JPS628425B2 - - 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 such substances, mention may be made in particular 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
Others Internat′l.Virol.、1st Int.Congr.Virol.
Helsinki 1968, S. Karger, New York, pp 100
-1, 1969). Recently, a relatively low molecular weight purely synthetic material, 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 is alkyl of 12 to 20 carbon atoms; R ₃ and R ₄ are hydrogen or hydroxyalkyl of 2 to 4 carbon atoms; n is an integer of 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 interferon in tissue culture, but occur only in vivo and can therefore be considered stimulants of host defense mechanisms. Furthermore, these compounds when administered alone and/or in highly polymerized yeast ribonucleic acid (Calbiochem 55712; Calbiochem, Los Angeles, Calif., USA).
These compounds, when administered in combination with inactive single-stranded ribonucleic acids, stimulate the animal body to produce interferons. When administered alone, these compounds that induce interferon are administered in much smaller doses when given in combination with single-stranded ribonucleic acid. 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 teaches 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 this patent. However, despite disclosing polyoxylated aliphatic amines extremely broadly, in fact endlessly, that patent makes no reference to specific compounds encompassed by the above general formula.
Combined with the vagueness of the disclosed manufacturing method, the technical content of the above-mentioned patent is unlikely to suggest a specific compound to those skilled in the art due to the countless possibilities. The method of the present invention is as follows. general formula is reacted with an alkylating agent of the general formula R ₁ -halo, where halo is chlorine or bromine, and, if desired, the product is reacted with a suitable acid to form a pharmaceutically acceptable acid. General formula, characterized by the formation of addition salts and methods for producing their nontoxic acid addition salts. However, in each of the above formulas, R ₁ is alkyl of 12 to 20 carbon atoms; R ₃ and R ₄ are each hydroxyalkyl of 2 to 4 carbon atoms; n is an integer of 2 to 4; be. A suitable method for preparing compounds of the above general formula is described by Zook and Wagner in ``Synthetic Organic Chemistry''.
(Synthetic Organic Chemistry, John Wiley &
Sons, Inc., New York, USA) 1953, p. 666670. 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 evaporation or precipitation by adding a non-solvent to the salt. Manufactured in 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-mentioned compounds was determined by the following method. In the first method, the test compound is administered to mice by the intraperitoneal route 18-24 hours before 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 circulating 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 will be rapidly imparted. 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 preferred range is from about 5 mg to about 100 mg per kg of body weight, and a more preferred range is from about 5 mg to about 50 mg per kg of body weight. The dosage will, of course, depend on the animal being treated and the particular amine used;
determined by individual response to its 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 water, aqueous ones such as 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" or mixed with a solid carrier and dispersed into the carrier means 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. Materials of the invention that are water-insoluble, including those with low solubility in water and/or those that are sparingly soluble in water, can be formulated to allow formulation of particle sizes of about 20 microns or less for optimal results. , 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 the amount that causes 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 animals is 10 to 25 times the LD ₅₀ (dose that causes 50% mortality in animals). For the measurement of antiviral activity, 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 can be administered to the test animals at various intervals, e.g., 6, 36, 48, and 72 hours prior to challenge, and by other parenteral routes, e.g., intramuscular and subcutaneous administration. do. The production of interferon is demonstrated in the following manner. N,N-dioctadecyl- as an inducer
N',N'-bis(2-hydroxyethyl)-1,3
- A typical formulation containing propanediamine is shown as an example. A mixture of inducer (100 mg) and polysorbate 80 (Tween 80; 0.1 ml) is heated in a boiling water bath. This amine melts and is completely miscible with polysorbate 80. Add to this mixture the following composition, pre-warmed to about 55°C, while swirling vigorously. 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 pH 7.0 -
It is formulated by vigorous swirling of this salt in 0.01M sodium phosphate. Interferon production is determined using female white Swiss mice (Charles River) as test animals. They are housed in groups of 5 animals weighing 20-25 g and given food and water ad libitum. The test substance was evaluated at 5 mg/Kg and 50 mg/Kg body weight, a single intraperitoneal injection (0.5 ml) 18 to 20 hours before exsanguination.
give. Mice are bled from the arm artery under ether anesthesia, blood is collected into heparinized pipettes and test tubes, and pooled plasma from 5 mice is prepared by centrifuging the blood at 2.000 ppm. Dilutions of plasma were collected on sheets of L-929 mouse fibroblasts (Flow, Rockville, MD, USA).
Pipette into a plastic tube containing 100ml (manufactured by Laboratories). The above cells were grown in L-15 medium containing 10% fetal calf serum and antibiotics (Grand Island, NY, USA).
This is a 24-hour culture in Island Biological. Cultures are grown from 1 ml initial transplants of 100,000 cells/ml. After 24 hours of incubation with plasma, the cultures were washed with medium and a dilution of vesicular stomatitis virus titrated to result in complete death of the cell sheet between 24 and 48 hours.
Attack with 0.2ml. 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 incubation for 24 to 48 hours 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 20-25 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 the unprotected group and the group given 100 mg/Kg of pyran copolymer as experimental control. The water-soluble compounds of the invention are conveniently administered in phosphate buffered salt solution. Water-insoluble hydrates 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 1.00 g/CMC -70 10.00g/ Sodium chloride 9.00g/ Methyl p-hydroxybenzoate 1.80g/ Propyl p-hydroxybenzoate
0.20g/distilled water 984.00g/If agglomeration of drug particles occurs, ultrasonication can be used to obtain a homogeneous system. Example 1 N,N-dioctadecyl-N',N'-bis(2
-hydroxyethyl)-1,3-propanediamine octadecyl bromide (666 g, 2.0 mol),
N-(3-aminopropyl)diethanolamine (162 g, 1.0 mol) and potassium carbonate (276 g,
2.0 mol) mixture was stirred vigorously and gradually heated to 120°C.
and maintained at this temperature for 30 minutes. The mixture was allowed to cool to 70°C and then 500 ml of a 1:1 methylene chloride-water mixture of methylene chloride (9.75)-water (9.75) was added. The methylene chloride phase was separated after 15 minutes and the remaining aqueous phase was extracted with methylene chloride (4). The combined methylene chloride extracts were dried over anhydrous magnesium sulfate and then stripped to half volume under reduced pressure. This concentrate was then stirred with silicic acid (300 g) for 30 minutes to remove the silicic acid and
The clear solution was slowly poured into acetone (16) containing succinic acid (300 g). The mixture was gradually cooled to 10°C and the succinate was removed. 615
g (68% of theory); melting point 78-90°C. This was mixed with acetone-methylene chloride (2-
It was purified by recrystallization from 1). The free base was obtained by dissolving the succinate (420 g) in methylene chloride (4) and aqueous sodium hydroxide (2.5 of a 5% solution). The mixture was stirred for 15 minutes and the methylene chloride phase was separated and washed sequentially with aqueous sodium hydroxide (1 x 18 of a 5% solution), water (3 x 6) and saturated aqueous sodium chloride (1 x 6). This was then dried (MgSO ₄ ), filtered and evaporated to an oil in vacuo. This oil was dissolved in acetone (5) at 50°C and the solution was allowed to cool gradually to obtain a white precipitate (267g) with a melting point of 39-41°C. Additional product (20 g) was obtained by cooling the liquid to 0°C. The total yield was 667 g, 43% of theory. The dilactate salt was prepared by adding 2 equivalents of lactic acid in ether to an ethereal solution of the base and then evaporating the ether. It became viscous at a melting point of 50-52°C and melted at 60-62°C. The diphosphate was made by adding excess phosphoric acid to a solution of the base in hexane. This was recrystallized from a large amount of methanol. The melting point becomes a gel at 140℃, 175
It turned brown at -190°C and melted at 245-247°C. The dihydrochloride salt was prepared by bubbling dry hydrogen chloride gas into an ethereal solution of the base. The residue obtained after removing the ether was slurried in acetone, filtered, and recrystallized from ether containing some methanol. Melting point becomes gel at 180-182℃, 238-240
completely melted at °C. Example 2 N,N-dioctadecyl-N',N'-bis(2
-Hydroxyethyl)ethanediamine A mixture of octadecyl bromide (6.66 g, 0.02 mol), N-(2-aminoethyl)diethanolamine (1.48 g, 0.01 mol) and potassium carbonate (2.26 g, 0.02 mol) is refluxed under nitrogen atmosphere. The mixture was heated at the point for 2 hours. The reaction mixture was then cooled and diluted with aqueous sodium hydroxide (10% solution of
50ml). Ethyl acetate (50ml) was added, the mixture was thoroughly stirred, and the ethyl acetate was separated, washed with water and dried over anhydrous magnesium sulfate. Evaporation of the solvent gave the crude product, which was recrystallized from ethyl acetate or acetone. Melting point 33-34℃. Hydrochloride, phosphate, succinate and picrate salts were prepared by adding the above bases to ethyl acetate containing the stoichiometry of the respective acid. The salts were collected by filtration, washed with cold ethyl acetate and dried. Salt melting point (°C) Dihydrochloride 228-30 Diphosphate 102- 3 Disuccinate 155- 6 Dipicrate 84- 6 The above reaction can be carried out using a suitable (2-hydroxyalkyl) alkanediamine derivative and a suitable alkyl bromide. The method was repeated to produce the following compounds.

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Claims (1)

[Claims] 1. General formula is reacted with an alkylating agent of the general formula R ₁ -halo, where halo is chlorine or bromine, and, if desired, the product is reacted with a suitable acid to form a pharmaceutically acceptable acid. General formula, characterized by the formation of addition salts and methods for producing their nontoxic acid addition salts. However, in each of the above formulas, R ₁ is alkyl of 12 to 20 carbon atoms; R ₃ and R ₄ are each hydroxyalkyl of 2 to 4 carbon atoms; n is an integer of 2 to 4; be.