Treatment of mitochondrial disorders with sGC stimulators

Abstract

The present disclosure relates to the use of stimulators of soluble guanylate cyclase (sGC), pharmaceutically acceptable salts thereof, and pharmaceutical preparations or dosage forms containing them, alone or in combination with one or more additional agents, for the treatment of various mitochondrial diseases, in which increased sGC stimulation, or increased concentrations of nitric oxide (NO) or cyclic guanosine 3',5'-monophosphate (cGMP), or both, or upregulation of the NO-sGC-cGMP pathway is desired. Compounds useful in the methods of the present invention are of Formula I or pharmaceutically acceptable salts thereof:

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 417,120, filed October 18, 2022. The entire contents of the prior application are expressly incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present disclosure relates to the use of stimulators of soluble guanylate cyclase (sGC), pharmaceutically acceptable salts thereof, and pharmaceutical formulations or dosage forms containing them, alone or in combination with one or more additional agents, for the treatment of various mitochondrial diseases in which increased sGC stimulation, or increased concentrations of nitric oxide (NO) or cyclic guanosine 3',5'-monophosphate (cGMP), or both, or upregulation of the NO-sGC-cGMP pathway, is desired. [Background technology]

[0003] sGC is the primary receptor for NO in vivo. Upon binding to sGC, NO activates its catalytic domain, leading to the conversion of guanosine-5'-triphosphate (GTP) to the second messenger cGMP. Elevated levels of cGMP then regulate the activity of downstream effectors, including protein kinases, phosphodiesterases (PDEs), and ion channels. In the body, NO is synthesized from arginine and oxygen by various nitric oxide synthase (NOS) enzymes and by the sequential reduction of inorganic nitrate. 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 many diseases. sGC stimulators are heme-dependent agonists of the sGC enzyme that act synergistically with various amounts of NO to increase its enzymatic conversion of GTP to cGMP. sGC stimulators are clearly distinct from and structurally unrelated to another class of NO-independent, heme-independent agonists of sGC known as sGC activators.

[0004] Therapies that improve or restore sGC function would offer considerable advantages over current alternative therapies that either target the NO-sGC-cGMP pathway or benefit from upregulation of the NO-sGC-cGMP pathway. There is an urgent need to develop new and safe therapies for patients with a dysfunctional or downregulated NO-sGC-cGMP pathway.

[0005] Mitochondria are organelles that generate energy for cells through oxidative phosphorylation to produce adenosine triphosphate (ATP) and are required for normal cellular function. Proper mitochondrial function is therefore important for maintaining health and life.

[0006] Mitochondrial diseases are a group of rare genetic disorders that occur when mitochondria are unable to generate enough energy for the body to function properly. They manifest clinically in a heterogeneous manner. Mitochondrial diseases can result from mutations (acquired or inherited) in mitochondrial DNA or nuclear genes encoding mitochondrial components. These disorders can be present at birth or can develop later in life. Many of these diseases can manifest with central nervous system (CNS) dysfunction.

[0007] In addition to reduced ATP production in mitochondrial diseases, reduced pyruvate conversion to acetyl-CoA, decreased nitric oxide (NO) synthesis resulting in NO deficiency, increased cell damage due to increased reactive oxygen species, and lactic acidosis due to decreased vascular reactivity are also observed. These can result in debilitating physical, developmental, and cognitive disabilities 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 significantly reduced in patients with mitochondrial diseases. It is estimated that one in 4,000 people has a mitochondrial disease. Mitochondrial diseases are usually progressive, and there are currently no effective treatments or cures for these diseases. Management is primarily supportive and may include nutritional management, exercise, and / or vitamin or amino acid supplements.

[0008] sGC stimulators have been found to be useful in the potential treatment of 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 September 23, 2022).

[0009] sGC stimulators that can cross the blood-brain barrier (BBB) ​​and penetrate into the central nervous system (CNS) provide additional benefits for the treatment of mitochondrial diseases with CNS manifestations. Thus, the sGC stimulators described herein are generally useful for the treatment of mitochondrial diseases. In addition, they are useful for the treatment of CNS manifestations of mitochondrial diseases due to their ability to cross the BBB and activate targets in the brain. Because treatment options for mitochondrial diseases are very limited, there remains a need to develop new therapies that improve many of the clinical manifestations associated with these diseases, including but not limited to CNS manifestations. Summary of the Invention

[0010] The present invention is based on the discovery that the compounds disclosed herein are potent sGC stimulators and therefore may be useful for treating mitochondrial diseases. Additionally, the discovery that these compounds are not only potent sGC stimulators but also brain-penetrating agents makes them useful for treating the CNS manifestations of many mitochondrial diseases. Compounds with related structural features, particularly a 4-OH substituent on the pyrimidine ring, were previously known only as synthetic intermediates that could be used in the preparation of sGC stimulators with a 4-amino substituent on the pyrimidine ring. The sGC-promoting activity of these classes of compounds was previously unknown. Additionally, no medical uses have been found for previously disclosed compounds of this class. It has been unexpectedly discovered that the compounds disclosed herein possess potent sGC-promoting activity, penetrate the BBB, increase peripheral cGMP concentrations in the brain, and exhibit both peripheral and CNS activity in in vitro and in vivo assays.

[0011] In a first aspect, the present invention provides a method of treating a mitochondrial disease in a subject in need thereof, comprising administering a therapeutically effective amount of a compound of formula I:

[0012] [ka]

[0013] [In the formula, J C is hydrogen, halogen, C 1~6 C substituted with alkyl and 1 to 3 fluorine atoms 1~6 fluoroalkyl; X is N or C(J C1 ) and J C1 is hydrogen, halogen, C 1~6 C substituted with alkyl and 1 to 3 fluorine atoms 1~6 fluoroalkyl; each J B is hydrogen, halogen, C 1~6 C substituted with alkyl and 1 to 3 fluorine atoms 1~6 fluoroalkyl; J D is hydrogen, halogen, C 1~6 C substituted with alkyl and 1 to 3 fluorine atoms 1~6 fluoroalkyl; n is an integer selected from 0, 1, 2, 3, or 4. or a pharmaceutically acceptable salt thereof, alone or in combination therapy.

[0014] In a second aspect, the present invention is directed 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.

[0015] In a third aspect, the present invention is further directed 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, for the manufacture of a medicament for the treatment of a mitochondrial disease in a subject in need thereof.

[0016] In a fourth aspect, the present invention is further directed to a compound of Formula I, or a pharmaceutically acceptable salt thereof, a pharmaceutical composition or dosage form comprising a compound of Formula I or a pharmaceutically acceptable salt thereof, for use in the treatment of a mitochondrial disease in a subject in need thereof.

[0017] In some embodiments of the first through fourth aspects, the mitochondrial disease is a disease that presents with CNS dysfunction or CNS symptoms. In some embodiments of the first through fourth aspects, the compounds of Formula I are useful for treating CNS manifestations of mitochondrial disease. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 shows the effect of compound I-14 on the change from baseline in MAP (ΔBMAP) in male normotensive rats. [Figure 2] FIG. 2 shows the effect of compound I-20 on ΔBMAP in male normotensive rats. [Figure 3] Figure 3 shows the levels of compound I-14 in the STR and HIPP regions of the brain of adult male Sprague-Dawley rats following PO administration of compound I-14 (3 mg / kg) at T = 0. Data are presented as mean ± SEM, N = 5. [Figure 4] FIG. 4 shows the concentration of cGMP in the CSF of rats 1, 2 and 6 hours after administration of a single oral dose of compound I-20 (1 mg / kg, 3 mg / kg or 10 mg / kg). [Figure 5] FIG. 5 shows the concentration of cGMP in the CSF of rats 1, 2 and 6 hours after administration of a single oral dose of compound I-14 (1 mg / kg, 3 mg / kg or 10 mg / kg). [Figure 6]Figure 6 shows the cognitive effects of compound I-14 in a chronic low-dose MPTP-lesioned macaque model of cognitive impairment in Parkinson's disease. SDR performance was significantly impaired after chronic low-dose MPTP exposure (**P<0.01). SD (simple discrimination) and SDR (simple discrimination reversal) performance following vehicle administration were no different from MPTP baseline performance. SDR performance significantly improved after administration of compound I-14 (**P<0.01) and worsened during washout (**P<0.01 vs. drug performance). Figure 6A shows mean ± SEM performance; Figure 6B is a scatter plot showing individual data with mean ± SEM. N = normal, pre-MPTP; WO = washout. DETAILED DESCRIPTION OF THE INVENTION

[0019] Reference will now be made in detail to certain specific embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the invention as defined in the claims. The invention is not limited to the methods and materials described herein, but includes all methods and materials similar or equivalent to those described herein that may be used in the practice of the invention. In the event that one or more of the incorporated references, patents, or similar materials, including but not limited to defined terms, term use, described techniques, etc., differs from or contradicts this application, this application controls.

[0020] Compound definitions and general terminology For purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, and the Handbook of Chemistry and Physics, 75th Edition, 1994. Additionally, 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, M.B. and March, J., eds., John Wiley & Sons, New York: 2001, which are incorporated herein by reference in their entireties.

[0021] If one or more positions of 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 the same for each occurrence at each position, unless otherwise specified. For example, phenyl can be substituted with R 100 In the two cases, each R 100 is independently selected from halogen and methyl, this corresponds to R 100 each instance of is independently selected from halogen or methyl; for example, one R 100 This means that one can be fluoro, one can be methyl, or both can be chloro, etc. Similarly, when a substitutable atom is bonded to two or more hydrogens (e.g., CH or NH), the substituents can be "independently selected" to be equal or the same for each instance at each position, unless otherwise specified. For example, when methyl (e.g., CH) is bonded to R 100 In the two cases, each R 100 is independently selected from halogen and methyl, this corresponds to R 100 each instance of is independently selected from halogen or methyl; for example, one R 100This means that one can be fluoro, one can be methyl (e.g., CHF(CH3)), or both can be chloro (e.g., CHCl2), etc.

[0022] The selection of substituents and combinations envisioned by this disclosure are only those that result in the formation of stable or chemically viable compounds. Such choices and combinations will be apparent to those skilled in the art and can be determined without undue experimentation. The term "stable," as used herein, refers to compounds that are substantially unchanged when subjected to conditions that allow for their production, detection, and, in some embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. Chemically viable compounds are compounds that can be prepared by one skilled in the art based on the disclosure herein, supplemented as necessary by relevant knowledge in the art.

[0023] Unless otherwise stated, all tautomeric forms of the compounds of the present disclosure are also within the scope of the invention. In one embodiment, the present invention provides a method for converting hydrogen to deuterium (i.e. 2 H), which may be preferred in some circumstances because it may provide certain therapeutic benefits resulting in better metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements). Deuterium-labeled compounds of the present invention may generally be prepared by substituting a deuterated reagent for a non-deuterated reagent according to procedures similar to those disclosed in the following schemes and / or examples herein.

[0024] For example, the term "alkyl," as used herein, as in "alkyl chain" or "alkyl group," refers to a saturated, unbranched (e.g., straight) or branched, monovalent hydrocarbon radical. x Alkyl is an alkyl chain containing x carbon atoms, where x is an integer different from 0. x~yAn "alkyl" is an alkyl chain containing x to y (inclusive) number of carbon atoms, where x and y are two different integers and both are different from 0. For example, C 1~6 Alkyl is an alkyl as defined above containing any number of carbon atoms from 1 to 6. Examples of alkyl groups include, but are not limited to, methyl (i.e., C1 alkyl), ethyl (i.e., C2 alkyl), n-propyl (C3 alkyl), isopropyl (different C3 alkyl), n-butyl, isobutyl, s-butyl, t-butyl, pentyl, hexyl, heptyl, octyl, and the like. In certain embodiments, an alkyl group is C 1~4 In certain embodiments, the alkyl group is C 1~3 Alkyl or C 1~2 In yet other embodiments, the alkyl group is methyl or ethyl.

[0025] The term "fluoroalkyl," as used herein, refers to an alkyl group as defined above, in which one or more of the hydrogen atoms bonded to the chain carbon atoms are replaced with fluoro at any one or more carbon atoms of the alkyl group. For example, a fluoroalkyl substituted with 1 to 3 fluorine atoms is an alkyl group in which 1 to 3 hydrogen atoms are replaced with fluorine atoms at any position on either the same or different carbon atoms of the alkyl chain.

[0026] As used herein, the term "halogen" or "halo" means F, Cl, Br, or I. In certain embodiments, halo is F or Cl. In yet other embodiments, halo is F.

[0027] The term "hydroxyl" or "hydroxy" refers to --OH. The compounds of the invention are defined herein by their chemical structures and / or chemical names. Where 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 is determinative of the compound's identity.

[0028] Compound and Composition Embodiments The present invention is directed to the medical use of a compound of formula I, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof, according to the first to fourth aspects discussed above.

[0029] [ka]

[0030] [In the formula, J C is hydrogen, halogen, C 1~6 C substituted with alkyl and 1 to 3 fluorine atoms 1~6 fluoroalkyl; X is N or C(J C1 ) and J C1 is hydrogen, halogen, C 1~6 C substituted with alkyl and 1 to 3 fluorine atoms 1~6 fluoroalkyl; each J B is hydrogen, halogen, C 1~6 C substituted with alkyl and 1 to 3 fluorine atoms 1~6 fluoroalkyl; J D is hydrogen, halogen, C 1~6 C substituted with alkyl and 1 to 3 fluorine atoms 1~6 fluoroalkyl; and n is an integer selected from 0, 1, 2, 3, or 4.

[0031] 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 is a halogen, C 1~6 C substituted with alkyl and 1 to 3 fluorine atoms 1~6fluoroalkyl, all other carbon atoms of the phenyl ring are unsubstituted, and the remaining variables are as defined above.

[0032] 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 hydrogen, halogen and C 1~6 alkyl; J C1 is hydrogen, halogen and C 1~6 alkyl, and each J B is hydrogen, halogen and C 1~6 alkyl; J D is hydrogen, halogen and C 1~6 alkyl, and the remainder of the variables are as defined above for Formula I in the first aspect or embodiment.

[0033] In a third embodiment of the first, second, third and fourth aspects, the compound of formula I has formula IA:

[0034] [ka]

[0035] 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. In a fourth embodiment of the first, second, third and fourth aspects, for a 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 first, second or third embodiment.

[0036] In a fifth embodiment of the first, second, third and fourth aspects, for a compound of formula IA or a pharmaceutically acceptable salt thereof, J C1is H; the remaining variables are as defined in the first aspect or any one of the first through fourth embodiments.

[0037] In a sixth embodiment of the first, second, third and fourth aspects, for a compound of formula IA or a pharmaceutically acceptable salt thereof, J C1 is F; the remaining variables are as defined in the first aspect or any one of the first through fifth embodiments.

[0038] In a seventh embodiment of the first, second, third and fourth aspects, the compound of formula IB has the formula IB:

[0039] [ka]

[0040] 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. In an eighth embodiment of the first, second, third and fourth aspects, for compounds of formula I, IA or IB or a pharmaceutically acceptable salt thereof, n is 2 or 3; the remaining variables are as described in the first aspect or the first, second, third, fourth, fifth, sixth or seventh embodiment.

[0041] In a ninth embodiment of the first, second, third, and fourth aspects, for compounds 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 It is alkyl.

[0042] 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 C1~4 and 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 are independently F or C 1~4 It is alkyl.

[0043] In an eleventh embodiment of the first, second, third and fourth aspects, for compounds of formula I, IA or IB or a pharmaceutically acceptable salt thereof, n is 2 or 3; B is independently F or methyl; and the remaining variables are as described in the first aspect or the first, second, third, fourth, or fifth, sixth or seventh embodiment.

[0044] In a twelfth embodiment of the first, second, third and fourth aspects, for compounds of formula I, IA or IB or a pharmaceutically acceptable salt thereof, n is 2; B are both F or J B is 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, one J B is F and the other is methyl.

[0045] In a thirteenth embodiment of the first, second, third and fourth aspects, for compounds of formula I, IA or IB or a pharmaceutically acceptable salt thereof, n is 3; B two of 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 is H.

[0046] In a fourteenth embodiment of the first, second, third and fourth aspects, for compounds of formula I, IA or IB or a pharmaceutically acceptable salt thereof, n is 1; 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.

[0047] In a fifteenth embodiment of the first, second, third and fourth aspects, for compounds of formula I, IA or IB or a pharmaceutically acceptable salt thereof, n is 0; the remaining variables are as described in the first aspect or the first, second, third, fourth, fifth, sixth or seventh embodiment.

[0048] 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 remainder of the 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.

[0049] 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.

[0050] In an eighteenth 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; 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] [Table 1-1]

[0055] [Table 1-2]

[0056] [Table 1-3]

[0057] [Table 1-4]

[0058] 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 represented by the following formula:

[0059] [ka]

[0060] The compound I-14 is the sodium salt of the compound I-14 represented by the formula: 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 I-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 represented by the following formula:

[0061] [ka]

[0062] The compound I-20 is the sodium salt of the compound I-20. In a twenty-fourth embodiment of the first, second, third and fourth aspects, the compound of the invention has the formula IC:

[0063] [ka]

[0064] or a pharmaceutically acceptable salt thereof, wherein X is N or C(J C1 ) and X is C(J C1 ) is represented by C in the table below; variables X, J C1 and J. B are defined in the table below; furthermore, Me represents a methyl group, and Me-F represents a fluorinated methyl group substituted with 1 to 3 fluorine atoms (i.e., -CHF, -CHF, or -CF):

[0065] [Table 2-1]

[0066] [Table 2-2]

[0067] [Table 2-3]

[0068] [Table 2-4]

[0069] [Table 2-5]

[0070] [Table 2-6]

[0071] [Table 2-7]

[0072] Pharmaceutically acceptable salts of the present invention "Pharmaceutically acceptable salts" of the compounds described herein include those derived from the compounds when mixed with inorganic or organic acids or bases. In some embodiments, salts can be prepared in situ during the final isolation and purification of the compounds. In other embodiments, salts can be prepared from the free form of the compounds in a separate synthetic 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 that can be used in medicine. However, non-pharmaceutically acceptable salts may be useful in the preparation of compounds of Formula I or their pharmaceutically acceptable salts.

[0073] 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, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc, and the like. Particular embodiments include ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable non-toxic organic bases include salts derived from primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, arginine, betaine, caffeine, choline, N, N 1 - Including salts of dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc.

[0074] 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, calcium, magnesium, potassium, or sodium salt. In other embodiments, the salt is a sodium salt.

[0075] When the compound of Formula I is a base, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids, such as 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, gentisinate, gluconate, gluconic acid, glucuronate, glutamate, glutamic acid, hydrobromide, hydrochloric acid, iodide, isethionate, isothioate, methylisothiazolinone ... Acids include nicotinate, lactate, 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, saccharate, salicylate, succinic acid, succinate, sulfuric acid, sulfate, tannate, tartrate, tartaric acid, p-toluenesulfonate, p-toluenesulfonic acid, etc. Particular embodiments include citric acid, hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid, and tartaric acid.

[0076] The compounds described herein, as well as their pharmaceutically acceptable salts, can also be utilized in compositions or dosage forms to treat or prevent the diseases identified herein. Pharmaceutical compositions, dosage forms and methods of administration The compounds disclosed herein and their pharmaceutically acceptable salts thereof can be formulated as pharmaceutical compositions or "formulations" for treatment and use according to the present invention.

[0077] A typical formulation is prepared by mixing a 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 materials, gelatin, oils, solvents, water, etc. The specific carrier, diluent, or excipient used depends on the means and purpose for which the compound of Formula I is formulated. Solvents are generally selected based on solvents recognized by those skilled in the art as safe (GRAS: Generally Regarded as Safe) for administration to mammals. Generally, safe solvents are non-toxic aqueous solvents, such as water and other non-toxic solvents that are soluble 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 that provide a quality presentation of the drug (i.e., a compound of Formula I or a pharmaceutical composition thereof) or aid in manufacturing a pharmaceutical product (i.e., a medicament), such as one or more buffering agents, stabilizers, antiadherents, surfactants, wetting agents, lubricants, emulsifiers, binders, suspending agents, disintegrants, fillers, sorbents, coatings (e.g., enteric or sustained release), preservatives, antioxidants, opacifying agents, glidants, processing aids, colorants, sweeteners, flavors, flavoring agents, and other known additives.

[0078] Acceptable diluents, carriers, excipients, and stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); 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 dextrin; 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™, PLURONICS™, or polyethylene glycol (PEG). Active pharmaceutical ingredients can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, for example, in hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively; colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's: The Science and Practice of Pharmacy, 21st ed., edited by University of the Sciences in Philadelphia, 2005 (hereinafter "Remington's").

[0079] Formulations can be prepared by conventional dissolving and mixing procedures.The term "therapeutically effective amount" as used herein means the amount of active compound or pharmaceutical agent that induces the biological or medical response in tissue, system, animal or human that is sought by researchers, veterinarians, physicians or other clinicians.The therapeutically effective amount of administered compound is determined by such considerations and is the minimum amount necessary to improve, cure or treat one or more of the disease or its symptoms.

[0080] The terms "administer," "administering," or "administration," with reference to a compound, composition, or dosage form of the invention, mean introducing a compound into the system of a subject or patient in need of treatment. When the compound of the invention is provided in combination with one or more other active agents, "administration" and variations thereof are each understood to include simultaneous and / or sequential introduction of the compound and the other active agents.

[0081] The compounds described herein can be administered systemically or locally, for example orally (in solid dosage forms including hard or soft capsules (e.g., gelatin capsules), tablets, pills, powders, sublingual tablets, troches, lozenges, and granules; and by inhalation (e.g., aerosols, gases, inhalers, nebulizers, etc.), otically (e.g., using ear drops), topically (e.g., in the form of creams, gels, inhalants, liniments, lotions, ointments, patches, pastes, powders, solutions, The compositions may be administered parenterally, including, but not limited to, liquid dosage forms including, but not limited to, pharmaceutically acceptable emulsions, microemulsions, aqueous or oily solutions, suspensions, syrups, and elixirs, or parenterally, such as via sprays, transdermal patches, ophthalmic solutions (e.g., eye drops, eye gels, eye ointments), rectally (e.g., enemas or suppositories), nasally, bucally, vaginally (e.g., douches, intrauterine devices, suppositories, vaginal rings, or tablets), ear drops, or via an implanted reservoir. The term "parenteral," as used herein, includes, but is not limited to, subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally, or intravenously.

[0082] Formulations of compounds intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions. In 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 starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants, such as glycerol; d) disintegrants, such as agar, calcium carbonate, jasmine, maltodextrin, niacin ... The tablet is mixed with potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, e) solution retarders such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof.The tablet can be uncoated or coated by known techniques, including microencapsulation, to mask unpleasant tastes or delay disintegration and absorption in the digestive tract, thereby providing a sustained effect over a longer period of time.For example, time-delay materials such as glyceryl monostearate or glyceryl distearate can be used alone or with wax.Water-soluble taste-masking materials such as hydroxypropyl-methylcellulose or hydroxypropyl-cellulose can be used.

[0083] In addition to the active compound, the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, 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 the inert diluent, the oral composition may also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents and perfumes.

[0084] Oral compositions (either solid or liquid) may also contain excipients and adjuvants, such as dispersing or wetting agents, for example, naturally occurring 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); emulsifying and suspending agents, for example, sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia; sweetening agents, flavorings, and fragrances; and / or one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, for example, sucrose or saccharin.

[0085] Pharmaceutical compositions can also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline, utilizing benzyl alcohol or other suitable preservatives, absorption enhancers to improve bioavailability, fluorocarbons, and / or other conventional solubilizers or dispersants. Formulations suitable for pulmonary or nasal administration have particle sizes ranging, for example, from 0.1 to 500 microns (including particles ranging between 0.1 and 500 microns in increments of microns, such as 0.5, 1, 30, 35 microns, etc.), which are administered by rapid inhalation through the nasal passages or through the mouth to reach the alveolar sacs.

[0086] The pharmaceutical compositions described herein can also be administered locally, especially when the target of treatment comprises areas or organs that are easily accessible by topical application, including diseases of the eye, ear, skin or lower intestinal tract.Suitable topical formulations can be easily prepared for each of these areas or organs.The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and, if necessary, any necessary preservative or buffer.

[0087] For topical application, pharmaceutical compositions can be formulated with a suitable ointment containing active ingredients 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 petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water.Alternatively, pharmaceutical compositions can be formulated with a suitable lotion or cream containing active ingredients suspended or dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2 octyldodecanol, benzyl alcohol and water.

[0088] Alternatively, the active ingredient can be formulated in a cream with an oil-in-water cream base. If desired, the aqueous phase of the cream base can contain a polyhydric alcohol, 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.

[0089] Topical formulations desirably include a compound that enhances 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 analogues.

[0090] The oily phase of emulsions prepared using the compounds of Table I can be composed of known ingredients in a known manner. While the phase may contain only an emulsifier (also known as an excretion enhancer), it is desirable to include a mixture of at least one emulsifier with a fat or oil, or both a fat and an oil. A hydrophilic emulsifier may be included along with a lipophilic emulsifier, which acts as a stabilizer. In some embodiments, the emulsifier includes both an oil and a fat. Together, the emulsifier, with or without a stabilizer, constitutes a so-called emulsifying wax, which, together with the oil and fat, constitutes a so-called emulsifying ointment base that forms the oily dispersed phase of a cream formulation. Excretion enhancers and emulsion stabilizers suitable for use in formulating compounds of Formula I include Tween™-60, Span™-80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate, and sodium lauryl sulfate.

[0091] In addition, the present invention contemplates the use of transdermal patches, which have the added benefit of providing controlled delivery of compounds to the body. Such dosage forms can be made by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

[0092] For ophthalmic use, the pharmaceutical composition may be formulated as a micronized suspension in pH-adjusted isotonic sterile saline, or preferably as a solution in pH-adjusted isotonic sterile saline, with or without a preservative, such as benzalkonium chloride. Alternatively, for ophthalmic use, the pharmaceutical composition may be formulated in an ointment such as petrolatum. For treatment of the eye or other external tissues, such as the mouth and skin, the formulation may be applied as a topical ointment or cream containing the active ingredient in an amount of, for example, 0.075 to 20% w / w. When formulated in an ointment, the active ingredient may be utilized with either an oil-based paraffinic or water-miscible ointment base.

[0093] 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, such as cocoa butter, beeswax, polyethylene glycol, or suppository wax, which are solid at ambient temperature but liquid at body temperature, so that they 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.

[0094] Sterile injectable forms of the compositions described herein (e.g., for parenteral administration) may be aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents (including those described in the preceding paragraphs). 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 conventionally used as solvents or suspending media. For this purpose, any non-irritating fixed oil, including synthetic monoglycerides or diglycerides, may be used. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectable solutions, including natural pharmaceutically acceptable oils, such as vegetable oils, such as peanut oil, olive oil, sesame oil, or coconut oil, especially their polyoxyethylated versions, or mineral oils such as liquid paraffin. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose, or similar dispersants commonly used in the preparation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Span, and other emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms, may also be used for the purpose of injectable formulations. Oil suspensions may contain thickening agents, such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners, such as those described above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as butylated hydroxyanisol or alpha-tocopherol.

[0095] In another embodiment, the compound of Formula I or its pharmaceutically acceptable salt can be formulated into a veterinary composition containing a veterinary carrier. The veterinary carrier is a material that is useful for administering the composition and can be a solid, liquid, or gaseous material that is otherwise inert. In the veterinary field, it is compatible with the active ingredient. These veterinary compositions can be administered parenterally, orally, or by any other desired route.

[0096] Definitions and general terminology regarding treatment methods The term "disease," as used herein, refers to any deviation from or disruption of the normal structure or function of any body part, organ, or system that is manifested by a characteristic set of symptoms and signs, and whose etiology, pathology, and prognosis may be known or unknown. The term disease encompasses disorders and conditions (or medical conditions) and other related terms such as syndromes, and is defined as a combination of symptoms attributable to a single cause or causes that commonly occur together to constitute a distinct clinical picture.

[0097] The term "mitochondrial disease" refers to a group of inherited conditions that affect mitochondria, the structures in each cell of the body responsible for generating energy. These disorders can manifest at any age in almost any affected organ, including the brain, muscles, heart, liver, nerves, eyes, ears, and kidneys. Some of these diseases affect only one organ or tissue, but many involve multiple organ systems, including the brain, muscles, heart, liver, nerves, eyes, ears, and / or kidneys. Mitochondrial diseases have heterogeneous manifestations.

[0098] Mitochondrial genetic disorders can result from mutations in either mitochondrial DNA or nuclear DNA, resulting in mitochondrial dysfunction and inadequate production of cellular ATP. Those resulting from mutations in mitochondrial DNA are inherited maternally, while those resulting from mutations in nuclear DNA can follow autosomal dominant, autosomal recessive, or X-linked patterns of inheritance (see https: / / rarediseases.info.nih.gov / diseases / 7048 / mitochondrial-genetic-disorders, last accessed June 3, 2022, the teachings of which are incorporated herein by reference).

[0099] Mitochondrial diseases contemplated throughout this disclosure are "primary mitochondrial diseases" or disorders. The term mitochondrial disease, as used herein, is equivalent to 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 accessed June 7, 2022).

[0100] Mitochondrial diseases are primarily manifested by a chronic loss of cellular ATP, which leads to a variety of clinical phenotypes and symptomatology. In addition to the ATP crisis, mitochondrial respiratory chain dysfunction also results in excessive ROS production and increased oxidative stress, leading to cellular and vascular damage and inflammation.

[0101] As used herein, "mitochondrial disease" is equivalent to the terms mitochondrial disorder or mitochondrial syndrome or mitochondrial condition. As used herein, the term mitochondrial disease and its equivalents refer to mitochondrial diseases of genetic origin and are the same as those referred to in the art as primary mitochondrial diseases.

[0102] "Treate," "treating," or "treatment," in reference to a disorder, disease, condition, symptom, or syndrome, refers to arresting or ameliorating the cause and / or effects (i.e., improvement in symptoms, physiological, physical, psychological, emotional, or any other clinical manifestation, observation, or measurement, or pathological assessment) of the disorder, disease, condition, or syndrome.

[0103] As used herein, the terms "treat," "treatment," or "treating" also refer to the delay, amelioration, or prevention of progression (i.e., known or expected progression of the disease), the severity and / or duration of the disease, or the delay, amelioration, or prevention of the progression of one or more symptoms, clinical manifestations, observations, or measurements, or the prevention or slowing of the negative progression of a pathological assessment (i.e., "managing" without "curing" the condition), resulting from the administration of one or more therapies.

[0104] As used herein, the terms "subject" and "patient" are used interchangeably. The terms "subject" and "patient" refer to an animal (e.g., a bird such as a chicken, quail, or turkey, or a mammal), and specifically, "mammal" includes non-primates (e.g., cows, pigs, horses, sheep, rabbits, guinea pigs, rats, cats, dogs, and mice), as well as primates (e.g., monkeys, chimpanzees, and humans), and more specifically, humans. In some embodiments, the subject is a non-human animal, such as a farm animal (e.g., horses, cows, pigs, and 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.

[0105] The term "biological sample," as used herein, refers to an in vitro or ex vivo sample, including, but not limited to, a cell culture or an extract thereof; a biopsy obtained from a mammal or an extract thereof; blood, saliva, urine, feces, semen, tears, lymph, ocular fluid, vitreous humor, cerebrospinal fluid (CSF), or other bodily fluid or an extract thereof.

[0106] Treatment method In a first aspect, the present invention is directed to a method of 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 through twenty-fourth embodiments above.

[0107] In a second aspect, the present invention is directed to a method of 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 through twenty-fourth embodiments above.

[0108] In a third aspect, the present invention is further directed 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, for the manufacture 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.

[0109] In a fourth aspect, the present invention is further directed 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 through twenty-fourth embodiments above.

[0110] Assessment of health status in patients with mitochondrial disease and the corresponding pathology underlying the observed dysfunction, decline, or symptoms can be performed using several different assessment tools or clinical measurements known and used in the art.

[0111] These range from imaging tools (e.g., magnetic resonance imaging (MRI), such as using arterial spin labeling (ASL) or functional fMRI-BOLD modalities), to laboratory measurements (e.g., fluid biomarkers measured in blood, cerebrospinal fluid (CSF), urine, plasma, serum, skin, saliva), to clinical outcome assessment tools or devices (e.g., patient- or clinician-reported outcome devices or performance outcome measures, such as cognitive assessments using PROMIS questionnaires, MFIS scoring, and others described herein or known in the art), digital assessments (e.g., those obtained with 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, clinic, or community settings. For example, the American Academy of Family Physicians (AAFP) lists and provides links to several possible cognitive assessment tools on its webpage, such as the MiniCog, MoCA, SLUMS test, CPCoG, MIS and MMSE, as well as others ( https: / / www.aafp.org / pubs / afp / issues / 2019 / 0115 / p101.html , last accessed June 3, 2022).

[0112] Some measurements are performed to aid in diagnosis and / or patient selection. Others are performed to aid in assessing prognosis. Others may be performed to assess pharmacological response to a particular intervention (pharmacodynamic or PD assessment) as described herein. Others may be performed to assess susceptibility to or risk of attenuation of or response to a particular intervention (e.g., assessment of genetic or other biomarkers), or to assess the progression of a patient's disease.

[0113] 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 patients with mitochondrial diseases as measured by ASL / MRI.

[0114] 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 diseases as measured by functional fMRI BOLD.

[0115] In another embodiment of the first, second, third and fourth aspects, the compounds disclosed herein are capable of improving cognition in patients with mitochondrial diseases as measured by one of the cognitive assessment tools known in the art.

[0116] In one embodiment of the first, second, third, and fourth aspects, the compounds disclosed herein are sGC stimulators that may be useful in the prevention and / or treatment of 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 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, as determined by changes in the level of a biomarker of inflammation.

[0117] In another embodiment of the first through fourth aspects of the present invention, the compounds disclosed herein are sGC stimulators that may be useful in the prevention and / or treatment of cardiovascular damage or dysfunction associated with mitochondrial disease. One embodiment of the present invention is a method of reversing or reducing cardiovascular damage or dysfunction, as determined by changes in the level of a biomarker of cardiovascular dysfunction in a subject with mitochondrial disease in need thereof, by administering to a 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.

[0118] One embodiment of the present invention, according to the first to fourth aspects, is a method for reducing the level of a biomarker associated with mitochondrial dysfunction in a patient with 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.

[0119] In some embodiments, the biomarker associated with mitochondrial dysfunction is selected from the group consisting of lactate, GDF-15, and FGF-21. In other embodiments, the biomarker for mitochondrial dysfunction is lactate. In other embodiments, it is selected from GDF-15 and FGF-21. In other embodiments, it is GDF-15. In still other embodiments, it is FGF-21.

[0120] Specific mitochondrial diseases that can be treated and / or prevented by administering a compound of formula I or a compound of 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: Alpers disease, autosomal dominant optic atrophy (ADOA), Barth syndrome / LIC (fatal infantile cardiomyopathy), beta-oxidation deficiency, long-chain fatty acid transport deficiency, coenzyme Q10 deficiency, complex I, II, III, IV, V deficiency, chronic progressive external ophthalmoplegia (CPEO), Friedreich's ataxia, Kearns-Sayre syndrome, leukodystrophy, Leigh's disease or syndrome, LHON, LHON Plus, MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like episodes), myoclonic epilepsy with ragged-red fibers (MERRF), mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial cytopathies, mitochondrial DNA depletion, mitochondrial encephalopathy, mitochondrial myopathy, multiple mitochondrial dysfunction syndrome, MNGIE (myoneurogenic gastrointestinal encephalopathy) 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.

[0121] In one embodiment, the mitochondrial disease is selected from Alpers, 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 complex I mitochondrial disease. In another embodiment, the mitochondrial disease is MELAS. In yet another embodiment, the mitochondrial disease is Leigh syndrome.

[0122] In another embodiment, 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 stimulating agents in biological samples is useful for various purposes known to those skilled in the art.Examples of such purposes include, but are not limited to, biological assays and biological specimen storage.

[0123] Combination therapy The compounds and pharmaceutical compositions described herein can be used alone or in combination therapy for the treatment of diseases mediated, modulated, or affected by sGC, cGMP, and / or NO.

[0124] As used herein, the terms "in combination" (as in the sentence "combination therapy") or "co-administration" can be used interchangeably to refer to the use of two or more therapies. The use of the terms does not restrict the order in which the therapies are administered to a subject.

[0125] The compounds and pharmaceutical compositions described herein can be used in combination therapy with one or more additional therapeutic agents.For the combined treatment with two or more active agents, when active agents are in separate dosage formulations, active agents can be administered separately or in combination.In addition, the administration of one element can be administered before, simultaneously with, or after the administration of other agents.

[0126] When used in combination therapy with other drugs, the "therapeutically effective amount" of the compounds and pharmaceutical compositions described herein and one or more other drugs depends on the type of drug used.Suitable dosages are known for approved drugs and can be adjusted by those skilled in the art according to the subject's condition, the type of condition to be treated, and the amount of the compounds described herein used.If the amount is not clearly indicated, an effective amount should be assumed.

[0127] In some embodiments, co-administration or combination therapy includes administration of a first and a second amount of a compound in an essentially simultaneous manner, for example, in a single pharmaceutical composition, such as a capsule or tablet having a fixed ratio of the first and second amounts, or in multiple separate capsules or tablets for each. In addition, such co-administration also includes use of each compound in a sequential manner, in any order.

[0128] When co-administration comprises separate administration of a first amount of the compound of Formula I and a second amount of additional therapeutic agent, the compounds are administered close enough in time to achieve desired therapeutic effect.For example, the period between each administration that can produce desired therapeutic effect can range from minutes to hours, and can be determined taking into account the properties of each compound, such as efficacy, solubility, bioavailability, plasma half-life and kinetic profile.For example, the compound of Formula I and the second therapeutic agent can be administered within about 24 hours of each other, within about 16 hours of each other, within about 8 hours of each other, within about 4 hours of each other, within about 1 hour of each other, or within about 30 minutes of each other, in any order.

[0129] Examples of other therapeutic agents that may be combined with the compounds of Formula I, or pharmaceutically acceptable salts thereof, either administered separately or in the same pharmaceutical composition, include, but are not limited to, the following: (1) Endothelium-derived releasing factor (EDRF) or NO gas.

[0130] (2) NO donors, including, but not limited to, nitrosothiols, nitrites, sydnonimines, NONOates, N-nitrosamines, N-hydroxylnitrosamines, nitrosimines, nitrotyrosines, diazetine dioxide, oxatriazole 5-imines, oximes, hydroxylamines, N-hydroxyguanidines, hydroxyureas, or furoxans. Some examples of these types of compounds include glyceryl trinitrate (GTN, also known as nitroglycerin, nitroglycerine, and trinitroglycerin), the nitrate ester of glycerol; sodium nitroprusside (SNP), in which a molecule of nitric oxide is coordinated to metallic iron forming a tetragonal bipyramidal complex; 3-morpholinosydnonimine (SIN-1), a zwitterionic compound formed by the combination of morpholine and sydnonimine; S-nitroso-N-acetylpenicillamine (SNAP), an N-acetylated amino acid derivative with a nitrosothiol functionality; diethylenetriamine / NO (DETA / NO), a compound of nitric oxide covalently bound to diethylenetriamine; and m-nitroxymethylphenyl ester of acetylsalicylic acid. Some more specific examples of these classes of NO donors include classical nitrovasodilators, such as organic nitrate and nitrite esters including nitroglycerin, amyl nitrite, isosorbide dinitrate, 5-isosorbide mononitrate, and nicorandil; isosorbide; 3-morpholinosydnonimine; linsidomine chlorohydrate ("SIN-1"); S-nitroso-N-acetylpenicillamine ("SNAP"); S-nitrosoglutathione (GSNO), sodium nitroprusside, S-nitrosoglutathione mono-ethyl-ester (GSNO-ester), 6-(2-hydroxy-1-methyl-nitrosohydrazino)-N-methyl-1-hexanamine, or diethylamine NONOate.

[0131] (3) Other substances that increase cGMP concentrations, including, but not limited to, protoporphyrin IX, arachidonic acid, and phenylhydrazine derivatives. (4) 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 (e.g., homo-Arg, homo-NOHA, N-tert-butyloxy- and N-(3-methyl-2-butenyl)oxy-L-arginine, canavanine, epsilon caproic acid, aguanine, Nitric oxide synthase substrates, including, but not limited to, agmatine, hydroxyl-agmatine, and L-tyrosyl-L-arginine; N-alkyl-N'-hydroxyguanidines (e.g., N-cyclopropyl-N'-hydroxyguanidine and N-butyl-N'-hydroxyguanidine), N-aryl-N'-hydroxyguanidines (e.g., N-phenyl-N'-hydroxyguanidine and its para-substituted derivatives having -F, -Cl, -methyl, -OH substituents, respectively); guanidine derivatives, such as 3-(trifluoromethyl)propylguanidine.

[0132] (5) Compounds that enhance eNOS transcription. (6) NO-independent, heme-independent sGC activators, including, but not limited to, 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-(thiomorpholine-4-sulfonyl)-phenyl)-benzamide (described in patent publications DE19830430 and WO2000002851); and HMR-1069 (manufactured by Sanofi-Aventis).

[0133] (7) YC-1 (see Patent Publications EP667345 and DE19744026); riociguat (BAY 63-2521, Adempas®, described in DE19834044); nerociguat (BAY 60-4552, described in WO2003095451); veruiciguat (BAY 1021189, described in US8420656); BAY 41-2272 (described in DE19834047 and DE19942809); BAY 41-8543 (described in DE19834044); etriciguat (described in WO2003086407); CFM-1571 (described in patent publication WO2000027394); A-344905, its acrylamide analog A-350619 and aminopyrimidine analog A-778935; Publications US20090209556, US8455638, US20110118282(WO2009032249), US20100292192, U S20110201621, US7947664, US8053455(WO2009094242), US20100216764, US8507512 (WO2010099054) US20110218202 (WO2010065275), US20130012511 (WO2011119518), US20130072492 (WO2011149921), US20130210798 (WO2012058132) and other sGC stimulators described in one of Tetrahedron Letters (2003), 44(48):8661-8663; and IW1973 (praliciguat), IW1701 (olinciguat) and CY6463 (formerly IW-6463). Heme-dependent NO-independent sGC stimulators, including but not limited to:

[0134] (8) Compounds that inhibit the degradation of cGMP and / or cAMP, including but not limited to: PDE1 inhibitors, PDE2 inhibitors, PDE-3 inhibitors such as amrinone, milrinone, enoximone, vesnarinone, pimobendan and olprinone, PDE4 inhibitors such as roflumilast, PDE5 inhibitors such as sildenafil and related drugs 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.

[0135] (9) Anticoagulants, including but not limited to: Coumarins (vitamin K antagonists), such as warfarin, acenocoumarol, phenprocoumon and phenindione, Heparin and derivatives, such as low molecular weight heparin, fondaparinux and idraparinux, Direct thrombin inhibitors, such as argatroban, lepirudin, bivalirudin, dabigatran and ximelagatran, and Tissue plasminogen activators, such as alteplase, which are used to dissolve clots and unclog arteries.

[0136] (10) Antiplatelet agents, including but not limited to clopidogrel, ticlopidine, dipyridamole, and aspirin. (11) Supplemental oxygen therapy.

[0137] (12) Alpha-1-adrenergic receptor antagonists, including, but not limited to, prazosin, indoramin, urapidil, bunazosin, terazosin, and doxazosin; atrial natriuretic peptide (ANP), ethanol, histamine inducers, tetrahydrocannabinol (THC), and papaverine.

[0138] (13) Bronchodilators, including but not limited to: short-acting beta-2 agonists, such as salbutamol or albuterol and terbutaline; long-acting beta-2 agonists (LABAs), such as salmeterol and formoterol; Anticholinergics, such as ipratropium and tiotropium; and theophylline, bronchodilators and phosphodiesterase inhibitors.

[0139] (14) Corticosteroids, including, but not limited to, beclomethasone, methylprednisolone, betamethasone, prednisone, prednisolone, triamcinolone, dexamethasone, fluticasone, flunisolide, hydrocortisone, and corticosteroid analogs such as budesonide.

[0140] (15) Omega-3 oils; folic acid, niacin, zinc, copper, Korean red ginseng root, ginkgo biloba, pine bark, Tribulus terrestris, arginine, oat (Avena sativa), horny goat weed, maca root, muira puama, saw palmetto, and Swedish flower pollen; vitamin C, vitamin E, vitamin K2; testosterone supplements, testosterone transdermal patches; dietary supplements, including, but not limited to, zoraxel, naltrexone, bremelanotide, and melanotan II.

[0141] (16) PGD2 receptor antagonist. (17) Immunosuppressants, including but not limited to cyclosporine, tacrolimus, rapamycin and other FK-506 type immunosuppressants, mycophenolate, mycophenolate mofetil.

[0142] (18) Nonsteroidal antiasthmatics, including but not limited to: beta-2 agonists, such as terbutaline, metaproterenol, fenoterol, isoetharine, albuterol, salmeterol, bitolterol, and pirbuterol; Beta-2 agonist-corticosteroid combinations, such as salmeterol-fluticasone, formoterol-budesonide, theophylline, cromolyn, cromolyn sodium, nedocromil, atropine, ipratropium, ipratropium bromide, and Leukotriene biosynthesis inhibitors, such as zileuton or veriflavone.

[0143] (19) Nonsteroidal anti-inflammatory drugs (NSAIDs), including but not limited to: Propionic acid derivatives such as alminoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid and tioxaprofen; Acetic acid derivatives, such as indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac, isoxepac, oxypinac, sulindac, tiopinac, tolmetin, zidometacin and zomepirac; Fenamic acid derivatives, such as flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid and tolfenamic acid, Biphenylcarboxylic acid derivatives, such as diflunisal and flufenisal, Oxicams, such as isoxicam, piroxicam, sudoxicam and tenoxicam, Salicylates, such as acetylsalicylic acid and sulfasalazine, and Pyrazolones such as apazone, bezpiperylon, feprazone, mofebutazone, oxyphenbutazone and phenylbutazone.

[0144] (20) 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, cyclooxygenase-2 (COX-2) inhibitors, including, but not limited to:

[0145] (21) Adrenergic neuron blocking agents, including but not limited to guanethidine and guanadrel. (22) Imidazoline I-1 receptor agonists, including but not limited to rilmenidine dihydrogen phosphate and moxonidine hydrochloride hydrate.

[0146] (23) Potassium channel activators, including but not limited to pinacidil. (24) Fenoldopam mesylate; other dopamine agonists, such as dopamine D1 agonists, including but not limited to ibopamine, dopexamine, and docarpamine.

[0147] (25) 5-HT2 antagonists, including but not limited to ketanserin. (26) Vasopressin antagonists, including but not limited to tolvaptan. (27) Calcium channel sensitizers, including but not limited to levosimendan or activators such as nicorandil.

[0148] (28) Adenylate cyclase activators, including but not limited to colforsin dalopate hydrochloride. (29) Positive inotropes, including but not limited to digoxin and methyldigoxin; metabolic inotropes, such as ubidecarenone; brain natriuretic peptides, such as nesiritide.

[0149] (30) Drugs used to treat erectile dysfunction, including but not limited to alprostadil, aviptadil, and phentolamine mesylate. (31) Drugs used to treat Alzheimer's disease and dementia, including but not limited to: Acetylcholinesterase inhibitors, such as galantamine, rivastigmine, donepezil and tacrine, and NMDA receptor antagonists, such as memantine, and 10. Oxidoreductase inhibitors, such as idebenone.

[0150] (32) Psychiatric medications, including but not limited to: Ziprasidone, risperidone, olanzapine, valproate, dopamine D4 receptor antagonists, such as clozapine; dopamine D2 receptor antagonists, such as nemonapride, mixed dopamine D1 / D2 receptor antagonists, such as zuclopenthixol; GABA A receptor modulators, such as carbamazepine, sodium channel inhibitors, e.g. lamotrigine, Monoamine oxidase inhibitors, such as moclobemide and indeloxazine, and Pimavanserin and perospirone.

[0151] (33) Drugs used to treat movement disorders or symptoms, including but not limited to: Catechol-O-methyltransferase inhibitors, such as entacapone, Monoamine oxidase B inhibitors, e.g. selegiline, dopamine receptor modulators, such as levodopa; dopamine D3 receptor agonists, such as pramipexole; decarboxylase inhibitors, e.g., carbidopa; Other dopamine receptor agonists, such as pergolide, ropinirole, cabergoline, ritigonide, istradefylline, talipexole, zonisamide and safinamide, and 10. Synaptic vesicular amine transporter inhibitors, such as tetrabenazine.

[0152] (34) Medications used to treat mood or affective disorders or OCD, such as the following types: tricyclic antidepressants, such as amitriptyline, desipramine, imipramine, amoxapine, nortriptyline, doxepin and clomipramine, selective serotonin reuptake inhibitors (SSRIs), such as paroxetine, fluoxetine, sertraline, trazodone, and citalopram; atypical antidepressants, such as agomelatine, Selective norepinephrine reuptake inhibitors (SNRIs), such as venlafaxine, reboxetine, and atomoxetine; dopaminergic antidepressants, such as bupropion and amineptine.

[0153] (35) Drugs used to enhance synaptic plasticity, including but not limited to: Nicotinic receptor antagonists, such as mecamylamine, and 10. Mixed 5-HT, dopamine and norepinephrine receptor agonists, such as lurasidone.

[0154] (36) Drugs used in the treatment of ADHD, such as amphetamines; 5-HT receptor modulators, such as vortioxetine, and alpha-2 adrenoceptor agonists, such as clonidine.

[0155] (37) Nitric oxide synthase cofactors, including but not limited to tetrahydrobiopterin, dihydrobiopterin, and sapropterin. (38) Blood glucose lowering drugs (also called blood glucose control drugs or antidiabetic drugs), including but not limited to: biguanides, e.g. metformin, sulfonylureas, such as glyburide, glibenclamide, glipizide, gliclazide, gliquidone, glimepiride, atorvastatin calcium in combination with glimepiride, meglinatide, tolbutamide, chlorpropamide, acetohexamide and tolazamide; alpha-glucosidase inhibitors, such as acarbose, epalrestat, voglibose and miglitol, insulin secretagogues, such as repaglinide, mitiglinide and nateglinide; Thiazolidinediones, such as rosiglitazone, troglitazone, ciglitazone, pioglitazone, englitazone, lobeglitazone sulfate and balaglitazone, DPP-4 inhibitors (or DPP-IV inhibitors), such as sitagliptin, vildagliptin, saxagliptin, alogliptin, linagliptin, alogliptin benzoate in combination with metformin or metformin hydrochloride, anagliptin, teneligliptin, 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), GLP-1 receptor agonists or incretin mimetics, such as exenatide, dulaglutide, liraglutide, semaglutide, lixisenatide, lixisenatide in combination with insulin glargine, albiglutide and pegapamozutide (TT-401), LY3298176 (a dual glucose-dependent insulinotropic polypeptide (GIP) and GLP-1 receptor agonist), SGLT2 inhibitors (SGLT2i), such as empagliflozin, empagliflozin in combination with linagliptin, empagliflozin in combination with metformin, ipragliflozin, ipragliflozin L-proline, tofogliflozin, sergliflozin etabonate, remogliflozin etabonate, ertugliflozin, ertugliflozin in combination with sitagliptin, ertugliflozin in combination with metformin, sotagliflozin, canagliflozin, canagliflozin in combination with metformin or metformin hydrochloride, dapagliflozin, dapagliflozin in combination with metformin or metformin hydrochloride and luseogliflozin, dapagliflozin in combination with saxagliptin, SGLT1 inhibitors or a combination of SGLT1 and SGLT2 inhibitors, such as sotagliflozin, Insulin therapy, such as one of many types of insulin, such as insulin glulisine, insulin degludec, insulin lispro, insulin aspart, insulin glargine, insulin detemir, isophane insulin, insulin Mixtard (human insulin containing both rapid-acting (soluble) and long-acting (isophane) insulin), insulin degludec in combination with insulin aspart, insulin human (rDNA origin) inhalation powder, recombinant human insulin, liver-directed endoplasmic reticulum insulin, insulin Tregopil (IN-105), insulin degludec in combination with liraglutide, insulin peglispro (LY-2605541) and nodulin, and Trimidone (lyn kinase activator).

[0156] (39) Blood pressure lowering drugs (also known as antihypertensives), including but not limited to: Diuretics, such as thiazide diuretics, chlorothiazide, chlorthalidone, hydrochlorothiazide, bendroflumethiazide, cyclopenthiazide, methyclothiazide, polythiazide, quinethazone, xipamide, metolazone, indapamide, cicletanine, furosemide, toresamide, amiloride, spironolactone, canrenoate potassium, eplerenone, triamterene, acetazolamide and carperitide; beta-blockers, such as acebutolol, atenolol, metoprolol and nebivolol; Angiotensin-converting enzyme (ACE) inhibitors, such as sulfhydryl-containing drugs (e.g., captopril, zofenopril), dicarboxylate-containing drugs (e.g., enalapril, quinapril, ramipril, perindopril, lisinopril, and benazepril), phosphonate-containing drugs (e.g., fosinopril), naturally occurring ACE inhibitors (e.g., casokinins, lactokineins, lactotripeptides Val-Pro-Pro and Ile-Pro-Pro), alacepril, delapril, cilazapril, imidapril, temocapril, moexipril, lisinopril, combinations of lisinopril with hydrochlorothiazide, trandolapril, and spirapril, Angiotensin II receptor blockers (ARBs), such as candesartan, losartan, losartan potassium-hydrochlorothiazide, valsartan, candesartan cilexetil, eprosartan, irbesartan, telmisartan, olmesartan medoxomil (or olmesartan), azilsartan medoxomil, azilsartan, amlodipine besilate in combination with irbesartan, azilsartan in combination with amlodipine besilate, cilnidipine in combination with valsartan, fimasartan rbesartan in combination with atorvastatin, irbesartan in combination with trichlormethiazide, losartan potassium in combination with hydrochlorothiazide and / or amlodipine besilate, atorvastatin calcium in combination with pratosartan, losartan potassium, nifedipine and candesartan cilexetil, sacubitril in combination with valsartan or LCZ-696, angiotensin AT2 antagonist and TAK-591 and olmesartan medoxomil, Endothelin receptor antagonists (ERAs), such as atrasentan, bosentan, sitaxsentan, ambrisentan, actelion-1 (macitentan), cyclo(D-trp-D-asp-L-pro-D-val-L-leu) (BQ-123), sparsentan, and tezosentan disodium; Electrocorticoid receptor antagonists (MRAs), such as spironolactone, amiloride hydrochloride in combination with spironolactone, aparalenone or MT-3995, eplerenone, and finerenone (BAY-94-8862); 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; Renin inhibitors, e.g. aliskiren, alpha-blockers, such as doxazosin and prazosin, alpha-beta blockers, such as carvedilol and labetalol; centrally acting agents, such as clonidine, guanfacine and methyldopa, vasodilators, such as nitroglycerin, hydralazine, and minoxidil, and Aldosterone antagonists such as finerenone, spironolactone and eplerenone.

[0157] (40) Antihyperlipidemic drugs, including but not limited to: statins, such as atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin; Combinations of statins with other drugs, such as amlodipine / atorvastatin, aspirin / pravastatin, ezetimibe / simvastatin, niacin / simvastatin, lovastatin / niacin, simvastatin / sitagliptin, and atorvastatin / ezetimibe; Fibrate or fibric acid derivatives. Examples include, but are not limited to, fenofibrate, gemfibrozil, bezafibrate, ciprofibrate, clinofibrate, and clofibrate.

[0158] Niacin (or nicotinic acid), bile acid ciquestrants, such as cholestyramine, colesevelam, colestilan, and colestipol; ezetimibe, lomitapide, phytosterols or orlistat, and PCSK9 inhibitors, such as alirocumab and evolocumab, (41) Sacubitril, or a combination of Sacubitril with Valsartan; a neprilysin inhibitor (also known as an endopeptidase inhibitor or NEP inhibitor or enkephalinase inhibitor), including but not limited to the investigational neprilysin inhibitors TD-1439 or TD-0714.

[0159] (42) Nephroprotective drugs, including but not limited to: bardoxolone, ACE inhibitors, e.g. captopril, ARBs, such as losartan or irbesartan, SGLT2 inhibitors, e.g. canagliflozin, GLP1 receptor agonists, MRAs, such as finerenone, ERAs, such as atrasentan, and Apoptosis signal-regulating kinase 1 (ASK1) inhibitors, such as selonsertib.

[0160] (43) Hydroxyurea (HU, hydroxycarbamide). (44) Anti-sickling agents, including but not limited to hydroxyurea, voxerotol, or GBT-440.

[0161] (45) Anti-adhesion therapies, including but not limited to blocking antibodies against P-selectin, E-selectin, VLA-4, and VCAM-1. (46) Glutamine.

[0162] (47) Erythropoietin (EPO) (also known as hematopoietin or hemopoietin), including all its forms, such as exogenous erythropoietin, recombinant human erythropoietin (rhEPO), or other erythropoiesis-stimulating agents (ESAs). Two examples are epoetin alfa and epoetin beta.

[0163] (48) Antibiotics, including but not limited to: Penicillin and its derivatives, including but not limited to penicillin, amoxicillin, ampicillin, azlocillin, cloxacillin, penicillin G, penicillin V, procaine penicillin or benzathine penicillin, among others.

[0164] cephalosporins, such as cephalexin, cefadroxil, cefaclor, cefuroxime and cefixime, macrolides, such as erythromycin, clarithromycin, azithromycin, and roxithromycin; Tetracycline and its derivatives, such as demeclocycline, doxycycline, minocycline, oxytetracycline and tetracycline, Sulfonamides, including but not limited to mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfasalazine, trimethoprim-sulfamethoxazole (cotrimoxazole), and sulfisoxazole; Quinolones, including but not limited to ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, ofloxacin, and nalidixic acid.

[0165] (49) FXR agonists, including but not limited to obeticholic acid, cenicriviroc, emricasan, GR-MD-02, selonsertib, and elafibranor. (50) Thyroid receptor-beta agonists, including but not limited to MGL-3196.

[0166] (51) Acetyl-CoA carboxylase inhibitors, including but not limited to GS-0976. (52) Treatment for mitochondrial disorders, including, but not limited to, mitochondrial cocktail ("mitococktail"), vitamins and supplements including coenzyme Q10, B complex vitamins, especially thiamine (B1) and riboflavin (B2); alpha-lipoic acid; L-carnitine (carnitol); creatine; citrulline; and L-arginine. As used herein, "mitococktail" refers to a combination of various vitamins and supplements, characterized in the appropriate field, commonly used by adults and children diagnosed with mitochondrial disease. The most common components of mitococktails include, but are not limited to, coenzyme Q10, complex vitamins (e.g., 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 (e.g., alpha-lipoic acid), L-carnitine, and creatine. The mitochondria cocktail may contain any one or more of the general ingredients listed above based on the patient's needs and can be determined by a physician.

[0167] (53) Treatment for epilepsy or seizures, including, but not limited to, phenytoin, valproic acid, phenobarbital, lamotrigine, carbamazepine, topiramate, oxcarbazepine, zonisamide, gabapentin, levetiracetam, pregabalin, clonazepam, lacosamide, rufinamide, and vigabatrin.

[0168] Packaging and Kits Pharmaceutical compositions (or formulations) for use can be packaged in a variety of ways depending on the method used to administer the drug. Generally, an article for distribution includes a container having deposited therein the pharmaceutical formulation 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 can also include a tamper-evident assembly to prevent inadvertent access to the contents of the package. In addition, the container has affixed thereto a label that describes the contents of the container. The label also includes appropriate warnings.

[0169] The compounds and pharmaceutical preparations described herein can be contained in a kit. The kit can include two or more drugs, each individually packaged or formulated, in single or multiple doses, or two or more drugs packaged or formulated in combination, in single or multiple doses. Thus, one or more drugs can be present in a first container, and the kit can optionally include one or more drugs in a second container. One or more containers can be placed in a package, and the package can optionally include administration or administration instructions. The kit can include additional components, such as a syringe or other means for administering the drug, as well as a diluent or other means for formulation. Thus, the kit can 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 can optionally include instructions describing how to use the pharmaceutical composition in one or more methods described herein (e.g., preventing or treating one or more of the diseases and disorders described herein). The kit may optionally include a second pharmaceutical composition containing one or more additional agents described herein for combination therapy, a pharmaceutically acceptable carrier, vehicle, or diluent. The pharmaceutical composition containing the compound described herein and the second pharmaceutical composition contained in the kit may optionally be combined in the same pharmaceutical formulation. [Example]

[0170] All references provided in examples are incorporated herein by reference.When used herein, all abbreviations, symbols and conventions are consistent with those used in modern 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.

[0171] Various embodiments of the present invention may be described below. Definitions of abbreviations used in the Examples section are provided in the table below.

[0172] [Table 3]

[0173] Synthesis Section Example 1 Synthesis of Compounds of Formula I The present invention also provides a method for synthesizing a compound of formula I, which represents another embodiment of the present invention. The compounds of formula I of the present invention can be prepared by the general and specific syntheses described herein, synthetic procedures reported in the chemical literature, or methods known to those skilled in the art. As will be understood by those skilled in the art, optimal reaction conditions can be determined experimentally, but may vary based on the type of reaction and the specific reagents used in the reaction. Thus, unless specifically specified, reaction conditions, such as pressure, temperature, relative ratios of reagents, solvents, and reaction times, can be easily selected and modified by those skilled in the art without undue experimentation.

[0174] The compounds and intermediates of the present invention can be purified 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). Purification by RP-HPLC 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 an additive 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 from a mixture of enantiomers by resolution using chiral HPLC. Reaction progress 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).

[0175] Starting materials used in the syntheses described herein are available 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.

[0176] 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 of the operability of the invention. As such, they are not intended to impose any limitations on the subject matter and scope of the claimed compounds of the invention.

[0177] All references provided in examples are incorporated herein by reference.When used herein, all abbreviations, symbols and conventions are consistent with those used in modern scientific literature.See, for example, GM Banik, G.Baysinger, PV Kamat, NJPienta (eds.), 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.

[0178] Example 1 Compound synthesis The compounds disclosed herein can be made, for example, from the corresponding nitrile intermediate using the general procedure described below (General Procedure C).

[0179] General Procedure C

[0180] [ka]

[0181] The compounds of the present invention can be prepared by following procedures similar to those described herein through the corresponding nitriles. Nitriles with different substitution patterns can be prepared by following the procedures described in WO2015187470, WO2016081668, WO2017197555, WO2017200825, WO2018 / 045276A1 and WO2019 / 126354A1.

[0182] The following nitrile intermediates were prepared according to literature procedures described in WO2018 / 045276A1 and WO2019 / 126354A1. Reaction conditions (e.g., reagent ratios, temperature, and reaction time) and purification methods were modified as necessary.

[0183] 8-benzylimidazo[1,2-a]pyrazine-6-carbonitrile, 8-(3-fluorobenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile, 8-(2-fluorobenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile, 8-(2,3-difluorobenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile, 8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile, 8-(3-fluoro-4-methylbenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile, 8-(3,5-difluorobenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile, 8-(3,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile, 8-(2,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile, 8-(2,5-difluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazine-6-carbonitrile, 8-(3-fluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazine-6-carbonitrile, 8-(3,5-difluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazine-6-carbonitrile, 8-(2,3-difluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazine-6-carbonitrile, 8-(2,5-Difluoro-4-methylbenzyl)-[1,2,4]triazolo[1,5-a]pyrazine-6-carbonitrile The synthesis procedures for compounds I-1 to I-20 are described below. The synthesis procedures for compounds I-20 to I-26 are described in patent application publication WO2019 / 126354.

[0184] General Procedure A Applied to the Synthesis of Compound I-1 The title compound was synthesized in two steps. Step 1: Synthesis of 8-(3-fluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide

[0185] [ka]

[0186] 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 a solution of 0.50 N sodium methanolate in methanol (0.17 mL, 0.087 mmol, 0.10 equiv.) (Note: A stoichiometric or excess amount of sodium methanolate can 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 stirred for 16 h. The reaction mixture was concentrated in vacuo, diluted with half-saturated NaHCO3 solution (20 mL), and extracted with 2 × 20 mL of CHCl2 / iPrOH (5:1). The combined organic phases were dried over sodium sulfate, filtered, and concentrated to give the crude carboximidamide product as a tan foamy solid. It was used in the next step without further purification. LC / MS ES + m / z=270.2[M+H] + .

[0187] Step 2: Synthesis of 5-fluoro-2-(8-(3-fluorobenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol

[0188] [ka]

[0189] 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 in a sealed vial at 90 °C 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), adjusted to pH 6 with saturated NaHCO solution, and extracted with 2 × 50 mL of CHCl / iPrOH (5:1). The combined organic phase was dried over sodium sulfate, filtered, and concentrated. The crude material was purified via silica gel chromatography (0-15% acetonitrile / methanol (7:1) in CH2Cl2) to deliver the title compound (180 mg, 64% yield over two steps) as a light tan solid. 1 H NMR (500 MHz, DMSO-d6) δ (ppm) 13.1–12.5 (one pair of s, 1 H, tautomers), 9.46 (s, 1 H), 8.30 (s, 1 H), 8.26–8.00 (one pair of s, 1 H, tautomers), 7.90 (s, 1 H), 7.50 (m, 1 H), 7.41 (m, 1 H), 7.32 (m, 1 H), 7.02 (apparent t, 1 H), 4.53 (s, 2 H). Compound I-2

[0190] [ka]

[0191] 2-(8-Benzylimidazo[1,2-a]pyrazin-6-yl)-5-fluoropyrimidin-4-ol (compound I-2) was synthesized as a white solid (25 mg, 14% overall yield) by general procedure A. Reaction conditions (e.g., reagent ratio, temperature, and reaction time) and purification methods were modified as necessary. 1 H NMR (500 MHz, methanol-d4) δ (ppm) 9.30 (s, 1 H), 8.09 (s, 1 H), 7.99 (d, 1 H), 7.80 (s, 1 H), 7.40 (d, 2 H), 7.17 (t, 2 H), 7.07–7.11 (m, 1 H), 4.52 (s, 2 H). Compound I-4

[0192] [ka]

[0193] 2-(8-(2,3-Difluorobenzyl)imidazo[1,2-a]pyrazin-6-yl)-5-fluoropyrimidin-4-ol (compound 4) was synthesized as a light tan solid (150 mg, 67% overall yield) by general procedure A. Reaction conditions (e.g., reagent ratio, temperature, and reaction time) and purification methods were modified as necessary. 1 H NMR (500 MHz, acetone-d6) δ (ppm) 10.6 (s, 1 H), 9.34 (s, 1 H), 8.16 (s, 1 H), 7.92 (s, 1 H), 7.78 (s, 1 H), 7.20 (t, 1 H), 7.09 (q, 1 H), 7.00 (q, 1 H), 4.60 (s, 2 H). Compound I-6

[0194] [ka]

[0195] 5-Fluoro-2-(8-(3-fluoro-4-methylbenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol (compound I-6) was synthesized as a pale yellow solid (200 mg, 54% overall yield) by general procedure A. Reaction conditions (e.g., reagent ratio, temperature, and reaction time) and purification methods were modified as necessary. 1 H NMR (500 MHz, DMSO-d6) δ (ppm) 12.9 (br. s, 1 H), 9.44 (s, 1 H), 8.29 (s, 1 H), 8.16 (br. s, 1 H), 7.89 (s, 1 H), 7.39 (d, 1 H), 7.27 (d, 1 H), 7.17 (t, 1 H), 4.48 (s, 2 H), 2.14 (s, 3 H). Compound I-7

[0196] [ka]

[0197] 2-(8-(3,5-Difluorobenzyl)imidazo[1,2-a]pyrazin-6-yl)-5-fluoropyrimidin-4-ol (compound I-7) was synthesized by general procedure A as a yellow solid (190 mg, 57% overall yield). Reaction conditions (e.g., reagent ratio, temperature, and reaction time) and purification methods were modified as necessary. 1 H NMR (500 MHz, DMSO-d6) δ (ppm) 13.0 (br. s, 1 H), 9.47 (s, 1 H), 8.31 (s, 1 H), 8.22 (br. s, 1 H), 7.91 (s, 1 H), 7.36 (br. s, 2 H), 7.07 (t, 1 H), 4.53 (s, 2 H). Compound I-3

[0198] [ka]

[0199] 5-Fluoro-2-(8-(3,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol (compound I-3) was synthesized as a white solid (67 mg, 34% overall yield) by general procedure A. Reaction conditions (e.g., reagent ratio, temperature, and reaction time) and purification methods were modified as necessary. 1 H NMR (500 MHz, chloroform-d) δ (ppm) 11.1 (br. s, 1 H), 9.14 (s, 1 H), 8.00-7.91 (m, 2 H), 7.87 (s, 1 H), 7.00 (d, 2 H), 4.56 (s, 2 H), 2.14 (s, 3 H). Compound I-14 The title compound was synthesized in two steps.

[0200] Step 1: Synthesis of 8-(2,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide

[0201] [ka]

[0202] 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 a solution of 0.50 N sodium methanolate in methanol (18 mL, 8.9 mmol, 1.0 equiv.). After stirring at ambient temperature for 4 h, an additional portion of 0.50 N sodium methanolate in methanol (5.3 mL, 2.7 mmol, 0.3 equiv.) was added, and stirring was continued for an additional 2 h. Ammonium chloride (470 mg, 8.9 mmol, 1.0 equiv.) was then added. After 16 h, the reaction mixture was concentrated in vacuo, suspended in saturated aqueous NaHCO3, and stirred for 20 min. The solid was collected by filtration and washed with 3 volumes of water and 2 volumes of ether. The crude product was resuspended in 100 mL of hot acetonitrile, diluted with ether, and filtered. The filter cake was washed with 3 volumes of ether and dried to give a tan solid (2.2 g, 83% yield) which was used in the next step without further purification. LC / MS ES + m / z=302.1[M+H] + .

[0203] Step 2: Synthesis of 5-fluoro-2-(8-(2,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol

[0204] [ka]

[0205] To 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-en-1-olate (2.9 g, 19 mmol, 3.0 equiv.). The solution was heated in a sealed vessel at 90° C. for 18 hours. After cooling to ambient temperature, 2.5 N ethanolic HCl (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 slight cooling, ether (100 mL) was added and the mixture was stirred for 10 minutes. The solid was collected by filtration and washed with 3 volumes of ether. The resulting solid was resuspended in water, stirred for 1 hour, and filtered. The crude material was purified via preparative reverse-phase HPLC (10-70% acetonitrile / water with 0.1% trifluoroacetic acid as an additive). Impure fractions were repurified via preparative reverse-phase HPLC (10-50% acetonitrile / water with 0.1% trifluoroacetic acid as an additive) to deliver the title compound (840 mg, 37% yield) as an off-white solid. 1 H NMR (500 MHz, methanol-d4) δ (ppm) 9.43 (s, 1 H), 8.21 (s, 1 H), 8.08 (br. s, 1 H), 7.89 (s, 1 H), 7.09 (m, 1 H), 7.00 (m, 1 H), 4.63 (s, 2 H), 2.23 (s, 3 H). Na+ salt of compound I-14

[0206] [ka]

[0207] 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 under a nitrogen atmosphere was added a solution of 0.50 N sodium methanolate in methanol (54 mL, 27 mmol). After brief sonication, the resulting pale yellow solution was stirred at ambient temperature for 15 minutes and concentrated to dryness in vacuo. The solid was resuspended in 250 mL of ether with the aid of sonication and concentrated (twice). The resulting solid was resuspended in 650 mL of ether and stirred at ambient temperature for 3 hours. The solid was collected by vacuum filtration and washed with ether (3 x 100 mL). After drying on the filter overnight, the product salt was dried in a vacuum oven at 45°C for 4 days to give 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, D2O) δ (ppm) 8.92 (s, 1 H), 7.99 (d, 1 H), 7.97 (d, 1 H), 7.70 (d, 1 H), 6.98 (dd, 1 H), 6.86 (dd, 1 H), 4.48 (s, 2 H), 2.14 (s, 3 H). Compound I-11

[0208] [ka]

[0209] 5-Fluoro-2-(8-(3-fluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)pyrimidin-4-ol (compound I-11) was synthesized as a yellow-gold solid (61 mg, 23% overall yield) by general procedure A. Reaction conditions (e.g., reagent ratio, temperature, and reaction time) and purification methods were modified as necessary. 1H NMR (500 MHz, DMSO-d6) δ (ppm) 13.3 (br. s, 1 H), 9.60 (s, 1 H), 8.86 (s, 1 H), 8.24 - 8.27 (m, 1 H), 7.32 - 7.47 (m, 3 H), 7.03 - 7.06 (m, 1 H), 4.59 (s, 2 H). Compound I-13

[0210] [ka]

[0211] 2-(8-(3,5-Difluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)-5-fluoropyrimidin-4-ol (compound I-13) was synthesized by general procedure A as a brown solid (57 mg, 17% overall yield). Reaction conditions (e.g., reagent ratio, temperature, and reaction time) and purification methods were modified as necessary. 1 H NMR (500 MHz, DMSO-d6) δ (ppm) 13.2 (br. s, 1 H), 9.61 (s, 1 H), 8.87 (s, 1 H), 8.25 (s, 1 H), 7.33 (d, 2 H), 7.10 (t, 1 H), 4.60 (s, 2 H). Compound I-10

[0212] [ka]

[0213] 2-(8-(2,3-Difluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)-5-fluoropyrimidin-4-ol (compound I-10) was synthesized by general procedure A as a pale yellow solid (85 mg, 16% overall yield). Reaction conditions (e.g., reagent ratio, temperature, and reaction time) and purification methods were modified as necessary. 1H NMR (500 MHz, DMSO-d6) δ (ppm) 13.0 (br. s, 1 H), 9.62 (s, 1 H), 8.85 (s, 1 H), 8.23 ​​(s, 1 H), 7.29 - 7.37 (m, 2 H), 7.09 - 7.16 (m, 1 H), 4.68 (s, 2 H). Compound I-12

[0214] [ka]

[0215] 2-(8-(2,5-Difluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazin-6-yl)-5-fluoropyrimidin-4-ol (compound I-12) was synthesized by general procedure A as an off-white solid (75 mg, 57% yield). Reaction conditions (e.g., reagent ratio, temperature, and reaction time) and purification methods were modified as necessary. 1 H NMR (500 MHz, methanol-d4) δ (ppm) 9.67 (s, 1 H), 8.69 (s, 1 H), 8.12 (d, 1 H), 7.25 (m, 1 H), 7.13 (m, 1 H), 7.02 (m, 1 H), 4.73 (s, 2 H). Compound I-19

[0216] [ka]

[0217] 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) was synthesized by general procedure A as a tan solid (140 mg, 66% overall yield). Reaction conditions (e.g., reagent ratio, temperature, and reaction time) and purification methods were modified as necessary. 1H NMR (500 MHz, DMSO-d6) δ (ppm) 13.1 (br. s, 1 H), 9.61 (s, 1 H), 8.83 (s, 1 H), 8.26 (br. s, 1 H), 7.35 (br. s, 1 H), 7.17 (m, 1 H), 4.57 (s, 2 H), 2.18 (s, 3 H). General Procedure B Applied to the Synthesis of Compound I-16 The title compound was synthesized in two steps.

[0218] Step 1: Synthesis of 8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide

[0219] [ka]

[0220] 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 solution of 0.50 N sodium methanolate in methanol (3.6 mL, 1.8 mmol, 1.0 equiv.) (Note: A catalytic amount or excess of sodium methanolate can 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 diluted with EtOAc (20 mL) and 10% aqueous NaHCO3 (10 mL). After stirring for 15 minutes, the product was collected by filtration, washed with water (10 mL), and dried under vacuum to afford the title compound (420 mg, 80% yield) as an off-white solid. LC / MS ES + m / z=287.9[M+H] + .

[0221] Step 2: Synthesis of 5-chloro-2-(8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol

[0222] [ka]

[0223] 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 a solution of 0.50 N sodium methanolate in methanol (1.4 mL, 0.70 mmol). The reaction was heated at 65 °C for 2.5 h in a sealed vial. 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 extracted with 2 × 15 mL of CHCl / iPrOH (8:1). The combined organic phase was dried over sodium sulfate, filtered, and concentrated. The crude material was purified via silica gel chromatography (0-20% acetonitrile / methanol (7:1) in CHCl) and repurified via silica gel chromatography (20-100% EtOAc / CHCl) to deliver the title compound (37 mg, 28% yield) as an off-white solid. 1 H NMR (500 MHz, DMSO-d6) δ (ppm) 12.6 (br. s, 1 H), 9.54 (s, 1 H), 8.36 (br. s, 1 H), 8.32 (s, 1 H), 7.89 (s, 1 H), 7.45 (br. s, 1 H), 7.25 (m, 1 H), 7.12 (m, 1 H), 4.58 (s, 2 H). Compound I-17

[0224] [ka]

[0225] 5-Chloro-2-(8-(2,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol (compound I-17) was synthesized by general procedure B as a tan solid (5.2 mg, 2.2% overall yield). Reaction conditions (e.g., reagent ratio, temperature, and reaction time) and purification methods were modified as necessary. 1 H NMR (500 MHz, DMSO-d6) δ (ppm) 9.47 (s, 1 H), 8.45 (s, 1 H), 8.22 (d, 1 H), 7.94 (s, 1 H), 7.23 (dd, 1 H), 7.16 (dd, 1 H), 6.72 (d, 1 H), 4.56 (s, 2 H), 2.17 (br s, 3 H).LC / MS ES + m / z=388.0[M+H] + .

[0226] Compound I-15 The title compound was synthesized in two steps. Step 1: Synthesis of 8-(2,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide

[0227] [ka]

[0228] 8-(2,5-Difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide was synthesized by step 1 of general procedure A or B as a tan solid (840 mg, 76% yield). Reaction conditions (e.g., reagent ratio, temperature, and reaction time) and purification methods were modified as necessary. LC / MS ES + m / z=302.0[M+H] + .

[0229] Step 2: Synthesis of 2-(8-(2,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol

[0230] [ka]

[0231] 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 (1.4 mL, 3.6 mmol) and concentrated to dryness. The crude material was purified via silica gel chromatography (0–20% acetonitrile / methanol (7:1) in CHCl) and repurified via silica gel chromatography (0–15% MeOH / CHCl) to deliver the title compound (120 mg, 28% yield) as a tan solid. 1 H NMR (500 MHz, DMSO-d6) δ (ppm) 11.9 (br. s, 1 H), 9.52 (s, 1 H), 8.31 (s, 1 H), 8.07 (br. d, 1 H), 7.89 (s, 1 H), 7.37 (dd, 1 H), 7.16 (dd, 1 H), 6.38 (br. d, 1 H), 4.54 (s, 2 H), 2.18 (s, 3 H). Compound I-8 The title compound was synthesized in two steps.

[0232] Step 1: Synthesis of 8-(2-fluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide

[0233] [ka]

[0234] 8-(2-Fluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide was synthesized by step 1 of general procedure A or B as a cream-colored solid (5.1 g, 91% yield). Reaction conditions (e.g., reagent ratio, temperature, and reaction time) and purification methods were modified as necessary. LC / MS ES + m / z=270.2[M+H] + .

[0235] Step 2: Synthesis of 2-(8-(2-fluorobenzyl)imidazo[1,2-a]pyrazin-6-yl)-5-methylpyrimidin-4-ol

[0236] [ka]

[0237] 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 reaction was heated to reflux for 2 hours. After cooling to ambient temperature, water was added, and the product was collected by filtration and dried to deliver the title compound as a cream-colored solid (410 mg, 82% yield). 1 H NMR (500 MHz, DMSO-d6) δ (ppm) 11.6 (br. s, 1 H), 9.48 (s, 1 H), 8.30 (s, 1 H), 7.94 (br. s, 1 H), 7.88 (s, 1 H), 7.48 (apparent t, 1 H), 7.29 (m, 1 H), 7.19 (m, 1 H), 7.11 (apparent t, 1 H), 4.60 (s, 2 H), 1.98 (s, 3 H). Compound I-9

[0238] [ka]

[0239] 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 treated with sodium bicarbonate (84 mg, 1.0 mmol) and Selectfluor™ (350 mg, 1.0 mmol) and heated at 50° C. Additional portions of sodium bicarbonate (42 + 28 mg) and Selectfluor™ (180 + 120 mg) were added over the course of the experiment. 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 via silica gel chromatography (0–20% acetonitrile / methanol (7:1) in CHCl) and repurified via preparative reverse-phase HPLC (15–65% acetonitrile / water with 0.1% formic acid as an additive) to deliver the title compound (23 mg, 9.7% yield) as a tan solid. 1 H NMR (500 MHz, DMSO-d6) δ (ppm) 12.6 (br. s, 1 H), 8.98 (s, 1 H), 8.19 (br. s, 1 H), 7.74 (d, 1 H), 7.48 (apparent t, 1 H), 7.28 (m, 1 H), 7.19 (m, 1 H), 7.10 (apparent t, 1 H), 4.56 (s, 2 H). Compound I-5

[0240] [ka]

[0241] 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 treated with sodium bicarbonate (94 mg, 1.1 mmol) and Selectfluor™ (400 mg, 1.1 mmol) and heated at 50° C. Additional portions of sodium bicarbonate (3 × 47 mg) and Selectfluor™ (3 × 200 mg) were added over the course of the experiment. 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 extracted with 2 × 40 mL of CHCl / iPrOH (6:1). The combined organic phases were dried over sodium sulfate, filtered, and concentrated. The crude material was purified by silica gel chromatography (0–20% acetonitrile / methanol (7:1) in CHCl), preparative reverse-phase HPLC (10–70% acetonitrile / water with 0.1% TFA as an additive), and final column chromatography (20–100% EtOAc / hexanes) to deliver the title compound (24 mg, 11% yield) as a white solid. 1 H NMR (500 MHz, DMSO-d6) δ (ppm) 12.8 (br. s, 1 H), 8.99 (s, 1 H), 8.20 (br. s, 1 H), 7.75 (d, 1 H), 7.42 (m, 1 H), 7.25 (m, 1 H), 7.13 (m, 1 H), 4.54 (s, 2 H). Compound I-18 The title compound was synthesized in five steps.

[0242] Step 1: Synthesis of 6,8-dibromo-3-fluoroimidazo[1,2-a]pyrazine

[0243] [ka]

[0244] 6,8-Dibromoimidazo[1,2-a]pyrazine (2.4 g, 8.7 mmol) in 40 mL of acetonitrile was treated with Selectfluor™ (4.6 g, 13 mmol) and heated at 50 °C. After 22 h, the reaction was cooled to ambient temperature, poured into 150 mL of half-saturated NaHCO solution, and extracted with 2 × EtOAc (400 mL total). The combined organic phases were dried over sodium sulfate, filtered, and concentrated. The crude material was purified by silica gel chromatography (0-20% EtOAc / hexanes) to deliver the title compound (580 mg, 23% yield) as an orange solid.

[0245] Step 2: Synthesis of 6-bromo-8-(2,5-difluoro-4-methylbenzyl)-3-fluoroimidazo[1,2-a]pyrazine

[0246] [ka]

[0247] A suspension of dry zinc powder (240 mg, 3.7 mmol) in THF (3.0 mL) was treated with 1,2-dibromoethane (30 mL, catalytic) and the resulting mixture was heated at 50 °C. Chlorotrimethylsilane (30 mL, catalytic) was then added. After 15 min, the mixture was cooled to ambient temperature. Dry lithium chloride (170 mg, 3.9 mmol) was added, followed by the dropwise addition of a solution of 1-(bromomethyl)-2,5-difluoro-3-methylbenzene (480 mg, 2.2 mmol) in THF (2.0 mL) (Caution: Exothermic reaction). The mixture was stirred at ambient temperature for 1 h. Meanwhile, a slurry of 6,8-dibromo-3-fluoroimidazo[1,2-a]pyrazine (580 mg, 2.0 mmol) and Pd(PPh)Cl (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, rinsing with 2 × 0.5 mL of THF to ensure complete transfer. The resulting mixture was stirred at ambient temperature for 1 hour and 20 minutes, then at 40 °C for 4 hours. After cooling to ambient temperature, the reaction was quenched with 4 mL of saturated NH Cl solution. The organic layer was concentrated, diluted with CHCl (10 mL), and filtered through a bed of Celite. The filtrate was concentrated to give a brown residue that was purified by silica gel chromatography (loaded with CH2Cl2 and eluted with 0-10% EtOAc / hexanes) to deliver the title compound (440 mg, 63% yield) as a yellow solid.

[0248] Step 3: Synthesis of 8-(2,5-difluoro-4-methylbenzyl)-3-fluoroimidazo[1,2-a]pyrazine-6-carbonitrile

[0249] [ka]

[0250] A reaction mixture consisting of 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(dba) (46 mg, 0.050 mmol), and 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 treated with CHCl (50 mL), water (40 mL), and 28% ammonium hydroxide solution (4.0 mL). The aqueous layer was extracted with CHCl (50 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated to give a brown oil that was purified by column chromatography (0-20% EtOAc / hexanes gradient) to give the title compound as a light tan solid (310 mg, 81% yield). LC / MS ES + m / z=302.8[M+H] + .

[0251] Step 4: Synthesis of 8-(2,5-difluoro-4-methylbenzyl)-3-fluoroimidazo[1,2-a]pyrazine-6-carboximidamide

[0252] [ka]

[0253] 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 a solution of 0.50 N sodium methanolate 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, treated with 10% aqueous NaHCO3 (10 mL), and sonicated to give a suspension. After stirring for 1 hour, the product was collected by filtration, washed with water (10 mL), and dried under vacuum to give the title compound (170 mg, >100% yield) as a light tan solid. It was used in the next step without further purification. LC / MS ES + m / z=319.7[M+H] + .

[0254] Step 5: Synthesis of 2-(8-(2,5-difluoro-4-methylbenzyl)-3-fluoroimidazo[1,2-a]pyrazine-6-fluoropyrimidin-4-ol

[0255] [ka]

[0256] 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-en-1-olate (310 mg, 2.0 mmol). The reaction was heated in a sealed vial at 90 °C for 16 hours. After cooling to ambient temperature, the mixture was diluted with water (7.5 mL) and adjusted to pH 4 with 1 N aqueous HCl. The resulting tan solid was collected by filtration, washed with water (50 mL) and ethyl ether (30 mL), and dried to give the title compound (140 mg, 71% yield over two steps) as a brown solid. 1H NMR (500 MHz, DMSO-d6) δ (ppm) 12.8 (br. s, 1 H), 8.98 (s, 1 H), 8.22 (br. s, 1 H), 7.74 (d, 1 H), 7.35 (br. s, 1 H), 7.15 (m, 1 H), 4.50 (s, 2 H), 2.18 (s, 3 H). Compound I-20 The title compound was synthesized in two steps.

[0257] Step 1: Synthesis of 8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide

[0258] [ka]

[0259] 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 solution of 25 wt% sodium methanolate 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, diluted with half-saturated NaHCO3 solution (20 mL) and 1.0 N sodium hydroxide solution (2.0 mL), and extracted with 2 x 20 mL of 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] + .

[0260] Step 2: Synthesis of 5-fluoro-2-(8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol

[0261] [ka]

[0262] 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 sodium (Z)-3-ethoxy-2-fluoro-3-oxoprop-1-en-1-olate (820 mg, 5.2 mmol). The reaction was 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 with 0.1% TFA as an additive) afforded the title compound (200 mg, 28% yield over two steps) as a yellow solid. 1 H NMR (500 MHz, DMSO-d6) δ (ppm) 12.6 (br. s, 1 H), 9.49 (s, 1 H), 8.32 (s, 1 H), 8.19 (br. s, 1 H), 7.89 (s, 1 H), 7.43 (s, 1 H), 7.25 (m, 1 H), 7.13 (m, 1 H), 4.58 (s, 2 H). Na+ salt of compound I-20

[0263] [ka]

[0264] To a light tan 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 under a nitrogen atmosphere was added a solution of 0.50 N sodium methanolate in methanol (57 mL, 28 mmol). After brief sonication, the resulting pale orange solution was stirred at ambient temperature for 15 minutes and concentrated to dryness in vacuo. The solid was resuspended in 200 mL of ether with the aid of sonication and concentrated (twice). The resulting solid was resuspended in 500 mL of ether and stirred at ambient temperature for 3 hours. The solid was collected by vacuum filtration and washed with ether (3 x 100 mL). After drying on the filter overnight, the product salt was dried in a vacuum oven at 45°C for 5 days to give 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 tan solid. 1 H NMR (500 MHz, D2O) δ (ppm) 8.90 (s, 1 H), 7.98 (d, 1 H), 7.95 (d, 1 H), 7.70 (d, 1 H), 7.10 (m, 1 H), 6.98-6.89 (m, 2 H), 4.53 (s, 2 H). Compound I-27 Compound I-27 was synthesized based on the synthetic scheme shown below.

[0265] [ka]

[0266] Compound 27a was reduced to compound 27b using BH in THF, followed by reaction with PBr to form compound 27c. Compound 27c was converted to the corresponding organozinc compound using zinc, dibromoethane, and TMSCl. The organozinc intermediate was subjected to a Negishi coupling with dibromo compound 27e, followed by cyclization of compound 27d with 3-bromo-1,1,1-trifluoropropan-2-one to give compound 27f. Compound 27f was further reacted in the presence of zinc, zinc cyanide, and Pd(dba) to give cyano compound 27g. The nitrile of compound 27g was reacted with ammonium chloride to give compound 27h. The amidine of compound 27h was condensed with compound 27i using General Procedure A to give cyclized compound I-27.

[0267] Compound I-28 Compound I-28 was synthesized based on the synthetic scheme shown below.

[0268] [ka]

[0269] Compound 28a was methylated to form compound 28b, which was then brominated with NBS to give compound 28c. Compound 28c was converted to 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 further reacted in the presence of zinc, zinc cyanide, and Pd2(dba)3 to give cyano compound 28f. The nitrile of compound 28f was reacted with ammonium chloride to give compound 28g. The amidine of compound 28g was condensed with compound 28h by general procedure A to give cyclized compound I-28.

[0270] Compound I-29 Compound I-29 was synthesized by general procedure B.

[0271] [ka]

[0272] Biological Section Evaluation of the Biological Properties of Compounds of Formula I The present invention also provides for the evaluation of the biological properties of compounds of Formula I. Representative compounds of the present invention are tested in vitro for their activity as sGC stimulators in various cells and assays, and in vivo for their ability to reduce blood pressure in animals. Reduction in blood pressure is used as an indicator of the compound's ability to engage peripheral targets in vivo. Further testing is used as an indicator of the ability of these compounds to cross the BBB, engage targets in the CNS, increase cGMP levels in the CNS, and consequently cause a functional response in animals. These biological properties represent another embodiment of the present invention.

[0273] Example 2 cGMP GloSensor cell-based assay for biological activity measurement in a 384-well format The activity of test compounds was evaluated using human embryonic kidney cells (HEK293), GloSensor™ 40F cGMP (Part Number: CS182801, Promega) expressing cells. A luminescent biosensor (genetically engineered luciferase) incorporated into these cells detects cGMP formed by compounds that stimulate the sGC enzyme and emits luminescence.

[0274] cGMP GloSensor cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS, final 10%) and hygromycin (200 μg / ml). The day before the assay, cells were grown at 1.5 × 10 in poly-D-lysine-coated 384-well white flat-bottom plates (Corning Cat. No. 35661). 4Cells were plated at a density of 10 cells / well in a volume of 50 μL in DMEM containing 10% FBS. Cells were incubated overnight at 37°C in a humidified chamber with 5% CO2. The following day, the medium was removed and the cells were replaced with 40 μl / well of GloSensor™, 2 mM (Promega catalog number E1291). Cells were treated for 90 minutes at 25°C to allow the substrate to equilibrate on the cells. Test compounds and diethylenetriamine NONOate (DETA-NONOate) were diluted to 3 mM (20x) in serum-free, CO2-independent medium and serially diluted at 4x dilutions to create a 5x dose curve, and 10 ul was added to each well (x μM concentration for the test compound solution and 10 μM concentration for the DETA-NONOate solution; 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, luminescence was measured immediately per well for 0.2 seconds using an Envision (PerkinElmer). For endpoint SAR screening, data was collected after 55 minutes of incubation at room temperature.

[0275] Concentration-response data were analyzed using a four-parameter fit (log(agonist) vs. slope of response-variable). EC 50 is interpolated from the curve fit and defined as the concentration at which the compound elicits a 50% maximal response. If experiments were performed multiple times for a given compound, the geometric mean of all experiments is reported.

[0276] Table A below shows the EC values ​​in the Glo assay for compounds of the present invention. 50 Summarize the values ​​of

[0277] [Table 4]

[0278] Example 3 Biological activity measurement using a cGMP neuronal cell-based assay Primary rat neurons were isolated from 18-day-pregnant Sprague-Dawley female fetuses. The fetuses were harvested in Hank's balanced salt solution (HBSS), and the brains were rapidly removed. The brain hippocampi were isolated and mechanically fragmented. Further tissue sublimation was performed with 0.25% (wt / vol) trypsin solution in Ca2+- and Mg2+-free HBSS for 15 minutes at 37°C. After trypsinization, the cells were washed and resuspended in Neurobasal medium supplemented with 0.5 mM L-glutamine, 12.5 μM glutamic acid, 2% B-27, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were plated at 26 × 10 cells / well in a poly-D-lysine-coated 384-well clear flat-bottom plate (Corning catalog number 354662). 3 or 3×10 4 or 4×10 4 Cells were plated at a density of 100 cells / well. Cells were incubated at 37°C in a humidified chamber with 5% CO2 for 6-7 days. The medium was removed, and cells were washed 1x with HBSS containing Ca2+ and Mg2+. Then, cells were replaced with 40 μL of HBSS containing 0.5 mM IBMX and incubated at 37°C for 15 minutes. 10 μL of a 5x stock of test compound, including diethylenetriamine NONOate (DETA-NO), was added. The final concentration of DETA-NO was either 10 μM or 30 μM. Cells were incubated at 37°C for 20 minutes. The medium was removed, and 50 μL 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 pellet cell debris, the supernatant was aspirated into a clear plate, and samples were analyzed for cGMP content. cGMP concentrations were determined from each sample using LC-MS / MS.

[0279] Concentration-response data were analyzed using a four-parameter fit (log(agonist) vs. slope of response-variable). EC 50is interpolated from the curve fit and defined as the concentration at which the compound elicits a 50% maximal response. If experiments were performed multiple times for a given compound, the geometric mean of all experiments is reported.

[0280] Table B below shows the EC values ​​in the neuronal assay for compounds of the present invention. 50 Summarize the values ​​of

[0281] [Table 5]

[0282] Example 4 Biological activity measurements with human α2β1 sGC isozyme stably expressed in CHO-K1 cells.

[0283] 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 in DMSO, followed by dilution to the appropriate concentration in assay buffer.

[0284] CHO-K1 cells stably transfected with human α2β1 sGC isozyme (generated by GenScript for Ironwood) were cultured in F-12K medium (ATCC catalog no. 30-2004) containing 10% fetal bovine serum, 4 μg / mL puromycin (Gibco catalog no. A11138-03), and 0.4 mg / mL geneticin (Gibco catalog no. 10131-027) in a humidified atmosphere of 95% CO2 in air at 37°C. For GC activity assays, cells were cultured at 3 × 10 4 cells / well or 15 x 10 3 Cells were seeded at a density of 100 cells / well in either 50 μL or 70 μL of medium into poly-D-lysine-coated 384-well flat-bottom plates (Fisher Scientific No. 08-774-311). Cells were incubated at 37°C in a humidified chamber supplemented with 5% CO for 24 hours.

[0285] For each test concentration, the compound was diluted with 100% DMSO to 100 times its final assay concentration. Immediately before the assay, the solution was diluted 20-fold in HBSS containing calcium, magnesium, and 50 μM DETA-NONOate (5× final assay concentration). The medium was removed, and the cells were washed once with 40 μL of HBSS. The cells were then incubated with 40 μL of a solution containing 0.5 mM IBMX in HBSS for 15 minutes at 37° C. 10 μL was added from the sGC stimulant / HBSS / DETA-NONOATE plate to the cells, which were then incubated for an additional 20 minutes at 37° C. The final DMSO concentration was 1% and the final DETA-NONOate concentration was 10 μM; final compound concentrations were 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.

[0286] Following compound incubation, 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. Samples were incubated on ice for 30–60 minutes. After centrifugation at 1000 × g for 5 minutes at 4°C to pellet cell debris, the supernatant was transferred to a clear plate and the samples were analyzed for cGMP content.

[0287] Data were analyzed using GraphPad Prism software v.8 with a four-parameter fit (log(agonist) vs. slope of response-variable). EC 50 is interpolated from the curve fit and defined as the concentration at which the compound elicits a 50% maximal response. If experiments were performed multiple times for a given compound, the geometric mean of all experiments is reported.

[0288] Table C below shows the EC values ​​in the CHO assay for compounds of the present invention. 50 Summarize the values ​​of

[0289] [Table 6]

[0290] Example 5 Blood pressure effects in normotensive rats following acute dosing at multiple concentrations of representative compounds of the present invention a) Compound I-14 Male Sprague-Dawley normotensive rats were purchased from Charles River Laboratories. These rats had an indwelling femoral artery catheter installed. The animals were secured in a tethering system and connected to a pressure transducer to monitor cardiovascular (CV) parameters, specifically mean arterial pressure (MAP) and heart rate (HR). The animals were allowed to acclimate to the system overnight, and baseline CV parameters were collected. Conscious, freely moving rats were then administered a single oral dose of compound I-14 (derived from the sodium salt of compound I-14) in Milli-Q water at 1, 3, 10, and 30 mg / kg. Blood samples were collected from each animal through the catheter line pre-dose and 2 hours post-dose for quantification of compound concentrations. Hemodynamic measurements were recorded for 10 hours post-dose. Fifty-four male rats were used in these studies and were ordered to be dosed within a weight range of 250-275 grams. They were individually housed under controlled conditions of temperature (21±1°C) and relative humidity (36±1%) in a room with a 12-hour light-dark cycle (lights on at 6:00 AM, lights off at 6:00 PM) at the SmartLabs Animal Facility (21 Erie Street, Cambridge, MA) under protocol MIL-110. Animals had ad libitum access to food (LabDiet Prolab Isopro RMH 3000, St. Louis, MO) and water. Two sets of studies were performed. For the first set of studies, compound I-14 was formulated at 0.3 and 1.0 mg / ml in Milli-Q water and frozen at -20°C. For the second set of studies, the sodium salt of compound I-14 was weighed out by Cyclerion Therapeutics and reconstituted at SmartLabs to provide solutions of 0.1, 0.3, 1.0, and 3.0 mg / ml of compound I-14 in Milli-Q water. The prepared formulations were either thawed >4 hours prior to dosing and stored at room temperature, or made >2 hours prior to dosing and stored at room temperature.

[0291] The study was conducted over six separate sessions. The total number of subjects and treatment assignments are listed in the table below. Animals were initially used within 3 days of receipt and, if the catheter remained patent, were reused once after a 6-7 day washout period. No animals were used more than once.

[0292] [Table 7]

[0293] Measuring blood pressure This study utilized ADInstruments LabChart (v8) to collect hemodynamic data from conscious, freely moving rats tethered to a blood pressure transducer (Harvard Apparatus catalog number APT300). After overnight acclimation to the tethering and pressure transducer, animals were dosed following 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 post-dose.

[0294] Hemodynamic data were monitored and exported using ADInstruments LabChart (v8). Blood pressure and heart rate were continuously monitored, and data were collected at 1,000 data points per second and then averaged over a 10-minute window for analysis. Changes from baseline MAP (ΔBMAP) and HR (ΔBHR) were calculated using pre-dose baselines averaged over the hour prior to dosing using Microsoft Excel for Microsoft 365. This 10-minute window of data was used to determine peak ΔVMAP, time-peak ΔVMAP, peak ΔVHR, and time-to-peak ΔVHR. Data sets were further consolidated into 1-hour windows for MAP and HR plots, and analysis of ΔBMAP, ΔBMAP, and ΔBHR. Definitions of these terms / abbreviations are summarized below.

[0295] [Table 8]

[0296] Statistical analysis was performed with Graphpad Prism (v8). Significance for ΔBMAP and ΔBHR data compared to vehicle-treated rats was determined by two-way repeated measures ANOVA followed by Dunnett's multiple comparison test; when there were missing data points, a mixed-effects analysis was utilized. Vehicle-adjusted MAP (ΔVMAP) was calculated by subtracting the ΔBMAP of the vehicle group from the ΔBMAP of each dose group at each time point. Vehicle-adjusted HR (ΔVHR) was calculated in a similar manner to ΔVMAP.

[0297] The significance of AOC data was determined by one-way ANOVA followed by Dunnett's multiple comparison test compared to vehicle. Some test data were removed prior to analysis. Data collected at 130 and 140 minutes post-dose were removed due to 2-hour blood sample collection. Several time points for one rat at 1 mg / kg were removed due to signal loss during the experiment, which began at 470 minutes and continued until the end of the study (600 minutes). Five-minute entire time courses were removed from all data sets for various reasons, including being outliers for a particular analysis, or due to signal loss resulting in anomalous results.

[0298] Blood pressure changes The change from baseline MAP (ΔBMAP) is shown graphically in Figure 1. There was a greater reduction in MAP (assessed by the change from baseline MAP, ΔBMAP) in compound I-14-treated rats than in vehicle-treated rats. The ΔBMAP data set was significant by two-way ANOVA (p<0.0001 for treatment and time, p=0.045 for the treatment x time interaction). Dunnett's multiple comparison test for the main effect of treatment yielded p=ns for the 1 mg / kg dose, p=0.0034 for the 3 mg / kg dose, and p<0.0001 for the 10 and 30 mg / kg doses compared to vehicle-treated rats. Separate Dunnett's multiple comparison tests of simple effects at each time point and each dose relative to vehicle-treated rats showed significant decreases in ΔBMAP throughout 6 hours post-dose in rats treated with 10 and 30 mg / kg compound I-14; 1, 2, and 3 hours post-dose in rats treated with 3 mg / kg compound I-14; but not in rats treated with 1 mg / kg compound I-14.

[0299] The maximum effect of compound I-14 on ΔVMAP was calculated by using a 10 minute range of data sets and is shown in Table D below.

[0300] [Table 9]

[0301] There is no effective dose at ΔBMAP of 1 mg / kg as assessed by the primary effect analysis, simple effect analysis, and AOC. conclusion Compound I-14 reduced MAP from baseline and adjusted from vehicle at 3, 10 and 30 mg / kg.

[0302] b) Compound I-20 A similar study to that described above was conducted with compound I-20. Conscious, freely moving rats were administered a single oral dose of compound I-20 in Milli-Q water at 1, 3, 10, and 30 mg / kg (doses prepared from the sodium salt of compound I-20). Blood samples were collected from each animal through a catheter line before and 2 hours after dosing for quantification of compound concentrations. Hemodynamic measurements were recorded for 10 hours after dosing.

[0303] Blood pressure changes Baseline MAP (Δ B The change from baseline MAP (ΔMAP) is shown graphically in Figure 2. The change from baseline MAP (ΔMAP) was significantly greater in rats treated with compound I-20 than in vehicle-treated rats. B (evaluated by change from MAP) significantly decreased. B The MAP data set was significant by two-way ANOVA (p<0.0001 for treatment and treatment × time interaction; p=0.022 for time). Dunnett's multiple comparison test for the main effect of treatment yielded p=0.055 (ns) for the 1 mg / kg dose, p=0.0001 for the 3 mg / kg dose, and p<0.0001 for the 10 and 30 mg / kg doses compared to vehicle-treated rats. Separate Dunnett's multiple comparison tests for the simple effects at each time point and dose relative to vehicle-treated rats show greater reductions in MAP through 6 hours post-dosing in rats treated with 3, 10, and 30 mg / kg compound I-20; at 1, 2, 3, 4, and 5 hours post-dosing in rats treated with 1 mg / kg compound I-20.

[0304] Δ V The maximal effect of compound I-20 on MAP (vehicle-adjusted MAP) was calculated by using a 10-minute range of data sets and is shown in Table E below.

[0305] [Table 10]

[0306] conclusion Compound I-20 reduced MAP at 1, 3, 10 and 30 mg / kg both from baseline and when adjusted from vehicle.

[0307] c) Other BP measurements In a study similar to that described above, compound I-4, formulated in PEG400 and dosed at 10 mg / kg, produced a maximum MAP reduction (Δ) from baseline of 20 mmHg 50 minutes after dosing. B In a study similar to that described above, compound I-20, formulated with PEG 400 and dosed at 10 mg / kg, exhibited a peak ΔP of -26 mmHg 42 minutes after dosing. B In another study, compound I-20 was tested at 1, 3, or 10 mg / kg and formulated in methylcellulose, and the compound was able to reduce MAP from baseline at all doses tested.

[0308] Example 6 sGC stimulators induced CREB phosphorylation in rat primary neurons the purpose 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). CREB has a well-documented role in neuroplasticity and long-term memory formation in the brain, and has been shown to be integrated in the formation of spatial memory (see Silva AJ et al., Annual Review of Neuroscience, 1998;21:127-148). The CREB protein is activated by phosphorylation at 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 Ca2+ / 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 be therapeutically beneficial for diseases in which cognition, neuroplasticity, and / or neuronal function are impaired.

[0309] Rat primary neuron culture Neurons were isolated from Sprague Dawley rat embryos at embryonic day 18 (E18). Approximately 10 embryos were obtained from each rat, and the whole brain was isolated from the embryos. The hippocampus and cortex were dissected from the brain under a stereomicroscope using two fine forceps. The meninges were carefully removed. After dissection, the tissue was minced and placed in a 15 mL conical tube with 10 mL of CaCl2+. 2+ and Mg 2+The tissue was gently washed once with HCl-free Hank's Balanced Salt Solution (HBSS, Corning catalog no. 21-022-CM). After washing, 5 mL of a solution of 0.25% trypsin (Invitrogen catalog no. 15090-046) and 0.1% deoxyribonuclease I (DNase I, Sigma catalog no. DN-25) was added to the tissue, followed by incubation at 37°C for 15 minutes. The tissue was then washed three times with ice-cold HBSS, and 3 mL of a solution of 0.1% DNase I was added. The tissue was then gently pipetted 12 times using a glass Pasteur pipette, followed by centrifugation at 500 x g for 10 minutes. The cell pellet was resuspended in culture medium (Neurobasal medium, Gibco catalog no. 21103-049), 2% B27 supplement (Gibco catalog no. 17504-044), 0.5 mM L-glutamine (Corning catalog no. 25-005-Cl), 25 μM L-glutamic acid (Sigma catalog no. G1251), and 1% penicillin / streptomycin (Gibco catalog no. 15070-063). The cell suspension was then plated at 100,000 cells / well into poly-L-lysine-coated 96-well plates. Twenty-four hours after plating, half of the culture medium was removed and replaced with the culture medium described above but without glutamic acid. Cells were maintained in a humidified incubator at 37°C with 5% CO2 and used in assays between 6 and 10 days after harvest.

[0310] Assay conditions For each test concentration, compounds were diluted in 100% DMSO to 100x their final assay concentration. Immediately prior to the assay, compounds were diluted 1 / 10 in HBSS (containing calcium and magnesium) (10x final assay concentration) containing 100 μM DETA-NONOate (10x final assay concentration). The medium was removed, and cells were washed once with 90 μL of HBSS (Corning catalog number 21-023-CV). Cells were then incubated in 90 μL of HBSS for 30 minutes at 37°C. 10 μL from the test article / HBSS / DETA-NONOATE plate was added to the cells and incubated for an additional 30 minutes at 37°C. The final DMSO concentration was 1%, and the final DETA-NONOate concentration was 10 μM; 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 by Cisbio protocol (phospho-CREB (Ser133) Cat. No. 64CREPEG). The plate was read using an Envision instrument (PerkinElmer).

[0311] Data analysis pCREB was determined for each well and analyzed with a three-parameter fit (log(agonist) vs. slope of response-variable) using GraphPad Prism software v.8. EC 50 is interpolated from the curve fit and is defined as the concentration at which the sGC stimulator compound elicits a 50% maximal response. Experiments were performed multiple times 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.

[0312] Table F below shows the EC values ​​in the pCREB assay for compounds of the present invention. 50 Summarize the values ​​of

[0313] [Table 11]

[0314] Example 7 Pharmacokinetic properties of rat cerebrospinal fluid (CSF) protocol PK in rats was determined following oral dosing. For oral (PO) experiments, groups of six male Sprague-Dawley rats with indwelling catheters placed in the cisterna magna were used. The PO groups were dosed with 3.0 or 10 mg / kg of compound formulated as a solution in PEG400 or a suspension of 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. Following administration of the oral dose, the gavage tube was flushed with approximately 0.5 mL of water to ensure complete delivery of the entire dose.

[0315] Plasma samples were collected as follows: CSF and blood samples were collected 1 hour, 2 hours, and optionally 4 hours after dosing. CSF samples (0.05 mL) were collected through an intracisternal catheter. Blood samples (0.25 mL) were collected via tail nick sampling. These samples were kept on ice until processed for plasma. Blood samples were centrifuged at 3200 rpm for 5 minutes at approximately 5°C within 1 hour of collection. Plasma was transferred directly to individual Eppendorf tubes (0.125 mL). Plug caps were placed on the tubes, and the tubes were frozen at approximately -70°C and stored until analysis. Plasma and CSF were collected and analyzed for the presence of compound.

[0316] Compound quantification The compound of interest and internal standard were extracted from plasma by precipitation and from CSF by either precipitation or dilution. Samples were analyzed using liquid chromatography (LC) with tandem mass spectrometry detection (MS / MS) using electrospray ionization. The standard curve ranged from 0.1 to 1000 ng / mL. Results for the compounds described herein in this assay are shown in Table G below (for a 10 mg / kg dose and / or 3 mg / kg of compound). Compound concentrations from several animals were combined to obtain a geometric mean for each specific dose and time point.

[0317] Kp,uu is defined as the concentration ratio of unbound drug in CSF to unbound drug in plasma. The unbound drug in plasma (or free plasma concentration) is calculated by multiplying the total plasma concentration by the unbound fraction determined from the binding plasma protein. The CSF concentration is then divided by the free plasma concentration to determine Kp,uu. (See, e.g., Di et al., J. Med. Chem., 56, pp. 2-12 (2013)).

[0318] [Table 12]

[0319] Example 8 Microdialysis experiments in rat brain the purpose The purpose of this study was to evaluate the levels of sGC stimulators of the present invention in hippocampal and striatal interstitial fluid (ISF) and circulating plasma after administration to male Sprague-Dawley rats. To do this, rats were implanted with hippocampal and striatal microdialysis probes and jugular vein cannulae (JVC). After collecting one pre-dose ISF sample, the animals were dosed with the sGC stimulator. Samples were collected through the hippocampal and striatal probes for 24 hours, and serial plasma samples were collected through the JVC after administration. All collected samples were stored at -80°C pending analysis of compound levels in the dialysate.

[0320] material and method animal Five Sprague-Dawley rats, pre-cannulated with jugular vein cannulas, were used for this study. Upon arrival, rats were group-housed in polycarbonate cages (2–3 rats / cage) and allowed to acclimate for at least 3 days before the study began. Animals were housed on a 12-hour light / dark cycle at room temperature maintained at 22 ± 2°C and approximately 50% humidity, and provided with food and water ad libitum. Rats were tracked via unique identification numbers. Experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Charles River Laboratories, South San Francisco.

[0321] Formulation and Dosing sGC stimulators were freshly formulated on the day of treatment and administered to animals as follows: Compound I-14 was dosed as its sodium salt at a dose of 3 mg / kg, at a concentration of 3 mg / mL, and formulated in MilliQ water.

[0322] In vitro experiments In vitro Metaquant microdialysis (MQ-MD) experiments were performed to test compound recovery through the probe membrane. To do this, MQ probes (polyacrylonitrile, 3 mm membrane) were connected by inlet PEEK tubing to a microperfusion pump (Harvard PHD 2000 syringe pump, Holliston, MA, or similar). The probes were individually placed in a bath of artificial CSF (aCSF) + 0.2% bovine serum albumin (BSA) containing 50 ng / mL of 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), using a carrier flow of ultrapure water + 0.2% BSA. The flow rates were 0.15 μL / min for slow flow and 0.8 μL / min for carrier flow. The probe outlet was connected by outlet PEEK tubing to an automated fraction collector (820 Microsampler, Univentor, Malta, or similar). After perfusion stabilized, samples were collected for 20 min into polypropylene vials. In separate vials, 150 μL samples of the bath contents were taken at the beginning and end of the experimental sampling period. In vitro dialysate and bath samples were analyzed for compound levels. Probe recovery was calculated as the ratio of compound concentration in the dialysate sample to the bath concentration and expressed as percent recovery.

[0323] Microdialysis procedure Rats were anesthetized using isoflurane (2%, 800 mL / min O2). Bupivacaine was used for local anesthesia, and carprofen was used for perioperative and postoperative analgesia. Animals were placed in a stereotaxic frame (Kopf Instruments, USA). Metaquant microdialysis probes (polyacrylonitrile; 3 mm exposed membrane) were then implanted into the striatum (STR) and hippocampus (HIPP). The probe tip positions in the STR were anterior-posterior (AP) = +0.9 mm from the parietal, lateral (L) = +3.0 mm from the midline, and ventral (V) = -7.0 mm from the dura, with the toothbar set at -3.3 mm. A second probe was then implanted into the hippocampus (HIPP). The positions for the probe tip in HIPP were anterior-posterior (AP) = -5.3 mm from the crown, lateral (L) = -4.8 mm from the midline, and ventral (V) = -7.0 mm from the dura, with the dental bar set at -3.3 mm. After surgery, animals were housed individually in cages and provided with food and water ad libitum.

[0324] Microdialysis experiments were performed the day after surgery. The microdialysis probe was connected to a microperfusion pump (Harvard PHD 2000 Syringe Pump, Holliston, MA) via flexible PEEK tubing. The microdialysis probe was perfused with aCSF containing 147 mM NaCl, 3.0 mM KCl, 1.2 mM CaCl, and 1.2 mM MgCl and 0.2% β-cyclodextrin at a slow flow rate of 0.15 μL / min, and a carrier of HO + 0.2% BSA at a flow rate of 0.8 μL / min. Microdialysis samples were collected for 30 min using an automated fraction collector (820 Microsampler, Univentor, Malta) into 300 μL polypropylene minivials containing 15 μL of 0.02 M formic acid + 0.04% ascorbic acid in ultrapure water. After stabilization, one baseline sample was taken, and the sGC stimulator was administered orally (PO) at T = 0, with samples collected continuously for 24 hours (collected at 1, 2, 3, 4, 5, 6, 8, 12, and 24 hours). Samples were aliquoted for sGC stimulator analysis. All ISF samples were stored at -80°C until analysis.

[0325] In addition to ISF collection, blood samples were collected via JVC into K2 + EDTA vials at T = -0.5, 0.5, 1, 2, 3, 4, 5, 6, 8, 12, and 24 hours after treatment. Blood was stored on ice until processed for plasma (centrifugation at 2,500 g for 10 minutes at 4°C). Plasma was aliquoted into 1.5 mL Eppendorf vials and stored at -80°C pending analysis.

[0326] Postmortem tissue collection After completion of microdialysis, animals were euthanized via CO2 asphyxiation. Brains were harvested in 10% neutral buffered formalin for confirmation of probe placement.

[0327] Bioanalytical Methods Measurement of compound levels in dialysate and plasma The concentrations of compound I-14 in dialysate and plasma samples were quantified by ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS / MS) detection in multiple reaction monitoring mode (MRM).

[0328] Plasma samples were first mixed with a solution of acetonitrile containing 100 ng / mL dexamethasone (internal standard) for protein precipitation. After 5 min of incubation at room temperature, the samples were centrifuged for 5 min (1300 rpm, 4°C), and the supernatant was diluted 100-fold with ultrapure water containing 0.1% formic acid.

[0329] Undiluted dialysate 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) containing 0.1% formic acid before analysis.

[0330] The undiluted dialysate sample and diluted plasma supernatant were injected into a Shimadzu system (Shimadzu, USA) using an automatic 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 35°C. Mobile phase A consisted of ultrapure water containing 0.1% formic acid. Mobile phase B was acetonitrile containing 0.1% formic acid.

[0331] Acquisition was achieved in positive ionization mode using a QTrap® 5500 mass spectrometer (Applied Biosystems, USA) equipped with a Turbo Ion Spray interface. The ion spray voltage was set at 5.5 kV, and the probe temperature was 600°C. The collision gas (nitrogen) pressure was maintained at medium. The following MRM transition was used for quantification: m / z 372.0 / 352.0. Suitable in-run calibration curves were fitted using weighted (1 / x) regression, and sample concentrations were determined using these calibration curves. Accuracy was confirmed with quality control samples after each sample series. Data were calibrated and quantified using the Analyst™ data system (Applied Biosystems, version 1.5.2).

[0332] Data evaluation Data were plotted using Prism 8 for Windows (GraphPad Software, Inc.). Reported dialysate concentrations of compound I-14 were corrected based on probe recovery. The mean recovery was 14.7% (SEM: 0.59).

[0333] result Five animals were successfully dosed and completed the study. One animal's JVC was blocked at 4 hours and no further blood was drawn. No obvious side effects of the treatment were observed.

[0334] Effects of Compound I-14 Administration on Striatal and Hippocampal ISF; Ratio of Compound I-14 in STI / HIPP vs. Plasma FIG. 3 shows the levels of compound I-14 in the STR and HIPP portions of ISF dialysates in adult male Sprague-Dawley rats following administration of compound I-14 (3 mg / kg; PO) at T=0 min.

[0335] Microdialysis allows for sampling of compound concentrations in the interstitial fluid of brain tissue. Given that the tissue at the sampling site has different rates of blood perfusion, physiological structure, and clearance mechanisms compared to cerebrospinal fluid (CSF), differences in distribution and measured concentrations between microdialysis and CSF-PK studies in the same animal could be expected. These differences were observed in the time to maximum concentration, measured concentrations, and ratios calculated from the measured concentrations (Nagaya Y, Nozaki Y, Takenaka O, et al., Investigation of the 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).

[0336] Example 9 Concentrations of cGMP are altered in rat cerebrospinal fluid (CSF) after a single administration of sGC stimulators (CSF Biomarker Measurement) This experiment was carried out to determine the effect of different doses of sGC stimulator compounds of the present invention on cGMP levels in rat CSF.

[0337] protocol Rats were administered a single dose of either 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 one or more times over three days between doses. The day before the experiment, rats were fasted overnight but had free access to water.

[0338] On the day of the experiment, concentrations of compound and cyclic guanosine monophosphate (cGMP) in rat CSF were determined following oral dosing. Male CD rats implanted with intracisternal cannulas (250–275 g) were used for these studies. Rats were housed singly under controlled conditions of temperature (21 ± 1°C) and relative humidity (36 ± 1%) in a room with a 12-h light / dark cycle (lights on at 6:00 AM, lights off at 6:00 PM) at the SmartLabs Animal Facility (21 Erie Street, Cambridge, MA) under protocol MIL-110. Animals had access to food (LabDiet Prolab Isopro RMH3000, St. Louis, MO) and water ad libitum. Rats were dosed with 0mg / kg (vehicle), 1mg / kg, 3mg / kg, 10mg / kg, or 30mg / kg of the compound of the present invention, which was formulated as a suspension in 0.5% methylcellulose, 0.5% Tween 80, or as a solution in MilliQ water.The formulation was prepared and stored frozen at -20 ℃, and thawed at room temperature for 1 hour before administration, or prepared and used within 2 hours, or prepared the day before administration and kept stirring at room temperature overnight until administration.PO dose was administered by oral gavage, and was delivered to the stomach using a syringe and a gavage tube.Following the administration of the oral dose, the gavage tube was flushed with approximately 0.5mL of water to ensure complete delivery of the entire dose.

[0339] 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 through an intracisternal catheter. Approximately 20 μL of CSF was collected and discarded (this included a syringe dead volume of 14–16 μL); then, approximately 50 μL of CSF was drawn into an Eppendorf tube containing 5 μL of glacial acetic acid. CSF samples were frozen by immersion in liquid nitrogen. Next, while the animals remained anesthetized, blood samples were obtained via tail nick and stored in K-EDTA tubes. These samples were kept on ice until processed for plasma. Blood samples were centrifuged at 3200 rpm for 10 minutes at approximately 5°C within 1 hour of collection. Plasma was transferred directly to tubes (0.125 mL) in a 96-well plate. Plug caps were placed on the tubes, and the tubes were frozen at approximately -70°C and stored until analysis. Plasma and CSF were collected and analyzed for the presence of compound.

[0340] Compound and cGMP quantification Compounds of the present invention, cGMP, and internal standards were extracted from plasma and CSF by precipitation. Samples were analyzed using liquid chromatography (LC) with tandem mass spectrometry detection (MS / MS) using electrospray ionization. The standard curve range for compounds was 0.1–1000 ng / mL. The standard curve range for cGMP was 0.01–40 ng / mL. CSF cGMP data were graphed using Graphpad Prism, version 8.4.3, and are expressed as mean ± SEM. Data were analyzed by analysis of mixed effects followed by Dunnett's multiple comparison test, compared to vehicle-treated rats within time points. Significance was set at p<0.05.

[0341] result At 1 and 2 hours after dosing, rats treated with 1, 3, and 10 mg / kg of compound I-20 (administered as a suspension in Tween / MC) showed no significant change in cGMP concentrations in the rat CSF compared with vehicle-treated rats. However, at 6 hours after dosing, rats treated with 10 mg / kg of compound I-20 had significantly higher cGMP levels in the CSF than vehicle-treated rats (see Figure 4).

[0342] Rats treated with compound I-14 (administered as the sodium salt) had higher levels of cGMP in the CSF at all doses tested, but not at all time points tested, compared with vehicle-treated rats. 1 and 2 hours after dosing, rats administered 3 mg / kg of compound I-14 had significantly higher concentrations of cGMP in the CSF. 2 hours after dosing, rats treated with 1 mg / kg of compound I-14 had significantly higher cGMP in the CSF compared with vehicle-treated rats. Rats administered either 10 or 30 mg / kg of compound I-14 had significantly higher cGMP in the CSF at 1, 2, and 6 hours after dosing (see Figure 5).

[0343] Example 10A Cerebrospinal fluid (CSF) pharmacokinetic properties in non-human primates (Study A) protocol The PK of NHPs was determined following oral dosing (PO). Groups of four 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 formulations were shipped frozen on dry ice, thawed, and thoroughly mixed before dosing. PO doses of 1 mg / kg for compound I-14 and 0.3 mg / kg for compound I-20 were administered by oral gavage.

[0344] Plasma and CSF samples were collected as follows: CSF samples were collected 3 and 24 hours after PO dosing. CSF samples (0.125 mL) were collected in the cisterna magna via direct puncture with direct dilution. Blood samples (0.8 mL) were collected from a peripheral vein at 0, 0.25, 0.5, 1, 2, 3, 6, 8, 2, 24, 32, and 48 or 72 hours. These samples were kept on ice until processed for plasma. Blood samples were protein precipitated using acetonitrile. Plasma was transferred directly to individual tubes (0.125 mL), and K2EDTA was used as an anticoagulant. Plug caps were placed on the tubes, and the tubes were frozen at approximately -70°C and stored until analysis. Plasma and CSF were collected and analyzed for the presence of compound.

[0345] Compound quantification Plasma and CSF samples were analyzed using liquid chromatography (LC) with tandem mass spectrometry detection (MS / MS) using positive ion electrospray ionization. The standard curve range was 0.1–1000 ng / mL.

[0346] The geometric mean of the values ​​obtained in the four animals in each compound study was obtained for concentrations in CSF and plasma, respectively. Kp,uu is defined as the concentration ratio of unbound drug in CSF to unbound drug in plasma. The unbound drug in plasma (or free plasma concentration) is calculated by multiplying the total plasma concentration by the unbound fraction determined from the plasma protein to which it binds. The CSF concentration is then divided by the free plasma concentration to determine Kp,uu (see, e.g., Di et al., J. Med. Chem., 56, pp. 2-12 (2013)).

[0347] The results for the compounds of the present invention are summarized in Table H below.

[0348] [Table 13]

[0349] Example 10B Cerebrospinal fluid (CSF) pharmacokinetic properties in non-human primates (Study B) The objective of this study was to investigate the pharmacokinetics of compound I-14 after administration of a single intrathecal bolus or oral gavage dose to cynomolgus monkeys on day 1, and to evaluate differences in pharmacokinetic values ​​when comparing CSF collected at the cisterna magna (Example 10A) versus lumbar sites.

[0350] Study design

[0351] [Table 14]

[0352] Test System / Method Species: Cynomolgus macaque (Macaca fascicularis) Strain: Cynomolgus monkey Number of females: 4 Age: Adult Weight: 2.5-4kg Animals were housed in stainless steel cages with stainless steel mesh floors and automatic water valves. Primary inclusions were as described in the Guide for the Care and Use of Laboratory Animals (National Research Council (NRC). Guide for the Care and Use of Laboratory Animals. Washington, DC: National Academy Press, 8th ed., 2011; Office of Laboratory Animal Welfare. Public Health Services Policy on Humane Care and Use of Laboratory Animals. Bethesda, Maryland: National Institutes of Health, revised 2015). These housing conditions were maintained unless deemed inappropriate by the study director and / or clinical veterinarian. When possible, animals were socially housed except when separated for designated study procedures / activities.

[0353] A single dose of Compound I-14 was administered intravenously as a bolus injection of solution at 0.15 mg / kg and orally by gavage as a suspension at 0.5 mg / kg to one group of 4 female cynomolgus monkeys in a crossover design. Plasma samples were collected at 0.25 (15 minutes), 0.5 (30 minutes), 1, 2, 3, 6, 8, 12, 24, 32, and 48 hours following IV and PO doses. CSF samples were collected at 3 and 24 hours for all animals from the cisterna magna in Groups 1 and 2 and from the lumbar spine in Groups 3 and 4. Sample extracts were prepared by protein precipitation and Compound I-14 concentrations were measured using LC-MS / MS. Pharmacokinetic parameters were calculated.

[0354] Summary / Conclusion Overall, the mean pharmacokinetic parameters between IV (Groups 2 and 4) and PO (Groups 1 and 3) were within each other's standard deviations.

[0355] Compound I-14 was observed in CSF at 3 and 24 hours following both IV and PO administrations using both cisterna magna and lumbar sampling. Three hours after IV and oral dosing, the geometric mean ratios of CSF concentration to unbound concentration in plasma were within Range C as described above, following both cisterna magna sampling and lumbar sampling, i.e., included in 2 - 3 (NHP CSF PK, Kp,uu≤1 = A; 1 < Kp,uu≤2 = B; 2 < Kp,uu≤3 = C, 3 < Kp,uu = D). Twenty-four hours after IV and oral dosing, the geometric mean ratios of CSF concentration to unbound concentration in plasma were included in Ranges B and C, respectively, following cisterna magna sampling and in B and C, respectively, following lumbar sampling.

[0356] Example 11 Evaluation of the Compounds of the Invention in a Novel Object Recognition (NOR) Model of Memory Enhancement Objective To evaluate the efficacy of CNS-penetrating sGC stimulators of the present invention in reversing memory disruption induced by MK-801 using the novel object recognition (NOR) test in male Long Evans rats.

[0357] Introduction The NOR is a test of cognitive learning and memory retrieval that exploits rodents' spontaneous preference to investigate novel objects compared to familiar ones. The NOR test is widely used to evaluate the potential cognitive-enhancing properties of novel test compounds. Because the NOR paradigm does not involve rewarding or aversive stimuli, it is less susceptible to confounding variables when translated into similar tests conducted in human clinical trials.

[0358] This study used a memory sparing model. MK-801 (dizocilpine), a non-competitive NMDA receptor antagonist, was used to induce recognition memory deficits. sGC stimulators were evaluated for their efficacy in preventing MK-801-induced memory impairment. The reference compound, galantamine, at 1 mg / kg (ip) significantly reversed the cognitive deficits induced by MK-801 at 0.1 mg / kg (ip), suggesting the validity of the study.

[0359] material and method animal Adult male Long-Evans rats (275-299 grams upon arrival from Envigo, Indianapolis, IN) were used in this study. Rats were introduced into the laboratory and assigned a unique identification number (marked on the tail). Rats were housed two per cage in filter-top polycarbonate cages and allowed to acclimate for at least 7 days before testing. The animal room was maintained at 22±1°C, approximately 50% relative humidity, and on a 12 / 12-h light / dark cycle (lights on at 7:00 EST). Food and water were provided ad libitum. All animals were examined, handled, and weighed prior to the study to ensure proper health and minimize nonspecific stress associated with testing. Each animal was randomly assigned to a treatment group. Experiments were performed during the animal's light cycle phase.

[0360] Test Compounds and Drugs The following compounds and drugs were used in these studies: MK-801 (0.1 mg / kg; Sigma-Aldrich) was dissolved in saline and IP injected 15 min before NOR training. The administration volume was 1 ml / kg.

[0361] Galantamine (1 mg / kg; Tocris) was dissolved in saline and injected IP 15 min before training. The administration volume was 1 ml / kg. Compound I-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 in ultrapure water) and orally administered 60 min before NOR training in a dosing volume of 2 ml / kg.

[0362] The following groups were tested, N=16 each (one rat was removed from the Compound I-20-1 mg / kg-MK-801 group prior to the start of the study 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.

[0363] Compound I-14 (0.01, 0.1, and 1 mg / kg) was formulated as its Na+ salt in MilliQ water and stored as frozen aliquots. Compound solution and compound vehicle aliquots were stored at -80°C and freshly thawed on each test day. Compound and vehicle were orally administered 60 minutes before NOR training. The administration volume was 10 ml / kg.

[0364] The following groups were tested against Compound I-14, with N=16 each: 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.

[0365] Experimental procedure The NOR test was conducted in an open-field arena (40 × 40 cm) placed in a dimly lit, sound-attenuating room. Each rat was tested separately, and care was taken to remove olfactory / gustatory cues between trials by cleaning the arena and test objects with 70% alcohol. All training and testing sessions were videotaped and scored by an observer blinded to the treatments.

[0366] On days 1 and 2, rats were allowed to freely explore the arena (without any objects inside) for a 5-minute habituation period. On day 3 (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 the injection and the start of NOR training. Each animal was placed in the test arena containing two identical objects. Each rat was placed in the arena in the same position and facing the same direction, and the time it took to actively explore the objects during the 3-minute training period (T1) was recorded. Rats were returned to their home cages after training. The NOR test (T2) was conducted 1 hour after T1. Each rat was returned to the test arena containing one familiar object and one novel object for 5 minutes, and the time it took to explore both objects was recorded. The order and position (left / right) of object presentation in T2 were randomized between rats to prevent bias from order or position preference.

[0367] Tissue collection Ten minutes after T2 (135 minutes after drug administration), trunk blood was collected into microcentrifuge tubes containing K2EDTA. The blood tubes were placed on ice for short-term storage. Within 15 minutes, the tubes were centrifuged at 10,000 RPM in a refrigerated centrifuge for 10 minutes. Plasma was extracted, and the samples were stored at -80°C until shipped to the sponsor.

[0368] statistical analysis Data from the NOR test (T2) are expressed as a recognition index, defined as the ratio of the time spent exploring the novel object to the total time spent exploring both objects during the test session (novel / (familiar + novel) × 100%).

[0369] For compound I-14, data were analyzed separately in two batches. Batch 1 included a saline-MK-801 group and a galantamine-MK-801 group. Data were analyzed using t-tests to assess the validity of the study. Batch 2 included five treatment groups containing the 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 an "sGC stimulator group" containing compound I-14 1 mg / kg-MK-801. Data from batch 2 were analyzed separately for time ranges of 0-1, 0-3, and 0-5 minutes using one-way ANOVA followed by Fisher's LSD post-hoc comparisons. Significance was set at P<0.05. Nineteen animals with recognition indices above 90% or below 30% were excluded because they suggested a strong (non-memory) bias between the two objects. Two rats with total exploration times to both objects that were 10 seconds shorter than 5 minutes were also excluded due to unreliable results (this is the standard criterion for our NOR test). One rat was excluded due to questionable drug exposure based on plasma analysis feedback. Statistical outliers that were within two standard deviations of the mean were then excluded from further analysis. Based on these criteria, one to six rats were excluded from each experimental group (originally N = 16) and were excluded from statistical analyses for all time ranges (0-1, 0-3, and 0-5 minutes).

[0370] For compound I-20, data were analyzed separately for the time ranges 0-1, 0-3, and 0-5 min using one-way ANOVA followed by Fisher's LSD post-hoc test; significance was set at P<0.05. Animals with recognition indices greater than 90% or less than 30% were excluded because they suggested a strong (non-memory) bias between the two objects. Statistical outliers within two standard deviations from the mean were then excluded from further analysis. Based on these criteria, 2-4 rats were removed from each experimental group (originally N=16) and excluded from statistical analysis for all time ranges (0-1, 0-3, and 0-5 min).

[0371] result a) Compound I-14 The rats in this study showed no obvious side effects at any dose, and maintained normal levels of alertness, activity, and goal-seeking.

[0372] In the 0-1 min time range, t-tests showed a significant difference between the saline-MK-801 and galantamine-MK-801 groups (P<0.01), demonstrating the validity of the assay. ANOVA for compound groups revealed a significant main effect of treatment on the recognition index [F(4,56)=4.698, P<0.01]. Fisher's LSD post-hoc comparisons 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 (P<0.05 and P<0.01, respectively), suggesting that 0.1 mg / kg MK-801 induced a significant memory deficit and that 1 mg / kg compound I-14 reversed the deficit.

[0373] In the 0-3 min time range, t-tests showed a significant difference between the saline-MK-801 and galantamine-MK-801 groups (P<0.001), demonstrating the validity of the assay. ANOVA for compound groups showed a significant main effect of treatment on the recognition index [F(4,56)=5.113, P<0.01]. Post-hoc comparisons showed that in this time range, the vehicle-saline group showed a significant difference from the vehicle-MK-801 group (P<0.01), suggesting that 0.1 mg / kg of MK-801 induced significant memory deficits. In this time range, compound I-14 at 1 mg / kg showed a tendency to reverse MK-801-induced memory deficits (P<0.10).

[0374] In the 0-5 min time range, t-tests showed a significant difference between the saline-MK-801 and galantamine-MK-801 groups (P<0.001), demonstrating the validity of the assay. ANOVA for compound groups showed a significant main effect of treatment on the recognition index [F(4,56)=2.847, P<0.05]. Post-hoc comparisons showed that compound I-14 at 1 mg / kg had a significantly higher recognition index than the vehicle-MK-801 group in this time range (P<0.05).

[0375] b) Compound I-20 The rats in this study showed no obvious side effects at any dose, and maintained normal levels of alertness, activity, and goal-seeking.

[0376] ANOVA showed a significant main effect of treatment on the cognitive index in the 0-1 min time range [F(5,77) = 3.379, P < 0.01]. Post-hoc 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-20 1 mg / kg-MK-801 group and the galantamine-MK-801 group showed significantly higher cognitive indices than the vehicle-MK-801 group (P < 0.01). Typically, data from the 0-1 min time range are relatively unstable; data from the 0-3 min and 0-5 min time ranges provide more reliable results.

[0377] In the 0-3 min time range, ANOVA found a significant main effect of treatment on the recognition index [F(5,77) = 3.922, P < 0.01]. Post-hoc tests showed that MK-801 0.1 mg / kg caused significant memory deficits 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 (P < 0.001). Compound I-20 0.3 mg / kg-MK-801 group also showed a tendency to reverse MK-801-induced memory deficits in this time range (P < 0.10).

[0378] In the 0-5 min time range, ANOVA revealed a significant main effect of treatment [F(5,77) = 5.219, P < 0.001]. Post-hoc tests showed that MK-801 at 0.1 mg / kg caused significant memory deficits, with recognition indices approaching chance levels (50%). Galantamine (1 mg / kg), compound I-20 at 0.3 mg / kg, and compound I-20 at 1 g / kg significantly reversed the MK-801-induced memory deficits (P < 0.05, P < 0.01, and P < 0.001, respectively, compared with the vehicle-MK-801 group). The compound I-20 0.03 mg / kg-MK-801 group also showed a tendency to reverse the MK-801-induced memory deficits in this time range (P < 0.10).

[0379] Example 12 Sleep-wake pharmacological EEG assessment in telemetered rats This study was conducted at PsychoGenics, Inc. Procedures were approved by the Institutional Animal Care and Use Committee in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

[0380] the purpose The present study was designed to evaluate the sleep stage-specific pharmaco-EEG (electroencephalography) signature of compound I-14 in wirelessly implanted young adult male SD rats.

[0381] material and method animal Young adult male Sprague-Dawley (SD) rats (approximately 275–325 g upon arrival) from Envigo (Indianapolis, IN) were used in this study. Upon receipt, rats were assigned unique identification numbers and housed three rats per cage in polycarbonate cages with filter-top microisolators. All rats were examined and weighed prior to the start of the study to ensure appropriate health and suitability. A 12 / 12 light / dark cycle was maintained for the duration of the study. Room temperature was maintained at 20–23°C, and relative humidity was maintained at approximately 50%. Food and water were provided ad libitum for the duration of the study. Following surgery, rats were housed singly. After a recovery period (7–10 days), animals were moved to the EEG recording room and placed in a DSI receiver for recording.

[0382] Test Arm a. Vehicle A i. Route: PO ii. Volume: 10ml / kg iii. Formulation: Water b. Compound I-14 at 3 mg / kg i. Route: PO ii. Volume: 10ml / kg iii. Formulation: 3mg / kg in water c. Compound I-14 at 10 mg / kg i. Route: PO ii. Volume: 10ml / kg iii. Formulation: 10 mg / kg in water d. Compound I-14 at 30 mg / kg i. Route: PO ii. Volume: 10ml / kg iii. Formulation: 30 mg / kg in water Surgical procedure Briefly, animals were implanted with a DSI telemetry device (F50-EET) in a three-channel (lead pair) configuration. Note the positive and negative lead attachment at each location. Each of the EEG channels on the DSI transmitter served as a differential input, which measured the difference between the positive and negative leads. Frontal / Parietal (Right): (+) 2 mm anterior, 2 mm lateral; (-) 4 mm posterior, 2 mm lateral; Frontal / Frontal (Bihemispheric): (+) 2 mm anterior, 2 mm lateral (Right); (-) 2 mm anterior, 2 mm lateral (Left); and Cervical EMG.

[0383] Briefly, animals were anesthetized with 4-5% isoflurane and 1 L / min oxygen for preoperative induction and maintained during surgery with a mixture of 1-2.5% isoflurane and 1 L / min oxygen. Depth of anesthesia was determined and monitored using the hindlimb withdrawal reflex and respiratory rate. The isoflurane level was continuously adjusted to maintain anesthesia throughout the surgical procedure. Ophthalmic lubricant was applied to the eyes using a sterile cotton swab. The surgical site was prepared for aseptic surgery by shaving the hair and cleaning the skin three times with chlorhexidine scrub and alcohol swabs. The abdomen, neck, and head were shaved and disinfected with three alternating preparations of chlorhexidine scrub and alcohol (2% chlorhexidine gluconate and 4% isopropyl alcohol). The anesthetized animal was then placed on a heating pad with circulating warm water for surgical implantation of the telemetry device.

[0384] Telemetry implant A 3-4 cm incision was made in the midline of the upper skull, extending approximately 1 cm posterior to the midpoint of the eye, down to the base of the skull, and into the dorsal mid-lateral cervical region. Blunt dissection was used to dislodge connective tissue, creating a subcutaneous pocket from the posterior end of the incision to the right lateral flank region. The pocket was irrigated with sterile saline. A transmitter was then placed within the pocket, with the leads extending outside the cervical incision. One lead connected to the transmitter was measured, cut to size, inserted into one of the dorsal cervical muscles, and secured in place with 5-0 silk sutures. Two leads were placed in a row along the same muscle bundle, spaced 2-4 mm apart, to provide an electromyography (EMG) signal. The periosteum was then removed to reveal the skull landmarks, parietal and lambda. If necessary, the skull area was wiped with sterile saline and then dried using gauze or sterile Q-tips. Stereotaxic locations for the regions of interest (posterior and parietal cortex) were used to mark the electroencephalography (EEG) leads. For each region of interest, one to two holes were drilled through the skull to ensure exposure of the dura. Dual-connection screws were inserted into the holes, and EEG leads were wrapped around them (maximum of four holes). These screws must contact the dura to detect EEG brain signals. Penetration of the dura does not cause adverse events and can improve EEG signals (compared to metal contact with the brain). The screws and surrounding skull surface were then permanently sealed using FLOW-It ALC composite. After the cement had completely dried, the skin over the skull was closed using suture staples.

[0385] Exam Schedule The study followed a dosing / recording schedule on Mondays and Thursdays, where after a 2-hour baseline EEG was recorded, rats received a first dose of Compound I-14 or vehicle A (all orally) at 0.3, 3, or 10 mg / kg between 7:50 and 8:00 AM, followed by a second dose of the same approximately 12 hours later, after which EEG was continued for 12 hours.

[0386] EEG recording begins at 6:00 AM (lights on). Medication is administered at 7:50 AM (1 hour and 50 minutes after lights are turned on) The second dose (same compound) will be given at 7:50 PM (1 hour and 50 minutes after lights out). EEG recording will continue until 7:50 AM the following day (24 hours after the first dose and 12 hours after the second dose). Data were recorded using a Data Sciences International (DSI) data collection platform from freely moving rats in their home cages. Recordings were conducted during the light-dark cycle. Lights were turned on at 6:00 AM and off at 6:00 PM for the duration of the test. Animals were habituated to medication (vehicle medication) prior to data collection.

[0387] Animals were tested in a crossover design with a period of at least 72 hours between doses. On each test day, animals were orally administered compound or vehicle just under 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.

[0388] EEG signal evaluation EEG was ensured to be free of line noise (50 or 60 Hz) or any continuous non-physiological frequency patterns that could be considered noise from external sources. All EEG recordings were within the normal range of motion (i.e., EEG signals were within the normal amplitude of motion, typically greater than 100 microvolts and less than 500 microvolts, without loss of signal fidelity, and typically observed EEG dips of <100 μV).

[0389] Sleep scoring Raw EEG recordings were manually scored using Neuroscore software (Data Sciences International) to identify sleep stages: active wake, quiet wake, NREM, and REM. Artifacts were removed from the data offline using NeuroScore (DSI), and sleep stages were manually assigned to each 10-s epoch using EEG, EMG, and locomotion activity (LMA) according to a previously described method using frontoparietal EEG, LMA, and EMG (Ivarsson et al., 2005; Parmentier-Batteur et al., 2012; Leiser et al., 2014, 2015): active wake (irregular, small amplitude EEG, high EMG and LMA activity); quiet wake (irregular, small amplitude EEG, low EMG, and no LMA activity); NREM (high amplitude irregular waves, mainly delta (1–4 Hz), low EMG, and no LMA); and REM sleep (stable, small amplitude waves dominated by theta (4–8 Hz), little EMG, and no LMA).

[0390] Sleep stage data were exported from the Neuroscore reporting template for sleep state time every 15 minutes (2 hours before dosing to 4 hours after dosing). The first sleep and first REM occurrences were also reported directly from the Neuroscore reporting template. Latency was calculated as the time from the first REM occurrence following the occurrence of NREM (i.e., T REM -T NREM = REM latency). Hypnograms were constructed using the percent time spent in each sleep stage per hourly range. Time in each sleep state was calculated as a percentage of total time in each sleep state (mean ± standard error of the mean, SEM). Data for individual animals were arranged by treatment group, sleep state, and range and exported to GraphPad PRISM for statistics and graphing.

[0391] spectroscopic analysis Spectroscopic analysis was performed using Matlab. Time-domain signals collected for multiple channels were collected in DSI / Neuroscore, and then the EDF files were imported into Matlab. Excel files, also marked with specific timestamps for baseline and post-dose, were imported into Matlab to lock the time. In Matlab, power spectral densities (PSDs) were computed using Welch's method. Both raw and relative spectral power were then computed for each of six frequency bands (delta, theta, alpha, beta, low gamma, and high gamma) and each 1 Hz subband. Data recorded for 2 hours before compound administration were pooled and defined as "baseline." Percent changes from baseline were calculated for each channel, subject, dose level, and spectral band and time segment. Average raw, relative, and percent change values ​​for each frequency band were calculated for each group. Spectroscopic analysis involved quantifying raw, relative, and percent change spectral power for conventionally defined EEG bands (delta: 0.5-3.9 Hz, theta: 4-7.9 Hz, alpha: 8-11.9 Hz, beta: 12-29.9 Hz, low gamma: 30-49.9 Hz, and high gamma: 50-100 Hz) for each recording per rat. In addition, EEG spectra from 1 to 100 Hz were presented as line graphs. These spectral plot data were provided to clients separately, but due to the wide range of graphs, not all are necessarily included. For clarity, percent change for each band is presented across time for the post-drug period.

[0392] result sleep Hypnograms of compound I-14 showed a significant decrease in REM and NREM in the high-dose (30 mg / kg) treatment group compared to the vehicle (A) group. An increase in quiet wakefulness was observed in the highest-dose compound group. The onset of NREM, REM, and REM latency was delayed in this treatment group. The most significant effects of compound I-14 occurred at the high dose, with effects on quiet wakefulness (increase), REM (decrease), and NREM (decrease) lasting for several hours after administration.

[0393] spectroscopic analysis Compound I-14 (3 mg / kg) increased low gamma from 0 to 180 minutes post-dosing, while the 30 mg / kg dose increased low gamma from 0 to 240 minutes post-dosing and high gamma from 0 to 180 minutes post-dosing in QW. At 30 mg / kg, the compound decreased delta, theta, and alpha and increased low and high gamma from approximately 0 to 300 minutes post-dosing in NREM.

[0394] Example 13 Evaluation of the cognitive effects of compound I-14 in a chronic low-dose MPTP-lesioned macaque model of cognitive impairment in Parkinson's disease This study was a non-GLP study in a chronic low-dose MPTP-lesioned macaque model of Parkinson's disease. This model has been described in the literature (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3282499 / ). This study was designed to evaluate the effects of compound I-14 administered as monotherapy on the performance of the following cognitive tasks: variable delayed response (VDR), sustained performance test (CPT), visual discrimination reversal learning (SDR), and object search (OR). This study was conducted in animals that had been pretreated with low doses of MPTP and were known to have difficulty performing the above-mentioned tasks. These studies utilized four male cynomolgus monkeys with MPTP-induced cognitive impairment.

[0395] This study had four parts. The first part of the study involved collecting baseline data from four animals on the four tasks to be studied. The second part of the study involved administering vehicle (sterile, filtered, distilled water) for 16 days and collecting vehicle data on four tasks once a week for two weeks. In the third part of the study, compound I-14 was administered orally daily for five days, followed by data collection on four tasks once a week for two weeks, while continuing to administer compound I-14 (3.0 mg / kg) daily two hours before testing each day. In the final part of the study, after a nine-day washout period, data were collected on four tasks once a week for two weeks. One dose of compound I-14 was evaluated as monotherapy (3.0 mg / kg) with five days of pre-dose followed by two weeks of daily dosing. The effects of treatment on cognitive performance were assessed two hours after daily drug administration and during the drug washout period.

[0396] MPTP-HCl was administered by intravenous injection at doses ranging from 0.05 mg / kg to 0.30 mg / kg, two to three times weekly for several months. MPTP administration was continued until cognitive impairment accompanied by minimal / mild parkinsonian movement disorders was observed. Animals were considered "cognitively impaired" if their cognitive task performance showed at least a 15% decline from pre-MPTP baseline levels. Because the MPTP response is somewhat idiosyncratic, MPTP was administered to target effects (i.e., the onset of cognitive impairment followed by the onset of movement disorders) rather than a specific duration of MPTP exposure or a specific cumulative dose.

[0397] The SDR represents a test of cognitive flexibility. In this task, three stimuli are presented simultaneously on a screen. One of these stimuli is arbitrarily designated as the positive (rewarded) stimulus; touching it results in a positive tone and reward, whereas touching the negative (non-rewarded) stimulus results in a different tone and the screen turns white. The location of the stimuli on the screen varies pseudorandomly from trial to trial. A maximum of 300 trials were presented in each session. Measures recorded in each session were: 1) the total number of trials required to learn the initial discrimination (i.e., achieve a specific criterion of 14 / 16 correct responses); and 2) the total number of trials required to learn the reversal (i.e., achieve criterion) in which the previously negative (non-rewarded) stimulus is now the positive (rewarded) stimulus (i.e., achieve the same criterion as described above). The number of trials required to reach criterion for learning the discrimination reversal was taken as a measure of cognitive flexibility.

[0398] Compound I-14 monotherapy significantly improved performance in the SDR paradigm. CY3018 significantly reduced the number of trials required to reach the criterion for learning discrimination reversal, indicating an improvement in at least one aspect of cognition in these animals. Simple discrimination performance was not significantly altered by vehicle or compound I-14 administration. However, administration of compound I-14 resulted in a significant improvement in discrimination reversal learning performance (Figure 6).

[0399] In addition, during baseline, vehicle, and washout testing, some animals were completely unable to learn the discrimination reversal, but when tested with compound I-14, no animals were unable to learn the reversal, indicating that the compound had a positive effect on all animals tested.

Claims

1. 1. A method of treating a mitochondrial disease in a subject in need thereof, comprising administering a therapeutically effective amount of a compound of Formula I: 【Chemistry 1】 [In the formula, J Ｃ is hydrogen, halogen, C １～６ C substituted with alkyl and 1 to 3 fluorine atoms １～６ fluoroalkyl; X is N or C(J Ｃ１ ) and J Ｃ１ is hydrogen, halogen, C １～６ C substituted with alkyl and 1 to 3 fluorine atoms １～６ fluoroalkyl; Each J Ｂ is hydrogen, halogen, C １～６ C substituted with alkyl and 1 to 3 fluorine atoms １～６ fluoroalkyl; J Ｄ is hydrogen, halogen, C １～６ C substituted with alkyl and 1 to 3 fluorine atoms １～６ fluoroalkyl; n is an integer selected from 0, 1, 2, 3, or 4. or a pharmaceutically acceptable salt thereof to a subject.

2. J Ｃ is hydrogen, halogen and C １～６ is selected from the group consisting of alkyl, X is N or C(J Ｃ１ ) and J Ｃ１ is hydrogen, halogen and C １～６ is selected from the group consisting of alkyl, Each J Ｂ is hydrogen, halogen and C １～６ independently selected from the group consisting of alkyl, J Ｄ is hydrogen, halogen and C １～６ is selected from the group consisting of alkyl, n is an integer selected from 0, 1, 2, 3 or 4; The method of claim 1.

3. The compound has formula IA: 【Chemistry 2】 or a pharmaceutically acceptable salt thereof.

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

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

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

7. The compound has formula IB: 【Transformation 3】 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. Each J Ｂ are independently H, F or C １～４ The method according to any one of claims 1 to 9, wherein the alkyl is alkyl.

11. Each J Ｂ The method of claim 10, wherein is independently F or methyl.

12. n is 2, and J Ｂ are both F, or J Ｂ 9. The method of claim 8, wherein one of is F and the other is methyl.

13. n is 3, and J Ｂ 9. The method of claim 8, wherein two of are F and the other is methyl.

14. n is 1, and J Ｂ The method of claim 9 , wherein is F.

15. 10. The method of claim 9, wherein n is 0.

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

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

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

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

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

21. Mitochondrial diseases include Alpers disease, autosomal dominant optic atrophy (ADOA), Barth syndrome / LIC (fatal infantile cardiomyopathy), beta-oxidation deficiency, long-chain fatty acid transport deficiency, coenzyme Q10 deficiency, complex I, II, III, IV, V deficiency, chronic progressive external ophthalmoplegia (CPEO), Friedreich ataxia, Kearns-Sayre syndrome, leukodystrophy, Leigh disease or syndrome, LHON, LHON 21. The method of any one of claims 1 to 20, wherein the mitochondrial cytopathies are selected from the group consisting of: mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like episodes, myoclonic epilepsy with ragged-red fibers (MERRF), mitochondrial recessive ataxia syndromes (MIRAS), mitochondrial cytopathies, mitochondrial DNA depletion, mitochondrial encephalopathy, mitochondrial myopathy, multiple mitochondrial dysfunction syndromes, MNGIE (muscle neurogastrointestinal 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. 22. The method of 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 mutations.

23. 22. The method of claim 21, wherein the mitochondrial disease is a complex I mitochondrial disease.

24. 22. The method of claim 21, wherein the mitochondrial disease is MELAS.

25. 22. The method of claim 21, wherein the mitochondrial disease is Leigh syndrome.

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

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

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

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

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

31. 31. The method of any one of claims 1 to 30, wherein treatment improves cognition in the subject.

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

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

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

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

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

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

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

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

Images