CS208156B2 - Method of making the new amino-derivatives of the propandioles - Google Patents

Description

(54) A process for producing novel amine derivatives of propanediols

The present invention relates to a process for the preparation of novel amine derivatives of propanedioles having antiviral activity.

Viral infections that attack mammals, including humans, are contagious and can cause major health problems and economic losses. Unfortunately, the discovery of antiviral compounds is far more complex and difficult than is the case with bacterial and fungal active compounds. Probably, the structural kinship of viruses and some essential cell components, such as ribonucleic acid and delsoxyribonucleic acid, is close to the barrier to research. However, many anti-viral agents of non-viral origin have been discovered, i.e., compounds that can prevent or treat viral infections, or materials that significantly increase antibody production, improve its efficacy, increase non-specific resistance, accelerate recovery or reduce symptoms. [Heirrman et al., Prcc. Soc. Exptl. -Biol. Med., 103, 625 (1960)]. Antiviral agents include, for example, interferon and synthetic materials such as aimantadhydrohydrohalide, pyrimidines, trguanides, guapidine, piperidines and methisazone. Since only a few viral infections can be treated with each of these agents, new synthetic anti-viral agents must be found to better prevent and treat viral infections.

Mammalian cells produce a substance in a viral infection that allows the cell to inhibit the reproduction of the virus. These compounds are called interferons. Interferons are glycoproteins, which may differ in their physiological and microtic properties but possess the same biological properties by inhibiting a wide variety of unrelated viruses. These compounds do not have toxic or other adverse effects on the cells and are specific within one species. (Lockairt, Frontiers of Biology, Vol. 2, "Interferons", edited by Finiteir, W. B. Saun.de, rs Co., Philadelphia, 19'Q6, pp. 19-20).

To date, no economical and practicable method has been proposed for the production of exogenous interfarons for common chemical applications. Efforts have therefore been made in a different direction to be able to administer to animals or humans to be protected against infection a non-viral substance that promotes or induces the production of interferon in cells. The interferome obtained by this method is called endogenous interferoin.

U.S. Patent No. 2,733,351 discloses compounds of the general formula

R1-X-CH2

CH — Z — ALK — B,

R 2 - Y - CH 2 where denote

R 1 and R 2 alkyl, aralkyl, aryl, cylcloalkyl, aryl substituted with nitro or halogen, optionally alkyl or alkoxy,

X, Y and Z are oxygen, sulfur or eulfoinyl,

Straight or branched chain alkyl of 1 to 6 carbon atoms; and

B bi (lower jalkylamino, piperiidine, morpholine, pyprolidine, (lower) alkylpyrrolidine, N'-alkylpiperazine or pipecoline) are local anesthetics. on page 11, lines 57-70 of the patent disclose intermediates of the above formula wherein B is amino or lower alkylamino, but none of the compounds has a higher alkyl group than n-pemtyl in the meaning of R 1 or R 2. R 1 and R 2 are both alkyl and X and Y are both oxygen.

Japanese Patent No. J7-6042-177 discloses insecticidal and miticidal compounds of the general formula:

R1-CH2

CH— (CH2) _q —A,

Rz — CH2 where they are

R 1 and R 2 include, but are not limited to, lower alkylthio, q 0 to 5 a

And, inter alia, 1-piperidine or di, (lower alkyl amino).

It has now been found that some novel amine derivatives of di-O- (n-higher alkyl and alkenyl) glycerols and -proipanedioles are substances that can serve to combat viral infections in mammals.

The novel amine derivatives of glyceirols and propanedioles having an antiviral activity of the general formula I,

Acid addition salts of these compounds with acids, wherein

R1 and R2 are straight chain alkyl of 12 to 20 carbon atoms or alkenyl of 12 to 20 carbon atoms, with the exception of the radicals having a double bond at the 1-position, are produced by the process according to the invention which consists in reducing the compound vzoirce II,

where

R 1 and R 2 are as defined above, and the resulting compound of formula (I) is optionally converted to a pharmaceutically acceptable acid addition salt thereof.

The novel compounds produced by the process of the invention have a direct antiviral effect on a variety of viruses in vivo in mammals and in vitro in tissue cultures of mammalian cells. An essential part of this effect is that the compounds of the present invention induce inteirphirone thyroid in the cells, i.e. endogenous interferon.

The term "pharmaceutically acceptable" acid addition salts refers to salts which are nontoxic in the doses used. These are water soluble and insoluble salts such as hydrochlorides, hydrobromides, phosphates, nitrates, sulfates, acetates, hexafluorophosphates, citrates, gluconates, benzoates, propionates, butyrates, sulfosalicylates, maleates, laurates, malate, fumarates, fumarates, , am, sonates (4,4'-diaminosilbein-2,2'-disulfonates), pamoates (1,1'-methyl-bls-2-thiroxy-3-naphthoates), stearates, 3-hydroxy-2-maftoates, p-toluene, sulfonates, methanesulfonates, lactates and salts with suramine.

A preferred group of compounds of formula I are the hydrochlorides of these compounds.

Other preferred groups of compounds of formula I are:

substances in which R1 and R2 are straight chain alkyl of 15 to 18 carbon atoms;

compounds wherein R 1 and R 2 are straight chain alkyl of 14 to 18 carbon atoms, both groups being identical; substances wherein R1 and R2 are n-hexadecyl.

Of particular interest are the following compounds and their pharmaceutically acceptable acid addition salts:

1- [2,3-di- (n-octadeicyloxypropyl) -4-aminomethyl-4-phenylpiperidine as well as pharmaceutically acceptable208156

1- [2,3-di- (n-hexadecyloxypropyl) -4-aiminomethyl-4-phenylpiperidine; and

1- [2,3-di- (1-tetradecyloxy) ipropyl] -4-aminomethyl-4-phenylpiperidine.

The compounds of formula (I) may be obtained from the appropriate 1,2-di-O- (n-higher alkyl or alkenyl) glyceirol by known methods. For example, the tosyl derivative of the starting material can be reacted with 4-cyano-4-phenynylpiperidine hydrochloride followed by reduction of the compound thus obtained.

Acid addition salts of the compounds of formula (I) may be prepared by conventional means, for example by mixing an amine derivative in a suitable solvent with the desired acid and isolating the salt by evaporation or precipitation after addition of a solvent in which the salt does not dissolve. Hydrochlorides can be readily prepared by passing hydrogen chloride through a solution of the amine derivative in an organic solvent. It will be understood that many of the hydrochlorides or dihydrochlorides of the compounds of formula I contain some water after isolation. It is not known whether it is crystalline water or the formation of true hydrates, but these salts can also be formulated and administered without prior dehydration.

The starting 1,2-di-O- (n-higher alkyl) glyceirols can be obtained according to the method of Kateis M. et al., Biochemistry, 2, 394 (1963). higher alkyl J glycerols can be obtained according to the method of Darnico R. R. et al., J. Lipid Res., 8, 63 (1967) The starting 1,2-al, 3-di-O- (n-higher alkenyl) glycerols are: can be obtained according to the method of Bauiman, W.I. and Mangold, K., Org. Chem., 31, 498 (1966).

The iProtiviral activity of the compounds of the invention was determined by two basic independent procedures. In the first procedure, the test substance is administered to the mouse intraperitoneally 18 to 24 hours prior to administration of a lethal dose of encephalomyociarditis virus (EMC). Ten days thereafter, the number of surviving mice is examined and compared to the number of surviving mice without treatment. The procedure is pirated such that the test compound is administered far from the site where the virus is administered, so that the interaction between the active agent and the virus can be avoided and only substances which actually increase the resistance to the virus can be detected.

In the second procedure, simple cell layers of human carrier polyips are grown on small-sized plates. These cells are then contacted with the test compounds approximately 18 hours before the lethal dose of vesicular stomatitis virus (VSV) is added to the cells. The test substance is washed out of the tissue culture prior to virus application. The fluid that is extracted from the fence, ekci infection, is titrated to detect the virus content of L-929 mouse fibrobilaist cultures. The values determined are compared to those from tissue cultures not contacted with the compounds of the invention.

In addition, a number of compounds of the invention have been investigated for their ability to enhance the known antiviral activity of polyinoteinoic and polycytidylic acids. In addition, some compounds were investigated for their ability to induce interferon formation in mice following parenteral administration as described by Hoffman, W. W., et al., Antiimicirobial Agents and Chemotheirapy, 3, 498-501 (1973).

Parenteral, topical or intirainasal administration of the above-mentioned amines or amidines to mammals prior to virus infection rapidly results in virus resistance. Preferably, administration of the active ingredient should occur at least 2 days or deine prior to infection, although this value varies somewhat depending on the type of virus and the species of animal to which the compound is administered.

The compounds of the invention are most readily and economically administered in dispersed form together with a suitable carrier. With respect to the concept of dispersed foirties, it is meant that the particles are retained by the individual molecules in the right solution in a suitable solvent, furthermore they may be colloidal particles which optionally form a suspension or an emulsion. They may also be particles dispersed in the solid carrier to form a powder. The compositions thus prepared are suitable for use as sprays in the form of solutions, suspensions or emulsions of the active compounds according to the invention.

For parenteral, i.e. subcutaneous, intramuscular or intraperitoneal, administration of the compounds of the invention, doses of 1 to 250 mg / kg body weight, preferably 5 to 100 mig / ikg, especially 5 to 50 mg / ikg, are used. Of course, the dose will depend on the mammalian dirus and the amine and must be determined by the result obtained on the first administration. Usually, small doses are administered initially and gradually increased until optimal effect is achieved.

For carriers for parental administration, these may be aqueous liquids, such as water, isotonic sodium chloride solution or dextrose, Ringer's solution, or non-aqueous liquids, for example animal or vegetable oils such as flaxseed oil, peanut , corn and sesame oil, however, any solvent which does not interfere with the efficacy of the compounds of the present invention and that is not toxic may be used. They are, for example, glycerol, ethanol, piropylene glycol or sorbitol. Liquid preparations containing the compounds of the invention may thus also contain a mixture of various cepaline diluents, such as propylene glycol, diethyl carbonate, glycerol or sorbitol.

Any method by which the antiviral compound is contacted with the mammalian respiratory system can be used for intranasal administration of the compounds of the invention. Effective methods are, for example, administration of nasal drops or administration of drops directly into the nasopharynx, inhalation and nebulization in the aerosol form. These routes are particularly important because they are easy, safe and effective routes of administration. For intranasal administration, the compound of the invention is usually administered together with the carrier at a concentration of 1 to 10 mg / ml of carrier. A concentration of 30 to 50 mg / ml is generally best.

For topical administration, the compound ¹ of the invention also preferably used together with a suitable carrier, especially · in order to ensure a perfect absorption. In this case a concentration of 1 to 250 mg / ml is used. Usually, a total of 1-250 mg / kg of a compound of the invention is administered in the above two routes of administration, preferably. 5 to 50 mg / kg.

The compounds of the present invention may be used as such or in admixture with one or more excipients or other drugs such as analgesics, anesthetics, antiseptics, vascular blocking agents, antibiotics, waicins, buffers and inorganic salts. In addition, the compounds may be administered in admixture with hyialuronidase in order to avoid or reduce irritation to a minimum and increase the absorption rate of the compound of the invention. Said enzyme is used at a dose of at least 150 units, although in exceptional cases lower or higher doses may be used.

The compounds of the invention which are insoluble in water or those whose water solubility is low are preferably administered in suspensions or emulsions which allow the formation of particles less than 20 microns in size. This is because the particle size influences the biological activity of the compounds according to the invention, which is probably based on a better absorption of the active substance. Various surfactants and protective colloids may also be used in the formulations. Suitable wetting agents are, for example, the partial esters of conventional aliphatic acids, such as lauric, oleic or stearic acid, with sorbitol-derived hexylic anhydrides and the polyoxyethylene derivatives of these ethers. These compounds are commercially available under the tradenames "Spalns" and "Tweenls" and are available from ICI United States Inc., Wilmington, Del. Cellulose ethers, in particular cellulose meithyl ether (imethocel, Dow Chemical Co., Midland, Mich.), Are highly effective as protective cools for use in emulsions containing the compounds of the invention.

When the compounds of the invention are water-soluble, it is most advantageous to administer them in the form of aqueous solutions, in particular in physiological saline containing phosphoireenan buffer. The water-insoluble compounds are preferably administered in formulations of the above type. A suitable solvent for water-insoluble compounds is, for example, dimethylsulfoxide. Such a preparation contains, for example, 25 to 100 mg of the selected active ingredient in the form of an emulsion in a mixture with an equal amount of Polyscrbate 80 in a mixture with glycerine, to which water at 80 ° C is added under vigorous stirring. Sodium chloride to a final concentration of 0.14 M and sodium phosphate at pH 7 to a final concentration of 0.01 M are added to the concentrated solution.

mg / ml active ingredient 50.0

Polysoiribate 80 50.0 glycerin 50.0 monohydrogen phosphate, hydrated 1.4 sodium chloride 7.9 water 842.0

1001.3

In those cases where the active compound particles are agglomerated, ultrasound can be used to obtain a homogeneous system.

The invention will be illustrated by the following examples.

Example 1 a d 1

1- [2,3-di- (n-hexadecyloxy jpropyl] ^{-4-aminoimethyl-4-phenylpiperidin!} Peridmdihydr ochlorid

A. 1- [2,3-Di- (n-ihexadecyloxypropyl) -4-cyano-4-phenylpiperidine

A mixture of 6.96 g (10 mmol) of 1,2-di-O- (n-hexadecyl) -3-O- (n-itosyl) glyceirol, 2.23 g (10 mmol) of 4-cyan-4-yl enylpiperidine dihydrochloride, 2 ml of triethylamine and 40 ml of N, N-dimethylformamide were stirred for 16 hours at 95-100 [deg.] C. The reaction mixture was then cooled, diluted with 200 ml of water and extracted three times with 150 ml of ethyl acetate. It was dried over magnesium sulfate, filtered, and evaporated to dryness in vacuo to give 6 g of an oily liquid which was purified by column chromatography eluting with a mixture of benzene and ethyl acetate.

Spectrum, Infrared chloroform Vmax 2220 om ^first

B. Resulting substance

A solution of 2.5 g (3.6 mmol) of 1- [2,3-di- (n-hexadecyloxypropyl) -4-cyan-4-phenylpiperidine in 100 ml of ether is mixed with 0.4 g (10, The reaction mixture was carefully diluted with water (100 mL) and extracted with ethyl acetate (3.times.100 mL). The ether extracts were combined, dried (MgSO4), filtered and evaporated in vacuo to an oil. The residue is purified by chromatography on silica gel, eluting the column with a mixture of benzene and ethanol, the resulting product

Examples 2 to 4

The following compounds of the corresponding 1,2-di-O- (n-alkyl or alkenyl) -3-O- (p-tosyl) glycerol are prepared by the method of Example 1:

The material was dissolved and treated with hydrogen chloride gas, then the solution was evaporated in vacuo. A solid is obtained which is recrystallized from ethyl acetate. Yield: 1.1 g of solid, m.p. 132 DEG-134 DEG C., which contains approximately 3/4 mol of water per mole of product.

Elementary analysis:

calculated:

H, 11.53; N, 3.50. found ·:

70.74 _0/0 C, 11.34% H, 3.40% N.

R-O-CH.

R-O-CH

HgC 1 x phenyl

Example R number Molecular formula Melting point (° C) Elemental analysis calculated (%) C found (° / o) H N 2 n-tetradeeyl C13H80O21N2.2HCl 140-142 69.46 11.25 3.75 69.45 11.00 3.45 3 n-octadeeyl C51H96O2N2.2HC.1 115-117 71.95 11.60 3.29 71.67 11.19 3.38 4 n-octadec-9-enyl C51H92O2N2.2HCl 118-120 71.17 11.23 3.25 71.06 10.84 3.09

Example 5

Efficacy of 1,3-di-O- (n-hexadecyl) -2-O- (3-aminopropyl) glycerol hydrochloride against EMC virus in vivo

The antiviral activity is expressed as the relative survival time (S _r ) in the experimental groups as compared to the control groups at day 10 after virus injection. This value is defined as follows:

The emulsion formulation was prepared by melting and mixing equal parts of the active ingredient, polysorbate 80, and glycerin followed by dispersion of the mixture in hot water with vigorous stirring. The concentration of the mixture was then adjusted to 0.14 M sodium chloride with the addition of 0.01 M sodium phosphate pH 7. Further dilution was performed with ¹ buffer consisting of a mixture of 0.14 M sodium chloride and 0.01 M sodium phosphate pH 7.

Three. groups of isamic white mice weighing 20 to 25 g were given 1.5, 5 and 15 mg of active ingredient per kg body weight as an intraperitoneal injection of volume. 0.5 million Four-control group of 10 mice were left untreated. 18 to 24 hours after the injection, all four groups of 0.2 ml were injected subcutaneously with an amount equal to 20 times the LD 50, a dose that causes 50% of the mice to die within 10 days, using eincephalomyocarditis virus. (EMC). The number of surviving mice was recorded over 10 days and the mean survival time (S _r ) was recorded.

^s x

Sr =

100 4-100

XL v = 1á.z10 č = 1a.z10 x100 i, d.i10 where is

S _r relative survival time,

With _x percent survival after 10 days in the experimental group, χ, the number of surviving mice on day "i" in the experimental group and ei the number of surviving mice in dein "i" in the control group.

Examples 6 to 10

In the same manner: as in Example 5, efficacy against EMC virus in vitro was determined for the compounds of the following table.

Dose of active substance S _r (average of 7 experiments)

15 mg / ikg 61 5 mg / kg 45 1.5 mg / kg 24

0 81S 6

S _{r of} active ingredient (img / kg). 5 1,5 0,5

Example number The compound produced according to example number

43

44

45

46

47

77 58 26 _ 76 85 42 - 52 42 32 - 79 72 28 -. 99 26 54 -

Example 1 a> d 11

Reduction of iv polyp yield in human polyp cells in ivitro using 1,3-di-O- (n-hexadecyl) -2-O- (3-aminoimidopropyl) glycerol hydrochloride

The growth medium was prepared by adding 100 µl of Eagl-1's base environment with 2 µl of 10 times concentrated solution for the growth of the material to determine efficacy 8 with antibiotic antibiotic, 1 ml (200 mimoles) glutamime solution, 1 ml 100torate neeseinciálních concentrated solution of amino acids, 1 ml of solution of sodium ipyrohrozmanu 100 Immola and 10 o / _o fetálního- calf serum inaktivoyaného heat. 96-well micirotitration plates were used, each of which 50,000 human nasal polyp cells were suspended in 0.2 ml growth medium. The plates were then incubated for 8-10 days at 37 ° C in an atmosphere of 5% carbon dioxide to form continuous single cell layers.

At the end of the 8 to 10 days of growth, the cell layers thus obtained were washed four times with physiological saline containing phosphate buffer, and 0.2 ml of maintenance medium containing 10, 5.0, 1.0, 0 was added to each well. , 5, 0.1 and 0 µg / ml of active ingredient. The maintenance medium was the same composition as the growth medium except that it contained only 2 ‘o / o fetal calf serum. The plates were incubated for an additional 18 hours at 37 ° C, after which the cell layers were again washed four times with saline.

EXAMPLE The compound produced according to the example number of phosphate / buffer containing the active ingredient was removed and a dose corresponding to 1000 times the TCOD50, the dose which caused 50% infection in unprotected cultures, was fed into the wells. Vesicular stomatitis virus (VCV) was used, the culture was left in the cultures for 2 hours at 37 ° C, then the cultures were washed four times with phosphate-buffered saline to remove unadracted virus particles and each well was again 0.2 µL of maintenance medium was added. Plates were then incubated for 7 hours at 37 ° C, after which (each plate was removed, the liquid was stored in a frozen state in tubes and titrated to the amount of infectious virus using higher fibroblasts L- 929. These mouse cell cultures were analyzed after 3-4 days, and the following table shows decreases in virus yield versus control, determined for five concentrations of the above active ingredient.

Reduction of virus yield in%; Concentration of the active substance in (µg / m -1)

5.0 1.0 0.5 0.1% 90% 84% 75% <68%

Examples 12 to 16

The method of Example 11 was determined to reduce virus in vitro yields of human β-olipt cells as well as the compounds shown in the following table:

% reduction in virus yield ^and concentration (pg / nil)

5.0, 1.0 0.5 0.1

12 421 13 43 14 44 , 15 45 16 46

+ + - - ND + ± - - ND + + - - ND + + - - ND, + - - - ND

a) ξ => 680/0 reduction, ± = ~ 68% reduction, - with <68% reduction, NR ξ = not performed /

Example 17

The ability of 1,3-di-O- (n-hexadecyl) -2-O- (3-aminopropyl) glycerol hydrochloride to produce circulating interferon

Simes equal parts by weight of the active ingredient, the addition of polysorbate 80 and glycerol are melted and then homogenized in hot 0.14 M sodium chloride solution containing 0.01 M sodium phosphate pH 7 (PBS). The resulting oil in water emulsion is further diluted with PBS prior to administration.

Swiss female mice weighing 20-25 g are administered an intraperitoneal injection of 0.5 mil. Amount of diluted emulsion containing the active ingredient at a dose of 25 mg / kg body weight. At 8, 12, 16 and 20 hours post injection, plastic visoirs were removed from four mice and pooled. Serial dilutions in L-15 (Leibovitz medium) containing 5 θ / θ fetal calf serum were incubated in microtitration overnight plates at 37 ° C for continuous individual layers of L-929 mouse fibiroblasts. The layers were then washed in a protein-free environment, and then contacted with a dose equivalent to 10 times the TCID 50, a dose that causes 50% infection in unprotected cultures. For infection, vesicular stomatitis virus (VSV) was used, which was in contact with the culture for 1 hour at 37 ° C, the unadsorbed virus was washed out, and the cells were re-contacted with L-15 containing 5% fetal calf serum. and incubated again at 37 ° C for 48 hours. L-929 cultures were then examined microscopically for cytopathological changes caused by the virus, showing an inverse relationship between plasma interferon levels and fibiroblaist damage. At the same time, plasma dilution was determined, which can guarantee 50% protection of simple L-929 cell layers.

A second experiment was performed in a similar manner except that mice were injected with 10 µg of active ingredient / fcg body weight and then peritoneal fluid samples were collected from four mice and pooled. Samples were again taken 6, 9, 12, 15 and 18 hours after injection. Samples were taken by injecting 1 ml of Hank's Balanced Solution containing 100 units of penicillin / ml and 100 stireptomycin / ml after opening the abdominal cavity, then massaging the abdomen briefly and then removing liquid from the peritoneal cavity.

The results obtained from both experiments are shown in the following table:

Source of interferon 6 8

Interfeirone Source Interferon level (units / ml) after injection at (h)

12 15 16

20 May

plazima perito- neální - 34 - 67 - 52 - 40 liquid 16 - 768 320 448 - 448 -

Example 18 In a similar way as in the ability to induce interfeiroin formation for a compound from the following example 17 bulky: Example number The compound was prepared according to the method of Example number Interferon level (units / ml j ^and time after injection (hj) Interferon level (units / ml) ^b time after injection (hj 6 9 12 15 18 18 43 70 ^c 100 120 ^d 100 ^e 13 109 284 312 224

(Interfiriron-plasma source (b) Source of interferon-plasma (c) <20 units / ml after 6 h, 70 units / ml after 9 h (dj 15 h (e) 18 h

Examples 19-22 of said acids for cellular resistance to viral infection for compounds

An increase in the effect of both cheeses, as shown in the following table, was determined:

1S

Increased effect of polyinosinic acid and polycytidyloic acid at a concentration of? G / ml

4,0 0,8

- _ _ + + - + + ± = <· 5x increase, ND = not performed '

20 & 1S6

Example Compound Prepared according to Example Number

19 Dec 43 20 May 44 21 45 22nd 46

(a) + ξξ> 5x increase, + ξ = ~ 5x increase, -

Claims (4)

SUBJECT A process for the preparation of novel amine derivatives of piropanidioles having antiviral activity of the general formula (I) as well as pharmaceutically acceptable acid addition salts of these compounds, wherein R 1 and R 2 are straight chain alkyl of 12 to 20 carbon atoms or aliphenyl of 12 to 20 atoms except for residues having a double bond at the 1-position, characterized in that the compound of formula (II) is reduced, wherein R 1 and R 2 are as defined above, and the resulting compound of formula I is optionally converted to its pharmaceutically acceptable acid addition salt. 2. A method according to claim 1, wherein the compound of formula (II) wherein R1 and R2 are straight chain alkyl of from about 14 to about 18 carbon atoms is used. 3. A process according to claim 2, wherein R @ 1 and R @ 2 are n-hexadeicyl. 4. A process according to claim 2, wherein the compound of formula (II) wherein both R 1 and R 2 zinaimenate is n-of-octadecyl.