WO2001010882A1

WO2001010882A1 - Depot forms of modified nucleoside 5'-hydrogenphosphates as inhibitors of reproduction of human immunodeficiency virus and human hepatitis b virus

Info

Publication number: WO2001010882A1
Application number: PCT/CA2000/000914
Authority: WO
Inventors: Elena A. Shirokova; Anastasiya L. Khandazhynskaya; Inna L. Karpenko; Natalya B. Tarussova; Alexander A. Krayevsky
Original assignee: Adani, Alexander
Priority date: 1999-08-10
Filing date: 2000-08-10
Publication date: 2001-02-15
Also published as: AU6551400A; WO2001010882A9

Abstract

The present invention is directed to novel compounds exhibiting a selective inhibition of the reproduction of HIV and HBV and possessing low toxicity. The present compounds are VI-VIII of formulae (VI, VII and VIII) wherein: Nucleosides of D and L-series; B = thymine, adenine, guanine, cytosine, uracyl, 5-fluorouracyl or 5-ethyluracyl; R = N3, NH2, F, H or F; X = S or O; R = adamantyl-1, bicyclo[6,5,1] heptyl-1 or tert.-butyl. These compounds being depot forms of 5'-hydrogenphosphonates of modified nucleosides are able to inhibit the reproduction of HIV and HBV and are less toxic as compared to the prior art compounds.

Description

DEPOT FORMS OF MODIFIED NUCLEOSIDE 5'-HYDROGENPHOSPHATES AS INHIBITORS OF REPRODUC^¬ TION OF HUMAN IMMUNODEFICIENCY VIRUS AND HUMAN HEPATITIS B VIRUS

FIELD OF THE INVENTION

The present invention relates to the art of molecular biology and virology. More specifically, in one of its aspects, the present invention relates to novel depot forms of 5 modified 5'-hydrogenphoshonates as selective inhibitors of the reproduction of Human Immunodeficiency Virus (HIV) and Human Hepatitis B Virus (HBV) in cell cultures . In another of its aspects, the present invention relates to a process for producing depot forms of modified 5'-hydrogenphoshonates.

0 DESCRIPTION OF THE PRIOR ART

It is known in the art that various 5'-hydrogenphosphonates of 3'-azido-3'- deoxythymidine (phosphazide) [1-3] and other modified nucleosides [4-6] inhibit the reproduction of the HIV. While somewhat effective, these compounds are comparatively hydrophilic because of the presence of the unprotected phosphonate group thereby 5 decreasing diffusion thereof into cells.

To the knowledge of the present inventors, only one attempt has been made to prepare a depot form of phosphazide [7], but in human serum these compounds are hydrolyzed to 3'-azido-3'-deoxythymidine rather than to phosphazide. This pathway dominates over the other due to hydrolysis of the nucleoside-phosphonate bond 0 proceeding more rapidly than the elimination of the phosphonate protective group. An example of a depot form of 2',3'-dideoxy-2',3'-didehydrothymidine hydrogenphosphonate has been published [8] , but the structure of this compound was not taught.

5 SUMMARY OF THE INVENTION

We have discovered a principal for the design of depot forms of nucleoside 5'- hydrogenphosphonates and a group of depot forms of modified nucleoside 5'- hydrogenphosphonates having anti HIV and HBV activity. The compounds according to the present invention, viz. depot forms of modified nucleoside 5'-hydrogen-

30 phosphonates, are able to inhibit selectively the reproduction of HIV and HBV in cell cultures. According to the synthetic pathway set out in Figure 1, prodrug I can be hydrolyzed in two possible ways: to nucleoside 5'-hydrogenphosphonate (II, Route a) or nucleoside III (Route b). The route of choice, inter alia, depends on the stability and steric availability of the corresponding bonds in I. Our study of several groups of prodrugs in human serum showed that most compounds were hydrolyzed via Route b except for those bearing tertiary alkyl esters, which were hydrolyzed via Route a.

The third possibility is the preliminary oxidation of I to IV via Route c with subsequent hydrolysis either to V (via Route d) or to III (via Route e). It is known that oxidation proceeds much faster for hydrogenphosphonate diesters than for hydrogenphosphonate monoesters.

The study of diesters of nucleoside hydrogenphosphonates in human serum showed that most compounds were hydrolyzed via Route b except for those bearing tertiary alkyl esters, which were hydrolyzed via Route a.

Prodrug forms of 5 '-hydrogenphosphonates of modified nucleosides hydrolyzed to the corresponding nucleoside 5 '-hydrogenphosphonates include:

wherein: nucleosides of D and L-series,; B = thymine, adenine, guanine, cytosine, uracyl, 5-fluorouracyl or 5-ethyluracyl;

R = N₃, H or F; X = S or O; and R' = adamantyl-1, bicyclo[2,2,l]heptyl-l, tert.-butyl.

BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the present invention will be described with reference to the accompanying drawing, in which:

Figure 1 illustrates a synthetic pathway, inter alia, for the production of prodrug I.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The synthesis of VI-VIII with thymine and adenine bases may be achieved according to the following reaction scheme (referred to hereinbelow as Scheme 2):

HO- O- NUCLEOSIDE

wherein:

NUCLEOSIDE = nucleoside component; R'- = adamantyl-1, bicyclo[2,2,l] heptyl-1 or tez-t.-butyl; and

R" - COC(CH₃)₃, -SO₂CH₃ or -SO₂CF₃.

The synthesis of VI-VIII with thymine, adenine, and guanine bases may be achieved according to the following reaction scheme (referred to hereinbelow as Scheme

ΉO-NUCLEOSΓDE o

II R'O- P — O- NUCLEOSIDE I H

wherein:

NUCLEOSIDE = nucleoside component; and

R' = adamantyl-1, bicyclo[2,2,l]heptyl-l or tert.-butyl.

The synthesis of VI-VIII with different nucleic bases may be achieved according the following reaction scheme (referred to hereinbelow as Scheme 4):

PhO- -

O

II PhO — P- O- NUCLEOSIDE I H

O

II R'OH

RΌ — P-O-NUCLEOSΓDE

H

wherein:

NUCLEOSIDE = nucleoside component;

R' = adamantyl-1, bicyclo[2,2,l] heptyl-1 or tert.-butyl.

Embodiments of the present invention will be described with reference to the following Examples which are provided for illustrative purposes only and should not be use to construe or limited the scope of the present invention.

[Al EXAMPLES OF THE SYNTHETIC PROCEDURE

EXAMPLE 1: P-fAdamantyl- l ) - 3 -azido-2 '.3 '-dideoxythvmidine 5'-hvdro- genphosphonate (Via. B=Thy, R=N₃, R'=adamantyl-1 - Scheme 2

A water solution (0.5 ml) of 3 '-azido-2 ',3 '-dideoxythymidine 5 '-hydrogenphosphonate (Phosphazide) Na-salt (88 mg, 0.25 mmol) was passed through a Dowex-50 (Py⁺) column (5 x 1 cm), the column washed with water until no UV- adsorption at 260 nm was observed. The eluate was evaporated to dryness and coevaporated with pyridine (3 x 2 ml). The residue was dissolved in MeCN (5 ml), cooled to -20°C and adamantanol-1 (58 mg, 0.4 mmol) was added under stirring. Pyridine (0.5 ml) and pivaloyl chloride (90 mg, 91 ml, 0.75 mmol) were added, cooling was removed and the reaction mixture was stirred for 10 min. The mixture was diluted with chloroform (10 ml) and washed with cooled saturated sodium bicarbonate (5 ml) and water (3 x 3 ml). The organic solution was dried with Na_jSO^ evaporated, and reevaporated with toluene. The product was purified on a Kieselgel 60 column (15 x 2.5 cm) eluting with chloroform : methanol 95 : 5. The target fractions were evaporated to give compound Via. The yield and physicochemical data are given in Tables 1-3.

Example 2: E-(Adamantyl-π 3 '-azido-2 '.3 '-dideoxythymidine 5'-hvdro- genphosphonate (Via. B=Thy. R=N₃. R^,=:adamantyl-1) - Scheme 3

A solution of PC1₃ (75 mg, 44 ml, 0.5 mmol) in dichloromethane (2 ml) was cooled to 1-2°C, adamantanol-1 (79 mg, 0.5 mmol) and pyridine (40 ml, 0.5 mmol) were added for 20 min at 4-5°C under stirring. A solution of 3 '-azido-2 ',3 '-dideoxythymidine prepared from Na-salt (65 mg, 0.25 mmol) as in the example 1 in pyridine (2 ml) was dropped for 3 h. The reaction mixture was stirred for 3 h at 18°C and then quenched with chloroform (5 ml) and a cooled solution of 1.5 M NH₄HCO₃ (3 ml), the organic layer was washed with water, dried with sodium sulfate, and evaporated. The residue was purified on a Kieselgel 60 column (2 x 25 mm) eluting with CHCl₃-tert.-BuOH 9 : 1 to give compound Via. The yield and physicochemical data are given in Tables 1-3.

Example 3: E-tgrt.-Butyl 3 '-azido-2 '.3 '-dideoxythymidine 5'-hydrogenphosphonate (VIb. B=Thv. R=N_;. R'=tert. -butyl) - Scheme 2

Phosphazide Na-salt (88 mg, 0.25 mmol) was coupled with tert. -butanol (24 mg, 35 ml, 0.38 mmol) under the conditions of example 1. For the yield and physicochemical data, see Tables 1-3.

Example 4: E-(Adamantyl-l) 2 ',3 ^{^}-dideoxythymidine 5 ^{^}-hydrogenphosphonate (Vic, B=Thv. R=H. R =adamantyl-1) - Scheme 2.

2 ',3 '-Dideoxythymidine 5 '-hydrogenphosphonate Na-salt (78 mg, 0.25 mmol) was coupled with adamantanol-1 (58 mg, 0.4 mmol) under the conditions of Example 1 to give Vic. For the yield and physicochemical data, see Tables 1-3.

Example 5: P-(Adamantyl-l) 2'.3 '-dideoxythymidine 5 '-hydrogenphosphonate (Vic, B=Thv. R=H. R^'=adamantyl-1 ) - Scheme 4.

A solution of 2 ',3 '-dideoxythymidine 5 '-hydrogenphosphonate (65 mg, 0.25 mmol) in dichloromethane (1 ml) and pyridine (1 ml) was added to the solution of diphenylphosphite (97 mg, 65 ml, 0.5 mmol) in acetonitrile (2 ml) at 0°C and, after 30- min stirring, adamantanol-1 (58 mg, 0.4 mmol) was added to the reaction mixture under stirring. After 1 h, the mixture was diluted with triethylamine - water 1 : 1, evaporated, the residue was diluted with chloroform (3 ml), washed with water (3 x 1 ml), and the residue chromatographed on a Kieselgel 60 column (2 x 25 mm) eluting with CHC1₃- ethanol 9 : 1 to give compound Vic. The yield and physicochemical data are given in Tables 1-3.

Example 6: E-(Adamantyl-l 2'.3'-dideoxycvtidine 5 '-hydrogenphosphonate (VId. B=Cvt. R=H. R'=adamantyl-n. Scheme 4

2 ',3 '-Dideoxycytidine (105 mg, 0.5 mmol), adamantanol-1 (116 mg, 0.8 mmol), and diphenylphosphite (194 mg, 130 ml, 1 mmol) were coupled under the conditions of example 5. For the yield and physocochemical data of VI d, see Tables 1-3.

Example 7: P-(Adamantyl-l) 3 '-fluoro-2 '.3 '-dideoxythymidine 5 '-hvdro- genphosphonate (Vie. B=Thy, R=F, R'^adamantyl-1 - Scheme 2

3 '-Fluoro-2 ',3 '-dideoxythymidine 5 '-hydrogenphosphonate Na-salt (82 mg, 0.25 mmol) was converted to Vie as described in example 1. The yield and physicochemical data are given in Tables 1-3.

Example 8: E-(Adamantyl-l 2^{^},3 "-dideoxyadenosine 5 ^{^}-hydrogenphosphonate CVIf, B=Ade, R=H. R =Adamantyl-l - Scheme 4

2 ',3 '-Dideoxyadenosine (118 mg, 0.5 mmol) was converted to Vlf under the conditions of example 6. The yield and physicochemical data of Vlf are given in Tables 1-3.

Example 9: P-tert. -Butyl 2 '.3 '-dideoxyadenosine 5 '-hydrogenphosphonate (Vlg.

Scheme 4.

2 ',3 '-Dideoxyadenosine (118 mg, 0.5 mmol) was converted to Vlg under the conditions of example 6. The yield and physicochemical data are given in Tables 1-3.

Example 10: E-(Adamantyl-l) 2 '.3 '-dideoxyguanosine 5 '-hydrogenphosphonate (Vlh. B=Gua. R=H. R^adamantyl-1) - Scheme 4

2', 3 '-Dideoxyguanosine (125 mg, 0.5 mmol) was converted to Vlh. as in the Example 6. The yield and physicochemical data are given in Tables 1-3.

Example 11: E-(Adamantyl-l 2'.3 '-dideoxy-2'.3'-didehvdrothvmidine 5 '-hydrogenphosphonate (Vila. B=Thy. R =adamantyl-1) - Scheme 2

2 ',3 '-Dideoxy-2 ',3 '-didehydrothymidine 5 '-hydrogenphosphonate Na-salt (78 mg, 0.25 mmol) was converted to Vila under the conditions of example 1. The yield and physicochemical data are given in Tables 1-3.

Example 12: E-(Adamantyl- l 2'.3 '-dideoxy-2 '.3 '-didehydrocvtidine 5 '- hvdrogenphosphonate (VHb B=Cvt R'=adamantyl-1) - Scheme 4

VHb was synthesized from 2',3'-dideoxy-2',3'-didehydrocytidine (125 mg, 0.5 mmol) under the conditions of Example 6. The yield and physicochemical data are given in Tables 1-3.

Example 13: E-(Adamantyl-0 L-3 '-thiocytidine 5 '-hydrogenphosphonate (Villa. B=Cvt. R =adamantyl-l^') - Scheme 3

Compound Villa was synthesized from L-3 '-thiocytidine (120 mg, 0.5 mmol) under the conditions of example 3. The yield and physicochemical data are given in Tables 1-3.

Example 14: P-tgrt.-Butyl L-3 '-thiocytidine 5 '-hydrogenphosphonate (VIHb. B=Cyt. R'=tert. -butyl) - Scheme 4

Compound VHIb was synthesized from L-3 '-thiocytidine (120 mg, 0.5 mmol) according to Example 3. The yield and physicochemical data are given in Tables 1-3.

TABLE 1 Yields and physicochemical characteristics

TABLE 2. 'H NMR spectra of VI-VIII, 200 MHz; CD₃CN; δ, ppm; (J, Hz):

TABLE 3

'H NMR spectral data for "depot" groups in VI-VIH, 200 MHz; CD₃CN; δ, ppm; (J, Hz) and ³¹P NMR spectra of VI-VIII, 81 MHz, CD₃CN; δ, ppm, (J, Hz)

rBl STABILITY IN THE HUMAN SERUM

The compounds according to the present invention are slowly hydrolyzed to corresponding nucleoside 5" -hydrogenphosphonates in human blood serum.

The hydrolysis of the compounds according to the present invention was performed in human blood serum. The assay mixture containing 0.5 ml of 100 μM solution of the compound of the invention in formamide and 99.5 μl of 100% human fetal serum was incubated at 37°C. After certain intervals, the 5-μl samples were mixed with 45 μl of 10% trifluoroacetic acid, centrifuged for 10 min at 12,000 rpm, and the

10 supematants were concentrated to 100 μl and analyzed by HPLC on a Silasorb C7 column (4 x 150 mm, 13 m) with a linear gradient of methanol from 0 to 80% in 0.05 M potassium phosphate buffer (pH 6.0) for 40 min. The flow rate was 0.5 ml/min. The extent of hydrolysis was assessed by measuring the amount of the product.

The results of the tests are shown in Table 4 hereinbelow.

15 TABLE 4

Hydrolysis of 50% of the compounds under the invention to corresponding nucleoside

5 '-phosphonates

As seen in Table 4, in most cases the half-lives of the compounds according to the present invention are rather high.

TABLE 5 Concentration of the compounds under the invention inhibiting the HIV reproduction

AZT - 3 '-azido-2', 3 '-dideoxythymidine, FLT - 3 '-fluoro-2 ',3 '-dideoxythymidine

The data presented in Table 5 demonstrate that antiviral activity of the compounds according to the present invention is comparable to that of AZT and other nucleosides and is sometimes higher.

rci ANTI-HIV ACTIVITY OF THE INVENTED COMPOUNDS

The data presented in Table 5 demonstrate antiviral activity and toxicity in HIV-infected MT4 cell culture of the compounds according to the present invention. Antiviral activity and toxicity were studied according to [4,6]. REFERENCES

The following publications, patents and patent applications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety:

1. Tarussova N.B., Khorlin A. A., Krayevsky A.A., Korneyeva M.N., Nosik D.N., Kruglov IN., Galegov G.A., Beabealashvilli R.Sh., Inhibition of human immunodefifciency virus in cell culture by 3'-azydo-2',3'-dideoxynucleosides, ϊϊl.Biol. Russian, 1989, 23, Ν6, 1716-1724.

2. Tarussova N.B., Kukhanova M.K., Krayevsky A.A., Karamov EN., Lukashov V.N., Kornilayeva G.N., Rodina M.A., Galegov G.A., Inhibition of human immunodeficiency virus (HIN) production by 5'-hydrogenphospho-nates of 3'-azido- 2',3'-deoxynucleosides, Nucleosides & Nucleotides, 1991, 10, Νl-3, 351-354.

3. Khorlin A.A., Tarusova N.B., Dyatkina N.B., Krayevsky A.A., Beabealashvilli R.Sh., Galegov G.A., Zhdanov V.M., Korneeva M.S., Nosik D.N., Maiorova S.N., Shobukhov V.M., Patent RF 1 548 182, 16.02.1992, US Patent 5,043,437, 27.8.1991; European Patent 0-354-246, 16.03.1994; Japan Patent N 0- 354-246 Bl, 08.25.1995; Corean Patent 106,957, 12.01.96.

4. Atrazheva E.D., Lukin M.A., Jasko M.V, Shushkova TV, Tarussova N.B., Krayevsky A. A., Balzarini J., De Clercq E., 2',3'-O-Cyclic derivatives of ribonucleosides and their 5 '-phosphonates: synthesis and anti-HIV activity, Med.Chem.Res., 1991, 1, 155-165.

5. Karamov EN., Lukashov V.V, Gorbachova A.P., Kornilayeva GV, Tarussova Ν.B., Krayevsky A.A., Inhibition of HIV reproduction in cell cultures by 2',3'-dideoxynucleoside 5 '-phosphites, ϊil.Biol. Russian, 1992, 26, Nl, 201-207). 6. Krayevsky A.A., Tarussova N.B., Zhu Q.-Y, Vidal P., Chou T.-C, Baron P., Polsky B., Jiang X.-Y, Matulic-Adamic J., Rosenberg I., Watanabe K.A., 5'- Hydrogenphosphonates and 5'-methylphosphonates of sugar modified pyrimidine nucleosides as potential anti-HIV agents. Nucleosides & Nucleotides, 1992, 11, N2-4, 177-196.

7. Mc Guigan C, Bellevergue P., Jones B.C.N.M., Mahmood N., Hay A.J., Petrik J., Karpas A., Alkyl hydrogen phosphonate phosphonate derivatives of anti- HIV agent AZT may be less toxic than parent nucleosides, Antiviral Chem., Chemother., 1994, 5, N4, 271-277.

8. Cardona V.M.F., Ayi A.L., Aubertin A.M., Guedj R., Anti-HIV activity of new compounds: Prodrug of D4T, Antiviral Res., 1998, 37, N3 A52.

Claims

What is claimed is:

1. Adamantyl esters of 5 '-O-hydrogenphosphonates of 3 '-azido-2 ',3' dideoxythymidine (phoshazide), 2 ',3 '-dideoxythymidine, 3 '-amino-2',3 ' dideoxythymidine, 3 '-fluoro-2 ',3 '-dideoxythymidine, 2 ',3 '-dideoxy-2 ',3 ' didehydrothymidme, 2 ',3 '-dideoxycytidine, 2 ',3 '-dideoxyadenosine, 2 ',3 ' dideoxyguanosine, L-3 '-thiocytidine.

2. tert. -Butyl esters of 5 '-O-hydrogenphosphonates of 3 '-azido-2 ',3' dideoxythymidine (phoshazide), 2 ',3 '-dideoxythymidine, 3 '-amino-2',3 ' dideoxythymidine, 3 '-fluoro-2 ',3 '-dideoxythymidine, 2 ',3 '-dideoxy-2 ',3 ' didehydrothymidme, 2 ',3 '-dideoxycytidine, 2 ',3 '-dideoxyadenosine, 2 ',3' dideoxyguanosine, L-3 '-thiocytidine.

3. Bicyclo[2,2,l] heptyl esters of 5 '-O-hydrogenphosphonates of 3 '-azido-2 ',3' dideoxythymidine (phoshazide), 2 ',3 '-dideoxythymidine, 3 '-amino-2',3 ' dideoxythymidine, 3 '-fluoro-2 ',3 '-dideoxythymidine, 2 ',3 '-dideoxy-2 ',3 ' didehydrothymidme, 2 ',3 '-dideoxycytidine, 2 ',3 '-dideoxyadenosine, 2 ',3 ' dideoxyguanosine, L-3 '-thiocytidine..

4. Compounds of the foimulae VI-VIII:

VI VII VIII

wherein:

Nucleosides of D and L-series; B = thymine, adenine, guanine, cytosine, uracyl, 5-fluorouracyl or 5-ethyluracyl;

R = N₃,NH₂,F,HorF;

X = S or O;

R = adamantyl-1, bicyclo[6,5,l] heptyl-1 or tert.-butyl.