CROSS-REFERENCE TO RELATED APPLICATIONS

This application claiins the benefit of U.S. Provisional Application Serial No. 62/898,679, filed on Septeinber 11, 2019, which is incorporated herein by reference in its entirety

FIELD OF INVENTION

This invention relates to antiviral coinpounds and coinpositions useful for the treatinent of acquired iininunodeficiency syndroine (AIDS).

BACKGROUND

The coinpound EFdA (MK-8591 ) is a nucleoside analog known to be effective as an inhibitor of the enzyine reverse transcriptase ( Current Opinion in HIV and AIDS 2018, 13, 294-299)

Certain EFdA analogs are described in a U.S. application published as US2019/185508, including phosphates, esters, carbonates, carbainates where at least one of the substitutions in the glycosyl donor ring contain a "D" atoin (D ^:::: deuteriuin ^:::: 2H).

Reverse transcriptase inhibitors can be effective in the treatinent of viral infections caused by viruses where reverse transcriptase function is essential for viral replication and production of viral proteins, such as HIV (Huinan Iininunodeficiency Virus) and HBV (Hepatitis B Virus). In the case of HIV, the viins is an RNA virus that uses reverse transcriptase to synthesize DNA reverse transcripts of the vital genoine which are translated by the host to provide the viral proteins. In the case of HBV, a DNA viins, the DNA viral polyinerase also has a reverse transcriptase function, generating viral DNA froin a viral RNA interinediate during replication. An antiviral exainple of a coinpound that hits both HIV and HBV is lainivudine.

SUMMARY

The invention provides, in various einbodiinents, (1) novel coinpositions of cheinical inatter coinprising bioactive prodrugs of MK-8591 or pharinaceutically acceptable salt of these prodrugs, and (2) novel forinulations of these prodrugs providing therapeutic and prophylactic treatinent of patients against viral infections of viruses such as HIV and HBV, wherein inhibition of a reverse transcriptase enzyine (RNA-directed DNA polyinerase) slows or blocks the viral infection. The route of adininistration for these treatinents can include, but not liinited to, oral, parenteral and iinplants (coinposition and device). The forinulations of this invention provide for slow or controlled or sustained release of EFdA froin these prodrugs when injected as an aqueous suspension forinulation.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows an X-ray Powder Diffractograin (XPRD) of EFdA.

Figure 2 shows data provided by Differential Scanning Caloriinetry (DSC) and Therino- Graviinetric Analysis (TGA) of EFdA.

Figure 3 shows DSC and TGA data for coinpound 2.

Figure 4 shows XPRD data for coinpound 3.

Figure 5 shows DSC data for coinpound 3.

Figure 6 shows TGA data for coinpound 3.

Figure 7 shows XPRD data for coinpound 5.

Figure 8 shows DSC data for coinpound 5.

Figure 9 shows TGA data for coinpound 5.

Figure 10 shows in graphic forin the data provided in Table 2.

Figure 11 shows in graphic forin the data provided in Table 3.

Figure 12 shows in graphic forin the data provided in Table 4.

DETAILED DESCRIPTION

The invention provides, in various einbodiinents, a coinpound of Forinula (I)

wherein

R ¹ is H or is X-L _m , wherein in = 1 or 2, and wherein X-L _m is -C(=O)L, -C(=O)OL, -C(=O)NH(L), -C(=O)NH(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)(NL ₂ ), or -R(=O)(NT ₂ ) ₂ , and each independently selected L is (C _1-22 )alkyl, (C _3-22 )alkenyl wherein the alkenyl can coinprise 1-6 unsaturations, (C _3-7 )cycloalkyl, (CHR) _n -phenyl wherein n=O or 1, or -CHR-N(R) ₂ , or R is -OCH(R)OP(=O)(OH) ₂ or R ¹ is a phosphate residue or its derivative residue coinprising a inonophosphate, a diphosphate, a triphosphate, a phosphonate, a phosphate polyester, a phosphate ainidate (inono and di), a phosphorothioate, a phosphoroseienoate, or a phophoroboranoate

R is H, (C _1-22 )alk\d, or (C _3-22 )alkenyl wherein the alkenyl can coinprise 1-6 unsaturations, or is (C _3-7 )cycloalkyl;

R ² is H or is X-L _m , or R ² is -O CH(R)OP(=O)(0H) ₂ or R ² is a phosphate residue or its derivative residue coinprising a inonophosphate, a diphosphate, a triphosphate, a phosphonate, a phosphate polyester, a phosphate ainidate (inono and di), a phosphorothioate, a phosphoroseienoate, or a phophoroboranoate; excluding following coinbinations :( a) R ^{1 :::} R ^{2 :::} acetyl, (b) R ¹ = R ² = H, and (c) R ^{2 :::} H when R ¹ is a phosphate residue or its derivative residue coinprising a inonophosphate, a diphosphate, a triphosphate, a phosphonate, a phosphate polyester, a phosphate ainidate (inono and di), a phosphorothioate, a phosphoroseienoate, or a phophoroboranoate; or a pharinaceutically acceptable salt thereof.

In various einbodiinents, a coinpound of Forinula (I) can be any of the specific coinpounds 2 - 51, shown in Table 1.

The invention further provides, in various einbodiinents, a inethod of inhibiting viral reverse transcriptase bioactivity, coinprising contacting a viins expressing an enzyine with reverse transcriptase bioactivity with an effective ainount or concentration of a coinpound of Forinula (I).

The invention further provides, in various einbodiinents, a inethod of prophylaxis of vireinia or treatinent of a viral infection in a patient wherein inhibition of a reverse transcriptase is inedically indicated, coinprising adininistering to the patient an effective ainount or concentration of a coinpound of Forinula (I). More specifically, the coinpound of Forinula (I) can be adininistered in a forinulation that provides for slow or controlled or sustained release of EFdA froin these prodrugs. More specifically, the coinpound of Forinula (I) can be forinulated as aqueous suspension, solutions, and can be encapsulated in particles for slow-release including PLGA and those known in the art. More specifically, the viral infection can be caused by HIV or HBV. The routes of adininistration for these prodrugs can include, but not liinited to, oral, parenteral and iinplants (drug delivery coinposition and device). In the inethod for the treatinent or prevention of the viral infection, the inethod inay further coinprise an additional anti-HIV and/or anti-HBV agent including but not liinited to, cabotegravir, dolutegravir, doravirine, elvitegravir, lersiverine, tenofovir disoproxil fuinarate, tenofovir alafenainide fuinarate or lainivudine.

Table 1: Coinpounds of the Invention

EXAMPLES

Abbreviations

The following abbreviations are used: tetrahy drofuran (THF), dichloroinethane (DCM), Acetonitrile (MeCN), N.N-diinethylforinainide (DMF), diinethylsulfoxide (DMSO), trifluoroacetic acid (TEA), triethylainine (TEA), diisopropylethylainine (DIPEA), inethanol (MeOH), Ethyl acetate (EtOAc), 4-Diinethylaininopyridine (DMAP), N-(3- Diinethylaininopropyl)-N'-ethylcarbodiiinide hydrochloride (EDC _. HC1), N,N'- Dicyclohexylcarbodiiinide (DCC).

Genera! Exainples for the Preparation of Coinpounds of the Invention

The starting inaterials and interinediates for the coinpounds of this invention inay be prepared by the application or adaptation of the inethods described below, their obvious cheinical equivalents, or, for exainple, as described in literature such as The Science of Synthesis, Voluines 1-8. Editors E. M. Carreira et al. Thieine publishers (2001-2008). Details of reagent and reaction options are also available by structure and reaction searches using coininercial coinputer search engines such as Scifinder (www.cas.org) or Reaxys (www . reaxys . coin) .

Part I: Preparation of Interinediates and EFdA (MK-8591) Exainple 1

Synthesis of intermediate-A, -B and Compound I (EFdA):

(2R,3S,5R)-5-(6-ainino-2-fluoro-9H-pnrin-9-y1)-2-(((tert- butyldiphenylsiIyl)oxy)inethyl)- 2-ethynyItetrahydrofuran-3-yl acetate, A: Interinediate A is prepared according to the reported procedure in the literature (Org. Lett. 2011, 13, 5264-5266.). LC-MS (ES1+): rn/z 574.19 jVi i ij .

(2R,3S,5R)-5-(6-ainino-2-fluoro-9H-purin -9-yl)-2-(((tert-bntyldiphenyIsilyl)oxy)inethyl)- 2-ethynyItetrahydrofuran-3-ol, B: To a solution of interinediate A (500 ing, 0.87 ininol) in MeOH (lOinL) was added inethanolic aininonia (lOinL) at rt and stirred for 12h. The reaction inixture was concentrated under reduced pressure to obtained ciude inaterial and the resulting crude inaterial was purified by silica-gel coluinn chroinatography using 2% MeOH in DCM to obtain the interinediate B (400 ing, 86.33%). LC-MS (ESI+): m/z 532.4 [M+H] ⁺ .

(2R,3S,5R)-5-(6-ainino-2-fluoro-9H-purin-9-yI)-2-ethynyI- 2-(hydroxyinetliyI) tetrahydrofuran-3-ol, 1: Coinpund 1 (EFdA) is prepared according to the reported procedure in the literature (Org. Lett. 2011, 13, 5264-5266.). 'H NMR (400 MHz, DMSO- dc) d 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 (in, 1H), 5.35 - 5.25 (in, 1H), 4.56 (q, J= 6.4 Hz, 1H), 3.67 - 3.60 (in, 1H), 3.60 - 3.49 (in, 2H), 2,74 - 2.63 (in. 111). 246 - 2.36 (in, 111). LC-MS (ESI+): m/z 294.2 | M 111

Part P: Preparation of Exainple Coinpounds Exainple 2

(2R,3S,5R)-5-(6-ainino-2-fluoro-9H-purin-9-yl)-2-ethynyI- 2-((isobutyryloxy)inethyl) tetrahydrofuran-3-yl isobntyrate, 2: To a inixture of EFdA (3 g, 6.8 ininol, 1 equiv.), DMAP (499 ing, 2 73 ininol, 0.4 equiv.) in anhydrous DMF (100 inL) was added isobutyric acid (8.4 g, 27.3 ininol, 6 equiv.) dr op wise at ainbient teinperature. The reaction stirred for 5 h at rt. The reaction is inonitored by LCMS due to the occasional alkylation of NH ₂ group observed for elongated reaction tiine. The reaction inixture was then filtered to reinove byproduct urea. Acetonitrile was used to rinse the reaction inixture. Thereafter, the reaction was washed twice with water and once with brine and then the solvent was dried, filtered and evaporated under reduced pressure. The resulting crude inaterial was purified by silica-gel coluinn chroinatography using 60-70%% EtOAc in Hexanes to obtain the coinpound 2 as a glassy solid. The obtained solid was dispersed in ininiinuin ainount of isopropanol followed by its rotatory evaporation to obtain pure coinpound as a white solid (2.5 g, 85% yield). LC- MS (ESI : in/z 434.49 [ ]+ Exainple 3

(2R,3S,5R)-5-(6-ainino-2-fluoro-9H-piirin-9-yl)-2-(((2-et hylbutanoyl)oxy)inethyl)-2- ethymltetrahydrofuran-3-yl 2-ethylbutanoate, 3: To a inixture of EFdA (1 g, 3.4 ininol, 1 equiv.), 2-ethylbutanoic anhydride (4.4 g, 20.4 ininol, 6 equiv.), TEA (3.8 inl, 27.2 ininol, 8 equiv.) in anhydrous MeCN (43 inL) and cooled to 0 °C was added DMAP (83 ing, 0 68 ininol, 0.2 equiv.) at 0 °C. The reaction stirred for 0.5 h at 0 °C and then to 5 h at rt. The reaction is inonitored by LCMS due to the occasional alkylation of NH ₂ group observed for elongated reaction tiine. The reaction was further quenched with inethanol and solvent was evaporated under reduced pressure. The resulting crude inaterial was purified by silica-gel coluinn chroinatography using 60-70%% EtOAc in Hexanes to obtain the coinpound 3 as a glassy solid. The obtained solid was dispersed in ininiinuin ainount of isopropanol followed by its rotatory evaporation to obtain pure coinpound as a white solid (1 33 g, 80% yield). LC-

MS (ESI+): in/z 490.56 [M+H]+. Exainple 4

(2R,3S,5R)-5-(6-ainino-2-fluoro-9H-purin-9-yl)-2-(((eydop entanecarbon )oxy)inethyl)- 2-ethynyItetrahydrofuran-3-yI eydopentanecarboxylate, 4 : Coinpound 4 was prepared by using the procedure followed for the coinpound 2. LC-MS (ESI+): m/z 486.44 [M+H] ⁺ .

Exainple 5

(2R,3S,5R)-5-(6-ainino-2-fluoro-9H-purin-9-yl)-2-ethynyI- 2-((2-phenylaeeioxy)inethyl) tetrahydrofuran-3-yl 2-phenylacetate, 5: To a inixture of EFdA(499 ing, 1.7 ininol, 1 equiv.), 2-phenylacetic anhydride (2.6 g, 10.2 ininol, 6 equiv.), TEA (1.9 inl, 13.6 ininol, 8 equiv.) in anhydrous MeCN (22 inL) and cooled to 0 °C was added DMAP (42 ing, 0.34 ininol, 02 equiv.) at 0 °C. The reaction stirred for 0.5 h at 0 °C and then to 3 h at rt. The reaction is inonitored by LCMS due to the occasional alkylation of NH ₂ group observed for elongated reaction tiine. The reaction was further quenched with inethanol and solvent was evaporated under reduced pressure. The resulting crude inaterial was purified by silica-gel coluinn chroinatography using 60-70%% EtOAc in Hexanes to obtain the coinpound 5 as a glassy solid. The obtained solid was dispersed in ininiinuin ainount of isopropanol followed by its rotatory evaporation to obtain pure coinpound as a white solid (694 ing, 77% yield). LC-MS (ESI+): in/z 530.52 [M+H]+.

(2R,3S,5R)-5-(6-ainino-2-fluoro-9H-purin-9-yl)-2-ethynyI- 2-((((S)-2- phenylpropanoyl)oxy) inethyl)tetrahydroftiran-3-yl (S)-2-phenyIpropanoate, 6: Coinpound 6 was prepared by using the procedure followed for the coinpound 2 with following differences: a) 4 equiv of corresponding acid used instead of 6; b) After reinove urea via filtration, the solvent was evaporated under reduced pressure and resulting white solid was then suspended in ininiinuin ainount of isopropanol and stirred for an hour. The suspension was then filtered to obtain pure coinpound as a white solid in 75 % yield. LC-MS (ESI+): m/z 558 65 [M+H]+

(2R,3S,5R)-5-(6-ainino-2-fluoro-9H-purin-9-yl)-2-((benzoy Ioxy)inethyI)-2-ethynyI tetrahydrofuran-3-yI benzoate, 7: Coinpound 7 was prepared by using the procedure followed for the coinpound 2 using 4.5 equiv of corresponding acid. LC-MS (EST+): m/z 502.41 [M+H]+

Exainple 8

(2R,3S,5R)-5-(6-ainino-2-fluoro-9H-purin-9-yl)-2-((((4Z,7 Z,10Z,13Z,16Z,19Z)-docosa-

4,7,10,13,16,19-liexaenoyI)oxy)inethyl)-2-ethynyItetrahyd rofuran-3-yl (4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoate, 8: To a inixture of EFdA (117 rng, 0.4 ininol, 1 equiv.), Docosahexaenoic Acid (0.39 g, 1.2 ininol, 3 equiv.), DMAP (4.9 rng, 0.04 ininol, 0.1 equiv.), EDOHC1 (307 ing, 1.6 ininol, 4 equiv.) in anhydrous DMF (4 inL) and cooled to 0 °C was added DIPEA (0.3 niL, 1.6 ininol, 4 equiv.) at 0 °C. The reaction stirred for 8 h at rt. The reaction is inonitored by LCMS due to the occasional alkylation of NH ₂ group observed for elongated reaction tiine. Thereafter, the reaction was diluted with EtOAc and washed thrice with 2.5% NaCl, thrice with buffer pH~7 solution and once with brine and then the solvent was dried, filtered and evaporated under reduced pressure. The resulting crude inaterial was purified by silica-gel coluinn chroinatography using 5% DCM in MeOH to obtain the coinpound 8 as gel-like seini solid (183 ing, 50% yield). LC-MS (ESI+): in/z 457.23 |M/2+H]+ . Exainple 9

(2R,3S,5R)-5-(6-ainino-2-fluoro-9H-purin-9-yl)-2-ethynyI- 2-((heptadeca!ioyIoxy)ineihyI) tetrahydroftsran-3-yI heptadecanoate, 9: Coinpound 9 was prepared by using the procedure followed for the coinpound 2 using 8 equiv of corresponding acid and 6 equiv of DCC. ¹ H NMR (400 MHz, Chloroforin-d) d 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 id. ,/ 12.1 Hz, 1H). 2.99 (d, j =

13.5, 6.7 Hz, 1H), 2.75 - 2.64 (in, 2H), 2.41 (t, .j = 7 5 Hz, 2H), 2.34 (q, ./= 3.8 Hz, 1H), 1.71 - 1.57 (in, 9H), 1.41 - 1.17 (in, 48H), 0.88 (t, J = 6.8 Hz, 6H). LC-MS (ESI+): m/z no signal observed.

Exainple 10

(2R,3S,5R)-5-(6-ainino-2-fluoro-9H-purin-9-yl)-2-ethynyI- 2-((pa!initoyIoxy)inethyl) tetraliydrofuran-3-yI palinitate, 10: To a flask containing EFdA (202 ing, 0.7 ininol, 1 equiv.) was added anhydrous pyridine (7 inL) The inixture was stirred for 1-2 inin and cooled to 0 °C. To this inixture was added palinitoyl chloride (1.3 inL, 4.2 ininol, 6 equiv.) dropwise ai 0 °C. The reaction stirred for overnight at rt. The reaction is inonitored by LCMS due to the occasional alkylation of NH ₂ group observed for elongated reaction tiine.

Thereafter, the reaction was diluted with EtOAc and washed thrice with water, once with NaHCO ₃ and once with brine and then the solvent was dried, filtered and evaporated under reduced pressure. The resulting crude inaterial was purified by silica-gel coluinn chroinatography using 60-70%% EtOAc in Hexanes to obtain the coinpound 10 as a glassy solid. The obtained solid was dispersed in ininiinuin ainount of isopropanol followed by its rotatory evaporation to obtain pure coinpound as a white solid (372 ing, 70% yield). ¹ H NMR (400 MHz, DYSSO-d ₆ ) d 8.34 (s, 1H), 7.91 ( (d, j = 30.8 Hz, 211). 6.34 (t, ./= 6 9 Hz, 1H), 5.70 (t, J= 6.4 11/.. 1H), 4.39 (d, J= 11.6 Hz, 1H), 4.22 (d, J= 11.9 Hz, 1H), 3.81 - 3.73 (in, 1H), 3.14 (dt, J= 14.1, 6.9 Hz, 1H), 2.60 (dt, J= 12.9, 6.0 Hz, 1H), 2.39 (td, J= 7.3, 2.4 Hz, 2H), 2.32 - 2.17 (in, 2H), 1 64 - 1.52 (in, 2H), 1.51 - 1.39 (in, 2H), 1 38 - 1.11 (in, 48H), 0.92 - 0.78 (in, 6H). LC-MS (ESI+): m/z no signal observed.

Exainple 11

(2R,3S,5R)-5-(6-ainino-2-fluoro-9H-purin-9-yl)-2-ethynyI- 2-((heptanoyloxy)inethyl) tetrahydrofuran-3-yI heptanoate, 11: Coinpound 11 was prepared by using the procedure followed for the coinpound 2 using 8 equiv of corresponding acid and 6 equiv of DCC. LC MS (ESI+): rn/z 518.25 [M+H]+ . Exainple 12

((2R,3S,5R)-3-((D-vaIyl)oxy)-5-(6-ainino-2-fluoro-9H-puri n-9-yl)-2- ethymitetrahydrofuran-2-y!)inet!iyl D-valinate, 12: Coinpound 12 was prepared by using the procedure followed for the coinpound 2 using 7 equiv of corresponding boc protected ainino acid and 7 equiv of DCC followed by typical deprotection of Boc group using HC1 in dioxane. LC-MS (ESI+): m/z 492.64 [M+H]+ .

Exainples 13-15

Coinpounds 13-15 were prepared by using the procedure followed for the coinpound 12. Exainples 16-23

Coinpounds 16-23 were prepared by using the procedure followed for the coinpound 24 described below in Exainple 24.

Exainple 24

(2R,3S,5R)-5-(6-ainino-2-fluoro-9H-purin-9-yl)-2-ethynyI- 2-(hydroxyinetliyI)tetra hydrofuran-3-yI heptanoate, 24:

Step-1: To a solution of interinediate B (400 ing, 0.75 ininol, 1 equiv.) in anhydrous MeCN (12 inL) was added DIPEA (0.6 inL, 3.77 ininol, 5 equiv.) and DMAP (18.4 ing, 0.15 ininol, 0.2 equiv.). To this stirrd solution at 0 °C was added dropwise heptanoic anhydride (365 ing, 1.5 ininol, 2 equiv.) in MeCN (5 inL). The resulting inixture was then stirred at rt for 1 h.

The reaction is inonitored by LCMS After coinpletion of reaction, reaction inixture was quenched in ice cold water (20inl) and was extracted with ethyl acetate (3 X 20 inL). The coinbined organic layer was dried, filtered and evaporated under reduced pressure. The resulting crude inaterial was purified by silica-gel coluinn chroinatography using 1.5% DCM in MeOH to obtain the interinediate C (430 ing, 88.8% yield). LC-MS (ESI+): in/z 645.00 |M+Hj+.

Step-2: To a solution of interinediate C (430 ing, 0.67 ininol, 1 equiv.) in anhydrous MeOH (5 inL) was added NH4F (495 inL, 13 35 ininol, 20 equiv.). The resulting inixture was then stirred at rt for 12 h. The reaction is inonitored by LCMS. After coinpletion of reaction, reaction inixture was quenched in ice cold water (20inl) and was extracted with ethyl acetate (3 X 20 inl). The coinbined organic layer w¾s dried, filtered and evaporated under reduced pressure. The resulting crude inaterial was purified by silica-gel coluinn chroinatography using 2.5% DCM in MeOH to obtain the coinpound 24 as an off-white solid (187 ing, 69.1% yield). LC-MS (ESI+): in/z 406.55 [M+H]+·

Note: If reaction not coinpleted after 12h Stirling at rt then stirred for additional 2 h at 60 °C for other coinpounds including coinpound 24.

Exainples 25-26

Coinpounds 25-26 were prepared by using the procedure followed for the coinpound 24 described above in Exainple 24.

Exainples 27-38

Coinpounds 27-38 were prepared by using the procedure followed for the coinpound 39 described below in Exainple 39.

Exainple 39

((2R,3S,5R)-5-(6-ainino-2-fluoro-9H-purin-9-yl)-2-etnhyyl -3-hydroxytetrahydrofuran-2- yl)inethyl dodecanoate, 39: To a flask containing EFdA (997 ing, 3.4 ininol, 1 equiv.) was added anhydrous pyridine (34 inL). The inixture was stirred for 1-2 inin and cooled to 0 °C. To this inixture was added lauroyl chloride (2.0 inL, 8.5 ininol, 2.5 equiv.) dropwise at 0 °C.

The reaction stirred for overnight at rt. The reaction is inonitored by LCMS due to the occasional alky lation of Nl¾ group observed for elongated reaction tiine. Thereafter, the reaction was diluted with EtOAc and washed thrice with water once with NaHCO ₃ and once with brine and then the solvent w'as dried, filtered and evaporated under reduced pressure. The resulting crude inaterial was purified by silica-gel coluinn chroinatography using using 1.5% DCM in MeOH to obtain the coinpound 39 as an off-white solid (1.0 g, 65% yield).

LC -MS (ESI+): in/z 476.66 [M+H]+ .

Exainples 40-42

Coinpounds 40-42 were prepared by using the procedure followed for the coinpound 39 described above in Exainple 39.

Exainples 43-50

Coinpound 43 can be prepared by using the procedure followed for the coinpound 39 using the acid chloride of known acid dihexadecylglycine.

Coinpound 44 can be prepared by the reaction between EFdA and the corresponding chloroforinate in the pyridine as the solvent.

Coinpounds 45-50 can be prepared following procedures siinilar to those of the other exainples described herein.

Synthesis of Stable Crystalline Forins

Exainple 51

Coinpound 2 (-200 ing) was coinpletely dissolved in ininiinuin quantity of acetone with stirring. This was followed by slow evaporation of the solvents at ainbient teinperature. This resulted in a reciystallized sainple as a white powder. Other solvents including EtOAc, MeOH and THE can also be used.

All forinulation protocols generated a stable aqueous suspension with 26-gauge syringeability.

Exainple 52

Coinpound 1 (EFdA) was grinded and sieved through #80. 300 ing of 1 was taken in suitable container and a solution of preforined 0.25% sodiuin carboxyinethyl cellulose (Sodiuin CMC) and 0.1% polyoxyethlene (20) sorbitan inonooleate (TWEEN-80) was added to obtain 1 grain (300 ing of 1 + ~700 ing of polyiner solution) of the final forinulation (~ 300 ing/g). The suspension was then bath sonicated for 10 inin in an Ice bath. The density of forinulation of is 1 064 g/inL. Henceforth, the above forinulation suspension will be 319 2 ing/inL concentration of coinpound 1 (EFdA).

Exainple 53

Recrystallized Coinpound 2 was grinded and sieved through #80. 250 ing of 1 was taken in suitable container and a solution of preforined 0.25% Sodiuin CMC and 0.1% TWEEN-80 was added to obtain 1 grain (250 ing of 1 + ~ 750 ing of polyiner solution) of the final forinulation (~ 250 ing/g). The suspension was then probe sonicated for 5 inin in an ice bath (Sonication tiine: 5 inin; Pulse Ainplitude: 20; Pulse on Tiine: 30 sec; Pulse off tiine: 20 sec).

Exainple 54

Coinpound 3 was grinded and sieved through #80. 250 ing of 3 was taken in suitable container and a solution of preforined 0.25% Sodiuin CMC and 0.5% TWEEN-80 was added to obtain 1 grain (250 ing of 3 + ~750 ing of polyiner solution) of the final forinulation (~

250 ing/g). The suspension was then probe sonicated for 5 inin in an ice bath (Sonication tiine: 5 inin; Pulse Ainplitude: 20; Pulse on Tiine: 30 sec; Pulse off tiine: 20 sec). The density of forinulation of is 1.004 g/inL. Henceforth, the above forinulation suspension will be 250.88 ing/inL concentration of coinpound 3 (~ 150 ing/inL of EFdA).

Exainple 55

Coinpound 5 was grinded and sieved through #80. 200 ing of 5 was taken in suitable container and a solution of preforined 0.25% Sodiuin CMC and 0.5% TWEEN-80 was added to obtain 1 grain (200 ing of 5 + ~800 ing of polyiner solution) of the final forinulation (~

200 ing/g). The suspension was then probe sonicated for 5 inin in an ice bath (Sonication tiine: 5 inin; Pulse Ainplitude: 20; Pulse on Tiine: 30 sec; Pulse off tiine: 20 sec). The density of forinulation of is 1.047 g/inL. Henceforth, the above forinulation suspension will be 209.4 ing/inL concentration of coinpound 5 (~ 115.97 ing/inL of EFdA). Pharinacokinetic Studies 1. Aniinals

Aniinals (Male SD rats ~ 200-250 g and Male rhesus inacaques ~2-3 kg) were obtained froin an approved vendor (SLAC Laboratory Aniinal Co. Ltd., Shanghai, China and/or Topgene Biotechnology, Wuhan city, Hubei Province, China).

Acclimation/Quarantine: Following arrival, aniinals were assessed as to their general health by a ineinber of the veterinary' staff or other authorized personnel. Aniinals were accliinated for at least 3 days before being placed on study.

Animal Husbandry : Aniinals were group housed during accliination and individually housed during the study. The aniinal rooin environinent will be controlled (target conditions: teinperature 18 to 26°C, relative huinidity' 30 to 70%, 12 hours artificial light and 12 hours dark). Teinperature and relative huinidity' were inonitored daily.

Animal Cannulation: No.

Aniinals were fasted at least 12 hours prior to the adininistration. All aniinals had access to Certified Rodent and non-Rodent Diet (Catalog # M01-F, SLAC Laboratory Aniinal Cl. Ltd., Shanghai, China) ad libitum 4 hours post dosing.

Water was autoclaved before provided to the aniinals ad libitum. Periodic analyses of the water was perforined and the results archived. There are no known containinants in the diet or water that, at the levels of detection, is expected to interfere with the purpose, conduct or outcoine of the study.

2. Dose Forinulation

SC Formulation: The forinulations were prepared according to the procedure given in Exainples 52-55 and Table 2-4. The forinulations were prepared on the day of dosing.

Aniinals were dosed within four hours after the forinulation is prepared. Two 20 mL aliquots of each forinulation were reinoved froin each of the forinulation solutions, transferred into 1.5 inL of polypropylene inicrocentrifuge tubes and run dose validation by LC/UV or LC- MS/MS.

3, Dose Adininistration

For SC dosing, the dose forinulation was adininistered via subcutaneous injection following facility' SOPs. 4, Sainple Collection

Approxiinately 200 mL blood was collected froin saphenous vein at each tiine point for rats and 0.5 inL for rhesus inacaques. All blood sainples were transferred into inicrocentrifuge tubes containing 4pL of K2EDTA (0.5M) as anti-coagulant and placed on wet ice until processed for plasina.

5, Blood/Plasina processing

Blood: Blood sainples were processed for plasina by centrifugation at approxiinately 4 °C, 3000 g 15 inin within half an hour of collection. Plasina sainples was stored in polypropylene tubes, quick frozen over dry ice and kept at -70+10 °C until LC/MS/MS analysis.

6, Sainple Analysis

Dose formulation concentration verification

* Aliquots of the forinulations were collected in the iniddle position of each dose forinulation in duplicate

* The concentrations of the test coinpound in dose forinulati on sainples were deterinined by the LC/UV or LC/MS/MS inethod Bioanalytical method and sample analysis LC -MS/MS inethods for the quantitative deterinination of test coinpound in corresponded biological inatrix was developed under non-GLP coinpliance.

* A calibration curve with 8 non-zero calibration standards was applied for the inethod including LLOQ.

* A set of QC sainples consi sting of low, iniddle, and high concentration was appli ed for the inethod.

* Tiie study sainple analysis will be perforined concurrently with a set of calibration standards and two sets of QC sainples using the LC-MS/MS inethod (If sainple nuinbers were inore than 48, then two calibration curves with 2 sets of QC sainples were applied).

* Acceptance criteria:

Linearity; a ininiinuin of 6 calibration standards was back calculated to within ±20% of their noininal values in plasina

Accuracy: A ininiinuin of 4 out of 6 QC sainples was back calculated to within ±20% of their noininal values in plasina.

Specificity: The inean calculated concentration in the single blank inatrix should be 0.5 tiines the LLOQ. Sensitivity: the LLOQ will be tried to target 1~3 ng/inL.

Carry over: the inean calculated carry -over concentration in the single blank inatrix iininediately after the highest standard injection should be £ LLOQ. If the carryover couldn’t ineet the criteria, then the percent of carryover should be estiinated following in-house bioanalytical SOP.

7. Data Analysis

Plasina concentration versus tiine data was analyzed by non-coinpartinental approaches using the Phoenix WINNONLIN 6.3 software prograin. C _max, T _max , T _½ , AUC _(0-t) , AUC _(0-inf), MRT _{(0- t)} , MRT _(0-inf), %F and graphs of plasina concentration versus tiine profile were reported.

Exainple 56

Several prodrugs including EFdA (l) as a control were subjected to a single dose rat PK studies via subcutaneous route of adininistration. All were injected with equivalent doses of 10 ing/kg and concentration of 4 ing/inL of EFdA as an aqueous suspension forinulation derived froin 0.5% CMC-Na and 0.5% TWEEN-80. While siinilar exposure wns observed, all prodrugs 2, 3 and 5 exhibited plasina levels of EFdA above LLOQ for inore than a week with C _max inuch lower than EFdA itself.

Table 2 shows the rat PK data for coinpounds 1, 2, 3 and 5 following SC adininistration at 10 ing/kg equivalent dose of EFdA. The data are shown in graphic forin is shown in Figure 10.

Tab!e 2

Exainple 57

After a series of forinulation optiinization, SC rat PK studies were perforined again with high equivalent dose of 100 ing/kg at high equivalent concentration of 120 ing/inL and 319 ing/inL of EFdA for prodrug 3 and EFdA respectively. Prodrug 3 provides a delayed and 100-fold lower C _max than EFdA. Enhanced half life and inean residence life were also observed in case of prodrug 3 inaking it suitable for prophylaxi s.

Table 3 shows the rat PK data for coinpounds 1 and 3 following SC adininistration at 10 ing/kg and 100 ing/kg equivalent dose of EFdA at high concentration forinulation. The data are shown in graphic forin in Figure 11.

Exainple 58

After rat PK analysis, the focus was shifted to non-rodents i.e. rhesus inacaque. Prodrug 5 and EFdA were subjected to a single dose rhesus inacaque PK studies via subcutaneous route of adininistration with equivalent doses of 50 ing/kg of EFdA. The aqueous suspension forinulations were derived froin 0.25% CMC-Na and Q.1%/0.5% TWEEN-80 with equivalent concentration of 116 ing/inL and 319 ing/inL of EFdA for prodrug 5 and EFdA respectively. Prodrugs 5 exhibited plasina levels of EFdA above LLOQ for inore than a inonth with 24-fold lower C _max than EFdA itself.

Table 4 shows the Rhesus PK data for coinpounds 1 and 5 following SC adininistration at 50 ing/kg equivalent dose of EFdA at high concentration forinulation. The data are shown in graphic forin in Figure 12.