IE860630L

IE860630L - Heterocyclic disulphides

Info

Publication number: IE860630L
Application number: IE860630A
Authority: IE
Original assignee: Hoechst Ag
Priority date: 1985-03-12
Filing date: 1986-03-11
Publication date: 1986-09-12
Also published as: NO860908L; PH22275A; DE3508666A1; DK165181C; KR940001773B1; ATE58293T1; ES8801624A1; IL78091A; KR860007246A; IL78091A0; EP0194571B1; DK108786A; PT82166A; AR245113A1; NZ215424A; DE3675561D1; EP0194571A1; GR860649B; ZA861780B; IE58729B1

Abstract

The disulphides have the formula heterocycle-S-S-heterocycle and are used for immunostimulation, immunorestoration and cytostatic treatment in the form of pharmaceutical agents. [EP0194571A1]

Description

It is known that the defense mechanisms of the living body, which are briefly termed the humoral immunity and cellular immunity, cooperate to neutralize and eliminate foreign bodies which may give rise to pathogenetic changes and be injurious, principally microorganisms or neoplastic cells.

Immunological investigations have shown that there are connections between a decrease in immunological activity provoked by internal or external factors and an increase in the infectious or tumorous diseases. In addition, other diseases arise owing to changes in the functioning of the immune system. These include, for example, autoimmune diseases or disorders caused by immune complexes. Thus, there has for a long time been a search for immuno-stimulants, that is to say substances which are able to modify the immunological activity of the recipient, preferably to increase it, and which allow, by reason of their high efficacy and good tolerability, wide use to support the body's defenses. Examples which have been tested for their stimulation of immunity are BCG and C. parvum, as well as extracts of M. tuberculosis and of the Brucellae.

However, at the concentrations at which these substances are used they produce marked side effects such as, for example, local granulomas to varying extents. The lack of knowledge of the exact nature of these substances makes it difficult to carry out a systematic investigation with reasonable reproducibility of the clinical results. Thus, in this connection there is a requirement for new immuno-stimulants which are chemically defined substances and have low toxicity, such as, for example, bestatin which is an immunostimulant of low molecular weight, is undergoing intensive investigation at present and is, in general, a scientific reference substance.

US-A-4,378,364 describes certain bis(hetero)disulfides whose heterocyclic ring can also be substituted by 3 carboxyl groups. Administration of these compounds to cancer patients after surgery is said to lead to an improvement in wellbeing, in activity, in appetite, in weight gain and to a reduction in pain. This is shown in 5 various examples with 6,6'-dithiodinicotinic acid (CPDS).

It has now been found, surprisingly, that the compounds according to the invention have a potent immuno-stimulating and immunorestorative action, as is expressed, for example, in the DTH reaction on sheep 10 erythrocytes, in the activation of mononuclear phagocytes and in a pronounced CSF activity. These immunostimulating effects can also be observed, for example, in an increase in the power of resistance to infections. Furthermore, the compounds according to the invention have, sur-15 prisingly, cytostatic activity, for example against B16 melanoma of the mouse.

The invention thus describes a class of substances which have immunopharmacological and cytostatic activity, are chemically defined, have low toxicity and are, as such 20 or combined with other active compounds, valuable medicaments . The compounds according to the invention have a LDS0 value above 1,000 mg/kg on intravenous injection in mice. The effective immunomodulatory and cytostatic amount for vertebrates, preferably warm-blooded mammals 25 is in the range from about 0.5 to about 100 mg/kg of body weight for each parenteral or oral dose, and this shows no toxic side effects and is thus very well suited for the treatment of diseases of the immune system.

"Thus the invention relates to disulfides of the general 30 formula I' Het'-S-S-Het' (I') in which Het' means 1,3-thiazolyl which is substituted by Cj-Ce-alkyl; by carboxy-C^-Cg-alkyl; by carboxyl or by C2-Cj-acylamino, where acyl is the radical of an aliphatic 35 dicarboxylic acid; - 4 1,2,4-triazolyl, which is substituted by hydroxyl and phenyl; 1,3,4-thiadiazolyl, which is substituted by carboxy-C,-C4-alkylthio, dihydro-1,2,4-triazinyl which is substituted by Ci-Cg-alkyl, halogeno-C2-C Suitable Ci-Cg-alkyI substituents are straight-chain or branched alkyl groups with 1-6, preferably 1-3, carbon atoms, such as, for example, methyl, ethyl, n- or i-propyl, n- or tert.-butyl, preferably methyl, which can optionally in turn be substituted by carboxyl.

Examples which may be mentioned of a C2-C5-acylamino group in which acyl is the radical of an aliphatic dicarboxylic acid are 3-carboxy-propionyl and 4-carboxy-butyryl.

Examples of C^-C^-alkylthio radicals substituted by carboxyl which may be mentioned are methylthio and ethylthio and of halogeno-Cz-C^-alkenyl radicals are vinyl or allyl which are substituted by, for example, chlorine or bromine.

The heterocycle Het' can be substituted once or several times, for example once to three times. However, preference is given to those compounds which carry one or 2 substituents in the heterocyclic ring or in the carbo-cyclic ring which is fused on. At least one of these substituents must have an acid group. Thus, either one 5 substituent can carry a carboxyl group such as, for example, carboxyalkyl or carboxyalkylthio, or it can also be bonded as carboxyl or hydroxyl group directly to the heterocycle Het'. 5 Particularly preferred according to the invention are compounds in which Het' is a thiazole residue which is substituted by a carboxyl or a carboxymethyl group.

Since the compounds of the general formula I' carry acid groups, such as, in particular, a carboxyl group, they 10 can also be in the form of their physiologically tolerated salts, for example as alkali metal and alkaline-earth metal salts such as, preferably, the Na, K, Ca or Mg salts or, for example, ammonium salts or substituted ammonium salts such as, for example, NH4®, 15 ethanolammonium, diethanolammonium, trialkylammonium, such as, for example, triethylammonium, tetraalkyl-ammonium, or salts with basic amino acids such as, for example, lysine or arginine.

The invention also relates to a process for the 20 preparation of the compounds, according to the invention, of the general formula I'.

The process according to the invention comprises conversion of a compound of the general formula II Het'-SH (II) 25 in which Het' has the meaning detailed above, by reaction with an oxidizing agent, into the compounds according to the invention.

Examples of suitable oxidizing agents which may be mentioned are oxygen, hydrogen peroxide, where appro-30 priate with addition of iron salts such as, for example, Mohr's salt, or with the addition of bases such as, for example, sodium hydroxide, potassium hydroxide, sodium G bicarbonate or potassium bicarbonate, organic peracids such as, for example, peracetic acid, perbenzoic acid or m-chloroperbenzoic acid, elementary iodine or bromine, where appropriate with the addition of bases such as, for 5 example, sodium hydroxide or potassium hydroxide, FeCl3 or K3(Fe(CN)6) .

The oxidizing agents which may be mentioned as preferred are: oxygen, hydrogen peroxide, organic peracids such as, for1 example, peracetic acid, perbenzoic acid and m-10 chloro-perbenzoic acid, or elementary iodine. The oxidation is preferably carried out in water or an organic solvent such as, for example, methanol, ethanol, isopropanol, ethyl acetate and halogenated hydrocarbons such as, for example, dichloromethane and chloroform. It 15 is also possible to use a mixture of these solvents. When working with oxygen, hydrogen peroxide and peracids, the use of solvents which are known to be able to form explosive peroxides, such as, for example, ether, tetra-hydrofuran, dioxane and acetone, methyl ethyl ketone, 20 isopropanol, etc. should be avoided.

The compounds according to the invention can also be prepared by reaction of a compound of the general formula III Het'-X (III) 25 in which Het' has the meaning detailed above, and X represents a reactive leaving group such as halogen, preferably chlorine or bromine, such as -0S02R, R preferably meaning methyl, trifluoromethyl, phenyl, tolyl or naphthyl, or such as -OPO(OR'2)/ R' preferably meaning 30 phenyl, with Me2S2, Me representing an alkali metal, preferably sodium.

Furthermore, the compounds according to the invention can be prepared by reaction of a compound of the general formula IV 7 Het' -S-S02Me (IV) in which Het' and Me have the meanings detailed above, with iodine in aqueous medium.

The reaction can be carried out in a temperature range 5 between about -40 °C and the boiling point of the solvent or mixture of solvents, preferably between about -10°C and about +40°C.

The active compound can be administered alone or combined with one or more, preferably one other medicament having 10 favorable effects on infections, caused, for example, by bacteria, fungi, or viruses, and on tumorous diseases. According to the invention, the active compounds can be administered both parenterally and orally. Suitable for parenteral administration are solutions or suspensions of 15 the active compound in a pharmaceutically tolerated vector, preferably a vegetable oil such as, for example, peanut oil or sesame oil, and alcoholic solutions of the active compound, for example, in ethanol, propanediol or glycerol or in mixtures of the abovementioned solvents. 20 To prepare aqueous solutions, the active compound is preferably used in the form of physiologically tolerated salts which are soluble in water. It is possible for the formulations to contain the customary auxiliaries and excipients. Examples of these are fillers, emulsifiers, 25 lubricants and buffers and flavor-modifying agents.

The invention therefore likewise relates to pharmaceutical agents for immunostimulation, immunorestoration and cytostatic treatment which contain a compound of the general formula I'. 30 In the text which follows, the action of thecompounds on the immune response of the mouse and their immuno-stimulant activities in various in vivo standard methods are illustrated by way of example. The various test methods em-ployed are known to be particularly well 35 suited for the assessment of iramunostimulants and their quality of action.

Experiment 1 Effect on the cellular immunological reaction, of the delayed type, to sheep erythrocytes fdelayed type hypersensitivity. DTEH Either 10s or 10s red blood cells from sheep were administered intravenously to each animal in groups of 5 female NMRI mice weighing 18 - 20 g. Sheep erythrocytes are regarded in immunology as a standard test substance (antigen) for the induction of cellular and humoral immune reactions. In particular, this test provides information about the functioning of the T-cell-dependent component (T-helper cells) of the immune system. The test substance obtained as in exemplary embodiment 8 (bis(5-carboxymethyl-4-methyl-2-thiazolyl) disulfide) was administered intraperitoneally twice a day in the concentrations 20 mg/kg, 30 mg/kg and 40 mg/kg in physiological saline solution on days -3, -2, -1 and 0. After 5 days, each animal received 2 x 108 sheep erythrocytes injected into the underside of the foot and, 24 hours later, the swelling of the foot was measured. The foot swelling is induced by a skin reaction of the delayed type (delayed type hypersensitivity, DTH) and is, as is known to the expert, a measure of the cellular immune response (Collins, F.M. and Mackaness, G.B., J. Immunol. 101, 830-845, 1968). The results compiled in Table 1 illustrate that there is an increase in the cellular immune response owing to the administration of the substance obtained according to the invention, for example after immunization with 106 sheep erythrocytes. A maximum stimulation can be observed in this experimental design on administration of 30 mg/kg test substance. 9 Table 1 Immunization of mice with sheep erythrocytes-action on the cellular immune response (DTH reaction) 2x/day i.p. administration % foot swelling with on days -3, -2 -1 and 0 of 10® erythrocytes PBS* 24.3 ± 1.3 20 mg/kg 28.3 ± 6.7 Test substance 30 mg/kg 36.5 ± 6.5 40 mg/kg 31.4 ± 8.7 *PBS = phosphate-buffered saline (NaCl:8,000 mg/1, KC1: 200 mg/1, Na2HP04. 2H20: 1,440 mg/1, KH2P04 : 200 mg/1 Experiment 2 Effect on the stimulation of non-specific immunity activation of mononuclear phagocytes This entailed investigation of the effect of the test substance obtained as in exemplary embodiment 8 on the stimulation of peritoneal macrophages in 6-8 week-old NMRI mice. Female mice received the test substance in parenteral or oral doses of 25 mg/kg, 50 mg/kg, 100 mg/kg and 200 mg/kg. Buffered saline solution was administered to a control group. The mice were sacrificed three days after the injections, and the peritoneal macrophages of the animals were examined for their state of activation. The secretion of the lysosomal enzymes (/S-galactosidase, ^-glucuronidase, and N-acetyl-^-D-glucosaminidase) was determined as one measure of macrophage activation. In addition, it was possible to examine the pinocytosis in comparable macrophage cultures by the uptake of colloidal gold (198Au) - as is known to the expert. The level of oxidative metabolism in macrophages is taken as another measure of their state of activation. This activity is measured with the assistance of a biolumate by determination of the chemiluminescence. 1 0 For this purpose, either 3 x 106 macrophages were cultured with 1 ml of TC 199 culture medium in Petri dishes of diameter 30 mm or 106 macrophages were cultured with 100 pi of medium in round-bottomed polyethylene tubes (for determination of the chemiluminescence) , with 5% C02 and at 37°C.

After incubation for one hour, the cultures were washed to' remove floating cells. The chemiluminescence (tube culture) was then determined directly, whereas the Petri dishes were incubated for a further 24 hours at 37°C and then the enzyme and pinocytosis activities in the cultures were determined. The following results were obtained.

Table 2 Action on the oxidative metabolism in mouse peritoneal macrophages (chemiluminescence in RLU*/15 minutes). 1 x administration intraperitoneal oral of PBS 2.97 ± 0.28 x 105 3.65 ± 0.81 x 105 Test 25 mg/kg 7.87 ± 0.28 x 105 6.41 ± 0.42 x 105 substance 50 mg/kg 9.72 ± 0.82 x 105 8.99 ± 0.39 x 105 100 mg/kg 12.85 ± 2.37 x 105 11.85 ± 0.92 x 105 200 mg/kg 24.40 ± 3.39 x 105 15.60 ± 1.98 x 105 *RLU = relative light units Both parenteral and oral treatment of NMRI mice with the test substance prepared as in Example 8 stimulate macrophage activity and thus have an immunity-stimulating action. Thus, the oxidative metabolism of macrophages, with the generation of oxygen radicals and the measurable light associated with this, is markedly raised. At dosages of 25 iug/kg upwards, there is a dose-dependent increase in the macrophage activity with both parenteral 11 and oral administration.

It can be seen from Table 3 that macrophages from control mice release only small amounts of lysosomal enzymes (£-glucuronidase, yS-galactosidase and N-acetyl-^-D-glucos-aminidase) into the culture supernatant. Mononuclear phagocytes from mice which have been treated parenterally or orally with the test substance for 72 hours secrete markedly more of the abovementioned acid hydrolases (p-Glu, £-Gal and N-Ac-Glu), and thus show a dose-effect curve which demonstrates a superiority over the controls for all the enzymes measured. It is evident that the test substance has a stimulant action on macrophage activity and contributes to an increase in enzyme release.

Table 3 Effect of the test substance on the release of the lysosomal hydrolase enzymes from mouse peritoneal macrophages . 1 x i.p./p.o. £-Glu /9-Gal N-Ac-Glu Administration mU/ml mU/ml mU/ml PBS Test substance 25 mg/kg 50 mg/kg 100 mg/kg 200 mg/kg 755/ 484 1001/897 1370/1133 1791/1593 2136/1886 1306/ 1702 2584/ 4917 11058/ 9179 17596/13195 22351/17357 1238/1168 2786/1947 3315/2862 4305/3676 6548/5621 The quantitative determination of the pinocytosis activity of mononuclear phagocytes was carried out by the method of Davies et al. (Davies, P., Allison, A.C. and Haswell, A.D.; Biochem. Biophys. Res. Com. 52, 627, 1973). Radioactive colloidal gold (198Au) with a particle size of 20 nm and a specific activity of 4-12 mCi/mg of Au was used for this. The results in Table 4 illustrate the effect of the test substance obtained as in Example 8 on the efficiency of endocytosis. The pinocytosis of colloidal gold (19!au) by mouse peritoneal macrophages from animals treated with the compound according to the invention is significantly and dose-dependently higher than by macrophages from untreated animals.

Table 4 The effect of the test substance of the efficiency of pinocytosis of mouse macrophages. 1 x administration of intraperitoneal oral PBS 0.286 x 103 cpm 0 . 198 x 103 cpm 25 mg/kg 0.341 " " 0.272 Test 50 mg/kg 0.396 " " 0.358 substance 100 mg/kg 0.462 " " 0.416 " 200 mg/kg 0.587 " " 0.506 Experiment 3 Increase in the resistance of Balb/c mice to infection with Candida albicans a) Prophylactic treatment: Balb/c mice were treated intraperitoneally with the test substance (compound of exemplary embodiment 8) at a dose of 2 x 60 mg/kg/day for 4 days. 24 hours after the last administration of the test substance, these animals and the control animals, which had received administrations of physiological saline solution in the same volumes and time intervals, are infected intravenously with Candida albicans (5 x 105 CFU/mouse) . The relevant mean survival times can be calculated from the death rate after the infection. 50% of the animals in the control group die after 9.7 days, while the group treated with the test substance showed a mean survival time of 16.1 days. With the selected administration regimen and with these doses (prophylactic administration), the test substance induces a significant increase in the resistance of the Balb/c mouse to Candida albicans.

J 3 Table 5 Mean survival times after C. albicans infection (5 x 105 CFU) Substance Mean Confidence limits 2 x 60 mg/kg/ survival day i.p. time (days) 95% 99% PBS 9.7 8.4-10.6 7.8-10.9 Test substance 16.1 14.8-17.4 14.4-17.8 b) Therapeutic treatment: 10 For the therapeutic treatment of a chronic Candida albicans infection, female Balb/c mice (15/group) were infected intravenously on day 0 with Candida albicans (1 x 10s CFU/mouse). After the infection had taken place, the animals were treated intraperitoneally on each of 8 15 consecutive days (days 3 - 10) with 60 mg/kg of the test substance (compound of exemplary embodiment 8) . The control animals received injections of physiological saline solution. Urine was collected from the animals on days 8, 14 and 21, and the organism count was determined. 20 On day 30, the animals were sacrificed and the organism count and the formation of necroses in the kidneys were determined. Table 6 shows that the test substance on therapeutic administration markedly reduces all the parameters of chronic Candida albicans infection 25 (organism count in the urine and kidneys and formation of necroses) and thus exerts a therapeutic effect on the disease. Whereas organisms are detectable in the urine of a high percentage of the control animals, there is a significant reduction in the organism count in the 30 treated animals.

In addition, the treatment reduces the colonization of the kidneys by the organism from 63% to 27% with, at the same time, a reduction in the formation of necroses from 87% to 10%. 11 Table 6 Therapeutic treatment of a chronic Candida albicans infection (1 x 105 CFU) Substance Animals with Kidneys with Necrotic 60 mg/kg positive findings positive kidneys i.p. organism findings Day 3-10 Day 8 Day 14 Day 21 Day 30 Day 30 PBS 4/15 8/15 10/15 19/30 26/30 (27%) (53%) (67%) (63%) (87%) Test 1/15 1/15 4/15 8/30 3/30 substance ( 7%) ( 7%) (27%) (27%) (10%) Experiment 4 Stimulation of the DTH reaction bv the compounds according to the invention NMRI mice were treated with the substances according to the invention as described in experiment 1.

The DTH reaction was checked as a test to determine the immunostimulation.

Table 7 shows the relative activity of the test substance, related to the compound from Example 8 whose maximal activation corresponds to 100% (difference between control and stimulation) . It can be seen from the table that the DTH reaction in the animals pretreated with the test substances is more pronounced than in the corresponding control animals.

Table 7 Compound from Dose Administration DTH reaction Example No. (mg/kg) (SRBC) 2 20 1 x i.p. day 0 88% 5 200 it 61% 6 100 ii 100% 8 100 II 100% 10 10 tt 112% 12 100 H 96% 13 200 fl 147% 14 100 It 118% Experiment 5 Stimulation of macrophage activity by the compounds according to the invention: NMRI mice were treated with the compounds according to the invention as described in experiment 2.

The macrophage function (chemiluminescence and enzyme activity) was checked as a test to determine the immuno-stimulation.

Table 8 shows the relative activity of the test substances, related to the compound from Example 8 whose maximal activation (difference between control and stimulation) corresponds to 100%. It can be seen from the table that in comparison with macrophages from untreated animals, the chemiluminescence reaction of these cells was also greatly stimulated by all the test substances, and their content of lysosomal enzymes was markedly raised. 5 6 Table 8 Compound from Macrophage activity Example No. Chemiluminescence Exocytosis (100 mg/kg i.p.) 1 30% 48% 2 72% 53% 5 37% 45% 6 26% 33% 8 100% 100% 10 66% 21% 12 39% 54% 13 81% 77% 14 97% 93% Experiment 6 Stimulation of the defense against infection bv Candida albicans bv prophylactic administration of the compounds according to the invention Balb/c mice were treated with the compounds according to the invention as described in experiment 3a.

As Table 9 shows, the mean survival time of the mice after C. albicans infection increases significantly compared with an untreated control group. 1 7 Table 9 Example Dose Relative prolongation of the No. (mg/kg) mean survival times* 1 x i.p. day after infection 1 1 144 2 1 123 5 10 142 6 10 145 8 1 130 10 10 135 12 10 205 14 10 107 *Mean survival time of the control group = 100 Experiment 7 Effect on the stimulation of defense against the B16 melanoma tumor Primary tumor growth was induced with 2 x 105 live B16 melanoma cells in female C57B1/6 mice (10 animals/group) weighing 18 - 20 g. After development of a particular tumor size (0.65 cm in diameter), the primary tumor was removed. The untreated animals then died of metastases in the lung. After the tumor induction, the animals were treated intraperitoneally on days 3, 5, 7, 9, 11 and 13 after amputation of the primary tumor with 50, 100 and 200 mg/kg of the test substance obtained as in Example 8. The relevant mean survival times can be calculated from the death rate after amputation of the primary tumor owing to the development of pulmonary metastases. According to this, 50% of the animals in the control group die after 26 days. The groups treated with the test substance showed (cf. Table 10^ a significant increase in the mean survival time to 43, 40 and 35 days with the appropriate dosages. i 8 Table 10 Therapeutic treatment of B16 melanoma tumor Administration 6 x i.p. (mg/kg) Mean survival time day 3, 5, 7, 9, 11, 13 (days) 5 PBS 26 50 43 Test substance 100 40 200 35 Experiment 8 1 o Effect on formation of bone marrow colonies This examined the effect of the test substance obtained as in Example 8 on the stimulation of bone marrow colonies in 6 - 8 week-old B2D2F1 mice. Female mice received the test substance intraperitoneally in the 15 doses 2.5 and 5 mg/kg. The animals were sacrificed one day later, and the bone marrow cells were isolated and cultured by generally known methods (Metcalf, Immunology 21. 427, 1971 and Stanley et al., J. Exp. Med. 143, 631, 1979). As is customary, L-cell supernatant (15%) was used 20 as CSF source (colony stimulating factor) for the development of the bone marrow colonies. The colonies were counted 8 days after the start of culturing. As can be seen in Table 11, a single dose of 2.5 or 5 mg of the test substance results in a marked increase in the 25 formation of bone marrow cell colonies both with and without addition of CSF (colony stimulating factor) in vitro. 1 9 Table 11 In vivo-effect on the formation of bone marrow colonies Test substance Number of bone marrow colonies (day 8) 1 x i.p. (mg/kg) with CSF (15%) without CSF (15%) PBS 41 ± 6 0 Test 2.5 94+11 24+2 substance 5.0 163±8 44 ±5 The examples which follow illustrate the invention.

Example 1 Bis(3-hydroxy-1-phenyl-1.2,4-triazol-5-vl^ disulfide 2.9 g (15 mmol) of 3-hydroxy-1-pheny1-5-mercapto-1,2,4-triazole are initially introduced into 100 ml of methanol and, at room temperature, 3.2 ml (33 mmol) of 35% strength hydrogen peroxide in methanol are added dropwise. The solution initially turns yellow and, after 20 min, the compound slowly precipitates out. After 4 hours, the precipitate is filtered off with suction, washed with methanol and dried.

Yield 2.1 g = 72% of theory Melting point: 232°C decomposition NMR (d6-DMS0) S = 11.58 ppm, s, 2H, OH 7.38 ppm, m, 10H, aromat. H Example 2: Bis(5-carboxybenzimidazol-2-vl) disulfide Preparation in analogy to Example 1 from 2-mercapto- 2 0 benzimidazole-5-carboxylic acid Yield 70% of theory Melting point: 235-38°C NMR (dg-DMS0) 5=9.4 ppm, d, NH 5 7.4-8.4 ppm, m, aromat. H Example 3: Bis F4-12-chloroallvH-6-hvdroxv-5-oxo-4,5-dihvdro-l,2 . 4-triazin-3-vl1 disulfide 2.2 g of 4-(2-chloroallyl)-6-hydroxy-3-mercapto-5-10 oxo-4,5-dihydro-l,2,4-triazine were dissolved in 25 ml of methanol and, at room temperature, 0.86 ml of 35% H202 was added dropwise, and the mixture was then stirred for 1 hour during which the 15 product crystallized out. It was filtered off with suction, washed with methanol and dried in air.

Yield 1.2 g Melting point: 204-05°C (decomposition) 20 NMR (d6-DMS0) 5 = 12.7 ppm (br. s., OH) 5.5 ppm (dd, = CH2) 4 . 8 ppm ( s , CH2N) Example 4: Bis ( 6-hvdroxv-2-methyl-5-oxo-2 , 5-dihvdro-l. 2 . 4-triazin-25 3-vH disulfide 1.59 g of 6-hydioxy-3-mercapto-2-methyl-5-oxo-2,5-di-hydro-1,2,4-triazine were dissolved in 2 1 30 ml of methanol and, at room temperature, 0.43 ml of 35% H202 was added dropwise, and the mixture was then stirred for 30 minutes. The solution was clarified with charcoal, evaporated to 5 dryness in a rotary evaporator, and the residue was triturated with ice-water. The precipitate was filtered off, washed with ice-water, dried in air, suspended in methanol, filtered off, washed with a little methanol, and dried in 10 * vacuo.

Yield 0.8 g Melting point: 220°C (decomposition) NMR (d6-DMS0) 5 = 3.87 ppm (s, CH3) Example 5: 15 Bis f 6-hvdroxv-4-methvl-5-oxo-4 ,5-dihydro-l, 2 ,4-triazin- 3-vl^ disulfide 3.7 g of 6-hydroxy-3-mercapto-4-methyl-5-oxo-4,5- dihydro-l,2,4-triazine were dissolved in 400 ml of methanol and 20 38 ml of H20. At room temperature, 2.5 ml of 35% H202 were added dropwise. After stirring the mixture for 30 minutes, it was concentrated to 100 ml, and the precipitate was filtered off with suction and dried. 25 * Yield 1.5 g Melting point: NMR (CF3C02D): 237 °C 5 = 3.67 ppm s, 2H, OH 3.8 ppm, s, 6H, CH3 2 2 Example 6: Bis(5-carboxvmethvl-4-methvl-l,3-thiazol-2-vl) disulfide 2 3.62 g of 5-carboxymethyl-4-methyl-2-mercapto-l,3-thiazole were dissolved in 5 250 ml of methanol, and the mixture was filtered off and, while cooling in a water bath, 12.5 ml of 35% H202 were added slowly. The mixture was then stirred in the water bath for 30 minutes and at room temperature for 2 hours. After 10 filtration and washing with methanol, 22.8 g of the title compound were isolated.

Melting point: 162°C NMR (CF3C02D) : S = 2.6 ppm, s, 6H, CH3 4.2 ppm, s, 4H, CH2 15 Example 7: Bis(5-carboxvmethvl-l.3-thiazol-2-vl) disulfide 0.88 g of 2-mercapto-l,3-thiazol-5-ylacetic acid is dissolved in about 10 ml of methanol and, while stirring at room temperature, 20 0.5 ml of 33% H202 solution is added. After 0.5 h, the suspension of crystals was cooled, filtered, and the residue from filtration was dried. Yield 0.6 g of decomposition point 150°C. Comparison by thin-layer chromatography on 25 Merck silica gel plates shows reaction complete (Rf: 0.3; mobile phase: ethyl acetate: 65, ethanol: 25, water: 10, formic acid: 1) 2^ Example 8: Bis(4-carboxvmethvl-l,3-thiazol-2-vl1 disulfide When 1.75 g of 4-carboxymethyl-2-mercapto-l,3-thiazole is reacted in analogy to Example 7, then 0.95 g of the title compound is obtained.

Melting point: 163-65°C NMR (DMSO-d6) : s ■■ = 3 .7 ppm, s, 4h, ch2 7 .6 ppm, s, 2h, ch 10 9 .4 ppm, s, 2h, co2h Example 9: Bis(2-carboxymethvlthio-l,3,4-thiadiazol-5-vl) disulfide 1.4 g of (2-mercapto-l,3,4-thiadiazol-5-yl)mercapto- acetic acid are dissolved in 10 ml of methanol 15 and, while cooling in ice, 0.65 ml of 35% H202 is added dropwise. The mixture is then stirred at room temperature for 1 hour and filtered. The crystals are washed with a little methanol and are dried in vacuo. 20 Yield: 1.3 g Melting point: 179°C (decomposition) NMR (DMSO-d6): 5 = 13 ppm, broad, C02 4.2 ppm, s, CH2 2 4 Example 10: Bis(5-carboxv-1,3-thiazol-2-vl^ disulfide 21 g of 2-mercapto-l,3-thiazole-5-carboxylic acid are dissolved in 5 100 ml of methanol by warming gently. While cooling in ice, 10.7 ml of 35% H202 are added slowly, and the mixture is then stirred at room temperature for 45 minutes. The solid is filtered off with suc-10 tion, washed with methanol and dried in vacuo.

Yield: 21.5 g Melting point: 280°C (decomposition) NMR (DMSO-d6): S = 8.48 ppm, s, thiazole-H Example 11: 15 Bis(5-carboxv-4-methvl-l,3-thiazol-2-vl) disulfide 1.75 g (10 mmol) of 4-methyl-2-mercapto-l,3-thiazole-5-carboxylic acid are suspended in 50 ml of methanol and, while cooling in ice, 0.86 ml of 35% H202 is added. The solution is then 20 stirred at 0°C for 1/2 hour and at room temperature for 1 hour. The crystals which have formed are filtered off with suction, washed with a little methanol and dried in vacuo.

Yield 1.52 g; Melting point: 240-41°C (decompos-25 ition) NMR (DMSO-d6):5 = 2.6 ppm, s, CH3 25 Example 12: Bis(4-carboxvmethvl-5-methvl-l,3-thiazol-2-vl) disulfide Stage 1 Methyl 4-bromopropionvlacetate 5 22 g of methyl propionylacetate are dissolved in 60 g of CH2C12, and 8 . 7 ml of Br2 in 30 ml of CH2C12 are added dropwise. The mixture is then stirred at room temperature for 1 1/4 h, 10 a further 0.66 ml of Br2 in 5 ml of CH2C12 is added, and the mixture is stirred for a further 1 1/4 h.

It is washed with 15 3 x 50 ml of H20 each time, 1 x 20 ml of saturated NaHC03 and 2 x 50 ml of H20 each time, dried over Na2S04 and evapora ted. 38.6 g of oil are obtained.

Stage 2 20 Methyl (2-mercapto-5-methvl-l,3-thiazol-4-vl)acetate 30.1 g of the oil from stage 1, dissolved in 80 ml of ethanol, are added dropwise to a solution of 24 g of freshly prepared ammonium dithiocarbamate in 200 ml of ethanol and 25 200 ml of H20 at room temperature. The mixture is then stirred for 2 1/2 h and 7.5 ml of trifluoroacetic acid are added. After having been stirred for 1 hour, the mixture is evaporated to dryness and 30 200 ml of CHC13/H20 (1:1) are added. After two further washings with H20, the solution is dried over Na2S04 and evaporated. The solid residue is 2 6 suspended in ether and filtered off.

Yield 8.9 g Melting point: 149°C (decomposition) NMR (d6-DMSO): S = 11 ppm, broad, SH 5 3.7 ppm, s, OCH3 3 . 5 ppm, s , CH2 2.1 ppm, s, CH3 Stage 3 (2-Mercapto-5-methvl-l,3-thiazol-4-vl)acetic acid 10 1.98 g of stage 2 were dissolved, with warming in 20 ml of methanol. The solution was then stirred at room temperature for 40 min, 50 ml of H20 were added, and the mixture was acidified to pH 1.0 with concentrated HC1. After stirring 15 for 1 h while cooling in ice, the solid was filtered off with suction and washed with ice-water until free of chloride. Drying in vacuo over P205 produced 1.67 g. i2H 2 NMR (d6-DMS0): S = 13.9 ppm, broad, CO 20 3.5 ppm, s, CH 2.08 ppm, s, CH3 Stage 4 Bis(4-carboxvmethvl-5-methvl-l.3-thiazol-2-vl) disulfide 430 mg of stage 3 were suspended in 25 20 ml of methanol and dissolved by heating. At room temperature 0.25 ml of 35% H202 was added dropwise, and the mixture was then stirred for 1 h. After having been concentrated to about 5 ml, the mixture was A. ( filtered, and the solid was washed with a little ice-cold methanol.

Yield: 280 mg NMR (ds - DMSO): 5=3.65 ppm, s, CH2 5 2.37 ppm, s, CH3 Example 13: Bis ( 5-carboxvmethvl-l,3-thiazol-2-vl) disulfide 0.8 g of 2-mercapto-5-carboxymethyl-l, 3-thiazole were dissolved in 10 ml of methanol and, while 10 stirring, 0.5 ml of 33% strength H202 solution was added dropwise. The mixture was allowed to stir overnight, and the product which had crystallized out was filtered off.

Yield 0.6 g of melting point 150°C (decomposition), 15 homogeneous by thin-layer chromatography (Rf = 0.3; silica gel; mobile phase: ethyl acetate, ethanol, water, formic acid (60:25:15:1) IR (KBr disk): 7 - 1710 cm (COOH).

^-NMR (d6-DMS0): S (ppm): 3.8 (s, CH2-thiazole, 2H) , 20 7.6 (s, thiazole-4H, 1H).

Example 14: Bis(4-carboxy-l,3-thiazol-5-vl) disulfide 5 g of bis(4-methoxycarbonyl-l,3-thiazol-5-yl) disulfide were suspended in 100 ml of methanol. 25 To this was added a solution of 1.5 g of NaOH in 20 ml of H20, and the mixture was heated under reflux for 1 h. After the solution had been cooled, methanol was removed in a rotary 2 S evaporator, the aqueous phase was extracted once with a little ethyl acetate and acidified with 2N HC1. The product was filtered off, dried and recrystallized from ethyl acetate. 2 5 g of melting point 154"C were obtained.

IR (KBr disk) : 7 = 1680 cm"1 (COOH) ^-NMR (d6-DMSO): S (ppm), 8.6 (s, thiazole-2H) .

Examples 15 to 17 were carried out as described in Example 1. 10 Example 15: Bis(5-qlutaromonoamido-l,3-thiazol-2-vH disulfide 1H-NMR (ds - DMSO): 5 = 1.6 - 2.6 ppm (m, 12H, six CH2 groups), 7.50 ppm (s, 2H, thiazole-H), 12.63 ppm (br s, 2H, -NH-), 13.05 ppm 15 (br s, 2H, -C02H) Example 16: Bis 15-succinomonoamido-l,3-thiazol-2-vl) disulfide 1H-NMR (d6-DMSO): S = 2.4 - 2.7 ppm (m, 8H, -CH2) , 7.50 ppm (s, 2H, 20 thiazole-H), 12.66 ppm (br s, 2H, -NH-), 13.15 ppm (br s, 2H, -C02H) Example 17: Bis r 5-(2-carboxvethvl)-4-methvl-l,3-thiazol-2-vl] disulfide 25 1H-NMR (d6-DMS0): 5 = 2.27 ppm (s, 6H, -CH3) , 2.55 ppm (d, 4H, thiazole-CH2) , 2.96 ppm (t, 4H, CH2-C02-)

Claims

9 Patent claims:

1. A disulfide of the formula I' Het'-S-S-Het' (I') in which Het' means 1,3-thiazolyl which is substituted by 5 Cj-Cg-alkyl; by carboxy-C^-Cg-alkyl; by carboxyl or by C2- C5-acylamino, where acyl is the radical of an aliphatic dicarboxylic acid; 1,2,4-triazolyl, which is substituted by hydroxyl and phenyl; 10 1,3,4-thiadiazolyl, which is substituted by carboxy-Cj- C<,-alkylthio; dihydro-1,2,4-triazinyl which is substituted by C^-Cg-alkyl, halogeno-C2-C4-alkenyl, oxo or hydroxyl or benzimidazolyl which is substituted by carboxyl, where 15 Het' is substituted by at least one carboxyl group which is bonded directly or to a substituent or by one directly bonded hydroxyl group, it also being possible for these two groups to be in the form of a physiologically tolerated salt. 20

2. A disulfide of the formula I' Het'-S-S-Het' (I') in which Het' means 1,3-thiazolyl which is substituted once or twice by Cj-C3-alkyl; by carboxy-C1-C3-al]cyl; by carboxyl or by C2-C5-acylamino, where acyl is the radical 25 of an aliphatic dicarboxylic acid, where Het' is sub stituted by at least one carboxyl group which is bonded directly or to a substituent and can also be in the form of a physiologically tolerated salt.

3. A process for the preparation of disulfides of the 30 formula I', which comprises a) converting a compound of the formula II Het'-SH (II) 3 0 in which Het' has the abovementioned meaning, by reaction with an oxidizing agent into a compound of the formula I', or b) reacting a compound of the formula III Het'-X (HI) in which het' has the abovementioned meaning, and X is a reactive group which can easily be eliminated, with Me2S2, where Me is an alkali metal, or c) reacting a compound of the formula IV Het' -S-S02Me (IV) in which Het' and Me have the abovementioned meanings, with iodine in aqueous medium.

4. A pharmaceutical agent which contains a compound of the formula I'.

5. A compound according to claim 1, substantially as hereinbefore described and exemplified.

6. A process for the preparation of a compound according to claim 1, substantially as hereinbefore described and exemplified.

7. A compound according to claim 1, whenever prepared by a process claimed in a preceding claim.

8. A pharmaceutical agent according to claim 4, substantially as hereinbefore described. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS.