GB2111053A - Di- and tri-sulphides - Google Patents

Abstract

Hydroxyalkyl, hydroxycycloalkyl-, alkoxyalkyl, alkoxycycloalkyl-, aminoalkyl-, aminocycloalkyl or epoxyalkyl-di- and -tri-sulfides as well as physiologically -hydrolysable and -acceptable esters and pharmaceutically acceptable acid addition salts thereof as pharmaceuticals in particular immunostimulants. Certain of the subject di- and tri-sulfides, typically those of formula <IMAGE> wherein R3 and R4 = H, c1-5 alkyl or acyl, x = 2 or 3, n = 2 to 8 and p = 1 to 3 are novel compounds and are claimed per se.

Description

SPECIFICATION Di- and tri-sulfides including novel (cycloalkyl)-(alkyl)-di- and -tn-sulfide derivatives for pharmaceutical, in particular immunostimulant, use The present invention relates to a new use, in particular a new pharmaceutical use, for certain di- and tri-sulfides as well as to certain novel (cycloalkyl)-(alkyl)-di- and -tri-sulfide derivatives per se.

In accordance with the present invention it has now surprisingly been found that certain di- and tri-sulfides, in particular alkyl- and cycloalkyl-di- and -tri-sulfides in which the alkyl or cycloalkyl moiety has a hydroxy, alkoxy or amino substituent or, in the case of alkyl moieties, an epoxy substituent, have valuable pharmaceutical, in particular immunostimulant, activity.

More particularly it has been found that such di- and tri- sulfides as aforesaid stimulate not only nitrogen responsive lymphoproliferation (primary immune response) but, most surprisingly, that they also stimulate anti-body production as well as cell-mediated immuno response (secondary immune response). It has further been found that these activities are evidenced not only in vitro but also in vivo.

By the finding that the said bis- and tri-sulfides stimulate both primary and secondary immune response, these compounds are now shown to be effective agents for the stimulation of antigenic immuno response and thus therapeutically useful as immunostimulants suitable for the treatment of a variety of morbid as well as non-morbid conditions e.g. as hereinafter discussed.

In accordance with the foregoing the present invention provides a) A hydroxyalkyl-, hydroxycycloalkyl-, alkoxyalkyl-, alkoxycycloalkyl-, aminoalkyl-, aminocycloalkyl- or epoxyalkyl- -di- or -tri-sulfide, or physiologically-hydrolysable and -acceptable ester or carbamate of a hydroxyalkyl- or hydroxycycloalkyl -di- or -tri-sulfide, or pharmaceutically acceptable acid addition salt of an aminoalkyl- or aminocycloalkyl- -di- or -tri-sulfide, for use as an immunostimulant.

In a more specific aspect the present invention also provides b) A di- or tri-sulfide as defined under a) above of formula R1 - (S)x - R2 (I) wherein R1 is hydroxyalkyl, hydroxycycloalkyl, alkoxyalkyl, alkoxycycloalkyl, aminoalkyl, aminocycloalkyl or epoxyalkyl, R2 is alkyl or cycloalkyl or has the meanings given for R1 and x is2or3, or physiologically-hydrolysable and -acceptable ester or carbamate of such a di- or tri-sulfide wherein R1 and/or R2 is hydroxyalkyl or hydroxycycloalkyl, or pharmaceutically acceptable acid addition salt of such a di- or -tri-sulfide wherein R1 and/or R2 is aminoalkyl or aminocycloalkyl, for use as an immunostimulant; as well as c) A compound as defined under b) above wherein, in formula I R1 is hydroxyalkyl wherein the hydroxy group is in the 2-position or is terminal, 2-hydroxy-cycloalkyl, 2-alkoxyalkyl, 2-alkoxycycloalkyl, 2-aminoalkyl or 2-epoxyalkyl, and R2 is alkyl or cycloalkyl or has the meanings given for R1.

In a further aspect the present invention also provides d) A hydroxyalkyl-, hydroxycycloalkyl-, alkoxyalkyl-, alkoxycycloalkyl-, aminoalkyl-, aminocycloalkyl- or epoxyalkyl- -di- or -tri-sulfide in which the hydroxy, alkoxy, amino or epoxy residue is the sole substituent on the alkyl or cycloalkyl moiety or in which the alkyl or cycloalkyl moiety is further substituted by one or more members selected from the group consisting of phenyl, cycloalkyl, halogen, hydroxy, alkoxy and amino and, in the case of cycloalkyl, alkyl and in which any alkyl moiety as aforesaid is uninterrupted, or physiologically-hydrolysable and -acceptable ester or carbamate of such a hydroxyalkyl- or hydroxycycloalkyl- -di- or -trisulfide, or pharmaceutically acceptable acid addition salt of such an aminoalkyi- or aminocycloalkyl- -di- or -tri-sulfide, for use as a pharmaceutical, in particular for use as an immunostimulant; and more especially e) A di- or tri-sulfide as defined under d) above of formula las illustrated under b) above wherein R1 is hydroxyalkyl, hydroxycycloalkyl, alkoxyalkyl, alkoxycycloalkyl, aminoalkyl, aminocycloalkyl or epoxyalkyl, the structure of said groups being limited as defined under d) above.

R2 is alkyl or cycloalkyl or has the meanings given for R1, and K is2or3, or physiologically-hydrolysable and -acceptable ester or carbamate of such a di- or tri-sulfide wherein R1 and/or R2 is hydroxyalkyl or hydroxycycloalkyl, or pharmaceutically acceptable acid addition salt of such a di- or tri-sulfide wherein R1 and/or R2 is aminoalkyl or aminocycloalkyl, for use as a pharmaceutical, in particular for use as an immunostimulant; as well as f) A compound as defined under e) above wherein, in formula 1, R1 is hydroxyalkyl wherein the hydroxy group is in the 2-position or is terminal, 2-hydroxycycloalkyl, 2-alkoxyalkyl, 2-alkoxycycloalkyl, 2-aminoalkyl or 2-epoxyalkyl, as defined under d) above, and R2 is alkyl or cycloalkyl or has the meanings given for R1 and g) A compound as defined under e) above wherein, in formula I, R1 is hydroxyalkyl wherein the hydroxy group is in the 2-position or is terminal, 2-hydroxycycloalkyl, 2-alkoxyalkyl, 2-alkoxycycloalkyl, 2-aminoalkyl or 2-epoxyalkyl in which the hydroxy, alkoxy, amino or epoxy residue is the sole substituent on the alkyl or cycloalkyl moiety and in which any alkyl moiety as aforesaid is uninterrupted; or hydroxyalkyl in which hydroxy group is in the 2-position or is terminal and in which the alkyl moiety bears one further substituent selected from the group comprising cycloalkyl, hydroxy and halogen and is uninterrupted, and R2 is alkyl or has the meanings given for R1.

In the above definitions a) to g), alkyl groups as R2 as well as the alkyl moiety of hydroxyalkyl, alkoxyalkyl, aminoalkyl and epoxyalkyl groups, e.g. as R1 or R2, may be branched or straight chained. Preferably they are straight chained.

Unless otherwise specified such alkyl groups and moieties may be substituted or further substituted, for example by 1, 2 or 3 substituents e.g. selected from phenyl, cycloalkyl and halogen or, in the case of alkyl moieties, further hydroxy, alkoxy and amino groups. Preferred substituents or further substituents are cycloalkyl, halogen and hydroxy.

In the case of alkoxy-, amino- and epoxy-alkyl groups as aforesaid, the alkoxy, amino or epoxy residue is preferably the sole substituent, i.e. the alkyl moiety is otherwise unsubstituted. In the case of hydroxyalkyl groups as aforesaid preferably the hydroxy residue is the sole substituent or the alkyl moiety is further substituted by one substituent selected from the group comprising cycloalkyl, halogen and hydroxy. Most preferably however the hydroxy group is the sole substituent.

In the above definitions a) to g) unless otherwise specified cycloalkyl groups as R2 as well as the cycloalkyl moiety of hydroxycycloalkyl, alkoxycycloalkyl and aminocycloalkyl groups, e.g. as R1 or R2, may also be substituted or further substituted for example by 1,2 or 3 substituents e.g. selected from phenyl, alkyl, cycloalkyl and halogen or, in the case of cycloalkyl moieties, further hydroxy, alkoxy and amino groups.

Preferably however cycloalkyl groups as aforesaid are unsubstituted and in the case of hydroxy-, alkoxy- and aminocycloalkyl groups as aforesaid the hydroxy, alkoxy or amino residue is the sole substituent.

It will of course be appreciate that in the above definitions d) to g), where particular substitution is specified no further substitution is possible.

In the above definitions a) to g), unless otherwise specified the alkyl moiety of hydroxyalkyl, alkoxyalkyl, aminoalkyl and epoxyalkyl groups e.g. as R1 or R2 may also be interrupted, e.g. by one or more sulphur or oxygen atoms or, preferably, uninterrupted.

In the above definitions a) to g), alkyl groups as R2 as well as the alkyl moiety of hydroxyalkyl, alkoxyalkyl, aminoalkyl and epoxyalkyl groups, e.g. as R1 and R2, preferably contain 2 to 8, more preferably 2 to 4 carbon atoms. Especially preferred are alkyl groups containing 2 to 4 carbon atoms which are unsubstituted and uninterrupted and hydroxyalkyl, alkoxyalkyl, aminoalkyl or epoxyalkyl groups containing 2 to 4 carbon atoms and wherein the hydroxy, alkoxy, amino or epoxy residue is the sole substituent.

In the above definitions a) to g) cycloalkyl groups as R2 as well as the cycloalkyl moiety of hydroxycycloalkyl, alkoxycycloalkyl or aminocycloalkyl groups e.g. as R1 or R2 preferably contain 3 to 8, more preferably 5 to 7, most preferably 5 or 6 carbon atoms. Especially preferred are unsubstituted cyclohexyl and hydroxycyclohexyl in which the hydroxy residue is the sole substituent, the latter being most preferred.

Alkoxy groups referred to above, including the alkoxy residue of alkoxyalkyl and alkoxycycloalkyl groups, preferably contain 1 to 4 carbon atoms, methoxy being most preferred. Amino groups referred to above, including the amino residue of aminoalkyl and aminocycloalkyl groups, may be optionally substituted e.g.

mono- or di-C1~4 alkyl substituted. Preferred amino groups as aforesaid are unsubstituted amino and methylamino.

The oxygen or nitrogen atom of hydroxy-, alkoxy-, amino- and epoxy-alkyl as well as of hydroxy-, alkoxyand amino-cycloalkyl groups as described above may not be attached to the carbon atom adjacent the sulphur bridge. Preferably it is in the 2-position (i.e. separated from the sulphur bridge by 2 carbon atoms). In the case of hydroxyalkyl groups the hydroxy group may also suitably be in a terminal position on alkyl moiety.

Preferred compounds in accordance with the invention are di-sulphides, i.e. in the case of definitions b), c) and e) tog) above compounds, of formula I wherein Xis 2.

Particularly preferred compounds in accordance with definitions b), c) and e) to g) above are those wherein R1 is hydroxyalkyl, hydroxycycloalkyl or alkoxyalkyl, especially hydroxyalkyl or hydroxycycloalkyl, and R2 is alkyl, hydroxyalkyl, hydroxycycloalkyl or alkoxyalkyl, especially hydroxyalkyl.

By the term "a physiologically-hydrolysable and -acceptable ester or carbamate" as used throughout the present specification and claims is meant esters and carbamates wherein a hydroxyalkyl or hydroxycycloalkyl group is acylated or carbamoylated and wherein the acyl or carbamoyl residue is removable by hydrolysis under physiological conditions to yield an acid which is itself physiologically acceptable, e.g.

non-toxic, at the dosages to be administered. Suitable esters include e.g. C1-4 alkanoyl and benzoyl esters.

Suitable acid addition salts as defined under a) to g) above, as well as elsewhere in the present specification and claims include e.g. the hydrochlorides and maleates.

Further compound groups in accordance with the invention comprise: h) A disulphide of formula I' P1, -S-S - R2, (I') wherein R1 and R2 each independently of the other is hydroxyalkyl, for use as an immunostimulant; I) A disulfide offormula I' as illustrated above wherein R; or P2 each independently of the other is hydroxyalkyl in which the hydroxy residue is the sole substituent on the alkyl moiety or in which the alkyl moiety is further substituted by one or more members selected from the group consisting of phenyl and hydroxy and in which the alkyl moiety is uninterrupted, for use as a pharmaceutical, in particular for use as an immunostimulant;; j) A di- or tri-sulfide of formula I" Rí(S)xR2 (1") wherein R'1 is hydroxyalkyl, alkoxyalkyl, aminoalkyl or epoxyalkyl, P2 is alkyl or has the meanings given for R'1 and x is2or3, or phsyiologically-hydrolysable and -acceptable ester or carbamate of such a di- or tri-sulfide wherein R and/or R2 is hydroxyalkyl, or pharmaceutically acceptable acid addition salt of such a di- or tri-sulfide wherein R1 and/or R2 is aminoalkyl, for use as an immunostimulant; and k) A di- or tri-sulfide of formula I" as illustrated above wherein R'1 is hydroxyalkyl, alkoxyalkyl, aminoalkyl or epoxyalkyl as defined under d) above, and R2 is alkyl or has the meaning given for P'1, or phsyiologically-hydrolysable and -acceptable ester or carbamate of such a di- or tri-sulfide wherein R and/or R2 is hydroxyalkyl, or pharmaceutically acceptable acid addition salt of such a di- or tri-sulfide wherein R1 and/or R2 is aminoalkyl, for use as a pharmaceutical, in particular for use as an immunostimulant.

Preferred groups and significances for the above definitions h) to k) are as hereinbefore discussed in relation to definitions a) to g).

EHydroxy-, amino- or alkoxy-cycloalkyl]-[hydroxy-, amino-, alkoxy- or epoxy-alkyl]-di- and -tri-sulfides as described above are novel per se and together with their esters and acid addition salts as herein defined, comprise a part of the present invention on their own behalf, i.e. independent of their pharmaceutical utility.

Accordingly in a further aspect the present invention also provides I) A [hydroxy-, alkoxy- or amino-cycloalkyl]-[hydroxy-, alkoxy-, amino- or epoxy-alkyl]-di- or -tri-sulfide, or physiologically-hydrolysable and -acceptable ester orcarbamate of a hydroxycycloalkyl and/or hydroxyalkyl-di or -tri-sulfide as aforesaid, or pharmaceutically acceptable acid addition salt of an aminocycloalkyl and/or aminoalkyl-di- or -tri-sulfide as aforesaid; especially m) A [2-hydroxy- or 2-alkoxy-(C3~8cycloalkyl)]-[2-hydroxy- or 2-alkoxy-(C28alkyl)]-di- or -trisulfide, or physiologically-hydrolysable and -acceptable ester or carbamate of a hydroxycycloalkyl and/or hydroxyalkyl di- or tri-sulfide as aforesaid.

Especially preferred -di- and tri-sulfides and esters according to m) above are n) those of formula I"'

wherein R3 and R4 each independently of the other is hydrogen, Alkyl or a physiologically-acceptable and -hydrolysable acyl residue, xis2or3, n is an integer from 2 to 8 and p is an integer from 1 to 3.

The hydroxy, alkoxy, amino or epoxy residue of hydroxy-, alkoxy- and amino-, -alkoxy and -cycloalkyl groups and of epoxyalkyl groups specified under I) and m) above is preferably the sole substituent on the alkyl or cycloalkyl moiety. However, in the case of I) or m) [but not n)j further substituents may if desired be present as hereinbefore described for such groups with reference to definitions a) to g) above. Similarly the alkyl moiety of hydroxy-, alkoxy- or amino-alkyl groups as specified under I) and m) may be uninterrupted or interrupted as described with reference to definitions a) to g) above. Preferably however they are uninterrupted.

Preferred alkoxy residues are C1~4alkoxy. Preferred amino residues are unsubstituted amino.

Alkyl moieties may be branched or straight chained. Preferably they are straight chained. Most preferably they contain 2 to 4 carbon atoms.

Cycloalkyl moieties preferably contain 5 to 7 carbon atoms, cyclohexyl moieties being the most preferred.

Di-sulfides are preferred to tri-sulfides.

When P3 or R4 in formula I"' is alkyl this is preferably methyl. Most preferably R2 or R4 are each hydrogen. x is preferably 2. p is preferably 2. n is preferably 2 to 4.

By the term "a physiologically-hydrolysable and -acceptable acyl residue" is meant an acyl residue which is removable by hydrolysis under physiological conditions to yield an acid which is itself physiologically acceptable.

In one group of compounds of formula I"' xis 2 and n is 2 or 3.

The present invention also provides a compound in accordance with any one of the definitions 1), m) or n) for use as a pharmaceutical, in particular for use as an immunostimulant.

Except for the compounds of definitions I) to n) above the compounds for use in accordance with the present invention are in general known or may be prepared analogously to known compounds, for example by oxidation of the corresponding mercaptans, e.g. in relation to definition b) above, of formula R1SH and R2SH, or by reaction of a corresponding mercaptan or hydrogen di-sulfide with an appropriate thiohalide, e.g. in relation to definition b) above, by reaction of a compound of formula R1SH or R1SSH with a compound of formula R2SCI or by reaction of a compound of formula R2SH or R2SSH with a compound of formula R1SCI. These reactions may be carried out in accordance with known techniques.

As already observed compounds as defined under I) to m) above are novel compounds and in accordance with a further aspect the present invention also provides a process for the production of such compounds which process comprises; o) reacting a [hydroxy-, alkoxy- or amino-cycloalkyl]- mercaptan or hydrogen disulfide or reactive derivative thereof, for example a [2-hydroxy- or 2-alkoxy-(C38cycloalkyl)j- mercaptan or hydrogen disulfide or reactive derivative thereof, or a physiologically-hydrolysable and -acceptable ester or carbamate of a hydroxycycloalkyl-mercaptan or reactive derivative as aforesaid, for example a compound of formula 11

wherein P3 and p have the meanings given for formula I"' and x' is 1 or 2, or a reactive derivative thereof, with a reactive derivative of a [hydroxy-, alkoxy-, amino- or epoxy-alkyli-mercaptan, for example a reactive derivative of a [2-hydroxy- or 2-alkoxy-(C2~8alkyl)]-mercaptan or a physiologically-hydrolysable and -acceptable ester or carbamate of a reactive derivative of a hydroxyalkylmercaptan as aforesaid, for example a compound of formula Ill Z - S - (CH2)n - OP4 (Ill) wherein R4 and n have the meanings given for formula 1"' and Z is a leaving group, and p) reacting a [hydroxy-, alkoxy, or amino-cycloalkyl]-mercaptan, for example a [2-hydroxy- or 2-alkoxy (C3-8)- cycloalkyl)j-mercaptan or a physiologically-hydrolysable and -acceptable ester or carbamate of a hydroxycycloalkyl mercaptan as aforesaid, for example a compound of formula II as illustrated above wherein P3 and p have the meanings given for formula 1"' and x' is I, with a [hydroxy-, alkoxy-, amino- or epoxy-alkyl]-mercaptan, for example a [2-hydroxy- or 2-alkoxy-(C28alkyl)]-mercaptan or a physiologicallyhydrolysable and -acceptable ester or carbamate of a hydroxy-alkylmercaptan as aforesaid, for example a compound offormula IV HS - (CH2)n - OR4 (IV) wherein R4 and n have the meanings given for formula 1"' under oxidising conditions, and recovering the obtained compound in free or acid addition salt form.

Reactive derivatives of cycloalkylmercaptans and esters thereof, e.g. compounds of formula II, for use in the method of process o) include, for example, the corresponding alkali-metal thiolates, for example, the corresponding sodium thiolates. Reactive derivatives of alkylmercaptans and esters thereof for use in the method of process o) include, for example, the corresponding halides. Suitable leaving groups as Z are accordingly chlorine and bromine, especially chlorine. The reaction is suitably carried out in the presence of a base, e.g. an alkali metal hydroxide ortriethylamine.

Suitable oxidising agents for use in accordance with process f) include e.g. iodine or potassiumhexacyanoferrate (III), in the presence of an inorganic base, in particular an alkali metal hydroxide, e.g. as recited above for process o).

Reactions o) and p) are conveniently carried out in the presence of an inert solvent or diluent such as H20 or an alkanol, such as methanol or mixtures thereof, at a temperature of from e.g. -5 C to reflux.

The starting materials for use in the above processes e.g. the compounds of formula 11,111 and IV are known or may be produced analogously to the known compounds. For example the compound 2-mercaptocyclohexanol of formula il, as well as its synthesis is described in J. Chem. Soc. 75, pp 748-754.

Esters and carbamates of definitions 1) to n) may be obtained directly in accordance with the above described processes o) and p), or they may be derived from initially obtained compounds having a free hydroxy group by acylation or alkanoylation in accordance with known techniques.

It will be appreciated that the starting materials for processes o) and p) containing a cycloalkyl moiety, e.g.

the compounds of formula II and lil as well as the products of e.g. formula I"', possess two asymmetric centres e.g. at the 1 - and 2-positions, whereby the substituents at these centres may have the cis- or trans-configuration relative to each other. These compounds accordingly exist in two diastereomeric and four optically active isomeric forms. Compounds e.g. of formula 1"' may be obtained in the form of individually optically active isomers, either by use of starting materials e.g. of formula II or III in stereospecific form (such forms being known and obtainable in accordance with known procedures) or by resolution of initially obtained isomeric, e.g. diesteriomeric or racemic mixtures, e.g. using fractional crystallisation or chromatographic techniques.The present invention is to be understood as embracing individual optically active isomeric forms of the compounds of formula I as well as racemic and diastereomeric mixtures thereof.

Naturally where further ring substituents are present additional optically active centres will be introduced, increasing the number of diastereomeric and racemic mixtures available.

For pharmaceutical purposes the use of racemic mixtures (e.g. free of diastereomeric contaminants) or individual isomers is preferred.

Throughout the present specification and claims racemic mixtures are indicated by one of the two enantiomers present followed by the designation "(Racemate)".

Examples of specific compounds defined under a) to n) above which are of particular use in accordance with the present invention are as follows: i) HO-(CH2)2-S-S-(CH2)2-OH.

ii) HO-(CH2)3-S-S-(CH2)3-OH.

iv) CH3O-(CH2)2-S-S-(CH2)2-OCH3.

RF = 0.67*

RF = 0.46**

ix) CH3O-CH2-C(CH)2-S-S-C(CH3)2-CH2-OH. RF =0.25*

xi) CH3-NH-(CH2)2-S-S-(CH2)2- NH-CH3.

xii) i-C3H7-S-S-(CH2)2-OH.

xiii) t-C4H9-S-S-(CH2)2-OH.

xviii) i-C3H7-CH(i-C3H7)-S-S-(CH2)2-OH, RF = 0.19* xix) CH3-NH-CO-O-(CH2)2-S-S-S-(CH2)2-O-CO-NH3-

(Racemate)

(Racemate). RF = 0.35 * = Silicagel (Merck5714/0100) : ethylether/hexane(1:1) ** = Silicagel (Merck 5714/0100) : ethylether/methylene chloride (1 :1) *** = Silicagel (Merck 5714/0100) : methylenechloride/methanol (9:1) Compounds v), vi), ix), xvi), xvii) and xviii) above are new and also form part of the present invention as novel compounds per se. They may be produced in accordance with known methods, e.g. analogously to methods disclosed in the art for the production of the remaining compounds i) through xx), all of which are known.

Compound xxi) is a compound in accordance with definition n) above and is also new and also forms part of the present invention as a novel compound per se. This compound may be produced e.g. in accordance with the following Example, which is illustrative of the procedures of processes o) and p) hereinbefore described.

EXAMPLE 1: Preparation of (2-h ydroxyetn yI)-(flR,2R1-2-n ydroxycyclohexyl)-disulphide {Racemate) 1 g 2-Mercaptocyclohexanol and 5.3 ml mercaptoethanol are dissolved with stirring at room temperature in a solvent medium comprising 20 ml water and 50 ml ethanol. 29.6 g potassium hexacyanoferrate (III) in 250 ml water and 5 g KOH in 30 ml water are then added simultaneously and dropwise to the obtained clear yellow solution until the pH reaches 8 to 10. The yellow suspension is stirred for a further 1 hour at room temperature and is extracted 4 times with 150 ml ether. The combined ether extracts are washed twice with 250 ml, % NaCI solution, dried on magnesium sulphate,filtered and concentrated by evaporation to yield a cloudy oil.The product is chromatographed on silicagel using methylene chloride/methanol (9:1) as eluant to yield the title product as a yellowish oil.

For use in accordance with the present invention, in particular for immunostimulant use, the subject compounds, i.e. the compounds in accordance with definitions a) to n) above, e.g. compounds i) through xxi) above, are preferably substantially free of corresponding mercaptans, i.e. from compounds of formula R1SH or R2SH, in particular from such compounds wherein R1 and/or R2 are hydroxyalkyl. Desirably they contain less than 5 %, preferably less than 2 %, most preferably less than 1% of such contaminants, in particular hydroxyalkylmercaptan contaminants.

Utility of the compounds in accordance with the invention i.e. as effective immunostimulant agents as hereinabove described, e.g. for increasing the antigenic immune respone of mammals, is shown in standard tests both in vitro and in vivo, e.g. as follows: In vitro TEST I: MishelilDutton-Test- generation of humoral response by primary immunisation of mouse spleen cells in suspension cultures to heterologous red blood cells [Science 153, 1004 (1966) and J. Exp. Med. 126, 423 (1967)].

Mouse spleen cells are cultured for 3 to 4 days in the presence of antigen (sheep erythrocytes, SE) and test substance. The cells are harvested, washed and plated with fresh antigen (SE) in semi-solid agar. After incubating for 60 minutes, complement is added and incubation continued for a further 90 minutes.

Sensitisation of mouse lymphocytes to the antigen during primary culture results in antibody release. In the presence of complement and the secreted specific antibody to SE, the sheep erythrocytes will consequently be lysed (plaque formation). Stimulation of plaque forming cells is observed using compounds in accordance with the present invention at a concentration of from ca. 0.1 Rg)ml-1 to ca. 10.0 u/ml1.

TEST II: Mixed Lymphocyte Reaction -[Bach et al., J. Exp. Med. 136, 1430 (1972)].

The reaction (i.e. proliferation and differentiation) of lymphocytes [mouse (Balb/c) spleen cells] on co-incubation for 5 days, with allogenic spleen cells from irradiated mice (CBA fi; ) is measured in the presence and absence of test-substance. Reaction in the absence of test-substance serves as control and is taken as 100 %. Reaction in the presence of test-substance is expressed as the % change compared with the 100 % control reaction. Stimulation of reaction is observed using compounds in accordance with the present invention at a concentration of ca. 0.1 Fg)ml~l to ca. 10 g/ml1.

TEST Ill: SecondaryHumorallmmunoresponse to the T-cell-specificantigen dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH) Three weeks after immunisation with DNP-KLH, mice receive a booster injection of the same antigen. The spleens are removed 1 to 4 weeks after "challenge" and a cell culture prepared. The specific anti-bodies developed in response to DNP-KLH antigen, are recovered from the supernatant and measured by ELISA-technique. Test substance is added at varying concentrations during in vitro incubation of the cell culture. Antibody development in the absence of test substance is used as control and is taken as 100 % reaction. Reaction in the present of test-substance is expressed as the % change in reaction compared with the 100 % control reaction.

Stimulation of reaction (immuno response) is observed using compounds in accordance with the present invention at a concentration of ca. 0.1 Zg/ml- to ca. 10 Fg/ml-1.

TEST IV: Stimulation of antigen-induced immune interferon production Lymphocytes [mouse (Balb/c) spleen cells] are co-incubated for 5 days with allogenic spleen cells from irradiated mice (CBA g ) analogously to test II above, in the presence or absence of test substance. The supernatants are collected and tested for levels of immune-interferon (protection of virus infected L929 fibroblasts), units of interferon measured being standardized using a human leucocyte interferon preparation of known potency.

Stimulation of immune interferon production is observed in the above test-method, using compounds in accordance with the present invention at a concentration of ca. 0.1 g/ml1 to ca. 5.0 Fg/ml-l.

In vivo TEST V: Test for Delayed- Type Hypersensitivity Reaction (cell mediated immunity) - fDietrich et al., In t.

Arch.Allergy andApplied Immunology 38,246(1970)1.

Groups of 10 or more mice are sensitised topically by painting the abdomen with antigen (oxazolone) on day 0. The test compound is administered i.p. or orally on each of the following 5 days. The challenging dose (antigen) is applied on day 9 by painting of the right ear. Skin thickness of both the right and untreated left ear are measured with a microcaliper after a further 24 hours. The mean difference in ear thickness between the two ears is taken as the parameter for evaluating the reaction. Pronounced stimulation of delayed-type hypersensitivity reaction is observed in healthy, mature mice having a normal immune response on adminstration of compounds in accordance with the present invention at a dosage of from ca. 0.1 mg/kg to ca. 50 mg/kg daily for 5 days i.p. or orally.

Particularly interesting activity was evidenced in the above test using compound i) above. Results obtained for this compound were as follows: Dose mg/kg Percentage increase of ear swelling (number of animals) 0.001 10% (10) 0.01 10% (10) 0.1 49-65% (40) 1.0 39-51% (40) 10 25-44% (20) 25 46% (10) The physiological tolerability of compound i) above is demonstrated in 72 hours acute toxicity trials employing groups of 10 mice. Administration at a dosage of 0.1 g/kg i.p. caused zero deaths, whereas administration at a dosage of 1 .Og/kg i.p. caused 10 deaths out of 10 animals. The compound shows good physiological tolerability at dosages required to elicit strong immunostimulant effect.

The tolerability of other compounds in accordance with the invention, e.g. of compounds ii) to xxi) above has been found to be of similar order to or better than that of compound i).

Compounds of the invention may also be employed in pharmaceutically acceptable acid addition salt form. Such salt forms also exhibit activity and tolerability of similar order to that of the free compounds.

The compounds of the invention, e.g. as defined under a) tog) above, including ester, carbamate and acid addition salt forms are accordingly useful as immuno-stimuiants, e.g. as immunological adjuvants, as systemic immunopotentiators and as stimulators of non-specific host resistance. The compounds of the invention I are thus indicated e.g. the tretment or supportive treatment (i.e. in combination with other specific or supportive therapy) of conditions associated with impaired immune response, especially impaired humoral response and/or delayed-type hypersensitivity and of conditions where elevation of the immune response is otherwise required. in particular, the compounds of the invention are indicated for the treatment or supportive treatment of morbid conditions arising from idiopathic immune deficiencies or as occurring in geriatric patients and patients with severe burns or general infections. The compounds of the invention are also indicated for the treatment or supportive treatment of viral illnesses (such as disseminated herpes, progressive vaccinia and disseminated varicella) as well as of Hodgkins Disease and other malignant tumors.

For the above use an indicated dosage is in the range offrom about 0.1 mg to about 350 mg, e.g. about 0.1 mg to 70 mg, administered once for adjuvant effect, e.g. in supportive treatment, or daily. For daily administration the compond is conveniently given in divided doses 2 to 4times a day or in sustained release form, and dosage forms suitable for oral administration comprise from about 0.025 mg to about 150 mg e.g.

to about 35 mg or, in the case of single dosages, up to 350 mg e.g. up to 70 mg of active ingredient admixed with a solid or liquid pharmaceutical carrier.

Having regard to their utility as immunostimulants, compounds of formula I are also indicated for use as adjuvants for vaccines. For such use an indicated dosage for oral administration comprises from about 0.5 mg to about 100 mg or, preferably, about 70 mg active ingredient.

In accordance with the foregoing the present invention also provides a compound in accordance with anyone of the definitions a) to n) above, e.g. any one of compounds i) to xxi) hereinabove, for use as an immunological adjuvant, as a systemic immuno-potentiator or for use as a stimulator of non-specific host resistance, e.g. as hereinabove described, or for use as a vaccine adjuvant, or in the treatment of conditions associated with impaired immune response.

In a further aspect the present invention also provides a method of stimulating the immune response, in particular the antigenic immune response, of a subject in need of such treatment which method comprises administering an effective amount of a compound as hereinabove defined with reference to anyone of definitions a) to n) above, for example, anyone of the compounds i) to xxi) hereinabove, as well as a method of administering a vaccine to a subject which comprises administering said vaccine together with an amount of a compound as hereinabove defined with reference to anyone of definitions a) to n) above, for example, anyone of the compounds i) to xxi) hereinabove, in an amount sufficient to induce an immuno adjuvent effect.

In yet a further aspect the present invention also provides a pharmaceutical composition comprising a compound in accordance with anyone of the definitions a) to n) above, in particular definitions d) to n) above, together with a pharmaceutically acceptable diluent or carrier therefor.

Pharmaceutical compositions in accordance with the invention may be prepared employing standard galenical techniques, e.g. by admixture with conventional pharmaceutically acceptable diluents, carriers or other excipients. Such formulations are conveniently compounded, e.g. in tablet or capsule form, or in form suitable for injection.

The following Examples illustrate compositions in accordance with the invention useful as immunostimulants.

EXAMPLE 2: Production of solid pharmaceutical compositions.

Tablets may contain the active agent in admixture with conventional pharmaceutically acceptable excipients, e.g. inert diluents, such as calcium carbonate, sodium carbonate, lactose and talc, granulating and disintegrating agents, e.g. starch and alginic acid, flavouring, colouring and sweetening agents, binding agents, e.g. starch, gelatin and acacia, and lubricating agents, e.g. magnesium stearate, stearic acid and talc.

The tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a long period.

For the manufacture of tablets, the active ingredient, e.g. compound of formula I can be mixed with lactose and granulated with water, 0.5% sodium alginate or 1% gelatine solution. The dried granulate is compressed into tablets in the presence of about 5% of talcum, 5% of corn starch and 0.1% of magnesium stearate. In this way, there are obtained, e.g. tablets of the following composition: Compound of formula I, e.g.

di-2-hydroxyethyl disulphide 20mg Lactose 70 mg Corn Starch 5 mg Talcum 5 mg Magnesium stearate 0.1 mg These tablets, which are provided with a crackline, can be administered orally in a dosage of one tablet two to four times per day.

Capsules may contain the active agent alone or admixed with an inert solid diluent, for example as mentioned above.

Capsules containing the ingredient indicated below may be prepared by conventional techniques and are administered at a dose of one capsule 2 to 4 times a day.

Ingredient Weight (mg) Capsule Compound of formula I, e.g. di-2-hydroxy ethyl disulphide 20 Inert solid diluent (Starch, caolin, calcium phosphate or carbonate, lactose etc.) 20 EXAMPLE 2: Production of liquid pharmaceutical compositions.

Solutions, suspensions, emulsions, dispersions, syrups and elixirs may contain the compound of formula I as the active agent in the form as described in the proceeding example in admixture with any of the conventional excipients utilized for the preparation of such compositions, e.g. suspending agents (methyl-cellulose, tragacanth and sodium alginate), wetting agents (lecithin, polyoxyethylene stearate and polyoxyethylene sorbitan monoleate) flavouring, colouring and sweetening agents and preservatives (ethyl-p-hydroxybentoate).

The following pharmaceutical compositions are formulated with the indicated amount of active agent using conventional techniques. The injectable suspension and the oral liquid suspension represent formulations useful as unit doses. The injectable suspension is suitable for administration once a day, whereas the oral liquid suspension is suitably administered 2 to 4 times per day for this purpose.

Ingredient Sterile injectable Weight (mg) suspension Oral liquid suspension Compound of formula I, e.g. di-2-hydroxyalkyl disulphide 20 20 Sodium carboxy methyl cellulose U.S.P. 1.25 12.5 Methyl cellulose 0.4 Polyvinylpyrrolidone 5 Lecithin 3 Benzyl alcohol 0.01 Magnesium aluminium silicate Flavour --- q.s.

Colour --- q.s.

Methyl paraben, U.S.P. --- 4.5 Propyl paraben, U.S.P. --- 1.0 Polysorbate 80 (e.g.

Tween (RTM 80), U.S.P. --- 5 Sorbitol solution, 70% U.S.P. --- 2.500 Buffer agent to adjust pH for desired stability q.s. q.s.

Water For injection q.s.to1 ml q.s. to 5 ml.

European Patent Publication No.0 058 134 Al (Application No.82810038.1: filed February 4, 1982) as well as the corresponding U.S. Patent Application No. 344,175 describes and claims novel bis- and poly-disulphides of formula A, R1O(CH2)m-StS-(CH2)p-SiXS(CH2)nOR2 (A) wherein R1 and R2 are, independently, hydrogen, Alkyl or a physiologically-hydrolysable and -acceptable acid residue, m and n are, independently, 2 or 3, p is an integer of from 2 to 6, and x xis1,2Or3, whereby when x is 2 or 3, each p may be the same or different.

In accordance with the teachings of said applications, these compounds possess immunostimulant activity and are useful, inter al. in the same indications as the subject compounds of formula I. No claim is made in the present application to the compounds of formula A above orto their use as immunostimulants, to their use in any method of treatment orto any pharmaceutical composition containing a compound of formula A above.

Claims (22)

1. A hydroxyalkyl-, hydroxycycloalkyl-, alkoxyalkyl-, alkoxycycloalkyl-, aminoalkyl-, aminocycloalkyl- or epoxyalkyl- -di- or -tri-sulfide, or physiologically-hydrolysable and -acceptable ester or carbamate of a hydroxyaikyl- or hydroxycycloalkyl- -di- or -trisulfide, or pharmaceutically acceptable acid addition salt of an amino-alkyl- or aminocycloalkyl- -di- or -trisulfide, for use as an immunostimulant.

2. A di- ortri- sulfide according to claim 1 of formula I P1 - (S)x P2 (I) wherein R1 is hydroxyalkyl, hydroxycycloalkyl, alkoxyalkyl, alkoxycycloalkyl, aminoalkyl, aminocycloalkyl or epoxyalkyl, R2 is alkyl or cycloalkyl or has the meaning given for R1 and x is2or3, or physiologically-hydrolysable and -acceptable ester or carbamate of such a di- or tri-sulfide wherein R1 and/or R2 is hydroxyalkyl or hydroxycycloalkyl, or pharmaceutically acceptable acid addition salt of such a di- or tri-sulfide wherein R1 and/or R2 is aminoalkyl or aminocycloalkyl, for use as an immunostimulant.

3. A compound according to claim 2 wherein, in formula I, R1 is hydroxyalkyl wherein the hydroxy group is in the 2-position or is terminal, 2-hydroxy-cycloalkyl, 2-alkoxyalkyl, P-alkoxycycloalkyl, 2-aminoaikyl or 2-epoxyalkyl, and R2 is alkyl or cycloalkyl or has the meaning given for R1.

4. A hydroxyalkyl-, hydroxycycloalkyl-, alkoxyalkyl-, alkoxycycloalkyl-, aminoalkyl-, aminocycloalkyl- or epoxyalkyl- -di- or -tri-sulfide in which the hydroxy, alkoxy, amino or epoxy residue is the sole substituent on the alkyl or cycloalkyl moiety or in which the alkyl or cycloalkyl moiety is further substituted by one or more members selected from the group consisting of phenyl, cycloalkyl, halogen, hydroxy, alkoxy and amino and, in the case of cycloalkyl, alkyl and in which any alkyl moiety is aforesaid is uninterrupted, or physiologically-hydrolysable and -acceptable ester or carbamate of such a hydroxyalkyl- or hydroxycycloalkyl- -di- or -tri-sulfide, or pharmaceutically acceptable acid addition salt of such an aminoalkyl- or aminocycloalkyl- -di- or -tri-sulfide, for use as a pharmaceutical.

5. A di- or tri-sulfide according to claim 4 of formula las illustrated in claim 2, wherein R1 is hydroxyalkyl, hydroxycycloalkyl, alkoxyalkyl, alkoxycycloalkyl, aminoalkyl, aminocycloalkyl or epoxyalkyl as defined in claim 4, R2 is alkyl or cycloalkyl or has the meaning given for R1, and x is2or3, or physiologically-hydrolysable and -acceptable ester or carbamate of such a di- or tri-sulfide wherein R1 and/or R2 is hydroxyalkyl or hydroxycycloalkyl, or pharmaceutically acceptable acid addition salt of such a di- or tri-sulfide wherein R and/or R2 is aminoalkyl or aminocycloalkyl, for use as a pharmaceutical.

6. A compound according to claim 5 wherein, in formula I, R1 is hydroxyalkyl wherein the hydroxy group is in the 2-position or is terminal, 2-hydroxycycloalkyl, 2-alkoxyalkyl, 2-alkoxycycloalkyl, 2-aminoalkyl or2-epoxyalkyl as defined in claim 4, and R2 is alkyl or cycloalkyl or has the meaning given for R.

7. A compound according to claim 5 wherein, in formula R1 is hydroxyalkyl wherein the hydroxy group is in the 2-position or is terminal, 2-hydroxycycloalkyl, 2-alkoxyalkyl, 2-alkoxycycloalkyl, 2-aminoalkyl or2-epoxyalkyl in which the hydroxy, alkoxy, amino or epoxy residue is the sole substituent on the alkyl orcycloalkyl moiety and in which any alkyl moiety as aforesaid is uninterrupted; or hydroxyalkyl in which hydroxy group is in the 2-position or is terminal and in which the alkyl moiety bears one further susbstituent selected from the group comprising cycloalkyl, hydroxy and halogen and is uninterrupted, and R2 is alkyl or has the meaning given for R.

8. A compound according to any one of claims 4to 7 wherein R2 is unsubstituted alkyl or has the meaning given for R.

9. Adisulfide according to claim 1 of formula I' P1, - S - S - P2, (I') wherein R, and R2 each independently of the other is hydroxyalkyl.

10. A disulfide according to claim 4 of the formula I' as illustrated in claim 9, wherein R; and R2 each independently of the other is hydroxyalkyl in which the hydroxy residue is the sole substituent on the alkyl moiety or in which the alkyl moiety is further substituted by one or more members selected from the group consisting of phenyl and hydroxy and in which the alkyl moiety is uninterrupted.

11. A compound according to claim 1 which is a di- or tri-sulfide of formula I" R1"(S)xR"2 wherein P'1, is hydroxyalkyl, alkoxyalkyl, aminoalkyl or epoxyalkyl, R"2 is alkyl or has the meaning given for P2 and x is2or3, or physiologically-hydrolysable and -acceptable ester or carbamate of such a di- or tri-sulfide wherein R1 and/or R2 is hydroxyalkyl, or pharmaceutically acceptable acid addition salt of such a di- or tri-sulfide wherein R1 and/or R2 is aminoalkyl.

12. A compound according to claim 4 which is a di- ortri-sulfide of formula I" as illustrated in claim 11 wherein Rn' is hydroxyalkyl, alkoxyalkyl, aminoalkyl or epoxyalkyl as defined in claim 4, and R2 is alkyl or has the meaning given for R; or physiologically-hydrolysable and - acceptable ester or carbamate of such a di- or tri-sulfide wherein R1 and/or R2 is hydroxyalkyl, or pharmaceutically acceptable acid addition salt of such a di- or tri-sulfide wherein R1 and/or R2 is aminoalkyl.

13. A compound according to claim 12 selected from the group consisting of: i) HO-(CH2)2-S-S-(CH2)2- OH; ii) HO-(CH2)3-S-S-(CH2)3-OH;

iv) CH30-(CH2)2-S-S-(CH2)2-OCH3;

ix) CH30-CH2-C(CH3)2-S-S-C(CH3)2-CH2-OH;

xi) CH3-NH-(CH2)2-S-S-(CH2)2-NH-CH3; xii) i-C3H7-S-S-(CH2)2-OH; xiii) t-C4H9-S-S-(CH2)2-OH;

xviii) i-C3H7-CH(i-C3H7)-S-S-(CH2)2-OH; xix) CH3-NH-CO-O-(CH2)2-S-S-S-(CH2)2-O-CO-NH- and

(Racemate)

14.A [hydroxy-, alkoxy- or amino-cycloalkyl]-[hydroxy-, alkoxy-, amino- or epoxy-alkyl]-di- or -tri-sulfide, or physiologically-hydrolysable and -acceptable ester or carbamate of a hydroxycycloalkyl and/or hydroxyalkyl-di- or -tri-sulfide as aforesaid, or pharmaceutically acceptable acid addition salt of an aminocycloalkyl and/or aminoalkyl-di- or -tri-sulfide as aforesaid.

15. A compound according to claim 14 which is a [2-hydroxy- or 2-alkoxy-(C57cycloaIkyI)]-[2-hydrnxy- or 2-aikoxy-(C2~8alkyl)]-di- or -tri-sulfide, or physiologically-hydrolysable and -acceptable ester or carbamate of a hydroxycycloalkyl and/or hydroxyalkyl di- ortri-sulfide as aforesaid.

16. A compound according to claim 15 of formula jZD

wherein R3 and R4 each independently of the other is hydrogen, C15alkyl or a physiologically acceptable and hydrolysable acyl residue, x is2or3, n is an integer from 2 to 8 and p is an integer from 1 to 3.

17. A compound according to claim 16 wherein xis 2 and n is 2 or 3.

18. A compound according to claim 17 which is (2-hydroxyethyl)-([1 P,2P]-2-hydroxycyclohexyl)-disulfide (Racemate).

19. A compound selected from the group consisting of the compounds of formulae v), vi), ix), xvi), xvii) and xviii) as illustrated in claim 13.

20. A compound according to any one of claims 14to 19 for use as a pharmaceutical.

21. A compound according to claim 20 for use as an immunostimulant.

22. A pharmaceutical composition comprising a compound as claimed in any one of claims 1 to 21, together with a pharmaceutically acceptable diluent or carrier therefor.