Cyclobutyl (S)-2-[[[(R)-2-(6-aminopurin-9-yl)-1-methyl-ethoxy]methyl-phenoxy-phosphoryl]amino]-propanoates, and production process and application thereof

Abstract

The present invention relates to chemotherapeutic agents for the treatment of viral and cancerous diseases. Said compounds are prodrugs of the inhibitors of human immunodeficiency virus (HIV) and hepatitis B virus (HBV) of DNA polymerase and are intended for the treatment of human immunodeficiency virus, hepatitis B and co-infections HIV/HCV, HIV/HBV, HIV/HCV/HBV, and HCV/HBV.

Claims

1. Cyclobutyl (S)-2-[[[(R)-2-(6-aminopurin-9-yl)-1-methyl-ethoxy]methyl-phenoxy-phosphoryl]amino]propanoate of general formula 1, cyclobutyl (S)-2-[(S)[[(R)-2-(6-aminopurin-9-yl)-1-methyl-ethoxy]methyl-phenoxy-phosphoryl]amino]propanoate of formula 1.1, and cyclobutyl (S)-2-[(R)[[(R)-2-(6-aminopurin-9-yl)-1-methyl-ethoxy]methyl-phenoxy-phosphoryl]amino] propanoate of formula 1.2, and isotopically enriched analogs, pharmaceutically acceptable salts, hydrates, solvates, crystalline or polycrystalline forms thereof ##STR00009##

2. A compound according to claim 1 is represented by fumarate, hemifumarate, dichloroacetate, or hydrochloride of formula 1.1 ##STR00010##

3. A pharmaceutical composition for the combination therapy and prophylaxis of viral infections in the form of tablet, capsules, or injections placed in pharmaceutically acceptable package comprising the compound of general formula 1, or a stereomer thereof, or an isotopically enriched analog, a pharmaceutically acceptable salt, hydrate, solvate, or crystalline or polymorphic form thereof in a therapeutically effective amount.

4. The pharmaceutical composition according to claim 3 containing fumarate, or hemifumarate, or dichloroacetate, or hydrochloride of the compound of formula 1.1 or an isotopically enriched analog, hydrate, solvate, or a crystalline or polymorphic form thereof.

5. The pharmaceutical composition according to claim 3 or 4 additionally including one or more pharmaceutically acceptable fillers.

6. The pharmaceutical composition according to any of claim 3 or 4 additionally comprising one or more therapeutic agents selected from the group consisting of inhibitors of the protease of human immunodeficiency virus (HIV), nonnucleoside inhibitors of reverse HIV transcriptase, nucleoside inhibitors of reverse HIV transcriptase, nucleotide inhibitors of reverse HIV transcriptase, HIV-interase inhibitors, and CCR5 inhibitors.

7. A method for the combination therapy of human immunodeficiency virus (HIV) including the administration to the subject in need thereof of a therapeutically effective amount of the compound of general formula 1, or a stereomer thereof, or an isotopically enriched analog, a pharmaceutically acceptable salt, hydrate, solvate, or crystalline or polymorphic form thereof.

8. The method for combination therapy according to claim 7, wherein the stereomer is the compound of formula 1.1, or a stereomer thereof, or an isotopically enriched analog, a pharmaceutically acceptable salt, hydrate, solvate, or a crystalline or polymorphic form thereof.

9. The method for combination therapy according to claim 7, wherein the salt is fumarate, or hemifumarate, or dichloroacetate, or hydrochloride of the compound of formula 1.1 or an isotopically enriched analog, hydrate, solvate, or a crystalline or polymorphic form thereof.

10. The method for combination therapy of human immunodeficiency virus (HIV) including the administration of a therapeutically effective amount of the pharmaceutical composition according to claim 3 or 4 to subject in need thereof.

11. The method for combination therapy according to any of claim 7 or 8 including the administration to a subject of one or more additional therapeutic agents selected from the group consisting of the inhibitors of human immunodeficiency virus (HIV) protease, inhibiting compounds, nonnucleoside inhibitors of reverse HIV transcriptase, nucleoside inhibitors of reverse HIV transcriptase, nucleotide inhibitors of reverse transcriptase, HIV-interase inhibitors, and CCR5 inhibitors.

12. The method for combination therapy of hepatitis B virus (HBV) including the administration to a subject in need thereof of a therapeutically effective amount of the compound of general formula 1, or a stereomer thereof, or their isotopically enriched analog, pharmaceutically acceptable salt, hydrate, solvate, or crystalline or polymorphic form.

13. The method for combination therapy according to claim 12, wherein the stereomer is the compound of formula 1.1, or an isotopically enriched analog, a pharmaceutically acceptable salt, a hydrate, a solvate, or a crystalline or polycrystalline form thereof.

14. The method for combination therapy according to claim 12 or 13 wherein the salt is fumarate, or hemifumarate, or dichloroacetate, or hydrochloride of the compound of formula 1.1 or their isotopically enriched analog, hydrate, solvate, or crystalline or polycrystalline form.

15. The method for the combination therapy of hepatitis B virus (HBV) including the administration to a subject in need thereof of a therapeutically effective amount of the pharmaceutical composition according to claim 3 or 4.

16. The method for combination therapy according to any of claim 12 including the administration to a subject in need thereof of one or more additional therapeutic agents selected from the group consisting of the human immunodeficiency virus (HIV) protease inhibitors, inhibiting compounds, nonnucleoside inhibitors of reverse HIV transcriptase, nucleoside inhibitors of reverse HIV transcriptase, nucleotide inhibitors of reverse transcriptase, HIV-interase inhibitors and CCR5 inhibitors.

17. The method for the combination therapy of human immunodeficiency virus (HIV) according to any of claim 7 or 10 including the administration to the subject in need thereof of one or more doses of the compound of general formula 1, or a stereomer thereof or the pharmaceutical composition according to claim 3 or 4.

18. The method for the combination therapy of hepatitis B virus (HBV) according to any of claims 12-16 including the administration to the subject in need thereof of one or more doses of the compound of general formula 1, or a stereomer thereof or the pharmaceutical composition according to claim 3 or 4.

19. A process for the preparation of cyclobutyl (S)-2-methyl-phenoxy-phosphoryl]amino]propanoate of general formula 1, cyclobutyl (S)-2-[(S)-methyl-phenoxy-phosphoryl]amino]propanoate of formula 1.1, cyclobutyl (S)-2-[(R)-methyl-phenoxy-phosphoryl]amino]-propanoate of formula 1.2, as well as isotopically enriched analogs, pharmaceutically acceptable salts, hydrates, solvates, or crystalline or polycrystalline forms thereof, including the use of L-alanine cyclobutyl ester of formula 2 and the compound of general formula 3 ##STR00011##

Description

BEST EMBODIMENT

[0047] The present invention will now be described in terms of certain embodiments which are not intended to limit its scope. On the contrary, the present invention covers all alternatives, modifications, and equivalents that can be included within the scope of the claims. Thus, the following examples, which include specific embodiments, will illustrate this invention without limiting it.

Example 1

[0048] General synthetic protocol for the synthesis of the prodrugs of general formula 1 (Scheme 1).

##STR00008##

[0049] Thionyl chloride (3 ml, 40 mmol) was added dropwise with stirring to a suspension of [(R)-2-(6-amino-purin-9-yl)-1-methyl-ethoxymethyl]phosphonic acid monophenyl ether (3.63 g, 10 mmol) (4) [WO 2013116720] in sulfolane (14 ml) and dichloromethane (12 ml). The mixture was refluxed at 50-55 C. under low Ar flow for 15 h. Then, vacuum (via a membrane pump) was delivered into the flask to remove volatile components for 2 h at 50-55 C. The reaction mixture was cooled down to 30 C., and a mixture of dichloromethane (10 ml) and dry acetonitrile (40 ml) was added with stirring. The reaction mixture containing chloride (3) was cooled down to (60)-(50) C., and a solution of L-alanine cyclobutyl ester (2) (1.546 g, 12 mmol) and triethylamine (4.172 ml, 30 mmol) in 6 ml of acetonitrile was added. The mixture was slowly heated to room temperature, diluted with dichloromethane (100 ml) and the solution was spread onto about 100 ml of silica gel on a glass filter. The product was extracted by dry flash chromatography eluating first with dichloromethane, then with a 30% solution of acetone in dichloromethane, and finally with pure tetrahydrofurane to afford 2 g of the compound of general formula 1, wherein the proportion of stereomers 1.1 and 1.2 was 2:3. The stereomers of formulas 1.1 and 1.2 were separated by HPLC on a Phenomenex Amylose-2 AXIA-Pac 25021.20 MM optical column in an isocratic system of AcCN:EtOH:HCOOH 200:20:0.5 (flow rate 20 ml/min) with a 254-nm UV detector.

[0050] The fumarates of stereomers 1.1 and 1.2 were obtained by crystallization with an equimolar amount of fumaric acid from 100 ml of acetonitrile. The resulting products were fumarate (S)-cyclobutyl 2-((S)(((R)-1-(6-amino-9H-purin-9-yl)propan-2-yloxy)methyl)(phenoxy)-phosphorylamino)-propanoate (1.1), LC-MS (ESI) 489 (M+H).sup.+, .sup.1H NMR (DMSO-d.sub.6, 300 MHz) 8.14 (s, 1H), 8.10 (s, 1H), 7.30 (m, 2H), 7.19 (s, 2H), 7.14 (m, 1H), 7.06 (m, 2H), 6.63 (s, 2H), 5.64 (t, J=11.1 Hz, 1H), 4.86 (p, J=7.2 Hz, 1H), 4.27 (dd, J.sub.r=14.4 Hz, J.sub.2=3.0 Hz, 1H), 4.14 (dd, J.sub.1=14.4 Hz, J.sub.2=6.6 Hz, 1H), 3.85 (m, 4H), 2.23 (m, 2H), 1.94 (m, 2H), 1.72 (m, 1H), 1.59 (m, 1H), 1.13 (d, J=6.9 Hz, 3H), 1.07 (d, J=6.6 Hz, 3H). .sup.31P NMR (DMSO-d.sub.6, 121.5 MHz) 22.05 and fumarate (S)-cyclobutyl 2-((R)(((R)-1-(6-amino-9H-purin-9-yl)propan-2-yloxy)methyl)(phenoxy)phosphorylamino)propanoate (1.2), LC-MS (ESI) 489 (M+H).sup.+, .sup.1H NMR (DMSO-d.sub.6, 300 MHz) 8.14 (s, 1H), 8.12 (s, 1H), 7.34 (m, 2H), 7.21 (s, 2H), 7.15 (m, 1H), 7.11 (m, 2H), 6.63 (s, 2H), 5.53 (dd, J.sub.1=12.0 Hz, J.sub.2=10.5 Hz, 1H), 4.82 (p, J=7.5 Hz, 1H), 4.29 (dd, J.sub.1=14.4 Hz, J.sub.2=3.6 Hz, 1H), 4.20 (dd, J.sub.r=14.4 Hz, J.sub.2=5.7 Hz, 1H), 3.98 (m, 1H), 3.86 (m, 3H), 2.21 (m, 2H), 1.91 (m, 2H), 1.69 (m, 1H), 1.57 (m, 1H), 1.13 (d, J=6.9 Hz, 3H), 1.05 (d, J=6.3 Hz, 3H). .sup.31P NMR (DMSO-d.sub.6, 121.5 MHz) 22.86.

Example 2

[0051] Preparation of a pharmaceutical composition in the form of tablet. Starch (1600 mg), ground lactose (1600 mg), talk (400 mg), and a salt of the prodrug of formula 1.1 (1000 mg) were mixed together and pressed into bar. The resulting bar was comminuted into granules and sifted through a sieve to collect granules of 14-16 mesh. The granules thus obtained were shaped into tablets of suitable form weighing 200 or 400 mg each.

Example 3

[0052] Preparation of a pharmaceutical composition in the form of capsules. The salt of the prodrug of formula 1.1 and lactose powder were carefully mixed in a ratio of 2:1. The resulting powdery mixture was packed into gelatin capsules of suitable size each weighing either 150 or 300 mg.

Example 4

[0053] Preparation of a pharmaceutical composition in the form of compositions for intramuscular, intraperitoneal, or hypodermic injections. The salt of the prodrug of formula 1.1 (500 mg), chlorobutanol (300 mg), propylene glycol (2 ml), and injectable water (100 ml) were mixed together. The resulting solution was filtered, placed into 5 ml ampoules, and sealed.

Example 5

[0054] Assessment of the metabolic parameters of prodrugs 1.1 and 1.2 and the TAF prototype in human peripheral blood mononuclear cells (PBMCs).

[0055] Generic solutions of tested compounds of formulas 1.1 and 1.2 and TAF were prepared in DMSO (Sigma) and kept at 20 C. The PBMCs (kept in liquid nitrogen before use) were extracted from human blood by means of Ficoll-Paque Premium (GE Healthcare) gradient centrifugation. The PBMCs were placed in 24-well plates (Greiner Bio-one), 1.5 mln cells per well (4.2 mln/ml), in the RPMI-1640 medium containing L-glutamine (2 mM), sodium private (0.11 mg/ml), essential and nonessential amino acids, penicillin 50 Un/ml, streptomycin (50 g/ml) (all reagents by PanEco), and 5% HI (Heat Inactivated) fetal bovine serum (HyClone). The cells were incubated overnight at 37 C. and 5% CO.sub.2. The next day, tested and reference compounds in a final concentration of 30 M were added to the cells. The cells and compounds were incubated at 37 C. and 5% CO.sub.2. After 2, 4, 8, 24, 48, and 72 hours of incubation, nonadherent cells were together with the medium transferred into 1.5 ml test tubes (Eppendorf) and centrifuged for 5 minutes at 1000 g to remove the medium. The cells were washed with 1 ml of a phosphate buffer (Gibco) and lysed with 200 l of 70% methanol cooled to 20 C. The cells that were adherent to the wells were washed with 1 ml of PBS (Gibco) and lysed with 200 l of 70% methanol cooled to 20 C. The lysates of adherent and nonadherent cells from respective wells were combined and stirred.

[0056] The content of tenofovir (TFV) and diphosphate tenofovir (DP-TFV) in the cell lysates was determined by UPLC-MS/MS using a 1290 UPLC System (Agilent) chromatograph and a QTrap5500 System (AB Sciex) mass spectrometer with a triple quadrupole. The analytes were separated on a Thermo Hypercarb (503.0 mm, 5 m, Thermo Scientific) column in a mobile phase comprising A0.5% ammonia in 25 mM of ammonium acetate and B0.5% ammonia in 25 mM of ammonium acetate:2-propanol:methanol (1:1:3) at a flow rate of 0.8 ml/min. Electrospraying (TurbolonSpray) in a negative ion detection mode was used as an ion source. Analytes were detected in an MRM mode with transitions for TFV: 286>107, 286>79, 286>63 m/z and for DP-TFV: 446>348, 446>176, 446>158, 446>79 m/z. Chromatograms were analyzed using the Analyst 1.5.2 Software (AB Sciex). The concentrations of TFV and diphosphate TFV in cell lysates were estimated from calibration curves obtained using reference samples of TFV and DP-TFV in 70% methanol. The results are given in Table 1.

Example 6

[0057] Evaluation of anti-HIV activity of the prodrugs of general formula 1 and the prototype (TAF). The antiviral activity of tested compounds was evaluated on e T-lymphocytes line, SupT1. The cells were infected with HIV strain NL4.3 carrying a gene encoding the green fluorescent protein (NL4.3-GFP). A virus preparation was obtained by means of transfection of 293T cells of antiviral DNA. After 48 hours of transfection, the preparation was frozen and stored until being used. To increase the efficiency of infection, the suspension of SupT1 cells was sedimented from the infection mixture by centrifugation. Tested compounds were added to the cells immediately before adding the virus. After 2 hours of incubation, the infection mixture was replaced by a fresh culture medium with tested compounds. The efficiency of infection was evaluated after 45 hours by computing the percentage of fluorescence-bright cells against uninfected cell cultures. Concurrently, the cytotoxicity of tested compounds was evaluated in the same, but uninfected, cell line SupT1 using the XTT reagent. To determine antiviral activity and cytotoxicity, serial tenfold dilutions of the preparations were used (starting with 10 M for antiviral activity and from 100 M for cytotoxicity). DMSO (0.1%) was used for negative control. The values of EC.sub.50, CC.sub.50 and SI (selectivity index) were found. The quality of tests was evaluated based on the following controls: signal to background ratio, integrase inhibitor raltegravir (1 M), and reproducibility of test results. Emetine (0.03, 0.09, and 0.2 M) was used as the reference for cytotoxicity evaluation. The results are summarized in Table 2.

INDUSTRIAL APPLICABILITY

[0058] The invention could be used in medicine and veterinary.