EP1102585A2

EP1102585A2 - USE OF CYCLOPENTABENZOFURAN-DERIVATIVES FOR COMBATING NF-$g(k)B-DEPENDENT DISEASES

Info

Publication number: EP1102585A2
Application number: EP99944303A
Authority: EP
Inventors: Matthias Gehling; Jörg Baumgarten; Axel Kretschmer; Horst-Peter Antonicek; Peter Proksch; Bambang W. Nugroho; Frank Bohnenstengel
Original assignee: Bayer AG
Current assignee: Bayer AG
Priority date: 1998-08-05
Filing date: 1999-07-29
Publication date: 2001-05-30
Also published as: WO2000007579A3; US6518274B1; WO2000007579A2; DE19835325A1; AU5729099A

Abstract

The invention relates to the use of cyclopentabenzofuran derivatives for the preparation of a medicament for the treatment of nuclear factor kappa B-dependent diseases.

Description

Use of cyclopentabenzofuran derivatives to combat ΝF-κB dependent diseases

The invention relates to the use of cyclopentabenzofuran derivatives

Manufacture of a drug for the therapy of NF-κB dependent diseases.

Extracts from the Aglaia elliptifolia plant show anti-leukemic properties. A dihydrocyclopenta- benzofuranol derivative called rocaglamide was identified as the first active compound (J. Chem. Soc, Chem. Commun. 1982, 1150; US

4 539 414). Thereupon several works appeared about finally also successful attempts at synthesis. It was only 10 years after the isolation of rocaglamide that its insecticidal properties were described (Pestic. Sei 36, 53 (1992); Phytochemistry 32, 67 (1993)) and then in another species, Aglaia odorata, three more, only in one substituent differentiating derivatives found (Phytochemistry 32, 307

(1993)).

Later e.g. first isolated four fused derivatives of Rocaglamid from the species Aglaia roxburghiana (WO 96/04 284), then numerous other new derivatives and their pharmacological properties are described (see e.g. J. Nat. Prod 59, 650

(1996); Tetrahedron 52, 6931 (1996); Phytochemistry 44, 1455 (1997); Phytochemistry 45, 1579 (1997); Z. Naturforsch., C: Biosci. 52, Tetrahedron 52, 17625 (1997); B.W. Nugroho, dissertation, Bayer. Julius-Maximilian Univ. Würzburg, 1997, WO 97/08 161 AI, J. Nat. Prod. 6_, 1482 (1998), Tetrahedron 53, 17625 (1997).).

An important step in many inflammatory processes is the translocation of the

Protein "Nuclear Factor kappa B", NF-κB for short, in the cell nucleus and the resulting stimulation of the expression of the genes, the products of which are responsible for inflammatory reactions (Trends Pharmacol. Sci. J_8, 46 (1997)). wise in asthma, the non-useful, excessive (non-self-limiting) production of these proteins is responsible for the enhancement and maintenance of the inflammatory process and the associated unpleasant to life-threatening symptoms of this disease. Because the long-term treatment with glucocorticoids corresponding to the current state of the art has some disadvantages, NF-κB is seen as an imperative target for the development of new anti-inflammatory agents against asthma.

It has now been found that the cyclopentabenzofuran derivatives of the formula (I)

in which

[A] R ^{1 represents} hydrogen,

R ^{2 represents} methoxy,

R ^{3 represents} hydrogen or

R and R together represent -OCH ₂ O-,

R ^{4 represents} methoxy,

R ^{5 represents} hydroxy, OCHO or acetoxy, R ⁶ and R ⁷ each represent hydrogen or

R ⁵ and R ⁶ together represent oxygen (oxo) or hydroxyimino,

R ^{8 represents} -COOR ¹² or -CONR ¹³ R ' ⁴ , wherein

R ¹² and R ^{13 represent} hydrogen or methyl and

R ^{14 represents} hydrogen, methyl, 4-hydroxybutyl or 2-tetrahydrofuryl,

or

R ^δ for a residue of the formula

stands,

R ⁵ and R ⁸ together for a group of foπneln (a) or (b)

stand, wherein the linking point adjacent to the N atom corresponds to R ⁵ and, moreover, R ⁶ and R ⁷ together stand for a direct bond,

R ^{9 represents} phenyl,

R ^{10 represents} methoxy,

R "represents hydrogen, hydroxyl, methoxy or 2-rhamnosyl, or

R ¹⁰ and R "are adjacent and together represent -OCH ₂ O-, or

R ^{15 represents} hydroxy, methoxy or ethoxy,

R ^{16 represents} hydrogen, hydroxy or methoxy,

[B] R ¹ , R ³ and R ⁸ each represent hydrogen,

R ² and R ⁴ each represent methoxy,

R ^{5 represents} hydroxy,

R ⁶ and R ⁷ each represent hydrogen or

R ⁵ and R ⁶ together represent oxygen (oxo group),

R ^{9 represents} phenyl,

R ^{10 represents} methoxy,

R "represents 2-methoxy or 2-rhamnosyl, or R and R are adjacent and together represent -OCH ₂ O-,

Suitable as inhibitors are the nuclear factor kappa B (NF-κB) -interrupted gene expression for the therapy of pathophysiological processes.

The substances that can be used according to the invention are from o.a. Literature known.

Examples of the substances of the formula (I) which can be used according to the invention are the compounds (1-1) to (1-45), which are listed below.

Table 1

(1-27) (1-28)

(1-29)

Table 2

Table 3

*) Cui et al., Tetrahedron 53_, 17625 (1997)

Table 4

*) Brader et al., J. Nat. Prod. 61, 1482 (1998).

Of the above-mentioned substances of the general formula (I) are the compounds

Il, 1-2, 1-3, 1-4, 1-5, 1-6, 1-8, 1-10, 1-12, 1-13, 1-15, 1-19, 1-20, 1-22, 1-26, 1-28. 1-32, 1-33, 1-42 preferred. The substances which can be used according to the invention are low molecular weight inhibitors which selectively inhibit nuclear factor kappa B (NF-κB) -mediated pathophysiological processes. NF-κB-mediated processes occur in inflammatory diseases, immunological diseases, septic shock, graft rejection, radiation damage, reperfusion injuries after ischemia, thrombosis or in complex, chronic inflammatory diseases such as arteriosclerosis.

The pharmacological effects of inhibitors of the nuclear factor kappa B

Nuclear factor kappa B (NF-κB) is a dimeric protein complex found in many tissue cells and especially in blood cells. NF-κB plays a special role in controlling the expression of genes which have an NF-κB binding sequence in their promoter sequence (5'-GGGPuNNPyPyCC-3 '). In this respect, NF-KB is a transcription factor. However, the physiological activity of NF-κB to control gene expression is subject to a regulatory principle in which NF-κB is released from a complex with the protein IκB in order to translocate as a transcription factor into the cell nucleus for gene activation. The regulatory principle for the release of active NF-κB from a complex with the protein IκB is not yet known in detail.

It is also not known how homodimeric and heterodimeric NF-KB protein complexes are formed. NF-κB acts as a dimeric transcription factor on gene activation. The dimerization can take place under the structurally related transcription factors Rel A, Rel B, c-Rel, p50 or p52, which form a family of transcription factor proteins. Also, in the dimerization of the subunits to the NF-κB, there can already be a regulatory principle for controlling the genes described in more detail later, which is not yet known. A key feature of NF-κB over other transcription factors is that NF-κB is a primary transcription factor. Primary transcription factors are already in the cell in an inactive (mostly complex-bound) form and are released after a corresponding stimulus in order to be able to develop their effects very quickly. Primary transcription factors are not only formed by activating the associated gene and subsequent transcription and translation.

This property of NF-κB, the formation of homodimeric or heterodimeric Rel proteins and the formation of an inactive protein complex with an IκB

Protein, offer completely different targets for pharmacologically active substances than the targets of de novo biosynthesis of transcription factors. For the sake of completeness it should be mentioned that the genes for the formation of NF-κB (genes of the Rel family) and the genes for the formation of the IκB proteins (gene family comprising the genes for IκB-α, IκB-β, pl05 / IκB-γ, plO0 / IκB-δ, IκB-ε and others) are of course also subject to regulation, which can be points of attack for pharmaceutically active substances. It is known, for example, that the expression of the consumed IκB proteins pl05 and plOO is increased by stimuli which also activate NF-κB, e.g. Tumor necrosis factor-α (TNF-α) or phorbol myristate acetate (PMA).

A regulatory mechanism is described in the literature, in which it is shown that the overexpression of IκB binds active NF-κB and thus inactivates it. This also applies if the NF-κB has already complexed with the DNA (PA Baeuerle, T. Henkel, Annu. Rev. Immunol. 12, 141-179, 1994). It can be concluded that there are several specific targets in the biochemical function of NF-κB and IκB proteins, which should make it possible to selectively inhibit undesired, pathophysiological, NF-κB-dependent gene activation. A chemical compound that selectively inhibits the function of NF-κB or enhances the function of IκB proteins or IκB genes should be used as a pharmaceutical for the suppression of NF-κB-mediated disease processes.

NF-κB can primarily promote all pathophysiological processes in which genes are involved which have the NF-κB binding sequence in their promoter. These are genes that play a decisive causal role in immunological complications, inflammatory diseases, autoimmune diseases, septic shock, transplant rejection, thrombosis or even in chronic inflammatory diseases such as arteriosclerosis, arthritis, rheumatism and psoriasis.

NF-κB binding sequences contain e.g. the promoters of lymphoid cell receptors (T cell receptors), of MHCI and MHCII genes, of cell adhesion molecules (ELAM-1, VCAM-1, ICAM-1), of cytokines and growth factors (see also the table below). Furthermore, NF-κB binding sequences can be found in the promoters of acute phase proteins (angiotensinogen, complement factors, etc.).

A chronically increased or acutely excessive activation of the genes mentioned leads to a variety of pathophysiological processes and syndromes.

The rapid and excessive production of cytokines of the inflammatory reaction (TNFα, Interleukin-2, Interleukin-6, Interleukin-8 and others) and the adhesion molecules (ELAM-1, ICAM-1, VCAM-1) in leukocytes, especially in Macrophages and also in endothelial cells, a causal characteristic of processes that are often fatal in septic shock; or leads to significant complications in radiation damage and graft rejection. Inhibitors that prevent NF-κB-mediated gene expression intervene very early in the manifestation of pathophysiological changes in some diseases and can therefore be very represent effective therapeutic principle. NF-κB inhibitors for diseases that are due to overexpression of acute phase proteins are also an example. Unwanted overexpression of acute phase proteins can cause a complex general reaction, in which tissue damage of various types, fever and local symptoms such as inflammation and necrosis can occur. Mostly the blood picture is changed. For example, NF-κB strongly induces the serum amyloid A precursor protein in the liver by inducing acute phase proteins.

For example, NF-κB-mediated gene expression of interleukin-2

(Il-2) Gens are inhibited.

Interleukin-2 is a cytokine that i.a. as a hematopoietic growth factor plays a central role in various inflammatory processes (Annu. Rev. Immunol 1994, 12: 141-79). The promoter of the interleukin-2 gene is dependent on NF-κB. A

Inhibition of NF-κB stimulation thus opens up the possibility of preventing oversupply of 11-2 production and thus of treating inflammatory processes.

In other clinical pictures such as tissue damage after reperfusion or cirrhosis of the liver, inhibitors of NF-κB-mediated gene expression can also represent a significant therapeutic advance. There is evidence that NF-κB-controlled genes are induced by oxidation reactions that lead to oxidative stress after reperfusion of ischemic tissue. In this way, an overexpression of cytokines and cell adhesion molecules in the ischemic tissue triggers an excessive recruitment of infiltrating lymphocytes. The recruited lymphocytes cause tissue damage.

The involvement of NF-κB-controlled gene expression in several neurodegenerative diseases is also evident. The selective inhibition of genes with NF-κB binding sequence becomes therapeutic in particular in the case of nerve diseases in which the redox state of cells of the neuronal tissue is disturbed Attributed benefits. A disrupted redox state of neuronal cells is assumed in amytrophic lateral sclerosis and in Down syndrome.

It is known that NF-κB is a common transcription factor in neuronal tissue and that NF-κB is a redox potential-controlled transcription factor in the brain (PA Bauerle, T. Henkel, Annu. Rev. Immunol. 12, 141-179, 1994 ). A listing of the genes induced by NF-κB is given in Table 2.

Table 2

Genes Induced by NF-κB (P.A. Bauerle, T. Henkel, Annu. Rev. Immunol. 12, 141-179, 1994)

Immunoreceptors Immunoglobulin K light chain

T cell receptor ß

T cell receptor α chain (human)

Major histocompatibility complex class I

(H-2K) ß ₂ -microglobulin

Invariant chain I

Tissue factor- 1

Cell adhesion molecules Endothelial leukocyte adhesion molecule 1

(ELAM-1)

Vascular cell adhesion molecule 1

(VCAM-1)

Intercellular cell adhesion molecule 1

(ICAM-1) *

Cytokines and growth factors ß-interferon

Granulocyte / macrophage colony-stimulating factor (GM-CSF)

Granulocyte colony-stimulating factor

(G-CSF)

Macrophage colony-stimulating actor

(M-CSF)

Melanoma growth stimulating activity

(groα-γ / MGSA)

Interleukin-2

Interleukin-6

Interleukin-8

TNFα

Lymphotoxin (TNF-ß)

Proenkephalin

MPC-l / JE *

Acute phase proteins angiotensinogen

Serum amyloid A precursor

Complement factor B

Complement factor c4

Urokinase-type plasminogen activator *

* The binding of NF-κB to the promoter of the gene mentioned has not yet been conclusively proven experimentally. In addition to the genes already mentioned, whose activity is controlled with the release of NF-κB and which play a role particularly in inflammatory processes, septic shock and graft rejection, NF-κB-controlled genes in viruses and those which cause oncogenic cellular changes are also mentioned cause

(Oncogenes such as c-myc, c-rel, Melanoma Growth Stimulating Activity MGSA). With these genes too, selective inhibition of NF-κB binding represents a promising, therapeutically usable concept. The gene expression of lymphotropic viruses such as HIV, HTLV and Epstein-Barr virus is activated either directly or by NF-κB or in the infected host cell NF-κB is induced, which is conducive to virus replication. In addition to HIV, NF-κB has a positive effect on gene expression in the cytomegalovirus (CMV) and adenovirus. Antiviral effects with NF-κB inhibitors are also conceivable here.

The use of the substances which can be used according to the invention can be seen from the examples below.

Application examples

Example A

Inhibition of NF-κB-mediated gene expression of the tumor necrosis factor-α (TNFα) gene in human monocytes

The promoter of the human TNFα gene contains three NF-κB binding sequences, which are designated as kl, k2 and k3. The NF-κB binding sequences are found in the promoter of the TNFα gene at the nucleotide positions kl = -587 to -577, k2 = -210 to -202 and k3 = -98 to -87 and these DNA sequences bind NF-κB specifically ( AE

Goldfield et al., Proc. Natl. Acad. Scri. USA 87, 9769-9773, 1990). Lipopolysaccharide or phorbol ester (such as phorbol myristate acetate) induce NF-κB release (M.J. Lenardo et al., Cell 58, 227-229, 1989).

The following biological test is therefore carried out to detect the inhibition of NF-κB-mediated TNFα gene expression by the substances described here.

Donor blood mononuclear cells are isolated using Vacutainer CPT (™) tubes (Becton Dickinson and Company, Franklin Lakes, NJ. 07417-1885) according to the manufacturer's instructions. The Vacutainer CPT tubes contain 1.0 ml of phosphate-buffered saline with 120 USP units of sodium heparin over 3.0 g of a polyester gel that covers 2.0 ml of a Ficoll solution. After centrifugation of the donor blood, the monocytes are taken from a zone above the polyester gel and ind with a cell density of 250 x 10 ⁵ cells per well

96-well microtiter plates sown for cell culture.

The cells are kept in medium RPMI-1640 (Gibco BRL, Life

Technologies GmbH, Dieselstr. 5, 76344 Eggenstein). The culture supernatant is then aspirated, medium RPMI-1640 is added again and the substances to be tested are usually added in concentrations between 0 μM (negative control) and 20 μM. Bacterial lipopolysaccharide (LPS), from Sigma-Aldrich Chemie GmbH, Grünwalder Weg 30, 82039 Deisenhofen, order no .: L 4391, in a concentration of 125 ng / ml for stimulating NF-κB-mediated TNFα is immediately followed Gene expression added. After a further incubation of 18 hours at 37 ° C. in a 5% CO ₂ atmosphere, culture supernatant is removed from the microtiter plates and the TNFα content is quantified therein using commercially available enzyme-linked immunosorbent assays (ELI SA).

TNFα ELISA for concentration determination are e.g. from Sigma-Aldrich Chemie GmbH, Grünwalder Weg 30, 82039 Deisenhofen under the name Human TNFα ELISA Kit, order no .: CKH-200A. According to the manufacturer's instructions for use, the concentration of the TNFα formed in the culture supernatant of the monocyte culture after LPS stimulation with and without inhibitor substance is determined quantitatively.

The results of the TNFα concentration determination are plotted against each other in an x-y-axis diagram. The graph of the y coordinates (TNFα concentration in the culture supernatant) and the x coordinates (concentration of the used

Inhibitors) allows the inhibition of NF-κB-mediated TNFα synthesis to be read as a function of the concentration of the inhibitor substance. In this way, the active substance concentration of the inhibitor added which, for example, inhibits TNFα synthesis by 50% can be read off from the diagram. This active ingredient concentration, which causes 50% inhibition, is called the effective inhibitor concentration for 50% inhibition (IC ₅₀ ).

For example, the concentrations of the half-maximum TNFα synthesis inhibition (IC ₅₀ ) of compound 1-2 (from Table 1) are 0.3 μM, for compound 1-15 1.0 μM and 0.5 μm for connection 1-21. The compounds thus represent potent inhibitors of NF-κB-mediated TNFα synthesis.

Example B Inhibition of NF-κB-Mediated Gene Expression in Human Tissue

Factor gene in monocytes

Tissue factor is a membrane-bound protein that is the primary initiator of the blood coagulation cascade and a key function in cardiovascular diseases such as unstable angina pectoris, acute implications after plaque

Rupture, vascular occlusion of various etiology, arteriosclerotic processes and other diseases such as septic shock or cancer. The tissue factor gene is particularly induced in monocytes and endothelial cells by NF-κB activation. The promoter of the human tissue factor gene contains an NF-κB binding site which contributes decisively to the activation of the promoter (P. Oeth et al. Arteriosclerosis, Thrombosis, and Vascular Biology 17, 365-374, 1997).

The following biological test was therefore carried out to further demonstrate the inhibition of NF-κB-mediated gene expression by the inhibitors according to the invention:

The promoter fragment of the human tissue factor gene, which contains the NF-κB binding sequence, was with oligonucleotide primers of the sequence 5'-TCC CTC GAG ATC TCC CAG AGG CAA ACT GCC AGA T-3 '(5' primer of the position -925) and 5 '-TCC TCG AGC CAT GGC TAC CAG TTG GGC GGC GAG ATC-3' (3 'primer containing the ATG start codon of the coding sequence of the tissue factor gene) by the polymerase chain reaction (PCR) and fused by Ncol / Xhol cloning with the luciferase start codon in the plasmid pGL3-Basic Vector (Promega Corp. 2800 Woods Hollow Road, Madison, WI 5371 1-5399 USA). Thereby the expression of the luciferase in the resulting recombinant Plasmid regulated by the human tissue factor promoter. This expression construct was subjected to the analysis of DNA sequencing and transfected into the monocyte cell line RAW 264.7 (American Type Culture Collection 12301 Parklane Drive, Rockville, Maryland 20852, USA). The transfection, selection and clone analysis were carried out according to standard methods as described (see

Transfection of Mammalian Cells in Culture, L.G. Davis et al. Basic Methods in Molecular Biology, Elsevier Sei. Publishing Co., New York 1986). After selecting RAW clones that had the expression construct stably integrated into the genome, one of these transfectants, called RAW-A3, was selected for the test of the inhibitors.

Test execution:

106 RAW-A3 cells are sown in each well in 12-well microtiter plates. The serum concentration in RPMI medium is gradually reduced from 10% fetal calf serum to 0.5% and 0.1% serum content in three days in order to minimize the serum-dependent tissue factor promoter activation. After 24 hours of culture in medium with 0.1% serum, the NF-κB inhibitor is added and then serum is added up to a concentration of 15% in the medium for promoter induction.

After a further 6 hours, the culture supernatant is aspirated and the cells are used to measure the luciferase activity in accordance with the "Luciferase Assay System" (Technical Bulletin from Promega, 2800 Woods Hollow Road, Madison, WI 5371 1-5399 USA) E4030, E1483, E1501) The cell lysate is incubated with the luciferase assay substrate and in a luminometer

Measure the measurement of the emitted light. When plotted in an xy-axis diagram with the respective emitted light (specified in relative light units) as the y coordinate and the inhibitor concentrations as the x coordinate, a graph for f (x) results, in which the half-maximum y value of an inhibitor - concentration x is to be assigned, which corresponds to the inhibitory concentration IC ₅₀ in this test. For example, the IC _J0 value of compound 1-2 (from Table 1) is 0.4 μM and compound 1-21 4.0 μM. The NF-κB-mediated expression of luciferase by the inhibitors according to the invention with a concentration in the lower micromolar or in the submicromolar range indicates the high effectiveness of the

Inhibition of NF-κB-mediated gene expression.

Example C

Inhibition of NF-κB-mediated gene expression of the tumor necrosis factor-α (TNFα) gene in human monocytes as a function of three different TNFα synthesis stimuli

The NF-κB-mediated induction of TNFα synthesis is independent of the different stimuli used by LPS, opsonized zymosan or phorbol myristate acetate (PMA) for the activation of the TNFα promoter

(A. Baldwin, Annual Rev. Immunology 14, 649, 1996). Therefore, the use of the inhibitors according to the invention should also lead to a comparably strong inhibition of TNFα synthesis regardless of the type of stimulation of TNFα synthesis.

The tests for this detection of the stimulus-independent inhibition of TNFα synthesis were carried out in the manner of the test execution exactly as described under Example A, with the difference that in addition to LPS as a stimulus, cell culture batches were also stimulated with 100 nM PMA or with 100 μg / ml opsonized zymosan . Zymosan and phorbol myristate acetate (PMA) can be obtained from Sigma,

Grünwalder Weg 30, 82041 Deisenhofen, Germany under the order numbers. Z4250 and P8139 are available. Zymosan is opsonized in human serum.

The NF-κB inhibitors according to the invention inhibit TNFα in a comparable manner regardless of the stimulus with IC ₅₀ values in the submicromolar range Synthesis in human monocytes as shown in Example A. For compound 1-2, the IC ₅₀ value is 0.4 μM after PMA stimulus and 0.4 μM after zymosan stimulus.

Example D

Inhibition of NF-κB Mediated Gene Expression of the Adhesion Protein ELAM-1 on Human Umbilical Cord Endothelial Cells (HUNEC)

The recruitment of leukocytes from the blood circulation into the extravascular space is essential for inflammatory responses and for the repair of

Damaged. The process of leukocyte immigration involves several steps in series. The initial interaction between leukocytes and the endothelium of the blood vessels is mediated by TΝF and II-1-dependent expression of the adhesion protein ELAM-1 on the endothelium. It mediates the so-called rolling of the leukocytes along the blood vessel wall. The transcriptional regulation of the ELAM-1 expression depends on the nuclear factor-κB (NF-κB) activation and binding to the ELAM-1 promoter as well as on the "AP-1 binding site." MA. Read et al., J. Biol. Chem. Vol. 272, 2753-2761, (1997).

The influence of compound 1-2 on the expression of ELAM-1 was checked in two different test approaches. The adhesion of human neutrophils to TNF-α stimulated HUVEC cells was measured in a functional approach. The expression of ELAM-1 on the surface of the HEVECs was determined using a fluorescence-labeled ELAM-1-specific monoclonal antibody by means of FACS (cell sorting) analysis. Test procedure: ELAM-1 dependent neutrophil adhesion

Endothelial cells

The neutrophils were isolated from human blood (100 ml). For this purpose, 3.5 ml of polyprep was placed in a centrifuge beaker and carefully overlaid with 5 ml of blood. After centrifuging at 2100 min ^{-1 for} 30 minutes, the neutrophil band was sucked off in the middle of the centrifuge cup. . After 1: 2 dilutions in Endothelial Cell Basal Medium (EBM) Fa Clonetics again for 20 minutes at 1000 has been min ^- centrifuged ¹ ml and then taken up to a cell concentration of 10 ⁶ cells /.

The umbilical cord endothelial cells were grown to confluence in 96-well microtiter plates in EBM + 10% FCS. At the start of the test, the medium was exchanged for EBM without FCS and the test substances were then used. After 20 minutes, the HUVECs were stimulated with 10 nM TNF-α. After 4 hours of incubation, 200 μl / well of the neutrophil suspension were exchanged. The neutrophils were previously labeled with a 25 μM BCESP fluorescent dye solution for 20 minutes. After 30 minutes of incubation, the BCECP neutrophil solution was drawn off with excess neutrophils and exchanged for 200 μl / well of 0.5% NaOH solution. The

Fluorescence of the adhering neutrophils was then measured in the fluorescence photometer.

Both the adhesion of the neutrophils to TNF-α stimulated HUVECs and the expression of ELAM-1 on the cell surface could be inhibited by 50%. The maximum inhibition of cell adhesion was determined to be 10 nM for compound 1-2. The maximum 50% inhibition of ELAM-1 expression is in good agreement with the NF-κB and AP-1-dependent regulation of the ELAM-1 promoter. Carrying out the experiment: Quantitative measurement of the TNF-induced expression of ELAM-1 in HUVECs:

Umbilical cord endothelial cells (HUVEC) were cultured as described above and incubated for 4 hours in the presence or absence of 10 ng / ml TNF in the presence or absence of test substances. The cells were extracted from the microtiter plates by incubation with 5 mM EDTA in PBS, centrifuged for 5 minutes at 1000 min ^-1 , and in 100 μl PBS plus 1% bovine serum albumin (BSA, Sigma-Aldrich GmbH, order no. A7906) in an anti-EL AM 1 antibody (monoclonal antibody, from Becton and Dickinson,

Erembodegem, Belgium, order no. 550 023, 30 μg / ml) at RT. After 15 minutes the cells were centrifuged again, the supernatant was discarded and the cells were washed in PBS plus 1% BSA. After renewed centrifugation, the cell pellet obtained was in PBS plus 1% BAS and a goat anti-mouse antibody (Dianova, Raboisen 5, Hamburg, Order No. 1 15-096-062; 30 μg / ml) for the duration incubated for 15 minutes. After centrifuging and washing again, the cells were taken up in 1 ml of PBS plus 1% BSA and measured in a flow cytometer (from Becton and Dickinson) at 488 nm. With this method, the intensity of the fluorescence is measured as a function of bound anti-ELAM-1 antibody per cell. 5000 cells were measured for each value.

HUVECs stimulated with TNF for 4 hours showed significantly stronger fluorescence signals after incubation with anti-ELAM-1 antibodies than cells, on the other hand, which were not incubated with TMF. Compound 1-2 was able to induce this induced expression of ELAM-1 in a concentration range between

Significantly inhibit 0.05 μM and 5 μM, however, the expression of ELAM-1 was not completely inhibited at any of the concentrations examined. Example E

Inhibition of interleukin-2 synthesis

To test the inhibitory effect of cyclopentabenzene derivatives on interleukin-2 synthesis, a reporter gene cell line was used which contains the interleukin-2 promoter coupled to the luciferase gene. The promoter contains the DNA sequence from -480 to +4. The vector used is pGL3; the starting cell line into which the entire construct was stably transfected is SS-1. The culture medium for this cell line was RPMI 1640 (Gibco, Rockille). It also contained: 100 μg / ml streptomycin, 100 U / ml penicillin, 2 mM L-glutamine, 10% heat-inactivated FBS and

800 μg / ml G418 sulfate.

Test execution

The reporter gene test cell line was added to phenol red-free RPMI

1 x 10 ⁶ cells per well were seeded in 96-well plates and with phorbol 12-myristat 13-acetate (PMA; 5 ng / ml) and ionomycin (10 M; 0.4 μg / ml) for 24 hours at 37 ° C incubated in an atmosphere of 5% CO2, 95% air. The test substances were added simultaneously with PMA.

Measurement of luciferase activity:

To generate the luminescence, LucLite ™ solution (Packard, Meriden, CT) was added to a concentration of 100 μl / well and the luminescence was measured in the luminometer (Luminoskan, Labsystems) immediately after the addition.

Claims

claims

1. Use of cyclopentabenzofuran derivatives of the formula (I)

in which

[A] R ^{1 represents} hydrogen,

R ^{2 represents} methoxy,

R ^{3 represents} hydrogen or

R ² and R ³ together represent -OCH ₂ O-,

R ^{4 represents} methoxy,

R ^{5 represents} hydroxy, OCHO or acetoxy,

R ⁶ and R ⁷ each represent hydrogen or

R ⁵ and R ⁶ together represent oxygen (oxo) or hydroxyimino,

R ^{8 represents} -COOR ¹² or -CONR ¹³ R ¹⁴ , wherein R ¹² and R ' ^{3 represent} hydrogen or methyl and

R ^{14 represents} hydrogen, methyl, 4-hydroxybutyl or 2-tetrahydrofuryl or

or

R ⁸ for a radical of the formula

stands,

R ⁵ and R ⁸ together for a group of the formulas (a) or (b)

stand, the linking point adjacent to the N atom corresponding to R ⁵ and, moreover, R ⁶ and R ⁷ together standing for a direct bond,

R ^{9 represents} phenyl,

R ^{10 represents} methoxy, R ¹ 'represents hydrogen, hydroxy, 2-methoxy or 2-rhamnosyl, or

R ¹⁰ and R ^{11 are} adjacent and together represent -OCH ₂ O-, or

R ^{15 represents} hydroxy, methoxy or ethoxy,

R ^{16 represents} hydrogen, hydroxy or methoxy,

[B] R ¹ , R ³ and R ⁸ each represent hydrogen,

R ² and R ⁴ each represent methoxy,

R ^{5 represents} hydroxy,

R ⁶ and R ⁷ each represent hydrogen or

R ⁵ and R ⁶ together represent oxygen (oxo group),

R ^{9 represents} phenyl,

R ^{10 represents} methoxy,

R "represents 2-methoxy or 2-rhamnosyl, or

R ¹⁰ and R '' are adjacent and together represent -OCH ₂ O-,

for the manufacture of a medicament for the treatment of Nuclear Factor KB-dependent diseases. O 00/07579

- J 1 -

2. Use according to claim 1, characterized in that a medicament for the treatment of inflammatory diseases, immunological diseases, septic shock, transplant rejection, radiation damage, reperfusion injuries after ischemia, thromboses or complex, chronic inflammatory diseases such as arteriosclerosis is produced.