Deoxynucleoside prodrugs for treating mitochondrial diseases caused by imbalanced nucleotide pools

Abstract

Provided herein are deoxynucleotide prodrugs and methods of use thereof for the treatment of diseases characterized by imbalanced nucleotide pools, such as MPV17 and deoxyguanosine kinase deficiencies.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 212,468, filed June 18, 2021, which is incorporated by reference in its entirety herein.

[0002] The present invention generally relates to prodrugs for delivery of single nucleosides or their prodrugs, and their use in the treatment of mitochondrial DNA depletion syndrome diseases, such as MPV17 and dGK deficiency disorders, optionally in combination with other nucleosides and their prodrugs to balance the nucleotide replicative pool. [Background technology]

[0003] Mitochondria have their own DNA (mtDNA) that codes for many important proteins for the assembly and activity of mitochondrial respiratory complexes (OXPHOS complexes). The mtDNA is filled by many proteins to form nucleoids that are evenly distributed within the mitochondrial matrix, which is essential for mitochondrial function. Maintenance of mitochondrial DNA (mtDNA) depends on several nuclear gene-encoded proteins, including a set of enzymes that form the replisomes necessary to synthesize mtDNA. These enzymes need to be present in balanced amounts to function properly. Furthermore, mtDNA synthesis requires a balanced supply of nucleotides, which is achieved by nucleotide recycling within the mitochondria and uptake from the cytosol. Mitochondrial DNA maintenance defects are a group of diseases caused by pathogenic variants in nuclear genes involved in mtDNA maintenance that result in impaired mtDNA synthesis leading to quantitative (mtDNA depletion) and qualitative (multiple mtDNA deletions) defects in mtDNA. Defective mtDNA leads to organ dysfunction due to insufficient synthesis of proteins encoded in mtDNA, resulting in insufficient energy production to meet the needs of the affected organ. MDS are inherited as autosomal recessive or dominant traits and are associated with a wide phenotypic spectrum ranging from mild adult-onset ophthalmoplegia to severe, lethal infantile liver failure.Mitochondrial diseases are clinically diverse disorders due to defects in oxidative phosphorylation that disrupt the mitochondrial respiratory chain (RC) and the biochemical pathway that converts electron energy into adenosine triphosphate (ATP) occurring in various, usually intense, energy-requiring organs.

[0004] The respiratory chain is composed of four multi-subunit enzymes (complexes I-IV) that transfer electrons to generate a proton gradient across the inner membrane of mitochondria, and this proton flow drives ATP synthesis via complex V (Non-Patent Documents 1-2). 10 (CoQ 10) is an essential molecule that shuttles electrons from complexes I and II to complex III. The respiratory chain is unique to eukaryotes, e.g., mammalian cells, because it is controlled by two genomes: mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). As a result, mutations in either genome can cause mitochondrial disease. Most mitochondrial diseases affect multiple body organs and are usually fatal in the bifocal form. There is no proven effective treatment for mitochondrial disease, and CoQ 10 Supportive care includes the administration of TK2 and its analogues to enhance respiratory chain activity and neutralize reactive oxygen species (ROS), which are toxic by-products of dysfunctional respiratory chain enzymes. A combination of deoxycytidine and deoxythymidine is currently in clinical trials in several countries for the treatment of TK2 deficiency disorder. (ClinicalTrials.gov Identifier: NCT03845712) Limited oral bioavailability of purine nucleosides limits the usefulness of direct deoxyguanosine and / or deoxyadenosine supplementation for other MDS.

[0005] Mitochondrial DNA depletion syndromes (MDS), a subgroup of mitochondrial diseases, are the cause of many severe childhood encephalomyopathy cases, characterized by a reduction in mitochondrial DNA (mtDNA) copy number in tissues, or by the accumulation of mtDNA depletion in tissues and insufficient synthesis of mitochondrial RC complexes (Non-Patent Document 3). Mutations in several nuclear genes, such as TK2, DGUOK (or dGK, as used in some examples herein, to be distinguished from DGK, which indicates diacylglycerol kinase), 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).

[0006] Deoxyguanosine kinase (dGK: dG (deoxyguanosine) kinase) is an essential rate-limiting component of the mitochondrial purine nucleotide salvage pathway, encoded by the nuclear gene encoding deoxyguanosine kinase (DGUOK). Mutations in DGUOK typically result in mtDNA depletion in the liver and brain, causing a hepatocerebral phenotype, and it is one of two mitochondrial deoxynucleoside salvage pathway enzymes involved in precursor synthesis for mtDNA replication. dGK is responsible for the initial rate-limiting phosphorylation of purine deoxynucleosides, using nucleoside triphosphates as phosphate donors. Mutations in the DGOUK gene are associated with hepatospecific and hepatocerebral forms of MDS (mtDNA depletion syndrome) disease. The deoxymonophosphates are then converted to diphosphates and triphosphates for incorporation into mtDNA. Previously, it was shown that in cultured cells, supplementation with 50 μm deoxyguanosine alone added to the culture medium was sufficient to increase mtDNA copy number in fibroblasts derived from patients with the DGUOK mutation (Non-Patent Document 23). More recently, a zebrafish mutant dGK model was generated, and these homozygous mutants showed a characteristic reduction in mtDNA amount but had no visible phenotype. A possible explanation for this could be compensation by the cytoplasmic enzyme deoxycytidine kinase (dCK). Similar to the experiments performed in fibroblast cultures, attempts to increase mtDNA levels in dGK mutant fish by adding only deoxyguanosine reduced mtDNA levels in the model. It was suggested that a concentration of only one of the substrate nucleosides introduced an imbalance in the dNTP pool, thereby disrupting mtDNA replication and, as a result, reducing mtDNA copy number. However, in further experiments in which dGK mutant fish were supplemented with both purine nucleosides, a significant increase in liver mtDNA copy number was detected (Non-Patent Document 24).

[0007] dGK clinical symptoms: The disease primarily presents in the neonatal, infancy, or childhood period. It is an autosomal recessive disorder that results in mitochondrial depletion. The disease manifests with symptoms of hepatopathy and encephalopathy. Patients usually first present with hypoglycemia and lactic acidosis. In addition, elevated serum concentrations of tyrosine or phenylalanine are evident on newborn screening in affected newborns, and hepatic transaminases, gamma glutamyl transferase (GGT), and conjugated hyperbilirubinemia are typically elevated. Patients have a variable neuromuscular phenotype, but symptoms may include developmental regression, hypotonia, severe myopathy, rotational nystagmus, and oculomotor ataxia.

[0008] Mpv17 was identified by disease-segregating mutations in three families of hepatocerebral MDS, and it was demonstrated that MPV17 is a mitochondrial inner membrane protein, the absence or dysfunction of which causes oxidative phosphorylation (OXPHOS) failure and mtDNA depletion not only in affected individuals but also in Mpv17- / - mice (Non-Patent Document 25). More recent reports have shown that MPV17 is a protein involved in the import of deoxynucleotides into mitochondria, the dysfunction of which causes disease in both infantile-onset and, more rarely, adult-onset forms. Infantile-onset forms are characterized as severe hepatocerebral MDS, while adult-onset forms are characterized by neuromuscular or multiorgan phenotypes with deletions (Non-Patent Document 26).

[0009] There is also a need for treatment of many forms of MDS and other diseases characterized by imbalanced nucleotide pools, as described, for example, in US Pat. No. 5,399,633. For example, some Mendelian genetic diseases with mtDNA depletion or multiple deletions, or both, are characterized by imbalanced deoxynucleotide triphosphate pools leading to defects in mtDNA replication. Other nuclear genes that disrupt mitochondrial dNTP pools include TK2, TYMP, RRM2B, SUCLA2, SUCLG1, and POLG. Treatments that restore dNTP pool balance would be useful to treat these disorders as well, and in particular, administration of a single prodrug agent would provide advantages in dosing and patient compliance. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Pat. No. 10,471,087 [Non-patent literature]

[0011] [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; [Non-Patent Document 7] Mandel, et al. 2001; [Non-Patent Document 8] Naviaux and Nguyen 2004; [Non-Patent Document 9] Ostergaard, et al. 2007; [Non-Patent Document 10] Saada, et al. 2003; [Non-Patent Document 11] Sarzi, et al. 2007; [Non-Patent Document 12] Spinazzola, et al, 2006 [Non-Patent Document 13] Behin, et al. 2012; [Non-Patent Document 14] Garone, et al. 2012; [Non-Patent Document 15] Longley, et al. 2006; [Non-Patent Document 16] Nishino, et al. 1999; [Non-Patent Document 17] Paradas, et al. 2012; [Non-Patent Document 18] Ronchi, et al. 2012; [Non-Patent Document 19] Spelbrink, et al. 2001; [Non-Patent Document 20] Tyynismaa, et al. 2009; [Non-Patent Document 21] Tyynismaa, et al. 2012; [Non-Patent Document 22] Van Goethem, et al. 2001 [Non-Patent Document 23] Camara, et al. 2014 [Non-Patent Document 24] Munro, et al. 2019 [Non-Patent Document 25] Spinazzola, et al. 2006 [Non-Patent Document 26] El-hattab, et al. 2018 [Summary of the Invention] [Problem to be solved by the invention]

[0012] The present invention generally relates to prodrugs of deoxyguanosine for delivery of deoxynucleosides or deoxynucleotides to tissues affected by dGK or MPV17 deficiency, optionally including one or more prodrugs of other nucleosides / nucleotides intended to balance the nucleotide pool. Surprisingly, it has been found that treatment with (dG) deoxyguanosine alone, or dG administered alone as a prodrug according to the present invention, is sufficient to increase mtDNA copy number in dGK or MPV17 deficiency disorders. [Means for solving the problem]

[0013] In one aspect, the present invention provides a compound of formula I for supplementing nucleosides for the treatment of mitochondrial depletion syndrome: [ka] 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 3-6Cycloalkyl, 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 Cycloalkenyl, NR 20 R 21 , an optionally substituted N-linked amino acid, and 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 C1-24 alkyl and optionally substituted aryl, optionally substituted -O-C1-24 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 1-24independently selected from alkyl, and optionally substituted aryl; R 16 and R 17 are -CN, optionally substituted C 2-8 Organyl carbonyl, C 2-8 Alkoxycarbonyl and C 2-8 is 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 cycloalkenyl; 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.

[0014] In another embodiment, the prodrug is a nucleoside / mononucleotide compound of formula II: [ka] 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 1-6Alkoxy, [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.

[0015] In yet another embodiment, the prodrug is a compound of formula III: [ka] 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; lipids; phospholipids; amino acids; carbohydrates; peptides and cholesterol.

[0016] In one embodiment, the base in each of formulas I, II, and III refers to guanine.

[0017] In one embodiment, the base in each of formulas I, II, and III refers to guanine with an amine protecting group.Examples of amino protecting groups include, but are not limited to, carbamate protecting groups such as 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide protecting groups such as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamide protecting groups such as 2-nitrobenzenesulfonyl; and imine and cyclic imide protecting groups such as phthalimide and dithiasuccinoyl.

[0018] In yet another aspect, the present invention further generally relates to a method of treating a disease or disorder characterized by an imbalanced nucleotide pool 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.

[0019] Suitable diseases or disorders include, but are not limited to, MPV17 deficiency and dGK mutation.

[0020] Administration can be via any route, including, but not limited to, oral, gastric feeding tube, intrathecal, parental, mucosal, and transdermal.

[0021] The dosage of at least one prodrug or a composition comprising same can be from about 15 mg / kg / day to about 500 mg / kg / day. [Brief description of the drawings]

[0022] [Figure 1A-1C]The levels of nucleosides in MPV17 knockout mice and normal controls in the liver, kidney, and brain, respectively, are compared. Figure 1A shows a marked depletion of dTTP and dGTP in liver mitochondria. The results further show that liver mitochondria (Figure 1A) but not kidney mitochondria (Figure 1B) or brain mitochondria (Figure 1C) of MPV17- / - mice reveal a deficiency of dGTP and dTTP deoxynucleotides. [Diagram 2] We present the results of a previous study using nucleoside supplementation in MPV17-deficient human fibroblasts. Figure 2 shows various combinations of nucleosides added to the cell culture broth. The abbreviation GdR corresponds to dG, TdR corresponds to dT, and A / CdR corresponds to a mixture of dA and dC. [Diagram 3] The synthesis scheme for 3'Val-dG (MT101G) is shown and further described in the Examples below. [Figure 4] The synthetic scheme for 3'Val-dA (MT101A) is shown and further described in the Examples below. [Diagram 5] The synthesis scheme for 3'Val,5'isobutryl dG (MT104G) is shown and further described in the Examples below. [Figure 6A] The starting material, yield, and structure of 3',5' diisobutyryl dG (MT104G) are shown. [Figure 6B] The starting material, yield, and structure of 3',5' diisobutyryl dA (MT104A) are shown, both of which are further described in the Examples below. [Figure 7] 1 shows a bar graph of the results of qPCR measurement of mtDNA copy number in liver MPV17- / - mice treated and untreated. Graphs represent the mean ± SEM for each experimental group. All treatments represent the molar equivalent of 75 mg / kg dC or 75 mg / kg dG. [Figure 8A] Results of further studies on the effects of prodrug treatment of MPV17− / − mice are presented. [Figure 8B]Results of further studies on the effects of prodrug treatment of MPV17− / − mice are presented. [Figure 8C] The results of further studies on the effect of prodrug treatment of MPV17- / - mice are presented. Figure 8A, Figure 8B, and Figure 8C show that body weight increases similarly between experimental and untreated groups. Figure 8A shows a combination of knockout (KO) littermates and dosing regimens. Figure 8B and Figure 8C show plots of pup weight gain in treated and untreated groups. [Figure 8D] Bar graphs showing the results of qPCR measurements of mtDNA copy numbers in the liver of MPV17- / - mice per treatment group comparing wild-type, untreated MPV17- / - mice, and treated MPV17 mice treated with MT101 and MT104 containing a combination of dG and dC. Graphs represent the mean ± SD for each experimental group. Values ​​for each experimental group were compared to those obtained in untreated MPV17- / - animals using the Kruskal-Wallis test followed by Dunn's nonparametric test. P values ​​are *p<0.05 and ***p>0.001. 20% and 50% represent the molar equivalents of 75-200 mg / kg dG, respectively. [Fig. 8E-8G] 8A shows a bar graph of several OXPHOS (oxidative phosphorylation) component protein levels comparing wild type, untreated MPV17- / - knockout mice, and treated MPV17- / - mice treated with MT101 and MT104 of the present invention, which contain a combination of dG and dC. OXPHOS components of complex I (Ndufb8), complex III (Core2), and complex V (Atp5a) were determined for each dosing regimen, as shown in FIG. 8D. [Figure 9] 9 shows a bar graph of the results of qPCR measurement of mtDNA copy number in liver MPV17- / - mice. Figure 9 compares wild type, untreated MPV17- / - mice, and MPV17- / - mice treated with single nucleoside prodrugs and underivatized dG and dC. Graphs represent the mean ± SEM of each experimental group. All treatments represent the molar equivalent of 75 mg / kg dC and / or 75 mg / kg dG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] 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.

[0024] As used herein, "patient" refers to a human subject.In some embodiments of the present invention, "patient" is known or suspected to have mitochondrial disease, mitochondrial DNA depletion syndrome, or dGK or MPV17 deficiency, which are diseases or disorders characterized by unbalanced nucleotide pools.

[0025] As used herein, a "therapeutically effective amount" refers to an amount sufficient to cause an improvement in a clinically significant condition in a subject, or to delay or minimize or alleviate one or more symptoms associated with a disease or disorder, or to effect a desired physiologically beneficial change in a subject.

[0026] As used herein, "treat," "treatment," and the like refer to a measure that slows, alleviates, improves, or alleviates at least one symptom of a disease or disorder, or reverses a disease or disorder after onset.

[0027] 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.

[0028] As used herein, "in need thereof" refers to a subject known to or suspected of having a mitochondrial disease, such as MPV17 deficiency or dGK deficiency, mitochondrial DNA depletion syndrome, which is a disease or disorder characterized by imbalanced nucleotide pools.

[0029] 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 a deoxynucleoside. Prodrugs are often, but not necessarily, pharmacologically inactive until converted to an active form. Prodrugs can be obtained by attaching a promoiety to a drug, usually via a functional group.

[0030] As used herein, a "promoiety" refers to a group, typically a functional group, attached to a deoxynucleoside via a bond that is cleavable under certain 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, pH, or the like. The agent may be endogenous to the conditions of use, such as an enzyme present in the systemic circulation of the patient to whom the prodrug is administered, or to the acidic conditions of the stomach, or the agent may be provided exogenously.

[0031] As used herein, a "side effect" is an undesirable reaction caused by administration of a drug. In most cases, administration of deoxynucleosides has not caused any side effects. The most expected side effect is mild gastrointestinal intolerance.

[0032] 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 required for a particular purpose, such as pharmaceutical formulation. For example, "about" can mean within 1 or more than 1 standard deviation, according to the 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, preferably within 5-fold, and more preferably within 2-fold, of a value. When a particular value is described in the application and claims, unless otherwise indicated, the term "about" should be assumed to mean within an acceptable error range of the particular value.

[0033] Whenever a group is described as "optionally substituted," the group may be unsubstituted or substituted with one or more of the indicated substituents. Similarly, when "unsubstituted or substituted" is described, if the group is substituted, the substituents may be selected from one or more of the indicated substituents. When no substituents are indicated, the indicated "optionally substituted" or "substituted" group can be any of the following: 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, mono-substituted amino groups, and di-substituted amino groups, and protected derivatives thereof.

[0034] The term "protecting group" or "protective derivative" as used herein refers to a labile chemical moiety known in the art to protect reactive groups, including but not limited to hydroxyl, amino, and thiol groups, from undesired reactions, typically during synthetic procedures. Protecting groups are typically used selectively and / or orthogonally to protect sites during reactions at other reactive sites, and can then be removed to leave unprotected groups intact or available for further reactions. Protecting groups known in the art are generally described in Greene's Protective Groups in Organic Synthesis, 4th Edition, John Wiley & Sons, New York, 2007.

[0035] Groups can be selectively incorporated into the compounds provided herein as precursors. For example, amino groups can be placed into the compounds provided herein as azide groups that can be chemically converted to amino groups at the desired point in the synthesis. Generally, groups are protected or exist as precursors that are inert to reactions that modify other regions of the parent molecule to convert to the final group at the appropriate time. Further representative protecting groups or precursor groups are described in Agrawal et al., Protocols for Oligonucleotide Conjugates, Humana Press; New Jersey, 1994, 26, 1-72. Alternatively, protecting groups can remain as components of the final product.

[0036] Examples of amino protecting groups include, but are not limited to, carbamate protecting groups such as 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide protecting groups such as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamide protecting groups such as 2-nitrobenzenesulfonyl; and imine and cyclic imide protecting groups such as phthalimide and dithiasuccinoyl.

[0037] Examples of hydroxyl protecting groups include, but are not limited to, acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, bis(2-acetoxyethoxy)methyl (ACE), 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, [(triisopropylsilyl)oxy]ethyl, and the like. [oxy]methyl (TOM), benzoyl formate, chloroacetyl, trichloroacetyl, trifluoro-acetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMT), trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX). Here, the more commonly used hydroxyl protecting groups include, but are not limited to, benzyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, benzoyl, mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX).

[0038] Examples of protecting groups commonly used to protect phosphate and phosphorus hydroxyl groups include methyl, ethyl, benzyl (Bn), phenyl, isopropyl, tert-butyl, allyl, cyclohexyl (cHex), 4-methoxybenzyl, 4-chlorobenzyl, 4-nitrobenzyl, 4-acyloxybenzyl, 2-methylphenyl, 2,6-dimethylphenyl, 2-chlorophenyl, diphenylmethyl, 4-methylthio-1-butyl, 2-(S-acetylthio)ethyl (SATE), 2-cyanoethyl, 2-cyano-1,1-dimethylethyl (CDM), 4-cyano-2-butenyl, 2-(trimethylthio)ethyl (TDE ... Trimethylsilyl)ethyl (TSE), 2-(phenylthio)ethyl, 2-(triphenylsilyl)ethyl, 2-(benzylsulfonyl)ethyl, 2,2,2-trichloroethyl, 2,2,2,2-tribromoethyl, 2,3-dibromopropyl, 2,2-trifluoroethyl, thiophenyl, 2-chloro-4-tritylphenyl, 2-bromophenyl, 2-[N-isopropyl-N-(4-methoxybenzoyl)amino]ethyl, 4-(N-trifluoroacetylamino)butyl, 4-oxopentyl, 4-tritylaminophenyl, 4-benzylaminophenyl, and morpholino. Here, the more commonly used phosphate and phosphorus protecting groups include, but are not limited to, methyl, ethyl, benzyl (Bn), phenyl, isopropyl, tert-butyl, 4-methoxybenzyl, 4-chlorobenzyl, 2-chlorophenyl, and 2-cyanoethyl.

[0039] As used herein, "Ca-Cb", 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 "C1-C4 alkyl" group refers to all alkyl groups having 1 to 4 carbons, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)-, and (CH3)3C-. When "a" and "b" are not specified for an alkyl, alkenyl, alkynyl, cycloalkylcycloalkenyl, aryl, heteroaryl, or heterocyclyl group, the broadest range set forth in these definitions is assumed.

[0040] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that contains a fully saturated (no double or triple bonds) hydrocarbon group. An alkyl group may have 1 to 20 carbon atoms (wherever 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 may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., and may contain up to 20 carbon atoms, although this definition also applies when the term "alkyl" appears without a numerical range specified. An alkyl group may also be a medium-sized alkyl having 1 to 10 carbon atoms. An alkyl group may also be a lower alkyl having 1 to 6 carbon atoms. The alkyl groups of the compounds may be any of the following: A alkyl group may be designated as "C1-C4 alkyl" or a similar designation. 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.

[0041] As used herein, "alkenyl" refers to an alkyl group that contains one or more double bonds in a straight or branched hydrocarbon chain. Examples of alkenyl groups include allenyl, vinylmethyl and ethenyl. Alkenyl groups can be unsubstituted or substituted.

[0042] As used herein, "alkynyl" refers to an alkyl group that contains one or more triple bonds in a straight or branched hydrocarbon chain. Examples of alkynyl include ethynyl and propynyl. Alkynyl groups can be unsubstituted or substituted.

[0043] As used herein, "cycloalkyl" refers to a monocyclic or polycyclic hydrocarbon ring system that is fully saturated (having no double or triple bonds). When composed of two or more rings, the rings may be fused together. Cycloalkyl groups can contain 3 to 10 atoms in the ring, or 3 to 8 atoms in the ring. Cycloalkyl groups can be unsubstituted or substituted. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0044] 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 more than one ring, the rings may be fused together and connected. Cycloalkenyls may contain 3 to 10 atoms in the ring, or 3 to 8 atoms in the ring. Cycloalkenyl groups may be unsubstituted or substituted.

[0045] 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) with a fully 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.

[0046] As used herein, "heteroaryl" refers to monocyclic, bicyclic, and tricyclic aromatic ring systems (ring systems having a fully delocalized pi-electron system) containing one or more heteroatoms (e.g., 1-5 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 a heteroaryl group can vary. For example, a heteroaryl group can contain 4-14 atoms in the ring, 5-10 atoms in the ring, or 5-6 atoms in the ring. Additionally, 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, benzoisoxazole, 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.

[0047] As used herein, "heterocyclyl" or "heteroalicyclyl" refers to monocyclic, bicyclic, and tricyclic ring systems in which 3, 4, 5, 6, 7, 8, 9, 10, and up to 18 members of carbon atoms, together with 1-5 heteroatoms, constitute the ring system. Heterocycles may optionally contain one or more unsaturated bonds arranged in such a way 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. Heterocycles may further contain one or more carbonyl or thiocarbonyl functional groups, and thus this definition is meant to include oxo and thio systems, such as lactams, lactones, cyclic imides, cyclic thioimides, and cyclic carbamates. When composed of two or more rings, the rings may be fused together and joined. Additionally, the nitrogen of a heteroalicyclic 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-oxathiine, 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, 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 analogues (e.g., benzimidazolidinone, tetrahydroquinoline, and 3,4-methylenedioxyphenyl).

[0048] As used herein, "aralkyl" and "aryl(alkyl)" refer to an aryl group connected as a substituent through 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).

[0049] 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 heteroaryl(alkyl) may 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.

[0050] As used herein, "alkoxy" refers to the 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.

[0051] As used herein, "acyl" refers to 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.

[0052] 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. Preferred purine bases are adenine and guanine, including amine and / or enol protected adenine and guanine bases. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil, and 5-alkylcytosine (e.g., 5-methylcytosine). Preferred pyrimidine bases are cytosine and thymidine, including amine or enol protected cytosine and thymidine bases. 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 -Ethanocytosin, N 6 ,N 6Heterocyclic 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 bases may be optionally substituted with amine or enol protecting groups.

[0053] As used herein, "-N-linked amino acid" refers to an amino acid that is linked to the indicated moiety through a main chain amino or monosubstituted amino group. When an amino acid is linked 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 linked through a nitrogen. An N-linked amino acid may be substituted or unsubstituted.

[0054] 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)-. N-linked amino acid ester derivatives can be substituted or unsubstituted.

[0055] As used herein, "-O-linked amino acid" refers to an amino acid that is attached to the indicated moiety through a hydroxy 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 hydroxy from its backbone carboxylic acid group is not present, and the amino acid is attached through the oxygen. O-linked amino acids may be substituted or unsubstituted.

[0056] As used herein, the term "amino acid" refers to any amino acid (both standard and non-standard amino acids), 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.

[0057] The term "amino acid" includes natural and synthetic β, γ or δ amino acids, which may be in the D or L configuration. The amino acid may 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.

[0058] 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.

[0059] As used herein, the terms "deoxynucleoside prodrug" or "prodrug deoxynucleoside" refer to prodrug derivatives of deoxynucleosides and include prodrugs which have a phosphate group or a derivative thereof incorporated into their structure.

[0060] As used herein, the term "dNTP" refers to deoxyribonucleotide triphosphate. Each dNTP is composed 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 the purines, while dTTP (deoxythymidine 5'-triphosphate) and dCTP (deoxycytidine 5'-triphosphate) constitute the pyrimidines. The purine-characteristic bases adenine and guanine both have a double ring structure, while the purine-characteristic bases thymine and cytosine both have a single ring structure.

[0061] The term "mitochondrial DNA depletion syndrome" as used herein refers to a class of phenotypically diverse 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 may result from an imbalance in the mitochondrial nucleotide pool available for mtDNA replication, and from abnormalities in mitochondrial replication. Based on the age of onset, two subtypes are distinguished: congenital (or early onset) and infantile (or late onset). Although the later onset type results in a longer survival time, the syndrome is fatal in almost all patients, and no effective treatment currently exists.

[0062] II. Prodrugs The present invention provides deoxynucleoside prodrugs for use in the treatment of MDS. "Deoxynucleoside" refers to 2'-deoxynucleosides, such as 2'-deoxyguanosine (dG or deoxyguanosine as shown below), 2'-deoxythymidine (dT or thymidine), 2'-deoxyadenosine (dA or deoxyadenosine), and 2'-deoxycytidine (dC or deoxycytidine). Each full-length name and common abbreviation can be used interchangeably. [ka]

[0063] In certain embodiments, the bases are selected from cytosine, thymine, guanine, and adenine.

[0064] Thus, the prodrugs described herein are deoxycytidine prodrugs (dC prodrugs), deoxythymidine prodrugs (dT prodrugs), deoxyguanosine prodrugs (dG prodrugs), and deoxyadenosine prodrugs (dA prodrugs).

[0065] The prodrugs are preferably in the naturally occurring β-D-configuration of the base and deoxyribose moieties, and the attachment points of the pyrimidine and purine rings to Formula I are preferably the naturally occurring 1-position for pyrimidine bases and 9-position for purine bases, according to standard numbering conventions.

[0066] 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.

[0067] In one embodiment, the present invention provides a prodrug of formula I: [ka] 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 Each of X, Y, and Z is 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 3-6 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 Cycloalkenyl, NR 20 R 21 , an optionally substituted N-linked amino acid, and 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 13are each independently selected from hydrogen, optionally substituted C1-24 alkyl and optionally substituted aryl, optionally substituted -O-C1-24 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 R 17 are -CN, optionally substituted C 2-8 Organyl carbonyl, C 2-8 Alkoxycarbonyl and C 2-8 is 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 cycloalkenyl; 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.

[0068] In certain embodiments, the prodrug is a compound of formula Ia: [ka] 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 C 3-6 Cycloalkyl, optionally substituted C 3-6 Cycloalkenyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted aryl (C 1-6 ) alkyl.

[0069] In certain embodiments, R 1 is aryl, and R 2 is C 1-24 alkyl, and R 3 is C 1-24 It is an alkyl.

[0070] In another particular embodiment, R 2 is an amino acid side chain.

[0071] In more particular embodiments, the prodrug is selected from one of the following compounds: [ka]

[0072] In another embodiment, the present invention provides a prodrug of formula II: [ka] 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 R22 -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.

[0073] In certain embodiments, the prodrug is a compound of formula IIa: [ka] In the formula, R is C 1-4 It is an alkyl.

[0074] In yet another particular embodiment, the prodrug is selected from one of the following compounds: [ka]

[0075] In yet another particular embodiment, the prodrug is selected from one of the following compounds: [ka]

[0076] In yet another embodiment, the present invention provides a prodrug of formula III: [ka] 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; 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.

[0077] Suitable bases may be unprotected or may contain protected amino groups. Examples of protecting groups that can be added to form protected amino groups include, but are not limited to, carbamate protecting groups such as 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide protecting groups such as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamide protecting groups such as 2-nitrobenzenesulfonyl; and imine and cyclic imide protecting groups such as phthalimide and dithiasuccinoyl.

[0078] In one embodiment, R 2 is an amino acid, i.e., the compound of formula III is a 3'-amino acid ester, e.g., the compound of formula IIIa: [ka] where R is the side chain of an amino acid.

[0079] In certain embodiments, the amino acid is valine (i.e., R is CH(CH3)2).

[0080] In a more particular embodiment, the prodrug is a compound of formula IIIb: [ka] In the formula, base refers to an optionally substituted heterocyclic base or an optionally substituted heterocyclic base having a protected amino group.

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

[0082] In more particular embodiments, 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] 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.

[0084] 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).

[0085] In more particular embodiments, the prodrug is selected from one of the following compounds: [ka]

[0086] In another embodiment, the present invention provides a method for the preparation of 1 and R 2 are both substituted acyl groups, and the acyl substitutions can be C1-C6 alkyl groups.

[0087] In certain embodiments, R 1 and R 2 are both isopropyl, and the prodrug is selected from one of the following compounds: [ka]

[0088] 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.

[0089] In one embodiment, the prodrugs described above may be utilized in the present methods.

[0090] A prodrug can be administered by itself (i.e., alone) or in the form of a pharmaceutical composition. A pharmaceutical composition containing one or more prodrugs for administration can include 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 acceptable and do not normally produce allergic reactions or similar adverse reactions, such as acute gastric peristalsis, dizziness, etc., when administered to humans, and that are approved by federal or state government regulatory agencies or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, more specifically in humans. A "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as saline, and oils, including oils of petroleum, animal, vegetable, or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil. When the pharmaceutical composition is administered intravenously, saline is the 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 also contain small amounts of wetting or emulsifying agents, or pH buffering agents, if necessary.

[0091] In certain embodiments, the disclosed methods can be used to treat mitochondrial DNA (mtDNA) depletion syndrome (MDS). Each nucleated cell contains hundreds of mitochondria. Mitochondria are unique organelles that are under two strands of genome control. Although mitochondria contain their own DNA, mtDNA, most of the mitochondrial proteins, including all proteins required for mtDNA replication, transcription, and repair, are encoded by nuclear genes.

[0092] Maintenance of mtDNA requires proteins essential for mtDNA synthesis, maintenance of mitochondrial nucleotide pools, and mediating mitochondrial fusion. Enzymes that synthesize mtDNA require a balanced supply of intramitochondrial nucleotides. These are supplied through the mitochondrial nucleotide salvage pathway and through import of nucleotides from the cytosol via specific transporters. The amounts of these enzymes must be properly balanced to function properly in mtDNA synthesis. Proteins known to be required for mtDNA synthesis are encoded by nuclear genes. When pathogenic variants disrupt the function of any one of the proteins encoded by these genes, mtDNA synthesis is impaired, resulting in quantitative defects in mtDNA (mtDNA depletion) or qualitative defects in mtDNA (multiple mtDNA deletions). To date, pathogenic variants in more than 20 nuclear genes are known to be associated with defects in mtDNA maintenance. One cause of mtDNA deletions is an imbalanced nucleotide pool that can lead to replication errors. Although DNA polymerases distinguish between ribonucleotides and deoxynucleotides, the system is not perfect and the polymerase can misincorporate ribonucleotides, especially when pool imbalance is present.

[0093] 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 of free nucleotide concentration leads to the impairment of mtDNA replication, and as a result, leads to the reduction of mtDNA copy number.

[0094] In certain embodiments, the method of the present invention is useful for treating diseases or disorders caused by imbalance of deoxyribonucleoside triphosphate (dNTP) pool.dTNP 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.

[0095] Accurate DNA synthesis requires adequate amounts of each dNTP, and properly balanced dNTP pools. The size of the total cellular pools ranges from 10 to 100 pmol / million cells of each dNTP 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 times smaller in both the cytosol and mitochondria. Contrary to what would be expected on the basis that the four nitrogenous bases in DNA are in roughly equimolar amounts, the four dNTPs are present in the pools in different ratios, with pyrimidines often exceeding purines. Individual cell lines may exhibit different pool compositions, even when derived from the same animal species. An increase in the concentration of one dNTP usually leads to the depletion of another dNTP.

[0096] The mtDNA depletion syndrome may be associated with a particular tissue or organ, such as muscle, liver, brain, and / or gastrointestinal tract. In certain embodiments, the mtDNA depletion syndrome is a myopathy syndrome or a hepatocerebral syndrome.

[0097] Disorders characterized by unbalanced nucleotide pools include, but are not limited to, nuclear genes that disrupt mitochondrial dNTP pools, such as MPV17 and deoxyguanosine kinase (dGK), defects parallel to TK2 deficiency due to autosomal recessive mutations in DGUOK resulting in dGMP and dAMP deficiency, and mtDNA depletion syndrome typically manifests as early childhood onset hepatocerebral disease (Mandel et al., 2001). Disorders associated with these genes can also be treated with the methods herein. For example, for patients with dGK deficiency, prodrugs of dG and / or dA would be logically administered. In certain embodiments of the method, either dG or a prodrug of dG alone can be used to treat MPV17 or dGK deficiency disorders. Optionally, dG or a prodrug of dG can be supplemented with smaller amounts of dA, dC, or dT, and salts and prodrugs thereof intended to balance the nucleoside pools are useful in treating patients with MPV17 or dGK deficiency disorders.

[0098] Molecular genetic testing using a panel of genes known to cause mtDNA depletion syndromes can be performed (Chanprasert et al., 2012), which may be useful in identifying patients with diseases or disorders characterized by an imbalanced nucleotide pool. This testing may include sequence analysis of the entire coding region and exon / intron junction regions of TK2 relevant to the diagnosis of MDS for sequence variants and deletions / duplications. If compound heterozygous or homozygous deleterious mutations are identified in the sequence analysis, the diagnosis of TK2 deficiency is established and therefore the subject is likely to benefit from deoxynucleoside therapy. If the 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.

[0099] Administration can be via any route, including, but not limited to, intrathecal, parental, mucosal, and transdermal.

[0100] 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, semi-solid or liquid polyols. Solutions and syrups may contain water, polyols, and sugars. The prodrugs described herein may be added to any form of liquid ingested by a patient, including, but not limited to, cow's milk, breast milk, both cow's and human, infant formula, and water. The prodrugs may be coated with or mixed with materials that delay disintegration and / or absorption in the digestive tract. Thus, sustained release may be achieved over many hours.

[0101] In another embodiment, administration is intrathecal. Intrathecal administration involves injecting a drug into the spinal canal, more specifically into the subarachnoid space to reach the cerebrospinal fluid. This method is commonly used for spinal anesthesia, chemotherapy, and analgesics. Intrathecal administration can be done by lumbar puncture (bolus injection) or by a port catheter system (bolus or infusion). The catheter is most commonly inserted between the lamina of the lumbar spine and the tip is threaded into the intrathecal cavity to the desired level (usually L3-L4). Water or saline are most commonly used as excipients for intrathecal formulations, but EDTA and lipids have been used as well.

[0102] In yet another embodiment, the 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 parental 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) condition 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 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, since some patients may be enterally fed by the time treatment is initiated, the prodrug may be administered via a gastronomy feeding tube or other enteral feeding means.

[0103] Pharmaceutical compositions adapted for nasal and pulmonary administration may contain a solid carrier, such as a powder, which can be administered by rapid inhalation through the nose. Compositions for nasal administration may contain a liquid carrier, such as a spray or droplets. Alternatively, direct inhalation into the lungs can be achieved by inhaling deeply or by introducing through a mouthpiece. These compositions may contain aqueous or oily solutions of the active ingredient. Compositions for inhalation may be delivered by specially adapted devices, including but not limited to pressurized aerosols, nebulizers or inhalers, which may be constructed to provide a predetermined dose of the active ingredient.

[0104] 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.

[0105] 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.

[0106] The dosage of at least one prodrug or a composition comprising the same may be from about 15 mg / kg / day to about 500 mg / kg / day. More preferred dosages are in the range of from about 50 mg / kg / day to about 400 mg / kg / day. More preferred dosages are in the range of from about 100 mg / kg / day to about 350 mg / kg / day, for example, from about 150 mg / kg / day to about 300 mg / kg / day, from about 200 mg / kg / day to about 350 mg / kg / day, or from about 250 mg / kg / day to about 350 mg / kg / day.

[0107] In one embodiment, the dose is about 20% equimolar to about 100% equimolar to a standard nucleoside (dT, dC, dG, dA), for example, about 20% to about 80%, about 20% to about 50%, about 50% to about 80%, or about 50% to about 100%. In a particular embodiment, the dose is about 20% equimolar to a standard nucleoside. In another particular embodiment, the dose is about 50% equimolar to a standard nucleoside. In yet another embodiment, the dose is about 100% equimolar to a standard nucleoside.

[0108] Administration of at least one prodrug or composition comprising same can be once a day, twice a day, three times a day, four times a day, five times a day, up to six times a day, preferably at regular intervals. When administered intravenously or intrathecally, the dose may be reduced. A preferred dose range for such administration is about 5 mg / kg / day to about 200 mg / kg / day.

[0109] In embodiments where the composition comprises two or more prodrugs, the ratio of the prodrugs can vary. For example, if a dG prodrug is administered with a second deoxynucleoside (dN), the ratio of dG / dN can be 95 / 5, 90 / 10, 85 / 15, 80 / 20, 75 / 25, 70 / 30, 65 / 35, 60 / 40, 55 / 45, or 50 / 50.

[0110] In one embodiment, the method further comprises monitoring the subject for improvement of the subject's condition before increasing dosage.The subject's response to therapeutic administration can be monitored by observing the changes in the subject's muscle strength and control, and mobility, as well as 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.

[0111] In another embodiment, the method further comprises monitoring the subject for side effects prior to reducing the dosage. Exemplary side effects include, but are not limited to, diarrhea, abdominal bloating, and other gastrointestinal symptoms.

[0112] The prodrug of the present invention can also be co-administered with other drugs. Such drugs include therapeutic drugs for treating the symptoms of certain forms of MDS. In particular, in the case of MPV17 or dGK deficiency, other drugs include ubiquitous inhibitors of nucleoside catabolism, including, but not limited to, enzyme inhibitors such as tetrahydrouridine (an inhibitor of cytidine deaminase) and imucillin H (an inhibitor of purine nucleoside phosphorylase) and tipiracil (an inhibitor of thymidine phosphorylase). Such inhibitors are known and are used in the treatment of some cancers. EXAMPLES

[0113] Example 1: Prodrug Test Identification Number

number

[0114] The synthesis of the prodrugs was accomplished using protection / deprotection strategies known in the art for nucleosides and nucleoside derivatives. The synthesis was performed using commercially available chemical grade nucleoside starting materials. Such starting materials are available from a number of suppliers, for example, Hongene Biotech Corporation, Shanghai, China. The general schemes for the synthesis of the compounds of Examples 1A-1D are shown in Figures 3, 4, 5, 6A, and 6B.

[0115] 1A. Synthesis of MT101-G Step 1: Starting with deoxyguanosine, the 5'-hydroxyl group of deoxyguanosine was selectively protected by adding a TBDPS (t-butyldiphenylsilyl) group under the reaction conditions of t-butyldiphenylsilyl chloride (TBDPSCl) / dimethylaminopyridine (DMAP) in dimethylformamide (DMF) at room temperature.

[0116] Step 2: Esterification of the 3' hydroxyl group was achieved using (carbobenzoxy)Cbz-L-valine and dicyclohexylcarbodiimide (DCC) in CH2Cl2 (DCM) to give the protected intermediate T545-3-2, which was purified by silica column chromatography in 49% overall yield.

[0117] Step 3: The TBDPS group was then removed using n-Bu4NF (TBAF) in tetrahydrofuran (THF) at room temperature to give T545-3-3 in 75% yield after column purification.

[0118] Step 4: Hydrogenation with hydrogen gas in solution with palladium on carbon catalyst in the presence of L-tartaric acid gave MT101-G as the L-tartrate salt. 1B. Synthesis of MT101-A

[0119] Step 1: Starting from deoxyadenosine, the 5′-hydroxyl group was selectively protected by 4,4′-dimethoxytrityl (DMTr) group in pyridine at 0-10° C. to give T545-4-1 in 60% yield after column purification.

[0120] Step 2: Esterification of the 3'-hydroxyl group was achieved with Cbz-L-valine and DCC in DCM at room temperature as in Example 1A to give T545-4-2, which was purified by silica column chromatography in an overall yield of 84%.

[0121] Step 3: Removal of the DMTr group was achieved with 80% aqueous AcOH at room temperature. The product was purified by silica column chromatography in 74% yield.

[0122] Step 4: H3PO4 / DCM conditions were used for de-Boc protection, followed by a charge of L-tartaric acid to give MT101-A in 76% yield via crystallization as the L-tartrate salt. 1C. Synthesis of MT105-G

[0123] 3′5′-Isobutyryl dG was obtained by direct treatment of dG with isobutyric anhydride in 66% yield after column purification. 1D. Synthesis of MT105-A

[0124] 3′5′-Isobutyryl dA was obtained by direct treatment of dA with isobutyric anhydride in 50% yield after column purification.

[0125] Example 2: Deoxynucleoside supplementation study in MPV17- / - mice 2A. Two litters from a breeding pair of MPV17- / - mice (KO x KO) were used in the study. The experimental groups were as follows: 1.- Untreated KO (n=1) 2. KO treated with -109.35mg / kg MT102C + 104.2mg / kg MT102G (n=3) 3. -KO treated with 163.87mg / kg MT103C + 150.56mg / kg MT103G (n=3) 4.-KO treated with 75mg / kg dC and 75mg / kg dG (purchased from Sigma) (n=3) 5.-KO treated with 144.9 mg / kg MT101G (n=2)

[0126] Treatment began on day 7 and ended on day 30. Treatment was administered by oral delivery throughout the study. No changes in health or mortality were observed in any of the experimental groups. On day 30, animals were sacrificed and tissues were harvested. Liver, muscle, kidney, intestine, heart, and brain were snap frozen in liquid nitrogen. Blood was collected by cardiac puncture and the plasma fraction was stored at -80°C.

[0127] The results of qPCR measurements of mtDNA copy number in liver tissue from treated and untreated mice are shown in FIG.

[0128] 2B. To compare results, WT (n=5), KO untreated (n=4), and KO treated with 20% MT101 (n=4) and 20% MT104 (n=4) from previous experiments were also included in the analysis.

[0129] 2C. In further studies of nucleoside prodrug supplementation, three other litters from the KO x KO breeding pair were used: 1.- Litter 2 (L2): Born 02 / 09 / 20 2.- Litter 3 (L3): Born 05 / 09 / 20 3.- Litter 4 (L4): Born 08 / 09 / 20

[0130] The experimental groups were set up as shown in FIG. 8A, which shows the doses as well as the replicates and sexes of the test animals from different litters as described above.

[0131] Treatment began on day 7 and ended on day 30. Treatment was administered by intra-oral delivery throughout the study. No discomfort was noted, rather, after several days the animals became accustomed to the technique.

[0132] The weight of each animal was monitored daily to adjust the dosage and detect possible adverse effects of the compounds. No changes in health or mortality were observed in any of the experimental groups, and weight gain in treated pups was similar to untreated, as shown in Figures 8B and 8C for the prodrugs MT101 and MT104, respectively.

[0133] On day 30, the animals were sacrificed and tissues were harvested. Liver, muscle, kidney, intestine, heart, and brain were flash frozen in liquid nitrogen. Blood was collected by cardiac puncture and the plasma fraction was stored at -80 °C.

[0134] Liver and muscle are analyzed showing significant mtDNA depletion on day 6. The effect on hepatic mtDNA copy number is shown in Figure 8D. The effect of nucleoside supplementation on mitochondrial OXPHOS proteins is shown in Figures 8E, 8F and 8G.

[0135] Using the same procedure as in Example 2A, experimental groups were set up to receive different combinations of supplementation as follows: 1.KO untreated (n=1) 2. KO treated with 20% MT101G (n=2) 3. KO treated with 20% MT101C (n=4) 4. KO treated with 20% MT105G (n=4) KO treated with 5.75mg / kg dG (Sigma, D7145) (n=4) KO treated with 6.75 mg / kg dC (Sigma, D3897) (n=2) 20% represents the molar equivalent of 75 mg / kg dC or 75 mg / kg dG.

[0136] mtDNA copy number in liver was analyzed by RT-qPCR. To compare the results, WT (n=5), KO untreated (n=4), and KO treated with 20% MT101G (n=2) from previous experiments were also included in the analysis.

[0137] Example 3: Fibroblasts from a dGK-deficient patient The prodrugs were tested in fibroblasts 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 is spontaneously depleted without the addition of DNA damaging agents. It has been previously demonstrated that supplementing the cell culture medium with dGuo (50 μM) is sufficient to prevent this depletion. Cells were grown until confluent. 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 in the same conditions until day 18, with regular addition of fresh medium. The copy number of mtDNA was evaluated at various time points (days 4, 9, and 18) throughout the experiment. 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.

[0138] 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.

[0139] The above merely illustrates the principles of the present invention. It will be understood that those skilled in the art can devise various configurations that embody the principles of the present invention and are included within its spirit and scope, although not expressly described or shown herein. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts that the inventors have contributed to promoting the art, and should be interpreted as not being limited to such specifically recited examples and conditions. Furthermore, all descriptions herein reciting the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Moreover, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Thus, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

**Claim 1**: A medicament for treating mitochondrial depletion diseases or disorders in a subject in need thereof, comprising a therapeutically effective amount of a prodrug, wherein the prodrug is at least one compound of formula I: 【Chemical 1】 wherein: the 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, 【Chemical 2】 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 each represent 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 ３－６ cycloalkenyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryl(C １－６ )alkyl, [Chemical Formula 3] selected independently from, or R ２ and R ３ can together form an annular portion, R ５ 、 R ６ and R ７ are each independently selected from 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 , optionally substituted N-linked amino acids, optionally substituted N-linked amino acid esters, R ８ 、R ９ 、R １１ and R １２ are each independently selected from hydrogen, optionally substituted C1-24 alkyl, and optionally substituted aryl, R １０ and R １３ are each independently selected from hydrogen, optionally substituted C1-24 alkyl, and optionally substituted aryl, optionally substituted -O-C1-24 alkyl, optionally substituted -O-aryl, optionally substituted -O-heteroaryl, and optionally substituted -O-monocyclic heterocyclyl, R １４ 、 R １５ and R １９ each independently selected from hydrogen, optionally substituted C １－２４ alkyl, and optionally substituted aryl, R １６ and R １７ are each independently selected from -CN, optionally substituted C ２－８ organoylcarbonyl, C ２－８ alkoxycarbonyl and C ２－８ organoylaminocarbonyl, R １８ is selected from 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, R ２０ and R ２１ are each independently selected from 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 n, m and p are each independently selected from 0, 1, 2 or 3. **Claim 2**: The medicament according to claim 1, wherein the mitochondrial depletion disease or disorder is selected from MPV17 and dGK deficiency; and / or the base refers to guanine or guanine having a protected amino group. **Claim 3** The medicament according to claim 1 or 2, wherein the prodrug is a compound of formula Ia: 【Chemical 4】 wherein: R １ 、 R ２ and R ３ are each independently selected from 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 ３－６ cycloalkenyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted aryl(C １－６ )alkyl. **Claim 4** R １ is aryl, R ２ is C １－２４ alkyl, and R ３ is C １－２４ alkyl, the medicament according to claim 3. **Claim 5**: The medicament according to claim 1 or 2, wherein at least one of the prodrugs is 【Chemical 12】 (wherein R is cytosine, thymine, guanine, or adenine). **Claim 6**: A medicament for treating a mitochondrial depletion disease or disorder selected from MPV17 and dGK deficiency in a subject in need thereof, comprising a therapeutically effective amount of a prodrug, wherein the prodrug is at least one compound of formula II: 【Chemical Formula 5】 wherein: the base refers to guanine or guanine having a protected amino group, X is selected from S and O, and R ２２ is -O － , -OH, -O-alkyl, optionally substituted C １－６ alkoxy, 【Chemical Formula 6】 is selected from an optionally substituted N-linked amino acid and an optionally substituted N-linked amino acid ester. R ８ 、R ９ 、R １０ 、R １１ 、R １２ 、R １３ 、R １４ 、R １５ 、n, m, and p are defined as described above. **Claim 7** The medicament according to claim 6, wherein the prodrug is a compound of formula IIa: 【Chemical Formula 7】 In the formula, R is C １－４ alkyl. **Claim 8** The medicament according to claim 1, wherein the prodrug is a compound of formula III: 【Chemical Formula 8】 wherein: the 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 monophosphate, diphosphate, or triphosphate of formula I, and modified phosphate); linear, branched or cyclic alkyl; acyl; CO-alkyl, CO-alkoxyalkyl; CO-aryloxyalkyl, CO-substituted aryl, sulfonic acid ester; alkylsulfonyl; arylsulfonyl; aralkylsulfonyl; lipid; phospholipid; amino acid; carbohydrate; peptide and cholesterol. **Claim 9** The medicament according to claim 8, wherein the prodrug is a compound of formula IIIa: 【Chemical Formula 9】 wherein R is the side chain of an amino acid. **Claim 10** The medicament according to claim 8, wherein the prodrug is a compound of formula IIIb: 【Chemical 10】 wherein: the base refers to an optionally substituted heterocyclic base or an optionally substituted heterocyclic base having a protected amino group. **Claim 11** The pharmaceutical according to claim 1, 6 or 8, wherein the at least one prodrug is selected from the following deoxyguanosine: 【Chemical 11】

12. The pharmaceutical according to claim 11, wherein a second prodrug is administered, and the base of the second prodrug is cytosine.

13. The second prodrug is 【Chemical 13】 (wherein R is cytosine, thymine, guanine, or adenine). The pharmaceutical according to claim 12.

14. The weight ratio of the deoxyguanosine prodrug to the second prodrug is 95 / 5, 90 / 10, 85 / 15, 80 / 20, 75 / 25, 70 / 30, 65 / 35, 60 / 40, 55 / 45, or 50 / 50. The pharmaceutical according to claim 12.

15. The pharmaceutical according to claim 1 or 6, wherein the prodrug is administered in the form of a pharmaceutical composition.

16. The pharmaceutical according to claim 1 or 6, wherein the administration method is oral.

17. The pharmaceutical according to claim 1 or 6, wherein the administered dose is about 200 mg / kg / day to about 1,000 mg / kg / day.

18. The pharmaceutical according to claim 1 or 6, wherein the prodrug or a composition containing the same is administered at least once a day.

19. The pharmaceutical according to claim 1 or 6, wherein the base refers to guanine.

20. The pharmaceutical according to claim 1 or 6, wherein the base refers to guanine having a protected amino group.

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