RO125385A2 - 1,3-oxathiolane derivatives, pharmaceutical composition comprising the same and method for treating hiv infection in humans by administering the same - Google Patents

Abstract

The invention relates to beta(-)-D-2-hydroxymethyl-5-(5-fluorocytosine-1-yl)-1,3-oxathiolane. The 1,3-oxathiolane derivatives are used for treating the infections with the human immunodeficiency virus.

Description

STATE IOHCI FOR ΚορΤΠβ Patent Application

The invention relates to a process for separating a mixture of 1,3 oxatiolane derivatives used in the treatment of HIV infection.

It has been known since 1981 that acquired immunodeficiency syndrome (AIDS) is a disease that severely affects the human immune system and almost without exception leads to death. In 1983, the etiological cause of acquired immunodeficiency syndrome (AIDS) was determined to be the human immunodeficiency virus (HIV). In December 1990, the World Health Organization estimated that between 8 and 10 million people worldwide were infected with the immunodeficiency virus. of which between 10,000 and 1400,000 are from the United States.

In 1985, synthetic nucleoside 3'-azido-3'-deoxythymidine (AZT) was reported to inhibit human immunodeficiency virus replication. Since then, other synthetic nucleosides that have been shown to be effective against human immunodeficiency virus (HIV) have been introduced, including 2,3-dideoxyquinosine (DDI), 2 ', 3'-dideoxycytidine (DDC), and 3'-fluoro-3'd deoxythymidine. FLT) and 2 ', 3'-dideoxy-2', 3'-didehydrothymidine (D4T).

Other 2 ', 3'-dideoxynucleosides have also been shown to inhibit the in vitro development of a variety of these viruses. It appears that, after cellular phosphorylation to 5'-triphosphate by cellular kinases, these synthetic nucleosides are incorporated into the viral deoxyribonucleic acid (DNA) growth strand, thus stopping the chain due to the absence of the 3'-hydroxyl group.

The success of various 2 ', 3'-dideoxynucleosides in inhibiting the replication of human HIV immunodeficiency virus in vivo or in vitro has led to a number of research topics for finding and testing nucleosides that replace a heteroatom at the 3' carbon atom of the nucleoside. Norbeck and co-workers describe that (±) -1 - ((23,43) -2- (hydroxymethyl) -4-dioxolanyl) thymine (refers to the form of (±) -dioxolane-T) has a modest activity compared to of HIV immunodeficiency virus (EC ₅₀ of 20 pm in ATH 8 cells) and is non-toxic to uninfected control cells at a concentration of 200 μΜ, Tetrahedron Letters 30 (46), 6246, (1989). European Patent Application Publication WO / 0337713 and U.S. Patent No. 504,149 to IAF BioChem International, Inc. describe 2-substituted-4-substituted 1,3-dioxolanes having antiviral activity.

& US 5047407 and European patent application no. 0382526 also having

titular As the holder of IAF BioChem International, Inc., it describes a number of 2-substrutified-5-substituted 1,3-oxathiolane nucleosides with antiviral activity and specifically reports that the racemic mixture (approximately position C4 ') of the C1' isomer -β _a | 2-Hydroxymethyl-5- (cytosin-1-yl) -1,3-oxathiolane (hereinafter referred to as (±) -BCH-189) has approximately the same activity as the immunodeficiency virus as AZT and is not toxic at the tested levels (±) -BCH-189 was also found to inhibit the replication of the AZT-resistant HIV immunodeficiency virus that is isolated in vitro from patients who have been treated with the synthetic nucleoside 3'-azido-3'-deoxythymidine (AZT) for a long time. for more than 36 weeks.

Another virus that causes serious problems in human health is the hepatitis B virus (HBV). The hepatitis B virus is inferior only to tobacco as a cause of human cancer. The mechanism by which the hepatitis B virus induces cancer is unknown, so it is postulated that it may directly trigger tumor growth or may indirectly trigger tumor growth through chronic inflammation, cirrhosis, and cell regeneration associated with infection.

After an incubation period of two to six months in which the host is infected with the hepatitis B virus, the disease progresses to acute hepatitis and liver damage, which causes abdominal pain, jaundice and high levels of enzymes in the blood. The hepatitis B virus can cause fulminant hepatitis, with rapid progression, often fatal as a form of the disease, in which massive portions of the liver are destroyed.

Patients with acute hepatitis are often recovered. In some patients, however, high levels of viral antigen persist in the blood for a long or indefinite period, producing a chronic infection. Chronic infections can lead to persistent chronic hepatitis. Patients infected with the persistent hepatitis B virus are mostly found in developed countries. By mid-1991, there were approximately 225 million chronic carriers of the hepatitis B virus in Asia alone and nearly 300 million carriers worldwide. Persistent chronic hepatitis can cause exhaustion, cirrhosis of the liver and hepatocellular carcinoma, a primary liver cancer.

In Western industrialized countries, high-risk groups for hepatitis B virus infection include those who come in contact with carriers of the hepatitis B virus.

¢ -2 0 0 9 - 0 0 7 5 3 - 2 0 - 0 2 - 1 9 9 2 hepatitis B or with samples of their blood. The epidemiology of hepatitis B virus is very similar to that of acquired immunodeficiency syndrome, which explains why hepatitis B virus infection is common among patients with AIDS syndrome or the acquired immunodeficiency syndrome (AIDS) complex. However, hepatitis B virus is much more contagious than human immunodeficiency virus (HIV).

A human serum vaccine has been developed to immunize patients against hepatitis B virus. While it has been found to be effective, vaccine production is complicated because the supply of human serum from chronic carriers is limited and the purification process is long and expensive. Moreover, each series of vaccines prepared from different sera must be tested on chimpanzees for better safety. Vaccines were therefore produced by genetic engineering. Daily treatments with interferon alfa, a genetically engineered protein, have been shown to be promising. However, for dating, there is no known pharmaceutical agent that effectively inhibits virus replication.

In order to place a nucleoside on the market for pharmaceutical purposes, it must not only be effective, with low toxicity, but must also be cost-effective to manufacture. A major focus of research and development has been on new, low-cost processes for large-scale nucleoside production. 2,3-Dideoxynucleosides are commonly prepared by one of two methods: derivatizing an intact nucleoside or condensing a sugar fragment with a heterocyclic base. Although there are many disadvantages associated with obtaining new nucleoside analogues by modifying intact nucleosides, a major advantage of this process is that convenient absolute stereochemistry has already been established naturally. However, the process may not be used in the production of nucleosides containing either unnaturally produced bases or unnaturally produced carbohydrate fragments (which, for this reason, are not prepared from intact nucleosides), such as, for example, 1,3-oxathiolane nucleosides and 1,3-dioxolane nucleosides.

when a carbohydrate moiety or compound with a carbohydrate-like structure is condensed with a base to form a synthetic nucleoside, the resulting nucleoside has two chiral centers (at positions CT and C4 ')

2009-00755- “20 0 2 ~ 1 992-c and thus exists as a diastereomeric pair. Each diastereomer exists as a set of enantiomers. Therefore, the product is a mixture of four enantiomers.

Nucleosides with unnaturally produced stereochemistry in either the ΟΓ or C4 'position have often been found to be less active than the same nucleosides with naturally established stereochemistry. For example, Carter et al reported that the concentration of (-) - carbovir enantiomer (2 ', 3'-didehydro-2', 3'-dideoxyguanosine) in the cell culture required to reduce reverse transcriptase activity by 50% ( EC ₅₀ ) is 0.8 pM, while EC ₅₀ for the (+) - carbovir enantiomer is greater than 60 μΜ. Antimicrobial Agentsand Chemotherapy, 34: 6,1297-1300 (June 1990).

WO 91/11186, describes that 1,3-oxathiolane nucleosides can be prepared with a higher diastereoselectivity (higher percentage of nucleosides with configuration (3 at the bond between the C1 'carbon atom and the heterocyclic base), through a thorough selection of Lewis acid Condensation of 1,3-oxathiolane nucleoside with a base has also been found to occur with an almost complete specificity when the tin chloride is used as a condensation catalyst. Other Lewis acids cause a low (or no) d'-β selectivity or cannot catalyze reactions.

Given that the acquired immunodeficiency syndrome, the AIDS-related complex and the hepatitis B virus have worldwide epidemic levels and have tragic effects on infected patients, there is a great need to provide new effective pharmaceutical agents for the treatment of these diseases. have a low toxicity to the host organism.

There is also a need to find a commercially viable, cost-effective process for producing pharmaceutically important nucleosides that achieves β-stereospecificity at the C4 'position of synthetic nucleosides prepared by condensation of a fragment with a carbohydrate-like structure with a base.

The problem solved by the invention is to present a process for the separation of 1,3-oxathiolane derivatives.

The process for separating the 1,3-oxatiolane derivatives according to the invention eliminates the above disadvantages in that the racemic mixture of 2 hydroxymethyl-5- (5-

fluorocytosin-1-yl) -1,3-oxathiolane is subjected to a resolution step under the action of an enzyme selected from the group consisting of esterase, lipase, substylizine, α-chymotrypsin, cytidine-deoxycytidine deaminase, which preferentially catalyzes a reaction in one between enantiomers, followed by exposure of the resulting product to the action of a second enzyme selected from the group esterase, lipase, α-chemotrypsin subtilisin which intensifies the resolution by favoring the binding reaction of one of the enantiomers, recrystallization of the resolution product and treatment with a from a group consisting of malic acid, mandelic acid, 10-camphorsulfonic acid, 3-bromocamphor-8-sulfonic acid, di-benzoyl tartaric acid and di-para-toluoyl-tartaric acid.

By applying the invention, the following advantages are obtained:

- 1,3-oxathiolane derivatives with increased antiviral activity are obtained;

- this method is regioselective.

The process for the preparation of 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane consists in the reaction of 5-fluorocytosine, optionally protected with 1,3-oxathiolane having the formula s ----- ^u in which R _1a is a hydrogen atom or a hydroxy protecting group, an acyl group and L is a labile group; and optionally remove any hydroxy protecting group.

Another process for the preparation of 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane is to react with a compound of the following formula.

NHR _țt>

Ri. a

wherein R _1a is a hydroxy group protecting group and R _1b is an amino group protecting group, with a fluorinating agent introducing the fluorine atom into position 5 of the cytosine cycle and then optionally removing the protecting groups. .

Another process for the preparation of 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane is to react with a compound of the formula

A

F

R ,. °

wherein R _1a is a hydroxy protecting group, with an agent that converts the oxo group at position 4 of the uracil ring to the amino group and then the protecting groups are removed.

The 1,3-oxathiolane derivative is prepared by (i) ozonating the compound of the formula CH ₂ = CH-CH ₂ -OR or ROCH ₂ -CH = CHCH ₂ OR, wherein the radical R is a protecting group to give the compound with the formula

H

R.

(ii) reacting the compound obtained in phase (I) with mercaptoacetic acid to form the compound with the structure ^R 1a °

O (iii) reducing the compound obtained in step (ii) and then reacting it with carboxylic acid anhydride, an alkylating agent or a halogenating agent to give the compound of formula

wherein L is a labile group.

Another object of the present invention consists in a pharmaceutical composition containing as active compound a 1,3-oxathiolane derivative and a carrier for oral administration and is packaged in the form of a capsule or tablet.

^ -2009-00755--20-02-1992-ν | η, .Ου, ιί I

Therefore, an object of the present invention is a method and a composition for the treatment of human patients infected with the human immunodeficiency virus, HIV.

Another object of the present invention is to provide a method and composition for the treatment of human patients or other host animals infected with hepatitis B virus, HBV.

Another object of the present invention is also enantiomerically enriched nucleosides in 1,3-oxathiolane.

The present invention further provides a method of decomposing C4 'enantiomers of 1,3-oxathiolane nucleosides.

For the treatment of human immunodeficiency virus and hepatitis B virus, HBV, humans and other host animals according to the present invention, for the above-mentioned treatment, an effective amount of 2-hydroxymethyl-5- (5-fluorocytosine) is provided. 1-yl) -1,3-oxathiolane, a pharmaceutically acceptable derivative of this compound, including the 5 'or N ⁴ alkylated derivative or the 5' or N ⁴ acylated derivative or a salt of this compound in a pharmaceutically acceptable vehicle.

It has also been found that 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane (FTC) is surprisingly highly active against human immunodeficiency virus, with very low toxicity to of the host cell. It has also been found that the compound 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane (FTC) has a very significant activity against hepatitis B virus, HBV and therefore may be used to treat patients who have a variety of diseases associated with hepatitis B virus infection, HBV.

Toxicity and pharmacokinetic studies confirm the full use of 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane (FTC) as an antiviral agent for pharmaceutical administration. The FTC compound and its enantiomers are non-toxic to peripheral human bone marrow cells above 50 μΜ and other cell lines above 200 μΜ. FTC-TP is a major intracellular metabolite in PBMC and HepG2 cells. The FTC-TP competitively inhibits the human immunodeficiency virus HIV-1 as a reverse transcriptase (RT) with K1 for 0.2 μΜ using a tempered-primer poly (l) oligo (dC) primer. Using sequential analysis,

-------- _σ ----------- FTC-TP proves to be a potent terminator of the DNA deoxyribonucleic acid chain when using human immunodeficiency virus HIV-RT (Cstops) .

Chronic treatment with FTC is not toxic to rodents, even at oral doses of 85 mg per kilogram for one day for at least two months. Pharmacokinetic studies of 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxatiolane (FTC) in rhesus monkeys indicate high oral bioavailability (approximately 73 ± 6%) and a terminal plasma half-life of approximately 1 , 34 ± 0.18 (mean oral and intravenous administration).

This disclosure also provides a process for the decomposition of a racemic mixture of nucleoside enantiomers, including the racemic FTC mixture, which includes the step of exposing the racemic mixture to the action of an enzyme that preferentially catalyzes a reaction in one of the enantiomers. . The process can be used to solve a wide range of nucleosides, including pyrimidine nucleosides and purine nucleosides which are optionally substituted on the carbohydrate side or on the base side. The process can therefore be used to solve nucleoside derivatives containing additional heteroatoms in the carbohydrate part, for example (±) -FTC and (±) -BCH-189. Nucleoside resolution can be achieved on a large scale at moderate costs. Using the models described in the present invention, the FTC can be separated into its (+) - beta-D and (-) - beta-L enantiomers. The (-) - beta-L enantiomer appears to be much more active than the (+) - beta-D enantiomer against HIV, HBV and SIV. The (+) enatiomer of the FTC is therefore active against HIV, HBV and HIV.

As used herein, the term "enantiomerically enriched nucleoside" refers to a nucleoside composition, including at least 95% of a single enantiomer of that nucleoside.

As used herein, the term FTC refers to the compound 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane (racemic form or enantiomers), thus to the compound 2'-deoxy -5-fluoro-3'-thiacitidine.

As used herein, the term (±) -FTC refers to (±) beta-D, L-2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane.

As used herein, the term (-) - FTC refers to (-) - beta-

Ο Ο 9 Ο Ο 7 5 5 - -20-02-1992- “

7/3 ί '

L-2-hydroxymethyl-beta-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane.

As used herein, the term (+) - FTC refers to (+) beta-D-2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane.

As used herein, the terms FTC-MP, FTC-DP and FTC-TP refer to FTC monophosphate, FTC diphosphate and FTC triphosphate, respectively.

As used herein, the term BCH-189 refers to 2-hydroxymethyl-5- (5-cytosin-1-yl) -1,3-oxathiolane.

As used herein, the term "preferential enzymatic catalysis" refers to enzyme catalysis of one substrate in place of another substrate.

As used herein, a mobile group is a functional group that is formed at an early carbonation when separated from the molecule to which it is attached.

In conclusion, the present invention provides a process for separating a mixture of 1,3-oxatiolane derivatives, and the preparation of the compound 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane consists of:

(i) the reaction of the optionally protected 5-fluorocytosine with 1,3-oxathiolane of formula A:

wherein R _1a is hydrogen or a hydroxyl protecting group, including an acyl group, and L is a mobile group; and optionally remove any hydroxyl protecting group.

(ii) reaction of the compound of formula B:

L - ^ U. iL _y (wherein R _1a is as defined above and R _1b is an amino group protecting group), with a fluorinating agent involved in the introduction of a fluorine atom at position 5 of the cytosine ring; or (iii) the reaction of a compound of formula C:

(wherein the radical R _1a is as defined above), with an agent involved in the transformation of the oxo group at position 4 of the uracil ring into the amino group; any remaining protecting group can be removed to obtain the desired compound.

f) a process for the separation of a (-) or (+) enantiomer of 2-hydroxymethyl-5 (5-fluorocytosin-1-yl) -1,3-oxathiolane which consists of subjecting the compound or derivative (e.g. 5'- ester) of this compound in the form of a mixture of enantiomers (-) and (+) the respective conditions or the subject of the action of the reagents which serve to separate the enantiomers and, if necessary, the conversion of the resulting derivative into the compound of origin.

In the process, in step (i), the hydroxy protecting group includes the protecting groups described in detail below, including the acyl (e.g. acetyl), arylacyl (e.g. benzoyl or substituted benzoyl), trityl or monomethoxytrityl, benzyl groups. or substituted benzyl, trisubstituted silyl including trialkylsilyl (e.g., dimethyl-t-butylsilyl) or diphenylmethylsilyl. The 5-fluorocytosine compound may optionally be protected with trisubstituted silyl groups. Protection groups can be removed by any conventional method. Mobile group L is a typical mobile group known in the literature on nucleoside chemistry, for example, a halogen such as chlorine or bromine, alkoxy such as methoxy or ethoxy or acyl such as acetyl or benzoyl.

Reaction (s) may be carried out in an organic solvent (eg 1,2-dichloroethane or acetonitrile) in the presence of a Lewis acid, preferably stannic chloride or trimethylsilyl triphthalate.

Oh-2 009-00753-20-02-1992 '' -A

Compounds of formula A (wherein L represents an acyl group, for example an acetyl group), can be obtained by the reaction of the compound of formula D

(wherein R _1a is defined above) with a reducing agent, for example, lithium aluminum hydride, followed by treatment with a suitable conventional reagent to give the desired intermediate, for example, carboxylic acid anhydride , such as acetic anhydride, for acylation, chlorinating or brominating reagents for halogenation or alkylating reagents.

The compound of formula D can be prepared by the reaction of the compound of formula E

R $ ^B 1a with HSCH ₂ CO ₂ H at high temperature.

The compound of formula E may be prepared by ozonolysis starting from an allyl ether or allyl ester having the formula CH ₂ = C-CH ₂ -OR or a dieter or a 2-butene-1,3-diol diester having the formula RO-CH ₂ - CH = CH-CH ₂ -OR, wherein R is a protecting group, such as, for example, an alkyl, silyl or acyl group.

With respect to step (ii) of the procedure, the 5-fluorine substituent may be introduced by methods known in the literature (MJ Robins et al., In Nucleic Acid Chemistry, Part 2, LB Townsend and RS Tipson, editors, J. Wiley and Sons, New York, 895-900 (19/8) and references, R. Duschinsky in Nucleic Acid Chemistry, Part 1, LB Townsend and RS Tipson, editors, J. Wiley and Sons, New York 43-46 (1978) and references included). The fluorinating agent may be, for example, trimethylhypofluorite in fluorochloromethane.

With respect to step (iii) of the process, the compound of formula C may be treated with 1,2,4-triazole, together with 4-chlorophenyl dichlorophosphate, to form compound 4- (1,2,4-

ίΧ-2 Ο Ο 9 - Ο Ο 7 5 3 -2 Ο ~ Ο 2 ~ 1 9 9 2;] <) η triazoiIii) corresponding, which is then converted to the desired 4-amino compound (cytidine), for example by the reaction with methanol.

The starting materials of formulas B and C can be prepared, for example, by the reaction of a suitable base (optionally protected) with a compound of formula Ά in a manner similar to that described in step (i). The compounds 5-fluorouracil and 5-fluorocytosine are commercial compounds supplied by Aldrich Chemical Co., Milwaukee, Wl 53233, USA.

The resolution of the enantiomers (±) may be supplemented as specified in detail below.

The FTC compound can be converted to the pharmaceutically acceptable ester by reaction with a suitable esterifying agent, for example, an acid halide or an anhydride. The FTC compound or the pharmaceutically acceptable derivative of this compound can be converted to a pharmaceutically acceptable salt in a convenient manner, for example, by treatment with a suitable base. The ester or salt of the FTC compound can be converted to the FTC compound, for example by hydrolysis.

I. Preparation of active compounds.

The racemic mixture of FTC can be prepared by the method described in detail in PCT Application Publication, WO 91/11186, published August 8, 1991, by Emory University, or by the procedure described in Example 1. In general, the method includes ozonation or of an allylic ether or of an allylic ester having the formula CH ₂ = CH-CH ₂ -OR or of a dieter or diester of 2-butene-1,3-diol having the formula RO-CH ₂ -CH = CH-CH ₂ - OR, wherein the radical R is a protecting group, such as for example alkyl, silyl or an acyl group, to form a glycoaldehyde of the formula OHC-CH ₂ -OR; adding thioglycolic acid to glycoaldehyde to form a lactone of formula 2- (R-oxy) -methyl-5-oxo-1,3-oxathiolane; reduction of lactone to various compounds containing a mobile group at position 5 of the oxathiolane cycle; coupling of these compounds with 5-fluorocytosine-silylate in the presence of SnCl ₄ to form the beta isomer of the FTC; and optionally removal of protection groups.

The following are examples of embodiments of the invention in connection with Figures 112, which represent:

<Χ_-2 Ο Ο 9 - Ο Ο 7 5 3 - -

- 2 Ο - Ο 2 - 1 9 9 2 - - <. . --------- _ν -;

- figure 1, ο illustration of the chemical structure of the compound 2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane (FTC);

- figure 2, an illustration of the process for the preparation of the compound 2-hydroxymethyl5- (5-fluorocytosin-1-yl) -1,3-oxathiolane;

- Figure 3, a flow diagram of the specificity of alkaline phosphatase and phosphodiesterase in snake venom, for the (+) and (-) enantiomers of the FTC compound;

- Figure 4, a graph showing the evolution of lipase-catalyzed hydrolysis of the 5 ¹ butyryl ester of the FTC compound over time, using the enzymes Amano PS-800® (open square) and PLE (open dotted circle);

- Figure 5, a graph of the effect of the concentration (μΜ) of the FTC-enriched racemic compounds and enantiomers (prepared by the method in Example 4) on the percentage inhibition of HIV-1 infected human PBM cells (closed circle, (±) - FTC), (open circle, (-) - FTC), (closed area, (+) - FTC);

- Figure 6, a graph of the concentration of racemically and enantiomerically enriched FTC compound (μΜ) (prepared by the procedure in Example 3) on the percentage of inhibition of HIV-1 infected human PBM cells (closed circle, (±) -FTC), (open circle, (-) FTC), (closed square, (+) - FTC);

- Figure 7, a graph of the absorption of tritium-saturated compound (±) -FTC in human PBM cells (mean of two determinations) over time (hours) to pmol / 10 ⁶ cells;

- figure 8, a graph of the output of the radiolabeled compound (±) -FTC from human PBM cells, measured in hours against pmol / 10 ⁶ cells;

- Figure 9, the presence of the compound [ ³ H] (±) -FTC and its phosphorylated derivatives in human HepG-2 cells (mean of two determinations), incubated in a medium containing 10 μΜ [ ³ H] (±) -FTC, measured in pmol / 10 ⁶ cells over time;

- figure 10, output of compound [ ³ H] (±) -FTC and its phosphorylated derivatives in human HepG-2 cells in pmol / 10 ⁶ cells versus cells exceeded in time after cell pulsation by 10 μΜ [ ³ H] ( ±) -FTC (700 DPM / pmol) for 24 hours and assessment of compound concentration 24 hours after elimination;

- Figure 11, decrease in the combined concentration of [ ³ H] (±) -FTC and its phosphorylated derivatives in human HepG-2 cells after incubation with 10 μΜ [ ³ H] (±) -FTC (700 DPM / pmol) for 24 hours, in pmol / 10 ⁶ cells exceeded in time;

(2009-2009'00753--2 0 - 0 2 - 1 9 9 2 -

- Figure 12, a graph of the effect of FTC enantiomers on the formation of granulocyte-macrophage precursor cell colony, as measured, as a percentage of survival relative to the concentration in pM (-) - FTC, open circle; (+) - FTC closed circle; AZT, dark square.

Example 1. Preparation of (±) -beta-D, L-2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane

The process for preparing the racemic mixture of the FTC compound is illustrated in Figure 2 and is described in detail below.

Protection of 2-butene-1,4-diol in a 2-liter, 3-neck, dry, inert atmosphere, 100 grams (93.5 milliliters = 1,135 moles = 1.00 equivalents) 2-butene-1, 4-diol and 15 grams (approximately 0.1 equivalent) of DMAP (4-dimethylaminopyridine) are dissolved in 800 milliliters of dry pyridine and stirred while the reaction mixture is cooled to 0 ° C. Then add slowly, to prevent overheating, butyryl chloride (260 milliliters = 2.2 equivalents) and leave the mixture to stir for another hour. The reaction is completed by adding small amounts of ice water to the oil. The liquid is then decanted from the salt and evaporated in vacuo. The remaining salt is dissolved in water and the aqueous solution is extracted twice with ethyl ether. The other combined layers are washed once with a saturated solution of CuSO ₄ , twice with a saturated solution of NaHCO ₃ containing Norit® and then filtered under vacuum through a celite® filter.

The concentrated reaction mixture is dissolved in ether and washed, following the same procedure as above for the salt solution. The combined organic layers are concentrated on a rotary evaporator and then brought under vacuum. In order for this reaction to take place quantitatively, a closed environment is required. The vacuum can be increased as needed. The product, 1,4-dibutyryl-2-butene-1,4-diol, is colorless to slightly yellow in the form of a clear liquid.

Ozonolysis of protected diol

The compound 1,4-dibutyryl-2-butene-1,4-diol (1,365 mol) is dissolved in 4 liters of dry methylene chloride (CH ₂ Cl ₂ ) in a 5-neck, 5-liter dry flask. capacity, equipped with a wide drying tube and an open tube for gas introduction. The tube is a non-fried gas bubbling tube, which will be sealed by ον 2 0 0 9 - 0 0 7 5 3 -

- 2 0 - 0 2 - 1 9 9 2 - ^Ί '' - - -, --- exposure to the concentrated solution. The solution is then stirred and cooled to -70 ° C, while the inert gas is bubbled through the solution. The gas inlet is sealed while the solution is sufficiently cooled and the vial and shaker are transferred to the ozone generator. Oxygen is bubbled through the solution kept stirring for at least 20 minutes while remaining on an ice bath. A Cryocool device is ideal for maintaining a low temperature for a long lasting reaction. Ozone is then introduced at 0.56 kg / cm ² -0.60 kg / cm ² (8 to 8.5 psi). Upon completion, the flow of ozone is stopped and oxygen is bubbled through the solution for about half an hour before adding 3 equivalents of Me ₂ S. The vial is then removed from the ice bath and transported to a niche, where it is stirred for about 2 days to obtain a complete reduction. The solution is then evaporated and brought under vacuum for several hours.

This reaction typically produces about 95% protected aldehyde (2-butyryloxyacetaldehyde), a clear, colorless to yellow liquid.

Cyclization of aldehyde with mercaptoacetic acid

Aldehyde (1.0 equivalents) is dissolved in toluene to give a solution of 0.80 to 0.85 M, which is brought into a vial with a Dean Stark separator. Thioglycolic acid (1.1 equivalents) is added and the mixture is heated to reflux. The water is removed azeotropically with the separator. The reaction is complete in 3 hours and the mixture is kept cold at room temperature. The organic solution is washed twice with equal volumes of saturated aqueous sodium bicarbonate solution NaHCO ₃ and then washed once with water, dried over magnesium sulphate MgSO ₄ and Norit, then filtered under vacuum through celite, before to be evaporated in vacuo. The first aqueous solution of sodium bicarbonate NaHCO ₃ is extracted once with ether; the ether is washed once with water, dried over magnesium sulfate MgSO ₄ and Norit®, filtered through celite® under vacuum and evaporated together with other organic materials from toluene solution. The combined material is placed under vacuum until the next day.

The reaction usually yields 90% of the compound 2 (butyryloxy) -methyl-5-oxo-1,3-oxathiolane.

Reduction of lactone and conversion to acetate

The compound 2-butyryloxy-methyl-5-oxo-1,3-oxathiolane (1.00 equivalent) is dissolved in

6C-2 Ο Ο 9 - Ο Ο 7 5 3 '... ...' Lvv ^ U '. _ζ dry tetrahydrofuran (THF) to obtain a 0.23 M solution which is brought into a 3-necked dry flask fitted with a mechanical stirrer and kept in an inert atmosphere. The solution is stirred and cooled to 0 ° C, after the equivalent of 1.0 M Li (t-BuO) ₃ AIH in tetrahydrofuran (THF) was added via cannula. The reduction is complete in about three hours, as seen by TLC thin layer chromatography, using as solvent system, 2 parts ether and 1 part hexane and anisaldehyde as a dye.

Approximately 10 equivalents of Ac ₂ A freshly distilled is then added and stirred for 2 days to obtain the acetylated product. The reaction is completed by adding saturated sodium bicarbonate solution NaHCO ₃ , which is stirred until the next day. The solution is then evaporated and stirred with several NaHCO ₃ sodium bicarbonate solution overnight. This solution is then extracted with ether which is then washed (carefully) twice with saturated sodium bicarbonate solution NaHCO ₃ and once with water, then dried over magnesium sulphate MgSO ₄ and Norit®, after which it is filter through celite® under vacuum and evaporate. The product is a clear, dark yellow liquid. Gas chromatographic analysis (Initial temperature - 80 ° C; Initial time = 5 minutes; Progression - 10 / minute; Final temperature = 240 ° C) indicates a typical purity of about 70%.

Silylation of 5-fluorocytosine

5-Fluorocytosine (1.05 equivalents based on the amount of acetylated lactol obtained in the previous step using the gas chromatographic method to indicate purity) is silylated by refluxing into at least 10 equivalents of hexamethyldisilazane containing a catalytic amount of pure ammonium sulphate ( 0.05 to 0.10 equivalents) for two hours, after which the solution becomes clear. The vial is then tightly sealed and the solvent is removed using a vacuum pump with an auxiliary separator. The product, in white solid form, is left under vacuum until the next day until it is ready for use in the next coupling reaction phase.

Coupling of the silylated 5-fluorocytosine compound with acetylated lactol

The silylated 5-fluorocytosine compound (33.86 grams, 0.124 mol) in dry dichloromethane (350 milliliters) forms a solution to which is added a solution of SnCl ₄ tin tetrachloride (135.6 milliliters a 1 molar solution in methylene chloride CH ₂ CI2) in an atmosphere of

O- “2 0 0 9” 00 7 5 5 ^m - 2 0 - 0 2 - 1 9 9 2 - ^r od. 7 '' ------- “nitrogen. The solution is stirred for 15 minutes at room temperature. This solution is added to the lactol acetate solution through a thin tube (38 grams, 0.113 moles) in dichloromethane (400 milliliters) under a nitrogen atmosphere for over 30 minutes.

The reaction solution is stirred for 2 hours, at which point the reaction is complete, as indicated by thin layer chromatographic analysis. The reaction solution is then diluted with dichloromethane (500 milliliters) and the reaction is stopped by adding ammonium hydroxide solution. The ammonium hydroxide solution (100 milliliters) is added slowly keeping the reaction temperature below 30 ° C, resulting in the formation of a white precipitate. The mixture is then stirred for an additional 30 minutes and then passed through a silica gel stopper column (7 inches 17.78 cm in diameter and 2 inches 5.08 in height). The column is eluted sequentially with dichloromethane (2 liters), ethyl acetate (2 liters) and a mixture of 9 parts ethyl acetate and 1 part ethanol (4 liters). The eluent ethyl acetate and the mixture of ethyl acetate and ethanol contain the desired product. These solutions are combined and evaporated under reduced pressure. The residual sticky solid is then washed with dry ether (20 milliliters) to give a white solid (25.35 grams; 71%) of FTC-5'-butyrate.

FTC-5'-butyrate (8.74 grams; 0.026 mol) is dissolved in 250 milliliters of methanol. Sodium methoxide (2.85 grams; 0.052 grams) is added at room temperature. The reaction mixture was stirred for 1 hour, at which point the reaction was complete, as confirmed by TLC thin layer chromatographic analysis. The ammonium chloride solution NH ₄ Cl (10 milliliters) is added to quench the reaction and then the solvent is removed under reduced pressure. The residue is then absorbed onto silica gel (5 grams) and passed through a narrow column, using a mixture of 9 parts ethyl acetate and 1 part ethanol as eluent. The product-containing fractions are combined and evaporated when a sticky solid product is obtained which is washed with dry ether, when a white FTC solid product is formed (6 grams, 88%).

Nuclear magnetic resonance analysis has the following results:

( ¹ H NMR: (DMSO-d ₆ ) 8.18 (1H, d, H ₆ , J = 8.4 Hz), 7.81 & 7.57 (2H, extended, NH ₂ ), 6.12 ( 1H, dd, H _{1 (} J = 5.7 & 4.2 Hz), 5.40 (1H, t, OH, J = 5.7 Hz), 5.17 (1H, t, 1H ₄ , J = 3 -6 Hz), 3.74 (2H, m, 2H ₅ ), 3.41 (1H, dd, 1H ₂ , J = 5.7 & 11.7 Hz), 3.11 (1H, dd, 1H ₂₎ , J = 4.2% 11.7 Hz);

^ -2009-00753 “2 0“ 0 2 - 1 9 9 2 ^ Ugusi ν, ζ .''- '' m ') ¹³ C NMR: (DMSO-d ₆ ) 157.85 (d, J = 13, 4 Hz), 153.28, 136.12 (d, J = 241 Hz), 126.01 (d, J = 32.6 Hz), 86, 90, 86, 84, 62, 48, 37, 07; melting point 195-196 ° C.

II. Resolution of nucleoside enantiomers

One method is presented in the present invention for resolving racemic mixtures of nucleoside enantiomers, including but not limited to FTC (+) and (-) enantiomers. The method can therefore be used to separate racemic mixtures of carbohydrates or the carbohydrate-like portion, such as 1,3-oxathiolane and 1,3-dioxolane derivatives. The method involves using an enzyme that preferentially catalyzes an enantiomer reaction in a racemic mixture. The reacted enantiomer is separated from the unreacted enantiomer based on a new difference in physical structure. From the description given in the literature it appears the possibility to choose an enzyme that is selective for the nucleoside enantiomer of choice (or selective for the unwanted enantiomer, as a method of its elimination), by selecting one of the enzymes to be discussed. below or by systematic evaluation of other known enzymes. From the literature, it is therefore known how to modify the substrate as much as necessary to reach the desired resolution. By using chiral nuclear magnetic resonance agents, polarimetry, or high-performance chiral liquid chromatography, the optical coating of the separated ester can be determined. The following examples further illustrate the use of enzymes to separate racemic mixtures of enantiomers. Other known methods for resolving racemic mixtures may be used in combination with the method of resolution described in the present invention. All such modifications are deemed to be part of the scope of this invention.

Resolution based on hydrolysis of C5 'nucleoside esters

The method includes reacting the C5'-hydroxyl group of the mixture of nucleoside racemates with an acyl compound to form the C5 'ester in which the nucleoside is at the carbinol end of the ester. The racemic mixture of C5 'esters of nucleoside is then treated with the enzyme which cleaves or hydrolyzes preferentially to one of the enantiomers and not to the other over a period of time.

An advantage of this method is that the method can be used for ^ “2009’00753- 2 0 - 0 2 - 1 9 9 2 Cod.

separation of a wide variety of nucleosides, including pyrimidine nucleoside and purine nucleoside which are optionally substituted on the carbohydrate side or on the base side. The method can therefore be used to separate nucleoside derivatives containing additional heteroatoms on the carbohydrate side, for example FTC and BCH-189. The broad applicability of these methods lies in the fact that, although the carbinol portion of the ester plays a role in the ability of an enantiomer differentiating enzyme, the major recognition portion for these enzymes is the carboxylic acid portion of the ester. Moreover, the result obtained from one enzyme can be successfully extrapolated from the study on the substrate of another enzyme in a seemingly different system, ensuring that the carboxylic acid portions of the two substrates are the same or substantially similar.

Another advantage of this method is that this method is regioselective. Enzymes that typically hydrolyze esters do not catalyze foreign reactions in other parts of the molecule. For example, the enzyme lipase catalyzes the hydrolysis of 2-hydroxymethyl-5-oxo-1,3-oxathiolane ester without hydrolyzing internal lactone. This contrasts sharply with the chemical methods of hydrolysis of the ester.

Another advantage of this method is that the separation of the unhydrolyzed enantiomer and the hydrolyzed enantiomer from the reaction mixture is very simple. The non-hydrolyzed enantiomer is more lipophilic than the hydrolyzed enantiomer and can be recovered efficiently by simple extraction with one of a wide variety of non-polar organic solvents or solvent mixtures, including hexane and hexane and ester mixture. The more polar hydrolyzed enantiomer, the least lipophilic, can then be obtained by extraction with much more polar organic solvents, for example, ethyl acetate, or by lyophilization, followed by extraction with ethanol or methanol. Alcohol should be avoided during hydrolysis, as it can distort the enzymes under certain conditions.

Enzymes and substrates

With the right combination of enzyme and substrate, conditions can be established for the isolation of any nucleoside enantiomer. The desired enantiomer can be isolated by treatment of the racemic mixture with an enzyme that hydrolyzes the desired enantiomer (followed by extraction of the polar hydrolyzate with polar solvent) or by treatment with an enzyme that hydrolyzes the unwanted enantiomer (followed by removal

undesired enantiomer with a non-polar solvent).

Enzymes that catalyze the hydrolysis of esters include esterases, such as porcine liver esterase, lipases including porcine pancreatic lipase, and Amano PS-Boo, lipase, substillisin, and alpha-chymotrypsin.

Figure 3 is a flow chart for the specificity of alkaline phosphatase and phosphodiesterase in snake venom for the (+) and (-) enantiomers of the FTC compound. As indicated, alkaline phosphatase hydrolyzes the triphosphate of both enantiomers to the FTC compound and is therefore not effective as a separation agent. Phosphodiesterase I preferentially hydrolyzes the (+) isomer of the FTC compound into its monoester, which can then be exposed to 5'-nucleotidase to obtain the (+) - FTC compound.

The most effective acyl group for C5 'esterification of the nucleoside can be determined without improper experimentation by evaluating a number of homologues using a system of selected enzymes. For example, when 1,3-oxathiolane nucleosides are esterified with butyric acid, the resolutions of both porcine liver esterase and Amano PS-800 esterase result in higher enantioselectivity (94-100% enantiomeric excess) and opposite selectivity. . Pig liver esterase preferentially hydrolyzes the (+) enantiomer of the FTC compound and the enzyme Amano800C preferentially hydrolyzes the (-) enentermer of the FTC compound. The percentage of enantiomeric excess reported in Table 1 is the amount of purified butyrate ester, which remains in the enzymatically treated mixture (eg, enantiomer (-) butyric ester of FTC compound in pig liver esterase and enantiomer (+) butyric ester) of the FTC compound in the case of Amano PS-800).

Unlimited examples of acyl groups that can be evaluated for use with an enantiomeric mixture of nucleosides and especially enzymes include alkyl carboxylic acids and substituted alkylated carboxylic acids, including acetic acid, propionic acid, butyric acid and pentanoic acid. Along with certain enzymes, it is preferred to use an acyl compound that is significantly free of free electrons to facilitate hydrolysis by weakening the ester bond. Examples of acyl groups significantly devoid of free electrons include alpha-haloesters such as 2-chloropropionic acid, 2-chlorobutyric acid and 2-chloropentanoic acid. Alpha-haloesters are excellent substrates for lipases.

Resolution conditions

Enzymatic hydrolysis is usually performed with a catalytic amount of enzyme in an aqueous buffer solution which has the optimum pH close to the enzyme in question. As the reaction proceeds, the pH values result from the release of carboxylic acid. The aqueous base will be added to keep the pH close to the optimum value for the enzyme. The reaction process can be easily determined by monitoring the change in pH and the basic amount needed to maintain the pH. The hydrophobic ester (non-hydrolyzed enantiomer) and the much more polar alcohol (hydrolyzed enantiomer) can be sequentially and selectively extracted from the solution by a judicious choice of organic solvents. immobilized on a solid support Enzymatic hydrolysis carried out under heterogeneous conditions may suffer from reduced reproducibility, therefore it is preferable that the hydrolysis be carried out under homogeneous conditions Alcoholic solvents are not preferred as they may distort the enzymes. by using non-ionic surfactants, such as Triton X-100, however, the addition of these surfactants not only helps to dissolve the starting material, but also contributes to the water solubility of the product. it can occur much more efficiently by the addition of a nonionic surfactant than in the condition ii heterogeneous, the isolation of both the raw material and the resulting product can be made much more difficult. The product can be isolated by more convenient chemical and chromatographic processes (eg selective salt formation). Diacylated nucleosides can be used, but are often very lipophilic and strong, so that they can be dissolved in the environment used.

Example 2: Lipase-catalyzed enantioselective hydrolysis of the FTC compound

A number of 5'-O-acyl derivatives of the FTC compound are prepared by selective O-acylation of the N-hydrochloride salt (see Table 1 and Figure 4) of the (±) -FTC compound. The efficiency of lipase derivative hydrolysis was investigated. As shown in Table 1, porcine liver esterase (PLE) shows a higher level of selectivity for the hydrolysis of the (+) enantiomer ester of the FTC compound, by the predominant elimination of the enantiomer (-) butyrate of the FTC compound in the chromatographically analyzed mixture. high performance liquid. In contrast, the PS-800 enzyme hydrolyzes

'v —r- «) ester of the enantiomer (-) of the preferred FTC compound, by the predominant elimination of the butyrate of the enantiomer (+) compound of the FTC in the mixture analyzed by high performance liquid chromatography. The degree of hydrolysis was found to be dependent on the nature of the acyl group; the acetyl derivative is significantly more inactive than the butyryl derivative. It has now been found that the degree of hydrolysis of the propionic acid ester of the FTC compound is even faster than that observed for the butyrate derivative. The separation percentage and the enantiomeric excess percentage are both determined using HPLC high performance liquid chromatography. Therefore, ^{enantioselectivity} is ^exacerbated . Enriched with PLE liver enzyme-catalyzed hydrolysis, it undergoes enzymatic hydrolysis with PS-800.

Table 1

Comparison of the effect of ester on enzymatic hydrolysis

substrate %Separation % E.E. (s.m.) FTC Esters with PLE: (-) - FTC (butyrate) acetate 32.68 N.D. PROPIONATE 39.87 N.D. butyrate 48.00 98 butyrate 45.71 98.6 FTC Esters with PS-800: (+) - FTC (butyrate) acetate 73.17 N.D. PROPIONATE 52.67 ___________ N.D ._________ butyrate 58.34 ___________ N.D, ________ butyrate ________ 41.50 _________ ___________94___________

Example 3. Process for the preparation of (+) - and (-) - FTC by enantioselective hydrolysis of lipase-catalyzed FTC butyrate

The 5'-O-butyrate of the compound (±) -FTC (0.47 mmol, 149 milligrams) is dissolved in 16 milliliters of a 4-part solution · buffer solution and 1 part CH ₃ CN at pH = 8. The clear solution is stirred and treated with 26 milligrams of pig liver esterase (PLE-A). The progress of the reaction is monitored by high performance liquid chromatography (HPLC) (Figure 4). After 20 hours (52% conversion), the reaction mixture is extracted twice with 80 milliliters of CHCl ₃ chloroform and then with 80 milliliters of ethyl acetate. The organic layer of the extracts is combined, dried over anhydrous magnesium sulfate MgSO ₄ , then filtered and concentrated by rotary evaporation. The resulting residue is eluted on 2x1000 m pTLC discs using ethyl acetate as eluent (double elution) to obtain after isolation 53 milligrams (36% based on the starting material) of FTC butyrate which is determined by high liquid chromatographic analysis. HPLC performance as having an enantiomeric excess of 98%. Enantiomerically enriched butyrate is then treated with 1.6 milliliters of methanol and then with 0.38 mmol (20 milligrams) of sodium methoxide. The resulting mixture is stirred at room temperature and the progress of the reaction is monitored by high performance liquid chromatography, HPLC. The reaction is complete in 30 minutes. The solvent is removed by rotary evaporation to give a crude solid white product (76 milligrams) which is eluted on 1000 m pTLC using a mixture of 5 parts ethyl acetate and 1 part ethanol as eluent. The FTC enantiomer (-) is isolated as a white solid (33 milligrams; 82% yield). By high performance HPLC chromatographic analysis of the FTC compound as a 5'-O-acetate derivative, it shows 97% enantiomeric excess (ee): [a] ( ^2o , D) -12O, 5 ° (c = 0.88; in ethanol absolute). The emulsions in the processing phase must be avoided by adding chloroform HCCI ₃ to the reaction mixture to make up (which therefore serves to denature the enzyme), followed by stripping the solvents in vacuo and then extracting with chloroform HCCI ₃ .

Similarly, 1,2 mmol (375 milligrams) of (±) -FTC 5'-O-butyrate is dissolved in 40 milliliters of a 4 part buffer solution pH = 8 and 1 part CH ₃ CN. The clear solution is stirred and treated with 58 milligrams of pig liver esterase (PLEA). The evolution of the reaction is monitored by high performance liquid chromatography

0-1009-00755HPLC. After 90 minutes (38% conversion), the reaction mixture is added to 150 milliliters of chloroform HCCI ₃ . The layers are separated and the aqueous layer is dehydrated to remove the solvent. The white residue from lyophilization is extracted 3 times with 10 milliliters of absolute ethanol. The extracts are filtered, combined and concentrated in vacuo to give 179 milligrams of crude oil. The crude material is then eluted on a 45 x 30 mm silica gel column using a mixture of 3 x 75 milliliters of ethyl acetate and 5 parts of ethyl acetate with 1 part of ethanol as eluent. The (+) FTC product is isolated as a white solid (109 milligrams; 37% based on the initial butyrate). High performance HPLC chromatographic analysis of the compound (+) - FTC as a 5'-O-acetate derivative, shows 97.4% enantiomeric excess; [α] D (20, D) + 113.4 ° (c = 2.53; absolute ethanol).

A similar reaction is performed using 0.12 mmol (37 milligrams) of FTC 5'-Obutyrate and 7 milligrams of PS-800 in a 4.0 milliliter mixture of 4 parts buffer pH = 8 and 1 part methylcyan CH ₃ CN . The reaction is considerably weaker than that with PLE-A pig liver esterase and requires 74 hours for 59% conversion. Separate butyrate (11.4 milligrams; 31% of the initial amount) shows 94% enantiomeric excess ee, by high performance liquid chromatography HPLC.

The resolution of nucleoside enantiomers, alternatively with cytidine-deoxycytidine deaminase, cytidine-deoxycytidine deaminase is used for the separation of racemic mixtures of 2-hydroxymethyl-5- (cytosine-1-yl) -1,3-oxathiolane and its derivatives, including 2-hydroxy -5- (5-fluoro-cytosin-1-yl) -1,3-oxathiolane.

The enzyme catalyzes the deamination of half of the cytosine in an uracil. One of the enantiomers of 1,3-oxathiolane nucleoside has been found to be the preferred substrate for cytidine deoxycity and deaminase. The enantiomer, which is not converted to the uracil derivative (and is therefore still basic), is extracted from the solution with an acid solution. Care should be taken to avoid strongly acidic solutions (pH below 3.0), which may dissolve the oxatiolanic cycle. Cytidine deoxycytidine disaminase can be isolated from rat liver or human liver, or can be expressed from recombinant sequences in a prokaryotic system, such as, for example, Escherdchia Coli.

The method of resolving cytidine nucleoside enantiomers using cytidine deoxycytidine disaminase may be used as the sole resolution method or may be

ca-2 009-00753

-20-02-1992 used in combination with other methods of resolution, including the resolution by enzymatic hydrolysis of 5'-O-nucleoside esters, as described above.

The combination of enzymatic resolution and classical methods of resolution

The process described above for the separation of racemic mixtures of nucleoside enantiomers can be combined with other classical methods of enantiomeric separation to increase the optical purity of the finished product.

Classical separation methods include a variety of physical and chemical techniques. Often the most efficient and simplest technique is recrystallization, based on the principle that racemates are often much more soluble than the corresponding individual enantiomers. Recrystallization can be performed at any stage, including acylated compounds or the enantiomerically finished product. If the result is a complete success, this simple path is a method worth choosing.

When recrystallization does not result in a material with an acceptable optical purity, other methods may be evaluated. If the nucleoside is basic, (eg, a cytidine) chiral acids may be used to form diastereomeric mixtures which may have significantly different solubility properties. Non-limiting examples of chiral acids include malic acid, mandelic acid, dibenzoyl tartaric acid, 3-bromocamphor-8-sulfonic acid, 10-camphorsulfonic acid, and di-para-toluiltary acid (ric. chiral acid therefore results in the formation of diastereomeric mixtures whose physical properties may be different enough to allow separation.

Small amounts of enantiomerically enriched nucleosides can be obtained or purified by treating the racemic mixture with an HPLC column that has been designated for chiral separations, including cyclodextrin-linked columns, marketed by Rainin Corporation.

Example 4: Separation of denucleoside racemic mixtures by HPLC high performance liquid chromatography.

Resolutions of the C4 ¹ enantiomers of (±) -FTC are performed using a chiral column with cyclodextrin bonds (AC-I cyclo bond), obtained from Rainin Corporation (Woburn, MA). The conditions are as follows: 0.5% isocratic methanol in water; fluid flow rate of 1 milliliter per minute, ultraviolet detection at 262 nm. Purity methanol

HPLC is obtained from J.T. Baker (Phillipsburg, N J). Racemic mixtures are injected and fractions are collected. The fractions containing each of the enantiomers mentioned are collected, frozen and then lyophilized. The compounds are characterized by ultraviolet spectroscopy and their retention times by HPLC high performance liquid chromatography. In general, (-) enantiomers have shorter retention times than (+) enantiomers (see J. Liquid Chromatography, 7: 353-376, 1984). Compound concentrations are determined by ultraviolet spectroscopy using a stock solution of known concentration (15 μΜ) prepared in water for biological evaluation. Retention times for separate enantiomers are shown in Table 2.

Table 2

Retention times of FTC compound enantiomers

compound R _f (minutes) (-) - FTC 8.3 (+) - FTC 8.7

Example 5. Alternative methods for separating FTC enantiomers using a chiral column.

Using a Cyclobond l-Ac column (cyclically bonded) (5 pm, 25 centimeters x 4.6 milliliters, Rainin Corporation, Wobum, MA, Catalog no. AST-41049) with a fluid flow rate of 0.6 milliliters per minute 0.5% isocratic methanol (Fisher Scientific, Inc. HPLC grade catalog A-452-4 in water) and ultraviolet detection at 262 nm, FTC enantiomers have a retention time of 12.68 minutes ((-) - FTC) and 13.20 minutes ((+) - FTC).

Using a Chiralpak AS column (10 pm, 25 cm x 4.6 mm JTBaker Inc., Phillsburg, NJ, catalog no. 7406-00, serial no. 09-29-10320) with a fluid flow of 0.8 milliliters per minute of isopropyl alcohol (grade for high performance liquid chromatography HPLC, Fisher Scientific, Inc. cat no. A-451-4) and ultraviolet detection at 262 nm, the enantiomers of the FTC compound have a retention time of 5.9 minutes ((-) FTC) and 9.8 minutes ((+) - FTC)

III. Ability of 2-hydroxymethyl * -5- (5-fluorocytosin-1-yl) 1,3-oxathiolane

(FTC) to inhibit HIV replication

It is often desirable to screen a number of racemic mixtures of nucleosides as a preliminary step in determining whether further separation into enantiomerically enriched components and further evaluation of antiviral activity is warranted. The ability of nucleosides to inhibit the human HIV immunodeficiency virus can be measured by various experimental techniques. The techniques used in the present invention, which are described in detail below, measure the inhibition of viral replication in phytohemagglutinin (PHA) in HIV-1-infected phytohematoglutinin-stimulated (PBM) human peripheral blood mononuclear cells (PBM) (LAV strain). The amount of virus produced is determined by measuring the enzyme reverse transcriptase on the encoded virus. The amount of enzyme produced is proportional to the amount of virus produced. Table 3 shows the EC ₅₀ values (concentration of nucleoside that inhibits virus replication in 50% of stimulated human peripheral blood mononuclear cells (PBM) estimated with an error factor of 10%) and IC ₅₀ values (nucleoside concentration that inhibits mononuclear cell development by 50% mitogen-stimulated human peripheral blood (PBM) uninfected) of a number of (±) -1,3-oxathiolane and nucleosides.

Example 6: Anti-HIV activity of (±) -1,3-oxathiolane nucleosides.

A. Phytohemagglutinin-stimulated PBM cells, 3 days old (10 ⁶ cells per ml) from healthy seronegative donors to HIV-1 and hepatitis B virus, are infected with HIV-1 (LAV strain) at a about 100 times the dose that infects 50% of tissue culture (TICD) per ml and they are cultured in the presence and absence of different concentrations of antiviral compounds.

B. Approximately one hour after infection, the medium with the compound to be tested (twice the final concentration in the medium) or without the compound is added to the vials (5 milliliters, with a final volume of 10 milliliters). AZT is used as a positive control.

C. The cells are exposed to the virus (approximately 2 x 10 ⁵ dpm / milliliter, as determined by reverse transcriptase analysis) and then placed in a CO ₂ incubator. HIV-1 (LAV strain) is obtained from the Centers for Disease Control in Atlanta, Georgia. The methods used for culturing PBM cells, harvesting the virus and determining reverse transcriptase activity are those described by Mc Dougal and

Tl

0-1009-00755- “20-02-1992-colab., (J. Immun. Meth. 76, 171-183, 1985) and Spira et al., (J.CIin. Meth. 25,9799,1987) except that fungizone was not included in the medium (see Schinazi et al., Antimicrob. Chemother Agents. 32, 1784-1787 (1988); Id 34: 1061-1067 (1990)).

D. On day 6, the cells and supernatant are transferred to a 15 ml tube and centrifuged at approximately 900 g for 10 minutes. Five milliliters of supernatant are removed and the virus is concentrated by centrifugation at 40,000 rpm for 30 minutes (Beckman 70.1 Ti rotor). The solubilized virus pellet is processed to determine reverse transcriptase levels. The results are expressed in dpm / milliliter of supernatant sample. The virus in smaller supernatant volumes (1 milliliter) can therefore be concentrated by centrifugation before solubilization and determination of reverse transcriptase levels.

The effective median concentration (EC ₅₀ ) is determined by the median effect method (Antimicrob. Chemother Agents. 30, 491-498 (1986)). Briefly, the percentage inhibition of the virus, as determined by reverse transcriptase measurements, is plotted against the micromolar concentration of the compound. EC ₅₀ is the concentration of the compound at which there is a 50% inhibition of viral development.

E. MITogen-stimulated uninfected human PBM cells (3.8 x 10 ⁵ cells per milliliter) are cultured in the presence and absence of the drug under conditions similar to those used for the antiviral analysis described above. Cells are counted after 6 days, using a hemacytometer and the Trypan Blue exclusion method, as described by Schinazi et al., AntimicrobialAgents and Chemotherapy22 (3), 499 (1982). The IC 50 value is the concentration of the compound that inhibits 50% of normal cell growth.

Table 3

EC ₅₀ and IC ₅₀ at different 1,3-oxathiolane nucleoside analogues in human PBM cells

Code X or Y R Antiviral cytotoxicity EC ₅₀ , pM IC ₅₀ , pM DLS-009 X = O H > 100 > 100 DLS-010 X = O Me 64.4 > 100

9--1 009-00753- ~ 2O ~ O2-1 992 ~ “fy ² ) '- TlM. ,,;

DLS-027 x = o F > 100 > 100 DLS-028 x = o BUT 60.8 > 100 DLS-044 x = o Br > 100 > 100 DLS-029 x = o and > 100 > 100 DLS-020 y = nh ₂ H 0.02 > 100 DLS-011 y = nh ₂ Me > 10 > 100 DLS-022 y = nh ₂ F 0.1 > 100 DLS-023 y = nh ₂ BUT 38.7 > 100 DLS-021 y = nh ₂ Br 77.4 > 100 DLS-026 y = nh ₂ | 0.72 > 100 DLS-058 (-) y = nh ₂ F 0.008 > 100 DLS-059 (+) y = nh ₂ F 0.84 > 100 DLS-053 y = nh ₂ cf ₃ 60.7 > 100

As shown in Table 3, in general, substituted cytosine 1,3-oxathiolane nucleosides are much more active than the corresponding uracil nucleosides. Errors in EC ₅₀ and IC ₅₀ measurements are estimated at ± 10%.

One of the (±) -FTC compounds (referred to as compound 8 Ct 022) exhibits not only exceptional activity (approximately 10 nM in PBM cells) but also very low toxicity (> 100 pm in PBM, Vero and GEM cells). .

IC ₅₀ of (±) -FTC is above 100 pM, indicating that the compound is non-toxic in uninfected PBM cells evaluated up to 100 μM.

or

Example 7: Antiviral activity of the enantiomers of the FTC compound, separated by HPLC.

The enantiomers of the FTC compound are isolated by the method of Example 4 and the antiviral activity is evaluated by the method of Example 6. The results are shown in Table 4 and illustrated in Figure 5.

Table 4

The antiviral activity of the (+) and (-) enantiomers of the FTC

Treatment concentration pM DPM / ml % Inhibition (corrected) EC ₅₀ : pM FTC (+) 0.0001 73,755 26.6 0.018 0.005 83,005 16.3 0.01 60,465 41.3 0.05 34,120 70.4 0.1 14,160 92.4 0.5 18,095 88.1 1 7,555 99.7 5 7,940 99.3 10 5,810 101.7 FTC (-) 0.001 76,275 23.8 0.02 0.005 58,590 43.3 0.01 75,350 24.8 0.05 28,890 76.20

0 0 9 - 0 0 7 5 3 ”

20-02-1992 //

Cc 042

o, 1 13,175 93.5 0.5 9,485 97.6 FTC (+) 0.001 94,340 3.8 0.28 0.005 107,430 -10.6 0.01 99,465 -1.8 0.05 87,120 11.8 0.1 86,340 12.7 0.5 33,225 71.4

As shown in Table 4, in this experiment the enantiomer (-) of the FTC compound appears to be about one order of magnitude more active than the (+) enantiomer of the FTC compound and has approximately the same anti-HIV activity as the racemic mixture. Neither the enantiomers nor the racemic mixture are toxic, up to 100 pM as measured by the Tryptan Blue exclusion method in human PBM cells.

Example 8: Antiviral activity of FTC enantiomers separated by the method of Example 3.

The (±) -FTC enantiomers are therefore separated by the method of Example 3 and the antiviral activity is evaluated by the method of Example 6. The results are shown in Figure 6. As shown in Figure 6, EC ₅₀ of the racemic mixture of the FTC compound has the value of 0.017 pM, EC ₅₀ of compound (-) - FTC at 0.0077 pM and EC ₅₀ of compound (+) - FTC at 0.84 pM.

Example 9: Absorption (+) - FTC in human PBM cells.

Studies were performed using radiolabeled FTC compound on the following intracellular profiles of the parent drug and on metabolites detected in cells. All studies were conducted in duplicate. Human peripheral blood mononuclear cells (PBM cells) were suspended in RPML1640 medium containing 10% fetal calf serum and antibiotics (2 x 10 ⁶ cells per milliliter), 10 milliliters per fixed time and incubated with the addition of 10 pM FTC approximately 700 dpm / pmol) The cells are then exposed to the drug for 2,6,12 and 24 hours. At these set times, the environment is

(^ -2 0 0 9 - 0 0 7 5 3 - “2 0 - 0 2 - 1 9 9 2 -

separately and the cells are washed twice with a solution of salt, cooled, Hank, balanced. The extraction is carried out with the addition of 0.2 milliliters of 60% methanol / cold water and maintained at -70 ° C until the next day. The next morning, the suspensions are centrifuged and the extractions are repeated twice for 0.5 hours at -70 ° C. The total supernatants (0.6 milliliters) are lyophilized until the sec. The residues are resuspended in 250 microliters of water and aliquots between 50 and 100 microliters are analyzed by HPLC high performance liquid chromatography. Quantitative analysis of intracellular drug and metabolic derivatives is performed by HPLC high performance liquid chromatography. Because some of these compounds have potential acid lability, the buffer system close to physiological pH is used to separate metabolites.

Figure 7 is a graph of the presence (consumption) of tritiated (±) -FTC compounds in human PBM cells (mean of two determinations) over time (hours) versus pmol / 10 ⁶ cells. Consumption studies indicate that the radiolabeled FTC compound is already absorbed into human lymphocytes, which produce very large amounts of 5'-triphosphate derivative of the FTC compound.

Example 10. The antiretroviral activity of the FTC compound in different cell lines The antiretroviral activity of the FTC compound is measured in a number of cell lines using procedures that are similar but not identical to those mentioned in Example 6. The cell lines are obtained from either donors. either from the AIDS Research and Reference Reagent Program, NIH, Rockville, Maryland, ATCC or Red Cross. EMC thymidine kinase-deficient cells are prepared by sequential transition of EMC cells in the presence of 5-bromo-2'-deoxyuridine. The results are presented in Table 5.

Table 5

FTC antiretroviral activity in various cellular systems

Cellular system (virus strain) EC ₅₀ (pM (+) - FTC HIV-1 PBMC (LAV-1) 0.027 MT2 (HTLV ₁₁₁₈ ) _______________________ __________________ 0.89 _____________

Χ-2 0 0 9 - 0 0 7 5 3 -C

CEM (LAV-1) 0.08 CEM-TK ^W (LAV-1) 0.026 CEM (HTLV ₁₁₁₈ ) NTH 0.09 HIV-2 PBMC (ROD2) 0.0038 (±) -FTC 0.0007 (-) FTC 0.026 (+) - FTC hoary AA-2 (SIV251) 4.6 C-8166 (SIV251) <8.0 IVF CrFK (61E) <1

Example 11: Output of (±) -FTC compounds from human PBM cells.

Studies using the radiolabeled FTC compound were performed to monitor the intracellular profiles of the parent drug and the metabolites detected in the cell after incubation in the drug medium for 24 hours, after which the drug is removed. This study measures the time it takes for intracellular triphosphate levels to decrease. The studies are performed in duplicate. Uninfected cells (2 x 10 ⁶ milliliters) are suspended in an appropriate medium supplemented with serum (10 milliliters per fixed time) and incubated at 37 ° C in an incubator with 5% CO ₂ . The concentration of the radiolabeled FTC compound is 10 μΜ. After pulsing the cells with the labeled compound for 24 hours, the cells are washed thoroughly and then filled with fresh medium without antiviral drugs (0 hours). At 0, 2, 4, 6, 12, 24 and 48 hours (secondary incubation time), the cells are removed and then immediately extracted with a 60% cold methanol / water mixture. The extracts are obtained by centrifuging and removing the cell pellets. The extracts are lyophilized and then stored at -70 ° C. Prior to analysis, the material is resuspended in 250 microliters of HPLC buffer mixture. Quantitative analysis of intracellular drug and metabolic derivatives is performed by liquid chromatography

0 ^ ~ 2OO9-OO755-

-20-02-1992-

High-performance FPLC, using either a Micrometrics or a Hewlett-Packard 1090 HPLC system with a column of Partisil 10 SAX anion exchangers (Whatman Inc.) with a fluid flow rate of 1 milliliter per minute, at a pressure of 0.07 kg / cm ² (1 kpsi), with an ultraviolet detection at 262 nm. The mobile phase consists of nonionized water (A), 2 mM NaH ₂ PO _4/16 mM NaOAc (pH = 6.6) (B), 15 mM NaH ₂ PO ₄ / 120.2 mM NaOAc (pH = 6.6) (C) and 100 mM NaH ₂ PO ₄ 4/800 mM NaOAc (pH = 6.6) (D).

Separation method: isocratic for 5 minutes with A, followed by linear gradient 15 minutes to 100% B, followed by linear gradient 20 minutes to 100% C, followed by linear gradient 10 minutes to 100% D, followed by isocratic 30 minutes with 100% D.

Retention times (minutes) in human cells

compound Unchanged Monophosphate Diphosphate T rifosfat (±) -FTC 5.0 39.0 55.0 68.0

Figure 8 is a graphical representation of the (±) -FTC labeled output of human PBM cells, measured in hours after removal of the drug from the concentration (pmol / 10 ⁶ cells). As shown in this figure, FTC-triphosphate has an intracellular half-life of approximately 12 hours and can be easily detected intracellularly at concentrations of 1-5 pM 48 hours after removal of the extracellular drug which is the above EC ₅₀ value for this compound further affinity (K ¹ ) for (±) -FTC triphosphate using HIV RT is 0.2 μΜ, which is below the concentration level at 48 hours.

Example 12. Anti-HIV activity of pharmaceutically acceptable derivatives of (±) -FTC compounds.

a. The number of pharmaceutically acceptable derivatives of the compound (±) -FTC prepared by derivatizing the 5 'and N ⁴ positions is evaluated for anti-HIV activity in PBM cells, using a procedure similar to that described in Example 6. The results are as follows: it follows. The 5'-O-butyrate ester of (±) -FTC has an EC50 value of 0.0017. The N ⁴ -acetyl derivative of the compound (±) -FTC has an EC50 value of 0.0028. N ⁴ -5'-O-butyrate ester of (±) -FTC has an EC ₅₀ value of 0.0058.

^ -2009-00755

-2 0 - 0 2 - 1 9 9 2

η)

b. The anti-HIV activity of the 5'-O-butyrate ester of compound (±) -FTC in the MT4 system (EC ₅₀ ) is 0.04 pM. In the same assay, the uncoated (±) -FTC compound has an IC ₅₀ value of 0.52 μΜ. The IC ₅₀ value for AZT in this system is 0.09 μΜ.

V. Ability of FTC to inhibit HBV replication.

Example 13. Evaluation of FTC (+) and (-) enantiomers activity in cell cultures 2.2.15.

The ability of FTC enantiomers to inhibit virus growth in cell cultures 2.2.15 (hepatitis virion transformed HepG2 cells) is described in detail below. A summary and description of the analysis for antiviral effects in this culture system and the analysis of DNA deoxyribonucleic acid in hepatitis B virus (Korba and Milman, 1991, Antiviral Res., 15: 217) have been described. Antiviral tests are performed on two separate cell passages. All wells in all plates are sown with the same density and at the same time.

Analysis parameters. Due to the inherent variations in intracellular and extracellular HBV DNA levels, only depressions greater than 3.5-fold (for HBV virion deoxyribonucleic acid DNA) or 3.0-fold (for HBV deoxyribonucleic acid DNA replication intermediates) from The mean levels for these forms of HBV DNA, which form in untreated cells, are considered to be statistically significant. (P <0.05). The levels of deoxyribonucleic acid DNA integrated with HBV hepatitis B virus in each preparation of cellular DNA deoxyribonucleic acid (which remains constant, relative to a pair of cells, in these experiments) are used to calculate the levels of HBV intracellular deoxyribonucleic acid forms. Thus, equal amounts of deoxyribonucleic acid in cellular DNA were compared between the separate samples.

Typical values for extracellular HBV virion DNA deoxyribonucleic acid in untreated cells are between 50 and 150 pg / milliliter of culture medium (average of approximately 76 pg / milliliter). Deoxyribonucleic acid DNA replication intermediates to intracellular HBV virus in untreated cells are between 50 and 100 pg / pg deoxyribonucleic acid cellular DNA (average of approximately 74 pg / pg deoxyribonucleic acid cellular DNA). In general, decreases in the levels of intracellular HBV virus deoxyribonucleic acid DNA due to treatment with compounds

GL -2009-00753antivirals are less pronounced and occur much more slowly than decreases in DNA deoxyribonucleic acid levels in the HBV virion (Korba and Milman, 1991, Antiviral Res., 15: 217).

The way in which hybridization assays are performed for these experiments resulted in the equivalence of approximately 1.0 pg of intracellular HBV DNA deoxyribonucleic acid up to 2-3 genomic copies per cell and 1.0 pg / milliliter of HBV DNA deoxyribonucleic acid extracellular up to 3 x 10 ⁵ viral particles per milliliter.

Toxicity analysis. Toxicity tests are performed to determine if the observed antiviral effects are due to an overall effect on cell viability. The method used in the present invention was to measure the absorption of natural red coloration, a standard and widely used assay for cell viability in a variety of host systems for viruses, including HSV and HIV. Toxicity tests are performed on 96-well flat tissue culture plates. Cells for toxicity assays are cultured and treated with test compounds using the same regimen as described below for antiviral assays. Each compound is tested at 4 concentrations, each in triplicate cultures (wells A, B and C). The absorption of the natural red dye is used to determine the relative level of toxicity. The absorbance of the dye internalized at 510 nm (A _sin ) is used for quantitative analysis. The values are presented as a percentage of the average A _sin values in 9 separate cultures of untreated cells maintained on the same 96-well plate as test compounds. The absorption of the dye in the 9 control cultures on plate 5 is between 91.6% to 110.4% and on plate 6 from 96.6% to 109%. The results are given in Table 6.

Table 6

Toxicity analysis of test compounds in cells 2.2.15

disc Compound Conc. (μΜ) Dye absorption (% of control) Enjoy A Enjoy B Enjoy C 5 DMSO 10.0 * 0.7 1.6 0.9 3.3 55.9 68.7 61.7

Ο—- 2 Ο Ο 9 - Ο Ο 7 5 5 - “2 0 - 0 2 - 1 9 9 2 -

1.0 91.2 96.4 106.8 0.3 98.7 102.9 93.5 6 (-) - FTC 300 53.0 51.1 51.5 100 64.1 66.6 77.6 30 98.7 94.3 96.4 10 94.3 94.9 92.2 6 (+) - FTC 300 43.4 56.7 58.5 100 77.7 66.3 72.1 30 81.1 88.3 88.1 10 90.9 99.4 90.5

for DMSO, the concentrations are presented as percentages of the original stock solution.

Toxicity assessment. As indicated in Table 6, no significant toxicity (greater than 50% decrease in absorption levels observed in untreated cells) was observed for the detested compounds at the concentrations used for antiviral assays. Both test compounds, (-) - FTC and (+) - FTC appear to be toxic at higher concentrations used for toxicity tests (330 μΜ).

Antiviral evaluations.

Control.

Under normal variations, DNA deoxyribonucleic acid levels for HBV virion and intracellular HBV replication intermediates (HBV R1) remain constant in untreated cells over the test period. DMSO at 1% does not affect HBV replication levels in cell cultures 2.2.15.

Test compounds.

As shown in Table 7, both (-) - FTC and (+) - FTC significantly inhibit HBV replication at test levels. As indicated in Table 8, compound (-) - FTC still significantly inhibits DNA deoxyribonucleic acid synthesis in HBV virion and DNA deoxyribonucleic acid DNA synthesis in intracellular HBV at concentrations of 4.1 and 0.25 μΜ.

00σ2 009-0075320-02-1992Table 7

Effect of test compounds on HBV production in cell cultures 2.2.15.

well Treatment HBV virion DNA * (pg / ml culture medium) Intracellular HBV DNA (pg / pg cellular DNA) Day 0 Day 4 Day 9 MONEY R 7A Untreated cells 59 75 94 2.7 93 7B Untreated cells 47 64 88 2.5 93 8A Untreated cells 65 100 71 2.2 97 8B Untreated cells 77 65 110 2.4 62 7K DMSO @ 1.00% 100 50- 48 1.9 95 7L DMSO @ 1.00% 48 96 54 2.8 98 8K DMSO @ 1.00% 93 63 68 2.2 86 8L DMSO @ 1.00% 66 57 59 1.6 97 9U (-) - FTC @ 10 μΜ 120 36 1 1.1 14 9V (-) - FTC @ 10 pM 89 48 1 1.5 19 10U (-) - FTC @ 10 μΜ 58 41 0.1 1.9 13 10V (-) - FTC @ 10 pM 110 32 0.1 1.2 16 9W (+) - FTC @ 10 pM 88 42 0.1 0.8 14 9X (+) - FTC @ 10 μΜ 58 57 0.2 0.4 19 10W (+) - FTC @ 10 pM 69 55 0.1 0.7 17 10X (+) - FTC @ 10 pM 45 39 o, 1 0.4 15

* Separation sensitivity for HBV virion DNA is 0.1 pg / ml.

Intracellular HBV DNA was analyzed 24 hours after the 9th day of treatment. The levels of HBV DNA embedded in each cellular DNA preparation are used to calculate the levels of episodic HBV genomes 3.2 kb (MONO.) And deoxyribonucleic acid DNA replication intermediates (Rl) in the case of HBV virus.

¢ ^ 2009-00753 “20-02-1992

Table 8

Effect of test compounds on HBV production in cell cultures 2.2.15.

well Treatment HBV virion DNA * (pg / ml culture medium) Intracellular HBV DNA * (pg / pg cellular DNA) Day 0 Day 4 Day 9 MONEY. R 31 Untreated cells 64 54 65 2.8 65 31B Untreated cells 51 54 77 2.0 53 32 Untreated cells 100 76 56 3.5 81 32B Untreated cells 53 97 83 3.1 68 35 (-) - FTC @ 4 pM 74 27 > 0.1 1.4 1 35B (-) - FTC @ 4 pM 87 28 > 0.1 0.5 1 36 (-) - FTC @ 4 pM 120 20 1 0.9 1 36B- (-) - FTC @ 4 pM 59 16 0.2 0.2 2 35C (-) - FTC @ 1 pM 70 13 > 0.1 17 2 35D (-) - FTC @ 1 pM 62 15 > 0.1 1.2 3 36C (-) - FTC @ 1 pM 60 22 1 1.4 2 36D (-) - FTC @ 1 pM 89 28 0.3 1.5 4 35th (-) - FTC @ 0.25 pM 84 15 > 0.1 1.5 4 35F (-) - FTC @ 0.25 pM 89 16 4 2.2 4 36E (-) - FTC @ 0.25 pM 66 13 1 1.8 8 36F (-) - FTC @ 0.25 pM 49 19 0.1 0.3 9

* Separation sensitivity for deoxyribonucleic acid DNA in HBV virion is 0.1 pg / ml.

+ Intracellular HBV DNA analysis was performed 24 hours after the 9th day of treatment. The levels of HBV DNA embedded in each cellular DNA preparation are used to calculate the levels of episomal HBV genomes of 3.2 kb (MONO) and deoxyribonucleic acid DNA replication intermediates (Rl) in the case of HBV virus.

Example 14: Absorption of (±) -FTC in human liver cells: HBV activity on the FTC compound.

0 ^ -2 009-00753

The procedure of Example 9 is repeated with human liver cells (HepG2 cells, available from ATCC) to determine the absorption and metabolism of the FTC compound in these cells. As shown in Figure 9, compound (±) - FTC is absorbed by large amounts of HepG2 cells. These cells in the human liver metabolize a high percentage of (±) -FTC to (±) -FTC triphosphate.

These data, in conjunction with other data provided in the present invention, show that (±) -FTC as well as the (-) and (+) enantiomers of this compound are phosphorylated in liver cells. These cells can be transformed with the hepatitis B virus.

Example 15: Exit of FTC compound from human HepG2 cells.

Figure 10 shows the output of the compound [ ³ H] - (±) -FTC and its phosphorylated derivatives in human HepG2 in pmol / 10 ⁶ cells over time, after pulsing the cells with 10 pM [ ³ H] - (±) -FTC ( 700 DPM / pmol) for 24 hours and evaluation of the compound concentration 24 hours after separation.

Figure 11 illustrates the combined decrease in [ ³ H] - (±) -FTC and phosphorylated derivatives of this compound in human HepG2 cells after incubation with 10 pM [ ³ H] - (±) -FTC (700 DPM / pmol ) for 24 hours, in pmol / 10 ⁶ cells over time.

As shown, even at 48 hours, more than 1 pM of the active compound (which is significantly higher than EC ₅₀ for the compound) is still present in the cells.

V. Toxicity in granulocyte-macrophage precursor cells.

Example 16: Effect of FTC on granulocyte-macrophage precursor cell formation.

Figure 12 is a graphical representation of the effect of the (+) and (-) enantiomers of the FTC compound on the formation of granulocyte-macrophage precursor cell colony, as measured in survival rates relative to the pM concentration of (-) - FTC in the circle open; (+) - Closed Circle FTC; AZT closed square. As indicated, the (-) enantiomer of the FTC compound appears to be less toxic, for example it has a higher value for IC ₅₀ , either than the (+) enantiomer or AZT in this cell line.

VI. Pharmacokinetics of the FTC compound.

Example 17: FTC metabolism in rats.

o ^ l Ο Ο 9 - Ο 0 7 5 3 -2 0 - 0 2 - 1 9 9 2-

Co '- ..,

Compound (±) -FTC is administered intravenously at doses of 10.50 and 10O milligrams per kilogram in rats. The surface area was evaluated under the plasma drug concentration versus time (AUC), total clearance (CL _T ), steady-state volume of distribution (V _ss ), mean residence time (MRT), and half-life (t ₁ ). _{/ 2} ). The results are presented in Table 9.

Table 9

FTC pharmacokinetic parameters after intravenous administration of 10, 50,100 milligrams per kilogram in rats

Dose AUC CL _T V _ss MRT ^ 1/2 mg / kg mg h / L L / h / kg L / kg h h 10 9.65 0.988 0.758 0.768 0.757 50 57.11 0.874 0.699 0.800 0.815 100 120.72 0.830 0.663 0.798 0.969

* AUC = area under the plasma drug concentration curve relative to time; CL = total release; Vss = stationary volume of the distribution; MRT = average clearance and t _1/2 = half-life.

Example 18. Pharmacokinetic parameters for FTC after oral and intravenous administration of FTC compound.

Independent pharmacokinetic parameters are derived for (±) -FTC by administration (intravenous (i.v.) and oral (p.o.)) of 33.3 milligrams / kilogram to rhesus monkeys. The results are shown in Table 10. Importantly, the average bioavailability of the compound in monkeys is 73% (± 6%).

CL. ~ 29u9-uu755- 2 0 - 0 2 - 1 9 9 2 Table 10

Independent pharmacokinetic parameters derived for FTC after intravenous (i.v.) or oral (p.o.) administration of 33.3 milligrams / kilogram in rhesus monkeys *

The monkey AUC mg h / L CL _T L / h / kg v _ss L / kg MRT h ^ 1/2 h K _a h ¹ F % iv Ruh 19.14 1.74 2.71 1.56 1.28 Rmi 26.31 1.26 1.97 1.56 1.22 RJd 22.51 1.48 2.00 1.36 1.47 Mediate 22.65 1.49 2.23 1.49 1.32 ± S.D. 3.59 0.24 0.42 0.12 0.13 P.O. Ruh 13.21 2.07 1.58 0.48 71 Rmi 21.11 2.32 1.08 0.43 80 Rjd 15.29 3.23 1.47 0.31 68 Mediate 16.54 2.54 1.38 0.41 73.00 (± 6) S.D. 4.09 0.61 0.26 0.09 6.24

* AUC = area under the plasma drug concentration curve over time; CL = total release; Vss = stationary volume of the distribution; MRT = average residence time and t _1/2 = half-life; F = bioavailability; and K _a = first order constant of the absorption rate.

Table 11

CSF / serum ratio of FTC compound and deaminated metabolite of this compound 1 hour after treatment

The monkey Path FTC Metabolite (FTU) RUh iv 0.076 0.024 RMi iv 0.062 0.032

^ nna / 1

RJd iv 0.162 0.052 Mediate 0.100 0.036 ± S.D. 0.054 0.014 RUh P.O. 0.048 0.026 RMi P.O. 0.039 0.037 RJd P.O. 0.117 0.055 Mediate 0.068 0.039 ± S.D. 0.043 0.015

Example 19. CSF / serum ratio of FTC compound and its metabolites in rhesus monkeys.

The ability of (±) -FTC to cross the blood-brain barrier was assessed by administering 33.3 milligrams per kilogram of the active compound to Rhesus monkeys and by measuring the amount of (±) -FTC in cerebrospinal fluid (CSF) and serum. one hour after administration. The results are shown in Table 11. Data show that a significant amount of the active compound crosses the blood-brain barrier in this mammal.

ARE YOU COMING. Preparation of pharmaceutical compositions.

People suffering from HBV hepatitis B virus or HIV immunodeficiency virus infection can be treated by giving the patient an effective amount of the (±) -FTC compound or its (-) or (+) enantiomers, compound, or a pharmaceutically acceptable derivative or salt thereof, in the presence of a pharmaceutically acceptable vehicle or diluent. The active materials may be administered by any convenient route, such as oral, parenteral, intravenous, intradermal, subcutaneous or topical, in liquid or solid form.

The active compound is included in a vehicle or a pharmaceutically acceptable diluent in an amount sufficient to deliver to the patient a therapeutically effective amount of the compound that inhibits viral replication in vivo, especially in the case of replication of HIV viruses and HBV, without causing serious toxic effects to

O O 9 “O U 7 ο ο - -20-02-1992-, Μ the treated patient. Inhibitory amount means the amount of active ingredient that is sufficient to exert an inhibitory effect, as measured, for example, by the analysis performed as mentioned above.

A preferred dose of (-), (+) or (±) -FTC for all of the above conditions will be in the range of about 1 to 50 milligrams per kilogram, preferably 1 to 20 milligrams per kilogram body weight per day, more generally 0.1 to about 100 milligrams per kilogram body weight of the body receiving the dose, per day. The effective dosage range of the pharmaceutically acceptable derivatives can be calculated based on the weight of the parent nucleoside to be released. If the derivative itself has activity, the effective dosage can be estimated as above, using the weight of the derivative or by other means known in the literature.

The compound is conventionally administered in units of any suitable dosage form, including but not limited to 7 to 3000 milligrams, preferably 70 to 1400 milligrams of active ingredient per unit dosage form. unitary. An oral dosage of 50 to 1000 milligrams is usually convenient. Ideally, the active ingredient will be administered so as to obtain the peak concentration of the active compound in plasma from about 0.2 to 70 μΜ, preferably from about 1.0 to 10 μΜ. This can be achieved, for example, by intravenous injection of a 0.1% to 5% solution of the active compound, optionally in saline or administered as a bolus of active ingredient.

The concentration of the active compound in the composition of the drug will depend on the rates of absorption, inactivation and excretion of the drug, as well as other factors known in the literature. It should be noted that the dosage values will also vary with the severity of the condition that needs to be tamed. It is further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual needs and professional judgment of the person administering or supervising the administration of the compositions and that the dosing intervals set forth in the present invention should be given by way of example only. , without intending to limit the purpose or practice of the claimed composition. The active ingredient may be given at once or may be divided into a number of smaller doses

What is administered at different time intervals. A preferred mode of administration of the active compound is the oral route. Pharmaceutical compositions that can be administered orally will generally include an inert diluent or an edible vehicle. These compositions may be included in gelatin capsules or compressed into tablets. For the purpose of oral administration, the active compound may be incorporated with the excipients and used in the form of tablets, pills or capsules. Pharmaceutically compatible binders and / or adjuvants may be included as part of the composition. Tablets, pills, capsules, tablets and the like may contain any of the following ingredients or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; such as starch or lactose, a disintegrant such as alginic acid, primogel or cornstarch; a lubricant such as magnesium stearate or sterols; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin or a flavoring agent such as mint, methyl salicylate or orange flavoring. When the form of the dosage unit is a capsule, it may contain, in addition to the type of material mentioned above, a liquid vehicle such as a fatty oil. In addition, unit dosage forms may contain various other materials that alter the physical form of the dosage unit, such as sugar coatings, shaving shellac, or other enteric agents.

(±) -FTC or its (-) or (+) enantiomers or pharmaceutically acceptable salts or derivatives thereof may be administered as a component of elixir, suspension, syrup, biscuit, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose or a sweetener and certain preservatives, dyes, paints and flavorings.

(±) -FTC or enantiomers (-) or (+) or pharmaceutically acceptable derivatives or salts of this compound may also be mixed with other active materials that do not interfere with the desired activity or with other materials that supplement the action. such as antibiotics, antifungals, anti-inflammatory substances or other antiviral substances, including other anti-fungal

^ 2 0 0 9 - 0 0 7 5 5 --20-02-092- / 24 ⁽ _.)

HIV.

Solutions or suspensions used for parenteral, intradermal, subcutaneous or topical application may include the following components: a sterile diluent such as water for injections, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid, buffering agents such as acetates, citrates or phosphates, toning agents such as sodium chloride or dextrose. The original preparation may be included in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

If intravenous, vehicles with saline or phosphate buffered saline (PBS) are preferred. In a preferred embodiment, the active compounds are prepared with vehicles that will protect the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biocompatible, biodegradable polymers can also be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. The methods for preparing these formulations are obvious to those skilled in the art. The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc.

Liposomal suspensions (including liposomes targeted to cells infected with monoclonal antibodies to viral antigens) are also preferred as pharmaceutically acceptable carriers. These suspensions may be prepared by methods known to those skilled in the art, such as those described in U.S. Patent No. 4,822,811 (which are incorporated herein by reference in their entirety). For example, liposome formulations can be prepared by dissolving appropriate lipids (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent which is then evaporated and leaving a thin thread. of dry lipid on the surface of the container. An aqueous solution of the active compound or its monophosphate, diphosphate and / or triphosphate derivatives are then placed in the container. The container is then manually twisted to release the lipid material from the sides of the container and to disperse.

^ 2 0 0 9 - 0 0 7 5 3 - /7.Γ ~ 2 0-02-1992-C lipid aggregates, thus obtaining a liposomal suspension.

The monophosphate, diphosphate and triphosphate derivatives of the FTC compound can be prepared as described below.

Monophosphate can be prepared according to the procedure given by Imai et al., From J.Org.Chem. 34 (6), 1547-1550 (June 1969). For example, about 100 milligrams of the FTC compound and about 280 μl of phosphoryl chloride react with stirring in about 8 milliliters of dry ethyl acetate at about 0 ° C for about four hours. The reaction is stopped with ice. The aqueous phase is purified on a column of activated carbon and the elution is carried out with 5% ammonium hydroxide in a mixture of equal parts of ethanol and water. Evaporation of the eluent gives FTC-5 ammonium monophosphate.

Diphosphate can be prepared according to the procedure described by Davisson et al. In J. Org. Chem. 52 (9), 1794-1801 (1987). The diphosphate of the FTC compound can be prepared from the appropriate tosylate, which can be prepared, for example, by reacting the nucleoside with tosyl chloride in pyridine at room temperature for about 24 hours, then processing the product in the usual manner (eg by washing , drying and crystallization of the product).

The triphosphate can be prepared according to the procedure of Hoard et al., From J. Am. Chem. Shock. 87 (8), 1785-1788 (1965). The FTC compound is activated (converted to imidazolid according to methods known in the literature) and treated with tributyl ammonium pyrophosphate in DMF dimethylformamide. The reaction primarily leads to the formation of nucleoside triphosphate, along with small amounts of unreacted monophosphate and diphosphate. Purification by anion exchange chromatography on a DEAE column is followed by isolation of triphosphate, such as tetrasodium salt.

, d j

Claims (14)

-20’0 2 - 1 9 9 2 - CLAIMS 1. 1,3-Oxathiolane derivatives characterized in that they are (-) - pD-2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane or its physiologically acceptable derivative, or its physiologically acceptable salt. 2. 1,3-Oxathiolane derivatives characterized in that they are (-) - PD-2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane as an isolated enantiomer or its derivative physiologically acceptable, or its physiologically acceptable salt. 3. 1,3-Oxathiolane derivatives characterized in that they are (-) - pD-2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane in an enantiomerically enriched form , or its physiologically acceptable derivative, or its physiologically acceptable salt. 1,3-Oxathiolane derivatives according to Claim 1, characterized in that they have the following general structural chemical formula: 0 <-2 Ο Ο 9 Ο 0 7 5 3 - “20-02-1992-” / 23 wherein R 1 and R ₂ are independently alkyl, a carboxylic acid ester in which the non-carbonyl moiety of the ester group is selected from the group consisting of linear, branched or cyclic alkyl; alkoxyalkyl; aralkyl; aryloxyalkyl; aryls including optionally halogen-substituted phenyl; C 1 -C 4 alkyl or C 4 -C 4 alkoxy; sulfonated ester; alkyl or aralkyl sulfonyl; mono, di or triphosphate ester or amino acid ester; and one of R 1 or R ₂ may be hydrogen. 1,3-Oxathiolane derivatives according to Claim 4, characterized in that R 1 and R ₂ are independently selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec- butyl, t-butyl, isopentyl, amyl, t-pentyl, 3-methylbutyryl, hydrogen succinate, 3-chlorobenzoate, cyclopentyl, cyclohexyl, benzoyl, acetyl, pivaloyl, mesylate, propionyl, butyryl, valeryl, caproic, caprylic, capric, lauric , myristic, palmitic, stearic, oleic, amino acids m ^{49 V.} ^ · · 'Bj ·. '. · including, but not limited to, alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalananyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaoyl, histin and one between R 1 and R 2 may be hydrogen. 1,3-Oxathiolane derivatives according to Claim 5, characterized in that R 1 is n-propyl and R 2 is hydrogen. 7. A pharmaceutical composition characterized in that it comprises (-) PP-2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane or its physiologically acceptable derivative, or its physiologically acceptable salt, and a pharmaceutically acceptable carrier. 8. A pharmaceutical composition characterized in that it comprises (-) pP-2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane as an isolated enantiomer or its physiologically acceptable derivative, or its acceptable salt physiologically and a pharmaceutically acceptable carrier. 9. A pharmaceutical composition characterized in that it comprises (-) pP-2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane in an enantiomerically enriched form, or its physiologically acceptable derivative, or its physiologically acceptable salt and a pharmaceutically acceptable carrier. Pharmaceutical composition according to Claims 7-9, characterized in that the carrier is suitable for oral administration. Pharmaceutical composition according to Claims 7 to 9, characterized in that it is in the form of a capsule or tablet. Method for the treatment of HIV infection in humans, characterized in that it comprises the administration of the compound (-) - p-D-2-hydroxymethyl-5- (5- / 27 ο ^ -2 Ο Ο 9 - Ο Ο 7 5 3 - -20-02-1992-fluorocytosin-1-yl) -1,3-oxathiolane or its physiologically acceptable derivative, or its physiologically acceptable salt in an amount therapeutically effective. 13. A method for the treatment of HIV infection in humans, characterized in that it comprises the administration of the compound (-) - pD-2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane as an isolated enantiomer or its derivative physiologically acceptable, or its physiologically acceptable salt in a therapeutically effective amount. 14. A method for the treatment of HIV infection in humans, characterized in that it comprises the administration of the compound (-) - pD-2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1,3-oxathiolane in an enantiomerically enriched form , or its physiologically acceptable derivative, or its physiologically acceptable salt in a therapeutically effective amount.