DD260279A5 - PROCESS FOR PREPARING HETEROCYCLIC COMPOUNDS WITH A 2- (2- [N, N-BIS-12) 2-CHLOROETHYL) DIAMIDO-PHOSPHORYLOXY] ETHYL) REST - Google Patents

Abstract

The invention relates to processes for the preparation of heterocyclic compounds with a 2- (2- & N, N-bis (2-chloroethyl) -diamido-phosphoryl-oxy! Ethyl) radical. The invention provides a process for the preparation of novel heterocyclic compounds which, compared to the known cytostatic agent "cyclophosphamide", have a considerably greater therapeutic breadth. The object of the invention is to provide processes for the preparation of novel compounds which can be used as highly effective cytostatics. According to the invention, the object is achieved by preparing compounds of general formula I in which the substituents which have the meaning given in the description are prepared by reacting a compound of general formula II in which the substituents have the meaning given in the description, with a compound of the general formula III in which the substituents which have the meaning given in the description are reacted. Formulas I and II

Description

Characteristic of the known state of the art

The compounds of the invention are highly potent cytotoxic agents with very low general and local toxicity (for example, acute and subatctic toxicity). In particular, the compounds of the invention also show a higher selectivity of the cytotoxic effect on human tumor cells with lower bone marrow toxicity (ie, compared to bone marrow toxicity). They therefore have, for example, a considerably greater therapeutic width (3-4 times greater) than the known cytostatic "cyclophosphamide." The compounds according to the invention are, for example, also active locally or intraeavitarily.

In addition, the compounds according to the invention are also suitable for the treatment of AIDS diseases. The antitumor effect of the compounds according to the invention is shown, for example, on the following models:

Bladder sarcoma of female nude mice (nu / nu mice);

S180Ascites tumor / mouse;

Yoshida ascites tumor / rat;

P815 mouse mastocytoma;

human tumor cells in the clonogenic assay in vitro.

In contrast to the "cyclophosphamide" and other cytostatic oxazaphosphorines, the release of the alkylan surprisingly takes place much more slowly in the compounds according to the invention.

Object of the invention

The invention provides a process for the preparation of novel heterocyclic compounds with a 2-2- [N, N-bis (2-chloroethyl) -didamido-phosphoryl-oxy] ethyl radical which has been prepared in comparison to the known cytostatic agent "cyclophosphamide". have a significantly greater therapeutic range.

Explanation of the essence of the invention

The object of the invention is to provide processes for the preparation of novel compounds which can be used as highly effective cytostatics.

The compounds according to the invention cause an enhancement of the immune defense (they therefore represent a biological response modifier). This enhancement of the immune defense takes place at a very low dosage (for example 0.05-5 mg per kg / body weight).

If in formula I at least two of the radicals R ₁ , R ₂ , R ₃ and R _{4 are} 2-chloroethyl, 2-bromoethyl or 2-C _r C ₄ -alkanesulfonyloxyethyl, then in each case these radicals may be the same but also may be different, that is, for example, R ₃ may be 2-chloroethyl and R _{1 is} 2-methylsulfonyloxyethyl (R ₂ and R ₄ = hydrogen).

If the radicals R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R _{8 are} C ₁ -C ₆ -alkyl, these are preferably methyl groups. Alkyl, alkoxy and alkanoyl groups occurring in the formula I may be straight or branched. If R _{8 is} a C ₂ -C ₆ alkanoyl group, it is in particular acetyl.

Such an alkanoyl group may also be an additional carboxy group preferably located at the terminal C atom of the alkanoyl group (w-position) and an existing amino group, for example, in proximity to the carboxyl group or in proximity to the CO group

 Examples include: α and γ-glutamyl.

If R ₇ and / or R _{8 are} d-Ce-alkoxycarbonyl, the alkoxy group preferably consists of one or two C atoms. If R _{7 is} a C 1 -C ₆ -alkylaminocarbonyl group and this contains an additional carboxyl group, the carboxyl group is preferably in the 1-position of the alkyl radical. Alkyl radicals which are attached to an aminocarbonyl group of the formula I. are preferably composed of one or two carbon atoms.

Particularly advantageous effects are exhibited by compounds of the formula I in which Z is a sulfur atom or the group-SC (R ₅ I ₂ ) (R _{5 is} in particular hydrogen).

If R _{7 is} a carboxylic acid amide group and the amide moiety is the residue of a natural amino acid or of a natural dipeptide, this natural amino acid or the peptide is linked to the carboxylic acid group via an amino group or amino function of the amino acid (preferably via the α-amino group) ₇ then has in particular the structure -CO-NH-CH (R ₁₈ I-CO-R ₁₉ , in which R _{19 is} OH, C ₁ -C ₆ -alkoxy or the radical NH-CH (R ₁₈ ) -COR ₂₀ and R _{18 represents} hydrogen, a C ₁ -C ₁₀ alkyl group, or a C ₁ -C 4 alkyl group represented by a hydroxy group, a

d-co-alkoxy group, a mercapto group, a C 1 -C 6 -alkylthio group, a phenyl group, a hydroxyphenyl group, an aminoCrCe-alkylthio group, an amino-d-Ce-alkyloxy group, an amino group, an aminocarbonyl group, an ureido group (H 2 NCONH-), a Guanidino group, a carboxy group or a d-Ce-alkoxycarbonyl group, or Ri8 forms together with the structural part -NH-CH-CO-R19 the 2-carboxy-pyrrolidinyl-1-residue (prolinyl (1) radical) or the 4-hydroxy-prolinyl (1) radical. R ₂ o is OH or C ₁ -C ₆ -alkoxy.

If Ra is the residue of a natural amino acid or a natural dipeptide, then this or the peptide is linked via the carboxy group to the 3-nitrogen atom of the heterocyclic ring of the formula I. R ₈ then means in particular the radical CO-CH (R ₂₁ (-NHR ₂₂ in which R _{21 represents} hydrogen, a d-do-alkyl group or a d-do-alkyl group represented by a hydroxy group, a d-Ce-alkoxy group , a mercapto group, a d-Ce-alkylthio group, a phenyl group, a hydroxyphenyl group, an amino-d-alkylthio group, an amino-d-Ce-alkyloxy group, an amino group, an aminocarbonyl group, an ureido group (H ₂ NCONH-), a guanidino group R ₂ i is the residue -CO- (CH ₂ ) _n -CH (NHR ₂₂ ) -CO-OR 23, in which R _{22 is} hydrogen, the group CO _{2 is} C 1 -C 4 alkyl or a C 1 -C 6 alkoxycarbonyl group. CH (R ₂₁ ) NH ₂ or the group CO- (CH ₂ ) _n -CH (NH ₂ ) -CO-OR _{23 is} hydrogen or dC ₆ -alkyl, and η is the numbers 1,2 or 3.

Particularly suitable residues of such natural amino acids or dipeptides (racemates, D and L forms) are those derived from the following amino acids: glycine, proline, phenylalanine, tyrosine.

If R _{7 represents} a carboxy group, the compounds of the formula I according to the invention can also be present in the form of the corresponding salts with physiologically compatible inorganic or organic cations.

Suitable inorganic cations are, for example:

NH4 ⁺ , cations of alkali metals (Na, K), alkaline earth metals (Ca, Mg), bismuth, aluminum or iron. Suitable organic cations are, for example, the cations of the following amines:

Primary, secondary and tertiary C 1 -C 6 -alkylamines which may also contain a second amino group (such as, for example, ethylenediamine, Ν, Ν'-dibenzylethylenediamine), ethanolamines (ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, choline, novocaine), cyclic amines ( for example C ₅ -C ₆ -cycloalkylamines such as cyclohexylamines guanidine, piperidine, N-ethylpiperidine, piperazine, morpholine, N-ethyl-morpholine, nicotinamide, glucosamine, methylglucamine, theobromine, caffeine, purines, imidazoles, furthermore the cations of homocysteine thiolactone or a-amino-e-caprolactam or a basic compound of the formula

^R 15 -? ^R 14 - ^C0R 13

I iv

NR ₁₆ R ₁₇

in question. In formula IV: R _{13 is} a hydroxy group, an amino group or a d-Ce-alkoxy group, R 14 is hydrogen or a difluoromethyl group, R _{15 is} hydrogen, an indolyl (3) -methyl radical, imidazolyl (%) -methyl radical, a C ₁ -Ci ₀ - • alkyl group or a C _r C ₁₀ alkyl group represented by a hydroxy group, a Ci-Ce alkoxy group, a mercapto group, a CrCö alkylthio group, a phenyl group, a hydroxyphenyl group, an amino-Ci-Ce-alkylthio group , an AmJnO-C ₁ -C ₆ alkyloxy group, an amino group, an aminocarbonyl group, a ureido group (H ₂ NCONH-), a guanidino group, a carboxy group or a d-Ce-alkoxycarbonyl group, or R ₁₅ forms together with the structural part CR ₁₄ (NR ₁₆ Ri ₇ ) the pyrrolidinyl (2) radical, the 4-hydroxy-pyrrolidinyl (2) radical or the 2-oxo-3-amino-3-difluoromethyl-piperidine and the radicals R ₁₆ and R _n are hydrogen or Ci-Ce-alkyl radicals.

Particularly suitable basic salt components are compounds of the formula IV in which Ri _{3 is} the hydroxyl group, R ₁₄ , R ₁₆ and R _{17 are} hydrogen and Ri _{5 is} hydrogen or a CrQ-alkyl group which is also denoted by an amino group or a guanidino group may be substituted (preferably in γ or δ position.

The alkyl groups, alkoxy groups, alkylthio groups occurring in the formula IV may be straight or branched. The Ci-C-io-alkyl group preferably contains 1-6 carbon atoms. The alkoxy groups and alkylthio groups are preferably those having 1-4, in particular 1-2, carbon atoms; the same applies with respect to the radicals R ₁₆ and Ri ₇ , if these are alkyl radicals. If in formula IV Ri _{5 is} an alkyl group which contains an amino-C ₁ -C ₆ -alkylthio (C ₁ -C ₆ -alkoxy) group, these are preferably the following groups: H ₂ N-CH ₂ -CH ₂ - S-CH ₂ -OdCr H ₂ N-CH ₂ -CH ₂ -O-CH ₂ -.

The compounds of the formula IV are preferably those compounds where Ri _{3 is} a hydroxy group and the radicals R _u , Ri ₆ and R _{17 are} hydrogen and R ₁₅ can assume in particular the meanings given below.

Preferred basic compounds of the formula IV are, for example, those where R _{13 is} a hydroxyl group, R _{14 is} hydrogen or difluoromethyl, R _{15 is} a C 1 -C 6 -alkyl group, in particular a C 1 -C 6 -alkyl group which (preferably in 2, 3, 4, 5 or 6-position; counting always begins at the point of attachment of the alkyl radical to the residual molecule), an amino group (especially in the 3- or 4-position), an aminoCrQ-alkylthio group, an aminoC ₂ -C ₄ -alkoxy group, a guanidino radical an imidazolyl (4) methyl radical or an indolyl (3) methyl radical and R ₁₆ and R _{17 are} hydrogen or C ₁ -C ₄ alkyl radicals; or amino acid derivatives of the formula IV, where R _{13 is} an amino group or a C ₁ -C ₄ alkoxy group, R _{14 is} hydrogen, R _{15 is} hydrogen, a phenylmethyl group, a 4-hydroxyphenylmethyl group or a dC ₆ alkyl group which (preferably in 2 , 3-, 4-, 5- or 6-position), a hydroxy group, a mercapto group, a dC ₄ alkylthio group, an aminocarbonyl group, a d ^ alkoxycarbonyl group or ureido group and R ₁₆ and Ri _{7 is} hydrogen or C ₁ -C ₄ -AlkyIreste mean.

In the abovementioned amino acids or amino acid derivatives, for example, the salts are each formed from one mole of the compound I and one mole of the compound IV.

Further suitable basic compounds of the formula IV are, for example, those where Ri _{3 is} an amino group or a C 1 -C ₄ -alkoxy group, R _{14 is} hydrogen or difluoromethyl, R _{15 is} a C 1 -C 4 -alkyl group, in particular C 1 -C 6 -alkyl group which (preferably in the 2-, 3-, 4- or ω-position) an amino group, an amino-C ₂ -C ₄ -alkylthio group, an amino-C ₂ -C ₄ -alkoxy group, a guanidino group, an imidazolyl- (4) -methyl group or an indolyl (3) -methyl radical and R ₁₆ and R _{17 are} hydrogen or C 1 -C ₄ -alkyl radicals.

In the case of the last-mentioned amino acid derivatives, the salts are formed, for example, in each case from 2 mol of the compound I and 1 mol of the amino acid derivative of the formula IV.

Individual examples of compounds of formula IV are: aspartic acid diamide (DL-form), aspartic acid aspartate (L-form), citrullinamide (H ₂ N-CO-NH- (CH ₂ ) 3-CH (NH ₂ ) -CONH ₂ , L-form) , Ornithine ethyl ester (L-form), arginine, arginine amide (L-form), 4-thialysine (H ₂ N-CH ₂ -CH ₂ -S-CH ₂ -CH (NH ₂ I-COOH), 2,6-diamino -nonanic acid (ε-methyllysine), 4-oxalysin (H ₂ N-CH ₂ -CH ₂ -O-CH ₂ -CH (NH ₂ I-COOH), glycinamide, Ν, Ν-dimethylglycinamide and the corresponding methyl or ethyl ester , Prolinamide, hydroxyprolineamide, phenylalanine amide, the methyl or ethyl ester of alanine or phenylalanine, homocysteine thiolactone (DL-form), a-amino-e-caprolactam [D (+) - form], lysine (especially L-lysine), difluoromethylornithine (DL or L form), valine methyl ester (L form), threonine ethyl ester, histidine, histidine methyl ester, histidine amide, alaninamide, ornithine.

In the salts, in the case where in the compound IV R 13 is an amino group or an alkoxy group and otherwise a basic group is present or in the case where Ri _{3 is} the hydroxy group but otherwise two basic groups are present, Compound I (acid Component) and compound II (basic component) in a practical ratio of 1: 1. (This also applies if the basic component is homocysteine thiolactone or α-amino-s-caprolactam.) On the other hand, in the basic component IV, R _{13 is} an amino group or an alkoxy group and contains, in addition to the α-amino group, yet another basic group, then, in general, the ratio of compound I to compound IV is 2: 1. The salts according to the invention are the neutral salts. The pH values of such salts are for example between 6-9, in particular 6-8.

Also suitable for example as the basic salt component are: amines of the formula NR'R "R '", in which the radicals R', R "and R '" are identical or different and represent hydrogen or C 1 -C 4 -alkyl groups which may also be a hydroxy group may contain (for example, oxyethyl).

Specific examples of such amines are: methylamine, ethylamine, propylamine, isopropylamine, dimethylamine, diethylamine, triethylamine, trimethylamine, tripropylamine, methylethylamine, dimethylethylamine, diethylmethylamine, 2-hydroxyethylamine, bis (2-hydroxyethyl) methylamine, (2 Hydroxy-ethyl) -ethylamine, (2-hydroxyethyl) -diethylamine, bis (2-hydroxy-ethyl) -ethylamine, (2-hydroxy-ethyl-methyl-ethylamine, cyclic amines being, for example, suitable: morpholine, piperidine, Pyrrolidine, piperazine, as diamines ethylenediamine, nicotinamide.

The compounds of the formula I according to the invention can also be present in the form of salts with physiologically tolerated acids. Examples of such acids are: hydrohalic acids, sulfuric acid, organic mono-, di- or tricarboxylic acids of the aliphatic, acyclic, aromatic or heterocyclic series and sulfonic acids. Examples of these are: formic, acetic, propionic, succinic, glycolic, lactic, malic, tartaric, citric, ascorbic, maleic, fumaric, hydroxymaleic or pyruvic acid; Phenylacetic, benzoic, p-aminobenzoic, anthranilic, p-hydroxybenzoic, salicylic or p-aminosalicylic, embonic, methanesulfonic, ethanesulfonic, hydroxyethylsulfonic, ethylenesulfonic; Halogenobenzenesulfonic, toluenesulfonic, naphthalenesulfonic or sulfanilic acid or 8-chloro-theophylline. Depending on the process conditions and starting materials, the end products of the formula I are obtained in free form (as basic compound), as acid or in the form of their salts. The salts of the end products can be converted into the bases or compounds I having a free carboxyl group in a manner known per se, for example with alkali, acids or ion exchangers. Of the latter, salts can be obtained by reaction with organic or inorganic acids or corresponding basic compounds, in particular those which are suitable for the formation of therapeutically usable salts.

Thus, for example, the carboxylic acids are neutralized at 0 ° C-20 ° C in aqueous solution with a base, preferably sodium bicarbonate and the solution is freeze-dried.

Amino acid salts, for example lysine and arginine salts, can be obtained by adding the equivalent amounts of the free amino acid to aqueous solutions of compounds of formula I at a starting pH of 4-5 and room temperature followed by lyophilization.

However, the replacement of the basic component of a salt of the compound I with a basic component can also be carried out, for example, on acidic ion exchangers which are charged with a basic compound IV. Examples of suitable acidic ion exchangers are those whose polymeric matrix carries sulfonic acid groups or carboxylic acid groups. The matrix of the ion exchangers may, for example, consist of a polystyrene resin, optionally containing 2 to 16, preferably 4 to 8, of divinylbenzene or of a phenolic resin. The polystyrene ion exchanger is preferably gelatinous. The loading of the ion exchanger with the basic component can for example be carried out in the following manner: 150 ml of ion exchange resin 1.2mval / ml * in a column (diameter about 4cm) with cooling jacket are regenerated with hydrochloric acid, washed neutral with distilled water and chloride ion, with a 10 % aqueous solution of the basic compound (220 mmol) IV and washed neutral with distilled water. To the procedure:

The process is carried out in a solvent at temperatures between -7O ⁰ C and 100 ⁰ C, preferably -1O ⁰ C to 8O ⁰ C, in particular 5 ⁰ C to 6O ⁰ C, that is optionally with cooling, at room temperature or with heating. If X and Y of the starting compound II are alkoxy groups or together form an alkylenedioxy group, the reaction with the other starting component III is expediently carried out in the presence of an acid catalyst, such as an inorganic or organic acid, for example trichloroacetic acid, p-toluenesulfonic acid, formic acid, hydrochloric acid, trifluoromethanesulfonic acid. When X and R _{4 form} the 6-membered ring (oxazaphosphorinane ring) and Y is hydroxy, alkoxy, alkylthio or the group-S-alk-SO ₃ Z, the pH of the reaction is adjusted, for example pH 5-12 , in particular 7-9, for example by means of alkali lye (NaOH). Suitable solvents are, for example: water, alcohols, in particular alkanols having 1-6 C atoms, such as methanol, ethanol, n-propanol, isopropanol or isobutanol, alkyl ketones each having 1-4C atoms, in particular acetone, methyl ethyl ketone, aprotic solvents (for example, dimethylsulfoxide, acetonitrile, N-methylpyrrolidone, dimethylformamide, hexamethylphosphoric triamide), halogenated hydrocarbons having 1-3 C atoms such as chloroform, ethylene dichloride, saturated cyclic ethers such as tetrahydrofuran, dioxane, saturated lower aliphatic ethers such as diethyl ether or the like Solvent or mixtures of several such solvents.

* Manufacturers of ion exchangers specify the capacity of ion exchangers (that is, the amount of functional groups such as -SO ₃ H, -CO ₂ H in mval / ml or mval / g of the ion exchange resin.

The introduction of the radical R ₈ by acylation according to the preparation process is carried out by isocyanic acid, C ₁ -C ₆ -alkyl isocyanic acid or by an acid of the formula R-OH, wherein R is a CrCe alkoxycarbonyl group, an aminocarbonyl group, a CT-Ce-alkylaminocarbonyl group, a Di-C ₁ -C ₆ -alkylaminocarbonyl group or a C ₂ -C ₆ -alkarioyl group or the residue of a natural amino acid or of a natural dipeptide, respectively, and such an acid or the dipeptide is preferably activated. If such activated acid is used for the acylation, these are preferably compounds of the formula RW, wherein R has the meanings given above and W is halogen (for example chlorine or bromine), but also, for example, a group of the formula -OR ', -SR' or a group of the formula -OSO ₃ H or -OCO-R "and R 'is a Ci-C ₆ alkyl radical or a phenyl radical, one by nitro groups, C _r C ₄ alkoxy groups, dC ^ alkyl groups represents or halogen atoms (chlorine, fluorine, bromine), a cyanomethyl radical or a carboxymethyl radical and R "is a straight or branched C ₁ -C ₆ -AlkyIrest, a Ci-C ₆ -AIkOXyrest, a phenoxy or benzyloxy group, wherein R and the group COCI can be if W is halogen and in this case is followed by a subsequent reaction in the usual manner with NH ₃ , a C _r C ₆ alkylamine or a di-C _r C ₆ alkylamine. If W is a halogen atom, it is preferably chlorine, bromine or iodine; If R 'or R "are alkyl radicals, haloalkyl radicals or alkoxy radicals, these are preferably of low molecular weight and consist of 1-4 carbon atoms Frequently, especially when W is a halogen atom or the group -OCOR", the presence of an acid-binding substance such as alkali metal hydroxides , Alkali metal carbonates, alkali metal bicarbonates, alkali metal acetates, alkaline earth carbonates, tertiary amines (trialkylamines such as triethylamine, pyridine) or alkali hydrides and the like appropriate. The acid-binding agent can also be used simultaneously as a solvent alone or in admixture with other customary agents (for example pyridine).

If the free acid R-OH is used, its activation is due to the presence of condensing agents such as dicyclohexylcarbodiimide, tetraethylpyrophosphite, 5- (3'-sulfonphenyl) ethylisooxazole, sulfonated bis-alkylamides (for example, SO [N (CH ₃ ) ₂ ] 2, N, N'-carbonyldiimidazole and so on (Organic Reactions, Vol. 12, 1962, pp. 205 and 239).

If R _{8 is} a d-Ce-alkyl group, this may be introduced into such compounds of formula I, wherein R _{8 is} hydrogen, by alkylation. Likewise, in compounds of the formula I in which Z is the group NH or -NH-C (R ₅ I ₂ -), the hydrogen of this basic nitrogen atom is replaced by alkylation by a C 1 -C 6 -alkyl group.

This alkylation is carried out, for example, by reaction with compounds of the formula Ci-Ce-alkyl-Hal ArSO ₂ O-C ₆ - alkyl and SO ₂ (O-Ci-C ₆ alkyl) _2, wherein Hal is a halogen atom (in particular chlorine, Bromine or iodine) and Ar may be an aromatic radical (for example a phenyl or naphthyl radical optionally substituted by one or more lower alkyl radicals.) Examples are p-toluenesulfonic acid C 1 -C ₆ -alkyl esters, C 1 -C 4 -dialkyl sulfates, C 1 -C ₆ -alkyl halides and the like .. The alkylation reaction is conveniently carried out in the presence of customary acid-binding agents, for example, the acid-accepting agents are the same as those used for the acylation.

The acylation or alkylation is for example at temperatures from -60 to 220 ⁰ C, preferably -20 and 150 ⁰ C, in particular +20 ⁰ C and 110 ⁰ C in inert solvents or suspending agents. Suitable solvents or dispersants are, for example: aromatic hydrocarbons such as, for example, benzene, toluene, xylene; aliphatic ketones such as acetone, methyl ethyl ketone; halogenated hydrocarbons, such as chloroform, carbon tetrachloride, chlorobenzene, methylene chloride; aliphatic ethers, such as butyl ether; cyclic ethers, such as tetrahydrofuran, dioxane; Sulfoxides, such as dimethylsulfoxide; tertiary acid amides such as dimethylformamide, N-methylpyrrolidone, hexamethylphosphoric triamide; aliphatic alcohols such as methanol, ethanol, isopropanol, amyl alcohol, tert-butanol, cycloaliphatic hydrocarbons such as cyclohexane and the like. Aqueous mixtures of the solvents mentioned can also be used. Often one works at the reflux temperature of the solvents or dispersants used. Often, the alkylation reaction components are used in excess. The alkylation can also be carried out in the presence of tetraalkylammonium salts (in particular halides) in combinates with alkali hydroxides at temperatures between 0-100 ⁰ C, preferably 20-8O ⁰ C in an aprotic solvent or in chloroform or methylene chloride. Suitable aprotic solvents are, in particular: tertiary amides (dimethylformamide, N-methylpyrrolidone, hexamethylphosphoric triamide), dimethyl sulfoxide, acetonitrile, dimethyloxyethane, acetone, tetrahydrofuran. In the acylation and alkylation, it is also possible to prepare an alkali compound from the compound to be reacted by reacting it in an inert solvent such as dioxane, dimethylformamide, benzene or toluene with an alkali metal, alkali metal hydrides or alkali metal amides (in particular sodium or sodium compounds). or butyllithium at temperatures between 0 and 15O ⁰ C and then, for example, the acylating or alkylating agent (compound RW, W = halogen) inflicts. Instead of the alkylating and acylating agents mentioned, other chemically equivalent agents commonly used in chemistry can also be used (see, for example, LF and Mary Fieser "Reagents for Organic Synthesis", John Wiley and Sons, Inc., New York, 1967, Vol , Pages 1303-4 and Vol. 2, page 471).

The acyl groups in the compounds of the formula I can be cleaved off again by solvolysis, giving the corresponding compounds of the formula I in which R _{8 is} hydrogen. This solvolytic cleavage takes place for example by saponification with dilute acids or by means of basic substances (potash, soda, aqueous alkali solutions, NH ₃ ) at temperatures between 10 and 150 ⁰ C, in particular 20-100 ⁰ C. The conversion of the radical R ₇ in others for this purpose possible treatments can be carried out by the following reactions:

a) saponification

The saponification consists of converting compounds of the formula I in which R _{7 is} a d-Ce-alkoxycarbonyl group, an aminocarbonyl group or an alkylamino or dialkylaminocarbonyl group (in each case with alkyl radicals of 1-6 C atoms) into a compound of the formula I. wherein Ri is the carboxyl group. This saponification is carried out for example by water, dilute aqueous alkali solutions, alcoholic alkali or optionally by means of mineral acids such as hydrochloric acid or sulfuric acid in alcoholic or aqueous-alcoholic solution at temperatures between 20 and 200 ⁰ C, preferably 20-100 ° C or by NaCl / dimethyl sulfoxide , It is also possible to use further inert solvents, such as dioxane, tetrahydrofuran, dimethylformamide, dimethylacetamide.

-6- ZbU Z / S

b) esterification

The esterification can be carried out, for example, by reacting carboxylic acid salts of compounds of the formula I (R ₇ = COOH), in particular alkali salts or silver salts, with C ₁ -C ₆ -alkali halides (chlorides, bromides, iodides) or by treating the free acids with dC ₆ -diazoparaffins or Ci-Ce-aliphatic alcohols in the presence of acidic substances such as hydrochloric or sulfuric acid, chlorosulfonic acid, aromatic sulfonic acids (p-toluenesulfonic acid) or complexing agents such as boron trifluoride or zinc chloride or by the corresponding acid halides of the formula I (R ₇ = HaI = Cl, Br, J) or acid anhydrides of the formula I (R ₇ = Ca-Ce-alkanoyloxycarbonyl group) on C ₁ -C ₆ -alkyl, optionally in the presence of basic substances such as pyridine, triethylamine, alkali metal or alkaline earth metal hydroxides, alcoholates, carbonates or acetates to act. This esterification is carried out at temperatures between 20 to 250 ⁰ C, in particular 60 to 100 ⁰ C, with or without solvent. Examples of suitable solvents are: Aromatic hydrocarbons, such as benzene, toluene, lower aliphatic alcohols, dimethylformamide, cyclic ethers, dimethyl sulfoxide, N-methyl-pyrrolidone, sulfolane, chloroform, tetramethylurea.

The abovementioned halides (R ₇ = COHaI) can be synthesized, for example, by reaction with halogenating agents, such as phosphorus tris and pentahalides (chlorides, bromides, iodides), thionyl halides (chloride, bromide, iodide), phosphorus oxychloride, phosphorus oxybromide), suifuryl halides (sulfuryl chloride, Sulfuryl bromide, sulfuryl iodide) or phosgene. The reaction can be carried out with or without solvent at temperatures between 20-150 ⁰ C, preferably 30-100 ⁰ C, performed. Suitable solvents are inert agents such as dioxane, tetrahydrofuran, aromatic hydrocarbons (benzene, toluene), halogenated hydrocarbons, (chloroform, methylene chloride) into consideration. Often it is expedient to use the corresponding starting acids (R ₇ = CO ₂ H) in the form of their alkali salts (sodium, potassium salts or silver salts).

c) The amides are prepared by reacting compounds of the formula I in which R _{7 is} a carboxyl group or a C ₁ -C ₆ -alkoxycarbonyl group (optionally via the corresponding halides or anhydrides) with ammonia, C ₁ -C ₆ -alkylamines, dialkylamines Alkyl radicals of 1 to 6 carbon atoms, or the corresponding amino acids or dipeptides or correspondingly protected amino acids or protected dipeptides by the usual methods of peptide chemistry. The reaction is carried out, for example, in a customary solvent or suspending agent, with the solvents already mentioned being suitable. Also water can be used. If the starting materials used are compounds of the formula I in which R _{7 is} the group -COHaI, the presence of acid-binding substances (alkali metal hydroxides, alkali metal carbonates, alkali metal bicarbonates, alkali metal acetates, alkaline earth metal carbonates, trialkylamines, pyridine and the like) or else excess ammonia or amine is expedient. In this case, the acid-binding agent can also be used at the same time alone or in a mixture with other customary agents as a solvent (for example pyridine). If the starting substance used is a compound of the formula I in which R _{7 is} the carboxyl group, then their activation is in the presence of condensing agents such as dicyclohexylcarbodiimide, bisulfated sulphamides (for example SO [N (CH ₃ ) ₂ ] 2), Ν, Ν '-Carbonyldiimidazole and the like required (Organic Reactions Vol 12, 1962, pages 205 and 239).

If the preparation of the amides via the anhydrides, the latter can, for example, by reacting the salts (alkali metal salts, silver salts) of compounds of formula I, wherein R _{7 is} the carboxyl group, with the corresponding C ₂ -C ₆ alkanoyl halides (chlorides, bromides , Iodides) or by reacting compounds of the formula I, in which R _{7 is} -COCl, -COBr or -COJ, with alkali metal salts or silver salts of the corresponding C ₂ -C ₆ -alkanecarboxylic acids. These reactions are carried out, for example, in a solvent or suspending agent at temperatures between 20-250 ⁰ C.

If the starting compound of the formula III contains a carboxy group, this can also be used in the form of a corresponding customary salt. Optionally, in the reaction product, the cation is then exchanged in a known manner for a physiologically compatible cation.

A starting compound wherein X together with R _{4 represents} a single bond and Y is a hydroxy group (for example 4-hydroxycyclophosphamide) can also be prepared in situ from the corresponding compound wherein Y is hydrogen (for example cyclophosphamide) by known ozonization, for example, in water, aqueous tetrahydrofuran or aqueous alcohol, and then reacted further without isolation directly with a compound III. The ozonization is conveniently carried out in the absence of hydrogen peroxide.

The isolation of the process products is preferably carried out by chromatographic methods, in particular by chromatography on silica gel or Sephadex (see Example 1 c).

The products of the process can be stabilized by incorporation of thiols having free SH group, such as cysteine, cysteamine, 2-mercaptoethanol. This is done, for example, by dissolving the compounds of formula I in an aqueous solution of the thiol compound, and then subjecting this mixture to a freeze-drying. For such stabilization, for example, per mole of compound I, 0.02-0.05 mole of the thiol compound is used.

The starting materials of the formula II in which X and Y each represent an alkoxy group or an alkylenedioxyring are known, for example, from Japanese Patent Application 71/25140 (Japanese Publication 4738928) or can be obtained analogously to the method given there or as follows: Reaction of Phosphoroxychlorid with a corresponding acetal of the formula HO-C (Rs) ₂ -C (R ₅ J ₂ -CR ₅ XY (X, Y = Ci-C ₆ -alkoxy or 0- (CH ₂ I _n -O with η = 1 -5) in an inert medium at low temperature (for example -20 to +10 ⁰ C) in the presence of a tertiary amine and subsequent reaction of the resulting reaction product with the amines HNR ₁ R ₂ , HNR ₃ R ₄ or ammonia in one or two steps, optionally in the presence of a tertiary amine at temperatures between -20 and 15 ° C.

The starting materials of the formula II in which X and Y are each an alkylthio group or together are an alkylene dithioring can be prepared analogously by reacting phosphorus oxytrichloride with the corresponding thioacetals of the formula HO-C (Rs) ₂ -C (Rs) ₂ -CR ₅ XY ( X, Y = C ^ Ce-alkylthio or -S- (CH ₂ I _n -S with η = 1-5), and then with the amines HNR ₁ R ₂ , HNR ₃ R ₄ or ammonia in one step or two steps produce.

Starting materials of the formula II in which X and Y together represent an oxygen atom can be obtained, for example, from the corresponding hydroxy compounds (X =OH, Y =H) in a manner known per se by oxidation (cf.

Example analogous to D. Swern, J. Org. Chem. 43, 1978, page 2480; A. Myles et al, Tetrahedron Letters, 1977, page 2475; D. Swern, Synthesis, 1981, 165; G. Piancatelli et al, Synthesis, 1982, page 245).

Furthermore, such starting materials can be obtained from the corresponding thioacetals (X and Y = alkylthio or S- (CH ₂ J _n -S) in a manner known per se by thioether cleavage (for example analogously to EJCorey, J. Org. Chem. 36,3553 [1971]) or from the corresponding oximes (X and Y = = N-OH) in a manner known per se by oxime cleavage (for example analogously to JH Boyer, Chem. Rev. 80, 541 [1980], PJMattingly et al, J. Org. Chem., 45, 410 [1980] and Synthesis, 1976, 808. Unknown starting compounds of the formula III can be prepared by known processes For example, the esters of the carboxylic acids of the formula III are prepared by reacting the carboxylic acids with the corresponding alcohol in a hydrochloric acid medium N-acetyl compounds are obtained by the action of acetyl chloride or acetic anhydride on the amino compound For example, to produce N-acetyl cysteine, the cysteine is converted to Ν, Ν'-diacetyl-cystine with acetyl chloride and the reaction nsgemisch with zinc in acetic acid at 50-60 ⁰ C reduced. N-alkyl compounds are obtained, for example, by reduction of corresponding thiazolidine compounds. Thus, N-methyl-cysteine is obtained by the action of sodium on thiazolidine-4-carboxylic acid in liquid ammonia and hydrolysis of the reaction product with acid.

The process products of the formula I are understood to mean all possible stereoisomers and mixtures thereof. In particular, these are, for example, compounds which have different configurations on the phosphorus atom or on a C atom of the heterocycle.

Diastereomeric mixtures can be separated in a known manner, for example by fractional crystallization or by analytical and preparative high pressure liquid chromatography. The pure enantiomers can be obtained by the usual methods of racemate resolution, for example by fractional crystallization of diastereomeric salts. For racemate resolution, for example, 1-phenylethylamine, brucine, quinidine, strychnine and cinchonine, as well as other bases and methods which are described in "Optical Resolution Procedures for Chemical Compounds", Vol. 2, Paul Newman, 1981, Verlag Optical Resolution Information Center in Riverdale, USA For this purpose, for example, converts a racemic salt according to the invention in the manner already indicated in a salt with one of the aforementioned optically active bases, separating the enantiomers in a known manner.

embodiments example 1

2- (2- [N, N-bis (2-chloroethyl) -diamido-phosphoryl-oxy] ethyl) -thiazolidin-4-carbonsäüre

Wed ₂ _ S

(ClCH ₂ CH ₂ ) ₂ UP-O-CH ₂ -CH ₂ -CH

a) Preparation of a compound II wherein X together with R _{4 represents} a single bond and Y is a hydroxy group: 5.0 g (18 mmol) of 4-hydroxycyclophosphamide and 2.4 g (20 mmol) of L-cysteine in 12 mL of distilled water mixed with 20 ml 1 N sodium hydroxide solution. The reaction mixture is stirred for 3 hours under N ₂ atmosphere, concentrated below ⁰ C in a high vacuum, the residue extracted with methanol / dichloromethane (3: 2) were ground, the insoluble portion filtered off with suction and the solution several times (on silica gel with methanol / dichloromethane 3 : 2) elutes.

The pure fraction with R _f = 0.38 (free carboxylic acid) was concentrated in vacuo, the residue treated twice with dry ether and finally dried to a glassy powder under high vacuum. Yield: 4.0 g (58% of theory).

The sodium salt can be obtained, for example, as follows: 1 g of the carboxylic acid D18038 (cipher designation for the free acid according to the above formula) is taken up in a little water, mixed with the equimolar amount of sodium bicarbonate and freeze-dried to give a powder. Yield: 1.1 g (100% of theory).

Unless otherwise stated, the Rf values are determined in a trough chamber with chamber saturation at room temperature:

Stationary phase: aluminum foil coated with cellulose; the cellulose contains a fluorescent indicator.

Applied substance quantity: approx. 5μΙ of a solution of 10mg / ml in methanol.

Eluent: water-acetonitrile 1: 2

Running distance of the solvent: 10cm

Identification was by the following specific staining reagents: 4- (4-nitro-benzyl) -pyridine (reagent on alkylating groups) and ninhydrin spray test.

b) Preparation of a compound II, wherein X together with R _{4 represents} a single bond and Y is a C ₂ alkoxy group (ethoxy group):

To a solution of 4,6 g (15 mmol) 4-ethoxycyclophosphamide in 10 ml of ethanol is added a solution of 1.9 g (16.5 mmol) of L-cysteine in 24 ml of distilled water and 16 ml of 1 N sodium hydroxide solution. After 3 hours worked up as indicated above.

Yield: 2.8 g (48% of theory)

Rf = 0.38 (free acid).

c) Preparation of a compound II, wherein X and Y are each an ethoxy group:

1.05 g (3 mmol) of O- (3,3-diethoxy-propyl) - [N, N-bis (2-chloroethyl)] - phosphoric acid diamide are added to a solution of 544 mg (4.5 mmol) of L-cysteine in approx 20 ml of 0.01 N HCl and heated to 6O ⁰ C until a clear solution is formed (about 75 minutes). After adjusting to pH 7 with NaOH, the reaction solution is purified by gel filtration over a Sephadex G-10 column

-8- Z60Z79

separated with 0.07 M phosphate buffer as eluent (Sephadex is a modified dextran medium for gel filtration in aqueous and polar medium).

The fractions containing the reaction product are collected and also desalted by separation over a Sephadex G-10 column with eluent H ₂ O.

This gives an aqueous solution of chromatographically pure reaction product D18038 which, frozen at -20 ⁰ C, is stable for several days.

The concentration of the alkylane in the solution is determined with the NBP assay (NBP = 4- (4-nitrobenzyl) -pyridine) and averages 5 mg / ml, giving a total of about 400 mg reaction product D18038 per batch corresponds (yield

35% of theory). ·

Another procedure with subsequent stabilization by addition of cysteine is the following:

3mmol (1050mg) of CMS ^ -diethoxy-propylHKN-bis-β-c-Morethyl OJ-phosphoric acid diamide (aldophosphamide diethyl acetal) are dissolved together with 4.5mmol (544mg) L-cysteine in 25ml 0.01N formic acid. After 55 minutes at 57 ° C, shaken three times with 25 ml of chloroform. The chloroform residues are removed in a water-jet vacuum (15 minutes). The aqueous solution is frozen and freeze-dried overnight. This gives about 1 g of lyophilisate, which is extracted with 60 ml of peroxide-free tetrahydrofuran (purification on an alumina column immediately before use) at 4 ° C (cold room) (shake for 1 minute, then 1 minute ultrasonic bath).

After centrifugation (Sorvall RC5C centrifuge, 40000 g, 15 ° C 4 ° C) and concentration of the tetrahydrofuran supernatant to dryness, the residue is dissolved in a 0.32 mg / ml aqueous L-cysteine solution using as much aqueous L-cysteine solution in that the concentration of compound I is 20 mg per ml, and this solution is freeze-dried.

The preparations thus prepared contain 5 mol% L-cysteine.

Yield about 200 mg of substance D18038 (yield about 18% based on the Aldophosphamiddtethylacetal).

The reaction product obtained by the methods a) -c) is obtained as a mixture of diastereomers.

The O- (3,3-diethoxy-propyl) - [N, N-bis (2-chloroethyl)] phosphoric acid diamide, which is used as starting material according to procedure c), can be obtained, for example, as follows:

3.6 g (23 mmol) of phosphorus oxychloride in 25 ml of dry methylene chloride are added dropwise at 0 ° to 5 ° C. within 2 hours while stirring with a solution of 3.4 g (23 mmol) of 3-hydroxypropanal diethyl acetal in 10 ml of methylene chloride and 3.3 ml of triethylamine , It is stirred at ^{0 0} C for one hour. Subsequently, 4.2 g of bis (2-chloroethyl) amine hydrochloride are added and added dropwise at 5-100 ⁰ C 7 ml of triethylamine in 10 ml of methylene chloride. The reaction mixture is stirred for one hour at 5 ° C and allowed to stand at 4 ° C. The next day, 2.8 g of liquid ammonia in 5 ml of methylene chloride are added dropwise with stirring. The temperature is allowed to rise to 20 ° C, stirred for 3 hours, washed with water, the organic phase is dried over sodium sulfate and concentrated. The residue is chromatographed on silica gel using chloroform / methanol (10: 1). Yield: 3.9 g; Rf value: 0.6; (Eluent: benzene / chloroform / methanol = 2: 1: 1, chamber saturation, run height 15 cm, staining: iodine vapor, development at room temperature).

Preparation of the lysine salt:

For salt formation, the amount of L-lysine is added, which is equimolar to the concentration of the substance D18038, and the solution is lyophilized in the freeze dryer. The resulting colorless lyophilisate is highly hygroscopic, but, in contrast to the free substance D18038, can be kept unchanged at +4 ⁰ C for weeks without exclusion of moisture.

The lysine salt still contains small amounts of the solvent (water) and is present as a mixture of diastereomers.

Thin-layer chromatography of the lysine salt (in thin-layer chromatography, the salt is separated into L-lysine and the substance D18 038, therefore the Rf values of these two components are obtained in this method): aluminum foil coated with cellulose (0.1 mm layer thickness);

Eluent water / acetonitrile 1: 2

Substance D18038: Rf = 0.68 L-lysine: R _f = 0.08

Stability:

Even after weeks of storage at -20 ° C, the lysine salt shows no changes in the chromatogram.

Production of injection solutions:

Solutions of 2-2- [N, N-bis (2-chloroethyl) -diamido-phosphoryl-oxy] -ethyl-thiazolidine-4-carboxylic acid are treated immediately before use by incubating 4-hydroxy-cyclophosphamide with a 10% solution for 45 minutes Excess of L-cysteine in 0.07 M phosphate buffer pH 7 at 37 ^° C.

Example 2

2- {2- [N, N-bis (2-chloroethyl) -diamido-phosphoryl-oxy] ethyl} thiazolidine-4-carboxylic acid ethyl ester

a) Preparation of a compound II wherein X together with R _{4 represents} a single bond and Y is a hydroxy group: 5.0 g (18 mmol) of 4-hydroxycyclophosphamide and 3.4 g (18 mmol) of L-cysteine ethyl ester hydrochloride are dissolved in 18 ml Water and 18ml 1 N sodium hydroxide solution for 4 hours under a nitrogen atmosphere. The reaction solution is extracted by shaking with dichloromethane and the organic phase is washed with a little 0.1N sulfuric acid and 3 times with water, dried over sodium sulfate and concentrated. The residue is treated with dry ether and concentrated in a high vacuum to a thin layer chromatography uniform oil.

Rf value = 0.83 (conditions as in Example 1). Yield: 4.8 g (65% of theory).

b) Preparation of a compound wherein X and Y are each an ethoxy group:

10 mg of O- (3,3-diethoxy-propyl) - [N, N-bis (2-chloro-ethyl)] -phosphoric acid-diamide and 8 mg of cysteine ethyl ester hydrochloride are heated in 1 ml of 0.01 HCl to 56 ° C.

After 60 minutes, the acetal has completely disappeared and a new peak (R _f value: 0.83) was obtained in a thin-layer chromatogram. The work-up of the reaction solution is carried out as indicated under al.

The reaction product obtained according to a) or b) consists in each case of a mixture of diastereomers.

Example 3

3N-Acetyl-2- {2- [N, N-bis (2-chloroethyl) -diamido-phosphoryloxy] ethyl} -thiazolidine-4-carboxylic acid 10mg of 0- (3,3-diethoxy-propyl) - [N, N-bis (2-chloroethyl)] - phosphoric acid diamide and 7.5 mg LN-acetyl-cysteine are heated to 56 ° C in 0.01 N HCl. It is extracted by shaking with methylene chloride, the methylene chloride phase dried with sodium sulfate, the methylene chloride removed, the residue taken up in methanol and chromatographed on a Sephadex column LH 20 with methanol as eluent (Sephadex LH 20 is a three-dimensionally crosslinked with epichlorohydrin dextran with hydroxypropyl groups, for the Gel filtration in organic solvents is used).

Example 4

Methyl 2- {2- [N, N-bis (2-chloroethyl) -diamido-phosphoryl-oxy] ethyl} -thiazolidine-4-carboxylate 1 g (~ 3 mmol) of O- (3,3-diethoxy-propyl ) - [N, N-bis- (2-chloffthyl)] - phosphoric acid diamide and 770mg (~ 4.5 mmol) of L-Cysteinmethylesterhydrochlorid are heated in 20 ml of 0.01 N HCI75 minutes at 56 ⁰ C. Subsequently, the reaction solution is neutralized with NaOH. Then shaken with 3 χ 50 ml of methylene chloride and the combined organic phase dried over sodium sulfate. After concentrating the organic phase, the yellow oil is taken up in water / methanol 1: 1 and separated on a Sephadex G-10 column with 0.07 M phosphate buffer pH 7.

The eluate, which contains the reaction product, is extracted by shaking with 3 × 250 ml of methylene chloride and the methylene chloride phase is concentrated. The residue is taken up in methanol and chromatographed on Sephadex LH 20 with methanol as the eluent. The fraction containing the reaction product is concentrated and dried under high vacuum. This gives a chromatographically pure yellowish oil which dissolves well in polar organic solvents, less well in apolar solvents and water. The reaction product is a mixture of diastereomers.

Yield: 200 mg (17% of theory)

Example 5

2- {2- [N, N-Bis- (2-chloroethyl) -diamido-phosphoryl-oxy] ethyl} -4-oxo-thiazolidine 1g (~ 3mmol) 0- (3,3-diethoxy-propyl) - [ N, N-bis- (2-chloroethyl) -phosphoric acid diamide and 730 mg (4.5 mmol) of N- (2-mercaptopropionyl-glycine are heated to 56 ° C. in 20 ml of 0.01 N HCl for 60 minutes x 50 ml of methylene chloride and the combined organic phase dried over sodium sulfate After concentration of the organic phase, the residue is taken up in methanol and chromatographed on a Sephadex LH 20 column with methanol to obtain a fraction containing two newly formed strongly UV-active peaks Concentration of the eluate gives a yellow oil which is poorly soluble in water and apolar solvents.

Yield: 230 mg (19% of theory).

Example 6

3N-L-y-glutamyl-2- {2- [N, N-bis (2-chloroethyl) -diamidophosphoryl-oxy] ethyl} thiazolidine-4-carboxylic acid (carboxymethylamide)

NH ₂ (ClCH-CH ₀ ) -NPO-CH ₀ -CH,

Δ Δ Δ w Δ Δ

CH-CO-NH-CH ₂ -CO ₂ H

CH-NH ₀

I ²

CO ₂ H

3mmol (1.09g) of O- (3,3-diethoxy-propyl) - [N, N-bis (2-chloroethyl) -phosphoric acid diamide are mixed with 4.5mmol (1.4g) of L-glutathione in 25ml 0.01N Formic acid heated to 56 ° C for 60 minutes. The reaction mixture is then extracted with 3 × 25 ml of chloroform and lyophilized. The separation is carried out on a silica gel column, wherein the silica gel with octadecanyl groups is added (C ^ -Reversed phase column), wherein the first water is separated with glutathione. The mixture is then eluted with methanol to give the glutathione-thiazolidone derivative.

After removal of the methanol, the reaction product is precipitated from water / ethanol 1: 1: Yield: about 200 mg; R f value:

Example 7

2- {1,1-dimethyl-1- [N, N-bis (2-chloroethyl) -diamidophosphoryl-oxy] ethyl} thiazolidine-4-carboxylic acid

NH ₀ CH-.

ι 2 ι 3

(ClCH-CH ₉ ), -PO-CH ₉ -C

Il

CH-CO ₂ H

3g 0 - [(3,3-Diethoxy-2,2-dimethyl-propyl- (1)] - [N, N-bis-2-chloroethyl)] -phosphoric acid diamide are suspended in a solution of i ^ gL-cysteine, mmoDinca.oOmlO ^ in formic acid (8,5mmol). The incubation time is 30 minutes at 80 ⁰ C. Thereafter, the aqueous phase in vacuo from chloroform residue is washed 3 times by shaking with 50 ml of chloroform, removed, and freeze-dried.

There remains a colorless solid which contains essentially only the thiazolidine reaction product and L-cysteine. To isolate the process product, the lyophilizate is digested twice with 30 ml of absolute tetrahydrofuran. The tetrahydrofuran extract gives 1.35 g of relatively clean reaction product (> 95%), corresponding to 38% of the theoretical yield. For final purification, the substance may be applied to a reverse phase column (for example, silica gel crosslinked with octadecanyl groups).

The column can be rinsed with H ₂ O without washing off the thiazolidine reaction product. The clean product can then be isolated with methanol-water (at least 20% methanol content) or with pure methanol. The analytical data of the thus purified process product are:

Elemental analysis H N C 5.88 10.29 calculated 35.29 5.74 10.01 found 34.99

High pressure liquid chromatography (HPLC)

Column C ₁₈ Novo-Pak

Eluent methanol / 0.02N phosphate buffer pH7 35:65

One peak after approx. 15 minutes.

TLC

Cellulose on aluminum foil Eluent: acetonitrile / H ₂ O 2: 1 Rf = 0.89.

Example 8

2- {2- [N, N-bis (2-chloroethyl) -diamido-phosphoryl-oxy] ethyl} -homothiazolidin-4-carboxylic acid

(ClCH ₂ CH ₂ ) 2N-PO-

1.05g (3mmol) of O- (3,3-diethoxy-propyl) - [N, N-bis (2-chloro-ethyl)] -phosphoric acid diamide and 4.5mmol (0.61g) of DL-homocysteine are heated in 25 ml of 0.01 N formic acid at 56 ° C for 60 minutes. Then, the reaction mixture is lyophilized and the lyophilizate taken up in 25 ml of tetrahydrofuran. The suspension is centrifuged and the supernatant is concentrated in vacuo.

The residue is taken up in tetrahydrofuran and treated with equimolar amount of concentrated hydrochloric acid. After addition of ether, the product of the process (DL-form) crystallizes as the hydrochloride at -20 ⁰ C. Melting point 136-138 ° C (decomposition).

Rf value: 0.83 (stationary phase cellulose).

Example 9

2- {2- [N, N'-bis (2-chloroethyl) -diamido-phosphoryl-oxy] -ethyl} thiazolidine-4-carboxylic acid

NH-CH ₂ CH ₂ Cl

ClCH ₉ CH ₀ -NH P ₀ -CH ₀ -CH ₀ -CH

2 2 u - 2 2,

O NH CH-CO ₂ H

4.2 g (12 mmol) of N, N'-bis (2-chloroethyl) -phosphoramide-3,3-diethoxy-propyl ester are incubated with 1.5-fold molar amount of L-cysteine in 100 ml of 0.01 N formic acid (1 hour at 60 ^° C.).

The reaction mixture is extracted by shaking with chloroform and concentrated on a lyophilizer.

The colorless powder, which consists of approximately equal parts of the thiazolidine compound and L-cysteine, is extracted with peroxide-free tetrahydrofuran (about 100 ml of tetrahydrofuran / 1 g of thiazolidine compound).

After centrifuging off the residue and concentrating the tetrahydrofuran solution, the thiazolidine compound remains as a colorless powder.

Thin layer chromatography on cellulose plate

Eluent: CH ₃ CIWH ₂ O 2: 1

The starting material N, N'-bis (2-chloroethyl) -phosphoric acid diamide-3,3-diethoxy-propyl ester can be obtained, for example, as follows:

In a solution of 34.5 g (13OmMoI) of freshly prepared phosphoric acid-S ^ -diethoxy-propyl ester dichloride in 120 ml of dry CH ₂ Cl ₂ , the double molar amount of chloroethylamine hydrochloride is suspended. At O ⁰ C is added dropwise 4 equivalents (based on one mole of phosphoric acid-S ^ -diethoxy-propyl ester dichloride) triethylamine in CH ₂ Cl ₂ TO and allowed to 16 hours at ca.

Stir 5 ° C.

The precipitated hydrochloride is removed by shaking with ice water, the organic phase dried over sodium sulfate and extracted by shaking with charcoal.

For further purification, the product can be passed through a short silica gel column: eluent methylene chloride.

Example 10

2- {2- [N, N'-bis (2-chloroethyl) -diamido-phosphoryl-oxy} ethyl] 1,5-perhydrothiazin (homothiazolidin) -4-carboxylic acid

-P-OCH ₀ CH ₀ -CH 2 2 2 \ w

4.2 g (12 mmol) of N, N'-bis (2-chloroethyl) -phosphoric acid-1,3-diethoxy-propyl ester are incubated with 1.5-fold molar amount of DL-homocysteine in 10 μl of 0.01 N formic acid (1 hour at 60 ^° C.).

The reaction mixture is extracted by shaking with chloroform and concentrated on a lyophilizer.

The colorless powder, which consists of approximately equal parts of the perhydrothiazinyl compound and DL-homocysteine, is extracted with peroxide-free tetrahydrofuran (about 100 ml tetrahydrofuran / 1 g reaction product).

After centrifuging off the residue and concentrating the tetrahydrofuran solution, the perhydrothiazinyl compound remains as a colorless powder.

Thin layer chromatography on cellulose plate

Eluent: CH ₃ CN / H ₂ O 2: 1

Examples of pharmaceutical preparations

Lyophilisate of the compound according to Example 1

100 g of the compound of Example 1 and 160 g of mannitol are dissolved under nitrogen and light in 4 liters of water for injection. The solution is sterile filtered through a suitable membrane filter and filled under aseptic conditions in proportions of 20ml in 50ml injection bottles. The injection bottles are provided with freeze-drying stoppers, the contents are freeze-dried. Subsequently, the freeze-drying plant is aerated with dry nitrogen and sealed the injection bottles in the system.

The residual water content of the lyophilizate must not exceed 0.5%. To prepare the solution for injection, the contents of the bottles are dissolved in 20 ml of water for injections. The solution for injection is isotonic.

TmI solution for injection contains 25mg active substance.

Lyophilisate of the compound according to Example 1 with 5 mol% cysteine

100 g of compound according to Example 1,1,6g cysteine and 160g mannitol are dissolved under nitrogen and light in 4 liters of water for injections. The solution is sterile filtered through a suitable membrane filter and filled under aseptic conditions in proportions of 20 ml in 50 ml injection bottles. The injection bottles are provided with freeze-drying stoppers, the contents are freeze-dried. Subsequently, the freeze-drying plant is aerated with dry nitrogen and sealed the injection bottles in the system. The residual water content of the lyophilizate must not exceed 0.5%. To prepare the solution for injection, the contents of the bottles are dissolved in 20 ml of water for injections. The solution for injection is isotonic. 1 ml solution for injection contains 25 mg active substance and 0.4 mg cysteine.

Claims (1)

A process for the preparation of heterocyclic compounds with a 2- (2- [N, N-bis (2-chloroethyl) -diamidophosphoryl-oxy] ethyl) radical of general formula I. MR 0 CR ₅ R ₅ - CR ₅ R ₅ in which the radicals R 1, R _2, R ₃ and R _{4 are} identical or different and are hydrogen, C ₁ -C ₄ -alkyl, 2-chloroethyl, 2-bromoethyl or 2-C ₁ -C ₄ -alkanesulfonyloxyethyl and at least two of these radicals are 2-chloroethyl, mean 2-bromoethyl or 2-C ₁ -C ₄ alkanesulfonyl-oxyethyl, R ₈ is hydrogen, C _r C ₆ alkyl, CrC ₆ -A! koxycarbonyl, C ₂ -C ₆ alkanoyl, which is also an additional carboxyl and or amino group, or is aminocarbonyl, which may contain one or two C ₁ -C ₆ -alkyl radicals on the nitrogen atom, the radicals R ₆ and R _{7 are} hydrogen or together form an oxygen atom or in which R _{6 is} hydrogen and in this case R _{7 is} a carboxyl group, a C ₁ -C ₆ alkoxycarbonyl group, an aminocarbonyl group, a C ₁ -C ₆ alkylaminocarbonyl group, which may also contain an additional carboxyl group, or a di-C ₁ -C ₆ -alkylaminocarbonyl group, Z is a sulfur atom, an oxygen atom, the group NRNR ₅ Grupe-SC (Rs) ₂ -, - 0-C (Rs) ₂ -or -NR ₅ -C (Rs) ₂ , and the radicals R _{5 are} identical or different and represent hydrogen or C ₁ -C ₆ -alkyl, and their salts with physiologically tolerated acids or cations and optionally the customary pharmaceutical application forms, characterized in that a compound of the general formula M ₃ R ₄ .CR R CR ₅ Rt wherein R _1, R _2, R _3, R ₄ and R ₅ have the meanings indicated and either X or Y depending on a C ₁ -C ₆ alkoxy or -C ₆ alkylthio group, or together an oxygen atom, a saturated alkylenedioxy ring of 1 to 5 represents C atoms, a saturated alkylenedithio ring of 1 to 5 carbon atoms or a hydroxyimino group, or in which X together with the radical R _{4 represents} a simple bond to form the 6-membered ring and in this case Y is a hydroxy group, a C ₁ -C ₆ alkoxy group or the group S-alk-SO ₃ Z wherein alk represents a C ₂ -C ₆ alkylene and Z is hydrogen or a cation, or wherein Y is a benzylthio group, a -C ₆ alkanoylthio group or an optionally by a carboxy group, a hydroxy group or C ₁ -C ₆ -Carbalkoxygruppe substituted CrC ₁₀ -Alkylthiogruppe is, with a compound of the general formula HZ-CR ₅ R ₅ -CR ₆ R ₇ -NHR ₈ III wherein Z, R ₅ , R ₆ , R ₇ and R _{8 have} the meanings given, and optionally in a compound of formula I, wherein R _{8 is} hydrogen, and the remaining symbols have the meanings indicated, by alkylation or acylation the R ₈ is introduced, wherein R ₈ has the meanings given apart from hydrogen, optionally the group R cleaves _8, possibly Z, if Z is the group NH or-NH-C (R ₅₎ -is _2, by alkylation of a CRCE alkyl group optionally, in a compound of formula I, the radical R ₇ , if this is not a hydrogen atom, transferred another possible meaning, and optionally converting the compounds obtained into the salts with physiologically acceptable cations or anions and optionally the compounds of formula I. processed with conventional galenic auxiliaries and / or carriers to conventional pharamazeutischen application forms. Field of application of the invention The invention relates to a process for the preparation of compounds of the formula I according to claim and to medicaments containing compounds of the formula I as active compounds.