WO2002014343A1 - Bufadienolide derivatives and use as immunosuppressive, antiinflammatory and analgesic agents - Google Patents

Abstract

The present invention relates to a novel use of bufadienolide derivatives, as such or in the form of pharmaceutical compositions, for immunomodulation, especially for immunosuppression, for antiinflammatory and/or for antitumor action, as especially for the use in the treatment (including therapy and/or prophylaxis) of conditions in warm-blooded animals that are responsive to said actions; to the use of said derivatives for the manufacture of a pharmaceutical preparation for the treatment of a disease or condition that responds to said actions; to processes for the manufacture of pharmaceutical compositions for this novel use; to pharmaceutical compositions comprising the steroids and/or to a method of treatment comprising administering such a steroid; the invention also relates to novel steroids of that type, to those novel compounds for use in a method for the diagnostic or therapeutic treatment of the human or animal body, and to processes for the preparation of these compounds and to novel intermediates useful for their preparation.

Description

IMMUNOSUPPRESSrVE, ANΉINFLAMMATORY AND ANALGETIC COMPOUNDS

The present invention relates to a novel use of bufadienolide derivatives, as such or in the form of pharmaceutical compositions, for immunomodulation, especially for immunosup- pression, for antiinflammatory and/or, in the case of non-cardioactive bufadienolides, for antitumor action, as especially for the use in the treatment (including therapy and/or prophylaxis) of conditions in warm-blooded animals that are responsive to this type of immunomodulation, antiinflammatory and/or , in the case of non-cardioactive bufadienolides, for antitumor action; to the use of said derivatives for the manufacture of a pharmaceutical preparation for the treatment of a disease or condition that responds to this type of immunomodulation, antiinflammatory and/or, in the case of non-cardioactive bufadienolides, for anti- tumor action; to processes for the manufacture of pharmaceutical compositions for this novel use; to pharmaceutical compositions comprising the steroids and/or to a method of treatment comprising administering such a steroid; the invention also relates to novel steroids of that type, to those novel compounds for use in a method for the diagnostic or therapeutic treatment of the human or animal body, and to processes for the preparation of these compounds and to novel intermediates useful for their preparation.

Background of the invention

Autoimmune diseases, rheumatic and rheumatoid diseases and many other conditions like allergy, septic shock or a disturbed activity of the immune system lead to pathogenic changes, causing the respective disease symptoms. Also in the field of transplantation, after transplantation of organs, tissues or cells from a related or unrelated donor (especially allo- or xenotransplantation), reactions of the immune system result in graft rejection.

Examples of autoimmune diseases include but are not limited to: Hashimoto's thyroiditis, primary myxoedema, Grave's disease, pernicious anaemia, autoimmune atrophic gastritis, Ad- dison's disease, ovarian failure, premature menopause, insulin-dependent diabetes mellitus, Goodpasture's syndrome, myasthenia gravis, Lambert-Eaton syndrome, pemphigus, bulous pemphigoid, psoriasis, Crohn's disease, sympathetic ophthalmia, autoimmune uveitis, multiple sclerosis, autoimmune hemolytic anaemia, autoimmune thrombocytic purpura, autoimmune neutropenia, primary biliary cirrhosis, chronic active hepatitis, cryptogenic cirrhosis, ulcerative colitis, Sjόgren's syndrome, antiphospholipid syndrome, mixed connective tissue disease, vasculitis, vitiligo, glomerulonephritis, etc. Examples of proliferative diseases are especially tumor diseases, e.g. mammary carcinoma, head and neck cancer, tumors of lungs and mediastinum, like bronchial carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, or other lung tumors, urogenitary tumors, like cervical carcinoma, vaginal carcinoma, renal, prostate or testis carcinoma, tumors of the gastrointestinal tract, like hepatoma, oesophageal tumors, pancreas tumors or colon tumors, oto-rhino-laryngologic tumors, like nasopharyngeal tumors, tumors of endocrine organs, like thyroid, adrenal or carcinoid tumors, bone- or soft tissue-sarcomas, like bone or Kaposi's sarcoma, skin tumors, like melanoma, tumors of the central nervous system, like gliomas, meningeomas, medulloblastomas or pituitary adenomas, or especially lymphomas and leu- kemias, like AML, ALL, PML, CMML, CML, CLL, Morbus Hodgkin, Non-Hodgkin Lymphoma, hairy cell leukemia, plasmocytoma, Burkitt Lymphoma or cutaneous T-cell lymphoma.

Examples of rheumatic or rheumatoid diseases include but are not limited to: Diffuse diseases of connective tissue (e.g. systemic lupus erythematosus, nephritis, systemic sclerosis etc.), rheumatoid arthritis and inflammatory polyarthritis, osteoarthritis, spine disorders (e.g. ankylosing spondylitis), arthropathy, internal derangement of knee, joint disorders, symptoms involving skin and integumentary tissue, tendinitis and bursitis, disorders of synovium and of tendon and bursitis, disorders of muscle, ligament and fascia, like polymyositis, disorders of soft tissue, crystal arthropathies, disorders of plasma protein metabolism, other inflammatory diseases (e.g. psoriatic arthritis, Lyme disease, Reiter's disease etc.).

In these areas, immunosuppressive and antiinflammatory therapeutics help to suppress the disease symptoms or rejection of grafts. The most important immunosuppressives are

- Glucocorticoids, like cortisone, triamcinolon, prednisolone or prednisone;

- cytostatics, like cyclophosphamide, methotrexat and azathioprine;

- cyclosporine A; and

- antibodies against cells of the immune system, especially antilymphocyte globulin (monoclonal antibodies or antilymphocyte sera).

Glucocorticoids work at a very early stage of the immune reaction. However, serious side effects are observed as the glucocorticoids also have strong metabolic effects leading to the symptoms of Cushing syndrome, ulcera in the gastro-intestinal tract, delayed wound healing, atrophy of muscles and skin, diabetogenic effects etc. These usually require periodic inter- ruption of treatment, that is, insertion of glucocorticoid free intervals in order to alleviate the side effects. Cytostatics, due to their general antiproliferative effects, lead to severe side effects such as bone marrow suppression, disturbed hematopoiesis, gastrointestinal symptoms, loss of appetite etc. Therefore, they cannot be used for longer periods. Cyclosporin A, a hydrophobic cyclic polypeptide produced by the fungus Tolypocladium inflatum, depresses both the humoral as well as the cellular immune response, mainly by inhibition of IL-1 secretion by monocytes and IL-2 secretion by T-helper cells in the early phase of the immune response. A relevant adverse drug reaction is the dose dependent damaging of the kidney; further reversible disturbance of liver function, cardiotoxicity, tremor, hirsutism, hypertrophy of the gingiva and edemas are frequently observed. Antilymphocyte globulins, like monoclonal antibody Muromonab-CD3 or antilymphocyte sera such as Pressimmun^® or Lymphoglobulin Institut Merieux^®, show side effects including fever, shivering-fit, dyspnoea and gastrointestinal symptoms. In addition, in cases where the antibodies are not of human origin, such as the mouse antibody Muromonab-CD3, loss of efficiency due to the formation of anti-antibodies and, if used repeatedly, hypersensitivity reactions may occur.

For antiproliferative, especially cancer/tumor treatment, numerous classical cytostatics (e.g. adriamycine, cisplatin), as well as more specific inhibitors e.g. of tyrosine kinases (like STI 571 (Gleevec^®)) or of microtubule disassembly (e.g. paclitaxel (Taxol^®) are used. Common to all these therapeutics are often considerable adverse drug reactions and more or less limited range of specificity against certain tumor types.

Especially the side-effects show that there is an urgent need for identification of novel classes of compounds with advantageous properties, especially in immunosuppression, inflammation and antitumor treatment in order to add to the existing assortment of therapeutics.

Bufadienolides from both plant and animal sources have been described [for a review, see Krenn et al., Phytochemistry 48(1), 1-29 (1998), which describes 267 substances of this class)]. In recent years, some evidence has accumulated indicating that in mammals, including man, compounds belonging to the class of bufadienolides can be found. Whereas the biological activities of cardenolides have been subject to extensive studies, bufadienolides are less well characterized. So far, they have been shown to have impact on cardiac muscle performance, renal sodium excretion, blood pressure, and to act on the nervous system. For bufalin, a cardioactive and cardiotoxic bufadienolide, antitumor effects have been shown against leukemia, colon adenocarcinoma, and melanoma cells (see N. Kawazoe et al., J. Biochem. 126, 278-86 (1999); Y. Jing et al., J. Cancer Res. 85, 645-651 (1994)Y. Jing et al., Anticancer Res. 14, 1193-8 (1994); Y. Masuda et al., Leukemia Res. 19(8), 549-56 (1995) and M. Watabe et al., J. Biol. Chem. 271(24), 14067-73 (1996)).

Summary of the Invention

Surprisingly, it has now been found that the class of bufadienolide steroids and analogues thereof ("bufadienolides" hereinafter) is capable of strong immunosuppression, antiiflamma- tory and/or, where non-cardiotoxic bufadienolides are concerned, antitumor action and thus represents a new class of compounds useful in the therapy of diseases that can be treated by compounds with the activities mentioned above. Thus, in particular the compounds of formula I defined below show very favorable and valuable pharmaceutical characteristics, especially with regard to the therapeutic and/or prophylactic treatment of diseases characterized by immunological dysregulation, inflammation and/or (where non-cardioactive bufadienolides are concerned) tumor growth and persistence. More specifically, the bufadienolides (especially compounds of formula I defined below) have been found to suppress the activity of cells belonging to the immune system of warm-blooded animals (preferably humans), especially T-and B-lymphocytes and of tumor cells,. They also suppress the cytokine production found in inflammation, stimulate the production of suppressive cytokines and inhibit the maturation of dendritic cells. The bufadienolides, especially compounds of the formula I, thus are appropriate for the treatment of conditions in warm-blooded animals that are responsive to these effects. Due to the properties of these compounds with an incomparable therapeutic potential to complement or replace existing, specific or less specific im- munosuppressive, antiinflammatory and/or (in the case of non-cardioactive bufadienolides, especially of formula I) antitumor treatment, the use of bufadienolides is of particularly high value for the treatment of various diseases or conditions that are positively influenced by or require immunomodulating, especially immunosuppressive, antiinflammatory and/or antitumor treatment. Especially surprising is the fact that the non-cardiotoxic bufadienolides show antitumor activity.

Detailed description of the invention

A compound to be used according to the invention is a bufadienolide (in the case of antitumor action, a non-cardiotoxic bufadienolide), defined as a steroid with a moiety of the formula lb,

or (in a broader, less preferred aspect of the invention) a carboxy or -COOR** wherein R** is hydrocarbyl; or is hydroxymethyl; in position 17 of the steroid ring system; or where the carbon in position 17 of the steroid ring system is bound to the carbon at position 14 via a bridge -C(=O)-C-(=O)-O-, where the right hand O is bound to position 14 and the left hand carbonyl to position 17 of the steroid ring system; especially a compound of the formula I,

wherein

R is hydrogen, hydroxy, amino, substituted amino, amino-lower alkoxy, N-substituted ami- no-lower alkoxy, azido, halo, acyloxy or the radical of a mono- or oligosaccharide bonded (preferably O-glycosidically) via an oxygen atom to the rest of the molecule of formula I;

Ri* is hydrogen, hydroxy, halo, amino or acyloxy; or RT and Ri* together are oxo (=O) or hydroxyimino (=NOH);

R₂ is hydrogen, hydroxy, halo, acyloxy or the radical of a monosaccharide bonded O- glycosidically via an oxygen atom to the rest of the molecule of formula I; any radical other than hydrogen being preferably in β-position;

R₂* is hydrogen and R₂** is hydrogen, hydroxy (preferably in β-position), halo or acyloxy (preferably β-acyloxy); or R₂* and R₂** together form oxo (=O); or R₂ and R₂*^* together with the bond between the carbon atoms that carry R₂ and R₂ form a double bond while R₂ ^* is hydrogen;

R₃ is lower alkyl, hydroxymethyl, acyloxymethyl, formyl (-C(=O)H), carboxy or hydrocarbyloxycarbonyl;

R₄ is hydrogen, hydroxy, halo or acyloxy (if other than hydrogen, preferably in β-position), and

R₅ is hydrogen or hydroxy (preferably in β-position),

R * and R₄** each is hydrogen; or

R₄ is acyloxy and R₄* and R₄*^* together with the adjacent bond connecting the carbon atoms that carry R * and R ** form a double bond; or R₄ is hydrogen and R₅ and R₄ ^** together form oxa in β-position ( — -O"— ):

R₆ is hydrogen, α-hydroxy or α-acyloxy, and R₆* is hydrogen; or R₆ and R₆* together are oxo (=O); or or R₆ and R₃ together form a group of the formula (la),

wherein R* is hydrogen or methyl and where the oxygen atom is bound in the position of R₆, the carbon atom in the position of R₃; R₇ is hydrogen or hydroxy in α-position;

R₈ is hydrogen or hydroxy in β-position, with the proviso that R₇ and R₈ are not simultaneously hydroxy ; or R₇ and R₈ together are oxo (=O);

R₉ is hydrogen or α-hydroxy or more preferably β-hydroxy and R₁₀ is hydrogen, or R₉ and R₁₀ together with the bond between the carbon atoms that carry

R₉ and R₁₀ form a double bond or oxa in β-position ( — -O_*— ) (non-cardioactive), or R₉ and R₁₃ taken together form a bridge -C(=O)-C(=.O)-O- where the O is bound in the position of R₉ and the left carbonyl is bound in the position of R₁₃, both preferably in the β- position (non-cardiotoxic, thus also appropriate for antiproliferative action);

Rn is hydrogen, hydroxy, especially β-hydroxy, halo or acyloxy, especially β-acyloxy;

R₁₂ is methyl, especially β-methyl;

R₁₃ is a moiety of the formula lb,

or, in a broader aspect of the invention (less preferred as far as antiinflammatory and/or im- munosuppressive effects are concerned, but in a preferred aspect as far as antiproliferative action is concerned), is carboxy or -COOR** wherein R** is hydrocarbyl (non cardiotoxic); or is hydroxymethyl (non-cardiotoxic); and each of R₁₄ and R₁₅ is hydrogen, or, if R₂** and R₂ together with the bond between the carbon atoms that carry R₂ and R₂** form a double bond and if R^ and Ri* together form oxo, R₁₄ and R₁₅ together with the bond between the carbon atoms that carry R₁₄ and R₁₅ form a double bond; a tautomer thereof, or a salt thereof, if a salt-forming group is present.

Unless otherwise indicated, the general terms and names used in the description of the present invention preferably have the following meanings (which can be used instead of more general terms in order to define preferred embodiments of the invention):

The bufadienolides, especially compounds of formula I, can exist as isomers or mixtures of isomers; thus, if one or more asymmetric carbon atoms are present, they can be in the (R)-, (S)- or (R,S)-configuration, if double bonds are present, they may be present in the E-, the Z- and/or the E,Z-form, each time independent of one another. Thus isomeric mixtures, e.g. mixtures of enantiomers, e.g. racemates, or diastereomeric mixtures, or pure enantiomers or diastereomers, or any combination thereof where more than one type of isomerism (E,Z- isomerism, chirality) is possible can be obtained, depending on the number of asymmetric carbon atoms and/or the number of double bonds and on whether isomers or isomeric mixtures are present. Preferred are pure isomers (enantiomers or diastereomers).

Where "bufadienolides" or "compounds of formula I" are mentioned herein, this also refers to their tautomers or their (especially pharmaceutically acceptable) salts, where appropriate.

The term "lower" defines a moiety with up to and including maximally 7, especially up to and including maximally 4, carbon atoms, said moiety being branched or straight-chained. Lower alkyl, for example, is methyl, ethyl, n-propyl, sec-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl or n-heptyl. Substituted amino is especially amino substituted by one or two moieties selected from lower alkyl, phenyl-lower alkyl, phenyl, lower alkanoyl, phenyl-lower alkanoyl or benzoyl, each of which may be unsubstituted or substituted, e.g. by one or more moieties independently selected from halogen, hydroxy, amino, carboxy, lower alkoxycarbonyl, cyano, sulfo, carbamoyl and sulfamoyl.

In amino-lower alkoxy, the amino group is preferably bound in the ω-position; preferred is ω- amino-C₁-C₄-alkyl, especially 2-aminoethoxy (from 2-ethanolamine). N-substituted amino-lower alkoxy is preferably lower alkoxy carrying substituted amino as defined above.

Halo is preferably fluoro or especially chloro, bromo or iodo.

Acyl in acyloxy is preferably the moiety of an organic carbonic or sulfonic acid having up to 20, especially 1 to 12 carbon atoms in an unbranched or branched chain or a ring, and may be substituted by one or more, especially one or two, moieties independently selected from the group consisting of halogen, hydroxy, amino, substituted amino (especially as defined above), carboxy, lower alkoxy-carbonyl, cyano, sulfo, imidazolyl, especially 4-imidazolyl, N- methylimidazolyl, especially 1-methyI-imidazol-4-yl or 3-methyI-imidazol-4-yl, guanidino, mer- capto, carbamoyl and sulfamoyl, more preferably one or two moieties selected from carboxy, amino, carboxy-lower alkanoylamino and 1-methyl-imidazol-4-yl; preferably, acyloxy is hemi- succinoyloxy, -glutaryloxy, -adipoyloxy, -pimeloyloxy or -suberoyloxy (each with one free carboxy group); lower alkanoyl, especially formyl, acetyl, propionyl, isobutyryl or hexanoyl;, O- suberoyl-, O-succinoyl-, O- adipoyl- or O-glutaroyl-arginine ester, or O-hemisuberoyl-1- or - 4-methylhistidyl; or is the moiety of a sugar acid, especially glucuronidoyl, e.g. α-D-glucuro- nidoyl.

A radical of a mono- or oligosaccharide bonded O-glycosidically via an oxygen atom to the rest of the molecule is preferably a mono- or oligosaccharoidyl moiety of one or more sugars (unsubstituted or acetylated or methylated one or more times) selected from 2-deoxy-α-D- ribofuranose, xylose, glucose, rhamnose, galactose, mannose, fucose, digitalose, gluco- methylose (Glum) and thevetose (Thev) as monosaccharides, or forming disaccharides or branched or unbranched oligosaccharides; preferred examples are rhamnosyl, e.g. α-L- rhamnosyl, xylosyl, glucomethylosyl (6-deoxyglucosyl), glucosyl, especially β-D-glucosyl, thevetosyl (especially α-L-Thev-), digitalosyl (6-deoxy-3-O-methyl-galactosyl), rhamnosyl- glucosyl, especially α-L-Rha-β-D-GIc-, cellobiosyl (D-Glc(1->4)-D-Glc-), thevetosyl, gen- tiotriosyl (D-Glc(1->6)-D-Glc(1->6)-D-Glc-), gentiobiosyl (D-Glc(1->6)-D-Glc-), rhamnosyl- rhamnosyl, β-D-Glc-α-L-(2', 3^'-(di-O-acetyl)-Rha-, β-D-Glc-β-D-GIc-, 4'-β-D-Glc-3'-α-L-Rha- L-Rha-, 3"-β-D-Glc-β-D-Glc-α-L-Rha-, Glc-Glc-Rha- (z.B. β-D-Glc-β-D-Glc-Rha-), 6"-β-D- Glc-D-Glc-α-L-Rha-, (e.g. 3"-) β-D-Glc-β-D-Glc-α-L-(2',3'-diacetoxy)-Rha-, β-D-Glc-β-D- Glc-α-L-Glum-, β-D-Glc-L-Glum-, Rha-(2'-O-acetyl)-Thev-, Rha-(2'-O-acetyl)-Glc-, Xyl(1- >4)Rha-, Glc(1->3)-(4-O-acetyl)-Rha-, Glc-Glc-Thev-(especially β-D-Glc-β-D-Glc-α-L-Thev-), Glc-Thev- (especially β-D-Glc-α-L-Thev) or β-(each of these oligo- or monosaccharide moieties bound via an O-atom to the steroid moiety);

Acyloxymethyl preferably carries an acyl moiety as defined above, and is especially lower alkanoyloxymethyl, preferably acetyloxy methyl.

Hydrocarbyl in hydrocarbyloxycarbonyl is especially an organic moiety having 1 to 20, preferably 1 to 12 carbon atoms within an unbranched or branched alkyl chain or a ring or any combination thereof, that is unsubstituted or substituted by one or more, especially one or two, moieties independently selected from halogen, hydroxy, amino, carboxy, lower al- koxycarbonyl, cyano, sulfo, carbamoyl and sulfamoyl; preferred is unsubstituted C C₁₂-alkyl, especially lower alkyl.

Tautomers may, for example, be present if any one of R^ and R₂ is N-alkylamidino [the possible tautomers being -C(=NH)-NH-lower alkyl <→- -C(=N-lower alkyl)-NH₂], N-lower alkylgua- nidino [the possible tautomers being selected from -NH-C(=NH)-NH-lower alkyl <→- -N=C(NH₂)-NH-lower alkyl -> -NH-C(NH₂)=N-lower alkyl], or N,N-di-lower alkylguanidino [the possible tautomers being selected from -NH-C(=NH)-N(lower alkyl)₂ - -N=C(NH₂)-N(lower alkyl)₂]. The presence and possible conditions for the existence of these tautomers or further tautomers are known to the person having skill in the art. All these tautomers are comprised by the definition of bufadienolides, especially compounds of formula I.

"Diseases" or "conditions" is not only limited to natural diseases but is intended to mean all pathological conditions, including graft-versus-host and/or host-versus-graft reactions after transplantation and the like, especially the diseases or conditions mentioned below References cited herein are herewith incorporated by reference, including all of their disclosure, especially that of relevance to the context of their quotation within the present text.

Salts are especially pharmaceutically acceptable salts of bufadienolides, especially compounds of formula I, if those have salt-forming groups. Such salts are formed, for example, from compounds of formula I having an acid group, for example a carboxy group, a sulfo group, or a phosphoryl group substituted by one or two hydroxy groups, and are, for example, salts thereof with suitable bases, such as non-toxic metal salts derived from metals of groups la, lb, lla and lib of the Periodic Table of the Elements, especially suitable alkali metal salts, for example lithium, sodium or potassium salts, or alkaline earth metal salts, for example magnesium or calcium salts, also zinc salts or ammonium salts, as well as salts formed with organic amines, such as unsubstituted or hydroxy-substituted mono-, di- or tri- alkylamines, especially mono-, di- or tri-lower alkylamines, or with quaternary ammonium compounds, such as tetrabutylammonium salts. The bufadienolides, especially compounds of formula I, having a basic group, for example an amino group, can form acid addition salts, for example with inorganic acids, e.g. hydrohalic acids, such as hydrochloric acid, sulfuric acid or phosphoric acid, or with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, fumaric acid, malic acid, tartaric acid, citric acid or nicotinic, as well as with amino acids, especially gluta- mic acid and aspartic acid, or with methanesulfonic acid, ethanesulfonic acid or N-cyclohe- xylsulfamic acid (forming cyclamates), or with other acidic organic compounds, e.g ascorbic acid. Bufadienolides, especially compounds of formula I with acid and basic groups can also form internal salts. For isolation or purification purposes, it is also possible to use pharmaceutically unacceptable salts, e.g. perchlorates or picrates.

Where hereinafter "compounds of (the) formula I" are mentioned, this term can, in a more general aspect of the invention, be replaced by the more general term "bufadienolides". In other words, "compounds of (the) formula I" stands for "bufadienolides, especially compounds of the formula I", where there is no explicit reference to compounds of the formula I only, e.g. by reference to a substituent displayed only in said formula.

The bufadienolides, especially compounds of formula I, have useful, in particular pharmacologically useful, properties. Surprisingly, it has been found that they are able to modulate, especially to suppress, the response of the immune system. This can especially be shown by their effect on human T cells (T lymphocytes). a) Especially, in one experiment, human T cells are stimulated in vitro with mitogens or alloantigens in the presence of compounds of formula I. The compounds of the formula I induce moderate to strong inhibition in the range of 100 nmol/10⁵ cells to 0.01 pmol/10⁵ cells, especially complete inhibition relatively to controls without added test compound of formula I in the range from 100 nmol/10⁵ cells to 0.5 pmol/10⁵ cells. In the same experimental setup, cortisol, cyclosporin A or tacrolimus (FK 506) suppress T cells at concentrations of 12 500, 195.2 or 195.2 pmol, respectively. In another experiment, preactivated T cells are downre- gulated and, most importantly, suppressed viable T cells cannot be restimulated. The results here show that 24 h preincubation with a compound of the formula I in a concentration higher than 0.1 pmol/10⁵ cells renders the cells unresponsive. If cells are incubated with a bufadienolide and ConA, they already become unresponsive after 12 h. In detail, the experiments are carried out as follows:

- Preparation of bufadienolide solutions: 1 to 5 mg of each bufadienolide tested (preparation or origin as described below) are dissolved in 0.1 ml of methanol and 0.1 ml of chloroform for 15 min, and DMSO is added in an amount leading to a final concentration of 50 mM bufadienolide (0.02 to 0.5 ml). Methanol and chloroform are evaporated by vacuum centrifugation. Stock solutions (50 mM) are stored at 4 °C and working solutions are prepared in RPMI cell culture medium (Gibco BRL, Paisley, UK).

- Cell cultures: b) Mitogen stimulation: Peripheral blood mononuclear cells (PBMC) of healthy volunteers are prepared by density gradient centrifugation (2000 g, 12 min) on lymphocyte separation medium (Eurobio, France) as previously described [Kanof, M.E., et al., Preparation of human mononuclear cell populations and subpopulations, in: Coico, R. et al. (eds.), Current Protocols in Immunology, Unit 7.1.1-7.1.7, Wiley & Sons, Inc., NY (1996)]. The cells (10⁵ /200 μl well in triplicate) are incubated for 48 hours with Concanavalin A (ConA) (2.5 μg/well) or phytohaemagglutinin (PHA) (12.5 μg/well) in culture plates with RPMI supplemented with 10% Fetal Calf Serum (FCS), 100 U/ml penicillin, 100μg streptomycin, 2mM glutamine (this is the medium used in all cell cultures described herein). c) Alloantigen Stimulation: For stimulation with allogenic cells, PBMC (1.3 x 10⁵ /well) of 3 healthy donors with different HLA phenotypes are prepared and irradiated with 30 Gy. These stimulator cells are coincubated with responder cells (10⁵ PBMC/200 μl/well) for 5 days (see James, S.P., "Culture for one-way mixed lymphocyte reaction", in: Coico, R. et al. (eds.), Current Protocols in Immunology, Wiley & Sons, Inc., NY (1996)), Unit 7.10.1.- 7.10.3.).

For a), b) and c), test compounds are added in dilution series (50 000 - 0.75 pmol/10⁵ cells) along with mitogen or allogenic stimulator cells on day 0. ³[H]thymidine incorporation (cpm) is measured after 48 h or 5 days by adding 1 μCi (= 3.7 x 10⁴ Bq) ³[H]-thy- midine (Amersham, Pharmacia Biotech, Braunschweig, Germany) per 10⁵ cells. Mean values of counts per minute (cpm) are determined in "Automatic Filter Counting System, InotecAG, Dottikon, Switzerland). In a parallel experiment, cortisol [hydrocortison-21- hydrogensuccinate (Rotexmedia GmbH, Trittau, Germany), cyclosporin A (Sandimmun, Novartis, Switzerland), or tacrolimus (FK506; Prograf, Fujisawa, Japan)] are added in dilution series (50 000 - 0.75 pmol/10⁵ cells/well) to the cell cultures. The positive control are ConA-, PHA- or alloantigen-stimulated cells without addition of test compounds, and the negative control consists of non-stimulated cells. DMSO controls at the same concentrations as those used for dissolving the test compounds of formula I are included in each experiment. d) Suppression of stimulated T cells: For suppression of activated T cells, each test compound of formula I (781 pmol/10⁵ cells/well) is added 24 h after initial stimulation with ConA (2.5 μg/10⁵ cells/well). e) Restimulation of suppressed T cells: For restimulation, the cells are incubated for 3, 12 or 24 h with (A) test compound of formula I (781 pmol/10⁵ cells/well) or (B) test compound of formula I (781 pmol/10⁵ cells/well) and ConA (2.5 μg/10⁵ cells/well), then washed, and the number of viable T cells is determined in a Fluorescence Activated Cell Sorter (FACS) (Becton Dickinson, San Jose, CA, USA) using double staining with anti- CD3-FITC antibody (Becton Dickinson) and 7-amino-actinomycin D (7-AAD) (Sigma- Aldrich GmbH, Germany) as described below. All samples are adjusted to the same number of viable T cells and then stimulated with ConA (2.5 μg/10⁵ cells/well). T cell proliferation is determined by ³[H]-thymidine incorporation (12 h) (see above) and mean values of counts per minute (cpm) are calculated. The positive control is referred to as 100 % stimulation. The results show that 24 h preincubation with a compound of the formula I renders the cells unresponsive. If cells are incubated with a compound of the formula I and ConA, they already become unresponsive after 12 h.

- FACS analysis of T cell viability: PBMC (10⁶/ml) are incubated for 3, 12 or 24 h with test compounds of formula I only (e.g. hellebrin or proscillaridin A; 781 pmol/10⁵ cells per test compound) or test compound of formula I with ConA. The following controls are used: me- dium only, medium with ConA (negative controls), 6 % ethanol in medium only, 6 % ethanol in medium with ConA (positive controls). Thereafter, the cells are stained with anti-CD3-FITC lgG1 mouse monoclonal antibody (Becton Dickinson), irrelevant lgG1 mouse monoclonal antibody (negative control) (Becton Dickinson) and extensively washed. After adding 50 ng 7-AAD/10⁵ cells, the suspension is incubated for 20 min, spun down, washed with PBS and analyzed within 30 min [see Fetterhof et al., Cytometry 14, 27 (1993) and Schmid, Cytometry 13, 204-208 (1992)]. The percentage of dead cells is determined by FACS. Only viable T cells are considered for restimulation in experiment (e). After 3, 12 and 24 h preincubation with a compound of formula I, e.g. with hellebrin or proscillaridin, 20 % or less, preferably 11 % or less, of the cells are dead, indicating that lack of restimulation upon pretreatment with a compound of formula I, especially hellebrin or proscillaridin A, in experiment e) described above is not due to T cell death but to a state of unresponsiveness. - Statistical analysis and data representation: Single values shown in tables represent the arithmetic mean of ³[H]-thymidine incorporation (triplicates of cpm) and are given in percentage of positive control (= 100 %). Statistical significance is calculated using the Mann-Whit- ney-U-test with two-tailed P values. "Complete suppression" is defined as stimulation value not statistically significantly different from the negative control. The degree of suppression (= activity factor) is defined as log 4 of the active ingredient dilution step required to suppress the cells.

Inhibition of Dendritic cells (DCs) by bufadienolides: Human monocytes are differentiated "in vitro" with the help of GM-CSF and IL-4 into immature DCs and thereafter with IL-4 and TNF- alpha into mature DCs. Viable mature DCs are incubated with allogeneic T cells and cell proliferation is measured by ³[H]-thymidine uptake as described above. Negative controls consist of T cells only. In one experiment (no. 3.1) given here as example, human monocytes are used for generation of mature DCs as described. In a parallel experiment (no. 3.2), the monocytes are cultivated in the presence of 1-100 pmol Proscillaridin A per 10⁵ cells under indentical conditions. After 7 days of culture, the cells are analyzed (i) with regard to their morphology under the microscope, (ii) for expression of surface markers with the help of FITC-labelled specific antibodies in a FACS device, and (iii) for their capacity to stimulate allogeneic T cells. The results show:

(i) The morphology of cells in experiment 3.2 is completely impaired, (ii) Surface marker analysis shows in experiment no. 3.1: weak expression of CD 14, significant expression of DR, high expression of CD83 (typical DC marker), and 0.4 % dead ceils; in experiment no. 3.2: no expression of CD14, weak expression of DR, no expression of CD83, and 42 % dead cells, (iii) The capacity of viable DCs to stimulate allogenic T cells is abrogated by pretreatment with Proscillaridin A (= ProA): with ProA 1 '900 cpm, without ProA 31O00 cpm, negative control: 1700 cpm (experiment no. 3.2). Interpretation: Treatment with bufadienolide, here ProA, compromises the maturation of DCs. The cells present impaired morphology, impaired surface marker expression and loss of T-cell stimulatory capacity. It is known that allogeneic transplants stimulate the immune system of the recipient mainly by DCs (Haeney, J. Antimicrob. Chemother. 36, Suppl. B, 1-9, 1995). Removal of DCs from the transplant was shown to abrogate its immunigenicity [Hart and McKenzie, Int. Rev. Immunol. 6(2-3), 127, 1990]. In clinical transplantation, ProA can be used for impairing the function of donor DCs and thus for reducing the immunogenicity of the graft. This can be reached by treating the donor with ProA before retrieval of the organ or by perfusing the explanted organ with ProA before implantation. It is also possible to treat the graft recipient after transplantation with ProA, or other bufadienolides.

Suppression of B cell activity can be demonstrated with bufadienolides as described for Proscillaridin A in Example 6 below.

Cytokine production in peripheral T-cells of individuals can be detected according to Jung et al. (J. Immunol. Methods 159, 197 , 1993). In the presence of bufadienolides (e.g. Proscillaridin A at 1-200 pmol/10⁵ cells), a significant decrease of IL-2, IL-4, IL-6, IL-13, interferon gamma and increase of IL-10 can be observed. This shows the antiinflammatory action of bufadienolides, making them useful for treatment e.g. of diseases like rheumatism and autoimmune diseases.

As bufadienolides suppress inflammatory cytokine production, they can also be used against postoperative shock and sepsis - this can be shown in guinea pigs or other animals using animal models described previously (Gadina et al., J. Exp. Med. 173, 1305, 1991; Dunn et al., J. Surg. Res. 34, 479, 1983; Chusid et al., . Lab. Clin. Med. 101, 441, 1983).

Using transplantation models in animals, the use of the bufadienolides in graft rejection inhibition can be shown. For example, human skin is grafted onto humanized severe combined immunodeficiency (SCID) mice (e.g. C.B-17 SCID, C.B-17 SCID/beige, SCID/NOD etc.) ac- cording to a previous protocol (Murray, A., et al., Am. J. Pathol. 153, 627, 1998). After that, allogeneic human peripheral blood mononuclear cells are injected intraperitoneally. A skin transplant in a humanized SCID mouse is usually rejected within 1 to 4 weeks. In order to suppress the rejection, the recipient is treated every 1^st to 3^rd day with 1 μg - 1g of bufadienolide, depending on the activity of the respective bufadienolide (e.g. in the case of Proscillaridin A 0.1 μg to 100 μg). Transplantation in minipigs, pigs and dogs: Organs like kidney, heart, lung, pancreas, liver, small bowel etc. are transplanted into allogeneic recipients as described (Zhu et al., Transplant. Proc. 28, 2725, 1996; Gruessner et al., J. Surg. Res. 49, 366, 1990; Kikuchi et al., Transplant. Proc. 30, 2987, 1998; Kitabayashi et al., Eur. Surg. Res. 26, 54, 1994). The recipients are treated every 1^st to 3^rd day with ProA (1 μg - 100 μg/kg body weight) or with another bufadienolide (1 μg - 1 g/kg, depending on its activity).

Treatment of pain and rheumatism in humans can be examined using the following protocol: In a clinical trial, patients with inflammatory forms of rheumatism receive injections of Proscillaridin A (0.1 - 3 mg) or other less active bufadienolides (e.g. 5 mg to 100 g) into the inflamed joint or tissue ("loco dolenti" infiltration). Reduction of inflammation as well as pain will occur within minutes or hours, and articular motility will improve. In animals, arthritis induced in guinea pigs, rats, cats or other animals can be induced by injecting carrageenan, complete Freund's adjuvant, ureate crystals or the like into the foot pad or into the joint of a limb and pain and inflammation are monitored as previously described (Idarola et al., Brain Res. 455. 202, 1998; Idarola et al., Pain 45, 313, 1988; Schaible et al., Exp. Brain Res. 66, 498, 1987; or Okuda et al., Pain 18, 287, 1984).

Cardiac activity, especially toxicity, can be tested in vivo or on vitro. This serves especially to find bufadienolides with low or no cardiotoxicity (which are especially preferred in the present invention as far as immunosuppressive and/or antiinflammatory action are concerned; and which are part of the present invention as far as antitumor action is concerned), allowing for a broader therapeutic window. Guinea pigs (Knaffl-Lenz, J. Pharmacol. Exp. Ther. 29, 407, 1926), cats (van Wijngaarden, Arch. Exp. Path. Pharmak. 113, 40, 1926), dogs (Dόrner, Arch. Exper. Path. Pharmakol. 226, 152, 1955), minipigs (http:/www.panlabs.com, Abadie et al) or other animals with hearts metabolically similar to human hearts can be used. In principle, the animals are anesthesized and fixed on a surgery table. A tracheal cannula is inserted, ECG and blood pressure are recorded, and the test preparation is infused into the jugular vein. The endpoint is cardiac arrest which should be reached within 30 to 60 min by ap- propriate adjustment of the dose of the infused test compound. In vitro testing is possible using human heart muscle as follows: (i) Membrane binding assay: Human heart membrane preparation is obtained as previously described (Schwinger et al. (I), J. Cardiovasc. Pharmacol. 35, 700, 2000). To that preparation, ³[H]-Ouabain is added along with increasing concentrations of the bufadienolide to be tested and radioactivity is measured. The stronger the binding of a bufadienolide to human heart muscle, the lower the radioactivity. This method allows to compare the binding activity of various bufadienolides to human heart muscle (Schwinger et al. (I), loc. cit.). (ii) Contraction experiments: The experiments are performed on isolated electrically driven left ventricular papillary muscle strips according to Schwinger et al. (II) (Am. Heart J. 131. 988, 1996). Test substances at increasing concentrations are added to the bathing solution and mechanical effects and force-frequency relation are measured (Schwinger et al. (II), loc. cit.). Where bufadienolides show a cardiotoxicity significantly lower than classical bufadienolides (e.g. hellebrin or proscillaridin A), they are considered non-cardiotoxic for the purposes of the present invention.

The compounds of formula I are able to kill human tumor cells without harming normal cells at the same concentration. Other mechanisms than the killing of cells are not excluded here. One mechanism of tumor cell killing, which is not intended to exclude other mechanisms, by bufadienolides is due to apoptosis, another to necrosis, or any combination of these two. The selective destruction of cancer cells, especially of cells responsible for proliferative diseases as defined above, allows therapeutic applications in which the tumor is destroyed but healthy tissues are preserved. The novel steroid compounds of formula I given below derived from bufadienolides are generated by omitting, modifying or removing the chemical groups responsible for cardioactivity: The 14-OH-group, the lactone ring of formula lb given above and below, both; or in addition annealing of rings C and D of the steroid ring system in trans configuration. The anti-tumoral effect of these novel non-cardioactive derivatives whose chemical structure strongly differs from that of the other bufadienolides is analysed. The results show that, in spite of the large structural change when compared with the cardio- active bufadienolides, the non-cardiotoxic derivatives are able to kill cancer cells. The antiproliferative effect of the non-cardioactive bufadienolides is time- and dose-dependent, whereas there is an optimal concentration beyond which the effect becomes weaker. A test system for the antiproliferative effect based on lymphoma cells activity is shown below in the examples. Other cancer cell lines that represent different types of tumors can be used instead, e.g. those of the NCI (USA) for screening against tumors; possible examples are breast tu- mor: MCF-7; lung tumor: NCI H596: colon tumor: HCT 116; prostate tumor: PC 3 cell lines, human colon carcinomas SW 620 and LS 174T, breast carcinoma MDA-MB-435S, human epithelial cell line A-431; the MDA-MB 468 breast adenocarcinoma cell line; the MDA-MB 231 breast adenocarcinoma cell line; the Colo 205 colon carcinoma cell line; and the DU145 prostate carcinoma cell line DU 145; all obtainable from the American Type Culture Collection (ATCC), Rockville, MD, USA.

The antitumor efficacy of the non-cardiotoxic compounds of formula I, especially the novel compounds of formula I, can be demonstrated in vivo as follows:

In vivo activity in the nude mouse xenotransplant model: female BALB/c nude mice (8-12 weeks old, for example Novartis Animal Farm, Sisseln, Switzerland) are kept under sterile conditions with water and feed ad libitum. Tumours are induced by subcutaneous injection of tumour cells (human epithelial cell line A-431 ; American Type Culture Collection (ATCC), Rockville, MD, USA, Catalogue Number ATCC CRL 1555; cell line from an 85-year-old woman; epidermoid carcinoma cell line) into carrier mice. The resulting tumors pass through at least three consecutive transplantations before the start of treatment. Tumor fragments (about 25 mg) are implanted subcutaneously in the left flank of the animals using a 13-gau- ge trocar needle under Forene^® anaesthesia (Abbott, Switzerland). Treatment with the test compound is started as soon as the tumor has reached a mean volume of 100 mm³. Tumor growth is measured two to three times a week and 24 hours after the last treatment by determining the length of two perpendicular axes. The tumor volumes are calculated in accordance with published methods (see Evans et al., Brit. J. Cancer 45, 466-8 [1982]). The antitumor effect is determined as the mean increase in tumour volume of the treated animals divided by the mean increase in tumour volume of the untreated animals (controls) and, after multiplication by 100, is expressed as T/C%. Tumour regression (given in %) is reported as the smallest mean tumour volume in relation to the mean tumour volume at the start of treatment. The test compound is administered daily by gavage. Instead of the A-431 cell line, other tumor cell lines, e.g. those mentioned above, or lymphoma cells can be used.

The bufadienolides, especially compounds of formula I, can thus be used in the treatment (including therapy and/or prophylaxis) of conditions in warm-blooded animals that are responsive to immunomodulation, especially immunosuppression, antiinflammatory action and/or (for non-cardioactive bufadienolides) antitumor action; among those conditions are especially one or more selected from the group consisting of transplant rejection, autoimmune disease, rheumatic (including rheumatoid) diseases (especially those with immuno- logical and/or inflammatory components), allergy, postoperative and septic shock, sepsis, and the like, preferably as defined above, for autoimmune diseases.rheumatic diseases and proliferative diseases, especially those mentioned under "Background of the Invention".

The bufadienolides, especially the compounds of the formula I, (or salts thereof) can be used alone or in combination with one or more other bufadienolides (or salts thereof) and/or other drugs, especially with other immunosuppressive, especially antiproliferative, drugs, preferably cytotoxics, such as azathioprine, cyclophosphamide or methotrexate; cyclosporin A or appropriate analogs thereof; glucocorticoids, such as cortisol or prednisone, antibodies [e.g. against lymphocytes, especially T-cells or B-cells, macrophages, monocytes, granulo- cytes (especially mast cells), natural killer cells, dendritic cells and the like], or with other antitumor agents, e.g. cytostatics, kinase inhibitors, or the like; or salts of any of these, where at least one salt-forming group is present; either as fixed combination or in a chronically coordinated way, e.g. simultaneously or chronologically staggered. In addition (in view of the possible sensitization towards infectious diseases), they may be used in combination with pathogen combating drugs, such as antibiotics or antivirals. In case of allergy, drugs binding at the mast cell, e.g. isoprenaline or sodium cromoglycate, or especially anti-histamines, such as loratidine, may be used in combination with a bufadienolide, especially a compound of the formula I, a tautomer thereof, or a salt thereof.

In general, the present invention relates also to the use of a compound of formula I in im- munomodulation (either in vitro or in vivo), preferably immunosuppression, in inhibition of inflammation and/or of proliferative diseases (in the case of non-cardiotoxic bufadienolides), especially of diseases and conditions as mentioned above.

Where the phrase "use according to the invention " is used, this relates to use in the processes, methods and/or pharmaceutical preparations or their preparation as defined above and below, especially the novel use of the compounds of formula I, as such or in the form of pharmaceutical compositions, for immunomodulation, especially for immunosuppression, inhibition of inflammation and/or (in the case of non-cardioactive compounds of the formula I) of proliferative (especially tumor) diseases, especially for the use in the treatment (including therapy and/or prophylaxis) of conditions in warm-blooded animals that are responsive to those effects; to processes for the manufacture of pharmaceutical compositions for this novel use, to pharmaceutical compositions comprising the steroids and/or to a method of treatment comprising administering such a steroid, in general or as defined by more specific definitions within this specification.

The invention preferably relates to the immunosuppressive, antiinflammatory and/or antitumor use according to the invention of a compound of formula I (due to their especially low cardiotoxicity) wherein each of R_{1 (} RΛR₂ , R₂*, R₂**, R₃, R₄, R₅, R₄*, R₄ ^**, R₆, R₆*, R₇, R_β, R₁₁. i_2> R₁3, R₁₄ and R-|₅ has the meanings given above or below for compounds of the formula I, and

R₉ and R₁₀ form a double bond or preferably oxa in β-position ( — ()•— ); a tautomer thereof, or a salt thereof, if a salt-forming group is present.

The invention preferably also relates to the immunosuppressive and/or antiiflammatory use according to the invention of a compound of formula I either

(i) wherein each of R_{1 t} RΛR₂ , R₂ ^*, R₂ ^**, R₃, R₄, R₅, R₄ ^*, R₄ ^**, R₆, R₆ ^*, Ry, R₈, R₉, R10, Rn, Rι_2l Ri₃. R₁₄ and R₁₅ has the meanings given above or below for compounds of the formula I, and the rings C and D are present in cis junction; a tautomer thereof, or a salt thereof, if a salt-forming group is present; or (ii) wherein each of R_{1 (} RΛR₂ , R₂*, R₂ ^**, R₃, R , R₅, R₄*, R ^**, R₆, R₆*, R?, R₈, R₉, R10, Rn, R₁₂, R₁ and R₁₅ has the meanings given above or below for compounds of the formula I, and R₁₃ is a moiety of the formula lb as shown above; a tautomer thereof, or a salt thereof, if a salt-forming group is present; or (iii) wherein each of R₂ , R₂ ^*, R₂ ^**, R₃, R4, R₅, R₄*, R₄ ^**, R₆, R₆ ^*, R₇, Rs, Rg, R10, Rn, R12, R13, R₁₄ and R₁₅ has the meanings given above or below for compounds of the formula I, and each of Ri and R^ is hydrogen, lower alkanoyloxy, especially acetyloxy, amino (especially preferred is R-_t = amino), amino-lower alkoxy, especially β-aminoethyloxy, or the radical of a mono- or oligosaccharide bonded O-glycosidically via an oxygen atom to the rest of the molecule of formula I, especially Rha-, such as α-L-Rha, or Glc-Rha-, such as β-D-Glc-α-L- Rha-, each bonded O-glycosidically via an oxygen atom to the rest of the molecule of formula I; with the proviso that at least one of R^ and Ri* is hydrogen ; or Ri and R-,* together form oxo (=O); a tautomer thereof, or a salt thereof, if a salt-forming group is present. As non-cardioactive bufadienolides (especially for antiproliferative action, but also preferred for immunosuppressive and/or antiinflammatory action) the compounds of formula I wherein R₁₃ is other than of the formula lb, especially those, wherein

(a) R₁₃ is carboxy or COOR^**, wherein R* is hydrocarbyl; or is hydroxymethyl; and wherein each of RL RΛ R₂, R₂ ^*, R₂ ^**, R₃₎ R₄, R₅, R₄*, R₄**, R₆, R₇, R₈, R₁₀, R_{11 (} R₁₂, R₁₄ and R₁₅ has the meanings given above or below for compound of the formula I;

(b) R₉ and R₁₃ taken together form a bridge -C(=O)-C-(=O)-O- where the O is bound in the position of R₉ and the left carboxy is bound in the position of R₁₃, both preferably in the β- position, and wherein each of R_{1 t} RΛ R₂, R₂*, R₂**, R₃, R₄, R₅, R₄*, R₄*^*, R₆, R₇, R₈, R_10l R₁₁. R₁₂, ι₄ and R15 has the meanings given above or below for compound of the formula I;

(c) R₉ and R₁₀ together form a double bond or preferably oxa in β-position, while each of R^ ι*ι R2. R₂*ι R2**_> R3> R₄, R5. R4*ι R₄**ι R6. R7, Rs, R-n, Ri2> R13, R14 and R15 has the meanings given above or below for compound of the formula I;

(d) R₉ is hydrogen and each of R_{1 (} R R₂, R₂ ^*, R₂**, R₃, R₄, R₅, R₄*, R₄ ^**, R₆, Rr, Re, R10, R₁₁, R₁₂, R₁₃, Ri₄ and R₁₅ has the meanings given above or below for compound of the formula I;

(e) Rg is hydrogen, rings C and D of the steroid ring system are annealed in cis-configurati- on, and R₁₃ is is carboxy or COOR**, wherein R* is hydrocarbyl; or is hydroxymethyl; and wherein each of R_{1 f} RΛ R₂, R2*, R2^**, R₃, R», Re, R4*, R4*^*, Re, R₇, Re, R10, Rn, R12, R13, R₁₄ and R₁₅ has the meanings given above or below for compound of the formula I; or

(f) Rg and R₁₀ together form a double bond or preferably oxa in β-position and R₁₃ is carboxy or COOR**, wherein R* is hydrocarbyl; or is hydroxymethyl; and wherein each of Ri, RΛ R2, R2^*, R₂**, R3, R4, R5, R4*, R4^**, Re, R7, Re, R11, Rι₂, R14 and R15 has the meanings given above or below for compound of the formula I; or salts thereof where salt-forming groups are present.

These compounds, as well as their salts, are also as such embodiments of the invention, except for Resibufogenin, Resibufogenin-O-hemisuberate and Mannobufagin.

Even more preferred is the use of a compound of formula i wherein the moieties Ri, RΛ R₂, R₂ ^*', R₂ ^**> R3, R4> R₄*, R *^*, R5, Re, Re^*, R7, Re, 9, R10, Rn, Rι₂, R13, R14 and R15 have the meanings defined as being preferred above, preferably in the preceding four paragraphs, especially wherein (if not indicated otherwise especially in the preceding four paragraphs) Ri is hydrogen, hydroxy, amino, azido, Rha-O-, Glc-Rha-O, lower alkanoyl(especially ace- tyl)oxy, carboxy-lower alkanoyl (especially suberoyl)oxy, O-suberoyl-metHis-O- or amino- lower alkoxy (especially β-aminoethyloxy);

Ri^* is hydrogen, hydrox , amino or lower alkanoyl(especially acetyl)oxy; or Ri and Ri* together form =O or =NOH; R₂ is hydrogen or hydroxy; each of R₂* and R₂** is hydrogen; or R₂ and R₂** together with the bond between the carbon atoms that carry R₂ and R₂** form a double bond while R₂* is hydrogen; R₃ is lower alkyl, especially methyl, formyl or hydroxymethyl; R₄ is hydrogen or lower alkanoyloxy, especially hydrogen or acetyloxy (especially in β- position);

R₄* and R₄** each are hydrogen; R₅ is hydrogen;

R₆ is hydrogen, α-hydroxy or α-lower alkanoyl(especially acetyl)oxy and R₆* is hydrogen, or R₆ and R₆* together are oxo (=O); R₇ is hydrogen or α-hydroxy;

R₈ is hydrogen or β-hydroxy, with the proviso that R₇ and R₈ are not simultaneously hydroxy; or R₇ and R₈ together are oxo (=O); R₉ is hydrogen or α-hydroxy or preferably β-hydroxy and R₁₀ is hydrogen, or R₉ and R_i0 together with the bond between the carbon atoms that carry

R₉ and R₁₀ form a double bond or oxa in β-position ( — O"— ), or R₉ and R₁₃ taken together form a bridge -C(=O)-C(=O)-O- where the O is bound in the position of R₉ and the left carbonyl is bound in the position of R₁₃, both preferably in the β- position (preferred non-cardiotoxic compounds); R₁₁ is hydroxy, β-hydroxy or β-lower alkanoyl(especially acetyl)oxy; R₁₂ is methyl, especially β-methyl;

R₁₃ is a moiety of the formula lb given above or (less preferably with respect to antiinflammatory or immunosuppressive action, more preferably with regard to antiproliferative action due to non-cardioactivity) is carboxy, lower alkoxycarbonyl or hydroxymethyl; and each of R₁₄ and R₁₅ is hydrogen, or, if R₂ ^** and R₂ together with the bond between the carbon atoms that carry R₂ and R₂** form a double bond and if R and R-,* together form oxo, R₁₄ and R₁₅ together with the bond between the carbon atoms that carry R₁₄ and R₁₅ form a double bond; a tautomer thereof, or a salt thereof, if a salt-forming group is present.

Specifically, the use of bufadienolides, especially compounds of formula I, described above or below other than hellebrin or hellebrigenol, more preferably other than steroids obtained from Helleborus species, is preferred, in the case of antiprotumor activity the use of bufadienolides other than bufalin, more preferably other than bufalin and cinobufagin.

Most preferred is the use of a compound of formula I or a novel compound of formula I mentioned in the examples, a tautomer thereof, and/or a (preferably pharmaceutically acceptable) salt thereof, most especially the use of proscillaridin A or hellebrin, as far as immunosuppressive and/or antiinflammatory action is concerned. With regard to antitumor activity, 3β-hydroxy-14β,15β-epoxy-aetianic acid methylester or 3β-acetoxy-14β,15β-epoxy-aetianic acid are especially preferred.

The invention relates also to novel compounds of formula I and their salts and to those compounds for use or the use of those compounds in the processes, methods and/or pharmaceutical compositions mentioned hereinbefore and hereinafter, and to pharmaceutical compositions comprising those compounds, especially the compounds mentioned below: Preferred among these compounds according to the invention is a compound of formula I selected from the group consisting of compounds mentioned in the examples as compound No. 2, 7, 8, 10, 12, 14, 15, 19, 20, 22, 23, 24, 25, 29, 34, 35, 39, 40, 42, 43, 57, 66, 67, 68, 74, 81, 83, 91 , 92 and 100, a tautomer thereof, or a salt thereof; especially from the group of compounds 2, 24, 42, 43, 54 and 93, a tautomer thereof, or a salt thereof.

The bufadienolides, especially compounds of the formula I, tautomers thereof, and salts thereof, and methods for their isolation and/or synthesis are known, or they can be obtained in analogy to methods that are known or are commercially available. For example, they can be synthesized and/or isolated as described in Krenn et al., Phytochemistry 48(1), 1-29 (1998), L. Fieser and M. Fieser, "Steroide", Verlag Chemie, Weinheim 1961; G. Baumgarten, "Die herzwirksamen Glykoside - Herkunft, Chemie und Grundlagen ihrer pharmakologischen und klinischen Wirkung, VEB Georg Thieme, Leipzig 1963; or the references quoted therein. For example, they can be obtained according to the following procedures. A) Isolation from animal and plant sources: For the isolation of bufadienolides the classic methods of extraction, partition and column chromatography are followed by efficient chromatographic techniques like HPLC or DCCC and/or crystallization for the purification of fractions in most cases. In column chromatography silica gel and Sephadex^®- LH 20 as stationary phases are very successful, but also neutral alumina or dry CC on silica gel can be employed. Polar substances like bufotoxins from different Bufo species are isolated by fractionation of polar extracts on Amberlite^® XAD-2 or XAD-4. For the purification of bufotoxins HPLC on RP-18 phases with methanol-water or acetonitrile- water is applicable. HPLC on RP-18 is also useful in the isolation of bryophillin A and bersaldegenin-3-acetate from Bryophyllum pinnatum, etc. Other useful separation steps employ RP-8 HPLC materials and acetonitrile- or methanol-water mixtures. References for these and comparable methods can be found in Krenn et al., Phytochemistry 48(1), 1-29 (1998), for example J. Org. Chem. 36(18), 2611 (1971) (extraction from Bersama abyssinica), Chem. Pharm. Bull. 22(7), 1673-4 (1974) (from Bufo vulgaris formosus Boulenger), Tetrahedron Lett. 54, 5673-6 (1968) (from Ch'an Su), J. Nat. Products 48(1), 159 (1985) (from Bufo asper Gravenhorst), Chem. Pharm. Bull. 38(7), 2767-71 (1985) (from Bufo bufo gargarizans CANTOR), Chem. Pharm. Bull. 32(11), 4396-4401 (1984) (from Bufo melanosticus Schneider), Tetrahedron Lett. 32, 2767-2768 (1974) and Chemistry and Industry, 3432-3 (April 1974) (from Bufo vulgaris formosus Boulenger), Tetrahedron Lett. 5, 467-8 (1974) (from Bufo vulgaris Boulenger), Chem. Pharm. Bull. 27(8), 1881-6 (1979) (from Bufo marinus (L.) Schneider), Chem. Pharm. Bull. 24, 1118-1120 (1976) and Chem. Pharm. Bull. 25(4), 714-730 (1977) (from Bufo vulgaris formosus Boulenger), Chem. Pharm. Bull. 35(6), 2300-2304 (1987) (from Bufo banko- rensis Borbour), Tetrahedron Lett. 25(5), 551-2 (1984) (from Bufo melanostictus Schneider), J. Nat. Products 52(5), 1071-9 (1989) (from Bryophyllum pinnatum or tu- biflorum), Helvetica Chimica Acta 69, 359 (1986) (from Kalanchoe daigremontiana), Phytochemistry 42(2), 513-22 (1996) (from Urginea maritima from Egypt), J. Nat. Prod. 59, 612-3 (1996) (from Urginea maritima), Planta Medica 56, 193 (1990) (from Urginea pancration), Planta Medica 55, 107 (1989) (from Helleborus niger roots), Planta Medica, 152-3 (1986) (from Helleborus odorus), Acta cryst. C43, 922-6 (1987) and Helv. Chim. Acta 53, 1993-2002 (from toad Ch'an Su), Fitoterapia 71. 126-129 (2000) (from Urginea maritima bulbs), Planta Med. 57, 560 (1991) (from Urginea maritima sensu strictu), Helv. Chim. Acta 50, 1659-70 (1957) and Pharm. Acta Helv. 24, 222-246 (1949) (from Bufo mauretanicus L.), see also K. Meyer and H.H.A. Linde: Collection of Toad Venoms and Chemistry of the Toad Venom Steroids, in: Venomous Animals and their Venoms, Vol. 2, Chapter 40, Academic Press Inc, New York/London, 1971 ; further bufadienolides have been isolated from snakes (e.g. Rhabdophis tigrinus), arthropodes (Photinus), and from various plants, e.g. in the six families Crassulaceae, Hyacinthaceae, Iridaceae, Melianthaceae, Ranunculaceae and Santalaceae; etc. For example, bufadienolides are isolated from secreting cells in the ear area of Japanese (Senso) and Chinese toads, from dried skin, or by squeezing out secretions from glands, e.g. from the parotid salivary gland. Useful separation materials and methods using HPLC and tic can be found, e.g., in Deutsche Apothekerzeitung 40, 2175, 1990.

Chemical Modification of bufadienolides. Natural or already modified bufadienolides can be modified by a variety of chemical reactions. Thus, hydroxy groups can be acylated by standard coupling or acylation procedures, e.g. acetylated by acetic acid or by acetic anhydride in pyridine or the like (primary and secondary hydroxy groups mainly), acylated by dicarbonic acid anhydrides to form hemiesters, sulfated by chlorosulfonic acid in pyridine acid or sulfuric acid in the presence of N,N'-dicyclohexylcarbodiimide, or sulfo- nated by other sulfonic acid derivatives, such as p-toluolsulfonic acid. Formiate can be obtained by heating hydroxy precursors in formic acid. Hydroxy can also be oxidized to oxo, e.g. by manganese dioxide or chromium trioxide, or (via the halogen or tosyl intermediate) converted into amino-lower alkoxy, e.g. with 2-ethanolamine. Carboxy groups can be alkylated, e.g. methylated by treatment with diazomethane or after formation of active esters (e.g. 4-nitrophenyl esters) and/or in presence of coupling agents, such as N,N'-dicyclohexyl carbodiimide, or they can be amidated by customary coupling reactions, such as those just mentioned, e.g. with moieties of amino acids, such as arginine or histidine or methylhistidine. Glycosidic moieties can be introduced by standard sugar coupling reactions, or they can be removed by hydrolysis, for example enzymatically, e.g. in case of removal of rhamnoside by hydrolysis with naringinase (α-rhamnosidase), in case of β-glucosides with β-glucuronidase (see B. Kopp, Dissertation, University of Vienna (1972), or H. Kirchner, Dissertation, Univ. Vienna, 1976), or in the presence of an acid, e.g. 5 % HCI. Oxo groups can be converted into oximes by reaction with hy- droxylamine, e.g. in the presence of acid, or they can be reduced (e.g. with sodium bo- rohydride, for example in isopropanol) to hydroxy. α-Substituents, such as α-hydroxy, can be converted into β-substituents (inversion, Walden inversion) and vice versa by appropriate bimolecular nucleophilic displacement reactions - for example, α-hydroxy can be converted into β-hydroxy (or vice versa) by halogenation, e.g. with PCI₅ or SOCI₂, and subsequent hydrolysis of the halogenated product e.g. with potassium acetate, or acylation with p-toluolsulfonylchloride and subsequent hydrolysis under S_N2 conditions. Azido and amino groups can be introduced by nucleophilic displacement of nucleofugic groups, such as tosyloxy or halo, especially chloro, e.g. with sodium azide or ammonia. Acyl moieties, such as amino acyl moieties or O-dicarbonic acid-N-aminoacid moieties, can be removed by hydrolysis, e.g. with 6 N HCI or enzymatically, e.g. with pancreas lipase preparations, acetyl by hydrolysis at alkaline pH, e.g. in sodium hydrogencarbona- te or 25 % potassium hydroxide solution. Guanidino groups, e.g. in arginyl moieties, can be converted into 4,6-dimethyl-pyrimidin-2-ylamino for example by treatment with acetylacetone-potassium hydrogen carbonate. Many of these reactions can be found in the references cited in Phytochemistry 48(1), 1-29 (1998) or the references cited above under A). Ketolactones can be obtained by treatment of compounds with the ring of formula lb in position 17 with potassium permanganate, they can then be esterified, e.g. to the methyl ester, reduced to the triols (e.g. with lithium aluminum hydride, LiAIH₄; see Helv. Chim. Acta 42, 807 (1959); Helv. Chim. Acta 42, 1457; Experientia 15, 238 (1958); or Helv. Chim. Acta 50, 1994-2008 (1967). Double bonds can be obtained from hydroxy precursors by dehydratation with SOCI₂ in pyridine; the resulting double bonds can be reduced to single bonds by hydrogenation in the presence of catalysts, such as PtO₂. Reduction of 3-keto to 3-hydroxy is possible with Na-amalgam or by catalytic hydrogenation (e.g. with Pd/H₂ in acetic acid, with NaBH₄ the 3α-OH is formed. Carboxy can be reduced to hydroxymethyl with complex hydrides, e.g. LiAIH .

Furthermore, some of them can be synthesized totally or starting from known starting materials, or in analogy to known procedures, or they are commercially available.

For example, compounds 3, 6, 4, 5, 11, 64, 65, 70, 89 and 90 mentioned in the Examples can be obtained as described in Phytochemistry 48, 1-29 (1998), as well as compounds 16, 54, 63, 69, 75 and 93; the aglyka 4, 6 and 90 by enzymatic cleavage of the respective gly- cosides (5, 89, Scillirosid) with α-rhamnosidase and/or β-glucuronidase, acetylation leads to compounds 1, 2, 12, 18, 29, 59, 66, 74, 80 and 92; the preparation of 7 is possible by dissolving 100 mg of acetylated 12 in 5 ml acetone, portionwise addition of 100 mg of finely ground KMnO₄, stirring at room temperature and further addition of the permanganate after 30 and 75 min. After 2 h the reaction is complete (tic) and yields 22 mg of 7 and 28 mg of ketolactone 10 which can be further purified on a tic plate (silica gel 0.5 mm, mobile phase chloroform:methanol = 99:1) and recrystallization from ether/petrolether. From 7, the methylated intermediate ("8a") can be obtained with methanolic diazomethane at room temperature. This way, also compounds 67, 68, 91, 34 and 35 can be obtained. 8a allows for synthesis of 8 by dehydration: 20 mg of 8a (etianic acid methyl ester) are dissolved in 0.2 ml of pyridine, cooled down to 0 °C and combined with a solution of 0.15 ml pyridine/SOCI 1:1 (0 °C), the mixture is kept for 2 h at 0 °C and then 30 min at + 23 °C, ice and water are added and the product is worked up with chloroform. Tic-purification (mobile phase chloroform: methanol = 99:1) yields the 14,15-en intermediate 8b (< 10 mg). This is then hydrated in the presence of 5 mg PtO₂ in acetic acid for 5 h, the resulting solution is evaporated and the product recrystallized from ether/petrol ether to yield 8. Compounds 2, 55 and the derivatives thereof (3-epi-compounds) can be obtained by reaction of the precursor bufadienolides with PCI₅ or SOCI₂, yielding the respective steroid chloride; hydrolysis with potassium acetate leads to 3α:3β in 85:15 ratio, separable by preparative tic. 3-keto compounds, e.g. 17, can be obtained by oxidation of 3-OH in benzene in presence of aqueous potassium permanganate solution or with CrO₃ in dimethylformamide (with some drops of cone. Sulfuric acid). 19 and 20 can be obtained by reduction of etianic acid with LiAIH₄. 15 can be obtained from 14 with hydroxylamine/pyridine. 22 can be obtained by heating of the hydroxy precursor 69 in 88% formic acid and customary purification. 14 can also be obtained from e.g. 50 mg bufo- talin in 0.5 ml acetone at 0 °C with addition of 2 drops of Kiliani solution (J. Chem. Soc. 1946, 1949), reaction for ten minutes, then addition of 0.5 ml methanol and 0.5 ml water, concentration under vacuum, 3 x extraction with chloroform, washing of the organic phase with water, drying over sodium sulfate, filtration and evaporation and final purification via preparative tic (benzene:acetone:cyclohexane 1:1:1 on silica gel).

Confirmation of the structure of the isolated or modified compounds makes use of standard procedures, e.g. elemental analysis, partial synthesis, NMR methods (especially ¹H-NMR, ¹³C-NMR) using modern pulse techniques like J-modulation, INEPT, DEPT and APT, or especially two-dimensional methods, particularly ¹H,¹ H-correlation spectroscopy, ¹³C,¹H-COSY and ¹³C,¹H-long range COSY; NOE difference spectroscopy, X ray crystallography, FAB mass spectroscopy, El- or FD-mass spectroscopy or DCI-MS, GC-mass spectroscopy, 2D- NOESY or other methods known to the person skilled in the art. In addition to these transformation/conversion reactions, other step can be conducted, e.g. for transforming a salt of an obtainable compound of formula I into the free compound or a different salt or an obtainable free compound of formula I into a salt, and/or separating obtainable mixtures of isomers of compounds of formula I into the individual isomers.

The invention relates also to those forms of the processes in which a compound obtainable as intermediate at any stage of the process is used as starting material and the remaining process steps are carried out, or in which a starting material is formed under the reaction conditions or is used in the form of a derivative, for example in protected form or in the form of a salt, or a compound obtainable by the process according to the invention is produced under the process conditions and processed further in situ. In the process of the present invention there are preferably used those starting materials which result in the compounds of formula I described at the beginning as being especially valuable. Special preference is given to reaction conditions and processes of manufacture that are analogous to those mentioned in the Examples.

Pharmaceutical compositions, the preparation thereof, and the use according to the invention of a bufadienolide, especially a compound of formula 1 and compositions comprising compounds of formula I as active ingredient

The present invention relates also to pharmaceutical compositions that comprise (preferably a novel or a preferred) compound of formula I, a tautomer or a salt thereof, as active ingredient and that can be used especially in the treatment of a disease mentioned at the beginning. Special preference is given to compositions for enteral, such as nasal, buccal, rectal (e.g. suppositories) or, especially, oral and/or parenteral, such as intravenous, intramuscular or subcutaneous, or local (topical, not systemic, e.g. to skin, eyes) administration to warm-blooded animals, especially humans. The compositions comprise the active ingredient on its own or, preferably, together with a pharmaceutically acceptable carrier.

The dose of active ingredient depends on the disease to be treated, on the species, its age, weight and individual condition, on individual pharmacokinetic conditions, and on the mode of administration. The preferred dose to be administered to warm-blooded animals, for example humans of approximately 70 kg body weight, is in the range of approximately 0.1 mg to approximately 50g, preferably from approximately 0.5 mg to approximately 3 g, most pre- ferably from approximately 1 mg to approximately 1.5 g, for example approximately from 5 mg to 500 mg or in acute cases (e.g. of transplant rejection) from 10 to 2000 mg per person per day, divided preferably into 1 to 3 single doses which may, for example, be of the same size. Usually, children receive half of the adult dose. "Approximately" preferably stands for a deviation in the range of ± 10 %, more preferably of + 2 % from the given numeric value; most preferably, the given number is meant.

The invention relates also to pharmaceutical compositions comprising a bufadienolide, especially compound of formula I, a tautomer or a pharmaceutically acceptable salt thereof (preferably a compound of the formula I, a tautomer or a pharmaceutically acceptable salt thereof mentioned above or below as being preferred, or a novel compound of the formula I, a tautomer or a pharmaceutically acceptable salt thereof), for use in a method for the prophylactic or, especially, therapeutic treatment of the human or animal body; to a process for the preparation thereof; and/or to a method of treating the conditions mentioned above or below, comprising administering a compound of the formula I, a tautomer thereof, or a pharmaceutically acceptable salt thereof, to a warm-blooded animal, especially a human in need of such treatment; especially for the treatment (including therapy and/or prophylaxis) of conditions in warm-blooded animals that are responsive to immunomodulation, especially immunosuppression, inhibition of inflammation and/or (in the case of non-cardioactive bufadienolides) of proliferative (especially tumor) diseases; among those conditions are especially one or more selected from the group described above.

Preference is given to a pharmaceutical composition that is suitable for administration to a warm-blooded animal, especially a human or a (especially commercially useful) mammal, suffering from a condition or disease as described above that is responsive to a bufadienolide, most especially a disease or condition as described as preferred above, said composition comprising a bufadienolide, especially a compound of the formula I, a tautomer thereof or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier.

The invention relates also to processes for, and to the use of bufadienolides, especially (preferably the novel) compounds of formula I, a tautomer thereof, or a salt thereof, in the preparation of pharmaceutical compositions that comprise a compound of formula I, a tautomer thereof, or a salt thereof, as active component (active ingredient), preferably to treat one or more of the conditions mentioned above and below.

Preferred as active ingredient is always a novel compound of formula I, a tautomer thereof or a pharmaceutically acceptable salt thereof (especially if preferred) as defined above.

The pharmacologically acceptable active ingredients of the present invention may be used, for example, for the preparation of pharmaceutical compositions that comprise an effective amount of the active ingredient together or in admixture with a significant amount of inorganic or organic, solid or liquid, pharmaceutically acceptable carriers.

The pharmaceutical compositions comprise from approximately 1 % to approximately 95% active ingredient, dosage forms that are in single dose form preferably comprising from approximately 20% to approximately 90% active ingredient, and dosage forms that are not in single dose form preferably comprising from approximately 5% to approximately 20% active ingredient. Unit dose forms are, for example, dragees, tablets, ampoules, vials, suppositories or capsules. Other dosage forms are, for example, ointments, creams, pastes, foams, tinctures, lipsticks, drops, sprays, dispersions, etc.. Examples are capsules comprising from approximately 0.05g to approximately 1.0g of the active ingredient.

The pharmaceutical compositions of the present invention are prepared in a manner known per se, for example by means of conventional mixing, granulating, confectioning, dissolving or lyophilising processes.

There are preferably used solutions of the active ingredient, additionally also suspensions or dispersions, especially isotonic aqueous solutions, dispersions or suspensions, which, for example in the case of lyophilised compositions comprising the active ingredient on its own or together with a carrier, e.g. mannitol, may be prepared before use. The pharmaceutical compositions may be sterilised and/or may comprise excipients, for example preservatives, stabilisers, wetting agents and/or emulsifiers, solubilisers, salts for regulating the osmotic pressure and/or buffers, and are prepared in a manner known per se, for example using conventional dissolving or lyophilising processes. The said solutions or suspensions may comprise viscosity increasing substances, such as sodium carboxymethylcellulose, carboxy- methylcellulose, dextran, polyvinylpyrrolidone or gelatin, or also solubilisers, e.g. ^Tween 80 [polyoxyethylene(20) sorbitan monooleate; trademark of ICl Americas, Inc, USA].

Suspensions in oil comprise as the oil component the vegetable, synthetic or semisynthetic oils customarily used for injection purposes. There may be mentioned especially liquid fatty acid esters which contain as acid component a long-chain fatty acid having from 8 to 22, especially from 12 to 22, carbon atoms, for example lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, arachidic acid, behenic acid or corresponding unsaturated acids, for example oleic acid, elaidic acid, erucic acid, brassidic acid or linoleic acid, where appropriate with the addition of antioxidants, for example vitamin E, β-carotene or 3,5-di-tert-butyl-4-hydroxytoluene. The alcohol component of those fatty acid esters has not more than 6 carbon atoms and is a mono- or poly-valent, for example mono-, di- or tri-valent, alcohol, for example methanol, ethanol, propanol, butanol or pen- tanol or their isomers, but especially glycol and glycerol.

The preparation of the injection compositions is carried out in customary manner under sterile conditions, as well as the introduction, for example, into ampoules or vials and the sealing of the containers.

Pharmaceutical compositions for oral administration (which are especially preferred) can be obtained, for example, by combining the active ingredient with one or more solid carriers, granulating a resulting mixture, where appropriate, and processing the mixture or granules, if desired, where appropriate with the addition of additional excipients, to form tablets or dragee cores. It is also possible for them to be incorporated into plastics carriers that allow the active ingredients to diffuse or be released in measured amounts.

Suitable carriers are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalci- um phosphate or calcium hydrogen phosphate, and alsσ binders, such as starches, for example corn, wheat, rice or potato starch, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, such as the above-mentioned starches and also carboxymethyl starch, cross-linked polyvinylpyrrolidone, or alginic acid or a salt thereof, such as sodium alginate. Additional excipients are especially flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof.

Dragee cores can be provided with suitable, where appropriate enteric coatings, there being used inter alia concentrated sugar solutions, which may comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents or solvent mixtures or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropylmethyl- cellulose phthalate. Colourings or pigments may be added to the tablets or dragee coatings, for example for identification purposes or to indicate different doses of active ingredient.

Pharmaceutical compositions for oral administration are also hard gelatin capsules, and soft sealed capsules consisting of gelatin and a plasticiser, such as glycerol or sorbitol. The hard gelatin capsules may comprise the active ingredient in the form of granules, for example in admixture with fillers, such as corn starch, binders and/or glidants, such as talc or magnesium stearate, and, where appropriate, stabilisers. In soft capsules the active ingredient is preferably dissolved or suspended in suitable liquid excipients, such as fatty oils, paraffin oil or liquid polyethylene glycols or fatty acid esters of ethylene glycol or propylene glycol, it likewise being possible to add stabilisers and detergents, for example of the polyoxyethylene sorbitan fatty acid ester type.

Other oral dosage forms are, for example, syrups prepared in customary manner that comprise the active ingredient, for example, in suspended form and in a concentration of approximately from 5% to 20%, preferably approximately 10% or in a similar concentration that provides a suitable single dose when administered, for example, in a measure of 5 or 10ml. Also suitable are, for example, powdered or liquid concentrates for the preparation of shakes, for example in milk. Such concentrates may also be packed in single dose quantities.

For parenteral administration there are suitable, especially, aqueous solutions of an active ingredient in water-soluble form, for example in the form of a water-soluble salt, or aqueous injection suspensions that comprise viscosity-increasing substances, e.g. sodium carboxy- methylcellulose, sorbitol and/or dextran, and, if desired, stabilisers. The active ingredient, where appropriate together with excipients, can also be in the form of a lyophilisate and be made into a solution prior to parenteral administration by the addition of suitable solvents. Preferred preservatives are, for example, antioxidants, such as ascorbic acid, or micro bicides, such as sorbic acid or benzoic acid.

Other possible formulations include ointments (oil-in-water emulsions), fatty ointments, creams (oil-in-water emulsions that comprise more than 50% water), pastes (creams and ointments having secretion-absorbing powder constituents), nasal sprays and the like.

The invention relates also to a process or a method for the treatment of a disease or condition mentioned above, said method comprising administering a bufadienolide, especially a compound of the formula I, a tautomer thereof, or a pharmaceutically acceptable salt thereof, especially a preferred compound of the formula I, a tautomer thereof, or a pharmaceutically acceptable salt thereof, to a warm-blooded animal, especially a human, suffering from a condition as defined above, preferably as defined as being preferred, and especially being in need of such treatment, in a dose sufficient to cause immunomodulation, especially immunosuppression, or (in case of non-cardioactive bufadienolides) antitumor activity; especially in each case causing response against such condition. Response is especially meaning amelioration of the symptoms of the condition, most especially removal to near removal of the symptoms, and treatment may be palliative or curative. The compounds of formula I, tautomers thereof or pharmaceutically acceptable salts thereof may be administered prophy- lactically or therapeutically as such or in the form of pharmaceutical compositions, preferably in an amount that is effective against the mentioned diseases, to a warm-blooded animal, for example a human, requiring such treatment, preferably applying the compounds in the form of pharmaceutical compositions.

Examples Embodiments of the invention are described in the following specific examples which are not to be construed to limit the scope of the invention in any way, but serve merely for illustration. In mixtures, relations of parts of solvent or eluent or reagent mixtures in liquid form are given as volume relations (v/v), if not indicated otherwise. Described compounds can be isolated or synthesized according to methods outlined above, or they are bought from commercial suppliers (e.g. proscillaridin A from Sigma-Aldrich, St. Louis, USA).

The following table (table A) describes the formulae and numbers of the compounds useful in the present invention (compounds marked with an asterisk (*) are novel and they, tau- tomers thereof or salts thereof, as such are part of the invention). In the following Examples, "Compound No." refers to the compound numbers (Cpd. No.) mentioned in Table A.

Table A:

Formula A:

Formula B:

Formula C:

Formula D (non-cardioactive):

Formula E (non-cardioactive):

Formula F (non-cardioactive):

Formula G (non-cardioactive):

3β, 11 α-Diacetoxy-12-keto-14-hydroxy-20-keto-5β, 14β-pregnane-21 -azidolactone(21 → A) (Compound No. 10*) Formula H (non-cardioactive):

3β,11α-Diacetoxy-12-keto-5β,14β-aetianic acid-methylester (Compound No. 8*)

Abbreviations: Ac = acetyl, sub = suberoyl, Rha = rhamnosyl, Glc = Gucosyl, His = histidine. 65 and 70 are isolated from Helleborus species; compounds 4,5,6, 64, 89 and 90 are isolated from Urginea species. Compounds 2, 3, 11, 12, 13, 14, 15, 17, 22, 23, 24, 25, 26, 27, 29, 39, 40, 42, 43, 54, 55, 57, 61, 66, 69, 72, 73, 74, 75, 81, 92 and 93 are isolated from toads, with compounds 2, 12, 14, 15, 22, 23, 24, 25, 29, 39, 40, 42, 43, 57, 66, 74, 81 and 92 in addition being chemically slightly modified. 7, 19, 34, 35, 67, 68, 83, 91 and 100 are isolated from toad venom and partially chemically modified. 10 and 8 are isolated from toad venom and strongly modified.

In the following Examples, "Compound No." refers to the compound numbers (Cpd. No.) mentioned in Table A:

Example 1: Inhibition of Concanavalin A-stimulated T cells

Human peripheral blood mononuclear cells (PBMC) are stimulated with ConA [details see general description above under a) and b)] and increasing dilutions of test compounds of the formula I are concomitantly added to the cell cultures [details see general description above under c)]. The majority of substances inhibit ConA-induced T cell-activation, the most potent ones being active at concentrations of 3 pmol per 10⁵ cells (Table 1). In parallel cell culture experiments cortisol, cyclosporin A or tacrolimus (FK 506) suppress T cells at concentrations of 12500, 195.2 and 195.2 pmol, respectively. Positive control consists of Con-A-stimulated cells (24700 + 2543 cpm = 100 + 10.3 %), negative control of unstimulated cells (440 + 223 cpm = 1.8 + 0.9 %). Concentrations of test compounds at which complete suppression is found are given in the table as "Complete Suppression Concentration". In the last column of Table 1 , the relative suppressive activity ("Activity Factor") is indicated as follows: The weakest substances (active at > 50000 pmol/10⁵ cells) are denoted with "1", the strongest (active at 0.75 pmol/10⁵ cells) with "9". The Activity Factor reflects the degree of suppression and represents the log4 of successive dilution steps.

Table 1 : (CSC = Complete Suppression Concentration)

Example 2: Suppression of stimulated (preactivated) T cells

In this experiment [details see general description above under a) an d)] the question is addressed whether already activated T cells can be downregulated by bufadienolide test compounds. 24 h after activation by Con A, test compounds (781 pmol/10⁵ cells) are added to the cell cultures. As shown in Table 2, pre-activated T cells can be inhibited. ³[H]-Thymidine incorporation is measured after 24 h. Stimulated cells serve as positive, unstimulated cells as negative control. Values are expressed as percentage of positive controls.

Table 2: Suppression of stimulated T cells

Example 3: Suppression of phytohaemagglutinin- and alloantigen-stimulated T cells To ensure that the immunosuppressive effect is not limited to ConA-stimulation, 5 of the most active substances are tested in PHA- or alloantigen-stimulated cell cultures [details see general description under a), b) and c)]. Increasing amounts of bufotalin (No. 54), hellebrin (No. 65), proscillaridin A (No. 89), scillorosidin (No. 90) or telocinobufagin (No. 93) are added and ³[H]-thymidine incorporation is measured after 2 and 5 days, respectively. Stimulated cells in medium with or without DMSO serve as positive control (= 100 % stimulation) and unstimulated cells (1.5 %) as negative control. Values are expressed as percentage of positive control. As shown in Table 3, they suppress the T cells response at concentrations similar to those required in the ConA experiments.

Table 3:

3A) Stimulation by phytohaemagglutinin:

Example 4: Lack of restimulation of suppressed T cells

T cells are preincubated for 3, 12 or 24 h with bufadienolide test compounds (here hellebrin = No. 65 or proscillaridin = No. 89 only) only, washed and then stimulated with ConA (Table 4). For details, see general description above under a) and e). After restimulation for 24 h with ConA, ³[H]-thymidine incorporation is measured. ConA-stimulated cells serve as positive (= 100 % stimulation), unstimulated cells as negative control. Values represent cpm per 10^s viable cells and are expressed as percentage of positive control. In a second experiment, the cells are incubated with test compounds and ConA, washed and then stimulated with ConA (Table 5). The results show that 24 h preincubation with compounds of test compound renders the cells unresponsive. If the cells are concomitantly incubated with test compound and ConA, however, they become unresponsive already after 12 h.

Table 4: Restimulation of bufadienolide-treated T cells

Example 5: T cell viability upon treatment with compounds of the formula I For details, see general description under a) and "FACS analysis of T cell viability. The un- responsiveness upon preincubation with hellebrin (No. 65) or proscillaridin A (No. 89) described in Example 4 could have been caused by cell death. In order to exclude that possibility, only viable T cells as shown by vital staining and FACS analysis are considered for restimulation. Moreover, in the experiment of which the results are shown in Table 6, hellebrin and proscillaridin A are analysed with regard to their capacity to induce T cell death. After 3, 12 and 24 h preincubation with the respective compound of formula A or with the compound plus ConA, the T cells remain viable (less than 11 % dead cells), indicating that the lack of restimulation upon pre-treatment with hellebrin or proscillaridin in Example 4 is not due to cell death but to a state of unresponsiveness. Negative controls consist of pretreatment with medium or medium together with ConA, positive controls of treatment with 6 % ethanol.

Table 6: Viability of bufadienolide-treated T cells 6A) Bufadienolide only

6B) Bufadienolide plus ConA:

Example 6: Suppression of B-cell activity

B cells of patients with rheumatoid arthritis are collected from synovial fluid of the inflamed joint and immortalized by EBV transformation. It is known that such B cells produce high amounts of rheumatoid factors (IgM-anti-lgG antibody and related antibodies) which are involved in the pathogenesis of the disease. In one experiment the B ceils are kept overnight in cell culture medium without (i) or with (ii) addition of Proscillaridin A (= ProA) (3, 12, 48, 192 pmol/10⁵ cells, respectively). Then antibody production of the B cells is measured in a plaque-forming cell assay as described in the literature (Weimer et al., Transplantation 48, 569, 1989). Briefly, the B cells are included in agarose along with sheep red blood cells (SRBC) coated with an anti-immunoglobulin antibody and with rabbit complement. Antibody- secreting B cells destroy the neighbouring SRBC generating so-called "plaques". The number of plaques reflects the number of antibody-producing B cells. The number of plaques per 10⁵ B cells is in experiment (i) 149+38, in experiment (ii) 155 (at 3 pmol ProA), 149 (12 pmol ProA), 124 (48 pmol ProA), 30 (192 pmol ProA). The findings show that ProA is able to suppress the antibody production of B cells from the inflamed joint of a patient with rheumatoid arthritis.

Example 7: Killing of tumor cells with bufadienolide and novel non-cardiotoxic derivatives Human lymphoma cells (Jurkat cells, ATCC) are incubated at 37 °C in RPMI and 10 % Fetal calf serum for 14-24 h with bufadienolide (no. 65 above) or strongly modified bufadienolide derivatives (no^'s. 35, 83 above) at various concentrations. The compounds no. 35 and 83 do not present cardiotoxic activity. Control experiments consist of (a) lymphoma cells incubated with cortisol at the same (10μM, 100μM) - or (b) higher concentrations (100000 μM)(positive control), (c) normal lymphocytes incubated with bufadienolide, and (d) lymphoma cells incubated in culture medium only (negative control). The percentage of apoptotic cells is defined by detecting DNA fragmentation (Nicoletti test, see Nicoletti et al., J. Immunol. Methods 139, 271 (1991)) in FACScan. The results show (see Table 7) a significant percentage of apoptotic tumor cells following incubation with bufadienolide or the novel derivatives. Cortisol induces a significantly lower degree of apoptosis at the same concentrations. At 100000μM cortisol, however, a percentage of 66% apoptotic cells is noted (data not shown). Most importantly, normal human lymphocytes do not present a significant percentage of apoptosis under identical bufadienolide treatment as that used for tumor cells.

Table 7: Apoptosis of tumor cells induced by bufadienolides and derivatives

Example 8: Enhancement of apoptosis by increasing the incubation time Human lymphoma cells (see Example 7) are incubated under the conditions described in Example 7 with 10μM hellebrin (no. 65) for 14, 36, and 60 hours and apoptosis is measured as mentioned. Cortisol at the same concentration serves as control. The rate of apoptosis increases with incubation time (s. Table 8). At 100000 μM cortisol (positive control) 66% apoptotic cells are obtained after 36 h incubation.

Table 8: Apoptosis of tumor cells induced by bufadienolides is time dependent

Example 9: Sterile dry substance for i.m. or i.v. injection:

5 mg of one of the compounds of formula I mentioned in Table A, especially proscillaridin A, compound 35 or compound 83, as active ingredient are dissolved in 1 ml of an aqueous solution with 20 mg of mannitol and 20 % cyclodextrins as solubilisers. The solution is sterile- filtered and introduced under aseptic conditions into a 2 ml ampoule, deep-frozen and lyo- philised. Before use, the lyophilisate is dissolved in 1 ml of distilled water or 1 ml of a physiological saline solution.

Example 10: Film-coated tablets

The following ingredients are used for the preparation of 10000 tablets each containing 100 mg of active ingredient (one of the compounds mentioned in Table A, especially proscillaridin A, compound 35 or compound 83): active ingredient 1000 g; corn starch 680 g; colloidal silica 200 g; magnesium stearate 20 g; stearic acid 50 g; sodium carboxymethyl starch 250 g; water quantum satis . A mixture of the active ingredient, 50 g of corn starch and the colloidal silica is processed with a starch paste, made from 250 g of com starch and 2.2 kg of de- mineralised water, to form a moist mass. This is forced through a sieve having a mesh size of 3 mm and dried at 45° for 30min in a fluidised bed drier. The dry granules are pressed through a sieve having a mesh size of 1 mm, mixed with a pre-sieved mixture (1 mm sieve) of 330 g of corn starch, the magnesium stearate, the stearic acid and the sodium carboxymethyl starch, and compressed to form slightly biconvex tablets.

Claims

What is claimed is

1. The use of bufadienolide, a tautomer thereof, or a salt thereof, for the manufacture of a pharmaceutical preparation for immunomodulation, antiinflammatory and/or, in the case of non-cardioactive bufadienolides, antiproliferative action.

2. The use according to claim 1 , where the bufadienolide has the formula I,

wherein Ri is hydrogen, hydroxy, amino, substituted amino, amino-lower alkoxy, N-substituted amino-lower alkoxy, azido, halo, acyloxy or the radical of a mono- or oligosaccharide bonded (preferably O-glycosidically) via an oxygen atom to the rest of the molecule of formula I;

Ri* is hydrogen, hydroxy, amino, halo or acyloxy; or Ri and Ri* together are oxo (=O) or hydroxyimino (=NOH);

R₂ is hydrogen, hydroxy, halo, acyloxy or the radical of a monosaccharide bonded O- glycosidically via an oxygen atom to the rest of the molecule of formula I; any radical other than hydrogen being preferably in β-position;

R₂* is hydrogen and R₂** is hydrogen, hydroxy (preferably in β-position), halo or acyloxy (preferably β-acyloxy); or R₂* and R₂** together form oxo (=O); or R₂ and R₂** together with the bond between the carbon atoms that carry R₂ and R₂** form a double bond while R₂* is hydrogen; R₃ is lower alkyl, hydroxymethyl, acyloxymethyl, formyl (-C(=O)H), carboxy or hydrocarbyloxycarbonyl;

R₄ is hydrogen, hydroxy, halo or acyloxy (if other than hydrogen, preferably in β-position), and

R₅ is hydrogen or hydroxy (preferably in β-position),

R₄* and R₄** each is hydrogen; or

R₄ is acyloxy and R₄* and R₄** together with the adjacent bond connecting the carbon atoms that carry R₄* and R ** form a double bond; or R is hydrogen and R₅ and R₄** together form oxa in β-position ( — o_*— );

R₆ is hydrogen, α-hydroxy or α-acyloxy, and R₆* is hydrogen; or R₆ and R₆* together are oxo (=O); or or R₆ and R₃ together form a group of the formula (la),

wherein R* is hydrogen or methyl and where the oxygen atom is bound in the position of R₆; the carbon atom is bound in the position of R₃; R₇ is hydrogen or hydroxy in α-position);

R₈ is hydrogen or hydroxy in β-position, with the proviso that R₇ and R₈ are not simultaneously hydroxy ; or R₇ and R₈ together are oxo (=O);

R₉ is hydrogen or α-hydroxy or more preferably β-hydroxy and R₁₀ is hydrogen, or Rg and R₁₀ together with the bond between the carbon atoms that carry

R₉ and R10 form a double bond or oxa in β-position ( — « o— ), defining a non- cardioactive bufadienolide class, or Rg and R_i3 taken together form a bridge -C(=O)-C(=O)-O- where the O is bound in the position of R₉ and the left carbonyl is bound in the position of Rι₃, both preferably in the β- position, defining a non-cardioactive bufadienolide class;

R_ϋ is hydrogen, hydroxy, especially β-hydroxy, halo or acyloxy, especially β-acyloxy;

R₁₂ is methyl, especially β-methyl;

R₁₃ is a moiety of the formula lb,

or, thus defining a non-cardioactive class of bufadienolides,is carboxy or -COOR** wherein R** is hydrocarbyl; or hydroxymethyl; and each of Rι and R_i5 is hydrogen, or, if R₂** and R₂ together with the bond between the carbon atoms that carry R₂ and R₂** form a double bond and if Ri and Ri* together form oxo, Rι₄ and R₁₅ together with the bond between the carbon atoms that carry R₁₄ and R₁₅ form a double bond; a tautomer thereof, or a salt thereof, if a salt-forming group is present.

3. The immunosuppressive, antiinflammatory and/or antitumor use according to claim 2 of a compound of formula I wherein each of Ri, RΛR₂ , R₂ ^*, R₂ ^**, R₃, R₄, Rs, RΛ R ^**, Re, Re^*, R₇, R₈, Rn, R_i2, R₁₃, R₁₄ and Rι₅ has the meanings given in claim 2, and

R₉ and Rio form a double bond or preferably oxa in β-position ( -— O"— ); a tautomer thereof, or a salt thereof, if a salt-forming group is present.

4. The immunosuppressive and/or antiinflammatory use according to claim 2 of a compound of formula I wherein each of R_{1 f} RΛR₂ , R2^*, R₂ ^**, R3, R Re, R4*, R4*^*, Re, Re^*, R₇. Rs, Rg, R₁₀, R₁₁, Rι₂, R₁3, R₁₄ and R₁₅ has the meanings given in claim 2, and the rings C and D are present in cis junction; a tautomer thereof, or a salt thereof, if a salt-forming group is present.

5. The immunosuppressive and/or antiinflammatory use according to claim 2 of a compound of formula I wherein each of R,, RΛR₂ , R₂ ^*, R₂**, R₃, R₄, R₅, R₄ ^*, R₄*^*, R₆, R₆*, R₇. Rs, Rg, R₁0, R₁₁, Rι₂, R₁₄ and R₁₅ has the meanings given in claim 2, and R₁₃ is a moiety of the formula lb as shown above; a tautomer thereof, or a salt thereof, if a salt-forming group is present.

6. The immunosuppressive and/or antiinflammatory use according to claim 2 of a compound of formula I wherein each of R₂ , R₂ ^*. R₂ ^**, R_3f R₄, Rs, RΛ R₄ ^**, Re, Re^*, R7, Rs, Rg, R10, R11, Ri₂, R₁₃, R₁₄ and R₁5 has the meanings given in claim 2, and each of Ri and Ri^* is hydrogen, lower alkanoyloxy, especially acetyloxy, amino (especially preferred is Ri = amino), amino- lower alkoxy, especially β-aminoethyloxy, or the radical of a mono- or oligosaccharide bonded O-glycosidically via an oxygen atom to the rest of the molecule of formula I, especially Rha-, such as α-L-Rha, or Glc-Rha-, such as β-D-Glc-α-L-Rha-, each bonded O- glycosidically via an oxygen atom to the rest of the molecule of formula I; with the proviso that at least one of Ri and Ri* is hydrogen ; or Ri and Ri* together form oxo (=O); a tautomer thereof, or a salt thereof, if a salt-forming group is present.

7. The use according to any one of claims 3 to 6 of a compound of formula I wherein the moieties Ri, RΛ R₂, R₂ ^*', R₂ ^**, R3, R₄, R₄ ^*, R4^**, Rs, Re, Re^*, R₇, Re, R9, R10, R11, R12, R13, Rι and R₁₅ have the meanings defined in the respective claim and, if not defined otherwise, Ri is hydrogen, hydroxy, amino, azido, Rha-O-, Glc-Rha-O, lower alkanoyl(especially ace- tyl)oxy, carboxy-lower alkanoyl (especially suberoyl)oxy, O-suberoyl-metHis-O- or amino- lower alkoxy (especially β-aminoethyloxy);

Ri* is hydrogen, hydroxy , amino or. lower alkanoyl(especially acetyl)oxy; or Ri and Ri* together form =O or =NOH; R₂ is hydrogen or hydroxy; each of R₂* and R₂** is hydrogen; or R₂ and R₂** together with the bond between the carbon atoms that carry R₂ and R₂** form a double bond while R₂* is hydrogen; R₃ is lower alkyl, especially methyl, formyl or hydroxymethyl;

R₄ is hydrogen or lower alkanoyloxy, especially hydrogen or acetyloxy (especially in β- position);

R * and R ** each are hydrogen; R₅ is hydrogen;

R₆ is hydrogen, α-hydroxy or α-lower alkanoyl(especially acetyl)oxy and R₆* is hydrogen, or R₆ and R₆* together are oxo (=O); R₇ is hydrogen or α-hydroxy;

R_s is hydrogen or β-hydroxy, with the proviso that R₇ and R₈ are not simultaneously hydroxy; or R₇ and R₈ together are oxo (=O); R₉ is hydrogen or α-hydroxy or preferably β-hydroxy and R₁₀ is hydrogen, or R₉ and R₁₀ together with the bond between the carbon atoms that carry

R₉ and R™ form a double bond or oxa in β-position ( ^— o»— ), defining a non- cardioactice class of bufadienolides, or, defining non-cardioactive compounds, R₉ and R_i3 taken together form a bridge -C(=O)-

C(=O)-O- where the O is bound in the position of R₉ and the left carboxy is bound in the position of Rι₃, both preferably in the β-position;

RH is hydroxy, β-hydroxy or β-lower alkanoyl(especially acetyl)oxy;

Rι₂ is methyl, especially β-methyl;

Rι₃ is a moiety of the formula lb given above or (defining non-cardiotoxic compounds) is carboxy, lower alkoxycarbonyl or hydroxymethyl; and each of R and R_i5 is hydrogen, or, if R₂** and R₂ together with the bond between the carbon atoms that carry R₂ and R₂** form a double bond and if Ri and Ri* together form oxo, R ₄ and R₁₅ together with the bond between the carbon atoms that carry Rι₄ and R₁₅ form a double bond; a tautomer thereof, or a salt thereof, if a salt-forming group is present.

8. The use according to claim 2 of non-cardiotoxic bufadienolides, or salts thereof, especially wherein R_i3 is other than of the formula lb and the remaining radicals have the meanings given in claim 2, for immunosuppressive, antiinflammatory and/or (especially) antiproliferative action.

9. The use according to claim 8 of non-cardioactive bufadienolides of formula I, or salts thereof, wherein a) Rι₃ is carboxy or COOR**, wherein R* is hydrocarbyl; or is hydroxymethyl; and wherein each of Ri, RΛ R₂, R₂*, R₂**, R₃, R₄, R5, RΛ R₄ ^**, Re, R₇, Rs, R10, Rn, Rι₂, R-ι₄ and R15 has the meanings given above or below for compound of the formula I; or b) R₉ and Rι₃ taken together form a bridge -C(=O)-C-(=O)-O- where the O is bound in the position of R₉ and the left carboxy is bound in the position of Rι₃, both preferably in the β- position, and wherein each of Ri, RΛ R₂, R₂ ^*, R₂ ^**, R₃, R , R₅, RΛ R ^**, R₆, R₇, Rs, R10, R₁₁, Rι₂, R₁₄ and R₁₅ has the meanings given above or below for compound of the formula I; c) R₉ and R₁₀ together form a double bond or preferably oxa in β-position, while each of Ri, RΛ 2, R2*, R2^**, R3, R₄, Rs, R4*, R ^**, Re, R₇, Rs, Rn, R12, R13, R14 and R15 has the meanings given above or below for compound of the formula I; d) R₉ is hydrogen and each of R RΛ R₂, R R2^**, R3, R₄, Rs, R4^*, R₄ ^**, Re, R₇, Rs, R10, R₁₁, R₁₂, R₁₃, R₁₄ and R₁5 has the meanings given above or below for compound of the formula I; e) R₉ is hydrogen, rings C and D of the steroid ring system are annealed in cis-configurati- on, and Rι₃ is is carboxy or COOR^**, wherein R^* is hydrocarbyl; or is hydroxymethyl; and wherein each of R_{1 f} R R₂, R₂*, R₂**, R_3l R R₅, R₄*, R₄**, R₆, R₇, R_βl R₁₀₎ R_{11 (} R₁₂, R₁₃, Rι and R_i5 has the meanings given above or below for compound of the formula I; or f) Rg and Rι₀ together form a double bond or preferably oxa in β-position and Rι₃ is carboxy or COOR**, wherein R* is hydrocarbyl; or is hydroxymethyl; and wherein each of R-i, RΛ R₂, R2*, R2 *, R3, R₄> Rs, R *, R₄ ^* , Re, R₇, Rs, R-11, R12, Rι₄ and R15 has the meanings given above or below for compound of the formula I; or of salts thereof where salt-forming groups are present.

10. The use of a bufadienolide according to any one of claims 1 to 9 for the manufacture of a pharmaceutical preparation for the therapy or prophylaxis of conditions in warm-blooded animals that are responsive to immunomodulation, especially immunosuppression; antiinflammatory action; and/or (as far as non-cardioactive bufadienolides are concerned) antitumor action; one or more diseases or conditions selected from the group consisting of transplant rejection; autoimmune disease; rheumatic diseases, especially those with immunological and/or inflammatory components; allergy; postoperative and septic shock; sepsis, or, in the case of tumor diseases, especially mammary carcinoma, head and neck cancer, tumors of lungs and mediastinum, urogenitary tumors, tumors of the gastrointestinal tract, oto-rhino- laryngologic tumors, tumors of endocrine organs, sarcomas, skin tumors, tumors of the central nervous system, or especially lymphomas or leukemias.

11. A compound of the formula I given in claim 1 , wherein a) Rι₃ is carboxy or COOR**, wherein R* is hydrocarbyl; or is hydroxymethyl; and wherein each of Ri, RΛ R₂, R₂*, R₂ ^**, R3, R₄, Rs, R₄ ^*, R4^**, Re, R₇, Rs, R10, R11, R12, R14 and R15 has the meanings given above or below for compound of the formula I in any one of claims 2 to 7; or b) R₉ and R₁₃ taken together form a bridge -C(=O)-C-(=O)-O- where the O is bound in the position of Rg and the left carboxy is bound in the position of Rι₃, both preferably in the β- position, and wherein each of Ri, RΛ R₂, RΛ R₂*^*, R3, R4, Rs, R4^*, R4^**, Re, R₇, Rs, R10, R₁₁, Rι₂, R₁₄ and R ₅ has the meanings given above or below for compound of the formula I in any one of claims 2 to 7; c) R₉ and Rι₀ together form a double bond or preferably oxa in β-position, while each of Ri, RΛ R∑, R2*, R₂**> R3, R4, Rs, R4^*, R₄ ^**, e, R₇, Rs, R11, Rι₂, R13, Rι₄ and R15 has the meanings given above or below for compound of the formula I; d) R₉ is hydrogen and each of R_{1 (} RΛ R₂. R2*, R₂ ^**, R₃, R Rs, R₄*, R₄ ^**, Re, R₇, Rs, R10, R₁₁, R₁₂, R₁₃, R₁₄ and R₁5 has the meanings given above or below for compound of the formula I; e) R₉ is hydrogen, rings C and D of the steroid ring system are annealed in cis-configurati- on, and Rι₃ is is carboxy or COOR**, wherein R* is hydrocarbyl; or is hydroxymethyl; and wherein each of Ri, RΛ R₂. R₂ ^*. R₂**, R3, R Rs, R₄ ^*, R₄*^*, Re, R₇, Re, R10, Rn, R12, R13, R_i4 and R₁₅ has the meanings given above or below for compound of the formula I; or f) R₉ and R₁₀ together form a double bond or preferably oxa in β-position and Rι₃ is carboxy or COOR**, wherein R* is hydrocarbyl; or is hydroxymethyl; and wherein each of Ri, RΛ R₂, R₂ ^*, R₂ ^**, R₃, R₄, Rs, RΛ R₄ ^**, Re, R₇, Rs, R11. Rι₂, Rι₄ and R15 has the meanings given above or below for compound of the formula I; or salts thereof where salt-forming groups are present, except for Resibufogenin, Resibufogenin-O-hemisuberate and Mannobufagin.

12. A compound of formula I given in claim 2 selected from the group consisting of areno- bufagin-3,11-diacetate, bufalon, bufalonoxim, 3α-aminobufaIin, 3β-aminobufaIin, 3β-azido- bufalin, bufotalin-acetate, gamabufotalin-diacetate, telocinobufagin-3-O-acetate, 3-epi-ci- nobufagin-3-O-acetate, resibufogenin-3-O-formiate, 3α-azido-cinobufagin, 3β-aminoetha- nolo-cinobufagin, 3β-aminocinobufagin, 3-keto-cinobufagin, marinobufagin-3-O-acetate, 3β- azido-resibufagenin, 3β,11α-diacetoxy14β-hydroxy-12-ketoaetianic acid, 3β-acetoxy-14β,20- dihydroxynorpregnane, 3β,14β,20-trihydroxynorpregnane, 3β,11α-diacetoxy-14β-hydroxy- aethianic acid methylester, 3β,14β-dihydroxyaeticanic acid-methyl ester, 3β-acetoxy-5β,14β- dihydroxyaetianic acid-methylester, 3β-hydroxy-Δ^5,δ-aetianic acid, 3β,16β-diacetoxy-14β,15β- epoxy-aetianic acidmethylester, 3β-hydroxy-14β,15β-epoxy-aetianic acid-methylester, 3β- acetoxy-14β,15β-epoxy-aetianic acid, 3β,11α-diacetoxy-12-keto-14-hydroxy-20-keto-5β,14β- pregnane-21-azidolactone(21→14) and 3β,11α-diacetoxy-12-keto-5β,14β-aetianic acid- methylester, a tautomer thereof, or a salt thereof.

13. A compound of formula I according to any one of claims 11 and 12 for use in the diagnostic or therapeutic treatment of a warm-blooded animal, especially a human.

14. A pharmaceutical composition for immunomodulation, antiinflammatory action and/or (in the case of non-cardioactive bufadienolides) antitumor action in a warm-blooded animal, said composition comprising a compound of formula I according to any one of claims 11 and 12 and a pharmaceutically acceptable carrier material.

15. The use of a compound, a tautomer thereof, or a salt thereof, according to any one of claims 11 and 12 for the manufacture of a pharmaceutical composition for immunomodulation, antiinflammatory and/or antitumor action.