Deoxynucleoside prodrugs for treating diseases caused by imbalanced nucleotide pools

Abstract

To provide pharmaceutical compositions for treating mitochondrial DNA depletion syndromes.SOLUTION: The pharmaceutical compositions contain deoxynucleotide prodrugs represented by formula III. In the formula, the base refers to an optionally substituted heterocyclic base or a protected-amino-group-containing, optionally substituted heterocyclic base; the heterocyclic base is selected from among a purine base, a pyrimidine base and a triazole base; R1 and R2 are independently selected from among hydrogen, linear, branched or cyclic alkyl, acyl, CO-alkyl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, and the like.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 656,861, filed April 12, 2018, which is incorporated herein by reference in its entirety.

[0002] The present invention relates generally to prodrugs for delivering deoxynucleosides and their use in treating diseases caused by imbalanced nucleotide pools, including mitochondrial DNA depletion syndrome. [Background technology]

[0003] Mitochondrial diseases are a clinically diverse group of disorders caused by defects in the mitochondrial respiratory chain (RC) and oxidative phosphorylation, a biochemical pathway that converts electron energy into adenosine triphosphate (ATP). The respiratory chain is composed of four multisubunit enzymes (complexes I-IV) that transport electrons and generate a proton gradient across the inner mitochondrial membrane. This proton flow drives ATP synthesis via complex V (Non-Patent Documents 1 and 2). Coenzyme Qio (CoQio) is an essential molecule that shuttles electrons from complexes I and II to complex III. The respiratory chain is unique to eukaryotic cells, such as mammalian cells, because it is controlled by two genomes: mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Consequently, mutations in either genome can cause mitochondrial disease. Most mitochondrial diseases affect multiple organs and are usually fatal in childhood or early adulthood. There are no proven effective treatments for mitochondrial diseases, and only supportive care is available, such as administering CoQio and its analogues to enhance respiratory chain activity and neutralize reactive oxygen species (ROS), the toxic by-products of dysfunctional respiratory chain enzymes.

[0004] Mitochondrial DNA depletion syndrome (MDS), a subgroup of mitochondrial diseases, is a common cause of severe childhood encephalomyopathies characterized by reduced mitochondrial DNA (mtDNA) copy number in tissues and insufficient synthesis of the mitochondrial RC complex (Non-Patent Document 3). Mutations in several nuclear genes, including TK2, DGUOK, POLG, POLG2, SCLA25A4, MPV17, RRM2B, SUCLA2, SUCLG1, TYMP, OPA1, and ClOorfl (PEOl), have been identified as causes of infantile MDS (Non-Patent Documents 4-12). Furthermore, mutations in these nuclear genes can also cause multiple deletions of mtDNA, with or without mtDNA depletion (Non-Patent Documents 13-22).

[0005] One of these genes is TK2, which encodes thymidine kinase (TK2), a mitochondrial enzyme required for the phosphorylation of pyrimidine nucleosides (thymidine and deoxycytidine) to generate deoxycytidine monophosphate (dTMP) and deoxycytidine monophosphate (dCMP) (Non-Patent Document 23). Mutations in TK2 impair the mitochondrial nucleoside / nucleotide salvage pathway required for the synthesis of deoxynucleotide triphosphates (dNTPs), components of mRNA replication and repair.

[0006] TK2 deficiency was first described in 2001 by Saada and colleagues in four affected children from four different families with severe, devastating myopathy (24). After normal early development, between 6 and 36 months of age, patients developed hyperCKemia, severe hypotonia, and subsequently loss of spontaneous activity. The disease progressed rapidly, with two One patient was placed on a ventilator within three years, while the other two patients had already died at the time of reporting.

[0007] Since the initial description, 60 additional patients have been reported in the literature, and at least 26 additional patients have been diagnosed but not reported (25-45), resulting in a total of 90 patients, 53 males and 37 females. The 26 recently diagnosed patients were identified by next-generation DNA sequencing. This large number of newly identified cases suggests that TK2 deficiency is an underdiagnosed disorder.

[0008] TK2 deficiency displays a wide clinical and molecular genetic spectrum, with the majority of patients presenting with a devastating clinical course in early childhood, while others present with slowly progressive wasting over decades.

[0009] Like most MDS and mitochondrial disorders, treatment for TK2 deficiency has been limited to supportive care. Administration of deoxythymidine monophosphate (dTMP) and deoxycytidine monophosphate (dCMP) has been shown to improve the condition of both TK2 knock-in mutant mice and human patients with TK2 deficiency (Patent Document 1, the entirety of which is incorporated herein), as has administration of deoxynucleosides (e.g., deoxythymidine (dT) or deoxycytidine (dC) or mixtures thereof) (Patent Document 2, the entirety of which is also incorporated herein). However, there remains a need for additional therapeutic interventions for TK2 deficiency.

[0010] Additionally, there is a need for treatments for other forms of MDS and other diseases characterized by imbalanced nucleotide pools. For example, several Mendelian genetic disorders involving mtDNA depletion and / or multiple deletions are characterized by imbalanced deoxynucleotide triphosphate pools, leading to defective mtDNA replication. One such disorder, the DGUOK mutation, impairs the mitochondrial enzyme deoxyguanosine kinase, which normally phosphorylates the deoxypurine nucleosides deoxyguanosine and deoxycytidine to produce deoxyguanosine monophosphate (dGMP) and deoxycytidine monophosphate (dCMP). Other nuclear genes that disrupt mitochondrial dNTP pools include TYMP, RRM2B, SUCLA2, SUCLG1, and MPV17. Therapies that restore dNTP pool balance would be similarly useful in treating these disorders. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent Application Serial No. 15 / 082,207 [Patent Document 2] International Publication No. 2016205671 Brochure [Non-patent literature]

[0012] [Non-Patent Document 1] DiMauro and Schon 2003; [Non-patent document 2] DiMauro and Hirano 2005 [Non-patent document 3] Hirano, et al. 2001 [Non-patent document 4] Bourdon, et al. 2007 [Non-patent document 5] Copeland 2008; [Non-patent document 6] Elpeleg,et al.2005;

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[0013] [Means for solving the problem]

[0014] The present invention relates generally to prodrugs for the delivery of deoxynucleosides. In one aspect, the present invention provides a compound of formula I: [ka]

[0015] wherein base refers to an optionally substituted heterocyclic base or an optionally substituted heterocyclic base having a protected amino group; R 1 is an optionally substituted acyl, an optionally substituted O-linked amino acid, [ka] wherein X, Y and Z are each independently selected from O and S; R 2 , R 3 and R 4 are each hydrogen, optionally substituted C 1-24 Alkyl, optionally substituted C 2-24 Alkenyl, optionally substituted C 2-24 Alkynyl, optionally substituted C 3-6 Cycloalkyl, optionally substituted C 3-6Cycloalkenyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryl (C 1-6 ) alkyl, [ka] or R 2 and R 3 and can be taken together to form a cyclic moiety, R 5 , R 6 and R 7 are each optionally substituted C 1-24 Alkyl, optionally substituted C 2-24 Alkenyl, optionally substituted C 2-24 Alkynyl, optionally substituted C 3-6 Cycloalkyl, optionally substituted C 3-6 Cycloalkenyl, NR 20 R 21 , an optionally substituted N-linked amino acid, an optionally substituted N-linked amino acid ester; R 8 , R 9 , R 11 and R 12 are each independently selected from hydrogen, optionally substituted C alkyl, and optionally substituted aryl; R 10 and R 13 are each independently selected from hydrogen, optionally substituted C alkyl and optionally substituted aryl, optionally substituted —O—C alkyl, optionally substituted —O-aryl, optionally substituted —O-heteroaryl, optionally substituted —O-monocyclic heterocyclyl; R 14 , R 15 and R 19 are each hydrogen, optionally substituted C 1-24 independently selected from alkyl, and optionally substituted aryl; R 16 and R17 are -CN, optionally substituted C, respectively. 2-8 Organyl carbonyl, C 2-8 Alkoxycarbonyl and C 2-8 independently selected from organylaminocarbonyl; R 18 is hydrogen, optionally substituted C 1-24 Alkyl, optionally substituted C 2-24 Alkenyl, optionally substituted C 2-24 Alkynyl, optionally substituted C 3-6 Cycloalkyl, and optionally substituted C 3-6 cycloakenyl; R 20 and R 21 are each hydrogen, optionally substituted C 1-24 Alkyl, optionally substituted C 2-24 Alkenyl, optionally substituted C 2-24 Alkynyl, optionally substituted C 3-6 Cycloalkyl, and optionally substituted C 3-6 cycloalkenyl, and Each of n, m, and p is independently selected from 0, 1, 2, or 3.

[0016] In another embodiment, the prodrug is a compound of formula II: [ka]

[0017] wherein base refers to an optionally substituted heterocyclic base or an optionally substituted heterocyclic base having a protected amino group; X is selected from S and O, and R 22 -O - , -OH, -O-alkyl, optionally substituted C 1-6 Alkoxy, [ka] , optionally substituted N-linked amino acids, and optionally substituted N-linked amino acid esters, wherein R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , n, m and p are defined as above.

[0018] In yet another embodiment, the prodrug is a compound of formula III: [ka]

[0019] wherein base refers to an optionally substituted heterocyclic base or an optionally substituted heterocyclic base having a protected amino group; R 1 and R 2 are independently selected from hydrogen, phosphate (including monophosphates, diphosphates, or triphosphates of Formula I, and modified phosphates), linear, branched, or cyclic alkyl, acyl, CO-alkyl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate ester, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, lipid, phospholipid, amino acid, carbohydrate, peptide, and cholesterol).

[0020] In yet another aspect, the present invention further generally relates to a method of treating a disease or disorder characterized by imbalanced nucleotide pools in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one prodrug of the present invention. The prodrug can be administered per se (i.e., alone) or in the form of a pharmaceutical composition.

[0021] Suitable diseases or disorders include, but are not limited to, TK2 deficiency, RRM2B deficiency, TYMP mutation, SUCLA2 deficiency, SUCLG1 deficiency, MPV17 deficiency, and DGUOK mutation.

[0022] Administration can be via any route, including, but not limited to, intrathecal, parental, mucosal, and transdermal.

[0023] The dosage of the at least one prodrug or composition comprising same can be from about 25 mg / kg / day to about 1,000 mg / kg / day. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description of the Invention I. Definition As used herein, "subject" refers to a mammal. Mammals include dogs, cats, rodents, cattle, horses, pigs, sheep and primates. Thus, the present invention can be used in veterinary medicine, for example, to treat companion animals, livestock, zoo laboratory animals, and wild animals. The present invention is particularly desirable for human medical use.

[0025] As used herein, "patient" refers to a human subject. In some embodiments of the present invention, a "patient" is a person suffering from a disease or disorder characterized by an imbalanced nucleotide pool, Known or suspected to have a mitochondrial disease, mitochondrial DNA depletion syndrome, or TK2 deficiency.

[0026] As used herein, a "therapeutically effective amount" refers to an amount sufficient to cause a clinically significant improvement in a subject's condition, or to delay, minimize, or alleviate one or more symptoms associated with a disease or disorder, or to effect a desired physiologically beneficial change in a subject.

[0027] As used herein, "treat," "treatment," and the like refer to a means of slowing, alleviating, ameliorating, or alleviating at least one symptom of a disease or disorder, or reversing a disease or disorder after onset.

[0028] As used herein, "prevent," "prevention," and the like refer to acting prior to the onset of an overt disease or disorder to prevent the onset of the disease or disorder, minimize the extent of the disease or disorder, or slow the course of its development.

[0029] As used herein, "in need thereof" refers to a subject known or suspected to have a mitochondrial disease, mitochondrial DNA depletion syndrome, or TK2 deficiency, which are diseases or disorders characterized by imbalanced nucleotide pools.

[0030] As used herein, "prodrug" refers to a derivative of a deoxynucleoside that is converted under conditions of use, such as in the body, to release the deoxynucleoside. Prodrugs are often, but not necessarily, pharmacologically inactive until converted to their active form. Prodrugs can be obtained by attaching a promoiety to a drug, usually via a functional group.

[0031] As used herein, a "promoiety" refers to a group, typically a functional group, attached to a deoxynucleoside via a bond that is cleavable under specific conditions of use. The bond between the drug and the promoiety may be cleaved by enzymatic or non-enzymatic means. Under conditions of use, for example, after administration to a patient, the bond between the drug and the promoiety may be cleaved to release the parent drug. Cleavage of the promoiety may proceed spontaneously, such as via a hydrolysis reaction, or may be catalyzed or induced by another agent, such as an enzyme, light, acid, or by a change in, or exposure to, a physical or environmental parameter, such as a change in temperature or pH. The agent may be endogenous to the conditions of use, such as an enzyme present in the systemic circulation of a patient to whom the prodrug is administered, or to the acidic conditions of the stomach, or the agent may be supplied exogenously.

[0032] As used herein, a "side effect" refers to an undesirable reaction caused by the administration of a drug. In most cases, the administration of deoxynucleosides does not cause side effects. The most expected side effect is mild gastrointestinal intolerance.

[0033] As used herein, "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of precision needed for a particular purpose, such as pharmaceutical formulation. For example, "about" can mean within 1 or more than 1 standard deviation, according to practice in the art. Alternatively, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of the value, preferably within 5-fold, and more preferably within 2-fold. When specific values ​​are described in the application and claims, unless otherwise specified, the term "about" means It should be assumed to mean within a certain tolerance of the value.

[0034] Whenever a group is described as "optionally substituted," the group can be unsubstituted or substituted with one or more of the indicated substituents. Similarly, when described as "unsubstituted or substituted," if the group is substituted, the substituents can be selected from one or more of the indicated substituents. If no substituents are specified, the indicated "optionally substituted" or "substituted" group can be any of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, aryl(alkyl), heteroaryl(alkyl), heterocyclyl(alkyl), hydroxy, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C- It means that the amino group may be substituted with one or more groups individually and independently selected from amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, azido, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, amino, monosubstituted amino and disubstituted amino groups, and protected derivatives thereof.

[0035] As used herein, "C-C," where "a" and "b" are integers, refers to the number of carbon atoms in an alkyl, alkenyl, or alkynyl group, or the number of carbon atoms in a ring of a cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl group. That is, the alkyl, alkenyl, alkynyl, cycloalkyl ring, cycloalkenyl ring, aryl ring, heteroaryl ring, or heterocyclyl ring contains from "a" to "b" carbon atoms. Thus, for example, a "C-C alkyl" group refers to all alkyl groups having 1 to 4 carbons, i.e., CH—, CH—CH—, CH—CH—CH—CH—, (CH)—CH—, CH—CH—CH—CH—CH—(CH)—, and (CH)—C—. When "a" and "b" are not specified for an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl group, the broadest range described in these definitions is assumed.

[0036] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain containing a hydrocarbon group that is fully saturated (having no double or triple bonds). An alkyl group can have 1 to 20 carbon atoms. (Whenever it appears herein, a numerical range such as "1 to 20" refers to each integer within the given range. For example, "1 to 20 carbon atoms" means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 20 carbon atoms, although this definition also applies when the term "alkyl" appears without a specified numerical range.) An alkyl group can also be a medium-sized alkyl having 1 to 10 carbon atoms. An alkyl group can also be a lower alkyl having 1 to 6 carbon atoms. The alkyl groups of a compound A alkyl group may be designated as "C1-C4 alkyl" or similar designations. By way of example only, "C1-C4 alkyl" indicates that there are 1 to 4 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, and hexyl. Alkyl groups may be substituted or unsubstituted.

[0037] As used herein, "alkenyl" refers to a straight or branched hydrocarbon chain having one or more alkyl groups. It refers to an alkyl group containing one or more double bonds. Examples of alkenyl groups include allenyl, vinylmethyl, and ethenyl. Alkenyl groups can be unsubstituted or substituted.

[0038] As used herein, "alkynyl" refers to an alkyl group containing one or more triple bonds in a straight or branched hydrocarbon chain. Examples of alkynyl include ethynyl and propynyl. An alkynyl group can be unsubstituted or substituted.

[0039] As used herein, "cycloalkyl" refers to a monocyclic or polycyclic hydrocarbon ring system that is fully saturated (no double or triple bonds). If the group consists of two or more rings, the rings may be fused together. A cycloalkyl group can contain 3 to 10 atoms in the ring, or 3 to 8 atoms in the ring. A cycloalkyl group can be unsubstituted or substituted. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0040] As used herein, "cycloalkenyl" refers to a monocyclic or polycyclic hydrocarbon ring system containing one or more double bonds in at least one ring, provided that if there are more than one, the double bonds cannot form a completely delocalized pi-electron system throughout all rings (otherwise the group would be "aryl" as defined herein). When composed of two or more rings, the rings may be fused together and connected. A cycloalkenyl can contain 3 to 10 atoms in the ring, or 3 to 8 atoms in the ring. A cycloalkenyl group can be unsubstituted or substituted.

[0041] As used herein, "aryl" refers to a carbocyclic (all carbon) monocyclic or polycyclic aromatic ring system (including fused ring systems in which two carbon rings share a chemical bond) having a completely delocalized pi-electron system throughout all rings. The number of carbon atoms in an aryl group can vary. For example, an aryl group can be a C6-C14 aryl group, a C6-C10 aryl group, or a C6 aryl group. Examples of aryl groups include, but are not limited to, benzene, naphthalene, and azulene. An aryl group can be substituted or unsubstituted.

[0042] As used herein, "heteroaryl" refers to monocyclic, bicyclic, and tricyclic aromatic ring systems (ring systems with fully delocalized pi-electron systems) containing one or more heteroatoms (e.g., 1 to 5 heteroatoms), i.e., elements other than carbon, such as, but not limited to, nitrogen, oxygen, and sulfur. The number of atoms in the rings of heteroaryl groups can vary. For example, heteroaryl groups can contain 4 to 14 atoms in the ring, 5 to 10 atoms in the ring, or 5 to 6 atoms in the ring. Furthermore, the term "heteroaryl" includes fused ring systems in which two rings share at least one chemical bond, such as at least one aryl ring and at least one heteroaryl ring, or at least two heteroaryl rings. Examples of heteroaryl rings include, but are not limited to, furan, furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole, 1,2,3-thiadiazole, 1,2,4-thiazole, benzothiazole, imidazole, benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole, benzisoxazole, isothiazole, triazole, benzotriazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, purine, pteridine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline, and triazine. Heteroaryl groups can be substituted or unsubstituted. may be.

[0043] As used herein, "heterocyclyl" or "heteroalicyclyl" refers to monocyclic, bicyclic, and tricyclic ring systems of 3, 4, 5, 6, 7, 8, 9, 10, or up to 18 members, in which carbon atoms, together with 1 to 5 heteroatoms, constitute the ring system. Heterocyclic rings may optionally contain one or more unsaturated bonds arranged in such a manner that a completely delocalized pi-electron system does not occur throughout all rings. Heteroatoms are elements other than carbon, such as, but not limited to, oxygen, sulfur, and nitrogen. Heterocyclic rings may further contain one or more carbonyl or thiocarbonyl functional groups; thus, this definition includes oxo and thio systems such as lactams, lactones, cyclic imides, cyclic thioimides, and cyclic carbamates. When composed of two or more rings, these rings may be fused together. Furthermore, the nitrogen of a heteroalicyclic ring may be quaternized. A heterocyclyl or heteroalicyclic group can be unsubstituted or substituted.Examples of such "heterocyclyl" or "heteroalicyclyl" groups include, but are not limited to, 1,3-dioxin, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane, 1,3-dioxolane, 1,4-dioxolane, 1,3-oxathiane, 1,4-oxathiin, 1,3-oxathiolane, 1,3-dithiol, 1,3-dithiolane, 1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, trioxane, and hexahydro-1,3,5-triazine. , imidazoline, imidazolidine, isoxazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline, thiazolidine, morpholine, oxirane, piperidine N-oxide, piperidine, piperazine, pyrrolidine, pyrrolidone, pyrrolidione, 4-piperidone, pyrazoline, pyrazolidine, 2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiopyran, thiamorpholine, thiamorpholine sulfoxide, thiamorpholine sulfone, and their benzo-fused analogs (e.g., benzimidazolidinone, tetrahydroquinoline, and 3,4-methylenedioxyphenyl).

[0044] As used herein, "aralkyl" and "aryl(alkyl)" refer to an aryl group connected as a substituent via a lower alkylene group. The lower alkylene and aryl groups of the aryl(alkyl) may be substituted or unsubstituted. Examples include, but are not limited to, benzyl, 2-phenyl(alkyl), 3-phenyl(alkyl), and naphthyl(alkyl).

[0045] As used herein, "heteroaralkyl" and "heteroaryl(alkyl)" refer to a heteroaryl group connected as a substituent via a lower alkylene group. The lower alkylene and heteroaryl groups of a heteroaryl(alkyl) can be substituted or unsubstituted. Examples include, but are not limited to, 2-thienyl(alkyl), 3-thienyl(alkyl), furyl(alkyl), thienyl(alkyl), pyrrolyl(alkyl), pyridyl(alkyl), isoxazolyl(alkyl), imidazolyl(alkyl), and their benzo-fused analogs.

[0046] As used herein, "alkoxy" refers to a group of formula -OR, where R is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaryl(alkyl), or heterocyclyl(alkyl), as defined herein. A non-limiting list of alkoxy is methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, phenoxy, and benzoxy. Alkoxy may be substituted or unsubstituted. That's fine.

[0047] As used herein, "acyl" refers to a hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaryl(alkyl), or heterocyclyl(alkyl) connected as a substituent through a carbonyl group. Examples include formyl, acetyl, propanoyl, benzoyl, and acryl. Acyl may be substituted or unsubstituted.

[0048] As used herein, the term "heterocyclic base" refers to an optionally substituted nitrogen-containing heterocyclyl that can be attached to an optionally substituted pentose moiety or a modified pentose moiety. In some embodiments, the heterocyclic base can be selected from an optionally substituted purine base, an optionally substituted pyrimidine base, and an optionally substituted triazole base (e.g., 1,2,4-triazole). The term "purine base" is used herein in its ordinary sense as understood by those skilled in the art and includes its tautomers. Similarly, the term "pyrimidine base" is used herein in its ordinary sense as understood by those skilled in the art and includes its tautomers. A non-limiting list of optionally substituted purine bases includes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g., 7-methylguanine), theobromine, caffeine, uric acid, and isoguanine. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil, and 5-alkylcytosine (e.g., 5-methylcytosine). An example of an optionally substituted triazole base is 1,2,4-triazole-3-carboxamide. Other non-limiting examples of heterocyclic bases include diaminopurine, 8-oxo-N 6 -Alkyl adenines (e.g., 8-oxo-N 6 -methyladenine), 7-deazaxanthine, 7-deazaguanine, 7-deazaadenine, N 4 ,N 4 -Ethanocytocin, N 6 ,N 6 Heterocyclic bases include -ethano-2,6-diaminopurine, 5-halouracils (e.g., 5-fluorouracil and 5-bromouracil), pseudoisocytosine, isocytosine, isoguanine, and other heterocyclic bases described in U.S. Patent Nos. 5,432,272 and 7,125,855, which are incorporated herein by reference for the limited purpose of disclosing additional heterocyclic bases. In some embodiments, the heterocyclic base may be optionally substituted with an amine or enol protecting group.

[0049] As used herein, "-N-linked amino acid" refers to an amino acid that is attached to the indicated moiety via a main chain amino or monosubstituted amino group. When an amino acid is attached to an -N-linked amino acid, one of the hydrogens that is part of the main chain amino or monosubstituted amino group is absent, and the amino acid is attached via the nitrogen. An N-linked amino acid may be substituted or unsubstituted.

[0050] As used herein, "-N-linked amino acid ester" refers to an amino acid in which the backbone carboxylic acid group has been converted to an ester group. In some embodiments, the ester group has a formula selected from alkyl-OC(=O)-, cycloalkyl-OC(=O)-, aryl-OC(=O)-, and aryl(alkyl)-OC(=O)-. A non-limiting list of ester groups includes substituted and unsubstituted versions of the following: methyl-OC(=O)-, ethyl-OC(=O)-, n-propyl-OC(=O)-, isopropyl-OC(=O)-, n-butyl-OC(=O)-, isobutyl-OC(=O)-, tert-butyl-OC(=O)-, neopentyl-OC(=O)-, cyclopropyl-OC(=O)-, cyclobutyl-OC(=O)-, cyclopentyl-OC(=O)-, cyclohexyl-OC(=O)-, phenyl-OC(=O)- , benzyl-OC(=O)- and naphthyl-OC(=O)-. The N-linked amino acid ester derivatives may be substituted or unsubstituted.

[0051] As used herein, "-O-linked amino acid" refers to an amino acid that is attached to the indicated moiety via a hydroxyl from its backbone carboxylic acid group. When an amino acid is attached to an -O-linked amino acid, the hydrogen that is part of the hydroxyl from its backbone carboxylic acid group is absent, and the amino acid is attached through the oxygen. The O-linked amino acid may be substituted or unsubstituted.

[0052] As used herein, the term "amino acid" refers to any amino acid (both standard and non-standard), including, but not limited to, α-amino acids, β-amino acids, γ-amino acids, and δ-amino acids. Examples of suitable amino acids include, but are not limited to, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. Additional examples of suitable amino acids include, but are not limited to, ornithine, hypusine, 2-aminoisobutyric acid, dehydroalanine, γ-aminobutyric acid, citrulline, beta-alanine, alpha-ethyl-glycine, alpha-propyl-glycine, and norleucine.

[0053] The term "amino acid" includes natural and synthetic β, γ or δ amino acids, and amino acids may be in the D or L configuration. The amino acid can be a derivative of alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleucinyl, β-prolinyl, β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl, β-serinyl, β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl, β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl, or β-histidinyl.

[0054] As used herein, the term "deoxynucleoside" refers to any nucleoside that contains a deoxy sugar, i.e., any compound formally derived from a sugar by replacing a hydroxy group with a hydrogen atom, e.g., deoxyribose. As used herein, the term "dNTP" refers to deoxyribonucleotide triphosphate. Each dNTP consists of a phosphate group, a deoxyribose sugar, and a nitrogenous base. There are four different dNTPs, which can be divided into two groups: purines and pyrimidines. dATP (deoxyadenosine 5'-triphosphate) and dGTP (deoxyguanosine 5'-triphosphate) constitute purines, while dTTP (deoxythymidine 5'-triphosphate) and dCTP (deoxycytidine 5'-triphosphate) constitute pyrimidines. The purine-characteristic bases adenine and guanine both have double-ring structures, while the purine-characteristic bases thymine and cytosine both have single-ring structures.

[0055] As used herein, the term "mitochondrial DNA depletion syndrome" refers to a phenotypically diverse class of diseases and disorders characterized by a severe reduction in mitochondrial DNA (mtDNA) content in affected tissues and organs, e.g., muscle, liver, brain, and / or gastrointestinal tract. The reduction or depletion can result from an imbalance in the mitochondrial nucleotide pool available for mtDNA replication and from abnormalities in mitochondrial replication. Based on age of onset, two subtypes are distinguished: congenital (or early-onset) and infantile (or late-onset). Although the later-onset form is associated with a longer survival time, the syndrome affects almost all It is fatal in patients and currently there is no effective treatment.

[0056] I. Prodrugs The present invention provides deoxynucleotide prodrugs. "Deoxynucleoside" refers to 2'-deoxynucleosides, such as deoxycytidine (dC, shown below), deoxythymidine (dT), deoxyadenosine (dA), and deoxyguanosine (dG). The full-length names and common abbreviations of each are used interchangeably.

[0057] In certain embodiments, the bases are selected from cytosine, thymine, guanine, and adenine.

[0058] Thus, the prodrugs described herein are deoxycytidine prodrugs (dC prodrugs), deoxythymidine prodrugs (dT prodrugs), deoxyguanosine prodrugs (dG prodrugs) and deoxyadenosine prodrugs (dA prodrugs).

[0059] [ka]

[0060] The prodrug is preferably in the natural β-D-configuration.

[0061] In one embodiment, the prodrug strategy involves masking reactive groups, such as charged -OH and phosphate groups, in vivo to allow crossing of cell membranes.

[0062] In one embodiment, the present invention provides a prodrug of Formula I: [ka]

[0063] wherein base refers to an optionally substituted heterocyclic base or an optionally substituted heterocyclic base having a protected amino group; R 1 is an optionally substituted acyl, an optionally substituted O-linked amino acid, [ka] is selected from the group consisting of X, Y, and Z are each independently selected from O and S; R 2 , R 3 and R 4 are each hydrogen, optionally substituted C 1-24 Alkyl, optionally substituted C 2-24 Alkenyl, optionally substituted C 2-24Alkynyl, optionally substituted C 3-6 Cycloalkyl, optionally substituted C 3-6 Cycloalkenyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryl (C 1-6 ) alkyl, [ka] or R 2 and R 3 and can be taken together to form a cyclic moiety, R 5 , R 6 and R 7 are each optionally substituted C 1-24 Alkyl, optionally substituted C 2-24 Alkenyl, optionally substituted C 2-24 Alkynyl, optionally substituted C 3-6 Cycloalkyl, optionally substituted C 3-6 Cycloal Kenil, N.R. 20 R 21 , an optionally substituted N-linked amino acid, an optionally substituted N-linked amino acid ester; R 8 , R 9 , R 11 and R 12 are each independently selected from hydrogen, optionally substituted C alkyl, and optionally substituted aryl; R 10 and R 13 are each independently selected from hydrogen, optionally substituted C alkyl and optionally substituted aryl, optionally substituted —O—C alkyl, optionally substituted —O-aryl, optionally substituted —O-heteroaryl, optionally substituted —O-monocyclic heterocyclyl; R 14 , R 15 and R 19 are each hydrogen, optionally substituted C1-24 independently selected from alkyl, and optionally substituted aryl; R 16 and R 17 are -CN, optionally substituted C, respectively. 2-8 Organyl carbonyl, C 2-8 Alkoxycarbonyl and C 2-8 independently selected from organylaminocarbonyl; R 18 is hydrogen, optionally substituted C 1-24 Alkyl, optionally substituted C 2-24 Alkenyl, optionally substituted C 2-24 Alkynyl, optionally substituted C 3-6 Cycloalkyl, and optionally substituted C 3-6 cycloakenyl; R 20 and R 21 are each hydrogen, optionally substituted C 1-24 Alkyl, optionally substituted C 2-24 Alkenyl, optionally substituted C 2-24 Alkynyl, optionally substituted C 3-6 Cycloalkyl, and optionally substituted C 3-6 cycloalkenyl, and Each of n, m, and p is independently selected from 0, 1, 2, or 3.

[0064] In certain embodiments, the prodrug is a compound of formula Ia: [ka]

[0065] In the formula, R 1 , R 2 and R 3 is hydrogen, optionally substituted C 1-24 Alkyl, optionally substituted C 2-24 Alkenyl, optionally substituted C 2-24 Alkynyl, optionally substituted C3-6 Cycloalkyl, optionally substituted C 3-6 Cycloalkenyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted aryl (C 1-6 ) alkyl.

[0066] In certain embodiments, R 1 is aryl and R 2 is C 1-24 alkyl, and R 3 is C 1-24 It is alkyl.

[0067] In another particular embodiment, R 2 is an amino acid side chain.

[0068] In more particular embodiments, the prodrug is selected from one of the following compounds: R: [ka]

[0069] In another embodiment, the present invention provides a prodrug of formula II: [ka]

[0070] wherein base refers to an optionally substituted heterocyclic base or an optionally substituted heterocyclic base having a protected amino group; X is selected from S and O, and R 22 -O - , -OH, -O-alkyl, optionally substituted C 1-6 Alkoxy, [ka] , optionally substituted N-linked amino acids and optionally substituted N-linked amino acid esters, wherein R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , n, m and p are defined as above.

[0071] In certain embodiments, the prodrug is a compound of formula IIa: [ka]

[0072] where R is C 1-4 It is alkyl.

[0073] In yet another particular embodiment, the prodrug is selected from one of the following compounds: [ka]

[0074] In yet another embodiment, the present invention provides a prodrug of formula III: [ka]

[0075] wherein base refers to an optionally substituted heterocyclic base or an optionally substituted heterocyclic base having a protected amino group; R 1 and R 2are independently selected from hydrogen, phosphate (including monophosphates, diphosphates, or triphosphates of Formula I and modified phosphates), linear, branched, or cyclic alkyl, optionally substituted acyl, CO-alkyl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate ester, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, lipids, phospholipids, amino acids, carbohydrates, peptides, and cholesterol).

[0076] In one embodiment, R 2 is an amino acid, i.e., the compound of formula III is a 3'-amino acid ester, e.g., a compound of formula IIIa: [ka]

[0077] where R is the side chain of the amino acid.

[0078] In certain embodiments, the amino acid is valine (i.e., R is CH(CH3)2).

[0079] In a more particular embodiment, the prodrug is a compound of formula IIIb: [ka]

[0080] wherein the base is an optionally substituted heterocyclic base or a base having a protected amino group. refers to an optionally substituted heterocyclic base comprising:

[0081] In even more particular embodiments, the prodrug is selected from one of the following compounds: [ka]

[0082] In yet another particular embodiment, the prodrug is selected from one of the following compounds: [ka]

[0083] In yet another embodiment, the prodrug is a compound of formula IIIc: [ka]

[0084] wherein base refers to an optionally substituted heterocyclic base or an optionally substituted heterocyclic base having a protected amino group; Ra is a straight, branched, or cyclic alkyl, and R is the side chain of an amino acid.

[0085] In certain embodiments, Ra is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl, and R is CH(CH3)2).

[0086] II.How to use The present invention provides methods for treating a disease or disorder characterized by imbalanced nucleotide pools in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one prodrug described herein.

[0087] In one embodiment, the prodrugs described above can be utilized in the present methods.

[0088] Prodrugs can be administered per se (i.e., alone) or in the form of a pharmaceutical composition. Pharmaceutical compositions containing one or more prodrugs for administration can contain a therapeutically effective amount of the prodrug and a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable, do not normally produce allergic reactions or similar adverse reactions, such as acute gastric peristalsis or dizziness, when administered to humans, and are approved by federal or state regulatory agencies or listed in the United States Pharmacopoeia or other generally recognized pharmacopeias for use in animals, more specifically in humans. A "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as saline, and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. When a pharmaceutical composition is administered intravenously, saline is a preferred carrier. Physiological saline and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, especially for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc.The composition can optionally contain small amounts of wetting or emulsifying agents, or pH buffering agents. It can also include.

[0089] In certain embodiments, the disclosed methods can be used to treat mitochondrial DNA (mtDNA) depletion syndrome (MDS). Each nucleated cell contains hundreds of mitochondria, which are unique organelles under the control of two genomes. Although mitochondria contain their own DNA, mtDNA, most mitochondrial proteins, including all proteins required for mtDNA replication, transcription, and repair, are encoded by nuclear genes.

[0090] Maintenance of mtDNA requires proteins essential for mtDNA synthesis, maintaining the mitochondrial nucleotide pool, and mediating mitochondrial fusion. The enzymes that synthesize mtDNA require a balanced supply of intramitochondrial nucleotides. These are supplied through the mitochondrial nucleotide salvage pathway and through the import of nucleotides from the cytosol via specific transporters. Proper function in mtDNA synthesis requires the appropriate balance of these enzymes. Proteins known to be required for mtDNA synthesis are encoded by nuclear genes. Pathogenic variants that disrupt the function of any one of the proteins encoded by these genes impair mtDNA synthesis, resulting in mtDNA quantitative defects (mtDNA depletion) or mtDNA qualitative defects (multiple mtDNA deletions). To date, pathogenic variants in more than 20 nuclear genes are known to be associated with defects in mtDNA maintenance.

[0091] In one embodiment, mtDNA depletion syndrome is nuclear DNA-based mtDNA depletion syndrome.In certain embodiments, nuclear DNA encodes the protein involved in nucleotide metabolism.The imbalance in free nucleotide concentration leads to mtDNA replication failure, resulting in a reduction in mtDNA copy number.

[0092] In certain embodiments, the method of the present invention is useful for treating diseases or disorders caused by the imbalance of deoxyribonucleoside triphosphate (dNTP) pool.dNTP is the precursor used by DNA polymerase for the replication and repair of nuclear and mitochondrial DNA in animal cells.The concentration of dNTP depends on the balance of synthesis, consumption and degradation.

[0093] Accurate DNA synthesis requires adequate amounts of each dNTP and a properly balanced dNTP pool. The total size of the cellular pools ranges from 10 to 100 pmol of each dNTP per million cells during S phase, with the mitochondrial pool accounting for up to 10% of the total. In quiescent or differentiated cells, the pools are approximately 10-fold smaller in both the cytosol and mitochondria. Contrary to what would be expected based on the roughly equimolar amounts of the four nitrogenous bases in DNA, the four dNTPs exist in different ratios in the pools, with pyrimidines often exceeding purines. Individual cell lines, even those derived from the same animal species, may exhibit different pool compositions. Increasing the concentration of one dNTP typically depletes another dNTP.

[0094] The mtDNA depletion syndrome may involve specific tissues or organs, such as the muscle, liver, brain, and / or gastrointestinal tract. In certain embodiments, the mtDNA depletion syndrome is a myopathy syndrome or a hepatocerebral syndrome.

[0095] Exemplary disorders characterized by imbalanced nucleotide pools include, but are not limited to, TK2 deficiency, deficiency associated with RRM2B (encoding p53R2, the p53-inducible small subunit of ribonucleotide reductase, RNR), and TYMP (coding thymidine phosphorylase, TP) causing mitochondrial neurogastrointestinal encephalomyopathies (MNGIE). Mutations in genes that disrupt mitochondrial dNTP pools include, but are not limited to, SUCLA2, SUCLG1, and MPV17. Parallel defects in deoxyguanosine kinase (dGK) due to autosomal recessive mutations in DGUOK, accompanied by dGMP and dAMP deficiencies, cause mtDNA depletion, usually manifesting as hepatocerebral disease with early childhood onset (Mandel, et al. 2001). Disorders associated with these genes can also be treated using the methods herein.

[0096] In certain embodiments, the disorder is thymidine kinase 2 (TK2) deficiency. TK2 is a mitochondrial enzyme involved in the salvage of deoxyribonucleotides required for mitochondrial DNA (mtDNA) replication. TK2 catalyzes the first rate-limiting step of the deoxypyrimidine salvage pathway. Autosomal recessive TK2 mutations cause a spectrum of infantile to adult-onset diseases that primarily manifest as myopathy.

[0097] As the mechanisms of other forms of MDS and other disorders are elucidated, the appropriate deoxynucleoside for treatment can be determined by the skilled practitioner.

[0098] For example, a patient with a TK2 deficiency is administered a prodrug of dC and / or dT. In another example, a patient with a DGUOK deficiency is administered a prodrug of dG and / or dA.

[0099] In one embodiment, this method further comprises identifying the patient with disease or disorder characterized by imbalanced nucleotide pool.In one embodiment, this disease or disorder is TK2 deficiency.Patients who show the above-mentioned phenotype of TK2 deficiency, including the most typical symptoms of progressive muscle disease, characterized by generalized hypotonia, proximal muscle weakness, loss of previously acquired motor skills, poor feeding, and respiratory distress, can be tested to confirm the diagnosis of disease.

[0100] Molecular genetic testing using a panel of genes known to cause mtDNA depletion syndromes should be performed (Chanprasert, et al. 2012) and can also be used to identify patients with diseases or disorders characterized by imbalanced nucleotide pools.

[0101] The TK2 gene is the only gene known to cause TK2-related mitochondrial DNA depletion syndrome (MTD). This testing can include sequence analysis of the entire coding region and exon / intron junction regions of TK2 for sequence variants and deletions / duplications. If compound heterozygous or homozygous deleterious mutations are identified by sequence analysis, a diagnosis of TK2 deficiency is established, and the subject is therefore likely to benefit from deoxynucleoside therapy. If sequence analysis does not identify two compound heterozygous or homozygous deleterious mutations, deletion / duplication analysis should be considered to determine and / or establish the diagnosis of TK2 deficiency.

[0102] Further testing to determine and / or establish a diagnosis of TK2 deficiency includes serum creatine kinase (CK) concentration, electromyography, skeletal muscle histopathology, mitochondrial DNA (mtDNA) content (copy number), and skeletal muscle electron transport chain (ETC) activity. TK2 deficiency is determined and / or established if these tests reveal one or more of the following: Elevated CK concentrations compared to healthy controls may indicate TK2 deficiency. A skeletal muscle biopsy can be performed, followed by skeletal muscle mtDNA content analysis. If the skeletal muscle biopsy reveals significant variation in fiber size, variable sarcoplasmic vacuoles, variable increased connective tissue, and irregular red fibers, as well as increased succinate dehydrogenase (SDH) activity and low to absent cytochrome c oxidase (COX) activity, then the CK concentration may be elevated. If the mtDNA copy number is significantly reduced (typically less than 20% of age- and tissue-matched healthy controls), a diagnosis of TK2 deficiency can be determined and / or established (Chanprasert, et al. 2012).

[0103] Furthermore, TK2 deficiency is inherited in an autosomal recessive manner, so siblings of affected individuals can be tested as soon as possible after birth to diagnose the disease.

[0104] Administration can be via any route, including, but not limited to, intrathecal, parental, mucosal, and transdermal.

[0105] In one embodiment, administration is oral. Exemplary oral dosage forms include, but are not limited to, capsules, tablets, powders, granules, solutions, syrups, suspensions (in non-aqueous or aqueous liquids), or emulsions. Tablets or hard gelatin capsules may contain lactose, starch or derivatives thereof, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, stearic acid, or salts thereof. Soft gelatin capsules may contain vegetable oils, waxes, fats, semisolid or liquid polyols. Solutions and syrups may contain water, polyols, and sugars. The prodrugs described herein can be added to any form of liquid consumed by patients, including, but not limited to, cow's milk, both bovine and human breast milk, infant formula, and water. The prodrugs can be coated with or mixed with a material that delays disintegration and / or absorption in the digestive tract. Thus, sustained release can be achieved over many hours.

[0106] In another embodiment, administration is intrathecal. Intrathecal administration involves injecting a drug into the spinal canal, more specifically the subarachnoid space, to reach the cerebrospinal fluid. This method is commonly used for spinal anesthesia, chemotherapy, and analgesics. Intrathecal administration can be achieved by lumbar puncture (bolus injection) or by a port-catheter system (bolus or infusion). The catheter is most commonly inserted between the lumbar lamina and the tip is threaded into the intrathecal cavity to the desired level (usually L3-L4). Water or saline is most commonly used as an excipient for intrathecal formulations, although EDTA and lipids have also been used.

[0107] In yet another embodiment, administration is parenteral, including intravenous administration. Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile solutions or suspensions for injection, which may contain antioxidants, buffers, bacteriostats, and solutes that render the composition substantially isotonic with the subject's blood. Other components that may be present in such compositions include water, alcohol, polyols, glycerin, and vegetable oils. Compositions adapted for parenteral administration may be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile carrier immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Suitable vehicles that can be used to provide parenteral dosage forms of the present invention are well known to those skilled in the art. Examples include: Water for Injection USP; aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as ethyl alcohol, polyethylene glycol and polypropylene glycol; and non-aqueous vehicles such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Additionally, because some patients may be receiving enteral nutrition by the time treatment begins, the prodrug can be administered via a gastronomy feeding tube or other enteral feeding means.

[0108] Pharmaceutical compositions adapted for nasal and pulmonary administration may include solid carriers such as powders, which The compositions can be administered by rapid inhalation through the nose. Compositions for nasal administration can contain a liquid carrier, such as a spray or droplets. Alternatively, direct inhalation into the lungs can be achieved by deep inhalation or introduction through a mouthpiece. These compositions can contain an aqueous or oily solution of the active ingredient. Compositions for inhalation can be delivered by specially adapted devices, including but not limited to pressurized aerosols, nebulizers, or inhalers, which can be configured to provide a predetermined dose of the active ingredient.

[0109] Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas. Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

[0110] Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.

[0111] The dosage of at least one prodrug or a composition comprising the same can be about 25 mg / kg / day to about 1,000 mg / kg / day. More preferred dosages are in the range of about 200 mg / kg / day to about 800 mg / kg / day. More preferred dosages are in the range of about 100 mg / kg / day to about 600 mg / kg / day, for example, about 250 mg / kg / day to about 500 mg / kg / day, about 300 mg / kg / day to about 500 mg / kg / day, or about 400 mg / kg / day to about 500 mg / kg / day.

[0112] In one embodiment, the dosage is about 20% to about 100% equimolar to a standard nucleoside (dT, dC, dG, dA), e.g., about 20% to about 80%, about 20% to about 50%, about 50% to about 80%, or about 50% to about 100%. In a specific embodiment, the dosage is about 20% equimolar to a standard nucleoside. In another specific embodiment, the dosage is about 50% equimolar to a standard nucleoside. In yet another embodiment, the dosage is about 100% equimolar to a standard nucleoside.

[0113] Administration of at least one prodrug or a composition containing the same can be once daily, twice daily, three times daily, four times daily, five times daily, or up to six times daily, preferably at regular intervals. For intravenous or intrathecal administration, the dose may be reduced. A preferred dose range for such administration is about 50 mg / kg / day to about 500 mg / kg / day.

[0114] In embodiments where the composition comprises two or more prodrugs, the ratio of the prodrugs can vary. For example, when two prodrugs are administered, they can be in a 50 / 50 ratio, or in ratios of about 5 / 95, 10 / 90, 15 / 85, 20 / 80, 25 / 75, 30 / 70, 35 / 65, 40 / 60, 45 / 55, 55 / 45, 60 / 40, 65 / 35, 70 / 30, 75 / 25, 80 / 20, 85 / 15, 90 / 10, and 95 / 5.

[0115] In one embodiment, the method further comprises monitoring the improvement of the subject's condition before increasing dosage.The response of the subject to therapeutic administration can be monitored by observing the changes in the subject's muscle strength and control, mobility, and height and weight.If one or more of these parameters increase after administration, treatment can be continued.If one or more of these parameters remain the same or decrease, dosage can be increased.

[0116] In another embodiment, the method further comprises monitoring the subject for side effects before reducing the dosage. Exemplary side effects include, but are not limited to, diarrhea, bloating, and other gastrointestinal symptoms.

[0117] The prodrugs of the present invention can also be co-administered with other drugs. Such drugs include therapeutic agents for treating the symptoms of certain forms of MDS. In particular, in the case of TK2 deficiency, other drugs include ubiquitous inhibitors of nucleoside catabolism, including enzyme inhibitors such as tetrahydrouridine (an inhibitor of cytidine deaminase), imucillin H (an inhibitor of purine nucleoside phosphorylase), and tipiracil (an inhibitor of thymidine phosphorylase). Such inhibitors are known and are used to treat some cancers. [Example]

[0118] Example 1: Mouse model of TK2 deficiency The efficacy of the prodrugs described herein was assessed through a mouse model. The homozygous Tk2 H126N knock-in mutant (Tk2) exhibits a phenotype strikingly similar to human infantile encephalomyopathies. - / - ) mice have been reported previously (Akman, et al. 2008). Between 10 and 13 days after birth, Tk2 - / - Mice rapidly develop a fatal encephalomyopathic disease characterized by reduced locomotion, unsteady gait, coarse tremor, growth retardation, and rapid progression to early death at 14–16 days of age. Molecular and biochemical analysis of mouse models indicates that the pathogenesis of the disease is due to a loss of enzyme activity and an imbalance in dNTP pools, with reduced dTTP levels in the brain and reduced levels of both dTTP and dCTP in the liver, leading to mtDNA depletion and defects in respiratory chain enzymes, including mtDNA-encoded subunits, most notably in the brain and spinal cord.

[0119] The prodrug was administered daily by oral gavage in 50 μl of Esbilac formula for small pets (Pet-Ag) to Tk2 H126N knock-in mice (Tk2 - / - ) and age-matched controls (Tk2 + A 50 / 50 mixture of 3 and 4 was administered at 580 mg / kg / day. A 50 / 50 mixture of 5 and 6 was administered at 120 mg / kg / day to 430 mg / kg / day.

[0120] All treatments were administered from postnatal day 4 to 29. At 21 days of age, mice were separated from their mothers and continued treatment by oral administration. + The mice were weighed and closely observed for comparison.

[0121] Mice were followed and weighed daily (it has previously been observed that the inability to gain weight is the first sign of illness).

[0122] The prodrug mixture extended survival of tk2- / - mice in a manner comparable to standard nucleosides and showed non-statistically significant improvements in body weight.

[0123] Example 2: Fibroblasts derived from a dGK-deficient patient The prodrugs were tested in fibroblasts derived from dGK-deficient patients to determine their effect on mtDNA copy number. The advantage of using dGK-deficient fibroblasts is that after quiescence is induced, mtDNA spontaneously depletes without the addition of DNA-damaging agents. It has previously been demonstrated that supplementing the cell culture medium with dGuo (50 μM) is sufficient to prevent this depletion. Cells were grown to confluence. Quiescence was induced by reducing FBS in the medium to 0.1%. After 3 days (day 0), we supplemented the cell culture medium with 50 μM dGuo (deoxyguanosine) or the prodrugs of the present invention. Cells were maintained under the same conditions until day 18, with regular addition of fresh medium. mtDNA copy number was assessed at various time points throughout the experiment (days 4, 9, and 18). Results are expressed as the mean + SD of duplicate experiments and plotted as mtDNA / nDNA ratio relative to the mean value obtained for three untreated healthy controls cultured in parallel.

[0124] Equimolar concentrations of dGuo and other available dG prodrugs (50 µM) were tested in parallel under similar conditions. Results showed that a 50 / 50 mixture of 7 and 8 at 50 µM prevented mtDNA depletion.

Claims

1. 1. A pharmaceutical composition for treating mitochondrial DNA depletion syndrome, said pharmaceutical composition comprising a prodrug, said prodrug being a compound of Formula IIIc, and said treatment comprising administering a therapeutically effective amount of said prodrug in a subject in need thereof: 【Chemistry 12】 During the ceremony, the bases are selected from optionally substituted cytosine, thymine, guanine, and adenine, or optionally substituted cytosine, thymine, guanine, and adenine with a protected amino group; R a is straight chain, branched chain, or cyclic alkyl; and R is the side chain of the amino acid.

2. The pharmaceutical composition of claim 1, wherein Ra is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl.

3. The pharmaceutical composition of claim 1, wherein R is CH(CH3)2.

4. The pharmaceutical composition of claim 1, wherein R is isopropyl.

5. 10. The pharmaceutical composition of claim 1, wherein the treatment comprises the administration of at least two prodrugs.

6. 6. The pharmaceutical composition of claim 5, wherein the weight ratio of one prodrug to another prodrug is 50 / 50.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the mitochondrial DNA depletion syndrome is caused by a mutation in a nuclear gene selected from the group consisting of TK2, RRM2B, TYMP, SUCLA2, SUCLG1, MPV17, POLG, POLG2, and DGUOK.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the administration is oral.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the dose of the prodrug administered is from about 200 mg / kg / day to about 1,000 mg / kg / day.

10. The pharmaceutical composition according to any one of claims 1 to 9, which is administered at least once a day.

11. 11. The pharmaceutical composition of any one of claims 1 to 10, wherein the base in the prodrug is selected from the group consisting of cytosine, thymine, guanine, and adenine.

12. A pharmaceutical composition described in any one of claims 1 to 6, wherein the mitochondrial DNA depletion syndrome is caused by a mutation in POLG.

13. A pharmaceutical composition described in any one of claims 1 to 6, wherein the mitochondrial DNA depletion syndrome is caused by a mutation in POLG2.

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