GB2285805A - Esters - Google Patents

Description

2285805 MARIGEN S.A.

CASE 160 ESTERS with DICARBONIC ACIDS having ANTITUMOUR PROPERTIES INTRODUCTION

The present invention concerns new esters having antitumor activity, of saturated and unsaturated dicarbonic acids with sterols, vitamin-D and vitamin-E compounds, processes for their production as well as for the preparation of spontaneously dispersible concentrates and pharmaceutical compositions containing these compounds, and their use for the treatment of tumors.

Surprisingly it has been found that the newly synthetized esters of saturated and unsaturated dicarbonic acids with sterols, vitamin-D and vitamin-E compounds possess outstanding antitumour properties, particularly if these compounds are being incorporated into spontaneously dispersible concentrates.

DESCRIPTION OF THE INVENTION

The new esters of saturated and unsaturated dicarbonic acids with sterols, vitamin-D and vitamin-E compounds correspond to the general formulae (1) to (V[):

R3 00C --(C1-6) - COOR3 n (1) R30OC---(CH2)-COOF n (11) %o0C - CH - COOR3 R2 R300C - C ' CH - COOR, 1 (111) F2 (IV) --2-- "H3 O H3 oo COOR3 R300C CH3 CH3 (V) H3 H3 COOR 3 R300C r CH3 CH3 (V&) whereby in the formulae (1) to (11) the letter n stands for the numbers 1 to 18, and in the formulae (1) to (V1) Ri designates a Cl to C20 alkyl or C2 to C20 alkenyl group, R2 means hydrogen or methyl and R3 represents one of the radicals of the following formulae:

R4 H3 4 H3 H (V11) (VIll) H H3 R4 AH H3 R4 H (1x) (X) H3 R4 H 1 A 1H3 R4 4H3 H C14_: CH - 3 CH3 (xl) (X11) H3 AH r 4 14; c A CH3 H AH3 IdH3 R4 :5 H (X111) (XV) 1H3 ooR4 4H:5 H H3 (XIV) H3 R4 H3 1 A (XVI) whereby in the formulae (V11) to (XVI) the radical R4 stands for a Cl to Clo alkyl or a C2 to Clo alkenyl group, R5 3 3 R6 CH3 7 (XVII) kCH3 H3 3 H3 ^JH F1r, C rh (XVIII) --4-- CH3 #04 CH H 3 3 CH3 CH2 c R4 & CH3 (XIX) H3 H3 CH3 CH3 a',,Z CH3 CH X "\\C H 3 3 C H 3 H 31 CH3 c CH2 1 W.0o& 4 (XX) 3 1 CH3 0010& CH3 CH3 (XXI) (XXII) and where in the formulae (XVII) and (XVIII) the radicals R5, R6 und R7 represent hydrogen or methyl, CR3 R8 -tCH= CI 1 c m Re-tCH-CH- f H3 (XXIII) Chtt-+CH CI 1 CH Cj- CH = CH m 1 m CH,9 (XXIV) whereby in the formulae (XXill) and (XXIV) the letter m means the numbers 1, 2, 3, 4 or 5 and R8 stands for one of the radicals of formulae:

__5 H3 CH & CH3 H3 CH3 )&OCH3 CH3 H3 OCH3 CH3 15 CH3 H3 H CH3 3 CH3 OH H3 0 0 CHj 0CO-CH3 CH3 H3 CHA CH3 H3 CH36 CH3 3 CH3 C CH3 H3 6 3 CH3 CH3 CH3:a0H CH3 H3 CW3 H H3 CH3CH3 '&c CH3 C(>-CR, CH CH3 3 3 H or CO OH 1 \ CH3 CH3 The alkyl and alkenyl groups at R1 and R4 can be straight-chained or branched. At R1 the alkyl groups preferably have 1 to 4 carbon atoms.

At R4 in the formulae (V11) to (XVI) the alkyl and alkenyl groups normally have 8 to 10 carbon atoms. Examples of such compounds are, inter alia: 1 H3 Y H3 CHd-f H-CH-CHi-Ti CHSf H-CHi.CHi-CH= CH3 CK, --6-- 2 v H3 H3 CHSH-CH27CH-( f CH3 3 v H3 H3 CH:i. 7 H-CH2-CH,CH-T CH3 4 H3 CH,l H-CH=CH-CH2T CH3 f H3 v H3 H3 CHd. 7 H-CH=C-CH-Y CH3 6 7 2H5 f H3 CHd. f H-CHi-CHi-C-.

f H3 H3 CHi.fH-CH=CH-CI± v CH3 CHd-f W(CH2) &T' CH F H3 3 Y2H5 H3 CH3 1 H-CH=CH-CH-Y CH3 v H3 5H H3 CH3 1 H-CHi-CH2-CH-TI CH3 J2H5 H3 CH,S-VH-CHi-CH-CH-V CH3 Y2H5 f H3 CHd.JH-CH27CH27CH- CH3 CH2 In accordance with the starting products maleic acid and fumaric acid respectively, the compounds of formulae (111) and (IV) have either a cis- or a trans-form.

The class of the vitamin-D compounds comprises the following chemical compounds: Cholecalciferol: (5Z, 7E)-(3S)-9,10-seco-5,7,10 (19)cholestatrien3-ol A --7-- 25-hydroxycholecalciferol [Calcidioll: (SZ, 7E)-(3S)-9,10-seco-5,7,10 (19)-cholestatrien-3,25-diol la-dehydroxy-cholecalciferol [Calcitrioll: (SZ, 71E)-(lS, 3R)-9,10-seco-5,7,10 (19)-cholestatrion-1,3,25-triol Ergocalciferol [Calcioll: (SZ, 7E, 22E)-(3S)-9,10-seco-5,7,10 (19),22ergostatetraen-3-ol Tachysterol: (6E)-(3S)-9,10-seco-5, (10)-6,8cholostatrien-3-ol Dehydrotachysterol: (5E, 71E)-(3S, 1OS)-9,10-seco-5,7cholestatrien-3ol.

The class of the vitamin-E compounds comprises all tocol- and tocotrienolcompounds, such as, e.g.: Tocol [2-methy]-2(4,8,12trimethyltridecyi)chroman-6-ol] a-Tocopherol [5,7,8-trimethyitocol], which can take on the following configurations: 12,12,12-a-tocopherol 2epi-a-tocop hero 1 2-ambo-a-tocop hero 1 al I-rac-a-toco p hero 1 4-a mbo8-ambo-a-toco p hero 1 P-Tocopherol [5,8-dimethyltocol] y-Tocopherol [7,8dimethyitocol] 8-Tocopherol [8-methyitocol] Tocotrienol-2-methy]-2-(4,8, 12-trimethyideca-3,7,11trianyi)chroman-6-ol] pl- oder p2-Tocopherol [5,7, 8-trimethyltocotrienol] and c-Tocopherol [5,8-dimethyitocotrienol or P-tocotrienol] The most important radical according to formula (M11) is characterized by formula (XXV):

4H3 H3 H3 3 H3 (XXV) --a-- This compound can be present in diverse stereoisomeric forms, such as e.g. all trans, 9-cis or 13-cis.

Of particular importance are compounds of the formulae (1) to (V1), in which the letter n represents the numbers 1 to 8, R1 stands for methyl or ethyl, R2 signifies hydrogen and R3 One of the radicals of the formulae (XXV) to (XXX) H3 H3 H3 :3 H3 CH3J CH3 'CH3 H3 AH39 H3 1 CH3 CH3 CH3 H3 AH H2 H3 2H5 P CH3 'CH3 H3 H3 H,, 3 00.1:::

(XXV) (XXVI) (XXVII) (XXVIII) H3 H3 2H5 CH3 CH3 H3 (XXIX) CH3 H3 CH3 I Oj (CHr-CH2 -CH2-CH)-CH3 CH,2 v H3 (XXX) Examples of inventive new double esters according to formulae (1) to (V1) are, inter alia: Malonic acid b i s-Sti 9 masteryl ester Succinic acid b i s-Sti 9 mastery] ester Glutaric acid bis-Stigmasterylester Adipic acid bis-Stigmasteryiester Pimelic acid bis-Stigmasteryiester Suberic acid bis- Stigmasteryiester Azelaic acid bis-Stigmasteryiester Sebacic acid b i s- Sti g masteryl ester Azelaic acid b i s-P-S ito steryl ester Sebacic acid bis-P- Sitosteryiester Azelaic acid bis-Ergosterylester Sebacic acid bis- Ergosteryiester Azelaic acid bis-Cholesteryiester Sebacic acid b is- Cholesteryl ester Maleic acid bis-Stigmasteryiester Fumaric acid bis-Stigmasteryiester Maleic acid bis-P-Sitosteryiester Fumaric acid bis-P-Sitosteryiester Maleic acid bis-Ergosterylester Fumaric acid bis-Ergosteryiester Maleic acid bis-Cholesteryiester Fumaric acid bis-Cholesteryiester Azelaic acid Ca 1 cif eryl-di ester Sebacic acid Calciferyl-diester Azelaic acid Cholecalciferyl-diester Sebacic acid Cholecalciferyi-diester Azelaic acid DL-a-Tocopheryl-diester Sebacic acid DL-a-Tocopheryi-diester Glutaric acid ethyl-Calciferylester Glutaric acid Calciferyi-mothylester Fumaric acid ethyl-Calciferylester Furnaric acid Calciferyi-methylester Azelaic acid ethyl-Cal cif erylester Azelaic acid Ca lcif eryl-methyl ester Azelaic acid ethyl-Cholecalciferyiester Azelaic acid Cholecalciferyi-methylester Sebacic acid ethyl-Calciferylester Sebacic acid Cal cif eryl-methyl ester Sebacic acid ethyl-Cholecalciferyiester Sebacic acid Cholecalciferyl-methylester -- 11 -- PROCESSING The compounds according to formulae (1), (111), (V) and (V1) can be manufactured according to the following procedures, which are known per se: a) Reaction of a compound of formulae (XXXI) to (XXXIV) HOOC---+ CF-- COOH HOOC-C-':: CH - COOH 1 R2 H3 H3 COOH H0Or CH3 CH3 H3 H3 H 00 C CH3 CH3 (XXXI) (XXX 11) (XXX 111) COOH (xxXIV) in which the letter n represents the numbers 1 to 18 and R2 means hydrogen or methyl, with 1,1'-carbonyidiimidazole (C131) at 25 to 70 'C under addition of a catalytic amount of an alcoholate in an indifferent solvent, such as tetrahydrofuran, benzene, toluene or chloroform, followed by alcoholysis of the imidazolides formed with a sterol, a vitamin-D or vitamin-E compound.

b) Formation of the dichloride or the dibromide of a compound of the formulae (XXX1) to (XXXIV) with a chlorination or bromination agent such as thionylchloride, oxalylchloride or oxalylbrornide, followed by the reaction of the dichloride or dibromide formed with a sterol, a vitamin-D or a vitamin-E compound at a temperature of between 40 and 80 C in an indifferent solvent such as toluene or tetrahydrofuran, and in the presence of a catalyst such as dimethylformarnide or pdimethylaminopyridine.

c) Procedure for the preparation of the compounds according to formulae (11) and (IV).

-- 12- Reaction of a compound of formulae (XXXV) and (XXXVI):

HALOGEN OC -4 CH24--- COOR, n HALOGEN OC-CCH -COOR, 1 (XxXV) 2 (XxXVI) in which the letter n determines the numbers 1 to 18. R1 stands for alkyl or alkenyl and R2 means hydrogen or methyl, with a sterol, a vitamin-D or a vitamin-E compound at a temperature of between 40 and 80 OC in an indifferent solvent such as toluene or tetrahydrofuran, and in the presence of a catalyst such as dimethyiformamide or pyridine.

The compounds of formulae (XXXV) and (XXXVI) are prepared in known manner by first producing the dihalogenide of the formulae (XXX) and (XXX1) respectively according to b) and then reacting the formed dihalogenides with a half-stechiometric amount of an alkyl or alkenyl alcohol in the presence of a catalyst such as e.g. dimethylformamide or pyridine, in order to obtain the halfester of the inventive compounds, followed by purification by means of distillation or chromatography.

The novel dicarbonic acid esters according to formulae (1) to (VI) surprisingly possess excellent antitumour activity. They are particularly effective against tumors of the skin and of epithelia[ tissues. most notably in case when these compounds have been incorporated into spontaneously dispersible concentrates. For this reason, spontaneously dispersible con-centrates made up with the now dicarbonic acid esters according to the formulae (1) to (VI) are also a subject matter of the present invention.

When treated with water, such concentrates render microemulsions having excellent phase stability as well as much improved membrane permeability and spreading properties. Control measurements, executed at the Swiss Federal Institute of Technology, Zurich (institute for Polymers, Biopolymers, Prof. Dr. Pier Luigi LUISI and Prof. Dr. Peter SCHURTENBERGER) showed that the generated micelles have a hydrodynamic radius of 1,2 to 2,4 nm.

All experimental observations gained with stable microemulsions of this kind can be uniformly interpreted by means of the assumption that the system produces in the aqueous phase organized aggregates, which are called All experimental observations gained with stable microemulsions of this kind can be uniformly interpreted by means of the assumption that the system produces in the aqueous phase organized aggregates, which are called MICELLES. These micelles possess a more or less globular shape, having a hydrodynamic radius of less than 10 nm. They are thermodynamically stable. The tensides and cotensides are capable at the phaseboundary of the microemulsion of hindering SELF-DIFFUSION. This means that no mixing takes place between the outer aqueous phase of the microemulsion and the inner oily phase, which contains the dicarbonic acid ester compounds solubilized in the coemulgator and/or blotenside solvent.

The micelles, which contain in their inner phase the solubilized antitumor substances, are coated by a tenside layer or bilayer and are thus enabled first to penetrate the human skin and then to penetrate through the plasma membrane of the turnour cell. This diffusion process takes place on account of thermal molecular movements (Brownian movement) exclusively.

The volume and the speed of substance transport across the cell membranes are dependent upon the differential in concentration existing between the extracellular outside and the inside of the individual tumour cell. The diffusion flow continues along the concentration gradient until it is consumed and an equal concentration of active substance [or of a therapeutic system] is reached in both compartments, the extracellular zone and the internal zone. Such diffusion processes occur independently of any energy input from outside into the interacting compartments. They can show slowrelease-effects. In biological systems they are not related to metabolic energy.

The speed of diffusion is governed by Fick's law of diffusion dm 1 dc dt q dx EQUATION (A) where dm signifies the amount in Mol of active substance molecules which penetrate a cell surface q (in cm2) per time-unit dt (in seconds). D is the coefficient of diffusion and dc the concentration differential over the distance dx.

---14 According to NERNST the diffusion coefficient is dependent on the absolute temperature and friction resistance D = R T = kT EQUATION (B) N f f Friction resistance is, according to STOKE's law, f = 6 7r il r EQUATION (C) a function of the viscosity of the diffusing solution and of the radius of the diffusing particles. By substituting f with f = 6 7c Tj r in the Nernst equation, one obtains the SUTHERLAND-EINSTEIN equation for the DIFFUSION COEFFICIENT D RT 1 kT N 6 r 6 n n r EQUATION (D) where k stands for the Boltzmann constant.

If for a particular diffusion process one assumes a regular reduction of concentration in the membrane of the tumor cell, then the expression dc A c in the diffusion law can be restated as - (= concentration dx X difference Ac over a membrane of thickness x). x is a constant value for a specific membrane. For this reason, it can be combined with the diffusion coefficient to express a new constant, the permeability coefficient P:

P = D X EQUATION (E) The expression drn 1 dt q in the diffusion equation is called FLUX J.

It has the dimension Mol per second per cm2. The negative sign on the right side of the equation indicates that the transport of the molecules of the active substance or the systems preparation containing active substances flows in the direction of the decreasing concentration.

Therefore, we have J FAc RT 1 -Wx 6 x n r AC = kT 1 -AC x 6 r 11 r EQUATION (F) It can be deduced from this equation that the velocity of the diffusion process across the cell membrane is governed by:

1) the concentration difference Ac in the two compartments 2) the radius of the particles of the diffusing active substance or system's preparation 3) the viscosity of the diffusing aqueous solution (emulsion) 4) the temperature __16--- The spontaneously dispersible concentrates prepared in accordance with the invention contain 0.001 to 25 % by weight of individual esters with di carbonic acids according to formulae (1) to (VI), and combinations of such compounds respectively 0.001 to 40 % by weight of a solvent or solvent mixture which is pharmaceutically acceptable and acts as a hydrotropib'or coemulsifier 0.001 to 90 % by weight of a pharmaceutically acceptable surfactant or surfactant mixture, and optionally up to 10 % by weight of a vitamin or provitarnin up to 10 % by weight of a free fatty acid, and if appropriate, customary excipients and/or diluents.

The surfactants or surfactant mixtures to be employed according to the invention can be anionic, cationic, amphoteric or non-ionic. Ideally, they are non-ionic and have an H1-13-value (i.e. a hydrophilic-lipophilic balance) of between 2 and 18; preferably, it is between 2 and 6 on the one hand and 10 and 15 on the other hand. HLB values describe the hydrophilic and lipophilic properties of an emulsifier. In this context see "HydrophileLipophile Balance: History and recent Developments" by Paul Becher in Journal of Dispersion Science and Technology, 5 (1), 81-96 (1984).

Suitable anionic surfactants can be both socalled water-soluble soaps and water-soluble synthetic compounds.

Suitable soaps are the alkali metal salts, alkaline earth metal salts or optionally substituted ammonium salts of higher fatty acids (C12 to C22), for example the natural Na or K salts of oleic or stearic acids, or of natural mixtures of fatty acids which can be obtained, inter alia, from coconut oil or tallow oil. Other surfactants which may be mentioned are fatty acid methyltaurine salts, and modified and non-modified phospholipids.

However, more frequently used surfactants are so-called synthetic surfactants, in particular fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylaryi-sulfonates.

The fatty sulfonates and fatty sulfates are usually present in the form of alkali metal salts, alkaline earth metal salts or optionally substituted ammonium salts and generally have an alkyl radical containing 8 to 22 C atoms, alkyl also encompassing the alkyl moiety of acyl radicals. Examples are the Na or Ca salt of ligninsulfonic acid, of dodecylsulfuric ester and sulfonic acids of fatty alcohol/ethylene oxide adducts. The sulfonated benzimidazole derivatives preferably contain two sulfonyl groups and one fatty acid radical containing about 8 to 22 C atoms. Alkylarylsulfonates are, for example, the Na, Ca or triethanolamine salts of dodecylbenzenesulfonic acid, of dibutyinaphthalene-sulfonic acid or of a naphthalenesulfonic acid/formaldehyde condensation product.

The non-ionic surfactants are mainly chosen from amongst the polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, saturated or unsaturated fatty acids and alkylphenols which can contain 3 to 30 glycol ether groups and 8 to 20 C atoms in the (aliphatic) hydrocarbon radical and 6 to 18 C atoms in the alkyl radical. Other suitable non-ionic surfactants are the water-soluble polyethyleneoxy-adducts onto polypropylene glycol and alkyl polypropylene glycol with 1 to 10 C atoms in the alkyl chain, which adducts contain 20 to 250 ethylene glycol ether groups and 10 to 100 propylene ether groups. The compounds which have been mentioned customarily contain 1 to 5 ethylene units per propylene glycol unit.

The following may be mentioned as examples of non-ionic surfactants: nonylphenol polyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxy-polyethoxyethanol, polyethylene-glycol and octylphenoxy-polyethoxyethanol. Moreover, fatty acid esters of polyoxyethylene-sorbitan, such as polyoxyethylene sorbitan trioleate, are also suitable.

The cationic surfactants are mainly quaternary ammonium salts which contain at least one alkyl radical having 8 to 22 C atoms as the Nsubstituent and which have lower, optionally halogenated alkyl radicals, benzyl radicals or lower hydroxyalkyl radicals as further substituents. The salts are mainly present in the form of halides, methyisulfates or ethyisulfates, for example stearyltrimethylammonium chloride or benzyidi(2chloroethyi)-ethyi-ammonium bromide.

When preparing the inventive spontaneously dispersible concentrates, special preference is given on the one hand to phosphoric acid ester tensides, such 2s:

-- 18- Tristyrylphenolpolyoxyethylene-1 8-monoldimethyi-phosphoric-acid-ester (SoprophorO FL, Rh8ne-Poulenc); 0 11 (-CH2 --CH2 -0),;-P-OCH3 0! CH3- Wi$i H-CH3 H-CH3 OH Soprophor FL (Rh8ne-Poulenc) No nyl p hen o 1-1 0-po Iyoxyethylene-mo no/d i methyl ph osp ho ric-acid- este r (DiphasolO 3873, CIBA-GEIGY); or the identical Sermule EA 188 (SERVO) 11 0 (-CH2CH2-0)2-1 - OCH3 C5-b 1 OCH3 (Tensid 508, C1BA-GEIGY); TinovetinOD JU (C1BA-GEIGY), a hydroxybiphenyi10-ethoxy-phosphoric acid ester Butyi-mono-4-ethoxy-phosphoric acid ester (ZerostatOD AT, C1BA- GEIGY), and 0 11 CH3-- CH2 b-fH-CH2-0 (-CH2-CH2---0)5-P-----OCH3 C2 H5 1 OCH3 (Zerostat OD AN, C1BA-GEIGY), respectively and on the other hand to betain compounds, i.e. amphoteric, salt- and waterfree imidazole derivatives, having an isoelectriclisoionic point near 7, such as e.g.

-- 19--- CH2 - CH2 - OH 1 N CH2 Rx-C N CH2 H [+1 CH2- CH2- COO Me in which Me[+] stands for hydrogen, alkali and/or earth alkali atoms, and Rx for a C1 to C32 alkyl or C2 to C32 alkenyl group.

Furthermore, so-called "multi-functional glucose derivatives", such as, e. g., Glucate@ SS (Methyl-glucose-sesquistearate) and Glucamate@ SSE-20 (PEG20-methyl-g I u co se-sesq u i stea rate) of Amerchol, Edison, N.J., are also being used.

The following compounds may be employed as the pharmaceutically acceptable solvent which acts as the hydrotropic, or coemulsifier, for example: esters of an aliphatic alcohol (C3 to C18) with an aliphatic carboxylic acid (C10 to C22), such as isopropyl laurate, hexyl laurate, decyl laurate, isopropyl myristate and lauryl myristate; hydrocarbons having a straight carbon chain (C12 to C32) which is substituted by 6 to 16 methyl groups and which can have up to 6 double bonds, examples which may be mentioned being terpenes, such as polymethylbutanes and polymethylbutenes.

Monoesters of ethylene glycol or propylene glycol with an aliphatic carboxylic acid (C6 to C22), such as propylene glycol monolaurate and propylene glycol monomyristate.

Esters of an aliphatic alcohol (C12 to C22) with lactic acid, such as, for example, myristyl lactate or, preferably, lauryl lactate. Monoesters or diesters of glycerol with an aliphatic carboxylic acid (C6 to C22), such as, for example, glyceryl caprylate or Miglyol@ 812 neutral oil (Oleum neutrale). Esters of a poly(2-7)ethylene glycol glycerolether having at least one free hydroxyl group with an aliphatic carboxylic acid (C6 to C22), such as, for example, aliphatic alcohols (C12 to C22), thus, inter alia, dodecanol, tetradodecanol, oleyl alcohol, 2-hexyldecanol and 2-octyl-decanol.

Esters containing at least one free hydroxyl group, of poly-(2-10)glycol with an aliphatic carboxylic acid (C6 to C22), monoethers of a polyethylene 4 with an aliphatic alcohol (C12 to C18), such as, for example, polyoxyethylene-(Cl 0) octylether. Heterocyclic compounds such as 1- methyl-2-pyrrolidone. Biotenside esters according to the general formula:

RZ-COO-O in which R2 stands for citronellyl, geranyl, farnesyl, phytyl or isophytyl and R3 means a Cl to C32 alkyl amd a C2 to C32 alkenylgroup respectively.

Before their application in the spontaneously dispersible concentrates all technical tensides have been cleaned by filtration or by chromatography over aluminum-oxide with an inert solvent as eluent, such as tetra-hydrofurane, ethyl alcohol or dichloromethane.

Suitable additives for the spontaneously dispersible concentrates according to the invention are vitamins and provitamins [such as, for example, vitamin A (retinoic acids), retinol, carotenes, tocopherols], and also free fatty acids, such as: valeric acid, isovaleric acid, sorbic acid, isocaproic acid, pelargonic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, hexacosanoic acid, octacosanoic acid, pentadecanoic acid, decenylic acid, undecylenic acid, dodecenylic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, erucic acid, etc.

The daily dose required for pharmaceutical administration is 0.001 to 25 mg/kg of body weight, if possible split into 2-3 individual doses. For this purpose, the new vitamin-sterol esters sterol phosphatides, or the spontaneously dispersible concentrates with these compounds, can be incorporated into the conventional pharmaceutical preparations and dosage forms, such as coated tablets, tablets, capsules, powders, granules, pellets, solutions, ampuls, emulsions, creams or suppositories together with the customary excipients and/or diluents and stabilizers.

The active substances or mixtures of active substances which form the subject-matter of the invention, and the spontaneously dispersible concentrates which contain these active substances or mixtures of active substances, can be administered to humans orally, by injection (intravenously, subcutaneously or intramuscularly) or in other ways. If they are presented as solid dosage forms for oral administration, this can be in the form of tablets, granules, pellets, powders or capsules, etc. The preparations can contain additives, for example a pharma-ceutical excipient, such as a saccharide or cellulose bass, a binder, such as starch paste or methylcel I u lose, a filler, or a disintegrant, etc., with additives being employed which are customarily used in the preparation of medicinal or pharmaceutical formulations. When the active substances or mixtures of active substances according to the invention are administered orally in the form of liquid dosage forms, they can be present in any form selected from amongst aqueous preparations for internal use, from suspensions, emulsions and syrups, etc., and they can also be present in the form of dried preparations which are dissolved or emulsified prior to use.

When the active substances or mixtures of active substances according to the invention are processed in the form of aqueous solutions, suspensions or oily or aqueous emulsions, preferably microemulsions, from the spontaneously dispersible concentrates according to the invention, they can also be injected. However, it is customary to prepare the injection solutions shortly before administration, by dissolving or suspending the extracts or concentrates in aqueous, liquid media, such as sterile water or physiological sodium chloride solution or glucose solution.

If required, conventionally used solvents, stabilizers, preservatives and additives for the preparation of isotonic solutions can be added to a preparation for injection. The preparations for injection obtained in this manner are administered intravenously, intramuscularly, subcutaneously or in any other suitable way.

The present invention also relates to pharmaceutical preparations which contain the active substances, or mixtures of active substances, or the spontaneously dispersible concentrates, which have been above described, for controlling the growth of turnour cells. The pharmaceutical preparations according to the invention are those which can be used for enteral (such as oral or rectal) or for parenteral or topical administration to warm-blooded animals, which preparations contain the spontaneously dispersible concentrate on its own or together with a pharmaceutically acceptable excipient.

The dosage of the concentrates according to the invention depends on the warm-blooded species, on the age and on the individual condition, and on the mode of administration. For example, doses in the range of about 0.1 50 mg/kg of body weight are administered subcutaneously, and doses in the range of 0.05 - 5 mg/kg of body weight are administered intraperitoneally to ---22--- warm-blooded animals having a low body weight, such as, for example, mice, rats and hamsters, to achieve an effect of tumour cell destruction.

The oral and rectal forms of the novel pharmaceutical preparations contain between I and 95 preferably between 10 and 95 %, and in particular between 20 and 95 of the spontaneously dispersible concentrate according to the invention. For example, they can be present in unit-type dosage forms, i.e., as coated tablets, micropellets, tablets, suppositories or ampuls and, in particular, as capsules.

Suitable pharmaceutically acceptable excipients for the oral forms are mainly fillers, such as sugars (for example lactose, sucrose, mannitol or sorbitol), cellulose preparations and/or calcium phosphates (for example tricalcium phosphate or calcium hydrogen phosphate), furthermore bin-ders, such as starch paste, with the use of, inter alia, corn starch, wheat starch, rice starch or potato starch, gelatine, tragacanth, methylcel I u lose, hyd roxymethylcel I u lose, sodium carboxy-methy1cellulose and/or polyvinylpyrrolidone and/or disintegrants (if desired), such as the above mentioned starches, furthermore carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, for example sodium alginate.

Examples of suitable flow-control agents are the polyethylene glycols Nos. 200 - 600 and above.

The gelatine capsules, which are still the preferred dosage form for humans, are provided with suitable coatings, concentrated sugar solutions [which can optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide], lacquer solutions (aqueous or those which have been prepared using organic solvents), or enteric coatings of solutions of suitable cellulose preparations, such as microcrystalline cellulose (Avice[O), acetyl-cellulose phthalate, hyd roxymethyleel I u losephthalate, MetoloseO, AQOATO or a copolymer, such as EudragitO L 30 D, being used, inter alia.

Pharmaceutical dosage forms for oral use which are particularly suitableaccording to the invention are two-piece gelatine capsules with a plasticizer, such as glycerol or sorbitol. The soft-gelatine or hardgelatine capsules and the capsules made of AQOATO (hydroxypropyl methylcellulose) respectively can contain the spontaneously dispersible concentrate according to the invention as a mixture with fillers, such as lactose, binders, such as starch, andlor glidants, such as talc or magnesium stearate, and, if appropriate, together with stabilizers and antioxidants, such as, for example, a-, g- or y-tocopherol. It may be expedient to employ suitable liquids, such as liquid polyethylene glycols Nos. 200 to 600 as diluents, to which stabilizers and antioxidants can also be added.

For parenteral administration, distilled water is added to the concentrates according to the invention. To the aqueous microemulsion for injection which then forms, there can be added viscosity-increasing substances, for example Na-carboxymethyl-cel I u lose, sorbitol, mannitol and/or dextran, and if appropriate also stabilizers and antioxidants.

The pharmaceutical preparations for parenteral administration preferably contain between 0.1 and 60 %, especially between 1 and 40 %, of the spontaneously dispersible concentrate according to the invention.

Suitable preparations for topical use, which are particularly suitable for the prophylaxis and the treatment of cancers of the skin, are, for example, creams, ointments, pastes, foams, tinctures and solutions, which contain between 0.001 and 70 % of the concentrate according to the invention.

Oily bases which are used for creams and oil-in-water emulsions which contain more than 60 % water, are mainly fatty alcohols, for example lauryl alcohol, cetyl alcohol or stearyl alcohol, waxes of liquid to solid consistency, for example isopropyl myristate, wool wax or beeswax and/or hydrocarbons, such as, for example, petroleum jelly (petrolatum) or paraffin oil. Substances which are mainly suitable for emulsifying these oily bases are surface-active, pharmaceutically acceptable substances having predominantly hydrophilic properties, such as, for example, non-ionic emulsifiers, in particular fatty acid esters of polyalcohols or ethylene oxide adducts (such as polyglycerol fatty acid esters or polyethylene sorbitan fatty acid esters) having an HLB value of less than 8. Additives which are added to the water phase are, inter alia, agents which prevent desiccation of the creams, for example polyalcohols, such as glycerol, sorbitol, propylene glycol and/or polyethylene glycols Nos. 200 to 600, and furthermore preservatives, odor-imparting substances, etc.

Ointments are water-in-oll emulsions which contain up to 70 %, but preferably between 20 and 50 %, water or aqueous phases.

Substances which are suitable as the lipid phase are mainly hydrocarbons, for example petroleum jelly, paraffin oil and/or solid paraffins, which ---24--- contain hydroxy compounds suitable for improving the water-binding capacity, for example fatty alcohols or esters, such as cetyl alcohol or wool wax alcohols.

In some cases, emulsifiers having an HLB-value of 8 to 16, such as, for example, sorbitan fatty acid esters (such as sorbitan isostearol) are also added. Additives which are added to the water phase are, inter alla, humectants, such as polyalcohols (glycerol, propylene glycol, sorbitol and/or polyethylene glycols No. 200, 400, 600); and furthermore preservatives, odor-imparting substances, etc.

Fatty ointments are anhydrous and chiefly contain hydrocarbons as the base, for example paraffin, petroleum jelly and/or liquid paraffins; moreover natural or partial ly-synthetic fats, such as, for example, coconut fatty acid triglyceride, furthermore: fatty acid partial esters of glycerol, such as, for example, the fatty alcohols, emulsifiers and/or additives which increase the water-absorption capacity, all of which have been mentioned in connection with the ointments.

Pastes are creams and ointments containing powder constituents which absorb secretions, such as, for example, metal oxides (such as titanium oxide or zinc oxide), and furthermore tale and/or aluminum silicates whose task it is to bind any moisture or discharge which may be present.

Foams are administered from pressurized containers and are oil-in-water emulsions of the spontaneously dispersible concentrates according to the invention which are present in aerosol form, with halogenated hydrocarbons (such as, for example, lower chloro-fluoroalkanes; such as dichlorodifluoromethane and dichlorotetra-fluorethane) being added as propellants. Other substances which may be added are the customary additives, such as preservatives, etc.

The present invention also relates to the use of the active substances, mixtures of active substances and spontaneuosly emulsifiable concentrates according to the invention for inhibiting the growth of tumour cells or as prophylactic agents against oncoses in humans and animals, administration preferably being carried out in the dosage forms which correspond to the pharmaceutical preparations described above.

Processing examples for inventive esters with dicarbonic acids according to formulae (1) to (V1).

1. Process for the preparation of azelaic acid bis ergosterylester a) To 9,5 9 azelaic acid in 100 m] toluene one adds dropwise at 10 OC 15 9 of oxaly] chloride (excess) in 50 m] toluene. Stand the solution at 20 OC during 12 hours. Evacuate subsequently the solvent as well as the excess of oxaly] chloride. One obtains 10,5 9 azelaic acid dichloride, with a boilingpoint of 243 - 248 OC.

b) To 800 mg ergosterol in 50 ml toluene add dropwise 250 mg azelaic acid dichloride, together with 200 mg dimethy1formamide. After heating for 2 hours to 60 OC, the solvent is distilled off. The residue is being recristallised in acetonitrile/chloroform 2:1.

The azelaic acid bis ergosterylester is obtained, as white crystals, with a melting point of 183,7 OC.

In a corresponding way, also the following compounds can be prepared:

Azelaic acid bis Cholersteryiester mp 138 - 139 OC IR 2942 cm-1 v(CH) 2868 v (CH) 1723 v (C=O) Ester 1467 1376 1188 1010 Sebacic acid bis Ergosteryiester RI Sebacic acid bis Cholesterylester Sebacic acid bis Sti 9 masteryl ester Glutaric acid bis Stigmasterylester Glutaric acid bis P-Sitosterolester Glutaric acid bis Cholesteryiester Glutaric acid bis Ergosterylester Adipic acid bis Cholesterylester Adipic acid bis Ergosteryiester Adipic acid bis Stigmasteryiester Fumaric acid bis Sti 9 masteryl ester Fumaric acid bis Cholesterylester Fumaric acid bis Ergosterylester 8 (C H) 8 (CH3) v (C-0) Ester v (C-O) 1.52514 m P 172,8 173,4 OC it 158,5 - 160,5 0C to 168,5 -C a@ 179,9 'C to U $R do H 31 11 11 196,8 'C 216,7 C 194,8 'C 222,5 - 223,5 'C 216,2 'C 277,5 - 278,5 'C 240, 0 - 240,5 'C 181,4 - 182,8 'C 2. Preparation of Azelaic acid Ergo ca Icife ryidies ter.

To 800 mg Ergocalciferol (Calciol) in 40 mi toluoene one adds dropwise at room temperature 250 mg azelaic acid dichloride and 300 mg pyridine in 25 mi toluene. After heating to 50 "C for 1 hour, the solvent is distilled off. The residue is chromatographed on a silicagel column with cyclohexanelethyl acetate (90:10) as eluent.

The azelaic acid ergocalciferyidiester is obtained, with a refractory index (R1) n20 of 1,51860. D Infrared spectrum:

IR 2932 cm-1 2870 1721 1630 1466 1370 1187 958 In a similar way, also the following compounds can be prepared:

Azelaic acid retinyl-diester R] 1.56858 (chromatographed with n-hexanelethyl acetate 80:20) Azelaic acid Cholecalciferyl-diester RI 1.54920 Succinic acid Cholecalciferyi-diester RI 1.53252 Fumaric acid Ergocalciferyi-diester Glutaric acid Ergocalciferyi-diester Azelaic acid Ergocalciferyi-diester Sebacic acid Ergocalciferyi-diester (CH) (CH) (C=O) Ester (C=C) 8 (CH) 8 (CH3) V (C-0) 8 (CH) trans C=C of (D3) RI RI RI RI 1.53774 1,54224 1.52340 1.53528 The characteristic ester bindings can be detected in the infrared spectrum at a wave length of ca. 1730 cm-1.

IR-SPECTRA, measured on a spectro photometer Perkin Elmer 983G:

Azelaic acid Erg ocalciferyl-diester IR 2958 cm-1 v (CH) 2930 " v (CH) 2870 " v (CH) 1723 1635 v (C=O) Ester v (C=C) of D2 1459 cm-1 1370 1185 973 M 2982 cm-1 2961 2865 1728 1653 1630 1605 1455 1377 1360 960 & (CH) 8 (CH3) v (C-0) Ester 8 (CH) trans v (CH) v (CH) v (CH) v (C=O) Ester V (C=C) v (C=C) V (C=C) 8 (CH) 8 (CH3) 6 (CH3) 8 (CH) of trans (C=C) Azelaic acid Retinyi-diester ad 01 11 at 91 H se 9 1 0 1 Structure of azelaic acid retinyi-diester confirmed by FT Raman- Spectroscopy. The ester-carbonyi-group is not visible.

Sebacic acid Ergocalciferyidiester IR 2957 cm-1 2871 1723 1459 1371 1183 973 (CH) (CH) (C=O) Ester 8 (CH) 8 (CH3) v (C-0) Ester 8 (CH) 3. Preparation of glutaric acid ethyl-Ergocalciforyiester To 400 mg Ergocalciferol (Calciol, Vitamin D2) in 30 mi toluene at room temperature one adds dropwise 200 mg (excess) glutaric acid- monoethylesterch to ride (bPO,07 68-70 "C) and 150 mg N,N- dimethylformarnide in 25 mi toluene. After heating for 2 hours to 60 OC, the solvent is distilled off. The residue is chromatographed on a silicagel column with cyclohexanel ethyl acetate (90: 10) as eluent. One obtains the Glutaric acid ethyl-Ergocalciferyiester RI 1.51656 In a corresponding manner, also other (asymmetric) dicarbonic acid methyland ethyidiesters with sterols and vitamin-compounds can be prepared, such as e.g.: Azelaic acid Erg oste ryl-methylester Azelaic acid Cholecalciferyl-methylester Azelaic acid ethyl-Cholecalciferyi-ester R] 1.49260 RI 1.49418 RI 1.52722 Composition examples of spontaneously dispersible agents which contain as substances possessing antitumour activity new dicarbonic acid ester compounds according to the formulae (1) to (111):

a) 0,5 to 25 % by weight of one or several of the dicarbonic acid ester compounds of the formulae (1) to (VI) 0,1 to 40 % by weight of isopropylmyristate, isopropylpalmitate or MiglyolO 812 (Dynamit Nobel) 20 to 45 % by weight of emulsifier mixture Diphasole 3873 (CIBA-GEIGY) or the identical product Sermule EA 188 (SERVO) to 45 % by weight of InvadinO JFC 800 % (CIBA-GEIGY) 0 to 60 % by weight multifunctional glucose derivatives, such as Glucate(P SS and Glucamatee SSE-20 (AMERCHOL) b) 0,5 to 25 % by weight of one or several of the dicarbonic acid ester compounds of the formulae (1) to (VI) 0,1 to 40 % by weight of isopropy1myristate, isopropylpalmitate or Miglyole 812 (Dynamit Nobel) to 45 % by weight of Invadin@ JFC 800 % (CIBA-GEIGY) 20 to 45 % by weight of SoprophorQD FL (Rh6ne-Poulenc) 0 to 50 % by weight multifunctional glucose derivates, such as Glucatee SS and Glucamate@ SSE-20 (AMERCHOL) c) 2,5 to 25 % by weight b) 0,5 to 26 % by weight of one or several of the dicarbonic acid ester compounds of the formulae (1) to (VI) 0,1 to 40 % by weight of isopropy1myristate, isopropylpalmitate or Miglyole 812 (Dynamit Nobel) to 60 % by weight of lnvadinO JFC 800 % (CIBA-GEIGY) to 40 % by weight of Amphonyle CAA and/or CA-AA (Oranienburger Chemikalien A.G., agent: Christ A.G., Aesch/ BL) MiglyolO 812 is a neutral oil (oleum neutrale) of Dynamit Nobel, which is a triacylglycerol of the coconut fatty acids, the so-called fractionated, middle chained (C8 to Clo) compounds [i.e. a caprylic/capric triglyceride in the CTFA classification].

DiphasolO 3873 (C1BA-GEIGY) (which is equivalent to SERMULC EA188) is a surfactant mixture consisting of the following two compounds (50:50):

0 11 CgH19 -0 (-CH2,CH2-0)-j -OP--OCH3 -0 1 Uri 0 11 C9H19 (-CH2-CH20 1 MOP-OCH3 Uk;r13 DiphasolO 3873 (C1BA-GEIGY) Invadin00 JFC 800 % (C1BA-GEIGY) is a tort. octyl phenyl polyoxyethyleneether with 9 to 10 oxyethylene groups SoprophorO FL (Rh6ne- Poulenc) is a tristyrylphenolpolyoxy-ethylene-18monoldimethyl- phosphoracidester. with formula:

0 11 (-CH2 --CH2--0)1-P-OCH3 % 8 1 CH3- 1+ do! H-CH3 H-CH3 OH Soprophor FL (Rh6ne-Poulenc) Amphonyle CAA and CA-AA respectively (Oranienburger Chemikalien A.G.) is an amphoteric, salt and waterfree imidazole derivative of coconut fat, with formula:

CH2 - CH2 'OH 1 N CH2 COCOYL- C 1+] 1 CH2 CH2- CH2 - Coo 1-1 Example for the pharmaceutical production of a system's preparation containing the inventive concentrates in the form of "multiple units". a) Granulation (granules and pellets) MetoloseO 90 SH-4000 (Shin-Etsu Chemical) AvicelC PH-101 Inventive MARIGENOLO-CONCENTRATE Aerosi10 200 71 90.09 80,39 139,49 80,39 390.09 Granulation in the high speed mixer or the fluidized bed, with the addition of 110 9 ethanol, sieving on a 18 to 42 mesh screen with crushing, drying for 24 h at 40 "C.

b) Enteric and sustained release coating In the fluidized bed with A00ATO AS-HG (Shin-Etsu Chemical) and Talc c) Composition of finished granules or micropellets Core Material Inventive MARIGENOLOD-CONCENTRATE Enteric coating E 44 % by weight % " go 31 % " U 100% N.B. The pellets or granules according to a) can also be filled without prior coating into capsules which are made of AQ0ATO (HPIOC-AS-M or H13MC- ASN), have been seated with acetonelethanol 1:1 and can thus perform the functions of pH-control and slow release.

P.S.: MARIGENOLOD is a trade-mark (Tm) of MARIGEN S.A., RIEHEN.

BIOLOGICAL ASSAYS.

The antiturnour activity of spontaneously dispersible concentrates containing active substances prepared according to the examples No. 1 to 4 is confirmed by the following test results:

1. In-vitro assays using suitable tumour cell lines A biological assay system using microtiter plates and serial dilutions has been developed. Batches of 104 tumour cells per mi were set up in culture medium RPM] 1640 and inactivated with 10 % of fetal calf serum (GIBCO); they are spread at a density low enough to enable them to grow during the assay, in so-called non-confluent monolayers. Samples are added after 6 24 hours, with 100 pi per row, to which 100 pi of medium are added in the first well. Half of this mixture is withdrawn, transferred into the next well and again treated with 100 p] of medium, etc. This results in an W/2 geometrical serial dilution.

In the plaque assay, the samples are incubated at 37 C for 3 to 5 days under 31/2 % Of C02. They are then stained and fixed using 0.1 % crystal violet (Fluka, Buchs) in a solution of 70 % of methanol, 1 % of formaldehyde and 29 % of water. The samples are evaluated under the microscope, magnification 300x. The greatest cytotoxic dilution is determined. The samples can also be evaluated quantitatively by means of scanning and absorption measurement in a spectro p h oto meter.

EVALUATION OF RESULTS PY6 polyoma virus PY6 polyoma virus TUMOR CELL LINE transformed 3T3 transformed 3T3 mouse FIBROBLASTS mouse FIBROBLASTS PREPARATION (CONCENTRATE with) In dilution active up to In dilution active up to EXPOSURE 40 h 64 h FUMARIC ACID ERGO- 9,6 Mio. 19,2 Mio.

STERYL-DIESTER MALONIC ACID ERGO- 1,0 Mio. 2,0 Mio.

STERYL-DIESTER AZELAIC ACID CHO- 19,2 Mio. 19,2 Mio.

LESTERYL-DI ESTER SEBACIC ACID ERGO- 4,8 Mio. 4,8 Mio.

STERYL-ESTER AZELAIC ACID bis- 9,6 Mio. 9,6 Mio.

CALCIFERYL-ESTER GLUTARIC ACID bis- 2,2 Mio. 4,4 Mio.

CALCIFERYL-ESTER AZELAIC ACID 2,0 Mio. 4,0 Mio.

RETINYL-DIESTER AZELAIC ACID 1,0 Mio. 2,0 Mio.

CHOLECALCIOL MONOESTER SEBACIC ACID 1,0 Mio. 2,0 Mio.

CALCIOL-DIESTER AZELAIC ACID ERGO- 640'000 STERYL-METHYLESTER AZELAIC ACID CHOLE- 640'000 CALCIFERYL-M ETHYL ESTER CONTINUATION PY6 Polyoma-Virus.

TUMOR CELL LINE transformed 3T3 Mouse FIBROBLASTS PREPARATION In dilution active up (CONCENTRATE with) to 1 EXPOSURE 6 days CROTONIC ACID-133-DIESTER 2'660'000 AZELAIC ACID-D3-DIESTER Vl 28'000 MALONIC ACI D-ERGOSTERYL-DI ESTER 40'000'000 DOSE-EFFICACY ASSAYS in-vivo (mouse) The assay was conducted with old reproduction animals Balb-c fem. (Charles River, Calco, Italy) Tumor-Cell-Line TSA (spontaneous murine mammalian adenocarcinoma), subcutaneously positioned with 4 x 104 cells.

The active substance was administered by syringe as aqueous microemulsion 1:100'000 (= 10 ppm active substance content), dose: 0,5 ml/per os 1 times/day, respectively 2 times/day.

Groups of 5 animals each. (This kind of tumor "catches" almost without exception and grows regularly as a subcutaenous mass). This fact allows to keep the statistical classes relatively small.

As a guiding substance the crotonic acid Cho lecalcif eryi-Ester (C 4:1133) was administered in the form of an inventive concentrate and of the aqueous microemulsion prepared therewith.

Measurement: palpation of the solid tumor mass: 1 [1/2 length + 1/2 breadth] in mm RESUL T In- vivo per os Duration of Controls D3-Crotonate D2-Oleate treatment (C4:1-D3) (C18:1-D2) 1 X 0,5 mildie 0 days 0 0 0 0 0 0 0 0 0 0 days 0 0 0 0 2 0 0 0 0 0 days 0 0 0 0 6 0 0 5 5 6 days 0 0 7 14 0 0 7 12 13 days 0 6 20 0 0 15 20 20 2 x 0,5 mildle 2 x 0,5 mildie 0 days 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 days 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 days 0 0 0 0 6 0 0 0 0 0 0 0 0 days 0 0 7 14 0 0 0 0 0 0 0 0 days 0 6 20 0 0 0 0 0 2 3 8 Assays made under the guidance of Prof. Dott. Guido FORM, Universitb degli Studi di TORINO, Scuola di Medicina, Istituto di Microbiologia.

The adenocarcinoma TSA is a very aggressive tumor line. It is being used as standard for evaluating cytostatica.

Action pattern of MARIGENOLO-concentrates in topical administration In order to gain first insights into the action pattern of the inventive concentrates in topical administration tests were conducted with the naked mouse, using as key substances: Ergosteryl-Undecenoate and ErgosterylCrotonate (Topi nu/nu f., Charles River, Calco, Italy), which were immunodepressed by means of splenectomy and subletal irradiation 4,5 Gy Cs137 dose 0,5 Gy/min.

The human lung adenocarcinorna ADK-P (an extremely malignant cell line) was implanted 3 days later, subcutaneously into the flank with 106 cells. This sort of tumor is very reliable, it "catches securely and without exception. It grows regularly and forms a solid mass und the skin. Period of latency 10 2 days, subsequently growth of the solid mass from 3 to 10 mm within a period -- 35 -- of 61 5 days. Close observation revealed no detectable metastasis, no spontaneous recidivism. Killing of the animals when the turnor mass reaches 15 mm.

Injection of a microemulsion 1:100'000 containing 10 ppm active substance when the tumor mass reached 4 mm of size, with 1 ml microemulsion 2 - 3 times per day directly into the tumor mass and its surrounding.

RESULTS: Retardation of the tumor growth by ca. 15 days. ONE COMPLETE REGRESSION BY FOUR, after 20 days of treatment. Observation of the treated animals lin the ensuign period during further 90 days. There were no metastases, no relaps. Pathological control of organs normal. No neoplastic tissues formed.

This may allow the conclusion that the described results could be achieved without the immediate participation of or the mediation by the immune system.

FORMATION OF ANTIBODIES Tests were conducted on behalf of MARIGEN by Readysysteme A.G., Bad Zurzach/Gamma, University of Li6ge, Belgium, on rabbits, with the objective of ascertaining a possible reaction of the immune system when adminis-tering inventive sterolester compounds, in particular:

CALCIOL-LINOLEATE [C 18:2-132-Ester] as key substance, the coemulgator C 11:1-Citronellylester and the "combination" sterolester/terpenester 25/75. Analysis of the serum using the ELISA-method did not evidence any traces of antibodies in the form of immunoglobulines. There were no allergens becoming manifest.

BLOOD VALUES The surface tension was measured using human peripheral blood immediately after drafting and mixing with an inventive concentrate, from which a microemulsion was prepared by adding distilled water, as described above.

-- 36 -- FULL BLOOD FULL BLOOD BLOOD VALUES EDTA Na-Citricum PROBES MNm-1 MNffl-1 Plate Ring Plate Ring 1) Full blood (whole blood) 51,6 51,6 51,4 51,4 2) Full blood mixed 1: 0,5 with 47,9 45,7 Azelaic acid Ergosteryl-di ester-concentrate 1:1'000 (C) ppm active substance content 3) Full blood mixed 1: 1 with 45,4 45,4 44,8 44,1 Azelaic acid Ergosteryl-di ester-concentrate 1:1'000 (C) 4) Full blood mixed 1: 5 with 44,9 46,4 45,2 Azelaic acid Ergosteryl-di ester-concentrate IA'000 (C) 5) Full blood mixed 1: 5 with 38,3 39,6 39,8 Ergosteryl-Undecenoate-con centrate 1:1'000 (C) 6) Full blood mixed 1: 1 with 32,1 32,7 32,9 Cholecalciferyl-Undecenoate concentrate 1: 100 (C) = 1,000 ppm active substance content N.B.: Measurements conducted on a Kriiss Digital-Tensiometer K10T Vacutainer Becton Dickinson The surface tension of normal blood amounts to 56,2 mNm-1 (Average). CfGeigy Scientific Tables Volume 3, Physical Chemistry, Composition of Blood, Hematology, Somatometric Data, 8th ed., Basel, 1984, p. 69.

METABOLISM During 5 consecutive days, healthy persons took 2 capsules/day each with 600 mg of an inventive concentrate having a 10%-STEROLESTER CONTENT. (Concretely: RETINOIC ACID ERGOSTERYL-ESTER as analytically suitable -- 37--- guiding substance). By means of thin layer chromatography, it could be shown that:

a part of the sterolester was eliminated in the urine, i.e. in the ester form a part of the sterolester could be traced, equally unchanged, in the blood the blood status evidenced no changes whatsoever, which gives an important indication regarding the toleration of the micellar solubilization and transport system used.

It may be emphasized that the diffusion of the inventive concentrates and microemulsions obeys a general physical principle, which was taken as guideline when optimizing the composition of these inventive concentrates. The penetration of the plasma membrane of tumor cells (and the subsequent spreading in the cell plasma) is illustrated by means of EM- photographs, using as key substances a 2%-concentrate prepared with ERGOSTEROL-10UNDECENOATE and ERGOSTEROL-all trans-RETINATE respectively. Cf. Figures 1/4 and 2/4 in the Annexure.

The analytical proof of the penetration of the plasma membrane of the tumor cell can be furnished using as method the "Micellar Electrokinetic Capillary Chromatography - MECC." This requires the preparation of an appropriate buffer system and the careful definition of the conditions for the assess-ment of the electroosmotic flux. In both samples, the concentrated urine and the blood fraction, the sterolesters could clearly and unequivocally be traced. Cf. the figures 3/4 and 4/4 in the annexure.

Figure 1/4 demonstrates the penetration of the membrane of Py6-cells (Fibroblasts) with ERGOSTEROL- I O-UND EC ENOATE Figure 2/4 demonstrates the penetration of the membrane of Py6-cells (Fibroblasts) with ERGOSTEROL-all trans-RETINATE Figure 3/4 gives as reference the MECCdiagram of the control substance ERGOSTEROL-all trans-RETINATE Figure 414 shows the MECC-diagrams for the blood probe and the urine probe containing ERGOSTEROL-all trans-RETINATE.

Claims (10)

1. PATENT CLAIMS 1) Dicarbonic acid esters of the formulae (1) to (V1)

   FooC.fCH2)- COOR3 n R.0OC-----(CH2);7 COOR, R300C - C - CH - COOR3 1 R2 R300C C 1 R2 CH - COOR, (1) (11) (111) (IV) 000 0 00 COOR 3 R300C CH3 CH3 (V) H3 H3 COOR3 R300C CH3 CH3 (V[) whereby in the formulae (1) to (11) the letter n stands for the numbers 1 to 18, and in the formulae (1) to (V[) Ri designates a Cl to C20 alkyl or C2 to C20 alkenyl group, R2 means hydrogen or methyl and R3 represents one of the radicals of the following formulae:

   C1.6 R4 CH3 H (VII) rh 4 H3 j 5 (Vill) H3 R4 H3 R4 H H H H (1x) (X) 1::;::1H3 R4 H #H 3 rH 3 H3 R4 H Cr H- 3 CH3 (X11) H3.R4 H H3 R4 BH #H 3 z z a H3 CH 3 H H H3 (X111) (XIV) 3 1H 3 R4 r6H, H3 H3 R4 H (XV) (XVI) whereby in the formulae (V11) to (XVI) the radical R4 stands for a Cl to Clo alkyl or a C2 to Clo alkenyl group, H :00.,.3 R) JQ 6 M3 (XVII) ZH H H1 CH3 3 3 F4 00. CH3 7 CH3 AS CH3 H3 H' 3 CH3 H3 CH2 CH3 R'F 4 H3 ;.,, CH3 R4 oo& (111) CH3 CH3 (V) CH3 (XVIII) 1-, CH3 x\\C H 3 18% C H 3 CH3 CH2 CH3 Rfr 4 H3 3 H3 CH3 CH3 H 3 3 CF CH CH3 R4 CH3 (IV) CH3 (V]) and where in the formulae (XVII) and (XVIII) the radicals R5, R6 und R7 represent hydrogen or methyl, Ra --tCH == CH- CH3 1 H3 CHOY -n, (XXIII) == CH-CH= C.)- CH = CH R,B---tCH=CH- C"= CH-)-+CH..O.

   m 1 m CH3 (XXIV) whereby in the formulae (XXIII) and (XXIV) the letter m means the numbers 1, 2, 3, 4 or 5 and R8 stands for one of the radicals of formulae:

   H3 &O a CH3 CH3 CH3 &H3 CH3 1 n L4 H3 OCH3 CH3 CH3 H3 H5 CH3 CH3 OH H3 0 0 CH31: 0CO-CH3 CH3 "3 CH3i CH3 H3 6 3 CH3 CH3 3 H3 H3 H3 6 3 C 3 H3 H3 CH3&OH CH3 H3 C 3 H H3 CH3 CH3 H3 %".4 CH3 CH3 0CO-CH3 CH3 H or C040H CH3 CH3
2. 2. The compounds according to claim 1, whereby in the formulae (1) and (11) the letter n means the number 1 to 8, R1 stands for methyl or ethyl, R2 is hydrogen and R3 determines one of the radicals of formulae (XXV) to (XXX) H3 H3 H3 ZH3 H.
3. 3 CH3 CH3 H3 H3 H3 AHJ3 CH3 0 CH3 ' H3 H3 H3 F A J3 H, H3 H3 H3 2H5 CH3 CH3 H3 (XXV) (XXVI) (XXVII) (XXVIII) 2H5 CH3 'CH3 FH3 AH 3 H3 (XXIX) CH3 CH3 CH3 v H3 (CHi-CH2---CH-CH)-CH 2 3 3 HQ 3. The compounds according to claim 2 Malonic acid bis-Stigmasteryiester Succinic acid bis-Stigmasteryiester Glutaric acid bis-Stigmasterylester Adipic acid bis-Stigmasteryiester Pimelic acid bis-Stigmasteryiester Suberic acid bis-Stigmasteryiester Azelaic acid bis-Stigmasteryiester Sebacic acid bis-Stigmasteryiester Azelaic acid bis-b-Sitosteryiester Sebacic acid bis-b-Sitosteryiester Azelaic acid bis-Ergosterylester Sebacic acid bis-Ergosteryiester Azelaic acid bis-Cholesteryiester Sebacic acid bis-Cholesteryiester Maleic acid bis-Stigmasteryiester Fumaric acid bis-Stigmasteryiester Maleic acid bis-P-Sitosteryiester Furnaric acid bis-P-Sitosteryiester Maleic acid bis-Ergosteryiester (m) Fumaric acid bis-Ergosterylester Maleic acid bis-Cholesterylester Fumaric acid bis-Cholesterylester Azelaic acid Calciferyl-diester Sebacic acid Calciferyl-diester Azelaic acid Cholecalciferyl-diester Sebacic acid Cholecalciferyl-diester Azelaic acid DL-a-Tocopheryl-diester Sebacic acid DL-a-Tocopheryl-diester Glutaric acid ethyl-Calciferylester Glutaric acid Calciferyl-methylester Fumaric acid ethyl-Calciferylester Fumaric acid Calciferyl-methylester Azelaic acid ethyl-Calciferylester Azelaic acid Calciferyl-methylester Azelaic acid ethyl-Cholecalciferyiester Azelaic acid Cholecalciferyi- methylester Sebacic acid ethyl-Cal cif erylester Sebacic acid Calciferyl- methylester Sebacic acid ethyi-C ho leca lcif e ryl ester Sebacic acid Cholecalciferyl-methylester
4. 4. Procedure for the preparation of compounds described in the formulae (1), (111), (V) and (V1) according to claim 1, characterized by reacting a compound of the formulae (XXXI) to (XXXIV) HOOC---+ CH2)' COOH n HOOC-C-- CH - COOH 1 R2 H3 H 3 COOH HOnn OC CH3 CH3 (XXXI) (XXXII) (XXXIII) H3 H3 00 COOH HWC CH3 CH3 (xxXIV) in which the letter n represents the numbers 1 to 18 and R2 means hydrogen or methyl, with 1,11-carbonyidiimidazole at 25 to 70 OC under addition of a catalytic amount of an alcoholate in an indifferent solvent, such as tetrahydrofuran, benzene, toluene or chloroform, followed by alcoholysis of the imidazolides formed with a sterol, a vitamin-D or a vitamin-E compound.
5. 5. Procedure for the preparation of compounds described in the formulae (1), (111), (V) and (V1) according to claim 1, characterized by reacting a compound of the formulae (XXX1) to (XXXIV) HOOC-- CHr---COOH n (XXXI) HOOC--C-'-" CH - COOH 1 R2 (XXX 11) H3 H3 COOH HOOC- CH3 CH3 (XXX 111) H3 H3 COOH H OOC CH3 CH3 (xxXIV) in which the letter n means the number 1 to 18 and R2 stands for hydrogen or methyl, with a chlorination or bromination agent in oder to prepare the dichloride or the dibromide, followed by the reaction of the dichloride or dibromide formed with a sterol, a vitamin-D or a vitamin-E compound at a temperature of between 40 and 80 OC in an indifferent solvent.

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6. 6. Procedure for the preparation of compounds of formulae (11) and (IV) according to claim 1, characterized by reacting a compound of formulae (XXXV) or (XXXVI) HALOGEN OC ---(CH2-)-COOR, n HALOGEN OC-CCH -COOR, 1 (XxXV) R2 (XxXVI) in which the letter n determines the numbers 1 to 18, R, stands for alkyl or alkenyl and R2 means hydrogen or methyl, with a sterol, a vitamin-D or a vitamin-E compound at a temperature of between 40 and 80 "C in an indifferent solvent and in the presence of a catalyst.
7. 7. Spontaneously dispersible concentrate which contains as the antitumor and biotenside components 0.001 to 25 % by weight of individual esters with dicarbonic acids according to formulae (1) to (VI), and combinations of such compounds respectively 0.001 to 40 % by weight of a solvent or solvent mixture which is pharmaceutically acceptable and acts as a hydrotropic or coemulsifier 0.001 to 90 % by weight of a pharmaceutically acceptable surfactant or surfactant mixture, and optionally up to 10 % by weight of a vitamin or provitamin up to 10 % by weight of a free fatty acid, and if appropriate, customary excipients and/or diluents.
8. 8. A pharmaceutical composition comprising I to 95 % of the spontaneously dispersible concentrate according to claim 1, and which further comprises an amount up to 10 % by weight of a pharmaceutically acceptable excipient, diluent, stabilizer, or a combination thereof.
9. 9. A therapeutic system's preparation according to claim 8, which contains 44 parts of core material for granules or pellets, 25 parts of the spontaneously dispersible concentrate and 31 parts of enteric and slowrelease-coating on the basis of hydroxylpropyl-methylcelluloseacetatesuccinate.
10. 10. A therapeutic system's preparation according to claim 8, which contains 64 parts of core material for granules and pellets respectively, and 36 parts of the inventive concentrate and which is filled into capsules made from hydroxypropyi-methyi-cellutose-acetate-succinate.