GB2148296A - 1,2-dithiolan derivatives, process for their production pharmaceutical compositions containing them and their use - Google Patents

Abstract

Pharmaceutical composition containing 1,2-dithiolane-1-oxide carboxylic acids or esters, amides or salts thereof, especially compounds of formula I <IMAGE> wherein R1 represents hydroxy, lower alkoxy, amino, cycloalkylamino or mono- or di-lower alkylamino whereby alkyl moieties may be @@@ unsubstituted or substituted by aryl, heteroaryl or cycloalkyl and n is an integer from 0 to 10, possess immunestimulant activity.

Description

SPECIFICATION 1,2-Dithiolan derivatives, processes for their production, pharmaceutical compositions containing them and their use The invention relates to 1 ,2-dithiolane-1 -oxide-carboxylic acids and esters, amides and salts thereof having immunostimulant activity.

More particularly the invention relates to compounds offormula I

wherein R1 represents hydroxy, lower alkoxy, amino, cycloalkylamino or mono- or di-lower alkylamino whereby alkyl moieties may be unsubstituted or substituted by aryl, heteroaryl or cycloalkyl and n is an integer from 0 to 10, where appropriate in free form or in salt form.

Various free acids and alkyl esters and salts thereof are known e.g. from Tetrahedron Letters 13, 1073-5 (1973); Arch. Biochem. Biophys. 185,576-83, (1978); J. Org. Chem. 40,58-62 (1975); Arkiv. Kemi. 25 (14-4), 263-77 (1966); JP Kokai 49/117616 as is 6,8-dithiooctanoic amide monooxide cf. JP Kokoku.10931/64. There is no disclosure of pharmaceutical use for these compounds.

It has now surprisingly been found that 1,3-dithiolane oxide carboxylic acids and derivatives thereof possess pharmacological, in particular immunostimulant activity as hereinafter described. In a particular aspect therefore the present invention concerns pharmaceutical compositions containing a 1,2-dithiolane-1- oxide carboxylic acid or a pharmaceutically acceptable ester, amide or salt thereof and such compounds for use as pharmaceuticals, in particular as immunostimulants.

More particularly the invention concerns a pharmaceutical composition containing a compound of formula I as defined above or where appropriate a pharmaceutically acceptable salt thereof and such compounds for use as pharmaceuticals in particular as immunostimulants.

The compounds of formula Ip

wherein R; represents cycloalkylamino or mono- or di-lower alkylamino whereby alkyl moieties may be ubsubstituted or substituted by aryl, heteroaryl or cycloalkyl and n is an integer from 0 to 10, are new and also form part of the invention.

The compounds of formula Ip also comprise a preferred group of compounds of formula I.

The compounds of formula Ip may be obtained by oxidising the corresponding compound of formula llp

wherein R1' and n are as defined above.

This process according to the invention can be effected using conventional oxidation procedures e.g.

employing m-chloro perbenzoic acid. The reaction can be carried out in an inert solvent e.g. in a chlorinated hydrocarbon such as dichloromethane preferably at lower temperatures e.g. under ice-cooling. The final product can be isolated and purified in conventional manner.

The remaining compounds can be prepared analogously or e.g. as described in the above mentioned literature.

The compounds of formula I can occur in syn- and anti-forms and in the form of optical isomers. Mixtures obtained from reactions can be separated in conventional manner e.g. by chromatography. When compounds are in a particular isomericform this will be stated.

The compounds of formula llp are in part new (IlpA) and these compounds, which also form part of the invention, may be prepared as follows:

wherein R2 and R3 represent, independently, cycloalkyl or alkyl which may be unsubstituted or substituted by cycloalkyl, heteroaryl or aryl and one of R2 and R3 may additionally represent hydrogen.

These two processes may be carried out in conventional manner e.g. as hereinafter described in the examples. The compounds of formula Ill are also new and constitute a further aspect of the invention.

The remaining intermediates are either known or may be prepared analogously to known techniques.

Intermediates may be isolated and purified in conventional manner or where appropriate further reacted directly.

Lower alkyl moieties preferably contain 1 to 4 especially 1 or 2 carbon atoms. Aryl stands for e.g. phenyl which may be substituted or preferably unsubstituted. Heteroaryl preferably stands for a heterocycle which may optionally be fused to a benzene ring which itself may be unsubstituted or substituted. Examples of substituents are alkoxy. Cycloalkyl groups preferably contain 3 to 10 especially 6 to 8 ring members. n is preferably an even number especially 0, 2 or 4.

In accordance with the present invention it has now surprisingly been found that 1 ,2-dithiolane-1 -oxide carboxylic acids and pharmaceutically acceptable esters, am ides and salts thereof especially the compounds of formula I (hereinafter compounds of the invention) possess immunestimulant activity.

More particularly it has been found that the said compounds stimulate not only antigen responsive lymphoproliferation, but also antibody production (primary and secondary immune response) as well as cell-mediated immuneresponse.

The immunestimulant activity of the subject compounds may be shown in standard tests both in vitro and in vivo. Thus positive immunostimulant activity is shown for compounds of the invention e.g. in the following test methods: In vitro: Testy: Mishell/Dutton 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 of the invention at a concentration of from 0.01 to 10.0 pg/ml.

Test II: Mixed lymphocyte reaction - [Bach etal.,J. Exp. Med. 136,1430 (1972)].

The reaction (i.e. proliferation and differentiation of lymphocytes [mouse (Balbic) spleen cells] on co-incubation for days, with allogeneic spleen cells from irradiated mice (CBA ) 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 of the invention at a concentration of 0.4 to 10 I*g/ml-'.

Test III: Secondary humoral immuneresponse to the T-cell-specific antigen 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 antibodies developed in response to DNP-KLH antigen, are recovered from the supernatant and measured by the 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 presence of test-substance is expressed as the % change in reaction compared with the 100% control reaction.

Stimulation of the reaction (immuneresponse) is observed using compounds of the invention at a concentration of 0.4 to 10 Fgiml.

Test IV: Stimulation ofantigen-induced immune interferon production.

Lymphocytes [mouse (Balbic) spleen cells] are co-incubated for 5 days with allogenic spleen cells from irradiated mice (CBA ;4 ) analogously to test II above, in the presence or absence of test substance. The supernatants are collected and tested for levels of immuneinterferon (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 of the invention at a concentration of ca. 0.2 to 5.0 Fg/ml.

In vivo: Test V: Test for delayed-type hypersensitivity reaction (cell mediated immunity) - [Dietrich eta!., Int. Arch.

Allergy 38,246(1970)].

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 hour. 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 administration of compounds of the invention at a dosage of from 0.1 to 10 mg/kg daily for 5 days i.p. or orally.

Test Vl: Jerne Test-generation ofhumoralresponses (assay forhaemolytic plaque forming cells) - [Jerne et al., "Cell Bound Antibodies" (ed. Amos and Koprowski), Wistar Inst. Press., Philadelphia, U.S.A. pp.

109-1221].

Mice are sensitised by i.v. injection of sheep erythrocytes (SE) and the test substance administered i.p. on day 0. After 4 to 10 days the mice are sacrificed and a spleen cell suspension prepared. The cell suspension is plated, together with fresh antigen (SE), on semi-solid agar and incubated for 2.5 hrs. in the presence of complement. Sensitised lymphocytes secrete antibody in response to antigen and, in the presence of complement, the antigen (SE) is lysed (plaque formation).

Administration of compounds of the invention at a dosage of from 0.1 to 1.0 mg/kg results in an increase both of 1gM as well as 1gG antibodies.

Test VII: Activation ofnatural killer cells.

Test substance is administered i.p. or p.o. to nude (athymic) mice (Balbic or C57/BL). After 16 hours the spleens are removed and the spleen cells incubated for 4 hrs. with chromium labelled target cells (YAK-1).

Natural killer cells destroy the target cells, releasing labelled chromium into the supernatant. The supernatant is collected and the amount of chromium released measured by means of a scintillation counter.

Release from target cells incubated with spleen cells from untreated mice is used as control and is taken as 100% and release following administration of test substance expressed as the % change in release compared with the control.

Increase in chromium release is observed subsequent to administration of compounds of the invention at dosages of from 0.1 to 10.0 mg/kg.

The compounds of formula I are accordingly indicated for use as immunestimulants, e.g. as immunological adjuvants, as systemic immuno-potentiators and as stimulators of non-specific host resistance. Compounds of the invention are thus indicated for e.g. the treatment 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 indicated. 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 uses as indicated oral dose is from about 0.1 mg to about 70 mg, administered once for adjuvant effect, e.g. in supportive treatment, or daily, administration in the latter case conveniently being effected in divided doses administered 2 to 4 times a day or in sustained release form. Indicated unit dosage forms for oral administration accordingly contain from about 0.025 mg to about 35 mg compound of formula I, where daily administration is desired or up to 70 mg compound of formula I, where a single, adjuvant treatment is desired.

Having regard to their immunostimulant activity, compounds of formula I are also indicated as adjuvants for vaccines. For this use an indicated oral dose is from 0.5 mg to 100 mg, preferably about 70 mg, administered on the day of vaccination with an optional follow-up at the same dosage rate 2 to 4 weeks later.

The compounds of formula I, wherein R1 represent hydroxy or amino may be used in free form or in the form of pharmaceutically acceptable salts, whereby the latter exhibit the same order of activity as the free forms.

The compounds may be administered enterally e.g. orally or parenterally e.g. as injectables.

Pharmaceutical compositions comprising the compounds of the invention may be prepared in accordance with 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 forms suitable for injection.

In accordance with the foregoing the present invention also provides a compound of the invention as hereinbefore defined for use as a pharmaceutical, in particularfor use as an immunostimulant, especially for use in treatment or supportive treatment, e.g. of conditions associated with impaired immune response as hereinbefore set forth.

In a further aspect the invention also provides a method of stimulating the immune response of a subject in need of such treatment which method comprises administering an effective amount of a compound of the invention as herein before defined.

In a yet further aspect the invention also provides a pharmaceutical composition comprising a compound of the invention as hereinbefore defined, together with a pharmaceutically acceptable diluent or carrier therefor.

Within the compounds of the invention those of formula I are preferred. Particularly interesting are compounds of formula Ip.

Preferred substituent meanings are: R1 = a) OH, OAlk or NH2 b) NH Alkyl c) N(Alkyl)2 d) substituted monoalkylamino e) NH-CH2(1 or 2)-phenyl, -cycloalkyl, -bicyclic heteroaryl n = a) even number b)0,2or4 c) O position of COR1 = a)3or5 b)4.

A particular compound group is that of formula Ip, wherein R; represents monoalkylamino substituted by cycloalkyl, phenyl or bicyclic heteroaryl, the lattertwo of which may themselves be unsubstituted or substituted by alkoxy; or cycloalkylamino and n is 0.

A further compound group is that of formula I, wherein R1 represents hydroxy, amino, mono- or di-(lower)alkyl amino, lower alkoxy or MeO, wherein Me is a metal equivalent and alkyl moieties may be unsubstituted or substituted by aryl and n is an integer from 0 to 10. Within this group compounds are preferred wherein R1 is other than hydroxy, MeO, lower alkoxy or amino.

In each of the above compound groups the R1 bearing group is preferably in 4-position.

For the above mentioned use the compounds are preferably in (anti) form.

A particularly preferred single compound is (anti)-1,2-dithiolane-4-carboxylic acid cyclohexylmethylamide-S-oxide.

The following examples illustrate the invention, temperatures being in degrees centigrade.

Example 1: Syn- and anti- 1,2-dithiolan-4carboxylic acid-3,4-dimethoxybenzylamide- 1-S-oxide 0.3 g of asparagusic acid veratrylamide are taken up in 15 ml of dry dichloromethane and cooled to ice bath temperature. 0.21 g of m-chloroperbenzoic acid dissolved in 15 ml of dry dichloromethane are added.

After 1 hour the reaction mixture is shaken with sodium bicarbonate solution, dried overwater-free magnesium sulfate and concentrated by evaporation. The crystalline residue is taken up in a little dichloromethane and separated over a medium pressure column (Merck [RTM] LiChroSorb [RTM] Si60, size C; eluant: ethylacetate) syn-isomer: Rf = 0.23, m.p. = 164-166 anti-isomer: Rf= 0.35,m.p. = 191-193 The following compounds may be obtained analogously to Example 1. (The "position" column indicates the point of attachment of the side chain; Rf-values are measured in toluenelethylacetate = 111).

Ex. Rr Pos. n m.p. Rf: 2 -NH.CH2- /H) 4 0 syn 103-105" ~ anti 131-133" 3 -NH.CH2.C6H5 4 0 syn 99-103" anti 161-163" 4 -NH/ H /. 4 0 syn139-144" 0,18 anti 179-180" 0,25 5 -NH.CH2 . 4 0 syn Syrup 0,16 . ,. anti Syrup 0,28 6 -NH-CH2CH2 .~.o 3 4 4 0 syn 125-145 0,44 II ,1 anti198-201" 4 anti 198-201" 0,51 H 7 -NH.CH2H > 3 0 syn131-134" anti 139-141" The required starting materials may be prepared as follows.

A. Asparagusic acid veratrylamide a) N-Hydroxysuccinimide ester of aspargusic acid To 0.32 g asparagusic acid, dissolved in 20 ml of dry dichloromethane, are added 0.62 g of di-(N-succinimidyl)carbonate, 0.3 ml of pyridine and 1 g of water-free magnesium sulfate. The mixture is warmed to 50" under nitrogen flushing and refluxed with stirring for 3 hours. The reaction mixture is then allowed to cool, the magnesium sulfate filtered off and the filtrate concentrated by evaporation. The residue is purified over a medium pressure column (Merck LiChroSorb Si60, size C; eluant toluenelethylacetate = 1/1, Rf = 0.56). m.p. 129".

b) Asparagusic acid veratrylamide 0.062 g of the N-hydroxysuccinimide ester of asparagusic acid are dissolved in 5 ml of dry dichloromethane to which are then added at room temperature 0.122 g of veratrylamine. After 20 minutes the reaction mixture is concentrated by evaporation and purified over a medium pressure column (Merck LiChroSorb Si60, size A; eluant : toluenelethylacetate = 1/1; RF = 0.32), m.p. 154".

The following starting materials may be obtained in analogous manner (the "position" column indicates the point of attachment of the side chain).

R7 Pos. n m.p.

B NH.CH2- H ) 4 119-123 .~.

C NH.CH2.C6H5 4 0 120-123" D -NH/ H \. 4 0 159-160" H E NHCH21\ t , 4 0 73-74" s , F -NH.CH2CH2" H3 4 0 Syrup 'N ." G -NH-CH2~1 H ) 3 0 100-103" Further compounds which may be employed according to the invention are e.g. those of formula I described in the above-mentioned literature such as syn- and anti-l ,2-dithiolane-3-carboxylic acid-1-oxide (m.p. 115-7-and resp.) and syn- and anti-l .2-dithiolane-4-carboxylic acid-1-oxide (m.p. 130-140and 130-136- resp.)

Claims (19)

1. A pharmaceutical composition containing a 1,2-dithiolane-1-oxide carboxylic acid or a pharmaceutically acceptable ester, amide or salt thereof together with a pharmaceutically acceptable diluent or carrier therefor.

2. A pharmaceutical composition containing a compound of formula I

wherein R1 represents hydroxy, lower alkoxy, amino, cycloalkylamino or mono- or di-lower alkylamino whereby alkyl moieties may be unsubstituted or substituted by aryl, heteroaryl or cycloalkyl and n is an integer from 0 to 10, where appropriate in free form or in the form of a pharmaceutically acceptable salt together with a pharmaceutically acceptable diluent or carrier therefor.

3. A pharmaceutical composition according to Claim 2, wherein in formula I R1 represents cycloalkylamino or mono-adi-lower alkylamino whereby alkyl moieties may be unsubstituted or substituted by aryl, heteroaryl or cycloalkyl and n is an integer from 0 to 10.

4. A compound of formula Ip

wherein R; represents cycloalkylamino or mono- or di-lower alkylamino whereby alkyl moieties may be unsubstituted or substituted by aryl, heteroaryl or cycloalkyl and n is an integer from 0 to 10.

5. A compound according to Claim 4, wherein R1' is selected from a) NH Alkyl b) N(Alkyl)2 c) substituted monoalkylamino d) NH-CH2(1 or 2)-phenyl, -cycloalkyl, -bicyclic heteroaryl.

6. A compound according to Claim 4 or 5, wherein the position of the group containing R1, is selected from a)3or5 b)4.

7. A compound according to any one of Claims 4to 6, wherein n is selected from a) even number b)0,2or4 c) O.

8. A compound according to Claim 4, wherein R1' represents monoalkylamino substituted by cycloalkyl, phenyl or bicyclic heteroaryl, the latter two of which may themselves be unsubstituted or substituted by alkoxy; or cycloalkylamino and n isO.

9. A compound according to Claim 4, wherein R1' represents mono- or di(lower)alkylamino and alkyl moieties may be unsubstituted or substituted by aryl and n is an integer from 0 to 10.

10. A compound according to any one of Claims 4 to 10, wherein the R1' bearing group is in 4-position.

11. (Anti)-1 ,2-dithiolane-4-carboxylic acid cyclohexylmethylamide-1 -S-oxide.

12. A compound selected from (anti)-1 ,2-dithiolane-4-carboxylic acid-3,4-dimethoxylbenzylamide-1 -Soxide and (anti)-1 ,2-dithiolane-4-carboxylic acid benzylamide-1 -S-oxide.

13. A compound selected from (anti)-1 ,2-dithiolane-4-carboxylic acid cyclohexylamide-1 -S-oxide, (anti) 1 ,2-dithiolane-4-carboxylic acid cyclooctylamide-1 -S-oxide, (anti)-1 ,2-dithiolane-4-carboxylic acid-S- methoxy-indol-3-ylamide-1 -S-oxide and (anti)-1 ,2-dithiolane-3-carboxylic acid cyclohexylmethylamide-1-S- oxide.

14. A compound according to any one of Claims 11 to 13 in corresponding (syn)-form.

15. A compound according to any one of Claims 4to 14 in free form.

16. A compound according to any one of Claims 4 to 14, wherein R; represents cycloalkylamino or optionally substituted mono-(lower)-alkylamino in salt form.

17. A compound according to any one of Claims 4to 16 in antiform.

18. A process for preparing a compound according to Claim 4 and salts thereof which comprises oxidising the corresponding compound of formula llp

wherein R; and n are as defined above, and recovering the compound obtained in free form or salt form as appropriate.

19. A compound of formula IlpA or Ill as hereinbefore defined.