JPS5836960B2 - Method for producing 1-β-D-ribofuranosyl-1.2.4-triazole-3-carboxamide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION During the past decade, many nucleoside analogs have been found to exhibit good antitumor and antiviral activity.

The most important of the currently known nucleoside antivirals are generally 5-iodo-2-deoxyuridene (IDU), 9-β-D-arabinofuranosylatenine (ara-A) and It is believed to be 1-β-D-arabinofuranosylcytosine (ara-C).

However, these compounds are effective only against a limited spectrum of viruses that do not include bacteria that cause respiratory diseases in humans (influenza, colds).

A nucleoside analog recognized to be effective against respiratory viruses is 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, which is described in U.S. Patent Application No. 240,252. No. (19
(This is a continuation-in-part of the invention filed under the name 1,2,4-triazole nucleoside described in U.S. Patent Application No. 149017 on June 1, 1971).
・197 under the name 2,4-triazole nucleoside
It is stated in the specification filed on March 31, 2017.

Certain derivatives of the latter compounds have also been found to have significant effects against these viruses, and in addition certain triazole bases of such compounds, 1.2.4-triazole-3-carboxamide and 1.2・4-
Triazole/L/-3-thiocarpoxyamide similarly has excellent antiviral activity against these respiratory viruses.

The chemical structure and synthesis of each compound have been previously reported (Latvijas PSR Zinatnu
Akad. Vestis. Kim. Ser. 1 9
6 5 (2) 2 0 4 ~ 2 0 8 Che
m. Abst. 6 3 1 3 2 4 3 (19
65)].

By the way, we discovered that 1,2,4-triazole-3-carpoxyamide can be converted to 1-β-D-ribofuranosyl-1,2,4-1-lyazole-3-carboxamide through enzymatic conversion. , the nucleosides are important effective antiviral compounds.

As described below, the triazole base reacts with the enzyme nucleoside phosphorylase to effect the desired transformation.

Accordingly, the present invention relates to a process for the preparation of a compound used as an antiviral agent having the following structural formula:
In this case, the compound is 1,2,4-triazole-3-carpoxyamide) or S (the compound is 1,2,
4-triazole-3-thiocarpoxyamide)
and Y represents H, an alkali metal or an amine]
.

During the synthesis of 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxyamide, the triazole base can be reacted with the enzyme nucleoside phosphorylase under appropriate conditions described below.

Carboxamide compounds can be prepared by the methods described in the following examples, in which temperatures and melting points are expressed in degrees Celsius.

Example ■ 3-cyano 1,2,4-triazole triethyl orthoformate (150rrLl) and 1
-Cyanoformimidic acid hydrazide [K. Mazda (
Matsuda) and L. T. Morin
n) of J. Org, Chem. 26 3783 (1
A mixture of 961)) (25.2'?, 0.30 mol) was cooled to 0.degree. C. and a solution of dioxane (4.0 yd) saturated with dry hydrogen chloride gas was added under stirring.

The mixture was cooled in a water bath for 5 hours and kept stirring at 25° C. for 15 hours.

The mixture was evaporated to dryness and the residue was treated with ether (500 rIL).
l) was added.

The solution was filtered, washed with water and the organic phase was dried over magnesium sulphate.

The solution was filtered to remove ether. Crystallization of raw material from ethyl acetate gives a melting point of 185-187
3-cyano 1,2,4-triazole at 16. Cl
(56.8%) was obtained.

All properties of the compound can be found in Cipens
) and Grinsteins
Method C by Latvijas PSR Zinat
nu. Akad, Vestis. Kim. Ser. 19
65(2) 204-208Chem. AbSt.
13243 (1965)].

Analysis for C3H2N4: Calculated value C38.30 H2.14 N59.56
Actual value C38.29 Hl, 98 N59.16
Example (1) 1,2,4-Triazole-3-carboxyamide Methyl 1,2,4-triazole-3-carboxylate was heated with excess ammonia until the reaction was complete.

The mixture was cooled and the product was collected. By recrystallization from water, 1,2,4-triazole-3-carboxamide with a melting point of 313-315°C (decomposition) was obtained almost quantitatively.

Analysis value for C3H4N40: Calculated value C32.14 H3.60 N49.99
Actual value C32.37 H3.73 N50.09
Example ■ 1,2,4-triazole-3-thiocarpoxyamide Example ■ 3-cyano 1,2,4-triazole (4.7
P, 0.050 mol), ethanol (501 rLl) and triethylamine (8,Q ml) at room temperature.
The mixture was stirred for an hour while hydrogen sulfide gas was bubbled through the mixture.

Removal of the solvent and addition of water to the residue yields the product 2.7? was gotten.

Recrystallization from water gave 1,2,4-triazole-3-thiocarboxamide with a melting point of 350°C.

Analysis value for C3H4N4S: Calculated value C28.12 H3.15 N43.72
S 2 5. 0 2 Actual value C28.12 H3.18 N43.6O
S25.09 Example ■ 1,2,4-triazole-3-carboxamide sodium salt■,2,4-triazole-3-carboxamide (1
12P, 1 0. 0 mmol), sodium hydroxide (
0.4 (1, 10.0 mmol) and water (10.0 ml)
The solution was frozen and the sample was lyophilized.

The product was obtained as a hand hydrate of 1,2,4-triazole-3-carboxamide sodium salt having a melting point >320°C.

Analysis value for C3H3N4 0Na H i / 2 H2 0: Calculated value C25.18 H2.82
N39.16Na 1 6.0 7 Actual value C25.24 H2.78 N38.92
Na 1 6.0 0 Other biocompatible alkali metal and amine salts, such as chlorine salts, are similarly prepared.

1,2,4-triazole-3-carboxamide is
At a pH value in the range of about 5 to 9, at a temperature in the range of about 0 to 50°C, advantageously at a temperature of about 25 to about 35°C, about 0.015 to 0.
．． 751n9/Tnl can be converted to 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxyamide by reaction with the enzyme nucleoside phosphorylase, particularly at an enzyme concentration of about 0.15 μ/ml.

Satisfactory results were obtained when the triazole base was present in a concentration greater than 5×10 −5 M.

Generally 0.1 to 2 hours, especially about 0.5 to 1 hour, are required for this reaction.

The enzyme source may be animal, tissue or bacterial.

The main bacterial source is E. Coli and yeast, and various animal sources also exist, including bovine liver, rat liver, calf liver, calf thymus, bovine liver, monkey brain, horse liver, calf spleen, human red blood cells, Also includes fish skin and fish muscle.

This synthesis will be better understood from the following examples.

Example ■ ■ - β-D-Ribofuranosyl-1,2,4-triazole-3-carpoxyamide 1 - Purified calf spleen nucleoside phosphorylase from 2,4-triazole-3-carpoxyamide 1
Synthesis of β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide This incubated sample was prepared using Tris-HCI (pH 7.4) in a final volume of 0.135 ml.
) 50 μmol, ribose 1-phosphate 0,25
μmol, 1,2,4-thiazole-3-carboxamide (H”) 42 μC/μmol, 0.05 μmol and calf spleen nucleoside phosphorylase (Sigma Chemical Co., Ltd.
It contained 80 μl of Co, St, Lewis, Mo).

The reaction is stopped by incubating the sample at 25° C. for 5 minutes and then freezing in dry ice-isopropanol.

A small amount of the thawed sample was then dripped onto silica gel along with standard solutions of 1,2,4-triazole-3-carpoxyamide and 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, followed by isopropanol: NH40H:H20 (7:1:
2) Separate inside.

■1-β-D-ribofuranosyl-1,2,4-thiazole-3- of 1,2,4-triazole-3-carpoxyamide, excluding the region of the chromatogram that corresponds to 2,4-triazole-3-carboxamide. Measurements were taken to determine the conversion to carboxamide.

The results of the evaluation of nucleoside phosphorylase using 1,2,4-triazole-3-carpoxyamide The above results indicate that the conversion rate achieved increases as the enzyme concentration increases. That is clear.

When using either 1,2,4-triazole-3-carpoxyamide or 1,2,4-triazole-3-thiocarboxamide as an antiviral agent, the drug typically contains about 0.0% of the total weight of the drug. 0.001 to 5%, particularly in an amount of about 0.01 to 2.5%, in a suitable diluent;
That is, the actual amount used will depend on a number of factors, such as the severity of the infection, the general health, and the age of the host.

In any event, the actual amount should be sufficient to provide the parasite with a chemotherapeutically effective dose of the drug;
This can be easily determined by one skilled in the art.

In one form, the compound can be used as an aerosol nasal spray of the type described in U.S. Pat. It is described to contain an amount of an antiviral compound suspended in a fluorinated or fluorochlorinated lower alkane (available under the trade name Freon).

Generally, the propellant is a gas at room temperature and atmospheric pressure, and about 1
It has a boiling point below 8°C (65p) (under atmospheric pressure) and is of course non-toxic.

Particularly suitable such propellants include dichlorodifluoromethane (Freon 12), dichlorotetrafluoroethane (Freon 14) and trichloromonofluoromethane (Freon 14).
Freon 11).

When used in suspension, the antiviral agent should be subdivided, for example into diameters of less than 100 microns, especially less than 25 microns, and in particular about 0.5 to 4 microns.

Surfactants, especially esters or partial esters of nonionic fatty acids having 6 to 22 carbon atoms, such as caproic acid, caprylic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, etc., reduce powder agglomeration. It is advantageous to help.

Typically, relatively small amounts of surfactants are used, such as about 0.1 to 5% by weight, especially 0.25 to 1.0% by weight, although larger amounts can be used if desired.

Similarly, the carboxamide antiviral agents described above can be dissolved in a liquid propellant as a suitable solvent as described in US Pat. No. 2,868,691.

If desired, any antiviral agent can be injected into the parasite, in the form of a physiological saline solution or suspension containing about 10 to 500 μl of drug per ml of solution. It may be.

Antiviral agents can be applied as oral preparations in the form of capsules or tablets.

Tablets and capsules contain approximately 1 compound per tablet or capsule.
Contains 0 to 500 ■.

The desired dosage of the antiviral compound varies depending on the patient's condition as described above, but is usually in the range of about 10 to 2000 μl per day.

To effectively inhibit RNA or DNA viruses, a concentration of approximately 32 μ2 of compound per ml of serum is required.

The capsules are conventional gelatin capsules which, in addition to the above amounts of antiviral agent, contain small amounts, for example less than 5% by weight, in particular less than 1.0%, of magnesium stearate or other fluidizing agents such as Avicel ( Avicel
(carboxymethyl cellulose)].

The tablets contain the antiviral agents and binders mentioned above, such as gelatin solution, starch paste in water, polyvinylpyrrolidone, polyvinyl alcohol in water, with a typical sugar coating.

The antiviral agent can be applied topically as a topical ointment, cream, emulsion or topical solution depending on the condition of the viral skin infection to be treated, where the antiviral agent is added in the amount described above in standard excipients or It is formulated with other ingredients commonly used in such topical applications.

Ointments are therefore recommended for chronic conditions because of their oily base, while creams, emulsions and topical solutions are recommended for acute, subcutaneous conditions.

Unlike ointments, creams are generally water-soluble and exhibit disappearing properties.

Emulsions are used when treatment with multiple drugs needs to be indicated, one of which is insoluble and the other soluble in the vehicle, and multiple emulsified excipient phases are required for uniform dispersion. It is.

Topical solutions are solutions of the active ingredient in a solvent, with a viscosity intermediate between that of creams and easily evaporated solvents such as alcohols, to achieve a balance between spread and prolongation of action.

Examples of many commonly applied topical ointments, creams and solution formulations are compositions of the following materials: Ointment (a) Petroleum without adjuvants (b) Plastibase (Skip ■ Hydrocarbon gel obtained from nC. and polyethylene and mineral oil) Cream (a) Methyl Paraben (Methyl Par
aben) USP Probylparaben (Propy
Paraben) USP Spermaceti US
Sodium P Lauryl Sulfate USP Stearyl Alcohol USP Cetyl Alcohol USP Glycerin USP Mountain Stearate Propylene Glycol Sorbitan Monostearate and Oleate Polyoxyethylene Sorbitan Monostearate Methyl Citrate and Provil Paraben (C) Aqueous Base Potassium Methyl Sorbate and provilparabenglycerol monostearate squalane polysorbate 80 (USP) spermacetei stearoyl alcoholan rubital solution (d) polyethylene glycol 400 (USP)
Propylene Glycol Carboxymethylene Monoacynoleamine Titanium Dioxide Butylated Hydroxytoluene Topical Solution (a) Polyvinyl Alcohol-Water (b) Polyethylene Glycol 400 Therefore, topical vehicles typically contain emollients, saponifiers, and emulsifiers in addition to the base agent. , solvents, penetrants, pH regulators, plasticizers, softeners, preservatives, hardeners, pigments and perfumes, etc., are all known in the literature.

For use against vaginal infections, topical vehicles that produce maximum dispersion of the active agent are advantageous.

For example, vaginal cream (a) Criserol monostearate Corn oil Glycerin Benzoic acid Glutamic acid Water (b) Glycerin Etynole Alcohol Liquid Petroleum Polyethylene glycol ether: Fatty alcohol Complex Paraben Preservative Water Vaginal suppositories (a) Lactose Polyethylene glycol 400 Polysorbate 80 Polyethylene Glycol 4000 Glycerol Lactic Acid (b) Polyethylene Glycol Polyoxyethylene Palmitate Lactic Acid Topical vehicles for vaginal application are pH-adjusted to acidic conditions where normal bacteria thrive and do not weaken the body's defense mechanisms. .

Of course, those skilled in the art are well aware of this and other considerations involved in topical application of antiviral agents.

Topical formulations contain a proportion of the active substance that effectively inhibits the virus, ie from about 0.01 to 10% by weight of the total weight of the composition, especially 0.
．． 0.025 to 1% by weight, especially 0.025 to 0.1% by weight.

Up to about 10% by weight can be used to treat refractory conditions.

Of course, the amounts of other ingredients in such formulations will correspond to the amounts of such ingredients commonly used, and determination of appropriate formulations is readily within the scope of this description to those skilled in the art.

The manner in which antiviral agents are applied depends on the particular viral infection being treated.

For example, if the infection is caused by influenza or other respiratory viruses and it is evident that it is in the upper respiratory cavity, an advantageous treatment method is an aerosol nasal spray.

This is because this is the most effective way to reach the location of the infection.

Oral or injection treatment depends on the severity of the infection.

When the infection is clearly a lower respiratory infection or a viral infection of other systems, the preferred mode of treatment is oral or by injection.

If the infection is local, such as herpes labialis (herpes labialis, acute fever), genital herpes (viral infection of the penis or vagina), shingles, chickenpox (ch
ickenpox), exzema herpeticum, herpesdermatitis, etc., suitable application is by topical application as mentioned above and, if possible, also by oral or injectable treatments as mentioned above. be combined.

By way of illustration, the following formulations for topical application are shown: Ointment The detailed antiviral activity of each of the triazoles of the invention is shown.

These were tested in an activity test using virus-induced cytopathological effects (CPE) according to the method of Sidwell et al.
logic ) 2 2 : 7 9 7 ~ 801 (
(1971)].

In short, the CPE method involves vitamins, amino acids,
Also included is the dissolution of the antiviral agent into the cell culture medium consisting of serum, buffer, penicillin, streptomycin and indicator dye.

Virus suspended in this cell culture medium was added to the achieved monolayer of KB cells, followed by the same amount of antiviral agent within 15 minutes.

Infected and treated cells were evaluated by the following microscopic examination.

Controls for each experiment include cell controls (cells and cell media only), virus controls (cells and virus and cell media), and toxicity controls (cells and cell media).

The Sidwell et al. virus evaluation (VR) system described in Applied Microbiology, supra, was used to evaluate the extent of CPE inhibition.

A VR greater than 0.5 indicates significant antiviral activity, and a VR less than 0.5 indicates slight antiviral activity.

In experiments with measles virus, virus spots were discernible when the method described above was applied by coating infected cells with agar monoserum monobicarbonate containing antiviral compounds.

The spots were stained with a neutral red dye on day 4 to measure the reduction in the number of spots that occurred upon exposure to the antiviral compound.

The method for this experiment is described by Wear et al. (Exptl. and Molec.
ular pathol. 9: 4 0 5-4
17, (1968)].

The results of this antiviral experiment are shown in Table 1 below: Reference Example Each antiviral agent was used for influenza A in mice.
2 was tested for effectiveness against infection.

Young adult mice were exposed to a sufficient amount of influenza A2 virus aerosol to cause approximately 65% of the animals to die within 8-13 days.

Groups were initially pre-exposed to virus for 4 hours and treated intraperitoneally twice daily for 6-9 days with various concentrations of the compound suspended in saline.

Controls included viral controls (exposed to virus and treated with saline alone), toxicity controls (sham infected with virus dilution (MEM) and treated with test compound), and normal controls (uninfected and untreated). is included.

Animals that die during the experiment are observed daily, and on day 25, the lungs of surviving mice are removed and the extent of viral cirrhosis occurring is evaluated on a 0-40 scale.

The lungs were then pooled into appropriate groups and treated with phosphate-buffered saline (P
BS, pH 7.2, 0.2M PO4, 0.15MN
aCl) 1. Homogenized in 5 ml.

The homogenized lung preparation was incubated at 1500 r.p. p
．． rn. Centrifuged for 15 minutes at PBS and diluted 2-fold in an equal volume of 0.5% guinea pig red blood cells for each dilution in PBS.

The extent of red cell aggregation, a measure of virus in the lungs, was recorded after incubation for 45 minutes at room temperature.

This red cell aggregate is expressed as hemagglutinin per ITIll (
HA) value.

From Examples 1 to 2, both compounds 1 and 2 have a spectrum of antiviral activity and are particularly effective against influenza viruses.

Relative cytotoxicity study results showed slight changes in cell structure, with Compound 1 at 32-1000μ/TrLl and Compound 2 at 1μ? It is clear that concentrations down to /rul cause similar changes.

Each compound is relatively insoluble in the aqueous medium used and the antiviral activity may be only slightly localized within the cellular structure;
The relative loss in solubility did not unduly limit the antiviral efficacy in animal studies.

In animal toxicity experiments, compound 1 was administered at 1000 μ/ky
Compound 2 was harmless to mice when applied intraperitoneally twice daily for 9 days at a concentration of
There was moderate toxicity manifested by weight loss and death upon application of low concentrations of 0 m9/kg/day.

Therefore, it is also recognized that the antiviral activity of ■.2.4-triazole-3-carpoxyamide is due to its enzymatic conversion by the parasite to 1-β-D-ribofuranosyl-1.2.4-triazole-3-carpoxyamide. .

In any case, such enzymatic conversion provides a method for synthesizing 1-β-D-ribofuranosyl-1,2,4-triazole-3-carpoxyamide.

The enzyme may be a purified protein or may be present in actively metabolizing bacterial or fungal cells.

If present in active metabolizing bacterial or fungal cells, this means that culture methods using these cells or mutants of these cells can convert 1-β-D-ribofuranosyl from 1,2,4-triazole-3-carpoxyamide. -1・2・
It is expected that 4-triazole-3-carboxamide will be produced.

Claims (1)

[Claims] 1 In order to produce 1-β-D-ribofuranosyl-1,2,4-triazole-3-carpoxyamide, 1.
1-β- characterized in that the 2,4-triazole-3-carboxamide is reacted with the enzyme nucleoside phosphorylase in the presence of ribose-1-phosphate, a pH value of about 5-9 and a temperature of about 0-50°C. A method for producing D-ribofuranosyl-1,2,4-triiso-/1/-3-carboxamide.