JPS6240351B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing diamines. More particularly, the present invention relates to a method for producing substituted alkanediamines that induce interferon in animals by parenteral administration. The discovery of antiviral compounds is extremely complex and difficult than the discovery of antibacterial and antifungal drugs. One of the reasons for this is the close structural similarity between viruses and certain major cellular components such as ribonucleic acid and deoxyribonucleic acid, and the establishment of suitable test methods for evaluating antiviral agents. This is because it is difficult. However, despite these difficulties, many non-viral substances have been discovered that can stimulate or induce interferon formation in animals. Among these substances, mention may be made, inter alia, of bacteria, parasites, bacterial endotoxins, pyran copolymers, helenins, phytohemagglutinins, polyacrylic compounds, nucleic acids and polynucleotides. However, the use of these inducers is problematic for one or more reasons, such as toxicity, antigenicity, and infectivity, which have not led to their clinical use (Zhdanov
In addition, Internat, l.Virol., 1st Int.Congr.Virol.
Helsinki 1968, S. Karger, New York, pp100−
1, 1969). Recently, a pure synthetic product of relatively low molecular weight, 2,7-bis[2-(diethylamino)
(ethoxy)fluoren-9-one dihydrochloride has been reported to be an oral inducer of interferon in mice (Abstracts Federation
Proceedings, Volume 29, No. 2, Page 635, 1970;
Abstracts 2189 and 2190). In accordance with the present invention, it has been discovered that certain diamines are effective inducers of interferon in vertebrates by the parenteral route of administration. The compounds of this invention, many of which are new, have the following general formulas and their non-toxic acid addition salts. R ₁ and R ₂ are alkyl of 12 to 20 carbon atoms; R ₃ is hydrogen or hydroxyalkyl of 2 to 4 carbon atoms; R ₄ is hydroxyalkyl of 2 to 4 carbon atoms; Yes; n is an integer from 2 to 4. A "non-toxic" acid addition salt refers to a salt that is non-toxic at the dosage administered. Non-toxic acid addition salts of the above bases are hydrochloride, hydrobromide, phosphate, nitrate, sulfate, acetate, hexafluorophosphate, citrate, gluconate, benzoate, propionate, butyrate. , sulfosalicylate, maleate, laurate, malate, fumarate, succinate, oxalate, tartrate, amsonate (4,4'-diaminostilbene-2,2'-disulfonate), pamoate (1,1-methylene-bis- 2-hydroxy-3-naphthoate),
Stearate, 3-hydroxy-2-naphthoate, p-toluenesulfonate, picrate, lactate and suramin salts. The compounds described herein exhibit broad spectrum activity against a variety of viruses in vivo when administered due to their ability to induce endogenous interferon production in animals. Its utility lies primarily in prevention rather than therapeutic control of viral infections. These compounds do not produce interferons in tissue culture, only in vivo and can therefore be considered stimulants of host defense mechanisms. Additionally, these compounds can be used alone and/or in an inert, single-stranded form, such as yeast nucleic acid, highly polymerized yeast ribonucleic acid (Calbiochem 55712; Calbiochem, Los Angeles, California, USA). (single-stranded)
When administered in combination with ribonucleic acids, these compounds stimulate the animal body to produce interferons. These compounds that induce interferon when administered alone are administered in much smaller doses when given in combination with single-stranded ribonucleic acid. Preferred interferon-inducing agents when administered alone are those in which R ₃ and R ₄ are hydrogen or hydroxyalkyl of 2 to 4 carbon atoms. The compounds of the present invention, many of which are well known, are prepared by methods well known to those skilled in the art. For example, U.S. Pat. No. 3,235,501 describes polyoxyalkylation derived from at least several thousand primary and secondary mono- and diamines, theoretically considering all combinations and substitutions of several substituents. These compounds in which aliphatic amines are described are prepared by alkoxylation (ethoxylation or propoxylation) of primary and secondary mono- and diamines. In such cases, alkoxylation occurs indiscriminately, producing a mixture of alkoxylated compounds in which 1 to 25 alkylene oxide moieties may be present. Many of the compounds of the above general formula fall within the theoretical group of compounds covered by the patent. However, despite disclosing polyoxylated aliphatic amines extremely broadly, in fact endlessly, this patent makes no reference to the specific compounds encompassed by the above general formula.
The myriad possibilities of the technical content of the above patents, combined with the vagueness of the disclosed manufacturing methods, are not believed to suggest specific compounds to those skilled in the art. A suitable method for preparing compounds of the above general formula is described by Zook and Wagner in ``Synthetic Organic Chemistry''.
Organic Chemistry, John Wiley & Sons
(New York), 1953, pp. 666-670. Compounds of the formula according to the invention are prepared by the following method: General formula and an amine of formula R ₃ R ₄ NH and, if desired, contacting the product with a suitable acid to form their pharmaceutically acceptable acid addition salts, general formula and methods for producing their nontoxic acid addition salts. However, in each of the above formulas, R ₁ and R ₂ are alkyl of 12 to 20 carbon atoms; R ₃ is hydrogen or hydroxyalkyl of 2 to 4 carbon atoms; R ₄ is alkyl of 2 to 4 carbon atoms; atomic hydroxyalkyl; n is 2 to
It is an integer of 4. Acid addition salts of the compounds described herein can be prepared by conventional methods such as mixing the amine compound and the required acid in a suitable solvent and recovering the salt by vaporization or by adding a non-solvent to the salt and precipitating it. manufactured by the method. The hydrochloride salt is easily prepared by passing dry hydrogen chloride through a solution of the amine compound in an organic solvent such as ether. The antiviral activity of the above compounds was determined in the following manner. In the first method, test compounds are administered to mice by the intraperitoneal route 18-24 hours prior to challenging the mice with a lethal dose of encephalomyocarditis virus, and survival is determined 10 days after challenge. drugs 18-24
This method of administration in advance and at a site clearly different from the site of virus injection aims to avoid local effects of drug and virus and to select only compounds that produce a systemic interferon response. This is what I did. A second general method determines compounds that exhibited antiviral activity in the first method with respect to their ability to produce an antiviral state in mice, as demonstrated by their ability to stimulate circulation of interferon after parenteral administration. be. In both methods, test compounds were obtained alone and from about 2 to about 20 times (by weight) inactive (non-interferon-inducing and non-antiviral), single-component, highly polymerized yeast. Administered in combination with ribonucleic acid (yeast nucleic acid). If the above-mentioned amines are administered parenterally to animals, including humans, before they are exposed to infectious viruses, resistance to the virus is rapidly conferred. The resulting resistance is nonspecific and effective against a large number of viruses. Such administration is effective even when given long in advance, such as 7 days before exposure to the virus. However, it is preferred that administration occur from about 3 days to about 1 day before exposure to the virus, although this will vary somewhat depending on the particular species of animal and the particular infectious virus. When administered parenterally, the substances of the invention have approximately 1
It is used at levels of mg/Kg body weight to about 250 mg/Kg body weight. A good range is from about 5 mg to about 100 mg per kg of body weight, and a more preferred range is about 5 mg to about 100 mg per kg of body weight.
mg to about 50 mg. The dosage will, of course, depend on the animal treated and the particular amine used, and will be determined by the individual's response to the administration. Generally, a small amount is administered initially and the dosage is gradually increased until the optimal amount for the particular subject being treated is determined. Intraperitoneal administration is the preferred method of parenteral injection because it is simple, convenient, and the compound appears to be less toxic. Suitable vehicles for parenteral injection are aqueous, such as water, isotonic saline, isotonic dextrose, Ringer's solution, or vegetable oils (cottonseed, peanut, corn, sesame).
and other non-aqueous vehicles (glycerol, ethanol, propylene glycol, sorbitol) that do not interfere with the effectiveness of the formulation and are non-toxic in the amounts or proportions used. Additionally, compositions suitable for extemporaneous preparation of solutions prior to administration may be advantageously prepared. Such compositions may include diluents such as propylene glycol, diethyl carbonate, glycerol, and sorbitol. When administering the compounds of this invention, they are most easily and economically employed in the form of a dispersion in an acceptable carrier. Dispersing this material means that the molecules are in a molecular state in dimension and are maintained in a true solution in a suitable solvent, or that the particles are in a colloidal state in dimension and in the form of a suspension or emulsion. This means that it is dispersed in a liquid phase. The term "dispersed" also means mixed with a solid carrier and dispersed into the carrier so that the mixture may be in the form of a powder or a spray. For topical application, the inducing agent is most conveniently used in an acceptable carrier to facilitate control of application and to provide good absorption. Again, concentrations in the range of about 1.0 mg/ml to about 250 mg/ml are good. Generally, in the above two administration methods, the dosage ranges from about 1.0 mg/Kg to about 250 mg/Kg of body weight, preferably from about 5.0 mg/Kg to about 50 mg/Kg of body weight. . The compounds used in this invention can be used alone, i.e. without combination with other pharmaceuticals, or as a mixture of two or more of the compounds described herein, or as analgesics, anesthetics, bactericides, decongestants, etc. It can be used in combination with other pharmaceutical agents such as antibiotics, vaccines, buffers and inorganic salts to obtain the desired pharmacological properties. Formulations that allow the formulation of particle sizes of about 20 microns or less for optimal results for substances of the invention that are water-insoluble, including those with low solubility in water and/or those that are sparingly soluble in water. , eg, in suspension, emulsion. The particle size in the formulations clearly influences their biological activity by better absorption of the active substances. Various surfactants and protective colloids are used in formulating these materials. The water-soluble substances described herein give best results when administered as an aqueous solution. The production of interferon upon administration of the compounds described herein has been shown to protect against viral infections in animals, with mice being common early test animals. Encephalomyocarditis virus is a convenient test organism. Challenge virus is prepared by inoculating mice at least 5 passages with a neurotropic strain of encephalomyocarditis virus (infected mouse brain). A 10% suspension of infected brain tissue is prepared from infected mice and stored at −70°C until needed (Takano
et al., J.Bact. 90 , 1542, 1965). This is titrated to death within 5 to 7 days after challenge to unprotected animals. This is injected subcutaneously into the nape of the neck. The appropriate dose is contained in 0.1 ml.
Generally, the dose administered to an animal is LD ₅₀ (animal's 50
10 to 25 times the drug dose that causes 10% death. To measure the antiviral effect, 18
Mice are injected parenterally (intraperitoneally) with the test compound at levels of 5 or 10 mg/Kg and 50 mg/Kg body weight 24 hours prior to the test and the number of survivors determined 10 days after challenge. Interferon production was described by Wheelock in Proc.Soc.Exptl.Biol.Med. 124 , 855−85.
(1967) following injection of the test compound. Once interferon production is observed with a compound, this compound may be administered to test animals at various intervals, such as 6, 36, 48 and 72 hours prior to challenge, and by other parenteral routes, such as intramuscular and subcutaneous administration. to be administered. The production of interferon is demonstrated in the following manner. N・N-diotadecyl-N′・ as an inducer
A typical formulation containing N'-bis(2-hydroxyethyl)-1,3-propanediamine is shown as an example. Heat in a boiling water bath with a mixture of inducer (100 mg) and polysorbate 80 (Tween 80; 0.1 ml). This amine melts and is completely miscible with polysorbate 80. To this mixture is added the following composition, prewarmed to about 55° C., with vigorous swirling. Methocel-15 (manufactured by Dow Chemical Company) 0.50g Tween 80 1.00g CMC-70* 10.00g Sodium chloride 9.00g Distilled water 984.80g * Sodium carboxymethyl cellulose manufactured by Hercules Powder Company of Wilmington, Delaware, USA. Then 7.28 ml of a 0.14 M sodium chloride-0.01 M sodium phosphate solution, pH 7.0, warmed to 55° C. is added with continuous vigorous swirling. The formulation thus produced has a concentration of inducing agent per ml of suspension.
Contains 10mg. N・N-dioctadecyl-N′・N′-bis(2
-Hydroxyethyl)-1,3-propanediamine hydrochloride is 0.14M sodium chloride at pH7.0-
It is easily formulated by vigorously swirling the salt in 0.01M sodium phosphate. Interferon production is determined using female white Swiss mice (Charles River) as test animals. Groups of 5 mice weighing 20-25 g are housed and provided with food and water ad libitum. Test substances are evaluated at 5 mg/Kg and 50 mg/Kg body weight and given as a single intraperitoneal injection (0.5 ml) 18 to 20 hours before exsanguination. Mice are bled from the arm artery under ether anesthesia, blood is collected in heparinized pipettes and test tubes, and pooled plasma from 5 mice is prepared by centrifuging the blood at 2000 ppm. Dilutions of plasma are pipetted into plastic tubes containing sheets of L-929 mouse fibroblast cells (Flow Laboratories, Rockville, MD, USA). The above cells are
L-15 medium containing 10% fetal calf serum and antibiotics (Grand Island, NY, USA)
This is a 24-hour culture at Grand Island Biological. Culture lysates are grown from a 1 ml initial transplant of 100,000 cells/ml. After 24 hours of incubation with plasma, cultures are washed with medium and challenged with 0.2 ml of a dilution of vesicular stomatitis virus titrated to produce complete death of the cell sheet at 24 to 48 hours. . The culture is contacted with the virus dilution in protein-free medium for 1 hour to allow the cells to absorb the virus, and then the tubes are filled with 1 ml of complete medium. After 24 to 48 hours of incubation at 37°C, tubes are evaluated for cytopathic effects of the virus and compared to standard interferon samples. Interferon units are recorded as the reciprocal of the plasma concentration that gives 50% protection against the cell sheet. N・N-dioctadecyl-N′・N′-bis(2
The antiviral effect of -hydroxyethyl)-1,3-propanediamine is determined using female white Swiss mice (Charles River) as test animals. Mice weighing 25-25 g are housed in groups of 5 and given food and water ad libitum. 2 test substances
Seed dose levels (5mg/Kg and 50mg/Kg body weight)
administered as a single 0.5 ml intraperitoneal injection 18 to 20 hours before virus challenge. The next day (18 days after injection)
to 24 hours), challenge subcutaneously with a 0.2 ml injection of encephalomyocarditis virus at a dilution titrated to give a time point of death of 5 to 6 days in unprotected animals. After that, survival data for 10 days was recorded.
The number of survivors during this 10-day period is used as an index of efficacy. The validity of each test is confirmed by considering an unprotected group and a group receiving 100 mg/Kg of pyran copolymer as an experimental control. The water-soluble compounds of the invention are conveniently administered in phosphate buffered salt solutions. Water-insoluble compounds may be administered in formulations of the type described above, or in a variety of other formulations as described above. Dimethyl sulmaxide is a suitable vehicle for water-insoluble compounds. A typical formulation of such a compound consists of 25 to 100 mg of the selected drug, dimethyl sulfoxide (1 ml), polysorbate 80 (1 ml) and 8 ml of the following composition: Methocel-15 0.50 g/Polysorbate 80 g/CMC- 70 10.00g/ Sodium chloride 9.00g/ Methyl p-hydroxybenzoate 1.80g/ Propyl p-hydroxybenzoate 0.20g/ Distilled water 984.00g/ If drug particles are agglomerated, use ultrasonication to homogenize them. system can be obtained. Example 1 N-(2-hydroxyethyl)-N'·N'-dioctadecyl-1,3-propanediamine N·N-dioctadecyl-3-aminopropanol (1.16 g, 2 m
Methanesulfonyl chloride (0.285g, 2.5mM) was added to the stirred solution of M) and the mixture was stirred for 75 minutes. Ethanolamine (1.22g, 20mM) was added and the mixture was refluxed for 45 minutes, then cooled and
Diluted with chloroform (200ml). The chloroform solution was washed sequentially with aqueous sodium hydroxide (5%), water and saturated aqueous sodium chloride.
This was then dried (Na ₂ SO ₄ ) and concentrated to a waxy solid. Picrate: Free base in ethanol (100
ml) and a solution of picric acid (2 g) in ethanol (20 ml) was added. When this solution was cooled, salt precipitated. This was taken, washed with cold ethanol, and dried. 1.2g, melting point 149-151°C. Recrystallization from hot ethanol raised the melting point to 150-152°C. The following compounds were prepared in a similar manner using the appropriate reactant (HNR ₃ R ₄ ) in place of ethanolamine. The hydrochloride salt was prepared by bubbling excess dry hydrogen chloride gas into a solution of the free base in chloroform and recovering the salt by evaporating the solvent.

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Claims (1)

[Claims] 1. General formula and an amine of formula R ₃ R ₄ NH and, if desired, contacting the product with a suitable acid to form their pharmaceutically acceptable acid addition salts, general formula and methods for producing their nontoxic acid addition salts. However, in each of the above formulas, R ₁ and R ₂ are alkyl of 12 to 20 carbon atoms; R ₃ is hydrogen or hydroxyalkyl of 2 to 4 carbon atoms; R ₄ is alkyl of 2 to 4 carbon atoms; atomic hydroxyalkyl; n is 2 to
It is an integer of 4.