EP2101753A2 - Use of cytohesin inhibitors for chemically inducing longevity - Google Patents

Abstract

The invention relates to compounds selected from among the group comprising general formulas (1), (2), (3), and/or (4) and/or the enantiomers, diastereomers, and derivatives thereof, and the pharmaceutically acceptable salts thereof for producing a medicament used for therapeutically and/or preventively treating diseases and pathological conditions linked to regulation of the insulin and/or insulin-like growth factor (IGF) signaling pathway and/or for chemically inducing longevity.

Description

 Use of cytohesin inhibitors for chemical induction of longevity

The invention relates to the use of compounds according to the general formulas (1), (2), (3) and (4) for the treatment and / or prophylaxis of diseases and pathological conditions associated with regulation of insulin and / or IGF. (Insulin-like growth factor) signaling pathway, and / or for the chemical induction of longevity.

Compounds according to the general formulas (1), (2), (3) and (4) are known from the document DE 10 2004 055 998 A1.

Each cell function, including cell aging and cell death, is controlled by a variety of cell signaling pathways. The development of some diseases also depends on cell aging and the likelihood of dying from disease increases with age.

Another common problem associated with today's lifestyle is obesity, also called obesity. This is a key factor in the development of many chronic diseases as well as some cancers.

The object of the present invention was to provide compounds which overcome at least one of the disadvantages of the prior art. Especially The task was to provide means that allow influencing the insulin signaling pathway.

This object is achieved by the use of compounds selected from the group comprising the general formulas (1), (2), (3) and / or (4) as indicated below:

wherein:

R is the same or each independently selected from the group comprising hydrogen, OH, COOH, COO (Cl -C 10 -alkyl), CONH ₂ , CONH (Cl-ClO-alkyl), CON (Cl -C 10 -alkyl) ₂ , NHCO (C 1 -C 10 -alkyl), NHCOCHCl ₂ , halogen, preferably selected from the group comprising Cl, Br, F, CF ₃ , amine, C 1 -C 10 -alkyl, C ₁ -C 10 -alkoxy and / or a structural element ( Al), (B1), (C1), (D1), (E1), (F1), (G1), (H1), (II), (Y1), (L1), (M1) as indicated below: Wonn: - A -

R ₄ is the same or each independently selected from the group comprising hydrogen, OH, COOH, COO (C 1 -C 10 -alkyl), CONH ₂ , CONH (C 1 -C 10 -alkyl), CON (C 1 -C 10 -alkyl ) ₂ , NHCO (C 1 -C 10 -alkyl), NHCOCHCl ₂ , halogen, preferably selected from the group comprising Cl, Br, F, CF ₃ , amine, C 1 -C 10 -alkyl and / or C 1 -C 10 -alkoxy,

and / or their enantiomers, diastereomers, derivatives and their pharmaceutically acceptable salts for the preparation of a medicament for the therapeutic and / or prophylactic treatment of diseases and pathological conditions associated with regulation of insulin and / or IGF (insulin-like growth factor) Signal pathway and / or the chemical induction of longevity.

It has surprisingly been found that the compounds which can be used according to the invention can show an influence on the insulin and / or IGF (insulin-like growth factor) signaling pathway. In particular, it has surprisingly been found that the compounds useful in the invention in mice lead to insulin resistance, i. reduced activity of the insulin signaling pathway.

Without wishing to be bound by any particular theory, it is believed that the influence of the insulin and / or insulin-like growth factor (IGF) pathway and insulin resistance on an inhibition of cytohesins, in particular of cytohesin-1, cytohesin 2, cytohesin-3 and / or cytohesin-4, by the compounds used in accordance with the invention.

It has surprisingly been found that compounds selected from the group comprising the general formulas (1), (2), (3) and / or (4) can lead to a chemically induced longevity. It has further been found, surprisingly, that the compound according to the formula (9) can lead to a significantly improved chemically induced longevity, for example to a significant prolongation of the life span in the case of flies. In particular, it is advantageous that in particular the compound according to the formula (9) can cause an activation of the immune system.

The compounds which can be used according to the invention preferably have at least one, preferably two, more preferably three identical and / or different structural elements (A1), (B1), (C1), (D1) and / or (E1). In further preferred embodiments, the compounds which can be used according to the invention have at least one structural element selected from the group comprising (E1), (F1), (G1), (III), (II), (II), (C1), (II), ( Ml) and / or (Ol).

In preferred embodiments, the structural element R _{4 is the} same or each independently selected from the group comprising hydrogen, NHCOCHCb, Cl, CF ₃ , and / or F.

A particular advantage of the compounds which can be used according to the invention is that the compounds can also be used in particular for prophylactic use. This not only gives the possibility to use the compounds which can be used according to the invention for the treatment of already existing pathological conditions but also their prophylactic use in the prevention, for example of age-related cell damage or obesity.

For the purposes of the present invention, the term "prophylactic treatment" is understood in particular to mean that the compounds which can be used according to the invention can be administered prophylactically, for example, before age-related cell damage occur. In particular, it is advantageous that, for example, obesity can be prevented by a prophylactic treatment.

In preferred embodiments, the compounds are selected from the group comprising the general formulas (1), (2), (3) and / or (4) selected from the group comprising compounds of the general formulas (5), (6), (7 ) and / or (8) as indicated below and / or their enantiomers, diastereomers, derivatives and their pharmaceutically acceptable salts:

wherein:

Ri is the same or each independently selected from the group comprising R ₂ , R ₃ and / or a structural element (A2), (B2), (C2), (D2), (N2) as indicated below:

is the same or each independently selected from the group comprising R _h R ₃ and / or a structural element (E2), (F2), (G2), (H2), (12), (J2), (K2), (L2 ), (M2), (02) as indicated below:

R ₃ (H2)

R ₃ is the same or each independently selected from the group comprising Ri, R ₂ , and / or selected from the group comprising hydrogen, OH, COOH, COO (Cl -C 10 -alkyl), CONH ₂ , CONH (Cl -C 10-alkyl), CON (C 1 -C 10 -alkyl) ₂ , NHCO (C 1 -C 10 -alkyl), NHCOCHCl ₂ , halogen, preferably selected from the group comprising Cl, Br, F, CF ₃ , amine, C 1 -C 10 -Alkyl and / or Cl-Cl O-alkoxy.

Preferably, at least one or more of the structural elements Ri, R ₂ and / or R _{3 are the} same or in each case independently selected from the sulfur-containing structural elements (A2), (B2), (C2) and / or (D2). The compounds which can be used according to the invention can have a plurality of identical and / or different structural elements (A2), (B2), (C2) and / or (D2). The compounds which can be used according to the invention preferably have at least one, preferably two, more preferably three identical and / or different structural elements (A2), (B2), (C2) and / or (D2). More preferably, the compounds which can be used according to the invention have at least one Structural element selected from the group comprising (E2), (F2), (G2), (H2), (12), (J2), (K2), (L2), (M2) and / or (02). Preferably, the structural element R _{3 of} the structural elements (E2), (F2), (G2), (H2), (12), (J2), (K2), (L2), (M2) and / or (02) is hydrogen ,

In further preferred embodiments, the compounds which can be used according to the invention are selected from the group comprising compounds of the general formulas (5), (6), (7) and / or (8) as indicated below and / or their enantiomers, diastereomers, derivatives and their pharmaceuticals compatible salts:

wherein:

Ri is the same or each independently selected from the group comprising R ₂ , R ₃ and / or a structural element (A3), (B3), (C3), (D3), (N3) as indicated below:

is the same or each independently selected from the group comprising R _h R ₃ and / or a structural element (E3), (F3), (G3), (H3), (13), (J3), (K3), (L3 ), (M3), (03) as indicated below:

(H3)

R ₃ is the same or each independently selected from the group comprising Ri, R ₂ , and / or selected from the group comprising hydrogen, OH, COOH, COO (Cl -C 10 -alkyl), CONH ₂ , CONH (Cl -C 10-alkyl), CON (C 1 -C 10 -alkyl) ₂ , NHCO (C 1 -C 10 -alkyl), NHCOCHCl ₂ , halogen, preferably selected from the group comprising Cl, Br, F, CF ₃ , amine, C 1 -C 10 -Alkyl and / or Cl-Cl O-alkoxy.

Preferably, at least one or more of the structural elements Ri, R ₂ and / or R _{3 of} the compounds (5), (6), (7) and (8) is the same or in each case independently selected from the sulfur-containing structural elements (A3), (B3 ), (C3) and / or (D3). The compounds which can be used according to the invention can have a plurality of identical and / or different structural elements (A3), (B3), (C3) and / or (D3). The compounds which can be used according to the invention preferably have at least one, preferably two, more preferably three identical and / or different structural elements (A3), (B3), (C3) and / or (D3). In further preferred embodiments, the Compounds which can be used according to the invention are at least one structural element selected from the group comprising (E3), (F3), (G3), (H3), (13), (J3), (K3), (L3), (M3) and / or ( 03).

In preferred embodiments of the compounds which can be used according to the invention, at least one structural element R, R ₁ , R ₂ , R ₃ and / or R ₄ , preferably at least one structural element R of the compounds (1), (2), (3) and (4), in particular R _{3 of} the compounds (5), (6), (7) and (8), each independently selected from the group comprising C 1 -C 5 -alkyloxy, preferably selected from the group comprising -O-methyl, -O-ethyl, - O-isopropyl and / or -O-tert-butyl. Particularly preferred among the C 1 -C 5 alkoxy groups are methoxy and / or ethoxy groups, very particularly preferred are methoxy groups.

It has surprisingly been found that the compounds which can be used according to the invention have a significantly improved effect, the smaller the alkoxy groups are. Thus, it has surprisingly been found that a significant increase in the effectiveness of the use of the compounds can be achieved when the alkoxy group R ₃ , in particular the compounds (5), a Cl-C2-alkoxy group, and achieved a further increase in the effectiveness of the compound can be when the alkoxy group R _{3 is} a methoxy group.

In preferred embodiments, the compounds 1,2,4-triazoles which can be used according to the invention are selected from the group comprising compounds of the formulas (1) and / or (5). Preferably, Ri of the compounds according to formula (5) is a structural element (A3), (B3), (C3), (D3) or (N3), R _{2 is} a structural element (E3), (F3), (G3), (H3 ), (13), (J3), (K3), (L3), (M3) or (03) and / or R3 is a C 1 -C 5 alkoxy group, preferably a methoxy or ethoxy group. Ri of the compounds of the formula (5) is preferably a structural element (A3) or (B3), R _{2 is} a structural element (E3), (F3) or (K3) and / or R _{3 is} a methoxy or ethoxy group. In very particularly preferred embodiments, the compounds which can be used according to the invention are selected from the group comprising compounds (9), (10), (11), (22), (23) as indicated below and / or their enantiomers, diastereomers, derivatives and their pharmaceuticals compatible salts:

O ^' OHN _{^ 1}

O

N

(9)

(10)

It has surprisingly been found that, in particular 1,2,4-triazoles according to the general formulas (1) and / or (5), in particular according to formula (9), cause insulin resistance and / or lead to a chemically induced longevity , In other preferred embodiments, the compounds which can be used according to the invention are selected from the group comprising compounds (12), (14), (20), (21) as indicated below and / or their enantiomers, diastereomers, derivatives and pharmaceutically acceptable salts thereof:

Another embodiment of the compounds which can be used according to the invention and / or their enantiomers, diastereomers and pharmaceutically acceptable salts thereof has the following formula (25):

In further preferred embodiments, the compounds which can be used according to the invention may be derivatized, for example phosphorylated, glycosylated, acetylated, ubiquitinylated, farnesylated, palmitoylated, geranylgeranylated and / or biotinylated.

Preferred derivatives are biotinylated compound, particularly preferred is, for example, compound (24) as indicated below and / or their enantiomers, diastereomers and their pharmaceutically acceptable salts

(24).

An advantage of the compounds which can be used according to the invention can be realized by the fact that they can exert an inhibitory effect on cytohesin-dependent signal cascades of the insulin and / or IGF (insulin-like growth factor) signaling pathway. The compounds which can be used according to the invention can, for example, show in vivo in the mouse and in the fly that insulin resistance can be effected. The compounds which can be used according to the invention can furthermore be shown in in vitro experiments in human liver cells to also be able to develop insulin resistance. An insulin resistance can lead to an increase in longevity and longevity and there are applications in the treatment of age-related diseases.

A particular advantage of the compounds which can be used according to the invention can be provided by the fact that they can allow a chemically induced longevity in the context of a therapeutic and / or prophylactic treatment. The term "chemically induced longevity" in the context of this invention is understood to mean that the life of an organism and / or of a tissue or organ can be prolonged by the administration of the compounds which can be used according to the invention An extension of the life of an organism and / or a tissue or organ can advantageously be achieved by administering a compound which can be used according to the invention without the need for medical intervention or genetic modification of the organism, ie a longevity can be induced chemically, in particular by administering a substance.

The compounds which can be used according to the invention advantageously make it possible to influence the insulin and / or IGF (insulin-like growth factor) signaling pathway, and to use the compounds which can be used according to the invention for the therapeutic and / or prophylactic treatment of diseases and pathological conditions associated with regulation insulin and / or IGF (insulin-like growth factor) signaling pathway. The regulation of the insulin-like and / or IGF (insulin-like growth factor) signaling pathway is influenced by a large number of hormones and messenger substances that are in a complex equilibrium. A disturbed regulation can lead to a variety of diseases, such as obesity. Preferably, these are diseases and pathological conditions associated with a disorder of insulin and / or IGF (insulin-like growth factor) signaling pathway, particularly diseases and pathological conditions caused by an increase in insulin and / or insulin. or IGF signaling. In preferred embodiments, the compounds which can be used according to the invention can bring about an inhibition of the insulin and / or IGF (insulin-like growth factor) signaling pathway.

Preferred treatable diseases and pathological conditions associated with regulation of the insulin and / or IGF signaling pathways are selected from the group consisting of obesity, cell aging, cell damage caused by aging, especially in the liver and / or pancreas, age-related pathological conditions of liver - and / or pancreatic cells, age-related dysfunctions, such as a reduced ability to regenerate liver and / or pancreas, cell stress, especially oxidative stress, in particular by increased metabolism of sugar-induced stress, and / or apoptosis, in particular ß-cell apoptosis.

In particularly preferred embodiments, use of the compounds which can be used according to the invention can lead to the prolongation of the life of animals, for example mammals, in particular in humans.

According to the invention, the compound according to the formula (9) has been shown to have a positive effect on the life span in vivo. Thus, it was found experimentally that the compound according to the formula (9) was able to prolong the life span of flies in vivo. A chemically induced prolongation of life or of age is a very particular advantage that can be provided by the compounds useful in this invention.

Without being bound to a particular theory, it is further assumed that the compounds which can be used according to the invention, in particular the compound according to the formula (9), can have a positive effect on the immune system. In particular, it is assumed that the compounds which can be used according to the invention, in particular the compound according to the formula (9), can bring about an activation of the immune system.

Advantageously, the compounds which can be used according to the invention may have a low or negligible toxicity during administration. This allows, for example, long-term administration. This also allows prophylactic administration, especially to humans.

The compounds which can be used according to the invention can be administered in accordance with customary methods. Preferred is oral or parenteral administration, more preferred is oral administration. In preferred embodiments, the compounds useful in the invention are formulated for oral or intravenous administration. Preferred auxiliaries and / or solvents are selected from the group comprising DMSO (dimethyl sulfoxide), glycerol and / or oil. Preferably, solvents are selected from the group comprising DMSO and / or vegetable oil, in particular olive oil. An advantage of using oil, such as olive oil, is that it can provide an improvement in compatibility.

Examples which serve to illustrate the present invention are given below. material and methods

cell culture

Human HepG2 cells (ECACC) were cultured in EMEM medium (Cambrex) with the addition of 10% fetal calf serum. 10 ⁵ cells were seeded in 2 ml of medium in 6 well plates and cultured at 37 ° C in a humidified atmosphere with 5% CO ₂ for 1 to 3 days before being used for experiments.

mice

C57BL / 6 mice (Charles River Laboratories) were used which were maintained in a pathogen-free animal facility for a 12-hour light / dark cycle. The animals received standard mouse food (19% protein, 3.3% fat, 41.3% carbohydrates, ssniff Spezialdiäten GmbH) ad libitum. Male animals were used between the ages of 8 and 12 weeks.

fly

Drosophila flies (strain # 6326 from the Bloomington Public Collection, genotype: white ¹¹¹⁸ , URL http://flybase.Org/.bin/fbidq.html7FBst0006326) were used in groups of 20 flies with male to female ratios of 1 : 1 at 25 ° C on standard fly food (0.53% (w / v) Fadenagar (company "Gewürzmühle Brecht"), 1.1% (w / v) brewer's yeast (company "Gewürzmühle Brecht"), 5.43 % (w / v) maize flour; 6.6% (v / v) sugar beet syrup (Grafschafter); 0.13% (w / v) nipagin (methyl-4-hydroxybenoate sodium salt, Merck); 1.3% (v (v) Ethanol (Roth), 0.3% DMSO (Roth) Adult flies were used for the experiments zero to four hours after hatching, and the animals were given new food every third day.

example 1 Studies on the transcription of IGFBPl (insulin-like growth factor binding protein) for influencing the insulin signal pathway through the compound according to the formula (9) in liver cells

Human HepG2 cells (ECACC) were removed from the medium for 24 hours, cells were incubated for an additional 12 hours at concentrations of 1.5625 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM or 50 μM of the compound of the formula (9) and incubated with 10 nM insulin. Control cells received DMSO in the same concentration as the treated cells.

Total RNA was isolated using the Absolutely RNA kit (Stratagene) and the cDNA was prepared by reverse transcribing 2 μg of RNA using the High Capacity cDNA Archive kit (Applied Biosystems) according to the manufacturer's instructions. Quantitative PCR was performed in 10 μl batches in an iQ5 cycler (BioRad) using the TaqMan Gene Expression Assay (Applied Biosystems) using gene-specific primers (Applied Biosystems) according to the manufacturer's instructions. The data were normalized to β2-microglobulin expression.

It was found that the compound according to the formula (9) could almost completely block the insulin-dependent inhibition of the transcription of IGFBPl (insulin-like growth factor binding protein), a prototypical insulin-regulated gene. Thus, the expression significantly reduced by 10 nM insulin in the presence of 12.5 μM of the compound of the formula (9) almost reached the value of the untreated cells.

Example 2

Investigations of the phosphorylation of Akt and FoxOlA for influencing the insulin signaling pathway by the compound according to the formula (9) in liver cells The expression of IGFBPl (insulin-like growth factor binding protein) is controlled by insulin using the forkhead box transcription factors Ol A (FoxOlA) and 03A (FoxO3A). After insulin stimulation, the protein kinase B / Akt (PKB / Akt) is activated by phosphorylation as a function of the phosphoinositide 3-kinase (PI3K) into the nucleus where it phosphorylates the transcription factor Ol A (FoxOlA), which leads to a diminution of the Gene expression of, for example, IGFBPl results.

Serum HepG2 cells (ECACC) were removed from the medium for 90 minutes, cells were incubated for an additional 60 minutes with 5 μM, 10 μM and 15 μM of the compound of formula (9), 200 nM Wortmannin or DMSO (dimethylsulfoxide). incubated at the appropriate concentration before the cells were stimulated for 10 minutes with 100 nM insulin. The cells were then incubated in lysis buffer (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium vanadate, 1% Triton X. -100) and protease inhibitor mix HP (Serva).

Normalized levels of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes. For protein detection by immunoblotting, an antibody against the phosphorylated protein Akt, pAkt (Thr308) (cell signaling) was used. The visualization was performed by the Enhanced Chemieluminescence System (Millipore) using a VersaDoc 5000 CCD camera (BioRad) and the intensity of the bands was quantified using the QuantityOne (BioRad) software.

It could be stated that the compound according to the formula (9) inhibited the insulin-dependent phosphorylation of both proteins Akt and FoxOlA in a concentration-dependent manner. Thus, application of 10 μM resulted in a 50% reduction in Akt phosphorylation compared to phosphorylation following insulin stimulation, whereas 15 μM of Formula (9) compound reduced Akt phosphorylation to approximately 25% phosphorylation Insulin stimulation resulted. These examples show that the inhibition of cytohesins by the compound according to formula (9) in human liver cells leads to an inhibition of the insulin signaling pathway in the sense of insulin resistance.

Example 3

Studies on the generation of insulin resistance by the compound according to the

Formula (9) in vivo in the mouse

One group of 6 C57BL / 6 mice received standard mouse feed mixed with 0.9 μmol / g of compound of formula (9) for 3 days, while a control group of 6 animals received standard mouse feed. Subsequently, the animals were intraperitoneally injected with 100 μl of physiological saline (control) or saline with 40 μg of recombinant human insulin (Sigma). After 10 minutes, the mice were anesthetized, the liver was removed and placed in lysis buffer (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate , 1 mM sodium vanadate, 1% Triton X-100) and protease inhibitor mix HP (Serva).

Total RNA from 15 mg of mouse liver each was isolated by means of the Absolutely RNA kit (Stratagene) and the cDNA was prepared by reverse transcription of 2 μg of RNA using the High Capacity cDNA Archive Kit (Applied Biosystems) according to the manufacturer's instructions. Quantitative PCR was performed in 10 μl batches in an iQ5 cycler (BioRad) using the TaqMan Gene Expression Assay (Applied Biosystems) using primers (Applied Biosystems) against Igfbpl, Fbp2, Pckl, Pck2, G6pc, Hk2, PkIr, Gck, Gckr carried out according to manufacturer's instructions. The data were normalized to β2-microglobulin expression. The study examined the expression of several genes that are controlled by the PI3K-dependent pathway of the insulin receptor signaling pathway, the FoxO-regulated model gene Igfbpl, pyruvate carboxykinase 1 and 2 (Pckl, Pck2), fructose-l, 6-bisphosphatase 2 (Fbp2 ), and glucose-6-phosphatase (G6pc) involved in gluconeogenesis as well as the glycolytic enzymes glucokinase (Gck), its regulator (Gckr), liver pyruvate kinase (PkIr) and hexokinase 2 (Hk2).

Furthermore, normalized amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes by immunoblotting using an antibody against the phosphorylated protein Akt, pAkt (Thr308) (cell signaling). The visualization and evaluation was carried out as indicated in Example 2.

It was found that the expression of the examined insulin-suppressed gluconeogenetic genes was increased by the administration of the compound according to the formula (9), while the expression of insulin-induced glycolytic genes was statistically significantly reduced. In accordance with this result, it was also found that the insulin-stimulated phosphorylation of the protein Akt was inhibited by the administration of the compound of the formula (9) by about 40%.

This indicates that the compound of the formula (9) induces hepatic insulin resistance in vivo.

Example 4

Studies on the generation of insulin resistance by the compound according to the

Formula (9) in vivo in flies Three groups of 100 Drosophila larvae received 10 mg of the compound of the formula (9) mixed with 1 g of autoclaved and powdered rearing yeast (Brewferm) while three control groups of 100 animals received the ragged rearing yeast. This feed was administered on water-saturated filters (Macherey-Nagel) for 3 days at 25 ° C.

In each case more than 10 animals of the group were washed thoroughly with water, in lysis buffer (NucleoSpin RNA II kit, Macherey & Nagel) and homogenized for 1 minute at maximum speed (Ultra-Turrax T25basic). The total RNA was isolated using the NucleoSpin RNA II kit (Macherey & Nagel, including DNase I treatment on the column).

cDNA was prepared from 500 ng total RNA using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer's instructions including DNasel treatment. PCR was carried out in batches with a total volume of 25 μl (iQ5 Real-Time PCR Detection System, BioRad). The batches contained 1 .mu.l of the cDNA batch, 200 nM each and 3'- and 5'-primer (Metabion) 12.5 ul 2x iQ5 SYBR Green Supermix (BioRad).

The PCR program used consisted of 40 cycles with the following steps: 15 seconds denaturing at 95 ⁰ C, 30 sec annealing at 59 ⁰ C and 30 seconds extension at 72 ⁰ C. The analysis was performed using the iQ5 Optical System Software (Version 1.1. 1442. OCR, BioRad). Actin 5C (Act5C, act) and the ribosomal protein L32 (RpL32, rp49) served as reference genes.

It was found that the expression of the examined insulin-repressed genes InR (insulin receptor) and 4E-BP, a translation repressor, by the administration of the compound according to the formula (9) in the case of 4E-BP to more than four times and in the Case of InR has been increased to more than double. Example 5

Studies of the phosphorylation of Akt by the compound of formula (9) in the Drosophila S2 Schneider cell line

10 ⁶ Drosophila S2 cells (Schneider, 1972. J. Embryol.Exp.Molphol., 27, 353) were incubated in cell culture medium (PAN) with 10% fetal calf serum and 1% streptomycin, penicillin (Gibco) in 35 mm cell culture dishes (Nunc ) and cultured for 2 days at 25 ⁰ C up to 70-80% confluence. After 2 days, DMSO ad 0.5% (v / v) ad was added to the medium and, depending on the experiment for 2 hours before insulin stimulation, the compound according to the formula (9 ad 10 μM) In case of insulin stimulation, human insulin (Sigma) was added for 15 minutes After stimulation, the cells were washed with cold PBS in cold (4 ° C.) lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1.0% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM NaF, 100 μM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride and EDTA-free protease inhibitor (Roche) according to the manufacturer's instructions, and used for Western blot analysis.

Each 10 μg of protein was separated on a 12% SDS-polyacrylamide gel and transferred to 0.22 micron porous nitrocellulose membranes (Amersham). The membranes were blocked with 5% (w / v) milk powder (Roth) in TBST (150 mM NaCl, 50 mM Tris pH 7.4, 0.1% (v / v) Tween-20). Incubation with primary antibody was performed overnight in 5% (w / v) BSA in TBST. The primary antibodies Anti-Phospho Akt (Ser505) and Anti-Akt (Cell Signaling) were each used at a dilution of 1: 1000. Secondary antibody incubation was carried out with peroxidase-coupled goat anti-rabbit immunoglobulin (Santa Cruz, Serum ID sc-2004) or peroxidase-coupled donkey anti-mouse immunoglobulin (Santa Cruz, Serum ID sc-2314) in one Dilution of 1: 15000 performed. Visualization was performed using the ECL Kit (Enhanced Chemiluminescence, Amersham) using the VersaDoc 5000 Imaging System (BioRad) and the QuantityOne (BioRad) software.

In accordance with the result of Example 4, it was found that the insulin-stimulated phosphorylation of the protein Akt was reduced by half by the administration of the compound of the formula (9).

This shows that the compound according to the formula (9) induces insulin resistance in vivo in the fly, which may result in an increase in longevity.

Example 6

Studies on the influence of the insulin signaling pathway by the compound according to the

Formula (9) and the compound according to the formula (25) in vitro in cells of Drosophila S2

Schneider cell line

Drosophila Schneider 2 (S2) cells were cultured in Schneider's Medium (PAN) with 10% heat-inactivated fetal calf serum (FCS). 2 × 10 ⁶ cells were grown to 80% confluency in 35 mm tubes at 25 ° C, washed once in phosphate buffered saline (1 × PBS, Gibco), and transferred to Schneider's medium without FCS for 12 hours. The cells were then preincubated for two hours with Schneider's medium containing 0.5% DMSO without FCS with or without the compound according to formula (9) or formula (25), the final concentrations for the compounds according to formulas (9) and (25) were 1 μM, 10 μM and 100 μM. This was followed by 4-hour stimulation with 10 μg / ml human insulin (Sigma).

Subsequently, the total RNA was after washing the cells twice with PBS using the kit NucleoSpin RNA II (Macherey & Nagel) according to the manufacturer isolated from the cells. First strand cDNA synthesis was carried out with 500 ng RNA each using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer's instructions. The real-time PCR reaction was performed with the SYBR Green Supermix (BIO-RAD) according to the manufacturer's instructions in the iQ5 Real-Time PCR Detection System (BIO-RAD). In this case, the PCR was carried out in batches with a total volume of 25 .mu.l, wherein the approaches each 1 ul of the cDNA approach, 200 nM 3'- and 5'-primer (Metabion) and 12.5 ul 2x iQ5 SYBR Green Supermix (BioRad ) contained. The PCR program used comprised 40 cycles with the following steps: 15 seconds denaturation at 95 ⁰ C for 30 seconds annealing at 59 ⁰ C and 30 seconds extension at 72 ⁰ C. The evaluation of the real time data as per manufacturer's specifications with the BIO-RAD iQ5 Optical System Software (Version 1.1.1442.OCR).

The activity of the insulin signaling pathway was determined by the transcription rate of the insulin target gene Drosophila eukaryotic initiation factor 4E binding protein (d4EBP, Thor). The genes Actin 5C (Act5C, act) and Ribosomal protein L32 (RpL32, rp49) were used as reference genes. Table 1 shows the sequences of the oligonucleotides used for the real time analysis.

Table 1 Oligonucleotide sequences

Gene sequence forward primer sequence reverse primer (metabion) (metabion)

Act5C GTGCACCGCAAGTGCTTCTAA TGCTGCACTCCAAACTTCCAC (SEQ ID NO: 1) (SEQ ID NO: 2)

RpL32 GCTAAGCTGTCGCACAAATG GTTCGATCCGTAACCGATGT (SEQ ID NO: 3) (SEQ ID NO: 4) d4EBP CATGCAGCAACTGCCAAATC CCGAGAGAACAAACAAGGTGG (SEQ ID NO: 5) (SEQ ID NO: 6) It was found that in Drosophila Schneider 2 (S2) cells treated with insulin, d4EBP was transcriptionally inhibited to less than 35% of the transcription rate of the controls. This inhibitory effect was suppressed by the compounds according to the formula (9) and (25) at a concentration of 100 .mu.M, the transcription rate of d4EBP by incubation with 100 .mu.M of the compound according to the formula (9) back to about 85% and Incubation with 100 μM of the compound according to formula (25) increased to more than 95% of the untreated controls.

Thus, it could be shown that both compounds according to formulas (9) and (25) influenced the insulin signaling pathway at a concentration of 100 μM and led to insulin resistance in vitro.

Example 7

Studies to influence the lifespan of adult Drosophila melanogaster

Flying through the compounds according to the formulas (9) and (25)

fly

Investigations were carried out on isogenic flies (Drosophila melanogaster) of genotype white ¹¹¹⁸ , also called white ^" or w ^" , and mutants (w ¹¹¹⁸ , step ^k08110 / step ^SH0323 ) (strain # 10770 from the Bloomington strain, genotype: step ^k08U0 / CyO, URL http://flybase.bio.indiana.edu/reports/FBst0010770.html; Strain # FBst0103734 from the public Szeged strain collection, genotype: step ^SH0323 / CyO, URL http://flybase.bio.indiana.edu / reports / FBst0103734.html), in which the Drosophila cytohesin steppke mutates and is therefore defective.

By eight backcrossing of the two steppe alleles kO8110 and SH0323 with white - flies, an isogenic genetic background was created Life span between the genotypes of the type white and the mutants (w; step ^k08U0 / step ^SH0323 ) permits. The average lifespan of the wild type was about 25 days, that of the mutants (w ¹¹¹⁸ , step ^k08U0 / step ^SH0323 ) was about 30 days.

special food

For the preparation of the special diet, 32.5 g of autolyzed yeast (Fluka (Catalog No. 73145)) and 10 g of agar (Difco (Catalog No. 281230)) were demineralized with 300 ml of water (Roth (Catalog No. 3175) ) and autoclaved at 120 ^° C. for 20 minutes. The solution was cooled to 60 ^° C. and 32.5 g of α-D (+) - glucose monohydrate (Roth (Catalog No. 6887)) were added while stirring. Then, 15 ml of 10% (w / v) dissolved in 70% (v / v) ethanol (Roth (Catalog No. 9065)) of p-hydroxybenzoic acid methyl ester (Sigma (Catalog No. H3647)) solution and 1, 35 ml of dimethyl sulfoxide (DMSO, Roth (Catalog No. A994)) was added. Subsequently, this stock solution was made up to a volume of 450 ml with water and kept at a temperature of 60 ⁰ C. For the control diet, 50 ml of a 0.7% (v / v) DMSO solution in water was prepared. For the feed containing the compounds of formula (9) or (25), to 49.8 ml of a 0.3012% solution of DMSO in water was added 0.2 ml of a 25 mM solution of the compounds according to formula (9). or (25) in 100% DMSO.

These 50 ml of the control solutions and the solutions containing the compounds of the formula (9) or (25) were each mixed with 450 ml of the stock solution and 4 ml each in polystyrene vessels (height 9.5 cm, diameter 2.3 cm, closed with cotton wool) submitted to the attitude of the flies. After 24 hours, the cooled vessels were closed with cotton wool and stored at 4 ⁰ C.

The final concentration of feed components in the control diet was 6.5% (w / v) autolyzed yeast, 6.5% (w / v) α-D (+) - glucose monohydrate, 2% (w / v) agar, 0 , 3% (w / v) /> hydroxybenzoic acid methyl ester, 2.1% ethanol (v / v), 0.34% (v / v) DMSO. The final concentration of feed components in the feed containing the compound of formula (9) was 6.5% (w / v) autolyzed yeast, 6.5% (w / v) α-D (+) - glucose monohydrate, 2 % (w / v) agar, 0.3% (w / v) /> hydroxybenzoic acid methyl ester, 2.1% ethanol (v / v), 0.34% (v / v) DMSO, 10 μM compound of the formula ( 9).

The final concentration of feed components in the feed containing the compound of formula (25) 6.5% (w / v) autolyzed yeast, 6.5% (w / v) α-D (+) - glucose monohydrate, 2% (w / v) agar, 0.3% (w / v) /> hydroxybenzoic acid methyl ester, 2.1% ethanol (v / v), 0.34% (v / v) DMSO, 10 μM compound according to formula (25 ).

flying attitude

The larvae were grown on standard fly feed containing 1.1% (w / v) brewer's yeast (company "Gewürzmühle Brecht"), 5.43% (w / v) maize flour, 0.53% (w / v) Fadenagar (company " Gewürzmühle Brecht "), 6.6% (v / v) sugar beet syrup (Grafschafter), 1.3% (v / v) ethanol (company Roth), 0.13% (w / v) /> hydroxybenzoic acid methyl ester (Sigma).

Twenty-four hours after the hatching of the adult fly adults, females and males were separated by brief, less than 2 minute, CO ₂ stunning. During the 24 hours after hatching, the animals were kept on standard fly food. Females and males were then separated and the females in 10 groups of approximately 20 animals in polystyrene vessels, height 9.5 cm, diameter 2.3 cm, closed with cotton wool, to 4 ml of special feed with or without the Compounds according to the formulas (9) or (25) held. The special food was made fresh every second week. Every second or third day, the flies were switched to fresh special food, and the dead animals were counted. The analysis of the life span took place with the Kaplan Meier analysis with the help of a statistics software (XLSTAT).

The Kaplan-Meier analysis showed a significant prolongation of the mean survival time for both the control ^animals of wild-type w ¹¹¹⁸ and for the mutants (w ¹¹¹⁸ , step ^k08U0 / step ^SH0323 ) when fed with the compounds according to formula (9) as shown in Table 2.

Table 2: Genotypes, treatment and survival times

_W ni8 kosiio SH0323 10 μM _{20 ()} 29.3 (± 0.7) F * _ compound (25) ^v '

The values show the average survival time in days (± standard error). The

Survival time and significance was assessed by the Kaplan Meier analysis with the help of

Software XLSTAT Life calculates, where: f P <0.001 against wl 118 (without treatment).

* P <0.05 vs wl 118 (without treatment). t P <0.001 against wl 118; stepkO8110 / stepSH0323 (no treatment). Control animals fed the compounds according to formula (9) had a significant (P <0.05) 11.5% prolonged average survival time compared to untreated control animals. While the mutants survived 19% (significance P <0.001) longer than control animals, feeding with compounds according to formula (9) resulted in a further prolongation of the average survival time of 15.3% (significance P <0.001), so that these Mutants had a 37.3% longer life span than untreated control animals.

This shows that administration of the compounds according to formula (9) can lead to a significant extension of the life span in flies.

A further increase in lifespan could be achieved in flies in which the Drosophila cytohesin steppke mutated and the amount of Drosophila cytohesin steppke protein was reduced.

This shows that the compounds according to the formula (9) are suitable for achieving a further increased extension of the life span. In particular, this shows that the extension of the life span of the mutant flies can be attributed to an inhibition of the fly cytohesin steppke.

In contrast, with the compounds according to the formula (25), neither in control animals nor in the mutants could a significant change in life span be achieved.

Example 8

Measuring food intake of flies

To show that the increased lifespan of the flies fed with the compound according to the formula (9) shown in Example 7 was not due to these For experimental groups due to the taste or odor of the compound according to formula (9) consumed less food and therefore lived longer, the food intake of flies was measured using the Capillary Feeder Assay (CAFE) and statistically evaluated.

As in Ja et al. (Ja et al., Prandiology of Drosophila and the CAFE assay., PNAS 2007 May 15; 104 (20): 8253-6). Five groups - two female adult flies were exposed for ten days to different dietary conditions with or without the compound of formula (9). The measurement of food intake per hour and fly was made on day 1, 2, 3, 5, 6, 7, 8, 9 and 10 of the ten days.

The control diet contained 5% (w / v) autolyzed yeast, 5% (w / v) α-D (+) - glucose monohydrate, 0.3% (w / v) p-hydroxybenzoic acid methyl ester, 2.1% ethanol (v / v), 0.34% (v / v) DMSO. The feed with the compound of formula (9) contained 5% (w / v) autolyzed yeast, 5% (w / v) α-D (+) - glucose monohydrate, 0.3% (w / v) p- Hydroxybenzoic acid methyl ester, 2.1% ethanol (v / v), 0.34% (v / v) DMSO, 10 μM compound according to formula (9).

It was found that the animals fed with the compound according to the formula (9) did not eat significantly less at a time than control feed animals. Thus, the observed extension of the life span by the compound according to the formula (9) is not due to a smaller amount of food.

Claims

Use of compounds selected from the group comprising the general formulas (1), (2), (3) and / or (4) as indicated below: wherein: R is the same or each independently selected from the group comprising hydrogen, OH, COOH, COO (Cl -C 10 -alkyl), CONH ₂ , CONH (Cl-ClO-alkyl), CON (Cl -C 10 -alkyl) ₂ , NHCO (C 1 -C 10 -alkyl), NHCOCHCl ₂ , halogen, preferably selected from the group comprising Cl, Br, F, CF ₃ , amine, C 1 -C 10 -alkyl, C ₁ -C 10 -alkoxy and / or a structural element ( Al), (B1), (C1), (D1), (E1), (F1), (G1), (H1), (II), (Y1), (L1), (M1) as indicated below: Wonn: R ₄ is the same or each independently selected from the group comprising hydrogen, OH, COOH, COO (C 1 -C 10 -alkyl), CONH ₂ , CONH (C 1 -C 10 -alkyl), CON (C 1 -C 10 -alkyl ) ₂ , NHCO (C 1 -C 10 -alkyl), NHCOCHCl ₂ , halogen, preferably selected from the group comprising Cl, Br, F, CF ₃ , amine, C 1 -C 10 -alkyl and / or C 1 -C 10 -alkoxy, and / or their enantiomers, diastereomers, derivatives and their pharmaceutically acceptable salts for the preparation of a medicament for the therapeutic and / or prophylactic treatment of diseases and pathological conditions associated with regulation of insulin and / or IGF (insulin-like growth factor) Signal pathway and / or the chemical induction of longevity. 2. Use of compounds according to claim 1, characterized in that the compounds selected from the group comprising the general formulas (1), (2), (3) and / or (4) are selected from the group comprising compounds of the general formulas (5), (6), (7) and / or (8) as indicated below and / or their enantiomers, diastereomers, derivatives and their pharmaceutically acceptable salts: (V) (8) Wonn: R, is the same or each independently selected from the group comprising R ₂ , R ₃ and / or a structural element (A2), (B2), (C2), (D2), (N2) as indicated below: R ₇ is the same or each independently selected from the group comprising R _h R ₃ and / or a structural element (E2), (F2), (G2), (H2), (12), (J2), (K2), (L2), (M2), (02) as indicated below: (E2) (F2) R. is the same or each independently selected from the group comprising R ₁ , R ₂ , and / or selected from the group comprising hydrogen, OH, COOH, COO (C 1 -C 10 -alkyl), CONH ₂ , CONH (Cl - C 10-alkyl), CON (C 1 -C 10 -alkyl) ₂ , NHCO (C 1 -C 10 -alkyl), NHCOCHCl ₂ , halogen, preferably selected from the group comprising Cl, Br, F, CF ₃ , amine, Cl- C10 alkyl and / or Cl-Cl O-alkoxy. 3. Use of compounds according to claim 1 or 2, characterized in that the compounds are selected from the group comprising compounds of general formulas (5), (6), (7), (8) as indicated below and / or their enantiomers , Diastereomers, derivatives and their pharmaceutically acceptable salts: Wonn: R, is the same or each independently selected from the group comprising R ₂ , R ₃ and / or a structural element (A3), (B3), (C3), (D3), (N3) as indicated below: / ^c H * CI (N3) R? is identical or in each case independently selected from the group comprising R ₁ , R ₃ and / or a structural element (E3), (F3), (G3), (H3), (13), (J3), (K3), ( L3), (M3), (03) as indicated below: R ₃ is the same or each independently selected from the group comprising R ₁ , R ₂ , and / or selected from the group comprising hydrogen, OH, COOH, COO (Cl -C 10 -alkyl), CONH ₂ , CONH (Cl - C 10-alkyl), CON (Cl- C 10 alkyl) ₂ , NHCO (C 1 -C 10 alkyl), NHCOCHCl ₂ , halogen, preferably selected from the group comprising Cl, Br, F, CF ₃ , amine, C ₁ -C 10 -alkyl and / or Cl-Cl O-alkoxy. 4. Use of compounds according to one of the preceding claims, characterized in that R, R ₁ , R ₂ , R ₃ and / or R _{4 are the} same or each independently selected from the group comprising C 1 -C 5 -alkyloxy, preferably selected from Group comprising -O-methyl, -O-ethyl, -O-isopropyl and / or -O-tert-butyl. 5. Use of compounds according to one of the preceding claims, characterized in that the compounds are selected from the group comprising compounds (9), (10), (11), (22), (23) as indicated below and / or their Enantiomers, diastereomers, derivatives and their pharmaceutically acceptable salts: H ₃ C- (9) (22) 6. Use of compounds according to one of the preceding claims, characterized in that the compounds are selected from the group comprising compounds (12), (14), (20), (21) as indicated below and / or their enantiomers, diastereomers, Derivatives and their pharmaceutically acceptable salts: (12) (21). 7. Use of compounds according to one of the preceding claims, characterized in that the derivative is a biotinylated compound, preferably compound (24) as indicated below and / or their enantiomers, diastereomers and their pharmaceutically acceptable salts (24). 8. Use of compounds according to one of the preceding claims, characterized in that the diseases and pathological conditions associated with a regulation of the insulin and / or IGF signaling pathway are selected from the group comprising obesity, cell aging, aging-related cell damage , in particular in liver and / or pancreas, age-related pathological conditions of liver and / or pancreatic cells, age-related dysfunction of liver and / or pancreas, cell stress, in particular oxidative stress, especially by increased sugar metabolism-induced stress, and / or apoptosis, in particular beta-cell apoptosis. Use of compounds according to any one of the preceding claims for prolonging the age of mammals. Use of compounds according to any one of the preceding claims, characterized in that the medicament is formulated for oral or intravenous administration. 11. Use of compounds according to one of the preceding claims, characterized in that the solvent is selected from the group comprising DMSO, glycerol and / or oil, preferably selected from the group comprising DMSO and / or vegetable oil, in particular olive oil.