Antiviral prodrugs and formulations thereof

Abstract

To provide antiviral prodrugs and formulations thereof.SOLUTION: The disclosure provides compounds, compositions and use methods thereof for treating and / or preventing viral infections such as HIV and HBV by administering a specific ester and other derivatives or pharmaceutically acceptable salts of 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA).SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 898,6 79, filed on September 11, 2019, the entire disclosure of which is incorporated herein by reference .

[0002] The present invention relates to antiviral chemical compounds and compositions useful for treating acquired immunodeficiency syndrome (AIDS). .

Background Art

[0003] The compound EFdA (MK - 8591) is a nucleoside analog known to be effective as an inhibitor of the enzyme reverse transcriptase (Current Opinion in H IV and AIDS 2018, 13, 294 - 299).

[0004]

Chemical Formula

[0005] Certain EFdA analogs, which include phosphates, esters, carbonates, carbamates, where at least one of the substitutions within the glycosyl donor ring is an atom "D" (D = deuterium = 2H), are described in a U.S. application published as US2019 / 185508 .

[0006] Reverse transcriptase inhibitors can be effective in the treatment of viral infections caused by viruses such as HIV (human immunodeficiency virus) and HBV (hepatitis B virus ), where the function of reverse transcriptase is essential for viral replication and the production of viral proteins . In the case of HIV, ​​When the virus is an RNA virus, this RNA virus uses reverse transcriptase to synthesize a DNA reverse transcript of the important genome, which is translated by the host to provide viral proteins. In the case of HBV, a DNA virus, the DNA virus polymerase also has the function of reverse transcriptase and generates viral DNA from viral RNA intermediates during replication. An example of an antiviral compound that acts on both HIV and HBV is lamivudine.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Non-Patent Documents

[0008]

Non-Patent Document 1

Summary of the Invention

[0009] In various embodiments, the present invention provides (1) a novel composition of a chemical substance comprising a bioactive prodrug of MK-8591 or a pharmaceutically acceptable salt of these prodrugs, and and (2) a novel formulation of these prodrugs for providing therapeutic and prophylactic treatment of patients against viral infections such as HIV and HBV, where inhibition of reverse transcriptase ( RNA-directed DNA polymerase) delays or blocks viral infection. Routes of administration for these treatments include, but are not limited to, oral, parenteral, and implants (compositions and devices), etc. The formulations of the present invention are aqueous suspensions ​​​​​​​ When injected as a liquid formulation, it provides a sustained or controlled or continuous release of EFdA from these prodrugs. The present invention provides, for example, the following items. The present invention provides, for example, the following items. (Item 1) Formula (I) [Wherein, [Wherein, R 1 is H, or X-L m [where m = 1 or 2, and where X-L m is -C(=O)L, -C(=O)OL, -C(=O)NH(L), -C(=O)N(L)2, -CH(R)OC(=O)L, -C(=O)CH(R)-NH(L), -C(=O)CH(R)-N(L)2, -P(=O)(NHL)2, -P(=O)(NHL)(NL2) or -P(=O)(NL2)2, and each independently selected L is (C 1-22 )alkyl, (C 3-22 )alkenyl (where the alkenyl can contain 1 to 6 unsaturations), (C 3-7 )cycloalkyl, (CHR) n -phenyl (where n = 0 or 1) or -CHR-N(R)2], or R 1 is -OCH(R)OP(=O)(OH)2, or R 1 is a phosphate residue or its derivative residue (including monophosphate, diphosphate, triphosphate, phosphonate, phosphate polyester, phosphate amide (mono and di), phosphorothioate, phosphorosenoate or phosphoroboranoate); R is H, (C 1-22 )alkyl or (C 3-22 )alkenyl (where the alkenyl can contain 1 to 6 unsaturations), or (C 3-7 )cycloalkyl; R 2 is H, or X-L mor R 2 is -OCH(R)OP(=O)(OH)2, or R 2 is a phosphate residue or a derivative residue thereof (including monophosphate, diphosphate, triphosphate, phosphonate, phosphate polyester, phosphate amide (mono and di), phosphorothioate, phosphorosenoate or phosphoroboranoate); provided that the following combinations are excluded: (a) R 1 = R 2 = acetyl, (b) R 1 = R 2 = H, and (c) R 1 when R 2 is a phosphate residue or a derivative residue thereof (which includes monophosphate, diphosphate, triphosphate, phosphonate, phosphate polyester, phosphate amide (mono and di), phosphorothioate, phosphorosenoate or phosphoroboranoate), R = H〕 (Item 2) R 1 and R 2 are both -C(=O)L, the compound according to Item 1. (Item 3) R 1 and R 2 at least one of which is -C(=O)L, the compound according to Item 1. (Item 4) R 1 and R 2 one of which is H, the compound according to Item 1. (Item 5) R 1 and R 2 one of which is -C(=O)L, and R 1 and R 2 one of which is H, the compound according to Item 1. (Item 6) L is (C 1-22 ) alkyl, the compound according to any one of Items 2 to 5. (Item 7) L is (C 3-22 ) alkenyl, where the alkenyl can contain 1 to 6 unsaturations, the compound according to any one of items 2 to 5. (Item 8) L is (C 3-7 ) cycloalkyl, the compound according to any one of items 2 to 5. (Item 9) L is (CHR) n -phenyl, where n = 0 or 1, the compound according to any one of items 2 to 5. (Item 10) n = 0, the compound according to item 9. (Item 11) n = 1, the compound according to item 9. (Item 12) A preparation comprising the compound according to any one of items 1 to 11 suspended in an aqueous suspension containing 0.25% sodium carboxymethylcellulose and 0.1% polyoxyethylene (20) sorbitan monooleate. (Item 13) A preparation comprising the compound according to any one of items 1 to 11 suspended in an aqueous suspension containing 0.25% sodium carboxymethylcellulose and 0.5% polyoxyethylene (20) sorbitan monooleate. (Item 14) A pharmaceutical composition comprising an effective amount of the compound according to any one of items 1 to 11 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. (Item 15) A method for inhibiting viral reverse transcriptase bioactivity, comprising contacting a virus expressing an enzyme having reverse transcriptase bioactivity with an effective amount or effective concentration of the compound according to any one of items 1 to 11. (Item 16) A method for treating a viral infection in a patient in whom inhibition of reverse transcriptase is medically indicated, comprising administering to the patient an effective amount or effective concentration of the compound according to any one of items 1 to 11. (Item 17) A method for preventing viral infections, the method comprising administering to a patient an effective amount or effective concentration of the compound according to any one of items 1 to 11. (Item 18) The method according to any one of items 16 to 17, wherein the administration of the compound results in a sustained or controlled or continuous release of EFdA from the compound. (Item 19) The method according to any one of items 16 to 17, wherein the route of administration of the compound is selected from the group consisting of oral, parenteral, subcutaneous injection, intravenous, intramuscular, intrastemal injection, infusion, and release from an implant. (Item 20) The method according to any one of items 15 to 17, wherein the compound is formulated as an aqueous suspension, a solution, or encapsulated within sustained-release particles. (Item 21) The method according to any one of items 15 to 20, wherein the viral infection is caused by HIV. (Item 22) The method according to any one of items 15 to 20, wherein the viral infection is caused by HBV. (Item 23) The method according to any one of items 15 to 22, further comprising administering an additional anti-HIV drug and / or anti-HBV drug. (Item 24) The method according to item 23, wherein the additional drug is selected from the group consisting of cabotegravir, dolutegravir, doravirine, elvitegravir, raltegravir, tenofovir disoproxil fumarate, tenofovir alafenamide fumarate, and lamivudine. (Item 25) Use of the compound according to any one of items 1 to 11 for treating and preventing HIV or HBV viral infections.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Embodiments for Carrying Out the Invention

[0011] Among various embodiments, the present invention relates to formula (I)

[0012]

Chemical Formula

[0013] Among various embodiments, the compound represented by formula (I) can be any one of compounds 2 to 51 shown in Table 1.

[0014] The present invention further provides, among various embodiments, a method for inhibiting viral reverse transcriptase biological activity, wherein the method comprises contacting a virus expressing an enzyme having reverse transcriptase biological activity with an effective amount or effective concentration of a compound represented by formula (I).

[0015] The present invention further provides, among various embodiments, a method for preventing viremia or treating a viral infection in a patient in whom inhibition of reverse transcriptase is medically indicated, wherein the method comprises administering to the patient an effective amount or effective concentration of a compound represented by formula (I). More specifically, the compound represented by formula (I) can be administered in a formulation that provides a sustained or controlled or continuous release of EFdA from these prodrugs. More specifically, the compound represented by formula (I) is an aqueous suspension ​ 、 can be formulated as a solution and encapsulated within sustained-release particles containing PLGA and those known in the art. More specifically, viral infections can be caused by HIV or HBV. Routes of administration for these prodrugs include, but are not limited to, oral, parenteral, and implant (drug delivery compositions and devices), etc. In a method for treating or preventing a viral infection, the method may further include additional anti-HIV drugs and / or anti-HBV drugs (which include, but are not limited to, cabotegravir, dolutegravir, doravirine, elvitegravir, raltegravir, tenofovir disoproxil fumarate, tenofovir alafenamide fumarate, or lamivudine).

[0016]

Table 1

Example

[0017] Abbreviations The following abbreviations are used: tetrahydrofuran (THF), dichloromethane (DCM), acetonitrile (MeCN), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), trifluoroacetic acid (TFA), triethylamine (TEA), diisopropyl ethylamine (DIPEA), methanol (MeOH), ethyl acetate (EtOAc), 4-dimethylaminopyridine (DMAP), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC-hydrochloride), N,N’-dicyclohexyl carbodiimide (DCC).

[0018] General Examples for the Preparation of the Compounds of the Present Invention Starting materials and intermediates for the compounds of the present invention can be prepared by applying or adapting the methods described below, their obvious chemical equivalents, or methods described in the literature such as, for example, "The Science of Synthesis, Volumes 1-8. Editors E. M. Carreira et al. Thieme publishers (2001-2008)". Details of reagents and reaction options can also be obtained by searching for structures and reactions using commercially available computer search engines such as Scifinder (www.cas.org) or Reaxys (www.reaxys.com).

[0019] ​​​​​​​ Part I: Preparation of Intermediates and EFdA (MK-8591) Example 1 Synthesis of Intermediate-A, Intermediate-B and Compound 1 (EFdA):

[0020]

Chem.

[0021]

Chem.

[0022] (2R,3S,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl) -2-(((tert-butyldiphenylsilyl)oxy)methyl)-2-ethynyltet rahydrofuran-3-ol, B A solution of Intermediate A (500 mg, 0.87 mmol) dissolved in Me OH (10 mL) was added with methanol ammonia (10 mL) at room temperature and stirred for 12 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude substance. The obtained crude substance was purified by silica gel column chromatography using 2% MeOH in DCM to obtain Intermediate B (400 mg, 86.33%). LC-MS (ESI +): m / z 532.4 [M+H]+.

[0023]

Chem.

[0024] (2R,3S,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl) -2-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol, 1 Compound 1 (EFdA) is prepared according to the method reported in the literature (Org. Lett. 2011, 13, 5264- 5266). 1 1H NMR (400 MHz, DMSO-d6) δ 8.30 (s, 1H), 7.86 (d, J = 27.7 Hz, 2H) , 6.24 (dd, J = 7.4, 5.0 Hz, 1H), 5.63 - 5.54 (m, 1H) , 5.35 - 5.25 (m, 1H), 4.56 (q, J = 6.4 Hz, 1H), 3.67 -3.60 (m, 1H), 3.60 - 3.49 (m, 2H), 2.74 - 2.63 (m, 1H), 2.46 - 2.36 (m, 1H). LC-MS (ESI+): m / z 294. 2 [M + H] + .

[0025] Part II: Preparation of the Compounds of the Examples Example 2

[0026]

Chemical Structure

[0027] (2R,3S,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl) -2-ethynyl-2-((isobutyryloxy)methyl)tetrahydrofuran-3-yl isobutyrate, 2 A mixture of EFdA (3 g, 6.8 mmol , 1 equivalent) and DMAP (499 mg, 2.73 mmol, 0.4 equivalent) in anhydrous DMF (100 mL) was added dropwise with isobutyric acid (8.4 g, 27.3 mmol, 6 equivalents) at ambient temperature. The reaction mixture was stirred at room temperature for 5 h. Since alkylation of the NH2 group may be observed when the reaction time is prolonged, the reaction is monitored by LCMS. The reaction mixture was then filtered to remove the by-product urea. The reaction mixture was rinsed with acetonitrile.​ Subsequently, the reaction product was washed twice with water and once with brine, then the solvent was dehydrated and filtered and evaporated under reduced pressure. The resulting crude material was purified by silica gel column chromatography using 60 - 70% EtOAc in hexane, and Compound 2 was obtained as a glassy solid . The resulting solid was dispersed in the minimum amount of isopropanol and then subjected to rotary evaporation to obtain the pure compound as a white solid (2.5 g, 85% yield). LC - MS (ESI+): m / z 434.49 [M + H] . + .

[0028] Example 3

[0029] [Chemical formula]

[0030] (2R,3S,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl) -2-(((2-ethylbutanoyl)oxy)methyl)-2-ethynyltetrahydrofura n-3-yl 2-ethylbutanoate, 3 A mixture of EFdA ( 1 g, 3.4 mmol, 1 equiv) and 2 - ethylbutanoic anhydride (4.4 g, 20.4 mmol, 6 equiv) and TEA (3.8 mL, 27.2 mmol, 8 equiv) in anhydrous MeCN (43 mL) was cooled to 0 °C, and DMAP (83 mg, 0.68 mmol, 0.2 equiv) was added thereto at 0 °C . The reaction mixture was stirred at 0 °C for 0.5 h and then at room temperature for 5 h. Since alkylation of the NH2 group may be observed when the reaction time is prolonged, the reaction was monitored by LC - MS . The reaction mixture was further quenched with methanol and the solvent was evaporated under reduced pressure . The resulting crude material was purified by silica gel column chromatography using 60 - 70% EtOAc in hexane . Purified by ram chromatography, Compound 3 was obtained as a glassy solid. The obtained solid was dispersed in the minimum amount of isopropanol and then subjected to rotary evaporation to obtain the pure compound as a white solid (1.33 g, 80% yield). LC-MS (ESI+): m / z 490.56 [M+H] + .

[0031] Example 4

[0032]

Chemical formula

[0033] (2R,3S,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl) -2-(((cyclopentanecarbonyl)oxy)methyl)-2-ethynyltetrahydro furan-3-yl cyclopentanecarboxylate, 4 Compound 4 was prepared using a method analogous to that for Compound 2. LC-MS (ESI+): m / z 486.4 4 [M+H] + .

[0034] Example 5

[0035]

Chemical formula

[0036] (2R,3S,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl) -2-ethynyl-2-((2-phenylacetoxy)methyl)tetrahydrofuran-3- yl 2-phenylacetate, 5 A mixture of EFdA (499 mg, 1.7 mmol, 1 equiv), 2-phenylacetic anhydride (2.6 g, 10.2 mmo l, 6 equiv), and TEA (1.9 mL, 13.6 mmol, 8 equiv) in anhydrous MeCN (22 mL) was cooled to 0 °C, and DMAP (42 mg, 0.34 mmol, 0.2 equiv) was added thereto at 0 °C. ​The reaction was stirred at 0° C. for 0.5 h and then at room temperature for 3 h. In some cases, alkylation of the NH2 group may be observed, and the reaction is monitored by LCMS. The reaction was further quenched with methanol and the solvent was evaporated under reduced pressure. The resulting crude material was purified by silica gel column chromatography using 60-70% EtOAc in hexanes. Purification by chromatography afforded compound 5 as a glassy solid. was dispersed in a minimum amount of isopropanol and then rotary evaporated to give The pure compound was obtained as a white solid (694 mg, 77% yield). SI+): m / z 530.52[M+H] + .

[0037] Example 6

[0038] [ka]

[0039] (2R,3S,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl) -2-ethynyl-2-((((S)-2-phenylpropanoyl)oxy)methyl)tet rahydrofuran-3-yl (S)-2-phenylpropanoate, 6 Compound 6 is Prepared using a method similar to that for Compound 2 with the following differences: a) 4 equivalents of the corresponding acid were used instead of 6 equivalents; (b) the urea was removed by filtration Afterwards, the solvent was evaporated under reduced pressure and the resulting white solid was then washed with a minimum amount of isopropanol. The suspension was then filtered to obtain the pure compound, which was then dispersed in water and stirred for 1 h. Obtained as a solid (yield 75%). LC-MS (ESI+): m / z 558.65[ M+H] + .

[0040] Example 7

[0041]

Chem.

[0042] (2R,3S,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl) -2-((Benzyloxy)methyl)-2-ethynyltetrahydrofuran-3-yl benzoate, 7: Example 8 Compound 7 was prepared using a method analogous to the method for Compound 2 with 4.5 equivalents of the corresponding acid. LC-MS (ESI+): m / z 502.41 [M + H]+ + .

[0043] (2R,3S,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl)

[0044]

Chem.

[0045] -2-((((4Z,7Z,10Z,13Z,16Z,19Z)-Docosa-4,7,10 ,13,16,19-hexaenoyl)oxy)methyl)-2-ethynyltetrahydrof ran-3-yl (4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,1 0,13,16,19-hexaenoate, 8: Example 9 A mixture of EFdA (117 mg, 0.4 mmol, 1 equiv) and docosahexaenoic acid (0.39 g, 1.2 mmol, 3 equiv) and DMAP (4.9 mg, 0.04 mmol, 0.1 equiv) and EDC·HCl (307 mg, 1.6 mmol, 4 equiv) in anhydrous DMF (4 mL) was cooled to 0 °C, to which DIPEA (0.3 mL, 1.6 mmol, 4 equiv) was added at 0 °C. The reaction mixture was stirred at room temperature for 8 h. Since alkylation of the NH2 group is observed when the reaction time is prolonged, the reaction is monitored by LCMS. Thereafter, the reaction mixture was diluted with EtOAc 117 mg, 0.4 mmol, 1 equivalent) and docosahexaenoic acid (0.39 g, 1.2 mmol ol, 3 equivalents) and DMAP (4.9 mg, 0.04 mmol, 0.1 equivalent) and EDC·H Cl (307 mg, 1.6 mmol, 4 equivalents) in anhydrous DMF (4 mL) was cooled to 0 °C, and thereto, DI PEA (0.3 mL, 1.6 mmol, 4 equivalents) was added at 0 °C. The reaction mixture was stirred at room temperature for 8 h. Alkylation of the NH2 group is observed when the reaction time is prolonged, so the reaction is monitored by LCMS. Thereafter, the reaction mixture was diluted with EtOAc is observed, the reaction is monitored by LCMS. Thereafter, the reaction mixture was diluted with EtOAc ​​Then, it was washed three times with 2.5% NaCl, three times with a pH approximately 7 buffer solution, and once with brine, and then the solvent was dehydrated, filtered, and evaporated under reduced pressure. The resulting crude substance was purified by silica gel column chromatography using 5% DCM in MeOH to obtain Compound 8 as a gel-like semi-solid (183 mg, 50% yield). LC-MS (ESI I+): m / z 457.23 [M / 2 + H] + .

[0046] (2R,3S,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl)

[0047]

Chemical Structure

[0048] -2-ethynyl-2-((heptadecanoyloxy)methyl)tetrahydrofuran-3- yl heptadecanoate, 9: Example 10 Compound 9 was prepared using a method analogous to the method for Compound 2, using 8 equivalents of the corresponding acid and 6 equivalents of DCC. 1 1H NMR (400 MHz, chloroform-d) δ 7.92 (s, 1H), 6.40 (t, J = 6.4 Hz, 1H), 5.76 (brs, 2H), 5.65 (dd, J = 7.1, 5.3 Hz, 1H ), 4.49 (d, J = 12.0 Hz, 1H), 4.38 (d, J = 12.1 Hz, 1H ), 2.99 (dt, J = 13.5, 6.7 Hz, 1H), 2.75 - 2.64 (m, 2 H), 2.41 (t, J = 7.5 Hz, 2H), 2.34 (q, J = 3.8 Hz, 1H ), 1.71 - 1.57 (m, 9H), 1.41 - 1.17 (m, 48H), 0.88 (t , J = 6.8 Hz, 6H). LC-MS (ESI+): m / z signal was not observed .

[0049] ​​ (2R,3S,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl)

[0050]

Chem.

[0051] -2-ethynyl-2-((palmitoyloxy)methyl)tetrahydrofuran-3-yl palmitate, 10: Example 11 A flask containing EFdA (202 mg, 0.7 mmol, 1 equiv) was added pyridine anhydride (7 mL). The mixture was stirred for 1 - 2 minutes and cooled to 0 °C. To this mixture, palmitoyl chloride (1.3 mL, 4.2 mmol, 6 equiv) was added dropwise at 0 °C. The reaction was stirred overnight at room temperature. Since N - alkylation may be observed when the reaction time is prolonged, the reaction is monitored by LCMS. After that, the reaction was diluted with EtOAc, washed three times with water, once with NaHCO3, once with brine, then the solvent was dried, filtered, and evaporated under reduced pressure. The resulting crude material was purified by silica gel column chromatography using 60 - 70% EtOAc in hexane to give Compound 10 as a glassy solid. The resulting solid was dispersed in the minimum amount of isopropanol and then subjected to rotary evaporation to give the pure compound as a white solid (372 mg, 70% yield). 1 1H NMR (400 MHz, DMSO - d6) δ 8.34 (s, 1H), 7.91 (d, J = 30.8 Hz, 2H), 6.34 (t, J = 6.9 Hz, 1H), 5.70 (t, J = 6.4 Hz, 1H ), 4.39 (d, J = 11.6 Hz, 1H), 4.22 (d, J = 11.9 Hz, 1H ), 4.39 (d, J = 11.6 Hz, 1H), 4.22 (d, J = 11.9 Hz, 1H )、3.81 - 3.73 (m, 1H), 3.14 (dt, J = 14.1, 6.9 Hz, 1 H), 2.60 (dt, J = 12.9, 6.0 Hz, 1H), 2.39 (td, J = 7. 3, 2.4 Hz, 2H), 2.32 - 2.17 (m, 2H), 1.64 - 1.52 (m, 2H), 1.51 - 1.39 (m, 2H), 1.38 - 1.11 (m, 48H), 0.9 2 - 0.78 (m, 6H). LC - MS (ESI+): m / z signal was not observed at all.

[0052] (2R,3S,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl)

[0053] [Chemical formula]

[0054] -2-ethynyl-2-((heptanoyloxy)methyl)tetrahydrofuran-3-yl heptanoate, 11: Example 12 Compound 11 was prepared using a method similar to the method for Compound 2 with 8 equivalents of the corresponding acid and 6 equivalents of DCC. LC - MS (ESI+): m / z 518.25 [M + H] + .

[0055] (((2R,3S,5R)-3-((D-Valyl)oxy)-5-(6-amino-2-f

[0056] [Chemical formula]

[0057] luoro-9H-purin-9-yl)-2-ethynyltetrahydrofuran-2-yl)meth yl D-valinate, 12: Examples 13 - 15 Compound 12 was prepared using a method similar to the method for Compound 2 with 7 equivalents of the corresponding boc - protected amino acid and 7 equivalents of DCC, and then Subjected to typical deprotection of the Boc group using HCl in dioxane. LC-MS (ES I+): m / z 492.64 [M+H] + .

[0058] Examples 16 - 23 Compounds 13 - 15 were prepared using a method analogous to the method for compound 12.

[0059] Example 24 Compounds 16 - 23 were prepared using a method analogous to the method for compound 24 described in Example 24 below below.

[0060] (2R,3S,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl)

[0061]

Chemical Structure

[0062] -2-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-yl heptanoate, 24: Examples 25 - 26 Examples 27 - 38 Step - 1: To a solution of intermediate B (400 mg, 0.75 mmol, 1 equivalent) dissolved in anhydrous MeCN (1 2 mL), DIPEA (0.6 mL, 3.77 mmol, 5 equivalents) and DMAP (18.4 mg, 0.15 mmol, 0.2 equivalent) were added. While stirring this solution at 0 °C , heptanoic anhydride (365 mg, 1. 5 mmol, 2 equivalents) in MeCN (5 mL) was added dropwise. The resulting mixture was then stirred at room temperature for 1 hour . The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was quenched in ice - cold water (2 0 mL) and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were dried, filtered, and evaporated under reduced pressure. The resulting crude material was purified by 1.5% DCM in MeOH Purified by silica gel column chromatography using to obtain Intermediate C (430 mg, 8 8.8% yield). LC-MS (ESI+): m / z 645.00 [M+H] + .

[0063] Step - 2: NH4F (495 mL, 13.35 mmol, 20 equivalents) was added to a solution of Intermediate C (430 mg, 0.67 mmol, 1 equivalent) dissolved in anhydrous MeOH (5 mL). Then, the resulting mixture was stirred at room temperature for 12 hours. The reaction was monitored by LCMS . After the reaction was completed, the reaction mixture was quenched in ice-cold water (20 mL) and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were dehydrated, filtered, and evaporated under reduced pressure . The resulting crude material was purified by silica gel column chromatography using 2.5% DCM in MeOH to obtain Compound 24 as an off-white solid (1 87 mg, 69.1% yield). LC-MS (ESI+): m / z 406 .55 [M+H] . . .55 [M+H] + .

[0064] Note: If the reaction does not complete after stirring at room temperature for 12 hours, stir for an additional 2 hours at 60 °C for other compounds including Compound 24 .

[0065] Example 39 Compounds 25 - 26 were prepared using a method similar to the method for Compound 24 described in Example 24 above .

[0066] (((2R,3S,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl Compounds 27 - 38 were prepared using a method similar to the method for Compound 39 described in Example 39 below .

[0067] )-2-ethynyl-3-hydroxytetrahydrofuran-2-yl)methyldodecanoate, 39:

[0068]

Chem.

[0069] Examples 40 - 42 Examples 43 - 50 Synthesis of stable crystalline forms To a flask containing EFdA (997 mg, 3.4 mmol, 1 equiv) was added pyridine anhydride (34 mL). The mixture was stirred for 1 - 2 minutes and cooled to 0 °C. To this mixture, lauroyl chloride (2.0 mL, 8.5 mmol, 2.5 equiv) was added dropwise at 0 °C. The reaction was stirred overnight at room temperature. Since alkylation of the NH2 group may be observed when the reaction time is prolonged, the reaction was monitored by LCMS. Then, the reaction was diluted with EtOAc, washed three times with water, once with NaHCO3, once with brine, then the solvent was dried, filtered, and evaporated under reduced pressure. The resulting crude product was purified by silica gel column chromatography using 1.5% DCM in MeOH to obtain Compound 39 as an off - white solid (1.0 g, 65% yield). LC - MS (ESI+): m / z 476.66 [M + H] +

[0070] Example 51 Compounds 40 - 42 were prepared using a method analogous to the method for Compound 39 described in Example 39 above.

[0071] General examples regarding the formulation of the compounds of the present invention Compound 43 was prepared using a method analogous to the method for Compound 39 from the known acid dihexadecyl ​​​​​​​​​​​It can be prepared using the acid chloride of L - glycine.

[0072] Compound 44 can be prepared by the reaction between EFdA and the corresponding chloroformate in pyridine as a solvent.

[0073] Compounds 45 - 50 can be prepared in a manner similar to the methods of other examples described herein.

[0074] Example 52 Example 53 Compound 2 (about 200 mg) was completely dissolved in the minimum amount of acetone with stirring. Thereafter, the solvent was slowly evaporated at ambient temperature. As a result, a recrystallized sample was obtained as a white powder. Other solvents such as EtOAc, MeOH and THF can also be used.

[0075] Example 54 In all formulation protocols, a stable aqueous suspension with 26 - gauge syringeability was produced.

[0076] Example 55 Compound 1 (EFdA) was pulverized and sieved through #80. 300 mg of Compound 1 was placed in a suitable container, and a pre - formed solution of 0.25% sodium carboxymethylcellulose (CMC - sodium) and 0.1% polyoxyethylene (20) sorbitan monooleate (TWEEN (R) -80) was added to obtain a final formulation of 1 g (300 mg of Compound 1+about 700 m g of the polymer solution) (about 300 mg / g). Then, the suspension was placed in an ice bath. ​​​​It was sonicated in a bath for 10 minutes. The density of the formulation is 1.064 g / mL. Thereafter, the above The formulation suspension has a concentration of 319.2 mg / mL of Compound 1 (EFdA).

[0077] Pharmacokinetics study The recrystallized Compound 2 was pulverized and sieved through #80. 250 mg of Compound 1 was placed in an appropriate container, and a pre-formed solution of 0.25% sodium CMC and 0.1% TWEE N-80 was added to obtain a final formulation of 1 g (250 mg of Compound 1 + approximately 750 mg of polymer solution) (approximately 250 mg / g). Then, the suspension was probe sonicated in an ice bath for 5 minutes (sonication time: 5 minutes; pulse amplitude: 20; pulse on time: 30 seconds; pulse off time: 20 seconds).

[0078] 1. Animals Compound 3 was pulverized and sieved through #80. 250 mg of Compound 3 was placed in an appropriate container and a pre-formed solution of 0.25% sodium CMC and 0.5% TWEEN-80 was added to obtain a final formulation of 1 g (250 mg of Compound 3 + approximately 750 mg of polymer solution) (approximately 250 mg / g). Then, this suspension was probe sonicated in an ice bath for 5 minutes (sonication time: 5 minutes; pulse amplitude: 20; pulse on time: 30 seconds; pulse off time: 20 seconds). The density of the formulation is 1.004 g / mL. Thereafter, the above formulation suspension has a concentration of 250.88 mg / mL of Compound 3 (approximately 150 mg of EFdA / mL).

[0079] Acclimation / Quarantine: Compound 5 was pulverized and sieved through #80. 200 mg of Compound 5 was placed in an appropriate container Take it and add the pre - formed solution of 0.25% sodium CMC and 0.5% TWEEN - 80 to obtain a final formulation of 1 g (200 mg of compound 5+about 800 mg of polymer solution) ( about 200 mg / g). Then, this suspension was probe - sonicated in an ice bath for 5 minutes ( sonication time: 5 minutes; pulse amplitude: 20; pulse - on time: 30 seconds; pulse off time: 20 seconds). The density of the formulation is 1.047 g / mL. Thereafter, the above - mentioned formulation suspension has a concentration of compound 5 of 209.4 mg / mL (about 115.97 mg / mL of EFdA) and is .

[0080] Animal management: Animal cannulation: Animals (male SD rats, about 200 - 250 g, and male rhesus monkeys, about 2 - 3 kg) were obtained from approved suppliers (「SLAC Laboratory Animal Co., Ltd., Shanghai, China」, and / or 「Topgene Biotech nology, Wuhan city, Hubei Province, Chin a」).

[0081] 2. Dosage formulation After the animals arrived, their general health status was evaluated by veterinary staff or other approved personnel. The animals were acclimatized for at least 3 days before being used in the test . .

[0082] SC formulation: The animals were housed in groups during the acclimatization period and individually during the test period. The environment of the animal room was controlled (target conditions: temperature 18 - 26 °C, relative humidity 30 - 70%, 12 - hour artificial light, and 12 - hour darkness). Temperature and relative humidity were monitored daily.

[0083] 3. Dosage administration None. The animals were fasted for at least 12 hours before administration. All animals were given Certified Rodent and non-Rodent Diet (Catalog # M01-F, SLAC Laboratory Animal Cl. Ltd., Shanghai, China) ad libitum 4 hours after administration. tified Rodent and non-Rodent Diet (Catal og # M01-F, SLAC Laboratory Animal Cl. L td., Shanghai, China) ad libitum. Water was autoclaved and given to the animals ad libitum. Water was analyzed periodically and the results were stored. There were no known contaminants at detectable levels in the diet and water that would interfere with the objectives, conduct, or results of this study.

[0084] 4. Sample collection 5. Blood / plasma processing The formulation was prepared according to the procedures shown in Examples 52 - 55 and Tables 2 - 4. The formulation was prepared on the day of administration. After the formulation was prepared, it was administered to the animals within 4 hours. Two 20 μL aliquots of each formulation were taken from each formulation solution and transferred into 1.5 mL polypropylene microcentrifuge tubes, and dose verification was performed by LC / UV or LC-MS / MS.

[0085] Blood: For SC administration, the above administration formulation was administered by subcutaneous injection according to the facility's SOP.

[0086] 6. Sample analysis For rats, approximately 200 μL of blood was collected from the prostrate vein at each time point, and for cynomolgus monkeys, 0.5 mL of blood was collected. All blood samples were transferred into microcentrifuge tubes containing 4 μL of K2EDTA (0.5 M) as an anticoagulant and placed on wet ice until processed for plasma. ​​​​​​​​

[0087] Verification of the concentration of the dosage formulation ​ The blood sample was processed for plasma within 30 minutes after collection by centrifugation at approximately 4°C and 3000 g for 15 minutes. The plasma sample was stored in a polypropylene tube, rapidly frozen on dry ice, and maintained at -70 ± 10°C until LC / MS / MS analysis. .

[0088] ​ ​ · An aliquot of each dosage formulation was collected in duplicate at the central position of the formulation; · The concentration of the test compound in the dosage formulation sample was measured by LC / UV or LC / MS / MS methods; ; Biological analysis methods and sample analysis · An LC-MS / MS method for quantifying the test substance in the corresponding biological matrix was developed under non-GLP; ; · A calibration curve with eight non-zero calibration standards was applied to the method including the LLOQ; · A set of QC samples consisting of low, medium, and high concentrations was applied to the method; · The test sample analysis was performed simultaneously using the LC-MS / MS method with one set of calibration standards and two sets of QC samples (when the number of samples exceeded 48, two calibration curves with two sets of QC samples were applied); ; ; · Acceptance criteria: Linearity: Six or more calibration standards were back-calculated to be within ±20% of the nominal value in plasma; Accuracy: Four or more of the six QC samples were back-calculated to be within ±20% of the nominal value in plasma; ; Specificity: The average calculated concentration in a single blank matrix should not be more than 0.5 times the LLOQ; ; Sensitivity: Try to target the LLOQ at 1 - 3 ng / mL; Carry - over: The average calculated carry - over concentration in a single blank matrix immediately after the highest standard injection should be such that. If the carry - over cannot meet the criteria, the percentage of carry - over should be estimated according to the in - house adult analysis SOP.

Chem.

[0089] 7. Data analysis , T , AUC max , T m ax , T 1 / 2 , AUC (0-t) , AUC (0-inf) , MRT (0-t) , MRT (0-inf) , %F and graphs of plasma concentration versus time profiles were reported.

[0090] Example 56 Several prodrugs containing EFdA(1) as a control were subjected to single - dose rat PK tests by subcutaneous administration. All were injected at an equal dose of 10 mg / kg of EFdA and an equal concentration of 4 mg / mL as an aqueous suspension formulation derived from 0.5% CMC - Na and 0.5% TWEEN - 80. Similar exposures were observed, but prodrug groups 2, 3, and 5 showed plasma levels of EFdA above the LLOQ for more than one week at C much lower than that of EFdA itself. max

[0091] ​​​​ Table 2 shows the rat PK data for Compounds 1, 2, 3, and 5 after SC administration at a dose equivalent to 10 mg / kg of EFdA. The data presented in graphical form are shown in Figure 10.

[0092] [Table 2]

[0093] Example 57 After a series of formulation optimizations, for prodrug 3 and EFdA, SC rat PK studies were performed again at high equivalent doses of 100 mg / kg at high EFdA equivalent concentrations of 120 mg / mL and 319 mg / mL, respectively. For prodrug 3, Cmax was delayed and showed a 100-fold lower value compared to EFdA. Furthermore, for prodrug 3, an extended half-life and mean residence time were also observed, which is suitable for prophylaxis. max

[0094] Table 3 shows the rat PK data for Compounds 1 and 3 after SC administration at EFdA equivalent doses of 10 mg / kg and 100 mg / kg in high-concentration formulations. The data are shown in graphical form in Figure 11.

[0095] [Table 3]

[0096] Example 58 After rat PK analysis, the focus shifted to non-rodents, namely, cynomolgus monkeys. Prodrugs 5 and EFdA were administered as a single dose by subcutaneous injection at equal doses of 50 mg / kg of EFdA. ​​​​​​​​​​It was subjected to a PK test with rhesus monkeys. The aqueous suspension formulation was derived from 0.25% CMC-Na and 0. 1% / 0.5% TWEEN-80 and had equal concentrations of 116 mg / mL and 319 mg / mL for prodrug 5 and EFdA, respectively. Prodrug 5 showed plasma levels of EFdA above the LLOQ for over 1 month and C showed a value 24-fold lower than that of EFdA itself. max was E nd.

[0097] Table 4 shows the rhesus monkey PK data for compounds 1 and 5 after SC administration at an equal dose of 50 mg / kg of EFdA in the high-concentration formulation. The data is also shown in graphical form in Figure 12.

[0098]

Table 4

Claims

[Claim 1] The invention described in the specification.

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