The present invention relates to a pharmaceutical preparation comprising as an active ingredient a primary amide and/or a secondary amide. In particular, the present invention relates to the use of derivatives of primary amides and/or secondary amides for the treatment of autoimmune diseases, tumor diseases and/or for immunosuppression.

Cytohesins are small guanine nucleotide exchange factors (GEFs) that stimulate ADP ribosylation factors, ubiquitously expressed Ras-like GTPases, which control various cellular regulatory networks. Members of the cytohesin family include cytohesin- 1, cytohesin-2 (ARNO), cytohesin-3, also known as Grp-1 in humans and Steppke in Drosophila, and cytohesin-4. All four currently known cytohesins share a conserved multi-domain structure, i.e. a Sec7-domain harbouring the GEF activity, a pleckstrin-homology domain, and a coiled-coil domain.

Although small molecular probes that effectively interfere with different cytohesin functions are highly desirable, only very few effective inhibitory chemotypes have thus far been described, as described in German patent application DE 10 2004 055 998. However, these inhibitors show only minor effectiveness, the most effective inhibitory chemotype being a pan-active 1 -, 2-, 4-substituted triazole, which targets the Sec7 domain of cytohesins- 1, -2, and -3, and inhibits their GEF activity and cytohesin-associated functions including insulin signalling. Further, it would be desirable to have inhibitors showing selectivity in the interference with cytohesin functions.

Therefore, the objective underlying the present invention was to provide novel compounds being usable as inhibitors of the GEFs of the cytohesin family in pharmacological treatments. The problem is solved by a pharmaceutical preparation comprising as an active ingredient a primary amide according to the general formula (1) and/or a secondary amide according to the general formula (2) as indicated below and/or racemates, enantiomers, diastereomers, solvates, hydrates, and pharmaceutically acceptable salts and/or esters thereof:

wherein:

A is selected from the group comprising linear or branched Ci-Cio-alkyl, linear or branched C ₂ -Cio-alkenyl and/or structural elements (Al), (A2), (A3), (A4), (A5), (A6), (A7), (A8), (A9) and/or (AlO) as given as follows:

(A6) (A7) (A8)

is selected from the group comprising structural elements (Bl), (B2), (B3), (B4), (B5), (B6), (B7), (B8), (B9), (BlO), (BI l), (B12) and/or (B13) as given as follows:

(B5) (B6)

is selected from the group comprising structural elements (Dl), (D2), (D3), (D4) and/or (D5) as given as follows:

(Dl) (D2)

is selected from the group comprising linear or branched Ci-Cio-alkyl, linear or branched C ₂ -Cio-alkenyl and/or structural elements (El) or (E2) as given as follows:

is selected from the group comprising linear or branched Ci-Cio-alkyl, linear or branched C ₂ -Cio-alkenyl and/or structural elements (Gl), (G2) or (G3) as given as follows:

or

E and G together with the nitrogen atom to which they are attached form a 5- to 7- membered aromatic or non aromatic heterocycle selected from the group comprising structural elements (EGl) or (EG2) as given as follows:

or

D and G together with the nitrogen atom to which they are attached form a 5- to 7- membered aromatic or non aromatic heterocycle (DGl) as given as follows:

R ¹ is selected, the same or each independently of the others, from the group comprising

hydrogen, linear or branched Ci-Cio-alkyl and/or linear or branched C ₂ -Cio-alkenyl;

R ² is selected, the same or each independently of the others, from the group comprising linear or branched Ci-Cio-alkyl and/or linear or branched C ₂ -Cio-alkenyl;

R ³ is selected, the same or each independently of the others, from the group comprising linear or branched Ci-Cio-alkyl and/or linear or branched CpCio-alkoxy;

R ⁴ is selected, the same or each independently of the others, from the group comprising

hydrogen and/or halogen, preferably selected from the group comprising Cl, Br, F;

R ⁵ is selected, the same or each independently of the others, from the group comprising

hydrogen and/or linear or branched Ci-Cio-alkoxy.

It was surprisingly found that the primary amides according to the general formula (1) and the secondary amides according to the general formula (2) exhibit a stronger inhibition of the guanine nucleotide exchange factors of the family of cytohesins, show a higher ability to interfere with cytohesin-dependent insulin signalling and/or exhibit a better effectiveness to block cytohesin- mediated cell adhesion of human leukocytes compared to the pan-active 1-, 2-, 4-substituted triazole known in the state of the art.

The term "alkyl" according to the invention is to be understood as meaning straight-chain or branched alkyl groups. The term "Ci-Cio-alkyl" as used herein refers to straight-chain or branched alkyl groups having 1 to 10 carbon atoms. Preferred Ci-Cio-alkyl groups are selected from the group comprising methyl, ethyl and the isomers of propyl, butyl, pentyl, hexyl, heptyl or octyl, such as, for example, isopropyl, isobutyl, tert. -butyl, sec-butyl and/or isopentyl. Especially preferred are Ci-C ₅ -alkyl groups selected from the group comprising methyl, ethyl, isopropyl and/or tert. -butyl.

The term "alkenyl" according to the invention is to be understood as meaning straight-chain or branched alkyl groups having at least one or several double bonds. The term "C ₂ -Cio-alkenyl" as used herein refers to straight-chain or branched alkenyl groups having 2 to 10 carbon atoms and at least one or several double bonds. A preferred C ₂ -Ci ₀ -alkenyl is propen-1-yl.

The term "alkoxy" according to the invention is to be understood as meaning an alkyl group connected to the oxy connecting atom unless specifically stated otherwise. The term "Ci-Cio-alkoxy" as used herein refers to an alkyloxy group having 1 to 10 carbon atoms. CpCio-alkyloxy group are preferably selected from the group comprising methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, secondary-butoxy and/or tertiary-butoxy. Especially preferred CpCio-alkoxy groups are selected from the group comprising methoxy, ethoxy and/or isopropoxy.

The term "halogen" according to the invention is to be understood as meaning fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.

In preferred embodiments, the pharmaceutical preparation comprises primary amides according to the general formula (1), wherein the structural element B is selected from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9), (BlO) and/or (B 13), more preferred from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9) and/or (BlO). In preferred embodiments, the pharmaceutical preparation comprises primary amides according to the general formula (1), wherein the structural element B is selected from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9), (BlO) and/or (B13) and the structural element A is (Al). In more preferred embodiments, the pharmaceutical preparation comprises primary amides according to the general formula (1), wherein the structural element B is selected from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9) and/or (BlO) and the structural element A is (Al). Further preferred is a pharmaceutical preparation comprising primary amides according to the general formula (1), wherein a combination of the structural element (B 13) and the structural element (Al) is excluded.

In preferred embodiments, the pharmaceutical preparation comprises primary amides according to the general formula (1), wherein the structural element A is (Al) and the structural element B is selected from the group comprising (Bl), (B2), (B3) and/or (B4).

Advantageously, the primary amides according to the general formula (1), wherein the structural element A is (Al) and the structural element B is selected from the group comprising (Bl), (B2), (B3) and/or (B4) showed to be potent inhibitors in nucleotide exchange assays and blocking of cytohesin- mediated cell adhesion of human leukocytes.

In more preferred embodiments, the pharmaceutical preparation comprises primary amides selected from the group comprising the general formulas (3) and/or (4) as indicated below and/or racemates, enantiomers, diastereomers, solvates, hydrates, and pharmaceutically acceptable salts and/or esters thereof:

wherein: R ¹ is selected, the same or each independently of the others, from the group comprising hydrogen, linear or branched CpCio-alkyl and/or linear or branched C ₂ -Cio-alkenyl.

Preferably, the substituent R ¹ of the primary amides according to the general formulas (3) and/or (4) is hydrogen or a Ci-C ₅ -alkyl group selected from the group comprising methyl, ethyl, isopropyl and/or tert. -butyl. Most preferably, substituent R ¹ of the primary amides according to the general formulas (3) and/or (4) is hydrogen or tert. -butyl.

Advantageously, the primary amides according to the general formulas (3) and (4) can exhibit a good inhibition of the guanine nucleotide exchange factors of the family of cytohesins, a high ability to interfere with cytohesin-dependent insulin signalling, and a good effectiveness to block cytohesin- mediated cell adhesion of human leukocytes.

It was surprisingly found that the primary amides according to the general formulas (3) and (4) can have a significantly improved effect if the substituent R ¹ is a smaller alkyl group or hydrogen. Thus it was surprisingly determined that a significant increase in efficacy in the inhibitory effects of the primary amide according to the general formula (3) can be achieved when the substituent R ¹ is hydrogen.

In very especially preferred embodiments, the pharmaceutical preparation comprises primary amides selected from the group comprising the general formulas (5) and/or (6) as indicated below and/or racemates, enantiomers, diastereomers, solvates, hydrates, and pharmaceutically acceptable salts and/or esters thereof:

Advantageously, the primary amides according to the general formulas (5) and (6) can exhibit a stronger inhibition of the guanine nucleotide exchange factors of the family of cytohesins, a higher ability to interfere with cytohesin-dependent insulin signalling, and a better effectiveness to block cytohesin-mediated cell adhesion of human leukocytes compared to the pan-active 1-, 2-, 4-substituted triazole known in the state of the art.

In other preferred embodiments, the pharmaceutical preparation comprises primary amides according to the general formula (1), wherein the structural element A is selected from the group comprising branched CpCio-alkyl groups, (A2) and/or (A3), preferably branched CpCio-alkyl groups or (A3), and the structural element B is selected from the group comprising (B5) and/or (B6).

Advantageously, the primary amides according to the general formula (1), wherein the structural element A is selected from the group comprising branched Ci-Cio-alkyl groups and/or (A3), and the structural element B is selected from the group comprising (B5) and/or (B6) can exhibit a selective activity in down-regulating insulin signaling.

In other preferred embodiments, the pharmaceutical preparation comprises primary amides according to the general formula (1), wherein the structural element A is (A4), and the structural element B is

(B7).

Advantageously, the primary amides according to the general formula (1), wherein the structural element A is (A4), and the structural element B is (B7) can exhibit a selective activity in inhibiting cytohesin-mediated cell adhesion of human leukocytes and/or inhibition of the guanine nucleotide exchange factors of the family of cytohesins.

In other preferred embodiments, the pharmaceutical preparation comprises primary amides according to the general formula (1), wherein the structural element A is (A5), and the structural element B is selected from the group comprising (B8) and/or (BlO).

In other preferred embodiments, the pharmaceutical preparation comprises primary amides according to the general formula (1), wherein the structural element A is selected from the group comprising (A6), (A7) and/or (AlO), and the structural element B is (B9). In other preferred embodiments, the pharmaceutical preparation comprises primary and/or secondary amides selected from the group comprising compounds (7) to (30) as indicated below and/or racemates, enantiomers, diastereomers, solvates, hydrates, and pharmaceutically acceptable salts and/or esters thereof:

The primary and secondary amides according to the general formulas (1) to (30), especially according to the general formulas (5) to (30) can be synthesized according to standard chemical methods.

The compound according to formula (5) is denoted according to the IUPAC (International Union of Pure and Applied Chemistry) nomenclature 4-tert-butyl-N-[4-[[(E)-3-furan-2-ylprop-2- enoyl]amino]phenyl]benzamide, the compound according to formula (19) is denoted N-(5-tert-butyl-2- phenylpyrazol-3-yl)-2-[[5-[(4-methoxyphenyl)amino]-l, and the compound according to formula (20) is denoted 2-(4-oxo-5-phenyl-3-prop-2-enylthieno[2,3-d]pyrimidin-2-yl)s ulfanyl-N-(5-phenyl-l, 2,4- thiadiazol-3 -yl)acetamide, respectively.

Advantageously, the primary and secondary amides according to the general formulas (5) and (6) and (7) to (30) can exhibit a selective activity. The primary amides according to the general formulas (20), (5), and (6) showed to be potent inhibitors in nucleotide exchange essays, wherein the primary amide according to the general formula (20) showed to have a dual activity in cell adhesion and nucleotide exchange assays. The primary amides according to the general formulas (11) and (29) exhibited a dual activity in cell adhesion and nucleotide exchange assays.

Very advantageously, the primary amides according to the general formulas (8), (9), and (10) were selectively active in down-regulating insulin signalling, the primary amides according to the general formula (12) showed a selective inhibition of cell adhesion.

Further advantageously, the amides according to the general formulas (25), (12), (26), and (30) were identified being strong cell adhesion inhibitors, wherein in particular the amides according to the general formulas (25), and (30), were found to be strong inhibitors of cytohesin-mediated cell adhesion. This allows thus for further extending the potential to analyze cytohesin functions using small molecules.

A further aspect of the present invention relates to primary amides according to the general formula (1) and/or a secondary amides according to the general formula (2) as indicated above and/or racemates, enantiomers, diastereomers, solvates, hydrates, pharmaceutically acceptable salts and/or esters thereof for use as a medicament.

Preferred are primary amides according to the general formula (1), wherein the structural element B is selected from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9), (BlO) and/or (B13), more preferred from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9) and/or (BlO). More preferred are primary amides according to the general formula (1), wherein the structural element B is selected from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9), (BlO) and/or (B 13) and the structural element A is (Al). In more preferred embodiments, the pharmaceutical preparation comprises primary amides according to the general formula (1), wherein the structural element B is selected from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9) and/or (BlO) and the structural element A is (Al). Further preferred is a pharmaceutical preparation comprising primary amides according to the general formula (1), wherein a combination of the structural element (B 13) and the structural element (Al) is excluded.

Preferred are the primary amides according to the general formula (1), wherein the structural element A is (Al) and the structural element B is selected from the group comprising (Bl), (B2), (B3) and/or (B4).

Preferably, primary amides according to the general formula (1), wherein the structural element A is selected from the group comprising branched Ci-Cio-alkyl groups, (A2) and/or (A3), preferably branched CpCio-alkyl groups or (A3), and the structural element B is selected from the group comprising (B5) and/or (B6), and/or primary amides according to the general formula (1), wherein the structural element A is (A4), and the structural element B is (B7), and/or primary amides according to the general formula (1), wherein the structural element A is (A5), and the structural element B is selected from the group comprising (B 8) and/or (BlO), and/or primary amides according to the general formula (1), wherein the structural element A is selected from the group comprising (A6), (A7) and/or (AlO), and the structural element B is (B9) and/or racemates, enantiomers, diastereomers, solvates, hydrates, and pharmaceutically acceptable salts and/or esters thereof are usable as a medicament.

Preferably, amides selected from the group comprising primary amides according to the general formulas (3) and/or (4), especially formulas (5) and/or (6), primary and/or secondary amides selected from the group comprising amides according to the formulas (7) to (30) and/or racemates, enantiomers, diastereomers, solvates, hydrates, pharmaceutically acceptable salts and/or esters thereof are usable as a medicament. Preferably, the substituent R ¹ of the primary amides according to the general formulas (3) and/or (4) is hydrogen or a Ci-C ₅ -alkyl group selected from the group comprising methyl, ethyl, isopropyl and/or tert. -butyl. Most preferably, substituent R ¹ of the primary amides according to the general formulas (3) and/or (4) is hydrogen or tert. -butyl.

Another aspect of the present invention relates to the primary amides according to the general formula (1) and/or a secondary amides according to the general formula (2) as indicated above and/or racemates, enantiomers, diastereomers, solvates, hydrates, and pharmaceutically acceptable salts and/or esters thereof for use in the therapeutic and/or prophylactic treatment of a disease selected from the group comprising autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, Crohn's disease, allergies, mastocytosis, tumor diseases such as lung or bronchial cancer, colon, gastrointestinal, pancreatic or rectal cancer, prostate cancer, lymphatic cancer or leukemia, bladder or kidney cancer, breast and/or ovarian cancer, diseases and pathological conditions that are linked to a hyperfunction of cytohesin dependent receptor tyrosine kinases such as the ErbB receptors and other growth factor receptors selected from the group comprising developmental syndromes such as craniosynostosis, dwarfism, Hirschsprung disease, multiple endocrine neoplasia, vascular

dysmorphogenesis and/or disorders of the lymphatic system, diseases and pathological conditions that are linked to a regulation of the insulin and/or insulin- like growth factor (IGF) signalling pathway selected from the group comprising obesity, cell aging, age-related cell damage, especially in the liver and/or the pancreas, age-related pathological conditions of liver and/or pancreatic cells, age-related functional disorders in the liver and/or pancreas, cell stress, especially oxidative stress, especially stress induced as a result of increased sugar metabolization, and/or apoptosis, especially β-cell apoptosis, for immunosuppression, for example, in cases of organ transplants, and/or for inhibition of angiogenesis.

Preferred are primary amides according to the general formula (1), wherein the structural element B is selected from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9), (BlO) and/or (B13), more preferred from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9) and/or (BlO).

More preferred are primary amides according to the general formula (1), wherein the structural element B is selected from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9), (BlO) and/or (B 13) and the structural element A is (Al). In more preferred embodiments, the pharmaceutical preparation comprises primary amides according to the general formula (1), wherein the structural element B is selected from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9) and/or (BlO) and the structural element A is (Al). Further preferred is a pharmaceutical preparation comprising primary amides according to the general formula (1), wherein a combination of the structural element (B 13) and the structural element (Al) is excluded.

Preferred are the primary amides according to the general formula (1), wherein the structural element A is (Al) and the structural element B is selected from the group comprising (Bl), (B2), (B3) and/or (B4).

Preferably, primary amides according to the general formula (1), wherein the structural element A is selected from the group comprising branched CpCio-alkyl groups, (A2) and/or (A3), preferably branched CpCio-alkyl groups or (A3), and the structural element B is selected from the group comprising (B5) and/or (B6), and/or primary amides according to the general formula (1), wherein the structural element A is (A4), and the structural element B is (B7), and/or primary amides according to the general formula (1), wherein the structural element A is (A5), and the structural element B is selected from the group comprising (B8) and/or (BlO), and/or primary amides according to the general formula (1), wherein the structural element A is selected from the group comprising (A6), (A7) and/or (AlO), and the structural element B is (B9) and/or racemates, enantiomers, diastereomers, solvates, hydrates, and pharmaceutically acceptable salts and/or esters thereof are usable for the therapeutic and/or prophylactic treatment of the aforementioned diseases.

In preferred embodiments, amides selected from the group comprising primary amides according to the general formulas (3) and/or (4), especially formulas (5) and/or (6), primary and/or secondary amides selected from the group comprising amides according to the formulas (7) to (30) and/or racemates, enantiomers, diastereomers, solvates, hydrates, and pharmaceutically acceptable salts and/or esters thereof are usable for the therapeutic and/or prophylactic treatment of the aforementioned diseases. Preferably, the substituent R ¹ of the primary amides according to the general formulas (3) and/or (4) is hydrogen or a Ci-C ₅ -alkyl group selected from the group comprising methyl, ethyl, isopropyl and/or tert. -butyl. Most preferably, substituent R ¹ of the primary amides according to the general formulas (3) and/or (4) is hydrogen or tert. -butyl.

Preferred diseases are selected from the group comprising autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, Crohn's disease, allergies, mastocytosis, tumor diseases such as lung or bronchial cancer, colon, gastrointestinal, pancreatic or rectal cancer, prostate cancer, lymphatic cancer or leukemia, bladder or kidney cancer, breast and/or ovarian cancer, diseases and pathological conditions that are linked to a hyperfunction of cytohesin dependent receptor tyrosine kinases such as the ErbB receptors and other growth factor receptors selected from the group comprising

developmental syndromes such as craniosynostosis, dwarfism, Hirschsprung disease, multiple endocrine neoplasia, vascular dysmorphogenesis or disorders of the lymphatic system and/or obesity.

More preferred diseases are tumor diseases selected from the group comprising lung or bronchial cancer, colon, gastrointestinal, pancreatic or rectal cancer, prostate cancer, lymphatic cancer or leukemia, bladder or kidney cancer, breast and/or ovarian cancer.

Surprisingly it was found that the primary amides according to the general formula (1) and/or secondary amides according to the general formula (2) can be effective against cancer cells. Especially the primary amide according to formula (5) was found to effectively inhibit the growth of cancer cells.

Without being bound to a special theory, it is assumed that it is essential for the effectivity against cancer cells that the primary amides according to the general formula (1) and/or secondary amides according to the general formula (2) inhibit a class of proteins called cytohesins which are supposed to have a regulatory function on the family of ErbB receptors. Particularly, it was found that inhibitors of the family of proteins called cytohesins inhibit the autophosphorylation and function of the epidermal growth factor receptor.

Epidermal growth factor receptors are key regulators of cell differentiation, proliferation and migration. The epidermal growth factor receptor is a transmembrane glycoprotein whose activation or autophosphorylation triggers a signalling cascade correlating with increased proliferation of tumour cells. Anticancer drags which are called epidermal growth factor receptor (EGFR) kinase inhibitors affect the epidermal growth factor receptor and are a new and potent alternative to chemotherapy. Epidermal growth factor receptor inhibitors therefore have become an important class of drags used in cancer chemotherapy.

In also very preferred embodiments, the primary amides according to the general formula (1) and/or a secondary amides according to the general formula (2) are usable for immunosuppression, for example, in cases of organ transplants.

The present invention also relates to the use of primary amides according to the general formula (1) and/or secondary amides according to the general formula (2) as indicated above and/or racemates, enantiomers, diastereomers, solvates, hydrates, and pharmaceutically acceptable salts and/or esters thereof for the manufacture of a medicament.

Preferred are primary amides according to the general formula (1), wherein the structural element B is selected from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9), (BlO) and/or (B13), more preferred from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9) and/or (BlO).

More preferred are primary amides according to the general formula (1), wherein the structural element B is selected from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9), (BlO) and/or (B 13) and the structural element A is (Al). In more preferred embodiments, the pharmaceutical preparation comprises primary amides according to the general formula (1), wherein the structural element B is selected from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9) and/or (BlO) and the structural element A is (Al). Further preferred is a pharmaceutical preparation comprising primary amides according to the general formula (1), wherein a combination of the structural element (B 13) and the structural element (Al) is excluded.

Preferred are the primary amides according to the general formula (1), wherein the structural element A is (Al) and the structural element B is selected from the group comprising (Bl), (B2), (B3) and/or (B4). Preferably, primary amides according to the general formula (1), wherein the structural element A is selected from the group comprising branched CpCio-alkyl groups, (A2) and/or (A3), preferably branched CpCio-alkyl groups or (A3), and the structural element B is selected from the group comprising (B5) and/or (B6), and/or primary amides according to the general formula (1), wherein the structural element A is (A4), and the structural element B is (B7), and/or primary amides according to the general formula (1), wherein the structural element A is (A5), and the structural element B is selected from the group comprising (B 8) and/or (BlO), and/or primary amides according to the general formula (1), wherein the structural element A is selected from the group comprising (A6), (A7) and/or (AlO), and the structural element B is (B9) and/or racemates, enantiomers, diastereomers, solvates, hydrates, and pharmaceutically acceptable salts and/or esters thereof are usable for the manufacture of a medicament.

In preferred embodiments, the present invention relates to the use of amides selected from the group comprising primary amides according to the general formulas (3) and/or (4), especially formulas (5) and/or (6), primary and/or secondary amides selected from the group comprising amides according to the formulas (7) to (30) and/or racemates, enantiomers, diastereomers, solvates, hydrates, and pharmaceutically acceptable salts and/or esters thereof for the manufacture of a medicament.

Preferably, the substituent R ¹ of the primary amides according to the general formulas (3) and/or (4) is hydrogen or a Ci-C ₅ -alkyl group selected from the group comprising methyl, ethyl, isopropyl and/or tert. -butyl. Most preferably, substituent R ¹ of the primary amides according to the general formulas (3) and/or (4) is hydrogen or tert. -butyl.

A further aspect of the present invention relates to the use of primary amides according to the general formula (1) and/or a secondary amides according to the general formula (2) as indicated above and/or racemates, enantiomers, diastereomers, solvates, hydrates, and pharmaceutically acceptable salts and/or esters thereof for the manufacture of a pharmaceutical preparation for the therapeutic and/or preventive treatment of a disease selected from the group comprising autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, Crohn's disease, allergies, mastocytosis, tumor diseases such as lung or bronchial cancer, colon, gastrointestinal, pancreatic or rectal cancer, prostate cancer, lymphatic cancer or leukemia, bladder or kidney cancer, breast and/or ovarian cancer, diseases and pathological conditions that are linked to a hyperfunction of cytohesin dependent receptor tyrosine kinases such as the ErbB receptors and other growth factor receptors selected from the group comprising developmental syndromes such as craniosynostosis, dwarfism, Hirschsprung disease, multiple endocrine neoplasia, vascular dysmorphogenesis and/or disorders of the lymphatic system, diseases and pathological conditions that are linked to a regulation of the insulin and/or insulin- like growth factor (IGF) signalling pathway selected from the group comprising obesity, cell aging, age- related cell damage, especially in the liver and/or the pancreas, age-related pathological conditions of liver and/or pancreatic cells, age-related functional disorders in the liver and/or pancreas, cell stress, especially oxidative stress, especially stress induced as a result of increased sugar metabolization, and/or apoptosis, especially β-cell apoptosis, for immunosuppression, for example, in cases of organ transplants, and/or for inhibition of angiogenesis.

The term "prophylactic treatment" of a disease according to the invention is to be understood as meaning that the compositions according to the invention can be applied before symptoms of the disease are manifest. Especially, the term "prophylactic treatment" of a disease is to be understood as meaning a medical treatment.

One particular advantage of the amides that can be used according to the invention is that the amides can especially also be used in preventive applications. This makes it possible to use the amides not only to treat existing pathological conditions, but also for preventive applications, for example to prevent age-related cell damage or obesity. Of particular advantage is the fact that, for example, obesity can be avoided through preventive treatment.

Advantageously, the primary and/or secondary amides according to the general formulas (1) and (2) can be especially useful for the treatment of diseases for which an inhibitor of the GEFs of the cytohesin family is indicated, and especially diseases and pathological conditions that are linked to a hyperfunction of cytohesin dependent receptor tyrosine kinases such as the ErbB receptors and other growth factor receptors selected from the group comprising tumor diseases such as lung or bronchial cancer, colon, gastrointestinal, pancreatic or rectal cancer, prostate cancer, lymphatic cancer or leukemia, bladder or kidney cancer, breast and/or ovarian cancer, and/or developmental syndromes such as craniosynostosis, dwarfism, Hirschsprung disease, multiple endocrine neoplasia, vascular dysmorphogenesis and/or disorders of the lymphatic system.

More preferred diseases are tumor diseases selected from the group comprising lung or bronchial cancer, colon, gastrointestinal, pancreatic or rectal cancer, prostate cancer, lymphatic cancer or leukemia, bladder or kidney cancer, breast and/or ovarian cancer. Surprisingly it was found that the primary amides according to the general formula (1) and/or secondary amides according to the general formula (2) can be effective against cancer cells. Especially the primary amide according to formula (5) was found to be effective against the growth of cancer cells.

Preferred are primary amides according to the general formula (1), wherein the structural element B is selected from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9), (BlO) and/or (B13), more preferred from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9) and/or (BlO).

More preferred are primary amides according to the general formula (1), wherein the structural element B is selected from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9), (BlO) and/or (B 13) and the structural element A is (Al). In more preferred embodiments, the pharmaceutical preparation comprises primary amides according to the general formula (1), wherein the structural element B is selected from the group comprising (Bl), (B2), (B3), (B4), (B5), (B6), (B9) and/or (BlO) and the structural element A is (Al). Further preferred is a pharmaceutical preparation comprising primary amides according to the general formula (1), wherein a combination of the structural element (B 13) and the structural element (Al) is excluded.

Preferred are the primary amides according to the general formula (1), wherein the structural element A is (Al) and the structural element B is selected from the group comprising (Bl), (B2), (B3) and/or (B4).

Preferably, primary amides according to the general formula (1), wherein the structural element A is selected from the group comprising branched Ci-Cio-alkyl groups, (A2) and/or (A3), preferably branched CpCio-alkyl groups or (A3), and the structural element B is selected from the group comprising (B5) and/or (B6), and/or primary amides according to the general formula (1), wherein the structural element A is (A4), and the structural element B is (B7), and/or primary amides according to the general formula (1), wherein the structural element A is (A5), and the structural element B is selected from the group comprising (B 8) and/or (BlO), and/or primary amides according to the general formula (1), wherein the structural element A is selected from the group comprising (A6), (A7) and/or (AlO), and the structural element B is (B9) and/or racemates, enantiomers, diastereomers, solvates, hydrates, and pharmaceutically acceptable salts and/or esters thereof are usable for the manufacture of a pharmaceutical preparation for the therapeutic and/or prophylactic treatment of the aforementioned diseases.

In preferred embodiments, amides selected from the group comprising primary amides according to the general formulas (3) and/or (4), especially formulas (5) and/or (6), primary and/or secondary amides selected from the group comprising amides according to the formulas (7) to (30) and/or racemates, enantiomers, diastereomers, solvates, hydrates, and pharmaceutically acceptable salts and/or esters thereof are usable for the manufacture of a pharmaceutical preparation for the therapeutic and/or prophylactic treatment of the aforementioned diseases. Preferably, the substituent R ¹ of the primary amides according to the general formulas (3) and/or (4) is hydrogen or a Ci-C ₅ -alkyl group selected from the group comprising methyl, ethyl, isopropyl and/or tert. -butyl. Most preferably, substituent R ¹ of the primary amides according to the general formulas (3) and/or (4) is hydrogen or tert. -butyl.

In preferred embodiments the disease is selected from the group comprising autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, Crohn's disease, allergies, mastocytosis, tumor diseases such as lung or bronchial cancer, colon, gastrointestinal, pancreatic or rectal cancer, prostate cancer, lymphatic cancer or leukemia, bladder or kidney cancer, breast and/or ovarian cancer and/or diseases and pathological conditions that are linked to a hyperfunction of cytohesin dependent receptor tyrosine kinases such as the ErbB receptors and other growth factor receptors selected from the group comprising developmental syndromes such as craniosynostosis, dwarfism, Hirschsprung disease, multiple endocrine neoplasia, vascular dysmorphogenesis and/or disorders of the lymphatic system.

More preferred diseases are tumor diseases selected from the group comprising lung or bronchial cancer, colon, gastrointestinal, pancreatic or rectal cancer, prostate cancer, lymphatic cancer or leukemia, bladder or kidney cancer, breast and/or ovarian cancer. Surprisingly it was found that the primary amides according to the general formula (1) and/or secondary amides according to the general formula (2) can be effective against cancer cells. Especially the primary amide according to formula (5) was found to effectively inhibit the growth of cancer cells.

Further advantageously, the primary and/or secondary amides according to the general formulas (1) and (2) can be useful for the therapeutic and/or preventive treatment of diseases and pathological conditions that are linked to a regulation of the insulin and/or insulin- like growth factor (IGF) signaling pathway. In preferred embodiments the diseases or pathological condition that is linked to a regulation of the insulin and/or insulin- like growth factor (IGF) signaling pathway is obesity.

In also very preferred embodiments, the primary and/or a secondary amides according to the general formulas (1) and (2) are usable for immunosuppression, for example, in cases of organ transplants or for inhibition of angiogenesis.

A further aspect of the present invention relates to the use of primary amides according to the general formula (1) and/or a secondary amides according to the general formula (2) as indicated above and/or racemates, enantiomers, diastereomers, solvates, hydrates, and pharmaceutically acceptable salts and/or esters thereof for chemically inducing longevity or increasing the lifespan of mammals.

Especially primary amides according to the general formulas (3) and/or (4), especially formulas (5) and/or (6), primary and/or secondary amides selected from the group comprising amides according to the formulas (7) to (30) and/or racemates, enantiomers, diastereomers, solvates, hydrates, and pharmaceutically acceptable salts and/or esters thereof are usable for chemically inducing longevity or increasing the lifespan of mammals. The term "chemically induced longevity" within the context of this invention means that by administering the amides according to the invention, the lifespan of an organism and/or a tissue or organ can be extended. An extension of the lifespan of an organism and/or of a tissue or organ can advantageously be achieved by administering an amide without necessitating surgical treatment or a genetic alteration of the organism; in other words, longevity can be induced chemically by administering a substance.

Advantageously, the amides can have only a slight or negligible toxicity when administered. This enables their long-term use, for example. It also enables their administration for preventive purposes, especially in humans.

The pharmaceutical preparation can thus be administered with usual methods such as, for example, orally, dermally and/or intravenously. Oral or parenteral administration is preferred, with oral administration being especially preferred. In preferred embodiments, the pharmaceutical preparation is formulated for oral or intravenous administration.

The Examples which follow serve to illustrate the invention in more detail but do not constitute a limitation thereof.

The figures show:

Figure 1 the relative exchange rate from guanidine-diphosphate (GDP) to guanidine-triphosphate (GTP) in the presence of the amides according to formulas (5) to (30) in comparison to the exchange rate in the presence of the control SecinH3 (K);

Figure 2 Regulation of d4E-BP mRNA levels due to interfering with insulin signaling in Drosophila S2 cells with amides according to formulas (5) to (30) in comparison to the control SecinH3 (K); Figure 3 the Inhibition of cell adhesion of Jurkat E6 cells to an immobilized ICAM- 1-Fc-fusion protein after stimulation with the monoclonal antibody OKT3 and phorbolester (PMA) of the amides according to formulas (5) to (30) in comparison to the control SecinH3 (K);

Figure 4 the relative cell numbers normalized to the DSMO treated control after 72 h incubation with the amide according to formula (5) (Secinlό) in comparison to the control compound according to formula (40) (XHl 009) determined with a MTT proliferation assay;

Figure 5 surface plasmon resonance sensorgrams of the interaction of the amide according to formula (5) with immobilized Sec-7 domain of cytohesin-1 at concentrations of 100 μM, 50 μM, 10 μM, 5 μM, 2.5 μM, and 1 μM corresponding to the tracings from the top to the bottom. Shown are fitted binding and dissociation curves;

Figure 6 the complex formation between the amide according to formula (5) and the immobilized Sec- 7 domain of cytohesin-2 determined by microscale thermophoresis;

Figure 7 (A) A CFSE-based proliferation assay in human primary T cells treated with the amide according to formula (5). (B) IL-2 reporter gene assay with TAg Jurkat T cells treated with the amide according to formula (5). Mean + SD of triplicate samples are shown. * indicates P value less than 0.05;

Figure 8 (A) IFN gamma and IL-2 ELISA assays performed in murine CDl 1+ splenic DC and CD8+ T cells after incubation with the amide according to formula (5), and (B) IL-2, IL-4, IFNgamma and TNFalpha assays performed in human CD4 ⁺ T cells.

The compounds according to formulas (5) to (30) are commercially accessible and were purchased at the suppliers as indicated in the Table 1 below.

Table 1 : compounds and suppliers Compound Supplier

Formula (5) Asinex Ltd., Moscow, Russia

Formula (6) Vitas-M Laboratory Ltd., Moscow, Russia

Formula (19) Enamine Ltd., Ukraine

Formula (23) Enamine Ltd., Ukraine

Formula (24) Enamine Ltd., Ukraine

Formula (25) Enamine Ltd., Ukraine

Formula (26) Enamine Ltd., Ukraine

Formula (30) Vitas-M Laboratory Ltd., Moscow, Russia

Formula (12) Enamine Ltd., Ukraine

Formula (H) Enamine Ltd., Ukraine

Formula (29) Vitas-M Laboratory Ltd., Moscow, Russia

Formula (7) AMRI, Albany Molecular Research, Inc., USA

Formula (14) Asinex Ltd., Moscow, Russia

Formula (15) ChemDiv, San Diego, USA

Formula (16) ChemDiv, San Diego, USA

Formula (17) Enamine Ltd., Ukraine

Formula (18) Enamine Ltd., Ukraine

Formula (21) Enamine Ltd., Ukraine

Formula (22) Enamine Ltd., Ukraine

Formula (27) Life Chemicals Inc., Burlington, Canada

Formula (28) Vitas-M Laboratory Ltd., Moscow, Russia

Formula (20) Enamine Ltd., Ukraine

Formula (8) AMRI, Albany Molecular Research, Inc., USA

Formula (13) AMRI, Albany Molecular Research, Inc., USA

Formula (9) AMRI, Albany Molecular Research, Inc., USA

Formula (10) AMRI, Albany Molecular Research, Inc., USA

Example 1 The inhibition of the exchange activity of the sec7-domains of cytohesin-2 (ARNO) of the amides according to formulas (5) to (30) was determined by a guanine nucleotide exchange assay.

For this purpose, the sec7-domains of cytohesin-2 (ARNO-Sec7) and [Δ17]ARF1, a mutant of the ADP-ribosylation factor (ARF) protein, were subcloned into pET15 vectors (Novagen) as described in Hafner, M. et al. Nature 444, 941-944 (2006). N-terminal truncated [Δ 17] ARFl (amino acids 18-181), lacking the first 17 amino acids and ARNO-Sec7 (amino acids 50-255 of ARNO) were expressed in Escherichia coli and purified by Ni-NTA chromatography (Ni-NTA agarose, Qiagen).

The exchange from guanidine-diphosphate (GDP) to guanidine-triphosphate (GTP) induces a conformation change of the ribosylation factor, as a result of which the fluorescence of a tryptophan radical of the protein is increased. GDP/GTP exchange was measured on [Δ 17] ARFl by tryptophan fluorescence because a large increase in intrinsic fluorescence of ARF occurs upon exchange of GDP for GTP. All measurements were performed in PBS (137 mM NaCl, 2.7 mM KCl, 10.2 mM Na ₂ HPO ₄ , 1.8 mM KH ₂ PO ₄ ) pH 7.4, 3 mM MgCl ₂ at 37°C.

[Δ 17] ARFl (1 μM) in PBS without MgCl ₂ was preincubated with 80 μM GDP in the presence of 2 mM ethylenediaminetetraacetic acid (EDTA) for 15 minutes. The bound GDP was stabilized by addition Of MgCl ₂ at a final concentration of 3 mM and incubation for 5 minutes. For each exchange reaction 250 nM [Δ 17] ARFl was mixed with 10 nM ARNO-Sec7 (total volume 200 μl) in the absence or presence of inhibitoring amides according to formulas (5) to (30). The reaction was started by injection of 50 μM GTP.

The tryptophan fluorescence was measured at excitation and emission wavelength of 280 nm and 340 nm respectively. All fluorescent measurements were performed with a Varioskan microplate reader (Thermo Scientific), in 96-well plates. For analysis all data were fitted by linear regression. The pan-active cytohesin inhibitor "SecinH3", a 1-, 2-, 4-substituted triazole according to formula (9) as disclosed in DE 10 2004 055 998 served as the control (K). The relative exchange rate of the control SecinH3 (K) was set to be 1.

The relative exchange rate was determined by comparing the initial linear rise of the fluorescence signal (exchange rate) in the presence of 5μM of the amides according to formulas (5) to (30) with the exchange rate in the presence of 5μM control SecinH3 (K), whereas the latter was set to be 1.

As can be seen in Fig. 1, several amides especially according to formulas (5), (6), (7), (11), (14) to (18), (20) to (22), and (27) to (29) exhibited an inhibition of the sec7-domain of the isolated cytohesin- 2, which was better than that of the control SecinH3 (K).

Further, the IC ₅ O value inhibition of the sec7-domain of the isolated cytohesin-2 was determined. The IC ₅ O value was be determined by plotting the initial linear rise of the fluorescence signal against the added concentrations of the amide. The IC ₅ O value corresponds to the concentration of the amide at which the activity of the protein is reduced to half. The lower the IC ₅ O value, the more strongly the compound inhibits the sec7-domain.

The IC ₅₀ value of the amides according to formulas (20), (5), and (6) measured for the isolated Sec-7 domain of cytohesin-2 was 2.1 μM, 3.1 μM, and 8.0 μM, respectively, compared to 11.4 μM for the reference SecinH3 (K). With an IC ₅ O value of 2.1 μM, the amide according to formula (20) was the most active new inhibitor in nucleotide exchange assays. Both amides according to formulas (5) and (20) inhibition displayed a dose-response behaviour, indicating specificity of the interactions.

Example 2

Drosophila assays:

In order to evaluate potential interference of the amides according to formulas (5) to (30) with Drosophila insulin signaling, S2 (Schneider 2 insect cell line, Invitrogen) cells were grown under starvation conditions and subsequently treated with insulin. This triggers a strong activation of the insulin signaling cascade resulting in the nuclear exclusion of the forkhead-box transcription factor dFOXO, which in turn down-regulates the expression of the transcriptional repressor d4E-BP (eIF4E- binding protein). If compounds inhibit the GEF activity of Steppke, insulin signaling is blocked and dFOXO is translocated into the nucleus, leading to an up-regulation of d4E-BP transcription. This was measured by quantitative RT-PCR and compared to the effect of the control SecinH3 (K) in the same assay.

Cell culture and compound treatment:

0.5 x 10 ⁶ S2 cells were grown in 24-well dishes in medium (Schneiders Drosophila Medium, PAN Biotech) without FCS (heat-inactivated fetal calf serum) representing starvation condition. Six hours after seeding cells were treated for 18 hours with 10 μM of the respective amide according to formulas (5) to (30) or 10 μM SecinFB (K) as reference. 22 hours after seeding cells were treated with 5 μg/ml insulin (Sigma). Controls were performed without amide and/or without insulin treatment. The amides were dissolved in dimethyl sulfoxide (DMSO). Final DMSO concentration in the cell culture medium was 0.5 %.

RNA Isolation, cDNA synthesis and Real-Time PCR:

NucleoSpin 8 RNA Kit (Macherey-Nagel) was used for RNA preparation according to manufacturer's instructions. Genomic DNA digestion and first strand cDNA synthesis was carried out with 250 ng total RNA using QuantiTect Reverse Transkription Kit (Qiagen). Quantitative PCR was performed with the iQ5 Real-Time PCR detection system (Bio-Rad) and iQ SYBR Green Super Mix (Bio-Rad). Three reactions were done in parallel for each template. Real Time PCR was analysed using Bio-Rad iQ5 Optical System Software and Microsoft Excel.

The d4E-BP mRNA level after treatment of cells with the amides according to formulas (5) to (30) compared to the control SecinFB is shown in Fig. 2. The d4E-BP mRNA level after treatment of cells with the control SecinFB (K) was set to be 1.

It can be seen from Fig. 2 that in Drosophila S2 cells treated with several amides, especially according to formulas (5), (6), (8), (9), (10), (13), and (20), d4E-BP transcript levels were increased. Especially the amide according to formula (5) induced a ~20-fold increase in dFOXO-dependent d4E-BP transcript levels compared to the reference (K).

As the activity of the insulin signaling pathway can be determined from the transcription rate of the insulin target gene Drosophila eukaryotic initiation factor 4E binding protein (d4E-BP), these results show that the insulin signaling pathway is influenced by these amides in vitro.

Example 3

Cell adhesion assays

The ability of the amides according to formulas (5) to (30) to inhibit adhesion of the T cell line Jurkat E6 to an immobilized ICAM-I (Inter-Cellular Adhesion Molecule l)-Fc-fusion protein was tested. On the T cell side, this interaction is exclusively mediated by the β-2 integrin LFA-I that was enabled to bind to immobilized ICAM-I through stimulation of Jurkat cells with an anti-TCR (T cell receptor) antibody or with the phorbol ester PMA (Phorbol 12-myristate 13 -acetate). Cell adhesion to ICAM-Fc was carried out on 96-well dishes and was read out by fluorescence detection of total input versus bound cells which had been labeled with the DNA dye H33342.

Jurkat E6.1 cells were incubated in HBSS (Hank's BSS, PAA), 0.5% DMSO and, where indicated, 25 μM of the respective amide for one hour at 37°C, 5% CO ₂ . After 30 minutes the cells were labeled with the fluorochrome bisbenzimide trihydrochloride H33342 (Sigma-Aldrich) at 12 mg/ml for 30 minutes at 37°C. 2 x 10 ⁵ cells/ well were subsequently dispensed into a 96-well plate at lOOμl/well. Prior to use plates were coated with 12μg/ml goat anti-human IgG for 90 minutes at 21°C, blocked with 1% BSA (Bovine serum albumin) in PBS overnight and incubated for 60 minutes with culture supernatants from CV-I cells expressing an ICAM-I-Fc fusion protein.

Cells were either stimulated with 5 μg/ml purified anti CD3 antibody (OKT3, hybridome of ATCC, mAb OKT3 purified on protein A, according to standard methods from Current Protocols in

Immunology, 5th Ed., Coligan et al., Edts, Wiley 2004) or with 50 ng/ml PMA, respectively. Cells were allowed to adhere to the ICAM-I-Fc coated dishes for 15 minutes and unbound cells were washed off with Hank's BSS (PAA). Adherent cells were read out using a fluorescence plate reader (Synergy-HTl, Biotek) at 485 nm in triplicates.

As can be seen in Fig. 3, several amides especially according to formulas (5), (6), (11), (12), (19), (23), (24), (25), (26), (29), and (30) inhibited the adhesion of the Jurkat E6.1 cells to ICAM-I better than that of the control (K). "100% adhesion" corresponds to the mean of OKT3 and PMA stimulated samples treated with 25 μM control control Secin H3 (K) as reference.

These experiments show that especially the primary amides according to formulas (5) and (6) exhibit a stronger inhibition of the guanine nucleotide exchange factors of the family of cytohesins, a higher ability to interfere with cytohesin-dependent insulin signalling, and a better effectiveness to block cytohesin-mediated cell adhesion of human leukocytes compared to the control, the pan-active 1-, 2-, 4-substituted triazole known in the state of the art.

These experiments further show that the primary and secondary amides according to formulas (7) to (30) can exhibit a selective activity. Especially, the primary amides according to formulas (20), (5), and (6) showed to be potent inhibitors in nucleotide exchange essay, wherein the primary amide according to formula (20) showed to have a dual activity in cell adhesion and nucleotide exchange assays. The primary amides according to formulas (11) and (29) exhibited a dual activity in cell adhesion and nucleotide exchange assays.

Further, the primary amides according to the formulas (8), (9), and (10) were selectively active in down-regulating insulin signalling, and the primary amide according to formula (12) showed a selective inhibition of cell adhesion, while the amides according to formulas (25), (12), (26), and (30) were identified as being strong cell adhesion inhibitors, wherein in particular the amides according to the formulas (25), and (30), were found to be strong inhibitors of cytohesin-mediated cell adhesion.

Example 4

Proliferation assay The effect of the amide according to formula (5) on the proliferation of cancer cells was tested in the human lung adenocarcinoma-derived cell line H460 (ATCC).

Human NCI-H460 cells were purchased from ATCC. H460 cells were maintained in RPMI (PAA) supplemented with 10% fetal bovine serum (FBS) (Lonza) and 100 U/ml penicillin and streptomycin (PAA) and were grown at 3,TC, 5% CO ₂ .

3 x 10 ³ H460 cells per 96 well were seeded into a clear, flat bottom 96 well plate (TPP). After 24 hours the cells were washed twice in 1 x DPBS (PAA) and treated with 0.25 μM, 1 μM, or 5 μM, of the amide according to formula (5) (Secinl6), the control compound according to formula (40) (XHl 009) as indicated below:

in RPMI without FBS. Controls were treated with DMSO as solvent in an end concentration of 0.4 % (v/v). Medium was changed daily.

After 72 hours cell proliferation was quantified by using a commercially available 3-(4,5- dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay (Cell Titer 96® Non-Radioactive Cell Proliferation Assay, Promega) as described in the manufacture's protocol. Briefly, 15 μl of Dye solution was added directly into the 100 μl media of the cells. After 4 hours incubation at 37°C, 5 % CO ₂ , 100 μl solubilization solution was added to solubilize the formazan product and the absorbance at 570 nm was measured in a Varioscan microplate reader (Thermo Scientific). All assays were performed at least in triplicates. For calculation of the relative proliferation rate/cell number the mean absorbance in the solvent (DMSO) only treated cells was set as 1.

Results are given as the mean +/- standard error. Statistical analyses were performed by the two-tailed t-test or one-way- ANO VA testing using GraphPadPrism software (version 5). All data sets passed the Kolmogorov and Smirnov test for Gaussian distribution. Differences of means were considered significant at a significance level of 0.05.

The diagram of figure 4 shows the relative cell numbers normalized to the DSMO treated control after 72 h incubation with the respective the amide according to formula (5) (Secinlό), the control compound according to formula (40) (XHl 009) determined with a MTT proliferation essay. Values are given as means +/ s.e.m., n=9. 1: p<0.001, for comparison with the control.

It can be seen that the amide according to formula (5) (Secinlό) inhibited cell proliferation in a concentration dependent manner, wherein 0.25 μM reduced the cell number to 90 %, 1 μM reduced the cell number to 73 %, and 5 μM reduced the cell number to 39 % with a IC 50 of 1.43 μM.

This shows an inhibition of cell proliferation in the human lung adenocarcinoma- derived cell line H460 by the amide according to formula (5) (Secinlό).

Example 5

Clonogenic survival assay

To further investigate the effect of the amide according to formula (5) on cell proliferation, a

Clonogenic survival assay was used in the human lung adenocarcinoma-derived cell line H460.

H460 cells were maintained in RPMI (PAA) supplemented with 10% FBS (Lonza) and 100 U/ml penicillin and streptomycin (PAA) and were grown at 37°C, 5% CO ₂ . For the anchorage-dependent clonogenic growth assay H460 cells were seeded into a six-well plate at low density of 3000 cell/well. After 24 hours the cells were washed twice with PBS and incubated for 72 hours with 1 μM, or 2.5 μM of the amide according to formula (5) (Secinlό), the control compound according to formula (40) (XH1009) in RPMI without FBS. Controls were treated with DMSO as solvent in an end

concentration of 0.4 % (v/v). Medium was changed daily.

After 72 hours incubation the cells were trypsinized and replated in six-well plates and cultured in RPMI, 10 % FCS for 5-7 days. After 5-7 days colonies were fixed with 0.1 % Coomassie blue in 30 % methanol and 10 % acetic acid. Pictures were taken with a Odyssey scanner (Licor).

It was seen that pretreatment with the amide according to formula (5) almost completely blocked the growth of colonies, whereas the untreated or with the control compound treated cells formed big colonies. Remaining colonies were much smaller than colonies of untreated cells, indicating an irreversible or long-lasting inhibition of cell proliferation by the amide according to formula (5).

This experiment shows an inhibition of cell proliferation in the human lung adenocarcinoma-derived cell line H460 by the amide according to formula (5) (Secinlό).

Example 6

Surface plasmon resonance assays

For the amide according to formula (5) Sec-7 domain binding was confirmed by surface plasmon resonance. Experiments were performed measuring binding and dissociation of the amide according to formula (5) (Secinlό) on the immobilized cytohesin-1 Sec-7 domain at flow rates of 50 μl/min and different concentrations.

Surface plasmon resonance experiments were performed using a dual-channel SR7000DC system (Reichert Inc., USA). Recombinant cytohesin-1 Sec-7 domain was covalently immobilized on a HC 1500m chip (Xantec bioanalytics GmbH, Germany). The surface was activated with activation buffer (0.1 M NHS (N-hydroxysuccinimide), 0.7% EDC (N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide), 0.05 M MES (2-morpholinoethane sulfonic acid), pH 5.0) and the protein was applied at a concentration of 50 μg/ml in 5 mM acetic acid, pH 4.5 to sample channel, only. Unreacted residues on the chip surface were quenched with 1 M ethanolamine, pH 8.5. Binding and dissociation were performed in 1% DMSO containing PBS at a flow rate of 50 μl/min. Regeneration of the chip surface was achieved by injection of 10 mM glycine HCl, pH 3.0.

The netto sample channel response which was calculated by subtracting the response from the reference channel from that of the sample channel was corrected for blank buffer injection and DMSO injection. Each curve represents at least three experiments. Data processing and curve fitting was done using SPR V4.0.17 (Reichert Inc., USA) and Scrubber2 software (BioLogic Software, Australia).

Figure 5 shows the surface plasmon resonance sensorgrams of the interaction of the amide according to formula (5) (Secinlό) with immobilized Sec-7 domain of cytohesin-1 at concentrations of 100 μM, 50 μM, 10 μM, 5 μM, 2.5 μM, and 1 μM. Shown are fitted binding and dissociation curves. From the top to the bottom, tracings correspond to the following concentrations of the amide according to formula (5) (Secinlό): 100 μM, 50 μM, 10 μM, 5 μM, 2.5 μM, and 1 μM.

As shown in Figure 5, binding was concentration-dependent and saturation of binding was observed, resulting in an estimated KD of 7.5 μM. The Surface plasmon resonance experiments confirm the interaction of the amide according to formula (5) with the Sec7 domain of cytohesins.

Example 7

Microscale thermophoresis measurements

To further confirm the interaction of the amide according to formula (5) with the Sec7 domain by performing microscale thermophoresis measurements.

10 nM ARNO-Sec7 was incubated at room temperature for 5 min with the indicated concentrations of 300 nM, 600 nm, 1 μM, 2.5 μM, 2 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 6 μM, 7 μM, or 8 μM of the amide according to formula (5) (Secinlό), indicated as stars in figure 6. As a control 10 nM ARNO-Sec7 was incubated with 10 ^"5 M of the non-binding control compound according to formula (40) (XHl 009), indicated as a square in figure 6. As a further control, RABex, a control protein, which does not bind the amide according to formula (5) (Secinlό), was incubated with the indicated concentrations of 10 ^~6 M, 4 x 10 ^~6 M, and 8 x 10 ^~6 M of the amide according to formula (5) (Secinlό), indicated as single dots in figure 6.

Complex formation between compound and protein was determined by microscale thermophoresis as described by Duhr and Braun (Why molecules move along a temperature gradient, PNAS 103, 19678- 19682, 2006) and Baaske et al. (Optical Thermophoresis for Quantifying the Buffer Dependence of Aptamer Binding 13. Angewandte Chemie International Edition 49, 2238-2241, 2010) on a Monolith NT.015 (NanoTemper).

In good agreement with the Surface plasmon resonance data of Example 6, a KD of 5 μM +/- 1 μM for the binding of the amide according to formula (5) (Secinlό) to ARNO-Sec7 was obtained.

The microscale thermophoresis measurements confirm the interaction of the amide according to formula (5) with the Sec7 domain of cytohesins.

Example 8

The effect of the amide according to formula (5) on T cell proliferation was tested in a proliferation assay in human primary T cells.

Untouched CD4 ⁺ T cells were isolated via immunomagnetic separation. Peripheral blood lymphocytes (PBLs) were isolated from standard buffy coat preparations of healthy blood donors by Ficoll density gradient centrifugation. Briefly, blood was diluted 1 :3 with PBS/2mM EDTA and loaded on a pancoll gradient (Pan Biotech). After centrifugation for 800 x g, for 30 minutes interphase was collected, washed with PBS/2mM EDTA and centrifugated for 640 x g, 7 minutes. Washing was repeated for at least 4 times with decreasing centrifugation speed. Cells were seeded in VLE-RPMI 1640 (Biochrom) supplemented with 10% fetal calf serum and 1% Penicillin/Streptomycin with a density of 6*10 ⁶ cells/ml into six-well plates. After 1 hour incubation non-adherent cell population of PBLs was collected and used for further experiments. Untouched human CD4 ⁺ T cells were isolated from the PBMC fraction using the CD4 ⁺ T Cell Isolation Kit II (Miltenyi Biotec) by high-gradient magnetic cell sorting on LS Separation Columns (Miltenyi Biotec) according to the manufacturer's protocols.

CD4 ⁺ T cells were stained with 0.5 μM Carboxyfluorescein succinimidyl ester (CFSE) (Molecular Probes) and afterwards incubated with 5 μM or 10 μM of the amide according to formula (5)

(Secinlό) one hour prior to stimulation by bead-coupled anti-CD3 and anti-CD28 antibodies (artificial APC, aAPC). For the generation of aAPC for CD4 ⁺ T cell stimulation, magnetic beads (Dynal Biotech) were coated with the following antibodies: anti-CD3 (OKT3; Ortho Biotech), and anti-CD28 (gift of Drs. C. June and J. Riley). These aAPC were coated with suboptimal anti-CD3 antibody (5%), or suboptimal levels of anti-CD28 antibody constituting the remaining 81% of protein added to the bead, as previously described (Parry et al., MoI Cell Biol 25, 9543-9553, 2005). To induce proliferation 1 -2 x 10 ⁶ CD4 ⁺ T cells were stimulated for 5 days in 24-well plates with aAPC at a ratio of 1 :2 of cells to beads and proliferation was then analyzed by flow cytometry (BD, Canto II).

It was found that specific employment of the cytohesin inhibitor of the amide according to formula (5) (Secinlό), but not that of DMSO alone, strongly abrogated human CD4 ⁺ T cell proliferation induced by anti-CD3 and anti-CD28 coated beads/aAPC in a concentration dependent manner, as determined by the CFSE-labeling and dilution assay, as can be seen in Figure 7A). It can be taken from Figure 7A) that the amide according to formula (5) (Secinlό) inhibits T cell proliferation in a concentration dependent manner.

This shows that the amide according to formula (5) (Secinlό) inhibits cytohesin guanine nucleotide exchange factor activity which abrogates activation of human CD4+ T cells.

Example 9

IL-2 reporter gene assay with TAg Jurkat T cells treated with the amide according to formula (5).

TAg Jurkat T cells were transfected with 15μg IL-2 promoter lucif erase expression plasmid and 5μg nephrocystin-1-GFP (Nl :GFP). Cells were subcultivated 24 hours before transfection to a density of 0.2*10 ⁶ cells/ml. l*10 ⁷ cells per electr op oration were added to 20 μg of plasmid DNA. Additionally, for reporter gene assay 15 μg reporter plasmid and 5 μg Nl :GFP plasmid were added. After transfer into 10 ml RPMI 1640 10% FCS/lOμg/ml Gentamycin cells were left 16-18 hours for expression. Cells were subsequently electroporated in a Gene Pulser X Cell Electroporator + CE module. Pulse conditions were square wave, 300 V, 1 pulse and 20 ms pulse length. Electroporated T cells were placed in 10 ml RPMI 1640 medium containing 10% heat-inactivated FCS and lOμg/ml gentamycin.

After 24 hours transfected T cells were pre-treated with 0.5% DMSO or 10 μM of the amide according to formula (5) (Secinl6) one hour prior to stimulation with 2μg/ml anti-CD3 together with lμg/ml anti-CD28. After 7 hours of stimulation, cells were harvested and luciferase activity was measured using the Microlumat plus Luminometer. Luciferase activity was normalized to transfection efficiency and protein concentration. Transfection efficiency was monitored by analyzing expression level of GFP in an EPICS XL FACS (Beckman Coulter). Protein concentration of the lysates was determined by bicinchoninic acid protein assay reagent (Pierce).

It was found that the amide according to formula (5) (Secinl6) strongly inhibited the activation of the IL-2 promoter in the reporter assay as can be seen from Figure 7B). Mean + SD of triplicate samples are shown.

Statistical significance between data groups was determined by analysis of variance (ANOVA) with the Instat software (GraphPad, San Diego, CA) and considered to be significantly different at P values less than 0.05. The P values are depicted as asterisks in the figures and* indicates a P value less than 0.05. Similar results were obtained in 3 separate experiments.

Example 10

At the functional level the effect of the amide according to formula (5) on cytokines production in murine T cells was tested.

Murine CDl 1+ splenic DC and CD8+ T cells were isolated via immunomagnetic separation.

Transgenic OT-I T cells from spleen were incubated with 2.5 μM or 5 μM of the amide according to formula (5) (Secinl6) in 0.5 % DMSO for 1 hour and then activated by co-cultivation of OVA- peptide-preincubated splenic DC. Supernatants were collected after 24 and 48 hours, respectively and IFN gamma and IL-2 ELISA assays were performed.

Figure 8 A) shows that the amide according to formula (5) (Secinlό) strongly antagonized IL-2 and IFNgamma production of CD8+ T cells from OT-I mice, which had been stimulated by OVA-DC. Representative data of 2 independent experiments are shown.

Example 11

Further, the effect of the amide according to formula (5) on cytokines production in human T cells was tested.

Untouched CD4+ T cells were isolated from the blood of healthy donors by MACS sorting as described in example 8 and were pre-treated with 0.5% DMSO or 10 μM of the amide according to formula (5) (Secinlό) Ih prior stimulation with aAPC coated with anti-CD3/CD28 at a ratio of 1 :2 of cells to beads. Supernatants were collected 48 hours later and stained for IL-2, IL-4, IFNgamma and TNFalpha secretion, using the BD Human Thl/Th2 Cytokine Cytometric Bead Array. Individual cytokine concentrations were obtained by acquiring capture beads on the FACSCanto II and converting mean fluorescence intensities into concentration values by extrapolating from the standard curve using CBA software.

It was found that the production of IL-2, IFNgamma, TNFalpha and IL-4 was also strongly inhibited by the amide according to formula (5) (Secinlό) in human CD4+ T cells, which had been stimulated with anti-CD3/CD28 beads as can be seen from figure 8B). Representative data of 3 independent experiments are shown.

These experiments show that the amide according to formula (5) has an effect on the functional level on cytokines production in murine and human T cells. The results of experiments 8, 9, 10 and 11 show that the amide according to formula (5) is suitable for the treatment of autoimmune diseases and for immunosuppression, for example in cases of organ transplants since these diseases are subject to T cell activation.