MC1766A1 - PROCESS FOR THE PREPARATION OF 3'-AZIDO-NUCLEOSIDES AND PROCESS FOR THE PREPARATION OF PHARMACEUTICAL FORMULATIONS CONTAINING THEM - Google Patents

Description

The present invention relates to 3'-azido-nucleosides, their pharmaceutically acceptable derivatives, combinations containing them and their use in therapy, in particular for the treatment or prophylaxis of certain viral and bacterial infections.

In the field of chemotherapy, there are few drugs that effectively combat the virus itself, due to the difficulty of attacking the virus while leaving uninfected host cells intact. It was long assumed that, because of the highly parasitic nature of viruses, all the means necessary for viral replication were provided by the host cell. It has recently been established that certain stages of the viral life cycle, which vary from one species to another, are determined by the virus itself, and these stages can prove susceptible to attack if they differ sufficiently from any corresponding function of the host cell. However, because of the great similarity between the functions of the virus and the host, finding effective treatments has proven very difficult.

For this reason, compounds recognized as suitable for treating viral infections usually have some toxicity to the host. Thus, the ideal medication is non-toxic at concentrations effective against a virus, but in the absence of such treatment,

I

the compound must have a good therapeutic ratio, that is to say that the concentrations at which the treatment is toxic are much higher than those at which antiviral activity is observed.

One group of viruses that has recently gained particular importance is that of retroviruses. Retroviruses constitute a subgroup of RNA viruses that, in order to replicate, must first perform "reverse transcription" of their RNA genome into DNA (transcription conventionally refers to the synthesis of RNA from DNA). Once it is in DNA form, the genome

The viral DNA can be incorporated into the host cell's genome, allowing it to fully exploit the host cell's transcription/translation mechanisms for replication. Once incorporated, the viral DNA is virtually indistinguishable from the host DNA, and in this state, the virus can persist as long as the cell lives. Because it is practically impossible to attack in this form, any treatment must target another stage of the virus's life cycle and must necessarily be continued until all virus-infected cells are dead.

HTLV-I and HTLV-II are two retroviruses known to cause leukemia in humans. HTLV-I infections, in particular, are very widespread and cause numerous deaths worldwide each year.

A retrovirus species has also been reproducibly isolated from subjects with AIDS. Although it has been largely characterized, there is still some disagreement over giving the virus an internationally recognized name. This virus is commonly known as T-lymphocyte-associated virus (HTLV-III), AIDS-associated retrovirus (ARV), or lymphadenopathy-associated virus (LAV), but it is predicted that the internationally accepted name will be "human acquired immunodeficiency virus" (HIV). This virus (here referred to as HIV) has been shown to preferentially infect and destroy T lymphocytes bearing the OKT surface marker and is now generally recognized as the causative agent of AIDS. The patient progressively loses this group of T lymphocytes, which disrupts the overall balance of their immune system, reduces their ability to fight other infections, and predisposes them to portosystemic infections that are frequently fatal. Thus, the usual cause of death among AIDS victims is

Opportunistic infections, such as pneumonia or cancers, can be caused by a virus, but death is not necessarily a direct consequence of HIV infection. Other conditions associated with HIV infection include thrombocytopenic purpura and Kaposi's sarcoma.

Other tissue types, including T4-positive B lymphocytes, macrophages, and non-blood-associated tissue of the central nervous system, are affected. This central nervous system infection is not necessarily associated with classic AIDS and has been observed in patients with asymptomatic HIV infections. HIV infection of the central nervous system is associated with progressive demyelination, which leads to losses and symptoms such as encephalopathy, progressive dysarthria, ataxia, and disorientation. Other conditions associated with HIV infection include the healthy carrier state, progressive generalized lymphadenopathy (GLL), and AIDS-associated syndrome (AAS).

are now considered to be caused by retroviruses. These infections include, for example, multiple sclerosis in humans, caprine arthritic encephalitis virus 25 infections in goats, and Visna-Maedi infections in sheep.

compounds against various retroviruses, for example Friend leukemia virus (VLF), a murine retrovirus. By example, Krieg et al. (Exp. Cell Res., 116 (1978) 21-29) found that 3'-azido-3'-dideoxythymidine is active against VLF in in vitro experiments, and Ostertag et al. (Proc. Nat. Acad. Sci. (1974) 71, 4980-85) stated that, based on the relative antiviral activity against VLF and an absence of cellular toxicity, 3'-azido-3'-dideoxythymidine "might advantageously act against bromodeoxy-

Recently, AIDV was also found in

Some chronic neurological infections

Reports described the testing of

5"

uridine in the medical treatment of diseases caused by DNA viruses." However, De Clerq et al. (Biochem. Pharm. (1980) 29_, 1849-1851) established six years later that 3'-azido-31-deoxythymidine did not exhibit appreciable activity against any of the viruses used in their tests, including DNA viruses such as vaccine virus, HSVI and varicella herpesvirus.

Bacteria also present a problem in therapy because all organisms mutually employ extremely similar vital processes, meaning that a substance toxic to one will likely be toxic to another. Furthermore, experience has shown that, over time, strains of bacteria develop that are resistant to commonly used antibacterial agents.

It has now been discovered that certain 3'-azidonucleosides, as described below, are useful in the therapy of viral and bacterial infections, including retroviral infections, such as HIV infections, and infections caused by Gram-negative bacteria, including certain strains of Gram-negative bacteria resistant to commonly used antibacterial agents.

Thus, according to a first aspect of the present invention, a compound of formula is provided

25 wherein A is a purine or pyrimidine base other than thymine, linked to position 9 or 1, or a pharmaceutically acceptable compound of this compound, intended for use in human or veterinary therapy.

6

The compounds of formula (I) and their pharmaceutically acceptable derivatives are referred to herein as compounds according to the invention.

Retroviral infections frequently affect the central nervous system of the subject and, from this point of view, a particular advantage of the compounds according to the invention lies, as experience has demonstrated, in their ability to cross the blood-brain barrier in clinically effective quantities.

10 It has been observed that the compounds according to the invention possess particularly potent activity against retroviral and Gram-negative bacterial infections.

Therefore, it is provided (a) the compounds according to the invention for use in the treatment or prophylaxis of retroviral or Gram-negative bacterial infections and (b) the use of the compounds according to the invention in the manufacture of a drug for the treatment or prophylaxis of a retroviral or Gram-negative bacterial infection.

20 The expression "retroviral infections", as used here, refers to any virus that,

for an integral part of its life cycle, it uses reverse transcriptase.

Specific bacteria against which good activity has been observed include: Escherichia coli, Salmonella dublin, Salmonella typhosa, Salmonella typhimurium, Shigella flexneri, Citrobacter freundii, Klebsiella pneumoniae, Vibrio cholerae, Vibrio anquillarum, Enterobacter aeroquenes, Pasteurella multocida, Haemophilus influenzae, Yersinia enterocolitica, Pasteurella haemolytica, Proteus mirabilis and Proteus vulgaris. These microorganisms are responsible for diseases such as traveler's diarrhea, urinary tract infections, shigellosis, typhoid fever and cholera in humans, as well as animal diseases such as neonatal enteritis in calves, post-weaning enteritis in pigs and colisepticemia in chickens.

The compounds according to the invention exhibit particularly good activity against the following viruses: human T-lymphocyte-associated viruses (HTLV), including HTLV-I, HTLV-II, and HTLV-III (HIV); feline leukemia virus, equine infectious anemia virus, caprine arthritis virus, and other lentiviruses, as well as against other human viruses such as hepatitis B virus, Epstein-Barr virus (EBV), and the causative agent of multiple sclerosis (MS). The compounds according to the invention have also been found to be effective in the treatment of Kaposi's sarcoma (KS) and thrombocytopenic purpura (TP).

For these latter indications (SP, SK and PT) and the caprine arthritis virus, the present invention encompasses compounds of formula (I), in which A is thymine, intended to be used in their treatment or prophylaxis, as well as the use of such compounds in the manufacture of a drug for their treatment or prophylaxis.

The activity of the compounds according to the invention against such a wide range of viral and bacterial infections clearly constitutes a major advantage in medicine, and the novel mode of action allows the use of these compounds in combination therapy to reduce the chances of resistance developing. These combinations are particularly useful because 3'-azidonucleosides exhibit a surprising capacity for potentiation by other therapeutic agents, as described below.

It has been discovered that 3'-azidonucleosides cooperate synergistically with a wide range of other therapeutic agents, thereby increasing the therapeutic potential of both agents disproportionately. Significantly less of each compound is required for treatment, the therapeutic ratio is increased, and consequently, the risk of toxicity from either compound is reduced.

%

Therefore, according to another aspect of the present invention, a compound of formula is provided:

in which B is a purine or pyrimidine base linked to position 9 or 1 respectively, or a pharma-5 ceutically acceptable derivative of this compound, intended for use in combination therapy with at least one other therapeutic agent.

In particular, a compound of formula (I)A, or a pharmaceutically acceptable derivative of this compound, is provided, intended for use in combination therapy as described above, the two active agents being present in a potentiating ratio.

A "potentiating ratio" is the ratio of the compound of formula (I)A, or a pharmaceutically acceptable derivative of that compound, to the second therapeutic agent.

which provides a greater therapeutic effect than the sum of the therapeutic effects of the individual components.

It should be noted that, although there is usually an optimal ratio to ensure maximum potentiation, even an extremely small amount of one agent will suffice to potentiate the effect of the other to some degree, and therefore any ratio of two potentiating drugs will exhibit the required synergistic effect. However, the strongest synergy is observed when the two agents are present in a ratio of 500:1 to 1:500, preferably 100:1 to 1:100, especially 20:1 to 1:20, and even better 10:1 to 1:10.

The present invention therefore provides yet another therapeutic combination of a compound of formula (I)A,

*

or a pharmaceutically acceptable derivative of that compound, and at least one other therapeutic agent.

The associations described above are referred to herein as associations according to the invention.

5 Thus, it is further proposed an association according to the invention intended to be used in the treatment or prophylaxis of any of the infections or indications mentioned above, or the use of such associations in the manufacture of a drug for the treatment 10 or prophylaxis of such an infection or indication.

The combinations according to the invention can conveniently be administered together, for example in a unitary pharmaceutical formulation, or separately, for example in a combination of tablets and injections administered at the same time or at different times, in order to obtain the desired therapeutic effect.

In antiviral tests, it has been found that, for example, azidonucleosides are potentiated by various agents such as interferons, nucleoside trans-20 transport inhibitors, glucuronidation inhibitors, renal excretion inhibitors, and even by other therapeutic nucleosides that may not necessarily be active against the same microorganisms as compounds of formula (I)A. 25 Particularly preferred types of interferon are types a, β, and γ, while nucleoside transport inhibitors include agents such as dilazep, dipyridamole, 6-(4-nitrobenzoyl)thio7-9-(0-D-ribofurannosyl)pyrine, papaverine, mioflazine, hexobendine, lidoflazine, and their acid addition salts.

30 Probenecid is particularly useful in combination with 31-azidonucleosides of formula (I)A, as it possesses both renal excretion-inhibiting and glucuronidation-inhibiting activity. Examples of other compounds useful in this respect include 35 acetaminophen, aspirin, lorazepam, cimetidine, ranitidine, zomepirac, Clfibrate, indo-

I®

methacin, ketoprofen, naproxen and other compounds that compete in the glucuronidation mechanism or otherwise undergo significant glucuronidation.

5. 31-Azidonucleosides of formula (I)A and their pharmaceutically acceptable derivatives are potentiated by other therapeutic nucleoside derivatives as described above, including acyclic nucleosides of the type described, for example, in UK Patent No. 1,523,865, US Patent No. 4,360,522, European Patents Nos. 74,306, 55,239 and 146,516 and European Patent Applications Nos. 434,393 and 434,395, and in particular compounds of general formula:

z n

\

/ (HAS)

n /

I

chx çhch oh y in which Z represents a hydrogen atom or a hydroxyl or amino group; and (a) X represents an oxygen or sulfur atom or a methylene group and Y represents a hydrogen atom or a hydroxymethylene group, or (b) X represents an oxymethylene group (-O.CE^) and Y represents a hydroxyl group,

20 and pharmaceutically acceptable derivatives of these compounds.

Examples of the aforementioned derivatives are salts and esters, and these include basic salts, for example, salts of alkali metals (such as sodium) or salts of alkaline earth metals, and pharmaceutically acceptable salts of organic acids such as lactic, acetic, malic, and p-toluenesulfonic acids, as well as pharmaceutically acceptable salts of mineral acids.

n

10

15

20

25

30

The esters of compounds of formula (A) that can be conveniently used according to the present invention include those containing a formyl-oxy or alkanoyloxy ester group at C-C16 (e.g., acetoxy or propionyloxy), optionally substituted alkanoyloxy (e.g., phenyl (alkanoyloxy such as p-benylacetoxy), or optionally substituted aroyloxy (e.g., benzoyloxy or naphthoyloxy), present at one or both terminal positions of the side chain located at position 9 of the compounds of formula (A). The aforementioned alkanoyloxy and aroyloxy ester groups may be substituted, for example, by one or more halogen atoms (e.g., chlorine or bromine) or amino, nitrile, or sulfamido groups, the aryl fragment of the group advantageously containing 6 to 10 carbon atoms.

Particularly preferred examples of compounds of general formula (A) above, for use according to the present invention, include 9-[(2-hydroxy-1-hydroxymethylethoxy)methyl]guanine, 2-amino-9-(2-hydroxyethoxymethyl)purine, and in particular 9-(2-hydroxyethoxymethylguanine (acyclovir). The latter compound has been found to exert a particularly good potentiating effect, especially when the compound of formula I(A) is 3'-azido-3'-deoxythymidine, as described in the Examples.

In the field of antibacterials, a wide range of antibiotics have also been found to be effective in potentiating the activity of azidonucleosides. These antibiotics include various agents such as: benzylpyrimidines, for example 2,4-diamino-5-(3',4',5'-tri-methoxybenzyl)pyrimidine (Trimethoprim) and its analogues, for example as described in UK Patent No. 1,405,246; suifonamides, for example sulfadimidine; rifampicin; tobramycin; fusidic acid; chloramphenicol; clindamycin and erythromycin.

Thus, from another perspective, it is proposed that

associations according to the invention in which the second agent is at least one of the aforementioned antiviral or antibacterial agents or classes of agents.

Other combinations suitable for use according to the present invention include those in which the second agent is, for example, interleukin-II, suramin, phosphonoformate, HPA 23, 2',3'-dideoxynucleosides, for example 2',3'-dideoxycytidine and 2',3'-dideoxyadenosine, or drugs such as levamisole or thymosin so as to increase the number and/or enhance the function of lymphocytes, as appropriate.

It should also be noted that the compounds and combinations according to the invention can also be used in conjunction with other immune modulation therapies, including, for example, bone marrow transplantation and the administration of lymphocytes.

Some of the retroviral infections described above, for example AIDS, are commonly associated with opportunistic infections. Thus, it should be noted that, for example, a combination of 3'-azido-3'-deoxythymidine (AZT) and acyclovir is particularly useful in treating a patient with AIDS and an opportunistic herpes infection, while a combination of AZT and 9-C(2-hydroxy-1-hydroxymethylethoxy)methylguanine is useful in treating a patient with AIDS and an opportunistic cytomegalovirus infection.

Pyrimidines of formula (I) or (I)A generally preferred for use according to the present invention are those conforming to the formula:

/ n

w

10

15

20

25

30

ho

N3

in which R is a hydroxy, mercapto, amino, alkylthio, aralcoxy, alkoxy, cyano, alkylamino or dialkyl-amino group, the alkyl groups possibly being joined and thus forming a heterocycle,

R is hydrogen or an acyl, alkyl, aroyl or sulfonate group;

3

R is a hydroxy, mercapto, amxno, triazolyl, alkylamino, dialkylamino group, where the alkyl groups are possibly linked to form a heterocycle, aralkoxy, alkoxy or alkylthio;

R^ is an alkyl group, substituted alkyl, halogen, perha-

logenomethyl, hydroxy, alkoxy, cyano, nitro, alkenyl, alke-

substituted nyl, alkynyl, substituted alkynyl or hydrogen;

and their pharmaceutically acceptable derivatives.

In the general formula (II), the dashed lines at positions 2 to 6 are intended to indicate the presence of single or double bonds at these positions, the relative positions of the single or double bonds being

1 2

determined by the fact that the R and R substituents are tautomerism-capable groups, for example keto-enol.

Preferred classes of pyrimidine nucleoside according to the invention are cytidine derivatives, for example

3

Compounds of formula (II) in which R is an amino or alkylamine group, especially when the azido group at 3' is in the erythro ("azido down" configuration); thymidine and uridine derivatives (e.g., compounds of formula C11) in which R is something other than an azido group.

amino or alkylamine group) in which the azido group at 3' is in erythro or threo configuration ("azido on top"); and the unsaturated nucleosides between positions 5C and 6C.

Purines of formulas (I) and (I)A generally preferred for use according to the present invention correspond to the formula:

( II ) A

6 7

in which R and R may be identical or different and are chosen from among 11 hydrogen and the amino, hydroxy, mercapto, alkylthio, alkoxy, aralkoxy, cyano and alkylamino groups; and their pharmaceutically acceptable derivatives.

Preferred classes of purine nucleoside according to the invention are adenine derivatives, for example compounds of formula (II)A in which R is an amino group

15 or amino-substituted, and guanine derivatives, for example

* 6

compounds of formula (II)A in which R is as defined and is something other than an amino group or substituted amino group, 7

and R is an amino group or substituted amino group.

The aforementioned acyl groups include alkyl or aryl groups as described below. With regard to the compounds of formulas (II) and (II)A above, the aforementioned alkyl groups advantageously contain 1 to 8 carbon atoms, in particular 1 to 4 carbon atoms, for example, methyl or ethyl groups, possibly substituted by one or more substituents.

If appropriate, as described below. The aforementioned aryl groups comprising aryl fragments of groups such as aralkoxy are preferably phenyl groups optionally substituted by one or more appropriate substituents, as described below. The aforementioned alkenyl and alkynyl groups advantageously contain 2 to 8, particularly 2 to 4, carbon atoms, for example, ethenyl or ethynyl groups, optionally substituted by one or more appropriate substituents as described below.

Suitable substituents attached to the aforementioned alkyl, alkenyl, alkynyl, and aryl groups are advantageously chosen from among the halogen, hydroxy,

alkoxy in alkyl in C-C4, aryl in C-C2, alkoxy in cjrb°xyr, amino and substituted amino where the amino is

with single or double substitution by an alkyl group in Cet, when they are double substituted, the alkyl groups are eventually linked to form a heterocycle.

Other classes of preferred compounds of formula (II) include those in which one or more of r\ R^, R^ and R^ are as defined below, namely:

R^" is a hydroxy, mercapto, alkoxy group in C-^-C^ or amino;

R^ is hydrogen or a methyl, C-, -C9 alkanoyl or benzoyl group;

3

R is a hydroxy, mercapto, amino or amino substituted group;

4 3

R is hydrogen when R is an amino group or substituted amino group, and a halogen, perhalogenethyl, C2-C3 alkyl, C2-C3 alkyl, or substituted ethenyl group when R is something other than an amino group or substituted amino group.

and the 5' derivatives of these compounds, including straight-chain or branched-chain alkyl esters possibly substituted with carboxy groups (e.g. succinate), C-Cg thioesters, aryl esters possibly

if substituted, a mesylate, a glucuronide or mono-, di- or triphosphates.

Other classes of preferred compounds of formula (II)A include those in which: 5 is an amino, alkylamino group in C1-C4, mercapto, hydroxy or alkoxy group in C1-C4 and/or

R^ is an amino, alkylamino group in C1~C4 or hydrogen;

and 5' derivatives of such compounds, including 10 esters of straight-chain or branched-chain alkyls possibly substituted with carboxy groups (e.g. succinate), C^-Cg thio-esters, 10 esters of aryls possibly substituted, a mesylate, a glucuronide or mono-, di- or triphosphates.

15. Preferred compounds of formula (I) for use in the treatment or prophylaxis of viral infections are compounds of formula (II) in which:

R^" is a hydroxy, mercapto, alkoxy group in C^-Cg, amino, aralkoxy in Cg-.C^ or is a 20 O-anhydro bond linking positions 2 and 5';

2

R is a methyl or benzoyl group or hydrogen;

3

R is a hydroxy, mercapto, amino, substituted amino or aralkoxy group in Cg-C12;

25 R^ is as defined, provided that A in the formula

(I) not by a thymine residue;

and their pharmaceutically acceptable derivatives. The azido group is advantageously in the erythro configuration. Particularly preferred compounds are compounds such as de-

30 of the above, which differ only in one of the substi-1 4

R-R killers of the pyrimidine ring of those having a residue

1 3p of thymine (where R and R are hydroxyl groups, R is hydrogen, and R^ is a methyl group). Preferred compounds of formula (I) for use in the treatment or prophylaxis of viral infections are as described above regarding formula (I)—but encompass it

further target compounds of formula (I)A in which B is a thymine residue, and in particular when this compound is 1°L. 3'-azido-31-deoxythymidine.

The compounds of formula (I) that are preferred for use in the treatment or prophylaxis of bacterial infections are the compounds of formula (II) in which:

R"*" is oxygen, sulfur, or a benzyl-oxy or methoxy group;

2

R is hydrogen or the benzoyl group;

3

R is as defined above;

R is the methyl group or a halogen, provided that A in formula (I) is not a thymine residue; and their pharmaceutically acceptable derivatives.

The 3'-azido group can be in any configuration, but the erythro configuration is particularly preferred, and the preferred pharmaceutically acceptable derivatives are 5'-esters of simple organic acids, preferably C2-Ci8* acids

Particularly preferred compounds are those as described above that differ by only one of the 1 4

R-R substituents of the nucleus, of pyrimidine of those having a thymine residue (where R"'" and R^ are hydroxy groups,

2.. 4

R is the hydrogenated 1' and R is the methyl group).

The particularly preferred compounds of formula (I)A for use in the treatment or prophylaxis of bacterial infections are as described above with respect to formula (I) but also include compounds of formula (I)A in which B is a thymine residue, and especially when this compound is 3'-azido-3*-deoxythymidine.

The term "pharmaceutically acceptable derivative" means any pharmaceutically acceptable salt, ester, or salt of such an ester, or any other compound which, when administered to the recipient, is capable of providing (directly or indirectly) the original 3'-azidonucleoside or a

Al»

A therapeutically effective metabolite or residue thereof. An example of a compound that is not an ester is the derivative in which the 5'-C and 2-C atoms are linked by an oxygen atom to form an anhydrous group.

5 Preferred esters of compounds of formula (I)

These include carboxylic acid esters in which the non-carbonyl fragment of the ester group is selected from straight-chain or branched-chain alkyl groups, alkoxy-alkyl (e.g., methoxymethyl), aralkyl (e.g., benzyl), aryloxyalkyl (e.g., phenoxymethyl), aryl (e.g., phenyl, possibly substituted with a C2-C4' alkyl group, a C1-C1 alkoxy group, or a halogen); sulfonic esters such as those with alkylr- or aralkylsulfonylium groups (e.g., methanesulfonyl); and mono- to di- or triphosphoric esters. Any reference to any of the above compounds also includes a reference to a pharmaceutically acceptable salt of that compound.

With regard to the esters described above, unless otherwise specified, any alkyl fragment present advantageously contains 1 to 18 carbon atoms, in particular 1 to 4 carbon atoms. Any aryl fragment present in these esters advantageously comprises a phenyl group.

Examples of pharmaceutically acceptable salts of compounds of formula (I) and their pharmaceutically acceptable derivatives include base salts, derived, for example, from a suitable base such as alkali metal salts (e.g., sodium), alkali-earth metal salts (e.g., magnesium), ammonium salts, and NX^ (where X is an alkyl group).

C1~C4^ and '*"es se^s of mineral acids such as hydrochloride.

Specific examples of pharmaceutically acceptable derivatives of the compound of formula (I), which may be used according to the present invention, include the monosodium salt and the following 5'-esters: monophosphate; disodium monophosphate; diphosphate, triphosphate; ace-

H

tate; 3-methyl-butyrate; octanoate; palmitate; 3-chloro-benzoate; benzoate; 4-methyl-benzoate; hydrogen-succinate; pivalate; and mesylate.

Some of the compounds according to the invention are novel. Therefore, the present invention provides compounds with the following formulas:

in which C is a purine or pyrimidine base bonded to position 9 or 1 respectively, and their pharmaceutically acceptable derivatives, other than:

(a) compounds of formula (I)B in which C is an adenine, guanine, uridine, cytidine or thymine base, and their 5'-mono- or 51-triphosphoric esters;

(b) the 5'-O-acetate, 5'-O-trityl and 5'-O- derivatives

(4-methylbenzenesulfonate) of the compound of formula (I)B in

*

which C is a uridine base and the 3'-azido group is in erythro configuration;

(c) compounds of formula (I)B in which C is

(i) a residue of 5-bromovirtyluridine or 5-trifluoromethyluridine and the 3'-azido group is in erythro configuration;

(ii) a uridine residue and the 3'-azido group is in threo configuration; and (iii) a 5-iodo or 5-fluori-dine residue and the 3'-azido group is in erythro or threo configuration; and 5'-O-trityl derivatives of such compounds;

(d) compounds of formula (I)B in which C is (i) a residue of 5-bormovinyluridine or cytidine and the 3'-azido group is in threo configuration; (ii) a residue of 5-fluorocytidine and the 3'-azido group is in erythro configuration; or (iii) a residue of 5-methylcytidine and the 3'-azido group is in threo or erythro configuration;

(e) the 51-O-acetic esters of compounds of formula (I)B in which C is a 4-chloro-2(1H)pyrimidinone or a 4-(1H-1,2,4-triazole-1-yl)-2(1H)pyrimidinone (optionally substituted at position 5 by fluorine or a methyl group) and the 3'-azido group is in erythro configuration;

(f) the 5,-O-(4-methoxyphenyl)diphenylmethyl derivative of the compound of formula (I)B in which C is a cytidine residue and the 3'-azido group is in erythro configuration;

(g) the 5'-O-trityl derivative of the compound of formula (I)B in which C is an adenine residue and the 3'-azido group is in threo configuration.

The preferred compounds are those described above regarding therapy, other than those specifically excluded above.

The compounds of formulas (I) and (I)A, and their pharmaceutically acceptable derivatives, also referred to herein as active ingredients, may be administered for therapeutic purposes by any appropriate route, including oral, rectal, nasal, topical (including buccal and sublingual), vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, and intradermal). It should be noted that the preferred route differs depending on the recipient's condition and age, the nature of the infection, and the active ingredient chosen.

In general, an appropriate dose is 3.0 to 120 mg per kilogram of the recipient's body weight per day, preferably 6 to 90 mg per kilogram of body weight per day, and even better 15 to 60 mg per kilogram of body weight per day. The desired dose is preferably presented in two, three, four, five, or six or more subdoses, administered at appropriate intervals throughout the day. These subdoses may be administered in unit-dose forms, containing, for example, 10 to 1500 mg, preferably 20 to 1000 mg, and even better 50 to 700 mg of the active ingredient per unit-dose form.

Experiments conducted with 3'-azido-31-deoxythymidine suggest that a dose of

in order to achieve peak plasma concentrations of the active compound of approximately 1 to approximately 75 µM, preferably approximately 2 to 50 µM, and even better approximately 3 to approximately 30 pM. This can be achieved, for example, by intravenous injection of a 0.1 to 5% solution of the active ingredient, possibly in physiological saline, or by oral administration as an embolus containing approximately 1 to approximately 100 mg/kg of the active ingredient. Desirable blood levels can be maintained by continuous infusion at a rate of approximately 0.01 to approximately 5.0 mg/kg/hour or by intermittent infusions containing approximately 0.4 to approximately 15 mg/kg of the active ingredient.

The combinations according to the invention can be administered in a manner similar to that described above to provide the desired therapeutic dosages of the active ingredient and the other therapeutic agent concerned. The dosage of the combination depends on the condition being treated, the particular active ingredient and the other therapeutic agent concerned, as well as other clinical factors such as the patient's weight and condition, and the route of administration of the combination. As indicated above, the active ingredient and the other therapeutic agent can be administered simultaneously (e.g., in a unitary pharmaceutical formulation) or separately (e.g., in separate pharmaceutical formulations), and, in general, the combinations can be administered topically, orally, rectally, or parenterally (e.g., intravenously, subcutaneously, or intramuscularly).

In particular, the combinations according to the invention in which the second agent is a nucleoside transport inhibitor can be administered to the subject in a conventional manner. However, for oral administration, a dosage of 1 to 200 mg/kg/day, preferably 5 to 50 mg/kg/day, is generally sufficient. For parenteral administration, a dosage of the active ingredient of 1 to 100 mg/kg/day, preferably 5 to 50 mg/kg/day, is generally sufficient.

A dose of 2 to 30 mg/kg/day is generally sufficient. The amount of nucleoside transport inhibitor in the combination is independent of the amount of active ingredient specified above and is sufficient to effectively inhibit nucleoside transport and is preferably in the range of 0.1 to 100 mg/kg/day, and in particular in the range of 1 to 20 mg/kg/day.

The combinations according to the invention, in which the second agent is a renal excretion inhibitor or a glucuronidation inhibitor, or both, can be administered to the subject in a conventional manner. However, for oral administration, an active ingredient dosage of 1 to 200 mg/kg/day, preferably 5 to 50 mg/kg/day, is generally sufficient. For parenteral administration, an active ingredient dosage of 1 to 100 mg/kg/day, preferably 2 to 30 mg/kg/day, is generally sufficient. The amount of the excretion/glucuronidation inhibitor in the combination is independent of the amount of active ingredient and is sufficient to provide 4 to 100 mg/kg/day, preferably 5 to 60 mg/kg/day, and even better, 10 to 40 mg/kg/day.

The combinations according to the invention, in which the second agent is an interferon, advantageously contain the active ingredient in quantities suitable for providing 5 to

25,250 mg per kg per day; and the appropriate range of doses

* 6 6

The effective dose of interferon 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 per square meter per day. The active ingredient, interferon, should be administered in g

30 a ratio of approximately 5 mg per kg of active ingredient to 3 x 10⁶ IU per square meter of interferon to approximately 250 mg per kg per day of active ingredient to 10 x 10⁶ IU per square meter per day of interferon, preferably approximately 5 mg per kg per day of active ingredient to 4 x 10⁶ IU per square meter per day 35 of interferon to approximately 100 mg per kg per day of ingredient

/T

active at 6 x10 IU per square meter per day of interferon.

(I'

Interferon is preferably administered by injection (e.g., subcutaneous, intramuscular, or intravenous), while the active ingredient is preferably administered orally or by injection, but it may be administered by any of the methods described herein. Interferon and the active ingredient may be administered together or separately, and the daily dose of either may preferably be administered in divided doses.

For combinations according to the invention in which the second agent is another therapeutic nucleoside, the suitable range of effective oral doses of the active ingredient is 2.5 to 50 mg per kg of body weight per day, preferably 5 to 10 mg per kg per day; and the suitable range of effective doses

I

The oral dose of the second therapeutic nucleoside is 5 to 100 mg per kg of body weight per day, preferably 15 to 75 mg per kg per day. For oral administration, the ratio of the active ingredient to the second therapeutic nucleoside is preferably approximately 1:1 to 1:10, and even better, approximately 1:2 to 1:8.

The appropriate range of effective intravenous doses of the active ingredient is generally about 1.5 to 15 mg per kg per day, and the appropriate range of effective intravenous doses of the therapeutic nucleoside is generally about 5 to 30 mg per kg per day. Preferably, for intravenous administration, the ratio of the active ingredient to the therapeutic nucleoside should be about 2:1 to 1:20, and even better, about 1:2 to 1:10.

The two components of the combination are preferably administered orally or by injection and may be given together or separately. The daily dose of either component may be administered in divided doses.

For associations according to the invention in the-

f)

If the second agent is an antibacterial agent, the appropriate range of effective doses of the active ingredient is 2.5 to 50 mg/kg/day, preferably 5 to 10 mg/kg/day, and the appropriate range of effective doses of the antibacterial agent is 2 to 1000 mg/kg/day, preferably 50 to 500 mg/kg/day. The preferred ratios of the active ingredient to the antibacterial agent are 20:1 to 1:500, particularly 2:1 to 1:25.

It should be noted that, although combinations containing three or more therapeutic agents are not specifically described here, such combinations are part of the present invention. For example, a combination of 31-azido-31-deoxythymidine (AZT) with acyclovir and probenecid has the dual advantage of potentiating the activity of AZT and increasing its bioavailability. Similarly, combinations of compounds of formula (I)A with two or more of the same variety of the second therapeutic agent are also part of the invention, for example, AZT with suifanimidine and trimethoprim.

Although the active ingredient can be administered alone, it is preferable to present it as a pharmaceutical formulation. The formulations of the present invention comprise at least one active ingredient, as defined above, and one or more acceptable carriers for it, and optionally other therapeutic agents. Each carrier must be "acceptable" in the sense that it must be compatible with the other ingredients of the formulation and not be harmful to the patient. The formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal, or parenteral (including subcutaneous, intramuscular, and intradermal) administration. The formulations can be conveniently presented in unit-dose form and can be prepared by all methods well known in the pharmaceutical arts. These methods include the step of combining the active ingredients.

The active ingredient is combined with a carrier consisting of one or more secondary ingredients. Generally, formulations are prepared by uniformly and thoroughly combining the active ingredient with liquid or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the present invention that are suitable for oral administration may be presented as discrete units such as capsules, caplets, or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. The active ingredient may also be presented as a bolus, electuary, or paste.

A tablet can be manufactured by compression or molding, possibly with one or more secondary ingredients. Tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granules, possibly mixed with a binder (e.g., polyvinylpyrrolidone, gelatin, or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethylcellulose), a surfactant, or a dispersant. Molded tablets can be prepared by molding, in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and they may be formulated to effect a slow or controlled release of the active ingredient they contain, for example by using hydroxypropylmethylcellulose in various proportions to achieve the desired release profile.

K>

Formulations suitable for topical administration in the mouth include tablets that contain the active ingredient in a flavored base, usually sucrose and gum arabic or tragacanth; lozenges containing the active ingredient in an inert base such as gelatin or glycerin, or sucrose and gum arabic; and mouthwashes containing the active ingredient in a suitable liquid carrier.

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

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations, containing, in addition to the active ingredient, carriers such as are known in practice to be suitable.

Formulations suitable for parenteral administration include sterile isotonic aqueous and non-aqueous solutions for injection, which may contain antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with respect to the blood of the intended recipient; and sterile aqueous and non-aqueous suspensions, which may contain suspending agents and thickeners. The formulations may be presented in sealed single-dose or multi-dose containers, for example, ampoules and vials.

I

They can be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid carrier, such as water for injections, immediately before use. Extemporaneous solutions and suspensions for injection can be prepared from sterile powders, granules, and tablets of the type described above.

Preferred unit-dose formulations are those that contain one daily dose or unit, a

"r daily sub-dose, as indicated above, or an appropriate fraction thereof, of an active ingredient.

to be presented for use as veterinary formulations that can, for example, be prepared by conventional methods in practice. Examples of such veterinary formulations include those adapted for:

(a) oral administration, for example beverages (for example aqueous or non-aqueous solutions or suspensions); tablets or large pills; powders, granules, or lozenges to be mixed with food; and pastes for application to the tongue;

(b) parenteral administration, for example by subcutaneous, intramuscular or intravenous injection, for example as a sterile solution or suspension; or (if appropriate) by intramammary injection where a suspension or solution is introduced into the mammary gland through the nipple;

(c) topical application, for example in the form of a cream, ointment or spray applied to the skin; or

(d) intravaginal administration, for example in the form of a pessary, cream or foam.

It should be noted that the formulations as described above are also suitable for the presentation of the associations according to the invention, whether they are unitary or separate formulations, and can be prepared in a similar manner.

In addition to the particulars mentioned above, the formulations of the present invention may include other conventional agents in practice, given the type of formulation in question; for example, formulations suitable for oral administration may contain additional agents such as sweeteners, thickeners, and flavoring agents.

The compounds according to the invention can also

It goes without saying that in addition to the ingredients

n

Compounds of formula (I)A and their pharmaceutically acceptable derivatives can be prepared conventionally using techniques that are well known in practice, for example as described in: Synthetic Procedures in Nucleic Acid Chemistry (1, 321, (1968), T.A. Krenitsky et al.), J. Med. Chem. (26, 981 (1983)); Nucleic Acid Chemistry, Improved and New Synthetic Processes, Methods and Techniques (Parts 1 and 2, Ed. L.D. Townsend, R.S. Tipson, (J. Wiley) 1978); J.R. Horwitz et al. (J. Org. Chem. 29, (July 1964) 2076-78); M. Imazawa et al. (J. Org. Chem. 43^ (15) (19 78) 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)), or by employing techniques similar to those described here.

The present invention further encompasses a process for preparing a compound of formula (I) and pharmaceutically acceptable derivatives of this compound, which consists of reacting a compound of formula:

HAS

M

(where A is as defined above and M represents a precursor group for the 3'-azido group) or a derivative (e.g., an ester or salt) thereof, with an agent or under conditions used to transform said precursor group into the desired azido group;

(B) to react a compound of formula:

R

(IV a)

(IVb)

or ho

-p;

n_

1 a'

r:

r.

a',"a'„"a'„ 1 „2 3

K

6 7 *

R and R represent res-

(in which R respectively are the groups R"1", R precursor groups of these, provided that at least one of r

R6 and R7

or

Ra'

> 3 4

R and R„ in formula (IVa) or at least one of the 1 6 3 7

groups R_ and R= in formula (IVb) represents a precursor group (a) with an agent or under conditions used to transform the precursor group(s) into the corresponding desired groups;

(C)

to cause a compound of formula to react

(Go) or

(Vb)

10 (in which R, R, R, R, R and R are as defined above) or a functional equivalent thereof, with a compound for introducing the desired ribofurannosyl ring at position 1 of the compound of formula (IVa) or at position 9 of the compound of formula (IVb); or 15 (d) for the preparation of compounds of formula (II),

to react a purine of formula (Vb) with a pyrimidine ring of formula

11

I

$2

HO

py

(VI)

(in which Py represents a 1-pyrimidinyl group); and subsequently or concomitantly, to carry out one or more of the following optional transformations:

(i) when a compound of formula (I) is formed, transform that compound into one of its pharmaceutically acceptable derivatives; and

(ii) when a pharmaceutically acceptable derivative of a compound of formula (I) is formed, transform that derivative into the original compound of formula (I) or into another

10 pharmaceutically acceptable derivative of a compound of formula (I).

In the process according to the invention described above, it should be noted that the choice of precursor compounds in processes (A) to (D) is largely dictated by the particular compound one wishes to prepare, the aforementioned agents and conditions being chosen from those known in the art of nucleoside synthesis chemistry. Examples of such transformation procedures are described below by way of example, and it is understood that they can be modified conventionally according to the desired compound. In particular, for example, in the case where a transformation is described that would otherwise lead to the undesired reaction of labile groups, such groups can then be protected conventionally, the protecting groups being subsequently removed after the transformation is complete.

Thus, for example, with regard to process (A), the M group of the compound of formula (Illa) or (Illb) may represent, for example, a halogen (e.g., chlorine), or a hydroxy or organosulfonyloxy radical (e.g., tri-

fl

ff fluoromethylsulfonyloxy, methanesulfonyloxy or p-toluenesulfonyloxy).

For the preparation of compounds of formula (I)

In which the 31-azido group is in the threo configuration, a compound of formula (lira) or (Ilib) in which M is a hydroxy group in the erythro configuration (the 5'-hydroxy group being advantageously protected, for example, by a trityl group) can be treated with, for example, triphenylphosphine, carbon tetrabromide, and lithium azide. Alternatively, M can represent an organosulfonyloxy leaving group in the erythro configuration that can be transformed into an azido group in the threo configuration by treatment with, for example, lithium or sodium azide, the 5'-hydroxy group being protected as described above. The removal of the protecting 5'-trityl group can then be carried out, for example, by treatment under mild acidic conditions or with zinc bromide.

For the preparation of compounds of formula (I)

In which the 3'-azido group is in the erythroo configuration, a compound of formula (Illa) or (Ilib) in which the M group is a halogen group (e.g., chloro) in the threo configuration (the 5'-hydroxy group being advantageously protected, e.g., by a trityl group) can be treated with, for example, lithium or sodium azide. The 3'-threo-halogenated (e.g., chlorinated) starting material can be obtained, for example, by reacting the corresponding 3'-threo-hydroxylated compound with, for example, triphenylphosphine and carbon tetrachloride or, alternatively, by treating it with an organosulfonyl halide (e.g., trifluoromethanesulfonyl chloride) to form a 3'-threo-organosulfonyloxylated compound which is then halogenated, e.g., as described above. Alternatively, a 3'-threo-hydroxylated or organosulfonyloxylated compound of formula (Illa) or (Ilib)

can be treated, for example, with triphenylphosphine,

carbon tetrabromide and lithium azide, to form the corresponding 3'-erythro-azido compound.

With regard to process (B), the following represent examples of various methods by which the precursor groups of formula (IVa) can be transformed into the desired groups r\ R^, R"^ and R4:

(a) When R^" represents an alkoxy group (e.g., methoxy or ethoxy), these compounds can be prepared from the corresponding compounds of formula (IVa) in

10 in which R^ represents a 2,5'-O-anhydro bond, for example by treatment with a suitable nucleophile such as an alkoxide, conveniently in the presence of potassium carbonate;

(b) When R"'" represents a mercapto group, these compounds can be prepared from the corresponding compounds

15 members of formula (IVa) in which R"*" represents an alkoxy cl group (for example ethoxy), for example by treatment with hydrogen sulfide;

2

(c) When R represents an alkyl group, these compounds can be prepared from the corresponding compounds

2

20 formula weights (IVa) in which R represents an a

hydrogen atom, for example by treatment with an alkylating agent, such as N,N-dimethylformamide dimethylacetal;

(d) When R represents a mercapto group, these compounds can be prepared from the corresponding compounds

i 3

25 dants of formula (IVa) in which R represents a suitable leaving group, for example 1,2,4-triazolyl, by treatment with, for example an alkali metal mercaptan (for example sodium);

3

(e) When R represents an amino group, these compo-

30 sesame seeds can be prepared from the corresponding compounds

3

members of formula (IVa) in which R= represents a group

3.

hydroxy, by treatment with an asbestos-containing agent, for example 11hexamethyl-disilazane and ammonium sulfate, in a bomb;

35 (f) When R4 represents a halogen radical (e.g. chloro), these compounds can be prepared from

9

of corresponding compounds of formula (IVa) in which

4v

R represents a hydrogen atom, by treatment with a

3.

halogenated agent, for example m-chloroperbenzoic acid.

Similar methods can be used to perform the transformation of the precursor group of the

6 7

formula (IVb) in the desired R group or R, as well as the methods described for example in the aforementioned references.

With regard to process (C), it can for example be carried out by treating the appropriate pyrimidine or purine of formula (Va) or (Vb) or a protected salt or derivative thereof, with a compound of formula:

(where Y represents a leaving group, for example an acetoxy, benzoyloxy or halogen group such as chloro, and the 5'-hydroxyl group is optionally protected, for example by a p-toluenesulfonyloxy group), and then removing all protecting groups.

With regard to process (D), the reaction of the compounds of formulas (Vb) and (VI) is conveniently carried out in the presence of a phosphorylating enzyme and, if necessary, by separating the 3'-azido anomers in a conventional manner.

When a compound of formula (I) is formed, this compound can be transformed into a pharmaceutically acceptable phosphate or other ester by reacting the compound of formula (I) with, respectively, a phosphorylating agent such as POClg or a suitable esterifying agent such as an acid halide or anhydride. Compounds of formula (I), including their esters, can be transformed into their pharmaceutically acceptable salts in a conventional manner, for example by treatment with a suitable base.

pf

When a derivative of a compound of for- is formed

j mule (I), such a compound can be transformed back into the original compound, for example by hydrolysis.

The following non-limiting examples illustrate the present invention. The term "active ingredient",

as used in the Examples means a compound of formula (I) or (I)A or a pharmaceutically accepted derivative

tî

<

10

15

20

25

30

35

Example 1: Tablet Formulations?

The following formulations A and B are prepared by wet granulation of the ingredients with a povidone solution, followed by the addition of magnesium stearate and compression.

mq/tablet mq/tablet

Formulation A

(a) Active ingredient

(b) Lactose B.P. (British Pharmacopoeia)

(c) Povidone B.P.

(d) Starch-sodium glycollate

(e) Magnesium stearate

Formulation B

(a) Active ingredient

(b) Lactose 150

(c) Avicel PH 101

(d) Povidone B.P.

(e) Starch-sodium glycollate

(f) Magnesium stearate

Formulation C Active ingredient Lactose Starch Povidone

Magnesium stearate

250

210 15 20

5_

500

mq/tablet 250

60 15 20 5

250

26 9 12

3_

300

mq/tablet 250

26 9 12 3

500 mq/tablet 100 200 50 5

4

359

300

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

Formulation D mg/capsule

Active ingredient 250

Pregelatinized starch NF15 150

400

•i

Formulation E

/ n <

mq/capsule

Active ingredient 250

Lactose 150

5 Avicel 100

500

Formulation F (Formulation for Controlled Release)

The formulation is prepared by wet granulation of the ingredients (below) with a 10 solution of povidone, then addition of magnesium stearate and compression.

mg/tablet

(a) Active ingredient 500

(b) Hydroxypropylmethylcellulose

15 (Methocel K4M Premium) 112

(c) Lactose B.P. 53

(d) Povidone B.P.C. 28

(e) Magnesium stearate 7

700

20 Example 2: Capsule Formulations Formulation A

A capsule formulation is prepared by adding and mixing the ingredients of Formulation D from Example 1 above and filling a two-part hard gelatin capsule. Formulation B (below) is prepared in a similar manner.

Formulation B

mq/capsule

(a) Active ingredient 250

30 (b) Lactose B.P. 143

(c) Starch-sodium glycollate 25

(d) Magnesium stearate 2

420

Formulation C mq/capsule

(a) Active ingredient 250

(b) Macrogol 4000 BP 350

A ^ 600

yr

Capsules are prepared by melting Macrogol 4000 BP, dispersing the active ingredient in

_the mass, .melted and by introducing the molten mass into a two-part hard gelatin capsule.

5 Formulation D

mq/capsule

Active ingredient 250

Lecithin 100

Peanut oil 100

10,450

Capsules are prepared by dispersing the active ingredient in lecithin and peanut oil and introducing the dispersion into soft elastic gelatin capsules.

15 Formulation E (Controlled Release Capsule)

The following controlled-release capsule formulation is prepared by extruding ingredients a, b, and c using an extruder, then spheronizing the extrudate and drying it. The dried pellets 20 are coated with the controlled-release membrane (d) and a two-part hard gelatin capsule is filled with them.

mq/capsule

(a) Active ingredient 250

(b) Microcrystalline cellulose 125 25 (c) Lactose BP 125 125

(e) Ethyl-cellulose 13

513

Example 3: Formulation of Injectables Formulation A

30 Active ingredient 0.200 g

0.1M hydrochloric acid solution q.s. for pH 4.0 to 7.0; 0.1M sodium hydroxide solution q.s. for pH 4.0 to 7.0; sterile water q.s. to 10 ml

The active ingredient is dissolved in most of the water (35°-40°C) and the pH is adjusted between 4.0 and 7.0 with hydrochloric acid or sodium hydroxide.

n as required, The charge is then brought to volume with water and filtered on a sterile micropore filter into a sterile 10 ml amber glass bottle (type 1) and sealed with sterile closures and seals covering 5.

Formulation B

Active ingredient 0.125 g

Sterile, pyrogen-free phosphate buffer

at pH 7 q.s. up to 25 ml

10 Example 4: Intramuscular injection Weight (g)

Active ingredient 0.20

Benzyl alcohol 0.10

Glycofurol 75 1.45

Water for injection q.s. up to 3.00 ml

15. The active ingredient is dissolved in the glycofurol.

Next, benzyl alcohol is added and dissolved, and water is added up to 3 ml. The mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml amber glass bottles (type 1).

Example 5: Syrup

Formulation A Weight (g)

Active ingredient 0.2500

Sorbitol solution 1.5000

25 Glycerol 2.0000

Sodium benzoate 0.0050

Peach flavor 17.42.3169 0.0125 ml

Purified water q.s. for 5.0000 ml

The active ingredient is dissolved in a mixture of 30% glycerol and most of the purified water. Next, an aqueous solution of sodium benzoate is added to the mixture, followed by the sorbitol solution and finally the flavoring. The volume is then made up with purified water and the mixture is thoroughly blended.

35 When the active ingredient is poorly soluble,

we use the following formulation (B).

Formulation B Weight (y)

Active ingredient 0.250

Sorbitol solution. 1.500

Glycerol 0.005

Dispersible cellulose 0.005

Sodium benzoate 0.010 ml Flavoring

Purified water, up to 5,000 ml

Mix the sorbitol solution, 10% glycerol, and a portion of the purified water. Dissolve the sodium benzoate in the purified water and add the solution to the mixture. Add and disperse the dispersible cellulose and the flavoring. Add and disperse the active ingredient. Make up to volume with purified water.

15 Example 6: Suppository mg/suppository

Active ingredient (63 pm)* 250 Hard fat, BP (Witepsol H15 -

Dynamit NoBel) 1770

20 2020

* The active ingredient is used in powder form in which at least 90% of the particles have a diameter of 63 pm or less.

One fifth of the Witepsol H15 is melted in 25 a container lined with water vapor at a maximum of 45°C.

The active ingredient is sieved through a 200 µm sieve and added to the molten base, mixing with a Silverson apparatus fitted with a cutting head until homogeneous dispersion. Maintaining the mixture at 45°C, the remaining Witepsol H15 is added to the suspension and stirred to ensure homogeneous mixing. The entire suspension is then passed through a 250 µm stainless steel sieve and, while continuing to stir, allowed to cool to 40°C. At a temperature of 38°C to 35-40°C, 2.02 g of the mixture is introduced into suitable 2 ml plastic molds. The sup-

Positions: Cool to room temperature. Example 7: Pessaries mg/pessary

Active ingredient (63 µm) 250

5 Anhydrous dextrose 380

Potato starch 363

Magnesium stearate 7

1000

The above ingredients are mixed directly and pessaries are prepared by direct compression of the resulting mixture.

Examples 8 to 10 below illustrate the preparation of formulations containing a compound of formula (I)A (active ingredient) and another therapeutic nucleoside, as specified.

Example 8: Injection Formulation A

Weight (mg)

Acyclovir 400

20 Active ingredient 200

NaOH 1M as needed

Sterile water, up to 10 ml

Formulation B

Weight (mg)

25 2-amino-9~(2-hydroxyethoxymethyl)purine 400

Active ingredient 200

NaOH 1M as needed

Sterile water, up to 10 ml

For the above formulations, the therapeutic nucleoside 30 is added to the 1M NaOH solution and stirred until dissolved. The active ingredient is then added and dissolved. Sterile water is added up to 10 mL. Filter through a sterile filter and fill sterile vials. Lyophilize.

35 For the above formulations, the therapeutic nucleoside is added to the 1M NaOH solution and stirred

10

15

20

25

30

35

until dissolved. Add the active ingredient and

"

Dissolve it. Add sterile water up to 10 ml. Filter through a sterile filter and fill sterile vials. Lyophilize.

Example 9: Tablets Formulation A

Acyclovir Active ingredient Povidone

Sodium starch glycolate Magnesium stearate

Formulation B

Active ingredient

2-amino-9-(2-hydroxyethoxymethyl)purine Povidone

Sodium starch glycolate Magnesium stearate

Weight (mg) 500 125 14 25

6_

670

Weight (mg) 125 125 8 12 3

273

For formulations A and B above, the therapeutic nucleoside and the active ingredient are mixed with sodium starch glycolate and granulated with a povidone solution. After drying, the granules are kneaded with magnesium stearate and formed into tablets.

Example 10: Capsules

Formulation A Weight (mg)

Acyclovir 250

Active ingredient 62.5

Lactose 170.5

Starch-sodium glycollate 15

Magnesium stearate 2

5.00

Mix the ingredients and fill hard gelatin capsules.

kt^

Formulation B

f

Weight (mg)

Ingredient, active ingredient 125

2-amino-9-(2-hydroxyethoxymethyl)purine 125 5 Lactose 133

Starch-sodium glycollate 15

Magnesium stearate 2

400

Mix the ingredients and fill 10 hard gelatin capsules.

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

Example 11: Injection of 15 Interferon 3 megaunits

Active ingredient 200 mg

Sterile buffer pH 7, up to 50 ml

Dissolve the interferon and the active ingredient in sterile water. - Filter through a sterile filter and fill 20 sterile vials.

Examples 12 to 15 below describe the preparation of formulations containing a compound of formula I(A) (the Active Ingredient), for example 3'-azido-3'-deoxythymidine, in combination with a nucleoside transporter inhibitor, for example dipyridamole. Example 12: Tablet

Weight (mg)

Nucleoside Transport Inhibitor 300

Active ingredient 200

30 Lactose 105

Starch 50

Polyvinylpyrrolidinone 20

Magnesium stearate 10

Total weight 685

35 The active compounds are mixed with lactose and starch and granulated using a wet method with a

4? j solution of ,1a polyvinylpyrrolidinone. The granules are dried, sieved and mixed with magnesium stearate,- then compressed into tablets.

Example 13: Capsule

5 Weight (mg)

Nucleoside Transport Inhibitor 300

Active ingredient 100

Lactose 100

Starch-sodium glycollate 10

10 Polyvinylpyrrolidinone 10

Magnesium stearate 3

Total weight 523

The active compounds are mixed with lactose and sodium starch glycolate and the granules are moistened with a solution of polyvinylpyrrolidinone.

The granules are dried, sieved and mixed with magnesium stearate and introduced into hard gelatin capsules.

Example 14; Cream 20 Weight (g)

Nucleoside transport inhibitor

7.5

Active ingredient

5.00

Glycerol

2.00

Cetostearyl alcohol

6.75

sodium lauryl sulfate

0.75

Soft white paraffin

12.50

Liquid paraffin

5.00

Chlorocresol

0.10

Purified water, up to

100.00

30. The active compounds are dissolved in a mixture of purified water and glycerol and heated to 70°C. The other ingredients are heated together to 70°C. The two parts are combined and emulsified. The mixture is cooled and then filled into containers.

Vt

Example 15: Intravenous Injections

' / \ ' Quantity (mg)

(1) Active ingredient 200 Nucleoside transport inhibitor 300

Glycerol 200

Sodium hydroxide solution, q.s. pH 7.0-7.5 Water for Injections, up to 10 ml

Glycerol is added to a portion of the water for injection. The two active compounds are added, and the pH is adjusted to 7.0–7.5 with sodium hydroxide solution. The solution is then brought up to volume with additional water for injection. Under aseptic conditions, the solution is sterilized by filtration, filled into sterile ampoules, and the ampoules are hermetically sealed.

Quantity (mg)

(2) Active ingredient 100 Nucleoside transport inhibitor 150

Mannitol 125

Sodium hydroxide solution, q.s. pH 8.0-9.0; Water for injections, up to 2.5 ml

The active compounds and mannitol are dissolved in a portion of the water for injection. The pH is adjusted to 8.0–9.0 with sodium hydroxide solution, and the volume is made up to the required amount of water for injection. Under aseptic conditions, the solution is sterilized by filtration, transferred to sterile vials, and the water is removed by lyophilization. The vials are hermetically sealed under a nitrogen atmosphere with a sterile stopper and a metal band.

Examples 16 to 18 below describe the preparation of formulations containing a compound of formula I(A) (the Active Ingredient), for example 2'-azido-3'-

i deoxythymidine, in combination with a glucuronidation/renal excretion inhibitor, for example probene-

cide.

Example 16: Tablet

Weight (mg)

Active ingredient 100 Glucuronidation inhibitor/renal excretion 200

Lactose 105

Starch 50

Polyvinylpyrrolidinone 20

Magnesium stearate 10

Total weight 485

The active compounds are mixed with lactose and starch, and the granules are moistened with a polyvinylpyrrolidinone solution. The granules are dried, sieved, mixed with magnesium stearate, and then compressed into tablet form.

Example 17: Capsule

Weight (mg)

Active ingredient 100 Glucuronidation inhibitor/renal excretion 100

Lactose 100

Starch-sodium glycollate 10

Polyvinylpyrrolidinone 10

Magnesium stearate 3_

Total weight 323

The active compounds are mixed with lactose and sodium starch glycolate, and the granules are moistened with a polyvinylpyrrolidinone solution. The granules are dried, sieved, mixed with magnesium stearate, and filled into hard gelatin capsules.

H

!

Example 18: Intravenous Injections ^

Quantity (mg)

(1) Active ingredient 200 Glucuronidation inhibitor/renal excretion 300

Glycerol 200

Sodium hydroxide solution, q.s. for pH 7.2-7.5

Water for injections, up to 10 ml

Glycerol is added to a portion of the water for injection. The two active compounds are added, and the pH is adjusted to 7.0–7.5 with sodium hydroxide solution. The solution is then brought to volume with additional water for injection. Under aseptic conditions, the solution is sterilized by filtration, transferred to sterile ampoules, and the ampoules are tightly sealed.

Quantity (mg)

(2) Active ingredient 100 Glucuronidation inhibitor/renal excretion 150

Mannitol 125

Sodium hydroxide solution, q.s. for pH 8.0-9.0

Water for injections, up to 2.5 ml

The active compounds and mannitol are dissolved in a portion of the water for injection. The pH is adjusted to 8.0–9.0 with sodium hydroxide solution, and the solution is made up to volume with additional water for injection. Under aseptic conditions, the solution is sterilized by filtration, transferred to sterile vials, and the water is removed by lyophilization. The vials are hermetically sealed under a nitrogen atmosphere with a sterile stopper and a metal band.

kr

Veterinary Formulations (Examples 19-22)

Example 19 v Small Animal Tablet ■

Per tablet

Active ingredient 120.0 mg

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 solution is added, with continuous stirring, to obtain a moist mass, which is then sieved to form granules. Magnesium stearate is applied to the granules by sieving. Tablets are produced by direct compression of the granules.

Example 20: Granules for medication in food

Weight (g)

Active ingredient 6.0

Povidone 1.0

Lactose 93.0

Aqueous alcohol, q.s.

The active ingredient is mixed with lactose. Aqueous alcohol containing dissolved povidone is added to this mixture. Sufficient aqueous alcohol is added to obtain a moist mass, which is then sieved to obtain granules that are subsequently dried.

Example 21: Paste for oral use

Weight (g)

Active ingredient 24

Xanthan gum 0.5

Methyl hydroxybenzoate 0.1

Polysorbate 80 0.1

Purified water, up to 100.0 ml

Dissolve polysorbate 80 and methyl hydroxybenzoate in water. Add xanthan gum and allow to swell. Add the active ingredient and disperse it, then dilute to volume.

X

H

i i

i

Example 22: Ointment j

Weight(q)

Active ingredient - 12

Soft white paraffin 88.0

The soft white paraffin is melted at 60°C. The active ingredient is added and dispersed, the mixture is allowed to cool, and then it is filled into compressible metal tubes.

Example 23: 3'-azido-3',5-dideoxy-5'-[(N,N-dimethyl-thiocarbamoyl)thio-thymidine]

(a) The 5'-hydroxyl group of 3.0 g (11.2 mmol) of 3'-azido-3'-deoxythymidine is mesylated by adding 2.7 mL of methanesulfonyl chloride to 20 mL of a solution of 3'-azido-3'-deoxythymidine in 20 mL of anhydrous pyridine. The reaction is allowed to proceed at 5°C for one hour, and then the reaction mixture is poured onto ice-cold water. The precipitate is collected by filtration. The desired product is obtained by reacting the 3'-azido-3'-deoxy-5'-mesylthymidine obtained in the first step with 0.78 g (5.6 mmol) of potassium carbonate in 75 mL of DMF. The reaction mixture is heated in an oil bath at 80°C for six hours, then poured into ice water. The product is extracted from the water with ethyl acetate. The solvent is expelled under vacuum, and the resulting oil is subjected to silica gel flash chromatography, eluting with CHCl₂KeOH (9:1 by volume). The product is obtained.

•s in a low yield. P.F.. = 184-186°C.

(b) 0.642 g (3.58 mmol) of the sodium salt of dimethyldithiocarbamic acid dihydrate and 3.58 mL of a tetrabutylammonium hydroxide (IN) solution in MeOH are added to 25 mL of DMF. The solution is brought to a boil to remove the water and MeOH. After cooling, 0.85 g (3.4 mmol) of 2,5'-O-anhydro-3'-azido-3*-deoxythymidine dissolved in 15 mL of DMF is added. The reaction mixture is heated in an oil bath to 55°C.

for approximately 16 hours. The reaction mixture is poured.

The precipitate is separated by filtration and placed in ice water. The product in the filtrate is extracted with ethyl acetate. The ethyl acetate is removed under vacuum, and the resulting oil is purified by instant chromatography on silica gel, eluting with CHCl₃MeOH (95:5 by volume). A second chromatography on silica gel was required. The second elution was performed with CHCl₃MeOH (98:2 by volume). A final purification was carried out by reversed-phase chromatography on C₃g, eluting with a water:methanol mixture (3:7). The yield was 2.5%. Example 24: 3'-azido-3'-deoxy-5'-0-acetyl-4-thio-thymidine

1.41 g (3.9 mmol) of 3'-azido-3'-deoxy-5'-0-acetyl-4-(1,2,4-triazole)thymidine (Lin et al., J. Med. Chem. 26, 1691 (1983)) is dissolved in 100 mL of acetone and 30 mL of H₂O, then treated with 0.39 g of NaSH·xHgO (Sung. J. Chem. Soc. Chem. Comm. 522, (1982)). The mixture is stirred for 30 minutes, the volume is reduced by half, and the solution is extracted with 200 mL of CHCl₃. The CHCl₃ is washed with 100 mL of HgO and dehydrated over NaSH·xHgO. The solvent is expelled under vacuum, and the resulting oil is placed on a silica gel pad (6.5 x 3 cm), followed by elution with 750 mL of CHClg. The solvent is expelled under vacuum to obtain a yellow oil, which is recrystallized in i-ProH to obtain 0.64 g (1.9 mmol; 48.7%); mol.P. = 75–78°C. Example 25 3'-azido-3'-deoxy-4-thiothymidine

-0.25 g (0.76 mmol, Example 21) of 3'-azido-3'-deoxy-5'-O-acetyl-4-thiothymidine is dissolved in a mixture of 5 mL of dioxane and 5 mL of concentrated NH₄OH and stirred for 18 hours. The solvent is driven off under vacuum, and the residue is applied to a silica gel column, eluting with CHCl₃/EtOAc (3:1 by volume). The appropriate fractions are collected, and the

solvent under vacuum to obtain a yellow oil that one

!

dissolved in EtgO, forming crystals by concentration: 0.16 g .{0.56 mmole ; . 74%) ; P.F.. = 116-118°C.

Example 26-: 3-N-methyl-3'-azido-3'-deoxythymidine

• 0.5 g (1.9 mmol) of 3'-azido-3'-deoxythymidine and 0.9 ml (7.5 mmol) of N-dimethyl-formamide dimethylacetal (Zemlicka, Coll. Czech. Chem. Comm. 3572 (1972)) are heated under reflux in 20 ml of CHCl₂ for 48 hours. The solvent is expelled under vacuum, and the material is placed on a silica gel column. Elution with EtOAc/CHCl₂ (1:1 by volume) gives the pure material as a viscous oil: 0.26 g (0.9 mmol, 47%). Example 27: 3'-azido-3'-deoxy-2-thiothymidine

The synthesis of 3'-azido-2-thio-thymidine is carried out by a five-step reaction sequence starting from 2,31-O-anhydro-5'-tritylthymidine (J.J. Fox. J. Org. Chem. 28, 936 (1963)).

10.5 g (22.4 mmol) of 2,3'-O-anhydro-5'-tritylthymidine is added to a 0.52 g (22.4 mmol) solution of sodium in 1.2 liters of anhydrous ethanol, and the reaction mixture is heated under reflux for six hours. The reaction mixture is cooled and neutralized with HClIN. The solvent is expelled under vacuum, and the resulting oil is purified by flash chromatography on silica gel, eluting with CHCl₂MeOH (96:4 by volume). A yield of 30% of 1-(2'-deoxy-5'-trityl-D-lyxofurannosyl)-2-ethoxythymine is obtained. 3.5 g (16.8 mmol) of the 2-ethoxythymidine derivative is dissolved in 35 ml of DMF containing 2.2 ml of triethylamine. The cold solution is saturated with h₂S. The reaction mixture is placed in a steel bomb and heated to 95°C. After twenty-seven hours, TLC (thin-layer chromatography) shows that no starting material remains. The reaction mixture is purged with N₂ for several hours and poured onto ice-cold water. The product is collected by filtration and purified by chromatography.

Instantaneous deposition on silica gel by eluting with CHCl₂:MeOH (97:3 by volume). A yield of 37% is obtained.

I

1-(2*-deoxy-5'-trityl-p-D-lyxofurannosy 1)-2-th-iothymine. The UV max spectrum at pH 1 of 277 nm and at pH 11 of 241 nm indicates the formation of 2-thiothymidine.

The 3'-hydroxyl group of the thiothymidine-type derivative is mesylated as follows: 665 mL (3.5 equivalents) of methanesulfonyl chloride are added, in four portions, over six hours, to a solution of 1.25 g of 1-(21-deoxy-5'-trityl-|3-D-lyxofurannosyl)-2-thiothymidine in 15 mL of anhydrous pyridine at 5°C. The reaction mixture is maintained at 5°C for approximately 16 hours. The reaction mixture is poured onto ice-cold water and the product is collected by filtration. Purification is carried out by flash chromatography on silica gel, eluating with a 1:1 (v/v) ethyl acetate:hexane mixture. The yield is 50%.

0.3 g (6 mmol) of lithium azide is dissolved in 20 mL of anhydrous DMF and 0.72 g (1.2 mmol) of 1-(2'-deoxy-3'-mesyl-5'-trityl-D-lyxofurannosyl)-2-thiothymine is added. The solution is heated in DMF to 85°C for 2.5 hours. The reaction mixture is poured onto ice-cold water and the product is collected by filtration. Purification is performed by flash chromatography on silica gel using CHClMeOH (98:2 by volume). The yield is 78%. A band in the IR spectrum at 2100 cm⁻¹ indicates the presence of an alkyl azide. The UV spectrum confirms the presence of 2-thiothymidine.

The final product is prepared by unblocking the 5'-hydroxyl group of 0.1 g of 3'-azido-3'-deoxy-2-thio-5'-tritylthymidine in 5 mL of 80% acetic acid in a water bath for 45 minutes. 0.021 g of 3'-azido-3'-deoxy-2-thiothymidine is obtained by silica gel chromatography, eluting with CHCl3:MeOH (96:4 by volume) in a yield of 3.7%.

did

\

Example 28: 3'-azido-31-deoxy-2-ethoxythymidine {

-31-Azido-3'-deoxy-2-ethoxythymidine is prepared by refluxing 2.6 g (7.5 mmol) of 3'-azido-3'-deoxy-5'-mesylthymidine in 25 mL of anhydrous ethanol with two equivalents of potassium carbonate (1.08 g, 7.5 mmol) for five hours. The solution is neutralized and resuspended under vacuum until an oil forms. The oil is purified by flash chromatography on silica gel by eluating with a mixture of acetate and ethylmethanol. The desired product is isolated in a yield of 39%; melting point = 98-100°C.

Example 29: 3'-azido-3'-deoxy-2-methoxythymidine

3'-Azido-3'-deoxy-2-methoxythymidine is prepared from 1.6 g (4.6 mmol) of 3'-azido-15,3'-deoxy-5'-mesylthymidine using the procedure in Example 25. The yield is 42%; PF = 47-51°C. Example 30: 3'-Azido-2',3'-dideoxy-5-methylisocytidine

0.35 g (1.4 mmol) of 2,5'-O-anhydro-3'-azido-3'-deoxythymidine is dissolved in 15 ml of MeOH previously saturated with ammonia and placed in a 77°C oil bath for 48 hours (Skaric and Matulic-Adamic, Helv. Chim. Acta. 63, 2179 (1980)). According to TLC (CHCl₂/MeOH 6:1 by volume), the reaction is incomplete. The solvent is expelled under vacuum and the resulting oil is placed on a silica gel column, then eluted with CHCl₃/MeOH (6:1 by volume). The appropriate fractions are collected to obtain 0.14 g (0.53 mmol; 38%) of the compound of titration; P.F. = 107-108°C.

Example 31: 3'-azido-"5-chloro-2',.3'-dideoxyuridine

...0.25 g (1 mmol) of 3'-azido-2',3'-dideoxyuridine is dissolved in 2 ml of anhydrous dimethylacetamide (DMAC), cooled to 0°C, and 2 ml of 0.5 M HCl is added to DMAC. 0.277 g (1.6 mmol) of m-chloroperbenzoic acid is added in two portions over a ten-minute period, and the mixture is allowed to reach room temperature. After two hours, 4 ml of H2O is added and...

M

30

35

*

The solution. The aqueous DMAC solution is extracted with 3 x 3 mL of Et2O and Et2O is evaporated under vacuum until an oil is obtained, which is then applied to a silica gel column. Elution with CHCl₃MeOH (15:1 by volume), collection of the appropriate fractions, and evaporation under vacuum yields an oil that is crystallized in Et2O to obtain 58.5 mg (0.2 mmol, 20%); P.F. = 169-170°C.

UV (nm): at pH 1 A max = 276 (£ = 7400), min = 239 (£ = 500); at pH 13 \max = 274 (l = 6400), A min = 249 U = 3800)

H1 NMR. (DMSO-dg) 5 8.29 (s,lH,H6), 6.04 (t,lH,Hl', J=5.5 Hz), 5.49-5.29 (m, 1H,5'-0H), 4.44-4.20 (m,lH,H3'), 3.88-3.71 (m, 1H,H4'), 3.71-3.53 (m,2H,H5'), 2.63-2.31 (m,2H,H2«);

Analysis for C^H^gN^O^Cl

Calculated • C 37.58, H 3.50, N 24.35, Cl 12.32 Found 0 37.67, H 3.54, N 24.39, Cl 12.40

Example 32: 3'-azido-5-bromo~2',3'-dideoxyuridine

3'-Azido-5-bromo-2',3'-dideoxyuridine is prepared from 0.827 g (3.3 mmol) of 3'-azido-2*,3'-dideoxyuridine (T.A. Krenitsky et al., J. Med. Chem., 26, 891 (1^SS)) by first acetylating the 5'-hydroxyl group with 15 mL of acetic anhydride and then brominating position 5 by adding 0.5 mL of acetic acid and 0.566 g of bromine. The reddish-brown solution is stirred at room temperature for two hours. The reaction mixture is then resuspended under vacuum until an oil is formed, which is triturated with ethyl ether. The oil is dissolved in a methanolic ammonia solution to remove the acetyl group. The desired product is isolated by silica gel chromatography using CHClg:MeOH (95:5 by volume). The yield is 32%; PF = 148-149°C.

Example 33: 3'-azido-2-,3'-dideoxy-5-iodouridine

*3'-azido-2',3'-dideoxy-5-iodouridine is prepared from 10 g (28 mmol) of 2'*3'-

<r

&

I

dideoxy-5-iodouridine by a reaction sequence to

J

four steps described in the literature (T.A. Krenitsky et al., J. Med. Chem., 26, 891. (1983)) P.F. = 126-130 Example 34-: 31-azido-2',3'-dideoxy-5-trifluoromethyluridine

3'-Azido-2',31-dideoxy-5-trifluoromethyluridine is prepared by the following four-step reaction sequence:

The 5'-hydroxyl group of 5.0 g (16.9 mmol) of 2',3'-dideoxy-5-trifluoromethyluridine is tritylated by adding 5.65 g (20.2 mmol) of triphenylmethyl chloride to the starting material in a suspension of 1.4 mL of dichloromethane, 70 mL of pyridine, and 55 g of 3A molecular sieve. The reaction mixture is stirred at room temperature for four days. After filtration and evaporation under vacuum, the oily product is chromatographed on silica gel by eluting with CHgClgrMeOH (95:5 by volume). The product fractions are collected, and the solvent is expelled under vacuum. The resulting oil is tritated with water, and the solid formed is collected by filtration. The 3'-hydroxyl group is chlorinated by dissolving 3.0 g of 5'-protected uridine in 30 mL of dimethylacetamide containing 3.27 g of triphenylphosphine and adding 51 mL of carbon tetrachloride. The reaction mixture is stirred at room temperature for approximately 16 hours. 1 mL of methanol is added. The reaction mixture is repeated until an oil forms, which is then analyzed by silica gel chromatography using Cl-Cl-Cl-EtOAc (9:1 by volume). The desired product is collected as an oil. The oil, 1-(3'-chloro-2'-deoxy-5'-trityl-threo--D-ribofuranno-syl).-5-trifluoromethyluracil, is released by dissolving it in 80 ml of nitromethane and adding 4.45 g of zinc bromide dissolved in 80 ml of nitromethane by the process of V. Kohli et al. (Tetrahedron Letters, _2jL, page 2683,

1980). The reaction mixture is stirred at room temperature for approximately 16 hours. A further 3.0 g of zinc bromide is added the following day, and the reaction is allowed to continue for approximately 16 hours. The final addition of 3.7 g of zinc bromide under gentle heating brings the reaction to a standstill. The reaction mixture is poured into 1M ammonium acetate. The product is extracted into dichloromethane. The dichloromethane is expelled under vacuum, and the resulting oil is chromatographed on silica gel, eluting with Cl-Cl-Cl-eOH (95:5 by volume). The final product is obtained by treating 0.48 g (1.53 mmol) of 1-(3'-chloro-2'-deoxy-p-Di-ribofuranno-syl)-5-trifluoromethyluracil with 0.19 g (3.8 mmol) of lithium azide in 4.8 mL of dimethylacetamide. The reaction mixture is heated at 90°C for four hours. The reaction mixture is repeated until an oil forms, which is then chromatographed on silica gel, eluting with CHCl-IMeOH (95:5 by volume). A second Chromatography is necessary. Elution on a 20% silica gel with C1C1eOH (97:3 by volume) yields a substantially pure product. Crystallization in toluene yields the pure product in a yield of

from 2.2 g (7.9 mmol) of 3'-azido-2',3'-dideoxyuridine in the form of HCl by the procedure of T.A. Krenitsky et al. (J. Med. Chem., 26, 891 (1983)). The yield is 40%; PF = 174.5-176.5°C.

Example 36: 3'-azido-2',3'-dideoxy-5-methylcytidine

10%.

25

Example 35: 3'-azido-2',3'-dideoxycytidine

3'-azido-2',3'-dideoxycytidine is prepared

..We prepare the-3'-azido-2',3'-dideoxy-5-

methylcytidine from 0.8 g (3.0 mmols) of 3'-azido-3'-deoxythymidine by the procedure of 1' Example..35. The yield is 19%.

35 Example 37: Threo-3'-azido-21,3'-dideoxycytidine

The synthesis of threo-3'-azido is carried out

i

2',3'-dideoxycytidine from 2'-deoxyuridine in| four steps.

__ The 5'-hydroxyl group of 2'-deoxyuridine is tritylated by the process described in Synthetic Procedures in 5 Nucleic Acid Chemistry, _1, 321 (1968).

Threo-3'-azido-2',3'-dideoxy-5'-trityluridine is prepared by reacting 5.0 g (10.6 mmols) of 2'-deoxy-5'-trityluridine with 3.07 g (11.7 mmols, 1.1 equivalents) of triphenylphosphine, 3.88 g (11.7 × 10⁻³ mmols, 1.1 equivalents) of carbon-tetrabromide and

5.21 g (10⁶ mmol, 10 equivalents) of lithium azide in 80 mL of DMF. Carbon tetrabromide is added last. The reaction is allowed to proceed at room temperature for approximately 16 hours. 5 mL of methanol is added. The solution is resuspended under vacuum until...

an oil is formed and subjected to instant chromatography on silica gel by eluting with ethyl acetate. The 5'-hydroxyl position is unblocked by heating in 80% acetic acid 20 in a water bath for twenty minutes. Upon cooling, tritylcarbinol precipitates and is separated by filtration. The filtrate is brought to dryness and diluted in ethyl ether. The product, namely threo-31-azido-2',3'-dideoxyuridine, is used without further purification. The final product, namely threo-3*-azido-21,3'-dideoxycytidine, is prepared in the form of HCl from the uridine analog by exactly the same procedure as that used for the preparation of the erythro risomer (T.A. Krenitsky et al., J. Med. Chem., 3° 26, j 891 (1983)). The quantity obtained is 0.021 g, representing a yield of 7%.

Example 38; 9-(31-azido-2',3'-dideoxy-α-D-ribofuranno-sy 1). adenine

9-(3'-azido-2',31-dideoxy-α-E)-35 ribofurannosyl)adenine is prepared in two steps starting from 2.0 g

(7.7 mmol) of Ng-octanoyladenine and 1.13 g (4.2 mmol)

&

î

of 3'-azido-3'-deoxythymidine by the procedure 5

described by M. Imazawa and F. Eckstein (J. Org. Chem.,

43, 3044 (1978); M.P. = 120-122°C.

UV pH l^max 258nm^.min 230 nm pH 13Amax 260nmAmin 229 nm

CHN for C10H12N8°2

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-21,3'-dideoxy-p-P-ribofuranno-syDadenine

9-(3'-azido-21,3 1-dideoxy-p-JD-ribofurannosyDadenine is prepared in two steps from 2^0 g (7.7 mmoles) of Ng-octanoyladenine and 1.13 g (4.2 mmoles) of 3'-azido-3'-deoxythymidine by the procedure described by M. Imazawa and F. Eckstein, J. Org. Chem., 43, 3044 (1978).

P.F. » 184-185°C.

UV pH Umax" 257nmXmin 230nm pH 13Amax 260nmAmin 228nm

CHN for C10H12N8O2 Calculated: C-43.48; H-4.38; N-40.56 Found: C-43.33; H-4.45; N-40.41

Example 40; 5'-acetyl-3'-azido-3-benzoy1-3'-deoxythymidine ..

0.75 g (2.4 mmol) of 5'-acetyl-3'-azido-3'-deoxythymidine is dissolved in 5 ml of pyridine and 1.4 ml (12 mmol, 5 equivalents) of chloride is added.

!

benzoyl at room temperature. The reaction mixture is stirred for approximately 16 hours, then poured onto 250 ml of ice-cold water. The pH of the aqueous solution is adjusted to 1. The product is extracted with chloroform. The organic phase is washed with water, dehydrated with MgSO₄, and filtered. The chloroform is removed, and the mixture is then filtered.

H

The oily product is subjected to instant chromatography on silica gel by eluting with chloroform. The product is collected in the form of an oil.

H1 NMR (DMSO-dg): 68.04-7.50 (m,6H;3N-benzoy!e and 6H), 66.12 (dd,lH, = 5.6Hz, Jlt 2fal = 6.7 Hz, 1 • H), 6 4.55-3.96 (m, 4H; 3'H,4'H,5H'), 62.62-2.38 (m,2H,2'H), 62.07 (s,3H, 5'-acetyl), 61.90 (d, 3H, J5^=l,0Hz, 5CH3)

CHN calculated for ci9Hi9N5°6 Calculated: C-55.20; H-4.63; N-16.94 Found: C-55.29; H-4.64; N-16.93

Example 41: Threo-3'-azido-5-bromo-2',3'-dideoxyuridine; We prepare threo-31-azido-5-bromo-2',3'-

dideoxyuridine from 2'-deoxyuridine by a five-step reaction sequence.'

The 5'-hydroxyl group of 2*-deoxyuridine is protected with a triphenylmethyl group in the usual manner. The 3'-hydroxyl group is mesylated. A 3'-azido group is introduced with the correct stereochemistry by adding 22 g (40 mmol) of 2'-deoxy-3'-mesyl-5'-trityluridine to a solution of 7.84 g (120 mmol, 3 equivalents) of sodium azide in 380 mL of dimethylformamide at 80°C. The reaction is carried out for 35 hours. The solution is poured onto 2 liters of ice water and the precipitate is collected by filtration. The product is isolated by silica gel chromatography by eluating with a chloroform-ethanol mixture (1:1 by volume).

in a 51% yield. The 5'-hydroxyl position is unblocked with 80% acetic acid in a water bath for 25 minutes. After cooling, the tritylcarbinol is separated by filtration. The filtrate is reduced under vacuum to a thick oil. The product is isolated by instant silica gel chromatography, eluting with a chloroform:methanol mixture (85:15 by volume). The 5'-position of threo-3'-azido-2',3'-dide-

oxyuridine by exactly the same operating procedure as that indicated for the bromination of erythro-3'-azido-2-3'-dideoxyuridine.

UV pH 1 Amax 280, t = 9400, <^min 244, [ = 2600 pH 13 A max 276 t = 6700, A min 251 £ = 3700

H1 NMR (DMSO-dg): 611.86 (s,lH,3-NH), 68.02 (s,lH,6H), 65.99 (dd,lH, 2a,

3.0 Hz;

Jl' 2b» = 7'5 Hz' rH)' 05.1 (t'1H' J5'CH 5'0H = 5'4 Hz' 5,QH)' ô4'49

(m,lH,3'H), 64.05 (m,lH,4'H), 63.71 (m,^H,5'H), 62.72 (m,lH,2b«), 62.18

(m,lH,2a')

CHN for CgH^QBrNgO^-0.25 HgO-Ojl C2H4O2 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 1-azido-2',3'-dideoxy-(3-D-erythropentofurannosyl)-2-(benzyloxo)-5-methyl-4-(1H)-pyrimidinone) 0.4 g (17.4 mmol, 2.6 equivalents) of sodium is reacted with 10 mL of anhydrous benzyl alcohol for one hour at room temperature. 1.65 g (6.6 mmol) of 2,5'-O-anhydro-3,-azido-3'-deoxythymidine is added. The reaction is allowed to continue for one hour. After pouring the reaction mixture onto 250 mL of ice water, the pH is adjusted to 7, and the aqueous phase is extracted with ethyl acetate. The organic phase is extracted four times with water.

After dehydration over MgSO₄, ethyl acetate is expelled under vacuum. The resulting oil is subjected to silica gel chromatography, first eluting with ethyl acetate, then with a 9:1 (v/v) ethyl acetate:methanol mixture. The chromatography is collected

J

The fractions containing the product are separated, and the solutes are expelled under vacuum to obtain an oil. The oil precipitates when covered with d-ethyl ether; P.F. = 125-126.5°C.

UV pH 1 unstable 5 pH 13 \max 256, A= 10700, £min 240, ^ = 8900

H1 NMR (DMSO-dg): 67.8 (d,lH,J65 = 1.2 Hz, 6H), 07.49-7.38 (m,5H,2-phenyl), 66.08 (dd,lH,J1, 2a, = 5.0 Hz; J1 2b, = 7.0 Hz, lH), 65.37 (s,2H,2CH2), 65.25 (t,lH,J5,CH 5.0H = 5.4 Hz, 5'OH) 64.36-4.32 (m,lH,3'H), 63.85-3.81 10 (m,lH,4'H), %3.7-3.58(m,2H,5'H), 62.53-2.34 (m,2H,2'H), 61.82 (d,3H,J5 =

1.0 Hz, 5CH3)

CHN for C17H19N504 Calculated: C-57.14; H-5.36; N-19.60 Found: C-57.02; H-5.43; N-19.53

Example 43: l-(3'-azido-21,3'-dideoxy-P-D-threo-pento-furanosil)-2-ethoxy-5-methyl-4-(1H)-pyrimidinone

20 1.08 g (3.13 mmols) of threo-3'- are dissolved

Azido-5'-O-mesylthymidine was prepared from threo-3'-azidothymidine in 100 ml of EtOH and treated with 0.26 g (3.13 mmol) of NaHCO3 under reflux for 18 hours. The reaction mixture was cooled and filtered. The solvents were driven off under vacuum, and the residue was placed on a silica gel column and then eluted with CHCl₃MeOH (9:1 by volume). By combining the appropriate fractions and driving off the solvents under vacuum, a quantity of 0.7 g (2.4 mmol, 75.7%) was obtained; 30 P.F. » 120–122°C.

UV (nrn)ï pH lrl max = 260 (t = 9300), A min = 237 (t = 5500), Ashoulderinent = 221 (i = 7500); at pH 13 A max = 256 (fc = 10000), Amin = 240 (t= 7700).

H1 NMR (DMSO-dg) 67.58 (s,lH,H6), 66.0 (dd,lH,Hl', J = 2.9, 4.56 Hz), 65.06 35 (t,lH,5'OH, J = 4.91 Hz), 64.51-4.47 (m,lH,H3'), 64.34 (q,2H,-OCH2-, J = 7.14 Hz),-64.10-4.05 (m,lH,H4'),6 3.73 (t,2H,H5', J = 5.62 Hz), 62.82-2.73 (m,lH,H2'b), 62.21-2.14 (m,lH,H2'a), 61.82 (s,3H,5CH3), 61.31 (t,3H,-CH2-CH3, 6,65 Hz).

e

H

Analysis for C12H17N5°4 •Calculated: 048.81, H 5.80, N 23.72

Found • : C 48.59, H 5.86, N 23.64 5 Example 44: Threo-3'-azido-23'-dideoxy-4-thiothymidine

5'-O-trityl-3'-deoxy-4-(1,2,4-triazole)thymidine (prepared according to the method of W. Sung, Nucleic Acids Research (1981), 9, 6139) in 100 ml of acetone and 30 ml of H₂O is treated with 0.22 g of Na₂SH·xH₂O. The mixture is stirred for 3 hours. The volume is reduced by half and extracted with 300 ml of CHCl₃. The CHCl₃ is washed with 50 ml of H₂O, dehydrated over Na₂SO₄, and then expelled under vacuum to obtain an oil. The 5'-O-trityl group is removed by dissolving this oil in 100 ml of 80% HOAc. The solution is heated in a water bath for 2 hours, then cooled, diluted with 100 ml of H2O, and filtered. The solvents are expelled under vacuum, and the oil is placed on a silica gel column. By eluting with CHCl₃MeOH (20:1 by volume), collecting the appropriate fractions, and then expelling the solvents under vacuum, a quantity of 0.18 g (0.62 mmol); 28%; Wp = 65-67°C is obtained.

UV (nm): at pH 1 Amax = 337 ( Σ = 20700), at min = 280 ( t = 1200), A shoulder = 238 25 Ct = 3400);

at pH 13 A max = 320 (t = 18400), 1 min = 257 (£ = 1700);

H1 NMR (DMSO-dJ 67.63 (s,lHfHl', J = 2.93, 4.89 Hz), 65.06 (s,lH,5'OH), 64.50-4HO •

4.46 (m,lH,H3'), 64.10-4.04 (m,lH,H4'), 63.73 (d,2H,H5', J = 5.61 Hz), 62.78-2.68 (m,lH,H2'b), 62.20-2.14 (m,lH,H2'a), 62.00 (s,3H,5CH3).

Analysis for C10H13N5O3SO,1 C2H5O-0,25 H2O Calculated: C 41.90, H 4.86, N 23.95, S 10.97 35 Found: C 41.99, H 4.73, N 23.88, S 10.91

1.25 g (2.2 mmol) of threo-3'-azido- are dissolved.

i

Example 45: 4-amino-3'-azido-5-bromo-2',3'-dideoxy-uridine

On-acetyl of 3'-azido-2',3'-dideoxyuridine and bromine according to the Visser process (Synthetic Procedures in Nucleic Acid Chemistry, Vol. 1, page 410)

to obtain 5'-acetyl-3'-azido-5-bromo-2',3'-dideoxyuridine. This material is reacted with 5 equivalents of 1,2,4-triazole and two equivalents of 4-chlorophenyl dichlorophosphate in anhydrous pyridine at room temperature for 7 days to obtain 5'-acetyl-3'-azido-5-bromo-4-(1,2,4-triazolyl)-2',3'-dideoxyuridine as a yellow oil, in moderate yield. By treatment at room temperature with ammonia-saturated methanol at 0°C for 18 hours, after recrystallization in ethyl acetate and filtration of the resulting crystals, 173 mg (0.5 mmole, 0.6.3%) of 4-amino-3'-azido-5-bromo-2',3'-dideoxyuridine is obtained; P.F. = 162-165°C (dec.).

UV (nm): at pH 1 A max = 300.215 (£ = 10700, 12100), A min = 253 (t = 1500); at pH 13 X max = 288 (i = 7300) X min = 260 (t = 3900)

H1 NMR (DMSO-dg) 5 8.3 (s, 1H, H6), 67.85 (large s, 1H, 4-NH2), 67.05 (large s, 1H, 4-NHz), 66.0 (t, 1H, Hl', 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, H4', H5'), 62.31 (t, 2H,H2', J = 6.27 Hz).

Analysis for C9"llN6°3Br

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: 3'-azido-5-bromovinyl-23'-dideoxyuridine

5-Bromovinyl-1,2'-deoxyuridine (BVDU) is synthesized using the method of Jones et al. (Tetrahedron Letters 45, 4415 (1979)) to obtain similar yields. BVDU is tritylated and mesylated by the method of Horwitz et al., J. Org. Chemfo 31, 205

*

i!

(1966). This product is treated with an equim-laden amount of sodium bicarbonate in refluxing methanol to obtain 3'-2-O-anhydro-5-bromovinyl-21-deoxyuridine. This product is treated with 3 equivalents of lithium azide in dimethylformamide/l%

water at 130°C. Silica gel chromatography of the crude material, followed by combination and evaporation of the appropriate fractions, gives 3'-azido-5-bromovinyl-2',3'-dideoxyuridine as a gold-colored oil.

UV (nm); at pH 1 A max = 292.247 A min = 270.238;

a pH 13 A max = 253, A min = 238, A shoulder: 284;

H1 NMR (DMSO-dg)ô Bf06(s, 1H, Hg), Ô7.25(d, 1H, -CH=CHBr, J=13.4 Hz) 66.85(d, 1H, =CHBr, J=13.7Hz) Ô6.07(t, 1H, Hp, J=6.3 Hz), 65.30(large s, 1H, 5"-OH), 64.42{q, 1H, Hy, 3=6.3, 6.1 Hz), 63.86-3.82(m, 1H, H4,), 63.68-3.59(m, 2H, H5,), 62.47-2.32(m, 2H,

Analysis for cnHi2N5°4Br Calculated: C, 36.89; H, 3.38; N, 19.55.

Found: 36.86; H, 3.41; N, 19.51

Example 47: Threo-3'-azido-5-chloro-28,3'-dideoxyuridine

The compound of the titration is prepared in a manner analogous to that used to prepare 3'-azido-5-chloro-2',3'-dideoxyuridine (Example 31) to obtain 0.15 g (0.5 mmole, 25%); P.F. = 65°C.

UV (nm); at pH lA max = 278.212 (i = 8900), Àmin = 240 (£ = 1600); at pH 13 Xmax = 275 (£ = 6600),7\min = 248 (i = 3300);

H1 NMR (DMSO-dg)S ll.89(s, 1H, NH), Ô7.94(s, 1H, Hg), Ô6.00(dd, 1H, H^, J=2.93, 4.64 Hz), 65.1 (t, 1H, 5'0H,J=5.1 Hz), 64.50-4.46(m, 1H, Hj,), 64.08-4.03(m, 1H, H4,), Ô3.71(t, 2H, H5„ J=5.32 Hz), S2.77-2.67(m, 1H, H2,b), 52.23-2.16(m, 1H, H2,a).

Analysis for c:gH^oN5O4C'1 0.1 H2O~0'1 C4H8°2 Calculated: C, 37.85; H, 3.72; N, 23.48; Cl, 11.89 h

Found: C, 37.94; H, 3.91; N, 23.23; Cl, 11.86

4

Example 48: 1-(3'-azido-2',3'-dideoxy-p-D-erythro-pentafurannosyl)-5-methyl-2-(pentyloxo)-4-(1H)-pyrimidinone 1-(3'-azido-2',3'-dideoxy-p-D-erythro-pentafurannosyl)-5-methyl-2-(pentyloxo)-4-(1H)-pyrimidinone is prepared by the process used to prepare 1-(31-azido-2',3'-dideoxy-p-D-erythro-pentafurannosyl)-2-(benzyloxo)-5-methyl-4-(1H)-pyrimidinone (Example 42). The final purification is carried out by high-performance liquid chromatography on C18 eluted with a water-ethanol mixture (3:7). The solvents are removed by evaporation to collect the product as an oil.

UV pHl ,\mav 258 nm £ = 9400, A . 232 nm £= 5900 iiiax min

PHi3Àmax 256 nm £ = 10600, A min 239 nm £= 8200 RMN:relevée dans.DMS0-d6.

NMR; 67.81(s,lH,6H), 56/04(t,lH,J1, 2,=6.7Hz, lH) 65.28(t,lH,J5,CH2 5.0H

64.27(t,2H,J2_Q(1CH 2CH )=6.6Hz,2-0(CH2)1)

63.88-3.84(m,lH,4lH^63/75-3.50(m,2H,5lCH2) 52.50-2.34(m,2H,2'H),

61.80(s,3H,5CH,), 61.72-1.68(m,2H,2-0(CH9)7), ôl,38-l,30(m,4H,2-0(CH9). and 4) ù£ c '

S0.92-0.87(m, 3H-, 2-0 (CH3) 5 >

CHN. for ^5H23^5^4 **2®

Calculated: C-52.70; H-6.93; N-20.48 Found: C-52.63; Hr6.92; N-20.48

Example 49: 3'-azido-3-benzoy1-3'-deoxy-5'-mesylthymidine

3'-Azido-3-benzoyl-3'-deoxy-5'-mesylthymidine is prepared from 3'-azido-3'-deoxy-5'-mesylthymidine by a process analogous to that used in Example 40 to prepare 5'-acetyl-3'-azido-3-benzoyl-3'-deoxythymidine from 5•-acetyl-3*-azido-3'-deoxythymidine; P.F. = 8b-88°C.

UV pHl S max 259 nm l= 21200, A min 230 nm t = 6400 pH13 >y max 265 nm fc = 9600, Amin 248 nm t= 8000

1 (

H NMR (DMS0-d6) 68.00-7.58 (m,6H,6H and phenyl), 66.15(t,lH,J^( 2,=£,1Hz,1'H) 64.55-4.47(m,3H; 3'H and 5'CH2), 64.12-4.10(m,lH,4'H)

53.28 (s^H^'-SO^CH-j), 62.65-2.4(m,2H,2'H),

61.89 (d,3H,J5>6=l,3Hz, 5CH3)

CHN for ^gH^NsOyS 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-3'-azido-2',3'-dideoxy-5-iodouridine 5-iodo-2'-deoxyuridine is tritylated and mesylated according to Horwitz et al. (J. Org. Chem., 31 (1966), 205). This product is heated with 3 equivalents of lithium azide in anhydrous dimethylformamide at 74°C for 48 hours. Silica gel chromatography of the reaction mixture with a CHClg/MeOH mixture (20:1 by volume), followed by collection and evaporation of the appropriate fractions, gives threo-3'-azido-2',3'-dideoxy-5-iodo-5'-trityluridine. The 5'-hydroxyl group is unblocked with a saturated zinc bromide/nitromethane solution at 0°C. Silica gel chromatography of the crude product using a CHCl₂/MeOH mixture (9:1 by volume), followed by collection and evaporation of the appropriate fractions, gives threo-3'-azido-2',3'-dideoxy-5-iodouridine as a solid; melting point = 80°C.

UV (nm); has ; pH 1 min = 247; at pH 13 * max = 277, * min = 252;

H1 NMR (DMSQ-dg) 6 8.37 (s, IH, He), 66.00 (t, IH, Hl', J = 6.17 Hz), 65.4-5.3

(m, 1H, 5' OH), 64.4-4;3 (m, 1H, H3'), 63.9-3.8 (m, 1H, H4'), 63.7-3.6 (m, 2H, H5').

1

Analysis for C9HioN5°4I 0.4 C4H8°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.

10

Example 51: 1-(5'-0-acety1-3'-azido-2',3'-dideoxy-^-

D-Erythropentafurannosyl)-5-methyl-4-(1,2,4-triazolyl-1)-2(IH)-pyrimidinone. 5'-Acetyl-3,1-azido-3'-deoxythymidine is reacted with 5 equivalents of 1,2,4-triazole and 2 equivalents of 4-chlorophenyl dichlorophosphate in anhydrous pyridine at room temperature for 10 days. Silica gel chromatography of the crude product using an EtOAc/hexane mixture (1:1 by volume) followed by collection and evaporation of the appropriate fractions yields an oil. Crystallization in EtOAc gives the compound as a solid weighing 2.7 g (7.5 mmol; 60%); mol.P. = 143–145°C.

15 UV (nm); at pH 1 Amax = 324.245.215 (t = 9300, 10000, 20500), *mip = 282.233

(U 2100.8200); at pH 13 XmQv s 276 (U 6000), = 242 (i= 2000). i nidA n a

H1 NMR (DMSO-dg) 69.34, 8.40 (2s, 2H, triazolyl), 68.23 (s, 1H, H6), 66.12 (t, 1H, Hl', J = 6.16 Hz), 64.48-4.17 (m, 4H, H3', H4', H5'), 62.35 (s, 3H, 5'-acetyl),62.07 20 (s, 3H, 5CH3).

Analysis for C14H16N8°4 Calculated ï C, 46.67; H, 4.48; N, 31.10.

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

^ Example 52: l-(3'-azido-21,3'-dideoxy-B-D-erythro-

(pentofurannosyl)-4-dimethylamino-5-methyl-2-(1H)-pyrimidinone. 5'-Acetyl-3'-azido-3'-deoxy-4-(1,2,4-triazolyl)thymidine is dissolved in anhydrous acetonitrile at room temperature under a nitrogen atmosphere. 20 equivalents of dimethylamine are added all at once, and the reaction mixture is stirred for 30 minutes. The solvents are driven off, the residue is dissolved in ammonia-saturated methanol, and the mixture is stirred.

35

The solution is left for 30 minutes. The solvents are removed, and the residue is dissolved in EtOAc. The compound is titrated

$

pr

4

?

precipitates slowly from the solution and is separated by filtration to obtain a white solid: P.F. = 157-

- --159°C."

UV(nm): at pH 1, ?^max=299.224 (£=14900.7900) At min=254 (t=2500);

5 at pH 13 A max=287 (t=14000), "A min=243 (£=6800).

HJ NMR (DMSO-d6) 6 7.63 (s,lH,H6), 5 6.06 (t,lH,Hl',J=6.53Hz), 55.22

(t,lH,5'OH,J=5.2Hz), 64.37-4.34 (m,lH,H30, 63.83-3.80 (m,lH,H4'), 63.65-3.60 (m,2H,H5'), 63.06 (s,6H,N(CH3)2), 62.29-2.23 (m,2H,H2'), 62.11 (s,3H,5-CH3).

10

Analysis for ci2H18N6°3 Calculated: C48.97, H6.16, N28.56 Found: C49.06, H6.20, N28.50

Example 53: 1-C3'~azido-23'-dideoxy-P-D-erythro-

pentofurannosyl)-5~methyl-2-(isopentyloxo)-4-(1H)-pyrimidinone The compound of titration is prepared by a process analogous to that used to prepare l-(3'-azido-2',3'-dideoxy-p-D-erythro-pentofurannosyl)-2-(benzyloxo)-5-methyl-4-(1H)-pyrimidinone (Example 42).

UV pHl(nm) A max 258 b=9000, Amin 237 £=6000 pH13 (nm) A max 255 l =11000, X min 239 £=8000

25 H1 NMR. (DMSO-d6): 67.79 (s,lH,6H), 6 6.02 (t,lH,Jr 2,=6.3 Hz, l'H), 65.23 (t,lH,J5,OHj5,H=5.2 Hz, 5'OH),

64.4-4.3 (m,3'H and 2-0-CH2-CH2CH(CH3)2),

63.9-3.8 (m,lH,4'H), 6 3.7-3.5 (m,2H,5'H), 6 2.5-2.3 (m,2'H)

61.78 (s,3H,5CH3), 6 1.7-1.5 (m,3H,2-OCH2-CH2-CH(CH3)2) 30 60.92 and 0.89 (d,6H,J=6.3 Hz,2-0(CH2)2CH(CH3)2)

CHN for . C'15H23N504

Calculated: C-53.40, H-6.87, N-20.76 Found: C-53.14, H-6.92, N-20.62

K

Example 54: 3--acetyl-3'-azido-5'-O-(3-chlorobenzoyl 1)-3'-deoxythymidine

^A mixture of 0.3 g (0.74 mmol) of 3'-azido-

5'-()-(4-chlorobenzoyl)-3'-deoxythymidine and 0.4 g (3.0 mmol) of silver cyanide in 20 mL of benzene are mixed. 2.6 g (33 mmol) of excess acetyl chloride is added in several portions. The mixture is stirred at room temperature until the starting material disappears (4 hours), as determined by TLC after eluting with a CHCl₂/MeOH mixture (20:1 by volume). The reaction mixture is filtered, and the filtrate is evaporated to dryness under reduced pressure. The resulting oily residue is chromatographed on silica gel, eluting first with a chloroform/hexane mixture (1:1 by volume), then with chloroform to obtain 0.21 g (64%) of the desired product as an oil.

UV (nrn): at pH 1 \ max 273 (£=9000), At min 251 (t=6200);

pH 13 A max 266 (£=8800), A min 247 U=7000)

H1 NMR (DMSO-dg) 5 1.68 (s, 3H, 5-CH3), 52.47 (s, 3-COCH3), 52.3-2.5 (m, 2'H), 64.1-4.2 et. 4.4-4.7 (m, 4H, 3'H, 4«H and 5'H), 66.1 (t, IH, Jr 2,=6.3 Hz, l'H), 57.5-8.0 (m, 5H, 6H and phenyl)

Analysis for G19H^gN50gCl-0.2 CHCl^

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: 3'-azido-5-cyano-2',3'-dideoxyruridine

•5-Iodo-231-dideoxyuridine is reacted with 2.1 equivalents of acetic anhydride in pyridine, excluding moisture, at room temperature for 2 hours to obtain 2',3*-dideoxy-3',51-diacetyl-5-iodouridine. 1 equivalent of the nucleoside, 1.3 equivalents of potassium cyanide, and 1.3 equivalents of potassium acetate are combined in anhydrous DMSO and heated at 97°C under nitrogen for 2 hours. The solvents are driven off under vacuum, and the residual oil is applied to a

A silica gel column is eluted with CHCl₂/EtOAc (1:1 by volume). The appropriate fractions are collected, combined, and evaporated to obtain 5-cyano-2',31-dideoxy-31,5'-diacetyluridine. The compound is dissolved in ammonia-saturated methanol and stirred at 0°C for 18 hours. The solvents are expelled under vacuum at room temperature to obtain crystals of the compound with a melting point of 160-162°C.

UV(nm): at pH 1 *max=276.215 (£=13500.11200), Amin=238 (t=1700); à-, pH 13 Àmax=276 (£=10100), Xmin=240 (t=3400)

H3 NMR(DMSO-d6) 58.81 (s,lH,H6), 66.00 (t,lH,Hl*, J=5.94Hz), 55.3-5.21

54.28-4.15 (m,lH,H3'), 53.82-3.77 (m,lH,H4'), 53.7-3.5 (m,2H,H5«), 62.18 (t,2H,H2«, 3=5.75 Hz).

Analysis for cioH11N3°5 H2°

Calculated: C 46.61, h 4.50, n 16.31 Found: C 46.67, h 4.71, n 16.54

Example 56: l-(3'-azido-21, S'-dideoxy-(3-D-threo-

pentafurannosyl)-5-methyl-2-pentyloxy-4-(1H)-pyrimidinone. 2,5,-0-anhydro-1-(3'-azido-2•,31-dideoxy-P-D-erythro-pentofurannosyl)-thymine is added to a 0.5 equivalent solution of potassium 1-butylate in pentane-1-ol. The reaction mixture is stirred for 2 hours at room temperature under nitrogen. The solvents are driven off under vacuum, and the residue is applied to a silica gel column. Elution with CHCl₂/MeOH (20:1 by volume), followed by combination and evaporation of the appropriate fractions, gives a clear oil that slowly crystallizes the compound at rest; P.F. = 110-111°C.

4

UV (nm): at pH 1 Àmax=255 (t=10200), Amin=237 (fc=6200), Xep.=222 (t=9000); at pH 13 \max=251.226 (1=11600.9600), ,\min=238 (t=8600)

H1 NMR (DMSO-dg) 67.58 (s,1H,H6), 65.98. (dd,lH,Hl,,J=2.98, 4.88Hz), 65.07 (t,lH,5'OH,J=5.42Hz), 64.5-4.47 (m,lH,H3'), 64.31-4.25 (m,2H,-OCH2-), 64.1-4.06 (m,lH,H4'), 63.73(t,2H,H5',J=5.62Hz), 62.82-2.72 (m,lH,H2'), 62.2-2.14 (m,lH,H2'), 61.82 (s,3H,5-CH3), 61.75-1.65 (m,2H,pentyl>,61.4-1.3 (m,4H,pentyl) 60.92-0.87 (m,3H,pentyl).

Analysis for ci5H23N4°4 Calculated: C53.40, H 6.87, N 20.76.

Found: C 53.31, H 6.90, N 20.74

Example 57: 1-(3'-azido-23'-dideoxy-p-D-threo-pento-furannosyl)-2-benzyloxy-5-methyl-4-(1H)-pyrimidinone The compound of the titer is prepared in a manner analogous to that described for 1-(3'-azido-2',3'-dideoxy-(3-JD-jchreo-pento-furannosyl)-5-methyl 1-2-penty 1-oxy-4-(1H)-pyrimidinone (Example 56) using benzyl alcohol instead of pentane-1-ol; P.F. = 137-139°C.

UV<nm): at pH lXmax-266 (t=9100), Amin=235 (fc=3000);

at pH 13 A max=256 ( £=11500), * min=240 ( j-=9600).

H1 NMR; (DMSO-d6) 67.6 (s,lH,H6), 67.49-7.37 (m,5H, phenyl), 66.01 (dd,lH,Hl', J=2#52}5f0Hz), 65.35 (s,2H,benzyl), 55.1-5.0 (m,lH,5'OH), 54.5-4.4 (m,lH,H3"), 54.1-4.0 (m,lH,H4'), 63.77-3.67 (m,2H,H5'), 62.85-2.70 (m,lH,H2'), 52.25-2.15 (m,lH,H2*) 51.83 (s,3H,5-CH3).

Analysis for C^yH^çjNijO^ 0.25 H20

Calculated: C 56.43, H 5.43, N 19.35.

Found: c 56.51, H 5.37, N 19.36.

Example 58: 1-(3'-azido—2',3'-dideoxy-P-D-erythro-

pentofurannosyl)-4-mebhoxy-5-methyl-2(IH)-pyrimidinone 5•-acetyl-3'-azido-3'-deoxy-4-(1,2,4-triazolyl)thymidine is treated at room temperature with 0.5N sodium methoxide in pen-methanol

1*

for 24 hours. The solution is neutralized at pH 7 with a sulfonic acid resin (DOW 50W, H+), the resin is separated by filtration, and the filtrate is evaporated under vacuum until a solid forms. This solid is dissolved in a small amount of CHCl₃, applied to a silica gel column, and elute with 2% MeOH in CHCl₃. The appropriate fractions are collected, combined, and evaporated to dryness to obtain a white solid; P.F. = 119-123°C.

10

UV(ntn): at pH 1 Amax=279 (£=7500), Amin=239 (t=1700);

at pH 13 Amax=279 (t=6400), ,\min=243 (£=1300).

H1 NMR (DMSO-dg) 58.02 (s,lH,H6), 66.08 (t,lH,Hl', J=5.86Hz), 55.29 (t,lH,5'OH, j=5.48Hz), 64.41-4.33 (m,lH,H3'), 63.9-3.86 (m,lH,H4'), 63.86 (s,3H,4-15 OCHj), 63.75-3.58 (m, 2H,H5'), 52.4-2.3 (m,2H,H2'), 51.89 (s,3H,5-CH3).

Analysis for C11H15N5O4

Calculated: C 46.97, H 5.38, N 24.90.

Found: C 47.06, H 5.40, N 24.86

20

Example 59: l-( 3' -azido-2' , 31 -didexoxy- |3~D-erythro-

(pentofurannosyl)-5-methyl-4-(1-pyrrolidinyl)-2-(IH)-pyrimidinone. 5'-Acetyl-3'-azido-3'-deoxy-4-25(1,2,4-triazolyl)thymidine is dissolved in anhydrous acetonitrile at room temperature under a nitrogen atmosphere. Five equivalents of pyrrolidine are added dropwise over 5 minutes, and the reaction mixture is stirred for 2 hours. The solvents are driven off under vacuum to obtain an oil. This oil is dissolved in ammonia-saturated methanol at room temperature and stirred for 4 hours. The solvents are driven off under vacuum, and the residue is applied to a silica gel column. Eluation with CHCl₂/MeOH 35 (20:1 by volume), followed by combination and evaporation of the appropriate fractions, gives the compound of

(4

titer in the form of a white solid; P.F. 158-171°C. ? UV(nm): at pH 1 Xmax=295, 233 (L=15700, 9500), A min=253 (1=2600); at pH 13 \max=285 (<t=1530a), Amin=241 (t=7900).

H1 NMR (DMSQ-d6) 67.57 (s,lH,H6), 66.02 (t,lH,Hl', J=6.5Hz), 65.22

(t,lH,5'OH,J=5.15Hz), 64.4-4.3 (m,lH,H3'), 63.84-3.77 (m,lH,H4'), 63.67-3.51 (m,6H,H5',4 H of pyrrolidine), 62.24 (t,2H,H2',J=6.08Hz) 62.14 (s,3H,5-CH3), 61.87-1.78 (m,4H,pyrrolidine).

Analysis for C4H2ON6°3 Calculated: C 52.49, H 6.29, N 26.23 Found: C 52.37, H 6.33, N 26.17

Example 60; 1-(3'-azido-2',31-dideoxy-P-D-erythropentofurannosyl)-4-benzyloxy-5-methyl-2-(IH)-pyrimidinone. 5'-acetyl-3'-azido-3'-deoxy-4-(1,2,4-triazolyl)thymidine, dissolved in anhydrous acetonitrile, is added dropwise at room temperature under nitrogen over 5 minutes to a suspension of 5 equivalents of benzyl alcohol and 2 equivalents of potassium t-butylate in anhydrous acetonitrile. The reaction mixture is stirred for one hour and the solvents are removed under vacuum. The residue is placed on a silica gel column and eluted with a CHClg/EtOAc mixture (3:1 by volume). The appropriate fractions are collected, combined and evaporated to obtain a solid. The solid is recrystallized in a 1:1 CHClg/EtOAc mixture (by volume) and the resulting crystals are separated by filtration to obtain the compound of the specified strength; P.F. 133-134°C.

UV(nm): at pH 1 A max=276 (fc=8000), A min=237 (t=2000);

at pH 13 Xmax=281 (£=8100), Amin=237 (£=1600).

H1 NMR (DMSO-dg) 68.05 (s, 1H,H6), 67.45-7.35 (m,5H,phenyl), 66.07 (t,lH,Hl', J=5.94Hz), 65.35 (s,2H,benzy!e), 65.27 (t,lH,5'OH,J=5.20Hz), 64.41-4.31 (m,lH,H3') 63.91-3.83 (m,lH,H4'), 63.7-3.6 (m,2H,H5'), 62.4-2.3 (m,2H,H2'), 61.9 (s,3H,5-CH3). ~

n

<

i

Analysis for c^gH2iN5°5 Calculated: C 57.14, H 5.36, N 19.60

Found: C 57.06, H 5.37, N 19.58.

Example 61: 5'-acetyl-3'-azido-5-bromo-23'-dideoxyuridine

3'-azido-23'-dideoxyuridine is acetyl and brominated according to the Visser process (Synthetic Procedures in Nucleic Acid Chemistry, _1_,' 410) to obtain the compound of titer; P.F. = 112-114°C.

UV(nm): at pH 1 Amax=279.210 (19500.10600), Amin=243 (£=2000); •at pH 13 Xmax=276 (£=700), \min=250 (1=4000).

NMR .(DMSO-dg) 611.88(s,lH,NH), 68.02(s,lH,H6), 66.08-6.01 (m,lH,HD, §4.47-4.40 (m,lH,H3'), 64.27-4.24 (m,2H,H5'), 64.03-4.00 (m,lH,H4!), 62.41-2.34 (m,2H,H2'), 62.09 (s,3H,acetyl) •

Analysis for C 5

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—(3 azido—21,3'—dideoxy—(3— D—threo—pento— f urannosyl-5-.(trifluoromethyl) uracil The 5'—hydroxyl group of 5—trifluoromethyl-2'-deoxyuridine is protected with a triphenylmethyl group, and the 3'-hydroxyl group is mesylated. 3.0 g (4.9 mmol) of the product is reacted at 70°C with 0.7 g of LiN in 50 mL of DMF for approximately 26 hours. The cooled reaction mixture is poured onto ice with stirring. The solid is isolated, washed with water, and purified by flash chromatography using a hexane/ethyl acetate mixture (2:1 by volume). By combining the appropriate fractions and removing the solvent, 0.83 g of the product is obtained. tritylated. It is dissolved in a saturated solution of zinc bromide in acetonitrile and shaken at 0°C

The mixture was left to stand for approximately 16 hours. After adding 100 mL of 1M ammonium acetate, the organic phase was separated and dried under vacuum. The residue was purified by flash chromatography using a 20:1 (v/v) chclg/ch^oh mixture. The fractions were combined and dried under vacuum. The yield of the product was 0.25 g, 0.8 mmol, 5.3%; mol/L at 118-120°C.

Analysis - for c10hioF3n5°4

Calculated: C 37.39, H 3.14, N 21.80, F 17.74

Found: C 37.31, H 3.23, N 21.75, F 17.60

Example 63: 1-(3'-azido-2',3'-didexoxy-P-D-threo-pento-furannosyl)uracil. The 5'-hydroxyl group of 2'-deoxyuridine is protected, and the 3'-hydroxyl group is mesylated. The 3'-mesyl group is displaced with inversion of configuration by heating in dimethylformamide at 80°C with 3 equivalents of lithium azide for 24 hours. The reaction mixture is poured into ice-cold water, and the product precipitates. After filtration, the wet product is removed. Final purification is carried out by silica gel chromatography, eluting with a chloroform/methanol mixture (95:5). The appropriate fractions are collected, and the solvents are removed to obtain the compound of specified titer as a solid; molten rock 142-145°C. Example 64: 1-(3'-azido-21,3'-dideoxy-ft-D-erythropentofurannosy 1)uracil The 5'-hydroxyl group of 30 g (0.13 mol) of 2'-deoxyuridine is tritylated by the method of Horowitz et al. (J.-Org. Chem., 31, 205 (196b)). The 3'-hydroxyl group (12.6 g, 0.027 mol) is chlorinated by the method of Example 62. The dimethylacetamide is driven off under vacuum and the thick oil is poured into 500 ml of water. The product is extracted three times with ether. The solvent is driven off and the resulting oil is silica gel chromatographed, first eluting with dichloromethane,

"

\

\

then with 1% methanol in dichloromethane.

"

The product fractions are collected and the solvents are expelled under vacuum. The 5'-hydroxyl group is then unblocked.

Without further purification, the mixture is heated in 80% acetic acid in a water bath for 20 minutes. Upon cooling, tritylcarbinol precipitates and is separated by filtration. The filtrate is concentrated under vacuum and analyzed by silica gel chromatography, eluting with ethyl acetate. The 3-chloro group is displaced by heating in HMPA with 3 equivalents of lithium azide at 90°C for approximately 16 hours. The reaction mixture is poured into water and treated with chloroform. The chloroform contains the product, and it is dehydrated over MgSO₄. Removal of the chloroform yields a solid, which is first recrystallized in an ethyl acetate/methanol mixture, and then with water. The recrystallized solids are dissolved in water and applied to a XAO column. After washing with water, the compound of the titer is elute with ethanol. The ethanol is driven off under vacuum to obtain a solid; P.F. = 168.5°C. Example 65: 1-13'-azido-2',3'-dideoxy-erythro-p-D-pentofurannosyl-5-ethyl)uracil. 5-Chloromercuri-2'-deoxyuridine is prepared from 10 g (0.044 mol) of 2'-deoxyuridine by the procedure of Bergstrom and Ruth (J. Carb. Nucl. and Nucl., 4, 257, (1977)). 2'-DesoAl-5-ethyluridine is prepared by the procedure of Bergstrom et al. (J. Am. Chem. Soc., 100, 8106 (1978)). The 5'-hydroxyl group is protected by the process of Example 1-2. The 5'-hydroxyl group is chlorinated, and the protection of the 5'-hydroxyl group is removed by the process of Example 64. The 3'-chloro group of threo-3'-chloroT-2,3-dideoxyuridine is displaced with inversion of configuration by heating in HMPA with 5 equivalents of lithium azide at 35-55°C for one hour. The compound is purified by silica gel chromatography by eluting with

HAS

.1

d-Ethyl acetate. Solvent removal from the appropriate fractions¹ gives the desired compound; P.F. = 112.5-115°C. Example: 1-(3*-azido-2•,3*-dideso-p-D-chreo-pento-furannosyl)-5-(2-bromovinyl)uracil → 5-Bromovinyl-2'-deoxyuridine is synthesized

(BVDU) by the method of Jones et al. (Tetrahedron Letters 45, 4415 (1979)) to obtain similar yields. BVDU and mesyl are tritylated by the method of Horowitz et al. (J. Org. Chem., 3JL, 205 (1966)). 4.25 g (6.5 mmols) of this product are dissolved in 100 ml of DMF containing 0.955 g (19.5 mmols) of Li₂ and heated at 74°C for 24 hours. The reaction mixture is poured onto 600 ml of ice with stirring. The solid that forms is isolated and purified by chromatography to obtain a gum (2.48 g). Treatment by dissolving the compound in 100 mL of 80% acetic acid and heating it in a water bath for 3 hours unlocks it. The cooled reaction mixture is diluted with water and filtered to remove tritylcarbinol. The filtrate is reduced to an oil, which is purified by flash chromatography in CHCl₂/CH₂OH (9:1 by volume). Collection of the appropriate fractions and removal of the solvent yields a solid, which is twice crystallized in aqueous methanol. Yield: 0.66 g, 1.8 mmol, 27.7%; mol/L 165–166°C.

Analysis for cnHi2BrH5°4 ~ H2°

Calculated values: 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

30

Example 67: l-(3'-azido-23'-dideoxy-p-D-threo-pentofurannosyl)-5-methylisocytosine 0.5 g (2 mmol) of l-(3'-azido-2',3'-dideoxy-p-D-threo-pentofurannosyl)-2-methoxy-5-35-methyl-4(H)-pyrimidinone is combined with 15 ml of saturated methanol

of ammonia in a can. After 6 days at the temperature

*

w

\

At ambient temperature, the bomb is heated in an oil bath at 5°C for 4 days. The reaction mixture is brought to dryness and purified by crystallization in a CHClg/CH₂OH mixture (9:1 by volume). The solid is washed with CHCl₂ and air-dried to obtain 0.16 g (0.6 mmol, 30%) of product; melting point 158-160°C.

Analysis - for <-ioH14N6°3 H2°

Calculated: C, 44.36; H, 5.40; N, 31.04

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

Example 68: 1-(3'-azido-2',3'-dideoxy-p-D-threo-pento-furannosyl)-3-methylthymidine. 1.0 g (1.95 mmol) of 51-trityl-3'-threo-3'-azido-3'-deoxythymidine and 0.93 g (7.8 mmol) of N,N-dimethylformamide dimethylacetal (Zemlicka, Coll. Czech. Chem. Comm., 35, 3572 (1972)) are refluxed in 50 mL of CHCl₂ for 96 hours. Upon removal of the solvent, an oil is obtained, which is further purified by flash chromatography using CHCl₂. Upon further removal of the solvent, 0.54 g of a foam is obtained. The foam is loosened by heating it in 50 mL of 80% acetic acid in a water bath for 2 hours. Tritylcarbinol is removed by filtration after diluting the reaction mixture with hydrogen peroxide (HgO). The filtrate is heated under vacuum to an oily state and purified by flash chromatography using CHCl₂/EtOAc (2:1 by volume). The solvent is then removed to obtain 0.20 g of the compound as an oil.

UV max (nm) at pH13 S max=267 ( £=3100, /\ ep.=209 (£.=8500) ; at pH13 ^nax=267(£ =8000).

H1 NMR(DMSO-d6): 7.56 (s,1H,H6); 6 6.07 (dd,lH,Hr); 6 3.17 (s,3H,N-gH3); 6 1.86 (s,3H,5-CH3).

Analysis for ^■^^•15^5^4 ~ H®Ac °'3 H2° '

Calculated: C 45.62, H 5.58, N 22.54

Found: C 45.67, H 5.60, N 22.57

Example 69: Acid (E)-3-|"l-(3'-azido-2',3'-dideoxy-(3-D-erythropentofurannosyl)-1,2,3,4-tetrahydro-2,4-dioxo-5-pyrimidinyl 1,2-propenoic The 2'-deoxyuridine is transformed into the derivative 5-chloromercury according to the process in the literature,

in a yield of 96% (Bergstrom and Ruth, J. Carb.

Nucl., 4, 267, (1977)). 50 g (1.04 mol) of this product is dissolved in 800 mL of anhydrous MeOH containing 104 g (1.04 mol) of ethyl acrylate and a 0.1 N solution of L⁻¹PdCl₂ in MeOH (1040 mL). The solution is stirred for 4 hours, then treated with H₂S for 2 minutes. The suspension is filtered through Celite buffer and the filtrate is evaporated to dryness under vacuum. The residue is trituned with MeOH to obtain a solid, which is filtered and dried to obtain 22 g of a white solid. This product is tritylated and mesylated by the process of Horowitz et al. (J. Org. Chem., 31, 205 (1966)). A 7.06 g (10.9 mmol) portion of this material is dissolved in 100 mL of anhydrous MeOH containing 0.92 g (10.9 mmol) of NaHCO3 and heated under reflux for 6 hours. The solvents are driven off under vacuum, the residue is swirled with H2O, then filtered and air-dried to obtain 6.1 g of a white solid. This product is dissolved in 50 mL of DMF/1 mL of H2O containing 1.6 g (33.2 mmol) of de-LiNg and heated at 125°C for 4 hours. The reaction mixture is poured onto 200 mL of ice, the precipitate is collected, and washed with H2O. This material is then dissolved in 100 mL of 80% HOAC.

I

and is heated at 100°C for 4 hours. The reaction mixture is diluted with H2O and the precipitate is separated by filtration. The filtrate is evaporated to dryness to obtain 800 mg of an oil. This oil is dissolved in 20 mL of 0.5 N NaOH and stirred at room temperature for

<

2 hours. The pH of the solution is adjusted to 3, the precipitate is separated by filtration and air-dried to obtain 550 mg (1.7 mmol) of the compound with a concentration of greater than 250°C.

UV (nm): at pHl^max = 300 (£ = 20400)J min = 230 (£ = 3900), ^ = 262 (£ = 13100)

at pH13^max = 297,266 (£= 15600, 14800),X min = 281, 288 (£= 13600, 8600);

H1 NMR (DMSO-d6) 68.37 (s,lH,H6), 67.30 (s, 1H,-CH=, 5=15.57Hz), 66.78 (d,lH, = CH-COOH, 5=15.93Hz), 66.1-6.06 (m,lH,HD, 65.41-5.37 (m, 1H,5'0H), 64.47-4.39 (m,lH,H3'), 63.87-3.83 (m,lH,H4), 63.74 3.58 (m, 2H,H5'), 62.54-2.81 (m,2H,H2*).

Analysis for G^2H13N5°6 Calculated: C 44.59, H 4.05, N 21.67

Found: C 44.45, H 4.06, N 21.60.

Example 70: Acid (E) ~ 3-[l-(3t-azido-2<,3'-dideoxy-(3-

D-threo-pentofurannosyl)~1,2,3,4-tetrahydro-2,4-dioxo-5-pyrimidinyl1—- 2-propenoic acid 1 transforms 2'-deoxyuridine into the 5-chloromercury derivative according to the process in the literature, in a yield of 96% (Bergstrom and Ruth, J. '.Carb.

Nucl» Nucl, 4, 257 (1977)). 50 g (1.04 mol) of this product is dissolved in 800 ml of anhydrous MeOH containing 10.4 g (1.04 mol) of ethyl acrylate and a 0.1 N solution of Li2PdCl in 10.40 ml of MeOH. The solution is stirred for 4 hours, then treated with H2S for 2 minutes. The suspension is filtered through Celite buffer and the filtrate is evaporated to dryness under vacuum. The residue is triturated with MeOH to obtain a solid, which is filtered and air-dried to obtain 22 g of a white solid. This product is tritylated and mesylated by the process of Horowitz et al. (J. Org. Chem., 31, 205 (1966)). A 2.7 g (4.2 mmol) portion of this material is dissolved in 55 ml of anhydrous DMF containing 0.62 g (12.7 mmol) of LiN₂ and heated at 70°C under N₂ for 24 hours. The reaction mixture is poured

AI

p

\

on 400 ml of ice, the precipitate is collected and

?

It is purified by flash chromatography. Elution with a CHCl₂/MeOH mixture (50:1 by volume) gives 1.48 g of the azido intermediate. This material is treated with 50% acetic acid (100 mL) at 100°C for 3 hours. By dilution with 0, filtration of the resulting suspension, and evaporation of the filtrate under vacuum,

I

710 mg of an oil is obtained. This oil is dissolved in 50 ml of NaOH and stirred at room temperature for 2 hours. The pH of the solution is raised to 3, the precipitate is separated by filtration and dried in air to obtain 240 mg (0.7 mmol, 50%) of the compound with a titer of 250°C.

UV{nm) at pH1 ^ max = 301 (λ=19500), } min = 230 (λ = 3600)&ép.=249 (λ = 12700)

at pH 13 ^ max = 299.267 (£= 1400.13200), ][ min = 232.239 (£ = 1200.7560] H18HN (DMSO-dg) 53.13 (s,7H,H6) 67.32 (d,lH,-CH=, 6 = 15.87 Hz) 66.78(d,lH, = CH-COOH, J=15.62Hz), 56.01-5.98 (m,lH,Hl'), 55.11-5.98 (m,lH,5'OH), 64.50-4.43 (m,lH,H3'), 54.13-4.08 (m,lH,H4'), 63.80-3.75 (m,2H,H5'), 62.77-3.67 (m,lH,H3"), 52.30-2.20 (m,lH,H2')

Analysis for C^2^13H5^6 ~ IjSHjO Calculated: C 41.15, H 4.60, N 19.99

Found: C 41.38, H 4.50, N 20.01.

Example 71: (E)-3-fl-(3'-azido-2',3'-dideoxy-3-D-erythropentofurannosyl)-1,2,3-,4-tetra-hydro-2,4-dioxo-5-pyrimidinyl]-2T-propanoic acid. 5'-trityl-31-mesyl-5-propenoate-2'-deoxyuridine is prepared according to the procedure in Example 69. This material is hydrogenated with 10% Pd/C in EtOH to obtain the propanoic derivative. This product is then treated in the manner described above to obtain the compound with the given titer; P.F. = 118-120°C.

\

<

UV (nm): at pHl/v max = 265 (λ = 9900), J/min = 235 (λ = 3100);

at pH13^ max = 265 (£=7400) J min - 247 (Ç. = 5400);

H1 NMR (DMSO-dg) ô 7.68 (s,lH,H6), 6 6.11-6.06 (m, 1H,H1'), 6 4.45-4.35 (m,lH,H3'), S 3.85-3.80 'm,lH,H4'),. 6 3.68-3.53 (m,lH, H5')

5 Analyzed for

Calculated: C 44.31, H 4.65, N 21.53

*

Example 72: Clinical study f;

Two AIDS patients were given 3'-azido-3'-deoxythymidine (AZT) orally at a dose of 2 mg/kg every 8 hours on days 1 and 2 of treatment.

On days 2 and 3, patients also received 500 mg of probenecid (PB) every 6 hours and a single dose of

AZT was administered to them on day 3. The maximum (Cmax) and minimum (Cmin) levels of AZT on day 3 (after probenecid administration) were significantly higher than the corresponding levels on day 1, resulting in a threefold reduction in total body clearance (Cl/F) and a prolongation of the mean half-life (tl/2) of AZT (0.88 to 1.73 hours) during PCB treatment. The mean AZT-glucuronide/AZT ratio in the urine was markedly reduced, from 7.3 to 2.4, after PB treatment. Table 1 summarizes the main pharmacokinetic parameters of AZT before and after co-administration of PB.

Table 1

Sick

AUC

Cmax

Cmin

Tmax

Cltot/F

tl/2

No.

(h *tim)

(pm)

(ym)

(h)

(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

921.00

1.61

AVERAGE

10.21

6.30

0.21

0.38

875.25

1.73

±Standard deviation

0.29

0.23

0.08

0.18

64.70

0.16

1. AZT

3.70

3.74

0.00

0.50

2333.80

0.87

2. AZT

3.04

2.51

0.00

0.31

3027.00

0.89

AVERAGE

3.37

3.13

0.00

0.41

2680.40

0.88

iStandard deviation

0.47

0.87

0.00

0.13

480.17

0.01

Example 73: Antiviral activity

Table 2 shows the enhancement of the anti-Friend leukemia virus (LFV) activity of 3'-azido-31-deoxythymidine (AZT) by dipyridamole, dilazep

tf

\

and 6-[(4-nitrobenzyl)thio]-9-3-D-ribofurannosyl)purine as nucleoside transport inhibitors. A plate was seeded with FG-10 cells one day prior to infection with FLV. One hour after infection, known concentrations of the test compounds/combinations were added. The plates were incubated for 3 days, the media were replaced with fresh McCoy Medium 5A, and the plates were incubated for another 3 days. The concentrations of the test compounds/combinations that provided 50% plaque inhibition were determined as shown in Table 2. Neither dipyridamole (10 pM) nor dilazep (5 µM) alone showed antiviral effect.

Table 2

DE^Q compound/association of azidothymidine (nM)

15 AZT 5

AZT + dipyridamole 1 juM 1

AZT + dipyridamole 5 pM 0.5

AZT + dipyridamole 10 pM 0.2

AZT + dilazep 5 pM 0.5

20 AZT + 6-C(4-nitrobenzyl)thio]-

9-g-D-ribofurannosyl)purine 5 pM 3

Example 74; Anti-PTI activity of 3'-azido-3'-deoxythymidine (AZT)

One patient was diagnosed whose number of

' —3

25 platelets, totaling 38,000 mm, were affected by purpura.

—3

thrombocytopenic (platelet count < 1000 mm³) and was treated for 6 weeks with 5 mg/kg of AZT intravenously every six hours and during this

— 3

During this period, his platelet count rose to 140,000 mm³. The treatment was then modified to 5 mg/kg every 4 hours orally for 4 weeks and discontinued for 4 weeks when a drop in platelet count was observed.

-3 -3

to 93,000 mm after 2 weeks, and to 70,000 mm after 4 weeks. Treatment was resumed at a dose of 5 mg/

35 kg/4 hours orally for 5 weeks and the number of

— 3

platelets amounted to 194,000 mm, a reduction to

B

2.5 mg/kg/4 hours orally without time limit resulted in a slight decline in platelet count, but not to the extent of a diagnosis of ITP.

Example 75: Treatment of Kaposi's Sarcoma with 3'-azido-

patients with Kaposi's sarcoma (KS) treated with AZT and the following effects were observed.

One patient experienced a complete cure. Ten patients showed regression of their lesions.

In 2 patients, SK remained stable.

In 3 patients, the lesions progressed.

The response at =* 50% is comparable to that obtained by treating with the current preferred therapy for SK, a recombinant 15α interferon.

Example 76: AZT/acyclovir combinations against HIV in vitro

By applying a method analogous to that of Example 79, combinations of AZT and acyclovir (ACV) were tested to determine their in vitro efficacy against HIV.

ACV alone showed little activity, with a concentration of 16 µg/ml (the highest tested) demonstrating less than 30% protection, whereas AZT at 8 µg/ml demonstrated 100% protection using this method. Table 3 shows the combinations of the two drugs required to achieve 100% protection.

5

deoxythymidine (AZT)

In this study, 9 diagnosed patients were treated.

Table 3

LCA (ug/ml)

AZT (UM) 0

0.5

30

8

2

1 4 8

1

0

35

These results show that ACV potentiates the antiviral activity of AZT by approximately 3 times.

Example 77: AZT/interferon combinations against HIV in vitro

Peripheral blood mononuclear cells (PBMCs) were obtained from HIV-negative, healthy volunteer donors by Ficoll-Hypaque sedimentation of heparinized blood. The cells were treated with 10 jug/ml of 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, Maryland). Starting 4 to 6 days after PHA exposure, the cells were dispensed at concentrations of 4 x 10⁶ cells/ml into 25 cm³ flasks containing 5 ml of medium and then exposed to the drugs and viruses detailed below. The day of virus inoculation is designated day 0. On day 4, fresh medium was added. Thereafter, every 3 to 4 days, a portion of the cell suspension was taken for analysis and replaced with cell-free medium. Experiments 2 and 4 were completed after 14 days, and experiments 1 and 3 after 16 days.

The virus reserves were cell-free supernatant fluids from HIV-infected H9 cells frozen in aliquots at -70°C. The infective dose

50% of tissue cultures (DICTcn) from the reserve b

The virus count was 10/ml.

Four separate experiments were performed using CMSP from different donors (Table 4).

In experiment 1, both drugs were added after the cells had been exposed to the virus. We put in g

suspension of 40 x 10 cell aliquot portions in

5

20 ml of medium containing 10 DICT^q of virus was incubated for 1 hour, then washed 3 times and resuspended in virus-free medium. In experiments 2 to 4, cells were incubated for 24 hours in medium containing or not containing recombinant α-interferon (rlFNaA), then exposed to AZT and the virus. Added

¥

The virus was introduced directly into the cultures in a small volume of medium and was not removed by washing. The viral inocula were 4 x 10³, 10³, and 2 x 10³ DICT₅Q in experiments 2, 3, and 4, respectively. The drug concentrations were adjusted at each change of medium so that the initial concentrations were maintained.

In all experiments, successive two-fold dilutions of a specific combination of rIFNaA and AZT were studied. Each concentration of rIFNaA and AZT used in combination was also studied individually to obtain reference points.

In all experiments, duplicates of the cultures were kept for each concentration and for the 15 infected and uninfected controls. In experiment 1, AZT 3.2 pM and 128 units/ml (U/ml) of rIFNaA were studied.

as well as 5 two-fold dilutions of this combination.

In experiments 2 and 3, AZT 0.16 pM and 128 U/ml of rIFNaA were used, along with 3-4 two-fold dilutions of this combination. In experiment 4, AZT 0.08 pM was used in conjunction with 128 U/ml of rIFNaA, along with 2 two-fold dilutions of this combination.

After approximately one week, the cultures were assessed every 3 to 4 days for the presence of the virus. The cells were then tested for HIV antigen.

by indirect immunofluorescence; the supernatants were evaluated for reverse transcriptase (TI), viruses produced and HIV p24 antigen by radioimmunoassay.

30 To calculate the effects of the associated drugs, we used the analysis of the effects of multiple drugs by Chou and Talalay (Advances in Enzyme Regulation, (1984), 22, 27-55).

The results were also evaluated using the isobologogram technique, a geometric method for assessing drug interactions. The concentration i

ri

The concentration of AZT producing a desired effect (e.g., 50% inhibitory) is plotted on the horizontal axis, and the concentration of rIFNaA producing the same degree of effect is plotted on the vertical axis. A straight line is drawn connecting these points, and the concentration of the agents in the combination that produces the same effect is plotted. If this point falls below the line, the combination is considered synergistic.

In Experiment 1, all AZT concentrations were completely inhibitory, making it impossible to assess combined effects. In Experiments 2–4, a clear synergistic interaction between the two agents was observed (Tables 4–9). Synergy was evident from all viral replication measures used and persisted even when the effect of rIFNaA alone was negligible. Synergy calculations were performed by applying the multiple drug effect analysis to the TI results obtained in Experiments 2–4, the virus production results obtained in Experiments 2 and 3, and the radioimmunoassay results obtained in Experiment 4. The isobologram method also indicated synergy.

Table 4

Method a

HIV

Timeline of 11

1 addition

added inoculation

drugs

HIV experience

(dict50)

rIFNaA

AZT

1

HAS

105

0

0

2

B

4 x 103

-24 hours

0

3

B

103

-24 hours

0

4

B

2 x 103

-24 hours

0

(a) Method A: 40 x 10 cells were suspended

5

in 20 ml of medium containing 10 DICT^q of HIV for 1 hour at 37 °C, then washed and resuspended.

Method B: The indicated quantity of virus was added to 2 x 10^ cells in medium; the cells were not washed afterwards.

H

Table 5

Experiment 2 - Day 10

Effects of rIFNaA and AZT on mean trans- values

(ules x 103)

rIFNaA (U/ml)

6 3

reverse criptase (CPM/10 cells x 10)

AZT (juM)

0

_8

1_6

32

64

128

0

205

173

150

193

169

151

o%

o

H

159

85

0.02

110

42

0.04

71

10

o%

o

00

31

4

0.16

7

1

Painting

6

Experiment 3 -

Day 13

Effects of rIFNaA

And

AZT

on the average values of TI

(CPM/106

cells

1

x

103)

rIFNaA

(U/ml)

AZT (lum)

0

16

32

6±

128

0

157

143

117

117

130

CM ©

*

O

51

10

0.04

10

0

00

o *

o

2

0

vo

H *

o

5

0

Table 7 Experiment 4 - Day 11 Effects of rIFNaA and AZT on mean TI values (CPM/10® cells x 103)

rIFNaA (U/ml)

AZT (uM) 0 32 64 128

0 32 5 4 2

0.0281

0.04 4 0

0.08 1 0

ff

Table 8

Experience

Day 3

13

Effects of rIFNaA and

AZT on virus production

(DICT50/ml)

5

!

rIFNaA (U/ml)

AZT (uM)

0

16

32

64

128

0

105'7

105'6

5 3 10 '

105'1

105'0

0.02

104'8

101'9

0.04

100'3

19

<10x'*

10

0.08

101.9

19

<10'L y

0.16

<101.9

<101.9

Table 9 Experiment 4 - Day 14

Effects of rIFNaA and AZT on HIV p24 3

15 rIFNaA (U/ml)

AZT (uM) 0 32 64 128

0 200-300 100-200 50-100 25-30 0.02 100-200 2.2

0.04 25-30 1.0

20 0.08 11.2 0.8

HIV p24 levels are presented in ng of protein/ml

Table 10

Association indices for AZT and rIFNaA, calculated from

25. The results concerning TI

Indices of association with different percentages of TI inhibition

Experience

A Day in Culture

50%

90%

95%

30

2

7

2.14

0.34

0.23

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

35

4

8

0.01

0.03

0.03

4

14

0.02

0.07

0.12

V

The I.A. values are determined by solving the equation for different degrees of TI inhibition. I.A. values less than 1 indicate synergy. The given I.A. values were obtained using the mutually non-exclusive form of the equation; the values obtained using the mutually exclusive form were always slightly lower.

Example 78: In vitro studies of antibacterial synergy

3'-Azido-3'-deoxythymidine and 10⁸ known antibacterial agents (listed in Table 11) were each treated with N,N-dimethylformamide for 30 minutes. Dilutions for microtiter were prepared using Wellcotest broth.

Prior to synergy testing, 15 minimum inhibitory concentration (MIC) determinations were performed for each individual compound against the test microorganism (E. coli CN314). Table 11 shows the MIC endpoints for each drug against E. coli CN314.

Successive dilutions of two 20-fold of the test drugs or of 3'-azido-3'-deoxythymidine were prepared in "flat-bottomed" microtiter plates or

if

"Transfer" plates were used, respectively. Plates containing the appropriate dilutions were combined, resulting in a series of 192 dilutions/combinations. Controls consisting of the compound alone were also included. The highest concentration of any of the drugs used was twice its MIC value (Table 11).

The test plates were inoculated with a bacterial culture containing approximately 5 x 10⁻⁵ colony-forming units (CFU)/ml and then incubated at 27°C for 18 hours. The reservoirs were rated for bacterial growth or lack thereof, and the MICs were determined. Fractional inhibitory concentrations (FICs) were calculated from the MIC values by dividing the MIC of the combination by the MIC of each isolated agent. The sum of the fractions (sum of the fractional inhibitory concentrations) was then calculated. A result of approximately 0.5 or less indicates synergy.

Table 11

Minimum inhibitory concentrations (MICs) of the investigational drugs alone

CIM compound (uq/ml)

Tobramycin 0.4

Fusidic acid 1000

Chloramphenicol 3.1

Clindamycin 100

Erythromycin 25

Rifampicin 6.2

3'-azido-3'-deoxythymidine 1.0

Trimethoprim 0.125

Sulfadimidine 32

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

JU

1

Table 12

}

Synergy studies: Combinations of AZT and other antibacterial agents

Optimal CIM (jug/ml):

Association

Medication/AZT

CIF 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

Trimethoprim/AZT

0.004/0.5

0.504

Suifadimidine/AZT

0.25/0.125

0.375

Example 79: Anti-HIV activity of 3'-azidonucleosides in vitro

The in vitro activity of 31-azidonucleosides (drugs) was tested in two cell lines, namely: H9 (OKT4+ T lymphocyte line, allowing HIV replication but partially resistant to the cytopathic effect of HIV) and TM3 (T lymphocyte clone, specifically directed against tetanus toxoid, immortalized by lethally irradiated HTLV-I and selected for rapid development and sensitivity to the cytopathic effect of HIV).

The inhibition assay was performed as follows: TM3 cells were stimulated with fresh, autologous, irradiated (4000 rad; 40 Gy) antigen-positive peripheral blood mononuclear lymphocytes (PBMLs) cultured in complete medium containing 15% by volume of interleukin-2 (IL-2, lectin-depleted; Cellular Products, Buffalo, New York) 6 days prior to the assay. Unstimulated ATH8 cells were used. After pre-exposure to 2 jugs of Polybrene per ml for 30 min, the targeted T lymphocytes (2 x 10⁻⁶) were pelleted, exposed to HIV for 45 min, resuspended in 2 ml of fresh medium, and incubated in culture tubes at 37°C in air.

Humidified cells containing CC>2 were treated in the same way.

'f //

controls, but without exposing them to the virus. The cells were continuously exposed to IL-2 and the drug. When ATH8 cells were used in this test system, five virus particles per cell constituted the minimum cytopathic dose of virus. In co-culture experiments

4

of cells, 5 x 10 H9 cells producing

5

HIV RF-II or uninfected H9 cells with 2 x 10 target T lymphocytes. At various times, the total viable cells were counted in a hemocytometer under the microscope using the tryptan blue exclusion method.

The results are shown in Table 13.

Table 13

DE5Q compound (juM)

3'-azido-21,3'-dideoxycytidine 10

3•-azido-5-bromo-21,31-dideoxyuridine 5

31-azido-5-bromo-2',3'-dideoxycytidine 5

(E)-3-[l-(31-azido-2',31-dideoxy-g-D-erythro-pentafurannosyl)-1,2,3,4-tetrahydro-2,4-dioxo-5-pyrimidinyl]-2-propenoic acid 100

Example 80: In vitro antibacterial activity of 3'-azidonucleosides

Table 14 shows the in vitro antibacterial activity of 3'-azidonucleosides in terms of Minimum Inhibitory Concentration (MIC) against various bacterial species.

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

!

i

Table 14

CIM (uq/ml)

Microorganism. Reference Compound

1

2

3

4

5

6

7

8

E. coli

0.1

>100

10

0.1

10

0.1

10

10

>100

Salmonella typhimurium

0.1

10

10

0.1

>100

10

10

>100

0.5

Salmonella typhosa

0.1

0.1

10

0.1

0.1

10

10

0.1

0.1

Entero. aeroqenes

0.1

10

>100

>100

100

10

10

>100

0.5

Citro. freundii

10

10

10

0.1

10

10

10

>100

0/5

The compounds used were:

1) 31-azido-31-deoxy-4-thiothymidine

2) 3'-azido-31-deoxy-5'-0-acetyl-4-thiothymidine

3) 3'-azido-31-deoxy-2-deoxy-2-thiothymidine

4) 51-acetyl-3'-azido-3-benzoyl-31-deoxythymidine

5) 1-(51-0-acetyl-3'-azido-21,31-deoxy-γ-D-erythropentofurannosyl)-5-methyl-4-(1,2,4-triazole-l-yl)-2(IH)-pyrimidinone

6) 1-(31-azido-21,31-dideoxy-B-D-erythropentofuran-nosyl)-2-(benzyloxo)-5-methyl-4-(IH)-pyrimidinone

7) 3'-azido-3'-deoxy-2-methoxythymidine

8) 31-azido-5-bromo-21,31-dideoxyuridine

<

Claims (27)

CLAIMS , , , 1. Process for preparing a compound of formula: C in which C is a purine or pyrimidine base bonded at position 9 or 1 respectively, and its pharmaceutically acceptable derivatives other than: (a) compounds of formula (I)B in which C is adenine, guanine, uridine, cytidine or thymine base, and their 5-mono- or 5-triphosphoric esters; 10 (b) 5'-O-acetate, 5'-O-trityl and 5'-O- derivatives (4-methylbenzenesulfonate) of the compound of formula (l)B in which C is a uridine base and the 3'-azido group is in erythro configuration; (c) compounds of formula (I)B in which C is 15 (i) a residue of 5-bromovinyluridine or 5-trifluoro- (ii) a methyluridine residue and the 3'-azido group is in the erythro configuration; (iii) a 5-iodo or 5-fluoruridine residue and the 3'-azido group is in the erythro or threo configuration; and the 5'-O-trityl derivatives of these compounds; (d) compounds of formula (I)B in which C is (i) a residue of 5-bromovinyluridine or cytidine and the 3'-azido group is in the threo configuration; (ii) a residue 25 of 5-fluorocytidine and the 3'-azido group is in erythro configuration; or (iii) a 5-methylcytidine residue and the 3'-azido group is in threo or erythro configuration; (e) the 51-O-acetic esters of compounds of formula (I)B in which C is a 4-chloro-2(lH)pyrimidinone or V\ ¥ 4-(1H-1,2,4-triazole-l-yl)-2(IH)pyrimidinone (possibly substituted at position 5 by fluorine or a methyl group) and the 3'-azido group is in the erythro configuration (f) the 5 1-O-[(4-methoxyphenyl)diphenylmethyl] derivative of the compound of formula (I)B in which C is a cytidine residue and the 3'-azido group is in the erythro configuration; and (g) the 5'-O-trityl derivative of the compound of formula (I)B in which C is an adenine residue and the 3'-azido group is in threo configuration; characterized in that it consists of either: (A) to react a compound of formula HAS (III) (in which A is a purine or pyrimidine base, other than thymine, bonded at position 9 or 1 respectively, and M represents a precursor group for the 3'-azido group) or a derivative of this compound, with an agent or under conditions for transforming said precursor group into the desired azido group; or (B) to react a compound of formula R4 a (IVa) or N3 (IVb) fr (in which R^, R^, R3, R4, R6 and R7 represent res- ^ a 1 a 2 3 3 4a g *7 JL' respectively the groups R,R,R,R,R and R, and R is a hydroxy, mercapto, amino, alkylthio, aralkoxy group, alkoxy, cyano, alkylamino, the alkyl groups being possibly- ✓ 2 actually linked to form a heterocycle; R is hydrogen or an acyl, alkyl, aroyl or sulfonate group; 3 R is a hydroxy, mercapto, amino, triazolyl, alkylamino, dialkylamino group, the alkyl groups possibly being linked to form a heterocycle, aralkoxy, 4 alkoxy or alkylthio; R is an alkyl group, substituted alkyl, halogen, perhalogenomethyl, hydroxy, alkoxy, cyano, nitro, alkenyl, substituted alkenyl, alkynyl, alkynyl 6 7 substituted or hydrogen; and R and R may be identical or different and are chosen from hydrogen and amino, hydroxy, mercapto, alkylthio, alkoxy groups, aralcoxy, cyano and alkylamino; or precursor groups 12 3 4 of these, provided that at least one of R_, R_, R_ and R_ a a a « a in formula (IVa) or at least one of the groups R_ and 7, , R in formula (IVb) represents a precursor group) a with an agent or under conditions used to transform said precursor group(s) into the corresponding desired groups; or (C) to react a compound of formula (in which R"*", R^, R3, R4, R® and R7 are as defined) or a functional equivalent of this compound, with a compound serving to introduce the desired ribofurannosyl ring at position 1 of the compound of formula (IVa) or at position 9 of the compound of formula (IVb); or (D) when A of the compound of formula (I) is a purine residue, to react a purine of formula (Vb) with a pyrimidine ring of formula: (in which Py represents a 1-pyrimidinyl group); and subsequently or concomitantly, to carry out one or more of the following optional transformations: (i) when a compound of formula (I)B is formed, transform that compound into one of its pharmaceutically acceptable derivatives; and (ii) when a pharmaceutically acceptable derivative of a compound of formula (I)Br is formed, transform that derivative into the original compound of formula (I)B or into another pharmaceutically acceptable derivative of a compound of formula (I)B. 2. A process according to claim 1, characterized in that a 3'-azido-3'-deoxypyrimidinenucleoside, or a pharmaceutically acceptable derivative thereof, is prepared, wherein: R is a hydroxy, mercapto, C2-C1 alkoxy or amino group; R is hydrogen or a methyl, C2-C1 alkanyl or benzoyl group; R3 is a hydroxy, mercapto, amino or amino-substituted group; and R4 is hydrogen when R is an amino or amino-substituted group, and a halogen or a perhalomethyl, C2-C3 alkyl alkenyl or ethenyl-substituted group when R3 is other than an amino or amino-substituted group. 3. A process according to claim 1, characterized in that a 3'-azido-3'-deoxypurinenucleoside is prepared, or a pharmaceutically acceptable derivative thereof, in 6 which: R is an amino, alkylamino group in Cj-C4» mer- 11 7'! capto, hydroxy or alkoxy in C^-C^; and R is a jt amino, alkylamino group in or hydrogen. 4. A method according to any one of claims 1 to 3, characterized in that the 3'-azido group is in erythro configuration. 5. A method according to any one of claims 1 to 4, characterized in that group C of formula (I)B is a derivative of guanine, adenine, thymine, uridine or cytidine. 6. A method according to any one of claims 1, 2, 4 and 5, characterized in that group C is a derivative of thymine or dluridine and in that the 3'-azido group is in threo configuration. 7. A process for preparing a pharmaceutical formulation, characterized in that it comprises combining a compound of formula (where B is a purine or pyrimidine base linked to position 9 or 1 respectively), or a pharmaceutically acceptable derivative of such a compound, as the first active ingredient, with at least one other therapeutic agent, as the second active ingredient, and a pharmaceutically acceptable carrier for these. 8. A process according to claim 7, characterized in that the two active ingredients are present in a potentiating ratio. 9. A process according to claim 7 or 8, characterized in that the second active ingredient is a glucuronidation inhibitor and/or a renal excretion inhibitor. 10. A method according to any one of claims 7 to 9, characterized in that the second ingredient ' active is a glucuronidation inhibitor. 11. A process according to any one of claims 7 to 10, characterized in that the second active ingredient is notably glucuronide in vivo. 12. A process according to any one of claims 7 to 11, characterized in that the second active ingredient is probenecid, aspirin, acetaminophen, lorazepam, cimetidine, ranitidine, zomepirac, clofibrate, indomethacin, ketoprofen or naproxen. 13. A process according to claim 7 or 8, characterized in that the second active ingredient is a nucleoside transport inhibitor. 14. A process according to claim 13, characterized in that the second active ingredient is dilazep, dipyridamole, the 6-/j4-nitrobenzoyl)thio/-9-(^ -D-ribofurannosyl)purine, papaverine, mioflazine, hexobendine, lidoflazine, and their acid addition salts 15. A process according to claim 7 or 8, characterized in that the second active ingredient is another therapeutic nucleoside. 16. A method according to claim 15, characterized in that the second therapeutic nucleoside corresponds to the formula H2N (HAS) CHX ÇHCH OH Y in which Z is hydrogen or a hydroxyl or amino group; and X is (a) an oxygen or sulfur atom or a methylene group and Y is a hydrogen atom or a group H hydroxymethylene, or (b) a methylenoxy group (-OCH„) and Y is a hydroxy group; or is a pharmaceutically acceptable derivative of such a nucleoside. 17. A process according to claim 16, characterized in that the second active ingredient is 9-[(2-hydroxy-1-hydroxymethylethoxy)methyl]guanine, 2-amino-9-(2-hydroxyethoxymethyl)purine or 9-(2-hydroxyethoxymethyl)guanine. 18. A process according to claim 7 or 8, characterized in that the second active ingredient is an antibacterial agent. 19. Process according to claim 18, characterized in that the second active ingredient is 2,4-diamino-5-(3',4',5'-trimethoxybenzyl)-pyrimidine or one of its analogues, suifadimidine, rifampicin, tobramycin, fusidic acid, chloramphenicol, clindamycin or erythromycin. 20. A method according to any one of claims 7 to 19, characterized in that, in the compound of formula (I)A or its pharmaceutically acceptable derivative, B is a thymine residue. 21. A process according to claim 7 or 8, characterized in that the compound of formula (I)A is 3'-azido-3'-deoxythymidine. 22. A process according to claim 21, characterized in that the second active ingredient is 9-(2-hydroxyethoxymethyl)guanine. 23. A method for preparing a pharmaceutical formulation, characterized in that it comprises combining a compound of formula (I)A, according to claim 7, or a pharmaceutically acceptable derivative of such a compound, with a pharmaceutically acceptable support for that compound or derivative. 24. A process according to claim 23, characterized in that the ingredients are sterile. 25. A method according to claim 24, characterized in that the formulation is suitable for administration by injection. |ol^ 26. A method according to claim 23, characterized in that the formulation is suitable for oral administration. 27. A method according to claim 26, characterized in that the formulation is in the form of a tablet or capsule. -28. A process according to any one of claims 7 to 27, characterized in that it comprises (i) preparing the compound of formula (I)A, or one of its pharmaceutically acceptable derivatives 10, by the process of any one of claims 1 to 6, and (ii) combining said compound or derivative with the second active ingredient and/or a pharmaceutically acceptable carrier for the compound or derivative and, if present, the second active ingredient 15.