CA1254903A

CA1254903A - Salts of oxazaphosphorine derivatives and their preparation

Info

Publication number: CA1254903A
Application number: CA000475401A
Authority: CA
Inventors: Jurgen Engel; Axel Kleemann; Ulf Niemeyer; Joerg Pohl; Peter Hilgard
Original assignee: Asta Pharma AG
Current assignee: Asta Medica GmbH
Priority date: 1984-03-01
Filing date: 1985-02-28
Publication date: 1989-05-30
Also published as: ES8607332A1; IE57950B1; FI850800A0; DE3569737D1; HUT36829A; KR850007067A; EP0158057A1; FI78705B; FI850800L; KR890003789B1; FI78705C; IE850510L; ES540777A0; IL74473A0; EP0158057B1

Abstract

ABSTRACT OF THE DISCLOSURE
There are provided new antitumor salts of oxazaphosphorine derivatives of the formula:

in which A is the group -S-alk-S03H and alk represents a C2-C6-alkylene residue with a basic compound of the formula:

II

wherein R4 is a hydroxy group or an amino group, R6 is hydrogen a C1-C6-alkyl group or a C1-C6-alkyl group which is substituted by an amino group, or a guanidino group.

Description

The present invention rela-tes to salts of oxya-zaphorphorine derivatives useful as antitumor agen-ts, their preparation and their use.

S In Belgian patent 892589 there are described oxazaphosphorin -4-thio-alkanesulfonic acids and specific salts thereof of the geneal formula:

e Y~

I~ / \ / ;

In the preceding formula Rl, R2 and R3 which can be -the same or differen-t stand for hydrogen, me-thyl, ethyl, 2-chloroethyl or 2-methanesulfonyloxyethyl, wherein at leas-t -two of those residues are 2-chloroethyl and/or 2-me-thanesulforlyloxyethyl, R4 is hydrogen or methyl, X is a straight or branched chain C2-C6-alkylene which can have a mercapto group on -the 1-,

2-, 3-, 4-, or 5- carbon atoms of -the alkylene chain, and Y~ is the hydrogen ca-tion, an alkali or alkaline earth ca-tion, the guandinium, morpholinium or cyclohexylammonium ca-tion or the cation which is derived from an amine of the formula NR5R6R7 where the residues R5 to R7 are -the same or -different and are hydrogen~ Cl-C2-alkyl groups or hydroxyethyl groups, or Y~ is the ethylenediammonium cation H3N-CH2 CH2-NH3 or the p.iperazinonium cation and z is 1 where Y~ is a monobasic cation, or z is 2 where Y~ is a dibasic cation of the cation of a compound having two monobasic cations.

Furthermore, the German published application 3133309 concerns 4-carbamoyloxy-oxaphosphorine derivatives of the general formula R5 ~XI
O - N - C - Z
Rl \ I3 N N ~ R4 /\ / \
R2 ~ ~ _ ~R4 R ~s wherein X is oxygen or sulfur, Rl, R2, and R3 can be the same or different and represent hydrogen, methyl, ethyl, 2-chloroethyl or 2-methanesulfonyloxyethyl, the residues R4 can be the same or di~ferent and represent hydrogen, methyl or ethyl, ~5 is hydrogen, Cl_4-alkyl, hydroxy-Cl_4-allcyl or phenyl and Z among others also is a Cl-C18-alkylamino group, which can contain different substituents, among others the carboxy group and their pharmaceuticlaly usable salts. In this German OS there is described as

3~3 example a compound wherein Z is the group -NH-CH2 - COOH and the corresponding cyclohexylamine salt.

The compounds of Belgian Patent 892589 and German published application 3133309 posses antitumor action as well as an immunosuppresive action.

According to the present invention there are provided salts of axazaphosphorine derivatives of the formula T
~J l~ /
(~1 C~12 C112 ) N ~ ~ N3 - _ Cll P ~ cl~2 - _ C112 in which A is the group -S-alk-SO3H and alk represents a C2-C6-alkylene residue unit a basic compound of the formula:

wherein R4 is a hydroxy group, or an amino group R6 is hydrogen a Cl-C6-alkyl group or a Cl-C6-alkyl group which is substituted by an amino group, or a guanidino group.

The present invention also provides a process for the production of salts of oxazaphosphorine derivatives of the general formula:
~/ A 7-2,) N ~ / N3~ ~ \
~ ~o7 ~
o ~ ~C112 ?3 in which A is the group -S- alk-SO3H and alk represents a C2-C6-alkylene residue a basic compound of the formula:

wherein R4 is a hydroxy group, or an amino group R6 is hydrogen a Cl-C6-alkyl group or a Cl-C6 -alkyl group which is substituted by an amino group, or a guandino group in which:
~ a) a compound of the formula e~ H
(~ CH2 CH2) N / N3~ -_ CH
P~ \

~ \0 ~CH2 CH2 wherein X is a hydroxy group or a ~ -C~-alkoxy group is reac-ted with a salt of th~ compound. Cl AH IV

wherein ~ is as above and the basic compund of formula II wherein the residues R4 and R6 are as above or first is reacted with the compound AH and subsequently with the basic compound of formula II or a compound of the formula III wherein X is benzylthio, Cl-C6-alkanoylthio, C1-C10-alkylthio, Cl-C10-alkylthio substituted by a carboxy group, a hydroxy group or a C1-C4-carbalkoxy group or wherein X is the group -N(OH)-CO-N~ and R is hydrogen, Cl-C6-alkyl, benzyl, phenyl, halogen -phenyl or alkyl-phenyl or wherein X is the group A and X can also exist in the salt form, is reacted with the excess of the compound oE the formula A'H V

~ 3 or a salt of the compound A~H with the basic compound of formula II wher~in the residues R4 and R6 are as above, wherein A' is different from A and has the definitions stated for A or (b) a compound of the formula I or the salt of compound of general formula I is reacted with a baslc compound of formula II wherein R~ and R6 are as above with formation of the corresponding salt and when the basic components in the compounds attained or the acid hydrog~n of the group A wherein ~ is no-t in lo the salt form are exchanged for another basic component.
The compounds of the invention possesses a strong antitumor activity and can be used especially to combat cancer.
Compared to the known compounds of Belgian patent 892589 and German published application 3133309 they have, for example, a reduced toxicity (for example reduced acute toxicity and leucotoxic action) and therefore have an improved therapeutic index and a better local and sys-temic as well as venous compa-tibility. Furthermore, the compounds of the invention possess a reduced irnmunosuppressive action, a reduced local tissue irritation and fre~uently lower haemolytic action.
Furthermore, the compounds of -the invention do not have or only have slight circulatory side effects (for example, sympathetic actions). Also the blood pressure is influenced less disturbingly.
The alkylene resude (Alk) of formula I can be straight or branched. Examples of these are methylene, dimethylene, trimethylene, or tetramethylene, resudues or also for example the 0 residues -CH-CH2 " -C(CH3)2-CH2- or CH2 CH2 1cH2 ;

the chain especially consists of 2 or 3 Carbon atoms when it is unbranched. In the event Alk is branched the portion which is attached to the acid group and the sulfur or nitrogen atom especially consists of 2 to 3 carbon atoms.

The alkyl groups occuring in formula II can be straigh-t or branched.

The compounds of formula II are preferably those compounds where R4 is a hydroxy group and R6 especially takes on the meanin~s which are stated below:

lo Preferred basic compounds of formula II are for example those where R4 is a hydroxy group, and R6 is a Cl-C6-alkyl group which contains (preferably in the 2-, 3-, 4-, 5-, or 6- position;
numbering always begin on the linkage point of the alkyl group with the rest of the molecule), an amino group (especially in the 3- or 4- position), or a guanidino residue, or aminoacid derivatives of formula II where R4 is an amino group and R6 is hydrogen.

In the previously mentioned aminoacids or aminoacid derivativ~s the salts for example are formed in each case ~rom one mole o oxazaphosphorine I and one mole of the compound II~

Furthermore, there can be used as basic compounds of formula II for example those compounds where R4 is an amino 2~ group, and R6 is a Cl-C6-alkyl group which contains, an amino group, or guanidino residue.

In the last mentioned aminoacid derivatives the salts are ~ormed for example in each case from 2 moles of oxazaphosphorine and I mole of the aminoacid derivative of formula II~

Specific examples of compounds of formula II are:
arginine arginiamide (L-form) 2,6-diamino enanthic acid ( -methyl lysine), glycinamide, lysine (especially L-lysine), alaninamide and ornithine.

In the salts of the invention in the event in compound II R4 is an amino group and otherwise no basic group is present or in the case where R4 ls the hydroxy group and otherwise a basic group is present, compound I ~acid-component) and Compound II (basic component) are present practically in the ratio 1:1.
On the other hand if in the basic component II R4 is an amino group and -this contains besides the ~ -amino group an additional basic group, then generally the ratio of compound I to compound II is 2:1. The salts of the invention are neutral salts. In the case of the sul~on1c acid salts the pII for example carboxyl~c acid the pH for example is between 6-9, especially 6-8.

In regard to Process (a): The process is carried out in a solvent at temperatures between -60C and +90C, preferably -30C to +60C, especially -20 -to +30C, that is in a given case with cooling, at room temperature or with heating. The reaction can be carried out in the presence of an acid catalyst, such as an inorganic or organic acld such as for example trichloroacetic acid, p-~, ~ 3 toluenesulfonic acid, trifluorom~thanesulfonic acid or even a Lewis acid such as AlC13, ZnCl2, TiC14, boron trifluoride etherate. The p~ is ad~usted to between 1 and 8, preferably between 2 and 6 for the reaction. This is especially true if the staring compounds are employed in form of the salts, optionally also if the free acid AH is employed and A contains the carboxy group.

As solvents there can be considered for example: water, alcohols, especially alkanols having 1-6 carbon atoms such as methanol, ethanol, propanol or isobutanol, hexanol, alkyl ketones containing l-4 carbon atoms in the alkyl groups, such as especially acetone or methyl ethyl ketone, aprotic solvents (for example dimethyl sulfoxide, acetoni-trile, N-methyl pyrrolidine, dimethyl formamide, hexameth~lphosphoric acid triamide), halogenated hydrocarbons haviny l-3 carbon atoms such as chloroform, ethylene dichloride, saturated cyclic ethers such as tetrahydrofuran, dioxane, sa-turated lower aliphatic ethers such as diethyl ether or similar solvents, respectively mixtures of several of such solvents.
In case in starting compound III the symbol X is the hydroxy or alkoxy group, the reaction can also be carried out in 2 steps, for example, by first reacting compound III with compound AH (without acidification~ and subsequently adding to the reaction mixture, optionally after concentration and addition of another of the solvents, mentioned, the basic component II in a solvent.

The use of a compound III wherein X is the hydroxy group or an alkoxy group, is especially suited in case the end product is obtained in crystalline form.

In the event that X is no-t hydroxy or alkoxy or X is in salt form, the compound A'H or compound A'H in salt form is employed in excess, for example, 1.5-10 molar, preferably 2-5 . . ~ , ~;~5~3 moles of the compound A'H or the salt of A'H per mole of compound III. The pH of the reaction solution is adjusted for example to 5.5-9, preferably 6.5-8 by means of alkali solution, e.g. aqueous sodium hydroxide, or with the amine which is already present as the basic component in the salt employed t under certain circumstances a pH up to 12 can also be favorable especially if the starting compounds are employed in salt form or in form of the free acids (for example if A contains the carboxy group).
The reaction temperature for example is 10-90%C, preferably 25-60C. The reaction time for example is several seconds toseveral hours. Subsequently for example the reaction solution is cooled to below 10C and brought to a pH between 4 to 5.5 or optionally to pH 7 with a mineral acid ~H2SO4, HCl, phosphoric acid), a sulfonic acid (for example mercapto-Cl-C6-alkanesulfonic acids, e.g. mercaptomethanesulfonic acid or mercaptohexanPsulfonic acid, or an ion exchanger l~) form).

The isolation of the product of the process for example can be carried out: by crystallization or by a chromatographic process, especially by preparative high pressure liquid chromatography, optionally once again wi-th subsequent reaction in the desired salt form on a correspondingly loaded cation exchanger.

In the event that the group A of formula III is present in salt form for example, there can be employed salts such as those described in German Offenlegungschrift 3133309 or Belgian patent 892589. For example there can be used the ammonium salt, the cyclohexylammonium salt or the guanidinium salt. It goes without saying that other customary salts can also be employed, for example, customary optical bases used for resolution o-f a racemate, which salts are producible in a manner analogous to the methods described there.

Starting compounds o~ formula III for example are also described in the following references or may be produced in a 9~3 manner analogous to the methods described there: Tetrahedron Letters Nr. 10 ~1979), pages 883-886, Cancer Treatment Reports 60, Nr. 4 (1976~, pages 429-435. If x is an optionally substituted Cl-C10-alkylthio group especially compounds of formula III are used wherein X has the followlng meanings: Cl-C6-alkylthio, the group -S-(CH2)n-CO2H (n = 1-6, especially 1-3~, the group -S-(CH2)n-OH (n = 2-6, especially 2-4) or the group -S-(CH2)n-COOC2H5 (n = 1-~), especially 1-3). If X is a Cl-C6-alkanoylthio group preferably acetylthio is used. If X is the 0 group -N(OH)-CO-NHR and R is Cl-C6-alkyl preEerably R consists of 1-4 especially 1-2 carbon atoms.

The production of the starting salts AH of formula IV, and the starting salts A'H of formula V can be carried out for example by reaction of a compound AH or A'H with a compound of formula II with or without a solvent at temperatures between 0 and ~0C. As solvents there can be used for example water, Cl-C6-alkanols (methanol~ ethanol)~ lower aliphatic ketones (acetone)~ cyclic ethers (dioxane)~ chlorinated hydrocarbon (methylene chloride, ethylene dichloride, chloroform, carbon tetrachloride)~ saturated lower aliphatic ethers (diethyl ether aprotic solvents (for example dimethylformamide, dimethylsulfoxide, acetonitrile) or mixtures of thess agents.

The production of this type of salt also can be carried ou-t for example in such manner that an alkali salt ~sodium salt) of the acid AH respectlvely A'H is dissolved in water (for example 1 to 20% solution; % = welght percent), this solution allowed to run through a column containing a strongly acid ion exchanger (H~ form, d fold excess) and the free acid in the eluate is neutralized with the basic component, the mixture concentrated in a vacuum, and optionally the residue recrystallized with a lower alcohol (methanol, ethanol), a lower ketone (acetone) or an ether (diethyl ether).

In Regard to Process (b): This process is carried out .; !

~ 3 at temperatures between 0-25C, preferably 0-5C. As solvents for this process there can be used for example: watsr, lower aliphatic alcohols, lower aliphatic ketones or mixtures of these agents, there is reacted 1 mole of component I wi-th 1 mole of component II. I-t is suitable to operate a pH range between 3 and In using compounds I wherein A is the S-alk-SO3H group (or a salt thereof) preferably there is employed a pH between 3-6, preferably 3.8-5, especially pH 4.

Frequently the addition of a buffer is favorable. As buffer systems with a pH range between 3.8 to 5.0 for example, there can be used: citric acid/sodium citrate; ace-tic acid/sod1um acetate; phosphoric acid/sodium hydrogen phosphate; tartaric acid/sodium tartrate; formic acid/sodium formate; sodium hydrogen phosphate/citric acid; succinic acid/sodium succinate; p~opionic acid/sodium propionate;aconitic acid/sodium aconitate; ~
dimethylglutaric acid and its sodium salt; maleic acid/sodium maleate; compound II/citric acid. As buffer for a pH range of 6 to 8 there can be used for example: sodium citrate/NaOH, trls-(hydroxymethyl)-aminome-thane-maleate/NaOH, KH2PO4/NaOH, KH2PO~, /Na2HP4 -Instead of sodium hydroxide solution to produce the buffer there can also bs used a basic compound of formula II so that the buffer already contains the basic component, which also is present in the end product of formula I.

The exchange of the basic component of a salt of compound I against a basic component of the invention can be carried out for example on acid ion exchangers which are loaded wlth the basic compound of formula II. In this case the basic compound II (which now is bound on the acid groups of the lon exchanger) is employed in excess (for example 2 to 10 moles, preferably 5 moles o~ component II to 1 mole oE component I). As acid ion exchanger there can be used for examples those whose polymeric matrix carries sulfonic acid groups or carboxylic acid ~;

~ 9 ~ 3 groups. The matrix of the ion exchanger for example can consists of a polystyrene resin op-tionally con-taining 2 to 16%, preferably

4 to 8% of divinyl benzene or a phenol resin. The polystyrene ion exchanger is preferably in gel form. The loading of the ion exchanger with the basic components for example can be carried out in the following manner: 150 ml of ion exchange resin 1.2 mval/ml. (The producer of the ion exchanger states the capacity of the ion exchanger (that is the amount of functional groups such as -SO3~, -C02H) in mval/ml or mval/g of the ion exchanger resin in a column (diameter about 4 cm~ with a cooling jacket is regenerated with hydrochloric acid, washed with dis-tllled water lo until neutral and free of chloride ions. Subsequen-tly the ion exchanger is treated with a 10% aqueous solution of the basic compound (220 moles) and washed with distilled water.
Additionally, the ion exchanger can be treated with a buffer (citric acid/citrate or acetic acid/acetate) of about pH 4 and subsequently the buffer again washed out. Furthermore, the ion exchanger also ca~ be loaded with neutral amino acid salts o~
formula II (for example the hydrochloride or hydrobromide).

In the event an ion exchanger is used it is favorable to add the bu~fer in the receiver for the eluate. In a few cases the common elution of the salt with the buffer and/or the salt of a mercapto-C2-C6-alkanesulfonic acid through the ion exchanger is also advantageous. For example, the startin~ compound, that is the compound I (A = S-alk-S03H~ or a customary known salt of compound I is dissolved in a buffer a-t pH 3.8 to 5.0, preferably 4.1 and this solution is passed over the ion exchange column and the eluate is caught in a corresponding buffer solution. The eluate or the lyophilizate produced therefrom then consists of the salt according to the invention and -the buffer and/or the salt of the mercapto-alkanesulfonic acid. Preferably the eluate, in a given case after dilution with water, is at -times immediately frozen or lyophilized.

However, hereby the procedure can also be that a , ~
-. ~

cus-tomary salt of compound I (for example an alkali salt) in aqueous solution is allowed to run through an acid lon exchanger as mentioned above in the H~ form and the compound I then neutralized in the eluate with the basic component of formula II.
This possible way of proceeding is especially suitable if the end product is produced n crystalline form.

As starting salts oE the compound of formula I there can be used for example those which are described ~n German Offenlegungschrift 3133309 or Belgian patent 892589. For example, there can be used the ammonium salt, the cyclohexylammonium salt or the guanidinium salt. However, there can also be employed other customary salts (for example salts with optically active bases which are customary for resolution of 1~ a racemate), which are producible in a manner analogous to the methods described there.

4~3 Included in the oxazaphosphorine derivatives of formula are all possible stereoisomers and mlxtures thereof. In detail for example i-t is a matter of cis-trans isomers on the oxazaphosphorine ring, that is cis-or-trans-position of the group A to the oxo-atom in the 2-position (phosphoryl oxygen). Thus for example, ;,~
~t ~ S'~9~;~

it is a matter of the cis isomers and the trans isomers (at times the racemate and the corresponding enantiomers), the separated cis isomers and the separated trans isomers.
Diastereomer salts (for example if a chiral amine is used for salt formation), can b~e separated in known manner, preferably through ~
crystallization. The pure enantiomers can be obtained according to the customary methods of resolving racemates, for example through fractional crystallization of the diastereomer salts from racemic acids of formula I and optically active bases or in a given case, through use of optically active starting materials according to formula III in the synthesisO

Generally in the synthesis cis/tran mixtures can be ,formed. Generally, mixtures are formed which consist of preponderantly the cis isomers and up to about 5-10% of the trans isomers. For example, the compounds according to Examples 1-5-consist of the cis isomers with less than 5-10% of the trans form.

With good crystallizing compounds there are obtained from such mixtures the crystallized cis or the trans form, especially the cis-form.
However, if the reaction is carried out in water free solvents or in solvents with a slight portion of water, there is obtained exclusively or in wide preponderance a single form, especially the cis-form. Thus there can be produced for example the pure cis-form,of a,noncrystallizing or poorly crystallizing c,ompo,u,n,d, of for,mu~a I by adding an acetonic solution of the compound of formula III

3~3 to an aqueous solution of the compound of formula IV or its salt at temperatures between -30 and ~20C and after the end of the reaction precipitating several times.

The starting compounds of ~ormula III can be employed as racemic cis and trans isomers (production of which are shown above), as optically active cis and trans forms and as mixtures thereof (in this connection see Belgian patent 892589 and German Offenlegungschrift 3133309, page 12).

For resolution of the racemate there can be employed for example as optically active bases phenylethylamine, brucine, ~ , strychnine and cinchonine as well as additional bases and methods which are described in "Optical Resolution Procedures for Chemical Compounds", volume 2~ Paul Newman, 19~1, Publisher Optical Resolution Information Center in Riverdale, USA. For this purpose there is converted for e~ample a racemic salt of the invention into a salt with one of the previously mentioned optically active bases in the manner already stated, the enantiomers separated in known manner and then the optically active base of the thus obtained enantiomers replaced again by a basic compound according to the invention.
However, the precedingly mentioned optically active bases can also be employed in the synthesis in process (a) in the reaction of a compound of formula III with a compound of formula IV or V or in process ~b) as basic salt component. In this case subsequently this optically active base is exchanged in customary manner against the basic ~2~ 3 salt component of the invention corresponding to the already given definition.

The compound of formula II can be employed as the racemeate or in the form of the pure enantiomers.

Generally the L-forms are preferred.

Included in the salts of the invention are all forms which result from the presence of various asymmetric carbon atoms, thus for exampler racemates, optically active forms, diastereomer forms.

It is recommended in the production to hold the salts of the invention in solution for as short a time as possible in order to prevent the hydrolysis to 4-hydroxyoxazaphosphorines and/or oxazaphosphorine ring (conversion of the cis-form into the trans-form~ or to hold the hydrolysis as low as possible. In the event that the salts of the invention are strongly contamina-ted (for e~ample contain large amounts of starting compounds)~ they can be obtained in pure form through customary chromatographic methods or preparative high pressure liquid chromatography~

rrhe salts of the inven-tion are stable, have storage stability ~especially at 40C), and can be readily processed galenically. For galenic preparations ~for example hydrolysis stable in~ection solutions, lyophilizates), especially in ~ .~, l~S~3 regard to storage stability it is also recommended to establish a pH range of about 3.5 7 with the sulfonic acid derivatives with the help of customary buffers (for example citrate buffer).
The optimum pH hereby is 4.0-4.3. In the event that the residue A is derived from the group N-(OH)-CO-NH-alk-CO2H, suitable a pH of 6.S-7.5 i5 established. These pH adjustments can be carried out both for solutions and suspension and also for solid galenic preparation.

Furthermore and independent of the addition of a buffer the addition of 0.5 to 5 equivalents of a salt (for example alkali salt, especially the sodium salt) of a mercapto2-C6-alkylsulfonic acid (for example a salt of 2-mercapto-ethane-sulfonic acid) and its disulfide and further thiols (for example cysteine) is also advantageously. Types of thiols and disulfides and the methods of their use are described in European patent application 83439. As this kind of salt there can be used for example the alkali salts (Na,K) or the salts with a basic component according to the invention (compound of formula II, homocysteinethiolactone, ~-amino-~-caprolactam).

The addition of the salt of mercapto-C2-C6-alkanesulfonic acid for example can be carried out by addition of an aqueous solution of the salt of the sulfonic acid (alkali salt, Eor example 20 weight percent) to an aqueous solution of the salt of the invention (preferably buffered to a pH
between 4 and 4.3). The thus obtained mixture then for example is lyophilized.

1;~5~

The advantages in addition of a mercaptoalkanesulfonic acid salt by this means consist of the following: improvement of the storage stability as well as of the stability in aqueous solution with the salts of the invention. (For example this is of significance in the production of the salts but also for example is useful in solution of lyophili2ates before use); improvement in chemotherapy of cancer illnesses by means of the salts of the invention especially in regard to toxic side effects tcf.
European patent application 83439 and Eurcpean Patent 2495.

The salts of the invention are suitable for the production of pharmaceutical compositions or preparatives. The pharmaceutical compositions or drugs contain one or more of the salts of the invention as active material, optionally in admixture with other pharmacologically or pharmaceutically active materials. The production of the drugs can be carried out using known and customary pharmaceutical carriers and adjuvants.

The drugs for example can be used enterally, parenterally, orally, perlingually or in the form of sprays. Dispensing can be carried out for example in the form of tablets, capsules, pills, dragees, plugs, liquids, or aerosols. As liquids there can be used for example: oily or aqueous solutions or suspensions, emulsions injectable aqueous or oily solutions or suspensions.

i4~33 - l9a -The compounds of the invention show a good cylostatic and curative effect in intravenous, intraperitoneal, or oral application with various experimental tumors of the rat and the mouse. For example there was produced, depending on the dosage, a curative effect with the compounds of the invention on the rat 5 days after intraperitoneal implantion of 105 cells of leukemia L5222 with different doses applied intravenously, intraperitoneally, or orally. As healing there is defined the recedive and metastasis free survival of tumor carrying animals after 90 days. From the frequency of healing obtained with the different dosages by means of test analysis according to R.
Fisher there was calculated as average curative dosage (DC 50) that dosage with which there could be healed 50% of the tumor carrying animals.

For example, the compounds of the invention were also dispensed at various doses intravenously, intraperitoneally, or orally one day after the ~lantation of 106 cells ;
of Yoshida-A~ites-Sarcol~ AH 13 and a curative effect produced depending on the dosaye.

Here also, the curative efect was defined as recedive and metastasis free survival of the tumor carrying animals for 90 days.

In corresponding manner by means of test analysis according to R. Fisher there was calculated as the average curative dosage (DC 50) that dosage at which 50% of the tumor carrying animals could be healed.

31~;~

- l9b -Furthermore, for example the compounds of the invention were dispensed with various doses intravenously, intraperitoneally, or orally once or multiple times (gx) on successive days after intraperitoneal implantation Of 106 cells of the mouse leukemia L1210 and a cytostatic effect produced.

The cytostatic effect is conceived as increase of the median survival time of the tumor bearing animals and is expressed as dosage dependent percent increa~e of the survival time compared to an untreated control group.

The a~erage curative dosage with the rat tumors is independent of the form of dispensation in the range of 0.1-10 mg/kg. With the same doses ther~ is produced an increase of the median survival time of 100~ with the mouse leukemia L1210.

Furthermore, the compounds of the invention stimulate the production of antibodies in a specific lower dosage range. This dosage range for example for the compound according to Example 5 is between 20-50 mg/kg rat (intravenously, intraperi-toneally). In comparison in the same dosage range the antibody production of the known antitumor agent cyclophosphamide is already suppressed.

Literature:
N. Brock: Pharmakologisch Grundlagen der Krebs Chenotherapie In: A. Georgu (Hrsg), Verhandlungender Deutschen Krebgesellschaft Volume 1, pages 15-42, Gustav Fischer Verlag, Stuttgart (1978).

., , :

-- ~.gc --This curative and cytostatic effect is comparable with the effect of the known medicines cyclophosphamide and ifosfamide.

The lowest curative or cytostatically effective dosage in the stated animal experiment for example is:
O.Ol mg.kg orally O.Ol mg/kg intraperitoneally O.Ol mg/kg intravenously As general dosage range for the curative and cytostatic action (animal experiments as above) there can be considered:
O.Ol-lO0 mg/kg orally, especially 0,1 -lO.0 mg/kg O.Ol-lO0 mg/kg intraperitoneally especially O.l-lO.0 mg/kg O.Ol-lO0 mg/kg intravenously, especially O.l-lO.0 mg/kg The compounds of the invention are indicated for use in malignant illnesses of men and animals.

The pharmaceutical preparations generally contain between l mg and l g, preferably lO0 to lO00 mg of the active component (or components) of the invention.

The dispensation can be carried out for example in the form of tablets, capsules, pills, dragees, plugs, salvesl gels, creams, powders, dusts, aerosols, or in liquid form. ~s liquid forms of use there can be employed for example: oily or ~Z~j~9~3 - l9d -alcoholic or a~ueous solutions as well as suspensions and emulsions. Preferred Eorms of use are tablets which contain between 10 and 200 mg or solutions which contain between 0.1 to 5~ of active material.

The individual dosage of the active components accordig to the invention for example can be (a) with oral Eorms of the medicine between 1-100 mg/kg, preferably 10-60 mg/kg (b) with parenteral forms of the medicine (for example intravenously, intramuscularly) between 1-100 mg/kg, preferably 10-60 mg/kg (c) with forms of the medicine for application rectally or vaginally between 1-100 mg/kg, preferably 10-60 mg/kgl (d) with forms of the medicine for local application to the skin and mucous membranes (for example in the form of solutions, lotions, emulsions, salves, etc.) between 1-100 mg/kg, preferably 10-60 mg/kg The doses in each case are based on the free base.

For example, there can be recommended ]-3 times daily 1 to 10 tablets having an active material content of 10 to 300 my or ~or example with intravenous injection 1 to 2 times daily one or more ampoules containing 1 to 10 ml containing 10 to 250 mg of material. The minimum daily dosage with oral dispension for example is 200 mg; the maximum daily i _ 19 e_ dosage with oral dispensation should not exceed 5000 mg.

There can also be recommended in individual cases dosages over 12 and more hours corresponding to continuous infusion.

The individual dosage in tre~ting dogs and cats orally generally is between 10 and 60 mg/kg bodyweight; the parenteral dosage is between about 10 and 60 mg/kg bodyweight.

The individual dosage in treating horses and cattle orally generally is between about 10 and 60 mg/kg bodyweight; the parenteral individual dosage is between about 10 and 60 mg/kg bodyweight.

The acute toxicity of the compounds of the invention on the mouse/expressed by the LD 50 mg/kg; :
method according to Miller and Tainter: Proc. Soc.
Exper. Biol~ a. Med. 57 (1944) 261), for example with oral application is between 100 and 1000 mg/kg (or about 1000 mg/kg).

The drug can be used in human medicine, veterinary medicine as well as in agriculture alone ~`
or in admixture with other pharmacologically active materials.

,:' ~5~ 3 Example 1 4-(2-Sulfo-ethylthio)-Cyclophosphamide-Glycinamide Salt (Cyclophosphamide is 2-[2-(bis-(2-chloroethyl)-amino)]-2-oxo-tetrahydro-2H-1,3,2-oxazaphosphorine) 4.0 grams (18 mmoles) of 4-hydroxy-cyclophoshpamide in 10 ml of distilled water were treated with S grams (18 mmoles) of 2-mercaptoethanesulfonic acid-glycinamide salt in 40 ml of acetone. The reaction solution was acidified with trichloroacetic acid to a pH of 4, kep for 2 hours at 5C adn for 20 hours at -25C. The salt separated out in crystalline form and was filtered off with suction, washed, dried, and recrystallized.from water/acetone.
M.P. 90-9BC; Yield 7.2 grams (76% of theory).
The salt contains about 1 equivalent of acetone.

Example.2 4-[1-Hydroxy-3-carboxymethyl-ureido-(1)]-cyclophosphamide-Lysine salt (L-Lysine) 7.5 grams (24.6 mmoles) of 4-ethoxycyclophosphamide and 3 grams (22.4 mmoles) of l-hydroxy-3-carboxymethyl-urea were kept in 50 ml of dry alcohol for 20 hours at 0C.
Subsequently the reaction solution was concentrated a.t 20C in a vacuum, the residue taken up in 200 ml of acetone, treated with 3.3 grams (22.6 mmoles) of L-lysine in 25 ml of methanol and the gelatinous precipitate centrifuged after standing.for;a.short time. The residue was dissblved:.in.water,-filtered, S~3 precipitated with acetone, filtered off with suction and washed with acetone) ether.
M.P. 125-128C; Yield: 6.5 grams (54% of theory~.

Example 3 4-~2-Sulfo-ethylthio)-cyclophosphamide-Glycinamide Salt A solution of 3.1 grams ~6.5 mmoles) of 4-(3-sulfopropylthio)-cyclophosphamide-guanidine salt and 4.2 grams (19.6 mmoles) of 2--mercaptoethanesulfonic acid-glycinamide salt in 15 ml of water was adjusted to pH 7.5 with aqueous sodium hydroxide and heated for 5 minutes at about 40C. Subsequently the reaction solution was cooled to 0C, adjusted to pH 4,5 with sulfuric acid, treated with 70 ml of acetone and kept for 3 days at 40C. The precipitate filtered off with suction and recrystallized from water/acetone.
M.P. from 85C (decompsition); YieldO 430 mg (14%
of theory3.

Example 4 4-(2-Sulfo-ethylthio)-Cyclophosphamide-Arginine Salt (L~Arginine) 37.2 grams ~74.5 mmoles) 0f 4-(2-sulfo-ethylthio)-cyclophosphamide-chclohexylamine salt were dissolved in 320 ml of distilled water at 5C
(pH 4.3) and passed over a column cooled to 4C
and containing 150 ml of cation exchanger. There was used a gel formed polystyrene resin containing 8% divinyl benzene and also containing sulfonic acid groups, which cation exchanger was loaded with arginineO Flow through velocity: 6 .` .~ 3 ml/minute. It was rinsed with 250 ml of water.
The eluate cooled to4C was diluted with cold water to a 5~ solution and subsequently lyophilized.
M.P. 85-90C (decomposition); Yield: 42.9 grams ~100~ of theory).

The addition of a buffer material and/or a mercaptoalkylsulfonic acid can be carried out as follows:
(a) Addition of Sodium Citrate Buffer The procedure is as stated above but the eluate is caught in sodium citrate buffer (pH 4.1~ and diluted for example subsequently to a solution which is 0.5 molar in sodium citrate and

5% in active material. Subsequently the solution was lyophilized.
Yield: 42.9 grams (100~ of theory), compound of Example 4 with sodium citrate buffer.

(b) Addition of Sodium Citrate Buffer And Sodium-2-mercapto-ethanesulfonate (Mesna) The procedure is as stated above except that the eluate is caught in a cooled sodium citrate buffer solution (pH 4.1~ which contains 8.2 grams (50 mmoles) Mesna and the solution is subsequently lyophilized.
Yield: 42.9 grams ~100% of theory) of compound of Example 4 with Mesna and sodium citrate buffer.

Example 5 4-~2-Sulfo-ethylthio)-Cyclophosphamide-Lysine Salt (L-Lysine) 37.3 grams (74.5 mmoles) of 4-(2-sulfo-~'~5~ 3 ethylthio)-cyclophosphamide-cyclohexylamine salt were eluted in a manner analogous to Example 4 on an ion exchanger (protonated lysine form) and subsequently lyophilized.
M.P. from 85C (decomposition); Yield: 40.8 grams (100~ of theory).

A different procedure for the example is the following:
82.4 grams (16.5 mmoles) of 4-(2-sulfo-ethylthio)-cyclophosphamide cyclohexylamine salt were dissolved in 850 ml of distilled water at 5C. The solution was adjusted to pH 4.1 with several particles of strongly acid ion exchanger (having sulfonic acid groups) and at a dropping speed of 50 ml/minute passed over a column cooled to 4C and containing 800 ml of ion exchanger (the same ion exchanger as in Example 4) containing 820 mmoles of sulfonic acid-L--lysine groups. The first 180 ml of the eluate were discarded and the following eluate continuously adjusted under stirring and ice water cooling to pH 4.1 with in all about 1.5 ml of strongly acid ion exchanger (having sulfonic acid groups). The ion exchanger was rinsed with 900ml of distilled water cooled to 0C. Subsequently the eluate was diluted with cold water to a 5% solution and immediately lyophilized.
M.P. ~ 85C; Yield: 90 grams (100~ of theory).

Compound According To Example 5 With Lysine-Citrate Buffer (Lysine -~ Citric Acid 7.2 grams (14.4 mmoles) of 4-(2-sulfo-ethylthio)-cyclophosphamide-cyclohexylamine salt ~;~5~3~3 were dissolved in 80 ml of 0.05 molar sodium citrate buffer;(pH 4.1) at 4C and passed over a column containing 40 ml of cation exchange resi.n (protonated lysine-formj:cooled:to 4C. It was rinsed with 80:ml:of buffer solution, the eluate was filled up to 150 grams of solution and lyophilized.
Yield: 7.9 grams (100~ of theory~ of the cornpound of Example 5 with lysine-citrate buffer.

Compound According To:Example 5 With 2-Mercaptoethanesulfonic:Acid-Lysine Salt And Lysine-itrate Buffer 7.~ grams (14~4 mmoles) of 4-~-sulfo-ethylthio~-cyclophosphamide-cyclohexylamine salt and 1.5 grams.(9.2 mmoles);of Mesna-.were dissolved in 80 ml of 0.05 molar~sodium c;trate bufer p~
4.1 and 4C and passed over a column containing 40 ml of cation exchange resin ~protonated lysine form) and cooled to 4C. It was rinsed with 50 and 3D ml of buffer solution, the cooled eluate ;11ed up to 150 grams of solution and lyophilized.
Yield: 7O9 grams (100% of theory) o the compound of Example 5 with 2.6 grams [100% of theory~ of 2- ;
mercaptoethanesulf~nic acid-lysine salt and lysine citrate buffer (pH 4.1~.

24 a Example 6 4-(2-sulfo-ethylthio)-cyclophosphamide-glycinamide salt 2,9 grams (8,6 mmoles) of 4-hydroxy-ethylthio)-cyclo-phosphamide and 5,6 grams (26 mmoles~ of 2-mercapto-ethanesulfonic acid-glycinamide salt were dissolved in 35 ml of distilled water. The pH was adjusted to 8 with glycinamide. The reaction solution was heated for about 4 minutes at about 40 C: Subsequently the reaction solution was cooled to 0 C, adjusted to pH 4,5 with 2-mercapto-ethanesulfonic acid and 500 ml of acetone were added. Then the reaction mixture was kept several aays at 4 C, the precipitate was filtered off with suction and recrystalliæed from water/acetone.
,~
,~ ` M.P. from 85 C; Yield 410 mg (9 % of theory).
The salt contains about 1 ~ ~ of acetone.

Examples of Pharmaceutical Preparations `
(a) Example For The Production Of A
Lyophilizate Of 4-Sulfo-ethylthio-cyclophosphamide-~ysine Salt 90 grams of 4-sulfo-ethylthio-.~ 3 cyclophosphamide-L-lysine salt were dissolved with stirring in 1500 ml of water suitable for injection purposes and c0012d to 4C.
Subsequently it was filled up to 1800 ml with water at 4C. The pH was adjusted to about 4.2 with several particles of regenerated cation exchanger (H~ form). The above solution was subjected to a sterile filtration in known manner to produce the lyophilizate. The receiving vessel was cooled. All of the processes subsequent to the sterile filtration were carried out under aseptic conditions.

The sterile solution was fi]led to 2 ml in a 10 ml injection flask. The active material content was 100 mg.

The flasks were provided with sterile freeze drying stoppers and lyophilized in a freeze drying unit. Subsequently the unit was deaerated with sterile, dried nitrogen and the ampoule flasks closed in the unit. For the production of an injection solution the contents of the flasks were dissolved in 5 ml of water suitable for injection purposes.

The lyophilizate is stored at 0-6C
(refrigerator).

(b) Example For The Production Of A
Lyophilizate Of 4-(2-Sulfo-ethylthio)-cyclophosphamide-Lysine Salt (L-Lysine~
With Citric Acid Lysine Buffer 1. An ion exchange column having a 1~5~V~3 cooling jacket was loaded with 1300 ml of an acid ion exchanger, regenerated with ~ liters of 10%
hydrochloric acid and washed with water suitable for injection purposes until neutral and free of chloride.

2. Subsequently the column was loaded with the help of 3 liters of a 10% lysine solution, free of excess lysine by washing with water suitable for injection purposes and washed until neutral.

3. There were passed over the column 1.4 liters of the following composition:
4-(sulfo-ethylthio3-cyclo-phosphamide cyclohexylamine salt 137.12 9 Citric Acid, water free 28.83 9 1 N NaOH 193.20 ml Water for injection purposes to 1.4 liter Active agents and adjuvant were dissolved in water of about 4C. The pH of the solution was 4.1. The cation exchange column was then also cooled to about 4C. The above solution was passed over the column. The flow through speed was 10 ml/minutes.

The eluate was caught in a cooled receiver, whereby the first 300 ml was discarded as forerunner. Subsequently the column was washed with water suitable for injection purposes and cooled to 4C and the entire volume of the eluate filled up to 3 liters.

lZS'~9(~3 The eluate which should be further processed to lyophilizate has the following composition:
4-(2-Sulfo-ethylthio~-cyclophosphamide Lysine salt (L-lysine) 150.00 g Citric Acid water free 28.83 g L-Lysine water free28.24 9 Water for injection purposes 2870.93 g 3078~00 9 = 3000 ml 4. For the production of the lyophilizate the above solution was subjected to a sterile filtration in known manner. The receiving vessel was oooled. All the procedures following the sterile filtration were carried out under aseptic conditions.

The sterile solution was filled into injection flasks as follows:
2 ml of solution in a lO ml injection flask.

The active material content is lO0 mg lO
ml of solution in a 30 ml injection flask.

The active material content is 5D0 mg.

The flasks were provided with sterile freeze drying stoppers and lyophilized in a freeze drying unit. Subsequently the unit was deaerated with sterile, dried nitrogen and the ampoule flasks closed in the unit.

~pp/~
For the production on~pp~ia~4-injection solution the contents of the flasks containing 100 mg of active material were dissolved in 5 ml of water suitable for injection purposes and the contents of the flasks containing 500 mg of active material were dissolved in 25 ml of such water.

(c) Example For The Production Of A
Lyophilizate Of 4-Sulfo-ethylthio-cyclophosphamide-Arginine Salt 90 grams of 4-sulfo-ethy~thio-cyclophosphamide-arginine salt ~ 135 grams of sodium-2-mercapto-ethanesulfonate were dissolved with stirring in 1500 ml of water, cooled to 4C
and suitable for injection purposes. After complete solution it was filled up to 1800 ml with water at 4C. The pH was adjusted to about 4.2 with several particles of regenerated cation exchanger (H+ form). For the production of the lyophilizate the above solution was subjected to a sterile filtration in known manner. The receiving vessel was cooled. All of the procedures subsequent to the sterile filtration were carried out under aseptic conditions. The sterile solution was filled to 2 ml in a 10 ml injection flask. The content of active material is lO0 mg. The flasks were provided with sterile freeze drying stoppers wnd lyophilized in a freeze drying unit. Subsequently the unit was deaerated with sterile, dried nitrogen and the ampoule flasks closed in the unit. For the production of an a ~o J, c C~b/~
hi~h~ injection solution the contents of the flasks were dissolved in 5 ml of water suitable for injection purposes.

l;~S4~3 The lyophilizate is stored at 0-6C
(refrigerator).

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. Pharamaceutically acceptable salts of oxazaphosphirine derivatives of the formula:

in which A is the group -S-alk-S03H and alk represents a C2-C6-alkylene residue with a basic compound of the formula:

II

wherein R4 is a hydroxy group or an amino group R6 is hydrogen a C1-C6-alkyl group or a C1-C6-alkyl group which is substituted by an amino group, or a guanidino group.

2. A salt according to claim 1, in which R4 is hydroxy and R6 is a C1-C6 alkyl group which is substituted by an amino or guanidino residue.

3. A salt according to claim 2, in which the alkyl group of R6 is substituted in the 2- to 6-position.

4. A salt according to claim 3, in which the substituent is in the 3- or 4-position.

5. A salt according to claim 1, in which R4 is an amino group and R6 is a C1-C6-alkyl group which contains an amino group, or a guanidino residue.

6. A salt according to claim 5, in which the alkyl group is substituted in the 2-, 3- or 4-position.

7. A salt according to claim 1, in which R4 is hydroxy.

8. A salt according to claim 1, in which the compound of Formula II is selected from arginine, arginine amide (L-form) 2,6-diamino enanthic acid (.epsilon. -methyl lysine), glycinamide, lysine, alaninamide and ornithine.

9. 4-(2-sulfo-ethylthio)-cyclophosphamide-glycinamide salt.

10. 4-[1-hydroxy-3-carboxymethyl-ureido-(l)]-cyclophosphamide-lysine salt (L-lysine).

11. 4-(2-sulfo-ethylthio)-cyclophosphamide-arginine salt (L-arginine).

12. 4-(2-sulfo-ethylthio)-cyclophosphamide-lysine salt (L-lysine).

13. A process for the production of salts of oxazaphosphorine derivatives of the general formula:

in which A is the group -S-alk-SO3H an dalk represents a C2-C6-alkylene residue a with a basic compound of the formula:

II

wherein R4 is a hydroxy group or an amino group R6 is hydrogen a C1-C6-alkyl group or a C1-C6-alkyl group which is substituted by an amino group, or a guanidino group in which:

(a) a compound of the formula III

wherein X is a hydroxy group or a C1-C4-alkoxy group is reacted with a salt of the compound AH IV

wherein A is as above and a basic compound of formula II wherein the residues R4 or R6 are as above or first is reacted with the compound AH and subsequently with the basic compound of formula II or a compound of the formula III wherein X is benzylthio, C1-C6-alkanoylthio, C1-C10-alkylthlo, C1-C10-alkylthio substituted by a carboxy group, a hydroxy group or a C1-C4-carbalkoxy group or wherein X is the group -N(OH)-CO-NHR and R is hydrogen, C1-C6-alkyl, benzyl, phenyl, halogen - phneyl or alkyl-phenyl or wherein X is the group A and X can also exist in the salt form, is reacted with the excess of the compound of the formula A'H V

or a salt of the compound A'H or with, the basic compound of formula II wherein the residues R4 and R6 are as above wherein A' is different from A and has the definitions stated for A or (b) a compound of the formula I or the salt of compound of general formula I is reacted with a basic compound of formula II wherein R4 and R6 are as above with formation of the corresponding salt and when the basic components in the compounds attained or the acid hydrogen of the group A when A is not in the salt form are exchange for another basic component.