated with DIC (65 ⁇ , 0.42 mmol) and HOBt monohydrate (64 mg, 0.42 mmol) in DMF (2.5 ml) overnight.
  • the resin was washed with DMF (3 x 3 ml). DMF (2.5 ml) was added followed by acetic anhydride (99 ⁇ , 1.05 mmol). The mixture was reacted for 1 h. Excess of the reagent was removed by filtration. The resin-bound intermediate was successively washed with DMF (3 x 3 ml), MeOH (3x3 ml), THF (3x3 ml), DCM (3x3 ml) and diethyl ether (3x3 ml). The resin was dried under reduced pressure.
  • Tritylchlorid-polystyrol resin 100-200 mesh (PepChem, PC-01-0011) (250 mg, 0.363 mmol) was treated with a solution of intermediate 59b (594 mg, 1.089 mmol) in dry DCM (2 ml) overnight. Excess of the reagent was removed by filtration. The resin-bound intermediate was successively washed with DMF (3x5 ml), MeOH (3x5 ml), THF (3x5 ml), DCM (3x5 ml) and diethyl ether (3x5 ml). The resin was dried under reduced pressure.
  • the product was purified by column chromatography.
  • Comparative Compound A is a synthetic derivative to the natural occurring CoQ10.
  • Comparative Compound C is a synthetic derivative to the natural occurring CoQ10.
  • Comparative Compound D is a synthetic derivative to Comparative Compound E.
  • Comparative Compound E Comparative Compound E is Idebenone which is first described in the specification of Japanese Patent Examined Publication No. 3134/1987 filed by Takeda Chemical Industries, Ltd.
  • Comparative Compound F is disclosed in WO-A-2006130775.
  • Mitochondria generate most of the cell's supply of adenosine triphosphate (ATP) which is used as a source of the chemical energy.
  • mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth.
  • the amount of mitochondria per cell varies widely and is also subject to modifications in response to physiological stimuli.
  • Mitochondria replicate independently of nuclear replication in response to low energy conditions via an AMPK driven pathway. Their degradation occurs via a specialized autophagy pathway called mitophagy. This pathway can serve multiple goals: first it is essential for maintaining mitochondrial homeostasis, but more importantly it can also be used to reduce mitochondrial mass. Under conditions of impaired mitochondrial function this is an essential, protective mechanism to prevent excessive buildup of radicals that would otherwise damage cellular integrity.
  • MitoTracker® is used to quantify the number of mitochondria in live cells. After passive diffusion into mitochondria, MitoTracker® Green FM reacts with thiols on proteins and forms a fluorescent conjugate. The method was performed as instructed by the manufacturer. Briefly, fibroblast cells seeded in a 96-well plate at a density of 10 4 cells per well are allowed to grow for 24 h. Cells were treated with 10 ⁇ compounds in growth medium for 72 h. Then, cells are washed with 100 ⁇ Hank's BSS. A working dye solution of MitoTracker® Green FM is prepared by diluting MitoTracker® Green FM stock solution 20000-fold in Hank's BSS.
  • a volume of 100 ⁇ /well of dye solution is added and plates are incubated for 15 min in the cell culture incubator. After washing twice with 50 ⁇ PBS, a volume of 50 ⁇ PBS is added and fluorescence is measured immediately (MitoTracker® Green FM: excitation: 490 nm; emission: 520 nm). After obtaining the MitoTracker signal, cells are fixed with PFA for 10 minutes at RT, wasched with 3x 100 ul PBS and nuclear DNA stained with DAPI solution (O.l Mg/ml DAPI in PBS) for 10 minutes at RT. After 3 additional washes with PBS, DAPI fluorescence is aquired (excitation: 350 nm); emission: 450 nm). Mitotracker signals are normalized to amount of nuclear DNA (equiv. of cell number).
  • Mitochondrial mass is a measure of the amount of mitochondria in cells. As a result of multiple studies, it is now generally accepted that upregulation of mitochondrial mass is beneficial to retain sufficient energy (ATP) levels in disorders where mitochondrial function is impaired. As can be taken from Table 8, while the comparison compound B slightly reduces mitochondrial mass, the compounds of the present invention significantly enhance mitochondrial mass.
  • Mitochondrial membrane potential is an important parameter of mitochondrial function used as an indicator of cell health. It is generated through proton pumps that are fuelled by the electrons donated from the citric acid cycle in form of NADH. These electrons are transferred between complexes I, II and III of the respiratory chain through coenzyme Q10.
  • the membrane potential is used in healthy cells to provide the electrochemical energy to drive mitochondrial ATP production. Under certain conditions however, high ⁇ can be the source of significant radical production by mitochondria.
  • the loss of mitochondrial membrane potential is a hallmark of apoptosis.
  • the mitochondrial permeability transition is an important step in the induction of cellular apoptosis. During this process, the electrochemical gradient across the mitochondrial membrane collapses and is accompanied by the release of cytochrome c into the cytoplasm.
  • the mitochondrial membrane potential is the H+-gradient over the inner mitochondrial membrane that is used for ATP production.
  • the compounds of the present invention significantly enhanced the mitochondrial membrane potential.
  • This assay investigates cytotoxicity which is defined as the cell-killing property of a chemical compound, independent from the mechanisms of cell death.
  • the HepG2 hepatoblastoma cell line is one of the most common human cell lines for hepatotoxicity studies. Even though the cells lack a part of the metabolizing enzymes present in fresh hepatocytes, they have been shown to be a useful tool for studying the toxicity of hepatotoxins.
  • This assay measures metabolic activity of living cells using the WST-1 cell proliferation reagent (Roche Diagnostics). The tetrazolium salt WST-1 is cleaved to water soluble formazan by cellular enzymes. An expansion in the number of viable cells results in an increase in the overall activity of mitochondrial dehydrogenases in the sample.
  • This augmentation in enzyme activity leads to an increase in the amount of formazan dye formed, which directly correlates to the number of metabolically active cells in the culture.
  • the formazan dye produced by metabolically active cells is directly quantified in a scanning multiwell spectrophotometer by measuring the absorbance of the formazan dye solution between 420 and 480 nm. Healthy HepG2 cells, when maintained in culture, continuously divide and multiply over time. A toxic chemical, regardless of site and mechanism of action, will interfere with this process and result in the reduction of the growth rate reflected in the cell number.
  • Hep-G2 cells are seeded into a 96-well microplate and maintained in culture for 24 hours. They are then exposed to the test compound over a range of eight concentrations. After 24 hours exposure, the cells are incubated in presence of reagent WST-1 for 30 min before measuring the absorbance of the formazan dye formed. Cytotoxicity is expressed as a concentration dependent reduction of the conversion formazan dye formation due to a decrease in cell proliferation as compared to untreated cells.
  • the TC 50 Total Concentration 50%
  • the TC 50 % values were calculated using the following formula:
  • Comparative compounds A and J are clearly associated with some level of in vitro toxicity, which can be interpreted as an indicator of expected hepatho-toxic liabilities in vivo, while compounds of the present invention clearly demonstrate no toxicity in the dose range evaluated.
  • Kinetic solubility determines the concentration above which a compound starts to precipitate and demonstrate turbidity (i.e. the solubility limit). Turbidity is measured using optical density.
  • Comparative Compounds F and G are practically insoluble in aqueous solutions which negatively affects the development of oral formulations
  • Comparative Compound I is already soluble enough to show oral bioavailability.
  • Table 11 the compounds of the present invention clearly demonstrate significantly improved solubility characteristics compared to the comparative compounds.
  • NAD7NADH ratio The balance between the oxidized and reduced forms of nicotinamide adenine dinucleotide is called the NAD7NADH ratio, which is an important component of what is called the redox state (RS), a measurement that reflects both the metabolic activities and the health of cells.
  • RS redox state
  • the effects of the NAD7NADH ratio are complex, controlling the activity of several key enzymes, including glyceraldehyde 3-phosphate dehydrogenase and pyruvate dehydrogenase.
  • estimates of the NAD7NADH ratio are typically around 700, thus it favours for oxidative reactions.
  • the NADP7NADPH ratio is normally about 0.005, so NADPH is the dominant form of this coenzyme.
  • Resazurin (7-Hydroxy-3 - -phenoxazin-3-one 10-oxide) is a blue dye used mainly as an oxidation-reduction indicator in the resazurin test for bacteria. It is also used as an indicator for cell viability in mammalian cell cultures. Blue colored Resazurin is reduced to the pink colored, fluorescent compound Resorufin in the presence of live cells. Resazurin has been used to quantify mitochondrial activity since it is considered to act as an intermediate electron acceptor in the electron transport chain between the final reduction of oxygen and cytochrome oxidase.
  • the cellular redox state is tightly controlled; therefore small changes can have big impacts.
  • the compounds of the invention have a similar or better effect than the comparative compounds, sometimes at even much lower concentrations. More signal in this assay reflects higher NADH levels and therefore better viability.
  • Electrons donated from the citric acid cycle in form of NADH are transferred between complexes I, II and III of the respiratory chain.
  • mitochondrial membrane potential is generated through proton pumps in the mitochondrial inner membrane.
  • This electrochemical gradient across the inner mitochondrial membrane is used in healthy cells to provide the energy to drive mitochondrial ATP production. Consequently, cellular ATP levels are a good indicator of mitochondrial function.
  • this mode of ATP production is impaired and alternative modes of ATP production are utilized that can be associated with toxic byproducts (i.e. lactic acidosis due to increased anaerobic glycolysis).
  • Immortalized lymphoblastoid cells BC1 LCL were used to characterize the effect of compounds on ATP levels under different glucose levels by an ATP dependent luciferase reaction. Briefly, cells were seeded in a 24-well plate at a density of 5 * 10 5 cells/ml with 1 ml per well in two different media containing 25 mM or no glucose, respectively. Both media were supplemented with 10% fetal bovine serum, 1% Pen/Strep, 200 mM L-glutamine.
  • Cells were treated with 0.1 % (v/v) of compounds (10 mM in DMSO; final concentration: 10 ⁇ ) and incubated for 72 h at 37 °C, 5% C0 2 , and 90% rH. The number of cells was counted and after brief washing in PBS and collecting through centrifugation (5 min; 200 x g) the cells were lysed in a volume of 0.5 ml lysis solution (4 mM EDTA, 0.2% Triton X-100) for 15 min on ice and 10 ⁇ of lysate was added into a white 96 well plate. In parallel, ATP standards (concentrations: 0, 1 , 2, 4, 6, 8, and 12 ⁇ in PBS) were also added into the 96 well plate.
  • the reaction was started by addition of 100 ⁇ reaction mix (300 ⁇ D-Luciferin, 5 ⁇ g/ml firefly luciferase, 75 ⁇ DTT, 25 mM HEPES, 6.25 mM MgCI 2 , 625 ⁇ EDTA and 1 mg/ml BSA) and the luminescence signal was quantified in a multimode plate reader (Tecan M1000 plate reader; luminescence integration time: 100 ms). The concentration of cellular ATP normalized to cell number is calculated for each well and triplicate measurements are averaged. The ATP levels are given in Table 13.
  • Lipid peroxidation is a well defined mechanism of cellular damage in both animals and plants that occurs during aging and in some disease states. This process proceeds by a free radical chain reaction mechanism. It most often affects polyunsaturated fatty acids, because they contain multiple double bonds in between which methylene-CH2- groups possess especially reactive hydrogens. If not terminated fast enough, lipid peroxidation damages cellular membranes, affect membrane fluidity and also mitochondrial function. In addition, end products of lipid peroxidation may be mutagenic and carcinogenic. For instance, the reactive end product of lipid peroxidation, malondialdehyde, directly causes DNA damage.
  • BODIPY® (4,4-difluoro-3a,4adiaza- s-indacene) fluorophore is an effective tracer of lipid trafficking, as well as being useful general- purpose membrane probes.
  • BODIPY 581/591-C11 can be used to measure antioxidant activity in lipid environments by exploiting its loss of fluorescence upon interaction with peroxyl radicals.
  • Primary human fibroblasts C4 (GM04545, Coriell) (passage ⁇ 12) were seeded at a concentration of 2000 cells/ well into black 96 well plates and incubated for 72 hours in DMEM in the presence of 10 ⁇ test compound or DMSO only.
  • 0.1 ml freshly prepared dye solution (HBSS, containing BODIPY dye 1 :1000 from stock solution) was added and cells were returned to the incubator for 30 min. After 2 brief washes with 0.1 ml warmed PBS fluorescence was measured in 50 ⁇ PBS. Fluorescence for 4 individual areas per well were individually quantified at two wavelengths (Ex,: 490, Enri!: 600; Ex 2 : 490, Em 2 : 530, bandwith 10nm, 50 flashes, 400Hz frequency, 20 ps integration time).
  • Altered mitochondrial function can lead to cellular damage via lipid peroxidation. Altering mitochondrial function through small molecules therefore also has the inherent risk of producing lipid peroxidation as observed for Comparative Compound A. In this context, all compounds that leave basal lipid peroxidation levels unchanged (at 100%) or even reduce them ( ⁇ 100%) do not show any inherent toxic liability or are even beneficial in the context of elevated levels of lipid peroxidation in a state of disease.
  • the compounds of present invention significantly reduce basal levels of lipid peroxidation while the comparative compounds increase basal levels significantly.
  • CI I Complex II
  • succinate dehydrogenase which catalyzes the conversion from succinate to fumarate.
  • FAD succinate dehydrogenase
  • FADH 2 Ackrell 2000
  • SURFE2R SSM sensors were coated with inner mitochondrial membranes according to the standard protocols. Briefly, sensors were filled with 50 ⁇ _ of SensorPrep A solution and incubated for 10-15 min. Afterwards the solution was removed, sensor were rinsed with deionized water three times dried in a stream of nitrogen gas and incubated for 15 min at room temperature to get rid of remaining solvents. 1.5 ⁇ _ of SensorPrep B1 solution were applied to the sensor and immediately covered with 50 ⁇ _ of the buffer (150 mM Na-gluconate, 30 mM Hepes pH 7.2/NMG, 10 mM MgCI2, 12.5 mM NaPi pH7.2, freshly added 0.2 mM DTT). Incubate the sensor for 15-60 min at 4°C.
  • the buffer 150 mM Na-gluconate, 30 mM Hepes pH 7.2/NMG, 10 mM MgCI2, 12.5 mM NaPi pH7.2, freshly added 0.2 mM DTT.
  • the biosensors were thawed immediately before the experiment and measured with the SURFE2R Workstation 50 or 500 devices.
  • the CII-CIII activity was studied by rapid exchange of a "non-activating" solution for an "activating" solution containing the 3 ⁇ oxidized cytochrome c. 2 s of non-activating buffer was followed by 1 s of activating buffer and then again 1 s non- activating buffer. Afterwards the sensor was rinsed 3 times with 1 mL non-activating buffer. A further activation with cytochrome c was performed after an incubation time of -11 min. Both buffers contained 1 mM succinate and 350 mg/L BSA to enhance the solubility of the test compounds.
  • Cll-lll For the activity of Cll-lll, the peak current (current amplitude) was evaluated.
  • the performed measurements of Cll-lll activities consisted of two parts. First the activity of Cll-lll was recorded in the absence of compound (instead 0.01% DMSO) for about 50 min to obtain a constant CII-CIII activity (constant peak current amplitudes). Afterwards, a test compound ( ⁇ 4 ⁇ ) was supplied to the non-activating and the activating solutions and the Cll-lll activity was recorded for further -50 min (110 min in total). For each sensor, all peak currents were normalized to the mean of the activities after 24, 36 and 48 min.
  • n (t 0-110) 2.
  • the CII- CIII activity was recorded for additional -50 min (up to 160 min in total).
  • a longer integration time just indicates that the compound is in principle capable of triggering higher complex II activities but in a cell free setting unable to quickly insert itself into the membrane via diffusion to affect complex ll-lll.
  • maximal concentrations in the membrane are reached at different speeds. This is an artefact of a cell free system where you rely on diffusion. In vivo, these compounds are likely transported in a coordinated fashion via lipid carrier, so this difference will not be seen.
  • Mitochondrial disorders are characterized by impaired mitochondrial function, which is usually displayed as lower mitochondrial synthesis of ATP. This energy crisis is seen as a major contributor for cellular impairment and ultimately cell death. Thus, improving the abberant energy status that is associated with impaired mitochondrial function is necessary to normalize cellular and tissue function.
  • Rat myoblast cells (L6) were seeded at a density of 5 * 10 3 cells per well in a 96-well plate and incubated for 24 hours in DMEM with 0.3 g/l glucose, 2% FBS and Penicillin-Streptomycin- Glutamine. Cells were treated with 1 ⁇ quinones in presence or absence of rotenone (1 ⁇ ), for 60 minutes in DMEM without glucose before ATP levels were quantified using luminescence from the ATP-dependent enzymatic oxidation of luciferin by luciferase. Cells were lysed in a volume of 200 ⁇ (4 mM EDTA, 0.2% Triton X-100) for five minutes.
  • ATP measurement buffer 25 mM HEPES pH 7.25, 300 ⁇ D-luciferin, 5 pg/ml firefly luciferase, 75 ⁇ DTT, 6.25 mM MgCI 2 , 625 ⁇ EDTA and 1 mg/ml BSA
  • Luminescence was quantified immediately using a multimode plate reader (Tecan M1000, Tecan iControl 1.6 software; Tecan Austria GmbH, Grodig, Austria).
  • ATP levels were standardized to protein levels using BCA assay (ThermoScientific, Rockford, IL, USA) and changes were calculated as percentage relative to levels of DMSO-treated control cells.
  • ATP rescue is defined as the percentage of quinone-induced increase in ATP levels in presence of rotenone relative to the ATP reduction by rotenone alone.
  • Altered mitochondrial function can lead to depleted cellular ATP levels.
  • altering mitochondrial function by small molecules of the current invention but not by Comparative Compound B alows the rescue of cellular energy status as observed here.
  • all compounds that counteract the lowered ATP levels associated with mitochondrial dysfunction can be seen as beneficial for the function and survival of the affected cells.
  • the compounds of present invention significantly rescue ATP levels under conditions of impaired mitochondrial function while the comparative compound B has no effect at all.

Abstract

The present invention relates to novel benzoquinone derivatives. The compounds are efficient as modulators of mitochondrial function and as such are useful for the treatment of pathological conditions where mitochondrial function is impaired.

Description

Novel Benzoquinone Derivatives and Use Thereof as Modulators of Mitochondrial Function
Field of the Invention
The present invention relates to novel benzoquinone derivatives. The compounds are efficient as modulators of mitochondrial function and as such are useful for the treatment of pathological conditions where mitochondrial function is impaired.
Background of the Invention
Mitochondria, sometimes described as "cellular power plants" because they generate most of the cell's supply of adenosine triphosphate (ATP), are essential to eukaryotic life. In addition to supplying cellular energy, mitochondria are also involved in a range of other processes, such as signalling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Mitochondria have been implicated in several human diseases, including mitochondrial disorders and cardiac dysfunction and may play a role in the aging process.
With their central place in cell metabolism, mitochondrial dysfunction is an important factor in a wide range of human diseases. Mitochondrial disorders are often present as neurological disorders, but can also occur as myopathy, diabetes, multiple endocrinopathy, or a variety of other systemic manifestations. These disorders can be caused by mutations of the mitochondrial DNA, by mutations of nuclear genes directly coding for oxidative phosphorylation enzymes and by defects in nuclear genes that are generally important for mitochondrial function. Environmental influences may also interact with hereditary predispositions and cause mitochondrial diseases. For example, there may be a link between pesticide exposure and the later onset of Parkinson's disease. Numerous other pathologies involving mitochondrial dysfunction include schizophrenia, bipolar disorder, autosomal dominant optic atrophy, epilepsy, stroke and autism (Jou et al. Chang Gung Med J. 2009; 32(4):370-9), dementia, Alzheimer's, Parkinson's and Huntington's disease (Yang et al. DNA Repair (Amst). 2008; 7(7): 1110-20), cardiovascular disease (Puddu et al. J Biomed Sci. 2009; 16:112), malignancies (Singh & Kulawiec; Methods Mol Biol. 2009; 471 :291-303), retinitis pigmentosa, metabolic syndrome, anorexia, obesity and diabetes mellitus (Burchell et al. Expert Opin Ther Targets 2010; 14(4):369-85; Pieczenik & Neustadt Exp Mol Pathol. 2007; 83(1):84-92).
Multiple sclerosis is the most common non-traumatic neurological disease in young adults, with a prevalence of 1:1000 in Northern Europe and North-America (Compston A Int MS J .2003. 10:29-31). At first, the disease course is generally episodic; exacerbations are followed by periods of remission which are characterized by focal infiltration of leukocytes and demyelination in the white matter. Gradually the disease becomes more progressive, where demyelination of the grey matter and axonal degeneration in the white matter become more prominent (Hafler DA J. Clin. Invest. 2004; 113:788-794). Available therapies are mainly immunomodulatory, which are effective in reducing the number of relapses. Disease progression, however, remains unchanged by these therapies (Filippini G et al. Lancet 2003; 361 :545-552.), demonstrating the need for novel therapeutic strategies oriented towards neuroprotection.
It is thought that one of the critical underlying defects of the disorders mentioned above is the deficiency of damaged mitochondria to provide sufficient energy to allow normal cellular functions and/or their characteristic to generate excess oxidative stress. As a consequence of this energy deprivation and macromolecular damage, cells die. This in turn impairs tissue function and causes disease pathology. This connection is most obvious in mitochondrial disorders that are characterized by pathologies in tissues with high energy demand such as brain and muscle cells, where cell loss or impaired cellular function in these tissues is intimately linked to the phenotype of the patient.
Although there are multiple therapeutic approaches for the indications listed above, only very few of those are addressing the underlying mitochondrial dysfunction. Thus, there still exists a need for novel therapeutic approaches for treating disorders caused by impaired mitochondria.
WO-A-2006130775 describes an attempt to treat or suppress mitochondrial diseases by modulating energy biomarkers such as lactic acid levels, levels of NAD, NADP, NADH and NADPH, and cytochrome C parameters. The compounds disclosed in the application have been tested for their ability to rescue human dermal fibroblasts from FRDA patients from oxidative stress. However, the data are not represented in the application.
It is therefore the object of the present invention to provide novel compounds which are effective as modulators of mitochondrial function. Surprisingly, it has been found that novel benzoquinone derivatives according to formula (I) shown below solve the object of the present invention.
Summary of the Invention
The present invention relates to benzoquinone derivatives of structural formula (I)
wherein R , R2, R3 and R4 are defined as described below.
The benzoquinone derivatives of structural formula (I) are effective as modulators of mitochondrial function and as such are useful for the treatment of pathological conditions where mitochondrial function is impaired. It has been found that the compounds of formula (I) are useful in the treatment of mitochondrial diseases such as Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy (DOA), macular degeneration, glaucoma, retinopathy, cataract, optic disc drusen (ODD), mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), myoclonic epilepsy with ragged red fibers (MERRF), myoneurogenic gastrointestinal encephalomyopathy (MNGIE), Kearns-Sayre syndrome, C0QIO deficiency, or mitochondrial complex deficiencies or neurodegenerative diseases such as Friedreich's ataxia (FRDA), amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, Stroke/Reperfusion Injury and Dementia or neuromuscular diseases such as Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Limb-Girdle muscular dystrophy (LGMD), X-linked dilated cardiomyopathy (XLDCM), Pantothenate kinase-associated neurodegeneration (PKAN), spinal muscular atrophy (SMA), multiple sclerosis and primary progressive multiple sclerosis (PP-MS), Kugelberg- Welander disease and Werdnig-Hoffmann disease or cancer or psychiatric disorders such as schizophrenia, major depressive disorder, bipolar disorder, and epilepsy or metabolic disorders such as ageing-related physical decline, obesity, overweight, type II diabetes, and metabolic syndrome or immune dysfunctions such as arthritis, psoriasis or rheumatoid arthritis. The present invention further relates to a method for treating pathological conditions where mitochondrial function is impaired, the method comprising administering an effective amount of a compound of formula (I) to a subject in need thereof.
The present invention also relates to pharmaceutical compositions comprising the compounds of the present invention and a pharmaceutically acceptable carrier.
Detailed Description of the Invention
The present invention relates to benzoquinone derivatives which are capable to modulate mitochondrial function.
The compounds of the present invention are represented by general formula (I)
and enantiomers, tautomers, solvates or pharmaceutically acceptable salts thereof,
wherein
a) R1 is a substituent represented by formula (II) Y1 (X1)a R5
(ID
wherein
X1 is -O- or -NR6-, wherein R6 is H or linear or branched d.6alkyl;
R5 is selected from the group consisting of
a linear or branched C1-6 alkyl which is unsubstituted or substituted with 1 to 5 substituents independently selected from halogen, OH, NH2 or CN, a 6-10 membered monocylic or bicyclic aryl group, optionally fused with a 5-6 membered heterocyclic or heteroaromatic group, the heterocyclic or heteroaryl group having 1 to 2 heteroatoms in the ring independently selected from N, O or S,
a 5-10 membered monocyclic or bicyclic heteroaromatic group having 1 to 4 heteroatoms independently selected from N, O or S,
a 5-7 membered monocyclic heterocyclic group having 1 to 4 heteroatoms in the ring independently selected from N, O or S, and
a 3-10 membered monocylic, bicylic or tricyclic cycloalkyl group, wherein each aryl, heteroaryl, heterocyclyl and cycloalkyl is unsubstituted or substituted with 1 to 5 substituents independently from each other selected from the group consisting of halogen, hydroxy, linear or branched Ci-6 alkyl, linear or branched halo-Ci.6 alkyl, Ci_6 alkoxy, -C(0)NH2, - NHC(0)C1-6 alkyl, 5-6 membered heteroaryl having in the ring 1 to 3 heteroatoms independently selected from N, O or S,
or
X1 and R5 together form a 4-7 membered monocyclic heterocyclic group having 1 or 2 heteroatoms in the ring independently selected from N or S and wherein the heterocycle is unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of a halogen, a hydroxyl group and a Ci-6 alkoxy group;
Y1 is a linear C8.i2 alkylene group or a linear C8-12 alkenylene group;
a is an integer of 0 or 1 ;
R2 is a Ci-6 linear or branched alkyl group;
R3 is a Ci-e alkoxy group; and
R4 is a C1-6 alkoxy group;
with the proviso that when a is 1 , X1 is -0-, and Y1 is C10 alkyl, then R5 is not a 2H-pyrane group;
or
R1 is a substituent represented by formula (III)
wherein
R7 and R8 independently from each other are hydrogen, linear or branched d. 6 alkyl or halogen;
R9 and R10 independently from each other are hydrogen, linear or branched C2.6alkenyl or linear or branched C2-6 alkinyl;
or R9 and R10 together are -(CH2)X- wherein x is an integer of 2 to 6;
with the proviso that R7, R8, R9 and R10 cannot simultaneously be a hydrogen atom;
Y2 is a linear C5.9 alkylene group or a linear C5.9 alkenylene group;
R2 is a linear or branched C1-6 alkyl group;
R3 is a Ci-6 alkoxy group; and
R4 is a Ci-6 alkoxy group;
or
R1 is a substituent represented by formula (IV)
(IV)
wherein
R11 and R12 independently from each other are
hydrogen,
linear or branched C1-6 alkyl,
6-10 membered monocyclic or bicyclic aryl group,
5-10 membered monocyclic or bicyclic heteroaromatic group having 1 to 4 heteroatoms in the ring independently selected from N, O or S, 4-10 membered monocylic heterocyclic group having 1 to 4 heteroatoms in the ring independently selected from N, O or S, or
3- 7 membered monocyclic cycloalkyi group,
wherein each alkyl, aryl, heteroaromatic, heterocyclic and cycloalkyi group is unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of halogen, hydroxy and Ci-6alkoxy;
or
R11 and R12 together with the nitrogen atom to which they are attached form a
4- 7 membered ring having in the ring 0 to 2 additional heteroatoms independently selected from N, O or S, wherein the ring is unsubstituted or substituted with 1 to 3 substituents selected from the group consisting of halogen, hydroxyl and C^alkoxy group;
Y3 is a linear C6.10 alkylene group or a linear C6-10 alkenylene group;
R2 is a linear or branched 01-6 alkyl group;
R3 is a group; and
R4 is a Ci.6alkoxy group;
or
R1 is hydrogen;
R2 is a substituent represented by X2-R13
wherein X2 is selected from the group consisting of -O- and -NR14-, wherein R14 is hydrogen or a d-e alkyl group, and
R13 is a C4-12 alkyl group, C4-12 alkenyl group, -(CH2)nCH2OH or -C2-10 alkenylene-CH2OH,
wherein n is an integer of 1 to 10,
or
wherein X2-R13 represents a piperidino group substituted in the 4-position with a C2-7 alkenylene-CH2OH group, a -(CH2)mCH2OH group wherein m is an integer of 1 to 7, a -C(0)-NH-C2-6 alkenylene-CH2OH group or a -C(0)-NH-(CH2)pCH2OH group wherein p is an integer of 1 to 6;
R3 is hydrogen; and R4 is OR15,
wherein R15 is a hydrogen atom or a d.2 alkyl group,
or R13 and R15 independently represent a linear -C2-7 alkenylene-CH2OH group or a -(CH2)mCH20H group wherein m is an integer of 1 to 7;
or
e) R1 is
Y4 is a linear C8-i2 alkylene group or a linear C8-i2 alkenylene group;
R2 is a linear or branched C1.6 alkyl group;
R3 is a Ci-e alkoxy group; and
R4 is a Ci.6alkoxy group;
or
f) R1 is
wherein
Y5 is a linear C8.i2 alkylene group or a linear C8-i2 alkenylene group; R2 is a linear or branched d-ealkyl group;
R3 is a hydroxy group or a Ci.6alkoxy group; and
R4 is a Ci-ealkoxy group;
R1 is
wherein
Y6 is a linear C6-io alkylene group or a linear C6-io alkenylene group; R2 is a linear or branched Ci.6alkyl group;
R3 is a d.6alkoxy group; and R4 is a Ci.6alkoxy group.
Preferred embodiments are set forth in the subclaims. Further preferred embodiments are described below.
In a preferred embodiment in combination with any of the above or below embodiments,
R1 has the formula (II);
a is 1 ;
X1 is an oxygen atom; and
R5 is selected from the group consisting of a 6 membered heteroaryl group having 1 to 2 nitrogen atom(s) in the ring and a phenyl group wherein the heteroaryl and the phenyl group are unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of halogen, linear or branched C1-6 alkyl, linear or branched halo On alkyl, C1-6alkoxy, -C(0)NH2, alkyl, 5-6 membered heteroaryl having in the ring 1 to 3 heteroatoms independently selected from N, O or S, and hydroxy.
In a preferred embodiment in combination with any of the above or below embodiments,
R1 has the formula (II);
a is 1 ;
X1 is a nitrogen atom (i.e., -NR6-); and
R5 is selected from the group consisting of
a 5-10 membered monocyclic or bicyclic heteroaryl group having 1 to 4 heteroatoms independently selected from N, O or S,
a phenyl group, and an adamantyl group,
wherein the heteroaryl group, the phenyl group and the adamantly group are unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of halogen, linear or branched Ci-6 alkyl, linear or branched halo Ci-6 alkyl, d.6 alkoxy, -C(0)NH2, -NHCiO^-e alkyl, 5-6 membered heteroaryl having in the ring 1 to 3 heteroatoms independently selected from N, O or S, and hydroxy or
X1 and R5 together form a 4-7 membered monocyclic heterocyclic group having 1 or 2 heteroatoms in the ring independently selected from N or S and whereby the heterocycle is unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of a halogen, a hydroxyl group and a Ci.6alkoxy group.
In a preferred embodiment in combination with any of the above or below embodiments,
R1 has the formula (II);
a is 0; and
R5 is a 5-10 membered monocyclic or bicyclic heteroaryl group having 1 to 4 heteroatoms independently selected from N, O and S, wherein the heteroaryl group is unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of halogen, linear or branched 01-6 alkyl, linear or branched halo C1-6 alkyl, d-6 alkoxy, - C(0)NH2, -NHC(0)Ci-6 alkyl, 5-6 membered heteroaryl having in the ring 1 to 3 heteroatoms independently selected from N, O or S, and hydroxy.
In a preferred embodiment in combination with any of the above or below embodiments,
R1 is hydrogen;
R2 is X2-R13 wherein X2 is -NR14 wherein R 4 is hydrogen or a d-e alkyl group, and R13 is a linear C4-12alkyl group, or -(CH2)nCH2OH wherein n is an integer of 1 to 10, or
wherein X2-R13 represents a piperidino group substituted in the 4-position with a - (CH2)mCH2OH group wherein m is an integer of 1 to 7 or a -C(0)-NH-(CH2)pCH2OH group wherein p is an integer of 1 to 6;
R3 is hydrogen; and
R4 is -O-R15 wherein R15 is hydrogen or C -2alkyl.
(i) In a preferred embodiment in combination with any of the above or below embodiments R1 is represented by formula (II). In a preferred embodiment in combination with any of the above or below embodiments,
R has the formula (II);
wherein
X1 is -O- or -NR6-, wherein R6 is H or linear or branched Ci-6alkyl;
R5 is selected from the group consisting of
a linear or branched d-6 alkyl which is unsubstituted or substituted with 1 to 5 substituents independently selected from halogen, NH2 or CN, a 6-10 membered monocylic or bicyclic aryl group, optionally fused with a 5-6 membered heterocyclic or heteroaromatic group, the heterocyclic or heteroaryl group having 1 to 2 heteroatoms in the ring independently selected from N, O or S,
a 5-10 membered monocyclic or bicyclic heteroaromatic group having 1 to 4 heteroatoms independently selected from N, O or S, a 5-7 membered monocyclic heterocyclic group having 1 to 4 heteroatoms in the ring independently selected from N, O or S, and a 3-10 membered monocylic, bicylic or tricyclic cycloalkyl group, wherein each aryl, heteroaryl, heterocyclyl and cycloalkyl is unsubstituted or substituted with 1 to 5 substituents independently from each other selected from the group consisting of halogen, linear or branched C1-6 alkyl, linear or branched halo-d-e alkyl, -C(0)NH2, -NHC(0)C^ alkyl, 5-6 membered heteroaryl having in the ring 1 to 3 heteroatoms independently selected from N, O or S,
or
X1 and R5 together form a 4-7 membered monocyclic heterocyclic group having 1 or 2 heteroatoms in the ring independently selected from N or S and wherein the heterocycle is unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of a halogen, a hydroxyl group and a Ci.6alkoxy group;
Y1 is a linear C8-12 alkylene group or a linear C8.12 alkenylene group;
a is an integer of 0 or 1 ; R2 is a C1-6 linear or branched alkyl group;
R3 is a d-ealkoxy group; and
R4 is a group;
with the proviso that when a is 1 , X1 is -0-, and Y1 is C10 alkyl, then R5 is not a 2H-pyrane group.
In a preferred embodiment in combination with any of the above or below embodiments,
R1 has the formula (II);
wherein
when R2 is methyl, R3 is methoxy, R4 is methoxy, and a is 0, then R5 is not a linear or branched Ci.6 alkyl which is substituted with one substituent OH, or a phenyl or C3.7 cylcoalkyl, wherein the phenyl or C3.7 cylcoalkyl is substituted with one substituent OH or with one substituent C1-4 alkoxy.
In formula (II) Y1 is a linear C8-i2 alkylene group or a linear C8-i2 alkenylene group. In a preferred embodiment in combination with any of the above or below embodiments, Y1 represents a linear C8.12 alkylene group, more preferably a linear C10 alkylene group.
In formula (II) the index a represents an integer selected from the values 0 and 1.
In a preferred embodiment in combination with any of the above or below embodiments, the index a assumes the value 0. If a is 0, it is preferred that R5 represents a 5-10 membered monocyclic or bicyclic heteroaryl group having 1 to 4 heteroatoms in the ring(s) independently selected from N, O or S, wherein the monocyclic heteroaryl group and one or both rings of the bicylic heteroaryl group is unsubstituted or substituted with 1 to 5 substituents independently from each other selected from the group consisting of halogen, linear or branched C1.6 alkyl, linear or branched halo-Ci-6 alkyl, C1.6 alkoxy, -C(0)NH2, -NHC(0)Ci.6 alkyl, 5-6 membered heteroaryl having in the ring 1 to 3 heteroatoms independently selected from N, O or S, and hydroxy. In a preferred embodiment in combination with any of the above or below embodiments, the heteroaryl moiety is unsubstituted or substituted with 1 to 3 C1-6 alkyl groups, more preferably 1 to 3 methyl groups. In a more preferred embodiment in combination with any of the above or below embodiments, R5 is a 9-10 membered, more preferably a 9 membered bicyclic heteroaryi group having 1 to 4 heteroatoms in the ring(s) independently selected from N, O or S, wherein the heteroaryi group is unsubstituted or substituted with 1 to 3 Ci-6alkyl groups. In an equally preferred embodiment in combination with any of the above or below embodiments the index a assumes the value 1.
If a is 1 , X1 represents -O- or -NR6- wherein R6 is H or linear or branched C1-6 alkyl, more preferably H or CH3.
In a preferred embodiment in combination with any of the above or below embodiments X1 is - 0-, R5 is a 5-10 membered monocyclic or bicyclic heteroaryi group having 1 to 4 heteroatoms in the ring(s) independently selected from N, O or S, wherein the monocyclic heteroaryi group and one or both rings of the bicylic heteroaryi group is unsubstituted or substituted with 1 to 5 substituents independently from each other selected from the group consisting of halogen, hydroxy, linear or branched d-e alkyl, linear or branched halo-Ci.6 alky Ci.6 alkoxy, -C(0)NH2, - NHCiOJCi-e alkyl, 5-6 membered heteroaryi having in the ring 1 to 3 heteroatoms independently selected from N, O or S.
In an equally preferred embodiment in combination with any of the above or below embodiments X1 is -0-, R5 is a 6-10 membered monocyclic or bicyclic aryl group which may be optionally fused with a 5-6 membered heterocylic or heteroaromatic group, wherein the heterocyclic or heteroaromatic group have 1 to 2 heteroatoms in the ring independently selected from N, O or S. Within the preceding preferred embodiment, the aryl, the heterocyclic and the heteroaryi group are unsubstituted or substituted with 1 to 5 substituents independently from each other selected from the group consisting of halogen, hydroxy, linear or branched C1.6 alkyl, linear or branched halo-C1-6 alkyl, C^ alkoxy, -C(0)NH2, -ΝΗΟ(0)01-6 alkyl, 5-6 membered heteroaryi having in the ring 1 to 3 heteroatoms independently selected from N, O or S. More preferably, R5 is a phenyl group which is unsubstituted or substituted with 1 to 3 substituents independently selected from - C(0)NH2, halogen, -NHC(0)Ci.6 alkyl, linear or branched halo-C1-6 alkyl and 5-6 membered heteroaryi having in the ring 1 to 3 heteroatoms independently selected from N, O or S. More preferably said 5-6 membered heteroaryi group is a 5 membered heteroaryi group having in the ring 1 to 3, preferably 2 to 3 nitrogen atoms.
In an alternative preferred embodiment in combination with any of the above or below embodiments X1 is -NR6-, R5 is preferably a 6-10 membered monocylic or bicyclic aryl group, optionally fused with a 5-6 membered heterocyclic or heteroaromatic group, the heterocyclic or heteroaromatic group having 1 to 2 heteroatoms in the ring independently selected from N, O or S; or a 5-10 membered monocyclic or bicyclic heteroaromatic group having 1 to 4 heteroatoms independently selected from N, O or; or a 5-7 membered monocyclic heterocyclic group having 1 to 4 heteroatoms in the ring independently selected from N, O or S, or a 3-10 membered monocylic, bicylic or tricyclic cycloalkyi group, wherein each aryl, heteroaryl, heterocyclyl and cycloalkyi is unsubstituted or substituted with 1 to 5 substituents independently from each other selected from the group consisting of halogen, hydroxy, linear or branched C1-6 alkyl, linear or branched halo-Ci.6 alkyl, C1-6 alkoxy, -C(0)NH2, -NHC(0)Ci-6 alkyl, 5-6 membered heteroaryl having in the ring 1 to 3 heteroatoms independently selected from N, O or S; or X1 and R5 together form a 4-7 membered monocyclic heterocyclic group having 1 or 2 heteroatoms in the ring independently selected from N or S and wherein the heterocycle is unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of a halogen, a hydroxyl group and a Ci.6 alkoxy group.
In a more preferred embodiment in combination with any of the above or below embodiments X1 is -NR6-, R5 is preferably a 5-10 membered monocyclic or bicyclic heteroaromatic group having 1 to 4 heteroatoms in the ring(s) independently selected from N, O or S, wherein the monocyclic heteroaryl group and one or both rings of the bicylic heteroaromatic group is unsubstituted or substituted with 1 to 5 substituents independently from each other selected from the group consisting of halogen, hydroxy, linear or branched C1-6 alkyl, linear or branched halo-C -6 alkyl, Ci-6 alkoxy, -C(0)NH2, -NHCiOJC^ alkyl, 5-6 membered heteroaryl having in the ring 1 to 3 heteroatoms independently selected from N, O or S.
(ii) In a preferred embodiment in combination with any of the above or below embodiments R1 is represented by formula (III).
In a preferred embodiment in combination with any of the above or below embodiments,
R1 has the formula (III);
wherein
when R2 is methyl, R3 is methoxy, R4 is methoxy, Y2 is a linear C5.9 alkylene group, R7 is hydrogen and R8 is hydrogen,
then R9 and R10 cannot simultaneously be a C1-4 alkyl group. In formula (III) Y2 is a linear C5-9 alkylene group or a linear C5.9 alkenylene group. In a preferred embodiment in combination with any of the above or below embodiments, Y1 represents a linear C5-9 alkylene group, more preferably a linear C7 alkylene group.
In a preferred embodiment in combination with any of the above or below embodiments R7 and R8 both represent hydrogen, R9 represents hydrogen and R10 represents a C2-6 alkinyl group, more preferably a C2 alkinyl group, or R9 and R10 together form a -CH2-CH2- bridge.
(iii) In a preferred embodiment in combination with any of the above or below embodiments R1 is represented by formula (IV).
In formula (IV) Y3 is a linear C6-i0 alkylene group or a linear C6-io alkenylene group. In a preferred embodiment in combination with any of the above or below embodiments, Y3 represents a linear C6-io alkylene group, more preferably a linear C8 alkylene group.
In a preferred embodiment in combination with any of the above or below embodiments R 1 and R 2 independently from each other represent hydrogen, linear or branched C1.6 alkyl, a 4-10 membered monocylic heterocyclic group having 1 to 4 heteroatoms in the ring independently selected from N, O or S, wherein each alkyl and heterocyclic group is unsubstituted or substituted with 1 to 5 substituents independently selected from halogen, hydroxy and C1 -6 alkoxy, or R 1 and R12 together with the nitrogen atom to which they are attached form a 4-7 membered ring having in the ring 0 to 2 additional heteroatoms independently selected from N, O or S, wherein the ring is unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of halogen, hydroxyl and C1.6 alkoxy group. More preferably, one of R 1 and R1 represents a hydrogen and the other represents a linear or branched C1 -6 alkyl, a 4-10 membered monocylic heterocyclic group having 1 to 4 heteroatoms in the ring independently selected from N, O or S, wherein each alkyl and heterocyclic group is unsubstituted or substituted with 1 to 5 substituents as defined above, or R11 and R12 together with the nitrogen atom to which they are attached form a 4-7 membered ring having in the ring 0 to 2 additional heteroatoms independently selected from N, O or S, wherein the ring is unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of halogen, hydroxyl and Ci.6 alkoxy group. (iv) In a preferred embodiment in combination with any of the above or below embodiments R1 and R3 represent hydrogen, R2 represents X -R13, wherein X2 and R13 are defined as above and R4 represents OR15, wherein R15 is defined as above.
In a further preferred embodiment in combination with any of the above or below embodiments X2 represents -NR14- wherein R14 is defined as above.
In a further preferred embodiment in combination with any of the above or below embodiments, R13 represents a C4-i2 alkyl group or -(CH2)nCH2OH wherein n is an integer of 1 to 10.
In an equally preferred embodiment in combination with any of the above or below embodiments X2 and R 3 combine to represent a piperidino group substituted in the 4-position with a - (CH2)mCH2OH group wherein m is an integer of 1 to 7 or a -C(0)-NH-(CH2)pCH2OH group wherein p is an integer of 1 to 6;
(v) In a preferred embodiment in combination with any of the above or below embodiments R1 is
wherein Y4 is preferably a C8 alkylene group.
In the above and the following, the employed terms have the meaning as described below:
Alkyl is a straight chain or branched alkyl having preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl or 2-ethylbutyl.
Alkenyl is a straight chain or branched alkyl having preferably 2, 3, 4, 5, 6 carbon atoms and which contains at least one carbon-carbon double bond, preferably one or two double bonds, most preferably one double bound. Preferred examples of a C2-6 alkenyl group are ethenyl, prop-
1- enyl, prop-2-enyl, isoprop-1-enyl, n-but-1-enyl, n-but-2-enyl, n-but-3-enyl, isobut-1-enyl, isobut-
2- enyl, n-pent-1-enyl, n-pent-2-enyl, n-pent-3-enyl, n-pent-4-enyl, n-pent-1 ,3-enyl, isopent-1- enyl, isopent-2-enyl, neopent-1-enyl, n-hex-1-enyl, n-hex-2-enyl, n-hex-3-enyl, n-hex-4-enyl, n- hex-5-enyl, n-hex-1 ,3-enyl, n-hex-2,4-enyl, n-hex-3,5-enyl, and n-hex-1 ,3,5-enyl. More preferred examples of an alkenyl group are ethenyl and prop-1-enyl.
Where indicated, the alkenyl group is exclusively a linear alkenyl group. In said cases, preferred examples of an alkenyl group have 5 to 12 carbon atoms and at least one double bond up to three double bonds depending on the length of the carbon chain. Generally, the double bonds occur in an isolated or a conjugated order.
Alkinyl is a straight chain or branched alkyl having preferably 2, 3, 4, 5, or 6 carbon atoms and which contains at least one carbon-carbon triple bond, preferably one or two triple bonds, most preferably one triple bond. Preferred examples of a C2-6 alkinyl group are ethinyl, prop-1-inyl, prop-2-inyl, n-but-1-inyl, n-but-2-inyl, n-but-3-inyl, n-pent-1-inyl, n-pent-2-inyl, n-pent-3-inyl, n- pent-4-inyl, n-pent-1 ,3-inyl, isopent-1-inyl, neopent-1-inyl, n-hex-1-inyl, n-hex-2-inyl, n-hex-3-inyl, n-hex-4-inyl, n-hex-5-inyl, n-hex-1 ,3-inyl, n-hex-2,4-inyl, n-hex-3,5-inyl and n-hex-1 ,3,5-inyl. More preferred examples of a C2-6 alkinyl group are ethinyl and prop-1-inyl.
The definition of the terms alkyl, alkenyl and alkinyl equally apply to the groups alkylene, alkenylene and alkinylene used throughout the invention.
Cycloalkyl is one, two or three alkyl rings having preferably 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms at the most. Preferred monocyclic examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl, more preferably cyclopropyl and cyclopentyl. Preferred bicyclic examples include decalinyl. Preferred tricyclic examples include adamantyl.
Heteroaryl is an aromatic moiety having in the ring at least one heteroatom independently selected from O, N or S. The heteroaryl group may be monocylic or bicyclic. Heteroaryl is preferably selected from thienyl, pyrrolyl, furanyl, imidazolyl, pyrazolyl, 1 ,2,3-triazolyl, 1 ,2,4- triazolyl, tetrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, thiazolyl, isothiazolyl, oxazolyl, isoxazyl, 1 ,2,3-oxadiazolyl, 1 ,2,4-oxadiazolyl 1 ,2,5-oxadiazolyl 1 ,3,4-oxadiazolyl indazolyl, quinolinyl, isquinolinyl, cinnolinyl, quinoxalinyl, quinazolinyl, phthalazinyl, pteridinyl, benzimidazolyl, indazolyl, indolyl, purinyl, imidazo[4,5-fc>]pyridinyl, imidazo[1 ,2-a]pyridinyl, and azaindazolyl, more preferably from imidazolyl, 1 ,2,4-triazolyl, tetrazolyl, 1 ,2,4-oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl and imidazo[4,5-b]pyridinyl.
Heterocyclyl is a saturated or partially unsaturated ring containing at least one heteroatom independently selected from O, N or S. Preferred examples include tetrahydrofuranyl, azetidinyl, pyrrolidinyl, pyrrolinyl, piperidinyl, piperazinyl, pyranyl, morpholinyl, thiomorpholinyl, 1 ,4-dioxanyl, more preferred examples include piperidinyl, piperazinyl and pyrrolidinyl.
Aryl is either a phenyl or a naphthalin group.
Halogen is a halogen atom selected from F, CI, Br and I, preferably from F, CI and Br. Salts
The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc and the like. Particularly preferred are the ammonium, calcium, lithium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine, caffeine, choline, Ν,Ν'-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylamino-ethanol, ethanolamine, ethylenediamine, N- ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.
When the compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, formic, furnaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, malonic, mucic, nitric, parnoic, pantothenic, phosphoric, propionic, succinic, sulfuric, tartaric, p-toluenesulfonic, trifluoroacetic acid and the like. Particularly preferred are citric, furnaric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric and tartaric acids. It will be understood that, as used herein, references to the compounds of formula (I) are meant to also include the pharmaceutically acceptable salts.
Administration
The compounds of formula (I) are preferably formulated into a dosage form prior to administration. Accordingly the present invention also includes a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof and a suitable pharmaceutical carrier.
The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art; e.g., in Remington's Pharmaceutical Sciences. The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.
Any suitable route of administration may be employed for providing a mammal, especially a human with an effective dosage of a compound of the present invention.
Suitable administration routes include oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intraarticular, and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including dermal, buccal, sublingual and intraocular) administration although the most suitable route may depend upon for example the condition and disorder of the recipient.
In a preferred embodiment in combination with any of the above or below embodiments, the administration route is oral.
In another preferred embodiment in combination with any of the above or below embodiments, the administration route is topical ocular. In another preferred embodiment in combination with any of the above or below embodiments, the administration route is an intraocular injection.
In another preferred embodiment in combination with any of the above or below embodiments, the administration route is an intraocular depot implant.
Formulation and Dose Ranges
The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association a compound of the present invention or a pharmaceutically acceptable salt or solvate thereof ("active ingredient") with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient. The active ingredient may further be presented as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.
Pharmaceutical preparations which can be used orally include tablets, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with binders, inert diluents, or lubricating, surface active or dispersing agents. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein. All formulations for oral administration should be in dosages suitable for such administration. The push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
The compounds of the present invention may be formulated for parenteral administration by injection, e.g. as intraocular, intraveneous, subcutaneous, intramuscular, or intraarterial injection. The injection may be administered by bolus injection or continuous infusion. The injection may be administered Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in powder form or in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or sterile pyrogen-free water, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Formulations for parenteral administration include aqueous and non-aqueous (oily) sterile injection solutions of the active compounds which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example intraocular or subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
For buccal or sublingual administration, the compositions may take the form of tablets, lozenges, pastilles, or gels formulated in conventional manner. Such compositions may comprise the active ingredient in a flavoured basis such as sucrose and acacia or tragacanth.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter, polyethylene glycol, or other glycerides.
Compounds of the present invention may be administered topically, that is by non-systemic administration. This includes the application of a compound of the present invention externally to the epidermis or the buccal cavity and the instillation of such a compound into the ear, eye and nose, such that the compound does not significantly enter the blood stream. In contrast, systemic administration refers to oral, intravenous, intraperitoneal and intramuscular administration.
Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of inflammation such as gels, liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.
In a topical ocular formulation, the amount of compound of formula (I) will generally be in the range of 0.001 to 10% weight/volume (%w/v). Preferred concentrations range from 0.1 to 5 %w/v. Topical administration to the eye is given one to six times per day. A typical formulation contains 0.1 - 5 % of compound of formula (I), 0.5 %w/v hydroxypropylmethylcellulose (HMPC), 0.8 %w/v sodium chloride, 0.28 %w/v sodium phosphate, 0.01 %w/v edetate disodium, 0.01 %w/v benzalkonium chloride. The pH is adjusted to 7.2 - 7.4. Purified water is added q.s. Gels for topical or transdermal administration of compounds of the subject invention may comprise a mixture of volatile solvents, nonvolatile solvents, and water. The volatile solvent component of the buffered solvent system may preferably include lower (CVCe) alkyl alcohols, lower alkyl glycols and lower glycol polymers. More preferably, the volatile solvent is ethanol. The volatile solvent component is thought to act as a penetration enhancer, while also producing a cooling effect on the skin as it evaporates. The nonvolatile solvent portion of the buffered solvent system is selected from lower alkylene glycols and lower glycol polymers. Preferably, propylene glycol is used. The nonvolatile solvent slows the evaporation of the volatile solvent and reduces the vapor pressure of the buffered solvent system. The amount of this nonvolatile solvent component, as with the volatile solvent, is determined by the pharmaceutical compound or drug being used. When too little of the non-volatile solvent is in the system, the pharmaceutical compound may crystallize due to evaporation of volatile solvent, while an excess will result in a lack of bioavailability due to poor release of drug from solvent mixture.
The buffer component of the buffered solvent system may be selected from any buffer commonly used in the art; preferably, water is used. There are several optional ingredients which can be added to the topical composition. These include, but are not limited to, chelators and gelling agents. Appropriate gelling agents can include, but are not limited to, semisynthetic cellulose derivatives (such as hydroxypropylmethylcellulose) and synthetic polymers, and cosmetic agents.
Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.
Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or nonaqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy base. The base may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap, a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil, wool fat or its derivatives or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or a macrogel. The formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as a sorbitan ester or a polyoxyethylene derivative thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.
Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions and may be prepared by dissolving the active ingredient in a suitable aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active agent.
Formulations for topical administration in the mouth, for example buccally or sublingually, include lozenges comprising the active ingredient in a flavored basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatine and glycerin or sucrose and acacia.
For administration by inhalation the compounds according to the invention are conveniently delivered from an insufflator, nebulizer pressurized packs or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetra-fluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Alternatively, for administration by inhalation or insufflation, the compounds according to the invention may take the form of a dry powder composition, for example a powder mix of the compound and a suitable powder such as lactose or starch. The powder composition may be presented in unit form, in for example, capsules, cartridges, gelatine or blister packs from which the powder may be with the aid of an inhalator or insufflator.
Preferred unit dosage formulations are those containing an effective dose, as herein below recited or an appropriate fraction thereof, of the active ingredient.
It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents. The compounds of the present invention may be administered via any route discussed above at a dose range for adult humans which is generally from 0.01 mg/kg/day to 60 mg/kg/day, more preferably from 0.01 mg/kg/day to 30 mg/kg day, most preferably from 0.01 mg/kg/day to 15 mg/kg/day.
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 treated and the particular mode of administration.
The compounds of the subject invention can be administered in various modes, e.g. orally, topically, or by injection. The precise amount of compound administered to a patient will be the responsibility of the attendant physician. 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, general health, sex, diets, time of administration, route of administration, rate of excretion, drug combination, the precise disorder being treated, and the severity of the indication or condition being treated. Also, the route of administration may vary depending on the condition and its severity.
Utility
The compounds according to formula (I) are for use in the treatment of a mitochondrial disease, a neurodegenerative disease, a neuromuscular disease, psychiatric disorders, metabolic disorders, cancer, or immune dysfunction.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of a mitochondrial disease.
The compounds of the present invention are modulators of mitochondrial function and as such are useful for the treatment of pathological conditions where mitochondrial function is impaired.
Mitochondria are e.g. critical for ocular function as they represent the major source of a cell's supply of energy and play an important role in cell differentiation and survival. Furthermore, the eye is one of the tissues with the highest energy consuption in the body (REF). Ocular mitochondrial dysfunction can occur as a result of inherited mitochondrial syndromes and mutations, such as Leber's hereditary optic neuropathy (LHON), dominant optic atrophy (DOA, mutations in OPA1) dominant optic atrophy with cataracts (mutations in OPA3), vitelliform macular dystrophy (VD), Jensen syndrome, optic atrophy of Leigh and Leigh-like syndrome, maculopathy of neuropathy-ataxia-retinitis-pigmentosa (NARP), retinopathy of mitochondrial- encephalomyopathy-lactic-acidosis-stroke (MELAS), chronic progressive external ophthalmoplegia (CPEO) and optic atrophy caused by mutations in the ND3 subunit of complex 1 (MTND3), which are also associated with migraine and encephalopathy. Furthermore, also in healthy individuals, mitochondrial haplogroups (Jones MM, Manwaring N, Wang JJ, Rochtchina E, Mitchell P, Sue CM. Arch Ophthalmol. 2007 Sep; 125(9): 1235-40) as well as certain mitochondrial gene variants were implicated in an elevated risk of developing ophtalmological pathologies (Kanda A, Chen W, Othman M, Branham KE, Brooks M, Khanna R, He S, Lyons R, Abecasis GR, Swaroop A. Proc Natl Acad Sci U S A. 2007 Oct 9; 104(41): 16227-32.) Thus, mitochondrial dysfunction, whether inherited or aquired is considered one of the main molecular pathologies involved in the initiation of ocular disorders (such as macular degeneration, glaucoma, retinopathy and cataracts) (reviewed by Jarrett SG, Lewin AS, Boulton ME. Ophthalmic Res. 2010;44(3): 179-90; Brennan LA, Kantorow M. Exp Eye Res. 2009 Feb;88(2): 195-203).
Although some macular dystrophies affecting younger individuals are sometimes described as macular degeneration, the term generally refers to age-related macular degeneration (AMD) and is a major cause of visual impairment in older adults (>50 years). Both for the inherited (VD) and acquired forms of macular degeneration, the major risk factors for this disorder are age, smoking, hypertension and diabetes. AMD, like LHON, is a medical condition which results in a loss of vision in the center of the visual field (the macula) because of damage to the retina. Although, changes in mitochondrial structure and function, such as loss of cristae and matrix density are a normal feature of ageing, these aberrations are significantly greater in AMD than in normal aging and also occur at an earlier time point (Feher J, Kovacs I, Artico M, Cavallotti C, Papale A, Balacco Gabrieli C. Neurobiol Aging. 2006 Jul;27(7):983-937), which would suggest that the severity of mitochondrial alterations are different between AMD and normal aging, and that the timing of damage to retinal cells may be critical for the development of AMD. Furthermore, AMD patients show increased mtDNA mutations selectively in the retina compared to blood cells while AMD- related changes in the mitochondrial proteome have also been reported. Consistent with mitochondrial involvement in the pathology of AMD, SanGiovanni et al. (2009) described multiple polymorphisms (SNPs) in mtDNA that were associated with AMD using a cohort of 215 patients with advanced AMD. These associations were driven entirely by the T2 haplogroup, and characterized by two variants in Complex I genes (A11812G of MT-ND4 and A14233G of MT-ND6), reminicent of mutations in Complex I that cause LHON. Consequently, treatment of AMD patients with compounds that alter mitochondrial function (mitotropic) has been attempted (Feher J, Papale A, Mannino G, Gualdi L, Balacco Gabrieli C. Ophthalmologica. 2003 Sep-Oct;217(5):351-7), although the effects so far remained only marginal.
Glaucoma (optic neuropathy) is a disease in which the optic nerve is damaged, leading to progressive, irreversible loss of vision, which is often, but not always, associated with increased pressure of the fluid in the eye. The nerve damage involves loss of retinal ganglion cells in a characteristic pattern. There are many different sub-types of glaucoma but they can all be considered a type of optic neuropathy. Raised intraocular pressure is a significant risk factor for developing glaucoma. Untreated glaucoma leads to permanent damage of the optic nerve and resultant visual field loss, which can progress to blindness. Mitochondrial involvement in the pathology of glaucoma is supported by work from Guo (Guo Y, Johnson EC, Cepurna WO, Dyck J, Doser T, Morrison JC. Invest Ophthalmol Vis Sci. 2010 Nov 4. [Epub ahead of print]), who showed selective downregulation of mitochondrial genes and genes related to energy production in a rat model of glaucoma. Furthermore, similar to results described for macular degeneration, a higher prevalence of mtDNA alterations were also reported for glaucoma patients. Finally, overexpression of the mitochondrial protein OPA1 , mutated in DOA, is protective in a mouse model of glaucoma (Ju WK, Kim KY, Duong-Polk KX, Lindsey JD, Ellisman MH, Weinreb RN. Increased optic atrophy type 1 expression protects retinal ganglion cells in a mouse model of glaucoma. Mol Vis. 2010 Jul 15;16:1331-42), which clearly demonstrates a causal relationship between mitochondrial dysfunction in aquired eye pathologies such as glaucoma. Mitochondrial dysfunction may either be due to nuclear or mitochondrial genes, by mechanical stress, insufficient blood supply, chronic hypoperfusion consequent to the commonly raised intraocular pressure in glaucomatous eyes, or by toxic xenobiotic).
Retinopathy is a general term that refers to some form of non-inflammatory damage to the retina of the eye. Next to the genetic or inherited forms of retinopathy, spontaneous forms can be induced by drugs, toxins and radiation (ionizing and ultra violet). Frequently, retinopathies are ocular manifestations of systemic diseases such as diabetes, hypertension, sickle cell disease or ciliopathy such as Bardet-Biedl syndrome. Similar to macular degeneration and glaucoma, mitochondrial involvement in retinopathy has been well described (reviewed by Jarrett SG, Lewin AS, Boulton ME. Ophthalmic Res. 2010;44(3): 179-90, Lee S, Van Bergen NJ, Kong GY, Chrysostomou V, Waugh HS, O'Neill EC, Crowston JG, Trounce IA. Exp Eye Res. 2010 Aug 4. (Epub ahead of print).
A cataract is a clouding that develops in the crystalline lens of the eye or in its envelope, varying in degree from slight to complete opacity and obstructing the passage of light. The gradual yellowing and opacification of the lens may reduce the perception of blue colours. Cataracts typically progress slowly to cause vision loss and are potentially blinding if untreated. The condition usually affects both the eyes, but similar to the pathology of LHON and VD, one eye is almost always affected earlier than the other. As for the pathologies above, mitochondrial involvement in cataract formation is clearly described. Furthermore, cataract formation is directly associated with some mitochondrial disorders such as dominant optic atrophy with cataracts, autosomal dominant progressive external ophthalmoplegia (PEOA3), PEOA2, mitochondrial myopathy caused by mutations of COX II or various others genes, Sengers syndrome, as well as mutations of mitochondrial genes such as mitochondrial tRNA-Ser (MTTS2), GFER, OPA3 but also large mitochondrial deletions.
Optic disc drusen (ODD) or optic nerve head drusen (ONHD) are globules of mucoproteins and mucopolysaccharides that progressively calcify in the optic disc. They are thought to be the remnants of the axonal transport system of degenerated retinal ganglion cells. ODD have also been referred to as congenitally elevated or anomalous discs, pseudopapilledema, pseudoneuritis, buried disc drusen, and disc hyaline bodies. They are associated with vision loss of varying degree occasionally resulting in blindness. Mitochondrial impairment due to Ca2+ overload has been suggested in the process of drusen formation (Tso MO. Ophthalmology. 1981 Oct;88(10): 1066-80). Furthermore, in the case of age-related maculopathy, higher risk for soft drusen were also found to be associated with mitochondrial haplotype J, whereas haplotype H was associated with significantly lower risk (Jones MM, Manwaring N, Wang JJ, Rochtchina E, Mitchell P, Sue CM. Arch Ophthalmol. 2007 Sep; 125(9): 1235-40).
Thus, Chrysostomou et al. (Chrysostomou V, Trounce IA, Crowston JG. Ophthalmic Res. 2010;44(3): 173-8) suggested that mitochondrial dysfunction, inherited or as a cause or consequence of injury, renders ocular cells (in particular retinal ganglion cells) sensitive to degeneration. Therapeutic approaches that target mitochondria should therefore provide a general means of protecting lens and retinal ganglion cells from degeneration, regardless of the etiology of the disease. A further indication which can be allocated as mitochondrial disease is autosomal dominant optic atrophy (DOA). Pathogenic OPA1 mutations cause autosomal dominant optic atrophy, a condition characterized by the preferential loss of retinal ganglion cells and progressive optic nerve degeneration. Approximately 20% of affected patients will also develop more severe neuromuscular complications, an important disease subgroup known as DOA(+). OPA1 was demonstrated to control both mitochondrial fusion and cristae morphology. In addition, OPA1 loss-of-function studies have shown that OPA1 also regulates apoptosis induction (Liesa M et al. Physiol Rev. 2009; 89:799-845). Although it is expressed in all the tissues assayed, OPA1 shows a specific tissue expression pattern, with the highest expression in the retina, brain, testis, liver, heart, skeletal muscle, and pancreas. Loss of OPA1 causes a marked reduction in mitochondrial membrane potential and a reduction in basal respiration and incapacity to enhance oxygen consumption in the presence of the uncoupler 2,4-dinitrophenol. Human fibroblasts from patients with certain OPA1 mutations (that cause autosomal dominant optic atrophy or ADOA), in addition to decreased rates of mitochondrial fusion, also show impaired ATP synthesis driven by complex I substrates. Due to their ability to enhance ATP synthesis the compounds of the present invention are useful in the treatment of DOA.
The compounds of the present invention are suitable for use in the treatment of ophthalmological mitochondrial diseases such as Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy (DOA), and ophtalmological disorders displaying mitochondrial dysfunction such as macular degeneration, glaucoma, retinopathy, cataract, optic disc drusen (ODD).
The compounds of the present invention are also suitable for use in the treatment of genetic neurodegenerative mitochondrial diseases with an ophthalmological component among the various symptoms such as MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms), MERFF (myoclonic epilepsy with ragged red fibers), MNGIE (myoneurogenic gastrointestinal encephalomyopathy), Kearns-Sayre syndrome, CoQ10 deficiency, or mitochondrial complex deficiencies (i.e. complex I, II, III, IV, V deficiency, and CPEO).
As used herein, the following diseases are designated as a "mitochondrial disease": Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy (DOA), macular degeneration, glaucoma, retinopathy, cataract, optic disc drusen (ODD), mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), myoclonic epilepsy with ragged red fibers (MERRF), myoneurogenic gastrointestinal encephalomyopathy (MNGIE), Kearns-Sayre syndrome, CoQ.10 deficiency, and mitochondrial complex deficiencies (1-5, CPEO).
As mentioned above, the compounds according to formula (I) are modulators of mitochondrial disorders and are thus useful in the treatment of mitochondrial diseases such as Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy (DOA), macular degeneration, glaucoma, retinopathy, cataract, optic disc drusen (ODD), mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), myoclonic epilepsy with ragged red fibers (MERRF), myoneurogenic gastrointestinal encephalomyopathy (MNGIE), Kearns-Sayre syndrome, CoQ10 deficiency, or mitochondrial complex deficiencies.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of a mitochondrial disease selected from Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy (DOA), macular degeneration, glaucoma, retinopathy, cataract, optic disc drusen (ODD), mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), myoclonic epilepsy with ragged red fibers (MERRF), myoneurogenic gastrointestinal encephalomyopathy (MNGIE), Kearns-Sayre syndrome, CoQ10 deficiency, or mitochondrial complex deficiencies (1-5, CPEO).
In another preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of a mitochondrial disease selected from Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy (DOA), macular degeneration, glaucoma, mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), or mitochondrial complex deficiencies (1-5, CPEO).
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of a Leber's hereditary optic neuropathy (LHON).
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of autosomal dominant optic atrophy (DOA). In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of macular degeneration.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of glaucoma.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of retinopathy.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of cataract.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of optic disc drusen (ODD).
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS).
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of myoclonic epilepsy with ragged red fibers (MERRF).
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of myoneurogenic gastrointestinal encephalomyopathy (MNGIE).
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of Keams-Sayre syndrome.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of CoQ10 deficiency. In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of mitochondrial complex deficiencies (1 -5, CPEO).
In another preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of a neurodegenerative disease.
The compounds of formula (I) can further be used to treat neurodegenerative diseases such as FRDA, ALS, Parkinson's disease, Alzheimer's disease, Huntington's disease, Stroke/Reperfusion Injury, or Dementia. Also associated with mitochondrial dysfunction are neuromuscular diseases such as DMD, BMD, LGMD, XLDCM, PKAN, SMA, Kugelberg- Welander disease, or Werdnig-Hoffmann disease which accordingly are also be treatable by using the compounds of formula (I).
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of a neurodegenerative disease selected from Friedreich's ataxia (FRDA), amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, stroke/reperfusion injury, or dementia.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of Friedreich's ataxia (FRDA).
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of amyotrophic lateral sclerosis (ALS).
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of Parkinson's disease.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of Alzheimer's disease.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of Huntington's disease. In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of stroke/reperfusion injury.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of dementia.
In another preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of a neuromuscular disease.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of a neuromuscular disease selected from Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Limb- Girdle muscular dystrophy (LGMD), X-linked dilated cardiomyopathy (XLDCM), Pantothenate kinase-associated neurodegeneration (PKAN), spinal muscular atrophy (SMA), multiple sclerosis and primary progressive multiple sclerosis (PP-MS), Kugelberg-Welander disease, and Werdnig- Hoffmann disease.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of Duchenne muscular dystrophy (DMD).
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of Becker muscular dystrophy (BMD).
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of Limb-Girdle muscular dystrophy (LGMD).
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of X-linked dilated cardiomyopathy (XLDCM). In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of Pantothenate kinase- associated neurodegeneration (PKAN).
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of spinal muscular atrophy (SMA).
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of multiple sclerosis.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of primary progressive multiple sclerosis (PP-MS).
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of Kugelberg-Welander disease.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of Werdnig-Hoffmann disease.
Regarding the treatment of multiple sclerosis (MS), the first connection between mitochondria and multiple sclerosis was established due to the finding of an MS-like disease in a subset of patients with Leber's hereditary optic neuropathy (LHON), a disease caused by mutations in mitochondrial (mt)DNA (Olsen NK, et al. Acta Neurol. Scand. 1995; 91 :326-329). Similar to findings from patients with mitochondrial disease, elevated levels of metabolites from extra- mitochondrial glucose metabolism have been found in the cerebrospinal fluid (CSF) of MS patients and correlated with disease progression (Regenold WT et al. J. Neurol. Sci. 2008; 275:106-112). As extra-mitochondrial glucose metabolism increases with impaired mitochondrial glucose metabolism (Seyama K et al. Acta Neurol. Scand. 1989; 80:561-568), this implicates mitochondrial dysfunction in MS disease progression. Moreover, mitochondria in neuronal cell bodies in non-demyelinated MS grey matter were found to have decreased activity of complexes I and III, and, as neurons partly provide their axon with mitochondria, this might further impair mitochondrial function in chronically demyelinated axons (Dutta, R. et al. Ann. Neurol. 2006; 59:478-489). Thus, the compounds of formula (I) according to the present invention are useful in the treatment of multiple sclerosis.
The vast majority of newly-diagnosed MS patients develop the relapsing-remitting form of the disease (RR-MS), in which periods of neurological worsening are followed by periods of spontaneous remission, at least at the beginning of the disease process. About 10-15% of patients develop primary progressive MS (PP-MS), characterized by progressive accumulation of neurological disability from the disease onset, without any superimposed worsening (i.e. relapses) or improvements (remissions).
Primary progressive MS (PP-MS) patients differ from RR-MS patients in several important characteristics: They tend to be older at the time of disease onset (mean 40 vs. 30 years); males and females tend to be affected equally; clinically there is a high prevalence of cortico-spinal dysfunction characterized by progressive weakness and spasticity; patients have more prominent involvement of the spinal cord and generally lower amount of distinct white matter lesions (i.e. plaques) in the brain and less evidence for brain inflammatory activity and, most importantly, PP-MS patients do not respond to immunomodulatory therapies with proven efficacy in RR-MS. Both new imaging modalities and pathological data suggest that in PP-MS, CNS pathology is more diffuse and occurs to some extent independently of focal lesions.
There are currently no treatments with proven therapeutic efficacy for PP-MS. Surprisingly, it has been found that the compounds of formula (I) which are effective as modulators of mitochondrial diseases are able to alleviate the symptoms of PP-MS.
In another preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of psychiatric disorders.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of a psychiatric disorder selected from schizophrenia, major depressive disorder, bipolar disorder, or epilepsy.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of schizophrenia. In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of major depressive disorder.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of bipolar disorder.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of epilepsy.
Altered mitochondrial activity is evident in multiple psychiatric diseases such as schizophrenia, major depressive disorder, bipolar disorder, epilepsy [reviewed by Rezin GT, Amboni G, Zugno Al, Quevedo J, Streck EL Neurochem Res. 2009 Jun;34(6):1021-9 and Shao L, Martin MV, Watson SJ, Schatzberg A, Akil H, Myers RM, Jones EG, Bunney WE, Vawter MP Ann Med. 2008;40(4):281-95].
For example, several studies have reported increased levels of oxidative damage [reviewed by Yao JK, Reddy RD, van Kammen DP CNS Drugs. 2001 ;15(4):287-310] and impaired complex 1 activity in patients with schizophrenia in distinct areas of the brain [Ben-Shachar D. J Neural Transm. 2009; 116(11): 1383-96]. In addition to deficiencies in respiratory chain activities, patients with autism also show metabolic markers indicative of altered mitochondrial respiration [Chugani DC, Sundram BS, Behen M, Lee ML, Moore GJ Prog Neuropsychopharmacol Biol Psychiatry. 1999;23(4):635-41]. Furthermore mitochondrial dysfunction has also been intimately linked to epilepsy [reviewed by Waldbaum S, Patel M Epilepsy Res. 2009 Oct 20 and Kudin AP, Zsurka G, Elger CE, Kunz WS Exp Neurol. 2009 Aug;218(2):326-32]. Therefore, the compounds of formula (I) are useful in the treatment of schizophrenia, major depressive disorder, bipolar disorder and epilepsy.
In another preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of metabolic disorders.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of a metabolic disorder selected from ageing-related physical decline, obesity, overweight, type II diabetes, or metabolic syndrome. In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of ageing-related physical decline.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of obesity.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of overweight.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of type II diabetes.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of metabolic syndrome.
In type II diabetes a clear connection between excess production of oxygen radicals, mitochondrial dysfunction and disease progression has been established [reviewed by Friederich M, Hansell P, Palm F Curr Diabetes Rev. 2009 May;5(2): 120-44], while in a wider sense most pathologies of metabolic syndrome such as heart failure can also be attributed to mitochondrial dysregulation [reviewed by Bugger H, Abel ED. Clin Sci (Lond). 2008 Feb;114(3):195-210].
Under conditions where cellular energy production is reduced, such as during ageing-related decline of mitochondrial function or during strenuous activity, it is beneficial to raise the production of ATP to counteract anticipated cell loss or toxicity from energy deprivation. Contrary to that, under special conditions, such as obesity, it can also be beneficial to restrain the mitochondrial capacity for ATP production to aide therapeutic weight loss efforts.
The compounds of the present invention show a beneficial effect in diabetes type II, obesity and metabolic syndrome.
In another preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of cancer. White modulators of mitochondrial function have been suggested as anti-cancer agents mainly via an apoptosis-inducing function, recent data also links the metastatic potential of tumours directly to mitochondrial DNA and mitochondrial radical production [reviewed by Ishikawa K, Koshikawa N, Takenaga K, Nakada K, Hayashi J Mitochondrion. 2008; 8(4): 339-44]. Therefore, modulating mitochondria in this context could not only be employed as anti-cancer strategy but also to prevent metastasis of malignancies which makes the compounds according to formula (I) useful as cancer therapeutics.
In another preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of immune dysfunction.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of an immune dysfunction selected from arthritis, psoriasis or rheumatoid arthritis.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of arthritis.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of psoriasis.
In a preferred embodiment in combination with any of the above or below embodiments, the compounds according to formula (I) are for use in the treatment of rheumatoid arthritis.
Altered mitochondrial metabolism is also linked to multiple dysfunctions of the immune system. In patients with systemic juvenile idiopathic arthritis for example, reduced expression of mitochondrial respiratory chain genes was observed [Ishikawa S, Mima T, Aoki C, Yoshio- Hoshino N, Adachi Y, Imagawa T, Mori M, Tomiita M, Iwata N, Murata T, Miyoshi M, Takei S, Aihara Y, Yokota S, Matsubara K, Nishimoto N Ann Rheum Dis. 2009;68(2):264-72]. Oxidative stress and mitochondrial pathology has been reported to be associated with sepsis [Victor VM, Espulgues JV, Hernandez-Mijares A, Rocha M Infect Disord Drug Targets. 2009;9(4):376-89]. Furthermore, targeting mitochondrial function has been suggested as treatment strategy for several autoimmune disorders such as arthritis and psoriasis [J.J. Bednarski, R.E. Warner, T. Rao, F. Leonetti, R. Yung, B.C. Richardson, K.J. Johnson, J.A. Ellman, A.W. Opipari Jr., and G.D. Glick Arthritis & Rheumatism, 2003;48, 757] and could be applied to modulate multiple immunological endpoints such as microbial infection [reviewed by Arnoult D, Carneiro L, Tattoli I, Girardin SE Semin Immunol. 2009;21 (4):223-32] and allergy [Chodaczek G, Bacsi A, Dharajiya N, Sur S, Hazra TK, Boldogh I. Mol Immunol. 2009;46(13):2505-14]. Thus, the compounds of formula (I) are also useful in the treatment of arthritis, psoriasis, sepsis, allergy and microbial infections.
Preparation of Compounds of the Invention:
In the schemes, preparations and examples below, various reagent symbols and abbreviations have the following meanings
AcOH acetic acid
Arg arginine
Boc tert-butoxycarbonyl
d day(s)
Dap 2,3-diaminopropionic aid
DCM dichloromethane
DEAD diethylazodicarboxylate
DIC diisopropylcarbodiimide
DMAP 4-(dimethylamino)-pyridine
DMF Ν,Ν-dimethylformamide
DMSO dimethylsulfoxide
EDC A/-ethyl-/V-(3-dimethylaminopropyl)carbodiimide
EtOAc ethyl acetate
Fmoc fluorenylmethoxycarbonyl
h hour(s)
HATU 0-(7-azabenzotriazol-1 -yl)-A/,A/,A/',A/'-tetramethyluronium hexafluorophosphate
HCI hydrochloric acid
HOBt 1 -hydroxybenzotriazole
MeOH methanol
MW molecular weight
NaOH sodium hydroxide
Phe phenylalanine
Pmc 2,2,5,7,8-pentamethyl-chromane-6-sulfonyl
PPh3 triphenylphosphine
RT room temperature Tcp trichloride-polystyrol
TFA trifluoroacetic acid
THF tetrahydrofurane
tR (min) HPLC retention time
Reaction Scheme 1 :
Mitsunobu reaction
As shown in Reaction Scheme 1 , idebenone can be reacted with optionally substituted alcohols in the presence of two reagents such as PPh3 and DEAD in an appropriate solvent such as THF to form the corresponding ether.
R in Reaction Scheme 1 is defined based on the corresponding moiety in the compounds of Examples 1 to 24.
Reaction Scheme 2:
Reductive amination
As shown in Reaction Scheme 2, idebenone can be reacted with a reagent such as Dess Martin reagent in an appropriate solvent such as DCM. The corresponding aldehyde can be reductively aminated. Reaction with an optionally substituted amine in the presence of a reagent like sodium cyano borohydride in appropriate solvents such as MeOH and AcOH yields the target compounds. R1 and R2 in Reaction Scheme 2 are defined like the corresponding moieties in the compounds of Examples 25 to 34.
Reaction Scheme 3:
Grignard reaction
As shown in Reaction Scheme 3, a benzoquinone aldehyde can be reduced with a Grignard reagent in a solvent such as THF to give the corresponding substituted benzoquinone alcohol. Alternatively, a hydroquinone methyl ester may be subjected to a Grignard reaction to yield the corresponding alcohol.
R in Reaction Scheme 3 is defined as in the corresponding moieties of Examples 35 and 36.
Reaction Scheme 4:
Kulinkovich reaction
A benzoquinone methyl ester is subjected to a Kulinkovich reaction as shown in Reaction Scheme 4. The benzoquinone methyl ester is reacted with Grignard reagent Et-MgBr in the presence of Ti(OiPr)4 in a solvent such as THF to yield the desired compound.
Reaction Scheme 5:
Oxidation
As shown in Reaction Scheme 5, a hydroquinone may be oxidised with a salt such as NO(S03K)2 in an aqueous solvent such as acetone and in the presence of a base such as KOH. Reaction Scheme 5 is defined applicable to the preparation of all compounds wherein the oxidation of a hydroquinone is required. Thus, R in Reaction Scheme 5 defines each possible moiety attached to the quinone as defined within the claims.
In order to facilitate legibility of Reaction Schemes 1 to 5, the alkyl chain attached to the quinone moiety is shown with a fixed length. However, the general procedures described in Reaction Schemes 1 to 5 are also applicable to a variable number of carbon atoms in the chain as disclosed within the application.
Analytical LC-MS
The compounds of the present invention were analyzed by analytical LC-MS. The conditions are summarized below.
Analytical conditions summary:
LC10Advp-Pump (Shimadzu) with SPD-M10Avp (Shimadzu) UV/Vis diode array detector and QP2010 MS-detector (Shimadzu) in ESI+ modus with UV-detection at 214, 254 and 275 nm,
Column: Waters XTerra MS C18, 3.5 μιη, 2.1 * 100 mm,
linear gradient with acetonitrile in water (0.15% HCOOH)
Flow rate of 0,4 ml/min;
Mobile Phase A: water (0.15% HCOOH)
Mobile Phase B: acetonitrile (0.15% HCOOH)
Methods are: A:
Polar QC linear gradient;
start concentration 1% acetonitrile
9.00 B.Conc 30
10.00 B.Curve 3
12.00 B.Conc 99
15.00 B.Conc 99
15.20 B.Conc 1
18.00 Pump STOP
B:
Medium QC linear qradient;
start concentration 10% acetonitrile
10.00 B.Conc 60
11.00 B.Curve 2
12.00 B.Conc 99
15.00 B.Conc 99
15.20 B.Conc 10
18.00 Pump STOP
C:
UnDolar QC linear qradient;
start concentration 15% acetonitrile
12.00 B.Conc 99
15.00 B.Conc 99
15.20 B.Conc 15
18.00 STOP 0
B. Curve Set the gradient curve of B (C, D) liquid in the gradient mode. The curve at
C. Curve 0<=t<=T can be calculated by the following equation, where c(t) is the
D. Curve concentration of the liquid B (C, D) at the relative time t in each interval, T is the gradient time, and a is the value for [B(C,D). Curve].
1. a>0 C(t)=C1+(C2-C1)*{ (exp(a*t/T)-1) / (exp(a)-1
2. a=0(line)
C(t)=C1+(C2-C1)*(t/T)
3. a<0
C(t)=C1+(C2-C1)*{ (exp{|a| (1-t T) }-1) / (exp|a|-1) }
C1 :lnitial value of [B(C,D).Conc]
C2:Final value of [B(C,D).Conc](To be set by LC Program) -10 - 10
D:
5 min. linear gradient; linear gradient from 5% to 95% acetonitrile in water (0.1% HCOOH)
0.00 min 5% B
5.00 min 95 % B
5.10 min 99 % B
6.40 min 99 % B
6.50 min 5 % B
8.00 min Pump STOP
The following tables describe detailed examples of the invention which can be prepared according to the Reaction schemes 1 to 5. These examples are, however, not construed to limit the scope of the invention in any manner.
Table 1 :
Table 2:
HPLC MS
No. salt R tR method MW [M+H]+
(min) (calc.) (found) free
base Table 3:
Table 4:
HPLC MS
No. R1 R2 tR (min) method MW [M+H]+
(calc.) (found) free base 37 H Me 7.82 C 352.46 353
38 Me Me 8.19 C 366.49 367
39 H F 6.89 C 356.43 357
Table 6:
Table 7:
HPLC MS
. R1 R2 tR method MW [M+H]+
(min) (calc.) (found) free
base 3.14 B 310.39 31 1
HO 5.08 B 295.37 296
HO 6.08 A 225.24 226
nd: not determined
General procedure (I):
Mitsunobu reaction
Under argon atmosphere, Idebenone (1 eq), PPh3 (1 eq) and an alcohol (1 eq) were dissolved in dry THF and a 40% solution of DEAD (1 eq) in toluene was added. The reaction was monitored either by LCMS and/or TLC. After the reaction was finished, the solvent was removed by evaporation and the residue was purified first with flash-chromatography (AcOEt/cyclohexane) and then with preparative HPLC-MS.
Examples 1 to 24 which were synthesized according to general procedure (I) are:
2,3-Dimethoxy-5-methyl-6-[10-(pyridazin-3-yloxy)-decyl]-[1 ,4]benzoquinone (1 )
2-[10-(3-Chloro-pyridin-4-yloxy)-decyl]-5,6-dimethoxy-3-methyl-[1 ,4]benzoquinone (2)
2,3-Dimethoxy-5-methyl-6-[10-(6-methyl-pyrimidin-4-yloxy)-decyl]-[1 ,4]benzoquinone (3) 2-[10-(2,6-Dimethyl-pyrimidin-4-yloxy)-decyl]-5,6-dimethoxy-3-methyl-[1 ,4]benzoquinone (4) 2-[10-(5-Chloro-pyridin-3-yloxy)-decyl]-5,6-dimethoxy-3-methyl-[1 ,4]benzoquinone (5)
2-[10-(2-Chloro-5-fluoro-pyridin-3-yloxy)-decyl]-5,6-dimethoxy-3-methyl-[1 ,4]benzoquinone (6) 2-[10-(2-Bromo-pyridin-3-yloxy)-decyl]-5,6-dimethoxy-3-methyl-[1 ,4]benzoquinone (7)
2-[10-(Benzo[1 ,3]dioxol-5-yloxy)-decyl]-5,6-dimethoxy-3-methyl-[1 ,4]benzoquinone (8)
2-[10-(6-Chloro-5-fluoro-pyridin-3-yloxy)-decyl]-5,6-dimethoxy-3-methyl-[1 ,4]benzoquinone (9) 2-[10-(4,5-Dimethoxy-2-methyl-3,6-dioxo-cyclohexa-1 ,4-dienyl)-decyloxy]-benzamide (10) 2-[10-(3-Chloro-4-f luoro-phenoxy)-decyl]-5,6-dimethoxy-3-methyl-[1 ,4]benzoquinone (11) N-{4-[10-(4,5-Dimethoxy-2-methy!-3,6-dioxo-cyclohexa-1 ,4-dienyl)-decyloxy]-phenyl}-acetam (12)
2-[10-(2,6-Dimethyl-pyridin-4-yloxy)-decyl]-5,6-dimethoxy-3-methyl-[1 ,4]benzoquinone (13) 2,3-Dimethoxy-5-methyl-6-[10-(2-methyl-pyridin-4-yloxy)-decyl]-[1 ,4]benzoquinone (14)
2-[10-(2-Chloro-pyridin-3-yloxy)-decyl]-5,6-dimethoxy-3-methyl-[1 ,4]benzoquinone (15)
2,3-Dimethoxy-5-methyl-6-[10-(pyridin-4-yloxy)-decyl]-[1 ,4]benzoquinone (16)
2,3-Dimethoxy-5-methyl-6-[10-(pyridin-2-yloxy)-decyl]-[1 ,4]benzoquinone (17)
2,3-Dimethoxy-5-methyl-6-[10-(pyridin-3-yloxy)-decyl]-[1 ,4]benzoquinone (18)
2-[10-(4-lmidazol-1-yl-phenoxy)-decyl]-5,6-dimethoxy-3-methyl-[1 ,4]benzoquinone (19)
2,3-Dimethoxy-5-methyl-6-[10-(4-[1 >2,4]triazol-1-yl-phenoxy)-decyl]-[1 ,4]benzoquinone (20) 2-[10-(2-Chloro-pyridin-4-yloxy)-decyl]-5,6-dimethoxy-3-methyl-[1 ,4]benzoquinone (21 )
2-[10-(4-Chloro-phenoxy)-decyl]-5,6-dimethoxy-3-methyl-[1 ,4]benzoquinone (22)
2,3-Dimethoxy-5-methyl-6-[10-(3-trif luoromethyl-phenoxy)-decyl]-[1 ,4]benzoquinone (23)
2,3-Dimethoxy-5-methyl-6-[10-(2-trifluoromethyl-pyrimidin-4-yloxy)-decy^ (24)
General procedure (II):
Reductive amination
Under argon atmosphere, 10-(4,5-dimethoxy-2-methyl-3,6-dioxo-cyclohexa-1 ,4-dienyl)-decanal (intermediate 35a, 1 eq) was dissolved in dry MeOH. An amine (3.0 eq), AcOH and NaBH3CN (1.5 eq) were added. The reaction mixture was stirred for 1 h and was monitored by LCMS. The solvents were removed by evaporation. The residue was dissolved in AcOEt and washed with water and brine, dried over sodium sulfate, filtered and evaporated. The obtained residue was dissolved in toluene and Ag2C03 (0.5 eq) was added. The reaction was monitored by LCMS. The reaction mixture was filtered over Celite and the solvent was removed by evaporation. The crude product was purified with preparative HPLC-MS. Examples 25 to 33 which were synthesized according to general procedure (II) are: 2,3-Dimethoxy-5-methyl-6-[10-(2H-tetrazol-5-ylamino)-decyl]-[1 ,4]benzoquinone (25)
2,3-Dimethoxy-5-methyl-6-[10-(methyl-pyridin-2-yl-amino)-decyl]-[1 ,4]benzoquinone (26)
2-[10-(3-Hydroxy-adamantan-1 -ylamino)-decyl]-5,6-dimethoxy-3-methyl-[1 ,4]benzoquinone (27) 2,3-Dimethoxy-5-methyl-6-[10-(pyridin-2-ylamino)-decyl]-[1 ,4]benzoquinone (28)
2,3-Dimethoxy-5-methyl-6-[10-(pyridin-3-ylamino)-decyl]-[1 ,4]benzoquinone (29)
2,3-Dimethoxy-5-methyl-6-(10-pyrrolidin-1 -yl-decyl)-[1 ,4]benzoquinone (30)
2,3-Dimethoxy-5-methyl-6-(10-phenylamino-decyl)-[1 ,4]benzoquinone (31 )
2,3-Dimethoxy-5-methyl-6-[10-(2,2,2-trif luoro-ethylamino)-decyl]-[1 ,4]benzoquinone (32)
2-[10-(4-Fluoro-piperidin-1 -yl)-decyl]-5,6-dimethoxy-3-methyl-[1 ,4]benzoquinone (33)
2,3-Dimethoxy-5-methyl-6-[10-(4-methyl-piperazin-1 -yl)-decyl]-[1 ,4]benzoquinone (34)
Synthesis of Example 35:
Intermediate 35a):
Under argon atmosphere, idebenone (4 g) was dissolved in dry dichloromethane (100 ml) and the solution was cooled to 0°C. Dess Martin reagent (6 g) was added and the reaction mixture was stirred at 0°C for 4h. The solution was diluted with dichloromethane (100 ml) and washed with a 1 M aqueous sodium thiosulfate solution (100 ml), saturated sodium bicarbonate solution (100 ml), water (100 ml) and brine (100 ml). The organic layer was dried over sodium sulfate, filtrated, and the solvent was removed under reduced pressure. The crude product was purified by flash-chromatography (AcOEt-cyclohexane).
Example 35:
Under argon atmosphere, intermediate 35a (200 mg) was dissolved in dry THF (6 ml) and the solution was cooled to -10°C. A 2M solution of ethinyl magnesium bromide in THF (0.35 ml) was added dropwise and the reaction mixture was stirred for 15 min. Then the solution was hydrolyzed at 0°C with a saturated ammonium chloride solution (25 ml) and water (5 ml). The solution was diluted with ethyl acetate (30 ml) and after phase separation, the aqueous layer was extracted with ethyl acetate (20 ml). The combined organic layer was washed with brine (2x20 ml), dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure. The crude product was purified first by flash-chromatography (AcOEt-cyclohexane) and then with preparative HPLC-MS.
Synthesis of Example 36:
Intermediate 36a):
Under argon atmosphere, methyl sebacoyl chloride (7.4 g) and 3,4,5-trimethoxytoluene (5.0) were dissolved in nitrobenzene (40 ml) and cooled to 0°C. Then aluminium chloride (8.9 g) was added portionwise. The reaction mixture was stirred 1 h at 0°C for 16h at room temperature. The aluminum chloride was carefully hydrolyzed with 60 ml of a 1 M aqueous hydrochloric acid solution to reach pH 1-2. The solution was stirred for 20 min, and after phase separation, the aqueous layer was extracted with diethyl ether (3x 100 ml). The combined organic layer was dried over sodium sulfate, filtrated and volatiles were removed under reduced pressure. The product was purified by flash chromatography (AcOEt-cyclohexane).
Intermediate 36b):
In a round bottomed flask, zinc (6.1 g), mercury chloride (0.6 g), water (11 ml) and cone, hydrochloric acid (0.32 ml) were strirred for 5 min and the solution was decanted. The zinc was washed with water (3x 10 ml) and transferred into a new clean round bottomed flask. To this amalgamated zinc was added water (1.5 ml), cone, hydrochloric acid (10 ml), toluene (10 ml) and intermediate 36a (3.1 g). The reaction mixture was stirred vigorously at reflux for 72h. The solution was decanted from the un-reacted zinc and was extracted with ether (2x 100 ml). The combined organic extract was washed with 0.2M aqueous hydrochloric acid solution (100 ml), dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure. The crude product was purified by flash chromatography (AcOEt-cyclohexane.
Intermediate 36c):
Under argon atmosphere, titanium (IV) isopropoxide (1.6 g) was added drop wise at -30°C to a 3M ethyl magnesium bromide solution in ether (4.6 ml) and THF (2 ml). Intermediate 36b (0.84 g) dissolved in THF (2.5 ml) was added dropwise at -30°C and the reaction mixture was stirred for 15 min, and was monitored by LCMS. Titanium (IV) isopropoxide (1.6 g), a 3M ethyl magnesium bromide solution in ether (4.6 ml) and THF (1 ml) were added and the reaction mixture was stirred for 15 min. at -30°C. The solution was hydrolyzed at 0°C with a saturated ammonium chloride solution and extracted with ethyl acetate (20 ml). After phase separation, the aqueous layer was extracted again with ethyl acetate (2 x 20 ml). The combined organic layer was washed with brine (25 ml), dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure. The crude product was purified by flash chromatography (AcOEt- cyclohexane).
Example 36:
Intermediate 36c (50 mg) was dissolved in a mixture of acetone (1 ml) and water (0.75 ml). Potassium nitrosodisulfonate (691 mg) and a 1% aqueous solution of potassium hydroxyde was added to reach a pH value of 8. The reaction mixture was stirred for 5h at room temperature and potassium nitrosodisulfonate (432 mg) was added. The pH was adjusted to 8 by adding a 1% aqueous solution of potassium hydroxyde and acetone (1.5 ml) was added. The reaction mixture was stirred for 3h at room temperature and potassium nitrosodisulfonate (432 mg) was added. The pH was adjusted to 8 by adding a 1% aqueous solution of potassium hydroxyde and acetone (1.5 ml) was added. The reaction mixture was monitored by LCMS. The reaction mixture was stirred for 14h at room temperature to obtain a complete conversion. A 1 M aqueous hydrochloride solution (10 ml) was added and the aqueous layer was extracted with ethyl acetate (3x10ml). The combined organic layer was dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure. The crude product was purified with preparative HPLC-MS.
Synthesis of Example 37:
Intermediate 37a):
Intermediate 36b (5 g) was dissolved in a mixture of acetone (100 ml) and water (75 ml). Potassium nitrosodisulfonate (25 g) and a 1% aqueous solution of potassium hydroxyde was added to reach a pH value of 8. The reaction mixture was stirred for 16h at room temperature and potassium nitrosodisulfonate (4.7 g) was added. The pH was adjusted to 8 by adding a 1% aqueous solution of potassium hydroxyde. The reaction mixture was stirred for 10h at room temperature and potassium nitrosodisulfonate (950 mg) was added. The pH was adjusted to 8 by adding a 1% aqueous solution of potassium hydroxyde. The reaction mixture was monitored by LCMS. The reaction mixture was stirred for 14h at room temperature to obtain a complete conversion. A 0.5M aqueous hydrochloride solution (300 ml) was added and the aqueous layer was extracted with ethyl acetate (4x200ml). The combined organic layer was dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure. The crude product was purified by flash-chromatography (AcOEt-cyclohexane).
Intermediate 37b):
Under argon atmosphere, intermediate 37a (6 g) was dissolved in dry THF (300 ml) and the solution was cooled to -60°C. A 2M solution of LDA in THF (31 ml) and iodomethane (6 ml) were added and the reaction mixture was stirred for 1 h. The reaction mixture was poured onto a saturated solution of ammonium chloride (300 ml) at 0°C. After phase separation, the aqueous layer was extracted with ethyl acetate (3x200 ml). The combined organic layer was washed with brine (200 ml), dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure. The crude product was purified by flash-chromatography (AcOEt-cyclohexane).
Example 37:
Under argon atmosphere, intermediate 37b (201 mg) was dissolved in dry THF (5 ml) and lithium aluminium hydride (40 mg) was added at 0°C. The reaction mixture was stirred at this temperature for 15 min. and for 1 h at room temperature. The solution was diluted with ethyl acetate (30 ml) and washed with a saturated ammonium chloride solution (30 ml). The aqueous layer was extracted with ethyl acetate (25 ml). The combined organic layer was washed with brine (30 ml), dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure. The crude product was dissolved in toluene and silver carbonate (64 mg) was added. The reaction mixture was stirred for 2h and filtrated over Celite. The solvent was removed under reduced pressure and the crude product was purified with preparative HPLC-MS.
Synthesis of Example 38:
Intermediate 38a):
Under argon atmosphere, intermediate 37b (1 g) was dissolved in dry THF (60 ml) and the solution was cooled to -60°C. A 2M solution of LDA in THF (5.5 ml) and iodomethane (1 ml) were added and the reaction mixture was stirred for 1h. The reaction mixture was poured onto a saturated ammonium chloride solution (50 ml) at 0°C. After phase separation, the aqueous layer was extracted with ethyl acetate (3x50 ml). The combined organic layer was washed with brine (100 ml), dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure. The crude product was purified by flash-chromatography (AcOEt-cyclohexane).
Example 38:
Under argon atmosphere, intermediate 38a (103 mg) was dissolved in dry THF (4 ml) and lithium aluminium hydride (15 mg) was added at 0°C. The reaction mixture was stirred at this temperature for 15 min. The solution was diluted with ethyl acetate (15 ml) and washed with a saturated solution of ammonium chloride (15 ml). The aqueous layer was extracted with ethyl acetate (15 ml). The combined organic layer was washed with brine (15 ml), dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure. The crude product was dissolved in toluene (3 ml) and silver carbonate (36 mg) was added. The reaction mixture was stirred for 2h and filtrated over Celite. The solvent was removed under reduced pressure and the crude product was purified with preparative HPLC-MS.
Synthesis of Example 39:
Intermediate 39a):
Under argon atmosphere, intermediate 37a (600 mg) was dissolved in dry THF (10 ml) and the solution was cooled to -78°C. A 2M solution of LDA in THF (3.3 ml) was added and the reaction mixture was stirred for 15 min. Then the solution was warmed up to -10°C and stirred for 30 min. The reaction mixture was again cooled to -78°C and N-fluorodibenzenesulfonimide (2.0 g) was added. The reaction mixture was stirred for 10 min. at -78°C for 14h at room temperature. The solution was diluted with ethyl acetate (120 ml) and washed with a saturated solution of ammonium chloride (100 ml). The aqueous layer was extracted with ethyl acetate (2x100 ml). The combined organic layer was washed with brine (100 ml), dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure. The crude product was purified by flash- ch romatog raphy ( AcO Et-cyclohexane) .
Example 39:
Under argon atmosphere, intermediate 39a (86 mg) was dissolved in dry THF (3.5 ml) and lithium aluminium hydride (25 mg) was added at 0°C. The reaction mixture was stirred at this temperature for 15 min. and 1 h at room temperature. The solution was diluted with ethyl acetate (15 ml) and washed with a saturated ammonium chloride solution (10 ml). The aqueous layer was extracted with ethyl acetate (15 ml). The combined organic layer was washed with brine (20 ml), dried over sodium sulfate, filtrated, and volatiles were removed under reduced pressure. The crude product was purified with preparative HPLC-MS.
Example 41 :
Intermediate 35a (100 mg) was dissolved in EtOH (1 ml) and 1,2-phenylendiamine (64 mg) was added. The reaction mixture was stirred at room temperature overnight. The resulting mixture was evaporated and the crude product purified by preparative HPLC.
Example 45:
Intermediate 45a):
Jones reagent was prepared by dissolving 670 mg of Cr03 in 1.25 ml dist. water. To this solution was added 0.58 ml of cone. H2S04 while cooling in an ice bath. After 5 min, the precipitating salts were brought into solution by dropwise addition of 0.15 ml of water.
Idebenone (508 mg) was dissolved in 20 ml of acetone and cooled to 0 °C in an ice bath. The Jones reagent was added dropwise and the mixture was stirred for 20 min at 0 °C. The reaction mixture was diluted with 200 ml of water and the suspension was extracted with CH2CI2 (3 x). The combined organic phase was extracted with 0.05 n HCI (2x), dried over MgS04, filtered and evaporated to yield a red oil. This was purified by column chromatography with hexane/EtOAc 5:1. The resulting red oil was dissolved in 3 ml of diethyl ether and precipitated by cooling the solution to -78 °C and addition of 3 ml of hexane. The precipitate was collected by filtration and dried in vacuo.
Example 45:
Intermediate 45a (30 mg) was dissolved in a mixture of dichloromethane (2 ml) and DMF (1 ml). Pyrrolidine (11 mg) was added followed by HATU (40 mg). The reaction mixture was stirred at room temperature overnight. The resulting mixture was evaporated and the crude product purified by preparative HPLC.
Examples 48 and 47:
2,5-dimethoxy-1 ,4-benzoquinone (500 mg) and piperidine-4-carboxylic acid (2-hydroxy-ethyl)- amide (170 mg) were suspended in a mixture of ethanol (2 ml) and chloroform (2 ml). The reaction mixture was heated in the microwave at 150°C for 400s and filtrated. The solvent was removed under reduced pressure and the residue was purified by flash chromatography. The mixture of compounds 48 and 47 was separated with preparative HPLC-MS.
Example 49:
2,5-dimethoxy-1 ,4-benzoquinone (500 mg) and decylamine (170 mg) were suspended in a mixture of methanol (2 ml) and chloroform (2 ml). The reaction mixture was heated in the microwave at 150°C for 400s and filtrated. The solvent was removed under reduced pressure and the crude product was purified with preparative HPLC-MS.
Example 50:
2,5-dimethoxy-1 ,4-benzoquinone (500 mg) and undecylamine (170 mg) were suspended in a mixture of methanol (2 ml) and chloroform (2 ml). The recation mixture was heated in the microwave at 150°C for 400s and filtrated. The solvent was removed under reduced pressure and the crude product was purified with preparative HPLC-MS. Examples 52 and 5
2,5-Dimethoxy-1 ,4-benzoquinone (500 mg) and 2-[(piperidin-4-ylmethyl)-amino]-ethanol (128 mg) were suspended in a mixture of ethanol (2 ml) and chloroform (2 ml). The recation mixture was heated in the microwave at 150°C for 400s and filtrated. The solvent was removed under reduced pressure and the residue was purified by flash chromatography. The mixture of compounds 52 and 51 was separated with preparative HPLC-MS.
Example 53:
2,5-Dimethoxy-1 ,4-benzoquinone (500 mg) and nonylamine (170 mg) were suspended in a mixture of methanol (2 ml) and chloroform (2 ml). The recation mixture was heated in the microwave at 150°C for 400s and filtrated. The solvent was removed under reduced pressure and the crude product was purified with preparative HPLC-MS.
Example 54:
2,5-Dimethoxy-1 ,4-benzoquinone (500 mg) and 10-amino-decan-1 -ol (170 mg) were suspended in a mixture of methanol (2 ml) and chloroform (2 ml). The reaction mixture was heated in the microwave at 150°C for 400s and filtrated. The solvent was removed under reduced pressure and the crude product was purified with preparative HPLC-MS.
Example 56:
2,5-dimethoxy-1 ,4-benzoquinone (100 mg) was dissolved in ethanol (3 ml). Potassium tert- butoxide (202 mg) and 5-amino-pentan-1-ol (0.2 ml) were added. The reaction mixture was stirred for 30 min. at room temperature, diluted with a 2M hydrochloride solution and extracted with DCM (2x20 ml). The combined organic extract was evaporated to give the crude product, which was purified with preparative HPLC-MS.
Example 57:
Intermediate 58a (100 mg) was dissolved in ethanol (6 ml). Potassium tert-butoxide (130 mg) and 5-amino-nonan-1 -ol (207 mg) were added. The reaction mixture was stirred for 30 min. at room temperature, diluted with a 2M hydrochloride solution and extracted with DCM (2x20 ml). The combined organic extract was evaporated to give the crude product, which was purified with preparative HPLC-MS.
Example 58:
Intermediate 58a):
2,5-dimethoxy-1 ,4-benzoquinone (1 g) was dissolved in a mixture of THF (30 ml) and methanol (30 ml). The reaction mixture was stirred for 2h at room temperature, diluted with a 2M hydrochloride solution and extracted with DCM (3x100 ml). The combined organic extract was dried over sodium sulfate and evaporated to give the crude product, which was used in the next step without any further purification.
Example 58:
Intermediate 58a (100 mg) was dissolved in ethanol (3 ml). Potassium terf-butoxide (67 mg) and 5-amino-pentan-1-ol (66 μΙ) were added. The reaction mixture was stirred for 2h at room temperature, diluted with a 2M hydrochloride solution and extracted with DCM (2x20 ml). The combined organic extract was evaporated to give the crude product, which was purified with preparative HPLC-MS.
Example 59:
Intermediate 59a):
To a stirring solution of Fmoc-Dap-OH (2500 mg, 7.66 mmol) and sodium carbonate (812 mg, 7.66 mmol) in acetonitrile/water 1 :1 (48 ml), isatoic anhydride (1499 mg, 9.19 mmol) in acetonitrile (24 ml) was added and the mixture was stirred at room temperature overnight. The mixture was acidified to pH 2 with 5% aqueous KHS04 and extracted three times with ethyl acetate. The combined organic layer was washed with brine, dried over sodium sulfate and the solvent was removed under reduced pressure.
Intermediate 59b):
Crude product from intermediate 59a (max. 7.66 mmol) was dissolved in water/dioxane (50 ml) and sodium bicarbonate (1094 mg, 13.02 mmol). Di-tert-butyl dicarbonate (2842 mg, 13.02 mmol) was added and the reaction mixture stirred overnight. The reaction mixture was diluted with water (150 ml) and was brought to acidic pH with 1 M aqueous HCI. The aqueous phase was extracted three times with ethyl acetate. The combined organic layer was washed with brine, dried over sodium sulfate and the solvent was removed under reduced pressure. The crude product was purified by preparative LC-MS.
Intermediate 59c):
Fmoc-Rink amide resin 200-400 mesh (PepChem, PC-01-0501) (273 mg, 0.21 mmol) was treated with a solution of 20% piperidine in DMF (3 ml) for 30 min. Excess of the reagent was removed by filtration. The resin was successively washed with DMF (3 x 3 ml), MeOH (3 x 3 ml) and DMF (3 x 3 ml) and treated with a solution of product from intermediate 59b (224 mg, 0.41 mmol) activated with DIC (65 μΙ, 0.42 mmol) and HOBt monohydrate (64 mg, 0.42 mmol) in DMF (2.5 ml) overnight. The resin was washed with DMF (3 x 3 ml). DMF (2.5 ml) was added followed by acetic anhydride (99 μΙ, 1.05 mmol). The mixture was reacted for 1 h. Excess of the reagent was removed by filtration. The resin-bound intermediate was successively washed with DMF (3 x 3 ml), MeOH (3x3 ml), THF (3x3 ml), DCM (3x3 ml) and diethyl ether (3x3 ml). The resin was dried under reduced pressure.
Kaiser test: dark blue after Fmoc-deprotection
slightly blue after overnight coupling
negative after capping with acetic anhydride
Intermediate 59d):
Intermediate 59c (500 mg, 0.385 mmol) was treated with a solution of 20% piperidine in DMF (3 ml) for 30 min. Excess of the reagent was removed by filtration. The resin-bound intermediate was successively washed with DMF (3x3 ml), MeOH (3x3 ml), THF (3x3 ml), DCM (3x3 ml) and diethyl ether (3x3 ml). The resin was dried under reduced pressure.
Kaiser test: dark blue after Fmoc-deprotection
Intermediate 59e): Boc
Intermediate 59d (0.21 mmol) was treated with a solution of Fmoc-Phe-OH (244 mg, 0.63 mmol) activated with DIC (98 μΙ, 0.63 mmol) and HOBt monohydrate (96 mg, 0.63 mmol) in DMF (2.5 ml) overnight. Excess of the reagents was removed by filtration. The resin was successively washed with DMF (3 x 3 ml), MeOH (3 x 3 ml), THF (3 x 3 ml), DCM (3 x 3 ml) and diethyl ether (3 x 3 ml). The resin was dried under reduced pressure.
Kaiser test: negative
Intermediate 59f):
Boc
Intermediate 59e (500 mg, 0.385 mmol) was treated with a solution of 20% piperidine in DMF (3 ml) for 30 min. Excess of the reagent was removed by filtration. The resin-bound intermediate was successively washed with DMF (3x3 ml), MeOH (3x3 ml), THF (3x3 ml), DCM (3x3 ml) and diethyl ether (3x3 ml). The resin was dried under reduced pressure.
Kaiser test: dark blue after Fmoc-deprotection
Intermediate 59g):
Intermediate 59f (0.21 mmol) was treated with a solution of Fmoc-D-Arg(Pmc)-OH (418 mg, 0.63 mmol) activated with DIC (98 μΙ, 0.63 mmol) and HOBt monohydrate (96 mg, 0.63 mmol) in DMF (2.5 ml) overnight. Excess of the reagents was removed by filtration. The resin was successively washed with DMF (3x3 ml), MeOH (3x3 ml), THF (3x3 ml), DCM (3x3 ml) and diethyl ether (3x3 ml). The resin was dried under reduced pressure.
Kaiser test: negative
Intermediate 59h):
Intermediate 59g (0.21 mmol) was treated with a solution of 20% piperidine in DMF (3 ml) for 30 min. Excess of the reagent was removed by filtration. The resin-bound intermediate was successively washed with DMF (3x3 ml), MeOH (3x3 ml), THF (3x3 ml), DCM (3x3 ml) and diethyl ether (3x3 ml). The resin was dried under reduced pressure.
Kaiser test: dark blue after Fmoc-deprotection
Intermediate 59i):
Intermediate 59h (0.10 mmol) was treated with a solution of Boc-Phe-OH (80 mg, 0.30 mmol) activated with DIC (46 μΙ, 0.30 mmol) and HOBt monohydrate (46 mg, 0.30 mmol) in DMF (1.5 ml) for 3 h. Excess of the reagents was removed by filtration. The resin was successively washed with DMF (3x2 ml), MeOH (3x2 ml), THF (3x2 ml), DCM (3x2 ml) and diethyl ether (3x2 ml). The resin was dried under reduced pressure.
Kaiser test: negative
Example 59:
Intermediate 59i (0.10 mmol) was treated with a solution of 20% TFA in DCM (2500 μΙ) for 1 h. The resin was removed by filtration. The resin was successively washed with 20% TFA in DCM (3 x 1 ml). The solvent was removed under reduced pressure. The crude product was purified by preparative LC-MS. Example 60:
Intermediate 60a):
Tritylchlorid-polystyrol resin 100-200 mesh (PepChem, PC-01-0011) (250 mg, 0.363 mmol) was treated with a solution of intermediate 59b (594 mg, 1.089 mmol) in dry DCM (2 ml) overnight. Excess of the reagent was removed by filtration. The resin-bound intermediate was successively washed with DMF (3x5 ml), MeOH (3x5 ml), THF (3x5 ml), DCM (3x5 ml) and diethyl ether (3x5 ml). The resin was dried under reduced pressure.
Intermediate 60b):
Intermediate 60a (250 mg, 0.363 mmol) was treated with a solution of 20% piperidine in DMF (3 ml) for 30 min. Excess of the reagent was removed by filtration. The resin-bound intermediate was successively washed with DMF (3x3 ml), MeOH (3x3 ml), THF (3x3 ml), DCM (3x3 ml) and diethyl ether (3x3 ml). The resin was dried under reduced pressure.
Kaiser test: dark blue after Fmoc-deprotection
Intermediate 60c):
Intermediate 60b (0.363 mmol) was treated with a solution of Fmoc-Phe-OH (422 mg, 1.089 mmol) activated with DIC (168 μΙ, 1.089 mmol) and HOBt monohydrate (167 mg, 1.089 mmol) in
DMF (1.5 ml) overnight. Excess of the reagents was removed by filtration. The resin was successively washed with DMF (3x3 ml), MeOH (3x3 ml), THF (3x3 ml), DCM (3x3 ml) and diethyl ether (3x3 ml). The resin was dried under reduced pressure.
Kaiser test: slightly blue. Therefore the reaction was completed (3 h coupling time).
Kaiser test: negative
Intermediate 60d):
Intermediate 60c (0.363 mmol) was treated with a solution of 20% piperidine in DMF (3 ml) for 30 min. Excess of the reagent was removed by filtration. The resin-bound intermediate was successively washed with DMF (3x3 ml), MeOH (3x3 ml), THF (3x3 ml), DCM (3x3 ml) and diethyl ether (3x3 ml). The resin was dried under reduced pressure.
Kaiser test: dark blue after Fmoc-deprotection
Intermediate 60e):
Intermediate 60d (0.363 mmol) was treated with a solution of Fmoc-D-Arg(Pmc)-OH (722 mg, 1.089 mmol) activated with DIC (169 μΙ, 1.089 mmol) and HOBt monohydrate (167 mg, 1.089 mmol) in DMF (1.5 ml) for 6 h. Excess of the reagents was removed by filtration. The resin was successively washed with DMF (3x3 ml), MeOH (3x3 ml), THF (3x3 ml), DCM (3x3 ml) and diethyl ether (3x3 ml). The resin was dried under reduced pressure.
Kaiser test: negative
Intermediate 60f):
Intermediate 60e (0.363 mmol) was treated with a solution of 20% piperidine in DMF (3 ml) for 30 min. Excess of the reagent was removed by filtration. The resin-bound intermediate was successively washed with DMF (3x3 ml), MeOH (3x3 ml), THF (3x3 ml), DCM (3x3 ml) and diethyl ether (3x3 ml). The resin was dried under reduced pressure.
Kaiser test: dark blue after Fmoc-deprotection
Intermediate 60g):
Intermediate 60f (0.363 mmol) was treated with a solution of Boc-Phe-OH (289 mg, 1.089 mmol) activated with DIC (169 μΙ, 1.089 mmol) and HOBt monohydrate (167 mg, 1.089 mmol) in DMF (1.5 ml) for 6 h. Excess of the reagents was removed by filtration. The resin was successively washed with DMF (3 x 3 ml), MeOH (3 x 3 ml), THF (3 x 3 ml), DCM (3 x 3 ml) and diethyl ether (3 x 3 ml). The resin was dried under reduced pressure.
Kaiser test: negative
Intermediate 60h):
Intermediate 60g (0.363 mmol) was treated with a solution of 20% TFA in DCM (2000 μΙ) for 30 min. The resin was removed by filtration. The resin was successively washed with DCM (3 x 1 ml). The solvent was removed under reduced pressure.
The residue was dissolved in a mixture of THF (10 ml) and 1 M aqueous NaOH solution (10 ml). Di-tert-butyl dicarbonate (277 mg, 1.271 mmol) was added and the reaction mixture stirred for 24 h. Additional di-tert-butyl dicarbonate (277 mg, 1.271 mmol) was added and the reaction mixture stirred for 3 d. The reaction mixture was diluted with water (150 ml) and was brought to acidic pH with 1 M aqueous HCI. The aqueous phase was extracted three times with DCM. The combined organic layer was washed with brine, dried over sodium sulfate and the solvent was removed under reduced pressure.
Intermediate 60i):
To a solution of idebenone (51 mg, 0.15 mmol) and intermediate 60h (136 mg, 0.15 mmol) in DCM (3 ml) was added EDC (44 mg, 0.23 mmol) and DMAP (4 mg, 0.03 mmol). The resulting solution was stirred at room temperature overnight. The reaction mixture was diluted with DCM and extracted twice with 5% citric acid and water. The combined aqueous phase was extracted twice with DCM. The combined organic layer was washed with brine, dried over sodium sulfate and the solvent was removed under reduced pressure. The crude product was purified by column chromatography.
Example 60:
A solution of intermediate 60i (109 mg, 0.075 mmol) in 20% TFA in DCM (5 ml) was stirred at room temperature overnight and the solvent was removed under reduced pressure. The crude product was purified by preparative LC-MS.
Example 61 :
Intermediate 61a):
Intermediate 59h (0.385 mmol) was treated with a solution of Fmoc-Phe-OH (447 mg, 1.155 mmol) activated with DIC (179 μΙ, 1.155 mmol) and HOBt monohydrate (177 mg, 1.155 mmol) in DMF (4 ml) overnight. Excess of the reagents was removed by filtration. The resin was successively washed with DMF (3x5 ml), MeOH (3x5 ml), THF (3x5 ml), DCM (3x5 ml) and diethyl ether (3x5 ml). The resin was dried under reduced pressure.
Kaiser test: negative
Intermediate 61b):
Intermediate 61a (0.385 mmol) was treated with a solution of 20% piperidine in DMF (5 ml) for 30 min. Excess of the reagent was removed by filtration. The resin-bound intermediate was successively washed with DMF (3x5 ml), MeOH (3x5 ml), THF (3x5 ml), DCM (3x5 ml) and diethyl ether (3x5 ml). The resin was dried under reduced pressure.
Kaiser test: dark blue after Fmoc-deprotection
Intermediate 61c):
Intermediate 61b (0.05 mmol) was treated with a solution of 10-(4,5-dimethoxy-2-methyl-3,6- dioxo-cyclohexa-1 ,4-dienyl)-decanoic acid (53 mg, 0.15 mmol) activated with DIC (23 μΙ, 0.15 mmol) and HOBt monohydrate (23 mg, 0.15 mmol) in DMF (750 μΙ) overnight. Excess of the reagents was removed by filtration. The resin was successively washed with DMF (3 x 1 ml), MeOH (3 x 1 ml), THF (3 x 1 ml), DCM (3 x 1 ml) and diethyl ether (3 x 1 ml). The resin was dried under reduced pressure.
Kaiser test: negative
Example 61 :
Intermediate 61c (0.385 mmol) was treated with a solution of 20% TFA in DCM (8 ml) for 1 h. The resin was removed by filtration. The resin was successively washed with 20% TFA in DCM (3 x 5 ml) and the combined filtrate was stored at room temperature overnight. The solvent was removed under reduced pressure. The crude product was purified by preparative LC-MS.
Example 62: Intermediate 62a):
Idebenone (1.65 g, 4.87 mmol) and methyl (S)-(-)-2-isocyanato-3-phenylpropionate (1.00 g, 4.87 mmol) were dissolved in toluene (50 ml) and stirred at 90°C for 1 d. The solvent was removed under reduced pressure.
The product was purified by column chromatography.
Intermediate 62b):
Intermediate 62a (2.61 g, 4.80 mmol) was dissolved in THF (50 ml) and cooled to 0°C. Lithium hydroxide monohydrate (0.60 g, 14.4 mmol) in water (10 ml) was slowly added and the reaction mixture was stirred at 0°C for 30 min and at room temperature overnight. The reaction mixture was concentrated and then partitioned between ethyl acetate and 1 N aqueous HCI. The aqueous layer was extracted twice with ethyl acetate. The combined organic layer was washed with brine, dried over sodium sulfate and the solvent was removed under reduced pressure. The product was purified by column chromatography followed by preparative LC-MS.
Intermediate 62c):
Intermediate 59h (0.385 mmol) was treated with a solution of intermediate 62b (220 mg, 0.415 mmol) activated with DIC (66 μΙ, 0.424 mmol) and HOBt monohydrate (65 mg, 0.424 mmol) in DMF (4 ml) overnight. Excess of the reagents was removed by filtration. The resin was successively washed with DMF (3 x 5 ml), MeOH (3 x 5 ml), THF (3 x 5 ml), DCM (3 x 5 ml) and diethyl ether (3 x 5 ml). The resin was dried under reduced pressure.
Kaiser test: negative
Example 62:
Intermediate 62c (0.385 mmol) was treated with a solution of 20% TFA in DCM (5000 μΙ) for 1 h. The resin was removed by filtration. The resin was successively washed with 20% TFA in DCM (3 x 2 ml). The filtrate was stored at room temperature overnight in order to cleave the PMC- group. The solvent was removed under reduced pressure. The crude product was purified by preparative LC-MS.
Physicochemical and pharmacological parameters of examples of the present invention were determined and compared to those determined for compounds A-F shown below which are state of the art and not part of the present invention.
Comparative Compound A
Comparative Compound A is a synthetic derivative to the natural occurring CoQ10.
Comparative Compound C (Q2)
Comparative Compound C is a synthetic derivative to the natural occurring CoQ10.
Comparative Compound D
Comparative Compound D is a synthetic derivative to Comparative Compound E.
Comparative Compound E Comparative Compound E is Idebenone which is first described in the specification of Japanese Patent Examined Publication No. 3134/1987 filed by Takeda Chemical Industries, Ltd.
Comparative Compound F
Comparative Compound F is disclosed in WO-A-2006130775. Biological Assays:
A. Mitochondrial Mass
Mitochondria generate most of the cell's supply of adenosine triphosphate (ATP) which is used as a source of the chemical energy. In addition, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. The amount of mitochondria per cell varies widely and is also subject to modifications in response to physiological stimuli. Mitochondria replicate independently of nuclear replication in response to low energy conditions via an AMPK driven pathway. Their degradation occurs via a specialized autophagy pathway called mitophagy. This pathway can serve multiple goals: first it is essential for maintaining mitochondrial homeostasis, but more importantly it can also be used to reduce mitochondrial mass. Under conditions of impaired mitochondrial function this is an essential, protective mechanism to prevent excessive buildup of radicals that would otherwise damage cellular integrity.
Experimental Procedure: The fluorescent dye, MitoTracker® is used to quantify the number of mitochondria in live cells. After passive diffusion into mitochondria, MitoTracker® Green FM reacts with thiols on proteins and forms a fluorescent conjugate. The method was performed as instructed by the manufacturer. Briefly, fibroblast cells seeded in a 96-well plate at a density of 104 cells per well are allowed to grow for 24 h. Cells were treated with 10 μητι compounds in growth medium for 72 h. Then, cells are washed with 100 μΙ Hank's BSS. A working dye solution of MitoTracker® Green FM is prepared by diluting MitoTracker® Green FM stock solution 20000-fold in Hank's BSS. A volume of 100 μΙ/well of dye solution is added and plates are incubated for 15 min in the cell culture incubator. After washing twice with 50 μΙ PBS, a volume of 50 μΙ PBS is added and fluorescence is measured immediately (MitoTracker® Green FM: excitation: 490 nm; emission: 520 nm). After obtaining the MitoTracker signal, cells are fixed with PFA for 10 minutes at RT, wasched with 3x 100 ul PBS and nuclear DNA stained with DAPI solution (O.l Mg/ml DAPI in PBS) for 10 minutes at RT. After 3 additional washes with PBS, DAPI fluorescence is aquired (excitation: 350 nm); emission: 450 nm). Mitotracker signals are normalized to amount of nuclear DNA (equiv. of cell number).
The results obtained with compounds of the invention and Comparative Compound B are depicted in Table 8.
Table 8
Compound Mitochondrial mass [%]
Comparative cpd B 95
Compound 34 170
Compound 24 122
Compound 17 120
Compound 39 117
Compound 45 113
Mitochondrial mass is a measure of the amount of mitochondria in cells. As a result of multiple studies, it is now generally accepted that upregulation of mitochondrial mass is beneficial to retain sufficient energy (ATP) levels in disorders where mitochondrial function is impaired. As can be taken from Table 8, while the comparison compound B slightly reduces mitochondrial mass, the compounds of the present invention significantly enhance mitochondrial mass.
B. Mitochondrial Membrane Potential
Mitochondrial membrane potential, Δψιη, is an important parameter of mitochondrial function used as an indicator of cell health. It is generated through proton pumps that are fuelled by the electrons donated from the citric acid cycle in form of NADH. These electrons are transferred between complexes I, II and III of the respiratory chain through coenzyme Q10. The membrane potential is used in healthy cells to provide the electrochemical energy to drive mitochondrial ATP production. Under certain conditions however, high Δψιη can be the source of significant radical production by mitochondria. In addition, the loss of mitochondrial membrane potential is a hallmark of apoptosis. The mitochondrial permeability transition is an important step in the induction of cellular apoptosis. During this process, the electrochemical gradient across the mitochondrial membrane collapses and is accompanied by the release of cytochrome c into the cytoplasm.
Experimental Procedure: Depolarization of the mitochondrial membrane is assayed using accumulation of the fluorescent dye 5,5',6,6'-tetrachloro-1 ,1',3,3'-tetraethyl benzimidazolylcarbocyanine iodide (JC-1 , Molecular Probes) in the mitochondrial membrane. The JC-1 emission wavelength shifts from 530 nm as monomer to 590 nm when aggregated, which is indicative of intact membrane potential. Likewise, a decrease of the red to green signal indicates depolarization of the mitochondrial membrane. Briefly, human fibroblasts seeded in a 96-well plate at a density of 104 cells per well were allowed to grow for 24 h. Cells were treated with 10 μΜ compounds in DMEM (stock 25 mM in DMSO) for 72 h before washed with 100 μΙ Hank's BSS. A volume of 100 μΙ/well of dye solution was added and plates were incubated for 15 min in the cell culture incubator. After washing, fluorescence was measured immediately (JC- 1 (green): excitation: 485 nm; emission: 535 nm; JC-1 (red): excitation: 550 nm; emission: 600 nm; Tecan M1000 plate reader and Tecan iControl software; Tecan Austria GmbH, Austria, Grodig, Austria). The extent of mitochondrial membrane depolarization was calculated by the ratio of red to green fluorescence.
Table 9
Compound Δψιπ [%]
Comparative cpd B 69
Compound 61 130
Compound 31 117
Compound 24 137
Compound 44 113
Compound 60 181
Compound 62 118
Compound 59 99
The mitochondrial membrane potential is the H+-gradient over the inner mitochondrial membrane that is used for ATP production. In theory, the higher the gradient the more force is available to make ATP. As can be taken from Table 9, the compounds of the present invention significantly enhanced the mitochondrial membrane potential. C. Cytotoxicity Assay
This assay investigates cytotoxicity which is defined as the cell-killing property of a chemical compound, independent from the mechanisms of cell death. The HepG2 hepatoblastoma cell line is one of the most common human cell lines for hepatotoxicity studies. Even though the cells lack a part of the metabolizing enzymes present in fresh hepatocytes, they have been shown to be a useful tool for studying the toxicity of hepatotoxins. This assay measures metabolic activity of living cells using the WST-1 cell proliferation reagent (Roche Diagnostics). The tetrazolium salt WST-1 is cleaved to water soluble formazan by cellular enzymes. An expansion in the number of viable cells results in an increase in the overall activity of mitochondrial dehydrogenases in the sample. This augmentation in enzyme activity leads to an increase in the amount of formazan dye formed, which directly correlates to the number of metabolically active cells in the culture. The formazan dye produced by metabolically active cells is directly quantified in a scanning multiwell spectrophotometer by measuring the absorbance of the formazan dye solution between 420 and 480 nm. Healthy HepG2 cells, when maintained in culture, continuously divide and multiply over time. A toxic chemical, regardless of site and mechanism of action, will interfere with this process and result in the reduction of the growth rate reflected in the cell number.
Experimental Procedure: Hep-G2 cells are seeded into a 96-well microplate and maintained in culture for 24 hours. They are then exposed to the test compound over a range of eight concentrations. After 24 hours exposure, the cells are incubated in presence of reagent WST-1 for 30 min before measuring the absorbance of the formazan dye formed. Cytotoxicity is expressed as a concentration dependent reduction of the conversion formazan dye formation due to a decrease in cell proliferation as compared to untreated cells. The TC50 (Toxic Concentration 50%), is defined as the concentration of tested compound required to reduce the cellular viability by 50% relative to the control culture. The lower the TC50 value, the higher is the cytotoxic potential of the test compound. The TC50% values were calculated using the following formula:
(Cone. > so - Cone < 5o) (% > so -50)
Cone. > so
(% > 50 - % < 50)
a) Cone. > 50 maximum measured concentration with the % of solvent control > 50 % b) Cone. < 50 minimum measured concentration with the % of solvent control < 50 % c) % > 50 relative absorbance at a) in %
d) % < 50 relative absorbance at b) in % Table 10
Compound TCso M]
Comparative cpd A 20.6
Comparative cpd F 40.7
Compound 32 >100
Compound 44 >100
Compound 56 >100
Compound 57 >100
As can be taken from Table 10, the Comparative compounds A and J are clearly associated with some level of in vitro toxicity, which can be interpreted as an indicator of expected hepatho-toxic liabilities in vivo, while compounds of the present invention clearly demonstrate no toxicity in the dose range evaluated.
D. Solubility Assay
This assay aims at investigating the kinetic solubility of chemical substances in aqueous solution. Kinetic solubility determines the concentration above which a compound starts to precipitate and demonstrate turbidity (i.e. the solubility limit). Turbidity is measured using optical density.
Experimental Procedure: Using a fully automated assay, compounds were serially diluted in DMSO. Aliquots of each dilution were transferred to a 384 well clear bottom clear plate in triplicate and diluted 50-fold with aqueous buffer: 2μΙ compound dilution plus 98μΙ PBS. The optical density was measured at four different wavelengths (450, 490, 550, 612 nm) after 1 hour incubation at room temperature. The sum of the 4 ODs (sumOD) was calculated and plotted against the compound concentration. The limit of solubility is defined as the concentration above which the sumOD increases above a threshold of 0.01.
Table 11
Compound solubility [μΜ]
Comparative cpd C 10
Comparative cpd D 6
Comparative cpd E 120
Compound 34 >200
Compound 44 >200
Compound 56 >200 Compound 39 >200
For oral bioavailability and tissue penetration it is essential to find a balance between the inherent hydrophobic nature of the compounds and modifications that render them more hydrophilic. While Comparative Compounds F and G are practically insoluble in aqueous solutions which negatively affects the development of oral formulations, Comparative Compound I is already soluble enough to show oral bioavailability. As can be taken from Table 11 the compounds of the present invention clearly demonstrate significantly improved solubility characteristics compared to the comparative compounds.
E. Cellular Redox State
The balance between the oxidized and reduced forms of nicotinamide adenine dinucleotide is called the NAD7NADH ratio, which is an important component of what is called the redox state (RS), a measurement that reflects both the metabolic activities and the health of cells. The effects of the NAD7NADH ratio are complex, controlling the activity of several key enzymes, including glyceraldehyde 3-phosphate dehydrogenase and pyruvate dehydrogenase. In healthy mammalian tissues, estimates of the NAD7NADH ratio are typically around 700, thus it favours for oxidative reactions. In contrast, the NADP7NADPH ratio is normally about 0.005, so NADPH is the dominant form of this coenzyme. These different ratios are key to the different metabolic roles of NADH and NADPH. To assess the redox state (RS) we used resazurin fluorescence. Resazurin (7-Hydroxy-3 - -phenoxazin-3-one 10-oxide) is a blue dye used mainly as an oxidation-reduction indicator in the resazurin test for bacteria. It is also used as an indicator for cell viability in mammalian cell cultures. Blue colored Resazurin is reduced to the pink colored, fluorescent compound Resorufin in the presence of live cells. Resazurin has been used to quantify mitochondrial activity since it is considered to act as an intermediate electron acceptor in the electron transport chain between the final reduction of oxygen and cytochrome oxidase.
Experimental Procedure: After harvesting lyphoblastoid suspension cells, resuspend in growth medium. SeedIO6 cells/ml into a 96-deep well plate. Add 0.5 μΙ of a 10 mM cpd solution in DMSO (final content of DMSO in assay 0.1%) to the cells. Mix the cells with the cpd and add 110 μΙ of the cells (total of 1.1x10s cells/well) into black 96-well plates and incubate for 20-24 h at 37 °C. Spin cells in the plate (5 min, 700 x g) and remove the growth medium by turning the plate. Wash the cells with 200 μΙ PBS and re-spin the cells. Remove the PBS and add 110 μΙ RPMI 1640 RPMI1640 w/ phenolred, w/o FCS. Remove 10 μΙ of each well and give to a new transparent flat bottom 96ell plate for protein determination. Add 11 μΙ 40 μΜ resazurin to a final concentration of 4 μΜ resazurin to each well. Measure the fluorescence change (ex = 530/544 nm, em = 590 nm) immediately and after 1 h, 2h, 3h and 4h. Add 70 μΙ BCA reagent mix to each well and incubate 1.5 h at 37 °C. Measure absorption at 562 nm and determine protein content of each well. Standardize resazurin turnover against protein content.
Data are given in Table 12 below.
Table 12
Compound Redox state [%] at cone. [M]
Comparative cpdA 96 ΐχΐσ10
Compound 31 116 ΐχΐσ11
Compound 20 128 ΐχΐσ10
Compound 30 132 ΐχΐσ12
Compound 56 120 ΐχΐσ11
Compound 44 103 ΐχΐσ13
The cellular redox state is tightly controlled; therefore small changes can have big impacts. Thus, it is notably that the compounds of the invention have a similar or better effect than the comparative compounds, sometimes at even much lower concentrations. More signal in this assay reflects higher NADH levels and therefore better viability.
F. ATP Assay
Electrons donated from the citric acid cycle in form of NADH are transferred between complexes I, II and III of the respiratory chain. In this process mitochondrial membrane potential is generated through proton pumps in the mitochondrial inner membrane. This electrochemical gradient across the inner mitochondrial membrane is used in healthy cells to provide the energy to drive mitochondrial ATP production. Consequently, cellular ATP levels are a good indicator of mitochondrial function. In multiple states of disease (i.e. Alzheimer's-, Parkinson's-, Huntington's-disease and mitochondrial disorders) this mode of ATP production is impaired and alternative modes of ATP production are utilized that can be associated with toxic byproducts (i.e. lactic acidosis due to increased anaerobic glycolysis). There is a general consensus that especially in tissues with high energy demand such as muscle and brain, chronic reduction of ATP levels ultimately leads to cell death. Therefore, compounds that significantly reduce ATP levels under normal growth conditions in vitro can be regarded as harbouring some potential for toxicity, whereas compounds that increase ATP could be beneficial in pathological conditions of low ATP levels. Experimental Procedure: In cellular assays one has to distinguish between mitochondrial and non-mitochondrial ATP production such as glycolysis. In conditions of low glucose, ATP is mainly produced by oxidative phosphorylation Therefore the assay was performed either in the presence (25mM) or absence of glucose in the medium. It has to be noted, however, that low levels of glucose contained in the serum result in estimated glucose concentrations of approximately 0.5mM. We therefore refer to this condition as "low glucose". Immortalized lymphoblastoid cells (BC1 LCL) were used to characterize the effect of compounds on ATP levels under different glucose levels by an ATP dependent luciferase reaction. Briefly, cells were seeded in a 24-well plate at a density of 5*105 cells/ml with 1 ml per well in two different media containing 25 mM or no glucose, respectively. Both media were supplemented with 10% fetal bovine serum, 1% Pen/Strep, 200 mM L-glutamine. Cells were treated with 0.1 % (v/v) of compounds (10 mM in DMSO; final concentration: 10 μΜ) and incubated for 72 h at 37 °C, 5% C02, and 90% rH. The number of cells was counted and after brief washing in PBS and collecting through centrifugation (5 min; 200 x g) the cells were lysed in a volume of 0.5 ml lysis solution (4 mM EDTA, 0.2% Triton X-100) for 15 min on ice and 10 μΙ of lysate was added into a white 96 well plate. In parallel, ATP standards (concentrations: 0, 1 , 2, 4, 6, 8, and 12 μΜ in PBS) were also added into the 96 well plate. The reaction was started by addition of 100 μΙ reaction mix (300 μΜ D-Luciferin, 5 μg/ml firefly luciferase, 75 μΜ DTT, 25 mM HEPES, 6.25 mM MgCI2, 625 μΜ EDTA and 1 mg/ml BSA) and the luminescence signal was quantified in a multimode plate reader (Tecan M1000 plate reader; luminescence integration time: 100 ms). The concentration of cellular ATP normalized to cell number is calculated for each well and triplicate measurements are averaged. The ATP levels are given in Table 13.
Table 13
Compound ATP levels [%]
Comparative cpd E 104
Comparative cpd A 6
Comparative cpd B 138
Compound 61 301
Compound 31 232
Compound 37 269
Compound 56 246
Compound 60 406
Compound 59 261 In general, more ATP is beneficial for the cell unlike the membrane potential (Δψητι) with its drawback of the risk of lipid peroxidation. Surprisingly, coupling as in compound 60 increases ATP levels significantly more compared to compound 61. Therefore, it appears to be of great relevance how the peptide (pep) is attached to the compound.
G: Lipid Peroxidation Assay
Lipid peroxidation is a well defined mechanism of cellular damage in both animals and plants that occurs during aging and in some disease states. This process proceeds by a free radical chain reaction mechanism. It most often affects polyunsaturated fatty acids, because they contain multiple double bonds in between which methylene-CH2- groups possess especially reactive hydrogens. If not terminated fast enough, lipid peroxidation damages cellular membranes, affect membrane fluidity and also mitochondrial function. In addition, end products of lipid peroxidation may be mutagenic and carcinogenic. For instance, the reactive end product of lipid peroxidation, malondialdehyde, directly causes DNA damage.
Experimental Procedure: The fluorescent BODIPY® (4,4-difluoro-3a,4adiaza- s-indacene) fluorophore is an effective tracer of lipid trafficking, as well as being useful general- purpose membrane probes. BODIPY 581/591-C11 can be used to measure antioxidant activity in lipid environments by exploiting its loss of fluorescence upon interaction with peroxyl radicals. Primary human fibroblasts C4 (GM04545, Coriell) (passage <12) were seeded at a concentration of 2000 cells/ well into black 96 well plates and incubated for 72 hours in DMEM in the presence of 10 μΜ test compound or DMSO only. After that, 0.1 ml freshly prepared dye solution (HBSS, containing BODIPY dye 1 :1000 from stock solution) was added and cells were returned to the incubator for 30 min. After 2 brief washes with 0.1 ml warmed PBS fluorescence was measured in 50 μΙ PBS. Fluorescence for 4 individual areas per well were individually quantified at two wavelengths (Ex,: 490, Enri!: 600; Ex2: 490, Em2: 530, bandwith 10nm, 50 flashes, 400Hz frequency, 20 ps integration time).
The extent of lipid peroxidation of Comparative Compounds A and J as well as of various compounds of the present invention is shown below in Table 14.
Compound Lipid peroxidation
Comparative cpd A 176
Comparative cpd F 118
Compound 61 88
Compound 45 87 Compound 21 81
Compound 12 75
Compound 46 87
Altered mitochondrial function, such as in mitochondrial disorders, can lead to cellular damage via lipid peroxidation. Altering mitochondrial function through small molecules therefore also has the inherent risk of producing lipid peroxidation as observed for Comparative Compound A. In this context, all compounds that leave basal lipid peroxidation levels unchanged (at 100%) or even reduce them (<100%) do not show any inherent toxic liability or are even beneficial in the context of elevated levels of lipid peroxidation in a state of disease.
As can be taken from Table 14, the compounds of present invention significantly reduce basal levels of lipid peroxidation while the comparative compounds increase basal levels significantly.
H: Measurement of mitochondrial complex II activity
Complex II (CI I) is an enzyme complex bound to the matrix face of the inner mitochondrial membranes. It consists of four subunits with several different enzymatic activities. One of these is the citric acid-cycle enzyme succinate dehydrogenase, which catalyzes the conversion from succinate to fumarate. In this reaction electrons are transferred from succinate to the prosthetic group FAD thus generating FADH2 (Ackrell 2000). These electrons are then transferred from the reduced FADH2 to ubiquinone, from ubiquinone to the reaction centers of complex III (CHI), and finally to the cytochrome c. During this electron translocation process, complex III pumps four protons from matrix to the intermembrane space. These proton fluxes can be detected as electrical currents on SSM-based SURFE2R sensors (http://www.sd-heidelberg.de/index.html). In the SURFE2R CII-CIII assay, both, CM and Clll are activated simultaneously by first equilibrating the sensors with succinate and then activating the CII-CIII reaction by oxidized cytochrome c. The resulting efflux of protons out from the membrane compartment induces negative currents, which are sensitive to the CM and Clll inhibitors malonate and antimycin A, respectively.
Experimental Procedure: SURFE2R SSM sensors were coated with inner mitochondrial membranes according to the standard protocols. Briefly, sensors were filled with 50 μΐ_ of SensorPrep A solution and incubated for 10-15 min. Afterwards the solution was removed, sensor were rinsed with deionized water three times dried in a stream of nitrogen gas and incubated for 15 min at room temperature to get rid of remaining solvents. 1.5 μΙ_ of SensorPrep B1 solution were applied to the sensor and immediately covered with 50 μΙ_ of the buffer (150 mM Na-gluconate, 30 mM Hepes pH 7.2/NMG, 10 mM MgCI2, 12.5 mM NaPi pH7.2, freshly added 0.2 mM DTT). Incubate the sensor for 15-60 min at 4°C. An aliquot (10 μΙ_) of inner mitochondrial membrane suspension (stored at - 80°C) were rapidly thawed, diluted with 190 μΙ_ of the sensor preparation buffer used above and sonicated in a 1.5 ml EppendorfO tube by applying 5 bursts with an amplitude of 30 % and a cycle time ratio of 0.5 (ultrasonic processor UP 50 H, Dr. Hielscher GmbH, Germany, equipped with MS 1 tip). 5 μΙ_ of the membrane suspension was applied onto the sensor surface, representing 4.3 μg total protein content per sensor. The sensors were centrifuged 45 min at 2500 x g, incubated at 4°C for 3-5h and frozen at -80°C.
The biosensors were thawed immediately before the experiment and measured with the SURFE2R Workstation 50 or 500 devices. The CII-CIII activity was studied by rapid exchange of a "non-activating" solution for an "activating" solution containing the 3 μΜ oxidized cytochrome c. 2 s of non-activating buffer was followed by 1 s of activating buffer and then again 1 s non- activating buffer. Afterwards the sensor was rinsed 3 times with 1 mL non-activating buffer. A further activation with cytochrome c was performed after an incubation time of -11 min. Both buffers contained 1 mM succinate and 350 mg/L BSA to enhance the solubility of the test compounds. For the activity of Cll-lll, the peak current (current amplitude) was evaluated. The performed measurements of Cll-lll activities consisted of two parts. First the activity of Cll-lll was recorded in the absence of compound (instead 0.01% DMSO) for about 50 min to obtain a constant CII-CIII activity (constant peak current amplitudes). Afterwards, a test compound (~4 μΜ) was supplied to the non-activating and the activating solutions and the Cll-lll activity was recorded for further -50 min (110 min in total). For each sensor, all peak currents were normalized to the mean of the activities after 24, 36 and 48 min. For each test compound at least two different biosensors were assayed for -110 min in total, indicated by n (t 0-110) = 2. For those test compounds, which activated Cll-lll by more than 30% at time 80 to 110 min, the CII- CIII activity was recorded for additional -50 min (up to 160 min in total).
Results of various compounds of the invention are reported in Table 15 below.
Table 15
Compound Complex 11-111 activity [%]
Comparative cpd A 97
Compound 38 110 (at 80-110 min)
Compound 21 144 (at 80-110 min)
Compound 20 145 (at 80-110 min) Compound 19 135 (at 80-110 min)
Compound 14 132 (at 80-110 min)
Compound 5 145 (at 80-110 min)
Compound 2 138 (at 80-110 min)
Compound 20 180 (at 130-160 min)
Compound 5 215 (at 130-160 min)
Many degenerative disorders are characterized by excess lipid accumulation which directly negatively affects tissue function and mitochondrial energy production. Lipids are normaly broken down during mitochondrial beta oxidation of fatty acids (FA), which gives rise to electron equivalents that have been proposed to enter the mitochondrial electron transport chain at the level of complex II (Bruss et al. Am J Physiol Endocrinol Metab 2010; 298:E108-16). If a compound is able to accelerate the rate of complex ll-lll function, it could be envisaged that more beta-oxidation and less lipid accumulation would occur. The last two compounds showed even higher activity after longer incubation periods (130-160 min) indicating that it takes longer for them to integrate into the membranes.
A longer integration time just indicates that the compound is in principle capable of triggering higher complex II activities but in a cell free setting unable to quickly insert itself into the membrane via diffusion to affect complex ll-lll. At a set concentration added to the cell free medium, maximal concentrations in the membrane are reached at different speeds. This is an artefact of a cell free system where you rely on diffusion. In vivo, these compounds are likely transported in a coordinated fashion via lipid carrier, so this difference will not be seen.
/: ATP Rescue Assay (under conditions of impaired mitochondrial function)
Mitochondrial disorders are characterized by impaired mitochondrial function, which is usually displayed as lower mitochondrial synthesis of ATP. This energy crisis is seen as a major contributor for cellular impairment and ultimately cell death. Thus, improving the abberant energy status that is associated with impaired mitochondrial function is necessary to normalize cellular and tissue function.
Experimental Procedure:
Rat myoblast cells (L6) were seeded at a density of 5*103 cells per well in a 96-well plate and incubated for 24 hours in DMEM with 0.3 g/l glucose, 2% FBS and Penicillin-Streptomycin- Glutamine. Cells were treated with 1 μΜ quinones in presence or absence of rotenone (1 μΜ), for 60 minutes in DMEM without glucose before ATP levels were quantified using luminescence from the ATP-dependent enzymatic oxidation of luciferin by luciferase. Cells were lysed in a volume of 200 μΙ (4 mM EDTA, 0.2% Triton X-100) for five minutes. In 96-well plates, 100 μΙ of ATP measurement buffer (25 mM HEPES pH 7.25, 300 μΜ D-luciferin, 5 pg/ml firefly luciferase, 75 μΜ DTT, 6.25 mM MgCI2, 625 μΜ EDTA and 1 mg/ml BSA) was combined with 10 μΙ lysate to start the reaction. Luminescence was quantified immediately using a multimode plate reader (Tecan M1000, Tecan iControl 1.6 software; Tecan Austria GmbH, Grodig, Austria). ATP levels were standardized to protein levels using BCA assay (ThermoScientific, Rockford, IL, USA) and changes were calculated as percentage relative to levels of DMSO-treated control cells. ATP rescue is defined as the percentage of quinone-induced increase in ATP levels in presence of rotenone relative to the ATP reduction by rotenone alone.
The extent of ATP rescue of Comparative Compound B as well as of various compounds of the present invention is shown below in Table 16.
Table 16
No Salt ATP rescue [%]
comparative 0 %
compound B
Compound 2 70 %
Compound 13 HCOOH 86 %
Compound 18 72 %
Compound 28 77 %
Compound 32 67 %
Compound 36 88 %
Compound 38 84 %
Compound 40 88 %
Compound 43 69 %
Altered mitochondrial function, such as in mitochondrial disorders, can lead to depleted cellular ATP levels. Thus, altering mitochondrial function by small molecules of the current invention but not by Comparative Compound B alows the rescue of cellular energy status as observed here. In this context, all compounds that counteract the lowered ATP levels associated with mitochondrial dysfunction can be seen as beneficial for the function and survival of the affected cells.
Conclusion:
As can be taken from Table 16, the compounds of present invention significantly rescue ATP levels under conditions of impaired mitochondrial function while the comparative compound B has no effect at all.

Claims

A compound represented by the general formula (I)
and enantiomers, tautomers, solvates or pharmaceutically acceptable salts thereof, wherein
a) R1 is a substituent represented by formula (II) Y1 (X1). R5
(II) wherein
X1 is -O- or -NR6-, wherein R6 is H or linear or branched C^ alkyl;
R5 is selected from the group consisting of
a linear or branched Ci.6 alkyl which is unsubstituted or substituted with 1 to 5 substituents independently selected from halogen, OH, NH2 or CN, a 6-10 membered monocylic or bicyclic aryl group, optionally fused with a 5-6 membered heterocyclic or heteroaromatic group, the heterocyclic or heteroaryl group having 1 to 2 heteroatoms in the ring independently selected from N, O or S,
a 5-10 membered monocyclic or bicyclic heteroaromatic group having 1 to 4 heteroatoms independently selected from N, O or S, a 5-7 membered monocyclic heterocyclic group having 1 to 4 heteroatoms in the ring independently selected from N, O or S, and a 3-10 membered monocylic, bicylic or tricyclic cycloalkyl group, wherein each aryl, heteroaryl, heterocyclyl and cycloalkyl is unsubstituted or substituted with 1 to 5 substituents independently from each other selected from the group consisting of halogen, hydroxy, linear or branched C1-6 alkyl, linear or branched halo-Ci-e alkyl, Ci-6 alkoxy, -C(0)NH2, - NHC(0)Ci-6 alkyl, 5-6 membered heteroaryl having in the ring 1 to 3 heteroatoms independently selected from N, O or S,
or
X1 and R5 together form a 4-7 membered monocyclic heterocyclic group having 1 or 2 heteroatoms in the ring independently selected from N or S and wherein the heterocycle is unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of a halogen, a hydroxyl group and a Ci-6 alkoxy group;
Y1 is a linear C8-12 alkylene group or a linear C8-12 alkenylene group;
a is an integer of 0 or 1 ;
R2 is a linear or branched alkyl group;
R3 is a C1 -6 alkoxy group; and
R4 is a Ci-6 alkoxy group;
with the proviso that when a is 1 , X1 is -0-, and Y1 is Cio-alkylene, then R5 is not a 2H-pyrane group;
or
R is a substituent represented by formula (III)
wherein
R7 and R8 independently from each other are hydrogen, linear or branched C^. 6 alkyl or halogen;
R9 and R10 independently from each other are hydrogen, linear or branched C2-6 alkenyl or linear or branched C2-6 alkinyl;
or R9 and R10 together are -(CH2)X- wherein x is an integer of 2 to 6; with the proviso that R7, R8, R9 and R10 cannot simultaneously be a hydrogen atom;
Y2 is a linear C5.9alkylene group or a linear C5.9alkenylene group;
R2 is a linear or branched d.6alkyl group;
R3 is a Ci.6alkoxy group; and
R4 is a Ci-e alkoxy group;
or
c) R1 is a substituent represented by formula (IV)
(IV) wherein
R11 and R12 independently from each other are
hydrogen,
linear or branched Ci.6alkyl,
6-10 membered monocyclic or bicyclic aryl group,
5-10 membered monocyclic or bicyclic heteroaryl group having 1 to 4 heteroatoms in the ring independently selected from N, O or S,
4-10 membered monocylic heterocyclic group having 1 to 4 heteroatoms in the ring independently selected from N, O or S, or
3- 7 membered monocyclic cycloalkyl group,
wherein each alkyl, aryl, heteroaryl, heterocyclic and cycloalkyl group is unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of halogen, hydroxy and Ci.6alkoxy;
or
R11 and R1 together with the nitrogen atom to which they are attached form a
4- 7 membered ring having in the ring 0 to 2 additional heteroatoms independently selected from N, O or S, wherein the ring is unsubstituted or substituted with 1 to 3 substituents selected from the group consisting of halogen, hydroxyl and Ci.6alkoxy group;
Y3 is a linear C6-ioalkylene group or a linear C6-i0alkenylene group;
R2 is a linear or branched C1-6 alkyl group;
R3 is a d-ealkoxy group; and
R4 is a Ci-6alkoxy group;
or
d) R1 is hydrogen;
R2 is a substituent represented by X2-R13
wherein X2 is selected from the group consisting of -O- and -NR14-, wherein R14 is hydrogen or a Ci-6 alkyl group, and
R 3 is a C4.12 alkyl group, C4-12 alkenyl group, -(CH2)nCH2OH or -C2-10 alkenylene-CH2OH
wherein n is an integer of 1 to 10,
or
wherein X2-R13 represents a piperidino group substituted in the 4-position with a C2.7 alkenylene-CH2OH group, a -(CH2)mCH2OH group wherein m is an integer of 1 to 7, a -C(0)-NH-C2.6 alkenylene-CH2OH group or a -C(0)-NH-(CH2)pCH2OI-l group wherein p is an integer of 1 to 6;
R3 is hydrogen; and
R4 is OR15,
wherein R15 is a hydrogen atom or a Ci.2 alkyl group,
or R13 and R15 independently represent a linear -C2-7 alkenylene-CH2OH group or a -(CH2)mCH2OH group wherein m is an integer of 1 to 7;
or
e) R is
wherein Y4 is a C8.i2alkylene group or a C8-i2alkenylene group; R2 is a linear or branched d-6alkyl group;
R3 is a Ci.6alkoxy group; and
R4 is a group;
or
R1 is
wherein Y5 is a C8-i2alkylene group or a C8-i2 alkenylene group; R2 is a linear or branched d.6alkyl group;
R3 is a hydroxy group or a C^alkoxy group; and
R4 is a Ci.6alkoxy group; or
g) R1 is
wherein Y6 is a C6-io alkylene group or a C6-io alkenylene group;
R2 is a linear or branched Chalky! group;
R3 is a Ci-6alkoxy group; and
R4 is a C!-ealkoxy group.
2. The compound according to claim 1 , wherein
R1 has the formula (II);
a is 1 ;
X1 is an oxygen atom; and
R5 is selected from the group consisting of a 6 membered heteroaryl group having 1 to 2 nitrogen atom(s) in the ring and a phenyl group wherein the heteroaryl and the phenyl group are unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of halogen, linear or branched C^.6 alkyl, linear or branched halo Ci-6 alkyl, Ci.6alkoxy, -C(0)NH2, alkyl, 5-6 membered heteroaryl having in the ring 1 to 3 heteroatoms independently selected from N, O or S, and hydroxy.
3. The compound according to claim 1 , wherein
R1 has the formula (II); a is 1 ;
X1 is a nitrogen atom; and
R5 is selected from the group consisting of
a 5-10 membered monocyclic or bicyclic heteroaryl group having 1 to 4 heteroatoms independently selected from N, O or S,
a phenyl group, and an adamantyl group,
wherein the heteroaryl group, the phenyl group and the adamantly group are unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of halogen, linear or branched C1-6 alkyl, linear or branched halo C1-6 alkyl, 0 -6 alkoxy, -C(0)NH2, -NHC(0)Ci.6 alkyl, 5-6 membered heteroaryl having in the ring 1 to 3 heteroatoms independently selected from N, O or S, and hydroxy
or
X1 and R5 together form a 4-7 membered monocyclic heterocyclic group having 1 or 2 heteroatoms in the ring independently selected from N or S and whereby the heterocycle is unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of a halogen, a hydroxyl group and a C^.e alkoxy group.
The compound according to claim 1 , wherein
R1 has the formula (II);
a is 0; and
R5 is a 5-10 membered monocyclic or bicyclic heteroaryl group having 1 to 4 heteroatoms independently selected from N, O and S, wherein the heteroaryl group is unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of halogen, linear or branched Ci_6 alkyl, linear or branched halo C1.6 alkyl, alkoxy, - C(0)NH2, -NHC(0)C1-6 alkyl, 5-6 membered heteroaryl having in the ring 1 to 3 heteroatoms independently selected from N, O or S, and hydroxy.
The compound according to claim 1 , wherein
R1 is hydrogen; R2 is X2-R13 wherein X2 is -NR14 wherein R14 is hydrogen or a C^6 alkyl group, and R 3 is a linear C4-12 alkyl group, or -(CH2)nCH2OH wherein n is an integer of 1 to 10, or
wherein X2-R13 represents a piperidino group substituted in the 4-position with a - (CH2)mCH2OH group wherein m is an integer of 1 to 7 or a -C(0)-NH-(CH2)PCH2OH group wherein p is an integer of 1 to 6;
R3 is hydrogen; and
R4 is -O-R15 wherein R15 is hydrogen or Ci-2 alkyl.
6. A pharmaceutical composition comprising the compound of any of claims 1 to 5.
7. The compound according to any of claims 1 to 5 as a medicament.
8. The compound according to any of claims 1 to 5 for use in the treatment of a mitochondrial disease, a neurodegenerative disease, a neuromuscular disease, psychiatric disorders, metabolic disorders, cancer, or immune dysfunction.
9. The compound according to claim 8 wherein the mitochondrial disease is selected from Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy (DOA), macular degeneration, glaucoma, retinopathy, cataract, optic disc drusen (ODD), mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), myoclonic epilepsy with ragged red fibers (MERRF), myoneurogenic gastrointestinal encephalomyopathy (MNGIE), Kearns-Sayre syndrome, CoQ.10 deficiency, or mitochondrial complex deficiencies.
10. The compound according to claim 8 wherein the mitochondrial disease is selected from Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy (DOA), macular degeneration, glaucoma, mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), or mitochondrial complex deficiencies (1-5, CPEO).
11. The compound according to claim 8 wherein the neurodegenerative disease is selected from Friedreich's ataxia (FRDA), amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, stroke/reperfusion injury, or dementia.
12. The compound according to claim 8 wherein the neuromuscular disease is selected from Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Limb-Girdle muscular dystrophy (LGMD), X-linked dilated cardiomyopathy (XLDC ), Pantothenate kinase-associated neurodegeneration PKAN,), spinal muscular atrophy (SMA), multiple sclerosis and primary progressive multiple sclerosis (PP-MS), Kugelberg-Welander disease, and Werdnig-Hoffmann disease.
13. The compound according to claim 8 wherein the psychiatric disorder is selected from schizophrenia, major depressive disorder, bipolar disorder, or epilepsy.
14. The compound according to claim 8 wherein the metabolic disorder is selected from ageing-related physical decline, obesity, overweight, type II diabetes, or metabolic syndrome.
15. The compound according to claim 8 wherein the immune dysfunction is selected from arthritis, psoriasis or rheumatoid arthritis.
16. Use of a compound according to any of claims 1 to 5 in the preparation of a medicament for the treatment of a mitochondrial disease, a neurodegenerative disease, a neuromuscular disease, psychiatric disorders, metabolic disorders, cancer, or immune dysfunction.
EP11746182.2A 2010-08-16 2011-08-16 Novel benzoquinone derivatives and use thereof as modulators of mitochondrial function Ceased EP2606022A2 (en)

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