Nucleoside LINE-1 inhibitors

Abstract

The present disclosure provides a method of treating or preventing a disease, disorder, or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a LINE-1 inhibitor.

Description

[Technical field]

[0001] Field The present disclosure provides LINE-1 inhibitors and methods of treating or preventing a disease, disorder, or condition caused by LINE-1 retrotransposition in a subject by administering to the subject a LINE-1 inhibitor or a pharmaceutical composition thereof or a tautomer thereof. [Background technology]

[0002] background Long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons form the only autonomously active family of transposable elements in humans. They are expressed and mobile in the germline, in embryonic stem cells, and in early embryos, but are silenced in most somatic tissues. LINE-1 plays an important role in individual genomic mutations through insertion mutations and sequence transfer, which can lead to inherited diseases and disorders. For example, many neurological disorders have increased activity of retrotransposable sequences. See, for example, Terry and Devine, Front. Genet., 08 January 2020, https: / / doi.org / 10.3389 / fgene.2019.01244 (Non-Patent Document 1) and WO 2020 / 154656 (Patent Document 1).

[0003] LINE-1 sequences encode two proteins, ORF1p and ORF2p, that are essential for their mobility. ORF1p is an RNA-binding protein with nucleic acid chaperone activity. ORF2p has endonuclease and reverse transcriptase activities. These proteins and LINE-1 RNA constitute the core of the retrotransposition machinery, the ribonucleoprotein particle (LINE-1 RNP). LINE-1 RNP mediates the synthesis of new LINE-1 copies upon cleavage of target DNA at the target site and reverse transcription of LINE-1 RNA. LINE-1 sequences benefit from cellular host factors to complete their life cycle, however, several cellular pathways also limit the intracellular accumulation of LINE-1 RNPs and their deleterious activities. See, for example, Pizarro and Cristofari (2016) Front. Cell Dev. Biol. 4:14. doi: 10.3389 / fcell.2016.00014. There is a need in the art for LINE-1 inhibitors that treat or prevent diseases, disorders, or conditions caused by LINE-1 retrotransposition in a subject. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO 2020 / 154656 [Non-patent literature]

[0005] [Non-Patent Document 1] Terry and Devine, Front. Genet., 08 January 2020, https: / / doi.org / 10.3389 / fgene.2019.01244 [Non-Patent Document 2] Pizarro and Cristofari (2016) Front. Cell Dev. Biol. 4:14. doi: 10.3389 / fcell.2016.00014 Summary of the Invention

[0006] overview In one aspect, the disclosure provides the compounds of Table 1, referenced below, and their pharma- ceutically acceptable salts or solvates, and / or their tautomers; the compounds of Table 2, referenced below, and their pharma- ceutically acceptable salts or solvates, and / or their tautomers; and the compounds of Table 3, referenced below, and their pharma- ceutically acceptable salts or solvates, and / or their tautomers. The compounds of Tables 1-3, and their pharma- ceutically acceptable salts or solvates, and / or their tautomers, are collectively referred to as "disclosed compounds" or individually referred to as "disclosed compounds."

[0007] In another aspect, the disclosure provides a method of treating or preventing a disease, disorder, or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a disclosed compound. Exemplary diseases, disorders, or conditions include, but are not limited to, neurodegenerative diseases, autoimmune diseases, and age-related diseases.

[0008] In another aspect, the present disclosure provides a method of treating or preventing a disease, disorder, or condition caused by a pathophysiological retrotransposon-associated process in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure.

[0009] In another aspect, the disclosure provides a method of treating or preventing a symptom of a disease, disorder, or condition caused by a pathophysiological retrotransposon-associated process in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a disclosed compound or a pharmaceutical composition thereof.

[0010] In another aspect, the disclosure provides a method of inhibiting a LINE-1 retrotransposition event, e.g., a somatic LINE-1 insertion, that causes a disease, disorder, or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a disclosed compound or a pharmaceutical composition thereof.

[0011] In another aspect, the disclosure provides a method of treating a disease, condition, or disorder in a subject in need thereof, the method comprising: (a) determining the presence or absence of overexpression of a biomarker, e.g., retrotransposon RNA, retrotransposon reverse transcriptase, or retrotransposon DNA, e.g., LINE-1 RNA, ORF1p, or ORF2p, in a biological sample obtained from the subject; and (b) administering a therapeutically effective amount of a compound of the disclosed compounds, or a pharmaceutical composition thereof, to the subject if overexpression of the biomarker is present in the biological sample.

[0012] In another aspect, the disclosure provides a method of identifying whether a subject having a disease, condition, or disorder is a candidate for treatment with a disclosed compound or pharmaceutical composition thereof, the method comprising: (a) determining the presence or absence of overexpression of a biomarker, e.g., retrotransposon RNA, retrotransposon reverse transcriptase, or retrotransposon DNA, e.g., LINE-1 RNA, ORF1p, or ORF2p, in a biological sample obtained from the subject; (b) identifying the subject as a candidate for treatment if overexpression of the biomarker is present; or (c) identifying the subject as not a candidate for treatment if overexpression of the biomarker is not present.

[0013] In another aspect, the disclosure provides a method of predicting a therapeutic outcome in a subject having a disease, condition, or disorder, the method comprising determining the presence or absence of overexpression of a biomarker, e.g., a retrotransposon RNA, a retrotransposon reverse transcriptase, or a retrotransposon DNA, e.g., LINE-1 RNA, ORF1p, or ORF2p, in a biological sample obtained from the subject, where (a) the presence of overexpression of the biomarker in the biological sample indicates that administration of the disclosed compound or pharmaceutical composition thereof to the subject is likely to cause a favorable therapeutic response; and (b) the absence of overexpression of the biomarker in the biological sample indicates that administration of the disclosed compound or pharmaceutical composition thereof to the subject is likely to cause an unfavorable therapeutic response.

[0014] In another aspect, the disclosure provides a method comprising administering a therapeutically effective amount of the disclosed compound or a pharmaceutical composition thereof to a subject in need thereof, where (a) the subject has a disease, disorder, or condition; and (b) the disease, disorder, or condition is characterized by having overexpression of a biomarker, e.g., a retrotransposon RNA, a retrotransposon reverse transcriptase, or a retrotransposon DNA, e.g., LINE-1 RNA, ORF1p, or ORF2p.

[0015] In another aspect, the disclosure provides a kit comprising a disclosed compound or a pharmaceutical composition thereof and instructions for administering the compound or composition to a subject having a disease, condition, or disorder caused by a pathophysiological retrotransposon-associated process.

[0016] In another aspect, the disclosure provides a compound of the disclosure or a pharmaceutical composition thereof for use in the treatment or prevention of a disease, disorder, or condition.

[0017] In another aspect, the disclosure provides a compound of the disclosure or a pharmaceutical composition thereof for use in the treatment or prevention of a disease, disorder, or condition caused by a pathophysiological retrotransposon-associated process.

[0018] In another aspect, the disclosure provides a compound of the disclosure or a pharmaceutical composition thereof for use in the treatment or prevention of a symptom of a disease, disorder, or condition caused by a pathophysiological retrotransposon-associated process.

[0019] In another aspect, the disclosure provides the use of a disclosed compound or a pharmaceutical composition thereof for the manufacture of a medicament for treating or preventing a disease, disorder, or condition.

[0020] In another aspect, the disclosure provides a use of a disclosed compound or a pharmaceutical composition thereof for the manufacture of a medicament for treating or preventing a disease, disorder, or condition caused by a pathophysiological retrotransposon-associated process.

[0021] In another aspect, the disclosure provides the use of a disclosed compound or a pharmaceutical composition thereof for the manufacture of a medicament for treating or preventing a symptom of a disease, disorder, or condition.

[0022] In another aspect, the disclosure provides a use of a disclosed compound or a pharmaceutical composition thereof for the manufacture of a medicament for treating or preventing a symptom of a disease, disorder, or condition caused by a pathophysiological retrotransposon-associated process. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description I. Disclosed Compounds In one embodiment, the disclosed compounds are those in Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0024] (Table 1) TIFF2025511024000001.tif155128TIFF2025511024000002.tif221122TIFF2025511024000003.tif225122 TIFF2025511024000004.tif221122TIFF2025511024000005.tif235122TIFF2025511024000006.tif130128

[0025] In another embodiment, the disclosed compounds are compounds of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0026] (Table 2) TIFF2025511024000007.tif57144TIFF2025511024000008.tif218144TIFF2025511024000009.tif223144TIFF2025 511024000010.tif232144TIFF2025511024000011.tif221144TIFF2025511024000012.tif237144TIFF20255110240 00013.tif226144TIFF2025511024000014.tif223144TIFF2025511024000015.tif209144TIFF2025511024000016.t if223144TIFF2025511024000017.tif214144TIFF2025511024000018.tif228144TIFF2025511024000019.tif234144 TIFF2025511024000020.tif221144TIFF2025511024000021.tif211144TIFF2025511024000022.tif228144TIFF202 5511024000023.tif217144TIFF2025511024000024.tif224144TIFF2025511024000025.tif217144TIFF20255110240 00026.tif224144TIFF2025511024000027.tif233144TIFF2025511024000028.tif213144TIFF2025511024000029.t if231144TIFF2025511024000030.tif233144TIFF2025511024000031.tif221144TIFF2025511024000032.tif101144

[0027] In one embodiment, the disclosed compounds are those in Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0028] (Table 3) TIFF2025511024000033.tif37141

[0029] Tables 1-3 provide chemical structures and associated chemical names generated by Chemdraw® Professional version 20.1.1.125. If there is any ambiguity between the chemical structures and the chemical names, the disclosed compounds are defined by their structures.

[0030] In another aspect, the disclosure provides a pharmaceutical composition comprising a disclosed compound, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof, and one or more pharma- ceutically acceptable excipients.

[0031] II. Treatment Methods and Uses The disclosed compounds inhibit LINE-1 retrotransposition and therefore may be used, for example, to treat diseases, disorders, or conditions in which LINE-1 retrotransposition plays a causative role.

[0032] The disclosed compounds or pharmaceutical compositions thereof may be administered to a subject in need thereof, e.g., a subject already suffering from a disease, condition, or disorder; a subject suspected of having a disease, condition, or disorder; or a subject at risk of acquiring a disease, condition, or disorder. When the disclosed compounds are administered to a subject at risk of acquiring a disease, condition, or disorder, the intention is to avoid the disease, condition, or disorder in the subject, e.g., by preventing or reducing the LINE-1 retrotransposition activity that causes the disease, condition, or disorder.

[0033] In one embodiment, the disclosure provides a method of treating or preventing a disease, disorder, or condition, and / or a symptom of a disease, disorder, or condition, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a disclosed compound or a pharmaceutical composition thereof.

[0034] In another aspect, the disclosure provides a method of treating a disease, disorder, or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a disclosed compound or a pharmaceutical composition thereof.

[0035] In another aspect, the disclosure provides a method of preventing a disease, disorder, or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a disclosed compound or a pharmaceutical composition thereof.

[0036] In another aspect, the disclosure provides a method of treating a symptom of a disease, disorder, or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a disclosed compound or a pharmaceutical composition thereof.

[0037] In another aspect, the disclosure provides a method of preventing a symptom of a disease, disorder, or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a disclosed compound or a pharmaceutical composition thereof.

[0038] In another aspect, the disclosure provides a compound of the disclosure or a pharmaceutical composition thereof for use in treating or preventing a disease, disorder, or condition in a subject, and / or treating or preventing a symptom of a disease, disorder, or condition.

[0039] In another aspect, the disclosure provides a compound of the disclosure or a pharmaceutical composition thereof for use in treating a disease, disorder, or condition in a subject.

[0040] In another aspect, the disclosure provides a compound of the disclosure or a pharmaceutical composition thereof for use in preventing a disease, disorder, or condition in a subject.

[0041] In another aspect, the disclosure provides a compound of the disclosure or a pharmaceutical composition thereof for use in treating a symptom of a disease, disorder, or condition in a subject.

[0042] In another aspect, the disclosure provides a compound of the disclosure or a pharmaceutical composition thereof for use in preventing a symptom of a disease, disorder, or condition in a subject.

[0043] In another aspect, the disclosure provides the use of a disclosed compound or a pharmaceutical composition thereof in the manufacture of a medicament for treating or preventing a disease, disorder, or condition in a subject and / or treating or preventing a symptom of the disease, disorder, or condition.

[0044] In another aspect, the disclosure provides the use of a disclosed compound or a pharmaceutical composition thereof in the manufacture of a medicament for treating a disease, disorder, or condition in a subject.

[0045] In another aspect, the disclosure provides the use of a disclosed compound or a pharmaceutical composition thereof in the manufacture of a medicament for preventing a disease, disorder, or condition in a subject.

[0046] In another aspect, the disclosure provides the use of a disclosed compound or a pharmaceutical composition thereof in the manufacture of a medicament for treating a symptom of a disease, disorder, or condition in a subject.

[0047] In another aspect, the disclosure provides the use of a disclosed compound or a pharmaceutical composition thereof in the manufacture of a medicament for preventing a symptom of a disease, disorder, or condition in a subject.

[0048] In another embodiment, the subject is (a) not infected with the HIV virus, (b) not suspected of being infected with the HIV virus, (c) not receiving treatment for the HIV virus, and / or (d) not receiving treatment to prevent the HIV virus.

[0049] In another aspect, the disclosure provides a method of inhibiting a LINE-1 retrotransposition event causing a disease, disorder, or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a disclosed compound.

[0050] III. Diseases, Disorders, and Conditions Without wishing to be bound by any particular theory, the disclosed compounds, because they inhibit LINE-1 retrotransposition, may be used to treat or prevent a disease, disorder, or condition in a subject.

[0051] In one embodiment, the disclosed compounds have a median inhibitory concentration (IC) of 1 μM or less in an in vitro HeLa cell-based dual luciferase assay, as described in Example 35, referenced below. 50 ) inhibits human LINE-1 retrotransposition activity. See also Jones et al., (2008) PLoS ONE 3(2): e1547. doi:10.1371 / journal.pone.0001547; Xie et al., (2011) Nucleic Acids Res. 39(3): e16. doi: 10.1093 / nar / gkq1076; Kopera et al., Methods Mol Biol 1400:139-156 (2016). In another embodiment, IC 50 In another embodiment, the IC 50 In another embodiment, the IC 50 In another embodiment, the IC 50 In another embodiment, the IC 50 In another embodiment, the IC 50 In another embodiment, the IC 50 is less than 0.005 μM.

[0052] In one embodiment, the disease, disorder, or condition, and / or its symptoms are caused by a pathophysiological retrotransposon-associated process, wherein the disease, disorder, or condition is not cancer and / or is not an infectious disease.

[0053] In another embodiment, the disease, disorder, or condition is a neurodegenerative disease. See, e.g., Dugger and Dickson, Cold Spring Harb Perspect Biol 2016;9:a028035. Exemplary neurodegenerative diseases include, but are not limited to, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), Huntington's disease, frontotemporal lobar degeneration (FTLD), mild cognitive impairment (MCI), corticobasal degeneration (CDB), progressive supranuclear palsy (PSP), Rett syndrome, peripheral degenerative disease, or Aicardi-Goutières syndrome (AGS). In another embodiment, the frontotemporal lobar degeneration is frontotemporal dementia. See, e.g., Mohandas and Rajmohan, Indian J Psychiatry 51(Suppl 1):S65-S69 (2009).

[0054] In another embodiment, symptoms of a neurodegenerative disease include, but are not limited to, memory loss, forgetfulness, apathy, anxiety, agitation, loss of inhibitions, or mood swings.

[0055] In another embodiment, the disease, disorder or condition is an autoimmune disease.See, for example, Wang et al., J Intern Med 278:369-395 (2016).Exemplary autoimmune diseases include, but are not limited to, lupus, rheumatoid arthritis (RA), Sjogren's syndrome, or multiple sclerosis (MS).

[0056] In another embodiment, symptoms of the autoimmune disease include, but are not limited to, fatigue, muscle pain, swelling and redness, low-grade fever, difficulty concentrating, numbness and tingling in the hands and feet, hair loss, or a skin rash.

[0057] In another embodiment, the disease, disorder, or condition is an age-related disease. See, for example, Franceschi et al., Front. Med. 5:61. doi: 10.3389 / fmed.2018.00061; De Cecco et al., Nature 5666:73-78 (2019); US2021 / 0106586. Exemplary age-related diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, atherosclerosis, osteoarthritis, osteoporosis, rheumatoid arthritis (RA), macular degeneration, peripheral degenerative disease, or skin aging. In one embodiment, the subject with the age-related disease is at least 40 years old. In one embodiment, the subject with the age-related disease is at least 45 years old. In one embodiment, the subject with the age-related disease is at least 50 years old. In one embodiment, the subject with age-related disease is at least 55 years old. In one embodiment, the subject with age-related disease is at least 60 years old. In one embodiment, the subject with age-related disease is at least 65 years old. In one embodiment, the subject with age-related disease is at least 70 years old. In one embodiment, the subject with age-related disease is at least 75 years old.

[0058] In another embodiment, the disease, disorder, or condition is autism spectrum disorder (ASD), cardiovascular dysfunction, hearing loss, hematopoietic stem cell function, pulmonary fibrosis, schizophrenia, or vision loss.

[0059] In another aspect, the disease, disorder, or condition is wound healing in a subject in need thereof.

[0060] In another aspect, the disease, disorder, or condition is tissue regeneration in a subject in need thereof.

[0061] In another embodiment, the disease, disorder, or condition is Alzheimer's disease.

[0062] In another embodiment, the symptoms of Alzheimer's disease are any one or more of memory loss, misplacing things, forgetting places and names of things, repeating questions, lack of flexibility, confusion, disorientation, obsessions, compulsions, delusions, aphasia, sleep disorders, mood swings, depression, anxiety, frustration, agitation, difficulty performing spatial tasks, lethargy, difficulty walking, weight loss, speech disorders, short-term memory loss, or long-term memory loss, and combinations thereof.

[0063] In another embodiment, Alzheimer's disease symptoms are determined using the Alzheimer's Disease Assessment Scale-cognitive (ADAS-cognitive), the Clinician Interview-Based Impression Scale (CIBIC-plus), or the Activities of Daily Living (ADL).

[0064] In another embodiment, the disease, disorder, or condition is Alzheimer's disease, and one or more optional therapeutic agents are administered to the subject.In another embodiment, the optional therapeutic agent is donepezil, galantamine, rivastigmine, memantine, bapineuzumab, ABBV-8E12, CTS-21166, verubecestat (MK-8931), lanabecestat (AZD3293), LY2886721, nicotinamide, MPT0G211, or adacanumab-avwa.

[0065] In another embodiment, the disease, disorder, or condition is amyotrophic lateral sclerosis.

[0066] In another embodiment, the disease, disorder or condition is amyotrophic lateral sclerosis, and one or more optional therapeutic agents are administered to the subject.In another embodiment, the optional therapeutic agent is edaravone, riluzole, raltegravir, curcumin, curcumin derivatives, chicoric acid, chicoric acid derivatives, 3,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid derivatives, aurintricarboxylic acid, aurintricarboxylic acid derivatives, caffeic acid phenethyl ester, caffeic acid phenethyl ester derivatives, tyrphostin, tyrphostin derivatives, quercetin, quercetin derivatives, S-1360, zintevir (AR-177), L-870812, and L-25 870810, MK-0518, BMS-538158, or GSK364735C.

[0067] In another aspect, the disease, disorder, or condition is ataxia-telangiectasia.

[0068] In another embodiment, the disease, disorder, or condition is age-related macular degeneration, systemic lupus erythematosus, IFN-related autoimmune disease, such as rheumatoid arthritis, psoriasis, vitiligo, hypothyroidism, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, myasthenia gravis, Addison's disease, celiac disease, polymyositis, or aggravated autoimmune hepatitis, Fanconi anemia, idiopathic pulmonary fibrosis, or cardiovascular disease.In another embodiment, the systemic disease is age-related macular degeneration.In another embodiment, the systemic disease is systemic lupus erythematosus.In another embodiment, the systemic disease is IFN-related autoimmune disease, such as psoriasis.In another embodiment, the systemic disease is Fanconi anemia.In another embodiment, the systemic disease is idiopathic pulmonary fibrosis.In another embodiment, the systemic disease is cardiovascular disease.

[0069] In another embodiment, the disease, disorder, or condition is Rett syndrome, AGS, ataxia-telangiectasia, ASD, schizophrenia, cocaine or methamphetamine abuse, FTLD, or ALS.See, e.g., Terry and Devine, Front. Genet., 08 January 2020, https: / / doi.org / 10.3389 / fgene.2019.01244.

[0070] In one embodiment, the disclosed compounds are administered as a single agent to a subject with a disease, disorder, or condition.

[0071] In another embodiment, the disclosed compound is administered to a subject having a disease, disorder, or condition in combination with one or more optional therapeutic agents.For optional therapeutic agents for treating neurodegenerative diseases, see, for example, Duraes et al., Pharmaceuticals 2018, 11, 44; doi:10.3390 / ph11020044.

[0072] In another embodiment, the disclosed compound is administered to a subject having a disease, disorder, or condition in combination with one optional therapeutic agent.In another embodiment, the disclosed compound is administered to a subject having a disease, disorder, or condition in combination with two optional therapeutic agents.In another embodiment, the disclosed compound is administered to a subject having a disease, disorder, or condition in combination with three optional therapeutic agents.

[0073] The disclosed compound and the optional therapeutic agent or agents may be administered in combination under one or more of the following conditions: at different cycles, for different durations, at different concentrations, by different routes of administration, etc.

[0074] In one embodiment, the disclosed compound and one or more optional therapeutic agents are administered to the subject in combination as part of a single pharmaceutical composition.

[0075] In another embodiment, the disclosed compound and one or more optional therapeutic agents are administered separately to the subject, for example, in combination as two or more separate pharmaceutical compositions.In this case, two separate pharmaceutical compositions are administered to the subject, one of which comprises the disclosed compound and the other of which comprises any therapeutic agent.The separate pharmaceutical compositions can be administered to the subject, for example, at different cycles, for different durations, or by the same or different administration routes, for example, the disclosed compound can be administered orally and any therapeutic agent can be administered intravenously.

[0076] In another embodiment, the disclosed compound is administered to the subject prior to administration of any one or more therapeutic agents, e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks prior to administration of any one or more therapeutic agents.

[0077] In another embodiment, the disclosed compound is administered to the subject after administration of any one or more therapeutic agents, e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks after administration of any one or more therapeutic agents.

[0078] In another embodiment, the disclosed compound and any one or more therapeutic agents are administered simultaneously.

[0079] In one embodiment, the disclosed compounds are administered to the subject according to a continuous dosing schedule.

[0080] In one embodiment, the disclosed compounds are administered to a subject according to an intermittent dosing schedule.

[0081] In one embodiment, the disclosed compounds are administered orally to a subject.

[0082] The therapeutic methods provided herein include administering the disclosed compounds to a subject having a disease, disorder, or condition in an amount effective to achieve its intended purpose. While individual needs vary, determining the optimal range of effective amounts of each component is within the skill of the art. Typically, the disclosed compounds are administered in amounts of about 0.01 mg / kg to about 500 mg / kg, about 0.05 mg / kg to about 100 mg / kg, about 0.05 mg / kg to about 50 mg / kg, or about 0.05 mg / kg to about 10 mg / kg. In one embodiment, the disclosed compounds are administered once a day. In another embodiment, the disclosed compounds are administered twice a day. In one embodiment, the disclosed compounds are administered three times a day. In one embodiment, the disclosed compounds are administered four times a day. These dosage amounts are exemplary, but there may be individual cases in which higher or lower dosage amounts are merited, and such are within the scope of the present disclosure. In practice, a physician will determine the actual dosage regimen which will be most suitable for an individual subject, and it may vary with the age, weight, and response of the particular subject.

[0083] A unit dose can contain from about 0.01 mg to about 1000 mg of a disclosed compound, such as from about 1 mg to about 500 mg, such as from about 1 mg to about 250 mg, such as from about 1 mg to about 100 mg. For example, a unit oral dose of a disclosed compound may be, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, 100 mg, 1 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, or 100 mg. The unit dose may be administered one or more times daily, for example, as one or more tablets or capsules. The unit dose may be administered to the subject by any suitable route, for example, orally, IV, inhalation or subcutaneously. In practice, a physician will determine the actual dosing regimen that will be most suitable for an individual subject, which may vary according to the age, weight and response of the particular subject.

[0084] In one embodiment, the disclosed compounds are administered to a subject in an amount of about 0.1 mg to about 100 mg once daily, twice daily, three times daily, or four times daily. In another embodiment, the disclosed compounds are administered to a subject in an amount of about 1 mg to about 50 mg per day.

[0085] In one embodiment, the disclosed compound is administered to the subject in a single dose.In another embodiment, the disclosed compound is administered to the subject in two separate doses.In another embodiment, the disclosed compound is administered to the subject in three separate doses.In another embodiment, the disclosed compound is administered to the subject in four separate doses.

[0086] The disclosed compounds can be administered to a subject in raw chemical form or as part of a pharmaceutical composition containing the disclosed compounds in combination with a suitable pharma- ceutically acceptable carrier. Such carriers can be selected from pharma- ceutically acceptable excipients, vehicles, and auxiliaries. The term "pharma- ceutically acceptable carrier", "pharma- ceutically acceptable vehicle", or "pharma- ceutically acceptable vehicle" includes any standard pharmaceutical carrier, solvent, surfactant, or vehicle. Suitable pharma- ceutically acceptable vehicles include aqueous and non-aqueous vehicles. Standard pharmaceutical carriers and their formulations are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 19th ed. 1995.

[0087] A pharmaceutical composition comprising a disclosed compound can contain about 0.01 to 99 percent by weight of the disclosed compound, e.g., about 0.25 to 75 percent by weight, e.g., about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% by weight of the disclosed compound.

[0088] The disclosed compounds or pharmaceutical compositions comprising the disclosed compounds may be administered to a subject by any suitable route, for example, oral, buccal, inhalation, sublingual, rectal, vaginal, intravesical, or intrathecal via lumbar puncture, transurethral, ​​nasal, percutaneous, i.e., transdermal, or parenteral (including intravenous, intramuscular, subcutaneous, intracoronary, intradermal, intramammary, intraperitoneal, intraarticular, intrathecal, retrobulbar, intrapulmonary injection and / or surgical implantation at a particular site) administration. Dosage forms vary depending on the route of administration. Dosage forms include, but are not limited to, tablets, dragees, delayed release troches, capsules, solutions, suspensions, oral / nasal sprays, transdermal patches, dissolving films, ointments, sustained or controlled release implants, mouth rinses and mouth washes, gels, hair rinses, hair gels, and shampoos, as well as suppositories, solutions suitable for administration by intravenous injection, and suspensions suitable for administration by subcutaneous injection, and powders suitable for reconstitution. Parenteral administration can be achieved by using needle and syringe or other techniques known in the art.In one embodiment, the disclosed compound is administered orally to the subject.In one embodiment, the disclosed compound is administered subcutaneously to the subject.In one embodiment, the disclosed compound is administered intravenously to the subject.

[0089] The disclosed compounds and pharmaceutical compositions comprising the disclosed compounds may be administered to any subject that may experience the beneficial effects of inhibiting LINE-1 retrotransposition activity. The term "subject" as used herein refers to any human or animal that may require or benefit from administration of the disclosed compounds. Foremost among such subjects are mammals, e.g., humans, although the methods and compositions provided herein are not intended to be so limited. Other subjects include veterinary animals, e.g., cows, sheep, pigs, horses, dogs, cats, etc. In one embodiment, the subject is a human. In one embodiment, the subject is an animal. In another embodiment, the subject is a human with a disease, condition, or disorder that responds to the inhibition of LINE-1.

[0090] The pharmaceutical preparations provided herein are prepared by conventional mixing, granulation, dragee making, dissolving or lyophilization process.Therefore, the pharmaceutical preparations for oral use can be obtained by combining active compound with solid excipient, optionally grinding the mixture obtained, and processing the mixture of granules to obtain tablets or dragee cores, after adding suitable auxiliaries as required or necessary.

[0091] Suitable excipients are in particular fillers such as sugars, for example lactose or sucrose, mannitol or sorbitol, cellulose preparations, and / or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and binders such as starch paste, for example with corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone. If desired, disintegrants such as carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate, can be added together with the above-mentioned starches. Auxiliaries can be suitable flow regulators and lubricants. Suitable auxiliaries include, for example, silica, talc, stearic acid or a salt thereof, such as magnesium stearate or calcium stearate, and / or polyethylene glycol. Dragee cores are, if desired, provided with suitable coatings that are resistant to gastric juices. For this purpose, gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or titanium dioxide, lacquer solutions and concentrated sugar solutions that may contain suitable organic solvents or solvent mixtures can be used.To produce coatings that are resistant to gastric juice, the solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate are used.For example, dyes or colorings can be added to tablets or dragee coatings for identification or to give different characteristics to combinations of active compound doses.

[0092] Other pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin and soft sealed capsules made of gelatin and plasticizers such as glycerol or sorbitol.Push-fit capsules can contain the active compound in granular form, which can be mixed with fillers such as lactose, binders such as starch, and / or lubricants such as talc or magnesium stearate, and optionally stabilizers.In soft capsules, the active compound is dissolved or suspended in a suitable liquid such as fatty oils or liquid paraffin in one embodiment.Furthermore, stabilizers can be added.

[0093] The possible pharmaceutical preparations that can be used rectally include, for example, suppositories, which are made up of a combination of one or more active compounds and a suppository base.Suitable suppository bases are, for example, natural or synthetic triglycerides or paraffin hydrocarbons.It is also possible to use gelatin rectal capsules, which are made up of a combination of active compounds and a base.Possible bases include, for example, liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.

[0094] Preparations suitable for parenteral administration include aqueous solutions of active compounds in water-soluble form, such as water-soluble salts and alkaline solutions.Furthermore, suspensions of the disclosed compounds can be administered to subjects.Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or polyethylene glycol 400.Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran.Optionally, suspensions can also contain stabilizers and other additives.

[0095] A therapeutically effective amount of the disclosed compounds, formulated according to standard pharmaceutical practice, is administered to a subject in need thereof. Whether such treatment is indicated will vary on a case-by-case basis and will depend on a medical evaluation (diagnosis) that takes into account the signs, symptoms, and / or dysfunctions present, the risk of developing the particular signs, symptoms, and / or dysfunctions, and other factors.

[0096] Pharmaceutical compositions include those in which the disclosed compounds are administered in an effective amount to achieve their intended purpose. The exact formulation, route of administration, and dosage are determined by the individual physician in consideration of the diagnosed condition or disease. The amount and interval of administration can be individually adjusted to provide a level of the disclosed compounds sufficient to maintain therapeutic effect.

[0097] The toxicity and therapeutic efficacy of the disclosed compounds can be determined by standard pharmaceutical methods in cell cultures or experimental animals, for example, to determine the maximum tolerated dose (MTD) of the compound, which is defined as the highest dose that does not cause toxicity in subjects.The dose ratio between the maximum tolerated dose and therapeutic effect is the therapeutic index.Dosage can vary within this range depending on the dosage form adopted and the route of administration used.Determining therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0098] The therapeutically effective amount of the disclosed compound required for use in therapy varies depending on the nature of the disease being treated, the length of time that activity is desired, and the age and condition of the subject, and is ultimately determined by the attending physician.For example, the amount and interval of administration can be individually adjusted to provide a plasma level of the disclosed compound that is sufficient to maintain the desired therapeutic effect.The desired dose can be conveniently administered in a single dose or as multiple doses administered at appropriate intervals, for example, as divided doses 1, 2, 3, 4 or more times per day.

[0099] IV. Kit In another aspect, the disclosure provides a kit comprising a disclosed compound or a pharmaceutical composition thereof and instructions for administering the compound or composition to a subject having a disease, disorder, or condition.

[0100] In another aspect, the disclosure provides kits containing the disclosed compounds or pharmaceutical compositions thereof packaged in a manner that facilitates their use to practice the disclosed methods.

[0101] In one embodiment, the kit comprises the disclosed compound or pharmaceutical composition thereof packaged in a container such as a sealed bottle or container, with a label attached to the container or included in the kit that describes the use of the compound or composition to carry out the disclosed method.In one embodiment, the compound or composition is packaged in a unit dosage form.The kit can comprise a single dose or multiple doses of the disclosed compound or pharmaceutical composition thereof.

[0102] In another embodiment, a kit comprises a disclosed compound or composition thereof and one or more optional therapeutic agents.

[0103] V. Biomarkers In another aspect, the disclosure provides a method of treating a subject having a disease, condition, or disorder, the method comprising the steps of: (a) determining the presence or absence of a biomarker in a biological sample obtained from the subject; and (b) administering a therapeutically effective amount of a disclosed compound to the subject if the biomarker is present in the biological sample.

[0104] The term "biomarker" as used herein refers to any biological compound, such as a gene, a protein, a fragment of a protein, a peptide, a polypeptide, a nucleic acid, or a chromosomal abnormality, such as a chromosomal translocation, that can be detected and / or quantified in a subject in vivo or in a biological sample obtained from a subject. A biomarker can be an entire intact molecule, or a part or fragment thereof. In one embodiment, the expression level of a biomarker is measured. The expression level of a biomarker can be measured, for example, by detecting the level of the protein or RNA, e.g., mRNA, of the biomarker. In some embodiments, a part or fragment of a biomarker can be detected or measured, for example, by an antibody or other specific binding agent. In some embodiments, a measurable aspect of a biomarker is associated with a given condition of a subject, such as the age of the subject. For biomarkers detected at the protein or RNA level, such measurable aspect can include, for example, the presence or absence or concentration of the biomarker, i.e., the expression level, in the subject or in a biological sample obtained from the subject. For biomarkers detected at the nucleic acid level, such measurable aspects may include, for example, the allelic form of the biomarker, or the type, rate, and / or degree of mutation of the biomarker, also referred to herein as mutation status.

[0105] For biomarkers detected based on protein or RNA expression levels, the expression levels measured between different phenotypic statuses may be considered different, for example, if the mean or median expression levels of the biomarker in different groups are calculated to be statistically significant. Common tests of statistical significance include t-tests, ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney, significance analysis of microarrays, odds ratios, among others. Biomarkers, alone or in combination, provide a measure of the relative likelihood that a subject belongs to one phenotypic status or another. Thus, they are useful, among other things, as markers of disease and as indicators that a particular treatment regimen is likely to result in a beneficial patient outcome. The term "overexpression" refers to an expression level of a biomarker in a subject having a disease, condition, or disorder that is higher than, for example, the mean or median expression level of the biomarker in normal disease-free subjects.

[0106] Biomarkers include, but are not limited to, retrotransposon RNA, retrotransposon reverse transcriptase, such as ORF1p, ORF2p, and / or retrotransposon DNA. In one embodiment, the measurable aspect of a biomarker is its expression status. In another embodiment, the measurable aspect of a biomarker is an increase in the level of a biomarker. In one embodiment, the measurable aspect of a biomarker is its mutation status.

[0107] In one embodiment, the biomarker is the expression level of LINE-1. Methods for determining the expression level of LINE-1 are described in US2020 / 0253888 and may include, for example, determining the level of ORF1p, determining the level of LINE-1 mRNA, determining the amount of LINE-1 in a cell sample of a subject, or determining the level of ORF2p, or a combination thereof.

[0108] In one embodiment, the biomarker is a retrotransposon RNA expression that is differentially present in a subject with one phenotypic status, such as a subject with an age-related disease or a neurodegenerative disease, compared to another phenotypic status, such as a normal disease-free subject, or a subject with a disease, disorder, or condition that does not have retrotransposon RNA overexpression.In one embodiment, the biomarker is retrotransposon RNA overexpression.

[0109] In one embodiment, the biomarker is LINE-1 RNA expression that is differentially present in subjects of one phenotypic status, e.g., subjects with an age-related disease or a neurodegenerative disease, when compared to another phenotypic status, e.g., normal disease-free subjects, or subjects with a disease, disorder, or condition without LINE-1 RNA overexpression. In one embodiment, the biomarker is LINE-1 RNA overexpression.

[0110] In another embodiment, the biomarker is a retrotransposon reverse transcriptase that is differentially present in subjects of one phenotypic status, e.g., subjects with an age-related disease or a neurodegenerative disease, when compared to another phenotypic status, e.g., normal disease-free subjects, or subjects with a disease, disorder, or condition that does not have overexpression of retrotransposon reverse transcriptase. In one embodiment, the biomarker is overexpression of retrotransposon reverse transcriptase.

[0111] In another embodiment, the biomarker is ORF1p expression that is differentially present in subjects with one phenotypic status, e.g., subjects with an age-related disease or a neurodegenerative disease, compared to another phenotypic status, e.g., normal disease-free subjects, or subjects with a disease, disorder, or condition that does not have ORF1p overexpression. In one embodiment, the biomarker is ORF1p overexpression.

[0112] In another embodiment, the biomarker is ORF2p expression that is differentially present in subjects with one phenotypic status, e.g., subjects with an age-related disease or a neurodegenerative disease, compared to another phenotypic status, e.g., normal disease-free subjects, or subjects with a disease, disorder, or condition that does not have ORF2p overexpression. In one embodiment, the biomarker is ORF2p overexpression.

[0113] Biomarker standards can be determined before, at the same time, or after obtaining biological samples from subjects.Biomarker standards for use with the methods described herein can include, for example, data from samples from subjects without neurodegenerative disease, data from samples from subjects with neurodegenerative disease.Comparisons can be made to establish predetermined threshold biomarker standards for different classes of subjects, for example, disease vs. disease-free subjects.Standards can be performed in the same assay, or can be known standards from previous assays.

[0114] In one embodiment, the biomarker is the retrotransposon DNA expression that is present differently in the subject of one phenotypic status, for example, the subject with age-related disease or neurodegenerative disease, when compared with another phenotypic status, for example, the normal disease-free subject, or the subject with disease, disorder, or condition that does not have retrotransposon DNA overexpression.In one embodiment, the biomarker is the overexpression of retrotransposon DNA.In another embodiment, the biomarker is the overexpression of retrotransposon nuclear DNA.In another embodiment, the biomarker is the overexpression of retrotransposon cytoplasmic DNA.

[0115] In one embodiment, the biomarker is LINE-1 DNA expression that is differentially present in subjects with one phenotypic status, e.g., subjects with age-related disease or neurodegenerative disease, when compared to another phenotypic status, e.g., normal disease-free subjects, or subjects with disease, disorder, or condition without LINE-1 DNA overexpression.In one embodiment, the biomarker is LINE-1 DNA overexpression.In another embodiment, the biomarker is LINE-1 nuclear DNA overexpression.In another embodiment, the biomarker is LINE-1 cytoplasmic DNA overexpression.

[0116] If the mean or median expression or mutation level of a biomarker is calculated to be different between groups, i.e., higher or lower, the biomarker is differentially present between different phenotypic status groups.Thus, the biomarker provides an indication that a subject, e.g., a subject with ALS, belongs to one phenotypic status or another.

[0117] The term "biomarker" as used herein is meant to include a group, set, or array of multiple biological compounds in addition to individual biological compounds, such as LINE-1 RNA. For example, the combination of overexpression of LINE-1 RNA and overexpression of ORF1p may constitute a biomarker, or the overexpression of LINE-1 RNA and LINE-1 DNA may constitute a biomarker. The term "biomarker" may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more biological compounds. In an embodiment, a biomarker includes 1, 2, or 3 biological compounds.

[0118] Determining the expression level or mutation status of a biomarker in a subject may be performed using any of many methods known in the art. Any method known in the art for quantifying a particular protein and / or detecting the expression of a biomarker, such as LINE-1 RNA expression, ORF1p expression, and / or ORF2p expression, or any other biomarker expression or mutation level, in a patient or biological sample may be used in the disclosed method. Examples include, but are not limited to, PCR (polymerase chain reaction), or RT-PCR, flow cytometry, Northern blot, Western blot, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), gene chip analysis of RNA expression, immunohistochemistry, or immunofluorescence. See, for example, Slagle et al. Cancer 83:1401 (1998). Certain embodiments of the disclosure include methods in which biomarker RNA expression (transcription) is determined. Another embodiment of the disclosure includes methods in which protein expression in a biological sample is determined. For example, see Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1988); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York 3rd Edition, (1995); Kamel and Al-Amodi, Genomics Proteomics Bioinformatics 15:220-235 (2017). For Northern blot or RT-PCR analysis, RNA is isolated from tissue samples using RNAse-free techniques. Such techniques are generally known in the art.

[0119] In one embodiment of the disclosure, a biological sample is obtained from a subject and the biological sample is assayed for determination of biomarker expression or mutation status.

[0120] In another form of disclosure, Northern blot analysis of biomarker transcription in tumor cell samples is performed. Northern analysis is a standard method for detecting and / or quantifying mRNA levels in a sample. First, RNA is isolated from the sample and assayed using Northern blot analysis. In the analysis, the RNA sample is first size-separated by electrophoresis in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane, cross-linked, and hybridized with a labeled probe. Typically, Northern hybridization involves the in vivo polymerization of radioactively or nonisotopically labeled DNA, or the generation of oligonucleotides as hybridization probes. Typically, the membrane holding the RNA sample is prehybridized or blocked before probe hybridization to prevent the probe from coating the membrane, thereby reducing nonspecific background signals. After hybridization, unhybridized probe is typically removed by washing with several changes of buffer. The stringency of the washing and hybridization conditions can be designed, selected, and performed by any person skilled in the art. Detection is achieved using detectably labeled probes and suitable detection methods.Radiolabeled and non-radiolabeled probes and their uses are well known in the art.The presence and / or relative level of expression of the assayed biomarker can be quantified, for example, by densitometry.

[0121] In another embodiment, biomarker expression and / or mutation status is determined using RT-PCR. RT-PCR can detect the progress of PCR amplification of target genes in real time. The design of primers and probes required to detect the expression and / or mutation status of the disclosed biomarkers is within the skill of the art. RT-PCR can be used to determine, for example, the level of RNA encoding the disclosed biomarkers in a tissue sample. In the disclosed embodiment, RNA from a biological sample is isolated under RNAse-free conditions and then converted to DNA by treatment with reverse transcriptase. Methods for reverse transcriptase conversion of RNA to DNA are well known in the art. PCR is described in the following references: Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263 (1986); EP 50,424; EP 84,796; EP 258,017; EP 237,362; EP 201,184; US Patent Nos. 4,683,202; 4,582,788; 4,683,194.

[0122] RT-PCR probes rely on the 5'-3' nuclease activity of the DNA polymerase used in PCR to hydrolyze oligonucleotides that hybridize to the target amplicon (biomarker gene). RT-PCR probes are oligonucleotides with a fluorescent reporter dye attached to the 5' end and a quencher moiety attached to the 3' end (or vice versa). These probes are designed to hybridize to an internal region of the PCR product. In the unhybridized state, the proximity of the fluorescent and quenching molecules prevents detection of the fluorescent signal from the probe. During PCR amplification, when the polymerase replicates the template to which the RT-PCR probe is bound, the 5'-3' nuclease activity of the polymerase cleaves the probe. This separates the fluorescent and quenching dyes and FRET no longer occurs. Thus, fluorescence increases with each cycle in a manner proportional to the amount of probe cleavage. The fluorescent signal emitted from the reaction can be measured or tracked over time using conventional and conventional techniques and commercially available instruments.

[0123] In another embodiment of the disclosure, the expression of the protein encoded by the biomarker is detected by Western blot analysis. Western blot (also known as immunoblot) is a method for protein detection in a given sample of tissue homogenate or extract. Gel electrophoresis is used to separate denatured proteins by mass. The proteins are then transferred from the gel to a membrane (e.g., nitrocellulose or polyvinylidene difluoride (PVDF)) where they are detected using a primary antibody that specifically binds to the protein. The bound antibody can then be detected by a secondary antibody conjugated with a detectable label (e.g., biotin, horseradish peroxidase, or alkaline phosphatase). Detection of the secondary label signal indicates the presence of the protein.

[0124] In another embodiment of the disclosure, expression of the protein encoded by the biomarker is detected by enzyme-linked immunosorbent assay (ELISA). In one embodiment of the disclosure, a "sandwich ELISA" includes the steps of coating a plate with a capture antibody; adding a sample where any antigen present binds to the capture antibody; adding a detection antibody that also binds to the antigen; adding an enzyme-linked secondary antibody that binds to the detection antibody; and adding a substrate that is converted by the enzyme on the secondary antibody into a detectable form. Detection of a signal from the secondary antibody indicates the presence of the biomarker antigen protein.

[0125] In another embodiment of the disclosure, expression of proteins encoded by the biomarkers, e.g., ORF1p, ORF2p, is detected by a single molecule array assay (Simoa™).

[0126] In another embodiment of the disclosure, the expression of the protein encoded by the biomarker, e.g., ORF1p, ORF2p, is detected by droplet digital ELISA (ddELISA). Using ddELISA, LINE-1 / ORF1p protein can be measured in serum. See Cohen et al., ACS Nano 14:9491-9501 (2020).

[0127] VI.Definitions The term "pathophysiological retrotransposon-associated process" as used herein refers to a deregulated physiological process associated with abnormal retrotransposition activity of at least one retrotransposon. Exemplary retrotransposons include, but are not limited to, LINE-1 and human endogenous retroviruses (HERVs), such as HERV-K and HERV-E. See, for example, Saleh et al, (2019) Front. Neurol. 10:894. doi: 10.3389 / fneur.2019.00894. Diseases, disorders, or conditions caused by pathophysiological retrotransposon-associated processes include, but are not limited to, neurodegenerative diseases, autoimmune diseases, age-related diseases, autism spectrum disorders (ASD), cardiovascular dysfunction, hearing loss, hematopoietic stem cell function, pulmonary fibrosis, schizophrenia, or vision loss.

[0128] As used herein, the term "pathophysiological LINE-1-associated process" refers to a deregulated physiological process associated with abnormal LINE-1 (L1) retrotransposition activity. See, e.g., Saleh et al, (2019) Front. Neurol. 10:894. doi: 10.3389 / fneur.2019.00894; Zhao et al., PLoS Genet 15(4): e1008043.<https: / / doi.org / 10.1371 / journal.pgen.1008043;> ” See Bundo et al., Neuron 81:306-313 (2014).

[0129] As used herein, the term "LINE-1 retrotransposition event causing a disease, disorder, or condition" refers to any causative factor associated with LINE-1 retrotransposition that results in or promotes a pathological condition, e.g., a disease or disorder, in a subject, e.g., transcriptional abnormalities, alternative splicing, insertional mutagenesis, DNA damage, chromosomal translocations, increased expression of LINE-1 RNA, ORF1p (a 40 kDa RNA-binding protein), ORF2p (a protein of approximately 150 kDa with endonuclease (EN) and reverse transcriptase (RT) activities). See, e.g., Beck et al., Annu Rev Genomics Hum Genet 12:187-215 (2011); Pizarro and Cristofari (2016) Front. Cell Dev. Biol. 4:14, "<https: / / doi.org / 10.3389 / fcell.2016.00014> " In one embodiment, the LINE-1 retrotransposition event is a somatic LINE-1 insertion. In another embodiment, the LINE-1 retrotransposition event is an increase in LINE-1 RNA expression in the subject.

[0130] The term "tautomer" as used herein refers to each of two or more isomers of a compound that exist together in equilibrium and are replaced by the movement of an atom, e.g., hydrogen, or group, in a molecule. Certain compounds of the disclosure may exist as tautomers. In situations where tautomers are possible, the present disclosure includes all tautomeric forms. For example, as shown in Chart 1, both lactim and lactam tautomers are included, and both amino and imino tautomers are included. Chart 1 TIFF2025511024000034.tif78128

[0131] Similarly, as shown in Chart 2, both lactim and lactam tautomers are included, and both amino and imino tautomers are included. Chart 2 TIFF2025511024000035.tif65128The equilibrium arrows in Charts 1 and 2 are not intended to indicate the location of equilibrium, but only that equilibrium exists between the two tautomeric forms.

[0132] The term "biological sample" as used herein refers to any tissue or bodily fluid from a subject suitable for detecting a biomarker. Examples of useful biological samples include, but are not limited to, biopsied tissue and / or cells, such as lymph nodes, inflamed tissue, tissue and / or cells involved in a condition or disease, blood, plasma, serous fluid, cerebrospinal fluid, saliva, urine, lymphatic fluid, cerebrospinal fluid, and the like. Other suitable biological samples are familiar to those skilled in the relevant field. The biological sample may be analyzed for expression levels of biological compounds, such as LINE-1 RNA, ORF1p protein, ORF2p protein, using any technique known in the art. Such techniques include, but are not limited to, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), or cytoplasmic light chain immunofluorescence combined with fluorescence in situ hybridization (cIg-FISH). The biological sample may be obtained using techniques well within the ordinary knowledge of a clinician. In one embodiment of the disclosure, the biological sample includes a tissue or blood sample.

[0133] The terms "a," "an," "the," and similar referents in the context of describing the disclosure (especially in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise indicated. The recitation of ranges of values ​​herein is intended only to serve as a shorthand method of individually referring to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually set forth herein. Any examples provided herein, or the use of exemplary language, such as "such as," are intended only to better illustrate the disclosure and are not limitations on the scope of the disclosure unless otherwise indicated. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0134] As used herein, the term "about" includes ±10% of the indicated number. Thus, "about 10" means 9 to 11.

[0135] As used herein, the terms "treat," "treating," "treatment," and the like refer to eliminating, reducing, or alleviating a disease, disorder, or condition, and / or symptoms associated therewith. Although not exclusive, treating a disease, disorder, or condition does not require that the disease, disorder, or condition, and / or symptoms associated therewith be completely eliminated. However, in one embodiment, administration of a disclosed compound results in the complete elimination of the disease and associated symptoms.

[0136] As used herein, the terms "prevent", "preventing", "prevention" and the like refer to a method of preventing the onset of a disease, disorder, or condition and / or symptoms associated therewith, or preventing a subject from acquiring a disease, disorder, or condition. The terms "prevent", "preventing" and "prevention" also include delaying the onset of a disease, disorder, or condition and / or symptoms associated therewith, as well as reducing the risk of a subject acquiring a disease, disorder, or condition. The terms "prevent", "preventing" and "prevention" also include "prophylactic treatment", which refers to reducing the likelihood of a disease, disorder, or condition recurring or recurring a previously controlled disease, disorder, or condition in a subject who does not have the disease, disorder, or condition recurrence or recurrence of a disease, disorder, or condition, but is at risk or susceptible to it. The terms "prevent", "preventing" and "prevention" also include delaying or reversing the progression of the underlying pathology of a disease, disorder, or condition, e.g., a mutation caused by a somatic LINE-1 insertion.

[0137] The term "therapeutically effective amount" as used herein refers to an amount of the disclosed compound and, optionally, one or more optional therapeutic agents sufficient to cause an improvement in one or more symptoms of a disease, disorder, or condition, or to prevent the progression of a disease, disorder, or condition, or to cause the regression of a disease, disorder, or condition. For example, a therapeutically effective amount refers to an amount of the disclosed compound that causes a therapeutic response, e.g., slows the progression of a disease, disorder, or condition in a subject by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% or more.

[0138] The term "container" refers to any container or packaging suitable for storing, transporting, distributing, and / or handling the disclosed compounds. Non-limiting exemplary containers include vials, ampoules, bottles, and syringes.

[0139] The term "package insert" means the information that accompanies a pharmaceutical product that provides instructions on how to administer the product, along with safety and effectiveness data necessary for physicians, pharmacists, and subjects to make informed decisions regarding the use of the product. Package inserts are generally considered the "label" for a pharmaceutical product.

[0140] In some embodiments, two or more therapeutic agents may have synergistic effects when administered in combination. As used herein, the terms "synergy", "synergistic", "synergistically", and their derivatives, such as in "synergistic effect" or "synergistic combination" or "synergistic composition", refer to a situation in which the biological activity of a combination of a drug and at least one additional therapeutic agent is greater than the sum of the biological activities of each drug when administered individually. For example, as used herein, the term "synergistically effective" refers to an interaction between a disclosed compound and another therapeutic agent that makes the overall effect of the drug greater than the sum of the individual effects of each drug. Berenbaum, Pharmacological Reviews 41:93-141 (1989).

[0141] Synergy may be expressed as a "synergy index (SI)," which may be determined by the method described in F. C. Kull et al. Applied Microbiology 9, 538 (1961) from a ratio determined by: Q a Q A + Q b Q B = Synergy Index (SI) During the ceremony, Q A is the concentration of component A acting alone that produced an endpoint associated with component A; Q a is the concentration of component A in the mixture that gave rise to the endpoint; Q B is the concentration of component B acting alone that produced an endpoint related to component B; and Q b is the concentration of component B in the mixture that gave rise to the endpoint.

[0142] In general, Q a / Q A and Q b / Q BWhen the sum of is greater than 1, antagonism is indicated. When the sum is equal to 1, additivity is indicated. When the sum is less than 1, synergy is exhibited. The lower the SI, the greater the synergy exhibited by that particular mixture. Thus, a "synergistic combination" has higher activity than can be expected based on the observed activity of each component when used alone. Furthermore, a "synergistically effective amount" of a component refers to the amount of the component required to elicit a synergistic effect, for example, with another therapeutic agent present in the composition.

[0143] As used herein, the terms "intermittent dosing," "intermittent dosing schedule," and similar terms refer to the administration of a disclosed compound to a subject that is not continuous.

[0144] Intermittent dosing of the disclosed compounds can maintain the efficacy achieved with continuous dosing, but with fewer side effects, e.g., less weight loss. Intermittent dosing regimens useful in the present disclosure include any discontinuous dosing regimen that provides a therapeutically effective amount of the disclosed compounds to a subject in need thereof. Intermittent dosing regimens can use doses of the disclosed compounds that are equal to, lower than, or higher than those that would be used in continuous dosing regimens. The advantages of intermittent dosing of the disclosed compounds include, but are not limited to, improved safety, reduced toxicity, e.g., reduced weight loss, increased exposure, increased efficacy, and / or improved subject compliance. These advantages can be realized when the disclosed compounds are administered as a single agent or when administered in combination with any one or more therapeutic agents. On the days that the disclosed compounds are to be administered to a subject, administration can be in single or divided doses, e.g., once a day, twice a day, three times a day, four times a day, or more. Administration can also be via any suitable route, e.g., orally, intravenously, or subcutaneously. In one embodiment, the disclosed compounds are administered once (QD) or twice (BID) to the subject on the days that the compounds are scheduled to be administered.

[0145] In one embodiment, the disclosed compounds are administered according to a continuous dosing schedule. In another embodiment, the disclosed compounds are administered according to an intermittent dosing schedule.

[0146] The term "in combination" used in connection with the administration of a disclosed compound and any one or more therapeutic agents to a subject means that the disclosed compound and any one or more therapeutic agents can be administered to the subject together, e.g., as part of a single pharmaceutical composition or formulation, or separately, e.g., as part of two or more separate pharmaceutical compositions or formulations. Thus, the term "in combination" used in connection with the administration of a disclosed compound and any one or more therapeutic agents to a subject is intended to encompass the administration of the disclosed compound and any one or more therapeutic agents in a sequential manner, where the disclosed compound and any one or more therapeutic agents are administered to the subject at different times, or simultaneously, or substantially simultaneously, e.g., less than 30 minutes apart. Simultaneous administration can be achieved, for example, by administering to the subject a single capsule having a fixed ratio of the disclosed compound and any one or more therapeutic agents, or multiple single capsules each of the disclosed compound and any one or more therapeutic agents. The sequential or substantially simultaneous administration of the disclosed compound and any one or more therapeutic agents can be achieved by any suitable route, including, but not limited to, oral, intravenous, subcutaneous, intramuscular, etc. The disclosed compound and the one or more optional therapeutic agents can be administered by the same route or by different routes.For example, the one or more optional therapeutic agents of the combination and the disclosed compound can be administered orally.Alternatively, for example, the disclosed compound can be administered orally, and the one or more optional therapeutic agents can be administered by intravenous injection.The disclosed compound and the one or more optional therapeutic agents can also be administered alternately.In one embodiment, the disclosed compound and the one or more optional therapeutic agents are administered separately to the subject, for example, as part of two or more separate pharmaceutical compositions or preparations.

[0147] VII. Specific Embodiments The disclosure provides the following specific aspects:

[0148] Embodiment 1. A method of treating or preventing a disease, disorder, or condition caused by a pathophysiological retrotransposon-associated process in a subject in need thereof, and / or treating or preventing a symptom of said disease, disorder, or condition, comprising: (i) a compound of Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; (ii) a compound of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; or (iii) A compound of Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof. The method comprises administering to the subject a therapeutically effective amount of.

[0149] Embodiment 2. A method of inhibiting a LINE-1 retrotransposition event that causes a disease, disorder, or condition in a subject in need thereof, comprising: (i) a compound of Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; (ii) a compound of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; or (iii) A compound of Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof. The method comprises administering to the subject a therapeutically effective amount of.

[0150] Embodiment 3. A method of treating a disease, condition, or disorder in a subject, comprising: (a) determining the presence or absence of overexpression of retrotransposon RNA, retrotransposon reverse transcriptase, or retrotransposon DNA in a biological sample obtained from the subject; and (b) if overexpression of retrotransposon RNA, retrotransposon reverse transcriptase, or retrotransposon DNA is present in the biological sample, (i) a compound of Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; (ii) a compound of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; or (iii) A compound of Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof. administering to a subject a therapeutically effective amount of The method comprises:

[0151] 4. A subject having a disease, condition, or disorder (i) a compound of Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; (ii) a compound of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; or (iii) A compound of Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof. 1. A method for identifying whether a patient is a candidate for treatment with (a) determining the presence or absence of overexpression of retrotransposon RNA, retrotransposon reverse transcriptase, or retrotransposon DNA in a biological sample obtained from the subject; (b) identifying the subject as a candidate for treatment if overexpression of retrotransposon RNA, retrotransposon reverse transcriptase, or retrotransposon DNA is present; or (c) identifying the subject as not being a candidate for treatment if there is no overexpression of retrotransposon RNA, retrotransposon reverse transcriptase, or retrotransposon DNA; The method comprises:

[0152] 5. A method of predicting a treatment outcome in a subject having a disease, condition, or disorder, comprising: The method includes determining the presence or absence of overexpression of retrotransposon RNA, retrotransposon reverse transcriptase, or retrotransposon DNA in a biological sample obtained from the subject; (a) the presence of overexpression of retrotransposon RNA, retrotransposon reverse transcriptase, or retrotransposon DNA in the biological sample; (i) a compound of Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; (ii) a compound of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; or (iii) A compound of Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof. indicates that administering to the subject is likely to result in a favorable therapeutic response; and (b) the absence of overexpression of retrotransposon RNA, retrotransposon reverse transcriptase, or retrotransposon DNA in the biological sample; (i) a compound of Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; (ii) a compound of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; or (iii) A compound of Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof. may cause an unfavorable therapeutic response in the subject, The method.

[0153] Embodiment 6. (i) a compound of Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; (ii) a compound of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; or (iii) A compound of Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof. Administering to a subject a therapeutically effective amount of (a) the subject has a disease, condition, or disorder, and (b) the disease, condition, or disorder is characterized by overexpression of retrotransposon RNA, retrotransposon reverse transcriptase, or retrotransposon DNA; The method.

[0154] Embodiment 7. The method of any of embodiments 1-6, comprising a compound of Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0155] Embodiment 8. The method of any of embodiments 1-6, comprising a compound of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0156] Embodiment 9 The method of embodiment 7, wherein the compound of Table 1 is Compound No. 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0157] Embodiment 10 The method of embodiment 7, wherein the compound of Table 1 is compound No. 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0158] Embodiment 11 The method of embodiment 7, wherein the compound of Table 1 is compound No. 8, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0159] Embodiment 12 The method of embodiment 7, wherein the compound of Table 1 is compound No. 9, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0160] Embodiment 13 The method of embodiment 12, wherein the compound of Table 1 is compound No. 4, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0161] Embodiment 14 The method of any of embodiments 1-6, comprising a compound of Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0162] Embodiment 15. The method of any of embodiments 1 to 14, for treating said disease, disorder, or condition in a subject.

[0163] Embodiment 16 The method of any of embodiments 1 to 14, for preventing said disease, disorder, or condition in a subject.

[0164] Embodiment 17 The method of any of embodiments 1-14, for treating said symptoms of a disease, disorder, or condition in a subject.

[0165] Embodiment 18 The method of any of embodiments 1-14, for preventing said symptoms of a disease, disorder, or condition in a subject.

[0166] Embodiment 19 The method of any one of embodiments 1 to 18, wherein the disease, disorder, or condition is a neurodegenerative disease.

[0167] The method of embodiment 19, wherein said neurodegenerative disease is Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Huntington's disease, frontotemporal lobar degeneration, mild cognitive impairment, corticobasal degeneration, progressive supranuclear palsy, Rett syndrome, peripheral degenerative disease, or Aicardi-Goutières syndrome.

[0168] Embodiment 21 The method of any of embodiments 1-18, wherein the disease, disorder, or condition is an autoimmune disease.

[0169] Embodiment 22 The method of embodiment 11, wherein the autoimmune disease is lupus, rheumatoid arthritis, Sjogren's syndrome, or multiple sclerosis.

[0170] Embodiment 23 The method of any one of embodiments 1 to 18, wherein the disease, disorder, or condition is an age-related disease.

[0171] Embodiment 24. The method of embodiment 23, wherein said age-related disease is Alzheimer's disease, Parkinson's disease, atherosclerosis, osteoarthritis, osteoporosis, rheumatoid arthritis, macular degeneration, peripheral degenerative disease, or skin aging.

[0172] Embodiment 25 The method of any of embodiments 1-18, wherein said disease, disorder, or condition is autism spectrum disorder (ASD), cardiovascular dysfunction, hearing loss, hematopoietic stem cell function, pulmonary fibrosis, schizophrenia, or vision loss.

[0173] Embodiment 26 The method of any one of embodiments 1 to 18, wherein the disease, disorder, or condition is progressive supranuclear palsy.

[0174] Embodiment 27 The method of any one of embodiments 1 to 18, wherein the disease, disorder, or condition is amyotrophic lateral sclerosis.

[0175] Embodiment 28 The method of any of embodiments 1 to 18, wherein the disease, disorder, or condition is Aicardi-Goutieres syndrome.

[0176] Embodiment 29 The method of any of embodiments 1-3 or 5-28, further comprising any one or more therapeutic agents for said subject.

[0177] Embodiment 30 The method of any of embodiments 1-29, wherein the subject (a) is not infected with the HIV virus, (b) is not suspected of being infected with the HIV virus, (c) is not receiving treatment for the HIV virus, and / or (d) is not receiving treatment to prevent the HIV virus.

[0178] Embodiment 31 The method of any of embodiments 1 to 30, wherein the compound inhibits human LINE-1 retrotransposition activity at a 50% inhibitory concentration of 1 μM or less in an in vitro HeLa cell-based dual luciferase assay.

[0179] Embodiment 32. (i) a compound of Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; (ii) a compound of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; or (iii) a compound of Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof, instructions for administering the compound to a subject having a disease, condition, or disorder; A kit for carrying out the method of any one of claims 1 to 31, comprising:

[0180] Embodiment 33 The kit of embodiment 32, comprising a compound of Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0181] Embodiment 34 The kit of embodiment 32, comprising a compound of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0182] Embodiment 35 The kit of embodiment 33, wherein the compound in Table 1 is Compound No. 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0183] Embodiment 36 The kit of embodiment 33, wherein the compound in Table 1 is compound No. 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0184] Embodiment 37 The kit of embodiment 33, wherein the compound in Table 1 is compound No. 8, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0185] Embodiment 38 The kit of embodiment 33, wherein the compound in Table 1 is compound No. 9, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0186] Embodiment 39 The kit of embodiment 33, wherein the compound in Table 1 is compound No. 4, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0187] Embodiment 40 The kit of embodiment 32, comprising a compound of Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0188] 41. For use in treating or preventing a disease, disorder, or condition caused by a pathophysiological retrotransposon-associated process in a subject in need thereof, and / or for treating or preventing a symptom of the disease, disorder, or condition. (i) a compound of Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof, or a pharmaceutical composition thereof; (ii) a compound of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof, or a pharmaceutical composition thereof; or (iii) A compound of Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof, or a pharmaceutical composition thereof.

[0189] 42. For use in inhibiting a LINE-1 retrotransposition event causing a disease, disorder, or condition in a subject in need thereof. (i) a compound of Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof, or a pharmaceutical composition thereof; (ii) a compound of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof, or a pharmaceutical composition thereof; or (iii) A compound of Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof, or a pharmaceutical composition thereof.

[0190] 43. For use in treating a disease, condition, or disorder characterized by overexpression of retrotransposon RNA, retrotransposon reverse transcriptase, or retrotransposon DNA. (i) a compound of Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof, or a pharmaceutical composition thereof; (ii) a compound of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof, or a pharmaceutical composition thereof; or (iii) A compound of Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof, or a pharmaceutical composition thereof.

[0191] Embodiment 44. The compound of any of Embodiments 41-43, comprising a compound of Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0192] Embodiment 45. The compound of any of Embodiments 41-43, comprising a compound of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0193] Embodiment 46 The compound of embodiment 44, wherein the compound in Table 1 is compound No. 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0194] Embodiment 47 The compound of embodiment 44, wherein the compound of Table 1 is compound No. 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0195] Embodiment 48 The compound of embodiment 44, wherein the compound in Table 1 is compound No. 8, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0196] Embodiment 49. The compound of embodiment 44, wherein the compound in Table 1 is compound No. 9, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0197] Embodiment 50. The compound of embodiment 44, wherein the compound in Table 1 is compound No. 4, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0198] Embodiment 51 The compound of any of Embodiments 41-43, comprising a compound in Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0199] Embodiment 52. A compound or pharmaceutical composition for use according to any of embodiments 41 or 44-51 for treating said disease, disorder, or condition in a subject.

[0200] Embodiment 53. A compound or pharmaceutical composition for use according to any of embodiments 41 or 44 to 51 for preventing said disease, disorder, or condition in a subject.

[0201] Embodiment 54. A compound or pharmaceutical composition for use according to any of embodiments 41 or 44-51 for treating said symptoms of a disease, disorder, or condition in a subject.

[0202] Embodiment 55. A compound or pharmaceutical composition for use according to any of embodiments 41 or 44-51 for preventing said symptoms of a disease, disorder, or condition in a subject.

[0203] Embodiment 56. A compound or pharmaceutical composition for use according to any of embodiments 41 to 55, wherein the disease, disorder, or condition is a neurodegenerative disease.

[0204] Aspect 57. A compound or pharmaceutical composition for use according to aspect 56, wherein the neurodegenerative disease is Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Huntington's disease, frontotemporal lobar degeneration, mild cognitive impairment, corticobasal degeneration, progressive supranuclear palsy, Rett syndrome, peripheral degenerative disease, or Aicardi-Goutières syndrome.

[0205] Embodiment 58. A compound or pharmaceutical composition for use according to any of embodiments 41 to 55, wherein the disease, disorder, or condition is an autoimmune disease.

[0206] Embodiment 59. The compound or pharmaceutical composition for use according to embodiment 58, wherein the autoimmune disease is lupus, rheumatoid arthritis, Sjogren's syndrome, or multiple sclerosis.

[0207] Embodiment 60. A compound or pharmaceutical composition for use according to any of embodiments 41 to 55, wherein the disease, disorder, or condition is an age-related disease.

[0208] Embodiment 61. The compound or pharmaceutical composition for use according to embodiment 60, wherein the age-related disease is Alzheimer's disease, Parkinson's disease, atherosclerosis, osteoarthritis, osteoporosis, rheumatoid arthritis, macular degeneration, peripheral degenerative disease, or skin aging.

[0209] Embodiment 62. A compound or pharmaceutical composition for use in any of embodiments 41 to 55, wherein the disease, disorder, or condition is autism spectrum disorder (ASD), cardiovascular dysfunction, hearing loss, hematopoietic stem cell function, pulmonary fibrosis, schizophrenia, or vision loss.

[0210] Embodiment 63. A compound or pharmaceutical composition for use according to any of embodiments 41 to 55, wherein the disease, disorder, or condition is progressive supranuclear palsy.

[0211] Embodiment 64. A compound or pharmaceutical composition for use in any of Embodiments 41-55, wherein the disease, disorder, or condition is amyotrophic lateral sclerosis.

[0212] Embodiment 65. A compound or pharmaceutical composition for use according to any of embodiments 41 to 55, wherein the disease, disorder, or condition is Aicardi-Goutieres syndrome.

[0213] Embodiment 66 A compound or pharmaceutical composition for use according to any of embodiments 41 to 55, wherein any one or more therapeutic agents are administered to the subject.

[0214] Embodiment 67. A compound or pharmaceutical composition for use in any of embodiments 41-66, wherein the subject (a) is not infected with the HIV virus, (b) is not suspected of being infected with the HIV virus, (c) is not receiving treatment for the HIV virus, and / or (d) is not receiving treatment to prevent the HIV virus.

[0215] Embodiment 68. A compound or pharmaceutical composition for use in any of embodiments 41 to 67, wherein said compound inhibits human LINE-1 retrotransposition activity with a 50% inhibitory concentration of 1 μM or less in an in vitro HeLa cell-based dual luciferase assay.

[0216] Embodiment 69. A compound or pharmaceutical composition for use according to embodiment 68, wherein said compound inhibits human LINE-1 retrotransposition activity with a 50% inhibitory concentration of 0.25 μM or less in an in vitro HeLa cell-based dual luciferase assay.

[0217] Embodiment 70. A compound for use according to any one of embodiments 41 to 69.

[0218] Embodiment 71. A pharmaceutical composition for use according to any one of embodiments 41 to 69.

[0219] 82. For the manufacture of a medicament for treating or preventing a disease, disorder, or condition caused by a pathophysiological retrotransposon-associated process in a subject in need thereof, and / or treating or preventing a symptom of the disease, disorder, or condition, (i) a compound of Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; (ii) a compound of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof, or a pharmaceutical composition thereof; or (iii) a compound of Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof, or a pharmaceutical composition thereof. Use of.

[0220] 83. For the manufacture of a medicament for inhibiting a LINE-1 retrotransposition event causing a disease, disorder, or condition in a subject in need thereof, (i) a compound of Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; (ii) a compound of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof, or a pharmaceutical composition thereof; or (iii) a compound of Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof, or a pharmaceutical composition thereof. Use of.

[0221] 84. A method for the manufacture of a medicament for treating a disease, condition, or disorder characterized by overexpression of retrotransposon RNA, retrotransposon reverse transcriptase, or retrotransposon DNA, comprising: (i) a compound of Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof; (ii) a compound of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof, or a pharmaceutical composition thereof; or (iii) a compound of Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof, or a pharmaceutical composition thereof; Use of.

[0222] Embodiment 85. The use of any of embodiments 82-84, comprising a compound of Table 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0223] Embodiment 86 The use of embodiment 85, comprising a compound of Table 2, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0224] Embodiment 87 The use of embodiment 86, wherein the compound of Table 1 is compound No. 1, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0225] Embodiment 88 The use of embodiment 87, wherein the compound of Table 1 is compound number 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0226] Embodiment 89 The use of embodiment 88, wherein the compound of Table 1 is compound number 8, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0227] Embodiment 90 The use of embodiment 89, wherein the compound of Table 1 is compound number 9, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0228] Embodiment 91 The use of embodiment 90, wherein the compound of Table 1 is compound number 4, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0229] Embodiment 92 The use of any of embodiments 82-84, comprising a compound of Table 3, or a pharma- ceutically acceptable salt or solvate thereof, or a tautomer thereof.

[0230] Embodiment 93. The use of any of embodiments 82 or 85-92 for treating a disease, disorder, or condition in a subject.

[0231] Embodiment 94. The use of any of embodiments 82 or 85-92 for preventing said disease, disorder, or condition in a subject.

[0232] Embodiment 95. The use of any of embodiments 82 or 85-92 for treating a symptom of a disease, disorder, or condition in a subject.

[0233] Embodiment 96 The use of any of embodiments 82 or 85-92 for preventing said symptoms of a disease, disorder, or condition in a subject.

[0234] Embodiment 97 The use of any of embodiments 82 to 96, wherein the disease, disorder, or condition is a neurodegenerative disease.

[0235] Aspect 98. The use of aspect 97, wherein the neurodegenerative disease is Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Huntington's disease, frontotemporal lobar degeneration, mild cognitive impairment, corticobasal degeneration, progressive supranuclear palsy, Rett syndrome, peripheral degenerative disease, or Aicardi-Goutières syndrome.

[0236] Embodiment 99. The use of any of embodiments 82 to 96, wherein the disease, disorder, or condition is an autoimmune disease.

[0237] Embodiment 100. The use of embodiment 99, wherein the autoimmune disease is lupus, rheumatoid arthritis, Sjogren's syndrome, or multiple sclerosis.

[0238] Embodiment 101 The use of any of embodiments 82 to 96, wherein the disease, disorder, or condition is an age-related disease.

[0239] Embodiment 102. The use of embodiment 101, wherein the age-related disease is Alzheimer's disease, Parkinson's disease, atherosclerosis, osteoarthritis, osteoporosis, rheumatoid arthritis, macular degeneration, peripheral degenerative disease, or skin aging.

[0240] Embodiment 103. The use of any of embodiments 82 to 96, wherein said disease, disorder, or condition is autism spectrum disorder (ASD), cardiovascular dysfunction, hearing loss, hematopoietic stem cell function, pulmonary fibrosis, schizophrenia, or vision loss.

[0241] Embodiment 104 The use of any of embodiments 82 to 96, wherein the disease, disorder, or condition is progressive supranuclear palsy.

[0242] Embodiment 105 The use of any of embodiments 82 to 96, wherein the disease, disorder, or condition is amyotrophic lateral sclerosis.

[0243] Embodiment 106 The use of any of embodiments 82 to 96, wherein the disease, disorder, or condition is Aicardi-Goutieres syndrome.

[0244] Embodiment 107 The use of any of embodiments 82 to 96, wherein any one or more therapeutic agents are administered to the subject.

[0245] Embodiment 108. The use of any of embodiments 82 to 107, wherein the subject (a) is not infected with the HIV virus, (b) is not suspected of being infected with the HIV virus, (c) is not receiving treatment for the HIV virus, and / or (d) is not receiving treatment to prevent the HIV virus.

[0246] Embodiment 109. The use of any of embodiments 82 to 108, wherein said compound inhibits human LINE-1 retrotransposition activity at a 50% inhibitory concentration of 1 μM or less in an in vitro HeLa cell-based dual luciferase assay.

[0247] Embodiment 110. The method of any one of embodiments 3 to 6, wherein the retrotransposon RNA is a LINE-1 RNA.

[0248] Embodiment 111 The method of any one of embodiments 3 to 6, wherein the retrotransposon reverse transcriptase is ORF2p.

[0249] Embodiment 112. The method of any one of embodiments 3 to 6, wherein the retrotransposon DNA is LINE-1 DNA.

[0250] Embodiment 113 The compound for use in embodiment 43, wherein the retrotransposon RNA is a LINE-1 RNA.

[0251] Embodiment 114 The compound for use in embodiment 43, wherein the retrotransposon reverse transcriptase is ORF2p.

[0252] Embodiment 115. The compound for use in embodiment 43, wherein the retrotransposon DNA is LINE-1 DNA.

[0253] Embodiment 116 The use of embodiment 84, wherein the retrotransposon RNA is a LINE-1 RNA.

[0254] Embodiment 117 The use of embodiment 84, wherein the retrotransposon reverse transcriptase is ORF2p.

[0255] Embodiment 118. The use of embodiment 84, wherein the retrotransposon DNA is LINE-1 DNA.

[0256] Embodiment 119 The method of any of embodiments 1 to 18, wherein the disease, disorder, or condition is ataxia-telangiectasia.

[0257] Embodiment 120 The method of any of embodiments 1 to 18, wherein the disease, disorder, or condition is age-related macular degeneration.

[0258] Embodiment 121 The method of any of embodiments 1-18, wherein the disease, disorder, or condition is systemic lupus erythematosus.

[0259] Embodiment 122 The method of any of embodiments 1 to 18, wherein the disease, disorder, or condition is an IFN-associated autoimmune disease.

[0260] Embodiment 123 The method of embodiment 122, wherein the IFN-associated autoimmune disease is psoriasis.

[0261] Embodiment 124 The method of any of embodiments 1 to 18, wherein the disease, disorder, or condition is Fanconi anemia.

[0262] Embodiment 125 The method of any of embodiments 1 to 18, wherein the disease, disorder, or condition is idiopathic pulmonary fibrosis.

[0263] Embodiment 126 The method of any one of embodiments 1 to 18, wherein the disease, disorder, or condition is a cardiovascular disease.

[0264] Embodiment 127 The compound for use in any of Embodiments 41 to 55, wherein the disease, disorder, or condition is ataxia-telangiectasia.

[0265] Embodiment 128 The compound for use in any of embodiments 41 to 55, wherein the disease, disorder, or condition is age-related macular degeneration.

[0266] Embodiment 129. A compound for use according to any of embodiments 41 to 55, wherein the disease, disorder, or condition is systemic lupus erythematosus.

[0267] Embodiment 130. A compound for use according to any of embodiments 41 to 55, wherein the disease, disorder, or condition is an IFN-associated autoimmune disease.

[0268] Embodiment 131 The compound for use of embodiment 130, wherein the IFN-associated autoimmune disease is psoriasis.

[0269] Embodiment 132 The compound for use in any of embodiments 41 to 55, wherein the disease, disorder, or condition is Fanconi anemia.

[0270] Embodiment 133. A compound for use according to any of embodiments 41 to 55, wherein the disease, disorder, or condition is idiopathic pulmonary fibrosis.

[0271] Embodiment 134 The compound for use according to any of embodiments 41 to 55, wherein the disease, disorder, or condition is a cardiovascular disease.

[0272] Embodiment 135 The use of any of embodiments 82 to 96, wherein the disease, disorder, or condition is ataxia-telangiectasia.

[0273] Embodiment 136 The use of any of embodiments 82 to 96, wherein the disease, disorder, or condition is age-related macular degeneration.

[0274] Embodiment 137. A compound for use according to any of embodiments 82 to 96, wherein the disease, disorder, or condition is systemic lupus erythematosus.

[0275] Embodiment 138 A compound for use according to any of embodiments 82 to 96, wherein the disease, disorder, or condition is an IFN-associated autoimmune disease.

[0276] Embodiment 139. The compound for use of embodiment 138, wherein the IFN-associated autoimmune disease is psoriasis.

[0277] Embodiment 140. A compound for use according to any of embodiments 82 to 96, wherein the disease, disorder, or condition is Fanconi anemia.

[0278] Embodiment 141 The compound for use according to any of embodiments 82 to 96, wherein the disease, disorder, or condition is idiopathic pulmonary fibrosis.

[0279] Embodiment 142 The compound for use according to any of embodiments 82 to 96, wherein the disease, disorder, or condition is a cardiovascular disease.

[0280] Embodiment 143. The method, kit, compound for use, or use of any of embodiments 1, 32, 41, or 82, wherein the disease, disorder, or condition is not (i) cancer or (ii) an infectious disease. EXAMPLES

[0281] The compounds in Tables 1 and 2 can be prepared as described in the Examples below and in, for example, Nomura et al., J. Med. Chem. 42:2901-2908 (1999); Ohrui et al., J. Med. Chem. 43:4516-4525 (2000), JP Patent No. 6767011, and / or US Patent No. 10,933,067.

[0282] The abbreviations in Table 4 may be used in the Examples.

[0283] (Table 4) TIFF2025511024000036.tif61139TIFF2025511024000037.tif232139TIFF2025511024000038.tif210139

[0284] The following LC-MS methods may be used in the examples.

[0285] Method A: UPLC-MS method: Waters Acquity UPLC CSH C18, 1.8 μm, 2.1 × 30 mm at 40 °C; 5% to 100% B in 5.2 min; hold 100% B for 1.8 min, run time = 7.0 min, flow rate 0.9 mL / min; Eluent; A = Milli-Q H2O + 10 mM ammonium formate pH = 3.8; B = MeCN. Waters Acquity UPLC system. UV detector = Waters Acquity PDA, 198-360 nm. MS detector = Waters SQD ESI.

[0286] Method B: Waters Acquity CSH C18, 3.5 μm, 4.6 × 30 mm at 40 °C; Iso 5% B for 0.5 min, 5%-100% B for 5 min; 100% B hold for 1.5 min, run time = 7.0 min, flow rate 0.9 mL / min; Eluent; A = Milli-Q H2O + 10 mM ammonium formate pH = 3.8; B = MeCN. Waters Alliance 2695 system. UV detection: Waters 2996 PDA, 198-360 nm. MS detector: Waters ZQ 2000, ESI.

[0287] Method C: SHIMADZU LC20-MS2010: MERCK, RP-18e 25-2mm at 50°C; 1.5ML / 4L TFA in water (solvent A) and 0.75ML / 4L TFA in acetonitrile (solvent B), elution gradient of 5%-95% (solvent B) over 0.7 minutes at a flow rate of 1.5ml / min, hold at 95% for 0.4 minutes, run time = 1.5 minutes. UV detector = 220nm, 254nm; MS detector = ESI.

[0288] Method D: LCMS-BT: SHIMADZU LC20-MS2020: X bridge Shield RP-18, 5μm, 2.1*50mm at 50℃; 0.8mL / 4L NH3·H2O aqueous solution (solvent A) and acetonitrile (solvent B), elution gradient from 0% to 30% (solvent B) over 2 minutes at a flow rate of 1ml / min, hold at 30% for 0.48 minutes, run time = 3 minutes; UV detector = 220nm, 254nm; MS detector = ESI.

[0289] Method E: LCMS-AN: Agilent LC1200-MS6110: Xtimate C18 2.1*30mm, 3μm at 50℃; 1.5ML / 4L TFA in water (solvent A) and 0.75ML / 4L TFA in acetonitrile (solvent B), elution gradient from 0% to 60% (solvent B) over 0.9 minutes at a flow rate of 1.2ml / min, hold at 60% for 0.6 minutes, run time = 2 minutes; UV detector = 220nm; MS detector = ESI.

[0290] Method F: LCMS-CI: Agilent LC1200-MS6110: Xbridge Shield RP-18,5um, 2.1*50mm; at 30℃; water (4L)+NH3·H2O (0.8mL) (solvent A) and acetonitrile (solvent B) with an elution gradient of 0%-60% (solvent B) over 2.0 min at a flow rate of 1ml / min, hold at 60% for 0.48 min, run time = 3.0 min; UV detector = 220nm, 254nm; MS detector = ESI.

[0291] Method G: UPLC-MS method: Waters Acquity UPLC CSH C18, 1.8 μm, 2.1 × 30 mm at 40 °C; 5% to 100% B in 2.0 min; 100% B held for 0.7 min, run time = 2.7 min, flow rate 0.9 mL / min; Eluent: A = Milli-Q H2O + 10 mM ammonium formate pH = 3.8; B = MeCN. Waters Acquity UPLC system. UV detector = Waters Acquity PDA, 198-360 nm. MS detector = Waters SQD ESI.

[0292] Method H: UPLC-MS method: Waters Acquity UPLC Agilent Poroshell 120 EC C18, 1.8 μm, 2.1 × 250 mm at 40 °C; 5% to 100% B in 5.0 min; 100% B held for 2.0 min, run time = 7.0 min, flow rate 0.5 mL / min; Eluent: A = Milli-Q H2O + 10 mM ammonium formate pH = 3.8; B = MeCN. Waters Acquity UPLC system. UV detector = Waters Acquity PDA, 198-360 nm. MS detector = Waters SQD ESI.

[0293] Example 1 Synthesis of 1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-vinyltetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (Compound No. 62) TIFF2025511024000039.tif251361-((2R,4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione was prepared according to WO 2007 / 038507 A2.

[0294] 1-((2R,4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (18.0 mg, 67.6 μmol) was dissolved in 9:1 EtOAc / pyridine (653 μl / 22.5 μl) in a 5 mL vial fitted with a rubber stopper. Solid Lindlar catalyst (18.0 mg, 67.6 μmol) was then added and the reaction mixture was flushed with H2 gas via a balloon for 30 minutes. The reaction was stirred until complete conversion to the title compound was observed by LC-MS. The reaction mixture was then filtered through Celite® and rinsed with EtOAc. The desired product was purified by silica gel flash chromatography (EtOAc) to give 1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-vinyltetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (11 mg, 58%). TIFF2025511024000040.tif34153

[0295] Example 2 Synthesis of 5-bromo-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-vinyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound No. 64) TIFF2025511024000041.tif25136 5-Bromo-1-((2R,4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione was prepared according to WO 2007 / 038507 A2.

[0296] 5-Bromo-1-((2R,4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (3.3 mg, 9.97 μmol) was dissolved in 9:1 EtOAc / pyridine (96.3 μl / 3.32 μl) in a 5 mL vial fitted with a rubber stopper. Solid Lindlar catalyst (3.30 mg, 9.97 μmol) was then added and the reaction mixture was flushed with H2 gas bubbled through a balloon for 30 minutes. The reaction was stirred until complete conversion to the title compound was observed by LC-MS. The reaction mixture was then filtered through Celite® and rinsed with EtOAc. The desired product was purified by silica gel flash chromatography (EtOAc) to give 5-bromo-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-vinyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (2.70 mg, 81%). TIFF2025511024000042.tif34153

[0297] Example 3 Synthesis of 1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-vinyltetrahydrofuran-2-yl)-5-methoxypyrimidine-2,4(1H,3H)-dione (Compound No. 6) TIFF2025511024000043.tif27142 Step 1. 1-((2R,4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methoxypyrimidine-2,4(1H,3H)-dione was prepared according to WO 2007 / 038507 A2.

[0298] To a mixture of [(2R,3S,5R)-2-ethynyl-5-(5-methoxy-2,4-dioxo-pyrimidin-1-yl)-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (15 mg, 28.93 μmol) in MeOH (2 mL) was added Lindlar's catalyst (7 mg) in one portion under H2 (15 psi) at 25° C. The resulting mixture was stirred at 25° C. for 1 h. The mixture was then filtered and the filtrate was concentrated to give the compound (15 mg, 99%) as a white solid.

[0299] Step 2. To a mixture of [(2R,3S,5R)-5-(5-methoxy-2,4-dioxo-pyrimidin-1-yl)-3-(4-methylbenzoyl)oxy-2-vinyl-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (15 mg, 28.82 μmol) in MeOH (5 mL) was added NaOMe (156 μg, 2.88 μmol) in one portion at 25° C. After stirring for 1 h at 25° C., the mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (basic) to give the title compound (5 mg, 62%) as a white solid. TIFF2025511024000044.tif27153

[0300] Example 4 Synthesis of 1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-vinyltetrahydrofuran-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (Compound No. 69) TIFF2025511024000045.tif251365-pyrimidinecarbonitrile was used to prepare 1-((2R,4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile according to WO 2007 / 038507 A2. 1-((2R,4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (8.9 mg, 32.1 μmol) was dissolved in MeOH (321 μl) in a 5 mL vial fitted with a rubber stopper. Solid Lindlar's catalyst (8.9 mg, 32.1 μmol) was then added and the reaction mixture was flushed with H2 gas bubbled through a balloon for 30 min. The reaction was stirred until complete conversion to the title compound was observed by LC-MS. The reaction mixture was then filtered through Celite® and rinsed with EtOAc. The desired product was purified by silica gel flash chromatography (EtOAc) to give 1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-vinyltetrahydrofuran-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (1.36 mg, 13%). TIFF2025511024000046.tif34153

[0301] Example 5 Synthesis of 4-amino-5-bromo-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-vinyltetrahydrofuran-2-yl)pyrimidin-2(1H)-one (Compound No. 90) TIFF2025511024000047.tif25140 4-Amino-5-bromo-1-((2R,4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one was prepared according to WO 2007 / 038507 A2.

[0302] 4-Amino-5-bromo-1-((2R,4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (64.0 mg, 194 μmol) was dissolved in MeOH (1.94 mL) in a 5 mL vial fitted with a rubber stopper. Solid Lindlar's catalyst (64 mg, 194 μmol) was then added and the reaction mixture was flushed with H2 gas bubbled through a balloon for 30 minutes. The reaction was stirred until complete conversion to the title compound was observed by LC-MS. The reaction mixture was then filtered through Celite® and rinsed with MeOH. The desired product was purified by silica gel flash chromatography using 0-100% 2-propanol in EtOAc to give 4-amino-5-bromo-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-vinyltetrahydrofuran-2-yl)pyrimidin-2(1H)-one (31.6 mg, 45%). TIFF2025511024000048.tif34153

[0303] Example 6 Synthesis of 2-amino-9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-vinyltetrahydrofuran-2-yl)-1H-purin-6(9H)-one (Compound No. 9) TIFF2025511024000049.tif24140 2-Amino-9-((2R,4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H-purin-6(9H)-one was prepared according to WO 2007 / 038507 A2.

[0304] 2-Amino-9-((2R,4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H-purin-6(9H)-one (125 mg, 429 μmol) was dissolved in EtOAc / pyridine (4.15 mL / 143 μl) in a 5 mL vial fitted with a rubber stopper. Solid Lindlar's catalyst (125 mg, 429 μmol) was then added and the reaction mixture was flushed with H2 gas bubbled through a balloon for 30 minutes. The reaction was stirred until complete conversion to the title compound was observed by LC-MS. The reaction mixture was then filtered through Celite® and rinsed with MeOH. The desired product was purified by using IC, 10 x 250 mm 5um column, and SFC purification using an isocratic gradient of 25% MeOH, 0.1% NH4OH, 75% CO2 at a flow rate of 10 mL / min and a run time of 20 minutes to give 2-amino-9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-vinyltetrahydrofuran-2-yl)-1H-purin-6(9H)-one (2.62 mg, 2%). TIFF2025511024000050.tif34153

[0305] Example 7 Synthesis of 4-amino-5-bromo-1-((4S,5R)-5-ethyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (Compound No. 70) TIFF2025511024000051.tif26140 4-Amino-5-bromo-1-((4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one was synthesized according to the procedure described in WO 2007 / 038507 A2.

[0306] 4-Amino-5-bromo-1-((4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (5.45 mg, 16.4 μmol) was dissolved in MeOH in a 5 mL vial fitted with a rubber stopper. Solid PtO2 (0.500 mg, 2.20 μmol) was then added and the reaction mixture was flushed with a H2 balloon for 30 minutes. The reaction was stirred until complete conversion to the title compound was observed by LC-MS. The reaction mixture was then filtered through Celite®, washed with MeOH and the solvent was removed under reduced pressure. The crude mixture was purified by silica gel column chromatography (EtOAc / hep 1:1 to 100% EtOAc) to give 4-amino-5-bromo-1-((4S,5R)-5-ethyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (0.750 mg, 13%) as a mixture of anomers as an off-white powder. TIFF2025511024000052.tif41154

[0307] Example 8 Synthesis of 1-((4S,5R)-5-ethyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methoxypyrimidine-2,4(1H,3H)-dione (Compound No. 68) TIFF2025511024000053.tif281361-((4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methoxypyrimidine-2,4(1H,3H)-dione was synthesized according to the procedure described in WO 2007 / 038507 A2.

[0308] 1-((4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methoxypyrimidine-2,4(1H,3H)-dione (7.20 mg, 28.7 μmol) was dissolved in MeOH in a 5 mL vial fitted with a rubber stopper. Solid Lindlar catalyst (7.20 mg, 28.7 μmol) was then added and the reaction mixture was flushed with a H2 balloon for 30 minutes. The reaction was stirred until complete conversion to the title compound was observed by LC-MS. The reaction mixture was then filtered through Celite®, washed with MeOH, and the solvent was removed under reduced pressure to give 4-amino-1-((4S,5R)-5-ethyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methoxypyrimidin-2(1H)-one (5.75 mg, 66%) as a mixture of anomers as a white powder. TIFF2025511024000054.tif34153

[0309] Example 9 Synthesis of (2R,3S,5R)-5-(6-(cyclopropylamino)-9H-purin-9-yl)-2-(hydroxymethyl)-2-vinyltetrahydrofuran-3-ol (Compound No. 94) TIFF2025511024000055.tif74145 N-Cyclopropyl-9H-purin-6-amine was used to synthesize ((2R,3S,5R)-5-(6-chloro-9H-purin-9-yl)-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-methylbenzoate according to the procedure described in WO 2007 / 038507 A2.

[0310] Step 1. To a mixture of 4-methylbenzoic acid [(2R,3S,5R)-5-(6-chloropurin-9-yl)-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (0.05 g, 94.17 μmol) and cyclopropanamine (8 mg, 141.25 μmol) in t-BuOH (2 mL) was added DIEA (24 mg, 188.34 μmol) in one portion at 25° C. under N2. The resulting mixture was stirred at 90° C. for 16 h. After cooling to room temperature, the mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (50% EtOAc in petroleum ether) to give the compound (45 mg, 87%) as a colorless oil. TIFF2025511024000056.tif34153

[0311] Step 2. To a solution of [(2R,3S,5R)-5-[6-(cyclopropylamino)purin-9-yl]-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (45 mg, 81.58 μmol) in MeOH (2 mL) was added Lindlar's catalyst (17 mg) in one portion at 25° C. under N2 (15 psi). The resulting mixture was stirred at 25° C. for 1 h. The mixture was filtered and directly concentrated to give the compound (45 mg, 99%) as a white solid.

[0312] Step 3. To a mixture of [(2R,3S,5R)-5-[6-(cyclopropylamino)purin-9-yl]-3-(4-methylbenzoyl)oxy-2-vinyl-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (45 mg, 81.29 μmol) in MeOH (5 mL) was added NaOMe (439 μg, 8.13 μmol) in one portion at 25° C. After stirring for 1 h at 25° C., the mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (acetonitrile 0-30% / 0.225% formic acid in water) to give the title compound (13.6 mg, 53%) as a white solid. TIFF2025511024000057.tif27153

[0313] Example 10 Synthesis of (2R,3S,5R)-5-(6-(cyclopropylamino)-2-fluoro-9H-purin-9-yl)-2-(hydroxymethyl)-2-vinyltetrahydrofuran-3-ol (Compound No. 5) TIFF2025511024000058.tif28148 N-Cyclopropyl-2-fluoro-9H-purin-6-amine was used to synthesize 4-methylbenzoic acid [(2R,3S,5R)-5-[6-(cyclopropylamino)-2-fluoro-purin-9-yl]-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl according to the procedure described in WO 2007 / 038507 A2.

[0314] Step 1. To a solution of 4-methylbenzoic acid [(2R,3S,5R)-5-[6-(cyclopropylamino)-2-fluoro-purin-9-yl]-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (50 mg, 87.78 μmol) in EtOAc (3 mL) was added Lindlar's catalyst (18 mg, 8.78 μmol, 10% purity) in one portion at 25° C. The mixture was degassed with H2 three times and stirred under H2 (15 psi) at 25° C. for 1 h. The reaction mixture was then filtered and the filtrate was concentrated to give the compound (46 mg, 92%) as a white solid. LCMS (ESI): m / z 594.1 (M+Na)+.

[0315] Step 2. To a solution of 4-methylbenzoate [(2R,3S,5R)-5-[6-(cyclopropylamino)-2-fluoro-purin-9-yl]-3-(4-methylbenzoyl)oxy-2-vinyl-tetrahydrofuran-2-yl]methyl (45 mg, 78.73 μmol) in MeOH (3 mL) was added NH3.H2O (1 mL, 6.49 mmol, 25% purity) at 25 °C. The mixture was stirred at 40 °C for 16 h. The reaction mixture was then concentrated. The residue was purified by reverse phase HPLC (1-27% acetonitrile / 0.225% formic acid in water) to give the title compound (14.1 mg, 53%) as a white solid. TIFF2025511024000059.tif27153

[0316] Example 11 Synthesis of (2R,3S,5R)-5-(2-amino-6-(cyclopropylamino)-9H-purin-9-yl)-2-(hydroxymethyl)-2-vinyltetrahydrofuran-3-ol (Compound No. 4) TIFF2025511024000060.tif84142N 6 -Cyclopropyl-9H-purine-2,6-diamine was used to synthesize (2R,3S,5R)-5-(2-amino-6-(cyclopropylamino)-9H-purin-9-yl)-2-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol according to the procedure described in WO 2007 / 038507 A2.

[0317] Step 1. To a mixture of 4-methylbenzoic acid [(2R,3S)-5-(2-amino-6-chloro-purin-9-yl)-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (0.20 g, 366.32 μmol) and cyclopropanamine (31 mg, 549.48 μmol) in t-BuOH (15 mL) at 25° C. under N2, DIEA (95 mg, 732.64 μmol) was added in one portion. The resulting mixture was stirred at 90° C. for 16 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (50% EtOAc in petroleum ether) to give the compound (0.04 g, 19%) as a colorless oil. TIFF2025511024000061.tif34153

[0318] Step 2. To a solution of 4-methylbenzoate [(2R,3S,5R)-5-[2-amino-6-(cyclopropylamino)purin-9-yl]-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (0.02 g, 35.30 μmol) in MeOH (2 mL) was added Lindlar's catalyst (7 mg) in one portion at 25° C. under H2 (15 psi). The resulting mixture was stirred at 25° C. for 1 h. The mixture was then filtered and concentrated to give the compound (0.02 g, 99%) as a white solid.

[0319] Step 3. To a solution of [(2R,3S,5R)-5-[2-amino-6-(cyclopropylamino)purin-9-yl]-3-(4-methylbenzoyl)oxy-2-vinyl-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (0.02 g, 35.17 μmol) in MeOH (5 mL) was added NaOMe (190 μg, 3.52 μmol) in one portion at 25° C. and stirred for 18 h. The mixture was then concentrated. The residue was purified by preparative HPLC (4-34% acetonitrile in water / 0.05% NH3.H2O) to give the title compound (14 mg, 53%) as a white solid. TIFF2025511024000062.tif27153

[0320] Example 12 Synthesis of 4-amino-5-fluoro-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-vinyltetrahydrofuran-2-yl)pyrimidin-2(1H)-one (Compound No. 8) TIFF2025511024000063.tif81143 (4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-one was synthesized according to the procedure described in WO 2007 / 038507 A2.

[0321] Step 1. To a solution of (4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-one (1 g, 6.4 mmol) and imidazole (1.31 g, 19.2 mmol) in DMF (30 mL) was added TBSCl (1.06 g, 7.1 mmol) at 20 °C. The resulting mixture was stirred at 20 °C for 16 h. The reaction was then diluted with EtOAc (80 mL) and washed with brine (50 mL x 2). The layers were separated and the organic layer was concentrated. The residue was purified by silica gel column chromatography (20%-30% EtOAc in petroleum ether) to give the compound (0.7 g, 40%) as a white solid. TIFF2025511024000064.tif12158

[0322] Step 2. To a solution of (4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-5-ethynyl-4 hydroxydihydrofuran-2(3H)-one (3 g, 11 mmol) in THF (50 mL) was added NaH (665 mg, 16 mmol, 60% dispersion in mineral oil) at 0 °C. After stirring at 0 °C for 10 min, MOMBr (2.77 g, 22 mmol) was added. The resulting mixture was stirred at 25 °C for another 3 h. Then, the reaction mixture was poured into water (50 mL) and extracted with DCM (50 mL x 2). The organic layer was washed with brine (50 mL x 2) and concentrated. The residue was purified by silica gel column chromatography (10% to 20% EtOAc in petroleum ether) to give the compound (2.1 g, 60%) as a colorless oil. TIFF2025511024000065.tif20158

[0323] Step 3. To a solution of (4S,5R)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-ethynyl-4-(methoxymethoxy)tetrahydrofuran-2-one (1.1 g, 3.5 mmol) in DCM (30 mL) was added DIBAL-H (4.2 mL, 4.2 mmol) dropwise at -70°C. The mixture was stirred for 30 min. Then the reaction was quenched with methanol (5 mL), washed with aqueous citric acid (10 wt%, 30 mL) and brine (30 mL x 2), and concentrated to give 1.1 g of crude (4S,5R)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-ethynyl-4-(methoxymethoxy)tetrahydrofuran-2-ol as a yellow oil, which was dissolved in DCM (20 mL). Et3N (457 mg, 4.52 mmol), DMAP (42 mg) and Ac2O (426 mg, 4.17 mmol) were added sequentially at 0 °C. After stirring at 25 °C for 1 h, the reaction mixture was diluted with MTBE (100 mL), washed with aqueous citric acid (10 wt%, 50 mL) and brine (50 × 2 mL), and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (10% to 30% EtOAc in petroleum ether) to give the compound (0.9 g, 72%) as a white solid.

[0324] Step 4. A mixture of N-(5-fluoro-2-hydroxy-pyrimidin-4-yl)benzamide (100 mg, 0.43 mmol) and BTMSA (219 mg, 1.29 mmol) in MeCN (10 mL) was stirred at 70° C. for 1 h, then cooled to 20° C. and TMSOTf (148 mg, 0.66 mmol) and a solution of acetic acid [(4S,5R)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-ethynyl-4-(methoxymethoxy)tetrahydrofuran-2-yl] (0.12 g, 0.33 mmol) in MeCN (5 mL) were added sequentially. After stirring at 20° C. for 3 h, the reaction mixture was poured into water (50 mL), extracted with EtOAc (50 mL×2), and concentrated. The residue was purified by preparative TLC (30% EtOAc in petroleum ether) to give the compound (40 mg, 22%) as a white solid. TIFF2025511024000066.tif35153

[0325] Step 5. A mixture of N-[1-[(2R,4S,5R)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-ethynyl-4-(methoxymethoxy)tetrahydrofuran-2-yl]-5-fluoro-2-oxo-pyrimidin-4-yl]benzamide (10 mg, 18 μmol) and Lindlar's catalyst (10 mg) in MeOH (5 mL) was stirred under H2 (15 psi) at 20° C. for 2 h. The reaction mixture was filtered and concentrated to give the compound (10 mg, 99%) as a white solid. LCMS (ESI): m / z 534.1 (M+H)+.

[0326] Step 6. To a solution of N-[1-[(2R,4S,5R)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-(methoxymethoxy)-5-vinyl-tetrahydrofuran-2-yl]-5-fluoro-2-oxo-pyrimidin-4-yl]benzamide (80 mg, 0.15 mmol) in MeOH (5 mL) was added acetyl chloride (118 mg, 1.5 mmol) at 20° C. The resulting mixture was stirred at 20° C. for 16 h. The reaction mixture was then filtered and concentrated. The residue was purified by preparative HPLC (FA) to give the title compound (6.7 mg, 16%) as a white solid. TIFF2025511024000067.tif27153

[0327] Example 13 Synthesis of (2R,3S,5R)-5-(6-(cyclopropylamino)-9H-purin-9-yl)-2-ethyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (Compound No. 96) TIFF2025511024000068.tif80158 N-Cyclopropyl-9H-purin-6-amine was used to synthesize ((2R,3S,5R)-5-(6-chloro-9H-purin-9-yl)-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-methylbenzoate according to the procedure described in WO 2007 / 038507 A2.

[0328] Step 1. To a mixture of 4-methylbenzoic acid [(2R,3S,5R)-5-(6-chloropurin-9-yl)-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (0.05 g, 94.17 μmol) and cyclopropanamine (8 mg, 141.25 μmol) in t-BuOH (2 mL) was added DIEA (24 mg, 188.34 μmol) in one portion at 25° C. under N2. The resulting mixture was stirred at 90° C. for 16 h. After cooling to room temperature, the mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (50% EtOAc in petroleum ether) to give the compound (45 mg, 87%) as a colorless oil. TIFF2025511024000069.tif34154

[0329] Step 2. To a mixture of (2R,3S,5R)-5-(6-(cyclopropylamino)-9H-purin-9-yl)-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-methylbenzoate (0.07 g, 126.91 μmol) in MeOH (5 mL) was added 10% palladium on carbon (2 mg) in one portion at 25° C. under H2 (15 psi). The resulting mixture was stirred at 25° C. for 1 h. The mixture was then filtered and directly concentrated to give the compound (0.07 g, 99%) as a colorless oil.

[0330] Step 3. To a mixture of [(2R,3S,5R)-5-[6-(cyclopropylamino)purin-9-yl]-2-ethyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (0.07 g, 125.98 μmol) in MeOH (5 mL) was added NaOMe (681 μg, 12.60 μmol) in one portion at 25° C. After stirring for 18 h at 25° C., the mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (5-35% acetonitrile in water / 0.05% NH3.H2O + 10 mM NH4HCO3) to give the title compound (10 mg, 49%) as a white solid. TIFF2025511024000070.tif34153

[0331] Example 14 Synthesis of (2R,3S,5R)-5-(2-amino-6-(cyclopropylamino)-9H-purin-9-yl)-2-ethyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (Compound No. 97) TIFF2025511024000071.tif80142N6-Cyclopropyl-9H-purine-2,6-diamine was used to synthesize (2R,3S,5R)-5-(2-amino-6-(cyclopropylamino)-9H-purin-9-yl)-2-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol according to the procedure described in WO 2007 / 038507 A2.

[0332] Step 1. To a mixture of 4-methylbenzoic acid [(2R,3S)-5-(2-amino-6-chloro-purin-9-yl)-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (0.20 g, 366.32 μmol) and cyclopropanamine (31 mg, 549.48 μmol) in t-BuOH (15 mL) at 25° C. under N2, DIEA (95 mg, 732.64 μmol) was added in one portion. The resulting mixture was stirred at 90° C. for 16 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (50% EtOAc in petroleum ether) to give the compound (0.04 g, 19%) as a colorless oil. TIFF2025511024000072.tif34153

[0333] Step 2. To a solution of 4-methylbenzoate [(2R,3S,5R)-5-[2-amino-6-(cyclopropylamino)purin-9-yl]-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (0.02 g, 35.30 μmol) in MeOH (5 mL) was added 10% palladium on carbon (24 mg) in one portion at 25° C. under H2 (15 psi). The resulting mixture was stirred at 25° C. for 1 h. The mixture was then filtered and concentrated to give the compound (0.02 g, 99%) as a white solid.

[0334] Step 3. To a solution of [(2R,3S,5R)-5-[2-amino-6-(cyclopropylamino)purin-9-yl]-2-ethyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (0.02 g, 35.05 μmol) in MeOH (5 mL) was added NaOMe (189 μg, 3.50 μmol) in one portion at 25° C. The resulting mixture was stirred at 25° C. for 18 h to give the title compound. TIFF2025511024000073.tif27153

[0335] Example 15 Synthesis of 1-((2R,4S,5R)-5-ethyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-hydroxy-2-oxo-1,2-dihydropyrimidine-5-carbonitrile (Compound No. 98) TIFF2025511024000074.tif31128 4-Methylbenzoic acid [(2R,3S,5R)-5-(5-cyano-2,4-dioxo-pyrimidin-1-yl)-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl was synthesized according to the procedure described in WO 2007 / 038507 A2.

[0336] Step 1. To a solution of 4-methylbenzoate [(2R,3S,5R)-5-(5-cyano-2,4-dioxo-pyrimidin-1-yl)-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (5 mg, 97.37 μmol) in MeOH (3 mL) was added 10% palladium on carbon (10 mg, 83.33 μmol) in one portion under H2 (15 psi) at 25° C. After stirring at 25° C. for 1 h, the mixture was filtered and concentrated to give the compound (25 mg, 50%) as a white oil.

[0337] Step 2. To a solution of 4-methylbenzoate [(2R,3S,5R)-5-(5-cyano-2,4-dioxo-pyrimidin-1-yl)-2-ethyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (50 mg, 96.61 μmol) in MeOH (5 mL) was added NaOMe (521.94 μg, 9.66 μmol) in one portion at 25° C. After stirring for 1 h at 25° C., the mixture was concentrated. The residue was purified by preparative HPLC (3-33% acetonitrile / 0.225% formic acid in water) to give the title compound (19 mg, 71%) as a white solid. TIFF2025511024000075.tif27153

[0338] Example 16 Synthesis of (9-((2R,4S,5R)-5-ethyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-ol (Compound No. 1) TIFF2025511024000076.tif331589 4-Methylbenzoate [(2R,3S,5R)-2-ethynyl-5-(6-hydroxypurin-9-yl)-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl was synthesized using H-purin-6-ol according to the procedure described in WO 2007 / 038507 A2.

[0339] Step 1. A mixture of [(2R,3S,5R)-2-ethynyl-5-(6-hydroxypurin-9-yl)-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (50 mg, 97 μmol) and Lindlar's catalyst (20 mg) in MeOH (5 mL) was stirred under H2 (15 psi) at 20° C. for 30 min. The reaction mixture was then filtered and concentrated to give the compound (40 mg, 79%) as a white solid.

[0340] Step 2. To a solution of 4-methylbenzoate [(2R,3S,5R)-2-ethyl-5-(6-hydroxypurin-9-yl)-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (40 mg, 77 μmol) in THF (3 mL) was added NaOMe (2 mg, 38 μmol) in MeOH (2 mL) at 0° C. The resulting mixture was stirred at 25° C. for 16 h. The reaction mixture was then concentrated. The residue was purified by preparative HPLC (basic) to give the title compound (4.3 mg, 20%) as a white solid. TIFF2025511024000077.tif19153

[0341] Example 17 Synthesis of (2R,3S,5R)-5-(6-(cyclopropylamino)-2-fluoro-9H-purin-9-yl)-2-ethyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (Compound No. 99) TIFF2025511024000078.tif87150 4-Methylbenzoic acid [(2R,3S,5R)-5-(6-chloro-2-fluoro-purin-9-yl)-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl was synthesized using N-cyclopropyl-2-fluoro-9H-purin-6-amine according to the procedure described in WO 2007 / 038507 A2.

[0342] Step 1. To a solution of 4-methylbenzoic acid [(2R,3S,5R)-5-(6-chloro-2-fluoro-purin-9-yl)-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (200 mg, 0.364 mmol) in t-BuOH (2 mL) was added DIPEA (63 μl, 0.364 mmol) and cyclopropanamine (22 mg, 0.383 mmol). The reaction mixture was stirred at 90 °C for 16 h. After cooling to room temperature, the mixture was diluted with water (10 mL), extracted with EtOAc (20 mL), washed with brine (20 mL × 2), dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (0% to 33% EtOAc in petroleum ether) to give the compound (140 mg, 67%) as a white solid. TIFF2025511024000079.tif35153

[0343] Step 2. To a mixture of [(2R,3S,5R)-5-[6-(cyclopropylamino)-2-fluoro-purin-9-yl]-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (90 mg, 158.01 μmol) in EtOAc (5 mL) was added palladium on carbon (25 mg, 47.40 μmol, 20% purity) in one portion at 25° C. under H2. The mixture was stirred at 25° C. under H2 (15 psi) for 16 h. The reaction mixture was then filtered and the filtrate was concentrated to give the compound (75 mg, 83%) as a white solid.

[0344] Step 3. To a solution of 4-methylbenzoate [(2R,3S,5R)-5-[6-(cyclopropylamino)-2-fluoro-purin-9-yl]-2-ethyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (70 mg, 122.03 μmol) in MeOH (3 mL) was added NH3.H2O (1 mL, 6.49 mmol, 25% purity) at 25 °C. The resulting mixture was stirred at 40 °C for 16 h. The reaction mixture was then concentrated under reduced pressure. The residue was purified by reverse phase HPLC (1-27% acetonitrile / 0.225% formic acid in water) to give the title compound (6.5 mg, 16%) as a white solid. TIFF2025511024000080.tif27154

[0345] Example 18 Synthesis of 9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-vinyltetrahydrofuran-2-yl)-9H-purin-6-ol (Compound No. 100) TIFF2025511024000081.tif741379 4-Methylbenzoate [(2R,3S,5R)-2-ethynyl-5-(6-hydroxypurin-9-yl)-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl was synthesized using H-purin-6-ol according to the procedure described in WO 2007 / 038507 A2.

[0346] Step 1. A mixture of [(2R,3S,5R)-2-ethynyl-5-(6-hydroxypurin-9-yl)-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (50 mg, 98 μmol) and Lindlar's catalyst (20 mg) in MeOH (20 mL) was stirred at 20° C. for 30 min under H2 (15 psi). The reaction mixture was then filtered. The filtrate was concentrated to give the compound (35 mg, 69%) as a white solid.

[0347] Step 2. To a solution of [(2R,3S,5R)-5-(6-hydroxypurin-9-yl)-3-(4-methylbenzoyl)oxy-2-vinyl-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (20 mg, 39 μmol) in THF (3 mL) was added NaOMe (1.1 mg, 19 μmol) in MeOH (2 mL) at 0° C. The resulting mixture was stirred at 25° C. for 16 h. The reaction mixture was then concentrated. The residue was purified by preparative HPLC (basic) to give the title compound (4 mg, 36%) as a white solid. TIFF2025511024000082.tif27153

[0348] Example 19 Synthesis of 5-bromo-1-((2R,4S,5R)-5-ethyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-hydroxypyrimidin-2(1H)-one (Compound No. 2) TIFF2025511024000083.tif811394-Methylbenzoate [(2R,3S)-5-acetoxy-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl was synthesized according to the procedure described in WO 2007 / 038507 A2.

[0349] Step 1. To a solution of [(2R,3S)-5-acetoxy-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (200 mg, 458.24 μmol) in MeOH (10 mL) was added 10% palladium on carbon (9.00 mg) in one portion under H2 (15 psi) at 25° C. The resulting mixture was stirred at 25° C. for 3 h. The reaction mixture was then filtered and concentrated to give the compound (0.2 g, 99%) as a white solid.

[0350] Step 2. To a solution of 5-bromo-1H-pyrimidine-2,4-dione (173.4 mg, 908.09 μmol) in MeCN (10 mL) was added BTMSA (309.5 mg, 1.82 mmol) in one portion under N2 at 25° C. The mixture was then stirred at 70° C. for 1 h. After cooling to 25° C., TMSOTf (131.2 mg, 590.26 μmol) and a solution of 4-methylbenzoate [(2R,3S)-5-acetoxy-2-ethyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (0.2 g, 454.05 μmol) in MeCN (5 mL) were added dropwise at 25° C. The resulting mixture was stirred at 25° C. for 1 h. The reaction was then quenched by the addition of 10 mL of aqueous citric acid (10 wt%) and then extracted with EtOAc (20 mL). The layers were separated and the organic layer was washed with brine (10 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by preparative TLC (50% EtOAc in petroleum ether) to give the compound (52 mg, 20%) as a colorless oil. TIFF2025511024000084.tif27153

[0351] Step 3. To a solution of 4-methylbenzoate [(2R,3S,5R)-5-(5-bromo-2,4-dioxo-pyrimidin-1-yl)-2-ethyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (0.02 g, 35.00 μmol) in MeOH (5 mL) was added NaOMe (189 μg, 3.50 μmol) in one portion at 25° C. The resulting mixture was stirred at 25° C. for 1 h. The mixture was then concentrated under reduced pressure. The residue was purified by preparative HPLC (acetonitrile 0-30% / 0.225% formic acid in water) to give the title compound (5.4 mg, 46%) as a white solid. TIFF2025511024000085.tif27154

[0352] Example 20 Synthesis of (2R,3S,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-2-(hydroxymethyl)-2-vinyltetrahydrofuran-3-ol (Compound No. 102) TIFF2025511024000086.tif301536-Methoxy-9H-purin-2-amine was used to prepare 4-methylbenzoate [(2R,3S,5R)-5-(2-amino-6-methoxy-purin-9-yl)-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl according to WO 2007 / 038507 A2.

[0353] Step 1. To a solution of [(2R,3S,5R)-5-(2-amino-6-methoxy-purin-9-yl)-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (20 mg, 36.93 μmol) in MeOH (2 mL) was added Lindlar's catalyst (8 mg) in one portion under H2 (15 psi) at 25° C. The resulting mixture was stirred at 25° C. for 1 h. The mixture was then filtered and the filtrate was concentrated to give the compound (20 mg, 99%) as a white solid.

[0354] Step 2. To a mixture of [(2R,3S,5R)-5-(2-amino-6-methoxy-purin-9-yl)-3-(4-methylbenzoyl)oxy-2-vinyl-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (17 mg, 31.27 μmol) in MeOH (5 mL) was added NaOMe (169 μg, 3.13 μmol) in one portion at 25° C. After stirring at 25° C. for 1 h, the mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (basic) to give the title compound (2.5 mg, 26%) as a white solid. LCMS (ESI): m / z 308.2 (M+H)+. TIFF2025511024000087.tif27153

[0355] Example 21 Synthesis of (2R,3S,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-2-ethyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (Compound No. 125) TIFF2025511024000088.tif301536-Methoxy-9H-purin-2-amine was used to prepare 4-methylbenzoate [(2R,3S,5R)-5-(2-amino-6-methoxy-purin-9-yl)-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl according to WO 2007 / 038507 A2.

[0356] Step 1. To a mixture of 4-methylbenzoate [(2R,3S,5R)-5-(2-amino-6-methoxy-purin-9-yl)-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (0.1 g, 184.65 μmol) in EtOAc (5 mL) was added Pd / C (60 mg, 10% purity) under H2 (15 psi). The mixture was stirred at 25° C. for 3 h. The mixture was then filtered and concentrated to give the title compound (80 mg, 79% yield) as a white solid.

[0357] Step 2. To a mixture of 4-methylbenzoate [(2R,3S,5R)-5-(2-amino-6-methoxy-purin-9-yl)-2-ethyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (80 mg, 146.63 μmol) in MeOH (5 mL) was added NaOMe (1 mg, 14.66 μmol) in MeOH (2 mL) at 0° C. The resulting mixture was stirred at 25° C. for 16 h. The reaction mixture was then concentrated. The residue was purified by preparative HPLC (basic) to give the title compound (7 mg, 15%) as a white solid. TIFF2025511024000089.tif20153

[0358] Example 22 Synthesis of 1-((2R,4S,5R)-5-ethyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methoxypyrimidine-2,4(1H,3H)-dione (Compound No. 103) TIFF2025511024000090.tif29130 4-Methylbenzoic acid [(2R,3S,5R)-2-ethynyl-5-(5-methoxy-2,4-dioxo-pyrimidin-1-yl)-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl was prepared according to WO 2007 / 038507 A2.

[0359] Step 1. To a mixture of [(2R,3S,5R)-2-ethynyl-5-(5-methoxy-2,4-dioxo-pyrimidin-1-yl)-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (15 mg, 28.93 μmol) in MeOH (5 mL) was added 10% palladium on carbon (9 mg) in one portion at 25° C. under H2 (15 psi). The resulting mixture was stirred at 25° C. for 1 h. The mixture was then filtered and the filtrate was concentrated to give the compound (15 mg, 99%) as a white solid.

[0360] Step 2. To a mixture of [(2R,3S,5R)-2-ethyl-5-(5-methoxy-2,4-dioxo-pyrimidin-1-yl)-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (15 mg, 28.71 μmol) in MeOH (5 mL) was added NaOMe (155 μg, 2.87 μmol) in one portion at 25° C. After stirring for 18 h at 25° C., the mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (basic) to give the title compound (2.3 mg, 28%) as a white solid. TIFF2025511024000091.tif19153

[0361] Example 23 Synthesis of 5-fluoro-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-vinyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound No. 17) TIFF2025511024000092.tif281285-Fluoropyrimidine-2,4(1H,3H)-dione was used to prepare 4-methylbenzoate [(2R,3S,5R)-2-ethynyl-5-(5-fluoro-4-hydroxy-2-oxo-pyrimidin-1-yl)-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl according to WO 2007 / 038507 A2.

[0362] Step 1. A mixture of 4-methylbenzoate [(2R,3S,5R)-2-ethynyl-5-(5-fluoro-4-hydroxy-2-oxo-pyrimidin-1-yl)-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (30 mg, 59.23 μmol) and Lindlar catalyst (12 mg) in EtOAc (2 mL) was stirred at 20° C. for 10 min under H2 (15 psi). The reaction mixture was then filtered and the filtrate was concentrated to give the compound (30 mg, 99%) as a white solid. LCMS (ESI): m / z 531.1 (M+Na)+

[0363] Step 2. A mixture of 4-methylbenzoate [(2R,3S,5R)-5-(5-fluoro-4-hydroxy-2-oxo-pyrimidin-1-yl)-3-(4-methylbenzoyl)oxy-2-vinyl-tetrahydrofuran-2-yl]methyl (30 mg, 59 μmol) and NaOMe (3.19 mg, 59 μmol) in MeOH (2 mL) and THF (3 mL) was stirred at 20° C. for 3 h. The reaction mixture was then concentrated to give the crude product, which was purified by preparative HPLC (basic) to give the title compound (4.3 mg, 27%) as a white solid. TIFF2025511024000093.tif19153

[0364] Example 24 Synthesis of 1-((2R,4S,5R)-5-ethyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-fluoropyrimidine-2,4(1H,3H)-dione (Compound No. 18) TIFF2025511024000094.tif281285-Fluoropyrimidine-2,4(1H,3H)-dione was used to prepare 4-methylbenzoate [(2R,3S,5R)-2-ethynyl-5-(5-fluoro-4-hydroxy-2-oxo-pyrimidin-1-yl)-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl according to WO 2007 / 038507 A2.

[0365] Step 1. A mixture of 4-methylbenzoic acid [(2R,3S,5R)-2-ethynyl-5-(5-fluoro-4-hydroxy-2-oxo-pyrimidin-1-yl)-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (50 mg, 98.72 μmol) and Lindlar catalyst (5 mg) in MeOH (5 mL) and EtOAc (5 mL) was stirred at 20° C. for 5 h under H2 (15 psi). The reaction mixture was then filtered and the filtrate was concentrated to give the compound (40 mg, 79%) as a white solid. LCMS (ESI): m / z 533.1 (M+Na)+.

[0366] Step 2. A mixture of 4-methylbenzoic acid [(2R,3S,5R)-2-ethyl-5-(5-fluoro-4-hydroxy-2-oxo-pyrimidin-1-yl)-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (40 mg, 78.35 μmol) and NaOMe (4.23 mg, 78.35 μmol) in MeOH (2 mL) and THF (3 mL) was stirred at 20° C. for 3 h. The reaction mixture was then concentrated to give the crude product, which was purified by preparative HPLC (basic) to give the title compound (5.8 mg, 27%) as a white solid. TIFF2025511024000095.tif19153

[0367] Example 25 Synthesis of 4-amino-1-((2R,4R,5S)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (Compound No. 77) TIFF2025511024000096.tif83145 Step 1. To a mixture of N-(2-oxo-1H-pyrimidin-4-yl)benzamide (148 mg, 687.36 μmol) in MeCN (15 mL) was added BTMSA (234 mg, 1.37 mmol). The resulting mixture was heated at 70° C. for 1 h. After cooling to ambient temperature, TMSOTf (99 mg, 446.78 μmol) and a solution of [(2S,3R)-5-acetoxy-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (150 mg, 343.68 μmol) were added sequentially. The resulting mixture was again heated at 70° C. for 1 h. After cooling to ambient temperature, the reaction mixture was poured into water (10 mL), extracted with EtOAc (20 mL×2) and concentrated. The residue was purified by SFC (DAICEL CHIRALPAK AD, (250 mm*30 mm, 10 um); supercritical CO2 / EtOH + NH4OH= 45 / 55; 80 mL / min) to give the compound (50 mg, 22%) as a white solid. TIFF2025511024000097.tif34153

[0368] Step 2. To a mixture of 4-methylbenzoate [(2S,3R,5R)-5-(4-benzamido-2-oxo-pyrimidin-1-yl)-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (50 mg, 84.52 μmol) in MeOH (2 mL) was added NaOMe (9 mg, 169.03 μmol) at 0° C. The resulting mixture was stirred at 25° C. for 16 h. The reaction mixture was then concentrated. The residue was purified by preparative HPLC (1-15% acetonitrile / 0.225% formic acid in water) to give the title compound (3.5 mg, 16%) as a white solid. TIFF2025511024000098.tif34153

[0369] Example 26 Synthesis of 2-amino-9-[(2R,4R,5S)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-1H-purin-6-one (Compound No. 79) TIFF2025511024000099.tif63135 Step 1. A solution of N-(6-hydroxy-9H-purin-2-yl)-2-methyl-propanamide (84 mg, 378.05 μmol) in MeCN (10 mL) was degassed with N2 three times. (1E)-N-trimethylsilylethanimidate trimethylsilyl (349 mg, 1.72 mmol) was then added. The resulting mixture was stirred at 70° C. for 1 h and then cooled to room temperature. Trimethylsilyl trifluoromethanesulfonate (88 mg, 395.23 μmol) was added, followed by [(2S,3R)-5-acetoxy-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (150 mg, 343.68 μmol) in acetonitrile (2 mL) in one portion at 25 °C under N2. The mixture was stirred at 70 °C for 16 h. After cooling to room temperature, the mixture was diluted with water (10 mL), extracted with EtOAc (20 mL), washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by preparative HPLC (54-85% acetonitrile / 0.225% formic acid in water) to give the compound (25 mg, 15%) as a white solid. TIFF2025511024000100.tif34153

[0370] Step 2. To a solution of 4-methylbenzoate [(2S,3R,5R)-2-ethynyl-5-[6-hydroxy-2-(2-methylpropanoylamino)purin-9-yl]-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (21 mg, 35.14 μmol) in MeOH (2 mL) was added ammonium hydroxide (30 mg, 105.42 μmol, 28 wt%). The solution was stirred at 60° C. for 16 h. After cooling to room temperature, the reaction mixture was concentrated. The residue was purified by preparative HPLC (1-20% acetonitrile / 0.225% formic acid in water) to give the title compound (3.0 mg, 29%) as a white solid. TIFF2025511024000101.tif19153

[0371] Example 27 Synthesis of (2R,3S,5R)-5-(4-amino-6-fluoro-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (Compound No. 75) TIFF2025511024000102.tif67140 3-Methyl-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine was used to prepare 4-methylbenzoate [(2R,3S,5R)-5-(4,6-diamino-3-methyl-pyrazolo[3,4-d]pyrimidin-1-yl)-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl according to WO 2007 / 038507 A2.

[0372] Step 1. To a mixture of 4-methylbenzoic acid [(2R,3S,5R)-5-(4,6-diamino-3-methyl-pyrazolo[3,4-d]pyrimidin-1-yl)-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (120 mg, 221.99 μmol) in MeOH (15 mL) was added NaOMe (24 mg, 443.98 μmol) in one portion at 0° C. under N2. The resulting mixture was stirred at 25° C. for 16 h. The reaction mixture was then concentrated. The residue was purified by preparative TLC (9% MeOH in dichloromethane (1% NH4OH)) to give the compound (60 mg, 89%) as a white solid. LCMS (ESI): m / z 327.1 (M+Na)+.

[0373] Step 2. To a mixture of (2R,3S,5R)-5-(4,6-diamino-3-methyl-pyrazolo[3,4-d]pyrimidin-1-yl)-2-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (60 mg, 197.17 μmol) in pyridine (2 mL) was added pyridine hydrogen fluoride (237 mg, 1.68 mmol, 70% purity) and tert-butyl nitrite (61 mg, 591.51 μmol) dropwise at 0° C. under N2. The resulting mixture was stirred at 0° C. for 1 h. The mixture was then diluted with DCM (15 mL) and H2O (5 mL). The layers were separated and the aqueous layer was extracted with DCM (10 mL×3). The combined organic extracts were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was further purified by preparative HPLC (10-40% acetonitrile / 0.225% formic acid in water) to give the title compound (1.58 mg, 3%). TIFF2025511024000103.tif19153

[0374] Example 28 Synthesis of (2R,3S,5S)-5-(4-amino-6-fluoro-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (Compound No. 3) TIFF2025511024000104.tif30156 3-Methyl-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine was used to prepare 4-methylbenzoate [(2R,3S,5S)-5-(4,6-diamino-3-methyl-pyrazolo[3,4-d]pyrimidin-1-yl)-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl according to WO 2007 / 038507 A2.

[0375] Step 1. To a mixture of 4-methylbenzoic acid [(2R,3S,5S)-5-(4,6-diamino-3-methyl-pyrazolo[3,4-d]pyrimidin-1-yl)-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (0.1 g, 184.99 μmol) in MeOH (15 mL) was added NaOMe (20 mg, 369.98 μmol) in one portion at 0° C. under N2. The mixture was stirred at 25° C. for 18 h. The reaction mixture was then concentrated and the residue was purified by preparative TLC eluting with 10% MeOH (1% NH4OH) in DCM (Rf = 0.3) to give the compound (50 mg, 89%) as a white solid.

[0376] Step 2. To a mixture of (2R,3S,5S)-5-(4,6-diamino-3-methyl-pyrazolo[3,4-d]pyrimidin-1-yl)-2-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (60 mg, 197.17 μmol) in pyridine (2 mL) was added HF-pyridine (237 mg, 1.68 mmol, 70% purity) and tert-butyl nitrite (61 mg, 591.51 μmol) dropwise at 0° C. under N2. The resulting mixture was stirred at 0° C. for 1 h. The mixture was then diluted with DCM (15 mL) and H2O (5 mL). The layers were separated and the aqueous layer was extracted with DCM (10 mL). The combined organic extracts were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by preparative HPLC (10-40% acetonitrile / 0.225% formic acid in water) to afford the title compound (5 mg, 8%) as a white solid. TIFF2025511024000105.tif19158

[0377] Example 29 Synthesis of 4-amino-1-[(2S,5S)-5-ethynyl-5-(hydroxymethyl)-2H-furan-2-yl]-5-fluoro-pyrimidin-2-one (Compound No. 74) TIFF2025511024000106.tif76131 Step 1. A solution of N-(5-fluoro-2-hydroxy-pyrimidin-4-yl)benzamide (59 mg, 253.94 μmol) in acetonitrile (3 mL) was degassed with N2 three times. (1E)-N-trimethylsilylethanimidate trimethylsilyl (129 mg, 634.85 μmol) was then added. The reaction mixture was stirred at 70° C. for 1 h and then cooled to room temperature. Trimethylsilyl trifluoromethanesulfonate (56 mg, 253.94 μmol) was added, followed by [(2S,4S,5R)-5-benzoyloxy-2-ethynyl-4-(p-tolylsulfanyl)tetrahydrofuran-2-yl]methyl benzoate (100 mg, 211.62 μmol) in acetonitrile (1 mL) under N2 at 25°C. After stirring at 70°C for 16 h, the reaction mixture was cooled to room temperature, diluted with water (10 mL), extracted with EtOAc (20 mL), washed with brine (20 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by preparative TLC (25% EtOAc in petroleum ether) to give the compound (21 mg, 17%) as a white solid. TIFF2025511024000107.tif34153

[0378] Step 2. To a solution of [(2S,4S,5S)-5-(4-benzamido-5-fluoro-2-oxo-pyrimidin-1-yl)-2-ethynyl-4-(p-tolylsulfanyl)tetrahydrofuran-2-yl]methyl benzoate (40 mg, 68.54 μmol) in MeCN (2 mL) was added [chloro(p-tolylsulfonyl)amino]sodium (22 mg, 95.95 μmol). The reaction mixture was stirred at 50° C. for 2.5 h. Water (10 mL) was added to the reaction mixture, which was extracted with EtOAc (20 mL), washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated. The residue was dissolved in n-BuOH (2 mL). The solution was then stirred at 90° C. for an additional 3 h. After cooling to room temperature, the reaction mixture was concentrated. The residue was purified by preparative TLC (25% EtOAc in petroleum ether) to give the compound (39 mg, 51%) as a white solid. LCMS (ESI): m / z 460.0 (M+H)+.

[0379] Step 3. To a solution of [(2S,5S)-2-(4-benzamido-5-fluoro-2-oxo-pyrimidin-1-yl)-5-ethynyl-2H-furan-5-yl]methyl benzoate (20 mg, 43.53 μmol) in MeOH (1 mL) was added MeONa (3 mg, 53.53 μmol) at 0° C. The resulting mixture was stirred at 25° C. for 3 h. The reaction was then concentrated. The residue was purified by preparative HPLC (1-27% acetonitrile / 0.225% formic acid in water) to give the title compound (5.93 mg, 19%) as a white solid. TIFF2025511024000108.tif19153

[0380] Example 30 Synthesis of 4-amino-1-((2S,4R,5S)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (Compound No. 76) TIFF2025511024000109.tif79140 Step 1. To a mixture of N-(2-oxo-1H-pyrimidin-4-yl)benzamide (148 mg, 687.36 μmol) in MeCN (15 mL) was added BTMSA (234 mg, 1.37 mmol). The resulting mixture was heated at 70° C. for 1 h. After cooling to ambient temperature, TMSOTf (99 mg, 446.78 μmol) and 4-methylbenzoate [(2S,3R)-5-acetoxy-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (150 mg, 343.68 μmol) were added sequentially. The resulting mixture was heated again at 70° C. for 1 h. After cooling to ambient temperature, the reaction mixture was poured into water (10 mL), extracted with EtOAc (20 mL×2) and concentrated. The residue was purified by SFC (DAICEL CHIRALPAK AD, (250 mm*30 mm, 10 um); supercritical CO2 / EtOH+NH4OH= 45 / 55; 80 mL / min) to give the compound (20 mg, 22%) as a white solid.

[0381] Step 2. To a mixture of 4-methylbenzoate [(2S,3R,5S)-5-(4-benzamido-2-oxo-pyrimidin-1-yl)-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl (0.02 g, 33.81 μmol) in MeOH (2 mL) was added NaOMe (4 mg, 67.61 μmol) at 0° C. The resulting mixture was stirred at 25° C. for 16 h. The reaction mixture was then concentrated. The residue was purified by preparative HPLC (1-17% acetonitrile / 0.225% formic acid in water) to give the title compound (3.8 mg, 16%) as a white solid. TIFF2025511024000110.tif27154

[0382] Example 31 Synthesis of 2-amino-9-((2S,4R,5S)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H-purin-6(9H)-one (Compound No. 78) TIFF2025511024000111.tif65139 Step 1. A solution of N-(6-hydroxy-9H-purin-2-yl)-2-methyl-propanamide (84 mg, 378.05 μmol) in acetonitrile (10 mL) was degassed with N2 three times. (1E)-N-trimethylsilylethanimidate trimethylsilyl (349 mg, 1.72 mmol) was then added. After stirring at 70 °C for 1 h, the reaction mixture was cooled to room temperature. Trimethylsilyl trifluoromethanesulfonate (88 mg, 395.23 μmol) was added, followed by [(2S,3R)-5-acetoxy-2-ethynyl-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (150 mg, 343.68 μmol) in acetonitrile (2 mL) in one portion at 25 °C under N2. After stirring at 70 °C for 16 h, the reaction mixture was cooled to room temperature, diluted with water (20 mL), extracted with EtOAc (30 mL), washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by preparative HPLC (54-85% acetonitrile / 0.225% formic acid in water) to give the compound (21 mg, 10%) as a white solid. TIFF2025511024000112.tif34153

[0383] Step 2. To a solution of [(2S,3R,5S)-2-ethynyl-5-[6-hydroxy-2-(2-methylpropanoylamino)purin-9-yl]-3-(4-methylbenzoyl)oxy-tetrahydrofuran-2-yl]methyl 4-methylbenzoate (21 mg, 35.14 μmol) in methyl alcohol (2 mL) was added ammonium hydroxide (300 mg, 1054.2 μmol, 28% purity). The solution was stirred at 60° C. for 16 h. The reaction was then cooled to room temperature and concentrated. The residue was purified by preparative HPLC (1-20% acetonitrile / 0.225% formic acid in water) to give the title compound (3.0 mg, 29%) as a white solid. TIFF2025511024000113.tif27153

[0384] Example 32 Synthesis of 1-((4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H-1,2,4-triazole-3-carboxamide (Compound No. 11) TIFF2025511024000114.tif70128 Step 1: (2R,3S)-5-(3-carbamoyl-1H-1,2,4-triazol-1-yl)-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-methylbenzoate. To a suspension of 1H-1,2,4-triazole-3-carboxamide (57.7 mg, 504 μmol) in MeCN (3.88 mL) was added N,O-bis(trimethylsilyl)acetamide (590 μL, 2.29 mmol) and the resulting mixture was heated to 70° C. with stirring for about 1 h. The mixture was then cooled to 0° C. and a solution of (2R,3S)-5-acetoxy-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-methylbenzoate (200 mg, 458 μmol) in MeCN (3.88 mL) was added dropwise with stirring, followed by trimethylsilyl trifluoromethanesulfonate (127 μL, 687 μmol). The reaction mixture was then warmed to 70° C. with stirring for approximately 18 hours, and then cooled to ambient temperature. The reaction was quenched with saturated aqueous NaHCO3 (10 mL), diluted with saturated aqueous NaCl (12 mL) and the organics extracted with EtOAc (3×30 mL). The combined organics were dried (anhydrous Na2SO4), filtered and concentrated in vacuo. The residue was purified by SiO2 flash chromatography (eluent: 50-100% EtOAc in heptane) to give the title compound as a mixture of anomers (132 mg, 270 μmol, 59%) as a yellow oil. TIFF2025511024000115.tif5128. For the less polar anomer, TIFF2025511024000116.tif5128.

[0385] Step 2: 1-((4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H-1,2,4-triazole-3-carboxamide. To a solution of (2R,3S)-5-(3-carbamoyl-1H-1,2,4-triazol-1-yl)-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-methylbenzoate (132 mg, 270 μmol) in MeOH (901 μmol) was added dropwise 28% aqueous NH4OH (60 μL) and the resulting mixture was stirred at ambient temperature for about 18 hours. After this time, an additional portion of 28% aqueous NH4OH (60 μL) was added dropwise and the mixture was heated to 45° C. with stirring for about 4 hours. The mixture was then cooled to ambient temperature and purified directly by C18 reverse phase chromatography (eluent: 0-100% MeCN in 10 mM aqueous ammonium formate) to give compound No. 11 (61.3 mg, 243 μmol, 90%) as a approx. 2:1 mixture of anomers as a yellow oil. TIFF2025511024000117.tif19153. For the minor anomer, TIFF2025511024000118.tif27153. TIFF2025511024000119.tif5128

[0386] Example 33 Synthesis of 4-amino-5-bromo-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-methyltetrahydrofuran-2-yl)pyrimidin-2(1H)-one (Compound No. 15) TIFF2025511024000120.tif96154 Step 1: (2R,3R,4S,5R)-2-(4-amino-5-bromo-2-oxopyrimidin-1(2H)-yl)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-3-yl acetate. To a mixture of 4-amino-5-bromo-1H-pyrimidin-2-one (0.38 g, 2.00 mmol) in MeCN (15 mL) at 25° C. under N2 was added N,O-bis(trimethylsilyl)acetamide (1.22 g, 6.00 mmol) in one portion. The mixture was then stirred at 70° C. for 1 h. After cooling to 25°C, TMSOTf (390 mg, 1.76 mmol) and (3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-2,3-diyl diacetate (0.5 g, 1.17 mmol) were added dropwise at 25°C. The resulting mixture was stirred at 25°C for 16 h. The reaction was then quenched by the addition of 10 wt% aqueous citric acid (10 mL) and extracted with EtOAc (20 mL). The organic layer was washed with brine (15 mL), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, DCM / MeOH = 50 / 1) to give the title compound (0.64 g, 98% yield) as a yellow oil. TIFF2025511024000121.tif34153

[0387] Step 2: 4-amino-1-((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3-hydroxy-5-methyltetrahydrofuran-2-yl)-5-bromopyrimidin-2(1H)-one. To a mixture of (2R,3R,4S,5R)-2-(4-amino-5-bromo-2-oxopyrimidin-1(2H)-yl)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-3-yl acetate (0.638 g, 1.14 mmol) in MeOH (15 mL) was added aqueous NaOH (2 M, 2 mL) in one portion at 25° C. The mixture was stirred at 25° C. for 30 min. The reaction mixture was then concentrated. The residue was purified by silica gel column chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, dichloromethane / MeOH = 100 / 0-50 / 1) to give the title compound (0.5 g, yield 85%) as a white solid. TIFF2025511024000122.tif27153

[0388] Step 3: O-((2R,3R,4S,5R)-2-(4-amino-5-bromo-2-oxopyrimidin-1(2H)-yl)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-3-yl) O-phenyl carbonothioate. To a mixture of 4-amino-1-((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3-hydroxy-5-methyltetrahydrofuran-2-yl)-5-bromopyrimidin-2(1H)-one (0.5 g, 968.27 μmol) in MeCN (15 mL) was added DMAP (355 mg, 2.90 mmol) followed by the dropwise addition of O-phenyl chloromethanethioate (251 mg, 1.45 mmol) at 25° C. under N2. After stirring at 25°C for 1 h, the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, DCM / MeOH = 100 / 0-99 / 1) to give the title compound (0.55 g, yield 87%) as a pale yellow solid. TIFF2025511024000123.tif27153

[0389] Step 4: 4-amino-1-((2R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-2-yl)-5-bromopyrimidin-2(1H)-one. To a mixture of carbonothioate O-((2R,3R,4S,5R)-2-(4-amino-5-bromo-2-oxopyrimidin-1(2H)-yl)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-3-yl) O-phenyl (0.55 g, 842.84 μmol) in toluene (12 mL) was added AIBN (69 mg, 421.42 μmol) and TTMSS (1.05 g, 4.21 mmol) in one portion at 25° C. under N2. The mixture was heated to 110° C. and stirred for 2 h. The reaction mixture was then cooled to 25° C. and concentrated. The residue was purified by preparative TLC (DCM / MeOH=15 / 1) to give the title compound (170 mg, 40% yield) as a colorless oil.

[0390] Step 5: 4-Amino-5-bromo-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-methyltetrahydrofuran-2-yl)pyrimidin-2(1H)-one (compound number 15). To a mixture of 4-amino-1-((2R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-2-yl)-5-bromopyrimidin-2(1H)-one (0.15 g, 299.77 μmol) in DCM (9 mL) was added BCl3 (1 M, 2.1 mL) dropwise at −78° C. under N2. The reaction mixture was stirred at −78° C. for 15 min, then warmed to −40° C. and stirred for an additional 0.5 h. The reaction was then quenched by the addition of MeOH (1 mL) and NH4OH (1 mL) at −40° C. After stirring at -40°C for an additional 10 min, the mixture was warmed to room temperature and stirred for 10 min. The reaction mixture was then directly concentrated. The residue was purified by preparative HPLC (basic) to give compound no. 15 (2.7 mg, 3% yield) as a white solid. TIFF2025511024000124.tif19158

[0391] Example 34 Synthesis of (2R,3S,5R)-5-(4-amino-5-bromo-2-oxopyrimidin-1(2H)-yl)-3-hydroxy-2-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (Compound No. 16) TIFF2025511024000125.tif93151 Step 1: (2R,3R,4S,5R)-2-(4-amino-5-bromo-2-oxopyrimidin-1(2H)-yl)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-cyanotetrahydrofuran-3-yl acetate. To a solution of 4-amino-5-bromo-1H-pyrimidin-2-one (400 mg, 2.1 mmol) in acetonitrile (15 mL) was added N,O-bis(trimethylsilyl)acetamide (1.6 mL, 6.3 mmol) and the reaction mixture was stirred at 70° C. for 1 h and cooled to room temperature. Trimethylsilyl trifluoromethanesulfonate (490 uL, 2.7 mmol) was added followed by (3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-cyanotetrahydrofuran-2,3-diyl diacetate (600 mg, 1.4 mmol) in acetonitrile (8 mL). After stirring at 105 °C for 16 h, the reaction was quenched with water (20 mL), extracted with EtOAc (30 mL), washed with brine (20 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel chromatography eluting with 0-3% MeOH in DCM to give the title product (302 mg, 0.53 mmol, 39% yield) as a brown oil. TIFF2025511024000126.tif20153

[0392] Step 2: (2R,3S,4R,5R)-5-(4-amino-5-bromo-2-oxopyrimidin-1(2H)-yl)-3-(benzyloxy)-2-((benzyloxy)methyl)-4-hydroxytetrahydrofuran-2-carbonitrile. To a solution of (2R,3R,4S,5R)-2-(4-amino-5-bromo-2-oxopyrimidin-1(2H)-yl)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-cyanotetrahydrofuran-3-yl acetate (300 mg, 526.9 μmol) in dioxane (8 mL) was added aqueous NaOH (1 M, 1.0 mL) at 25° C. The resulting reaction mixture was stirred at 25° C. for 1 h. The reaction was then quenched with water (5 mL), extracted with EtOAc (10 mL), washed with brine (10 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel chromatography eluting with 0-3% MeOH in DCM to give the title product (260 mg, 94% yield) as a white solid.

[0393] Step 3: A solution of (2R,3S,4R,5R)-5-(4-amino-5-bromo-2-oxo-pyrimidin-1-yl)-3-benzyloxy-2-(benzyloxymethyl)-4-hydroxy-tetrahydrofuran-2-carbonitrile (260.0 mg, 493.0 μmol) in acetonitrile (5 mL) was degassed with N2 three times. DMAP (78.3 mg, 640.9 μmol) and O-phenyl chloromethanethioate (170.2 mg, 986.0 μmol) were then added. The resulting reaction mixture was stirred at 25° C. for 1 h. The reaction mixture was then diluted with water (5 mL), extracted with EtOAc (10 mL), washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography eluting with 0-3% MeOH in DCM to afford the title product (251.0 mg, 378 μmol, 76% yield) as a brown oil. TIFF2025511024000127.tif5128

[0394] Step 4: (2R,3S,5R)-5-(4-amino-5-bromo-2-oxopyrimidin-1(2H)-yl)-3-(benzyloxy)-2-((benzyloxy)methyl)tetrahydrofuran-2-carbonitrile. A solution of (2R,3S,4R,5R)-5-(4-amino-5-bromo-2-oxo-pyrimidin-1-yl)-3-benzyloxy-2-(benzyloxymethyl)-4-hydroxy-tetrahydrofuran-2-carbonitrile (250 mg, 376.8 μmol) in toluene (5 mL) was purged with N2 for 5 min. Bis(trimethylsilyl)silyl-trimethyl-silane (281.1 mg, 1.13 mmol) and AIBN (29.0 mg, 176.8 μmol) were added. The reaction mixture was stirred at 110° C. for 2 h. The reaction mixture was concentrated, and the residue was purified by silica gel chromatography eluting with 0-3% MeOH in DCM to give the title product (95.0 mg, 186 μmol, 49% yield) as a white solid.

[0395] Step 5: (2R,3S,5R)-5-(4-amino-5-bromo-2-oxopyrimidin-1(2H)-yl)-3-hydroxy-2-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (compound number 16). A solution of (2R,3S,5R)-5-(4-amino-5-bromo-2-oxopyrimidin-1(2H)-yl)-3-(benzyloxy)-2-((benzyloxy)methyl)tetrahydrofuran-2-carbonitrile (95.0 mg, 186 μmol) in DCM (3 mL) was degassed three times with N2, cooled to -78 °C, and then BCl3 (1 M in DCM, 0.5 mL) was added dropwise. The resulting reaction mixture was stirred at -45 °C for 0.5 h. The reaction was then quenched with MeOH (1 mL) at -45 °C and warmed to 0 °C. The reaction was adjusted to pH > 7 with 1 mL of NH3.H2O (28% purity) and concentrated to a residue, which was purified by preparative HPLC (0-30% acetonitrile in water / NH3·H2O+NH4HCO3) to give the title compound (3.5 mg, 10.6 μmol, 6% yield) as a white solid. TIFF2025511024000128.tif27153

[0396] Example 35 Synthesis of 1-((2R,5R)-5-ethynyl-5-(hydroxymethyl)-2,5-dihydrofuran-2-yl)-5-fluoropyrimidine-2,4(1H,3H)-dione (Compound No. 19) TIFF2025511024000129.tif105143 Step 1: ((2R,4R,5R)-5-(benzoyloxy)-2-ethynyl-4-(p-tolylthio)tetrahydrofuran-2-yl)methyl benzoate. A solution of TBAF (1M in THF, 15.4 mL, 15.4 mmol) was added to a stirred solution of ((2R,4R,5R)-5-(benzoyloxy)-4-(p-tolylthio)-2-((trimethylsilyl)ethynyl)tetrahydrofuran-2-yl)methyl benzoate (7.01 g, 12.9 mmol) in THF (50.0 mL) at ambient temperature, and the resulting mixture was stirred at this temperature for about 1 h. The reaction was then quenched with 20% aqueous NHOAc, and the mixture was concentrated in vacuo. The residue was purified by flash chromatography on SiO2 (eluent: 0-30% EtOAc / hexanes) to afford the title compound (2.5 g, 5.30 mmol, 41%) as a white solid. TIFF2025511024000130.tif5128

[0397] Step 2: ((2R,4R,5R)-2-ethynyl-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(p-tolylthio)tetrahydrofuran-2-yl)methyl benzoate. A flame-dried round-bottom flask equipped with a magnetic stir bar was charged under nitrogen with 5-fluorouracil (231 mg, 1.78 mmol) and MeCN (3.00 mL). HMDS (201 μL, 1.78 mmol) and trimethylsilyl trifluoromethanesulfonate (511 μL, 2.79 mmol) were both added and the resulting mixture was stirred at ambient temperature for approximately 1 h. Solid ((2R,4R,5R)-5-(benzoyloxy)-2-ethynyl-4-(p-tolylthio)tetrahydrofuran-2-yl)methyl benzoate (600 mg, 1.27 mmol) was added in one portion and the resulting mixture was stirred at ambient temperature overnight. The reaction was quenched with a solution of K3PO4 (138 mg, 635 μmol) in water (1.00 mL). The organics were then extracted with EtOAc, dried (anhydrous Na2SO4), filtered, and concentrated in vacuo. The residue was purified by SiO2 flash chromatography (eluent: 0-90% EtOAc in hexanes) to give the title compound (510 mg, 1.06 mmol, 84%) as a white solid. TIFF2025511024000131.tif5128

[0398] Step 3: ((2R,5R)-2-ethynyl-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,5-dihydrofuran-2-yl)methyl benzoate. ((2R,4R,5R)-2-ethynyl-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(p-tolylthio)tetrahydrofuran-2-yl)methyl benzoate (510 mg, 1.06 mmol) was dissolved in MeCN (8.00 mL) with stirring, and both chloramine T trihydrate (366 mg, 1.27 mmol) and AcOH (6.14 μL, 106 μmol) were added. The reaction mixture was stirred at ambient temperature for approximately 2.5 h and then quenched by the addition of 10 wt % aqueous NHOAc (5 mL). The phases were separated and the organics were dried (anhydrous NaSO), filtered, and concentrated in vacuo. The residue was dissolved in n-BuOH (8.00 mL) and the resulting solution was heated to 95 °C for approximately 3 h. The reaction mixture was then cooled to ambient temperature, the volatiles were removed in vacuo, and the residue was purified by SiO flash chromatography (eluent: 0-95% EtOAc in hexanes) to give the title compound (200 mg, 0.56 mmol, 53%) as a clear oil. TIFF2025511024000132.tif5128

[0399] Step 4: 1-((2R,5R)-5-ethynyl-5-(hydroxymethyl)-2,5-dihydrofuran-2-yl)-5-fluoropyrimidine-2,4(1H,3H)-dione (compound number 19). ((2R,5R)-2-ethynyl-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,5-dihydrofuran-2-yl)methyl benzoate (200 mg, 561 μmol) was dissolved in MeOH (25 mL) with stirring at ambient temperature and DBU (4.3 μL, 28.1 μmol) was added. The resulting mixture was heated to 60° C. until the reaction was complete as judged by LCMS. The mixture was cooled to ambient temperature, adsorbed onto silica gel, and purified by SiO2 flash chromatography (eluent: 0-10% MeOH in DCM) to afford compound No. 19 (76 mg, 300 μmol, 54%) as a white solid. TIFF2025511024000133.tif34153

[0400] Example 36 Synthesis of 1-((2S,5R)-5-ethyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound No. 20) TIFF2025511024000134.tif102155 Step 1: (2R,3S)-5-(5-bromo-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-methylbenzoate. To a solution of N,O-bis(trimethylsilyl)acetamide (104 μL, 687 μmol) in MeCN (2.00 mL) was added 5-bromouracil (44.7 mg, 229 μmol) and the resulting mixture was heated to 70° C. with stirring for about 1 h. Trimethylsilyl trifluoromethanesulfonate (50.3 μL, 275 μmol) and (2R,3S)-5-acetoxy-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-methylbenzoate (100 mg, 229 μmol) were then added and the reaction mixture was stirred at 70 °C overnight before being cooled to ambient temperature. The reaction was partitioned between water and EtOAc. The combined organics were dried (anhydrous Na2SO4), filtered and concentrated in vacuo. The residue was purified by SiO2 flash chromatography (eluent: 0-90% EtOAc in heptane) to give the title compound as a mixture of anomers (121 mg, 213 μmol, 93%) as a clear oil. For the more polar anomer, TIFF2025511024000135.tif5128. For the less polar anomer, TIFF2025511024000136.tif5128.

[0401] Step 2: 5-Bromo-1-((2S,4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione. (2R,3S)-5-(5-bromo-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-methylbenzoate (454 mg, 213 μmol) was dissolved in MeOH (2.00 mL) and a solution of NaOMe (25 wt % in MeOH, 2.00 mL, 427 μmol) was added with stirring. The reaction mixture was stirred at ambient temperature until the reaction was judged complete by LCMS. The reaction mixture was adsorbed onto silica gel and purified by SiO2 flash chromatography (eluent: 0-20% MeOH in DCM) to afford the title compound (44.9 mg, 13.6 μmol, 64%) as a mixture of anomers as a white solid. Further purification by preparative HPLC afforded the title compound (6.00 mg) as a single stereoisomer. TIFF2025511024000137.tif27153

[0402] Step 3: 5-Bromo-1-((2S,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-vinyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione. In a glass vial equipped with a magnetic stir bar and capped with a rubber stopper, 5-bromo-1-((2S,4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (6.00 mg, 18.1 μmol) was dissolved in MeOH (181 μL) with stirring. Lindlar's catalyst (6.00 mg) was then added and the vessel was flushed with hydrogen gas from a balloon. The reaction mixture was then stirred under hydrogen (1 atm) at ambient temperature for about 3 h. The mixture was filtered through a short plug of Celite®, the filter cake was washed with MeOH, and the filtrate was concentrated in vacuo to give the crude title compound (4.85 mg, 14.5 μmol, 80%). TIFF2025511024000138.tif27154

[0403] Step 4: 1-((2S,4S,5R)-5-ethyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (compound number 20). In a glass vial equipped with a magnetic stir bar and capped with a rubber stopper, 5-bromo-1-((2S,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-vinyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (6.04 mg, 18.1 μmol) was dissolved in MeOH (181 μL) with stirring. PtO2 (2.06 mg, 9.06 μmol) was then added and the vessel was flushed with hydrogen gas from a balloon. The reaction mixture was then stirred under hydrogen (1 atm) at ambient temperature for about 4 h. The mixture was filtered through a short plug of Celite®, the filter cake was washed with MeOH, and the filtrate was concentrated in vacuo. Purification of the residue by SiO2 flash chromatography (eluent: 50-100% EtOAc in heptane, then 100% IPA) afforded compound no. 20 (2.45 mg, 9.6 μmol, 53%) as an amorphous white solid. TIFF2025511024000139.tif27153

[0404] Example 37 Synthesis of 4-amino-5-bromo-1-((2R,4S,5R)-5-(fluoromethyl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (Compound No. 117) TIFF2025511024000140.tif96153 Step 1: (2R,3R,4S,5R)-2-(4-amino-5-bromo-2-oxopyrimidin-1(2H)-yl)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-(fluoromethyl)tetrahydrofuran-3-yl acetate. To a mixture of 4-amino-5-bromo-1H-pyrimidin-2-one (0.26 g, 1.37 mmol) in MeCN (15 mL) was added (1E)-N-trimethylsilylethanimidate trimethylsilyl (835.14 mg, 4.11 mmol) in one portion at 25° C. under N2. The mixture was then stirred at 70° C. for 1 h. After cooling to 25°C, TMSOTf (373.37 mg, 1.68 mmol) and (2S,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-(fluoromethyl)tetrahydrofuran-2,3-diyl diacetate (0.5 g, 1.12 mmol) were added dropwise at 25°C. The resulting mixture was stirred at 25°C for 16 h. The reaction was then quenched by the addition of 10 wt% aqueous citric acid (10 mL) and then extracted with EtOAc (20 mL). The organic layer was washed with brine (15 mL), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel) eluted with 2% MeOH in dichloromethane to give the title compound (0.326 g, 51% yield) as a yellow oil. TIFF2025511024000141.tif20153

[0405] Step 2: 4-amino-1-((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-(fluoromethyl)-3-hydroxytetrahydrofuran-2-yl)-5-bromopyrimidin-2(1H)-one. To a mixture of (2R,3R,4S,5R)-2-(4-amino-5-bromo-2-oxopyrimidin-1(2H)-yl)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-(fluoromethyl)tetrahydrofuran-3-yl acetate (0.325 g, 563.83 μmol) in MeOH (15 mL) was added aqueous NaOH (2M, 2 mL) in one portion at 25° C. The mixture was stirred at 25° C. for 30 min. The reaction mixture was concentrated directly. The residue was purified by silica gel column chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel) eluting with 2% MeOH in DCM to give the title compound (0.29 g, 96% yield) as a white solid. TIFF2025511024000142.tif27154

[0406] Step 3: Carbonothioate O-((2R,3R,4S,5R)-2-(4-amino-5-bromo-2-oxopyrimidin-1(2H)-yl)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-(fluoromethyl)tetrahydrofuran-3-yl) O-phenyl. To a solution of 4-amino-1-((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-(fluoromethyl)-3-hydroxytetrahydrofuran-2-yl)-5-bromopyrimidin-2(1H)-one (0.29 g, 542.69 μmol) in MeCN (15 mL) was added DMAP (198.90 mg, 1.63 mmol), followed by dropwise addition of O-phenyl chlorothionoformate (141 mg, 814.04 μmol) under N2 at 25° C. After stirring for 1 h at 25° C., the mixture was directly concentrated. The residue was purified by silica gel column chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel) eluted with 1% MeOH in DCM to give the title compound (0.266 g, 73% yield) as a pale yellow solid. TIFF2025511024000143.tif20158

[0407] Step 4: 4-amino-1-((2R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-(fluoromethyl)tetrahydrofuran-2-yl)-5-bromopyrimidin-2(1H)-one. To a mixture of carbonothioate O-((2R,3R,4S,5R)-2-(4-amino-5-bromo-2-oxopyrimidin-1(2H)-yl)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-(fluoromethyl)tetrahydrofuran-3-yl) O-phenyl (0.266 g, 396.69 μmol) in toluene (12 mL) was added AIBN (33 mg, 198.35 μmol) and TTMSS (493.21 mg, 1.98 mmol) in one portion at 25° C. under N2. The mixture was heated to 110° C. and stirred for 2 h. The reaction mixture was then cooled to room temperature and directly concentrated. The residue was purified by preparative TLC (DCM / MeOH=15 / 1) to give the title compound (40 mg, 19% yield) as a colorless oil.

[0408] Step 5: 4-Amino-5-bromo-1-((2R,4S,5R)-5-(fluoromethyl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (compound number 117). To a mixture of 4-amino-1-((2R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-(fluoromethyl)tetrahydrofuran-2-yl)-5-bromopyrimidin-2(1H)-one (0.04 g, 77.16 μmol) in DCM (9 mL) was added BCl3 (1M in DCM, 0.5 mL) dropwise at −78° C. under N2. The reaction mixture was stirred at −78° C. for 15 min, then warmed to −40° C. and stirred for an additional 0.5 h. The reaction was then quenched by the addition of MeOH (1 mL) and NH4OH (1 mL) at -40°C. After stirring for an additional 10 min at -40°C, the mixture was warmed to room temperature and stirred for an additional 10 min. The reaction mixture was then directly concentrated. The residue was purified by preparative HPLC (basic) to give the title compound (1.6 mg, 6% yield) as a white solid. TIFF2025511024000144.tif19153

[0409] Example 38 Synthesis of 5-chloro-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-methyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound No. 120) TIFF2025511024000145.tif94144 Step 1: (2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-(5-chloro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-5-methyltetrahydrofuran-3-yl acetate. A mixture of 5-chloro-1H-pyrimidine-2,4-dione (0.22 g, 1.50 mmol) and BTMSA (767.51 mg, 4.50 mmol) in MeCN (10 mL) was stirred at 70° C. for 1 h, then the reaction mixture was cooled to room temperature. A solution of (0.5 g, 1.17 mmol) in MeCN (10 mL) and TMSOTf (520 mg, 2.34 mmol) was added and the mixture was stirred at 30° C. for 16 h. The reaction was then diluted with EtOAc (50 mL), washed with brine (30 mL×2), and concentrated to give the crude title compound (0.5 g, 83% yield) as a colorless oil. TIFF2025511024000146.tif5128

[0410] Step 2: 1-((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3-hydroxy-5-methyltetrahydrofuran-2-yl)-5-chloropyrimidine-2,4(1H,3H)-dione. A mixture of (2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-(5-chloro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-5-methyltetrahydrofuran-3-yl acetate (0.5 g, 970.96 μmol) in MeOH (10 mL) and NaOH (2M, 1 mL) was stirred at 20° C. for 0.2 h. The reaction was concentrated, diluted with EtOAc (50 ml), washed with brine (30 mL x 2), and then concentrated to give the crude title compound (0.4 g, 87% yield) as a white solid which was used directly in the next step.

[0411] Step 3: O-phenyl chlorothionoformate ((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-(5-chloro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-5-methyltetrahydrofuran-3-yl) O-phenyl. To a solution of 1-((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3-hydroxy-5-methyltetrahydrofuran-2-yl)-5-chloropyrimidine-2,4(1H,3H)-dione (0.2 g, 422.91 μmol) and DMAP (155 mg, 1.27 mmol) in MeCN (20 mL) was added O-phenyl chlorothionoformate (109 mg, 634.36 μmol), followed by stirring at 20° C. for 1 h. The reaction mixture was then diluted with EtOAc (50 mL), washed with brine (30 mL×2), and concentrated to give the crude product. The residue was purified by column chromatography (DCM / MeOH=100 / 1-100 / 5) to give the title compound (0.23 g, 89% yield) as a colorless solid.

[0412] Step 4: 1-((2R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-2-yl)-5-chloropyrimidine-2,4(1H,3H)-dione. A mixture of O-((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-(5-chloro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-5-methyltetrahydrofuran-3-yl) O-phenyl carbonothioate (0.33 g, 541 μmol), TTMSS (673.6 mg, 2.71 mmol) and AIBN (44 mg, 270 μmol) in toluene (15 mL) was stirred at 110° C. for 2 h. After cooling to 20° C., the reaction mixture was concentrated to give the crude product. The residue was purified by column chromatography (DCM:MeOH=100 / 1 to 20 / 1) to give the title compound (0.11 g, yield 44%) as a colorless oil. TIFF2025511024000147.tif5128

[0413] Step 5: 5-Chloro-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-methyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (compound number 120). A mixture of 1-((2R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-2-yl)-5-chloropyrimidine-2,4(1H,3H)-dione (50 mg, 109.43 μmol) and PdCl2 (9.7 mg, 54.71 μmol) in MeOH (10 mL) was stirred at 26 °C under H2 (15 Psi) for 20 min. The reaction was then filtered and the filtrate was concentrated to give the crude product. The residue was purified by preparative HPLC (acetonitrile 0-27% in water / FA-MeCN) to give compound No. 120 (13.9 mg, yield 46%) as a white solid. TIFF2025511024000148.tif19154

[0414] Example 39 Synthesis of 1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-methyltetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (Compound No. 122) TIFF2025511024000149.tif93151 Step 1: (2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyl-2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl acetate. To a mixture of 5-methyl-1H-pyrimidine-2,4-dione (0.2 g, 1.59 mmol) in MeCN (15 mL) was added N,O-bis(trimethylsilyl)acetamide (811 mg, 4.76 mmol) in one portion at 25° C. under N2. The mixture was then stirred at 70° C. for 1 h. After cooling to 25° C., TMSOTf (519 mg, 2.33 mmol) and (2S,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-2,3-diyl diacetate (0.5 g, 1.17 mmol) were added at 25° C. The resulting mixture was stirred at 25° C. for 16 h. The reaction was then quenched by the addition of 10 wt % aqueous citric acid (10 mL) and then extracted with EtOAc (20 mL). The organic layer was washed with brine (15 mL), dried over Na2SO4, filtered, and concentrated to give the title compound (0.5 g, 87% yield) as a yellow oil. TIFF2025511024000150.tif5128

[0415] Step 2: 1-((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3-hydroxy-5-methyltetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione. To a mixture of (2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyl-2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl acetate (0.5 g, 1.01 mmol) in MeOH (15 mL) was added aqueous NaOH (2M, 2 mL) in one portion at 25° C. The mixture was stirred at 25° C. for 30 min. The reaction mixture was concentrated directly to give the title compound (0.45 g, 98% yield) as a white solid.

[0416] Step 3: Carbonothioate O-((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyl-2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl) O-phenyl. To a mixture of 1-((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3-hydroxy-5-methyltetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (0.35 g, 773.48 μmol) in MeCN (15 mL) was added DMAP (283.5 mg, 2.32 mmol), followed by dropwise addition of O-phenyl chlorothionoformate (200 mg, 1.16 mmol) under N2 at 25 °C. After stirring at 25 °C for 1 h, the mixture was directly concentrated. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, DCM / MeOH = 99 / 1) to give the title compound (0.4 g, 88% yield) as a light yellow solid. TIFF2025511024000151.tif5128

[0417] Step 4: 1-((2R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione. To a mixture of O-((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyl-2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl) carbonothioate (0.2 g, 339.75 μmol) in toluene (12 mL) was added AIBN (28 mg, 169.87 μmol) and TTMSS (422 mg, 1.70 mmol) in one portion at 25° C. under N2. The mixture was heated to 110° C. and stirred for 2 h. The reaction mixture was then directly concentrated. The residue was purified by preparative TLC (DCM / MeOH=99 / 1) to give the title compound (100 mg, 67% yield) as a colorless oil. TIFF2025511024000152.tif5128

[0418] Step 5: 1-((2R,4S,5R)-4-Hydroxy-5-(hydroxymethyl)-5-methyltetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (compound no. 122). To a mixture of 1-((2R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (0.08 g, 183.28 μmol) in MeOH (2 mL) was added PdCl (3 mg) at 25° C. under H (15 psi). The mixture was stirred at 25° C. for 20 min. Then the mixture was filtered and concentrated directly. The residue was purified by preparative HPLC (FA) to give compound no. 122 (10.2 mg, 22% yield) as a white solid. TIFF2025511024000153.tif20153

[0419] Example 40 Synthesis of 4-amino-5-chloro-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-methyltetrahydrofuran-2-yl)pyrimidin-2(1H)-one (Compound No. 131) TIFF2025511024000154.tif95149 Step 1: (2R,3R,4S,5R)-2-(4-amino-5-chloro-2-oxopyrimidin-1(2H)-yl)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-3-yl acetate. To a mixture of 4-amino-5-chloro-1H-pyrimidin-2-one (0.21 g, 1.44 mmol) in MeCN (15 mL) was added N,O-bis(trimethylsilyl)acetamide (881 mg, 4.33 mmol) in one portion at 25° C. under N2. The mixture was then stirred at 70° C. for 1 h. After cooling to 25°C, TMSOTf (389 mg, 1.75 mmol) and (2S,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-2,3-diyl diacetate (0.5 g, 1.17 mmol) were added dropwise at 25°C. The resulting mixture was stirred at 25°C for 16 h. The reaction was then quenched by the addition of 10 wt% aqueous citric acid (10 mL) and then extracted with EtOAc (20 mL). The organic layer was washed with brine (15 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel) eluted with 2% MeOH in DCM to give the title compound (0.59 g, 98% yield) as a yellow oil. TIFF2025511024000155.tif5128

[0420] Step 2: 4-amino-1-((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3-hydroxy-5-methyltetrahydrofuran-2-yl)-5-chloropyrimidin-2(1H)-one. To a mixture of (2R,3R,4S,5R)-2-(4-amino-5-chloro-2-oxopyrimidin-1(2H)-yl)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-3-yl acetate (0.55 g, 1.07 mmol) in MeOH (15 mL) was added aqueous NaOH (2M, 2 mL) in one portion at 25° C. The mixture was stirred at 25° C. for 30 min. The reaction mixture was then concentrated. The residue was purified by silica gel column chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel) eluting with 2% MeOH in DCM to give the title compound (0.5 g, 99% yield) as a white solid.

[0421] Step 3: O-((2R,3R,4S,5R)-2-(4-amino-5-chloro-2-oxopyrimidin-1(2H)-yl)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-3-yl) O-phenyl carbonothioate. To a mixture of 4-amino-1-((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3-hydroxy-5-methyltetrahydrofuran-2-yl)-5-chloropyrimidin-2(1H)-one (0.5 g, 1.06 mmol) in MeCN (15 mL) was added DMAP (388 mg, 3.18 mmol) followed by the dropwise addition of O-phenyl chloromethanethioate (274 mg, 1.59 mmol) at 25° C. under N2. After stirring at 25° C. for 1 h, the mixture was concentrated directly. The residue was purified by silica gel column chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel) eluted with 1% MeOH in DCM to give the title compound (0.5 g, 78% yield) as a pale yellow solid. TIFF2025511024000156.tif27153

[0422] Step 4: 4-amino-1-((2R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-2-yl)-5-chloropyrimidin-2(1H)-one. To a mixture of carbonothioate O-((2R,3R,4S,5R)-2-(4-amino-5-chloro-2-oxopyrimidin-1(2H)-yl)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-3-yl) O-phenyl (0.5 g, 822.23 μmol) in toluene (12 mL) was added AIBN (68 mg, 411.11 μmol) and TTMSS (1.02 g, 4.11 mmol) in one portion at 25° C. under N2. The mixture was heated to 110° C. and stirred for 2 h. The reaction mixture was then concentrated directly. The residue was purified by preparative TLC (DCM / MeOH = 20 / 1) to give the title compound (80 mg, yield 21%) as a colorless oil.

[0423] Step 5: 4-Amino-5-chloro-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-methyltetrahydrofuran-2-yl)pyrimidin-2(1H)-one (compound number 131). To a mixture of 4-amino-1-((2R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-2-yl)-5-chloropyrimidin-2(1H)-one (0.02 g, 43.87 μmol) in MeOH (2 mL) was added PdCl2 (778 ug) under H2 (15 psi) at 25° C. The mixture was stirred at 25° C. for 0.3 h. The mixture was then filtered and concentrated directly. The residue was purified by preparative HPLC (acetonitrile 0-27 / 0.225% FA in water) to give Cpd. No. 131 (2.6 mg, 7% yield) as a white solid. TIFF2025511024000157.tif12154

[0424] Example 41 Synthesis of ((((3R,5R)-5-(6-amino-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)methyl)phosphonic acid (Compound No. 113) TIFF2025511024000158.tif146146 Step 1: (2R,3R,4S)-2-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl benzoate. A mixture of N-(9H-purin-6-yl)benzamide (1.0 g, 4.2 mmol) in acetonitrile (20 mL) was degassed with N2 three times. N,O-bis(trimethylsilyl)acetamide (2.2 g, 10.5 mmol) was then added. The resulting mixture was stirred at 70 °C for 1 h and then cooled to room temperature. Then, trimethylsilyl trifluoromethanesulfonate (873.0 mg, 3.9 mmol) was added, followed by (3R,4S)-2-acetoxy-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl benzoate (1.3 g, 3.4 mmol) in acetonitrile (10 mL). The reaction mixture was stirred at 75 °C for 16 h. After cooling to room temperature, the reaction mixture was diluted with water (30 mL), extracted with EtOAc (40 mL), washed with brine (30 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography eluting with 0-30% ethyl acetate in petroleum ether to give the title product (0.8 g, 42% yield) as a white solid. TIFF2025511024000159.tif19153

[0425] Step 2: (2R,3R,4S)-2-(6-benzamido-9H-purin-9-yl)-4-hydroxytetrahydrofuran-3-yl benzoate. To a solution of (2R,3R,4S)-2-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl benzoate (0.8 g, 1.4 mmol) in THF (10 mL) was added TBAF in THF (1 M, 1.4 mL) at 0° C. The reaction mixture was stirred at 25° C. for 1 h. The reaction mixture was then diluted with water (10 mL), extracted with EtOAc (20 mL), washed with brine (20 mL×2), dried over Na2SO4, and concentrated. The residue was purified by silica gel chromatography eluting with 0-3% MeOH in DCM to give the title product (0.35 g, 55% yield) as a white solid. TIFF2025511024000160.tif27153

[0426] Step 3: Diisopropyl ((((3S,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-hydroxytetrahydrofuran-3-yl)oxy)methyl)phosphonate. A solution of (2R,3R,4S)-2-(6-benzamido-9H-purin-9-yl)-4-hydroxytetrahydrofuran-3-yl benzoate (700.0 mg, 1.6 mmol) and diisopropoxyphosphorylmethyl trifluoromethanesulfonate (516.0 mg, 1.6 mmol) in THF (15 mL) was degassed three times with N2 and cooled to 0 °C. NaH (189.0 mg, 4.7 mmol, 60% purity) was added in several batches and the reaction mixture was stirred at 0 °C for 1 h. The reaction mixture was then quenched with saturated aqueous NH4Cl (10 mL) and extracted with EtOAc (20 mL). The organic layer was washed with brine (20 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel chromatography eluting with 0-3% MeOH in DCM to give the title product (350 mg, 43% yield) as a white solid. TIFF2025511024000161.tif27153

[0427] Step 4: O-((2R,3R,4S)-2-(6-benzamido-9H-purin-9-yl)-4-((diisopropoxyphosphoryl)methoxy)tetrahydrofuran-3-yl) O-phenyl carbonothioate. To a solution of ((((3S,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-hydroxytetrahydrofuran-3-yl)oxy)methyl)diisopropylphosphonate (350.0 mg, 673 μmol) in MeCN (10 mL) was added DMAP (82.0 mg, 674 μmol) and O-phenyl chlorothionoformate (151.0 mg, 876 μmol). The reaction mixture was stirred at 25° C. for 16 h. The reaction mixture was then diluted with water (10 mL), extracted with EtOAc (20 mL), washed with brine (20 ml), dried over Na2SO4, filtered and concentrated to give the crude title compound (180.0 mg, 41% yield) as a yellow solid.

[0428] Step 5: ((((3R,5R)-5-(6-benzamido-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)methyl)diisopropylphosphonate. A solution of O-((2R,3R,4S)-2-(6-benzamido-9H-purin-9-yl)-4-((diisopropoxyphosphoryl)methoxy)tetrahydrofuran-3-yl) O-phenyl carbonothioate (180.0 mg, 274 μmol) in toluene (6 mL) was purged with N2 for 5 min. AIBN (36.0 mg, 219.0 μmol) and TTMSS (341.0 mg, 1.37 mmol) were added. The reaction mixture was stirred at 110° C. for 2 h. After cooling to room temperature, the reaction mixture was concentrated. The residue was purified by silica gel chromatography eluting with 0-3% MeOH in DCM to give the title compound (90 mg, 65% yield) as a white solid. TIFF2025511024000162.tif5128

[0429] Step 6: Diisopropyl ((((3R,5R)-5-(6-amino-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)methyl)phosphonate. To a solution of diisopropyl ((((3R,5R)-5-(6-benzamido-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)methyl)phosphonate (90.0 mg, 179 μmol) in MeOH (2 mL) was added NH4OH (1 mL, 28% purity). The resulting reaction mixture was stirred at 25° C. for 16 h. The reaction mixture was then concentrated. The residue was purified by silica gel chromatography eluting with 0-3% MeOH in DCM to give the title compound (50 mg, 70% yield) as a white solid. TIFF2025511024000163.tif5128

[0430] Step 7: ((((3R,5R)-5-(6-amino-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)methyl)phosphonic acid (compound number 113). To a solution of ((((3R,5R)-5-(6-amino-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)methyl)diisopropylphosphonate (50.0 mg, 125 μmol) in MeCN (3 mL) was added 2,6-lutidine (107.0 mg, 1.00 mmol) at 25° C. The reaction mixture was cooled to 0° C. and TMSBr (153.0 mg, 1.00 mmol) was added. The reaction mixture was stirred at 0° C. for 2 h. The reaction was then quenched with 1 M TEAB solution (1 mL) and concentrated. The residue was purified by preparative HPLC (acetonitrile 0-15% / FA in water) to give Cpd. No. 113 (2.5 mg, 7% yield) as a white solid. TIFF2025511024000164.tif27153

[0431] Example 42 Synthesis of 5-(aminomethyl)-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-methyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound No. 140) TIFF2025511024000165.tif97149 Step 1: (2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-(5-cyano-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-5-methyltetrahydrofuran-3-yl acetate. A mixture of 2,4-dioxo-1H-pyrimidine-5-carbonitrile (0.25 g, 1.82 mmol) and BTMSA (932 mg, 5.47 mmol) in MeCN (10 mL) was stirred at 70° C. for 1 h, then the reaction was cooled to room temperature. A solution of (2S,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-2,3-diyl diacetate (0.5 g, 1.17 mmol) in MeCN (10 mL) and TMSOTf (520 mg, 2.34 mmol) was added and the mixture was then stirred at 30° C. for 16 h. The reaction was then diluted with EtOAc (50 mL), washed with brine (30 mL×2), and the organic layer was concentrated to give the crude title compound (0.55 g, 93% yield) as a colorless oil. TIFF2025511024000166.tif5128

[0432] Step 2: 1-((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3-hydroxy-5-methyltetrahydrofuran-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile. A mixture of (2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-(5-cyano-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-5-methyltetrahydrofuran-3-yl acetate (0.59 g, 1.17 mmol) in MeOH (10 mL) and NaOH (2M, 1.20 mL) was stirred at 20° C. for 0.2 h. The reaction was concentrated, diluted with EtOAc (50 mL), washed with brine (30 mL×2), and then concentrated to give the crude title compound (0.5 g, 92% yield) as a white solid, which was used directly in the next step.

[0433] Step 3: Carbonothioate O-((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-(5-cyano-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-5-methyltetrahydrofuran-3-yl) O-phenyl. To a solution of 1-((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3-hydroxy-5-methyltetrahydrofuran-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (0.5 g, 1.08 mmol) and DMAP (395.39 mg, 3.24 mmol) in MeCN (20 mL) was added O-phenyl chlorothionoformate (279 mg, 1.62 mmol), then stirred at 20 °C for 1 h. The reaction was diluted with EtOAc (50 mL), washed with brine (30 mL x 2), and then the organic layer was concentrated to give the crude product. The residue was purified by column chromatography (DCM / MeOH = 100 / 1 to 100 / 5) to give the title compound (0.47 g, 73% yield) as a colorless solid.

[0434] Step 4: 1-((2R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile. A mixture of 1-[(2R,3R,4S,5R)-4-benzyloxy-5-(benzyloxymethyl)-5-methyl-3-phenoxycarbothioyloxy-tetrahydrofuran-2-yl]-2,4-dioxo-pyrimidine-5-carbonitrile (0.47 g, 783.79 umol), TTMSS (974.48 mg, 3.92 mmol), and AIBN (64.35 mg, 391.89 umol) in toluene (15 mL) was stirred at 110° C. for 2 hours. The reaction was concentrated to give the crude product. The residue was purified by column chromatography (DCM:MeOH=100 / 1 to 20 / 1) to give the title compound (0.09 g, yield 26%) as a colorless oil. TIFF2025511024000167.tif5128

[0435] Step 5: 5-(aminomethyl)-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-5-methyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (compound number 140). A mixture of 1-((2R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (0.05 g, 111.74 umol) and PdCl2 (9.91 mg, 55.87 umol) in MeOH (10 mL) was stirred under hydrogen (15 psi) at 26° C. for 20 minutes. The reaction was filtered and concentrated to give the crude product. The residue was purified by preparative HPLC (acetonitrile 0-28% in water / FA-MeCN) to give Cpd. No. 140 (13.08 mg, yield 43%) as a white solid. TIFF2025511024000168.tif26154

[0436] Example 43 Synthesis of 4-amino-5-fluoro-1-((2R,5S)-5-(hydroxymethyl)-5-methyltetrahydrofuran-2-yl)pyrimidin-2(1H)-one (Compound No. 219) TIFF2025511024000169.tif112153 Step 1: (2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-5-methyltetrahydrofuran-3-yl acetate. To a solution of 5-fluoro-1H-pyrimidine-2,4-dione (0.9 g, 7.3 mmol) in MeCN (8 mL) was added N,O-bis(trimethylsilyl)acetamide (5.4 mL, 21.9 mmol). The mixture was stirred at 70° C. for 1 h under nitrogen atmosphere. The reaction mixture was then cooled to room temperature. Trimethylsilyl trifluoromethanesulfonate (1.4 mL, 7.9 mmol) and (2S,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-5-methyltetrahydrofuran-2,3-diyl diacetate (2 g, 4.7 mmol) in MeCN (20 mL) were added. The resulting reaction mixture was stirred at 25 °C under nitrogen atmosphere for 16 h. The reaction was quenched with water (20 mL), extracted with EtOAc (50 × 2 mL), washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated to give the crude product. The residue was purified by silica gel chromatography (solvent gradient: 0-3% MeOH in dichloromethane) to give the title product (2.3 g, 99% yield) as a white solid. TIFF2025511024000170.tif5128

[0437] Step 2: 1-((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3-hydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoropyrimidine-2,4(1H,3H)-dione. To a solution of (2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-5-methyltetrahydrofuran-3-yl acetate (2.3 g, 4.6 mmol) in THF (25 mL) was added aqueous NaOH (2 M, 6.9 mL). The mixture was stirred at 25° C. for 1 h. The reaction was then quenched with water (15 mL), extracted with EtOAc (50 mL), washed with brine (10 mL), dried over Na2SO4, filtered and concentrated to give the crude product, which was purified by silica gel chromatography (100-200 mesh silica gel, eluted with 0-50% ethyl acetate in petroleum ether) to give the title product (2.0 g, 98.8% yield). TIFF2025511024000171.tif5128

[0438] Step 3: 1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-5-methyltetrahydrofuran-2-yl)-5-fluoropyrimidine-2,4(1H,3H)-dione. A mixture of 1-((2R,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3-hydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoropyrimidine-2,4(1H,3H)-dione (1 g, 2.2 mmol) and PdCl2 (388.5 mg, 2.2 mmol) in MeOH (10 mL) was degassed and purged with H2 three times, then the mixture was stirred under H2 atmosphere (15 psi) at 25 °C for 1 h. The reaction mixture was then filtered and concentrated to give the crude material. This was purified by silica gel chromatography (100-200 mesh silica gel, eluted with 0-50% ethyl acetate in petroleum ether) to give the title compound (561.7 mg, 93% yield) as a colorless solid.

[0439] Step 4: 2-acetoxy-2-methylpropanoate ((2R,3R,4S,5R)-4-bromo-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-2-methyltetrahydrofuran-2-yl)methyl. To a solution of 1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-5-methyl-tetrahydrofuran-2-yl]-5-fluoro-pyrimidine-2,4-dione (200 mg, 724.1 umol) in MeCN (4 mL) was added acetate (2-bromo-1,1-dimethyl-2-oxo-ethyl) (1.6 g, 7.2 mmol) at room temperature. The reaction mixture was stirred at 50° C. for 16 hours. The reaction was then quenched with water (10 mL), extracted with EtOAc (20 mL), washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give the crude product. The residue was purified by silica gel chromatography (100-200 mesh silica gel, eluted with 0-25% ethyl acetate in petroleum ether) to give the title compound (262.5 mg, 95% yield) as a colorless oil. TIFF2025511024000172.tif5128

[0440] Step 5: ((2S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-methyl-2,5-dihydrofuran-2-yl)methyl 2-acetoxy-2-methylpropanoate. A mixture of ((2R,3R,4S,5R)-4-bromo-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-2-methyltetrahydrofuran-2-yl)methyl 2-acetoxy-2-methylpropanoate (40 mg, 78.5 umol), AcOH (44.9 uL, 785.4 umol), and Zn-Cu (30 mg, 232.7 umol) in MeOH (4 mL) was degassed and purged with N2 three times, then the mixture was stirred at 25° C. under N2 atmosphere for 16 h. The reaction was then quenched with water (5 mL), extracted with EtOAc (10 mL), washed with brine (10 mL), dried over Na2SO4, filtered and concentrated to give the crude product, which was purified by silica gel chromatography (solvent gradient: 0-3% MeOH in dichloromethane) to give the title product (25 mg, 85% yield). TIFF2025511024000173.tif5128

[0441] Step 6: (2S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-methyltetrahydrofuran-2-yl)methyl 2-acetoxy-2-methylpropanoate. To a solution of (2S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-methyl-2,5-dihydrofuran-2-yl)methyl 2-acetoxy-2-methylpropanoate (25 mg, 67.5 umol) in MeOH (4 mL) was added PdCl2 (12 mg, 67.5 umol). The mixture was stirred at 25° C. for 1 h under H2 (15 psi). The reaction mixture was filtered and concentrated to give the crude product. This was purified by silica gel chromatography (100-200 mesh silica gel, eluted with 0-25% ethyl acetate in petroleum ether) to give the title compound (24 mg, 95% yield) as a colorless oil. TIFF2025511024000174.tif5128

[0442] Step 7: (2S,5R)-5-(4-amino-5-fluoro-2-oxopyrimidin-1(2H)-yl)-2-methyltetrahydrofuran-2-yl)methyl 2-acetoxy-2-methylpropanoate. To a solution of (2S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-methyltetrahydrofuran-2-yl)methyl 2-acetoxy-2-methylpropanoate (50.0 mg, 134.0 μmol), EtN (60.0 μL, 402.8 μmol), and DMAP (16.0 mg, 134.0 μmol) in MeCN (3 mL) was added 2,4,6-triisopropylbenzenesulfonyl chloride (82.0 mg, 268.6 μmol). The reaction mixture was stirred at 25° C. for 16 h. A solution of NH3 in MeOH (7 M, 1 mL) was added. The reaction mixture was stirred at 25 °C for 16 h. The reaction mixture was quenched with water (10 mL), extracted with EtOAc (20 mL), washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated to give the crude product, which was purified by silica gel chromatography (solvent gradient: 0-3% MeOH in dichloromethane) to give the title product (30.0 mg, 80% yield) as a yellow solid. TIFF2025511024000175.tif5128

[0443] Step 8: 4-Amino-5-fluoro-1-((2R,5S)-5-(hydroxymethyl)-5-methyltetrahydrofuran-2-yl)pyrimidin-2(1H)-one (Cpd. No. 219). To a solution of ((2S,5R)-5-(4-amino-5-fluoro-2-oxopyrimidin-1(2H)-yl)-2-methyltetrahydrofuran-2-yl)methyl 2-acetoxy-2-methylpropanoate (30.0 mg, 80.8 μmol) in MeOH (2 mL) was added NaOMe (5.0 mg, 80.8 μmol). The reaction mixture was stirred at 25° C. for 16 h. The mixture was concentrated to give a residue which was purified by preparative HPLC (acetonitrile 0-30% in water / NH3.H2O+NH4HCO3) to give Cpd. No. 219 (9.4 mg, 48% yield) as a white solid. TIFF2025511024000176.tif27153

[0444] Example 44 Analytical properties for representative compounds of the disclosure are provided in Table 5.

[0445] (Table 5) TIFF2025511024000177.tif84154TIFF2025511024000178.tif226154TIFF2025 511024000179.tif216154TIFF2025511024000180.tif230154TIFF20255110240 00181.tif216154TIFF2025511024000182.tif226154TIFF2025511024000183.t if216154TIFF2025511024000184.tif226154TIFF2025511024000185.tif152154

[0446] Example 33 Human LINE-1 retrotransposition assay Representative disclosed compounds were tested for inhibition of human LINE-1 retrotransposition activity in HeLa cells according to the following procedure.

[0447] HeLa cervical cancer cells were cultured at 37°C in high-glucose Dulbecco's modified Eagle's medium (DMEM) with 4500 mg / L glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher) in a humidified 5% CO2 incubator.

[0448] The assay was performed using the reporter plasmid pYX017 as described (Xie, et al., 2011) with some modifications. Reporter assays were performed in 96-well white optical bottom plates. HeLa cells were seeded into wells 24 h prior to transfection and compound treatment so that cells were approximately 30% confluent on the day of transfection. Various cell plating densities were tested, ranging from 2 × 10 3 A cell density of 100 μg / ml was determined to be optimal.

[0449] Compounds were resuspended in DMSO. Serial dilutions (1:3) were prepared in DMSO. Media containing compounds at various concentrations were prepared by adding 2 μl of compound dilution to 1 ml of culture medium. The final concentration of DMSO in the medium was 0.2%.

[0450] Plasmids were transfected into cells using FuGENE® HD Transfection Reagent (Promega, E2311, Lot 382574 and Lot 397842). Transfection reagent:DNA mixtures were prepared according to the manufacturer's instructions in OpiMEM (Thermo Fisher). Various ratios of transfection reagent and DNA were tested and a 3:1 ratio was determined to be optimal. Culture medium was removed from cells and discarded. Transfection reagent:DNA mixtures (5 μl) were mixed with compound-containing medium (100 μl / well) and added to cells in each well. Cells were cultured at 37° C. / 5% CO2 for various incubation times. A 72 hour incubation time was determined to be optimal.

[0451] Luciferase reporter activity was quantified with the Dual-Luciferase® Reporter Assay System (Promega) according to the manufacturer's instructions for multiwell plates, except that cells were lysed directly on the multiwell plates with 30 μl of Passive Lysis Buffer (PLB) for 20 min at room temperature with gentle shaking to ensure complete cell lysis.

[0452] Firefly and Renilla luciferase signals were measured using a SpectraMax i3x multimode microplate reader. Integration times of 100ms and 10ms were used to measure firefly and Renilla signals, respectively. Relative L1 activity is calculated as firefly / renilla*1000 or firefly / renilla*10,000. Nonlinear regression was used to fit the dose-response inhibition data to a four-parameter logistic equation (using Graphpad Prism 8) to determine the IC of each inhibitor. 50 Values ​​were determined and the results are provided in Table 6.

[0453] Table 6. Inhibition of human LINE-1 activity TIFF2025511024000186.tif24674TIFF2025511024000187.tif23874TIFF2025511024000188.tif117128

[0454] Although the compounds, methods, kits, and compositions have been fully described herein, those skilled in the art will recognize that the same can be practiced within a wide and equivalent range of conditions, formulations, and other parameters without affecting the scope of the methods, compounds, and compositions provided herein or any aspect thereof. All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.

Claims

1. Compounds selected from the group consisting of the following: or a pharmaceutically acceptable salt or solvate thereof, or a tautomer thereof.

2. Compounds selected from the group consisting of the following: or a pharmaceutically acceptable salt or solvate thereof, or a tautomer thereof.

3. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt or solvate thereof, or a tautomer thereof, One or more pharmaceutically acceptable excipients and A pharmaceutical composition for use in the treatment of a disease, disorder, or condition, comprising: The disease, disorder, or condition is a neurodegenerative disease, autoimmune disease, age-related disease, autism spectrum disorder, cardiovascular dysfunction, hearing loss, hematopoietic stem cell dysfunction, pulmonary fibrosis, schizophrenia, visual impairment, progressive supranuclear palsy, amyotrophic lateral sclerosis, Eicardi-Goutier syndrome, ataxia telangiectasia, age-related macular degeneration, systemic lupus erythematosus, IFN-associated autoimmune disease, Fanconi anemia, idiopathic pulmonary fibrosis, or cardiovascular disease. Pharmaceutical composition.

4. The pharmaceutical composition according to claim 3, wherein the disease, disorder, or condition is a neurodegenerative disease.

5. The pharmaceutical composition according to claim 4, wherein the neurodegenerative disease is Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Lewy body dementia, multiple system atrophy, Huntington's disease, frontotemporal dementia (FTD), frontotemporal lobar degeneration, mild cognitive impairment, corticobasal degeneration, progressive supranuclear palsy, Rett syndrome, peripheral degenerative disease, or Eicardi-Goutier syndrome.

6. The pharmaceutical composition according to claim 3, wherein the disease, disorder, or condition is an autoimmune disease.

7. The pharmaceutical composition according to claim 6, wherein the autoimmune disease is lupus, rheumatoid arthritis, Sjögren's syndrome, or multiple sclerosis.

8. The pharmaceutical composition according to claim 4, wherein the disease, disorder, or condition is an age-related disease.

9. The pharmaceutical composition according to claim 8, wherein the age-related disease is Alzheimer's disease, Parkinson's disease, atherosclerosis, osteoarthritis, osteoporosis, rheumatoid arthritis, macular degeneration, peripheral degenerative disease, or skin aging.

10. The pharmaceutical composition according to claim 3, wherein the disease, disorder, or condition is autism spectrum disorder, cardiovascular dysfunction, hearing loss, hematopoietic stem cell dysfunction, pulmonary fibrosis, schizophrenia, or visual impairment.

11. The pharmaceutical composition according to claim 3, wherein the disease, disorder, or condition is progressive supranuclear palsy.

12. The pharmaceutical composition according to claim 3, wherein the disease, disorder, or condition is amyotrophic lateral sclerosis.

13. The pharmaceutical composition according to claim 3, wherein the disease, disorder, or condition is Aicardi-Gutierre syndrome.

14. The pharmaceutical composition according to claim 3, wherein the disease, disorder, or condition is ataxia telangiectasia, age-related macular degeneration, systemic lupus erythematosus, IFN-related autoimmune disease, Fanconi anemia, idiopathic pulmonary fibrosis, or cardiovascular disease.

15. The pharmaceutical composition according to claim 3 for treating the disease, disorder, or condition in the subject.

16. A pharmaceutical composition according to claim 3 for treating symptoms of a disease, disorder, or condition in the subject.

17. The pharmaceutical composition according to claim 3, wherein the treatment further comprises the step of administering one or more arbitrary therapeutic agents to the subject.

18. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt or solvate thereof, or a stereoisomer thereof, or a tautomer thereof, Instructions for administering the compound to the target of treatment of a disease, disorder, or condition and A kit that includes, The disease, disorder, or condition is a neurodegenerative disease, autoimmune disease, age-related disease, autism spectrum disorder, cardiovascular dysfunction, hearing loss, hematopoietic stem cell dysfunction, pulmonary fibrosis, schizophrenia, visual impairment, progressive supranuclear palsy, amyotrophic lateral sclerosis, Eicardi-Goutier syndrome, ataxia telangiectasia, age-related macular degeneration, systemic lupus erythematosus, IFN-associated autoimmune disease, Fanconi anemia, idiopathic pulmonary fibrosis, or cardiovascular disease. kit.

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