EP2101753A2 - Utilisation d'inhibiteurs de la cytohésine pour l'induction chimique de longévité - Google Patents

Utilisation d'inhibiteurs de la cytohésine pour l'induction chimique de longévité

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Publication number
EP2101753A2
EP2101753A2 EP07822588A EP07822588A EP2101753A2 EP 2101753 A2 EP2101753 A2 EP 2101753A2 EP 07822588 A EP07822588 A EP 07822588A EP 07822588 A EP07822588 A EP 07822588A EP 2101753 A2 EP2101753 A2 EP 2101753A2
Authority
EP
European Patent Office
Prior art keywords
group
alkyl
compounds
insulin
indicated below
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07822588A
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German (de)
English (en)
Inventor
Michael Famulok
Markus Hafner
Anton Schmitz
Bernhard Fuss
Ingo Zinke
Michael Hoch
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rheinische Friedrich Wilhelms Universitaet Bonn
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Rheinische Friedrich Wilhelms Universitaet Bonn
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Application filed by Rheinische Friedrich Wilhelms Universitaet Bonn filed Critical Rheinische Friedrich Wilhelms Universitaet Bonn
Publication of EP2101753A2 publication Critical patent/EP2101753A2/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4196—1,2,4-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • 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.
  • IGF Insulin-like growth factor
  • 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.
  • 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,
  • 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.
  • IGF insulin-like growth factor
  • IGF insulin-like growth factor
  • 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).
  • 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).
  • 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.
  • 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.
  • prophylactic treatment it is advantageous that, for example, obesity can be prevented by a prophylactic treatment.
  • 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:
  • 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:
  • R h R 3 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 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.
  • 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).
  • 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).
  • the structural element R 3 of the structural elements (E2), (F2), (G2), (H2), (12), (J2), (K2), (L2), (M2) and / or (02) is hydrogen ,
  • 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:
  • 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:
  • R h R 3 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:
  • 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.
  • 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).
  • 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).
  • 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.
  • the compounds which can be used according to the invention have a significantly improved effect, the smaller the alkoxy groups are.
  • 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.
  • 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).
  • 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.
  • 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:
  • 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:
  • 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.
  • biotinylated compound particularly preferred is, for example, compound (24) as indicated below and / or their enantiomers, diastereomers and their pharmaceutically acceptable salts
  • 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.
  • 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.
  • 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.
  • 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.
  • the compound according to the formula (9) has been shown to have a positive effect on the life span in vivo.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Human HepG2 cells 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) 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 Spezialdi decisiven GmbH) ad libitum. Male animals were used between the ages of 8 and 12 weeks.
  • 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 "GeBruzmühle Brecht"), 1.1% (w / v) brewer's yeast (company “GeBruzmühle 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
  • IGFBPl insulin-like growth factor binding protein
  • 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 ⁇ M, 3.125 ⁇ M, 6.25 ⁇ M, 12.5 ⁇ M, 25 ⁇ M or 50 ⁇ M 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.
  • IGFBPl insulin-like growth factor binding protein
  • Serum HepG2 cells (ECACC) were removed from the medium for 90 minutes, cells were incubated for an additional 60 minutes with 5 ⁇ M, 10 ⁇ M and 15 ⁇ M 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 ⁇ -glycerophosphate, 1 mM sodium vanadate, 1% Triton X. -100) and protease inhibitor mix HP (Serva).
  • lysis buffer (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphat
  • the compound according to the formula (9) inhibited the insulin-dependent phosphorylation of both proteins Akt and FoxOlA in a concentration-dependent manner.
  • application of 10 ⁇ M resulted in a 50% reduction in Akt phosphorylation compared to phosphorylation following insulin stimulation
  • 15 ⁇ M of Formula (9) compound reduced Akt phosphorylation to approximately 25% phosphorylation Insulin stimulation resulted.
  • mice received standard mouse feed mixed with 0.9 ⁇ mol / 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 ⁇ l of physiological saline (control) or saline with 40 ⁇ g of recombinant human insulin (Sigma).
  • 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 ⁇ -glycerophosphate , 1 mM sodium vanadate, 1% Triton X-100) and protease inhibitor mix HP (Serva).
  • 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 ⁇ -glycerophosphate , 1 mM sodium vanadate, 1% Triton X-100
  • protease inhibitor mix HP Serva
  • 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 ⁇ g of RNA using the High Capacity cDNA Archive Kit (Applied Biosystems) according to the manufacturer's instructions.
  • Quantitative PCR was performed in 10 ⁇ l 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 ⁇ 2-microglobulin expression.
  • Gck glucokinase
  • Gckr its regulator
  • PkIr liver pyruvate kinase
  • Hk2 hexokinase 2
  • 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.
  • 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 ⁇ l (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.
  • 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 ⁇ M)
  • human insulin Sigma
  • 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 ⁇ M sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride and EDTA-free protease inhibitor (Roche) according to the manufacturer's instructions, and used for Western blot analysis.
  • 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).
  • 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 ⁇ M, 10 ⁇ M and 100 ⁇ M. This was followed by 4-hour stimulation with 10 ⁇ g / ml human insulin (Sigma).
  • PAN Schneider's Medium
  • FCS heat-inactivated fetal calf serum
  • 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.
  • 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) ⁇ -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) ⁇ -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 ⁇ M 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) ⁇ -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 ⁇ M compound according to formula (25 ).
  • the larvae were grown on standard fly feed containing 1.1% (w / v) brewer's yeast (company “GeBruzmühle Brecht”), 5.43% (w / v) maize flour, 0.53% (w / v) Fadenagar (company “ GeBruzmühle 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).
  • the values show the average survival time in days ( ⁇ standard error).
  • 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.
  • Example 7 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
  • the food intake of flies was measured using the Capillary Feeder Assay (CAFE) and statistically evaluated.
  • CAFE Capillary Feeder Assay
  • the control diet contained 5% (w / v) autolyzed yeast, 5% (w / v) ⁇ -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) ⁇ -D (+) - glucose monohydrate, 0.3% (w / v) p- Hydroxybenzoic acid methyl ester, 2.1% ethanol (v / v), 0.34% (v / v) DMSO, 10 ⁇ M compound according to formula (9).

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Abstract

L'invention concerne les composés choisis dans le groupe contenant les formules générales (1), (2), (3) et/ou (4) et/ou leurs énantiomères, diastéréomères, dérivés et leurs sels pharmaceutiquement compatibles pour la production d'un médicament destiné au traitement thérapeutique et/ou prophylactique de maladies et d'états pathologiques qui sont en liaison avec une régulation de la voie de signalisation de l'insuline et/ou du facteur de croissance insulinomimétrique IGF et/ou pour l'induction chimique de longévité.
EP07822588A 2006-11-15 2007-11-14 Utilisation d'inhibiteurs de la cytohésine pour l'induction chimique de longévité Withdrawn EP2101753A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102006054205A DE102006054205A1 (de) 2006-11-15 2006-11-15 Verwendung von Cytohesin-Inhibitoren zur chemischen Induktion von Langlebigkeit
PCT/EP2007/062337 WO2008058995A2 (fr) 2006-11-15 2007-11-14 Utilisation d'inhibiteurs de la cytohésine pour l'induction chimique de longévité

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CA2693001A1 (fr) * 2008-04-16 2009-10-22 University Of Utah Research Foundation Compositions et methodes de traitement de l'angiogenese pathologique et de la permeabilite vasculaire
AU2011312128B2 (en) * 2010-10-07 2015-12-10 University Of Louisville Research Foundation, Inc. IGF-1 dependent modulation of VSELs
DK3186242T3 (da) 2014-08-29 2021-12-20 Tes Pharma S R L Alfa-amino-beta-carboxymuconsyre-semialdehyd-decarboxylasehæmmere
WO2019101647A1 (fr) 2017-11-21 2019-05-31 Bayer Aktiengesellschaft 2-phénylpyrimidine-4-carboxamides à utiliser en tant qu'inhibiteurs d'ahr

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BR0116792A (pt) * 2000-12-22 2004-02-17 Ortho Mcneil Pharm Inc Derivados de diamina de triazol substituìdos como inibidores de quinase
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DE102004055998A1 (de) * 2004-11-19 2006-05-24 Rheinische Friedrich-Wilhelms-Universität Bonn Niedermolekulare Inhibitoren von Guaninnucleotid-Austauschfaktoren der Cytohesin-Familie
WO2006087718A1 (fr) * 2005-02-17 2006-08-24 Yissum Research Development Company Of The Hebrew University Of Jerusalem Prolongement de duree de vie avec des medicaments
JP2006342116A (ja) * 2005-06-10 2006-12-21 Kyorin Pharmaceut Co Ltd ピリミジン−５−カルボキサミド誘導体

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DE102006054205A1 (de) 2008-05-29

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