JPS6352607B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an antiviral agent for vertebrates containing isoprenylamine or a physiologically acceptable acid addition salt thereof as an active ingredient. Substances that have been previously determined to have the effect of preventing or relieving diseases caused by viruses that host vertebrates, or that have been recognized as being able to significantly increase antibody activity and suppress symptoms. There are known substances. Reported antiviral substances include interferon, a substance that induces interferon (interferon inducer), and synthetic substances that directly act on virus proliferation, such as amantadine hydrochloride or metisazone. be. Interferon is a glycoprotein with antiviral properties that is produced by vertebrate cells themselves when they are infected with a virus, and is effective against a wide range of viruses. Inducers for inducing interferon in vertebrates by methods other than viral infection include natural polymeric substances such as the double-stranded ribonucleic acid of certain bacterial phages, or dioxylic acid such as polyinosinic acid-polycytidylic acid. Synthetic polymeric substances such as heavy chain ribonucleic acids, as well as small molecule inducers such as tyrolones, are known. However, interferon has problems in its purification, and a practically economical production method has not yet been developed. Furthermore, conventional interferon inducers have not been put to practical use mainly due to their toxicity. Synthetic antiviral agents currently available on the market that directly act on viral proliferation have a rather narrow range of viral infections that can be treated with them, so the emergence of new synthetic antiviral agents is always desired. For this reason, the present inventors have conducted various studies to find a compound that produces high titer interferon and also has antiviral effects at the animal level.
It has been found that the compound represented by the general formula below and its acid addition salt exhibit excellent interferon-inducing ability, and also have excellent antiviral activity in animal tests. The active ingredient in the antiviral agent according to the present invention has the general formula (wherein n is 9, 10 or 11). In order to produce isoprenylamine represented by the above general formula and its acid addition salt, for example, the general formula Starting from isoprenyl alcohols of the formula (wherein n is 9, 10 or 11), i.e. nonaprenol (solanesol), decaprenol or undecaprenol, the corresponding amines can be prepared by applying known amine synthesis methods. Examples include methods of manufacturing. Furthermore, if necessary, the obtained amine can be formed into a salt by a conventional method. More specifically, a suitable isoprenyl alcohol represented by the above general formula is converted into a halide or sulfonic acid ester, and then (i) it is reacted directly with ammonia or (ii) it is reacted with a protected amine (e.g. The desired amine can be produced by a method such as reaction with (potassium phthalimide) and subsequent removal of the protecting group, or (iii) hydrolysis of the salt obtained by reaction with hexamethylenetetramine or guanidine. The resulting acid addition salt of the amine is obtained by mixing the amine with the desired acid in a suitable solvent and crystallizing and recovering the salt by evaporation or other means. Examples of acid addition salts suitable as pharmaceuticals include salts of hydrochloric acid, acetic acid, citric acid, fumaric acid, lactic acid, and the like. Next, a method for producing the compound represented by the general formula and its acid addition salt will be exemplified. Production example 1 17.4 g of solanesyl bromide and 5.6 g of potassium phthalimide, 70 ml of N,N-dimethylformamide
was added, stirred for 2 hours, and further stirred at 60°C for 1 hour. Insoluble matter was separated and the liquid was concentrated under reduced pressure.
The residue was dissolved in ethyl acetate, washed sequentially with water and saturated brine, and dried over anhydrous sodium sulfate.
Concentrate under reduced pressure. The residue was purified by silica gel chromatography (eluting with a mixture of n-hexane and benzene) to give N-solanesylphthalimide.
12.8g (yield 67%, melting point 51-53°C) was obtained. Next, 5 g of this N-solanesyl phthalimide was added to a solution of 0.42 ml of hydrazine (hydrated, 85% or more) and 40 ml of 95% ethanol, and the mixture was heated under reflux for 2 hours with stirring in a nitrogen stream. After cooling, a solution of 1.9 g of potassium hydroxide dissolved in 11 ml of water was added to the mixture with stirring. The precipitated oil was extracted twice with ether. The ether layer was washed successively with water containing a small amount of potassium carbonate and saturated brine, dried over anhydrous sodium sulfate containing a small amount of potassium carbonate, and then concentrated under reduced pressure. The obtained oily powder was dissolved in 40 ml of acetone, and about 5N hydrogen chloride-ethanol solution was added under ice cooling with stirring until the mixture became slightly acidic. The precipitated crystals were collected and recrystallized from acetone containing a small amount of ethanol to obtain 2.2 g (50% yield) of solanesylamine hydrochloride. Melting point 57-59℃.
The elemental analysis values for C ₄₅ H ₇₆ NCl are as follows. C% H% N% Calculated acid value: 81.09 11.49 2.10 Actual value: 80.61 11.44 2.18 Production Example 2 Starting from decaprenyl bromide, decaprenylamine hydrochloride was obtained in the same manner as in Example 1. melting point
56-58℃. The elemental analysis values for C ₅₀ H ₈₄ NCl are as follows. C% H% N% Calculated value: 81.74 11.52 1.91 Actual value: 81.19 11.41 1.88 Production Example 3 Starting from undecaprenyl bromide, undecaprenylamine hydrochloride was obtained in the same manner as in Example 1. Melting point 55-57℃. The elemental analysis values for C ₅₅ H ₉₂ NCl are as follows. C% H% N% Calculated value: 82.29 11.55 1.74 Actual value: 81.70 11.64 1.75 Next, the physiological effects of the active ingredients used in the present invention will be explained in more detail. (1) Interferon-inducing ability test A solution of the test compound dissolved in surfactant-containing water is administered intraperitoneally to a group of 5 ICR female mice weighing approximately 25 g. After 20 hours, blood was collected and serum was separated to obtain serum interferon. To measure the titer of induced serum interferon, L-929 from mice previously cultured in monolayers.
Contact the cells with a 10-fold dilution of the test serum, and
After culturing in a carbon dioxide incubator at 37°C, the test dilution was removed, the cells were inoculated with vesicular stomatitis virus, and then overlaid with a tissue culture medium containing 1% agar. The cells were then cultured at 37° C. for 24 hours, and the cells were stained with neutral red diluted to an appropriate concentration, the number of plaques formed was counted, and the rate of inhibition of plaque formation was calculated relative to the group to which no test compound had been administered. Table 1 shows the plaque formation inhibition rate of each test compound.

[Table] Hydrochloride
40mg 83.0%
Undecaprenylua 40mg/Kg 70.3%
Min hydrochloride
(2) Effect on mice infected with vaccinia virus 0.1 ml of a diluted solution of vaccinia virus (DIE strain) was intravenously injected into a group of 10 female ICR mice weighing around 15 g at a distance of 2 cm from the base of the tail. Lesions that appeared on the surface of the tail 8 days after inoculation were stained with a 1% fluorescein-0.5% methylene blue solution and counted. The test compound was administered intraperitoneally, orally, or subcutaneously on the day before virus inoculation or for 6 days from the day of virus inoculation, and the antiviral effect was evaluated by the lesion inhibition rate compared to the test compound-free group. Table 2 shows the inhibition rate of each test compound.
Shown below.

[Table] (3) Effect on influenza virus infected mice 25g of influenza virus A/PR-8
A group of 10 female mice before and after ICR are infected by nasal spray. A surfactant-containing aqueous solution containing the test compound was added 24 hours and 3 hours before virus infection.
Then, from the second day after infection, intraperitoneal administration was administered five times every other day. Mice that remained alive for 21 days or more after virus infection were considered to be alive, and the survival rate was calculated using the following formula. Number of survivors/number of treatments x 100 = survival rate (%)

[Table] Salt-free administration - 10%
(4) Inducibility test for human interferon (in
(vitro) Human fibroblasts cultured in a monolayer are contacted with a 1% ethanol solution of the test compound diluted with PBS (-), and super induction (using cycloheximide and actinomycin D) is performed using a conventional method. and 37
After overnight culture at °C, the supernatant was used as an interferon sample. To measure the interferon titer, the same human fibroblast cells were contacted with the undiluted sample, and 37
After overnight incubation at ℃, the cells were inoculated with vesicular stomatitis virus, 3H-uridine was added, and the degree of cell disintegration was determined by the release of 3H-RNA from the cells. The cell lysis inhibition rate for the sample was calculated. Table 4 shows the cytolysis inhibition rate of each test compound.

[Table] Ruamine hydrochloride
(5) Toxicity To determine the acute toxicity of the active ingredient of the present invention, 50%
Percent lethal dose was determined using 20-25 g ddY male mice. The results are shown in Table 5, and it can be seen that intraperitoneal administration is quite safe.

[Table] As is clear from the above test results, the active ingredient of the present invention not only has the ability to induce interferon in vivo, but also has low toxicity and excellent antiviral action. Furthermore, since the interferon activity and individual antiviral effects of the active ingredient do not necessarily correlate, the antiviral effect of the active ingredient at the animal level is not necessarily limited to interferon, but also to other hosts. It is also possible that an intervening defense mechanism is involved. Many symptoms of diseases caused by viruses are known in humans, such as herpes infections such as cyst rash, influenza, and measles. Therefore, when the active ingredients of the present invention are used for the treatment of viral infections, they are administered orally, through the respiratory tract, and by subcutaneous, intramuscular and intravenous injections. The dosage ranges from 0.5 to 20 mg/Kg, preferably from 3 to 5 mg/Kg, and is used several times a day (2 to 4 times) depending on conditions such as the patient's age, symptoms, and route of administration. The active ingredients of the invention can be formulated into compositions for administration in any conventional manner, such as tablets, capsules, granules, powders, etc.
It can be prepared into oral solutions, ophthalmic solutions, suppositories, ointments, injections, etc. When the active ingredient of the present invention is orally administered, it may be formulated into tablets, capsules, granules, or powders. These solid preparations for oral administration contain commonly used excipients, such as silicic anhydride, magnesium aluminate metasilicate, synthetic aluminum silicate, lactose,
sugar, corn starch, microcrystalline cellulose, hydroxypropyl starch or glycine, binders such as gum arabic, gelatin, tragacanth, hydroxypropylcellulose or polyvinylpyrrolidone, lubricants such as magnesium stearate, talc or silica, disintegrants such as potato starch, It may contain calcium carboxymethyl cellulose, or wetting agents such as polyethylene glycol, sorbitan monooleate, polyoxyethylene hydrogenated castor oil, sodium lauryl sulfate, and the like. In particular, soft capsules can be prepared by dissolving or suspending them in polyethylene glycol or commonly used oil-based bases such as sesame oil, peanut oil, germ oil, and fractionated coconut oil such as miglyol. Tablets and granules may be coated in conventional manner. Oral liquid preparations are aqueous or oily emulsion solutions,
It may be made into a syrup or the like, or it may be a dry product which can be redissolved in a suitable vehicle before use. Such liquid preparations contain commonly used additives such as emulsifying aids such as sorbitol syrup, methyl cellulose, gelatin, hydroxyethyl cellulose, etc., and emulsifying agents such as lecithin, sorbitan monooleate, polyoxyethylene hydrogenated castor oil, non-aqueous Vehicles such as fractionated coconut oil, almond oil, peanut oil, preservatives such as methyl p-hydroxybenzoate, propyl p-hydroxybenzoate or sorbic acid may be added. Furthermore, these preparations for oral administration may contain preservatives, stabilizers, etc., if necessary. In addition, when the active ingredient of the present invention is administered in the form of a parenteral suppository, it can be prepared by a conventional method using a lipophilic base such as cacao butter, witepsol, or a hydrophilic base such as polyethylene glycol.
Alternatively, a mixture of polyethylene glycol, sesame oil, peanut oil, germ oil, fractionated coconut oil, etc. can be used as a rectal capsule wrapped in a gelatin sheet. The rectal capsule may be coated with a wax-like substance if desired. Next, when this compound is used as an injection, it may be in the form of an oil solution, emulsion, or aqueous solution.
These solvents may contain commonly used emulsifiers, stabilizers, etc. These compositions can contain the compound at one time depending on the method of administration.
% or more, preferably 5% to 50%. Next, examples of formulations of the present invention will be shown. Formulation example 1 Hard capsule for oral use Dissolve 25 g of decaprenylamine hydrochloric acid and 7.5 g of polyoxyethylene castor oil in acetone,
Next, 25 g of silicic anhydride is mixed. After evaporating the acetone, add 5 g of calcium carboxymethyl cellulose, 5 g of corn starch, 7.5 g of hydroxypropyl cellulose and microcrystalline cellulose.
Mix 20g, add 30ml of water, knead and granulate. This was granulated using a No. 24 mesh (BS) granulator equipped with a screen (Eck pelleter, manufactured by Fuji Paudal Co., Ltd.). The granules were dried to less than 5% moisture and sieved through a No. 16 mesh (BS) sieve. Next, these particles are packed in a capsule filling machine.
Filled to 190 mg per capsule. Formulation example 2 Soft capsule for oral use Undecaprenylamine hydrochloride 50g and polyethylene glycol (Macrogol-400) 130g
Mix to make a homogeneous solution. Separately gelatin 93
A gelatin solution was prepared with a composition of g, glycerin 19 g, D-sorbitol 10 g, ethyl paraoxobenzoate 0.4 g, propyl paraoxybenzoate 0.2 g and titanium oxide 0.4 g, and this was used as a capsule coating agent by manual plate punching. Contents 180Kg
A soft capsule containing the following was produced. Formulation Example 3 Injection 5 g of decaprenylamine hydrochloride, an appropriate amount of peanut oil and 1 g of benzyl alcohol are mixed, and the total amount is made up to 100 c.c. using peanut oil. Dispense 1 c.c. of this solution into ampoules using aseptic technique and melt and seal. Production example 4 Injection decaprenylamine hydrochloride 1.0g, Nitsukor
Mix 5.0 g of HCO60 [Nikkol HCO60 (trade name)] (hydrogenated castor oil polyoxyethylene-60-mol-ether), 20 g of propylene glycol, 10 g of glycerol, and 5.0 g of ethyl alcohol,
Add 100ml of distilled water to this and stir. Dispense this solution into 1.4 ml ampoules using aseptic technique and melt and seal.

Claims (1)

[Claims] 1. General formula (wherein n is 9, 10 or 11) and an acid addition salt thereof as an active ingredient. Viral agent.