Treatment of mitochondrial diseases using sGC stimulators

Abstract

The present invention relates to the use of stimulators of soluble guanylate cyclase (sGC), pharmaceutically acceptable salts thereof and pharmaceutical formulations or dosage forms comprising them, alone or in combination with one or more additional agents, for the treatment of various mitochondrial diseases wherein sGC stimulation is increased, or the concentration of nitric oxide (NO) or cyclic 3 ', 5'-guanosine monophosphate (cGMP), or both, is increased, or both, or both. Or an up-regulation of the NO-sGC-cGMP pathway is desirable. Compounds useful in the methods of the invention are compounds of Formula I or pharmaceutically acceptable salts thereof.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 417,120, filed on October 18, 2022. The entire contents of the foregoing application are expressly incorporated herein by reference. Technical Field

[0003] The present invention relates to stimulators of soluble guanylate cyclase (sGC), pharmaceutically acceptable salts thereof, and pharmaceutical preparations or dosage forms comprising the same, alone or in combination with one or more additional agents, for use in treating various mitochondrial diseases, wherein increased sGC stimulation, or increased concentrations of nitric oxide (NO) or cyclic 3',5'-monoguanosine phosphate (cGMP) or both, or upregulation of the NO-sGC-cGMP pathway is desired. Background Art

[0004] sGC is the main receptor for NO in the body. After binding to sGC, NO activates its catalytic domain and converts guanosine-5'-triphosphate (GTP) into the second messenger cGMP. Increased amounts of cGMP in turn regulate the activity of downstream effectors including protein kinases, phosphodiesterases (PDEs) and ion channels. In vivo, NO is synthesized from arginine and oxygen by various nitric oxide synthases (NOS) and by sequential reduction of inorganic nitrates. Experimental and clinical evidence indicates that reduced NO concentrations, reduced NO bioavailability and / or reduced responsiveness to endogenously produced NO contribute to the development of numerous diseases. sGC stimulators are heme-dependent agonists of the sGC enzyme that synergize with varying amounts of NO to increase its enzymatic conversion of GTP to cGMP. sGC stimulators are clearly distinguished from another class of NO-independent, heme-independent sGC agonists known as sGC activators and are structurally unrelated.

[0005] Therapies that improve or restore sGC function offer considerable advantages over existing alternative therapies that target the NO-sGC-cGMP pathway or otherwise benefit from upregulation of the NO-sGC-cGMP pathway. There is an urgent need to develop novel and safe therapies for patients with a dysfunctional or downregulated NO-sGC-cGMP pathway.

[0006] Mitochondria are organelles that provide energy to cells through oxidative phosphorylation to produce adenosine triphosphate (ATP), which is required for normal cell function. Therefore, normal mitochondrial function is essential for maintaining health and life.

[0007] Mitochondrial diseases are a group of rare, inherited disorders that occur when the mitochondria cannot produce enough energy for the body to function properly. They have varying clinical presentations. Mitochondrial diseases may be caused by mutations (acquired or inherited) in mitochondrial DNA or in nuclear genes encoding mitochondrial components. These disorders may be congenital or develop later in life. Many of these disorders may present as central nervous system (CNS) dysfunction.

[0008] In addition to the reduced ATP production in mitochondrial diseases, lactic acidosis due to reduced conversion of pyruvate to acetyl-CoA, NO deficiency due to reduced nitric oxide (NO) synthesis, increased cellular damage due to elevated reactive oxygen species, and reduced vascular reactivity are observed. They can lead to severe physical, developmental, and cognitive impairments, with symptoms including poor growth; loss of muscle coordination; muscle weakness and pain; seizures; vision and / or hearing loss; gastrointestinal problems; learning disabilities; and organ failure. Life expectancy is greatly reduced in patients with mitochondrial diseases. It is estimated that 1 in 4,000 people are affected by mitochondrial diseases. Mitochondrial diseases are usually progressive, and there is currently no effective treatment or cure. Treatment is primarily supportive, including nutritional management, exercise, and / or vitamin or amino acid supplementation.

[0009] sGC stimulators have been found to be useful for treating mitochondrial diseases (WO2020014504; https: / / www.globenewswire.com / en / news-release / 2022 / 06 / 17 / 2464653 / 0 / en / Cyclerion-Therapeutics-Announces-CY6463-Data-Demonstrating-Improved-Cellular-Energetics-in-Preclinical-Models-of-Mitochondrial-Disease.html; accessed on September 23, 2022).

[0010] sGC stimulators that can cross the blood-brain barrier (BBB) ​​and can penetrate into the central nervous system (CNS) provide additional benefits for treating mitochondrial diseases with central nervous system manifestations. Therefore, sGC stimulators described herein can generally be used to treat mitochondrial diseases. In addition, since they can cross the BBB and activate targets in the brain, they can be used to treat the central nervous system manifestations of mitochondrial diseases. The treatment options for mitochondrial diseases are still extremely limited, so it is still necessary to develop new therapies to improve many clinical manifestations associated with these diseases, including but not limited to central nervous system manifestations. Summary of the invention

[0011] The present invention is based on the discovery that the compounds disclosed herein are potent sGC stimulators and are therefore useful for treating mitochondrial diseases. In addition, these compounds are not only potent sGC stimulators, but also have brain permeability, which makes them useful for treating the central nervous system manifestations exhibited by many mitochondrial diseases. Compounds with relevant structural features, especially 4-OH substituents on the pyrimidine ring, were previously known only as synthetic intermediates that can be used to prepare sGC stimulators with 4-amine substituents on the pyrimidine ring. The sGC stimulating activity of such compounds was previously unknown. In addition, compounds of this category in previous disclosures do not have medical uses. It was unexpectedly found that the compounds of the present invention have potent sGC stimulating activity, and they are able to penetrate the BBB, increase cGMP concentrations in the brain and periphery, and show peripheral and central nervous system activity in in vitro and in vivo assays.

[0012] In a first aspect, the present invention relates to a method of treating a mitochondrial disease in a subject in need thereof, comprising administering a therapeutically effective amount of a compound represented by Formula I or a pharmaceutically acceptable salt thereof, alone or in combination therapy:

[0013]

[0014] in:

[0015] J C Selected from hydrogen, halogen, C 1-6 Alkyl and C substituted by 1 to 3 fluorine atoms 1-6 Fluoroalkyl;

[0016] X is N or C (J C1 );

[0017] J C1 Selected from hydrogen, halogen, C 1-6 Alkyl and C substituted by 1 to 3 fluorine atoms 1-6 Fluoroalkyl;

[0018] Each J B are independently selected from hydrogen, halogen, C 1-6- Alkyl and C substituted by 1 to 3 fluorine atoms 1-6 Fluoroalkyl;

[0019] J D Selected from hydrogen, halogen, C 1-6 Alkyl and C substituted by 1 to 3 fluorine atoms 1-6 fluoroalkyl; and

[0020] n is an integer selected from 0, 1, 2, 3 or 4.

[0021] In a second aspect, the present invention relates to a method of treating a mitochondrial disease in a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition or dosage form comprising a compound of Formula I or a pharmaceutically acceptable salt thereof, alone or in combination therapy.

[0022] In a third aspect, the present invention also relates to the use of a compound of formula I or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition or dosage form comprising a compound of formula I or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating a mitochondrial disease in a subject in need thereof.

[0023] In a fourth aspect, the present invention also relates to a compound of formula I or a pharmaceutically acceptable salt thereof, a pharmaceutical composition or a dosage form comprising the compound of formula I or a pharmaceutically acceptable salt thereof, for use in treating a mitochondrial disease in a subject in need thereof.

[0024] In some embodiments of the first to fourth aspects, the mitochondrial disease manifests as central nervous system dysfunction or central nervous system symptoms. In some embodiments of the first to fourth aspects, the compound of formula I can be used to treat central nervous system manifestations of mitochondrial diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Shown are the effects of Compound 1-14 on the change from baseline in MAP (ΔBMAP) in male normotensive rats.

[0026] Figure 2 Compound I-20 showed an effect on the Δ B The role of MAP.

[0027] Figure 3 Shown are the levels of compound 1-14 in the STR and HIPP regions of the brain of adult male Sprague-Dawley rats following PO administration (3 mg / kg) of compound 1-14 at T=0 min. Data are presented as mean±SEM, N=5.

[0028] Figure 4 Shown are the concentrations of cGMP in rat CSF 1, 2, and 6 hours after administration of a single oral dose of Compound 1-20 (1 mg / kg, 3 mg / kg, or 10 mg / kg).

[0029] Figure 5 Shown are the concentrations of cGMP in rat CSF 1, 2, and 6 hours after administration of a single oral dose of Compound 1-14 (1 mg / kg, 3 mg / kg, 10 mg / kg, or 30 mg / kg).

[0030] Fig. 6 shows the cognitive effect of compound I-14 in the Parkinson's disease cognitive deficit macaque model of chronic low-dose MPTP damage. After long-term low-dose MPTP exposure, SDR efficacy was significantly impaired (**P<0.01). After using vehicle, SD (simple discrimination) and SDR (simple discrimination reversal) efficacy were indistinguishable from MPTP baseline efficacy. SDR efficacy was significantly improved (**P<0.01) after compound I-14 was used and deteriorated during the removal period (**P<0.01 contrast drug efficacy). Fig. 6A Mean ± SEM performance is shown; and Figure 6B Scatter plots showing individual data with mean ± SEM. N = normal, before MPTP; WO = cleared. DETAILED DESCRIPTION OF THE INVENTION

[0032] Reference will now be made in detail to certain embodiments of the present invention, examples of which are illustrated in the accompanying structures and formulas. Although the present invention will be described in conjunction with the enumerated embodiments, it should be understood that it is not intended to limit the present invention to these embodiments. On the contrary, the present invention is intended to include all alternatives, improvements and equivalents, which are all included in the scope of the present invention defined by the scope of the claims. The present invention is not limited to the methods and materials described herein, but includes any methods and materials similar to or equivalent to those methods and materials shown in the descriptions herein that can be used for the practice of the present invention. In the case where one or more of the incorporated literature references, patents or similar materials (including but not limited to defined terms, term usage, described technology, etc.) are different from or conflict with the present application, the present application shall prevail.

[0033] Definitions and common terms related to chemical compounds

[0034] For purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements, CAS version, and Handbook of Chemistry and Physics, 75th Edition, 1994. In addition, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999 and "March's Advanced Organic Chemistry", 5th Edition, Smith, MB and March, J. Eds., John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

[0035] When one or more positions in a structure can be substituted with one or more substituents selected from a specified group or list, the one or more substituents at each position can be "independently selected" to be equal or identical at each position and for each occurrence, unless otherwise specified. For example, if a phenyl group is substituted with two R 100 Replace and each R 100 are independently selected from halogen and methyl, then R 100 Each occurrence of is independently selected from halogen or methyl; for example, one R 100 can be fluorine and one can be methyl, or both can be chlorine, etc. Similarly, if the substitutable atom is bound to more than one hydrogen (e.g., CH 3 or NH 2 ), substituents may be "independently selected" to be equal or identical at each position and for each occurrence, unless otherwise specified. For example, if a methyl group (e.g., CH 3 ) through two R 100 Replace and each R 100 are independently selected from halogen and methyl, then R 100 Each of R is independently selected from halogen or methyl; for example, one R 100 can be fluorine and one can be methyl (e.g., CHF (CH 3 ), or both may be chlorine (e.g. CHCl 2 ),etc.

[0036] The selection and combination of substituents envisioned by the present invention are only those selections and combinations that form stable or chemically feasible compounds. Such selections and combinations will be obvious to those skilled in the art and can be determined without excessive experimentation. As used herein, the term "stable" refers to a compound that does not substantially change when subjected to conditions that allow its production, detection, and in some embodiments, its recovery, purification, and for one or more purposes disclosed herein. Chemically feasible compounds are compounds that can be prepared by those skilled in the art based on the content disclosed herein (assisted by relevant knowledge in the art when necessary).

[0037] Unless otherwise stated, all tautomeric forms of the compounds of the invention are intended to be within the scope of the invention.

[0038] In one embodiment, the invention may include using deuterium (i.e. 2 H) to replace hydrogen, which may provide certain therapeutic advantages resulting from greater metabolic stability (e.g., increased half-life in vivo or reduced dosage requirements) and may therefore be preferred in some cases. Deuterium-labeled compounds of the invention can generally be prepared by following procedures similar to those shown in the schemes and / or examples below, by substituting a deuterated reagent for an undeuterated reagent.

[0039] As used herein, the term "alkyl" as in, for example, "alkyl chain" or "alkyl group" refers to a saturated unbranched (eg, straight chain) or branched monovalent hydrocarbon radical. x Alkyl is an alkyl chain containing x carbon atoms, where x is an integer other than 0. x-y "alkyl" (wherein x and y are two different integers, both of which are not 0) is an alkyl chain containing a number of carbon atoms between x and y (including the endpoints). For example, C 1-6 Alkyl is an alkyl group as defined above containing any number of carbon atoms between 1 and 6. Examples of alkyl groups include, but are not limited to, methyl (i.e., C 1 Alkyl), ethyl (i.e. C 2 alkyl), n-propyl (C 3 Alkyl), isopropyl (different C 3 In certain embodiments, alkyl is C 1-4 In certain embodiments, the alkyl group is C 1-3 Alkyl or C 1-2 In other embodiments, the alkyl group is methyl or ethyl.

[0040] As used herein, the term "fluoroalkyl" refers to an alkyl group as defined above, wherein at any one or more carbon atoms of the alkyl group, one or more of the hydrogen atoms attached to the chain carbon atoms have been replaced by fluorine. For example, a fluoroalkyl group substituted with 1 to 3 fluorine atoms is an alkyl group in which 1 to 3 hydrogen atoms at any position on the same carbon atom or different carbon atoms of the alkyl chain have been replaced by fluorine atoms.

[0041] As used herein, the term "halogen" or "halo" refers to F, Cl, Br, or I. In certain embodiments, halo is F or Cl. In other embodiments, halo is F.

[0042] The term "hydroxyl" or "hydroxy" refers to -OH.

[0043] The compounds of the present invention are defined herein by their chemical structure and / or chemical name. When a compound is referred to by both a chemical structure and a chemical name and the chemical structure and chemical name conflict, the chemical structure determines the identity of the compound.

[0044] Compound and Composition Embodiments

[0045] According to the first to fourth aspects discussed above, the present invention relates to the medical use of a compound of formula I, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising said compound or a pharmaceutically acceptable salt thereof.

[0046]

[0047] in:

[0048] J C Selected from hydrogen, halogen, C 1-6 Alkyl and C substituted by 1 to 3 fluorine atoms 1-6 Fluoroalkyl;

[0049] X is N or C (J C1 );

[0050] J C1 Selected from hydrogen, halogen, C 1-6 Alkyl and C substituted by 1 to 3 fluorine atoms 1-6 Fluoroalkyl;

[0051] Each J B are independently selected from hydrogen, halogen, C 1-6 Alkyl and C substituted by 1 to 3 fluorine atoms 1-6 Fluoroalkyl;

[0052] J D Selected from hydrogen, halogen, C 1-6 Alkyl and C substituted by 1 to 3 fluorine atoms 1-6 fluoroalkyl; and

[0053] n is an integer selected from 0, 1, 2, 3 or 4.

[0054] In a first embodiment of the first, second, third and fourth aspects, for compounds of formula I, n is an integer selected from 1, 2, 3 or 4, and each J B independently selected from halogen, C 1-6 Alkyl and C substituted by 1 to 3 fluorine atoms 1-6 fluoroalkyl, all other carbon atoms of the benzene ring are unsubstituted, and the remaining variables are as defined above.

[0055] In a second embodiment of the first, second, third and fourth aspects, for a compound of formula I or a pharmaceutically acceptable salt thereof, J C is selected from hydrogen, halogen and C 1-6 Alkyl; J C1 is selected from hydrogen, halogen and C 1-6 Alkyl; each J B are independently selected from hydrogen, halogen and C 1-6 Alkyl; J D is selected from hydrogen, halogen and C 1-6 alkyl; and the remaining variables are as defined above for Formula I in the first aspect or first embodiment.

[0056] In a third embodiment of the first, second, third and fourth aspects, the compound of formula I is represented by formula IA:

[0057]

[0058] or a pharmaceutically acceptable salt thereof, wherein the variables are as described above for Formula I in the first aspect or the first or second embodiment.

[0059] In a fourth embodiment of the first, second, third and fourth aspects, for the compound of formula IA or a pharmaceutically acceptable salt thereof, J C1 is H, F or Cl; and the remaining variables are as defined in the first aspect or the first, second or third embodiment.

[0060] In a fifth embodiment of the first, second, third and fourth aspects, for the compound of formula IA or a pharmaceutically acceptable salt thereof, J C1 is H; and the remaining variables are defined in the first aspect or any one of the first to fourth embodiments.

[0061] In a sixth embodiment of the first, second, third and fourth aspects, for the compound of formula IA or a pharmaceutically acceptable salt thereof, J C1 is F; and the remaining variables are defined in the first aspect or any one of the first to fifth embodiments.

[0062] In a seventh embodiment of the first, second, third and fourth aspects, the compound of formula I is represented by formula IB:

[0063]

[0064] or a pharmaceutically acceptable salt thereof, wherein the variables are as described above for Formula I according to the first aspect or any one of the first to sixth embodiments.

[0065] In an eighth embodiment of the first, second, third and fourth aspects, for a compound of Formula I, IA or IB, or a pharmaceutically acceptable salt thereof, n is 2 or 3, and the remaining variables are as described in the first aspect or the first, second, third, fourth, fifth, sixth or seventh embodiment.

[0066] In a ninth embodiment of the first, second, third and fourth aspects, for a compound of Formula I, IA or IB or a pharmaceutically acceptable salt thereof, n is 0 or 1, and the remaining variables are as described in the first aspect or the first, second, third, fourth, fifth, sixth or seventh embodiment. In some embodiments, each J B are independently halogen or C 1-6 alkyl.

[0067] In a tenth embodiment of the first, second, third and fourth aspects, for a compound of Formula I, IA or IB or a pharmaceutically acceptable salt thereof, each J B are independently H, F or C 1-4 alkyl; the remaining variables are as described in the first aspect or the first, second, third, fourth, fifth, sixth, seventh, eighth or ninth embodiment. In some embodiments, each J B Independently F or C 1-4 alkyl.

[0068] In an eleventh embodiment of the first, second, third and fourth aspects, for a compound of Formula I, IA or IB or a pharmaceutically acceptable salt thereof, n is 2 or 3; each J B is independently F or methyl; the remaining variables are as described in the first aspect or the first, second, third, fourth or fifth, sixth or seventh embodiment.

[0069] In a twelfth embodiment of the first, second, third and fourth aspects, for a compound of Formula I, IA or IB or a pharmaceutically acceptable salt thereof, n is 2; J B All are F, or J B One of J is F, the other is methyl; the remaining variables are as described in the first aspect or the first, second, third, fourth, fifth, sixth or seventh embodiment. In some embodiments, one J is B is F and the other is methyl.

[0070] In a thirteenth embodiment of the first, second, third and fourth aspects, for a compound of Formula I, IA or IB or a pharmaceutically acceptable salt thereof, n is 3; J B Two of them are F and the other is methyl; the remaining variables are as described in the first aspect or the first, second, third, fourth, fifth, sixth or seventh embodiment. In some embodiments, J D is H or F. In some embodiments, J D is F. In some embodiments, J D For H.

[0071] In a fourteenth embodiment of the first, second, third and fourth aspects, for the compound of Formula I, IA or IB or a pharmaceutically acceptable salt thereof, n is 1; J B is F; and the remaining variables are as described in the first aspect or the first, second, third, fourth, fifth, sixth or seventh embodiment.

[0072] In a fifteenth embodiment of the first, second, third and fourth aspects, for a compound of Formula I, IA or IB, or a pharmaceutically acceptable salt thereof, n is 0; and the remaining variables are as described in the first aspect or the first, second, third, fourth, fifth, sixth or seventh embodiment.

[0073] In a sixteenth embodiment of the first, second, third and fourth aspects, for a compound of Formula I, IA or IB or a pharmaceutically acceptable salt thereof, J D is hydrogen; and the remaining variables are as described in the first aspect or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth or fifteenth embodiment.

[0074] In a seventeenth embodiment of the first, second, third and fourth aspects, for a compound of Formula I, IA or IB or a pharmaceutically acceptable salt thereof, J D is F, Cl or methyl; and the remaining variables are as described in the first aspect or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth or fifteenth embodiment.

[0075] In an eighteenth embodiment of the first, second, third and fourth aspects, for the compound of Formula I, IA or IB or a pharmaceutically acceptable salt thereof, J D is F; and the remaining variables are as described in the first aspect or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth or fifteenth embodiment.

[0076] In a nineteenth embodiment of the first, second, third and fourth aspects, for a compound of Formula I, IA or IB or a pharmaceutically acceptable salt thereof, J C is H, Cl or F; and the remaining variables are as described in the first aspect or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth or eighteenth embodiment.

[0077] In a twentieth embodiment of the first, second, third and fourth aspects, for a compound of Formula I, IA or IB or a pharmaceutically acceptable salt thereof, J C is H; and the remaining variables are as described in the first aspect or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth or eighteenth embodiment.

[0078] In a twenty-first embodiment of the first, second, third and fourth aspects, the compound of formula I is a compound shown in Table I, or a pharmaceutically acceptable salt thereof.

[0079] Table I. Exemplary sGC stimulators.

[0080]

[0081]

[0082]

[0083] or a pharmaceutically acceptable salt thereof.

[0084] In a twenty-second embodiment of the first, second, third and fourth aspects, for the methods and uses of the present invention, the sGC stimulator is compound I-14, or a pharmaceutically acceptable salt thereof. In one embodiment, the pharmaceutically acceptable salt is a sodium salt. In another embodiment, the sGC stimulator is a sodium salt of compound I-14 represented by the following formula:

[0085]

[0086] In a twenty-third embodiment of the first, second, third and fourth aspects, for the methods and uses of the present invention, the sGC stimulator is compound 1-20, or a pharmaceutically acceptable salt thereof. In one embodiment, the pharmaceutically acceptable salt is a sodium salt. In another embodiment, the sGC stimulator is a sodium salt of compound 1-20 represented by the following formula:

[0087]

[0088] In a twenty-fourth embodiment of the first, second, third and fourth aspects, the compound of the invention is a compound of formula IC:

[0089]

[0090] or a pharmaceutically acceptable salt thereof, wherein X is N or C(J C1 ), where when X is C(J C1 ), which is represented by C in the following table; and the variables X, J C1 and J B The definitions are as follows; in addition, wherein Me represents a methyl group, and Me-F represents a fluorinated methyl group substituted with 1 to 3 fluorine atoms (i.e., -CH 2 F, -CHF 2 or -CF 3 ):

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099] The pharmaceutically acceptable salts of the present invention.

[0100] "Pharmaceutically acceptable salts" of the compounds described herein include those salts obtained when the compounds are mixed with inorganic or organic acids or bases. In some embodiments, the salts can be prepared in situ during the final isolation and purification of the compounds. In other embodiments, salts can be prepared from the compounds in free form in a separate synthesis step. The preparation of the pharmaceutically acceptable salts described above and other typical pharmaceutically acceptable salts is more fully described in Berg et al., "Pharmaceutical Salts", J. Pharm. Sci., 1977: 66: 1-19, which is incorporated herein by reference in its entirety. Pharmaceutically acceptable salts of compounds of formula I are those salts shown in medicines. However, pharmaceutically unacceptable salts may be suitable for preparing compounds of formula I or pharmaceutically acceptable salts thereof.

[0101] When the compound of formula I is acidic, suitable "pharmaceutically acceptable salts" refer to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic and organic bases. Salts derived from inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, trivalent manganic salts, divalent manganous salts, potassium salts, sodium salts, zinc salts and the like. Specific embodiments include ammonium salts, calcium salts, magnesium salts, potassium salts 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, arginine, betaine, caffeine, choline, N,N1-dibenzhydrylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, reduced glucosamine, glucosamine, histidine, hydramine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.

[0102] In some embodiments, the compound of formula I has an acidic OH group that can react with a base (e.g., a pharmaceutically acceptable non-toxic base) to form a salt (e.g., a pharmaceutically acceptable salt). In some embodiments, the salt is an ammonium salt, a calcium salt, a magnesium salt, a potassium salt, or a sodium salt. In other embodiments, the salt is a sodium salt.

[0103] When the compound of formula I is a base, salts can be prepared from pharmaceutically acceptable non-toxic acids (including inorganic and organic acids). Such acids include acetate, acetic acid, acid citrate, acid phosphate, ascorbate, benzenesulfonic acid, benzenesulfonate, benzoic acid, benzoate, bromide, hydrogen sulfate, hydrogen tartrate, camphorsulfonic acid, chloride, citrate, citric acid, ethanesulfonate, ethanesulfonic acid, formate, fumarate, fumaric acid, gentisate, gluconate, gluconic acid, glucuronate, glutamate, glutamic acid, hydrobromic acid, hydrochloric acid, iodide, isethionic acid, isonicotinoate, lactic acid , lactic acid, maleate, maleic acid, malic acid, mandelic acid, methanesulfonic acid, methanesulfonate, mucic acid, nitrate, nitric acid, oleate, oxalate, pamoic acid, pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)), pantothenic acid, pantothenate, phosphate, phosphoric acid, glucarate, salicylate, succinic acid, succinate, sulfuric acid, sulfate, tannate, tartrate, tartaric acid, p-toluenesulfonate, p-toluenesulfonic acid, and the like. Specific embodiments include citric acid, hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid, and tartaric acid.

[0104] In addition to the compounds described herein, pharmaceutically acceptable salts thereof may also be used in compositions or dosage forms to treat or prevent the diseases identified herein.

[0105] Pharmaceutical compositions, dosage forms and methods of administration.

[0106] The compounds disclosed herein and their pharmaceutically acceptable salts may be formulated into pharmaceutical compositions or "formulations" for use in the treatments and uses of the present invention.

[0107] Typical preparations are prepared by mixing the compound of formula I or a pharmaceutically acceptable salt thereof with a carrier, diluent or excipient. Suitable carriers, diluents and excipients are well known to those skilled in the art, and include materials such as carbohydrates, waxes, water-soluble and / or swellable polymers, hydrophilic or hydrophobic substances, gelatin, oils, solvents, water, etc. The carrier, diluent or excipient used will be used depending on the mode and purpose of formula I compound or its pharmaceutically acceptable salt. Solvents are generally selected based on solvents recognized by those skilled in the art as safe for administration to mammals (GRAS-generally considered safe (Generally Considered as Safe)). In general, safe solvents are non-toxic aqueous solvents, such as water and other non-toxic solvents that are soluble in water or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycol (e.g., PEG400, PEG300), etc. and mixtures thereof. The formulation may also include other types of excipients, such as one or more buffers, stabilizers, anti-adherents, surfactants, wetting agents, lubricants, emulsifiers, binders, suspending agents, disintegrants, fillers, adsorbents, coatings (e.g., enteric or slow release), preservatives, antioxidants, light shielding agents, glidants, processing aids, colorants, sweeteners, aromas, flavoring agents, and other known additives used to provide a medicament (i.e., a compound of Formula I or a pharmaceutical composition thereof) with an elegant appearance or to aid in the manufacture of a pharmaceutical product (i.e., a medicament).

[0108] Acceptable diluents, carriers, excipients, and stabilizers are those that are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-methyl paraben; phenol); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN TM 、PLURONICS TM Or polyethylene glycol (PEG). The active pharmaceutical ingredient can also be embedded in the prepared microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, by coacervation technology or by interfacial polymerization; embedded in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or embedded in macroemulsions. Such techniques are disclosed in Remington's: The Science and Practice of Pharmacy, 21st edition, University of the Sciences in Philadelphia, 2005 edition (hereinafter referred to as "Remington's").

[0109] The formulations can be prepared using conventional dissolution and mixing methods. As used herein, the term "therapeutically effective amount" refers to the amount of active compound or pharmaceutical substance that causes the biological or pharmaceutical response sought by the researcher, veterinarian, physician or other clinician in a tissue, system, animal or human. The therapeutically effective amount of the compound to be administered will be determined by such considerations and is the minimum amount required to improve, cure or treat one or more of the disease or its symptoms.

[0110] The term "administer / administering / administration" with respect to a compound, composition or dosage form of the invention refers to the introduction of a compound into the system of an individual or patient in need of treatment. When a compound of the invention is provided in combination with one or more other active agents, "administration" and variations thereof are each understood to include the simultaneous and / or sequential introduction of the compound and other active agents.

[0111] Depending on the severity and type of disease to be treated, the compositions described herein can be administered systemically or topically, for example, orally (including but not limited to solid dosage forms, including hard or soft capsules (e.g., gelatin capsules), tablets, pills, powders, sublingual tablets, troches, lozenges, and granules; and liquid dosage forms, including but not limited to pharmaceutically acceptable emulsions, microemulsions, aqueous or oily solutions, suspensions, syrups, and elixirs; by inhalation (e.g., using an aerosol, gas, inhaler, nebulizer, or the like); otically (e.g., using ear drops); topically (e.g., using creams, gels, inhalants, liniments, lotions, ointments, patches, etc.); The composition may be administered by injection orally (e.g., tablets, pastes, powders, solutions, sprays, transdermal patches, etc.); ocularly (e.g., using eye drops, ocular gels, ocular ointments); rectally (e.g., using enemas or suppositories); nasally; buccally; vaginally (e.g., using douches, intrauterine devices, vaginal suppositories, vaginal rings, or tablets, etc.); by ear drops; by implanted reservoirs or the like; or parenterally. As used herein, the term "parenteral" includes, but is not limited to, subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Preferably, the composition is administered orally, intraperitoneally, or intravenously.

[0112] Formulations of compounds intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions.

[0113] In such solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid, b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia, c) humectants such as glycerol, d) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption promoters such as quaternary ammonium compounds, g) wetting agents such as cetearyl alcohol and glyceryl monostearate, h) absorbents such as kaolin and bentonite, and / or i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. Tablets may be uncoated or may be coated by known techniques (including microencapsulation) to mask unpleasant taste or delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period of time. For example, time-delay materials such as glyceryl monostearate or glyceryl distearate used alone or with wax may be used. Water-soluble taste-masking materials such as hydroxypropyl methylcellulose or hydroxypropyl cellulose may be used.

[0114] In addition to the active compound, the liquid dosage form may contain inert diluents (e.g., water or other solvents), solubilizers and emulsifiers commonly used in the art, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and aromatics.

[0115] Oral compositions (solid or liquid) may also contain excipients and adjuvants such as dispersants or wetting agents, for example, natural phospholipids (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate); emulsifiers and suspending agents, for example, sodium carboxymethylcellulose, cross-linked carboxymethylcellulose, povidone, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic; sweeteners, flavoring agents and fragrances; and / or one or more preservatives (e.g., ethyl or n-propyl p-hydroxybenzoate), one or more coloring agents, one or more flavoring agents and one or more sweeteners (e.g., sucrose or saccharin).

[0116] The pharmaceutical composition can also be administered by nasal aerosol or inhalation. The composition is prepared according to the well-known technology in the field of pharmaceutical preparations and can be prepared as a saline solution, which uses benzyl alcohol or other suitable preservatives, absorption promoters (for enhancing bioavailability), fluorocarbons and / or other conventional solubilizers or dispersants. Preparations suitable for intrapulmonary or nasal administration have, for example, a particle size within the range of 0.1 micron to 500 microns (including a range of particles between 0.1 and 500 microns, in microns, increments of, for example, 0.5, 1, 30, 35 microns, etc.), and the preparation is administered by rapid inhalation through the nasal passage or by inhalation through the mouth to the alveolar sac.

[0117] The pharmaceutical compositions described herein can also be administered topically, especially when the therapeutic target includes areas or organs that can be easily reached by topical administration (including diseases of the eyes, ears, skin, or lower intestinal tract). Suitable topical formulations for each of these areas or organs are readily prepared. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any desired preservatives or buffers that may be required.

[0118] For topical administration, the pharmaceutical composition can be formulated in a suitable ointment containing an active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid paraffin, white soft paraffin, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water. Alternatively, the pharmaceutical composition can be formulated in a suitable lotion or cream containing an active component suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, hexadecyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0119] Alternatively, the active ingredients may be formulated in a cream using an oil-in-water cream base. If desired, the aqueous phase of the cream base may include a polyol, i.e., an alcohol having two or more hydroxyl groups, such as propylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400) and mixtures thereof.

[0120] Topical formulations may desirably include compounds that enhance the absorption or penetration of the active ingredient through the skin or other affected area. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogs.

[0121] The oil phase of the emulsion prepared using the compounds of Table I can be composed of known ingredients in a known manner. Although the phase may only contain an emulsifier (emulsifier) ​​(originally called an emulsifier (emulgent)), it ideally contains at least one emulsifier and a mixture of fat or oil or fat and oil. Hydrophilic emulsifiers and lipophilic emulsifiers used as stabilizers may be included. In some embodiments, the emulsifier includes both oil and fat. Emulsifiers containing or not containing stabilizers together constitute so-called emulsifying waxes, and the wax together with oil and fat constitute a so-called emulsified ointment base, which forms the oily dispersed phase of the cream formulation. Emulsifiers and emulsion stabilizers suitable for preparing compounds of Formula I include Tween TM -60, Span TM -80, cetearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.

[0122] In addition, the present invention includes the use of transdermal patches, which have the additional advantage of providing controlled delivery of the compound to the body. The dosage form can be prepared by dissolving or dispersing the compound in an appropriate medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate can be controlled by providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0123] For ophthalmic use, the pharmaceutical composition can be formulated as a micronized suspension in isotonic, pH-adjusted sterile saline, or preferably as a solution in isotonic, pH-adjusted sterile saline, with or without a preservative, such as benzalkonium chloride. Alternatively, for ophthalmic use, the pharmaceutical composition can be formulated in an ointment (e.g., paraffin). For treatment of the eye or other external tissues (e.g., mouth and skin), the formulation can be applied as a topical ointment or cream containing, for example, the active ingredient in an amount of 0.075 to 20% w / w. When formulated in an ointment, the active ingredient can be used with an oil-based paraffin or water-miscible ointment base.

[0124] Compositions for rectal or vaginal administration are preferably suppositories, which can be prepared by mixing the compounds described herein with suitable non-irritating excipients or carriers (e.g., cocoa butter, beeswax, polyethylene glycol or suppository wax), which are solid at ambient temperature but liquid at body temperature and thus melt in the rectum or vaginal cavity and release the active compound. Other formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams or sprays.

[0125] The sterile injectable form (e.g., for parenteral administration) of the composition described herein may be an aqueous or oily suspension. These suspensions (including those described in the above paragraphs) may be prepared using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspension media. For this purpose, any mild fixed oil may be used, including synthetic monoglycerides or diglycerides. Fatty acids (e.g., oleic acid and its glyceride derivatives) may be used to prepare injections, such as pharmaceutically acceptable natural oils, such as vegetable oils, such as peanut oil, olive oil, sesame oil, or coconut oil, especially in their polyoxyethylene forms, or mineral oils (e.g., liquid paraffin). These oily solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants, which are commonly used to prepare pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants (e.g., Tweens, Spans, and other emulsifiers or bioavailability enhancers commonly used to prepare pharmaceutically acceptable solids, liquids, or other dosage forms) may also be used for the purpose of injectable formulations. The oily suspension may contain thickeners, such as beeswax, hard paraffin, or cetearyl alcohol. Sweeteners (e.g., those described above) and flavorings may be added to provide a palatable oral formulation. These compositions may be preserved by adding antioxidants (e.g., butylated hydroxyanisole or alpha-tocopherol).

[0126] In another aspect, the compound of formula I or a pharmaceutically acceptable salt thereof can be formulated in a veterinary composition comprising a veterinary carrier. Veterinary carriers are materials that can be used for the purpose of administering the composition and can be solid, liquid or gaseous materials that are originally inert. In the veterinary field, they are compatible with the active ingredient. These veterinary compositions can be administered parenterally, orally or by any other desired route.

[0127] Definitions and general terms related to treatment methods

[0128] As used herein, the term "disease" refers to any deviation or interruption in the normal structure or function of any body part, organ or system, manifested by a characteristic set of symptoms and signs, whose cause, pathology and prognosis may be known or unknown. The term disease includes other related terms such as disorders, conditions (or medical conditions), and syndromes, which are defined as a combination of symptoms due to a single cause, or a combination of symptoms that usually occur together to constitute a unique clinical presentation.

[0129] The term "mitochondrial disease" refers to a group of genetic conditions that affect mitochondria, the structures in every cell of the body responsible for producing energy. These disorders can appear at any age and affect nearly all organs, including the brain, muscles, heart, liver, nerves, eyes, ears, and kidneys. Some of these diseases affect only one organ or tissue, and many involve multiple organ systems, including the brain, muscles, heart, liver, nerves, eyes, ears, and / or kidneys. Mitochondrial disease manifests itself in different ways.

[0130] Mitochondrial genetic diseases can be caused by mutations in mitochondrial DNA or nuclear DNA, resulting in mitochondrial dysfunction and insufficient cellular ATP production. Those diseases caused by mitochondrial DNA mutations are transmitted through maternal inheritance, while those caused by nuclear DNA mutations may follow autosomal dominant, autosomal recessive, or X-linked inheritance patterns. (See: https: / / rarediseases.info.nih.gov / diseases / 7048 / mitochondrial-genetic-disorders, last visited on June 3, 2022, the teachings of which are incorporated herein by reference).

[0131] The mitochondrial diseases contemplated in the present disclosure are "primary mitochondrial diseases" or disorders. The term "mitochondrial disease" as used herein is the same as the term "primary mitochondrial disease" sometimes used in the art. For definitions and distinctions between primary mitochondrial disorders or diseases and secondary mitochondrial dysfunction, see https: / / www.mitoaction.org / resources / primary-mitochondrial-disease-and-secondary-mitochondrial-dysfunction-importance-of-distinction-for-diagnosis-and-treatment / (last visited June 7, 2022).

[0132] Mitochondrial diseases are primarily characterized by chronic loss of cellular ATP, which results in a variety of clinical phenotypes and symptoms. In addition to ATP crisis, mitochondrial respiratory chain dysfunction can lead to excessive ROS production and increased oxidative stress, resulting in cellular and vascular damage and inflammation.

[0133] As used herein, "mitochondrial disease" is equivalent to the term "mitochondrial disorder" or "mitochondrial syndrome" or "mitochondrial disorder". As used herein, the term "mitochondrial disease" and its equivalents refer to mitochondrial diseases of genetic origin, which are the same as those diseases referred to in the art as primary mitochondrial diseases.

[0134] "Treatment" or "treatment" with respect to a disorder, disease, condition, symptom or syndrome means eliminating or ameliorating the cause and / or effects (i.e., symptoms, physiological, physical, psychological, emotional or any other clinical manifestation, observation or measurement, or improvement in pathological assessment) of the disorder, disease, condition or syndrome.

[0135] As used herein, the terms "treat," "treatment," and "treating" also refer to delaying or ameliorating or preventing the progression of a disease (i.e., known or expected disease progression), severity, and / or duration, or delaying or ameliorating or preventing the progression of one or more symptoms, clinical manifestations, observations, or measures, or preventing or slowing a negative progression of a pathological assessment (i.e., "managing" rather than "curing" a condition) by administering one or more therapies.

[0136] As used herein, the terms "subject" and "patient" are used interchangeably. The terms "subject" and "patient" refer to animals (e.g., birds such as chickens, quail or turkeys, or mammals), specifically "mammals", including non-primates (e.g., cattle, pigs, horses, sheep, rabbits, guinea pigs, rats, cats, dogs and mice) and primates (e.g., monkeys, chimpanzees and humans), more specifically humans. In some embodiments, the subject is a non-human animal, such as a farm animal (e.g., a horse, cattle, pig or sheep), or a companion animal or pet (e.g., a dog, cat, mouse, rat, hamster, gerbil, guinea pig or rabbit). In some embodiments, the subject is a human.

[0137] The term "biological sample" as used herein refers to an in vitro or ex vivo sample, including but not limited to cell cultures or extracts thereof; biopsy material obtained from a mammal or extracts thereof; blood, saliva, urine, feces, semen, tears, lymph, ocular fluid, vitreous fluid, cerebrospinal fluid (CSF) or other body fluids or extracts thereof.

[0138] Treatment

[0139] In the first aspect, the present invention relates to a method for treating a mitochondrial disease in a subject in need thereof, comprising administering a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, alone or in combination therapy. In certain embodiments, the compound of formula I is as described in any one of the first to twenty-fourth embodiments above.

[0140] In a second aspect, the present invention relates to a method for treating a mitochondrial disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition or dosage form comprising a compound of formula I or a pharmaceutically acceptable salt thereof, alone or in combination therapy. In certain embodiments, the compound of formula I is as described in any one of the first to twenty-fourth embodiments above.

[0141] In a third aspect, the present invention also relates to the use of a compound of formula I or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition or dosage form comprising a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a mitochondrial disease in a subject in need thereof. In certain embodiments, the compound of formula I is as described in any one of the first to twenty-fourth embodiments above.

[0142] In a fourth aspect, the present invention also relates to a compound of formula I or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition or dosage form comprising a compound of formula I or a pharmaceutically acceptable salt thereof, for use in treating a mitochondrial disease in a subject in need thereof. In certain embodiments, the compound of formula I is as described in any one of the first to twenty-fourth embodiments above.

[0143] A variety of different assessment tools or clinical measurements known and used in the art can be used to evaluate the health status of patients with mitochondrial disease and the corresponding pathology of observed dysfunction, decline, or symptoms.

[0144] These range from imaging tools (e.g., magnetic resonance imaging (MRI), such as using arterial spin labeling (ASL) or functional fMRI-BOLD patterns), laboratory measurements (e.g., fluid biomarkers measured in blood, cerebrospinal fluid (CSF), urine, plasma, serum, skin, saliva), clinical outcome assessment tools or instruments (e.g., patient- or clinician-reported outcome tools or performance outcome measures, such as cognitive assessments using PROMIS questionnaires, MFIS scores, and other methods described herein or known in the art), digital assessments (e.g., assessments obtained through wearable devices, sensor- or camera-based assessments), and electrophysiological assessments (e.g., EEG). These are known in the art and can be used in hospital, clinical, or community settings. For example, the American Association of Family Physicians (AAFP) describes and provides links to many potential cognitive assessment tools on its webpage, such as the MiniCog, MoCA, SLUMS Examination, CPCoG, MIS, and MMSE, among others (https: / / www.aafp.org / pubs / afp / issues / 2019 / 0115 / p101.html, last accessed June 3, 2022).

[0145] Some measurements are performed to help diagnose and / or select patients. Other measurements are performed to help assess prognosis. Other measurements can be used to assess the pharmacological response to a certain intervention described herein (pharmacodynamic or PD assessment). Other measurements can be used to assess sensitivity or decline or risk of response to a certain intervention (e.g., assessing genetic markers or other biomarkers) or assessing disease progression in patients.

[0146] In one embodiment of the first, second, third and fourth aspects, the compounds disclosed herein are capable of increasing cerebral blood flow (CBF) in the brain of a patient suffering from a mitochondrial disease as measured by ASL / MRI.

[0147] In another embodiment of the first, second, third and fourth aspects, the compounds disclosed herein are capable of increasing brain connectivity in patients with mitochondrial disease as measured by functional fMRI BOLD.

[0148] In another embodiment of the first, second, third and fourth aspects, the compounds disclosed herein are capable of improving cognitive abilities in a patient suffering from a mitochondrial disease as measured by one of the cognitive assessment tools known in the art.

[0149] In one embodiment of the first, second, third and fourth aspects, the compounds disclosed herein are sGC stimulators, which can be used to prevent and / or treat inflammation associated with mitochondrial disease. One embodiment of the present invention is a method of reducing inflammation in a subject with mitochondrial disease in need thereof, as determined by a change in the value of an inflammatory biomarker, by administering to the subject any one of the compounds of Formula I or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition or dosage form comprising it.

[0150] In another embodiment of the first to fourth aspects of the present invention, the compounds disclosed herein are sGC stimulators, which can be used to prevent and / or treat cardiovascular damage or dysfunction associated with mitochondrial disease. One embodiment of the present invention is a method for reversing or reducing cardiovascular damage or dysfunction, as determined by changes in the values ​​of biomarkers of cardiovascular dysfunction in a subject with mitochondrial disease in need thereof, by administering to the subject any one of the compounds of Formula I or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition or dosage form comprising it.

[0151] According to the first to fourth aspects, one embodiment of the present invention is a method for reducing the value of a biomarker associated with mitochondrial dysfunction in a patient with a mitochondrial disease by administering to the subject any one of the compounds of formula I or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition or dosage form comprising the same.

[0152] In some embodiments, the biomarker associated with mitochondrial dysfunction is selected from lactate, GDF-15, and FGF-21. In other embodiments, the biomarker of mitochondrial dysfunction is lactate. In other embodiments, it is selected from GDF-15 and FGF-21. In other embodiments, it is GDF-15. In other embodiments, it is FGF-21.

[0153] Specific mitochondrial diseases that can be treated and / or prevented by administering a compound of Formula I or any one of the first to twenty-fourth embodiments, or an equivalent amount of a pharmaceutically acceptable salt thereof, include, but are not limited to:

[0154] Alpers disease, autosomal dominant optic atrophy (ADOA), Bartter syndrome / LIC (lethal infantile cardiomyopathy), beta-oxidation defects, long-chain fatty acid transport defects, coenzyme Q10 defects, complex I, II, III, IV, V defects, chronic progressive lateral ophthalmoplegia (CPEO), Friedreich's ataxia, Kearns-Sell syndrome, leukodystrophy, Leigh disease or syndrome, LHON, LHON Plus, MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms), myoclonic epilepsy with rending red muscle fiber disease (MERRF), mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial cell disease, mitochondrial DNA depletion, mitochondrial encephalopathy, mitochondrial myopathy, multiple mitochondrial dysfunction syndrome, MNGIE (myoneurogenic gastrointestinal encephalopathy), NARP (neuropathy, ataxia, retinitis pigmentosa, and ptosis), Pearson syndrome (Pearson Syndrome), pyruvate carboxylase deficiency, pyruvate dehydrogenase deficiency, or pyruvate dehydrogenase complex deficiency (PDCD / PDH), and POLG mutations.

[0155] In one embodiment, the mitochondrial disease is selected from Alpers disease, Complex I, II, III, IV deficiency, CPEO, KSS, LCHAD, Leigh syndrome, leukodystrophy, LHON, MELAS, MEPAN, MERRF, MIRAS, mitochondrial DNA depletion, MNGIE, NARP, Pearson syndrome, and POLG mutation. In one embodiment, the mitochondrial disease is a Complex I mitochondrial disease. In another embodiment, the mitochondrial disease is MELAS. In another embodiment, the mitochondrial disease is Leigh syndrome.

[0156] In other embodiments, the present invention provides a method for stimulating sGC activity in a biological sample, comprising contacting the biological sample with a compound or composition of the present invention. The use of sGC stimulators in biological samples can be used for a variety of purposes known to those skilled in the art. Examples of such purposes include, but are not limited to, bioassays and biological sample storage.

[0157] Combination therapy

[0158] The compounds and pharmaceutical compositions described herein can be used alone or in combination therapy to treat diseases mediated, regulated or affected by sGC, cGMP and / or NO.

[0159] As used herein, the terms "combination" (as in "combination therapy") or "co-administration" are used interchangeably to refer to the use of more than one therapy. The use of the terms does not restrict the order in which the therapies are administered to a subject.

[0160] The compounds and pharmaceutical compositions described herein can be used in combination with one or more additional therapeutic agents. For combined treatments with more than one active agent (where the active agent is in a separate dosage formulation), the active agent can be administered alone or in combination. In addition, the administration of one element can be before, at the same time, or after the administration of another agent.

[0161] When used in combination therapy with other agents, a "therapeutically effective amount" of the compounds and pharmaceutical compositions described herein and the other drug or drugs will depend on the type of drug used. Suitable dosages are known for approved agents and can be adjusted by a skilled artisan based on the condition of the subject, the type of disorder being treated, and the amount of the compound described herein. In cases where an amount is not explicitly noted, an effective amount should be assumed.

[0162] In some embodiments, co-administration or combination therapy includes administering the first amount and the second amount of the compound in a substantially simultaneous manner, such as in a single pharmaceutical composition (e.g., a capsule or tablet having a fixed ratio of the first and second amounts) or in multiple separate capsules or tablets for each agent. In addition, the co-administration also includes the use of each compound in a sequential manner in any order.

[0163] When co-administration involves separate administration of a first amount of a compound of formula I and a second amount of another therapeutic agent, the compounds are administered close enough in time to have the desired therapeutic effect. For example, the time period between each administration that can produce the desired therapeutic effect can range from a few minutes to a few hours, and can be determined based on the properties of each compound (e.g., efficacy, solubility, bioavailability, plasma half-life, and kinetic characteristics). For example, the compound of formula I and the second therapeutic agent can be administered in any order within about 24 hours, within about 16 hours, within about 8 hours, within about 4 hours, within about 1 hour, or within about 30 minutes of each other.

[0164] Examples of other therapeutic agents that can be combined with a compound of Formula I or a pharmaceutically acceptable salt thereof, either alone or in the same pharmaceutical composition, include, but are not limited to:

[0165] (1) Endothelium-derived releasing factor (EDRF) or NO gas.

[0166] (2) NO donors, including but not limited to nitrosothiol, nitrite, sydnonimine, NONOate, N-nitrosamine, N-hydroxynitrosamine, nitrosimine, nitrotyrosine, diazetidin dioxide, oxatriazole 5-imine, oxime, hydroxylamine, N-hydroxyguanidine, hydroxyurea or furazan oxide. Some examples of these types of compounds include: glyceryl trinitrate (also known as GTN, nitroglycerin, nitroglycerin, and glyceryl trinitrate), which is a nitrate ester of glycerol; sodium nitroprusside (SNP), which is a tetragonal bipyramidal complex in which a nitric oxide molecule is coordinated to an iron metal; 3-morpholino-sidoneimine (SIN-1), which is a zwitterionic compound formed by the combination of morpholine and sidoneimine; S-nitroso-N-acetylopecicillamine (SNAP), which is an N-acetylated amino acid derivative containing a nitrosothiol functional group; diethylenetriamine / NO (DETA / NO), which is a compound in which nitric oxide is covalently linked to diethylenetriamine; and m-nitrosylmethylphenyl ester of acetylsalicylic acid. More specific examples of some of the described classes of NO donors include: classical nitrovasodilators, such as organic nitrates and nitrites, including nitroglycerin, amyl nitrite, isosorbide dinitrate, isosorbide 5-mononitrate, and nicorandil; isosorbide, 3-morpholino-sterone imine; linsidomine chlorohydrate ("SIN-1"); S-nitroso-N-acetylpenicillamine ("SNAP"); S-nitrosoglutathione (GSNO), sodium nitroprusside, S-nitrosoglutathione monoethyl ester (GSNO-ester), 6-(2-hydroxy-1-methyl-nitrosohydrazino)-N-methyl-1-hexylamine or diethylamine NONOate.

[0167] (3) Other substances that enhance cGMP concentration, including but not limited to protoporphyrin IX, arachidonic acid, and phenylhydrazine derivatives.

[0168] (4) nitric oxide synthase substrates, including but not limited to L-arginine, N-hydroxyguanidine-based analogs, such as N[G]-hydroxy-L-arginine (NOHA), 1-(3,4-dimethoxy-2-chlorobenzylideneamino)-3-hydroxyguanidine and PR5 (1-(3,4-dimethoxy-2-chlorobenzylideneamino)-3-hydroxyguanidine); L-arginine derivatives (such as homo-Arg, homo-NOHA, N-tert-butyloxy- and N-(3-methyl-2-butene-1-yl)-1-hydroxy-L-arginine); 1-(4-(6-nitro-1-yl)-1-hydroxy-2-nitro-4-ol)-1-nitro ...

[0169] (5) Compounds that enhance eNOS transcription.

[0170] (6) NO-independent, heme-independent sGC activators, including but not limited to:

[0171] BAY 58-2667 (described in patent publication DE19943635); HMR-1766 (ataciguat, described in patent publication WO2000002851); S 3448 (2-(4-chloro-phenylsulfonylamino)-4,5-dimethoxy-N-(4-(thiomorpholinethiomorpholine-4-sulfonyl)-phenyl)-benzamide (described in patent publications DE19830430 and WO2000002851); and HMR-1069 (from Sanofi-Aventis).

[0172] (7) Heme-dependent and NO-independent sGC stimulators, including but not limited to:

[0173] YC-1 (see patent publications EP667345 and DE19744026); riociguat (BAY 63-2521, described in DE19834044); nelociguat (BAY 60-4552, described in WO 2003095451); vericiguat (BAY 1021189, described in US8420656); BAY 41-2272 (described in DE19834047 and DE19942809); BAY 41-8543 (described in DE19834044); etriciguat (described in WO 2003086407); CFM-1571 (described in patent publication WO2000027394); A-344905, its acrylamide analog A-350619 and aminopyrimidine analog A-778935;

[0174] Other sGC stimulators described in any of the following publications: US20090209556, US8455638, US20110118282 (WO2009032249), US20100292192, US20110201621, US7947664, US8053455 (WO2009094242), US20100216764 , US8507512, (WO2010099054) US20110218202 (WO2010065275), US20130012511 (WO2011119518), US20130072492 (WO2011149921), US20130210798 (WO2012058132), and Tetrahedron Letters (2003), 44(48):8661-8663; and IW-1973 (praliciguat), IW1701 (olinciguat) and CY6463 (formerly IW-6463).

[0175] (8) Compounds that inhibit the degradation of cGMP and / or cAMP, including but not limited to:

[0176] PDE1 inhibitors, PDE2 inhibitors, PDE-3 inhibitors, such as amrinone, milrinone, enoximone, vesylarone, pimobendan and olprinone, PDE4 inhibitors, such as lumilast, PDE5 inhibitors, such as sildenafil and related agents, such as avanafil, lodenafil, mirodenafil, sildenafil citrate, tadalafil, vardenafil and udenafil; alprostadil; dipyridamole and PF-00489791; PDE6 inhibitors, PDE9 inhibitors, such as PF-04447943, PDE10 inhibitors, such as PF-02545920 (PF-10) and PDE11 inhibitors.

[0177] (9) Anticoagulants, including but not limited to:

[0178] Coumarins (vitamin K antagonists), such as warfarin, acenocoumarol, phenprocoumon, and phenindione;

[0179] Heparin and derivatives, such as low molecular weight heparin, fondaparinux, and idraparinux;

[0180] direct thrombin inhibitors, such as argatroban, lepirudin, bivalirudin, dabigatran, and ximelagatran; and

[0181] Tissue plasminogen activators, which are used to dissolve clots and unclog arteries, such as alteplase.

[0182] (10) Antiplatelet drugs, including but not limited to topidogrel, ticlopidine, dipyridamole and aspirin.

[0183] (11) Supplemental oxygen therapy.

[0184] (12) α-1-adrenoceptor antagonists, including but not limited to prazosin, indoramin, urapidil, bunazosin, terazosin and doxazosin; atrial natriuretic peptide (ANP), ethanol, histamine inducers, tetrahydrocannabinol (THC) and papaverine.

[0185] (13) Bronchodilators, including but not limited to:

[0186] Short-acting beta 2 agonists, such as albuterol or albuterol and terbutaline;

[0187] Long-acting beta 2 agonists (LABAs), such as salmeterol and formoterol;

[0188] anticholinergic drugs, such as pratropium and tiotropium; and

[0189] Theophylline, bronchodilators, and phosphodiesterase inhibitors.

[0190] (14) Corticosteroids, including but not limited to beclomethasone, methylprednisolone, betamethasone, prednisone, prednisolone, triamcinolone, dexamethasone, fluticasone, flunisolide, hydrocortisone, and corticosteroid analogs (e.g., budesonide).

[0191] (15) Dietary supplements, including but not limited to omega-3 oils; folic acid, niacin, zinc, copper, Korean red ginseng root, ginkgo, pine bark, Tribulus terrestris, arginine, Avenasativa, horny goat weed, maca root, muira puama, saw palmetto, and Swedish flower pollen; vitamin C, vitamin E, vitamin K2; testosterone supplements, testosterone transdermal patches; zoraxel, naltrexone, bremelanotide, and melanotan II.

[0192] (16)PGD2 receptor antagonist.

[0193] (17) Immunosuppressants, including but not limited to cyclosporine, tacrolimus, rapamycin and other FK-506 type immunosuppressants mycophenolate mofetil and mycophenolate mofetil.

[0194] (18) Non-steroidal anti-asthmatic drugs, including but not limited to:

[0195] beta2-agonists, such as terbutaline, metaproterenol, fenoterol, isoetharine, albuterol, salmeterol, bitolterol, and pirbuterol;

[0196] beta2 agonist corticosteroid combinations, such as salmeterol-fluticasone, formoterol-budesonide, theophylline, cromolyn, cromolyn sodium, nedocromil, atropine, ipratropium, ipratropium bromide; and

[0197] Leukotriene biosynthesis inhibitors such as zileuton or veliflapon.

[0198] (19) Nonsteroidal anti-inflammatory drugs (NSAIDs), including but not limited to:

[0199] Propionic acid derivatives, such as alminoprofen, benoxaprofen, buclofen, carprofen, fenbufen, fenoprofen, fluprofen, flubiprofen, ibuprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid and tioxaprofen;

[0200] Acetic acid derivatives, for example indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin and zomepirac;

[0201] fenamic acid derivatives, for example flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid and tolfenamic acid;

[0202] Biphenic acid derivatives, such as diflunisal and flufenisal;

[0203] Oxicams, such as isoxicam, piroxicam, sudoxicam, and tenoxican;

[0204] Salicylates, such as acetylsalicylic acid and sulfasalazine; and

[0205] Pyrazolones, including but not limited to apazone, bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, and phenylbutazone.

[0206] (20) Cyclooxygenase-2 (COX-2) inhibitors, such as celecoxib, rofecoxib, valdecoxib, etoricoxib, parecoxib and lumiracoxib; opioid analgesics, such as codeine, fentanyl, hydromorphone, levorphanol, meperidine, methadone, morphine, oxycodone, oxymorphone, propoxyphene, buprenorphine, butorphanol, dezocine, nalbuphine and pentazocine.

[0207] (21) Adrenergic neuron blocking agents, such as guanethidine and guanadrel.

[0208] (22) Imidazoline I-1 receptor agonists, including but not limited to rimenidine dihydrogen phosphate and moxonidine hydrochloride hydrate.

[0209] (23) Potassium channel activators, including but not limited to pinacidil.

[0210] (24) Dopamine D1 agonists, including but not limited to fenoldopammesilate; other dopamine agonists, such as ibopamine, dopexamine and docarpamine.

[0211] (25) 5-HT2 antagonists, including but not limited to ketanserin.

[0212] (26) Vasopressin antagonists, including but not limited to tolvaptan.

[0213] (27) Calcium channel sensitizers, including but not limited to levosimendan) or activators (e.g., nicorandil).

[0214] (28) Adenylate cyclase activators, including but not limited to colforsindapropate hydrochloride.

[0215] (29) Positive inotropic agents, including but not limited to digoxin and metildigoxin; metabolic cardiotonic agents, such as ubiquinone; brain natriuretic peptides, such as nesiritide.

[0216] (30) Drugs used to treat erectile dysfunction, including but not limited to alprostadil, aviptadil and phentolamine mesilate.

[0217] (31) Drugs used to treat Alzheimer's disease and dementia, including but not limited to:

[0218] Acetylcholinesterase inhibitors, such as galantamine, rivastigmine, donepezil, and tacrine; and

[0219] NMDA receptor antagonists, such as memantine; and

[0220] Oxidoreductase inhibitors, for example idebenone.

[0221] (32) Psychotropic drugs, including but not limited to:

[0222] Ziprasidone, risperidone, olanzapine, valproate;

[0223] Dopamine D4 receptor antagonists, such as clozapine;

[0224] Dopamine D2 receptor antagonists, such as nemonapride;

[0225] Mixed dopamine D1 / D2 receptor antagonists, such as zuclopenthixol;

[0226] GABA A receptor modulators, such as carbamazepine;

[0227] Sodium channel inhibitors, such as lamotrigine;

[0228] Monoamine oxidase inhibitors, such as moclobemide and indeloxazine; primavanserin and perospirone.

[0229] (33) Drugs used to treat movement disorders or symptoms, including but not limited to:

[0230] Catechol-O-methyltransferase inhibitors, such as entacapone;

[0231] monoamine oxidase B inhibitors, such as selegiline;

[0232] dopamine receptor modulators, such as levodopa;

[0233] dopamine D3 receptor agonists, such as pramipexole;

[0234] Decarboxylase inhibitors, such as carbidopa;

[0235] Other dopamine receptor agonists, such as pergolide, ropinirole, and cabergoline;

[0236] Ritigonide, istradefylline, talipexole; zonisamide and safinamide; and

[0237] Inhibitors of synaptic vesicle amine transporters, such as tetrabenazine.

[0238] (34) Medications used to treat mood or affective disorders or OCD, such as the following

[0239] tricyclic antidepressants, such as amitriptyline, desipramine, imipramine, amoxapine, nortriptyline, doxepin, and clomipramine;

[0240] Selective serotonin reuptake inhibitors (SSRIs), such as paroxetine, fluoxetine, sertraline, trazodone, and citalopram;

[0241] Atypical antidepressants, such as agomelatine;

[0242] Selective norepinephrine reuptake inhibitors (SNRIs), such as venlafaxine, reboxetine, and atomoxetine; dopamine antidepressants, such as bupropion and amineptine.

[0243] (35) Drugs used to enhance synaptic plasticity, including but not limited to:

[0244] Nicotinic receptor antagonists, such as mecamylamine; and

[0245] Mixed 5-HT, dopamine, and norepinephrine receptor agonists, such as lurasidone.

[0246] (36) Drugs used to treat ADHD, such as amphetamines; 5-HT receptor modulators, such as vortioxetine, and α-2 adrenergic receptor agonists (such as clothianidin).

[0247] (37) Nitric oxide synthase cofactors, including but not limited to tetrahydrobiopterin, dihydrobiopterin and sapropterin.

[0248] (38) Blood sugar lowering drugs (also known as blood sugar control drugs or anti-diabetic drugs) include but are not limited to:

[0249] Biguanides, such as metformin;

[0250] Sulfonylureas such as glyburide, glybenclamide, glipizide, gliclazide, gliquidone, glimepiride, atorvastatin calcium in combination with glimepiride, meglinatide, tolbutamide, chlorpropamide, acesulfame and tolazimide;

[0251] alpha-glucosidase inhibitors, such as acarbose, epalrestat, voglibose, and miglitol;

[0252] Insulin secretagogues, such as repaglinide, mitiglinide, and nateglinide;

[0253] Thiazolidinediones such as rosiglitazone, troglitazone, ciglitazone, pioglitazone, englitazone, lobeglitazone sulfate, and balaglitazone;

[0254] DPP-4 inhibitors (or DPP-IV inhibitors), such as sitagliptin, vildagliptin, saxagliptin, alogliptin, linagliptin, alogliptin benzoate in combination with metformin or metformin hydrochloride, anagliptin, teneliliptin, gliptin), atorvastatin calcium and glimepiride, empagliflozin in combination with linagliptin, gemigliptin, sitagliptin phosphate monohydrate in combination with pioglitazone hydrochloride, sitagliptin in combination with pioglitazone, sitagliptin in combination with atorvastatin calcium, and (2S,4S)-1-[2-(1,1-dimethyl-3-oxo-3-pyrrolidin-1-yl-propylamino)acetyl]-4-fluoro-pyrrolidine-2-carbonitrile (DBPR-108);

[0255] GLP-1 receptor agonists or incretin mimetics, such as exenatide, dulaglutide, liraglutide, semaglutide, lixisenatide, lixisenatide in combination with insulin glargine, albiglutide and pegapamodutide (TT-401), LY3298176 (dual glucose-dependent insulinotropic polypeptide (GIP) and GLP-1 receptor agonist);

[0256] SGLT2 inhibitors (SGLT2i) such as empagliflozin, empagliflozin in combination with linagliptin, empagliflozin in combination with metformin, ipragliflozin, ipragliflozin L-proline, tofogliflozin, sergliflozin etabonate, repagliflozin etabonate, ertugliflozin, empagliflozin in combination with sitagliptin, empagliflozin in combination with metformin, sotagliflozin, canagliflozin, canagliflozin in combination with metformin or metformin hydrochloride, dapagliflozin, dapagliflozin in combination with metformin or metformin hydrochloride and luseoglifozin, dapagliflozin in combination with saxagliptin;

[0257] SGLT1 inhibitors or a combination of SGLT1 and SGLT2 inhibitors, such as sogliflozin;

[0258] Insulin therapy, such as one of many types of insulin: glulisine, degludec, lispro, aspart, glargine, detemir, protamine insulin, insulin mixtard (human insulin containing both rapid-acting (soluble) and long-acting (protamine) insulin, degludec combined with aspart, human (rDNA-derived) inhaled powder insulin, recombinant human insulin, liver-directed vesicular insulin, insulin tregopi (IN-105), degludec combined with liraglutide, insulin peglispro (LY-2605541), and nodlin; and

[0259] Tolimidone (lyn kinase activator).

[0260] (39) Blood pressure lowering drugs (also known as antihypertensive drugs), including but not limited to:

[0261] diuretics such as thiazide diuretics, chlorothiazide, chlorthalidone, hydrochlorothiazide, benzflumethiazide, cyclopenthiazide, methylchlorothiazide, polythiazide, quinethazolone, xipamide, metolazone, indapamide, cicletanine, furosemide, toresamide, amiloride, spironolactone, canrenoate potassium, eplerenone, triamterene, acetazolamide, and carperitide;

[0262] beta-blockers, such as acebutolol, atenolol, metoprolol, and nebivolol;

[0263] Angiotensin-converting enzyme (ACE) inhibitors, such as thiol-containing drugs (e.g., captopril, zofenopril), diformate-containing drugs (e.g., enalapril, quinapril, ramipril, perindopril, lisinopril, and benazepril), phosphonate-containing drugs (e.g., fosinopril), naturally occurring ACE inhibitors of the invention (e.g., tyrosinase, lactokinin, lactotripeptide Val-Pro-Pro and Ile-Pro-Pro), alacepril, delapril, cilazapril, imidapril, temocapril, moexipril, lisinopril, lisinopril in combination with hydrochlorothiazide, trandolapril, and spirapril;

[0264] Angiotensin II receptor blockers (ARBs), such as candesartan, losartan, losartan potassium-hydrochlorothiazide, valsartan, candesartan cilexetil, eprosaran, irbesartan, telmisartan, olmesartan medoxomil (or olmesartan), azilsartan medoxomil, azilsartan, amlodipine besylate in combination with irbesartan besylate), azilsartan in combination with amlodipine besylate, cilnidipine in combination with valsartan, fimasartan, irbesartan in combination with atorvastatin, irbesartan in combination with triclomethasone, losartan potassium in combination with hydrochlorothiazide and / or amlodipine besylate, pratosartan, atorvastatin calcium in combination with losartan potassium, nifedipine and candesartan cilexetil, sacubitril in combination with valsartan or LCZ-696, angiotensin AT2 antagonists and TAK-591, and olmesartan medomide;

[0265] Endothelin receptor antagonists (ERAs) such as atrasentan, bosentan, sitaxentan, ambrisentan, actelion-1 (macitentan), cyclo(D-trp-D-asp-L-pro-D-Val-L-leu) (BQ-123), sparsentan, and tezosentan disodium.

[0266] Mineralocorticoid receptor antagonists (MRAs) such as spironolactone, amloprothione hydrochloride in combination with spironolactone, apararenone or MT-3995, eplerenone, and finerenone (BAY-94-8862);

[0267] Calcium channel blockers such as amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, diltiazem, efonidipine, felodipine, lacidipine, lercanidipine, manidipine ), nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, pranidipine, isradipine, verapamil, gallopamil, diltiazem, mibefradil, bepridil, fluspirilene, and fendiline;

[0268] Renin inhibitors, such as aliskiren;

[0269] alpha blockers, such as doxazosin and prazosin;

[0270] alpha-beta blockers, such as carvedilol and labetalol;

[0271] Central nervous system drugs, such as clonidine, guanfacine, and methyldopa;

[0272] Vasodilators, such as nitroglycerin, hydralazine, and minoxidil; and

[0273] Aldosterone antagonists, such as spironolactone, eplerenone, and spironolactone.

[0274] (40) Anti-hyperlipidemia drugs, including but not limited to:

[0275] statins, such as atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin;

[0276] a combination of a statin with another drug, such as amlodipine / atorvastatin, aspirin / pravastatin, ezetimibe / simvastatin, niacin / simvastatin, lovastatin / niacin, simvastatin / sitagliptin, and atorvastatin / ezetimibe;

[0277] Fibrates or fibric acid derivatives. Examples include, but are not limited to, fenofibrate, gemfibrozil, bezafibrate, ciprofibrate, clinofibrate, and clofibrate;

[0278] Niacin (or nicotinic acid);

[0279] Bile acid sequestrants, such as cholestyramine, colesevelam, colestilan, and colestipol;

[0280] Ezetimibe, lomitapide, plant sterols, or orlistat; and

[0281] PCSK9 inhibitors, such as alirocumab and evolocumab;

[0282] (41) Neprilysin inhibitors (also known as endopeptidase inhibitors or NEP inhibitors or enkephalinase inhibitors), including but not limited to sacubitril, or a combination of sacubitril and valsartan; enkephalinase inhibitors, TD-1439 or TD-0714 under development.

[0283] (42) Renal protective drugs, including but not limited to:

[0284] Bardoxolone;

[0285] ACE inhibitors, such as captopril;

[0286] ARBs, such as losartan or irbesartan;

[0287] SGLT2 inhibitors, such as canagliflozin,

[0288] GLP1 receptor agonists;

[0289] MRAs, such as feroxetine;

[0290] ERAs, such as atrasentan; and apoptosis signal-regulating kinase 1 (ASK1) inhibitors, such as selonsertib.

[0291] (43) Hydroxyurea (HU).

[0292] (44) Anti-sickling cell drugs, including but not limited to hydroxyurea, voxelotor or GBT-440.

[0293] (45) Anti-adhesion therapy, including but not limited to blocking antibodies that adhere to P-selectin, E-selectin, VLA-4, and VCAM-1.

[0294] (46) Glutamine.

[0295] (47) Erythropoietin (EPO), also known as hematopoietin / hemopoietin, includes all its forms, such as exogenous erythropoietin, recombinant human erythropoietin (rhEPO) or other erythropoiesis stimulating agents (ESAs), two examples of which are epoetin alfa and epoetin beta.

[0296] (48) Antibiotics, including but not limited to:

[0297] Penicillin and its derivatives include, but are not limited to, penicillin, amoxicillin, ampicillin, azlocillin, cloxacillin, penicillin G, penicillin V, procaine penicillin or benzathine penicillin, etc.

[0298] cephalosporins, such as cephalexin, cefadroxil, cefaclor, cefuroxime, and cefexime;

[0299] macrolides, such as erythromycin, clarithromycin, azithromycin, and roxithromycin;

[0300] Tetracycline and its derivatives, such as demeclocycline, doxycycline, minocycline, oxytetracycline and tetracycline;

[0301] Sulfonamides, including but not limited to mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadiazine, sulfamethoxazole, sulfamethoxazole, azole, sulfasalazine, trimethoprim-sulfamethoxazole Co-trimoxazole and sulfamethoxazole azoles; and

[0302] Quinolinones, including but not limited to ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, ofloxacin and nalidixic acid.

[0303] (49) FXR agonists, including but not limited to obeticholic acid, cenicriviroc, emricasan, GR-MD-02, selonsertib, and elafibranor.

[0304] (50) Thyroid receptor-β agonists, including but not limited to MGL-3196.

[0305] (51) Acetyl-CoA carboxylase inhibitors, including but not limited to GS-0976.

[0306] (52) Treatments for mitochondrial disorders include, but are not limited to, vitamins and supplements, including mitochondrial cocktail therapy (“mito cocktail”), coenzyme Q10, vitamin B complex, especially thiamine (B1) and riboflavin (B2); alpha lipoic acid; L-carnitine; creatine; citrulline and L-arginine. As used herein, “mito cocktail” refers to a combination of vitamins and supplements that are commonly used for adults and children diagnosed with mitochondrial disease and are characterized in the relevant field. The most common ingredients in Mito cocktails include, but are not limited to, coenzyme Q10, complex vitamins (such as vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B6 (pyridoxine), vitamin B12 (cobalamin), vitamin C, vitamin E, vitamin K1 or a combination thereof), other antioxidants (such as alpha lipoic acid), L-carnitine and creatine. Mito cocktails can contain one or more of the above common ingredients according to the patient's needs and are determined by the physician.

[0307] (53) Therapeutic agents for epilepsy or epileptic seizures, including but not limited to phenytoin, valproic acid, phenobarbital, lamotrigine, carbamazepine, topiramate, oxcarbazepine, zonisamide, gabapentin, levetiracetam, pregabalin, clonazepam, lacosamide, rufinamide, and vigabatrin.

[0308] Packaging and Kits

[0309] The pharmaceutical composition (or formulation) used can be packaged in a variety of ways depending on the method used to administer the drug. Typically, the articles for distribution include containers in which the pharmaceutical formulation is stored in an appropriate form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders, etc. The container may also include a tamper-proof assemblage to prevent inadvertent contact with the contents of the package. In addition, the container is provided with a label describing the contents of the container. The label may also include appropriate warnings.

[0310] The compounds and pharmaceutical compositions described herein may be included in a kit. The kit may include single or multiple doses of two or more medicaments, each packaged or formulated separately, or single or multiple doses of two or more medicaments packaged or formulated in combination. Thus, one or more medicaments may be present in a first container, and the kit may optionally include one or more medicaments in a second container. One or more containers are placed in a package, and the package may optionally include instructions for administration or dosage. The kit may include other components, such as a syringe or other devices for administering medicaments and diluents or other devices for preparation. Thus, the kit may include: a) a pharmaceutical composition comprising a compound described herein and a pharmaceutically acceptable carrier, vehicle or diluent; and b) a container or package. The kit may optionally include instructions for using the pharmaceutical composition in one or more methods described herein (e.g., preventing or treating one or more diseases and disorders described herein). The kit may optionally include a second pharmaceutical composition, which includes one or more other medicaments, pharmaceutically acceptable carriers, vehicles or diluents described herein for co-therapeutic use. Pharmaceutical compositions comprising compounds described herein and a second pharmaceutical composition contained in the kit may be optionally combined in the same pharmaceutical composition. Example

[0311] All references provided in the examples are incorporated herein by reference. All abbreviations, symbols and conventions used herein are consistent with those used in the current scientific literature. See, for example, Janet S. Dodd, ed., The ACS Style Guide: A Manual for Authors and Editors, 2nd edition, Washington, DC: American Chemical Society, 1997, which is incorporated herein by reference in its entirety.

[0312] Various embodiments of the invention are described below.

[0313] Definitions of abbreviations used in the Examples section are provided in the table below.

[0314]

[0315]

[0316] Synthesis

[0317] Example 1: Synthesis of Compounds of Formula I

[0318] The present invention also provides a method for synthesizing a compound of formula I, which represents another embodiment of the present invention. The compound of formula I of the present invention can be prepared according to the general and specific synthetic methods described herein, the synthetic operations reported in the chemical literature, or methods known to those of ordinary skill in the art. As can be understood by those of ordinary skill in the art, the optimal reaction conditions that can be determined during the experiment can be changed based on the specific reagents used in the reaction type and the reaction. Therefore, unless specifically described, reaction conditions such as pressure, temperature, relative ratios of reagents, solvents and reaction times can be easily selected and changed by those of ordinary skill in the art without excessive experiments.

[0319] The compounds and intermediates of the present invention can be prepared by purification methods known to those skilled in the art. These methods include, but are not limited to, silica gel chromatography, recrystallization, reverse phase HPLC (RP-HPLC) and supercritical fluid chromatography (SFC). RP-HPLC purification can be achieved on a suitable reverse phase column (e.g., Waters XBridge OBD C18, 5 μm, 19×150 mm) using a suitable gradient selected from 0% to 100% acetonitrile in water containing additives such as 0.1% TFA or FA. Diastereomers can be separated by silica gel chromatography, RP-HPLC or chiral HPLC. Discrete enantiomers can be obtained by using chiral HPLC to separate a mixture of enantiomers. The progress of the reaction can be monitored by methods known to those skilled in the art, such as thin layer chromatography, reverse phase HPLC or tandem reverse phase HPLC-mass spectrometry (LC-MS).

[0320] The starting materials used in the syntheses described herein are either purchased from commercial sources or can be prepared by one of ordinary skill in the art using methods reported in the chemical literature or referenced herein.

[0321] The general methods described herein can be used to prepare compounds of Formula I and compounds of Formula I. The general and specific methods described herein are provided as illustrations for implementing the present invention. Therefore, it is not intended to impose any limitation on the scope of the subject matter of the present invention and the compounds claimed.

[0322] All references provided in the examples are incorporated herein by reference. As used herein, all abbreviations, symbols and conventions are consistent with those used in contemporary scientific literature. See, for example, G. M. Banik, G. Baysinger, P. V. Kamat, N. J. Pienta, ed., The ACS Guide to Scholarly Communication, Washington, DC: American Chemical Society, 2020 (https; / / pub.acs.org / doi / book / 10.1021 / acsguide), which is incorporated herein by reference in its entirety.

[0323] Example 1: Compound Synthesis

[0324] The compounds disclosed herein can be prepared, for example, from the corresponding nitrile intermediates using the general method depicted below (General Method C):

[0325] General Method C

[0326]

[0327] The compounds of the present invention can be prepared by methods similar to those described herein by their corresponding nitrile. Nitriles with different substitution patterns can be prepared by methods described in the following WO2015187470, WO2016081668, WO2017197555, WO2017200825, WO2018 / 045276A1 and WO2019 / 126354A1.

[0328] The following nitrile intermediates were prepared according to the literature methods described in WO2018 / 045276A1 and WO2019 / 126354A1. Reaction conditions (such as reagent ratio, temperature and reaction time) and purification methods were modified as needed.

[0329] 8-Benzylimidazo[1,2-a]pyrazine-6-carbonitrile;

[0330] 8-(3-Fluorobenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile;

[0331] 8-(2-Fluorobenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile;

[0332] 8-(2,3-difluorobenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile;

[0333] 8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile;

[0334] 8-(3-Fluoro-4-methylbenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile;

[0335] 8-(3,5-difluorobenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile;

[0336] 8-(3,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile;

[0337] 8-(2,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile;

[0338] 8-(2,5-difluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazine-6-carbonitrile;

[0339] 8-(3-Fluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazine-6-carbonitrile;

[0340] 8-(3,5-difluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazine-6-carbonitrile;

[0341] 8-(2,3-Difluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazine-6-carbonitrile;

[0342] 8-(2,5-Difluoro-4-methylbenzyl)-[1,2,4]triazolo[1,5-a]pyrazine-6-carbonitrile.

[0343] The method for synthesizing compounds I-1 to I-20 is described below. The method for synthesizing compounds I-20 to I-26 is described in patent application publication WO2019 / 126354.

[0344] General method A for the synthesis of compound I-1

[0345] The title compound was synthesized in two steps:

[0346] Step 1: Synthesis of 8-(3-fluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide

[0347]

[0348] To a solution of 8-(3-fluorobenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile (220 mg, 0.87 mmol, 1.0 equiv) in methanol (5.0 mL) was added 0.50 N sodium methoxide in methanol (0.17 mL, 0.087 mmol, 0.10 equiv) (Note: stoichiometric or excess sodium methoxide may also be used). After stirring at ambient temperature for 6 h, ammonium chloride (280 mg, 5.2 mmol, 6.0 equiv) was added and the reaction was stirred for 16 h. The reaction mixture was concentrated in vacuo and washed with half-saturated NaHCO 3 The solution (20 mL) was diluted with 2×20 mL CH 2 Cl 2 / iPrOH (5:1). The combined organic phases were dried over sodium sulfate, filtered, and concentrated to give the crude formamidine as a light brown foamy solid. It was used in the next step without further purification. LC / MSES + m / z=270.2[M+H] + .

[0349] Step 2: Synthesis of 5-fluoro-2-(8-(3-fluorobenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol

[0350]

[0351] To a suspension of 8-(3-fluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide (210 mg, 0.79 mmol, 1.0 equiv) in ethanol (7.0 mL) was added sodium (Z)-3-ethoxy-2-fluoro-3-oxoprop-1-en-1-olate (490 mg, 3.1 mmol, 4.0 equiv). The reaction was heated at 90 °C in a sealed vial for 2.5 h. After cooling to ambient temperature, 1.0 N aqueous HCl (3.1 mL, 3.1 mmol, 4.0 equiv) was added. The resulting mixture was concentrated in vacuo, diluted with water (50 mL), and washed with saturated NaHCO 3 The solution was adjusted to pH 6 and washed with 2×50 mL of CH 2 Cl 2 The combined organic phases were dried over sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel chromatography (0 to 15% acetonitrile / methanol (7:1) in CH 2 Cl 2 The obtained product was purified by HPLC (HPLC-MS / MS) to give the title compound (180 mg, 64% yield over 2 steps) as a light brown solid. 1 H NMR (500 MHz, DMSO-d 6)δ(ppm)13.1-12.5(singlet pair, 1H, tautomer),9.46(s, 1H),8.30(s, 1H),8.26-8.00(singlet pair, 1H, tautomer),7.90(s, 1H),7.50(m, 1H),7.41(m, 1H),7.32(m, 1H),7.02(app.t, 1H),4.53(s, 2H).

[0352] Compound I-2

[0353]

[0354] 2-(8-Benzylimidazo[1,2-a]pyrazin-6-yl)-5-fluoropyrimidin-4-ol (Compound I-2) (25 mg, 14% overall yield) was synthesized as a white solid according to General Method A. Reaction conditions (such as reagent ratios, temperature and reaction time) and purification methods were varied as necessary. 1 H NMR (500 MHz, methanol-d 4 )δ(ppm)9.30(s,1H),8.09(s,1H),7.99(d,1H),7.80(s,1H),7.40(d,2H),7.17(t,2H),7.07-7.11(m,1H),4.52(s,2H).

[0355] Compound I-4

[0356]

[0357] 2-(8-(2,3-difluorobenzyl)imidazo[1,2-a]pyrazin-6-yl)-5-fluoropyrimidin-4-ol (Compound 4) (150 mg, 67% overall yield) was synthesized as a light brown solid according to General Procedure A. Reaction conditions (such as reagent ratios, temperature and reaction time) and purification methods were varied as necessary. 1 H NMR (500 MHz, acetone-d 6 )δ(ppm)10.6(s,1H),9.34(s,1H),8.16(s,1H),7.92(s,1H),7.78(s,1H),7.20(t,1H),7.09(q,1H),7.00(q,1H),4.60(s,2H).

[0358] Compound I-6

[0359]

[0360] 5-Fluoro-2-(8-(3-fluoro-4-methylbenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol (Compound I-6) (200 mg, 54% overall yield) was synthesized as a pale yellow solid according to General Method A. Reaction conditions (such as reagent ratio, temperature and reaction time) and purification methods were varied as needed. 1 H NMR (500 MHz, DMSO-d 6 )δ(ppm)12.9(br.s,1H),9.44(s,1H),8.29(s,1H),8.16(br.s,1H),7.89 (s,1H),7.39(d,1H),7.27(d,1H),7.17(t,1H),4.48(s,2H),2.14(s,3H).

[0361] Compound I-7

[0362]

[0363] 2-(8-(3,5-difluorobenzyl)imidazo[1,2-a]pyrazin-6-yl)-5-fluoropyrimidin-4-ol (Compound I-7) (190 mg, 57% overall yield) was synthesized as a yellow solid according to General Method A. Reaction conditions (such as reagent ratio, temperature and reaction time) and purification methods were varied as needed. 1 H NMR (500 MHz, DMSO-d 6 )δ(ppm)13.0(br.s,1H),9.47(s,1H),8.31(s,1H),8.22(br.s,1H),7.91(s,1H),7.36(br.s,2H),7.07(t,1H),4.53(s,2H).

[0364] Compound I-3

[0365]

[0366] 5-Fluoro-2-(8-(3,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol (Compound I-3) (67 mg, 34% overall yield) was synthesized as a white solid according to General Method A. Reaction conditions (such as reagent ratio, temperature and reaction time) and purification methods were varied as necessary. 1 H NMR (500 MHz, CHLOROFORM-d) δ (ppm) 11.1 (br. s, 1H), 9.14 (s, 1H), 8.00-7.91 (m, 2H), 7.87 (s, 1H), 7.00 (d, 2H), 4.56 (s, 2H), 2.14 (s, 3H).

[0367] Compound I-14

[0368] The title compound was synthesized in two steps:

[0369] Step 1: Synthesis of 8-(2,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide

[0370]

[0371] To a suspension of 8-(2,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile (2.5 g, 8.9 mmol, 1.0 equiv) in methanol (44 mL) was added 0.50 N sodium methoxide in methanol (18 mL, 8.9 mmol, 1.0 equiv). After stirring at ambient temperature for 4 h, another portion of 0.50 N sodium methoxide in methanol (5.3 mL, 2.7 mmol, 0.3 equiv) was added and stirring was continued for another 2 h. Then ammonium chloride (470 mg, 8.9 mmol, 1.0 equiv) was added. After 16 h, the reaction mixture was concentrated in vacuo and suspended in saturated NaHCO 3 The crude product was resuspended in 100 mL of acetonitrile under heating, diluted with ether and filtered. The filter cake was washed with 3 volumes of ether and dried to give a light brown solid (2.2 g, 83% yield). It was used in the next step without further purification. LC / MS ES + m / z=302.1[M+H] + .

[0372] Step 2: Synthesis of 5-fluoro-2-(8-(2,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol

[0373]

[0374] To a solution of 8-(2,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide (1.9 g, 6.2 mmol, 1.0 equiv) in ethanol (31 mL) was added sodium (Z)-3-ethoxy-2-fluoro-3-oxoprop-1-ene-1-olate (2.9 g, 19 mmol, 3.0 equiv). The solution was heated in a sealed container at 90 °C for 18 h. After cooling to ambient temperature, a 2.5 N ethanolic HCl solution (7.4 mL, 19 mmol, 3.0 equiv) was added. The resulting mixture was concentrated in vacuo and suspended in acetonitrile (100 mL) with heating. After cooling slightly, ether (100 mL) was added and the mixture was stirred for 10 min. The solid was collected by filtration and washed with 3 volumes of ether. The resulting solid was resuspended in water, stirred for 1 h and filtered. The crude material was purified by preparative reverse phase HPLC (10% to 70% acetonitrile / water containing 0.1% trifluoroacetic acid as an additive). Impure fractions were repurified by preparative reverse phase HPLC (10% to 50% acetonitrile / water containing 0.1% trifluoroacetic acid as an additive) to give the title compound (840 mg, 37% yield) as an off-white solid. 1 H NMR (500 MHz, methanol-d 4 )δ(ppm)9.43(s,1H),8.21(s,1H),8.08(br.s,1H),7.89(s,1H),7.09(m,1H),7.00(m,1H),4.63(s,2H),2.23(s,3H).

[0375] Na of compound I-14 + Salt

[0376]

[0377] To an off-white suspension of 5-fluoro-2-(8-(2,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol (Compound I-14, 10 g, 27 mmol) in 450 mL of anhydrous MeOH was added 0.50 N sodium methoxide in methanol (54 mL, 27 mmol) under a nitrogen atmosphere. After brief sonication, the resulting pale yellow solution was stirred at ambient temperature for 15 min and concentrated to dryness in vacuo. The solid was resuspended in 250 mL of ether with sonication and concentrated (twice). The resulting solid was resuspended in 650 mL of ether and stirred at ambient temperature for 3 h. The solid was collected by vacuum filtration and washed with ether (3×100 mL). After drying on the filter overnight, the product salt was dried in a vacuum oven at 45 °C for 4 days to afford sodium 5-fluoro-2-(8-(2,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-olate (11 g, 99% yield) as a white solid. 1 H NMR (500 MHz, D 2 O)δ(ppm)8.92(s,1H),7.99(d,1H),7.97(d,1H),7.70(d,1H),6.98(dd,1H),6.86(dd,1H),4.48(s,2H),2.14(s,3H).

[0378] Compound I-11

[0379]

[0380] 5-Fluoro-2-(8-(3-fluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)pyrimidin-4-ol (Compound I-11) (61 mg, 23% overall yield) was synthesized as a golden solid according to General Method A. Reaction conditions (such as reagent ratio, temperature and reaction time) and purification methods were varied as needed. 1 H NMR (500 MHz, DMSO-d 6 )δ(ppm)13.3(br.s,1H),9.60(s,1H),8.86(s,1H),8.24-8.27(m,1H),7.32-7.47(m,3H),7.03-7.06(m,1H),4.59(s,2H).

[0381] Compound I-13

[0382]

[0383] 2-(8-(3,5-difluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)-5-fluoropyrimidin-4-ol (Compound I-13) (57 mg, 17% overall yield) was synthesized as a brown solid according to General Method A. Reaction conditions (such as reagent ratio, temperature and reaction time) and purification methods were varied as needed. 1 H NMR (500 MHz, DMSO-d 6 )δ(ppm)13.2(br.s,1H),9.61(s,1H),8.87(s,1H),8.25(s,1H),7.33(d,2H),7.10(t,1H),4.60(s,2H).

[0384] Compound I-10

[0385]

[0386] 2-(8-(2,3-difluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)-5-fluoropyrimidin-4-ol (Compound I-10) (85 mg, 16% overall yield) was synthesized as a pale yellow solid according to General Method A. Reaction conditions (such as reagent ratio, temperature and reaction time) and purification methods were varied as needed. 1 H NMR (500 MHz, DMSO-d 6 )δ(ppm)13.0(br.s,1H),9.62(s,1H),8.85(s,1H),8.23(s,1H),7.29-7.37(m,2H),7.09-7.16(m,1H),4.68(s,2H).

[0387] Compound I-12

[0388]

[0389] 2-(8-(2,5-difluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)-5-fluoropyrimidin-4-ol (Compound I-12) (75 mg, 57% yield) was synthesized as an off-white solid according to General Method A. Reaction conditions (such as reagent ratio, temperature and reaction time) and purification methods were varied as necessary. 1 H NMR (500 MHz, methanol-d 4 )δ(ppm)9.67(s,1H),8.69(s,1H),8.12(d,1H),7.25(m,1H),7.13(m,1H),7.02(m,1H),4.73(s,2H).

[0390] Compound I-19

[0391]

[0392] 5-Fluoro-2-(8-(2,5-difluoro-4-methylbenzyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)pyrimidin-4-ol (Compound I-19) (140 mg, 66% overall yield) was synthesized as a light brown solid according to General Method A. Reaction conditions (such as reagent ratio, temperature and reaction time) and purification methods were varied as needed. 1 H NMR (500 MHz, DMSO-d 6 )δ(ppm)13.1(br.s,1H),9.61(s,1H),8.83(s,1H),8.26(br.s,1H),7.35(br.s,1H),7.17(m,1H),4.57(s,2H),2.18(s,3H).

[0393] General method B for the synthesis of compound I-16

[0394] The title compound was synthesized in two steps:

[0395] Step 1: Synthesis of 8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide

[0396]

[0397] To a suspension of 8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile (490 mg, 1.8 mmol, 1.0 equiv) in methanol (5.0 mL) was added a 0.50 N solution of sodium methoxide in methanol (3.6 mL, 1.8 mmol, 1.0 equiv) (Note: catalytic amounts or excess sodium methoxide may also be used). After stirring at ambient temperature for 3 hours and 45 minutes, ammonium chloride (970 mg, 18 mmol, 10 equiv) was added and the reaction was stirred for 20 hours. The resulting mixture was concentrated in vacuo to a volume of approximately 2 mL and washed with EtOAc (20 mL) and 10% NaHCO 3 After stirring for 15 min, the product was collected by filtration, washed with water (10 mL) and dried in vacuo to give the title compound (420 mg, 80% yield) as an off-white solid. LC / MS ES + m / z=287.9[M+H] + .

[0398] Step 2: Synthesis of 5-chloro-2-(8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol

[0399]

[0400] To a suspension of 8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide (100 mg, 0.35 mmol) and ethyl 2-chloro-3-oxopropanoate (110 mg, 0.70 mmol) in methanol (1.7 mL) was added 0.50 N sodium methoxide in methanol (1.4 mL, 0.70 mmol). The reaction was heated in a sealed vial at 65 °C for 2.5 h. After cooling to ambient temperature, the resulting mixture was concentrated in vacuo, diluted with water (10 mL), adjusted to pH 3 with 6.0 N aqueous HCl, and washed with 2×15 mL of CH 2 Cl 2 The combined organic phases were dried over sodium sulfate, filtered and concentrated. The crude product was purified by silica gel chromatography (0% to 20% acetonitrile / methanol (7:1) in CH 2 Cl 2 ) and purified by silica gel chromatography (20% to 100% EtOAc / CH 2 Cl 2 ) to afford the title compound (37 mg, 28% yield) as an off-white solid.

[0401] 1 H NMR (500 MHz, DMSO-d 6 )δ(ppm)12.6(br.s,1H),9.54(s,1H),8.36(br.s,1H),8.32(s,1H),7.89(s,1H),7.45(br.s,1H),7.25(m,1H),7.12(m,1H),4.58(s,2H).

[0402] Compound I-17

[0403]

[0404] 5-Chloro-2-(8-(2,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol (Compound I-17) (5.2 mg, 2.2% overall yield) was synthesized as a light brown solid according to General Method B. Reaction conditions (such as reagent ratios, temperature and reaction time) and purification methods were varied as necessary. 1 H NMR (500 MHz, DMSO-d 6)δ(ppm)9.47(s,1H),8.45(s,1H),8.22(d,1H),7.94(s,1H),7.23(dd,1H),7.16(dd,1H),6.72(d,1H),4.56(s,2H),2.17(br s,3H).LC / MS ES + m / z=388.0[M+H] + .

[0405] Compound I-15

[0406] The title compound was synthesized in two steps:

[0407] Step 1: Synthesis of 8-(2,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide

[0408]

[0409] 8-(2,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide (840 mg, 76% yield) was synthesized as a light brown solid according to step 1 of general method A or B. Reaction conditions (such as reagent ratio, temperature and reaction time) and purification methods were varied as necessary. LC / MS ES + m / z=302.0[M+H] + .

[0410] Step 2: Synthesis of 2-(8-(2,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol

[0411]

[0412] To a suspension of 8-(2,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide (360 mg, 1.2 mmol) and methyl 3-methoxyacrylate (0.39 mL, 3.6 mmol) in ethanol (6.0 mL) was added Hunig's base (0.63 mL, 3.6 mmol). The reaction was heated in a sealed vial at 90 °C for 3 h. After cooling to ambient temperature, the resulting mixture was treated with 2.5 N ethanolic HCl solution (1.4 mL, 3.6 mmol) and concentrated to dryness. The crude material was purified by silica gel chromatography (0% to 20% acetonitrile / methanol (7:1) in CH 2 Cl 2 ) and purified by silica gel chromatography (0 to 15% MeOH / CH 2 Cl 2 ) to give the title compound (120 mg, 28% yield) as a light brown solid. 1H NMR (500 MHz, DMSO-d 6 )δ(ppm)11.9(br.s,1H),9.52(s,1H),8.31(s,1H),8.07(br.d,1H),7.89(s, 1H),7.37(dd,1H),7.16(dd,1H),6.38(br.d,1H),4.54(s,2H),2.18(s,3H).

[0413] Compound I-8

[0414] The title compound was synthesized in two steps:

[0415] Step 1: Synthesis of 8-(2-fluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide

[0416]

[0417] 8-(2-Fluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide (5.1 g, 91% yield) was synthesized as a creamy yellow solid according to step 1 of general method A or B. Reaction conditions (such as reagent ratio, temperature and reaction time) and purification methods were varied as necessary. LC / MS ES + m / z=270.2[M+H] + .

[0418] Step 2: Synthesis of 2-(8-(2-fluorobenzyl)imidazo[1,2-a]pyrazin-6-yl)-5-methylpyrimidin-4-ol

[0419]

[0420] To a solution of 8-(2-fluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide (400 mg, 1.5 mmol) and ethyl 2-methyl-3-oxopropanoate (230 mg, 1.8 mmol) in t-BuOH (9.9 mL) was added potassium bicarbonate (220 mg, 2.2 mmol). The reactant was heated to reflux for 2 h. After cooling to ambient temperature, water was added and the product was collected by filtration and dried to give the title compound (410 mg, 82% yield) as a creamy yellow solid. 1 H NMR (500 MHz, DMSO-d 6 )δ(ppm)11.6(br.s,1H),9.48(s,1H),8.30(s,1H),7.94(br s,1H),7.88(s,1H),7.48(app.t,1H),7.29(m,1H),7.19(m,1H),7.11(app.t,1H),4.60(s,2H),1.98(s,3H).

[0421] Compound I-9

[0422]

[0423] A solution of 5-fluoro-2-(8-(2-fluorobenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol (230 mg, 0.67 mmol) in 9.0 mL of acetonitrile-THF (2:1) was stirred with sodium bicarbonate (84 mg, 1.0 mmol) and Selectfluor TM (350 mg, 1.0 mmol) and heated at 50°C. During the experiment, additional sodium bicarbonate (42 + 28 mg) and Selectfluor TM (180+120 mg). After a total of 49 h, the reaction was cooled to ambient temperature and 20 mL of water was added. The resulting mixture was acidified to pH 3 with 1.0 N aqueous HCl and extracted with 2×25 mL of EtOAc. The combined organic phases were dried over sodium sulfate, filtered, and concentrated. The crude material was purified by silica gel chromatography (0% to 20% acetonitrile / methanol (7:1) in CH 2 Cl 2 The product was purified by HPLC (15% to 65% acetonitrile / water containing 0.1% formic acid as an additive) to give the title compound (23 mg, 9.7% yield) as a light brown solid. 1 H NMR (500 MHz, DMSO-d 6 )δ(ppm)12.6(br.s,1H),8.98(s,1H),8.19(br.s,1H),7.74(d,1H),7.48(app.t,1H),7.28(m,1H),7.19(m,1H),7.10(app.t,1H),4.56(s,2H).

[0424] Compound I-5

[0425]

[0426] A solution of 5-fluoro-2-(8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol (200 mg, 0.56 mmol) in 10 mL of acetonitrile-THF (1:1) was stirred with sodium bicarbonate (94 mg, 1.1 mmol) and Selectfluor TM (400 mg, 1.1 mmol) and heated at 50°C. During the experiment, additional sodium bicarbonate (3 x 47 mg) and Selectfluor TM(3 x 200 mg). After a total of 74 h, the reaction was cooled to ambient temperature and 40 mL of water was added. The resulting mixture was acidified to pH 3 with 1.0 N aqueous HCl and washed with 2 x 40 mL of CH 2 Cl 2 The combined organic phases were dried over sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel chromatography (0 to 20% acetonitrile / methanol (7:1) in CH 2 Cl 2 Purification by HPLC (10% to 70% acetonitrile / water with 0.1% TFA as additive) and final column chromatography (20% to 100% EtOAc / hexanes) gave the title compound (24 mg, 11% yield) as a white solid. 1 HNMR (500 MHz, DMSO-d 6 )δ(ppm)12.8(br.s,1H),8.99(s,1H),8.20(br.s,1H),7.75(d,1H),7.42(m,1H),7.25(m,1H),7.13(m,1H),4.54(s,2H).

[0427] Compound I-18

[0428] The title compound was synthesized in 5 steps:

[0429] Step 1: Synthesis of 6,8-dibromo-3-fluoroimidazo[1,2-a]pyrazine

[0430]

[0431] A solution of 6,8-dibromoimidazo[1,2-a]pyrazine (2.4 g, 8.7 mmol) in 40 mL of acetonitrile was prepared by using Selectfluor TM (4.6 g, 13 mmol) and heated at 50° C. After 22 h, the reaction was cooled to ambient temperature and poured into 150 mL of half-saturated NaHCO 3 The solution was dissolved and extracted with 2 x EtOAc (400 mL total). The combined organic phases were dried over sodium sulfate, filtered and concentrated. The crude was purified by silica gel chromatography (0 to 20% EtOAc / hexanes) to give the title compound (580 mg, 23% yield) as an orange solid.

[0432] Step 2: Synthesis of 6-bromo-8-(2,5-difluoro-4-methylbenzyl)-3-fluoroimidazo[1,2-a]pyrazine

[0433]

[0434] A suspension of dry zinc powder (240 mg, 3.7 mmol) in THF (3.0 mL) was treated with 1,2-dibromoethane (30 mL, catalyst) and the resulting mixture was heated at 50 ° C. Chlorotrimethylsilane (30 mL, catalyst) was then added. After 15 minutes, the mixture was cooled to ambient temperature. Dry lithium chloride (170 mg, 3.9 mmol) was added, followed by a solution of 1- (bromomethyl) -2,5-difluoro-3-methylbenzene (480 mg, 2.2 mmol) in THF (2.0 mL) was added dropwise (note: exothermic reaction). The mixture was stirred at ambient temperature for 1 h. At the same time, 6,8-dibromo-3-fluoroimidazo [1,2-a] pyrazine (580 mg, 2.0 mmol) and Pd (PPh 3 ) 2 Cl 2 A slurry of 4-(41 mg, 0.059 mmol) in THF (3.0 mL) was degassed with nitrogen. The newly formed zincate solution was transferred to this slurry via syringe and rinsed with 2×0.5 mL THF to ensure complete transfer. The resulting mixture was stirred at ambient temperature for 1 hour 20 minutes and then at 40° C. for 4 h. After cooling to ambient temperature, the reaction was washed with 4 mL of saturated NH 4 The organic layer was concentrated and washed with CH 2 Cl 2 (10 mL) and filtered through a celite bed. The filtrate was concentrated to give a brown residue which was purified by silica gel chromatography (loaded with CH 2 Cl 2 The compound was purified by eluting with 0 to 10% EtOAc / hexanes to give the title compound (440 mg, 63% yield) as a yellow solid.

[0435] Step 3: Synthesis of 8-(2,5-difluoro-4-methylbenzyl)-3-fluoroimidazo[1,2-a]pyrazine-6-carbonitrile

[0436]

[0437] 6-Bromo-8-(2,5-difluoro-4-methylbenzyl)-3-fluoroimidazo[1,2-a]pyrazine (440 mmol, 1.2 mmol), zinc cyanide (100 mg, 0.87 mmol), Pd 2 (dba) 3 The reaction mixture of 1,1′-bis(diphenylphosphino)ferrocene (dppf) (41 mg, 0.075 mmol) in anhydrous DMF (5.0 mL) was degassed with nitrogen and then heated at 90° C. for 6 h. The reaction was cooled to ambient temperature and washed with CH 2 Cl2 (50 mL), water (40 mL) and 28% ammonium hydroxide solution (4.0 mL). The aqueous layer was washed with CH 2 Cl 2 (50 mL) was extracted. The combined organic layers were purified by Na 2 SO 4 Drying, filtration and concentration gave a brown oil which was purified by column chromatography (0 to 20% EtOAc / hexanes gradient) to give the title compound as a light brown solid (310 mg, 81% yield). LC / MS ES + m / z=302.8[M+H] + .

[0438] Step 4: Synthesis of 8-(2,5-difluoro-4-methylbenzyl)-3-fluoroimidazo[1,2-a]pyrazine-6-carboximidamide

[0439]

[0440] To a suspension of 8-(2,5-difluoro-4-methylbenzyl)-3-fluoroimidazo[1,2-a]pyrazine-6-carbonitrile (150 mg, 0.50 mmol) in methanol (6.0 mL) was added 0.50 N sodium methoxide in methanol (1.0 mL, 0.50 mmol). After stirring at ambient temperature for 4 hours and 30 minutes, ammonium chloride (270 mg, 5.0 mmol) was added and the reaction was stirred for 18 hours. The resulting mixture was concentrated in vacuo and washed with 10% NaHCO 3 Aqueous solution (10 mL) was treated and sonicated to give a suspension. After stirring for 1 h, the product was collected by filtration, washed with water (10 mL) and dried in vacuo to give the title compound (170 mg, >100% yield) as a light brown solid. It was used in the next step without further purification. LC / MS ES + m / z=319.7[M+H] + .

[0441] Step 5: Synthesis of 2-(8-(2,5-difluoro-4-methylbenzyl)-3-fluoroimidazo[1,2-a]pyrazine-6-fluoropyrimidin-4-ol

[0442]

[0443] To a suspension of 8-(2,5-difluoro-4-methylbenzyl)-3-fluoroimidazo[1,2-a]pyrazine-6-carboximidamide (0.50 mmol, theoretical amount from the previous step) in ethanol (5.0 mL) was added sodium (Z)-3-ethoxy-2-fluoro-3-oxoprop-1-ene-1-olate (310 mg, 2.0 mmol). The reaction was heated in a sealed vial at 90 °C for 16 h. After cooling to ambient temperature, the mixture was diluted with water (7.5 mL) and adjusted to pH 4 with 1N aqueous HCl. The resulting light brown solid was collected by filtration, washed with water (50 mL) and diethyl ether (30 mL), and dried to give the title compound (140 mg, 71% yield over 2 steps) as a brown solid. 1 H NMR (500 MHz, DMSO-d 6 )δ(ppm)12.8(br.s,1H),8.98(s,1H),8.22(br.s,1H),7.74(d,1H),7.35(br.s,1H),7.15(m,1H),4.50(s,2H),2.18(s,3H).

[0444] Compound I-20

[0445] The title compound was synthesized in two steps:

[0446] Step 1: Synthesis of 8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide

[0447]

[0448] To a solution of 8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile (500 mg, 1.9 mmol) in methanol (22 mL) was added a 25 wt% solution of sodium methoxide in methanol (2.1 mL, 9.3 mmol). After stirring at ambient temperature for 1 h, ammonium chloride (1.0 g, 19 mmol) was added and the reaction was stirred overnight. The reaction mixture was concentrated in vacuo and washed with half-saturated NaHCO 3 The solution was diluted with 1.0 N sodium hydroxide solution (2.0 mL) and extracted with 2×20 mL EtOAc. The combined organic phases were dried over sodium sulfate, filtered and concentrated to give the crude product as a brown solid. It was used in the next step without further purification. LC / MS ES + m / z=288.1[M+H] + .

[0449] Step 2: Synthesis of 5-fluoro-2-(8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol

[0450]

[0451] To a suspension of 8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide (500 mg, 1.7 mmol) in ethanol (9.0 mL) was added (Z)-3-ethoxy-2-fluoro-3-oxoprop-1-ene-1-ol sodium (820 mg, 5.2 mmol). The reactants were heated in a sealed vial at 90 °C for 2 h. After cooling to ambient temperature, concentrated HCl solution was added dropwise to acidify the mixture to pH 4. The resulting mixture was concentrated in vacuo. Purification by preparative reverse phase HPLC (acetonitrile-water gradient elution, containing 0.1% TFA as an additive) gave the title compound as a yellow solid (200 mg, 28% yield over 2 steps). 1 H NMR (500 MHz, DMSO-d 6 )δ(ppm)12.6(br.s,1H),9.49(s,1H),8.32(s,1H),8.19(br.s,1H),7.89(s,1H),7.43(s,1H),7.25(m,1H),7.13(m,1H),4.58(s,2H).

[0452] Na of compound I-20 + Salt

[0453]

[0454] To a light brown suspension of 5-fluoro-2-(8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol (Compound I-20, 10 g, 28 mmol) in 450 mL of anhydrous MeOH was added a 0.50 N solution of sodium methoxide in methanol (57 mL, 28 mmol) under nitrogen. After brief sonication, the resulting light orange solution was stirred at ambient temperature for 15 min and concentrated to dryness in vacuo. The solid was resuspended in 200 mL of ether with sonication and concentrated (twice). The resulting solid was resuspended in 500 mL of ether and stirred at ambient temperature for 3 h. The solid was collected by vacuum filtration and washed with ether (3×100 mL). After drying on the filter overnight, the product salt was dried in a vacuum oven at 45 °C for 5 days to afford sodium 5-fluoro-2-(8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-olate (11 g, 99% yield) as a light brown solid. 1 H NMR (500 MHz, D 2O)δ(ppm)8.90(s,1H),7.98(d,1H),7.95(d,1H),7.70(d,1H),7.10(m,1H),6.98-6.89(m,2H),4.53(s,2H).

[0455] Compound I-27

[0456] Compound I-27 was synthesized based on the synthesis scheme shown below.

[0457]

[0458] Using BH in THF 3 Compound 27a was reduced to compound 27b, which was then reacted with PBr 3 Compound 27c was formed by reaction. Compound 27c was converted to the corresponding organozinc compound using zinc, dibromoethane and TMSCl. The organozinc intermediate was subjected to Negishi coupling with dibromo compound 27e, which was in turn formed by cyclization of compound 27d with 3-bromo-1,1,1-trifluoropropane-2-one to give compound 27f. Compound 27f was synthesized in the presence of zinc, zinc cyanide and Pd 2 (dba) 3 Further reaction in the presence of 27g afforded cyano compound 27g. The nitrile of compound 27g reacted with ammonium chloride to afford compound 27h. The amidine of compound 27h was condensed with compound 27i according to general method A to afford cyclized compound I-27.

[0459] Compound I-28

[0460] Compound I-28 was synthesized based on the synthesis scheme shown below.

[0461]

[0462] Compound 28a was methylated to form compound 28b, which was brominated with NBS to give compound 28c. Compound 28c was converted into the corresponding organozinc compound using zinc, dibromoethane and TMSCl. The organozinc compound was subjected to Negishi coupling with compound 28d to give compound 28e. Compound 28e was reacted with zinc, zinc cyanide and Pd 2 (dba) 3 Further reaction in the presence of cyano compound 28f was obtained.

[0463] The nitrile of is reacted with ammonium chloride to give compound 28g. The amidine of compound 28g is condensed with compound 28h according to general method A to give cyclized compound I-28.

[0464] Compound I-29

[0465] Compound I-29 was synthesized according to general method B.

[0466]

[0467] Biological part

[0468] Evaluation of the biological properties of compounds of formula I

[0469] The present application also provides an evaluation of the biological properties of the compounds of Table I. Representative compounds of the present invention are tested in vitro for activity as sGC stimulators in various cells and assays, and in vivo for their ability to reduce blood pressure in animals. Blood pressure reduction is used to indicate the ability of the compound to engage the target peripherally in vivo. Other tests are used to indicate the ability of these compounds to cross the BBB, engage the target in the CNS, increase the level of cGMP in the CNS, and thereby stimulate the functional response of the animal. These biological properties represent another embodiment of the present invention.

[0470] Example 2: Bioactivity Measurement by cGMP Glo Sensor Cell-Based Assay, 384-Well Format

[0471] Expressing GloSensor TM Human embryonic kidney (HEK293) cells expressing 40F cGMP (Cat. No. CS182801, Promega) were used to assess the activity of test compounds. A luminescent biosensor (engineered luciferase) incorporated into these cells detects cGMP formed by compounds that stimulate the sGC enzyme and emits fluorescence.

[0472] cGMP GloSensor cells were maintained in Dulbecco's Modification of Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS, 10% final) and hygromycin (200ug / ml). The day before the assay, cells were plated at 1.5×10 4 Cells were plated at a density of 10 cells / well in a volume of 50 μL in DMEM with 10% FBS in a poly-D-lysine coated 384-well flat white bottom plate (Corning catalog number 35661). The cells were incubated at 37°C in an atmosphere of 5% CO 2 The cells were cultured overnight in a humidified chamber. The next day, the culture medium was removed and 40 μl / well of GloSensor TM , 2 mM (Promega, catalog number E1291) to replace the cells. The cells were treated at 25°C for 90 minutes to allow the matrix to equilibrate in the cells. The test compounds and diethylenetriamine NONO salt (DETA-NONO salt) were incubated in a serum-free CO 2The 5X dose curve was generated by diluting 10 μl of the solution to 3 mM (20×) in independent medium and serially diluting in 4× dilutions to generate a 5X dose curve, from which 10 μl was added to each well (x μM concentration for test compound solution and 10 μM concentration for DETA-NONO salt solution; where x is one of the following final concentrations: 30 μM, 7.5 μM, 1.9 μM, 469 nM, 117 nM, 29.3 nM, 7.3 nM, 1.83 nM, 0.46 nM, 0.11 nM, 0.03 nM). For kinetic studies, fluorescence was immediately measured at 0.2 sec / well using Envision (Perkin Elmer). For endpoint SAR screening, data were collected after 55 min of incubation at room temperature.

[0473] Concentration response data were analyzed using a 4-parameter fit (log(agonist) vs. response - variable slope). 50 is interpolated from the curve fit and is defined as the concentration of the compound eliciting 50% of its maximal response. When multiple experiments were performed for a given compound, the geometric mean of all experiments is reported.

[0474] Table A below summarizes the EC values ​​of the compounds of the invention in the Glo assay: 50 value.

[0475] Table A

[0476] Compound No. <![CDATA[Glo EC 50 (nM)]]> Compound No. <![CDATA[Glo EC 50 (nM)]]> I-21 B I-5 A I-2 B I-14* A I-1 B I-16 A I-4 B I-15 B I-23 B I-19 B I-20* A I-17 A I-11 B I-18 A I-13 B I-12 B I-10 B I-3 A I-6 A I-8 C I-7 B I-9 B

[0477] The sGC enzyme activity values ​​in HEK cells were determined by GloSensor assay. The sGC enzyme activity values ​​(expressed as EC 50 , which is defined as the concentration of a compound that elicits 50% of its maximal response) Code definition: EC 50 ≤100nM=A;100nM <EC 50 ≤1000nM=B;1000nM <EC 50 = C. *For compounds 1-20 and 1-14, both the free acid and the sodium salt were used and the results here are the average of all experiments run independently of the format.

[0478] Example 3. Bioactivity Measurement by cGMP Neuronal Cell-Based Assay

[0479] Rat primary neurons were isolated from fetuses of 18-day pregnant Sprague-Dawley female rats. The fetuses were collected in Hank's balanced salt solution (HBSS) and the brains were quickly removed. The hippocampus was isolated and mechanically fragmented. The cells were incubated at 37°C in the absence of Ca 2+ and Mg 2+Further tissue digestion was performed in HBSS with 0.25% (wt / vol) trypsin solution for 15 min. After trypsinization, the cells were washed and resuspended in neurobasal medium supplemented with 0.5 mM L-glutamine, 12.5 uM glutamate, 2% B-27, 100 U / mL penicillin and 100 μg / mL streptomycin. The cells were plated at 26 × 10 3 or 3×10 4 or 4×10 4 The cells were seeded at a density of 10 cells / well in a poly-D-lysine-coated 384-well flat clear bottom plate (Corning catalog number 354662). The cells were cultured at 37°C in an atmosphere of 5% CO 2 The cells were cultured in a humidified chamber for 6 to 7 days. The medium was removed and the cells were washed with water containing Ca 2+ and Mg 2+ The cells were washed 1× with 40uL of HBSS containing 0.5mM IBMX and replaced with 40uL of HBSS containing 0.5mM IBMX and incubated at 37°C for 15 minutes. 10uL of 5× test compound stock solution with diethylenetriamine NONO salt (DETA-NO) was added. The final concentration of DETA-NO was 10μM or 30μM. The cells were incubated at 37°C for 20min. The culture medium was removed, 50uL of ice-cold 10% acetic acid was added, and incubated at 4°C for 60 minutes. After centrifugation at 1000×g for 5 minutes at 4°C to precipitate cell debris, the supernatant was aspirated into a clean dish and the cGMP content of the sample was analyzed. The cGMP concentration was determined from each sample using LC-MS / MS.

[0480] Concentration response data were analyzed using a 4-parameter fit (log(agonist) vs. response - variable slope). 50 is interpolated from the curve fit and is defined as the concentration of the compound that elicits 50% of its maximal response. When multiple experiments were performed for a given compound, the geometric mean of all experiments is reported.

[0481] Table B below summarizes the EC values ​​of compounds of the invention in neuronal assays: 50 value.

[0482] Table B

[0483]

[0484] Neuron-based cell assays. EC 50 ≤100nM=A;100nM <EC 50 ≤1000nM=B;1000nM <EC 50 = C. *For compounds 1-20 and 1-14, both the free acid and the sodium salt were used and the results here are the average of all experiments run independently of the format.

[0485] Example 4: Measurement of biological activity of human α2β1 sGC isozyme stably expressed in CHO-K1 cells

[0486] sGC stimulators were dissolved in DMSO as 10 mM solutions and stored at -20° C. To achieve the desired test concentrations, stock concentrations were serially diluted into DMSO and then diluted to the appropriate concentration in assay buffer.

[0487] at 37°C in air containing 5% CO 2 CHO-K1 cells stably transfected with human α2β1sGC isozyme (generated by GenScript from Ironwood) were cultured in F-12K medium (ATCC catalog number 30-2004) with 10% fetal bovine serum, 4 μg / mL puromycin (Gibco catalog number A11138-03) and 0.4 mg / mL geneticin (Gibco catalog number 10131-027) in a 95% humidified atmosphere. For GC activity analysis, cells were plated at 3×10 4 cells / well or 15×10 3 The cells were seeded at a density of 50 μL or 70 μL in 384-well poly-D-lysine coated flat bottom plates (Fisher Scientific #08-774-311). The cells were incubated at 37°C in an atmosphere supplemented with 5% CO 2 The cells were cultured in a humid chamber for 24 h.

[0488] For each test concentration, the compound was diluted to one percent of its final assay concentration in 100% DMSO. Just prior to analysis, the solution was diluted 20-fold into HBSS containing calcium, magnesium, and 50 μM DETA-NONO salts (5× final assay concentration). The culture medium was removed and the cells were washed once with 40 μL HBSS. The cells were then incubated for 15 min at 37°C with 40 μL of a solution containing 0.5 mM IBMX in HBSS. 10 μL of the solution from the sGC stimulator / HBSS / DETA-NONO salts plate was added to the cells and incubated for another 20 min at 37°C. The final DMSO concentration was 1%, the final DETA-NONO salt concentration was 10 μM; and the final compound concentration was 30,000 nM, 6000 nM, 1200 nM, 240 nM, 48 nM, 9.6 nM, 1.92 nM, 0.384 nM, 0.077 nM, 0.015 nM, or 0.003 nM.

[0489] After incubation with compounds, the assay buffer was removed and 50 μL of ice-cold 10% acetic acid + 150 ng / mL internal standard (+3 cGMP) was added to each well. The samples were incubated on ice for 30 to 60 min. After centrifugation at 1000×g for 5 min at 4°C to pellet cell debris, the supernatant was transferred to a clean dish and the samples were analyzed for cGMP content.

[0490] Data were analyzed using GraphPad Prism software v.8 with a 4-parameter fit (log(agonist) vs. response - variable slope). 50 is interpolated from the curve fit and is defined as the concentration of the compound that elicits 50% of its maximal response. When multiple experiments were performed for a given compound, the geometric mean of all experiments is reported.

[0491] Table C below summarizes the EC values ​​of the compounds of the invention in the CHO assay. 50 value.

[0492] Table C

[0493]

[0494] CHO cell analysis. 50 ≤100nM=A;100nM <EC 50 ≤1000nM=B;1000nM <EC 50 = C. *For compounds 1-20 and 1-14, both the free acid and the sodium salt were used and the results here are the average of all experiments run independently of the format.

[0495] Example 5: Blood levels of representative compounds of the invention in normotensive rats after acute doses at various concentrations Pressure Impact

[0496] a) Compound I-14

[0497] Normal blood pressure male Sprague Dawley rats were purchased from Charles River Laboratory. These rats have been placed with indwelling femoral artery catheters. The animals were tied to a tether system and connected to a pressure sensor to monitor cardiovascular (CV) parameters, specifically mean arterial pressure (MAP) and heart rate (HR). Animals were adapted to the system overnight and baseline CV parameters were collected. Subsequently, 1, 3, 10 and 30 mg / kg single oral doses of Milli-Q water containing compound I-14 (doses produced by the sodium salt of compound 14) were administered to awake, freely moving rats. Blood samples were collected from each animal by catheter line 2 hours before and after administration for quantitative compound concentration. Hemodynamic measurements within ten hours after administration were recorded. Fifty-four male rats were used for these studies and sorted within 250 to 275 grams of body weight for accommodation. According to protocol MIL-110, they were housed individually under controlled conditions of temperature (21 ± 1 ° C), relative humidity (36 ± 1%) and placed in a 12-hour light-dark cycle (lights on at 6:00 AM and off at 6:00 PM) room in a SmartLabs breeding room (21 Erie Street, Cambridge, MA). Animals were allowed to access food (LabDiet Prolab Isopro RMH 3000, St. Louis, MO) and water ad libitum. Two sets of studies were conducted. For the first set of studies, compound I-14 was formulated in Milli-Q water at 0.3 and 1.0 mg / ml and frozen at -20 ° C. For the second set of studies, the sodium salt of compound I-14 was weighed at Cyclerion Therapeutics and reconstituted at SmartLabs to provide 0.1, 0.3, 1.0 and 3.0 mg / ml solutions of compound I-14 in Milli-Q water. The prepared formulation was thawed within 4 hours prior to dosing and stored at room temperature, or prepared within 2 hours prior to dosing and stored at room temperature.

[0498] The study was conducted over 6 separate phases. The total number of subjects and treatment assignments are listed in the table below. Animals were initially used within 3 days of receipt and once more after 6 to 7 days of cleaning if the catheter remained patent. No animal was used more than twice.

[0499] Groups Number of animals Therapeutic agents Dose volume Dose concentration 1 13 Medium 10mL / kg - 2 9 1 mg / kg Compound I-14 10mL / kg 0.1mg / mL 3 6 3 mg / kg Compound I-14 10mL / kg 0.3mg / mL 4 11 10 mg / kg Compound I-14 10mL / kg 1.0mg / mL 5 6 30 mg / kg Compound I-14 10mL / kg 3.0mg / mL

[0500] Blood pressure measurement

[0501] This study uses ADInstruments LabChart (v8) to collect hemodynamic data from awake, freely moving rats tethered to a blood pressure sensor (Harvard Apparatus catalog number APT300). After overnight adaptation to the tether and pressure sensor, the animals were dosed after a 1-hour baseline recording period. Animals were administered a single oral (PO) dose of the sodium salt form of compound I-14 or vehicle at a dose volume of 10 mL / kg. Data collection continued for 10 hours after dosing.

[0502] ADInstruments LabChart (v8) was used to monitor and export hemodynamic data. Blood pressure and heart rate were continuously monitored, and data were collected at 1000 data points / second, then averaged into 10-minute groups for analysis. The change in MAP relative to baseline (ΔBMAP) and the change in HR relative to baseline (ΔBHR) were calculated using Microsoft Excel of Microsoft 365 using the pre-dose baseline averaged over a 1-hour period before administration. The ΔVMAP peak, the time of the ΔVMAP peak, the ΔVHR peak, and the time of the ΔVHR peak were determined using this 10-minute grouping data set. The data set was further merged into a 1-hour grouping for MAP and HR graphs and analysis of ΔBMAP, ΔBMAP, and ΔBHR. The definitions of these terms / abbreviations are summarized below:

[0503] <![CDATA[Δ B MAP,dMAP]]> Change in mean arterial pressure from baseline <![CDATA[Δ B HR,dHR]]> Change in heart rate from baseline <![CDATA[Δ V MAP]]> Vehicle-adjusted mean arterial pressure <![CDATA[Δ V MAP]]> Vehicle-adjusted heart rate AOC Area on the curve

[0504] Statistical analysis was performed in GraphPad Prism (v8). The significance of ΔBMAP and ΔBHR data compared to vehicle-treated rats was determined by two-factor repeated measures ANOVA followed by Dunnett's multiple comparison test, and mixed effects analysis was used if there were missing data points. The vehicle-adjusted MAP (ΔVMAP) was calculated by deducting the ΔBMAP of the vehicle group from the ΔBMAP of each dosage group at each time point. The vehicle-adjusted HR (ΔVHR) was calculated in a manner similar to ΔVMAP.

[0505] The significance of AOC data compared to vehicle was determined by one-way ANOVA followed by Dunnett's multiple comparison test.

[0506] Some research data were removed before analysis. Due to 2-hour blood sample collection, data collected at 130min and 140min after administration were removed. Due to signal loss during the experiment starting at 470 minutes and continuing until the end of the study (600min), several time points under 1mg / kg were excluded for one rat. For various reasons (including that they were outliers for specific analysis or abnormal results caused by signal loss), the entire time course of 5 animals was excluded from all data sets.

[0507] Blood pressure changes

[0508] The change in MAP from baseline (ΔBMAP) is shown graphically in Figure 1 Rats treated with Compound 1-14 had a greater reduction in MAP (as assessed by the change in MAP from baseline, ΔBMAP) compared to vehicle-treated rats. The ΔBMAP data set was significant by two-way ANOVA (p<0.0001 for treatment and time, and p=0.045 for treatment×time interaction). Dunnett's multiple comparison test for the main treatment effect yielded: p=ns for 1 mg / kg, p=0.0034 for 3 mg / kg, and p<0.0001 for 10 and 30 mg / kg doses compared to vehicle-treated rats. Compared with vehicle-treated rats, separate Dunnett's multiple comparison tests for single effects at each time point and each dose showed that ΔBMAP was reduced more throughout the 6-hour period after dosing in rats treated with 10 and 30 mg / kg Compound 1-14; in rats treated with 3 mg / kg Compound 1-14, it was reduced more at 1-hour, 2-hour, and 3-hours after dosing; and in rats treated with 1 mg / kg Compound 1-14, it was not reduced.

[0509] The maximum effect of Compound 1-14 on ΔVMAP was calculated by using the 10-minute binned data set and is shown in Table D below:

[0510] Table D. Maximum Effect of Compound 1-14 on Vehicle-Adjusted MAP (ΔVMAP)

[0511]

[0512]

[0513] As determined by main effect analysis, single effect analysis and AOC assessment, the dose with no effect on ΔBMAP was 1 mg / kg.

[0514] in conclusion

[0515] Compound 1-14 reduced MAP at 3, 10 and 30 mg / kg relative to baseline and as adjusted from vehicle.

[0516] b) Compound I-20

[0517] A study similar to the study described above was conducted with compound 1-20. Single oral doses of 1, 3, 10 and 30 mg / kg containing compound 1-20 (doses prepared from the sodium salt of compound 1-20) in Milli-Q water were administered to awake, freely moving rats. Blood samples were collected from each animal through a catheter line 2 hours before and after dosing for compound concentration quantification. Hemodynamic measurements were recorded ten hours after dosing.

[0518] Blood pressure changes

[0519] The change in MAP from baseline (ΔBMAP) is shown graphically in Figure 2 Compared to vehicle-treated rats, rats treated with Compound 1-20 had a greater reduction in MAP (as measured by the change in MAP relative to baseline, Δ B MAP, to assess). By two-way ANOVA, Δ B The MAP data set was significant (p<0.0001 for treatment and treatment×time interaction; and p=0.022 for time). Dunnett's multiple comparison tests for the main treatment effect yielded p=0.055 (ns) for 1 mg / kg, p=0.0001 for 3 mg / kg, and p<0.0001 for 10 and 30 mg / kg doses compared to vehicle-treated rats. Separate Dunnett's multiple comparison tests for the single effects at each time point and dose showed that MAP was reduced more in rats treated with 3, 10, and 30 mg / kg of Compound 1-20 throughout the 6-hour post-dose period compared to vehicle-treated rats; and reduced more at 1-hour, 2-hour, 3-hour, 4-hour, and 5-hour post-dose in rats treated with 1 mg / kg of Compound 1-20.

[0520] The effect of compound 1-20 on Δ V The maximum effect of MAP (vehicle adjusted MAP) is shown in Table E below:

[0521] Table E. Maximum Effect of Compound 1-20 on Vehicle-Adjusted MAP (ΔVMAP)

[0522] Compound I-20 <![CDATA[Peak Δ V MAP (mm Hg)]]> Time to peak (min) 1mg / kg -14.3 20 3mg / kg -19.2 20 10mg / kg -20.2 30 30mg / kg -38.1 30

[0523] in conclusion

[0524] Compound 1-20 reduced MAP at 1, 3, 10 and 30 mg / kg relative to baseline and as adjusted from vehicle.

[0525] c) Other BP measurements

[0526] In studies similar to those described above, Compound I-4 formulated in PEG400 and administered at 10 mg / kg showed a maximal decrease in MAP from baseline (Δ B MAP) was 20 mm Hg. In studies similar to those described above, compound 1-20 formulated in PEG400 and administered at 10 mg / kg showed a peak Δ B MAP was -26 mm Hg. In another study where compound 1-20 was tested at 1, 3 or 10 mg / kg and formulated in methylcellulose, the compound was able to reduce MAP relative to baseline at all doses tested.

[0527] Example 6: sGC stimulator-induced CREB phosphorylation in rat primary neurons

[0528] Target

[0529] To evaluate the ability of the compounds of the present invention to activate cAMP response element binding protein (CREB) in rat primary neurons. CREB is a cellular transcription factor. It binds to a DNA sequence called cAMP response element (CRE) and regulates the transcription of downstream genes (see Bourtchuladze R, et al., Cell 1994; 79 (1): 59-68). There is sufficient evidence to prove that CREB plays a role in neuronal plasticity and long-term memory formation in the brain, and has been shown to be indispensable in spatial memory formation (see Silva AJ, et al., Annual Review of Neuroscience 1998; 21: 127-148). CREB protein is activated by phosphorylation of serine 133 by various kinases, including cAMP-dependent protein kinase or protein kinase A (PKA), cGMP-dependent protein kinase or protein kinase G (PKG) and Ca 2+ / calmodulin-dependent protein kinase. (See Shaywitz AJ and Greenberg ME, Annual Review of Biochemistry 1999; 68(1):821–861 and Wong JC, et al., J Cell Biochem 2012:113(11):3587-98). Stimulation of CREB may have therapeutic benefits for diseases in which cognition, neuronal plasticity and or neuronal function are impaired.

[0530] Rat primary neuron culture

[0531] Neurons were isolated from Sprague Dawley rat embryos at embryonic day 18 (E18). Approximately 10 embryos were obtained from each rat, and the entire brain was isolated from the embryo. Two fine forceps were used to peel the hippocampus and cortex from the brain under a stereomicroscope. The meninges were carefully removed. After peeling, the tissue was minced and washed with 10 mL of Ca-free water in a 15-mL conical tube. 2+ and Mg 2+ The tissue was gently washed once with 0.25% Hank's balanced salt solution (HBSS, Corning catalog number 21-022-CM). After washing, 5 mL of a solution of 0.25% trypsin (Invitrogen catalog number 15090-046) and 0.1% deoxyribonuclease I (DNase I, Sigma catalog number DN-25) was added to the tissue, followed by incubation at 37°C for 15 min. Next, the tissue was washed 3 times with ice-cold HBSS, 3 mL of 0.1% DNase I solution was added, and then the tissue was slowly aspirated 12 times using a glass Pasteur pipette, followed by centrifugation at 500×g for 10 min. The cell pellet was resuspended in culture medium (Neurobasal medium, Gibco catalog number 21103-049), 2% B27 supplement (Gibco catalog number 17504-044), 0.5 mM L-glutamine (Corning catalog number 25-005-Cl), 25 μM L-glutamate (Sigma catalog number G1251) and 1% penicillin / streptomycin (Gibco catalog number 15070-063), and then the cell suspension was plated at 100,000 cells / well in a poly-L-lysine-coated 96-well plate. Twenty-four hours after plating, half of the culture medium was removed and replaced with the culture medium described above but without glutamate. The cells were maintained in a 5% CO 2 The cells were placed in a 37°C humidified incubator and used for assays between days 6 and 10 after collection.

[0532] Analysis conditions

[0533] For each test concentration, the compound was diluted to 100× its final assay concentration in 100% DMSO. Just before the assay, the compound was diluted 1 / 10 into HBSS (containing calcium and magnesium) (10× final assay concentration) containing 100 μM DETA-NONO salt (10× final assay concentration). The culture medium was removed and the cells were washed once with 90 μL HBSS (Corining catalog number 21-023-CV). The cells were then incubated with 90 μL HBSS at 37°C for 30 min. Cells were added to a 10 μL test substance / HBSS / DETA-NONO salt culture plate and incubated for another 30 min at 37°C. The final DMSO concentration was 1%, the final DETA-NONO salt concentration was 10 μM; and the final compound concentrations were 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, 0.0001 μM, 0.00001 μM, and 0.0 μM. The medium was removed, the cells were lysed and the level of pCREB was determined according to the Cisbio protocol (phosphorylated CREB (Ser133) Cat. No. 64CREPEG). The plates were read using an Envision instrument (PerkinElmer).

[0534] Data analysis

[0535] pCREB was measured for each well and analyzed using GraphPad Prism software v.8 with a 3-parameter fit (log (agonist) vs. response - variable slope). 50 Interpolated from the curve fit and defined as the concentration of sGC stimulator compound at which 50% of its maximal response was elicited. Multiple experiments were performed for a given compound and the geometric mean of all experiments is reported. For both compounds 14 and 20, the free acid was used in these experiments.

[0536] Table F below summarizes the EC values ​​of compounds of the invention in the pCREB assay. 50 value.

[0537] Table F

[0538]

[0539] Example 7: Pharmacokinetic properties of rat cerebrospinal fluid (CSF)

[0540] plan

[0541] PK was determined in rats after oral dosing. For oral (PO) experiments, a group of 6 male Sprague-Dawley rats with indwelling catheters placed in the cisterna magna were used. The PO group was given 3.0 or 10 mg / kg of compound formulated as a solution in PEG400 or a suspension in 0.5% Tween 80 and 0.5% methylcellulose in water. The PO dose was administered by oral gavage and delivered to the stomach using a syringe and gavage tube. After oral dose administration, the gavage tube was flushed with approximately 0.5 mL of water to ensure complete delivery of the full dose.

[0542] Collect plasma samples as follows: collect samples of CSF and blood 1 hour, 2 hours and optionally 4 hours after administration. Collect CSF samples (0.05mL) through intracisternal catheter. Collect blood samples (0.25mL) through tail incision. Keep these samples on ice until processing plasma. Within 1 hour of collection, centrifuge blood samples at 3200rpm for 5 minutes at approximately 5°C. Transfer plasma directly to a separate Eppendorf tube (0.125mL). Place a stopper cap on the catheter and freeze the catheter at approximately -70°C and store until analysis. Collect plasma and CSF and analyze the presence of compound.

[0543] Quantification of compounds.

[0544] The compounds and internal standards discussed are extracted by precipitation from plasma and extracted from CSF by precipitation or dilution. The samples are analyzed using electrospray ionization using liquid chromatography (LC) and tandem mass spectrometry detection (MS / MS). The standard curve is within the range of 0.1 to 1000 ng / mL. The results of the compounds described herein in this assay are illustrated in Table G below (for 10 mg / kg dose and / or 3 mg / kg of the compound). The compound concentrations of several animals are combined to obtain the geometric mean at each specific dose and time point.

[0545] Kp,uu is defined as the ratio of the concentration of unbound drug in CSF to that in plasma. Unbound drug in plasma (or free plasma concentration) is calculated by multiplying the total plasma concentration by the unbound fraction as determined by plasma protein binding. Next, the CSF concentration is divided by the free plasma concentration to determine Kp,uu. (See, e.g., Di et al., J. Med. Chem., 56, 2-12 (2013))

[0546] Table G

[0547]

[0548] Rat CSF PK. Kp,uu≤1=A;1 <Kp,uu≤2=B;2<Kp,uu≤3=C,3<Kp,uu=D。

[0549] Example 8: Microdialysis Experiment on Rat Brain

[0550] Target

[0551] The goal of the current study was to evaluate the content of the sGC stimulant of the present invention in the interstitial fluid (ISF) of the hippocampus and striatum and in the circulating plasma after administration to Sprague Dawley male rats. To this end, microdialysis probes were implanted in the hippocampus and striatum and the jugular vein cannula (JVC) in the rats. After collecting a pre-dose ISF sample, the animals were given the sGC stimulant. After administration, samples were collected for 24 hours by the hippocampus and striatum probes, and continuous plasma sampling was collected by the JVC. All collected samples were stored at -80°C pending analysis of the compound content in the permeate.

[0552] Materials and methods

[0553] animal

[0554] Five Sprague Dawley rats pre-cannulated with jugular veins were used for this study. Upon arrival, rats were group housed in polycarbonate cages (2 to 3 / cage) and acclimated for at least 3 days prior to the start of the study. The animals were housed in a room maintained at 22 + Room temperature at 2°C and approximately 50% humidity in a 12-hour light / dark cycle with ad libitum access to food and water. Rats were tracked by unique identification numbers. Experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of Charles River Laboratories South San Francisco.

[0555] Preparation and administration

[0556] sGC stimulators were freshly prepared on the day of treatment and administered to the animals as follows: Compound 1-14 was given at a dose of 3 mg / kg as its sodium salt and at a concentration of 3 mg / mL in MilliQ water.

[0557] In vitro experiments

[0558] In vitro Metaquant microdialysis (MQ-MD) experiments were performed to test the recovery of compounds through the probe's membrane. To this end, the MQ probe (polyacrylonitrile, 3 mm membrane) was connected to a microperfusion pump (Harvard PHD 2000 syringe pump, Holliston, MA or similar) via an inlet PEEK catheter. The probe was placed individually in an artificial CSF (aCSF) + 0.2% bovine serum albumin (BSA) bath containing 50 ng / mL sGC stimulant. The bath contents were continuously stirred and maintained at 37°C. Each probe was perfused with aCSF + 0.2% β-cyclodextrin (β-CD, slow flow rate) and a carrier flow of ultrapure water + 0.2% BSA was used. The flow rate was 0.15 μL / min for slow flow rate and 0.8 μL / min for carrier flow. The outlet of the probe was connected to an automated fraction collector (820 microsampler, Univentor, Malta or similar) via an outlet PEEK catheter. After perfusion stabilized, samples were collected into polypropylene vials over 20-minute periods. In separate vials, 150 μL samples of the bath contents were collected at the beginning and end of the experimental sample collection period. In vitro dialysate and bath samples were analyzed for compound content. Probe recovery was calculated as the ratio of compound concentration in the permeate sample relative to bath concentration and expressed as percent recovery.

[0559] Microdialysis method

[0560] Use isoflurane (2%, 800 mL / min O 2 ) anesthetized rats. Bupivacaine was used for local anesthesia and carprofen was used for preoperative / postoperative analgesia. The animals were placed in a stereotaxic frame (Kopf Instruments, USA). Then, MetaQuant microdialysis probes (polyacrylonitrile; 3 mm exposure membrane) were implanted in the striatum (STR) and hippocampus (HIPP). The coordinates of the probe tip for STR were: anterior-posterior (AP) from bregma = +0.9 mm, lateral (L) from midline = +3.0 mm, and ventral (V) from dura = -7.0 mm, with a rack setting of -3.3 mm. Then a second probe was implanted in the hippocampus (HIPP). The coordinates of the probe tip for HIPP were: anterior-posterior (AP) from bregma = -5.3 mm, lateral (L) from midline = -4.8 mm and ventral (V) from dura = -7.0 mm, with a rack setting of -3.3 mm. After surgery, animals were housed individually in cages and provided food and water ad libitum.

[0561] Microdialysis experiments were performed one day after surgery. The microdialysis probe was connected to a microperfusion pump (Harvard PHD 2000 syringe pump, Holliston MA) using a flexible PEEK catheter. The microdialysis probe was perfused with a 147 mM NaCl, 3.0 mM KCl, 1.2 mM CaCl 2 and 1.2 mM MgCl 2 aCSF containing 0.2% β-cyclodextrin was slowly flowed at 0.15 μL / min and H 2 O + 0.2% BSA vehicle was perfused at a rate of 0.8 μL / min. Microdialysis samples were collected into 300uL polypropylene microbottles containing 15 μL 0.02M formic acid + 0.04% ascorbic acid / ultrapure water over a 30-minute period by an automated fraction collector (820 microsampler, Univentor, Malta). After stabilization, a baseline sample was collected, and sGC stimulants were administered orally (PO) at T = 0 and samples were collected continuously for 24 hours (collected at 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 12 h, and 24 h). Samples were aliquoted for analysis of sGC stimulants. All ISF samples were stored at -80 ° C until analysis.

[0562] In addition to ISF collection, blood samples were collected via JVC to K at T = -0.5h, 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 8h, 12h and 24h after treatment. 2 +EDTA vials. Blood was stored on ice until plasma processing (centrifugation at 2,500 g for 10 min at 4°C). Plasma was aliquoted into 1.5 mL Eppendorf vials and stored at -80°C pending analysis.

[0563] Postmortem tissue collection

[0564] After microdialysis, CO 2 Animals were euthanized by asphyxiation. Brains were collected in 10% neutral buffered formalin for probe placement verification.

[0565] Bioanalytical methods

[0566] Measurement of compound levels in permeate and plasma

[0567] The concentration of compound 1-14 in the permeate and plasma samples was quantified by ultra performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS / MS) detection in multiple reaction monitoring mode (MRM).

[0568] The plasma samples were first mixed with an acetonitrile solution containing 100 ng / mL dexamethasone (internal standard) for protein precipitation. After incubation at room temperature for 5 minutes, the samples were centrifuged for 5 minutes (1300 rpm, 4°C) and the supernatant was diluted 100 times in ultrapure water with 0.1% formic acid.

[0569] Prior to analysis, undiluted permeate samples (10 μL) were mixed with 4 μL of an internal standard solution containing 50 ng / mL dexamethasone (internal standard) dissolved in acetonitrile / ultrapure water (1:1) and 0.1% formic acid.

[0570] Undiluted permeate samples and diluted plasma supernatants were injected into a Shimadzu system (Shimadzu, USA) by an automated sample injector (Shimadzu Sil-30AC autosampler, Shimadzu, USA). Analytes were separated by liquid chromatography using a linear gradient of mobile phase B at a flow rate of 0.800 mL / min on a reversed phase XBridge BEH C8 column (2.1*50 mm, 2.5 μm particle size; Waters, USA) maintained at a temperature of 35°C. Mobile phase A consisted of ultrapure water with 0.1% formic acid. Mobile phase B was acetonitrile with 0.1% formic acid.

[0571] Use a turbo ion spray interface Acquisition was achieved with a 5500 mass spectrometer (Applied Biosystems, USA) in positive ionization mode. The ion spray voltage was set to 5.5 kV and the probe temperature was 600 °C. The collision gas (nitrogen) pressure was maintained at the medium. Quantification was performed using the following MRM transition zone: m / z 372.0 / 352.0. Appropriate import calibration curves were fitted using weighted (1 / x) regression. Sample concentrations were determined using these calibration curves. Accuracy was verified by quality control samples after each sample series. The Analyst TM The data were calibrated and quantified using the data system (Applied Biosystems, version 1.5.2).

[0572] Data evaluation

[0573] Data were plotted using Prism 8 for Windows (GraphPad Software, Inc.). The reported permeate concentration of compound 1-14 was corrected based on probe recovery. The average recovery was 14.7% (SEM 0.59).

[0574] result

[0575] All five were successfully dosed and completed the experiment. The JVC of one animal was blocked at the 4-hour time point and no additional blood was collected. No obvious side effects of the treatment were observed.

[0576] Effects of Compound I-14 Administration in Striatal and Hippocampal ISF; Ratio of STI / HIPP to Compound in Plasma Rate

[0577] Figure 3 Shown are the levels of Compound 1-14 in the STR and HIPP fractions in the ISF permeate of adult male Sprague-Dawley rats following administration (3 mg / kg; PO) of Compound 1-14 at T=0 min.

[0578] Microdialysis allows sampling of compound concentrations in the interstitial fluid between brain tissues. In view of the fact that the tissue at the sampling location has different blood perfusion rates, physiological composition and clearance mechanisms compared to cerebrospinal fluid (CSF), we expect that the distribution and measured concentrations between the microdialysis study and CSF-PK study in the same animal will be different. These differences are observed in the time to maximum concentration, the concentration measured and the ratio calculated by the measured concentration (Nagaya Y, Nozaki Y, Takenaka O, et al., Investigation of utility of cerebrospinal fluid drug concentration as a surrogate for interstitial fluid concentration using microdialysis coupled with cisternal cerebrospinal fluid sampling in wild-type and Mdr1a (- / -) rats. Drug Metab Pharmacokinet. 2016; 31 (1): 57-66).

[0579] Example 9: Changes in cGMP concentration in rat cerebrospinal fluid (CSF) after a single administration of sGC stimulators (CSF Biomarker Measurements)

[0580] This experiment was performed to determine the effects of different doses of the sGC stimulator compounds of the present invention on cGMP levels in rat CSF.

[0581] plan

[0582] Rats were administered a single dose of vehicle or sGC stimulator (1, 3, 10 or 30 mg / kg). One, two and six hours after administration, CSF samples were collected and analyzed to determine cGMP and compound concentrations, and plasma samples were collected and analyzed to determine compound concentrations. Each rat was sampled once or more, with an interval of 3 days or more between each administration. The day before the experiment, the rats were fasted overnight and water was available ad libitum.

[0583] On the day of the experiment, the concentration of compound and cyclic guanosine monophosphate (cGMP) in rat CSF was determined after oral administration. Male CD rats (250 to 275 g) implanted with intracisternal cannulae were used for these studies. According to protocol MIL-110, rats were individually housed under controlled conditions of temperature (21 ± 1 ° C), relative humidity (36 ± 1%) and placed in a 12-hour light-dark cycle (lights on at 6:00 AM and off at 6:00 PM) room in a SmartLabs breeding room (21 Erie Street, Cambridge, MA). Animals were allowed to access food (LabDiet Prolab Isopro RMH 3000, St. Louis, MO) and water ad libitum. Rats were given 0 mg / kg (vehicle), 1 mg / kg, 3 mg / kg, 10 mg / kg or 30 mg / kg of a compound of the invention formulated as a suspension in 0.5% methylcellulose, 0.5% Tween80 or a solution in MilliQ water. The formulation was prepared, stored frozen at -20°C and thawed at room temperature for 1 hour before dosing, or prepared and used within 2 hours, or prepared the day before dosing and kept stirred at room temperature overnight and until dosing. The PO dose was administered by oral gavage and delivered to the stomach using a syringe and gavage tube. After oral dosing, the gavage tube was flushed with approximately 0.5 mL of water to ensure complete delivery of the full dose.

[0584] Plasma and CSF samples were collected under isoflurane anesthesia as follows: CSF and blood samples were collected 1, 2, and 6 hours after dosing. CSF samples were collected via intracisternal catheters. Approximately 20 μL of CSF was collected and discarded (this included a syringe dead volume of 14 to 16 μL); approximately 50 μL of CSF was then drawn into an Eppendorf tube containing 5 μL of glacial acetic acid. CSF samples were flash frozen by immersion in liquid nitrogen. Next, the animals were kept under anesthesia and blood samples were obtained by tail incision and stored in K-EDTA tubes. These samples were kept on ice until plasma was processed. Blood samples were centrifuged at 3200 rpm for 10 minutes at approximately 5°C within 1 hour of collection. Plasma was transferred directly to 96-well coils (0.125 mL). A stopper cap was placed on the catheter and the catheter was frozen at approximately -70°C and stored until analysis. Plasma and CSF were collected and analyzed for the presence of compound.

[0585] Quantification of compounds and cGMP.

[0586] Compounds of the invention, cGMP and internal standards were extracted from plasma and CSF by precipitation. Samples were analyzed using electrospray ionization using liquid chromatography (LC) and tandem mass spectrometry detection (MS / MS). The standard curve range for the compound was between 0.1 and 1000 ng / mL. The standard curve range for cGMP was between 0.01 and 40 ng / mL. CSF cGMP data were plotted using GraphPad Prism, version 8.4.3 and expressed as mean ± SEM. Data were analyzed using mixed effects analysis, followed by Dunnett's multiple comparison test to compare with vehicle-treated rats within the time point. Significance was set to p<0.05.

[0587] result

[0588] One hour and two hours after dosing, rats treated with 1, 3, and 10 mg / kg of Compound 1-20 (given as a suspension in Tween / MC) had no significant changes in cGMP concentrations in rat CSF compared to vehicle-treated rats. However, at six hours after dosing, rats treated with 10 mg / kg of Compound 1-20 had significantly higher cGMP in CSF compared to vehicle-treated rats. (See Figure 4 )

[0589] Compared to vehicle-treated rats, rats treated with Compound 1-14 (administered as the sodium salt) had higher cGMP levels in CSF at all doses tested, but not at all time points tested. One and two hours after dosing, rats administered 3 mg / kg of Compound 1-14 had significantly higher cGMP concentrations in CSF. Two hours after dosing, rats treated with 1 mg / kg of Compound 1-14 had significantly higher cGMP in CSF compared to vehicle-treated rats. At 1, 2, and 6 hours after dosing, rats administered 10 or 30 mg / kg of Compound 1-14 had significantly higher cGMP in CSF (see Figure 5 ).

[0590] Example 10A: Non-Human Primate Cerebrospinal Fluid (CSF) Pharmacokinetic Properties (Study A)

[0591] plan.

[0592] PK in NHP was determined after oral administration (PO). A group of 4 female cynomolgus monkeys were used to study each compound. Compound I-20 sodium salt was formulated as a 0.06 mg / mL solution in MilliQ water. Compound I-14 sodium salt was formulated as a 0.2 mg / mL solution in MilliQ water. The formulation was shipped frozen on dry ice and then thawed and mixed thoroughly before administration. PO doses of 1 mg / kg of Compound I-14 and 0.3 mg / kg of Compound I-20 were administered by oral gavage.

[0593] Plasma and CSF samples were collected as follows: CSF samples were collected 3 and 24 hours after PO administration. CSF samples (0.125 mL) were collected in the cisterna magna by direct needle puncture by direct dilution. Blood samples (0.8 mL) were collected from a peripheral vein at 0, 0.25, 0.5, 1, 2, 3, 6, 8, 12, 24, 32, and 48 hours. These samples were kept on ice until the plasma was processed. Blood samples were subjected to protein precipitation using acetonitrile. Plasma was transferred directly into individual catheters (0.125 mL) and precipitated using K 2 EDTA served as anticoagulant. A stopper cap was placed on the catheter and the catheter was frozen at approximately -70°C and stored until analysis. Plasma and CSF were collected and analyzed for the presence of compound.

[0594] Quantification of compounds.

[0595] Plasma and CSF samples were analyzed using liquid chromatography (LC) and tandem mass spectrometry detection (MS / MS) using positive electrospray ionization. The standard curve range was 0.1 to 1000 ng / mL.

[0596] The geometric mean of the values ​​obtained for the four animals in each compound study are the concentrations in CSF and plasma, respectively.

[0597] Kp,uu is defined as the ratio of the concentration of unbound drug in CSF to that in plasma. The unbound drug in plasma (or free plasma concentration) is calculated by multiplying the total plasma concentration by the unbound fraction as determined by plasma protein binding. Next, the CSF concentration is divided by the free plasma concentration to determine Kp,uu. (See, e.g., Di et al., J. Med. Chem., 56, 2-12 (2013))

[0598] The results for compounds of the invention are summarized in Table H below.

[0599] Table H

[0600]

[0601] NHP CSF PK. Kp,uu≤1=A;1 <Kp,uu≤2=B;2<Kp,uu≤3=C;3<Kp,uu=D。

[0602] Example 10B: Non-Human Primate Cerebrospinal Fluid (CSF) Pharmacokinetic Properties (Study B)

[0603] The objectives of this study were to investigate the pharmacokinetics of Compound 1-14 following a single intrathecal bolus or oral gavage dose administered to cynomolgus monkeys on Day 1 and to assess differences in pharmacokinetic values ​​when comparing CSF collected from the cisterna magna (as in Example 10A) to that from the lumbar spine.

[0604] Study Design

[0605]

[0606] No. = number; F = female; CSF = cerebrospinal fluid; No = number a The same animals will be used in each group, and there will be a minimum 7-day washout period between dosing days.

[0607] Test system / method

[0608]

[0609] Animals were housed in stainless steel cages equipped with stainless steel shading and automatic water supply valves. The main enclosures were as described in the Guide to Laboratory Animal Management and Use (National Research Council (NRC). Guide for the Care and Use of Laboratory Animals. Washington, DC: National Academy Press, 8th Edition. 2011; Office of Laboratory Animal Welfare. Public Health Services Policy on Humane Care and Use of Laboratory Animals. Bethesda, Maryland: National Institutes of Health, 2015 revision). Unless the person in charge of the study and / or clinical veterinarian consider it inappropriate, these housing conditions are maintained. Animals were socially housed (when possible), except for the time when they were separated for the specified research method / activity.

[0610] In a crossover design, a single dose of Compound I-14 was administered intravenously as a bolus solution of 0.15 mg / kg and orally administered to a group of four cynomolgus macaques as a 0.5 mg / kg suspension by gavage. Plasma samples were collected at 0.25 (15 min), 0.5 (30 min), 1, 2, 3, 6, 8, 12, 24, 32, and 48 h after IV and PO doses. For all animals, CSF samples were collected from the cerebellomedullary cisterna in Groups 1 and 2 and from the lumbar vertebrae in Groups 3 and 4 at 3 and 24 h. Sample extracts were prepared by protein precipitation and Compound I-14 concentrations were measured using LC-MS / MS. Pharmacokinetic parameters were calculated.

[0611] Summary / Conclusion

[0612] Overall, mean pharmacokinetic parameters between IV (Groups 2 and 4) and PO (Groups 1 and 3) were within standard deviations of each other.

[0613] Compound I-14 in the CSF was observed using both cisterna magna and lumbar sampling at 3 and 24 hours after IV and PO administration. At 3 hours after IV and oral dosing, the geometric mean ratio of CSF concentration in plasma to unbound concentration decreased in both cases after cisterna magna sampling and after lumbar sampling, falling within range C as described above, i.e., between 2 and 3 (NHP CSF PK. Kp,uu≤1 = A; 1 < Kp,uu≤2 = B; 2 < Kp,uu≤3 = C; 3 < Kp,uu = D). At 24 hours after IV and oral dosing, the geometric mean ratio of CSF concentration in plasma to unbound concentration decreased after cisterna magna sampling, falling within ranges B and C respectively, and decreased after lumbar sampling, falling within ranges B and C respectively.

[0614] Example 11: Evaluation of compounds of the invention in the novel object recognition (NOR) model of memory enhancement

[0615] Objective

[0616] To evaluate the efficacy of the CNS penetrant sGC stimulant of the present invention in reversing MK-801-induced memory disruption, the novel object recognition (NOR) test was used in male Long Evans rats.

[0617] Introduction

[0618] NOR is a test for recognition learning and memory retrieval that takes advantage of the spontaneous preference of rodents for novel objects compared to familiar objects. The NOR test has been widely used to evaluate the potential recognition-enhancing properties of novel test compounds. Since the NOR paradigm does not involve rewarding or aversive stimuli, it provides fewer confounding variables when translated into similar tests conducted in human clinical trials.

[0619] In this study, a memory retention model was used. The non-competitive antagonist of the NMDA receptor, MK-801 (Dizocilpine), was used to induce a deficit in recognition memory. The efficacy of the sGC stimulant in preventing MK-801-induced memory impairment was evaluated. The reference compound, galantamine 1 mg / kg (intraperitoneal), significantly reversed the recognition deficit induced by MK-801 0.1 mg / kg (intraperitoneal), indicating the validity of the test.

[0620] Materials and Methods

[0621] animal

[0622] Adult male Long-Ivan rats (275 to 299 grams upon arrival from Envigo, Indianapolis, IN) were used in this study. Rats were placed in an experimental room and assigned a unique identification number (tail tag). Rats were housed 2 per cage in polycarbonate cages with filtered tops and acclimated for at least 7 days before testing. The animal room was maintained at a 12 / 12h light / dark cycle (lights on at 07.00 EST), 22±1°C, and approximately 50% relative humidity. Food and water were provided ad libitum. All animals were examined, handled, and weighed prior to the study to ensure adequate health and to minimize nonspecific stress associated with the test. Each animal was randomly assigned to the entire treatment group. Experiments were performed during the light cycle phase of the animal.

[0623] Test compounds and drugs

[0624] The following compounds and drugs were used in these studies:

[0625] MK-801 (0.1 mg / kg; Sigma-Aldrich) was dissolved in saline and injected IP 15 min before NOR training. The dose volume was 1 ml / kg.

[0626] Galantamine (1 mg / kg; Tocris) was dissolved in saline and injected IP 15 minutes before training. The dose volume was 1 ml / kg.

[0627] Compound 1-20 (0.03, 0.3 and 1 mg / kg) was formulated in vehicle (0.5% (w / w) methylcellulose and 0.5% (w / w) Tween 80 / ultrapure water) and orally administered at a dose volume of 2 ml / kg 60 min prior to NOR training.

[0628] The following groups were tested: N = 16 per group, (one rat was removed from the Compound I-20-1 mg / kg-MK-801 group prior to the start of testing due to health issues): 1) Vehicle-Saline; 2) Vehicle-MK-801 0.1 mg / kg; 3) Galantamine 1 mg / kg-MK-801; 4) Compound I-20, 0.03 mg / kg-MK-801; 5) Compound I-20, 0.3 mg / kg-MK-801; and 6) Compound I-20, 1 mg / kg-MK-801

[0629] Compound I-14 (0.01, 0.1 and 1 mg / kg) was formulated as a solution in MilliQ water in its Na+ salt form and stored in frozen aliquots. Aliquots of compound solution and compound vehicle were stored at -80°C and freshly thawed on each test day. Compounds and vehicles were orally administered 60 minutes before NOR training. The dose volume was 10 ml / kg.

[0630] The following groups of Compound I-14 were tested, where N = 16 for each group: 1) saline-MK-801 0.1 mg / kg; 2) galantamine 1 mg / kg-MK-801; 3) vehicle-saline; 4) vehicle-MK-801; 5) Compound I-14, 0.01 mg / kg-MK-801; 6) Compound I-14, 0.1 mg / kg-MK-801; and 7) Compound I-14, 1 mg / kg-MK-801

[0631] Experimental methods

[0632] The NOR test was conducted in an open field (40×40 cm) in a soundproof room placed under dim light. Each rat was tested individually, and care was taken to remove olfactory / taste cues by cleaning the field and test objects with 70% alcohol between trials and rats. All training and test trials were video recorded and scored by an observer blinded to the treatment.

[0633] On the 1st and 2nd day, rats were allowed to freely explore the place (without object inside) for a 5-minute adaptation phase. On the 3rd day (training and test day), rats were administered vehicle, saline and / or compound solution according to the corresponding pretreatment, which was defined as the time between injection and the start of NOR training. Each animal was placed in a test place where two identical objects were present. Each rat was placed in the same position in the place facing the same direction, and the time spent actively exploring the object during the 3-minute training phase (T1) was recorded. After training, the rats were returned to their breeding cages. NOR test (T2) was performed 1 hour after T1. Each rat was placed back in the test place where a familiar object and a novel object were present for 5 minutes, and the time spent exploring the two objects was recorded. The order and position of presentation of the objects (left / right) in T2 were random between rats to prevent the deviation caused by order or position preference.

[0634] Tissue collection

[0635] Approximately 10 minutes after T2 (135 minutes after drug administration), trunk blood was collected in microcentrifuge tubes containing K2EDTA. Blood tubes were kept on ice for short-term storage. Within 15 minutes, the tubes were centrifuged at 10,000 RPM for 10 minutes in a refrigerated centrifuge. Plasma was extracted and samples were stored in a -80°C freezer until shipped to the trial sponsor.

[0636] Statistical analysis

[0637] Data from the NOR test (T2) were expressed as a recognition index, defined as the ratio of time spent exploring the novel object relative to the total time spent exploring both objects during the test phase (novel / (familiar+novel)×100%).

[0638] For compound I-14, the data were analyzed separately in two batches. The first batch included a saline-MK-801 group and a galantamine-MK-801 group. The data were analyzed using a t test to assess the effectiveness of the test. The second batch included a "sGC stimulator group", which included 5 treatment groups containing a compound vehicle (MilliQ water): vehicle-saline, vehicle-MK-801, compound I-14 0.01 mg / kg-MK-801, compound I-14 0.1 mg / kg-MK-801, and compound I-14 1 mg / kg-MK-801. The second batch of data was analyzed by a single-factor ANOVA followed by a Fisher LSD post hoc comparison within the respective 0 to 1, 0 to 3, and 0 to 5 minute time ranges. Significance was set at P<0.05. Nineteen animals with a recognition index higher than 90% or lower than 30% were excluded because of a strong (non-memory) bias between the two subjects. Two rats with total exploration times less than 10 seconds within five minutes for both subjects were also excluded due to unreliable results (this is the standard deviation for our NOR test). Based on feedback from plasma analysis, one rat was removed due to suspected drug exposure. Subsequently, statistical outliers above or below two standard deviations from the mean were removed from further analysis. By these criteria, 1 to 6 rats were excluded from each experimental group (initial N=16) and from statistical analysis within all time ranges (0 to 1, 0 to 3, and 0 to 5 minutes).

[0639] For compound 1-20, data were analyzed in each of the 0 to 1, 0 to 3, and 0 to 5 minute time ranges using a one-way ANOVA followed by a Fisher's LSD post hoc test, with significance set at P < 0.05. Animals with a recognition index above 90% or below 30% were excluded because of a strong (non-memory) bias between the two subjects. Subsequently, statistical outliers above or below two standard deviations from the mean were removed from further analysis. By these criteria, 2 to 4 rats were excluded from each experimental group (initially N = 16) and from statistical analysis in all time ranges (0 to 1, 0 to 3, and 0 to 5 minutes).

[0640] result

[0641] a) Compound I-14

[0642] None of the rats in this study showed significant side effects at any dose. The rats maintained normal levels of alertness, activity, and exploration of objects.

[0643] In the 0 to 1 minute time range, the t test showed a significant difference between the saline-MK-801 group and the galantamine-MK-801 group (P < 0.01), indicating the validity of this analysis. The ANOVA for the compound groups showed a significant main treatment effect on the recognition index [F (4,56) = 4.698, P < 0.01]. Fisher's LSD post hoc analysis comparison showed that in this time range, both the vehicle-saline group and the 1 mg / kg compound I-14-MK-801 group showed significant differences from the vehicle-MK-801 group (respectively, P < 0.05 and P < 0.01), indicating that MK-801 0.1 mg / kg induced significant memory loss and 1 mg / kg of compound I-14 reversed the deficit.

[0644] In the 0 to 3 minute time range, the t test showed a significant difference between the saline-MK-801 group and the galantamine-MK-801 group (P < 0.001), indicating the validity of this analysis. The ANOVA of the compound group showed a significant main treatment effect on the recognition index [F (4,56) = 5.113, P < 0.01]. Post hoc analysis comparisons showed that within this time range, the vehicle-saline group showed a significant difference from the vehicle-MK-801 group (P < 0.01), indicating that MK-801 0.1mg / kg induced significant memory loss. Within this time range, 1mg / kg of compound I-14 showed a trend of reversing MK-801-induced memory loss (P < 0.10).

[0645] In the 0 to 5 minute time range, the t test showed a significant difference between the saline-MK-801 group and the galantamine-MK-801 group (P < 0.001), indicating the validity of this analysis. The ANOVA of the compound groups showed a significant main treatment effect on the recognition index [F (4,56) = 2.847, P < 0.05]. Post hoc analysis comparisons showed that in this time range, 1 mg / kg of compound I-14 had a significantly higher recognition index relative to the vehicle-MK-801 group (P < 0.05).

[0646] b) Compound I-20

[0647] None of the rats in this study showed significant side effects at any dose. The rats maintained normal levels of alertness, activity, and exploration of objects.

[0648] ANOVA showed a significant main treatment effect on the recognition index in the 0 to 1 min time range [F(5,77)=3.379, P<0.01]. Post hoc analysis tests showed that the vehicle-MK-801 0.1 mg / kg group and the vehicle-saline group did not show significant differences in this time range (P>0.05). The compound I-201 mg / kg-MK-801 group and the galantamine-MK-801 group showed significantly higher recognition indices than the vehicle-MK-801 group (Ps<0.01). In general, data in the 0 to 1 min time range are relatively unstable; treatments in the 0 to 3 min and 0 to 5 min time ranges provide more reliable results.

[0649] In the time range of 0 to 3 min, ANOVA found a significant main treatment effect on the recognition index [F(5,77)=3.922, P<0.01]. Post hoc analysis tests showed that MK-801 0.1 mg / kg caused significant memory loss in this time range (vehicle-saline group vs. vehicle-MK-801 group, P<0.05). Compound I-20 1 mg / kg-MK-801 group and galantamine-MK-801 group significantly reversed MK-801-induced memory deficits (Ps<0.001). Compound I-20 0.3 mg / kg-MK-801 group also showed a trend of reversing MK-801-induced memory deficits in this time range (P<0.10).

[0650] In the 0 to 5 min time range, ANOVA showed a significant main treatment effect [F(5,77)=5.219, P<0.001]. Post hoc analysis tests showed that MK-801 0.1 mg / kg caused significant memory loss, with the recognition index approaching the chance level (50%). Galantamine (1 mg / kg), compound I-20 0.3 mg / kg and 1 g / kg significantly reversed the MK-801-induced memory deficits (Ps<0.05, P<0.01 and Ps<0.001, respectively, compared with the vehicle-MK-801 group). The compound I-20 0.03 mg / kg-MK-801 group also showed a trend of reversing the MK-801-induced memory deficits in this time range (P<0.10).

[0651] Example 12: EEG Evaluation of Sleep-Wake Pharmacology in Telemeter-Implanted Rats

[0652] This study was performed at PsychoGenics, Inc. Methods were approved by the Institutional Animal Care and Use Committee in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

[0653] Target

[0654] This study was designed to evaluate the drug-specific EEG (electroencephalography) signature of Compound 1-14 during sleep phases in wirelessly implanted young adult male SD rats.

[0655] Materials and methods

[0656] animal

[0657] Young adult male Sprague-Dawley (SD) rats (approximately 275 to 325 grams upon arrival) from Envigo (Indianapolis, IN) were used in the study. Upon receipt, the rats were assigned unique identification numbers and housed in polycarbonate cages with micro-isolator filter tops at 3 rats per cage. All rats were checked and weighed before the start of the study to ensure adequate health and suitability. During the course of the study, a 12 / 12 light / dark cycle was maintained. The room temperature was maintained between 20°C and 23°C, with relative humidity maintained at approximately 50%. Food and water were provided ad libitum during the study time. After surgery, the rats were housed individually. After the recovery phase (7 to 10 days), the animals were transferred to an EEG recording room and placed on a DSI receiver for recording.

[0658] Research Group

[0659] a. Medium A

[0660] i. Route: PO

[0661] ii. Volume: 10ml / kg

[0662] iii. Preparation: Water

[0663] b. 3 mg / kg of compound I-14

[0664] i. Route: PO

[0665] ii. Volume: 10ml / kg

[0666] iii. Preparation: 3 mg / kg in water

[0667] c. 10 mg / kg of compound I-14

[0668] i. Route: PO

[0669] ii. Volume: 10ml / kg

[0670] iii. Preparation: 10 mg / kg in water

[0671] d. 30 mg / kg of compound I-14

[0672] i. Route: PO

[0673] ii. Volume: 10ml / kg

[0674] iii. Preparation: 30 mg / kg in water

[0675] Surgical method

[0676] Briefly, animals were implanted with a DSI telemetry device (F50-EET) in a 3 channel (lead pair) configuration. Note the placement of the positive and negative leads in each location. Each of the EEG channels on the DSI transmitter serves as a differential input whereby the difference between the positive and negative leads is measured. Frontal / parietal (right): anterior (+) 2 mm, lateral 2 mm; posterior (-) 4 mm, lateral 2 mm; frontal / frontal (bilateral hemispheric): anterior (+) 2 mm, lateral 2 mm (right); anterior (-) 2 mm, lateral 2 mm (left); and cervical EMG.

[0677] In summary, animals were anesthetized with 4% to 5% isoflurane and 1L / min oxygen before surgery for induction and maintained with 1% to 2.5% isoflurane / 1L / min oxygen mixture during surgery. The depth of anesthesia was measured and monitored using the hind limb withdrawal reflex and respiratory rate. The isoflurane content was continuously adjusted to maintain the surgical anesthesia plane throughout the surgical procedure. Ophthalmic lubricant was applied to the eyes with a sterile cotton swab. The surgical site was prepared for aseptic surgery by shaving and cleaning the skin three times with chlorhexidine scrubs and alcohol swabs. The abdomen, neck, and head were shaved and sterilized with 3 alternating operations of chlorhexidine scrubs and alcohol (2% chlorhexidine gluconate and 4% isopropyl alcohol). The anesthetized animals were then placed on a warm water circulating heating pad for surgical implantation of a telemetry device.

[0678] Telemetry Implant

[0679] A 3 to 4 cm incision is made in the midline on the top of the skull extending from about 1 cm posteriorly to the midpoint of the eye to the base of the skull and to the mid-dorsal neck region. Using blunt dissection, a subcutaneous pocket is created from the rear end of the incision by pushing alongside the connective tissue to the right ventral region. The pocket is rinsed with sterile saline and the transmitter is then placed in the pocket with the lead extending out of the neck incision. A wire connected to the transmitter is measured, cut to size and inserted into one of the dorsal neck muscles and secured in place with a 5-0 silk suture. This method is repeated for the second lead so that the two leads are placed in a straight line along the same muscle bundle and spaced 2 to 4 mm apart and provide electromyography (EMG) signals. The periosteum is then removed to reveal the skull landmarks, bregma and lambda. As needed, the skull area is wiped with sterile saline and then dried using gauze or a sterile cotton swab (Q-tip). Electroencephalogram (EEG) leads are marked using the stereotaxic coordinates of the region of interest (posterior and parietal cortex). For each region of interest, 1 to 2 holes are drilled on the skull to ensure exposure of the dura mater. The dura mater contact screw is inserted into the hole and the EEG leads are wound around it (up to 4 holes). These screws must be in contact with the dura mater to detect EEG brain signals. Penetration through the dura mater will not cause adverse events and can improve EEG signals (more brain and metal contact). Then use FLOW-It ALC compound to permanently seal screws and surrounding skull surface areas. After fully drying the adhesive, suture nails are used to close the skin covering the skull.

[0680] Test schedule

[0681] This study followed a Monday and Thursday dosing / recording schedule with a baseline EEG recording for 2 hours and then rats received a first dose of 0.3, 3, or 10 mg / kg of Compound 1-14 or Vehicle A (all oral) between 7:50 and 8:00 AM and a second dose of the same approximately 12 hours later, and then EEG was continued for 12 hours.

[0682] Start EEG recording at 6AM (when lights are turned on)

[0683] Administer the drug at 7:50 AM (1 hour and 50 minutes after lights on)

[0684] · Second dose (same compound) at 7:50 PM (1 hour and 50 minutes after lights out)

[0685] • Continue EEG recording until 7:50 AM the next day (24 hours after the 1st dose and 12 hours after the 2nd dose).

[0686] Data from rats moving freely in a cage were recorded using the International Data Science (DSI) data acquisition platform. Recording was performed during the light and dark cycle. During the study, the lights were turned on at 6AM and turned off at 6PM. Animals were adapted to dosing (vehicle dosing) before data collection.

[0687] Animals were tested in a crossover design with at least a 72-hour washout period between doses. On each test day, animals received compound or vehicle orally just 2 hours (7:50 AM) after lights on (6:00 AM). Data were recorded starting 2 hours before dosing and continued for 24 hours after dosing.

[0688] EEG signal evaluation

[0689] Ensure that the EEG does not contain line noise (50 or 60 Hz) or any continuous non-physiological frequency patterns that would be considered noise from external power supplies. All EEG recordings were within normal operating range (i.e., EEG signals were within normal operating amplitude range, typically greater than 100 microvolts and less than 500 microvolts, with no signal fidelity loss, typically observed by EEG reduction of <100 μV).

[0690] Sleep score

[0691] The raw EEG recordings were manually scored using neuroscore software (Data Science International) to identify sleep stages: active wakefulness, quiet wakefulness, NREM, and REM. Artifacts were removed offline from the data using neuroscore (DSI) and sleep stages were manually assigned to each 10-s period using preparietal EEG, LMA, and EMG by conventional methods as previously described: active wakefulness (less regular low-amplitude EEG, and high EMG and LMA activity); quiet wakefulness (less regular low-amplitude EEG, and low EMG and no LMA activity); NREM (consisting of high-amplitude irregular waves with predominant delta (1 to 4 Hz), low EMG, and no LMA); REM sleep (stable low-amplitude waves dominated by theta (4 to 8 Hz), almost no EMG, and no LMA).

[0692] Sleep stage data were derived from the neuroscore reporting template for sleep state time every 15 min (2 hours before dosing to 4 hours after dosing). The onset of the first sleep and first REM episodes was also reported directly from the neuroscore reporting template. The latency was calculated as the time period of the onset of the first REM episode after the NREM episode (i.e., T REM -T NREM =REM latency). Hypnograms were prepared using the percentage of time spent in each sleep stage for each hourly time interval. Time in each sleep state was calculated as the percentage of total time in each sleep state (mean ± standard error of the mean, SEM). Data for each animal were grouped by treatment group, sleep state, and frequency and exported to GraphPad PRISM for statistics and graphics.

[0693] Spectrum Analysis

[0694] Spectral analysis was performed using Matlab. The time domain signals collected for multiple channels were collected into DSI / neuro-scores and the EDF files were subsequently transferred to Matlab. Excel files with specific timestamps for baseline and post-dose were also transferred to Matlab for time locking. In Matlab, the power spectral density (PSD) was calculated using the Welch method. Then, the original and relative spectral power were calculated for each of the six frequency bands (δ, θ, α, β, low γ and high γ) and each 1 Hz sub-band. The 2-hour data recorded before compound administration were combined and defined as "baseline". The percentage change relative to baseline was calculated based on each channel, subject, dose level, spectral band and time segment. The average original, relative and percentage changes of each frequency band of each group were calculated. Spectral analysis includes the original spectral power, relative spectral power and spectral power change percentage of the EEG bands (δ-0.5 to 3.9 Hz, θ-4 to 7.9 Hz, α-8 to 11.9 Hz, β12 to 29.9 Hz, low γ30 to 49.9 Hz and high γ50 to 100 Hz) defined traditionally for each recording of each rat. In addition, 1 to 100 Hz EEG spectrum is represented by a line graph. These spectrogram data are provided to the client separately and not all data are included in this article, because the number of curve graphs is extensive. For clarity, the percentage change of each frequency band in the time period after administration is presented.

[0695] result

[0696] Sleep

[0697] The hypnogram of compound I-14 showed that REM and NREM in the high-dose (30 mg / kg) treatment group were significantly reduced compared to the vehicle (A) group. An increase in quiet wakefulness was observed in the highest dose compound group. In this treatment group, the onset of NREM, REM and REM latency was postponed. The most profound effects of compound I-14 appeared in high doses, where the effects on quiet wakefulness (increase), the effects on REM (decrease) and the effects on NREM (decrease) continued for several hours after administration.

[0698] Spectrum Analysis

[0699] In QW, compound I-14 (3 mg / kg) increased low gamma from 0 to 180 minutes after dosing, while at a 30 mg / kg dose, it increased low gamma from 0 to 240 minutes after dosing and increased high gamma from 0 to 180 minutes after dosing. At 30 mg / kg, in NREM, the compound decreased delta, theta, alpha and increased low and high gamma from approximately 0 to 300 minutes after dosing.

[0700] Example 13: Evaluation of the cognitive effects of compound I-14 in a macaque model of Parkinson's disease cognitive deficits induced by chronic low-dose MPTP

[0701] This study is a non-GLP study in the chronic low-dose MPTP lesion macaque model of Parkinson's disease. This model has been described in the literature (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3282499 / ). The study is designed to evaluate the effect of compound I-14 administered as a monotherapy on the performance of the following cognitive tasks: variable delayed response (VDR), persistence performance (CPT), visual discrimination reversal learning (SDR) and object retrieval (OR). The study was carried out in animals that had previously been treated with low-dose MPTP and were known to have difficulties in the performance of the tasks mentioned above. These studies utilized four male cynomolgus macaques with cognitive deficits induced by MPTP.

[0702] The study has 4 parts. The first part of the study is to collect baseline data about four tasks to be studied from four animals. The second part of the study is to apply vehicle (sterile filtered distilled water) for 16 days and collect vehicle data about four tasks once a week for two weeks. In the third part of the study, compound I-14 is orally administered daily for five days and then collects data about four tasks once a week for two weeks, wherein compound I-14 (3.0mg / kg) is continued to be administered daily 2 hours before testing every day. In the final part of the study, there is a 9-day clearance period and then collects data about four tasks once a week for two weeks. After pre-administration for five days and daily administration for two weeks, a dose of compound I-14 is evaluated in the form of monotherapy (3.0mg / kg). The effect of treatment on cognitive performance is evaluated 2 hours after daily drug administration and during drug clearance.

[0703] MPTP-HCl is administered by intravenous injection at a dosage in the range of 0.05mg / kg to 0.30mg / kg, 2 to 3 times per week for several months. MPTP administration continues until cognitive deficits occur, with minimal / mild Parkinson's disease motor impairment. If the animal shows at least 15% reduction in cognitive task performance relative to the baseline level before MPTP, it is considered as "cognitive impairment". Because the reaction to MPTP is somewhat special, MPTP is administered to play a role (that is, cognitive deficits occur and motor deficits occur subsequently), rather than given for a specific exposure duration or a specific MPTP cumulative dose.

[0704] SDR stands for a test of cognitive flexibility. In this task, three stimuli are presented on the screen simultaneously and one of these stimuli is arbitrarily designated as a positive (reward) stimulus, and contact with it produces a positive tone and a reward, while contact with a negative (non-reward) stimulus produces a different tone and a screen blank. The position of the stimulus on the screen is pseudo-randomly varied according to different trials. A maximum of 300 trials are displayed in each stage. The measurements recorded for each stage are: 1) the total number of trials required for learning the initial discrimination (i.e., reaching the specified 14 / 16 correct standard) and 2) the total number of trials required for learning (i.e., reaching the same criteria as mentioned above) reversal, where the previous negative (non-reward) stimulus is now a positive (reward) stimulus (i.e., reaching the standard). The number of trials required to reach the standard for learning the reversal of discrimination is considered a measure of cognitive flexibility.

[0705] Compound I-14 monotherapy significantly improves the efficacy in the SDR paradigm. CY3018 significantly reduces the number of trials required to reach the standard of learning discrimination reversal, which is intended to improve at least one aspect of these animals' cognition. Simple discrimination efficacy was not significantly changed by the administration of vehicle or compound I-14. However, the administration of compound I-14 significantly improved the discrimination reversal learning efficacy (Figure 6).

[0706] Additionally, during baseline, vehicle, and washout testing, some animals failed to successfully learn the discriminative reversal, however, when tested by compound 1-14, all of the animals learned the reversal, indicating that this compound had a positive effect on all animals tested.

Claims

1. A method for treating a mitochondrial disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof: in: J C Selected from hydrogen, halogen, C 1-6 Alkyl and C substituted by 1 to 3 fluorine atoms 1-6 Fluoroalkyl; X is N or C (J C1 ); J C1 Selected from hydrogen, halogen, C 1-6 Alkyl and C substituted by 1 to 3 fluorine atoms 1-6 Fluoroalkyl; Each J B are independently selected from hydrogen, halogen, C 1-6- Alkyl and C substituted by 1 to 3 fluorine atoms 1-6 Fluoroalkyl; J D Selected from hydrogen, halogen, C 1-6 Alkyl and C substituted by 1 to 3 fluorine atoms 1-6 fluoroalkyl; and n is an integer selected from 0, 1, 2, 3 or 4.

2. The method according to claim 1, in: J C is selected from hydrogen, halogen and C 1-6 alkyl; X is N or C (J C1 ); J C1 is selected from hydrogen, halogen and C 1-6 alkyl; Each J B are independently selected from hydrogen, halogen and C 1-6 alkyl; J D is selected from hydrogen, halogen and C 1-6 Alkyl; and n is an integer selected from 0, 1, 2, 3 or 4.

3. The method according to claim 1 or 2, wherein the compound is represented by formula IA: or a pharmaceutically acceptable salt thereof.

4. The method according to any one of claims 1 to 3, wherein J C1 is H, F or Cl.

5. The method according to any one of claims 1 to 3, wherein J C1 For H.

6. The method according to any one of claims 1 to 3, wherein J C1 For F.

7. The method according to claim 1 or 2, wherein the compound is represented by formula IB: or a pharmaceutically acceptable salt thereof.

8. The method according to any one of claims 1 to 7, wherein n is 2 or 3.

9. The method according to any one of claims 1 to 7, wherein n is 0 or 1.

10. The method according to any one of claims 1 to 9, wherein each J B are independently H, F or C 1-4 alkyl.

11. The method according to claim 10, wherein each J B is independently F or methyl.

12. The method according to claim 8, wherein n is 2 and J B All are F, or J B One is F and the other is methyl.

13. The method according to claim 8, wherein n is 3 and J B Two of them are F and the other one is methyl.

14. The method according to claim 9, wherein n is 1 and J B For F.

15. The method according to claim 9, wherein n is 0.

16. The method according to any one of claims 1 to 15, wherein J D For hydrogen.

17. The method according to any one of claims 1 to 15, wherein J D It is F, Cl or methyl.

18. The method according to any one of claims 1 to 15, wherein J D For F.

19. The method according to any one of claims 1 to 18, wherein J C It is H or F.

20. The method according to any one of claims 1 to 18, wherein J C For H.

21. The method according to any one of claims 1 to 20, wherein the mitochondrial disease is selected from the group consisting of Alpers disease, autosomal dominant optic atrophy (ADOA), Bartter syndrome / LIC (lethal infantile cardiomyopathy), beta-oxidation defects, long chain fatty acid transport defects, coenzyme Q10 defects, complex I, II, III, IV, V defects, chronic progressive lateral ophthalmoplegia (CPEO), Friedreich's ataxia, Kearns-Sayre syndrome, leukodystrophy, Leigh disease or syndrome, LHON, LHONPlus, MELAS (mitochondrial myopathy, encephalomyopathy, Lactic acidosis, stroke-like symptoms), myoclonic epilepsy with rending red muscle fiber disease (MERRF), mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial cytopathy, mitochondrial DNA depletion, mitochondrial encephalopathy, mitochondrial myopathy, multiple mitochondrial dysfunction syndrome, MNGIE (myoneurogenic gastrointestinal encephalopathy), NARP (neuropathy, ataxia, retinitis pigmentosa, and ptosis), Pearson syndrome, pyruvate carboxylase deficiency, pyruvate dehydrogenase deficiency, or pyruvate dehydrogenase complex deficiency (PDCD / PDH), and POLG mutations.

22. The method according to claim 21, wherein the mitochondrial disease is selected from the group consisting of Alpers disease, Complex I, II, III, IV deficiency, CPEO, KSS, LCHAD, Leigh syndrome, leukodystrophy, LHON, MELAS, MEPAN, MERRF, MIRAS, mitochondrial DNA depletion, MNGIE, NARP, Pearson syndrome and POLG mutation.

23. The method according to claim 21, wherein the mitochondrial disease is a Complex I mitochondrial disease.

24. The method according to claim 21, wherein the mitochondrial disease is MELAS.

25. The method according to claim 21, wherein the mitochondrial disease is Leigh's syndrome.

26. The method according to any one of claims 1-25, wherein the method further comprises administering to the subject an additional therapeutic agent.

27. The method according to any one of claims 1-26, wherein the treatment results in an increase in cerebral blood flow (CBF) in the brain of the subject.

28. The method according to claim 27, wherein the increase in cerebral blood flow is measured by ASL / MRI.

29. The method according to any one of claims 1-28, wherein the treatment results in increased brain connectivity in the subject.

30. The method of claim 29, wherein the increased brain connectivity is measured by functional magnetic resonance imaging (fMRI) BOLD.

31. The method according to any one of claims 1-30, wherein the treatment improves cognitive abilities of the subject.

32. The method according to any one of claims 1-31, wherein the treatment reduces inflammation in the subject.

33. The method according to claim 32, wherein the reduction in inflammation is determined by a change in the value of a biomarker associated with inflammation.

34. The method according to any one of claims 1-33, wherein the treatment reverses or reduces cardiovascular damage or dysfunction.

35. The method according to any one of claims 1 to 34, wherein the treatment reduces the value of a biomarker associated with mitochondrial dysfunction.

36. The method according to claim 35, wherein the biomarker associated with mitochondrial dysfunction is selected from the group consisting of lactate, GDF-15 and FGF-21.

37. The method according to claim 35, wherein the biomarker associated with mitochondrial dysfunction is selected from the group consisting of GDF-15 and FGF-21.

38. The method according to claim 35, wherein the biomarker associated with mitochondrial dysfunction is GDF-15.

39. The method according to claim 35, wherein the biomarker associated with mitochondrial dysfunction is FGF-21.

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