JPH0246574B2 - - 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 that viruses and certain major cellular components such as ribonucleic acid and deoxyribonucleic acid are structurally very similar, 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.
These have not yet been used clinically (Zhdanov et al. Internat'l. Virol. I, 1st Int. Congr.
Virol. Helsinki 1968, S. Karger, New York,
pp100-1, 1969). Only recently has a relatively low molecular weight, purely synthetic substance, 2,7-bis[2-(diethylamino)
[Ethoxy]fluorene-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; X ¹ is (—CH ₂ )— _o or

[Formula]; 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. Furthermore, these compounds when administered alone and/or in highly polymerized yeast ribonucleic acid, which is an enzymatic nucleic 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. 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 teaches that polyoxyalkyls derived from at least several thousand primary and secondary mono- and diamines can theoretically be used if all combinations and substitutions of several substituents are considered. 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, this patent makes no reference to the specific compounds encompassed by the above general formula.
Combined with the vagueness of the disclosed manufacturing method, the myriad possibilities of the above-mentioned technical content are not believed to suggest obvious compounds to those skilled in the art. The basic reaction is the alkylation of primary or secondary amines with, for example, alkyl halides or hydroxyalkyl halides, usually chlorides or bromides, in an organic solvent in the presence of a base or acid acceptor. Other alkalization methods can of course be used, such as the use of aluminum alkoxides, sulfuric acid and esters of p-toluenesulfonic acid. A suitable method for preparing compounds of the above general formula is described by Zook and Wagner in ``Synthetic Organic Chemistry,'' by John
Wiley & Sons, New York, USA) 1953, p.
666-670. The compound of the present invention () is produced by the following method: General formula or and, if desired, contacting the product with a suitable acid to form a pharmaceutically acceptable acid addition salt thereof. 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; X ¹ is (—CH ₂ )— _{o or}

[Formula]; n is an integer from 2 to 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 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, 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 exposure to infectious viruses, resistance to the virus will be 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, the administration is approximately 3 minutes before contact with the virus.
Preferably, this is done one day to about one day in advance, but this will vary slightly depending on the particular animal species 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, and will be determined by the individual's 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" 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. 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. Infected mouse brains prepared by inoculating mice at least 5 passages with a neurotropic strain of encephalomyocarditis virus as a challenge virus). 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 an animal is LD ₅₀ (50% of the animal
10 to 25 times the drug dose that causes death. 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 performed 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. 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. 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 carboxymethylcellulose 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 with a pH of 7.0 warmed to 55° C. is added with continuous vigorous swirling. The formulation thus produced contains 10 mg of inducing agent per ml of suspension. 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 centrifugation at 2000 ppm. Plasma dilutions were collected on sheets of L-929 mouse fibroblasts (Flow, Rockville, MD, USA).
Pivet into a plastic tube containing 100% Polymer Laboratories). The above cells were grown in L-15 medium containing 10% fetal calf serum and antibiotics (Grand Island, New York, USA).
This is a 24-hour culture in Island Biological (manufactured by 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. The antiviral effect is determined using female white Swiss mice (Charles River variety) as test animals. Groups of 5 mice weighing 20-25g were raised,
Provide food and water ad libitum. The test substance was evaluated at two dose levels (5 mg/Kg and 50 mg/Kg body weight), and a single dose was administered 18 to 20 hours before virus challenge.
Administer by 0.5 ml intraperitoneal injection. The next day (18-24 hours post-injection), challenge subcutaneously with a 0.2 ml injection of encephalomyocarditis virus at a titration to give a 5- to 6-day time point of death in unprotected animals. Survival data is then recorded for 10 days and 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 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 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-1,3-propanediamine In a 2-gallon autoclave, 3-(dioctadecylamino)propionitrile (100 g), ethanol (3750 ml) containing anhydrous ammonia (100 g) and Raney Nickel (on a dry basis)
20g) and purged with nitrogen and then with hydrogen. This is then sealed and hydrogen pressure is applied.
Increased to 250psi. Stir the autoclave;
The temperature was increased to 70°C and the mixture was maintained at this temperature for 1.5 hours, at which point hydrogen uptake ceased. The autoclave was cooled to 20°C, evacuated, and the contents removed. The catalyst was filtered off, washed with ethanol, and the combined washings and reaction mixture were concentrated in vacuo to a viscous green-yellow oil (82 g) that solidified on standing. Melting point 39-41
℃. Analytical value: Calculated value for C ₃₉ H ₈₂ N ₂ : C, 80.97; H, 14.17; N, 4.44% Actual value: C, 80.60; H, 14.17; N, 4.79% Octadecylamino)propionitrile was prepared by refluxing a mixture of dioctadecylamine (200 g) and acrylonitrile (1930.8 ml) for 18 hours.
The mixture was then concentrated to a waxy semi-solid, which was slurried in acetone;
It was air-dried overnight. Example 2 1-(Dioctadecyl)aminomethyl-4-aminomethylbenzene 1-(dioctadecylamino)methyl-4-cyanobenzene (11.9g, 18.7mM, see Example 8), dihydrobis-(2-methoxyethoxy) Sodium aluminate (11.0g of 70% reagent, 37.5mM)
and benzene (300ml) was refluxed under 1 atmosphere of nitrogen for 40 hours. This was cooled and sodium hydroxide solution (50ml of a 10% solution) was subsequently added.
The benzene phase was separated, washed with water (2 x 50ml) and dried (Na ₂ SO ₄ ). Concentration of the benzene solution gave the product as an oil that solidified on standing (8.0 g). The product was purified by chromatography on a silica gel column eluting with ethyl acetate. This product (500mg) was mixed with chloroform (20ml)
Addition of ethyl acetate dissolved in water and saturated with HCl (10ml) gave the dihydrochloride salt. The salt was filtered, washed with ether and air dried: 430 mg,
Melting point 208-210℃. The following compounds were similarly prepared from appropriate reactants:

[Table] * Shows the position of the aminomethyl group.

Claims (1)

[Claims] 1. General formula or and, if desired, contacting the product with a suitable acid to form a pharmaceutically acceptable acid addition salt thereof. and methods for producing their nontoxic acid addition salts. However, in each of the above formulas, R ₁ and R ₂ are alkyl having 12 to 20 carbon atoms; X ¹ is -(CH ₂ -) _o or [Formula]; n is an integer from 2 to 4; be.