FI90664B - Method for production of therapeutically applicable 3'- azidonucleosides - Google Patents

Description

1 90664

The present invention relates to a process for the preparation of a therapeutically useful compound of formula R3. This invention relates to a process for the preparation of a therapeutically useful 3'-azido nucleoside.

/ (I) B

"W

15 * 3 wherein R 1 is hydroxy or alkoxy; R 2 is hydrogen or acyl; R 3 is hydroxy, pyrrolidinyl or alkoxy; R 4 is alkyl or hydrogen; provided that when R 1 and R 3 are each hydroxy and R 2 is hydrogen, R 4 is not hydrogen or methyl; and pharmaceutically acceptable salts and organosulfonates thereof. The new compounds are particularly useful in the treatment and prevention of certain viral and bacterial infections.

In antiviral chemotherapy, there are few drugs that are effective against the virus itself due to the difficulty of attacking the virus without attenuating uninfected host cells. It was long assumed that due to the extreme parasitic nature of the viruses, the entire machinery required for virus replication was provided by the host cell. Recently, it has been established that certain stages of the viral life cycle, which vary from species to species, are determined by the virus itself, and these stages may be susceptible to attack when they differ sufficiently from any corresponding host cell function. However, due to the similarity of viral and 5 host cell functions, it has proven to be very difficult to find effective treatments.

Therefore, compounds found to be suitable for the treatment of viral infections are usually to some extent saturated with myr-10 in the host. Thus, the ideal drug treatment is non-toxic at concentrations that are effective against viruses, but in the absence of such treatment, the compound should have a good therapeutic ratio, i.e., concentrations at which the treatment is toxic-15 are significantly higher than those at antiviral levels. the effect is observed.

One group of viruses that has recently gained special importance is retroviruses. Retroviruses belong to a subset of RNA viruses that need to multiply. 20 first "reverse copy" their genome into RNA DNA (usually "copy" means the synthesis of RNA from DNA). In DNA form, the viral genome can join into the genome of the host cell, allowing it to take full advantage of the DNA of the host cell, it is virtually indistinguishable, and in this state the virus can survive as long as the cell lives. Because it is in fact invulnerable in this form, treatment must target some other stage of the viral life cycle and must necessarily be continued until all cells infected with the virus have died.

HTLV-I and HTLV-II are both retroviruses and are known to cause leukemia in humans. HTLV-I infections are particularly widespread and cause several deaths worldwide each year.

35 One type of retrovirus has also been repeatedly isolated from 3,90664 immunodeficient patients. Although the characteristics of the virus have been extensively studied, there is still some controversy over its internationally accepted name. It is currently known as either human T-cell lymphotropic-5 rus III (HTLV III), immunodeficiency retrovirus (ARV), or lymph node disease-associated virus (LAV), but it is assumed that the internationally accepted name is human immunodeficiency virus (HIV). ). This virus (referred to herein as HIV) has been shown to primarily infect and destroy T cells with the OKT4 surface marker and is now widely considered to cause immunodeficiency. The patient loses more and more of these T cells, upsetting the overall balance of the immune system and impairing the patient's ability to fight other infections and expose him to opportunistic infections, which often prove fatal. The common cause of death in immunocompromised victims is thus an opportunistic infection, such as pneumonia or cancers, which can be caused by viruses, and not necessarily directly HIV infection. Other conditions associated with HIV infection include thrombocytopenia purpura and Kaposi's sarcoma.

Recently, HIV has also been isolated from other tissue types, including B cells expressing the T4 marker, macrophages, and non-blood-associated central nervous system tissue.

This central nervous system infection is not necessarily associated with classical immune deficiency and has been observed in asymptomatic HIV-infected patients. HIV infection of the central nervous system is associated with progressive demyelination, leading to relapse and symptoms such as brain disease, worsening speech disorder, groping, and disorientation. Other conditions associated with HIV infection include asymptomatic carrier, progressive generalized lymph node disease (PGL), and immunodeficiency complex (ARO).

35 Today, some chronic neurological infections are considered to be caused by retroviruses. Such infections include, for example, human multiple sclerosis, and arthritic and encephalitis virus infections in goats and visna-Maedi infections in sheep.

5 Testing of compounds against various retroviruses, such as Friend's leukemia virus (FLV), a rat and mouse retrovirus, has been described. For example, Kriet et al. (Exp. Cell Res., 116 (1978) 21-29) found that 3'-azido-3'-deoxythymidine 10 was active against FLV in in vitro experiments, Os-tertag et al. (Proc. Nat. Acad. Sci. (1974) JX, 4980-85) stated that due to the antiviral activity and non-toxicity associated with FLV, 3'-azido-3'-dideoxythymidine "could advantageously replace bromideoxy-15 uridine DNA. in the treatment of diseases caused by viruses ". De Clerq et al. (Biochem. Pharm. (1980) 29, 1849-1851) confirmed, however, six years later that 3'-azido-3'-dideoxythymidine had no significant effect against the virus 20 used in their experiments, DNA viruses against vaccinia, HSVI and varicella-zoster virus (VZV).

Bacteria also cause treatment problems, because when the living processes of all living organisms are very similar, a substance 25 that is toxic to one organism is likely to be toxic to others. In addition, experience has shown that, over time, bacterial strains develop that are resistant to commonly used antibacterial agents.

It has now been found that certain 3'-azidonucleosides are useful in the treatment of viral and bacterial infections, including HIV infections and infections caused by gram-negative bacteria, including certain bacterial strains resistant to commonly used antibacterial agents.

Thus, the present invention relates to a process for the preparation of a therapeutically useful compound of formula R3 5 11 r * '' ^ Kr

/ (DB

y wherein R 1 is hydroxy or alkoxy; R 2 is hydrogen or acyl; R 3 is hydroxy, pyrrolidinyl or alkoxy; R 4 is alkyl or hydrogen; provided that when R 1 and R 3 are each hydroxy and R 2 is hydrogen, R 4 is not hydrogen or methyl; and pharmaceutically acceptable salts and organosulfonates thereof.

In the general formula above, the dashed lines between the 2- and 6-positions are intended to indicate the presence of single bonds and double bonds in the meantime, the relative positions of the single and double bonds being determined depending on whether the substituents R1 and R2 are capable of, for example, keto-enol tautomerism. .

Preferred pyrimidine nucleoside groups of the invention are cytidine derivatives in which the 3'-azido group is in ervtro form ("azido down"); thymidine and uridine derivatives in which the 3'-azido group is in either ervtro or threo ("azido up") form; and unsaturated nucleosides between the 5C and 6C positions.

The above-mentioned acyl groups preferably contain an alkyl or aryl group described below. Alkyl 6,90664 groups preferably have 1 to 8 carbon atoms, especially 1 to 4 carbon atoms, and are, for example, methyl or ethyl groups optionally substituted with one or more suitable substituents as described below. Aryl groups, including the aryl portions of aralkoxy-like groups, are preferably phenyl groups substituted with one or more suitable substituents as described below.

Suitable substituents for the above groups are preferably selected from halogen atoms, hydroxy, C 1-4 alkoxy, C 1-4 alkyl, C 1-4 aryl, C 1-4 aralkoxy, carboxy, amino and substituted amino groups, amino group being mono- or disubstituted with a C 1-4 alkyl group, and when the amino group is disubstituted, the alkyl groups are optionally joined together to form a heterocyclic ring.

The azido group is preferably in ervtro form. Particularly preferred are those compounds in which only one of the substituents R * -R4 on the pyrimidine-20 ring differs from that of the thymine group (of which R1 and R3 are hydroxy groups, R2 is a hydrogen atom and R4 is a methyl group).

Examples of pharmaceutically acceptable salts of the compounds of the invention include base salts, for example derivatives of a suitable base such as alkali metal (e.g. sodium), alkaline earth metal (e.g. magnesium) salts, ammonium and NXj (where X is C 1-4 alkyl). ) and mineral acid salts such as hydrochloride.

The compounds of the invention and their pharmaceutically acceptable derivatives can be prepared in a conventional manner using methods known in the art, for example, those described in Synthetic Procedures in Nucleic Acid Chemistry (1, 321 (1968)), TA Krenitsky et al. ). J. Med.

7 90664

Chem. (26, 981 (1983)); Nucleic Acid Chemistry Improved and New Synthetic Processes, Methods and Techniques (parts 1 and 2, eds. L.D. Townsend, R.S. Tipson, (J. Wiley) 1978); J.R. Horwitz et al. (J. Org. Chem. 29, (July 1964) 5 2076-78); M. Imazawa et al. (J. Org. Chem., 43. (15) (1978) 3044-3048); K. A. Watanabe et al. (J. Org. Chem., 45, 3274 (1980)); and R.P. Glinski et al. (J. Chem., Soc. Chem. Commun., 915 (1970)), which are incorporated herein by reference, or similar methods described herein.

The process according to the invention for the preparation of compounds of formula (I) B is characterized in that, (A) a compound of formula R3 15: (M)

; "" YJ

M

wherein R 1, R 2, R 3 and R 4 are as defined above and M is halogen, hydroxy or an organosulfonyloxy radical, is reacted with an alkali metal azide; (B) a compound of formula R3 a "2 I 4 30 a N, -] · ;. y R, N (IV) p 35 ^ n3 8 90664 where R1, R2. and R 3, respectively, represent R 1, R 2 and R 3 or their precursor groups, provided that at least one of R 1, R 2, and R 3 is a precursor group, and R 4 is as defined above, is reacted with a substance or under conditions in which said precursor group (s) are converted to the corresponding desired groups, wherein the substance or conditions are as follows: a) reacting a compound of formula IV wherein R 1 is a 2.5'-0-10 anhydro bond with an alkoxide in the presence of potassium carbonate to form the corresponding compound; wherein R 1 is C 1-8 alkoxy; b) reacting a compound of formula IV wherein R 2 is hydrogen with an acylating agent to form the corresponding compound wherein R 2 is an acyl group; c) reacting a compound of formula IV wherein R 3 is a 1,2,4-triazolyl group with an alkoxylating agent or pyrrolidine to form the corresponding compound wherein R 3 is an alkoxy group or pyr-, respectively. 20 rolidinyl group.

From the process of the invention described above, it should be noted that the choice of precursor compounds is determined. mainly according to the particular compound to be prepared, and the substances and conditions are accordingly selected from those known in the art of synthetic chemistry of nucleosides.

Examples of such conversion methods are provided below for guidance and it will be appreciated that they may be modified in the usual manner depending on the desired compound. In particular, if, for example, a version of con-30 has been described which would otherwise lead to undesired reactions of labile groups, then such groups can be protected in the usual way and the protecting groups removed later after conversion.

Thus, for example, in process (A), the group M of a compound of formula (III) 35 may represent, for example, a halo (e.g. chloro), hydroxy or organosulfonyloxy (e.g. trifluoromethylsulfonyloxy, methanesulfonyloxy or p-toluenesulfonyloxy) radical. .

5 For the preparation of compounds in which the 3 'azido group is in the threo form. a compound of formula (III) in which the group M is an hydroxy group in the ervtro form (in which the 5'-hydroxy group is preferably protected by, for example, a trityl group) may be treated, for example, with triphenylphosphine, carbon tetrabromide and lithium azide. Alternatively, M may represent a leaving group in the ervtro form which can be converted to an azido group in the threo form, for example by treatment with lithium or sodium azide, the 5'-hydroxy group being protected in the same manner as described above. The 5'-trityl protecting group can then be removed, for example, by mild acid or zinc bromide treatment.

For the preparation of compounds in which the 3'-azido group is in ervtro form. a compound of formula (III) wherein the group M is a halogen (e.g. chlorine) group in the threo form (wherein the 5'-hydroxy is preferably protected by, for example, a trityl group) may be treated with, for example, lithium or sodium azide. The 3'-threo-halogen (e.g. chlorine) starting material is obtained, for example, by reacting the corresponding 3'-ervertrohydroxy compound with, for example, triphenylphosphine and carbon tetrachloride, or alternatively by treatment with an organosulfonyl halide (e.g. trifluoromethanesulfonyl chloride) to give the corresponding 3 ' Alternatively, the 3'-threo-hydroxy- or organosulfonyloxy compound of formula (III) may be treated with, for example, triphenylphosphine, carbon tetrabromide and lithium-3-zid to give the corresponding 3'-threo-hydroxysulfonyloxy compound, which is then halogenated as described above. ervtro-atsidovh- 10 90664 dist.

The compounds of formula (I) B may be converted into their pharmaceutically acceptable salts in a conventional manner, for example by treatment with a suitable base.

Retroviral infections often affect the patient's central nervous system, and in this context the compounds of the invention have the particular advantage that, as experiments have shown, they are able to cross the blood-brain barrier in clinically effective amounts.

The compounds of the invention have been found to be particularly effective against retroviral infections and infections caused by gram-negative bacteria.

As used herein, the term "infections caused by retroviruses" refers to any virus that uses a reverse transcriptase enzyme during a substantial portion of its organ-15 circulation.

The bacteria against which the compounds have been found to be particularly effective are listed below.

Escherichia coli, Salmonella Dublin, Salmonella typhosa, Salmonella typhimurium, Shigella flexneri, Citrobacter freundii, Klebsiella pneumoniae, Vibrio Chloe-rae. Vibrio anquillarum, Enterobacter aerogenes, Pas-teurella multicoda, Haemophilus influenzae, Yersinia en-terocolitica, Pasteurella haemolytica, Proteus mirabilis 25 and Proteus vulgaris. gut inflammation, porcine post-weaning intestinal inflammation and chicken coliform septicemia.

The compounds of the invention are particularly effective against the following viruses: human T-cell lymphotropic viruses (HTLV), especially HTLV-I, HTLV-II and HTLV-III (HIV); feline leukemia virus, equine infectious anemia virus, goat arthritis virus and other lentiviruses, as well as other human viruses such as hepatitis B virus, Epstein-Barr virus (EBV) and multiple sclerosis (MS). The compounds of the invention have also been found to be effective in the treatment of Kapo-5 sin sarcoma (KS) and thrombocytopenia purpura (TP).

The effect of the compounds of the invention against so many bacterial and viral infections is undoubtedly of great medical benefit, and the new mode of action allows these compounds to be used in combination therapy, thereby reducing the potential for the development of resistance. However, these combinations are particularly useful because 3'-azidonucleosides have amazing potency when used in conjunction with other therapeutic agents, as described below.

It has been found that 3'-azidonucleosides act synergistically with many other therapeutic agents, thus disproportionately enhancing the therapeutic effect of both agents. Significantly less of each compound is required for treatment, the therapeutic ratio increases and thus the risk of toxicity for both compounds decreases.

Thus, the compounds of the invention are well suited for use in combination therapy with at least one other therapeutic agent.

In particular, the compounds according to the invention are suitable for use as described above in combination therapy in which the two active substances are present in a strengthening ratio.

A "potency ratio" is a ratio of a compound of the invention, or a pharmaceutically acceptable derivative thereof, to another therapeutic agent that provides a therapeutic effect greater than the sum of the therapeutic effects of the individual components.

12 90664

It should be noted that although there is generally an optimum ratio to achieve maximum enhancement, even a negligible amount of the second agent is sufficient to somewhat potentiate the effect of the other, and thus any relationship between the two enhancing drugs still has the desired synergistic effect. However, the greatest synergy is observed when the two substances are present in a ratio ranging from 500: 1 to 1: 500, preferably from 100: 1 to 1: 100, especially from 20: 1 to 10 1:20 and especially from 10: 1 to 1:10.

The combinations described above are also referred to herein as the combinations of the invention.

To achieve the desired therapeutic effect, the combinations of the invention may well be administered together, for example in a single pharmaceutical composition, or separately, for example as combinations of tablets and injections administered simultaneously or at different times.

In antiviral experiments, for example, it has been found that azidonucleosides are potentiated by various types of agents such as interferons, nucleoside transport inhibitors, glucuronidation inhibitors, renal secretion inhibitors, and even other therapeutic nucleosides of the invention which do not necessarily have the same effects.

Particularly preferred are interferons of the α, β and type, while nucleoside transport inhibitors include agents such as dilazep, dipyridamole, 6 - [(4-nitrobenzoyl) thio] -9- (β-D-ribofuranosyl) pu-30 rhin, papaverine, myoflazine, hexobendine, lidoblazine and their acid addition salts.

Probenecid is particularly useful in combination with the 3'-azidonucleosides of the invention because it inhibits both renal secretion and glucuronidation. Examples of other compounds useful in this regard include acetaminophen, aspirin, lorazepam, cimetidine, ranitidine, zomepirac, clofibrate, indomethacin, ketoprofen, naproxen, and other compounds that tend to be glucuronidated or otherwise glucuronidated.

The effect of the 3'-azidonucleosides of the invention and their pharmaceutically acceptable derivatives is enhanced by other therapeutic nucleoside derivatives as described above, including, for example, 10 GB Patent 1,523,865, U.S. Patent No. 4,360,522, EP 74,306,555 and 55,239. and EP patent applications 434 393 and 434 395 for image-type acyclic nucleosides, and in particular for the general formula z ioT \ '»- 20 / \ N /

H2N N I

CKX CHCH OH

: Wherein Z represents a hydrogen atom or a hydroxy or amino group; X represents (a) an oxygen or sulfur atom or a methylene group and Y represents a hydrogen atom or a hydroxymethylene group; or (b) methyleneoxy (-OCH 2) and Y represents a hydroxy group; and pharmaceutically acceptable derivatives thereof.

Examples of the above derivatives are salts and esters and their base salts, for example alkali metal (for example sodium) or alkaline earth metal salts and pharmaceutically acceptable salts of organic acids such as lactic, acetic, malic or p-toluenesulphonic acid. , as well as pharmaceutically acceptable salts of inorganic acids such as hydrochloric acid and sulfuric acid.

Esters of compounds of formula (A) which may be well used in accordance with this invention include formyloxy or C 1-6 (e.g. C 1-6) alkanoyloxy- (e.g. acetoxy or propionyloxy) optionally substituted aralkanoyloxy- (e.g. for example compounds containing a phenyl-CM-α1-10 kanoyloxy (such as phenylacetoxy) or optionally substituted aroyloxy (e.g. benzoyloxy or naphthyloxy) ester group at one or both endpoints of the 9-side chain of compounds of formula (A). The above-mentioned Aral-15 kanoyloxy and aroyloxy ester groups may be substituted, for example, by one or more halogen atoms (for example chlorine or bromine atoms) or aminonitrile or sulfamido groups, the aryl part of the group preferably containing 6-10 carbon atoms.

Particularly preferred compounds of formula (A) above for use herein are 9- [2-hydroxy-1-hydroxymethylethoxy) methyl] guanine, 2-amino-9- (2-hydroxyethoxymethyl) purine and especially 9- (2-hydroxyethoxymethyl) purine. -hydroxyethoxymethyl) guanine (acyclovir). After. The latter compound has been found to have a particularly good potentiating effect, especially when the combination contains 3'-azido-3'-deoxythymidine, as described in the examples.

With regard to antibacterial agents, it has been found that many antibiotics also potentiate the action of azidonucleosides. These substances include, but are not limited to, benzylpyrimidines, e.g., 2,4-diamino-5- (3 ', 4', 5'-trimethoxybenzyl) pyrimidine (trimethoprim), and derivatives thereof, as described, for example, in GB-pa-35 1,405,246; sulfonamides, for example is 90,664 sulfadimidine; rifampicin; tobramycin; fusidiinihap acid; chloramphenicol; clindamycin and erythromycin.

Other suitable uses for the uses described herein include combinations in which the second agent is, for example, interleukin-5-nin II, suramine, phosphonoformate, HPA 23, 2 ', 3'-dideoxynucleoside, for example 2', 3'-dideoxycytidine and 2 ', 3' -dideoxyadenosine, or a drug such as leveva misol or thymosin, which is used to increase the number of lymphocytes and / or to increase function in an appropriate manner.

It should further be noted that the compounds and combinations of the invention may also be used in conjunction with other therapies for the regulation of immune function, including bone marrow and lymphocyte transplants.

15 Some of the retroviral infections described above, such as immunodeficiency, are usually associated with opportunistic infections. Thus, it should be noted that, for example, the combination of 3'-azido-3'-deoxythymidine (AZT) and acyclovir would prove particularly useful in the treatment of an immunodeficient patient with opportunistic herpes infection, whereas AZT and 9 - [(2-hydroxy) The combination of -1-hydroxymethylethoxy) methyl] guanine would be useful in the treatment of an immunodeficient patient with an opportunistic cytomegalovirus infection.

.25 The compounds of the invention and their pharmaceutically acceptable salts, also referred to herein as the active ingredient, may be administered by any suitable route orally, anal, nasally, topically (including oral and sublingual), vaginally and parenteral administration (including subcutaneous, intramuscular, intravenous and intradermal administration). It will be appreciated that the preferred route of administration will vary with the condition and age of the recipient, the nature of the infection and the active ingredient selected.

90664 BC

In general, a suitable dose is between 3.0 and 120 mg per kilogram of body weight of the recipient and per day, preferably between 6 and 90 mg per kilogram of body weight per day, and most preferably between 15 and 60 mg per kilogram of body weight per day. The desired dose is preferably divided into two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage form, containing, for example, 10-1500 mg, preferably 20-1000 mg, and most preferably 50-700 mg of active ingredient per unit dose.

Experiments with 3'-azido-3'-deoxythymidine suggest that the dose should be administered so as to achieve a peak of about 1 to about 75 μΜ, preferably about 2 to 50 μΜ, most preferably about 3 to about 30 μΜ of active ingredient. plasma concentration. This can be accomplished by intravenous injection of a solution of 0.1 to 5% of the active ingredient, optionally in saline, or an oral pill of about 1 to about 100 mg of ak-20 active ingredient / kg. Desired blood concentration levels can be maintained by continuous infusion at a rate of about 0.01 to about 5.0 mg / kg per hour or by intermittent infusions of about 0.4 to about 15 mg of active ingredient / kg.

The combinations of the invention may be administered in a manner similar to that described above to achieve the desired therapeutic dosages of the active ingredient and the adjuvant therapeutic agent. The dosage of the combination will depend on the condition being treated, the active ingredient used and the additional therapeutic agent used, and other clinical factors such as the condition of the patient and the route of administration of the combination. As mentioned above, the active ingredient and the adjuvant therapeutic agent may be administered simultaneously (e.g., in a single pharmaceutical composition) or separately (e.g., 17,90664 in separate pharmaceutical compositions) and the combinations may generally be administered topically, orally, rectally, or parenterally (e.g., intravenously, subcutaneously or intramuscularly).

In particular, the combinations of the invention in which the second agent is an inhibitor of nucleoside transport may be administered to a patient in the usual manner. However, for oral administration, a dosage of 1-200 mg / kg / day, preferably 5-50 mg / kg / day, of the active ingredient is generally sufficient. For parenteral administration, a dosage of 1-100 mg / kg / day, preferably 2-30 mg / kg / day, of the active ingredient is generally sufficient. The amount of renal secretion / glucuronidation inhibitor in the combination is independent of the amount of active ingredient and a sufficient dose is in the range of 4 to 100 mg / kg / day, preferably 5 to 60 mg / kg / day, most preferably 10 to 40 mg / kg / day.

In the combinations according to the invention in which the second substance is an interferon, the active substance is preferably 5 to 250 mg / kg / day; and a suitable effective interferon dose is 3 x 10 6 to 10 x 10 6 IU per square meter of body surface area per day, preferably 4 x 10 6 to 6 x 10 6 IU / m 2 / day. The active substance and interferon should be administered in a ratio ranging from about 5 mg active substance / kg / 3 x 10 6 IU interferon / m to about 250 mg active substance / -25 kg / day / 10 x 10 6 IU interferon / m / day, preferably about 5 mg of active substance / kg / day / 4 x 10 6 IU interferon / m 2 / day - about 100 mg of active substance kg / day / 6 x 10 6 IU interferon / m 2 / day.

The interferon is preferably administered by injection (for example, subcutaneously, intramuscularly or intravenously), while the active ingredient is preferably administered orally or by injection, but may be administered by any of the methods described herein. The interferon and the active ingredient may be administered together or separately, and the daily dose of each may preferably be administered in divided doses of 90664.

For combinations of the invention in which the second agent is another therapeutic nucleoside, a suitable effective dose of orally administered active ingredient 5 is 2.5 to 50 mg per kilogram of body weight per day, preferably 5 to 10 mg / kg / day; and a suitable effective dose of the second oral therapeutic nucleoside is 5 to 100 mg per kilogram of body weight per day, preferably 15 to 75 mg / kg / day. It is preferred that for oral administration, the ratio of active ingredient to other therapeutic nucleoside is from about 1: 1 to 1:10, more preferably from about 1: 2 to 1: 8.

A suitable effective dose of intravenously administered active ingredient is generally about 1.5 to 15 mg / kg / -15 days, and a suitable effective dose of intravenously administered therapeutic nucleoside is generally about 5 to 30 mg / kg / day. It is preferred that for intravenous administration the ratio between the active ingredient and the second therapeutic nucleoside is between about 2: 1 and 1:20, more preferably between about 1: 2 and 1:10.

Both components of the combination are preferably administered orally or by injection and may be administered together or separately. The daily dose of each can be administered in divided doses.

For compositions according to the invention in which the second agent is an antibacterial agent, a suitable active ingredient dose is 2.5 to 50 mg / kg / day, preferably 5 to 10 mg / kg / day, and a suitable effective antibacterial dose is is 2-1000 mg / kg to 30 / day, preferably 50 to 500 mg / kg / day. The ratio between the active ingredient and the antibacterial agent is preferably between 20: 1 and 1: 500, in particular between 2: 1 and 1: 125.

It should be noted that although combinations containing three or more therapeutic agents are not specifically described herein, such combinations may also be prepared from the agents of the invention. For example, the combination of 3'-azido-3'-deoxythymidine (AZT), acyclovir and probenecid has the dual advantage of both enhancing the effect of AZT and improving its availability. Likewise, combinations of the compounds of the invention and two or more other therapeutic agents of the same type are possible, such as AZT in combination with sulfadimidine and trimethoprim.

Although it is possible to administer the active ingredient Yk-10 per se, it is preferred to use it as a pharmaceutical formulation. The formulations of this invention include at least one active ingredient as defined above in association with one or more carriers and, optionally, other therapeutic agents. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof, including oral, anal, nasal, topical (including oral and sublingual), vaginal or Formulations may well be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy, such methods comprising the step of bringing into association the active ingredient with the carrier which constitutes one or more carriers which represent one or more of the excipients in the form of a parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. Formulations are generally prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as an aqueous solution or suspension or as a solution or suspension in a liquid other than water; or as an oil-in-water liquid emulsion or a water-in-oil liquid-5 emulsion. The active ingredient may also be in pill, sieve or paste form.

A tablet may be made by compression or molding, optionally in combination with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine in a free-flowing form such as a powder or granules of an active ingredient optionally mixed with a binder (for example povidone, gelatin, hydroxypropylmethylcellulose), lubricant, inert diluent, preservative, preservative. (for example sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethylcellulose) surfactant or dispersant. Molded tablets may be made by molding in a suitable machine, a powdered mixture of the compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein by employing, for example, hydroxypropylmethylcellulose in various proportions to give the desired release profile.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in admixture with a flavored base, usually sucrose and acacia or tragacanth; pastilles in which the active ingredient is mixed with an inert base such as gelatin and glycerol or sucrose and acacia; and mouthwashes in which the active ingredient is mixed with a suitable liquid carrier.

2i 90 6 64

Formulations for rectal administration may be presented as a suppository with a suitable base such as cocoa butter or salicylate.

Formulations suitable for vaginal administration include isotonic sterile injectable solutions in water and non-water, which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and sterile suspensions in water and non-water, which may contain suspending agents and thickening agents. The formulations may be presented in sealed unit-dose or multi-dose containers, for example ampoules and vials, and may be stored in a lyophilized state, requiring only the addition of a sterile liquid carrier, for example water for injections, before use. Unprepared injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, or an appropriate fraction thereof, of the active ingredient.

The compounds of the invention may also be in the form of formulations for veterinary use, which may be prepared, for example, by conventional methods in the art. Examples of such veterinary drug formulations are suitable for the following routes of administration: (a) oral administration, for example compulsive drugs (for example solutions and suspensions in water or non-water); tablets or pills; powders, granules or pellets to be mixed with feed; pastes applied to the tongue; 22 90 664 (b) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection of a sterile solution or suspension; or (if appropriate) by intramammary injection, the suspension or solution being delivered to the udder via the nipple; (c) topical administration, for example as a cream, ointment or spray applied to the skin; or (d) a method of intravaginal administration, for example, as a pessary, ointment or foam.

It should be noted that single or separate formulations as described above are also suitable for the combinations of the invention and may be prepared in the same manner.

It should be noted that in addition to the substances specifically mentioned above, the formulations of this invention may contain other substances conventional in the art depending on the type of formulation, for example, formulations suitable for oral administration may contain additives such as sweeteners, thickeners and spices.

The biological activity of some 3'-azodinucleosides and combinations containing them is described below.

31-Azido-3'-deoxythymidine (AZT) was administered orally to two immunodeficient patients at doses of 2 mg / kg every 8 hours on days 1 and 2 of treatment. On days 2 and 3, patients were also given 500 mg probenecid (PB) every 6 hours and a single dose of AZT was given on day 3. The peak (C,) and basal (C.) Values of AZT on day 3 • 30 (after probenecid administration) were significantly higher than the corresponding levels on day 1, resulting in a 3-fold decrease in total body clearance (Cl / F) and AZT increase in mean mean half-life (tl / 2) (from 0.88 to 1.73 hours) during PB treatment. The mean AZT-glucuronide / AZT ratio decreased significantly from 7.3 to 2.4 after PB treatment. The values of the main pharmacokinetic parameters of AZT before and after co-administration of PB are summarized in Table 1.

23 90 664 from 7.3 to 2.4 after PB treatment. The values of the main pharmacokinetic parameters of AZT before and after co-administration of PB are summarized in Table 1.

Table 1 5

Patient AUC CmaxCmin T max Cltot / F tl / 2 No. (h * pm) (pm) (pm) Ch ') (ml / min / 70kg) (h) 1. AZT / PB 10.41 6.13 0.15 0.25 829.50 1.84 2. AZT / PB 10.00 6.46 0.27 0.50 92 ^ 00 1.61 ^ Average 10.21 6 ^ 30 0.21 0.38 875 .25 1.73 t SP 0.29 0; 23 0.08 0.18 64.70 0.16 1. OCT 3.70 3.74 0.00 0.50 2333.80 0.87 2. OCT 3 .04 2.51 0.00 0.31 3027.00 0.89 15 Average 3.37 3.13 0.00 0.41 2680.40 0.88 ± SP 0.47 0.87 0.00 0, 13 480.17 0.01

Antiviral activity Enhancement of the anti-leukemia virus (FLV) activity of 31-azido-31-deoxythymidine (AZT) by nucleoside transport inhibitors dipyridamole, dilazep and 6-ε- (4-nitrobenzyl) thi-7- ribofuranosyl-1β) purine is shown in Table 2. FG-10 cells were transferred to the plate the day before infection with FLV.

One hour after infection, test compounds or combinations were added to a certain concentration. The plate was incubated for three days, the medium was changed to fresh McCoy's 5A medium and incubated for another three days. Concentrations of test compounds / combinations that achieved 50% colony inhibition were determined as shown in Table 2. Dipyridamole (10 μM) and di-dilazep (5 μM) alone had no detectable antiviral activity alone.

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Table 2

Compound / Combination Azidothymidine ED. (NM) A2T 5 50 5 AZT + 1 pM dipyridamole 1 A2T + 5 μM dipyridamole A2T + 10 μM dipyridamole q 2 A2T + 5 μΜ dilazep g ^ 5 A2T + 5 μM 6 - [(^ - nitrobenzyl) thio [-10 9 - & - D - ribofuranosyl) purine 3 Anti-ITP activity of 31 31-azido-3'-deoxythymidine (AZT)

A patient with a platelet count of 33 38,000 nm was diagnosed with thrombocytopenia 15 purpura (platelet count <100,000 mm) and was treated with 5 mg / kg AZT intravenously every 6 hours for 6 weeks, during which time his platelet counts increased to 140,000, e.g. The treatment was then changed to 5 mg / kg / 4 hours orally for 4 weeks, discontinued for 20 to 4 weeks, at which time the platelet count was found to decrease to 93,000 mm after two weeks and to 70,000 mm after four weeks. Treatment was restarted at 5 mg / kg / 4 hours orally for five weeks and the platelet count increased to 194,000 mm 25 Reducing the dose to 2.5 mg / kg / 4 hours orally resulted in a small decrease in platelet count, but did not to the extent that it would have resulted in ITP diangoosis.

Treatment of Kaposi's sarcoma with 31-azido-31-deoxythymidine (AZT) • 30 In this study, 9 patients with diagnosed Kaposi's sarcoma (KS) were treated with AZT and the following effects were observed.

One patient recovered completely.

Injuries were reduced in three patients.

-35 KS in two patients remained stable.

The injuries of three patients worsened.

25 90 664

The response, which was -50%, is comparable to the results obtained with the currently preferred treatment for KS, recombinant α-interferon.

In vitro anti-HIV activity of AZT / acyclovir combinations Using a method similar to Example 61, the anti-HIV activity of AZT / acyclovir combinations was tested in vitro.

ACV alone had little effect, the highest tested concentration of 16 μg / ml protected less than 30%, while AZT achieved 100% protection at 8 μM by this method.

Table 3 shows the drug combinations that achieve 100% protection.

15 Table 3 ACV (μg / ml) AZT (μM) 0 8 0.5 20 2 1 1 4 0 8 These results show that ACV potentiates the antiviral effect of AZT by approximately 3-fold.

25 In vitro anti-HIV activity of AZT / interferon combinations

Peripheral mononuclear blood cells (PBMCs) from healthy HIV seronegative volunteer donors were obtained by Ficoll-.30 Hypaque sedimentation of heparinized blood. Cells were treated with 10 ug / ml phytohemagglutinin (PHA) and grown in RPMI 1640 medium supplemented with 20% fetal calf serum (FCS), antibiotics, 1-glutamine and 10% interleukin- 2 (IL-2) (Electronucleonics, Bethesda, MD). Four to five days after PHA challenge, 26 9 0664 5 3 cells were split at a concentration of 4 x 10 cells / ml into 25 cm flasks containing 5 ml of medium and the viruses were then allowed to act as described below. The date of virus inoculation is day 0. On the fourth day, 5 fresh media were added. Every 3 or 4 days thereafter, a portion of the cell suspension was removed for analysis and replaced with cell-free medium. Experiments 2 and 4 ended after 14 days and Experiments 1 and 3 after 16 days.

The virus stock was supernatant of cell-free HIV-infected H9 cells-10 frozen in batches at -70 ° C. The 50% infectious virus stock dose (TCID ^ q) of the tissue culture was 10 ^ / ml.

Four separate experiments were performed using PBMCs from different donors (Table 4). In Experiment 1, 15 molar drugs were added after cells were exposed to the virus. 40x10 batches of cells were suspended in 20 ml of 5 media containing 10 doses of TCID® virus for one hour, washed three times and resuspended in virus-free medium. In Experiments 2-4, cells in-20 were incubated for 24 hours in medium with or without recombinant interferon (rIFNoJA), and then allowed to be exposed to AZT and viruses. The virus was added directly to the cultures in a small culture volume and was not washed away. Virus transfers were 4x10, 10, and 25x10 doses of TCID1-g in Experiments 2, 3, and 4, respectively. Drug concentrations were adjusted with each medium change so that the original concentrations were maintained.

In all experiments, two-fold serial dilutions of a given combination of rIFNo (A and AZT) were examined. Each concentration of rIFNoA and AZT used in the combination was also examined alone to obtain control points.

In all experiments, duplicate cultures were maintained for each concentration and for the infected and uninfected control group. In Experiment 1, a combination of 35 3.2 μM AZT and 128 units of rIFNolA / ml (U / ml) was tested, as well as a 5-fold dilution of this combination.

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In Experiments 2 and 3, a combination of 0.16 μM AZT and 128 U / ml rIFN 2 A and 3-4 double dilutions of this combination were used. In Experiment 4, a combination of 0.08 μM AZT and 128 U / ml rIFNO was used, and 2 two-fold dilutions of this compound.

After about a week, the cultures were checked for viruses every 3 to 4 days. Cellular HIV antigens were determined by indirect immunofluorescence; the supernatant fluids were assayed for reverse transcriptase activity (RT activity), viral yield, HIV-p24 antigen by radioimmunoassay.

Chou and Talalay's multidrug effect analysis method (Advances in Enzyme Regulation, (1984), 22, 27-55) was used to evaluate the effects of drug combinations.

The results were also evaluated using the isobologram method, a geometric assay for drug interactions. The concentration of AZT that produces the desired (e.g., 50% inhibitory) effect is plotted on the horizontal axis and the concentration of rIFNoiA that produces the same effect is plotted on the vertical axis. Draw a line joining these points and mark the point corresponding to the combination of concentrations producing the same effect. If this point is below the line, the combination is considered sy-25 nergistic.

In Experiment 1, all AZT concentrations were completely inhibitory, so it was impossible to evaluate the interactions. In Experiments 2-4, a synergistic interaction of the agents was consistently observed (Tables 4-9). Synergy was evident in all viral replication assays used and was maintained even when the single effect of rIFNo (A was negligible. 35 virus yield results and RIA results from Experiment 4. The isobologram method also showed synergy.

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Table 4 HIV

inoculated HIV Timing of drug addition 5 Experiment method3 (TCID.-n) rIFNdA AZT

1 A 1CT 0 0 2 B 4x10 ^ -24 hours 0 3 B 10 ^ -24 hours 0 4 B 2x10 ^ -24 hours 0 10 (a) Method A: 4x10 cells were suspended in 20 ml of medium containing 103 TCID ^ dose of HIV, for one hour to 37 ° C, then the cells were washed and resuspended.

Method B: Said amount of viruses was added per 2x10 cells in the medium; the cells were not subsequently washed.

Table 5 Effects of 20 rIFN «<A and AZT on average reverse transcriptase activity assay results (cpm / 10 ^ cells x 103).

. . rlFNcxA (U / ml) "25 AZT (, μm) 0 8 16 32 64 128 0 205 173 150 193 169 151 0.01 159 85 ·· ': 0.02 110 42 0.04 71 10 '30 0.08 31 4 0.16 7 - | 29 9 O 66 4

Table 6 Experiment 3 - Day 13 Effects of rIFN (* A and AZT on mean RT values 5 (cpm / 106 cells x 10 ^).

rlFNoiA (U / ml) AZT (, μM) 0 16 32 6j4 U8 0 157 143 117 117 130 10 0.02 51 10 0.04 10 0 0.08 2 0 0.16 5 0 15 Table 7

Experiment 4 - day Ί 1 Effects of rIFN «A and AZT on mean RT values (cpm / 10 ^ cells x 10 ^) ^ rlFNaA (U / ml) AZT ^ M) 0 32 64 128 0 32 5 4 2 0.02 8 1 2 5 ° 704 4 0 ° r08 1 0 3ϋ 90664

Table 8 Experiment 3 - day 13 Effects of rlFNctfArn and AZT on virus yield (TCID ^ -g / ml) 5 rlFNaA (U / ml) ΑΖΤ (μΜ) 0 16 32 64 128 0 105'7 105? 6 105'3 ΙΟ5 / 1 105, ° 10, 02dfl ^ _μΐίώ! _ *, 04,10.3 .dO1'9, 08.10 W> L <l £ l! -►, 9

Table 9 15 Experiment 4 - Day 14 Effects of rIFNo * A and AZT on HIV-p24.

IFNαA (U / mi) ΑΖΤ (μΜ) 0 32 64 128 20 0 200-300 100-200 50-100 25-30 0; 02 100-200 2.2 0.04 25-30 1 ^ 0 0.08 11 , 0.28 25 aHIV-p24 concentrations are expressed as ng protein / ml 3i 90664

Table 10 Combined indices of AZT and rIFNoiA calculated from RT results

Recombinant indices corresponding to different degrees of RT inhibition 5

Cultivation-

Experiment day 50% 90% 95% 2 7 2.14 0.34 0.23 10 2 10 0.37 0.30 0.28 <3 6 0.26 0.73 1, .39 3 13 0.12 0 .15 0.17 3 16 <0.01 0.02 0.05 4 8 0.01 0.03 0.04 15 4 14 0.02 0.07 0.12

The values of the composite indices were determined by solving the equation corresponding to different degrees of RT inhibition. Values of composite indices <1 suggest synergy. The values of the composite-20 indices shown were obtained using a mutually non-exclusive form of the equation; the values obtained using the mutually exclusive form were always slightly lower.

In Vitro Antibacterial Synergy Studies 31-Azido-3'-deoxythymidine and 8 known 25 antibacterial agents (listed in Table 11) were each treated with N, N-dimethylformamide for 30 minutes. Microtiter dilutions were prepared using Wellcotest broth.

Prior to synergy testing, the MIC of each compound was determined separately against the test organism (E. coli CN314). Table 11 shows the MIC endpoints for each drug for E. coli CN314.

Two-fold serial dilutions of test drugs or 3'-azido-3'-deoxythymidine were prepared in 35 "flat bottom" or "transfer" microtiter plates, respectively.

32 90664

Plates containing appropriate dilutions were pooled to give a series of 192 dilutions. The highest concentration of any drug used was twice its MIC (Table 11). The test plates were inoculated with

E

5 rows of culture at about 5x10 CFU / ml were then incubated at 27 ° C for 18 hours. Wells were labeled according to whether bacteria grew in them or not and MIC values were determined. "Partial inhibition concentrations" (FIC values) were calculated from the MIC values by dividing the MIC value of the combination by the MIC value of each individual substance. The sum of the fractions (sum of partial inhibition concentrations) was then calculated. If the result is about 0.5 or less, it indicates synergy.

Table 11 15 Minimum Inhibitory Concentrations (MICs) of Test Drugs Used Alone

Compound MIC (μg / ml)

Tobramycin 0.4 Fusidic acid 1000

Chloramphenicol 3.1

Clindamycin 100

Erythromycin 25

Rifampicin 6.2 -25 3'-azido-3'-deoxythymidine 1.0

Trimethoprim 0.125

Sulfadimidine 32

The results of the synergy experiments are shown in Table 12.

30 33 90664

Table 12

Synergy studies: combinations of AZT and other antibacterial agents

Optimi-MIC values (yug / ml):

Combination Drug / AZT FIC Index

Tobramycin / AZT 0.2 / 0.125 0.25

Fusidic acid / AZT 250 / 0.03 0.28

Chloramphenicol / AZT 1.6 / 0.06 0.31

Clindamycin / AZT 12.5 / 0.25 0.375

Erythromycin / AZT 6.2 / 0.25 0.5

Rifampicin / AZT 3.1 / 0.06 0.56 15 Trimethoprim / AZT 0.004 / 0.5 0.504

Sulf adimidine / AZT 0.25 / 0.125 0.375 In vitro anti-HIV activity of 3'-azidonucleosides The in vitro activity of 3-azidonucleosides (drugs) was determined in two cell lines; H9 (OKT4 + T cell line, permeable to HIV proliferation but partially resistant to the cytopathic effect of HIV) and TM3 (T cell clone, specific for tetanus toxoid, immortalized with lethal irradiated HTLV-I, and selected for rapid growth and because it is sensitive to the cytopathic effect of HIV).

The inhibition assay was performed as follows: T3 cells were stimulated with antigen-plus, irradiated (4000 rad; -30 40 Gy) fresh autologous peripheral mononuclear blood cells (PBM) and cultured in complete medium with 15% (v / v) interleukin-2 (IL-2, lectin-free, Cellular Products, Buffalo, NY) six days before assay. ATH8 cells were used without antigen antigen-.35 simulation. After pre-exposure to polybrene (2 μg / ml, 30 min), the target T cells were pelleted, exposed to HIV for 45 minutes, resuspended in 2 ml of fresh medium, and incubated in culture tubes. At 5 ° C in air containing 5% CO 2. Control cells were treated similarly but were not exposed to virus. IL-2 and the drug were allowed to continuously act on the cells. When ATH8 cells were used in this assay, five virus particles per cell was the lowest cytopathic virus dose. In cell co-culture experiments, 5x10 4 lethal irradiated (10,000 rad) HIV-RF-II-producing H9 cells or uninfected H9 cells were added to 2x10 3 target T cells. The total number of cells living at different time points was counted on a hemocytometer under a microscope using the tryptan blue exclusion method.

The results are shown in Table 13.

15 Table 13

Compound ED 2 Q (μM) 31-azido-2 ', 3'-dideoxycytidine 3'-azido-5-bromo-21,3'-dideoxyuridine 3'-azido-5-bromo-2' , 3'-dideoxycytidine 5 (E) -3- [1- (3'-azido-2 ', 31-dideoxy- [3-p-erythro-pentofuranosyl) -1,2,3,4-tetrahydro-2, 4-Dioxo-5-pyrimidinyl-2,5H-2-propenoic acid 100 In vitro antibacterial activity of 31-azidonucleosides

Table 14 shows the antibacterial activity of 3'-azidonucleosides in vitro according to the minimum inhibitory concentration (MIC) against different bacterial species.

The standard used was trimethoprim (TMP) and the medium was Wellcotest susceptibility test agar supplemented with 7% lysed horse blood.

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Table 14 MIC (, ug / ml) Standard

Organism Compound__dard 1 2 3 4 3678 E.coli 0; 1 »> 100 10 0,1» 10 0fl »10 10> 100

Salmonella 0,1 »10 10 0,1»> 100 10 10> 100 0,3 typhimunum / '10 Salmonella o, l »0,1» 10 0,1 »0,1» 10 10 0,1 »0, 1 nitrogen; 7 'r'

Entero. 0 ^ 1 »10> 100> 100 100 10 10> 100 0; 5 aerogenes

Citro. f reundii 10 10 10 “f1» 10 10 10> 10 ° °, · 5 15 The compounds used were: 1) 3'-azido-3'-deoxy-4-thiothymidine 2) 3'-azido-3'-deoxy- 5'-O-acetyl-4-thiothymidine 3) 31-azido-3'-deoxy-2-deoxy-2-thiothymidine 4) 5'-acetyl-3'-azido-3-benzoyl-3'-deoxytyridine dine 5) 1- (5'-O-acetyl-31-azido-2 ', 3'-deoxy-N-D-erythro-pentofuranosyl) -5-methyl-4- (1,2,4-triazol-1 -yl) -2 (1H) -pyrimidinone 6) 1- (3 1 -azido-2 1,3'-dideoxy - N - D-erythropentofuranosyl) -2- (benzyloxo) -5- methyl-4- (1H) -pyrimidinone 7) 3'-azido-31-deoxy-2-methoxythymidine 8) 3'-azido-5-bromo-2 ', 31-dideoxyuridine

The following examples are for illustration only; , the invention and do not limit its scope in any way.

The term "active ingredient" as used in the examples means the 3'-azidonucleoside compound described herein or a pharmaceutically acceptable derivative thereof. It should be noted that not all of the compounds described in Examples 23-71 are novel compounds of the invention.

36 90664

Example 1: Tablet Formulations The following formulations A and B are prepared by wet granulating the ingredients with a solution of povidone followed by the addition of magnesium stearate and compression of the tablets.

5 Formulation A

mg / tablet mg / tablet (a) Active ingredient 250 250 (b) Lactose B.P. 210 26 (c) Povidone B.P. 15 9 10 (d) Sodium starch glycollate 20 12 (e) Magnesium stearate 5_ 3_ 500 300

Formulation B

15 mg / tablet mg / tablet (a) Active ingredient 250 250 (b) Lactose 150 (c) Avicel PH 101 60 26 (d) Povidone B.P. 15 9 20 (e) Sodium starch glycollate 20 12 (f) Magnesium stearate 5_ 3 500 300

Formulation C

25 mg / tablet

Active ingredient 100

Lactose 200 Starch 50

Povidone 5 30 Magnesium stearate 4 359

The following formulations, D and E, are prepared by direct compression of the mixture of ingredients. The lactose used in Formulation E is of the direct compression type (Dairy Crest - "Zeparox").

Formulation D

37 90 664 mg / capsule

Active ingredient 250

Pregelatinised starch NF15 150 5 400

Formulation E

mg / capsule

Active ingredient 250

Lactose 150 10 Avicel 100 500

Formulation F (formulation / controlled release) The formulation is prepared by wet granulation of the ingredients (below) with povidone solution, followed by the addition of magnesium stearate and the compression of the tablets.

mg / tablet (a) Active substance 500

(b) Hydroxypropylmethylcellulose (Methocel K4M

20 Premium) 112 (c) Lactose B.P. 53 (d) Povidone B.P.C. 28 (e) Magnesium stearate 7,700 25 Example 2: Capsule formulations

Formulation A

The capsule formulation is prepared by mixing the ingredients of Formulation D of Example 1 above and filling the two-part hard gelatin capsule with the mixture. Formula-30 thio (infra) is prepared in the same manner.

Formulation B

mg / capsule (a) Active ingredient 250 (b) Lactose B.P. 143 35 (c) Sodium starch glycollate 25 (d) Magnesium stearate 2,420

Formulation C

38 90664 mg / capsule (a) Active ingredient 250 (b) Macrogol 4000 BP 350 5 600

Capsules are prepared by melting Macrogol 4000 BP, dispersing the active ingredient in the melt and filling the two-part hard gelatin capsule with the melt. Formulation D

10 mg / capsule

Active ingredient 250

Lecithin 100

Peanut oil 100 450 15 Capsules are prepared by dispersing the active ingredient in lecithin and peanut oil and filling the soft, elastic gelatin capsules with a slurry.

Formulation E (Controlled Release Capsule) The following controlled release capsule formulation is prepared by injection molding ingredients a, b and c using an extruder, after which the extrudate is pelleted and dried. The dried pellets are coated with a controlled release film (d) and the two-part hard gelatin capsule is filled with them.

mg / capsule (a) Active ingredient 250 (b) Microcrystalline cellulose 125 (c) Lactose B.P. 125 30 (d) Ethylcellulose 13,513 • · 39 90,664

Example 3: Injectable Formulation Formulation A

Active ingredient 0.200 g

Hydrochloric acid solution, 0.1 M q.s. to pH 4.0 to 7.0 Sodium hydroxide solution, 0.1 M q.s. to a pH of 4.0 to 7.0

Sterile water q.s. To 10 ml

The active substance is dissolved in most of the water (35-40 ° C) and the pH is adjusted to between 4.0 and 7.0, either with chloro-10 hydrochloric acid or sodium hydroxide. The batch was filled to full volume with water and filtered through a sterile microporous filter into a sterile 10 ml brown flask (type 1) sealed with a sterile stopper and coating.

15 Formulation B

Active ingredient 0.125 g

Sterile, pyrogen-free phosphate buffer, pH 7, q.s. To 25 ml

Example 4: Intramuscular injection 20 Weight (g)

Active ingredient 0.20

Benzyl alcohol 0.10

Glycofurol 75 1.45

Water for injections q.s. To 3.00 ml 25 The active substance is dissolved in glycofurol. Bentyl alcohol is added and dissolved, and water is added to 3 ml. The mixture is filtered through a sterile microporous filter and sealed in a sterile 3 ml brown flask (type 1).

Formulation A

40 90664

Weight (g)

Active ingredient 0.2500

Sorbitol solution 1,5000 5 Glycerol 2,000

Sodium benzoate 0.0050

Flavor, peach 17.42.3169 0.0125 ml

Purified water q.s. To 5,000 ml

The active ingredient is dissolved in a mixture of Gly-10 serol and most of the purified water. An aqueous solution of sodium benzoate is added to the solution, a solution of sorbitol is then added, and finally a flavoring agent. Fill with purified water and mix well.

When the active ingredient is poorly soluble, the following formulation (B) is used.

Formulation B

Weight (g)

Active substance 0.250 20 Sorbitol solution 1,500

Glycerol 0.005

Dispersible cellulose 0.005

Sodium benzoate 0.010 ml

Flavor 25 Purified water to 5,000 ml

Mix the sorbitol solution, glycerol and some of the purified water. Dissolve sodium benzoate in purified water and add the solution to the mixture. Add and disperse the dispersible cellulose and flavor. Add and disperse the active ingredient. Fill with purified water.

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Example 6: Suppository mg / suppository k

Active ingredient (63 μm) 250

Hard grease, BP (Witepsol H15 - Dynamit 5 NoBel) 1770 2020 *

The active substance is used as a powder having at least 90% of the particles having a diameter of 63 μm or less.

One-fifth of Witepsol H15 is melted in a steam-jacketed pan at a maximum temperature of 45 ° C. The active ingredient is sieved through a 200 μm sieve and added to the molten stock with stirring using a silverson with a cutting blade until a uniform dispersion is obtained. Keeping the mixture at 45 ° C, the remainder of the Witepsol H15 is added to the suspension and mixed to ensure homogeneity of the mixture. The entire suspension is passed through a 250 stainless steel sieve and allowed to cool to 40 ° C with constant stirring. At a temperature of 38 ° C to 40 ° C, 2.02 g of the mixture is placed in a suitable 2 ml plastic mold. The suppositories are allowed to cool to room temperature.

Example 7: Pessaries mg / pessary 25 Active substance (63 μm) 250

Anhydrous dextrose 380

Potato starch 363

Magnesium stearate 7 1000 30 The above ingredients are mixed directly and the pessaries are prepared by pressing the resulting mixture immediately.

The following Examples 8-10 illustrate the preparation of a compound of formula (I) A (active ingredient) and another formulation containing a nucleoside mentioned in each connection.

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Example 8: Injection Formulation A

Weight (mg)

Acyclovir 400 5 Active substance 200 1-M NaOH required amount

Sterile water to 10 ml

Formulation B

Weight (mg) 10 2-amino-9- (2-hydroxyethoxymethyl) purine 400

Active ingredient 200 1 M NaOH required amount

Sterile water to 10 ml In the above formulations, the therapeutic nucleoside is added to 1 M NaOH solution and stirred until the substance dissolves. The active ingredient is added and dissolved. Add sterile water to 10 ml. Filter through a sterile filter and place in sterile bottles. Freeze-dried.

Example 9; tablets

Formulation A

Weight (mg)

Acyclovir 500

Active substance 125 25 Povidone 14

Sodium starch glycollate 25

Magnesium stearate 6 670

Formulation B

30 Weight (mg)

Active substance 125 2-amino-9- (2-hydroxyethoxymethyl) purine 125

Povidone 8 35 Sodium starch glycollate 12

Magnesium stearate 3 273 43 9 O 6 6 4

In formulations A and B above, the therapeutic nucleoside and active ingredient are mixed with sodium starch glucolate and the mixture is granulated with povidone solution. After drying, the granules are mixed with 5 magnesium stearate and the mixture is compressed.

Example 10; Capsules Formulation A

Weight / mg

Acyclovir 250 10 Active substance 62.5

Lactose 170.5

Sodium starch glycollate 15

Magnesium stearate 2,500 15 Mix the ingredients and fill the hard gelatin capsules with the mixture.

Formulation B

Weight / mg

Active substance 125 2 2-Amino-9- (2-hydroxyethoxymethyl) purine 125

Lactose 133

Sodium starch glycollate 15

Magnesium stearate 2,400 25 Mix the ingredients and fill the hard gelatin capsules with the mixture.

Example 11 describes a formulation containing interferon and a compound of formula (I) A (active ingredient).

Example 11; Injection

Interferon 3 mega units

Active ingredient 200 mg

Sterile buffer, pH 7 to 50 ml 44 90 664

Dissolve the interferon and active substance in sterile water. Filter through a sterile filter and place in sterile bottles.

The following Examples 12-15 illustrate the preparation of formulations containing a compound of formula (I) A (active ingredient), for example 3'-azido-3'-deoxythymidine, and a nucleoside transport inhibitor, for example dipyridamole.

Example 12: Tablet 10 Weight (mg)

Nucleoside transport inhibitor 300

Active ingredient 200

Lactose 105 Starch 50 15 Polyvinylpyrrolidone 20

Magnesium stearate 10

Total weight 685

The active compounds are mixed with lactose and starch and wet granulated with a solution of polyvinylpyrrole-20. The granules are dried, screened and mixed with magnesium stearate and then compressed into tablets.

Example 13; Capsule

Weight (mg) 25 Nucleoside transport inhibitor 300

Active ingredient 100

Lactose 100

Sodium starch glycollate 10

Polyvinylpyrrolidone 10 30 Magnesium stearate 3

Total weight 523

The active ingredient is mixed with lactose and sodium starch glycollate and wet granulated with a polyvinylpyrrolidone solution. The granules are dried, screened and mixed with magnesium stearate and filled into hard gelatin capsules.

45

Example 14: Cream

Weight (mg)

Nucleoside transport inhibitor 7.5

Active ingredient 5.00 5 Glycerol 2.00

Cetostearyl alcohol 6.75

Sodium lauryl sulphate 0.75

White soft paraffin 12.50

Liquid paraffin 5.00 10 Chlorocresol 0.10

Purified water to 100.00 ml

The active compounds are dissolved in a mixture of purified water and glycerol and heated to 70 ° C. The other ingredients are heated together at 70 ° C. The portions are combined and emulsified. The mixture is cooled and placed in containers. Example 15: Intravenous injections Amount (mg) (1) Active ingredient 200

Nucleoside transport inhibitor 300 20 Glycerol 200

Sodium hydroxide solution q.s. pH 7.0-7.5

Water for injections to 10 ml

Glycerol is added to a small amount of water for injections. The active compounds are added and the pH is adjusted to between 7.0 and 7.5 with sodium hydroxide solution. The solution is made up to volume with water for injections. The solution is sterilized by filtration under aseptic conditions, placed in sterile ampoules and sealed.

Amount (mg) 30 (2) Active substance 100

Nucleoside transport inhibitor 150

Mannitol 125

Sodium hydroxide solution q.s. pH 8.0 to 9.0 35 Water for injections to 2.5 ml 46 90 664

The active compounds and mannitol are dissolved in a portion of water for injections. The pH is adjusted to between 8.0 and 9.0 with sodium hydroxide solution and the solution is made up to volume with water for injection.

5 The solution is sterilized by filtration under aseptic conditions, placed in sterile bottles and freeze-dried. The bottles are sealed under a nitrogen atmosphere and closed with a sterile closure and a metal collar.

The following Examples 16-18 illustrate the preparation of formulations containing a compound of formula 1 (A) (active ingredient), for example 3'-azido-3'-deoxythymidine and a glucuronidation / renal excretion inhibiting agent, for example probenecid.

Example 16: Tablet 15 Weight (mg)

Active ingredient 100

Glucuronidation / renal secretion inhibitor 200

Lactose 105 20 Starch 50

Polyvinylpyrrolidone 20

Magnesium stearate 10

Total weight 485

The active compounds are mixed with lactose and starch and wet granulated with a solution of polyvinylpyrrolidone. The granules are dried, screened and mixed with magnesium stearate and then compressed into tablets.

Example 17: Capsule 30 Weight (mg)

Active ingredient 100

Glucuronidation / renal secretion inhibitor 100

Lactose 100 35 Sodium starch glycollate 10

Polyvinylpyrrolidone 10

Magnesium stearate 3

Total weight 323 47 90 664

The active compounds are mixed with lactose and sodium starch glycollate and wet granulated with a polyvinylpyrrolidone solution. The granules are dried, screened and mixed with magnesium stearate and placed in 5 hard gelatin capsules.

Example 18: Intravenous injections Amount (mg) (1) Active ingredient 200

Glucuronidation / renal secretion inhibitor 300

Glycerol 200

Sodium hydroxide solution q.s. pH 7.2-7.5

Water for injections to 10 ml

Glycerol is added to a small amount of water for injections.

The active compounds are added and the pH is adjusted to between 7.0 and 7.5 with sodium hydroxide solution. The solution is made up to volume with water for injections. The solution is sterilized by filtration under aseptic conditions, placed in sterile ampoules and sealed.

20 Amount (mg) (2) Active substance 100

Glucuronidation / renal secretion inhibitor 150

Mannitol 125 25 Sodium hydroxide solution q.s. to a pH of 8.0 to 9.0

Water for injections to 2.5 ml

The active compounds and mannitol are dissolved in a portion of water for injections. The pH is adjusted to 8.0 to 9.0 with sodium hydroxide solution and the solution is made up to volume with water for injection. The solution is sterilized by filtration under aseptic conditions, placed in sterile bottles and freeze-dried. The flasks are sealed under a nitrogen atmosphere and sealed with a sterile closure and a metal collar.

48 90664

Veterinary Drug Formulations (Examples 19-22)

Example 19: Tablets for small animal use

Per tablet

Active ingredient 120.0 mg 5 Corn starch 20.0 mg

Microcrystalline cellulose 100.0 mg

Magnesium stearate 1.5 mg

The active ingredient, microcrystalline cellulose and corn starch are mixed. Sufficient starch-10 solution is added with constant stirring until a moist mass is formed which is passed through a sieve to form granules. Magnesium stearate is screened on. Tablets are made by compressing the granules directly.

Example 20: Medicinal granules for use in feed 15 Weight (mg)

Active ingredient 6.0

Povidone 1.0

Lactose 93.0

Alcohol-water mixture q.s.

20 The active ingredient is mixed with lactose. To this is added an aqueous alcohol solution in which povidone has been dissolved. Sufficient aqueous alcohol is added until a moist mass is formed which is allowed to pass through a sieve to form granules which are then dried.

Example 21; Mouth paste

Weight (mg)

Active substance 24

Xanthan gum 0.5 30 Methyl hydroxybenzoate 0.1

Polysorbate 80 0.1

Purified water to 100.0 ml

Polysorbate 80 and methyl hydroxybenzoate are dissolved in most of the water. The xanthan gum is added, the dis-35 is dispersed and allowed to swell. The active ingredient is added and dispersed and the mixture is diluted to final volume.

49 90 664

Example 22: Ointment

Weight (mg)

Active substance 12

White soft paraffin 88.0 The white soft paraffin is melted at 60 ° C.

The active ingredient is added and dispersed, the mixture is allowed to cool and placed in folding metal tubes.

Example 23; 31-Azido-31,51-dideoxy-5 '/ (N, N-dimethylthiocarbamoyl) thio / thymidine 10 (a) 3'-Azido-3'-deoxythymidine (3.0 g, 11.2 mmol) 5 The '' hydroxyl group was mesylated by adding methanesulfonyl chloride (2.7 mL) to a solution of 3'-azido-31-deoxythymidine in 20 mL of dry pyridine. The reaction was allowed to proceed at 5 ° C for one hour, after which the mixture was poured into ice water. The precipitate was collected by filtration. The desired product was obtained by reacting 31-azido-3'-deoxy-5'-mesylthymidine from the first step with potassium carbonate (0.78 g, 5.6 mmol) in DMF (75 mL). The reaction mixture was heated at 80 ° C in an oil-20 bath for 6 hours and then poured into ice water. The product was extracted from water with ethyl acetate. The solvent was removed in vacuo and the resulting oil was flash chromatographed on silica gel eluting with CHCl 3: MeOH (9: 1 v / v). Product yield was low.

Mp = 184-186 ° C.

(b) Sodium dimethyldithiocarbamic acid hydrate (0.642 g, 3.58 mmol) and 3.58 mL of 1-N tetrabutylammonium hydroxide in MeOH were added to 25 mL of DMF. The solution was boiled to remove water and MeOH. After cooling, 2,51-O-anhydro-3'-azido-3'-deoxythymidine (0.85 g, 3.4 mmol) dissolved in 15 mL of DMF was added. The reaction mixture was heated at 55 ° C in an oil bath overnight. The reaction mixture was poured into ice water and the precipitate was removed by filtration. The product was extracted from the filtrate with ethyl acetate. The ethyl acetate was removed at 90664 in vacuo and the resulting oil was purified by flash silica gel chromatography eluting with CHCl 3: MeOH (95: 5 v / v). It was necessary to repeat the silica gel chromatography treatment. A second mixture of CHCl 3 / MeOH 5 (98: 2 v / v) was used as eluent. The product was finally purified by g-reverse phase chromatography eluting with aqueous methanol (3: 7). The yield was 2.5%.

Example 24: 31-Azido-3 * -deoxy-5'-O-acetyl-4-thiothymidine 3'-Azido-3'-deoxy-5'-O-acetyl-4- (1,2,4-tria sol) thymidine (Lin et al., J. Med. Chem. 26, 1691 (1983)) (1.41 g; 3.9 mmol) was dissolved in 100 ml of acetone and 30 ml of water, and then treated With 0.39 g of NaSHxf 2 Cha (Sung, J. Chem. Soc. Chem. Conan. 522, (1982)).

The mixture was stirred for 30 min, halved in volume and extracted with 200 mL of CHCl 3. The CHCl 3 was washed with 100 mL of water and dried over Na 2 SO 4. The solvent was removed in vacuo and the resulting oil was applied to a silica gel pad (6.5 x 3 cm) and eluted with 750 mL of CHCl 3.

The solvent was removed in vacuo to give a yellow oil which was recrystallized from i-PrOH to give 0.64 g of product (1.9 mmol, 48.7%); mp. = 75-78 ° C.

Example 25: 31-Azido-31-deoxy-4-thiothymidine 3'-Azido-3'-deoxy-5'-O-acetyl-4-thiothymidine 25 (0.25 g; 0.76 mmol, Example 21) was dissolved to a mixture of 5 ml of dioxane and 5 ml of concentrated NH 4 OH solution and the solution was stirred for 18 hours. The solvent was removed in vacuo and the residue was applied to a silica gel column and eluted with CHCl 3 / EtOAc (3: 1 v / v).

The appropriate fractions were combined and the solvent removed in vacuo to give a yellow oil which was dissolved in Et 2 O to give crystals on concentration: 0.16 g (0.56 mmol; 74%); mp. = 116-118 ° C.

si 90664

Example 26: 3-N-methyl-31-azido-31-deoxythymidine 3'-azido-3'-deoxythymidine (0.5 g; 1.9 mmol) and Ν, Ν-dimethylformamide dimethyl acetate (Zemlicka, 5 Coll. Czech Chem. Comm. 3-5 3572 (1972)) (0.9 mL; 7.5 mmol) was refluxed in 20 mL of CHCl 3 for 48 h. The solvent was removed in vacuo and the material was applied to a silica column. Elution with EtOAc / CHCl 3 (1: 1 v / v) gave pure material as a viscous oil: 0.26 g (0.9 mmol, 47%).

Example 27: 31-Azido-31-deoxy-2-thiothymidine 31-Azido-2-thiothymidine was synthesized in a five-step reaction sequence using 2,3'-O-anhydro-5'-tritylthymidine as starting material (JJ Fox, J. Org. Chem. 2 <8, 936 (1963)).

2,3'-O-Anhydro-5'-tritylthymidine (10.5 g, 22.4 mmol) was added to a solution of sodium (0.52 g, 22.4 mmol) in dry ethanol (1.2 L). and the reaction mixture was refluxed for six hours. The reaction mixture was cooled and neutralized with 1 N HCl. The solvent was removed in vacuo and the resulting oil was purified by flash silica gel chromatography eluting with CHCl 3 / MeOH (96: 4 v / v).

The yield of 1- (2'-deoxy-5'-trityl-D-lyxofuranosyl) -2-ethoxythymine was 30%. The 2-ethoxythymine derivative (3.5 g, 16.8 mmol) was dissolved in 35 mL of DMF with 2.2 mL of triethylamine. The cold solution was saturated with Na 2 SO 4. The reaction mixture was placed in a steel bomb and heated at 95 ° C. After twenty-seven hours, TLC analysis showed no starting material remaining. The reaction mixture was purified by reflux for several hours and poured into ice water. The product was collected by filtration and purified by flash silica gel chromatography eluting with CHCl 3 / MeOH (97: 3 v / v). The yield of 1- (21-deoxy-5'-trityl-N-D-lyxofuranosyl) -2-thiothymine was 37%. The UV absorption maxima at pH 1 at 277 nm and at pH 11 at 242 nmn indicated the formation of 2-thiothymidine.

52 90664

The 3'-hydroxyl group of the thiothymidine derivative was mesylated as follows: methanesulfonyl chloride (665 mL, 3.5 eq.) Was added in four portions over six hours to a solution of 1- (2'-deoxy-5'-trityl-SD-lysofuranosyl) -2-thiothymidine (1.25 g) in dry pyridine (15 ml) at 5 ° C. The reaction mixture was kept at 5 ° C overnight. The reaction mixture was poured into ice water and the product was collected by filtration. The product was purified by flash silica gel chromatography eluting with ethyl acetate: hex-10 (1: 1 v / v). The yield was 50%.

Lithium azide (0.3 g, 6 mmol) was dissolved in 20 mL of dry DMF and 1- (2'-deoxy-31-mesyl-5'-trityl-N-D-lyxofuranosyl) -2-thiothymine (0, 72 g, 1.2 mmol) was added. The DMF solution was heated at 85 ° C for 2.5 hours.

The reaction mixture was poured into ice water and the product was collected by filtration. The product was purified by flash silica gel chromatography eluting with CHCl 3: MeOH (98: 2 v / v). The yield f 1 was 78%. An IR spectral band at 2100 cm-1 indicated the presence of an alkyl azide. The UV spectrum confirmed the presence of 2-thiothymine-20 dine.

The final product was prepared by deprotection of the 5'-hydroxyl group of 3'-azido-31-deoxy-2-thio-5'-tritylthymidine (0.1 g) by keeping the compound in 80% acetic acid (5 ml) in a steam bath for 45 minutes. . 3'-Azido-3'-deoxy-2-thiothymidine (0.021 g) was obtained by silica gel chromatography (eluting with CHCl 3 -MeOH (96: 4 v / v)) in 37% yield.

Example 28: 31-Azido-31-deoxy-2-ethoxythymidine 3'-Azido-3'-deoxy-2-ethoxythymidine was prepared by refluxing 3'-azido-3'-deoxy-5'-mesylthymidine (2.6 g, 7.5 mmol) in dry ethanol (25 mL) with two equivalents of potassium carbonate (1.08 g, 7.5 mmol) for 5 h. The solution was neutralized and evaporated to an oil in vacuo, the oil was purified by flash silica gel chromatography eluting with ethyl acetate: methanol. The desired product was isolated and the yield was 39%; mp. = 98-100 ° C.

53 90664

Example 29: 31-Azido-31-deoxy-2-methoxy-thymidine 3'-Azido-31-deoxy-2-methoxythymidine was prepared from 31-azido-3'-deoxy-5'-mesylthymidine (1.6 g, 5 4 , 6 mmo]) by the method of Example 25. The yield was 42%; mp. - 47-51 ° C.

Example 30: 3'-Azido-21,31-dideoxy-5-methylisocytidine 2,5'-O-anhydro-31-azido-3'-deoxythymidine (0.35 g; 1.4 mmol) was dissolved in ml of MeOH pre-saturated with ammonia and bombarded at 77 ° C (oil bath) for 48 hours (Skaric and Matulic-Adamic, Helv. Chim. Acta 6J3, 2179 (1980)). TLC analysis (6: 1 v / v CHCl 3 MeOH) indicated that the reaction was not complete. The solvent was removed in vacuo and the resulting oil was applied to a silica gel column and eluted with CHCl 3 / MeOH (6: 1 v / v). The appropriate fractions were combined to give 0.14 g (0.53 mmol; 38%) of the title compound; mp. = 107-108 ° C.

Example 31: 3'-Azido-5-chloro-2 ', 31-dideoxyuridine 3'-Azido-21,31-dideoxyuridine (0.25 g; 1 mmol) was dissolved in 2 mL of dry dimethylacetamide (DMAC). , cooled to 0 ° C, and 2 mL of 0.5 M HCl in DMAC was added, m-chloroperbenzoic acid (0.277 g; 1.6 mmol) was added in two portions over ten minutes, and then the mixture was allowed to warm to ambient temperature. temperature. After 2 hours, 4 ml of water were added and the solution was filtered. The aqueous DMAC solution was extracted with Et 2 O (3 x 3 mL) and the Et 2 O was evaporated in vacuo to an oil which was applied to a silica gel column. Elution with CHCl 3 / MeOH (15: 1 v / v), combined appropriate fractions and evaporation in vacuo gave an oil which was recrystallized from Et 2 O to give 58.5 mg of product (0.2 mmol, 20%); mp. = 169-170 ° C.

54 90664 UV (nm): at pH 1 ") \ max. = 276 (£ = 7400), \ min. = 239 (£ = 500); at pH 1 3" X max. = 274 (£ = 6400) ), * X min. = 249 (6 = 3800).

1 H NMR (DMSO-d 6) δ 8.29 (s, 1H, H 6), 6.04 (δ, ΙΗ, ΗΓ, J = 5T5 Hz), 5.49-5.29 (m, 1H, 5'-OH ), 4.44-4.20 (m, 1H, H3 '), 3.88-3.71 (m, 1H, H4'), 3.71-3.53 (m, 2H, H5 '), Δ 2.63-2.31 (m, 2H, H 2 ');

Analysis for C 9 H 10 N 2 O 2 Cl

Calculated: C, 37.58; H, 3.50; N, 24.35; Cl, 12.32 Found: C, 37.67; H, 3.54; N, 24.39; Cl, 12.40

Example 32: 31-Azido-5-bromo-21,31-dideoxyuridine 3'-Azido-5-bromo-2 ', 3'-dideoxyuridine was prepared from the known 3'-azido-2', 3'-dideoxyuridine ( TA Krenitsky et al., J. Med. Chem., 26, 891 (1983)) (0.827 g, 3.3 mmol) by first acetylating the 5'-hydroxyl group with acetic anhydride (15 mL) and then brominating the 5-position. by adding acetic acid (0.5 ml) and bromine (0.566 g). The red-brown solution was stirred at room temperature for two hours. The reaction mixture was evaporated to an oil in vacuo and triturated with ethyl ether. the oil was dissolved in methano-20 li-amino acid to remove the acetyl group. The desired product was isolated by silica gel chromatography eluting with CHCl 3 / MeOH (95: 5 v / v). The yield was 32%; i sp. = 148-149 ° C.

Example 33: 31-Azido-21,3'-dideoxy-5-iodouri- '25 didine 3'-Azido-2', 3'-dideoxyiododiidine was prepared from 2 ', 3'-dideoxy-5-iodouridine (10 g, 28 mmol) with a four-step reaction sequence described in the literature (TA Krenitsky et al., J. Med. Chem., 26, 891 (1983)); mp. = '30 126-130 ° C.

Example 34: 31-Azido-2 ', 3'-dideoxy-5-trifluoromethyluridine 3'-Azido-2', 3'-dideoxy-5-trifluoromethyluridine was prepared by the following four step reaction sequence.

55,90664 The 5'-hydroxyl group of 2 ', 3'-dideoxy-5-trifluoromethyluridine (5.0 g, 16.9 mmol) was tritylated by adding triphenylmethyl chloride (5.65 g, 20.3 mmol) to the starting material in a suspension of dichloromethane. (1.4 ml), pyridin-5 (70 ml) and a 3A molecular sieve (55 g). The reaction mixture was stirred at room temperature for four days. After filtration and evaporation in vacuo, the oily product was treated by silica gel chromatography eluting with CH 2 Cl 2 / eOH (95: 5 v / v). The resulting oil was triturated with water and the resulting solid was collected by filtration.

The 31-hydroxyl was chlorinated by dissolving 5'-protected uridine (3.0 g) in dimethylacetamide (30 ml) with triphenylphosphine (3.27 g) and adding carbon tetrachloride (51 ml). The reaction mixture was stirred at room temperature overnight. One milliliter of methanol was added. The reaction mixture was evaporated to an oil and treated with silica gel chromatography eluting with Cl 2 Cl 2 EtOAc (9: 1 v / v). The desired product was collected as an oil, the oil, 1- (3'-chloro-2'-deoxy-5'-trityl-threo-β-D-ribofuranosyl) -5-trifluoromethyl-20-luracil was deprotected by dissolving it in nitromethane ( 80 ml) and adding zinc bromide (4.45 g) dissolved in nitromethane (80 ml) using V. Kohlin et al. method (Tetrahedron Letters, 2λ_, p. 2683, 1980). The reaction mixture was stirred at room temperature overnight. An additional 25 zinc bromide (3.0 g) was added the next day and the reaction was allowed to proceed overnight. A final addition of zinc bromide (3.7 g) and gentle heating completed the reaction. The reaction mixture was poured into 1-M ammonium acetate. The product was extracted into dichloromethane. The dichloromethane was removed in vacuo and the resulting oil was chromatographed on silica gel eluting with 3Η2 <2ΐ2: ΜβΟΗ (95: 5 v / v). The final product was obtained by treating 1- (3'-chloro-2 1-deoxy-β-D-ribofuranosyl). ) -5-trifluoromethyluracil (0.48 g, 1.53 mmol) with lithium azide (0.19 g, 3.8 mmol) in dimethyl-35-acetamide (4.8 mL) The reaction mixture was heated at 90 ° C 56 90664 for 4 h. The reaction mixture was evaporated to an oil and chromatographed on silica gel eluting with CHCl 3 / MeOH (95: 5 v / v). a pure product was obtained in a yield of 10%.

Example 35: 31-Azido-21,3 * -dideoxycytidine 31-Azido-2 ', 3'-dideoxycytidine was prepared from 3'-azido-21,31-dideoxyuridine (2.2 g, 7.9 mmol) as the HCl salt. . T.A. Krenitskyn et al. method (J. Med. Chem., 26, 891 (1983)). The yield was 40%, m.p. = 174.5 -176.5 ° C.

Example 36: 3'-Azido-21,31-dideoxy-5-methylcytidine 3'-Azido-2 ', 3'-dideoxy-5-methylcytidine was prepared from 3'-azido-3'-deoxythymidine (0.8 g, 3.0 mmol) by the method of Example 35. The yield was 19%.

Example 37: Threo-31-azido-21,31-dideoxycytidine

Threo-3'-azido-2 ', 3'-dideoxycytidine was synthesized from 2'-deoxyuridine in four steps.

The 5'-hydroxyl group of 2'-deoxyuridine was tritylated by the method described in Synthetic Procedures in Nucleic Acid Chemistry, 1, 321 (1968).

Threo-3'-azido-2 ', 3'-dideoxy-5'-trityluridine was prepared by reacting 2'-deoxy-5'-trityluridine (5.0 g, 10.6 mmol) with triphenylphosphine (3.07 g, 11 , 7 mmol, 1.1 eq.) And carbon tetrabromide (3.88 g, 11/7 mmol), 1.1 eq.) And lithium azide (5.21 g, 106 mmol, 10 eq.) In DMF ( 80 ml). Carbon tetrabromide was added last. The reaction was allowed to proceed at room temperature overnight. Methanol (5 mL) was added. The solution was evaporated to an oil in vacuo and treated with flash silica gel-35 chromatography eluting with ethyl acetate. The 5'-hydroxyl position was deprotected by heating in 80% 57 9 0 6 6 4 acetic acid in a steam bath for twenty minutes. On cooling, the tritylcarbinol precipitated and was filtered off. The filtrate was dried and slurried in ethyl ether. The product, threo-31-azido-2 *, 3'-dideoxyuridine 5 was used in the next step without further purification.

The final product The HCl salt of threo-31-azido-21,31-dideoxycytidine was prepared from the corresponding uridine compound using exactly the same method as used for the preparation of the erythro isomer (TA Krenitsky et al., J. Med. Chem., 1981, 891, (1983)). ). Yield 0.021 g, 7%.

Example 38: 9- (31-Azido-21,3'-dideoxy-α-D-ribofuranosyl) adenine 9- (31-azido-21,31-dideoxy-N-D-ribofuranosyl) adenine was prepared in two in step Ng-octanoyl adenyl-15 (2.0 g, 7.7 mmol) and 31-azido-3'-deoxythymidine (1.13 g, 4.2 mmol) by M. Imazawa and F. Eckstein (J. Org. Chem., 43, 3044 (1978)), m.p. = 120-12 2 ° C.

UV pH 1% n = 258 nm Tvmin = 230 nm pH 13 13, max. = 260 nm \ min. = 229 nm C.AH. »CHN distribution of No0„

Ί <J \ Z o Z

calculated; C, 43.48; H, 4.38; N, 40.56 Found; C, 43.28; H, 4.45; N, 40.38 Example 39: 9- (3'-azido-2 ', 3'-dideoxy-β-D-ribofuranosyl) adenine 9- (3'-azido-2', 3'-dideoxy- N-D-ribofuranosyl) adenine was prepared in two steps from Ng-octanoyladenine (2.0 g, 7.7 mmol) and 3'-azido-3'-deoxythymidine 30 (1.13 g, 4.2 mmol) M. Imazawan and F. Eckstein (J. Org.

Chem., £ 3, 3044 (1978)).

Sp. = 184-185 ° C.

UV pH 1 ^ max. = 257 nm \ min. = 230 nm pH 13 ^ max = 260 nm Xmin = 228 nm 35 ^ 10 ^ 12 ^ 8 ^ 2111 calculated CHN distribution

Calculated: C, 43.48; H, 4.38; N, 40.56 Found: C, 43.33; H, 4.45; N, 40.41 58 90664

Example 40: 5'-Acetyl-31-azido-3-benzoyl-31-deoxythymidine 5'-Acetyl-31-azido-31-deoxythymidine (0.75 g, 2.4 mmol) was dissolved in pyridine (5 mL) and benzoyl chloride. -5 ride (1.4 mL, 12 mmol, 5 eq) was added at room temperature. The reaction mixture was stirred overnight and then poured into ice water (250 mL). The pH of the aqueous solution was adjusted to 1. The product was extracted with chloroform. The organic phase was washed with water, dried over MgSO 4 and filtered. The chloroform was removed and the oily product was subjected to flash silica gel chromatography eluting with chloroform. The product was collected as an oil.

1 H NMR (DMSO-d 6): 8.04 - 7.50 (m, 6H; 3 N-benzoyl and 6 H), δ f12.12 (dd, 1H, J 1 I 2a, = 5.6 Hz, J 1, 2b , = 6> Ί Hz, 1Ή), J4.55 - 3.96 (m, 4H; 3'H, 4'H, 5 Ή), g 2.6 2 - 2.38 (m, 2H, 2Ή ), Δ 2.07 (s, 3H, 5'-acetyl CH-J, JT 1.90 (d, 3H, Jc. = 1.0 Hz, 5 CH 3)

j u b, b J

Calculated for CH 2 Cl 2 / CH 2 N 2 O 2 Calculated: C, 55.20; H, 4.63; N, 16.94 Found: C, 55.29; H, 4.64; N, 16.93 Example 41: Threo-31-azido-5-bromo-21,31-dideoxyuridine

Treo-3'-azido-5-bromo-21,31-dideoxyuridine was prepared from 2'-deoxyuridine in a five-step reaction sequence.

The 5'-hydroxyl group of 2'-deoxyuridine was protected with a triphenylmethyl group in the usual manner. The 3'-hydroxyl was mesylated. The stereochemically correct 3'-azido group was obtained by adding 2'-deoxy-3'-mesyl-5'-trityluridine (22 g, 40 mmol) to a solution of sodium 30 azide (7.84 g, 120 mmol, 3 eq.) In dimethylformamide (380 ml) at 80 ° C. The reaction was allowed to proceed for 35 hours. The solution was poured into ice water (21) and the precipitate was collected by filtration. The product was isolated by silica gel chromatography eluting with chloroform: methanol .35 (1: 1 v / v) in 51% yield. The 5'-hydroxyl position 59 90664 was deprotected with 80% acetic acid in a steam bath for 25 minutes. After cooling, the tritylcarbinol was filtered off. The filtrate was then evaporated to a thick oil in vacuo. The product was isolated by flash silica gel chromatography eluting with chloroform: methanol (85:15 v / v). Threo-31-azido-21,31-dideoxyuridine was brominated using exactly the same method as described for the bromination of erythro-31-azido-2 ', 3'-dideoxyuridine.

UV pH 1 / ln.2.2, λ = 9400, λτϋη.244, ε = 2600 δ, PH 13 /} max & 276 λ = 6700, λ min, 251 λ = 3700 1 H NMR (DMSO-d 6): 611.86 (s , 1H, 3-NH), 68.02 (s, 1H, 6H), 65.99 (dd, 1H, J 1 = x mL · a 3.0 Hz;

Jr, 2b '= 7.5 Hz »1Ή)» δ5> 1 J5'CH 5'H = 5'4 Hz> 5'OH) »δ4; 49 (m, 1H, 3'H), 64.05 ( πη, 1Η, 4Ή), 63.71 (m Jh, 5'H), 62.72 (m, 1H, 2b '), 62.18 (m, 1H, 2a') C9H1QBrN5O4-0.25 H2O-0 CH 1 Distribution Calculated: C, 32.25; H, 3.21; N, 20.44; Br, 23.32 Found: C, 32.17; H, 3.21; N, 20.33; Br, 23.19

Example 42: 1- (3'-Azido-21,3-dideoxy- [3-β-β-pentofuranosyl) -2- (benzyloxo) -5-methyl-4- (1H) -pyrimidinone

Sodium (0.4 g, 17.4 mmol, 2.6 eq) was reacted with dry benzyl alcohol (10 mL) for one hour at room temperature. 2,51-O-Anhydro-3'-azi-25-do'-deoxythymidine (1.65 g, 6.6 mmol) was added. The reaction was allowed to proceed for one hour. The mixture was poured into ice water (250 ml), the pH was adjusted to 7 and the aqueous phase was extracted with ethyl acetate. The organic phase was extracted with water (4 times). After drying over MgSO 4, the ethyl acetate was removed in vacuo. The resulting oil was subjected to silica gel chromatography eluting first with ethyl acetate and then with ethyl acetate: methanol (9: 1 v / v). Fractions containing product were collected and the solvents removed in vacuo to give an oil. the oil was crystallized after covering with ethyl ether; mp. = 125 - 126.5 ° C.

60 90 664 UV pH 1 unstable pH 13 "X max. = 256, £ = 10700, Xmin. = 240, £> 8900 1 H NMR (DMSO-d 6); 67.8 (d, 1H, J65 = 1.2) Hz, 6H), 67.49-7.38 (m, 5H, 2-phenyl), 66.08 (dd, 1H, Jr 2a, = 5.0 Hz; ^ 2b. = 7.0 Hz, ΓΗ) , 65.37 (s, 2H, 2CH 2), 65.25 δ (t, 1H, J 5, CH = 5.4 Hz, '5ΌΗ) 64.36-4.32 (m, 1H, 3'H), 63.85-3.81 (m, 1H, 4'H),% 3.7-3.58 (m, 2H, 5'H), 62.53-2.34 (m, 2H, 2'H), 61.82 (d, 3H, 35 6 = 1.0 Hz, 5CH 3) CH 2 Distribution of C 11 H 19 N 2 O 3 • Calcd: C, 57.14; H, 5.36; N, 19.60. C, 57.02, H, 5.43, N, 19.53

Example 43: 1- (3'-Azido-2 ', 3'-dideoxy-β-D-threopentofuranosyl) -2-ethoxy-5-methyl-4- (1H) -pyrimidinone Threo-31-azido- 51-O-Mesylthymidine (1.08 g, 3.13 mmol) prepared from threo-31-azidothymidine / dissolved in 100 mL of EtOH and treated with NaHCO 3 (0.26 g, 3.13 mmol). mmol) at reflux for 18 hours. The reaction mixture was cooled and filtered. The solvents were removed in vacuo and the residue was applied to a silica gel column and eluted with CHCl 3 / MeOH (9: 1 v / v). Combining the appropriate fractions and removing the solvents in vacuo gave 0.7 g of product (2.4 mmol, 75.7%), m.p. = 120-122 ° C.

UV (nm): at pH 1 X max. = 26 0 (£ = 9 3 0 0), "Amin. = 237 (€ = 5500), 1. Shoulder end = 221 (£ = 7500); at pH 1 3 Amaks. = 256 (£. = 10000),

Amine = 240 (£ = 7700).

25 H 1 NMR (DMSO-d 6) 67.58 (s, 1H, H 6), 66.0 (dd, 1H, H 1 ', J = 2.9, 4.56 Hz), 65.06 (t, 1H f 5) 'OH, 3 = 4r91 Hz), 64.51-4.47 (m, 1H, H3'), 64.34 (q, 2H, -OCH2-, 3 = 7.14 Hz), 64.10-4 .05 (m, 1H, H4 '), δ 3.73 (t, 2H, H5', 3 = 5.62 Hz), 62.82-2.73 (m, 1H, H2'b), -> 62.21-2.14 (m, 1H, H2'a), 61.82 (s, 3H, 5CH3), 61.31 (t, 3H, -CH2-CH3, 3 = 6.65 Hz).

Elemental Analysis for 2 H, J 3 Calculated: C, 48.81; H, 5.80; N, 23.72

Found: C, 48.59; H, 5.86; N, 23.64

Example 44: Threo-31-azido-21,31-dideoxy-4-thio-thymidine

Threo-3'-azido-5'-O-trityl-3'-deoxy-4- (1,2,4-triazole) thymidine (1.25 g, 2.2 mmol) (prepared not 90664 W. According to the method of Sung (Nucleic Acids Research (1981), E, 6139)) was dissolved in 100 ml of acetone and 30 ml of water and treated with 0.22 g of NaSHxH2O.

. The mixture was stirred for 3 hours. The mixture was halved and extracted with 300 mL of CHCl 3.

The CHCl 3 was washed with 50 mL of water, dried over Na 2 SO 4 and evaporated in vacuo to give an oil. The 5'-O-trityl group was removed by dissolving this oil in 100 mL of 80% HOAC. The solution was heated on a steam bath for 2 hours and 10 hours and cooled, diluted with 100 ml of water and filtered. The solvents were removed in vacuo and the oil was applied to a silica gel column. Elution with CHCl 3 / MeOH (20: 1 v / v), collection of the appropriate fractions and removal of the solvents in vacuo gave 0.18 g of product (0.62 mmol; 28%); mp. = 65-67 ° C.

UV (nm): at pH 1 ^ X max. = 337 (/ = 20700), Amin. = 280 (£ = 1200), Xolhead = 238 (/ = 3400); at pH 1 3 Amaks. = 320 (£ = 18400); Aunin. = 257 (£ = 1700); 1 H NMR (DMSO-d 6) 67.63 (s, 1H, H 1 ', J = 2.93, 4.89 Hz), 65.06 (s, 1H, 5'OH), 64.50-20 4.46 ( m, 1H, H3 '), 64.10-4.04 (m, 1H, H4'), 63.73 (d, 2H, H5 ', J = 5.61 Hz), 62.78-2.68 (m, 1H, H2'b), 62.20-2.14 (m, 1H, H2'a), 62.00 (s, 3H, 5CH3).

qH, J 1 N 2 O 2 S-O.1 C 2 H 9 O-0.25 H 2 O Elemental Analysis Calculated: C, 41.90; H, 4.86; N, 23.95; S, 10.97 Found: C, 41.99; H, 4.73; N, 23.88; S, 10.91 Example 45: 4-Amino-3'-azido-5-bromo-2 *, 3'-dideoxyuridine 3 * -Azido-21,3'-dideoxyuridine was acetylated and brominated by the Visser method ( Synthetic Procedures in Nucleis Acid Chemistry Vol. 1 p. 410) to give 5'-acetyl-3'-azido-5-bromo-2 ', 3'-dideoxyuridine. This material was reacted with five 1,2,4-triazole equivalents and two 4-chlorophenyl dichlorophosphate equivalents in dry pyridine at ambient temperature for 7 days to give 5'-acetyl-3'-azido-5-35 bromo-4- (1,2,4-triazolyl) -21,31-dideoxyuridine 62,90664 as a yellow oil in moderate yield. Treatment at ambient temperature with ammonia-saturated methanol at 0 ° C for 18 hours, after recrystallization from ethyl acetate and filtration of the product crystals, gave 4-amino-3'-azido-5-bromo-2 ', 3'-dideoxyuridine 173 rag ( 0.5 mmol, 6.3%); mp. = 162-165 ° C (decomposes).

UV (nm): at pH 1 X max. = 300.21 δ (, δ = 10700, 12100), Λ min. = 253 (£ = 1500), at pH 1 3 max. = 288 (£ = 7300), Άπιίη. = 260 10 (<f = 3900) 1 H NMR (DMSO-d 6) δ 8.3 (s, 1H, H 6), 67.85 (broad s, 1H, 4-NH 2), 67.05 (broad s, 1H , 4-NH 2), 66.0 (t, 1H, H1 ', J = 5.94 Hz), 65.33 (t, 1H, 5'-OH, J = 5.01 Hz), 64.4- 4.3 (m, 1H, H3 '), 63.9-3.5 (m, 3H, H41, H5'), 62.31 (t, 2H, H2 ', 3 = 6.27 Hz). C 9 H 18 N 2 O 2 Brin Elemental Analysis Calculated: C, 32.64; H, 3.35; N, 25.38; Br, 24.13

Found: C, 32.52; H, 3.41; N, 25.32; Br, 24.04

Example 46: 31-Azido-5-bromovinyl-2 ', 31-dideoxyuridine 5-Bromovinyl-21-deoxyuridine (BVDU) was synthesized using the method of Jones et al. method (Tetrahedron Letters 45, 4415 (1979)) in similar yields. BVDU was tritylated and me- ·; ·. was embraced by Horwitz et al. method (J. Org. Chem., 31, 205 (1966)). This product was treated with an equimolar amount of sodium bicarbonate at reflux in methanol to give 3 ', 2-O-anhydro-5-bromovinyl-2'-deoxyuridine. This product was treated with three equivalents of lithium azide in 1% water / dimethylformamide at 130 ° C. Combining the appropriate fractions from the silica gel chromatography of the crude product and evaporating to 30'-azido-5-bromovinyl-2,3,3-dideoxyuridine was obtained as a golden oil; UV (nm); at pH 1 Xmax = 292.247 A min = 270.238; - at pH 13 3 max max = 253, Xmin = 238, Xop = 284; H 1 NMR (DMSO-d 6) δ 8.06 (s, 1H, Hg), 67.25 ( d, 1H, -CH = CHBr, J = 13.4 Hz) 66.85 (d, 35 1H, = CHBr, J = 13.7 Hz) 66.07 (t, 1H, H 2, J = 6; 3 Hz), 65.30 (broad s, 1H, 5'-OH), 64.42 (q, 1H, Ηγ, 3 = 6.3, 6.1 Hz), 63.86-3.82 (m, 1H, H ^), 63.68-3 ^ 59 ^, 2H, H5I), 62.47-2.32 (m, 2H, H2,).

63 90664 C.JH1 2N504Br Elemental analysis Calculated: C, 36.89; H, 3.38; N, 19.55 Found: C, 36.86; H, 3.41; N, 19.51

Example 47: Threo-31-azido-5-chloro-21,3 * -dideoxy-5-siuridine

The title compound was prepared in a similar manner to 3'-azido-5-chloro-2 ', 3'-dideoxyuridine (Example 31) in 0.15 g (0.5 mmol, 25%); mp. = 65 ° C.

UV (nm); at pH 1 \ max = 278.21 2 (£ = 8900), 7 \ min. = 240 10 (£ = 1600); at pH 13 Λ max = 275 (£ = 6600), Λ min = 248 (£ = 3300); 1 H NMR / DMSO-d 6 M 11.89 (s, 1H, NH), 67.94 (s, 1H, Hfi) t 66.00 (dd, 1H, H 2, J = 2.93, Hz), 65.1 (t, 1H, 5'OH , J = 5.1 Hz), 64.50-4.46 (m, 1H, Hj), 64r08-4.03 (m, 1H, H4,), 63.71 (t, 2H, H5 J = 5.32 Hz) , 62.77-2.67 (m, 1H, H 2 Ib), 62.23-2.16 (m, 1H, H 2 I, a).

Elemental analysis for C 9 H 8 N 2 O 2 Cl 2 0.1 H 2 O-0.1 C 4 H 9 O 2 Calculated: C, 37.85; H, 3.72; N, 23.48; Cl, 11.89 Found: C, 37.94; H, 3.91; N, 23.23; Cl, 11.86

Example 48: 1- (3 1 -azido-2,3,1-dideoxy-β-D-erythro-20-pentofuranosyl) -5-methyl-2- (pentyloxo) -4- (1H) -pyrimidinone 1- (3'-Azido-2 ', 3'-dideoxy- [E-β-erythro-pentofuranosyl) -5-methyl-2- (pentyloxy) -4- (1H) -pyrimidinone was prepared by the method used in 1- For the preparation of (3'-azido-25,2,3'-dideoxy-N-D-erythro-pentofuranosyl) -2- (benzyloxo) -5-methyl-4- (1H) -pyrimidinone (Example 42). The product was finally purified by HPLC with CH 2 Cl 2, eluting with water: methanol (3: 7). The solvents were evaporated and the product was collected as an oil.

Δ UV pH 1, λ 258 nm λ = 9400, λ min 232 nm λ = 5900 pH 134, 256 nm 10600 'An 239 nm λ = 8200 NMR analysis performed in DMSO-d 6.

64 90664 NMR: 67.81 (s, 1H, 6H), 66.04 (t, 1H, J1 (? 2, = 6.7Hz, 1'H) 65.28 (t, 1H, J5, CH2.5, OH = 5.1Hz, 5OH) 64.43-4.37 (m, 1H, 3'H), 64.27 (t, 2H, J2 O (1CH2CH) = 6.6Hz, 2-0 (CH2) 1) δ 63.88-3.84 (πι, 1Η, 4Ή ?, ' δ3.7 ^ -3.50 (πΊ, 2Η, 5'ΟΗ2) 62.50-2.34 (m, 2H, 2'H), 61.80 (s, 3H, 5CH3), 61.72-1.68 (m, 2H, 2-0 (CH2) ) 2), 01.38-1.30 (m, 4H, 2-O (CH 2), ia) i0.92-0.87 (m, 3H, 2-O (C m) 5) CHN distribution 5 ^ 23N5 ^ 4 ^ 4 H 2 °

Calculated: C, 52.70; H, 6.93; N, 20.48 Found: C, 52.63; H, 6.92; N, 20.48 Example 49: 31-Azido-3-benzoyl-31-deoxy-51-mesylthymidine 3'-Azido-3-benzoyl-3'-deoxy-5'-mesylthymidine was prepared from 3'-azidoyl. From 31-deoxy-5'-mesylthymidine using a procedure similar to Example 40 for the preparation of 5'-acetyl-3'-azido-3-benzoyl-3'-deoxythymidine from 5'-acetyl-3'-azido-31-deoxythymidine ; mp. = 86-88 ° C.

UV PHI λmax 259 nm λ = 21200, λ max j 230 nm λ = 6400 PH 13 / λ max 6500, λ max 248 nm λ = 8000 δ H 1 NMR (DMSO-d 6) 68; 00-7; 58 (m, 6H, 6H and phenylD, ε and 1'CH2), δ) 64r55-4r47 (m, 3H; 3Ή and 5'CH2), 04.12-4.10 (m, 1H, 4 1 H) 63.28 (s, 3H, 5'-SO 2 CH 3), 62.65-2f 4 (m, 2H, 2'H), δ 1.89 (d, 3H, J 5> 6 = 1; 3Hz, 5CH3)

CHN distribution C 18 H 19 N 5 O 7 S

25 'Calculated: C, 48.10; H, 4.26; N, 15.58; S, 7.13 Found: C, 48.00; H, 4.23; N, 15.39; S, 7.10

Example 50: Threo-31-azido-21,31-dideoxy-5-iodouridine 5-iodo-21-deoxyuridine was tritylated and mesylated according to Horwitz et al. method (J. Org. Chem., 31 (1966), 205).

This product was heated with three lithium azide equivalents in anhydrous dimethylformamide at 74 ° C for 48 hours. Silica gel chromatography of the reaction mixture eluting with CHCl 3 / MeOH (20: 1 v / v), combining and evaporating the appropriate fractions afforded trco-3'-azido-21,3'-didoxy-3S 5-iodo-5'-trityluridine. The 5'-hydroxyl was deprotected with 65 90 664 saturated zinc bromide / nitromethane solution at 0 ° C. Chromatography of the crude product on silica gel using CHCl 3 / MeOH (9: 1 v / v) as eluent, combined and evaporation of the appropriate fractions afforded threo-31 -5-azido-2 ', 3'-dideoxy-5-iodouridine as a solid; mp. = 80 ° C.

UV (nm) at pH 1 "X min. = 247; at pH 13 X max. = 277, min. = 252; 1 H NMR (DMSO-d 6) δ 8.37 (s, 1H, H 6), 66, 00 (t, 1H, H1 ', J = 6.17 Hz), 65.4-5.3 (m, 1H, 5'OH), 54.4-4.3 (m, 1H, H3'), 63 , 9-3.8 (m, 1H, H4 '), 63.7-3.6 (m, 2H, H5').

Elemental analysis for C 9 H 10 N 5 O 4 0.4 C 4 H 9 O 2 Calculated: C, 30.73; H, 3.21; N, 16.90; I, 30.63 Found: C, 30.93; H, 3.09; N, 16.61; I, 30.94.

Example 51: 1- (51-O-acetyl-3'-azido-2 ', 3'-dideoxy-D-erythro-pentofuranosyl) -5-methyl-4- (1,2,4-triazol-1 -yl) -2 (1H) -pyrimidinone 5'-Acetyl-3'-azido-3'-deoxythymidine was reacted with five equivalents of 1,2,4-triazole and two equivalents of 4-chlorophenyl dichlorophosphate in dry pyridine at ambient temperature for 10 days. .

Chromatography of the crude product on silica gel using EtOAc / hexane (1: 1 v / v) as eluent, combination of the appropriate fractions and evaporation gave an oil. Crystallization from EtOAc gave 2.7 g (7.5 mmol; 60%) of the title compound as a solid. mp. = 143-145 ° C.

UV (nm) at pH 1> max. = 324.245.215 (£ = 9300.10000.20500), λ min = 282.233 (£ "= 2100, 8200); at pH 1 3 * A max = 276 (£ = 6000), Amin = 242 (ε = 2000) 1 H NMR (DMSO-d 6) g 9.34-8.40 (2s, 2H, triazolyl), δ 8.23 ”30 (s, 1H, H 6), £ 6.12 (t, 1H, H1 ', J = 6.16 Hz), δ 4.48-4.17 (m, 4H, H3', H4 ', H5'), <f2.35 (s, 3H, 5'-acetyl), 2.07 (s, 3H, 5CH 3).

Elemental analysis for C 14 H 16 N 8 O 4 Calculated: C, 46.67; H, 4.48; N, 31.10

Found: C, 46.58; H, 4.51; N, 31.02.

66 90 664

Example 52: 1- (31-Azido-21,31-dideoxy-β-D-erythro-pentofuranosyl) -4-dimethylamino-5-methyl-2- (1H) -pyrimidinone 51-acetyl-3 ' -Azido-3'-deoxy-4- (1,2,4-triazolyl-5H) thymidine was dissolved in dry acetonitrile at ambient temperature under a nitrogen atmosphere. 20 equivalents of dimethylamine were added in one portion and the reaction mixture was stirred for 30 minutes. The solvents were removed, the residue was dissolved in ammonia-saturated methanol, and the solution was stirred for 30 minutes. The solvents were removed and the residue was dissolved in EtOAc. The title compound slowly precipitated from solution and filtered off to give a white solid; mp. = 157-159 ° C.

UV (nm) at pH 1 Λ max. = 299.224 (£ = 14900.7900) Amine = 254 15 (£ = 2500); at pH 13 and max = 237 (£ = 14,000), Amin = 243 (£ = 6800).

1 H NMR (DMSO-d 6) δ 7.63 (s, 1H, H 6), δ 6.06 (t, 1H, H 1, J = 6.53Hz), 65.22 (t, 1H, 5OH, J = 5.2Hz) ), 64.37-4.34 (m, 1H, H3 '), 63.83-3.80 (m, 1H, H4'), 63.65-3.60 (m, 2H, H5 '), 63 ^ 06 (s, 6H, N (CH 3) 2), 62.29-2.23 (m, 2H, H 2 '), 62.11 (s, 3H, 5-CH 3).

Elemental analysis for 20 C12H18N6 ° 3

Calculated: C, 48.97; H, 6.16; N, 28.56

Found: C, 49.06; H, 6.20; N, 28.50

Example 53: 1- (31-Azido-2 ', 31-dideoxy-S-D-erythro-pentofuranosyl) -5-methyl-2- (isopentyloxy) -4- (1H) -pyrimidinone

The title compound was prepared using a procedure similar to 1- (31-azido-2 ', 31-dideoxy-N-D-erythro-pentofuranosyl) -2- (benzyloxo) -5-methyl-4- (1H) -pyrimidino- ni was used for the preparation (Example 42).

30 uv pH1 (nm) Araks.258, £ = 9000, irritin 237, £ = 6000 pH13 (nm) /? Tdcs.255, £ = 11000, ^ min 239, £ = 8000 67 90664 1 H NMR (DMSO-d 6) : 67.79 (s, 1H, 6H), δ 6.02 2, = 6.3 Hz, ΓΗ), 65.23 (t, 1H, J5, OHj5, H = 5.2 Hz, ΌΗΌΗ), 64 , .4-4.3 (m, 3'H j, a 2-O-CH2-CH2CH (CH3) 2), 63.9-3.8 (m, 1H, 4'H), δ 3.7 -3.5 (m, 2H, 5'H), δ 2.5-2.3 (m, 2'H) δ 61.78 (s, 3H, 5CH 3), δ 1.7 -], 5 ( m, 3H, 2-OCH 2 -CH 2 -CH (CH 3) 2) 60.92 and 0.89 (d, 6H, J = 6.3 Hz, 2-O (CH 2) 2 CH (CH 3) 2) C 15-23 ^ 5 CHN distribution of ^ 4 Calculated: C, 53.40; H, 6.87? N, 20.76 Found: C, 53.14; H, 6.92; N, 20.62 Example 54: 3-Acetyl-31-azido-51-O- (3-chlorobenzoyl) -31-deoxythymidine

To a mixture of 3'-azido-5'-O- (4-chlorobenzoyl) -31-deoxythymidine (0.3 g, 0.74 mmol) and silver cyanide (0.4 g, 3.0 mmol ) In 20 mL of benzene, an additional 15 mL of acetyl chloride (2.6 g, 33 mmol) was added in several portions. The mixture was stirred at room temperature until the starting material disappeared (4 hours) by TLC analysis (CHCl 3 / MeOH, 20: 1 (v / v)).

The reaction mixture was filtered and the filtrate was evaporated to dryness under reduced pressure. The resulting oily residue was treated with silica gel chromatography eluting with chloroform / hexane (1: 1 (v / v)) followed by chloroform to give. 0.21 g (64%) of the desired product is obtained in the form of an oil.

UV (nm): at pH 1 λ max = 273 (£ = 9000), "Amin. = 251 (£ = 6200); at pH 13 λ max = 266 (£ = 8800), \ min. = 247 (ε = 7000); 25 H 1 NMR (DMSO-d 6) δ 1.68 (s, 3H, 5-CH 3), 62.47 (s, 3-CGCH-j), 62.3-2, Δ (m, 2Ή), 64.1-4.2 and 4.4-4.7 (m, 4Η, 3Ή, 4Ή and 5Ή), 66.1 (t, 1H, Hz, ΓΗ), 67.5 -8.0 (m, 5H, 6H and phenyl) Elemental analysis for CH 2 Cl 2 O-CH 2 Cl 2 Calculated: C, 48.89; H, 3.89; N, 14.85? Cl, 12.03 Found: C, 49.05; H, 3.94; N, 14.79? Cl, 12.56

Example 55: 31-Azido-5-cyano-21,31-dideoxyuridine 5-Iodo-2 ', 31-dideoxyuridine was reacted with acetic anhydride (2.1 equivalents) in pyridine under dry conditions at ambient temperature for 2 hours 35 hours to give 2 ', 3'-dideoxy-3', 5'-diacetyl-5-jo-diuridiinia. Nucleoside (1 equivalent), potassium cyanide 68 90664 (1.3 equivalents) and potassium acetate (1.3 equivalents) were placed in dry DMSO and heated at 97 ° C under nitrogen for 2 hours. The solvents were removed in vacuo and the residual oil was applied to a silica gel column and eluted with CHCl 3 / EtOAc (1: 1 (v / v)). The appropriate fractions were collected, combined and evaporated to give 5-cyano-2 ', 31-dideoxy-3', 5'-diacetyluridine. The compound was dissolved in ammonia-saturated methanol, and the solution was stirred at 0 ° C for 18 hours. The solvent was removed in vacuo at ambient temperature to give crystals of the title compound; mp. = 160-162 ° C.

UV (nm): at pH 1 * \ niaks. = 276.215 (£ = 13500.1 1200), A min. = 238 (6 = 1700); at pH 1 3 Amaks. = 276 (£ = 1 01 00), Amin. = 240 (ε = 3400) δ H 1 NMR (DMSO-d 6) 68.81 (s, 1H, H 6), 66.00 (t.HH.Hl *, δ = 5.94.9), 65.3-5 , 21 (m, 2H, 5'OH, 3OH), 64.28-4.15 (m, 1H, H3 '), 63.82-3.77 (m, 1H, H4'), 63 / 7- 3.5 (m, 2H, H 5 '), 62.18 (t, 2H, H 2, J = 5.75 Hz).

Elemental analysis for C 20 H 18 N 2 O 2 • 0.25 H 2 O Calcd: C, 46.61; H, 4.50; N, 16.31 Found: C, 46.67; H, 4.71; N, 16.54

Example 56: 1- (3 1-Azido-21,3'-dideoxy-N-D-threo-pentofuranosyl) -5-methyl-2-pentyloxy-4- (1H) -pyrrolidinyl | Midinone 2,5'-O-anhydro-1- (3'-azido-2 ', 31-dideoxy-N-D-threo-25 pentofuranosyl) thyrine was added to a solution of potassium t-butoxide (0.5 equivalents). ) in pentan-1-ol. The reaction mixture was stirred for 2 hours at ambient temperature under a nitrogen atmosphere. The solvents were removed in vacuo and the residue was applied to a silica gel column. Eluting with CHCl 3 / MeOH (20: 1 (v / v)), combining the appropriate fractions and evaporating gave a clear oil which formed crystals of the title compound on standing; mp. = 110-11 ° C.

69 90664 UV (nm) at pH 1 Xmax. = 255 (£ = 1,0200), Amin. = 237 (£ = 6200), λορ = 222 (¢ = 9000); at pH 13 'λ max. = 251,226 (£> 1 1 600.9600),' Amin. = 238 (¢ = 8600) 1 H NMR (DMSO-d 6) 67.58 (s, 1H, H 6), 65.98 (dd, 1H, H1 ', J = 2.98, 4r88Hz), 65.07 δ (t, 1H, 5'OH, J = 5.42 Hz), 64.5-4.47 (m, 1H, H3 '), 64.31-4.25 (m, 2H, -OCH2-), 64.1-4.06 ( m, 1H, H4 '), 63.73 (t, 2H, H5', J = 5 / .62Hz), 62.82-2.72 (m, 1H, H2 '), 62.2-2.14 (m, 1H, H2') , 61.82 (s, 3H, 5-CH 2), 61.75-1.65 (m, 2H, pentyl), 61.4-1.3 (m, 4H pen style, 60.92-0.87 (m, 3H, pentyl).

Elemental analysis of C,, Η - ,, Ν, Ο,

Ib Δ0 4 4 10 Calculated: C, 53.40; H, 6.87; N, 20.76

Found: C, 53.31; H, 6.90; N, 20.74

Example 57: 1- (31-Azido-2 ', 31-dideoxy-) S-D-threo-pentofuranosyl) -2-benzyloxy-5-methyl-4- (1H) -pyrimidinone

The title compound was prepared in a similar manner to 1- (31-azido-2 ', 3'-dideoxy-β-D-threo-pentofuranosyl) -5-methyl-2-pentyloxy-4- (1H) -pyrimidinone (Example 56) using benzyl alcohol. in place of pentan-1-ol; mp. = 137-139 ° C.

20 UV (nm) at pH 1 ^ max. = 266 (£, = 9100), / min. = 235 (£ = 3000); pH 1 at 3 ^ max. = 256 (£ T = 1,500), / Vmin. = 240 (£ = 9600).

1 H NMR (DMSO-d 6) δ 7.6 (s, 1H, H 6), ε 7.49 - 7.37 (m, 5H, phenyl), 56.01 (dd, 1H, H 1 ', J = 2 , 52.5.0Hz), ¢ 5.35 (s, 2H, benzyl), ci 5.1-5.0 (m, 1H, 5 1 OH), ¢ 4.5-4.4 (m, 1H, H3 '), <4, 1 - 4.0: -, - 25 (m, 1H, H4 1), £ 3.77 - 3.67 (m, 2H, H5'), J 2.85 - 2 , 70: (m, 1H, H 2 '), J "2.25 - 2.15 (m, 1H, H 2 ·), δ 1.83 (s, 3H, 5-CH 3).

; Elemental analysis for C 17 H 18 N 4 O 0.25H 2 O.

Calculated: C, 56.43; H, 5.43; N, 19.35 Found: C, 56.51; H, 5.37; N, 19.36 Example 58: 1- (3 1-Azido-2 1,3'-dideoxy-R-D-erythro-pentofuranosyl) -4-methoxy-5-methyl-2 (1H) -pyrimidinone 5'-acetyl-3'-azido-3'-deoxy-4- (1,2,4-triazolyl) thymidine was treated at ambient temperature of 0.5-N. 35 with sodium methoxide in methanol for 24 hours. The solution was neutralized to pH 7 with sulfonic acid resin 70 90664 (DOW 50W, H +), the resin was filtered off and the filtrate was evaporated to a solid in vacuo. This solid was dissolved in a small amount of CHCl 3, applied to a silica gel column and eluted with 2% MeOH in CHCl 3.

The appropriate fractions were collected, combined and evaporated to dryness to give a white solid; mp. = 119-123 ° C.

UV (nm) at pH 1 Xmax. = 279 (£ = 7500), X min. = 239 (£ * = 1700); pH 1 at 3Xmax. = 279 (£ = 6400), i \ .min. = 243 (£ = 1300).

1 H NMR (DMSO-d 6) 58.02 (s, 1H, H 6), 56.08 (t, 1H, H 1 ', J = 5.86 Hz), 55.29 (t, 1H, 5'OH, J = 5.48 Hz), 54.41-4.33 (m, 1H, H3 '), 53.9-3.86 (m, 1H, H4') f 53.86 (s, 3H, 4-OCH3), 53.75-3.58 (m, 2H, H 5 '), 52.4-2.3 (m, 2H, H 2'), 51.89 (s, 3H, 5-CH 3).

Elemental Analysis for C 11 H 15 N 5 O 4 Calculated: C, 46.97; H, 5.38; N, 24.90 Found: C, 47.06; H, 5.40; N, 24.86

Example 59: 1- (31-Azido-21,31-dideoxy-β-D-erythro-pentofuranosyl) -5-methyl-4- (1-pyrrolidinyl) -2- (1H) -pyrimidinone 5'-acetyl-31 -azido-3'-deoxy-4- (1,2,4-triazol-20-yl) thymidine was dissolved in dry acetonitrile at ambient temperature under a nitrogen atmosphere. Five equivalents of pyrrolidine were added dropwise over 5 minutes and the reaction mixture was stirred for 2 hours. The solvents were removed in vacuo to give an oil. This oil was dissolved in methanol saturated with ammonia at ambient temperature and the solution was stirred for 4 hours. The solvents were removed in vacuo and the residue was applied to a silica gel column. Elution with CHCl 3 / MEOH (20: 1 (v / v)), combination of the appropriate fractions and evaporation gave the title compound as a white solid; mp. = 168-171 ° C.

UV (nm) at pH 1 ") \ max. = 295.233 (£ = 1 5700.9500)," \ min. = 253 (£ = 2600); at pH 13 * Xmax. = 28 δ (£ = 1,500), * X min. = 241 (£ = 7900).

Δ 90664 HXNMR (DMSO-d 6) 67.57 (s, 1H, H6), 66.02 (δ, ΙΗ, ΗΓ, J = 6.5Hz), 65.2 (t, 1H, 5'OH, J = 5; 15Hz), 64.4-4.3 (m, 1H, H3 '), 63.84-3.77 (m, 1H, H4'), 63.67-3.51 (m, 6H, H5 ', 4-pyrrolidine-Hs), 62r24 (t, 2H, H2, J = 6.08Hz) 62.14 (s, 3H, 5-CH3), 61.87-1.78 (m, 4H, pyrrolidine).

Elemental analysis of 5 ^ 14 ^ 20 ^ 6 ^

Calculated: C, 52.49; H, 6.29; N, 26.23 Found: C, 52.37; H, 6.33; N, 26.17

Example 60: 1- (31-Azido-2 ', 3 * -dideoxy-N-D-erythro-pentofuranosyl) -4-benzyloxy-5-methyl-2- (1H) -pyrim-10-midinone

5'-Acetyl-3'-azido-3'-deoxy-4- (1,2,4-triazolyl) thymidine dissolved in dry acetonitrile was added dropwise at ambient temperature under a nitrogen atmosphere over 5 minutes to a suspension of benzyl alcohol (5 equivalents). ) and potassium t-butoxide (2 equivalents) in dry acetonitrile. The reaction mixture was stirred for one hour and the solvents were removed in vacuo. The residue was applied to a silica gel column and eluted with CHCl 3 / EtOAc (3: 1 (v / v)). The appropriate fractions were collected, combined and evaporated to a solid. The solid was recrystallized from CHCl 3 / EtOAc (1: 1 v / v) and the resulting crystals were filtered off to give the title compound; mp. = 133-134 ° C.

UV (nm) at pH λmax = 276 (£ = 8000), λ min = 237 (£ = 2000); 25 at pH 13 λ max = 281 (£ = 81 00), Tyrnin. = 23 7 (£ = 1600).

1 H NMR (DMSG-d 6) 68.05 (s, 1H, H 6), 67.45-7-35 (m, 5H, phenyl, 66.07 (t, 1H, H 1 ', J = 5.94 Hz), 65.35 (s, 2H) benzyl, 65.27 (t, 1H, 5'GH, J = 5.20Hz), 64.41-4.31 (m, 1H, H3 ') 63.91-3.83 (m, 1H , H4 '), 63.7-3.6 (m, 2H, H5'), 62.4-2.3 (m, 2H, H2 '), 61.9 (s, 3H, 5-CH3).

Elemental analysis for 30 C 19 H 21 N 5 O 5

Calculated: C, 57.14; H, 5.36; N, 19.60 Found: C, 57.06; H, 5.37; N, 19.58

Example 61: 51-Acetyl-31-azido-5-bromo-21,31-dideoxyuridine 3'-Azido-2 ', 3'-dideoxyuridine was acetylated and brominated by the method of Visser (Synthetic Procedures in Nucleic Acis Chemistry, 1, 41 0) to give the title compound; mp. = 112-114 ° C.

72 90664 UV (nm) at pH 1 λπ ^ β. = 279.21 O (£ = 9500.10600), i \ min. = 243 (£ = 2000); at pH 13 'Xmax. = 276 (£ = 700), \ min. = 250 <£ = 4000). 1 H NMR (DMSO-d 6) 611.88 (s, 1H, NH), 68.02 (5.1H, H 6), 66.08-6.01 (πη, ΙΗ, ΗΓ), δ 64, / 17-4 .40 (m, 1H, H3 ') f 64.27-4.24 (m, 2H, H5'), 64.03-4.00 (m, 1H, H4 '), 62.41-2.34 (m, 2H, H 2 '), 62.09 (s, 3H, acetyl).

Elemental analysis of C ^ H ^ 2N ^ 0 ^ Br

Calculated: C, 35.31; H, 3.23; N, 18.72; Br 21.36 Found: C, 35.29; H, 3.25; N, 18.66; Br, 21.45 Example 62: 1- (31-Azido-21,31-dideoxy-N-D-threopentofuranosyl-5- (trifluoromethyl) uracil 5'-hydroxyl group of t-trifluoromethyl-2'-deoxyuridine was protected with a triphenylmethyl group and the 3'-hydroxyl group was mesylated The product (3.0 g, 4.9 mmol) was reacted at 0.7 ° C with 0.7 g of LiN 2 in 50 mL of DMF for about The cooled reaction mixture was poured onto ice with stirring, the solid was isolated, washed with water and purified by flash chromatography using hexane / ethyl (2: 1 v / v) as eluent to give the appropriate fractions and removal of the solvent to give 0.83. This was dissolved in a saturated solution of zinc bromide in acetonitrile and the solution was stirred overnight at 0. After the addition of 100 ml of ammonium acetate (1-M), the organic layer was separated and dried in vacuo. chromatography using CHCl 3 / CH 2 OH as eluent (20: 1 v / v) The fractions were combined and dried in vacuo. The yield of the title compound was 0.25 g, 0.8 mmol.1, 5.3%; mp. = 118-120 ° C.

Elemental analysis of 30 qH ^ qF ^ Nj-O ^

Calculated: C, 37.39; H, 3.14; N, 21.80; F, 17.74 Found: C, 37.3 L; H, 3.23; N, 21.75; F, 17.60

Example 63: 1- (31-Azido-21,31-dideoxy-N-D-tropopofuranosyl) uracil 35 The 5'-hydroxyl group of 2'-deoxyuridine was protected and the 3'-hydroxyl group was mesylated. The 31-mesyl group was removed by reshaping by heating 73,90664 in dimethylformamide at 80 ° C with lithium azide (3 eq.) For 24 hours. The reaction mixture was poured into ice water and the product precipitated. After filtration, the wet product was deprotected. Final purification was performed by silica gel chromatography-eluting with chloroform / methanol (95: 5). The appropriate fractions were combined and the solvents removed to give the title compound as a solid; mp. = 14 2-145 ° C.

Example 64: 1- (31-Azido-2 ', 3'-dideoxy-N-D-erythro-10-pentofuranosyl) uracil The 5'-hydroxyl group of 2'-deoxyuridine (30 g, 0.13 mol) was tritylated Horwitzin et al. by the method of J. Org. Chem., 3, 205, (1966). The 3'-hydroxyl group (12.6 g, 0.027 mol) was chlorinated by the method of Example 62. The dimethylacetamide was removed in vacuo and the thick oil was poured into water (500 The product was extracted with ether (3x) The solvent was removed and the resulting oil was chromatographed on silica gel eluting first with dichloromethane and then with 1% methanol in dichloromethane. Additional purification by heating in 80% acetic acid on a steam bath for 20 minutes Upon cooling, the tritylcarbinol precipitated and was filtered off, the filtrate was concentrated in vacuo and treated with silica gel chromatography eluting with ethyl acetate (3M). at 90 ° C overnight The reaction mixture was poured into water and extracted with chloroform. in a portion dried over MgSO 4.

Removal of chloroform gave a solid which was recrystallized first from ethyl acetate / methanol and then from water. The recrystallized solid was dissolved in water and applied to an XAO column. After washing with water, the product was eluted with ethanol. The ethanol was removed in vacuo to give a solid; mp = 166.5 - 168.5 ° C.

74 90664

Example 65: 1- (3, -Azido-2,3,3-dideoxy-erythro-β-D-pentofuranosyl-5-ethyl) uracil 5-Chloromercuric-2'-deoxyuridine was prepared from 2'-deoxyuridine (10 g, 0.044 mol) By the method of Bergstron and 5 Ruth (J. Carb. Nucl., And Nucl. E, 257, (1977)). 2'-Deoxy-5-ethyluridine was prepared according to Berstrom et al. method (J. Am. Chem. Soc., 100, 8106, (1978)). The 5'-hydroxyl group was protected by the method of Example 62. The 3'-hydroxyl group was chlorinated and the 5'-10 hydroxyl was deprotected by the method of Example 64.

The 3'-chloro of threo-31-chloro-2 ', 3 1-dideoxyuridine was removed by conversion to HMPA by heating in lithium azide (5 eq.) At 55 ° C for one hour. The title compound was purified by silica gel chromatography using ethyl acetate as eluent. Removal of the solvent from the appropriate fractions gave the desired compound; mp = 112.5-115 ° C.

Example 66; 1- (3 1-Azido-2 ', 31-dideoxy-5S-D-threo-pentofuranosyl) -5- (2-bromovinyl) uracil 5-Bromovinyl-2'-deoxyuridine (BVDU) was synthesized

Jonesin et al. method (Tetrahedron Letters £ 5, 4415 (1979)) in corresponding yields. BVDU was tritylated and mesylated according to Horowitz et al. method (J. Org. Chem., 31, 205 (1966)). This product (4.25 g, 6.5 mmol) was dissolved in 100 mL of DMF with 0.955 g (19.5 mmol) of LiN 2 and the solution was heated at 74 ° C for 24 h. The reaction mixture was poured into 600 ml of ice with stirring. The resulting solid was isolated and purified by chromatography. . cumin (2.48 g). Dissolve the product in 100 ml of 80% acetic acid and heat in a steam bath 3

the compound was deprotected for 1 hour. The cooled reaction mixture was diluted with water and filtered to remove tritylcarbinol. The volume of the filtrate was reduced to an oil which was purified by flash chromatography using CHCl 3 / CH 2 OH as eluent.

mixture (9: 1 v / v). Combining the appropriate fractions and removing 75,90664 of solvent afforded a solid which was crystallized twice from aqueous methanol. Yield 0.66 g, 1.8 mmol, 27.7%; mp. = 165-166 ° C.

C 1 H 12 Br 2 O 5 • 0.5.5 Elemental Analysis Calculated: C, 35.98; H, 3.57; N, 19.07; Br, 21.85 Found: C, 35.98; H, 3.57; N, 19.07; Br, 21.76

Example 67: 1- (3'-Azido-2,3,3-dideoxy-1H-threo-pentofuranosyl) -5-methylisocytosine 1- {3 * -azido-2 ', 3'-dideoxy D-threo-pentofurano-10-yl) -2-methoxy-5-methyl-4 (H) -pyrimidinone (0.5 g, 2 mmol) was combined with 15 mL of ammonia saturated methanol and placed in a bomb. After the bomb was at ambient temperature for six days, it was heated in an oil bath at 65 ° C for four days.

The reaction mixture was dried and purified by crystallization from CHCl 3 / CH 2 OH (9: 1 v / v). The solid was washed with CHCl 3 and air dried to give 0.16 g, 0.6 mmol (30%) of product; mp. = 158-160 ° C.

Elemental Analysis for C 18 H 18 N 2 O 3/4 H 2 O Calcd C, 44.36; H, 5.40; N, 31.04

Found: C, 44.42; H, 5.36; N, 31.04

Example 68: 1- (31-Azido-21,31-dideoxy-β-D-threo-pentofuranosyl) -3-methylthyrine 5'-Trityl-3'-azido-3'-deoxythymidine (1.0 g, 25 L , 95 mmol) and N, N-dimethylformamide dimethyl acetal (Zemlicka, Coll. Czech. Chem. Comm., 35-3572 (1972)) (0.93 g, 7.8 mmol) were refluxed in 50 mL of CHCl 3. For 96 hours. Removal of the solvent gave an oil which was further purified by flash chromatography using CHCl 3 as eluent. Removal of the solvent gave 0.54 g of foam. The protection was removed by heating the foam in 50 ml of 80% acetic acid in a steam bath for two hours. The tritylcarbinol was removed by filtration after diluting the reaction mixture with water. The filtrate was evaporated to an oil in vacuo and the oil 76 90664 was purified by flash chromatography using CHCl 3 / EtOAc (2: 1 v / v) as eluent. The solvent was removed to give the title compound as an oil, 0.20 g.

UV max (nm) at pH 1 λ max = 267 (£ = 8100), λορ = 209 δ (£ = 8500); at pH 13 Λ max = 267 (£ = 8000).

1 H NMR (DMSO-d 6): δ 7.56 (s, 1H, H 6); Δ 6.07 (dd, 1H, Hr); <£ 3.17 (s, 3H, N-CH 3); (£ 1.86 (s, 3H, 5-CH 3)).

Elemental Analysis for H 2 O 2 N 2 O 2 -0.4 HOAc-0.3 H 2 O Calcd C, 45.62; H, 5.58; N, 22.54

Found: C, 45.67; H, 5.60; N, 22.57

Example 69: (E) -3- [1- (3'-Azido-2,3,3'-dideoxy-N-D-erythro-pentofuranosyl) -1,2,3,4-tetrahydro-2,4-dioxo -so-5-pyrimidinyl] -2-propenoic acid 2'-Deoxyuridine was converted to the 5-chloromercuric derivative by the method described in the literature in 96% yield (Bergstrom and Ruth, J. Carb. Nucl. 4, 267, (1977)). This product (50 g; 1.04 mol) was dissolved in 800 ml of dry MeOH containing ethyl acrylate (104 g; 1.04 mol) and a 0.1 N solution of Li 2 PdCl 2 in 0.1 N MeOH-1 (1040 ml). ). The solution was stirred for four hours and then treated with H 2 S for two minutes. The suspension was filtered through a pad of celite and the filtrate was evaporated to dryness in vacuo. The residue was triturated with MeOH to give a solid which was filtered and dried to give 22 g of a white solid. This product was tritylated and mesylated according to Horowitz et al. method (J. Org. Chem., 31, 205 (1966)). A portion of this material (7.06 g; 10.9 mmol) was dissolved in 100% dry MeOH with NaHCO 3 (0.92 g, 10.9 mmol) and refluxed for 6 h.

The solvents were removed in vacuo and the residue was taken up. with water, filtered and air dried to give 6.1 g of a white solid. This product was dissolved in a mixture of 50 mL of DMF and 1 mL of water with 35 LiN 3 (1.63 g; 33.2 mmol) and the solution was heated at 125 ° C for 4 h. The reaction mixture was poured into 77,90664 200 ml of ice, the precipitate was collected and washed with water. This material was dissolved in 100 mL of 80% HOAc and the solution was heated at 100 ° C for four hours. The reaction mixture was diluted with water and the precipitate was filtered off. The filtrate was evaporated to dryness to give 800 mg of an oil, the oil was dissolved in 20 ml of 0.5 N NaOH and the solution was stirred at ambient temperature for 2 hours. The pH of the solution was adjusted to 3, the precipitate was filtered off and air dried to give 550 mg (1.7 mmol) of the ot-10 compound: m.p. > 250 ° C.

UV (nm) at pH 1Λ max. = 300 (£. = 20400), Amin. = 230 (£ = 3900), \> p = 262 (£ = 13100); at pH 13 Xmax = 297.266 (E = 15600.14800), A min = 281.288 (δ = 13600.8600); 1 HXNMR (DMSO-d 6) 68.37 (s, 1H, H 6), 67f30 (s, 1H, -CH =, δ = 15.57 Hz), 66.78 (d, 1H, = CH-COOH, δ = 15.93 Hz), 66.1-6.06 (ηη, ΙΗ, ΗΓ), 65.41.5.37 (m, 1H, 5'H), 64.47-4.39 (m, 1H, H3 '), 63 , 87-3.83 (m, 1H, H4), 63.74 3.58 (m, 2H, H5 '), 62.54-2.81 (m, 2H, H2').

2 ^ ^ 3Nc ° 6 * n Elemental analysis Calculated: C, 44.59; H, 4.05; N, 21.67

Found: C, 44.45; H, 4.06; N, 21.60

Example 70: (E) -3-Z1- (31-azido-2 ', 31-dideoxy-N- [D-threo-pentofuranosyl) -1,2,3,4-tetrahydro-2,4-dioxo-] -pyrimidinyl] -2-propenoic acid 2'-Deoxyuridine was converted to a 5-chloromercuric derivative by the method described in the literature in 96% yield (Bergstrom and Ruth, J. Carb. Nucl. Nucl., £ 257 (1977)). This product (50 g, 1.04 mol) was dissolved in 800 mL of dry MeOH with ethyl acrylate 30 (10.4 g, 1.04 mol) and a 0.1 N solution of Li 2 PdCl 3 in MeOH ( 10.40 ml). The solution was stirred for four hours and then treated with H 2 S for two minutes. The suspension was filtered through a pad of celite and the filtrate was evaporated to dryness in vacuo. The residue was triturated with MeOH to give a solid which was filtered and air dried to give 22 g of a white solid. This product was tritylated and mesylated according to Horowitz et al. method (J. Org. Chem., 31, 205 (1966)). A portion of this material (2.7 g; 4.2 mmol) was dissolved in dry DMF (55 mL) if there was LiN-α (0.62 g; 12.7 mmol) and the solution was heated to 70 ° C. in a nitrogen atmosphere for 24 hours. The reaction mixture was poured into 400 ml of ice, the precipitate was collected and purified by flash chromatography. Elution with CHCl 3 / MeOH (50: 1 v / v) afforded 1.48 g of the azido intermediate. These substances were treated with 50% acetic acid (100 ml) at 100 ° C for 3 hours. Dilution with water, filtration of the resulting suspension and evaporation of the filtrate in vacuo afforded 710 mg of an oil. This oil was dissolved in 50 mL of NaOH solution, which was stirred at ambient temperature for 2 hours.

The pH of the solution was adjusted to 3, the precipitate was filtered off and air dried to give 240 mg (0.7 mmol, 50%) of the title compound; mp. = 250 ° C.

UV (nm) at pH 1 Λ max = 301 (£ "= 19500), Amine = 230 (£ = 3600), λορ = 249 (£ = 12,700); pH 1 3: max. = 299,267 (£ = 1400.1 3200) min = 232.239 (E = 1200.7560) HXNMR (DMSO-d 6) 63.13 (s, 7H, H 6) 67.32 (d, 1H, -CH =, δ = 15.87 Hz ) 66.78 (d, 1H, = CH-COOH, J = 15.62Hz), 66.01-5.98 (m, ΙΗ, ΗΓ), 65.11-5.98 (m, 1H, 5 ' OH), 64.50-4.43 (m, 1H, H3 '), 64.13-4.08 (m, 1H, H4'), 63.80-3; 75 (m, 2H, H5 '), 62 Elemental analysis for C 12 H 13 H 5 O 6 · 1 • 5 H 2 O Calcd C, 41.15; H, 77-3.67 '25 (m, 1H, H 3') δ 6-30-2.20 (m, 1H, H 2 ·) 4.60; N, 19.99 Found: C, 41.38; H, 4.50; N, 20.01

Example 71: (E) -3- [1- (3'-Azido-2,3,3-dideoxy-30} -D-erythro-pentofuranosyl) - (2,3,4-tetrahydro-2,4- dioxo-5-pyrimidinyl-2-propanoic acid 5'-trityl-3'-mesyl-5-propenoate-2, -deoxyuridine was prepared according to the method of Example 69. This material was hydrogenated with 10% Pd / C in EtOH. This product was then treated as described in the above procedure to give the title compound, mp = 118-120 ° C.

UV (nm): at pH 1A max = 265 (5 = 9900), maternal. = 235 (δ = 3100); at pH 13 3 max = 265 (£ = 7400), min min = 247 (£ = 5400); Δ H 1 NMR (DMSO-d 6) δ 7.68 (s, 1H, H 6), δ 6.11-6.06 (m, ΙΗ, ΗΙ '), δ 4.45-4, .35 (m, 1H, H3 '), δ 3.85-3.80' m, 1H, H4 '), δ 3.68-3.53 (m, 1H, H5') 5N5 ° 6 Elemental analysis Calculated: C, 44.31 ; H, 4.65; N, 21.53 Found: C, 44.26; H, 4.68; N, 21.49

Claims (6)

R 4 is alkyl or hydrogen; provided that when R 1 and R 3 are each hydroxy and R 2 is hydrogen, R 4 is not hydrogen or methyl; and pharmaceutically acceptable salts and organosulfonates thereof, characterized in that the compound of formula (A) R 3 r 2 I A 30 I; : un) d 1 / R 2 H 0 0 ° / 35) M is not 90664 wherein R 1, R 2, R 3 and R 4 are as defined above and M is halogen, hydroxy or an organosulfonyloxy radical, is reacted with an alkali metal azide; (B) a compound of formula 5 R3 a R2 v. R4 ϋ y κ (IV) N3 wherein Rxa, R2a and R3a respectively represent R1, R2 and R3 or precursor groups thereof, provided that at least one of R1, , R 2a and R 3a are a precursor group, and R 4 is as defined above, is reacted with a substance or under conditions in which said precursor group (s) are converted into the corresponding desired groups, wherein the substance or conditions are as follows: a) according to formula IV a compound wherein R 3a is a 2,51-O-anhydro bond is reacted with an alkoxide in the presence of potassium carbonate to form the corresponding compound wherein R 1 is C 1-4 alkoxy; b) reacting a compound of formula IV wherein R 2a is hydrogen with an acylating agent to form a corresponding compound wherein R 2 is an acyl group; c) a compound of formula IV wherein R 3a is 1,2,4-. 30 triazolyl group, is reacted with an alkoxylating agent or pyrrolidine to form the corresponding compound, wherein R3 is an alkoxy group or a pyrrolidinyl group, respectively. 82 90664 For the preparation of therapeutically active compounds according to formula 5 or 'j) 1ST 10 / (I) B HOp / 15 in which R1 Sr hydroxyl or alkoxy; R2 is an acyl or acyl; R3 is hydroxy, pyrrolidinyl or alkoxy; R4 is alkyl or a residue; under the action of R1 and R3 are hydrogen and R2 are hydrogen, R4 is inert or methyl; and the pharmaceutical composition of the salt and organosulphonate of the compound, as defined in (A) and for the purposes of formula 25 R3 li: (III) 30. N ”γ. / 35 M 83 90664 and R1, R2, R3 and R4 are selected from the group consisting of halogen, hydroxy and organosulfonyloxy radicals, cyclically reacted with alkali metal alkaloids; (B) in the form of 5 R3 and „2 1 4 Ra R li 10 Ra N (IV) H0 "^ .o N3 15. a plurality of R3a, R2a or R3a is represented by a representative group R1, R2 and R3 or a precursor group under the conditions of the group R3a, R2a and R3a is a precursor group, and R4 is selected from the group consisting of reactants having an acid group or a mixture of 20 or more precursors of the same group of compounds, as defined above, and having the following precursors: Form IV, in which R is a 2, S 2 O-anhydrous compound, a ring which reacts with an alkoxide 25. A potassium carbonate for which a mixture is present, in which R 1 is a C 1-4 alkoxy; Form IV, wherein R2a is used, the ring is reacted with an acylated group to form a compound of formula, wherein R2 is an acyl group; 30 c) a compound of formula IV, wherein R3a is a 1,2,4-triazolyl group , the ring is reacted with an alkoxy-free medium or a pyrrolidine for In this case, R3 is an alkoxy group or pyrrolidinyl group. 35