DK149331B - METHOD OF ANALOGUE FOR PREPARING PIPERIDE INGREDIATES AFDI-0- (N-ALKYL OR N-ALKENYL) -PROPANDIOL OR PHARMACEUTICAL ACCEPTABLE ACID ADDITION SALTS THEREOF - Google Patents

Description

1A 9 3 31

The invention relates to an analogous process for the preparation of novel piperidine derivatives of di-O- (n-alkyl- or n-alkenyl) -propanediol of the formula ✓- / CH2NH2

h ^ OSL

Or pharmaceutically acceptable acid addition salts thereof, wherein R 1 and R are each selected from the group consisting of normal alkyl groups having from 12-20 carbon atoms and normal alkylene groups where the double bond is not in the 1-position, with from 12-20 carbon atoms, which method is peculiar to the characterizing part of claim 1. The compounds of this invention are useful for controlling viral infections in mammals.

Viral infections that attack mammals, including humans, are usually infectious diseases that can cause great human suffering and financial loss. Unfortunately, the discovery of antiviral compounds is much more complicated and cumbersome than the discovery of antibacterial and antifungal agents. This is partly due to the close structural similarity between viruses and the structure of certain essential cellular compounds such as ribonucleic acid and deoxyribo nucleic acid. However, several non-viral "antiviral compounds", i.e. compounds "which can produce either a protective or therapeutic effect for a clear detectable benefit to the virus-infected host, or a material that can significantly enhance antibody formation, improve antibody activity, improve non-specific resistance, effect faster healing, or inhibit symptoms", [Herrman et al., Proc. Soc. Exp. Biol. Med., 103, 625, (I960)]. The list of antiviral agents listed includes, to name a few, interferon and synthetic materials such as amantadine hydrochloride, pyrimidines, biguanides, guanidine, pteridines and methisazone. Due to the rather narrow range of viral infections that can be treated with any of the commercially available antivirals at present, new synthetic antivirals are always welcomed as potentially valuable enhancements to medical technology equipment.

In response to viral infections, mammalian cells produce a substance that allows cells to withstand the proliferation of a number of viruses. The antiviral or virus interfering substances are called "interferons". The interferons are glycoproteins that can differ in their physicochemical properties but all have the same biological properties, namely inhibition of a large number of unrelated viruses, have no toxic or other deleterious effect on the cells and are species specific (Lockart , Frontiers of Biology, Vol. 2, "Interferons," published by Finter, WB Saunders Co., Philadelphia, 1966, pages 19-20).

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No practical economic approach has yet been developed for the preparation of exogenous clinical interferons for viral infections. An alternative to the preparation of interferons has therefore been attempted, which includes administering to the animal to be protected or treated a non-viral substance that stimulates or induces the production of interferon in the cells. Interferon produced in this way is termed "endogenous" interferon.

U.S. Patent No. 2,738,351 discloses that compounds of the general formula R 2 X-CH 2

CH-Z-ALK-B

2) R -Y-CH 2 1 2 where R and R may be alkyl, aralkyl, aryl, cycloalkyl, nitrosubstituted aryl, halogen-substituted aryl, alkyl-substituted aryl or alkoxy-substituted aryl, X, Y and Z may be oxygen, sulfur or sulfonyl, ALK means a straight or branched alkylene group having from 1-6 carbon atoms and B can be di (lower) alkylamino, piperidino, morpholino, pyrrolidino, (lower alkyl) pyrrolidino, N'-alkylpiperazino or pipecolino , are locally anesthetic agents. Further described in the discussion of alternative synthetic pathways (see Columns 1, 11. 57-70 of the aforementioned patent) are intermediates of the aforementioned formula wherein B is amino and (lower alkyl) amino groups. However, none of the compounds specifically mentioned in the aforementioned patent contains an R or R alkyl group which is larger than n-pentyl. Furthermore, there are no compounds where both R and R are alkyl groups and both X and Y are oxygen atoms.

Insecticidal and miticidal compounds of formula R1-CH-I 2 CH- (CH-) -A 2 I 2 5.

R -CH2 2 wherein R and R are each e.g. may be lower alkylthio groups, q means 0-5, and A may be, inter alia, a 1-piperidino or di (lower alkylamino group is described in Japanese Patent Specification No. J7-6042-177.

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It has now been found that the novel compounds of formula I and the pharmaceutically acceptable acid addition salts thereof prepared by the analogous process of the invention exhibit anti-viral activity against a large amount of viruses in mammals and in vitro in mammalian cell cultures. At least a substantial amount of this effect results from the ability of said compounds to induce interferon production in the cells, i. endogenous interferon.

By "pharmaceutically acceptable" acid addition salts is meant those salts which are "non-toxic" at the doses administered. The pharmaceutically acceptable acid addition salts which may be used include such water-soluble and water-insoluble salts such as hydrochlorides, hydrobromides, phosphates, nitrates, sulfates, acetates, hexafluorophosphates, citrates, gluconates, benzoates, propionates, butyrates, sulfosalicylates, sulfosalicylates , laurates, malates, fumarates, succinates, ocalates, tartrates, amsonates (4,4'-diamino-stilben-2,2'-disulfonates), pamoates (1,1'-methylene-bis-2-hydroxy-3-naphtho). ater), stearates, 3-hydroxy-2-naphthoates, p-toluene sulfonates, methanesulfonates, lactates and suramin salts.

A preferred group of the compounds prepared by the process of the invention consists of the hydrochloride salts of the bases of formula I.

Another preferred group of the compounds of formula I consists of those compounds wherein R and R are each normal alkyl groups having from 14 to 18 carbon atoms.

Another preferred group of the compounds of formula I2 I consists of those compounds wherein R and R are each normal alkyl groups having from 14-18 carbon atoms and containing the same number of carbon atoms.

Another preferred group of compounds of formula I

1 2 consists of those compounds wherein R and R are each n-hexadecyl groups.

Particularly valuable are the following compounds and their pharmaceutically acceptable acid addition salts.

1- [2,3-di (n-octadecyloxy) propyl] -4-aminomethyl-4-phenyl-piperidine, 1- [2,3-di (n-hexadecyloxy) propyl] -4-aminomethyl-4-phenyl] piperidine and 1- [2,3-di (n-tetradecyloxy) propyl] -4-aminomethyl-4-phenyl-piperidine.

Acid addition salts of the bases of formula I can be prepared by conventional methods such as mixing the amine compound in a suitable solvent with the desired acid and recovering the salt by evaporation or precipitation after the addition of a solvent in which the salt is insoluble. Hydrochloride salts can be readily prepared by passing hydrogen chloride through a solution of the amine compound in an organic solvent. As can be seen from the following examples, many of the isolated hydrochloride or dihydrochloride salts of the bases of formula I tend to contain a significant amount of water.

It is not known whether this observed "trapped" water is arbitrarily contained during crystallization or corresponds to the formation of real molecular hydrates or arises from the presence of another phenomenon. In each case, the salts containing "trapped" water can be readily prepared and administered without prior dehydration.

The starting materials 1,2-di-O- (n-higher alkyl) glycerols can be prepared by the method of Kates, M. et al., Biochemistry, 2, 394 (1963). The starting materials 1,2-di-O- (n- higher alkenyl) glycerols can be prepared by Bauman, W.J. and Mangold, Η. K., J. Org. Chem., 31, 498 (1966).

The anti-viral effect of the compounds of the invention was determined using two independent methods. In the first, the test compound was administered to mice by intraperitoneal route 18 to 24 hours before administration of a lethal dose of encephalomyocarditis (EMC) virus. Survival data are listed 10 days after virus administration and compared with data from unprotected animals. The method by which the drug is administered 18 to 24 hours prior to and at a substantially separate site from the virus injection is intended to eliminate local drug-virus effects and identify only those compounds that provide a systemic anti-viral response.

In the second method, monolayers of human nasal polyp cells grown on microtiter plates are treated with the test compound for approx. 18 hours before treatment with a lethal dose of vesicular stoma-titis virus (VSV). The test compound is washed away from the monolayer prior to virus treatment. Culture fluid is extracted from the plates after an incubation period and titrated for the amount of infectious virus present in microtiter plates of L-929 mouse fibroblasts. Comparison is made with viral data from culture fluid extracted from unprotected polycells.

Furthermore, many of the compounds prepared according to the invention were tested for their ability to enhance the known antiviral action of polyinosine: polycytidylic acid. Finally, some of the compounds were tested for their ability to induce circulating interferon in mice after parenteral administration using that of Hoffman, WW, et al., Antimicrobial Agents and Chemotherapy, 3, 498-501 (1973). ) described procedure.

Parenteral, topical or intranasal administration of the present amines to mammals prior to exposure of the mammal to an infectious virus provides rapid resistance to the virus. Preferably, administration should take place from ca. 2 to approx. 1 day before exposure to virus, but this will vary somewhat with the particular animal species and with the special infectious virus.

When administering the compounds of the present invention, they are most readily and economically used in dispersed form in an acceptable carrier.

When said material is dispersed, it means that the particles may be of molecular size and kept in true solution in a suitable solvent, or that the particles may be of colloidal size and dispersed in a liquid phase in the form of a suspension or a emulsion. The term "dispersed" also means that the particles may be mixed with and dispersed in a solid support such that the mixture is in the form of a powder or dust. This term also includes mixtures suitable for use as spraying agents, comprising solutions, suspensions or emulsions of the compounds of the invention.

Administered parenterally (subcutaneously, intramuscularly, intraperitoneally), the subject compounds are used in an amount of from ca. 1 mg / kg body weight to approx. 200 mg / kg body weight. The preferred range is from approx. 5 mg / kg to approx. 100 mg / kg body weight, and the most preferred range from approx. 5 mg to approx. 50 mg / kg body weight.

The dose, of course, depends on the mammal to be treated and the particular amine compound used and is determined by the person responsible for the administration. Usually, small doses are administered initially with gradual increase of dose until the optimal dose level is determined for the particular subject under treatment.

The invention is further explained by the following examples.

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Example 1 1- [2,3-Di (n-hexadecyloxy) propyl] -4-aminomethyl-4-phenylpiperidine dihydrochloride.

A. 1- [2,3-Di (n-hexadecyloxy) propyl] -4-cyano-4-phenylpiperidine.

A mixture of 1,2-di-O- (n-hexadecyl) -3-O- (p-tosyl) glycerol (6.96 g, 10 mmol), 4-cyano-4-phenylpiperidine hydrochloride (2, 23 g, 10 mmol), triethylamine (2 ml) and Ν, Ν-dimethylformamide (40 ml) were stirred for 16 h to 95-100 ° C. The reaction mixture was then cooled, diluted with water (200 ml) and extracted with ethyl acetate (3 x 150 ml). The combined ethyl acetate extract was dried over MgSO 4, filtered and evaporated in vacuo to give an oil (6 g) which was obtained. purified by column chromatography (elution with benzene: ethyl acetate) (oil, ir (CHCl 3) 2220 cm -1).

B. The title compound.

A solution of 1- [2,3-di- (n-hexadecyloxy) propyl] -4-cyano-4-phenylpiperidine (2.5 g, 3.6 mmol) in ether (100 mL) was treated with lithium aluminum hydride (0 (4 g, 10.5 mmol) and the resulting mixture was stirred for 4 hours at room temperature. The reaction mixture was treated gently with water (100 ml) and extracted with ether (3 x 100 ml). The combined ether extract was dried over MgSO 4, filtered and evaporated in vacuo to an oil which was purified by chromatography on silica gel (eluting with benzene: ethanol) and then dissolved. The solution was treated with hydrogen chloride gas and then evaporated in vacuo to give a solid which was recrystallized from ethyl acetate (1.1 g of solid containing ca.

3/4 mol H2O per mole product, 40% yield, m.p. 132-134 ° C, elemental analysis calculated: 70.60% C, 11.53% H, 3.50% N, found: 70.74% C, 11.34% H, 3.40% N).

Example 2-4 In a similar manner as described in Example 1, the following compounds were prepared from suitable 1,2-di-O- (n-alkyl or alkenyl) -3-O- (p-tosyl) glycerols.

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Example 5

In vivo activity against EMC virus.

Preparation of an emulsion was done by melting and mixing equal parts of the indicated compound, polysorbate 80 and glycerine and subsequently dispersing the mixture in hot water with vigorous stirring. The preparation was then adjusted to a final concentration of 0.14M sodium chloride and 0.01M sodium phosphate, pH 7. Further dilutions were made with 0.14M sodium chloride, 0.01M sodium phosphate, pH 7, buffer solution.

3 Groups of 10 female albino mice (20-25 g body weight) were given 0.5 ml of intraperitoneal injections containing amounts of 1.5, 5, and 15 mg of the indicated compound / kg body weight. A fourth control group of 10 mice received no injection. 18-24 hours later, all 4 groups received 0.2 ml of subcutaneous injection containing 20 times the LD 1g dose, which causes 50% mortality in unprotected mice for 10 days, of encephalomyocarditis (EMC) virus. Survival data were collected over the next 10 days and relative survival (Sr) calculated.

Antiviral efficacy is expressed as the relative survival (Sr) in experimental groups compared to the control 10 days after infection. Sr is defined by the formula ζ "+> ---- x-i -> e ±

Sr = X i = l to 10 i = l. tili10 X 100 100 + .100 -5 ef - i = l to 10 where Sr = the relative survival

Sx = percent survival after 10 days in the experimental group = the number of survivors per day in the experimental group e ^ = the number of survivors per day in the control group.

149331 ro

Compound prepared - Sr at dose (mg / kg) readily in Example No. 15 5 1.5 1 77 58 26 2 76 85 42 3 52 42 32 4 79 72 28

Example 6

Reduction of virus yield on human polyp cells in vitro.

A culture medium was prepared by adding to Eagle's minimal essential medium (100 ml) 100X concentrated antibiotic-antimycotic solution (2 ml) 200 mM glutamine solution (1 ml) 100X concentrated solution of non-essential amino acids (1 ml) 100 mM sodium pyruvate solution (1 ml) and heat-inactivated fetal calf serum (10%). Each well in 96 well microtiter plates was seeded with ca. 50,000 human nasal polyp cells suspended in 0.2 ml of growth medium. The plates were then incubated for 8-10 days at 37 ° C in a 5% CO 2 atmosphere to form monolayers of the cells.

After the cell growth period of 8-10 days, continuous monolayers on the plates were washed several times with phosphate-buffered saline and immediately treated with 0.2 ml per well of maintenance medium containing 10, 5.0, 1.0, 0.5, 0.1 and 0 µg / ml of the indicated compound. The maintenance medium was identical to the growth medium described above with the exception that the amount of fetal calf serum is 5%. The plates were incubated for an additional 18 hours at 37 ° C, and the monolayers were then washed several times with phosphate-buffered saline to remove the indicated compound, infected with a material containing ca. 1000 times TCID ^ g, i.e. the dose causing 50% infection in 1 unprotected cultures of vesicular stomatitis virus (VSV) for a 2 hour adsorption period at 37 ° C, washed 4 times with phosphate buffered saline to remove non-adsorbed virus particles and provided with 0 , 2 ml per well of the maintenance medium. The plates were then incubated for 7 hours at 37 ° C and the culture fluid of 5-8 reproduced cells taken from each plate, stored frozen in test tubes and then filtered for the amount of infectious virus present in microtiter plates of L-929 mouse fibroblasts. The L-929 mu cultures were counted microscopically and analyzed for ca. 3-4 days later with the following decrease in virus yield (with respect to control) determined for the 5 concentrations of the test compounds indicated:

Compound Preparation% Reduction of Virus Yield Easily in Example No. _ Concentration µ / ml_ 10 5.0 1.0 0.5 0.1

1 + ± - - ND

2 + + - - ND

3 +++ - ND

4 + - - - ND

(a) + a> 68% Decrease, ± = ^ v68% Decrease - =. <68% decrease, ND = not determined

Example 7

Effect of 1- [2,3-di (n-hexadecyloxy) propyl] -4-aminomethyl-4-phenyl-piperidine dihydrochloride to induce circulating interferon.

A mixture of equal weight amounts of the indicated compound, polysorbate 80 and glycerol was pooled and then homogenized in hot 0.14M sodium chloride containing Ο, ΌΙΜ sodium phosphate, pH 7, (PBS). The resulting oil-in-water emulsion was readily diluted with PBS for administration.

Female mice (body weight 20 to 25 g) were injected intraperitoneally (0.5 ml) with an amount of the above-mentioned diluted emulsion containing 25 mg of the indicated compound per kg body weight. At 8, 12, 16 and 20 hours after injection, plasma samples were taken from 4 mice and collected. Serial dilutions in L-15 (Leibovitz) medium containing 5% fetal calf serum were incubated in microtiter plates overnight at 37 ° C on confluent monolayers of L-929 mouse fibroblasts. The monolayers were then washed with protein-free medium, infected with 10-fold TCID the dose causing 50% infection in unprotected cultures of vesicular stomatitis virus (VSV) for an adsorption period of 1 hour at 37 ° C, washed, again treated with L-15 medium containing 5% fetal calf serum and then incubated again for 48 hours at 37 ° C. The L-929 cultures were then counted microscopically for viral cytopathology and analyzed, and with the plasma interferon level the reciprocal of the plasma dilution causing 50% protection of the L-929 monolayers was determined.

Yet another test following the procedure mentioned above except that the mice were given 10 mg of the indicated compound per ml. body weight, and peritoneal wash samples were taken from 4 mice and collected 6, 9, 12, 15, and 18 hours after injection. The samples were taken by exposing the peritoneal membrane, injecting 1 ml of Hank's balanced saline solution containing 100 penicillin units / ml and 100 μg streptomycin / ml into the peritoneal cavity, brief abdominal massaging and subsequent aspiration of the peritoneal wash.

The following data were obtained from these two experiments

Interferon- Interferon amounts (unit / ml) source Time (hours) after injection_ 6 8 9 12 15 16 18

Plasma <20 70 70 100 120 - 100

Peritoneal wash <13 - 109 284 312 - 224

Example 8

Enhancement of cellular resistance to viral infections with polyinosine-polycytidylic acid tpoly- (I: C)].

Growth medium was prepared by adding to the Eagle's minimum essential medium (100 ml) 100X concentrated antibiotic-anti-mycotic solution (2 ml), 200 mM glutamine solution (1 ml) and heat inactivating a fetal calf serum (5%). Mouse L-929 fibroblasts were suspended in growth medium and each well in 96 well microtiter plates were seeded with 0.2 ml of the suspension containing 20,000 to 30,000 cells. The plates were incubated for 2-4 days at 37 ° C in a 5% CO 2 atmosphere to form monolayers of the cells. The plates were washed 4 times with phosphate-buffered saline immediately prior to treatment.

Poly (I: C) was prepared at concentrations of 5.0, 1.0, 0.2 and 0.04 µg / ml in the medium described above minus calf serum. 0.1 ml of each dilution was collected in a pond-breaking arrangement on the L-929 cell monolayers with 0.1 ml dilutions containing 20.0, 4.0, 0.8, 0.16 and 0.032 µg of the specified connection per. ml of serum-free medium. Control wells were given either poly (I: C) or the compound alone. The plates were incubated for 6 hours at 37 ° C in a 5% CO 2 atmosphere, washed 4 times with phosphate-buffered saline and provided with 0.1 ml per ml. deepening of growth medium containing 2% fetal calf serum. After an additional 18 hours of incubation, the plates were evaluated for toxicity and then infected with 0.1 ml / ml. deepening of vesicular stomatitis virus (VSV) suspension containing 10-30 g of TCID® q (tissue culture infected with dose causing 50% infection). The plates were incubated for an additional 3-4 days and then assessed microscopically for cytopathogenic effect (CPE). Cells protected from viral infection were free of CPE. The minimum protective dose (MPD) of poly (I: C) alone was noted and the amount of enhanced or enhanced anti-viral action caused by combination with the indicated compound listed for each dilution level of the compound.

Compound Preparation - Poly (I: C) Increase 3 Slightly in Example No. Concentration (µg / ml) 4.0 0.8 1 + + + 2 + + 3 + + ± 4 + - - (a) + => 5X increase, 1 = ^ 5X increase - = <5X increase, ND = not determined.