JPH047726B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to 3'-azido-3'-deoxythymidine and its use in the treatment or prevention of human retroviral infections. Retroviruses are a subgroup of RNA viruses that must first undergo 'reverse transcription'('transcription' is traditionally understood to mean the synthesis of RNA from DNA) of their genomic RNA into DNA in order to replicate. form. When the DNA of a tank is formed,
The viral genome integrates into the host cell genome and makes full use of the host cell's transcription/translation machinery for the purpose of replication. of the virus
Once DNA is incorporated, it becomes
Virtually indistinguishable from DNA, the virus
In this state, they can survive for as long as the cell's lifespan. Since the virus in this state is invulnerable to attack, any treatment must be directed to other states of the life cycle and must therefore continue until all virus-bearing cells are killed. There must be. Both HTLV- and HTLV- are retroviruses and are known to cause human leukemia. HTLV- infections are particularly widespread and cause many deaths worldwide each year. Retroviral species have now already been reproducibly isolated from patients with AIDS. Although the characteristics of this retrovirus are widely known, there is currently no consensus name for it as a virus, and recently human cell lymphotropic virus (HTLV
), AIDS-associated retrovirus (ARV), and lymphadenopathy-associated virus (LAV). Acquired immunodeficiency virus (AIDV) is expected to be the internationally agreed-upon name. This virus (hereinafter referred to as AIDV) is
preferentially infects T cells with ^OKT4 surface labeling,
It has been found to destroy this and is currently recognized as the cause of AIDS. As patients progressively lose this population of T cells, the overall balance of the immune system is reversed, its ability to fight other infections is diminished, and patients become susceptible to opportunistic infections that are often judged incurable. Thus, deaths in AIDS victims are usually due to opportunistic infections such as pneumonia or virus-induced cancers, and not as a direct result of AIDS infection. Recently, other types of tissues, such as B cells, macrophages, and non-blood related tissues of the central nervous system (CNS) expressing ^T4 labeling, have also been reported.
AIDV is being recalled. This latter infection has been found in patients with classic AIDS symptoms and is associated with progressive demyelination, leading to weakness, brain disease, progressive dysarthria, ataxia, and disorientation. You are experiencing symptoms. There are at least four clinical signs of AIDV infection. In the initial "carrier" state, the only indicator of infection is the presence of anti-AIDV antibodies in the bloodstream. It is believed that such "carriers" can become infected, for example, through blood transfusions, sexual intercourse, or used needles. This carrier state often does not progress to the second stage, which is characterized by persistent generalized lymphadenopathy (PGL). Recently, approximately 20% of PGL patients have
It is estimated that the disease will progress to a more advanced stage called ``AIDS-related syndrome'' (ARC). Physical symptoms associated with ARC include general discomfort, increased body temperature, and chronic infection. This condition usually progresses to the final, fatal AIDS condition, in which the patient completely loses the ability to fight infection. These and other retroviruses have only recently been recognized and the diseases they are associated with are life-threatening in nature, making it imperative to develop strategies to combat these viruses. ing. Currently, various drugs have been proposed as ``therapeutic drugs'' for AIDS. Drugs include antimoniotungstate, suramin, ribavirin, and isoprinosine, but they are either somewhat toxic or do not exhibit significant antiretroviral activity. Once infected, the AIDV genome is integrated into the host cell's DNA, and in this state cannot be damaged by attack, so it persists for as long as the host cell survives, eventually causing new infections. Therefore, any treatment for AIDS must be long-term, perhaps lifelong, and the treatment materials must therefore be toxicologically acceptable. The report describes testing of compounds against various retroviruses, such as the murine retrovirus Friend leukemia virus (FLV). For example, Krieg et al.
Res.), 116 (1978), 21-29) showed that 3-azido-3'-deoxythymidine was
Ostertag et al. (Proc. Nat. Acad. Sci. (1974), 71 , 4980) found that it was active against FLV.
Based on its poor FLV-related antiviral activity and cytotoxicity, 3'-azido-3-deoxythymidine has been shown to be “advantageous over bromodeoxyuridine for the medical treatment of diseases caused by DNA viruses.” It may be possible to replace it with but,
De Clerq et al. (Biochem.Pharm.)
(1980), 29, 1849-1851) six years later,
They determined that 3'-azido-3'-deoxythymidine had no appreciable activity against any of the viruses used in their tests, including cowpox, HSVI, and varicella virus (VZV). Glinski et al (Journal of Organ Chemistry (J.org.Chem.),
(1973, 38 , 4299-4305) discloses certain derivatives of 3'-azido-3'-deoxythymidine (described below) and their ability to inhibit mammalian exoribonuclease activity. The inventors have now demonstrated that 3'-azido-3'-deoxythymidine has surprising activity against human retroviruses, and is particularly effective against AIDV, as evidenced by the experimental data cited below. I discovered that it shows. This activity makes the compounds useful in the therapy of human retroviral infections. In a first aspect of the invention, a compound of formula (i.e. 3'-azido-3-deoxythymidine)
are proposed as compounds for use in the treatment or prevention of human retroviral infections. The compound of formula () is hereinafter referred to as a compound according to the present invention. 3'-azido-3- against human retroviruses
The activity of deoxythymidine was confirmed by various in vitro assay systems. For example, A.I.D.V.
Infection of the H9 human lymphoblastoid cell line by
Application of 3'-azido-3'-deoxythymidine at concentrations as low as 0.013 mcg/ml up to 20 hours after infection provides effective protection. AIDV infection of U937 human lymphoblastoid cells, PHA-stimulated leukocytes, and cultured peripheral blood lymphocytes is also prevented at similar low concentrations. Additionally, in a 10-day dosing experiment with up to 5000 AIDV virions per cell and cloned T4, tetanus-specific T Belper lymphocytes, cells treated with 3'-azido-3'-deoxythymidine decreased. On the other hand, the number of untreated cells decreased to 1/5. Transformed with HTLV-
Cytopathic effects in the same cell line co-infected with AIDV were also completely blocked. Other studies using AIDV reverse transcriptase have shown that
It has been found that the activity of this enzyme is inhibited by 3'-azido-3'-deoxythymidine triphosphate by a competitive inhibition mechanism. Phase 1 clinical trials have shown that 3'-azide-
It has been found that 3'-deoxythymidine has the ability to cross the blood/brain barrier in clinically effective amounts. This property is unusual and valuable for the treatment and prevention of CNS infections caused by human retroviruses. The ability of 3'-azido-3'-deoxythymidine to modify retrovirus-induced toxicity was demonstrated in a murine model. In this model, administration of 3'-azido-3'-deoxythymidine prevented splenomegaly caused by intravenous administration of Lauscher murine leukemia virus, the murine counterpart of HTLV. Further experiments showed that 3'-azido-3'-deoxythymidine was effective in HTLV at concentrations as low as 0.8 mcg/ml.
- was also found to inhibit the in vitro replication of. A more particularly preferred aspect of the invention thus proposes the use of the compounds according to the invention for the manufacture of medicaments for the treatment and prevention of human retroviral infections. The invention further provides a method for the treatment or prevention of AIDS in a patient, which comprises administering to the patient an effective amount of a compound according to the invention. The invention also comprises administering to a patient an effective antiviral amount of a compound according to the invention.
Also included are methods of treating or preventing infections caused by retroviruses in such patients. Furthermore, according to the present invention, AIDS as defined above
Also proposed are compounds according to the invention for use in the treatment or prevention of viral infections. Examples of human retroviral infections that can be treated or prevented according to the device include T-cell lymphotropic retroviruses (HTLV), particularly HTLV-, HTLV- and AIDV (HTLV-). Clinical situations that can be treated or prevented according to the invention include:
AIDS, AIDS-related syndromes, and HTLV-
I have positive leukemia and lymphoma. Patients suitable for treatment also include those with antibodies to AIDV, AIDV-CNS infections, PGL, and ARC. “Pharmaceutically acceptable derivatives are pharmaceutically acceptable salts, esters, salts of such esters, or other compounds which, when administered to a patient, contain 3'-azido-3'-deoxythymidine, or any compound thereof that can result in an antiretrovirally active metabolite. Examples of compounds other than esters include those in which the 5'-C and 2-C atoms are linked by an oxygen atom. Preferred esters of the compound of formula () include esters of carboxylic acids in which the moiety other than the carbonyl of the ester group is a linear or branched alkyl group. , alkoxyalkyl (e.g. methoxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxymethyl), aryl (e.g. halogen, C _1-4 alkyl, or
phenyl optionally substituted with C _1-4 alkoxy);
Sulfonic acid esters such as alkyl- or aralkylsulfonyl (methanesulfonyl); and mono-, di- or tri-phosphoric esters. With respect to the above esters, the alkyl moiety present in the ester is 1 unless otherwise specified.
-18 carbon atoms, especially 1-4 carbon atoms. The aryl moiety present in the ester preferably consists of a phenyl group. Reference to any of the above compounds also includes reference to the pharmaceutically acceptable salts thereof. Experiments have shown that 3'-azido-3'-deoxythymidine is converted to 5'-monophosphate by cellular enzymes in vitro. The monophosphate is then phosphorylated by other enzymes to form diphosphate and then triphosphate. Other studies have shown that AIDV appears to be an effective chain terminator in reverse transcription, as evidenced by its effects on avian osteoblastosis virus and Moloney murine leukemia virus. 3'-Azide-
The triphosphate form of 3'-deoxythymidine has been demonstrated. This form also inhibits AIDV reverse transcriptase in vitro, but
The effect on DNA polymerase activity is almost negligible. Examples of pharmaceutically acceptable salts of compounds of formula () include, for example, alkali metals (e.g. sodium), alkaline earth metals (e.g. magnesium)
salts, ammonium, and base salts derived from suitable bases, such as NX ⁺ ₄ (X is C _1-4 alkyl). The invention thus further provides novel pharmaceutically acceptable derivatives of compounds of formula (), comprising:
The following 5'-derivatives are monophosphate, disodium monophosphate, 2-cyanoethyl monophosphate, triphosphate, p-toluenesulfonate, acetate, triphenylmethyl and methanesulfonate, or the 5'-C is ,
Further provided are derivatives other than those that bind to another nucleotide or nucleoside derivative. Formula () which can be used according to the invention
Specific examples of pharmaceutically acceptable derivatives of the compounds include the monosodium salts, and the following 5'-esters: monophosphate, disodium monophosphate, diphosphate, triphosphate,
These include acetate, 3-methyl-butyrate, octanoate, palmitate, 3-chlorobenzoate, benzoate, 4-methylbenzoate, hydrogen succinate, pivalate, and mesylate. 3′-azido-3′-deoxythymidine, or
The pharmaceutically acceptable derivatives thereof (hereinafter referred to as active ingredients) may be used orally, anally, nasally, topically (including buccal, sublingually), vaginally, or in humans for the prevention or treatment of retroviral infections. Parenteral (subcutaneous, intramuscular,
Administration can be by any route, including intravenous and intradermal). The preferred route will vary depending on the condition and age of the recipient, the nature of the infection, and the active ingredient chosen. Generally, a suitable dosage is 6-90 mg per kg of patient body weight per day, most preferably 15-60 mg. The desired dosage is preferably
2, 3, 4 times at appropriate intervals during the day
It is preferable to administer the drug in 5, 6 or more divided doses. These sub-doses may be administered in the form of unit doses (e.g. containing 10-1500 mg, preferably 20-1000 mg, most preferably 50-700 mg of active ingredient per unit dose form).
Experiments with 3'-azido-3'-deoxythymidine have shown that administration results in plasma concentrations of the active compound of about 1 to ca.
It is estimated that it is best to reach a peak of 75 μM, preferably about 2-50 μM, most preferably about 3-30 μM. This administration may be accomplished, for example, by intravenous injection of the active ingredient as a 0.1-5% solution, optionally in saline, or by intravenous injection of the active ingredient as a 0.1-5% solution, optionally in saline.
- It can be administered orally as a large pill containing about 100 mg/Kg. The desired blood concentration is a continuous fluid that provides about 0.01 to about 5.0 mg/Kg per hour, or about 0.4 to about 15 mg/Kg of the active ingredient per hour.
It can be maintained by intermittent rinsing containing mg/Kg. If it is not possible to administer the active ingredient alone, it is preferable to present it as a pharmaceutical formulation. The formulations of the present invention contain at least one active ingredient as defined above, together with one or more acceptable carriers thereof, and optionally other therapeutic agents. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and must not be harmful to the patient. The formulation includes oral, anal, nasal, topical (cheek,
sublingual), vaginal or parenteral (subcutaneous, intravenous,
(including intramuscular and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the pharmaceutical arts. Such methods also include the means for bringing the active ingredient into association with the carrier which constitutes one or two accessory ingredients. Generally, formulations include the active ingredient homogeneously in a liquid carrier, a finely divided solid carrier, or both.
and in tight integration, if necessary by shaping the product. Suitable for oral administration, the formulations of the invention can be prepared as discrete units such as capsules, cachets, tablets, etc. containing a predetermined amount of the active ingredient, as powders or granules, as aqueous or non-aqueous solutions or suspensions. or as an oil-in-water or water-in-oil emulsion. The active ingredient can also be presented as a bolus, lozenge or hest. Oral flavorants may also contain sweeteners, flavoring agents,
Other materials known in the art may also be included, such as thickening agents. A tablet may be made by compression or molding, optionally with one or more accessory ingredients.
Compressed tablets contain the active ingredient in a fluid form, such as a powder or granules, with binders (e.g. povidone, gelatin, hydroxymethyl cellulose), lubricants, inert diluents, preservatives, disintegrants (e.g. sodium glycolate starch, cross-linking agents). Povidone, cross-linked carboxymethylcellulose sodium), surfactants, or dispersants may be optionally mixed and compacted in a suitable machine. Molded tablets may be made 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 may be formulated to provide slow and constant release of the active ingredient, eg with varying amounts of hydroxypropylmethylcellulose. Preparations suitable for topical administration in the mouth include a flavoring base, usually sucrose and acacia tragacanth, containing the active ingredients; gelatin and glycerin, or an inert combination such as sucrose and acacia. These include lozenges containing the active ingredient in a base; mouthwashes containing the active ingredient in a suitable liquid carrier. Formulations for rectal administration may be presented as a suppository with a suitable base containing, for example, cocoa butter or a salicylic acid agent. Formulations suitable for intracavitary administration may be presented as pet sarees, tampons, creams, gels, pastes, foams or spray formulations containing, in addition to the active ingredient, carriers recognized as appropriate in the art. Formulations suitable for parenteral administration may contain aqueous or non-aqueous isotonic agents that may contain antioxidants, buffers, bacteriostatic agents, and solutes that render the formulation isotonic with the blood of the intended recipient. Sterile injectable solutions include sterile aqueous or non-aqueous suspensions that may contain suspending agents and thickening agents. The preparations may be presented in unit-dose or multi-dose containers, such as ampoules and vials, to which a sterile liquid carrier, such as water for injection, need only be added immediately before use. It can be stored in a freeze-dried state. Solutions and suspensions for extemporaneous injection are:
It can be prepared from sterile powders, granules, tablets, etc. as described above. Preferred unit dose formulations contain 1 of the active ingredient.
daily dose or unit dose; daily subdose;
or a formulation containing an appropriate fractionated amount thereof. The component to be administered is, for example, 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]
Guanine, 2-(hydroxyethoxymethyl)guanine [acyclovir], 2-amino-9-(2-
In combination with other drugs such as hydroxyethoxymethyl) purines, interferons (e.g. alpha-interferon), interleukins, and phosphonoformates, or in combination with other drugs such as It can also be used therapeutically in conjunction with drugs such as levamisole and thymosin, which help increase the number and/or function appropriately. It is to be understood that, in addition to the ingredients specifically mentioned above, the formulations of the present invention may also include other materials common to the art of formulation. Compounds of formula (), and pharmaceutically acceptable derivatives thereof, may be prepared by conventional methods, such as those described in the following references, or analogous thereto: J.A. JRHorwitz and others, Journal of Organic Chemistry (J.Org.
Chem.), 29 (July 1964), 2076-78; M. Imazawa et al., Journal of
Organic Chemistry, 43 (15) (1978),
3044−3048; K.A. Watanabe (KA
Watanabe et al., Journal of Organic Chemistry, 45 , 3274 (1980); and RPGlinski, Journal of Organic Chemistry, 45, 3274 (1980);
Comiunication (J.Chem.Soc.Chem.
Commun., 915 (1970), as well as the methods described in the Examples. The present invention further provides the formula () consisting of the following reaction:
(A) a compound of the formula (wherein M represents a precursor group for the 3'-azido group), or a derivative thereof; (for example,
ester or salt) with or under conditions that aid in the conversion of the precursor group to the desired azide group; (B) an agent that aids in the conversion of a compound of the formula (wherein R represents a hydroxyl group, or a precursor group to a pharmaceutically acceptable derivative thereof) into the corresponding desired group; to react with or under conditions; (C) reacting a compound of formula (), or a functionally equivalent compound thereof, with a compound that serves to introduce the desired ribofuranosyl ring into the 1-position of a compound of formula (D) reacting a compound of the following formula, in which R ¹ is a hydroxyl group, or R as defined above, with an agent or under conditions that aid in the conversion of said compound to a compound according to the invention; and simultaneously and subsequently, optionally, one or two of the following conversions: (i) When 3'-azido-3'-deoxythymidine is formed, converting said compound into its pharmaceutical converting it into a derivative that is acceptable to the public. (ii) when a pharmaceutically acceptable derivative of 3'-azido-3'-deoxythymidine is formed;
Converting said derivative into a compound of formula () or another derivative thereof. In the aforementioned methods of the invention, it is appreciated that the aforementioned substances and conditions, as well as the precursor compounds of formulas () and (), are selected from those known in the art of nucleoside synthetic chemistry. Examples of such conversion procedures are provided below as a guide, but it should be understood that they may be modified in conventional manner depending on the desired compound of formula (). In particular, if a conversion is described that would lead to an undesirable reaction of an otherwise unstable group, the group is protected in a conventional manner and the protecting group is removed after the conversion is complete. be able to. Thus, in method (A), the group M of the compound of formula ()
is, for example, halogen (e.g. chlorine), hydroxy,
or an organic sulfonyloxy group (eg trifluoromethylsulfonyloxy, methanesulfonyloxy, or p-toluenesulfonyloxy group). For the preparation of compounds of formula (), the group M is
Compounds of formula () in which the halogen (e.g. chlorine) is in the threo configuration (the 5'-hydroxy group is advantageously protected e.g. by a trityl group) may be used, for example, with lithium azide or sodium azide. can be processed with. 3′−threo
- A halogen (e.g. chlorine) starting material can e.g.
For example, it can be obtained by reaction with triphenylphosphine and carbon tetrachloride or by treatment with an organic sulfonyl halide (e.g. trifluoromethanesulfonyl chloride), forming the corresponding 3'-erythro-organosulfonyloxy compound. and then halogenated. 3′-threo-hydroxy compounds of formula () can also be treated with, for example, triphenylphosphine, carbon tetrabromide and lithium azide to give the corresponding
A 3'-erythro azide compound can be formed. Any protecting groups can then be removed, eg, as described above. For method (B), R is a protected hydroxyl group, e.g.
represents an ester group of the type cited above in connection with, in particular acetoxy, or an ether group such as a trialkylsilyloxy group, for example tert-butyldimethylsilyloxy, or an aralkoxy group, for example triphenylmethoxy. Such groups can be converted, for example, into the desired hydroxyl group by hydrolysis or into 3′-hydroxyl groups by transesterification.
It can be converted to other ester groups of azido-3'-deoxythymidine. For process (C), this means, for example, that a suitable pyrimidine of formula (), or a salt or protected derivative thereof, is treated with a compound of formula It is done by folding. (wherein A is e.g. acetoxy, or benzoyloxy, or halogen (e.g. chlorine) and B is an optionally protected group (e.g. p-toluenesulfonyloxy group)) Regarding the method of (D) , R ¹ is a precursor group as described for R in formula (). 3'-azido-3'-deoxythymidine can then be advantageously obtained, for example, by reaction with an alkali metal azide (e.g. lithium azide) in a suitable solvent such as wet DMF, followed by mild It is preferably hydrolyzed by acids or bases under conditions. 3'-azido-3'-deoxythymidine can be converted into a pharmaceutically acceptable phosphate, respectively, by reaction with a phosphorylating agent, e.g. _POCl3 , or a suitable esterifying agent, e.g. an acid halide or acid anhydride. or can be converted into other esters. A compound of formula (), including its esters, can be converted into its pharmaceutically acceptable salts in a conventional manner, eg, by treatment with a suitable base. The ester or salt of 3'-azido-3'-deoxythymidine can be prepared by e.g.
It can be converted to the original compound. Examples are given below, but these are merely intended to illustrate the effects of the present invention, and are not intended to limit the scope of the present invention in any way.
The term "active ingredient" as used in the examples refers to a compound of formula () or a pharmaceutically acceptable derivative thereof. Example 1 Tablet Formulation The following formulations A and B were prepared by wet granulating the ingredients with a solution of povidone, then adding magnesium stearate and compressing.

【table】

【table】

Table: The following formulations D and E were 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 Ingredients 250 Pregelatinized Starch NF15 150 400 Formulation E mg /Capsule Active Ingredients 250 Lactose 150 Avicel 100 500 Formulation F (Controlled Release Formulation) mg/capsule This formulation was prepared by wet granulating the following ingredients with povidone solution, adding magnesium stearate and compressing. mg/tablet (a) Active ingredient 500 (b) Hydroxypropylcellulose (Methocel
K4M Premium (Methocel K4M Premium)
112 (c) Lactose BP 53 (d) Povidone BDC 28 (e) Magnesium Stearate 7 700 Drug release occurs over approximately 6-8 hours;
Completed after 12 hours. Example 2 Capsule Formulation Formulation A Formulation was prepared by mixing the ingredients of Formulation D of the above example and filling two-part hard gelatin capsules. Formulation B (below) was prepared similarly. Formulation B mg/capsule (a) Active ingredient 250 (b) Lactose BP 143 (c) Sodium glycolic acid starch 25 (d) Magnesium stearate 2 420 Formulation C mg/capsule (a) Active ingredient 250 (b) Macrogol 4000BP 350 600 Capsules are made by melting Macrogol 4000BP.
Two-part hard gelatin capsules were prepared by dispersing the active ingredient in the melt and filling the melt into two-part hard gelatin capsules. Formulation D mg/capsule Active ingredient 250 Lecithin 100 Peanut oil 100 450 Capsules were prepared by dispersing the active ingredient in lecithin and peanut oil and filling soft gelatin capsules with the dispersion. Formulation F (Control Release Capsules) A control release capsule formulation was then prepared by extruding components (a), (b) and (c) using an extruder, followed by spheronization and drying. The dried pellets were coated with a controlled release membrane (d) and filled into two-part hard gelatin capsules. mg/capsule (a) Active ingredient 250 (b) Microcrystalline cellulose 125 (c) Lactose BP 125 (d) Ethyl cellulose 13 513 Example 3 Injectable preparation Formulation A Active ingredient 200g 0.1M hydrochloric acid solution PH4.0− 0.1M sodium hydroxide solution adjusted to 7.0 PH adjusted to 4.0-7.0 Diluted to 10 ml sterile water Dissolve the active ingredient in most of the water at 35°-40°C.
Adjust the pH to 4.0− with an appropriate amount of hydrochloric acid or sodium hydroxide.
Adjusted to 7.0. The volume was made up to 10 ml by adding water, filtered through a sterile micropore filter, placed in a sterile amber glass bottle (type 1, 10 ml), sealed with a sterile bottle, and double-sealed. Formulation B Active ingredient 0.125 g Sterile, pyrogen-free PH7 buffer diluted to 10 ml Example 4 Intramuscular injection active substance 0.20 g Benzyl alcohol 0.10 g Glycofurol 75 1.45 g Water for injection to 3.00 ml Dilution Dissolve the active ingredient in glycofurol, add benzyl alcohol to dissolve, then add water to 3ml.
I made it. Filter the mixture through a sterile micropore filter into sterile amber glass bottles (Type 1, Type 3).
ml). Example 5 Syrup Active Ingredients 0.2500g Sorbitol Solution 1.5000g Glycerin 2.0000g Sodium Benzoate 0.0050g Fragrance, Peach 17.42.3169 0.0125ml Purified Water Diluted to 5.0000ml Active Ingredients in Glycerin and Most Purified Water Add an aqueous solution of sodium benzoate to the solution, then add a solution of sorbitol,
Finally, the fragrance was added. Purified water was added to bring the total volume to 5 ml, and the mixture was thoroughly mixed. Example 6 Suppository mg/Suppository active ingredient (63 μm) ^* 250 Hard fat, BP (Dynamit Nobel-Witepsol H15)
H15) 1770 2020 *At least 90% of the particles of the active ingredient have a particle size of 63μm
It was used as the following powder. One-fifth of the hard fat was melted in a pot with a steam canopy at a maximum temperature of 45°C. The active ingredients were sieved through a 200 μm sieve, added to the molten base and mixed using a Silverson with cutters until a smooth dispersion was obtained. While keeping the mixture at 45°C, the remaining hard fat was added to the suspension and stirred to obtain a homogeneous mixture. After filtering the entire suspension through a 250 μm stainless steel sieve, while stirring,
It was left to cool to 40°C. At 38°-40°C, 2.02 g of the mixture was filled into a suitable plastic mold (2 ml) and allowed to cool to room temperature to form suppositories. Example 7 Petusari mg/petusari Active ingredient (63 μm) 250 Anhydrous dextrose 380 Potato starch 363 Magnesium stearate 7 1000 Petusari was prepared by directly mixing the above ingredients and compressing the mixture directly. Example 8 3'-azido-3'-deoxy-5'-O-octanoylthymidine Octanoyl chloride (1.2 equivalents) was added to a pyridine solution (0°C) of 3'-azido-3'-deoxythymidine. . The reaction was allowed to proceed by increasing the temperature to room temperature. Upon completion of the reaction as indicated by tlc (CHCl ₃ :MeOH; 20:1 on silica gel), the solution was poured onto ice water and the resulting aqueous phase was decanted. The oil phase was chromatographed on silica gel eluting with _CHCl3 :MeOH. The title compound was obtained as an oil after evaporation of the solvent from the appropriate fractions. CHN: Calculated composition - C: 54.95, H: 6.92, N:
17.80; Measured composition - C: 54.82, H: 6.96, N:
17.66. δ7.46 (d, 1H, J _5,6 = 1Hz, 6H), δ6.13 (t,
1H, 1′H), δ4.5−4.2 (m, 3H, 3′H, 5′CH ₂ )
δ4.0−3.8 (m, 1H, 4′H), δ2.3−2.1 (m, 4H,
2'H, octanoyl (CH ₂ ) 1), δ1.81 (d, 3H,
J _5,6 = 1.0Hz, 5CH ₃ ), δ1.5-0.6 (m, 13H, 5'octanoyl (CH ₂ ) ₅ CH ₃ ) Example 9 5'-acetyl-3'-azido-3'-deoxy Thymidine Acetyl chloride (2.1 equivalents) was added to a solution of 3'-azido-3'-deoxythymidine (20 g) in pyridine (50 ml) at room temperature. The reaction mixture was stirred for 2 hours, left at 0-5°C for 20 hours, and then poured into ice water with stirring to decant the aqueous phase. Dissolve the oily product in water, water (5x), 0.5N hydrochloric acid, water (2x)
and dried over magnesium sulfate. The solution was filtered and evaporated in vacuo. The residual oil was dissolved in chloroform, applied to a silica gel column, and separated by flash chromatography using a 2% methanol solution in chloroform. The product-containing fractions were evaporated and the oil was chromatographed again using an ethyl acetate/hexane mixture (6:4 by volume). The fractions containing the product were evaporated in vacuo to give a white solid. Melting point 96-98℃. Calculated values - C: 46.60, H: 4.89, N: 22.65. Actual measurements - C: 46.67, H: 4.94, N: 22.59. Example 10 The following compounds were prepared from the appropriate acid halide or acid anhydride following the procedure of Example 8 or 9.
3'-azido-5'-O-benzoyl-3'-deoxythymidine Calculated values - C: 53.68, H: 4.77, N: 18.41. Actual measurements - C: 53.61, H: 4.72, N: 18.46. Melting point 54-59℃. 3'-azido-3'-deoxy-5-O-hyvaloylthymidine Calculated values - C: 51.27, H: 6.03, N: 19.93. Actual measurements - C: 51.07, H: 6.05, N: 19.83. Melting point: 99-100℃. 3'-azido-3'-deoxy5'-O-(3-methylbutyryl)thymidine Calculated values - C: 50.24, H: 6.13, N: 19.53. Actual measurements - C: 50.27, H: 6.11, N: 19.49. δ7.46 (d, lH, J _5,6 = 1.2Hz, 6H), δ6.13 (t, lH, 1′H) δ4.55−4.15 (m, 3H, 3′H, 5′CH ₂ ) , δ3.8−4.15 (m−1H, 4′H), δ2.4−1.78 (m, 3H, 2′H, 5′ methine), δ1.80 (d, 3H, J _5,6 = 1.2Hz , 5CH ₃ ), δ0.9 (d, 6H, J = 6.4Hz, 5'butyryl and methyl) 3'-azido-3'-deoxy-5'-O-palmitoylthymidine Calculated value -C: 61.67, H: 8.57, N: 13.85. Actual measurements - C: 61.85, H: 8.59, N: 13.75. δ7.45 (d, 1H, J _5,6 = 1.0Hz, 6H), δ6.12 (t, 1H, 1′H), δ4.5−4.05 (m, 3H,
3′H, 5′CH ₂ ), δ4.0−3.8 (m, 1H, 4′H) δ2.35−2.11 (m, 4H, 2′H, palmitoyl (CH ₂ ) 1), δ1.8 (d, 3H, J _5,6 = 1.0Hz, 5CH ₃ ) δ1.35−0.6 (m, 29H, palmitoyl (CH ₂ ) ₁₃ CH ₃ ) 3′-azido-3′-deoxy-5-O-tolyl Thymidine Calculated values - C: 56.10, H: 4.97, N: 18.17. Actual measurements - C: 55.88, H: 5.00, N: 18.09. Melting point: 73℃. NMR measurement with DMSO- _d6 . NMR−δ7.95−7.29 (m, 5H; bH, cH, 6H),
δ6.16 (t, 1H, 1′H), δ4.6−4.4 (m, 3H, 3′H, 5′H), δ4.2−4.0 (m, 1H, 4′H), δ2.39 (s, 3H, dCH ₃ ), δ1.63 (s, 3H, 5CH ₃ ) 3'-azido-3'-deoxythymidine-5'-O-
(Hydrogen succinate) Calculated values - C: 44.98, H: 4.83, N: 17.96. Actual measurements - C: 44.90, H: 4.77, N: 17.85. NMR measurement with DMSO- _d6 . NMR-δ7.46 (s, 1H, 6H), δ6.13 (m, 1H, 1'H), δ4.34-4.20 (m, 2H, 5'H), δ3.99-3.94 (m, 1H) , 4'H), δ1.78 (s, 3H, 5CH ₃ ) 3'-azido-3'-deoxy-5'-mesylthymidine Calculated value - C: 38.25, H: 4.37, N: 20.28, S: 9.28 . Actual measurements - C: 38.15, H: 4.38, N: 20.19, S: 9.30. Melting point: 253°C (decomposed). NMR measurement with DMSO- _d6 . NMR−δ7.49 (d, 1H, J _5,6 = 10Hz, 6H), δ6.15 (t, 1H, J ₁ ′ _,2 ′=6.6Hz, 1′H), δ4.54−4.41 (m , 3H; 3′H, 5′H), δ4.14−4.02 (m, 1H, 4′H), δ3.24 (s, 3H, 5′-mesyl CH ₃ ), δ1.79 (d, 3H , J _5,6 = 1.0Hz, 5CH ₃ ) 3'-azido-5'-O-(3-chlorobenzoyl)
-3'-Deoxythymidine Calculated values - C: 50.31, H: 3.97, N: 17.26, Cl: 8.74. Actual measurements - C: 50.16, H: 4.03, N: 17.13, Cl: 8.66. NMR measurement with DMSO. NMR−δ11.37 (s, 1H, 3-NH), δ7・98−7.43 (m, 5H; 5′-phenyl, 6H), δ6.17 (dd, 1H; J ₁ ′ _,2a ′=6.1Hz , J ₁ ′ _,2b ′=7.2
Hz,
1'H), δ4.68-4.48 (m, 3H; 3'H, 5'H), δ4.14-4.11 (m, 1H, 4'H), δ2.48-2.41 (m, 2H, 2 ′H), δ1.64 (d, 3H, J _5,6 = 1.2Hz, 5CH ₃ ) Example 11 2,5′-O-anhydro-3′-azido-3′-deoxythymidine Starting material dry pyridine Add methanesulfonyl chloride (2.7 mL) to the (2 mL) solution and add 3'-azido-3'-deoxythymidine (3.0 g, 11.2 m Mol).
was mesylated. After reacting at 5°C for 1 hour, the mixture was poured into ice, and the precipitate was collected by filtration. The product (3'-azido-5'-mesylthymidine) was reacted with potassium carbonate (0.78 g, 5.6 mmol) in DMF (75 mL). The reaction was heated in an 80° C. oil bath for 6 hours and then poured into ice water. The product was extracted from water with ethyl acetate, the solvent removed in vacuo and the residual oil subjected to flash chromatography on silica gel.
Elution was performed with a mixture of CHCl ₃ :MeOH (9:1 by volume). By evaporating the solvent from the appropriate fraction,
The title compound was obtained as a solid. Melting point 184-186°C Example 12 3'-azido-3'-deoxythymidine (a) 2,3-anhydrothymidine Dry 500 mL of thymidine (85.4 mg, 0.353 mol)
(2-chloro-1,1,2-trifluoroethyl)diethylamine (100.3 g,
0.529mol) (DE
Ayer), Journal of Medical Chemistry (J.Med.Chem.), 6 , 608 (1963)). The solution was heated to 70° C. for 30 minutes and then poured into 950 ml of ethanol with vigorous stirring. The precipitated product was filtered from the solution, and the ethanol supernatant was refrigerated and filtered to yield the title compound. Melting point 228-230℃. (b) 3'-azido-3'-deoxythymidine 2,3'-O-anhydrothymidine (25 g,
0.1115mol) and sodium azide (29g,
0.446 mol) was suspended in a mixture of 250 ml DMF and 38 ml water. After heating the mixture at reflux for 5 hours,
poured into the water. Aqueous solution in EtOAc (3 x 700 ml)
The extract was dried over _Na2SO4 , then filtered and the EtOAc was removed _in vacuo to give a thick oil.
This oil was stirred with 200 ml of water to precipitate the title compound as a solid, which was collected by filtration. Melting point 116−
118℃. Example 13 Monosodium salt of 3'-azido-3'-deoxythymidine Approximately 1 g of 3'-azido-3'-deoxythymidine was
Dissolve in 50 mL of distilled water and PH using 1N NaOH.
was adjusted to 12. Freeze-dry about half of the solution,
The title compound was obtained as a refrigerated dry powder. Analyzed as C ₁₀ H ₁₂ N ₅ NaO ₄ 6/10N ₂ O. Calculated value - C: 40.03, H: 4.43, N: 23.34, Na: 7.66 Actual value - C: 39.88, H: 4.34, N: 23.23, Na: 7.90 Example 14 3'-azido-3'-deoxythymidine Preparation of 5'-monophosphate 3'-azido-3'-deoxythymidine (0.5 g,
1.87 mmol) in 5 mL of triethyl phosphate,
The mixture was cooled to -5°C. Phosphorus oxychloride (0.685 mL, 7 mmol) was added to the solution in one portion under rapid stirring and then kept at −10° C. for 22 hours. A portion of the liquid was taken and added to concentrated ammonia water. This sample was subjected to thin layer chromatography (cellulose, n-
Analysis with PrOH/H ₂ O (3:7 vol.) confirmed that no starting material remained and showed simple fluorescent spots that were less mobile than the nucleosides.
Pour the reaction mixture into 20 mL of ice water, immerse it in an ice bath, and adjust the pH by adding 2N NaOH to the solution.
Adjusted to 7.5. The basic mixture was extracted once with chloroform and once with ether. water layer again
After adjusting the pH to 7.5, it was concentrated in vacuo to remove residual organic solvent. The material was stored at −10° C. and subjected to the next purification process. First, add coconut charcoal (50-200 mesh, 100g) to 1N HCl (500mL),
Deactivated charcoal was prepared by washing thoroughly with water (3 L), 3% toluene in 95% ethanol (35 mL), 95% ethanol (600 mL), and finally water. Deactivated charcoal (12 mL of precipitated wet charcoal) was added to the monophosphate solution (0.72 g, 1.8 mmol, 30 mL) with stirring. The supernatant was decanted and the charcoal was washed with 150 mL of water. Charcoal with ammonium hydroxide
Nucleotides were eluted by washing with 1.5M 50% ethanol solution. The solution was filtered through a 0.22 μ filter, concentrated in vacuo to 10 mL, and further filtered using Amicon Centriflo CF-25.
After filtering through a membrane, freeze-drying diammonium 3'-azido-3'-deoxythymidine-5'-
Monophosphate was obtained as a solid. This compound had the characteristics of a nucleoside 5-monophosphate, being converted to a nucleoside by 5'-nucleotidase. Example 15 3'-azido-5'-triphosphate-3'-deoxythymidine and 3'-azido-5'-diphosphate-3'-deoxythymidine (a) Bis(tri-n-butylammonium) pyrophosphate Dow 50 ( Dow50) [Dowex
Ion exchange resin - Dow Chemical Laboratories] Pour 40 mL of pyridinium resin into a tube with a diameter of 25 cm.
A resin column was prepared by filling the resin column with water and washing with water until no color was eluted. Phosphate decahydrate (1.12g, 2.51mM)
It was dissolved in 30 mL of water and added to the column. The column was eluted with water and a 125 mL fraction of the eluent containing UV absorbing material was collected. liquid
After concentration in vacuo to 10 mL, tri-n-butylamine (1.2 mL) was added. Further vacuum concentration
The resulting residue was co-evaporated with pyridine four times and dried. The product was stored in a -5°C freezer. (b) Hydrogen form of 3'-azido-5'-monophosphate-3'-deoxythymidine The ammonium salt obtained in Example 14 (0.1 g,
Dissolve 0.283mMol) in 6mL of water and dissolve 1.5mL (10
The hydrogen form of the monophosphate was prepared by passing it through a Dow 50H ⁺ column (equivalent). (c) Phosphoromorpholinate of 3'-azido-3'-deoxythymidine The hydrogen form of the monophosphate obtained in step (b) (0.283 mmol) was dissolved in 9 mL of water. After adding morpholine (99 μL, 1.13 mmol, 4 eq.), the solution was heated to reflux. Dicyclohexylcarbodiimide (0.234 g, 1.13 mmol, 4 eq.) dissolved in tert-butanol (5 mL) was added over 3 hours and the reaction mixture was heated at reflux overnight, then cooled to room temperature, filtered and the solvent was removed. Removed in vacuo. Ethanol was added four times and evaporated each time. The residue was dissolved in methanol and ether was added to precipitate the phosphoromorpholinate. The precipitate was triturated with ether four times and dried on a rotary evaporator to give the title compound. (d) The phosphoromorpholinate derivative obtained in step (c) was dried by removing the pyridine four times in vacuo. Bis(n) obtained in step (a)
-Bu) ₃ N pyrophonate was also dried by removing pyridine in vacuo. The phosphoromorpholinate was dissolved in 5 mL of pyridine and added to the container containing the pyrophosphate reagent. After the reaction mixture was left at room temperature overnight, the pyridine was removed in vacuo. Water was added to the residue and removed in vacuo three times.
The residue was frozen. The residue was then thawed and dissolved in 50 mL of water.
The solution was diluted with DEAE Saphadex A- equilibrated with 50mM ammonium bicarbonate.
25 columns (1 x 10 cm). Phosphate is 50-800mM ammonium bicarbonate.
Elution was performed with a 300 mL linear gradient. Fractions containing diphosphate nucleotides were pooled, as were triphosphate fractions. The pooled diphosphate and triphosphate fractions were each vacuum dried, redissolved in water, dried again, further dissolved in water, and freeze-dried. Example 16 Enzymatic synthesis of 3'-azido-5'-triphosphate-3'-deoxythymidine 5'-triphosphate was synthesized from 5'-diphosphate using pyruvate kinase and nucleoside diphosphate kinase. Reaction mixture was 6mM
3'-Azide TDP, 12mM adenosine triphosphate, 40mM _MgCl2 , 40mM potassium piperazine-N,N'-bis(2-ethanesulfonic acid)
PIPES buffer (PH 6.8), 30 mM phosphoenolpyruvate, 40 IU/ml nucleoside diphosphate kinase, and 100 IU/ml pyruvate kinase were contained in a final volume of 5 mL. The reaction mixture was incubated at 37° C. for 5 days and then applied to a column (2.5×10 cm) of DEAE Sephadex A-25 equilibrated with ammonium bicarbonate. The nucleotide is
Elution was with a gradient of 100-1000mM ammonium bicarbonate. Fractions containing triphosphate were pooled and dried by vacuum evaporation. The compound was prepared using a preparative HPLC column (Whatman, Magnum 9SAX).
using 10-100mM potassium phosphate (PH3.5)
Further purification was carried out by elution with a gradient of The resulting compound was further purified using the DEAE Sephadex A-25 column described above. Fractions containing tetraammonium 3'-azido-3'-deoxythymidine-5'-triphosphate were pooled, dried in vacuo, redissolved in water, and freeze-dried to yield the title compound. Example 17 Antiviral activity (a)(i) Retrovirus-induced toxicity 3′-azido-3′-deoxythymidine was used to infect 1.5×10 ⁴ Pfu of RVB3 strain of Rauscher Murine leukemia virus. was administered to BALB/c mice. Treatment started 4 hours after infection and every 8 hours.
It was administered intraperitoneally at 80 mg/Kg or orally in drinking water at concentrations of 0.5 or 1.0 mg/ml. These treatments were found to prevent infection of splenocytes, the subsequent development of splenomegaly, and also to suppress Viraemia. (ii) HTLV-TM- cells (a T cell clone susceptible to HTLV- infection) were co-cultured with irradiated HTLV-producing MJ tumor cells as follows. a) TM-cells alone b) TM-cells and MJ tumor cells c) TM-cells, MJ tumor cells and 3'-
Azido-3-deoxythymidine (3 μM) d) TM- cells, MJ tumor cells and 3-
Azido-3-deoxythymidine (9 μM) e) TM- cells, MJ tumor cells and 3-
Azido-3-deoxythymidine (27 μM) On day 18, total DNA was extracted from each culture medium.
Digestion with BamHI produced a fragment of the HTLV-genome with a standard molecular weight of 3.3 kD, independent of any host side order. Digest is
The probe was examined using radiolabeled λMT-2, a standard probe that recognizes BamHI of HTLV-. No hybridization was observed for a), indicating the absence of virus in the uninfected controls. A strong signal was observed in the untreated infected control in case b). In case c), a weak signal was observed, indicating incomplete extinction of the virus. No hybridization was observed in d) and e), indicating complete eradication of the virus. Each culture medium was also probed with a probe for T cell-receptive β-chain, and a strong signal was observed for all culture media, indicating that TM- continued to exist for the duration of the experiment. It was showing. (b) AIDV (i) Reverse transcriptase activity 3′-azido-5′-triphosphate-3′-
In vitro testing of deoxythymidine against AIDV reverse transcriptase (AIDV RT) was performed. AIDV RT was pelletized and extracted.
It was purified from AIDV by elution with a DEAE column and a phosphocellulose column. Enzyme activity was linear over 60 minutes and stable for at least 2 months when stored in 60% glycerol and 1 mg/ml bovine serum albumin. rA as template primer
Using −odT _(12-18) , AIDV RT is 7.0−
Optimal PH of 7.3, optimal _MnCl2 concentration of 0.3mM, and optimal _MgCl2 concentration of 5mM were maintained. 5mM
The activity in the presence of _MgCl2 is 0.3mM
The activity was 10 times stronger than in the presence of _MgCl2 .
Maximum enzyme activity is 80−140mM KCl, 60−
Seen in the presence of 100mM NaCl. [ ^3H ]
Incorporation of dTTP was linear with enzyme concentration. In tests, 3'-azido-5'-triphosphate-3'-deoxythymidine
It was found to be a competitive inhibitor of AIDV RT, and rA-
It showed a Ki of 0.04 μM when using odT _(12-18) . The enzyme has a Km of dTTP of 2.81 µM and 3'-azido-5'-triphosphate-
It was suggested that 3'-deoxythymidine was more tightly bound to this enzyme than to dTTP. Experiments with RT for avian myeloblastosis virus, Moloney murine leukemia virus, and AIDV have shown that
It was found that 3'-azido-5'-triphosphate-3'-deoxythymidine is a terminator for DNA chain elongation. (ii) In vitro anti-AIDV activity 3'-azido-3'-deoxythymidine has been tested and shown to be active in a number of in vitro assay systems. Drug efficacy was determined by reverse transcriptase (RT) activity in supernates obtained from infected, uninfected, and drug-treated cells. 3′-azido-3′-deoxythymidine is
At concentrations of 2.7−0.0013 mcg/ml, H9 and U937 human lymphoblastoid cell lines
Effectively blocked AIDV infection. Similarly, normal PHA infection stimulated leukocytes and cultured peripheral blood lymphocytes were inhibited even at drug concentrations as low as 0.013 mcg/ml. In experiments adding or subtracting drugs in H9 cells, the 3′-
Azido-3'-deoxythymidine is most effective when present when susceptible cells are infected with the virus, but its antiviral activity remains strong even when applied 20 hours after the initial AIDV infection. still retained most of it. Suppression of viral replication is achieved by drugs that inhibit viral absorption.
It was also clearly demonstrated when present in the medium only during 20 hours. The effect is 0.13 and
It was observed at a concentration of 0.013mcg/ml. 3'-azido-3'-deoxythymidine was purified
Direct resistance to AIDV virus
It showed no RT activity. Similarly, drugs are
It had little or no effect on virion production or release from chronically infected H9ADIV cell lines. (iii) Prevention of AIDV infection The ability of 3'-azido-3'-deoxythymidine to inhibit AIDV infection of cells was tested as follows. Cloned T4-positive tetanus-specific T helper lymphocytes were infected (at doses up to 5000 virions per cell) into pools of AIDV isolates and cell survival was monitored after infection.
After 10 days of culture, 3′-
No cytopathic effect of the virus was observed on infected T cells treated with azido-3'-deoxythymidine, and the number of untreated infected cells was reduced to one-fifth. Cell survival of HTLV-transformed, AIDV-coinfected cell lines derived from the above cells was also evaluated. In 7 days, 3'-azido-3'-deoxythymidine was 2.7, 0.27 and 0.13 mcg/ml.
The cytopathic effect was completely blocked at a concentration of . A protective effect was observed in infections induced by cell-free virions and cell-associated viruses. 3'-azido-3'-deoxythymidine, even at a concentration of 0.27 mcg/ml, effectively prevented the induction of cytopathic effects by the less related Haitian isolate of ATDV. Example 18 Toxicity Assay 3'-azido-3'-deoxythymidine was administered to mice and rats. The LD ₅₀ value was over 750 mg/Kg in both mice and rats.

Claims (1)

Claims: 1. A pharmaceutical composition for the treatment or prevention of human retroviral infections containing as active ingredients 3'-azido-3'-deoxythymidine and a pharmaceutically acceptable carrier. 2. The composition according to claim 1, wherein the carrier is other than water. 3. A sterile composition according to claim 1 or 2. 4 Claim 3 suitable for administration by injection
The composition described in Section. 5. The composition of claim 4, which is contained in a sealed vial. 6. The composition according to claim 1, wherein the carrier is sterile water. 7. A composition according to any one of claims 1 or 2, which is suitable for oral administration. 8. The composition according to claim 1, which is a tablet or capsule containing 3'-azido-3'-deoxythymidine. 9. The composition according to claim 8, wherein the active ingredient is capable of sustained release after oral administration. 10. The composition of claim 7, comprising a flavoring agent. 11. A composition according to any one of claims 1 to 10, containing the active ingredient in a unit dose or multiple doses thereof. 12. The unit dose of the active ingredient is 20 to 700 mg,
A composition according to claim 11. 13 Claims 1 to 1 for treatment or prevention of acquired immunodeficiency virus (AIDS) infection
The composition according to any one of Item 2. 14. A composition according to any one of claims 1 to 13 for the treatment or prevention of AIDS. 15. A composition according to any one of claims 1 to 13 for the treatment or prevention of HTLV-infection.