Antiviral prodrug and pharmaceutical composition thereof

Abstract

The invention relates to an antiviral prodrug and a pharmaceutical composition thereof. Diesters of 4, 4 '-ethynyl-2-fluoro-2'-deoxyadenosine, and aqueous non-gastrointestinal suspensions thereof, provide prolonged in vivo viral inhibition against human immunodeficiency virus (HIV).

Description

[0001] This application is a divisional application of the Chinese invention patent application with application date of September 11, 2020, application number 202080077922.6, and invention name “Antiviral Prodrugs and Pharmaceutical Compositions Thereof”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 898,679, filed September 11, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0004] The present invention relates to antiviral compounds and compositions useful in the treatment of acquired immunodeficiency syndrome (AIDS). Background Art

[0005] 4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) (MK-8591) represented by Formula I:

[0006]

[0007] It is a nucleoside analog that is a potent inhibitor of nucleoside reverse transcriptase (Current Opinion in HIV and AIDS 2018, 13, 294-299), and can be used as an antiretroviral drug for the treatment and pre-exposure prophylaxis of HIV-1 infection. EFdA is metabolized in cells to an active triphosphate anabolic metabolite (EFdA-TP), which inhibits HIV reverse transcriptase.

[0008] However, EFdA has a relatively high water solubility and a relatively short time course of blood concentration. As a result, when EFdA is administered to patients for the treatment of human immunodeficiency virus (HIV) infection or for pre-exposure prophylaxis, EFdA can only provide a limited duration of viral suppression. Therefore, there is a need for a formulation that can extend the duration of viral suppression after administration. The compounds and pharmaceutical preparations of the present invention meet this need. Summary of the invention

[0009] The antiviral compounds of the present invention are diesters of EFdA and have limited solubility in water. Although EFdA has a water solubility of 0.877 mg / mL at physiological pH, the EFdA diesters of the present invention have a water solubility of less than 0.03 mg / mL at physiological pH, preferably less than 0.002 mg / mL. These diesters of EFdA are crystalline, can be used to provide extended duration of HIV inhibition, and can be administered parenterally as a suspension in a pharmaceutically acceptable carrier. The preferred prophylactic dose for human subjects is an EFdA diester in the range of about 80 mg to about 800 mg, administered parenterally at intervals of about 6 months, in a dosage volume of about 0.5 mL to about 4 mL per dose. The preferred therapeutic dose for human patients is an EFdA diester in the range of about 80 mg to about 800 mg, administered parenterally at intervals of about 3 months, in a dosage volume of about 0.5 mL to about 4 mL per dose.

[0010] The parenteral preparation containing the antiviral compound of the present invention may be a dry preparation containing the antiviral compound and a pharmaceutically acceptable excipient or a stable suspension of the antiviral compound in an aqueous or oil-based medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In the accompanying drawings,

[0012] Figure 1 The X-ray powder diffraction pattern (XPRD) of EFdA is shown.

[0013] Figure 2 EFdA data provided by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) are shown.

[0014] Figure 3 DSC and TGA data of Compound 2 are shown.

[0015] Figure 4 XPRD data of compound 3 is shown.

[0016] Figure 5 DSC data of Compound 3 is shown.

[0017] Figure 6 TGA data of Compound 3 is shown.

[0018] Figure 7 XPRD data of compound 5 are shown.

[0019] Figure 8 DSC data of Compound 5 is shown.

[0020] Fig. 9 TGA data of Compound 5 is shown.

[0021] Fig.10 The data provided in Table 2 are shown in graphical form.

[0022] Fig.11 The data provided in Table 3 are shown in graphical form.

[0023] Fig.12 The data in Table 4 are shown in graphical form.

[0024] Fig.13 The data for Example 10 are shown in graphical form.

[0025] Fig.14 XPRD data for Compound 2 are shown.

[0026] Fig.15 XPRD data of compound 6 are shown. DETAILED DESCRIPTION

[0027] The diester represented by formula (II) achieves prolonged viral inhibition in vivo:

[0028]

[0029] Where R 1 and R 2 are independently -C(=O)R 3 , and R 3 It is a member of the group consisting of isopropyl, 3-pentyl, cyclopentyl and benzyl. The above diesters are prepared by reacting EFdA with the desired acid or anhydride and recovering the diester as a crystalline compound. The following examples illustrate the preparation of preferred diesters.

[0030] Preparation of (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-((isobutyryloxy)methyl)tetrahydrofuran-3-yl isobutyrate

[0031]

[0032] Isobutyric acid (8.4 g, 27.3 mmol, 6 equiv.)) was added dropwise to a mixture of EFdA (compound 1) (3 g, 6.8 mmol, 1 equiv.) and 4-dimethylaminopyridine (DMAP) (499 mg, 2.73 mmol, 0.4 equiv.) in anhydrous dimethylformamide (DMF) (100 mL) at room temperature. The reaction was stirred at room temperature for 5 h. Since occasional alkylation of the NH2 group was observed when the reaction time was extended, the reaction was monitored by LC-MS. The reaction mixture was then filtered to remove the by-product urea. Acetonitrile was used to rinse the reaction mixture. Thereafter, the reaction mixture was washed twice with water and once with brine, and the solvent was then dried, filtered and evaporated under reduced pressure. The crude material was purified by silica gel column chromatography using hexane containing 60-70% ethyl acetate (EtOAc) to obtain compound 2 as a glassy solid. The obtained glassy solid was dispersed in a minimum amount of isopropanol, which was then rotary evaporated to obtain pure compound 2 as a white solid (2.5 g, 85% yield). LC-MS (ESI+): m / z 434.49 [M+H] + .

[0033] About 250 mg / g of Compound 2 was suspended in aqueous 0.25% CMC-Na / 0.5% Tween-80 (26G injectable) and tested for stability at 40°C / 75% relative humidity for 2 weeks. Fig.14 XPRD data of compound 6 from incubated suspension are provided before (lower panel), after one week (middle panel), and after two weeks (upper panel) of incubation, indicating that the compound maintains good crystallinity in suspension.

[0034] Preparation of (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-(((2-ethylbutyryl)oxy)methyl)-2-ethynyltetrahydrofuran-3-yl 2-butyric acid ethyl ester

[0035]

[0036] A mixture of EFdA (1 g, 3.4 mmol, 1 equiv.), 2-ethylbutyric anhydride (4.4 g, 20.4 mmol, 6 equiv.), triethanolamine (TEA) (3.8 mL, 27.2 mmol, 8 equiv.) in anhydrous acetonitrile (MeCN) (43 mL) was cooled to 0 ° C, and 4-dimethylaminopyridine (DMAP) (83 mg, 0.68 mmol, 0.2 equiv) was added at 0 ° C. The resulting mixture was stirred at 0 ° C for 0.5 h and then at room temperature for 5 h. Since occasional alkylation of the NH2 group was observed when the reaction time was extended, the reaction was monitored by LC-MS. The reaction mixture was quenched with methanol and the solvent was evaporated under reduced pressure. The crude material was purified by silica gel column chromatography using hexane containing 60-70% ethyl acetate (EtOAc) to obtain compound 3 as a glassy solid. The obtained glassy solid was dispersed in a minimum amount of isopropanol, which was then rotary evaporated to obtain pure compound 3 as a white solid (1.33 g, 80% yield). LC-MS (ESI+): m / z 490.56 [M+H] + .

[0037] Preparation of (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-(((cyclopentanecarbonyl)oxy)methyl)-2-ethynyltetrahydrofuran-3-ylcyclopentanecarboxylate

[0038]

[0039] Compound 4 was prepared by following the procedure for Compound 2, except that cyclopentanoic acid was used instead of isobutyric acid. LC-MS (ESI+): m / z 486.44 [M+H] + .

[0040] Preparation of (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-((2-phenylacetoxy)methyl)tetrahydrofuran-3-yl 2-phenylacetate

[0041]

[0042] A mixture of EFdA (499 mg, 1.7 mmol, 1 equiv.), 2-phenylacetic anhydride (2.6 g, 10.2 mmol, 6 equiv.), triethanolamine (TEA) (1.9 mL, 13.6 mmol, 8 equiv.) in anhydrous acetonitrile (MeCN) (22 mL) was cooled to 0 ° C, and 4-dimethylaminopyridine (DMAP) (42 mg, 0.34 mmol, 0.2 equiv.) was added at 0 ° C. The reaction was stirred at 0 ° C for 0.5 h and then stirred at room temperature for 3 h. Since occasional alkylation of the NH2 group was observed when the reaction time was extended, the reaction was monitored by LC-MS. The reaction mixture was quenched with methanol and the solvent was evaporated under reduced pressure. The crude material was purified by silica gel column chromatography using hexane containing 60-70% ethyl acetate (EtOAc) to obtain compound 5 as a glassy solid. The obtained glassy solid was dispersed in a minimum amount of isopropanol, which was then rotary evaporated to obtain pure compound 5 as a white solid (694 mg, 77% yield). LC-MS (ESI+): m / z 530.52 [M+H] + .

[0043] Table 1 shows the compound characterization data obtained by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS).

[0044] (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-((benzoyloxy)methyl)-2-ethynyltetrahydrofuran-3-ylbenzoate, Compound 6

[0045] Compound 6 was prepared by using a procedure similar to that of compound 2 and using 4.5 equiv of the corresponding acid. LC-MS (ESI+): m / z 502.41 [M+H] + About 275 mg / g of Compound 6 was suspended in aqueous 0.25% CMC-Na / 0.5% Tween-80 (26G injectable) and tested for stability at 40°C / 75% relative humidity. Fig.15 XPRD data of compound 6 from incubated suspension are provided before (lower panel), after one week (middle panel), and after two weeks (upper panel) of incubation, indicating that the compound maintains good crystallinity in suspension.

[0046] Table 1: Preferred compounds

[0047]

[0048]

[0049]

[0050] The foregoing discussion and examples are illustrative and should not be considered restrictive. Other variations within the spirit of the invention are possible and will readily suggest themselves to those skilled in the art.

[0051] Synthesis of stable crystalline form

[0052] Example 1

[0053] Compound 2 (-200 mg) was completely dissolved in a minimum amount of acetone under stirring. The solvent was then slowly evaporated at room temperature. A recrystallized sample was obtained as a white powder. Other solvents including ethyl acetate, methanol and tetrahydrofuran may also be used.

[0054] General Examples of Formulations of Compounds of the Invention

[0055] All formulation regimens produced stable aqueous suspensions with 26G injectability.

[0056] EFdA preparations

[0057] EFdA was ground and sieved through a #80 sieve. A pre-made 0.25% sodium carboxymethylcellulose (Sodium CMC) and 0.1% polyoxyethylene (20) sorbitan monooleate (Tween-80) solution was added to about 300 mg of EFdA (Compound 1) to provide a final suspension formulation of about 1 g (300 mg Compound 1 + about 700 mg polymer solution) (about 300 mg / g). The suspension was then sonicated in an ice bath for 10 min. The density of the formulation was 1.064 g / mL and provided an EFdA concentration of 319.2 mg / mL.

[0058] Example 2

[0059] The recrystallized compound 2 was ground and sieved through a US Standard Sieve Series No. 80 sieve (nominal mesh size 0.180 mm) for wire knitted screen cloth. 250 mg of compound 2 was placed in a suitable container and a pre-made 0.25% sodium carboxymethylcellulose (CMC) and 0.1% Tween-80 solution was added to obtain a final formulation of 1 g (250 mg of compound 2 + about 750 mg of polymer solution) (~250 mg / g). The suspension was then probe-ultrasonicated in an ice bath for 5 min (ultrasonication time: 5 min; pulse amplitude: 20; pulse on time: 30 s; pulse off time: 20 s).

[0060] Example 3

[0061] Compound 3 was ground and sieved through a U.S. Standard Sieve Series No. 80 sieve (nominal mesh size 0.180 mm) for wire knitted screen cloth. 250 mg of compound 3 was placed in a suitable container and a pre-made 0.25% sodium CMC and 0.5% Tween-80 solution was added to obtain a final suspension of 1 g (250 mg of compound 3 + about 750 mg of polymer solution) (~250 mg / g). The suspension was then probe-ultrasonicated in an ice bath for 5 min (ultrasonication time: 5 min; pulse amplitude: 20; pulse on time: 30 s; pulse off time: 20 s). The density of the suspension was 1.004 g / mL. The suspension contained 250.88 mg / mL of compound 3 (~150 mg / mL EFdA).

[0062] Example 4

[0063] Compound 5 was ground and sieved through a US Standard Sieve Series No. 80 sieve (nominal mesh size 0.180 mm) for wire knitted screen cloth. 200 mg of compound 5 was placed in a suitable container and a pre-made solution of 0.25% sodium CMC and 0.5% Tween-80 was added to obtain a final suspension of 1 g (200 mg of compound 5 + about 800 mg of polymer solution) (-200 mg / g). The suspension was then probe-ultrasonicated in an ice bath for 5 min ((ultrasonication time: 5 min; pulse amplitude: 20; pulse on time: 30 s; pulse off time: 20 s). The density of the suspension was 1.047 g / mL. The suspension contained 209.4 mg / mL of compound 5 (-115.97 mg / mL EFdA).

[0064] Pharmacokinetic (PK) studies

[0065] Animals: Animals (male SD rats -200-250 g and male rhesus monkeys -2-3 kg) were obtained from approved suppliers (Slack Laboratory Animal Co., Ltd., Shanghai, China and / or Tianqin Biotechnology Co., Ltd., Wuhan, Hubei, China).

[0066] Acclimation / Isolation: Upon arrival, the general health of the animals shall be assessed by veterinary staff or other authorized personnel. Animals shall be acclimated for at least 3 days prior to study entry.

[0067] Animal housing: Animals were housed in groups during the acclimation period and individually during the study. The animal room environment was controlled (target conditions: temperature 18 to 26°C, relative humidity 30 to 70%, 12 h artificial light and 12 h artificial darkness). Temperature and relative humidity were monitored daily.

[0068] Animal Intubation: No

[0069] Animals were fasted for at least 12 h before drug administration. Four hours after drug administration, all animals had free access to certified rodent and non-rodent diets (Cat. #M01-F, Slack Laboratory Animal Co., Ltd., Shanghai, China).

[0070] Water was autoclaved before being provided to the animals ad libitum. Water was analyzed regularly and the results were archived. No known contaminants that could be expected to interfere with the purpose, conduct, or results of the study were detected in the diet or water.

[0071] 1. Dosage Formulation

[0072] SC Formulation: On the day of dosing, the suspension was prepared according to the procedures described in Examples 2-5 and Tables 2-4 above. It was administered to animals within 4 h of suspension preparation. Two 20 μL aliquots of each prepared suspension were transferred to 1.5 mL polypropylene microcentrifuge tubes and dose verification was performed by LC / UV or LC-MS / MS.

[0073] 2. Dosage Administration

[0074] The suspension was administered by subcutaneous injection (SC) according to the facility standard operating procedure (SOP).

[0075] 3. Sample Collection

[0076] At each time point, approximately 200 μL of blood was collected from the saphenous vein of rats and approximately 0.5 mL of blood was collected from rhesus monkeys. All blood samples were transferred to microcentrifuge tubes containing 4 μL of K2EDTA (0.5 M) as an anticoagulant and placed on wet ice until plasma was processed.

[0077] 4. Blood / Plasma Processing

[0078] Blood: Within half an hour after collection, blood samples were processed to obtain plasma by centrifugation at 3000 g for 15 min at approximately 4°C. Plasma samples were stored in polypropylene tubes, snap-frozen on dry ice and kept at -70 ± 10°C until LC / MS / MS analysis.

[0079] 5. Sample Analysis

[0080] Dose concentration verification

[0081] ■ Collect an aliquot of the suspension from the middle of each prepared suspension in duplicate.

[0082] ■ The concentration of active ingredient in each aliquot of suspension is determined by LC / UV or LC / MS / MS methods Bioanalytical methods and sample analysis

[0083] ■ The LC-MS / MS method for the quantitative determination of the active ingredients (tested compounds) in the respective biological matrices was developed under non-GLP compliance.

[0084] ■A calibration curve containing eight non-zero calibration standards was applied to the method including the lower limit of quantitation (LLOQ).

[0085] ■The method uses a set of quality control (QC) samples consisting of low, medium and high concentrations.

[0086] ■ Research sample analysis will be performed using LC-MS / MS methods in parallel with a set of calibration standards and two sets of QC samples

[0087] (If the number of samples exceeds 48, two calibration curves with 2 sets of QC samples are applied).

[0088] ■Acceptance criteria:

[0089] Linearity: At least 6 calibration standards back-calculated to within ±20% of their nominal values ​​in plasma

[0090] Accuracy: At least 4 of the 6 QC samples were back-calculated to within ±20% of their nominal values ​​in plasma.

[0091] Specificity: The average calculated concentration in a single blank matrix should be 0.5 times the LLOQ.

[0092] Sensitivity: The target lower limit of quantitation (LLOQ) is 1-3 ng / mL.

[0093] Carryover: The average carryover concentration calculated in the single blank matrix sample following the highest standard loading should be the LLOQ. If the carryover does not meet the specification, the percentage carryover should be estimated as per the internal bioanalytical SOP.

[0094] 6. Data Analysis

[0095] The plasma concentration versus time data were analyzed by noncompartmental methods using Phoenix WinNonlin 6.3 software program. max 、T max 、T 1 / 2 , AUC (0-t) , AUC (0-inf) , MRT (0-t) , MRT (0-inf) , %F and plasma concentration-time curves.

[0096] Embodiment 5:

[0097] Several prodrugs, including EFdA (1) as a control, were administered to rats via subcutaneous injection for a single-dose PK study. All animals were injected with a 10 mg / kg equivalent dose and 4 mg / mL concentration of EFdA as an aqueous suspension in 0.5% CMC-Na and 0.5% Tween-80. Although similar exposures were observed, all compounds 2, 3, and 5 showed plasma EFdA levels above the lower limit of quantification (LLOQ) for more than one week, with C max Much lower than EFdA.

[0098] Table 2 shows the PK data of rats after subcutaneous (SC) administration of compounds 1, 2, 3 and 5 at a 10 mg / kg equivalent dose of EFdA. The data are presented in graphical form. Fig.10 middle.

[0099] Table 2: PK in SC rats: EFdA, EFdA levels of compounds 1, 2, 3 and 5

[0100]

[0101] Embodiment 6:

[0102] After optimization, the PK study in SC rats was conducted again using a high equivalent dose of 100 mg / kg at the equivalent concentration of each compound, compound 3 at 120 mg / mL EFdA, compound 5 at 116 mg / mL EFdA, and EFdA at 319 mg / mL EFdA. max Prolonged and 100-fold lower than EFdA. Prolonged half-life and mean residence time were also observed, making Compounds 3 and 5 suitable for prophylaxis.

[0103] Tables 3A and 3B show the rat PK data following SC administration of compounds 1, 3, and 5. The data for compound 3 are shown graphically in Fig.11 middle.

[0104] Table 3A: PK in SC rats: EFdA and EFdA levels of Compound 3

[0105]

[0106] Table 3B: PK in SC rats: EFdA and EFdA levels of compound 5

[0107]

[0108] Embodiment 7:

[0109] After the PK analysis in rats, the focus shifted to non-rodents, namely rhesus monkeys. Compound 5 and EFdA were administered by subcutaneous injection at an equivalent dose of 50 mg / kg EFdA to conduct a single-dose rhesus monkey PK study. The aqueous suspension contained 0.25% CMC-Na with 0.1% / 0.5% Tween-80, and the equivalent concentrations of compound 5 and EFdA were 116 mg / mL and 319 mg / mL of EFdA, respectively. Plasma EFdA levels of compound 5 were observed to be above the LLOQ for more than one month, and C max 24 times lower than EFdA itself.

[0110] Table 4 shows the PK data of compounds 1 and 5 in rhesus monkeys after subcutaneous (SC) administration of EFdA at a dose equivalent to 50 mg / kg. The data are shown in graphical form. Fig.12 middle.

[0111] Table 4: PK in SC rhesus monkeys: EFdA plasma levels after administration of EFdA or Compound 5 in plasma

[0112]

[0113] The suspension medium of the compound of the present invention can be an aqueous carrier, such as water for injection (WFI) or a vegetable oil carrier, such as sesame oil, olive oil, etc. The suspension medium may also contain pharmaceutically acceptable excipients, such as nonionic surfactants, suspending agents or flocculants, preservatives, buffers, toxicity regulators, chelating agents, antioxidants, etc.

[0114] For prophylaxis, a preferred prophylactic dose for human subjects is in the range of about 80 mg to about 800 mg, with the antiviral compound administered parenterally at about 6-month (half-year) intervals in a dosage volume of about 0.5 mL to about 4 mL per dose.

[0115] For the treatment of human patients, the effective amount of the antiviral compound of the present invention is preferably about 80 mg to about 800 mg, with a dosage volume of about 0.5 mL to about 4 mL per dose, more preferably an effective amount of about 200 mg to about 400 mg, and a dosing regimen of 3 months. However, the dosing regimen may vary depending on the time interval between the doses administered in a specific dosing regimen.

[0116] The term "effective amount" as used in this specification and in the claims refers to an amount of an antiviral compound sufficient to inhibit HIV reverse transcriptase, inhibit HIV replication, exert a prophylactic effect and / or exert a therapeutic effect after administration.

[0117] The term "administering" and variations thereof, such as "administering a compound," with respect to the claimed methods of treatment means providing the antiviral compound to a patient and includes self-administration as well as administration of the compound to a patient by another person.

[0118] Preferably, parenteral suspensions suitable for injection contain about 3-45% by weight of the antiviral compound of the invention based on the weight of the suspension.Preferred particle sizes are no greater than about 50 microns (μm), more preferably the average particle size is in the range of about 6 μm to about 15 μm.

[0119] Preferred flocculants or suspending agents are linear polymers, especially substituted celluloses, such as methyl cellulose, carboxymethyl cellulose (CMC), hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and the like.

[0120] Preferred surfactants are nonionic surfactants. A particularly preferred surfactant is polyoxyethylene (20) sorbitan monooleate (Tween-80).

[0121] In addition to parenteral dosage forms, the compounds of the present invention may also be administered in oral dosage forms and as implants.

[0122] Dosage forms containing the compounds of the present invention may also include additional anti-HIV and / or anti-HBV agents, such as cabotegravir, dolutegravir, doravirine, elvitegravi, lersiverine, tenofovir disoproxilfumarate, tenofovir alafenamide fumarate, and lamivudine, among others.

[0123] In various embodiments, the present invention further provides a method for preventing viremia or treating viral infection in a patient, wherein the inhibition of reverse transcriptase is medically indicated, comprising administering to the patient an effective amount or concentration of a compound of formula (II). More specifically, the compound of formula (II) can be administered in the form of a formulation that provides a slow, controlled or sustained release of EFdA from these prodrugs. More specifically, the compound of formula (II) can be formulated into an aqueous suspension, solution, and can be encapsulated in particles for sustained release, including PLGA and other such materials known in the art. More specifically, viral infection can be caused by HIV or HBV. The routes of administration of these prodrugs may include, but are not limited to, oral, non-gastrointestinal, and release from implants (drug delivery compositions and devices). In the method for treating or preventing viral infection, the method may also include additional anti-HIV and / or anti-HBV agents, including but not limited to cabotegravir, dolutegravir, doravirine, elvitegravir, lecithin, tenofovir disoproxil fumarate, tenofovir alafenamide fumarate and lamivudine, etc.

[0124] Example 8

[0125] In the composition containing 400 mg / gm of micronized compound 5, the concentration of surfactant was optimized with 0.3% and 0.5% methylcellulose. The formulations were prepared with different concentrations of Tween-80 (0.1, 0.2 and 0.3%). After storage for 10 days, no significant effect of surfactant concentration on the viscosity, flow and resuspension time of the formulation was observed. The observations are shown in Table 5.

[0126] Table 5: Surfactant concentration optimization using 400 mg / gm of compound 5

[0127]

[0128]

[0129] Viscosity: ++++ slightly viscous, +++ viscous, ++ very viscous, + semi-solid

[0130] Flow rate: + no flow, ++ little flow, +++ good flow, ++++ very good flow √: injectable Example 9

[0131] A batch (40 gm) of Compound 5 suspension was prepared with 0.3% methylcellulose and 0.2% Tween-80 concentrations. Although the concentrations of polymer and surfactant were optimized with a drug concentration of 40 wt%, batches with a drug concentration of 35 wt% were also prepared during the accelerated and long-term stability studies. The composition of the formulation loaded in the stability chamber is given in Table 6. The detailed manufacturing procedures for formulation preparation and composition are given below.

[0132] Table 6: Preparation composition

[0133] Element Amount (mg / g) Quantity, g / 40g Compound 5 350 14 Methylcellulose 3 0.120 Twain-80 2 0.08 Water for Injection (WFI) 645 25.8

[0134] Methylcellulose was slowly added to the required amount of water for injection (WFI) in a glass bottle while stirring continuously. The resulting mixture was stirred using a magnetic stirrer until the solution was clear and free of any lumps. Tween-80 (0.08 g) was added to the obtained solution and stirred thoroughly.

[0135] Micronized compound 5 (14 g; average particle size 11 μm) was slowly added to the prepared polymer, surfactant solution using a magnetic stirrer under vigorous stirring at 1400 rpm. After complete addition of compound 5, the obtained suspension was stirred (600 rpm) for 20 minutes to evenly disperse the particles.

[0136] The prepared formulations were characterized for various physicochemical properties such as assay, pH, % purity, redispersibility, injectability, polymorphic form and particle size. The results are shown in Table 7.

[0137] Table 7: Physicochemical characterization of the prepared formulations

[0138] Test Parameters Appearance White dispersion pH 5.78 HPLC assay (% labeled amount) 108.8％ %purity 99.34％ Redispersibility have Injectability have Polymorphic Form Same as API Average particle size 14μm

[0139] Based on the properties shown in Table 7, the formulations shown in Table 6 were considered acceptable.

[0140] Example 10

[0141] EFdA and aqueous suspensions of compounds 3 and 5 were subcutaneously administered to rhesus monkeys, peripheral blood mononuclear cells (PBMCs) were collected regularly, and pharmacokinetic data were evaluated. The results observed for the two compounds are shown in Tables 8, 9 and 10, and the results observed for compound 5 are shown in Tables Fig.13 shown.

[0142] Table 8: PK in SC rhesus monkeys: PBMC levels of EFdA-TP after administration of Compound 5 or EFdA

[0143]

[0144]

[0145] Table 9: PK in SC rhesus monkeys: EFdA plasma levels and EFdA-TP PBMC levels after administration of Compound 3

[0146]

[0147] Table 10: PK of SC rhesus monkeys: EFdA levels of compounds 3 and 5 in plasma

[0148]

[0149]

Claims

1. A compound of formula (II): Where R 1 and R 2 are independently -C(=O)R 3 , R 3 Selected from the group consisting of isopropyl, 3-pentyl, cyclopentyl, phenyl and benzyl. 2 . The compound according to claim 1 , which is crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-((isobutyryloxy)methyl)tetrahydrofuran-3-yl isobutyrate.

3. The compound according to claim 1, which is crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-((2-ethylbutanoyl)oxy)methyl-2-ethynyltetrahydrofuran-3-yl 2-butyric acid ethyl ester.

4. The compound according to claim 1, which is crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-(((cyclopentanecarbonyl)oxy)methyl)-2-ethynyl-tetrahydrofuran-3-ylcyclopentanecarboxylate. 5 . The compound according to claim 1 , which is crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-((2-phenylacetoxy)methyl)tetrahydrofuran-3-yl 2-phenylacetate.

6. An aqueous parenteral suspension comprising a compound of formula (II): in, R 1 and R 2 are independently -C(=O)R 3 , R 3 selected from the group consisting of isopropyl, 3-pentyl, 1-ethoxypropyl, cyclopentyl and benzyl; Nonionic surfactants; Suspension concentrate; and water.

7. The aqueous parenteral suspension according to claim 6, wherein The compound is present in an amount of about 3% to 4.5% by weight of the suspension, the nonionic surfactant is present in an amount of about 0.1% to 0.5% by weight of the suspension, and the suspending agent is present in an amount of about 0.1% to 0.5% by weight of the suspension.

8. The aqueous parenteral suspension according to claim 6, wherein The nonionic surfactant is polyoxyethylene (20) sorbitan monooleate.

9. The aqueous parenteral suspension according to claim 6, wherein The suspending agent is selected from the group consisting of methylcellulose, carboxymethylcellulose, hydroxypropylcellulose and hydroxypropylmethylcellulose.

10. The aqueous parenteral suspension according to claim 6, wherein The compound is crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2((2-phenylacetoxy)methyl)-tetrahydrofuran-3-yl 2-phenylacetate.

11. The aqueous parenteral suspension according to claim 6, wherein The compound is crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-((2-ethylbutanoyl)oxy)methyl-2-ethynyltetrahydrofuran-3-yl 2-butyric acid ethyl ester.

12. A method for inhibiting human immunodeficiency virus in a patient comprising parenterally administering to the patient an effective amount of a compound of claim 1 in the form of an aqueous suspension.

13. The method according to claim 12, wherein: The administration was a three month dosing regimen.

14. The method according to claim 12, wherein: The effective amount is a dosage in the range of about 80 mg to about 800 mg.

15. The method according to claim 12, wherein: The effective amount is a dosage in the range of about 200 mg to about 400 mg.

16. The method according to claim 12, wherein: The compound is crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-((2-phenylacetoxy)methyl)tetrahydrofuran-3-yl 2-phenylacetate.

17. The method according to claim 12, wherein: The compound is crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-((2-ethylbutanoyl)oxy)methyl-2-ethynyltetrahydrofuran-3-yl 2-butyric acid ethyl ester.

18. A method for preventing HIV infection in an HIV-uninfected human subject comprising parenterally administering to the human subject an effective amount of a compound of claim 1 as an aqueous suspension on a semi-annual dosing schedule.

19. The method according to claim 18, wherein: The compound is administered in a unit dose ranging from about 80 mg to about 800 mg.

20. The method according to claim 18, wherein: The compounds are administered in a dosage volume ranging from about 0.5 mL to about 4 mL per dose.

21. The aqueous parenteral suspension according to claim 6 or 7, wherein The nonionic surfactant is polyoxyethylene (20) sorbitan monooleate.

22. An aqueous parenteral suspension according to any one of claims 6, 7 or 21, wherein The suspending agent is selected from the group consisting of methylcellulose, carboxymethylcellulose, hydroxypropylcellulose and hydroxypropylmethylcellulose.

23. An aqueous parenteral suspension according to any one of claims 6, 7, 21 or 22, wherein The compound is crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2((2-phenylacetoxy)methyl)-tetrahydrofuran-3-yl 2-phenylacetate or crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-((2-ethylbutanoyl)oxy)methyl-2-ethynyltetrahydrofuran-3-yl 2-butyric acid ethyl ester.

24. A method of inhibiting human immunodeficiency virus (HIV) in a patient comprising parenterally administering to the patient an effective amount of a compound of any one of claims 1 to 5 as an aqueous suspension.

25. The method according to claim 24, wherein: The administration was a three month dosing regimen.

26. A method for preventing HIV infection in an HIV-uninfected human subject comprising parenterally administering to the human subject an effective amount of a compound of any one of claims 1 to 5 as an aqueous suspension on a semi-annual dosing schedule.

27. The method according to any one of claims 24 to 26, wherein: The effective amount is a dosage in the range of about 80 mg to about 800 mg.

28. The method according to any one of claims 24 to 27, wherein: The compounds are administered in a dosage volume ranging from about 0.5 mL to about 4 mL per dose.

29. The method according to any one of claims 24 to 28, wherein: Administration of the compound provides a slow or controlled or sustained release of EFdA from the compound in vivo.

30. The method according to any one of claims 24 to 29, wherein: The route of administration of the compound is selected from the group consisting of oral, parenteral, subcutaneous, intravenous, intramuscular, intrasternal injection, infusion and release from an implant.

31. The method according to any one of claims 24 to 30, wherein: The compound is formulated as an aqueous suspension, solution, or encapsulated in particles for sustained release.

32. The method according to any one of claims 24 to 31, wherein: The viral infection is caused by HIV.

33. The method according to any one of claims 24 to 31, wherein: The viral infection is caused by HBV.

34. The method of any one of claims 24 to 33, further comprising administering an additional anti-HIV and / or anti-HBV agent.

35. The method of claim 34, wherein: The additional pharmaceutical agent is selected from the group consisting of cabotegravir, dolutegravir, doravirine, elvitegravir, lecithin, tenofovir disoproxil fumarate, tenofovir alafenamide fumarate, and lamivudine.

36. Use of the compound according to any one of claims 1 to 11 for treating and preventing HIV or HBV viral infection.

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