WO2005042442A2

WO2005042442A2 - 2-methylthio-1,4-naphthoquinone derivatives, methods for the production thereof, pharmaceutical compositions containing the same, and use thereof

Info

Publication number: WO2005042442A2
Application number: PCT/EP2004/010465
Authority: WO
Inventors: Werner E. G. MÜLLER; Narshinh L. Thakur; Arachana N. Thakur; Heinz C. SCHRÖDER; Gerhard Lang; Hideyuki Tsuruta; Gerhard Bringmann
Original assignee: Johannes Gutenberg-Universität Mainz; Julius-Maximilians- Universität Würzburg
Priority date: 2003-09-22
Filing date: 2004-09-17
Publication date: 2005-05-12
Also published as: WO2005042442A3; DE10343798A1

Abstract

Disclosed are the compounds 2-methylthio-1,4-naphthoquinone (MTN) and MTN derivatives of general formula (2) as well as methods for the production or isolation thereof. MTN and MTN derivatives have antitumor properties, especially antiangiogenetic properties in cell culture models while being provided with characteristics preventing/inhibiting neointimal proliferation.

Description

2-methylthio-1,4-naphthoquinone and 2-methylthio-1,4-naphthoquinone derivatives.

Process for their preparation, pharmaceutical compositions containing them and their use

The present invention relates to new bioactive compounds from a bacterium isolated from the marine sponge Dysidea avara. The substance was named 2-methylthio-1,4-Νaphthoquinone (MTN) due to its chemical structure. The invention further relates to a method for isolating this compound, medicaments containing it and their use in the treatment of diseases. Derivatives of MTN are also described. The compounds are highly bioactive and have a particularly selective inhibitory effect on the formation of blood vessels.

1. Background of the Invention Angiogenesis is the morphogenetic process in which new capillaries are formed which serve to supply the tissues with blood in (higher) multicellular animals. This process, which takes place both in physiological processes such as tissue formation and in pathophysiological changes such as the growth of tumors, is controlled via a number of signal transduction pathways (overview: Harris AL: Current Status of antiangiogenetic factors. Br J Haematol 109: 477-489 , 2000). Although the underlying mechanism has been known since 1971 (Folkman J: Tumor angiogenesis: tumor implications. N Engl J Med 285: 1182-1186, 1971), therapeutic strategies based on it have only been intensively developed in recent years (overview: Brower V : Tumor angiogenesis - new drugs on the block. Nature Biotechnology 17: 963-968, 1999). Today, influencing angiogenic processes is a preferred field of research for biotechnologically-oriented studies that aim to find new tumor therapeutics. A number of low molecular weight, mostly 'synthetically produced organic substances have an anti-angiogenic potency both in in vitro as well as in Jn-vz ^' vo. models and are therefore of therapeutic interest. The advantage of bioactive natural products, which are formed as so-called secondary metabolites, in particular by sponges - the most original animals - is obvious. As a sessile filter, sponges are particularly dependent on the formation of secondary metabolites. These animals use these substances not only for defense against microorganisms, but also for multicellular animals that form a blood vessel system and apparently also for morphogenetically controlling the formation of their own "nasalization system", the water channel system. The latter effect was surprising. Only after genes were found in sponges that code for ligands and their receptors, which can be involved in the regulation of angiogenesis in higher animals, could a rational screening for the discovery of such substances be started. Natural products (secondary metabolites) that show bioactive effects mostly seem to be superior to synthetically manufactured chemicals. In this way, the bioactive secondary metabolites of sponges, which were recommended over 800 million years ago, could be selected for optimal effect during this long period. Evolution has thus taken over the complex synthesis and selection processes required for the development of new lead structures using combinatorial chemistry. Angiogenesis is controlled by intracellular molecules / ligands that interact with their corresponding receptors. Examples include the proteins from the series of angiostatins (endogenous inhibitors of angiogenesis) which inhibit the function of growth factors (which bind, for example, to the NEGF / NEGF receptor), and the fibroblast growth factor (which binds to the fibroblast growth factor). Receptor interacts) or the vascular, endothelial growth factor (with the corresponding receptor). The coordinated interplay of these systems controls the physiological / pathophysiological development of the vessels and capillaries.

It is therefore an object of the invention to provide new bioactive substances which are suitable for use against tumors and / or for the prevention of angiogenesis in general, but in particular angiogenesis in tumors. This object is achieved by combining general formula 2:

wherein R ^{1 is} either H, an unsubstituted, monosubstituted or polysubstituted - C ₁₈ alkyl, where the alkyl can be straight, branched or cyclic, a monosubstituted or polysubstituted, straight, branched or cyclic Ci-C ₁₈ alkenyl or an acyl group such as eg formyl, acetyl, trichloroacetyl, fumaryl, maleyl, succinyl, benzoyl, branched or heteroatom or aryl-substituted acyl groups, and R to R independently of one another can be either H, an unsubstituted, monosubstituted or polysubstituted C 1 -C ₁₈ alkyl, the alkyl may be straight, branched or cyclic, alkenyl, an unsubstituted, monosubstituted or polysubstituted aryl or heteroaryl radical, an unsubstituted, monosubstituted or polysubstituted benzyl group, an acyl group, such as formyl, acetyl, trichloroacetyl, succyl or benzyl, maleyl, maleyl branched or heteroatom or aryl substituted acyl group, an alkoxy substituent such as -OM e, -OEt, -OraPr, -z'Pr, -OwBu, -Oz ^' Bu, -Osec u, - OtBu, whose alkyl group is branched, unbranched or cyclic, an alkyl group bonded via a sulfur atom, such as, for example, -SMe, SEt, or a sulfonyl group, such as. B. -SO ₃ H, -SO ₂ Me, -SO ₂ CF ₃ , -SO ₂ C ₆ H ₄ CH ₃ or SO ₂ C ₆ H ₄ CH ₂ Br, or a nitrogen substituent such as -NH ₂ , -NHR , -NRR '(with R, R' = alkyl, aryl, heteroaryl, alkenyl), -NC or -NO ₂ , or fluorine, chlorine, bromine, iodine, -CN or a hetero substituent, and pharmaceutically acceptable salts or solvates of the Connection.

In one embodiment, R i - =, CH ₃ and R -R = H

In another embodiment, R ¹ = CH ₃ , R ² = SCH ₃ and R ³ -R ⁶ = H. In a preferred embodiment, R ¹ = -CH ₂ -CH (CH ₃ ) ₂ and R ² -R ⁶ = H. In another preferred embodiment, R ¹ = -C (CH ₃ ) ₃ and R ² -R ⁶ = H.

The object is also achieved by a method for producing a compound according to the present invention comprising culturing a bacterium in culture medium, said bacterium being related to the alpha-proteobacterium MBIC3368, having 99% sequence identity with regard to the rDNA to the alpha-proteobacterium MBIC3368 and occurs associated with the marine sponge Dysidea avara, and isolating at least one compound according to any one of claims 1-5 from the culture medium and / or the bacterial biomass, the isolation comprising at least one of the following steps: a) extraction with an organic solvent, preferably one Ci - C ₆ alcohol, preferred n-butanol, b) gel filtration.

The method preferably further comprises a subsequent synthetic derivatization of the isolated compound.

In another embodiment, a compound according to the present invention is isolated from a sponge of the species Suberites domuncula, preferably by extraction in a manner known per se, or a substance according to the invention is obtained by culturing a bacterium which is associated with the sponge Suberites domuncula and then Extracting with an organic solvent and / or gel filtration isolated.

The object is also achieved by a compound according to the present invention for use as a medicament, and also by a pharmaceutical composition comprising a compound according to the invention according to one of claims 1-5 together with suitable additives or auxiliaries.

The compound is preferably in the form of a depot substance or as a precursor together with a suitable, pharmaceutically acceptable dilution solution or carrier substance.

In one embodiment, the composition is in the form of a dosage unit, a dosage unit being the compound according to one of the Contains claims 1-5 in an amount which is in the range from 5 ug to 50 ug, preferably from 10 ug to 40 ug, more preferably from 15 ug to 30 ug, most preferably about 20 ug.

In one embodiment, the compound is present in the composition in an amount sufficient to achieve a blood concentration in the range 0.3 to 3.0 μg / ml when administered to a mammal.

The pharmaceutical composition preferably contains further chemotherapeutic agents.

In one embodiment, the pharmaceutical composition is in the form of tablets, dragées, capsules, drip solutions, suppositories, injection or infusion preparations for oral, rectal or parenteral use.

The objects of the invention are also achieved by the use of a compound according to the present invention for the manufacture of a medicament for the treatment of tumor diseases or infections by gram-negative bacteria, as well as by the use of a compound according to the present invention for the manufacture of a medicament for the prevention or Inhibition of angiogenesis, it being preferred that angiogenesis is prevented or inhibited in the case of tumor diseases.

The objects of the invention are also achieved by the use of a compound according to the present invention for the manufacture of a medicament for preventing or inhibiting neointimal proliferation, with it being preferred that the compound be applied to a stent, preferably as a coating.

In one embodiment, the compound is in the form of a depot substance or as a precursor, together with a suitable, pharmaceutically acceptable diluent solution or carrier substance.

In one embodiment, the compound is used in an amount that in the Range is from 5 ug to 50 ug, preferably from 10 ug to 40 ug, preferably from 15 ug to 30 ug, most preferably about 20 ug.

In one embodiment, the compound is used in an amount sufficient to achieve a blood concentration in the range of 0.3 to 3.0 µg / ml when administered to a mammal.

The phylogenetically oldest ligand / receptor system that controls angiogenesis is the CD36 thrombospondin system (TSP system) (Calvo D; Dopazo J, Vega MA: The CD36, CLA-1 (CD36L1), and LIMPII (CD36L2 ) gene family: cellular distribution, chromosomal location, and genetic evolution. Genomics 25: 100-106, 1995). CD36, initially also called glycoprotein IV, was first detected on the platelet membranes. When the specific ligand, thrombospondin (TSP), binds to the CD36 receptor, angiogenesis is inhibited. With the help of molecular biological methods, the inventors were able to demonstrate that the sponges - as the phylogenetically oldest animals - already have a member of the CD36 receptor family. The sea swan m Suberites domuncula served as the investigation model. Based on this, a screening for the corresponding ligand was successfully carried out, which was then also found using the PCR technique. It could be shown that this ligand has the binding motif specific for the TSP. Functional analyzes and the use of a recombinant TSP-related polypeptide showed that this molecule in S. domuncula as a model system inhibits the formation of the channel system through which the surrounding sea water is conducted through the sponge. The chemical structure of this substance is a 2-methylthio-1,4-naphthoquinone (MTN). Building on this i -vzvo model, a natural substance has now been identified that is formed by a microorganism (bacterium) found in a sponge. It can be assumed that this bacterium co-evolved with. has gone through this original animal species (sponge). From this it can be deduced that MTN was selected as a functionally highly active and highly specific active ingredient.

MTN

After clarifying the mode of action of MTN in the homologous system (ie S. domuncula), it was checked whether MTN also has an effect in a heterologous model system; the chorioallantoic membrane (CAM) model of the chicken embryo (Auerbach R, Lewis R, Shinners B, Kubai L, Akhtar N: Angiogenesis assays: a critical overview.Clinical Chem. 49: 32-40, 2003 ) selected. Surprisingly, it was found here that the substances according to the invention, in particular MTN as well as DMTN and the derivatives of claims 4-5, inhibit the growth of the capillaries which originate from the central blood vessel even at very low doses. Furthermore, it was surprisingly found that the substances according to the invention, in particular MTN, DMTN and the derivatives of claims 4-5, inhibit the cell division of tumor cells at higher concentrations. This cell toxicity is even tumor-specific; lymphatic tumors are most sensitive to inhibition. Based on this data, MTN and its derivatives are very promising substances for the treatment of tumors, especially those that can only grow in size due to intensive vascularization. In particular, it can be expected that the growth of such tumors (eg carcinomas) is inhibited, which pass from the TNM / Tl phase to the TNM / T2 and in particular the TNM / T3 phase. Intensive vascularization occurs during this process. The vascularization-dependent inhibition of tumor growth is enhanced by the cytotoxic effect (inhibition of tumor cell proliferation occurring at higher doses) of MTN and its derivatives. A further application of this surprising inhibition of vascularization is the application in the coating of stents. The aim of a medicinal stent coating is to reduce neointimal proliferation inhibit to prevent this pathway from re-narrowing the blood vessels after implanting a (stainless steel) stent. The previous attempts at neointimal inhibition of proliferation by gold or other inorganic materials have proved unsuccessful. However, application of stent coatings with organic bioactive substances such as rapamycin appears to be potentially promising. The aim of the stent coating is to reduce platelet adhesion and to inhibit the proliferation of smooth muscle cells. Therefore, a way is also shown here to produce a coating for the stents by covalently connecting the substances proposed for patenting with polymers. For the covalent binding of the substances proposed for patent to the polymer. Carrier material, it is possible to reduce the connections to the ^'respective hydroquinone and to link by a Esterbindung with a long chain carboxylic acid via the 4-OH group (see illustration), the other end is connected to the polymer. The hydroquinone released in the body by lipases is spontaneously oxidized to naphthoquinone, the actual active ingredient. The rate of hydrolytic release of the active ingredient, and thus its concentration, can be regulated via the chain length of the carboxylic acid used as a "spacer".

An object of the present invention is to present the highly bioactive substance from a marine organism or an associated microorganism for the potential therapy of tumors. For this purpose, its manufacture and uses are explained.

2. MTN and MTN derivatives

According to the invention, the above-mentioned object is first achieved by the substance MTN and the MTN derivatives as bioactive substances. It is surprising that in the tumor systems used here were able to demonstrate such a pronounced antitumor activity as discussed below. It was also unknown that MTN or its descendants have anti-angiogenic activity. This effect surprisingly occurs even at very low doses or when using small amounts in the range from 0.2 ng - 2 ng, which locally already inhibit or completely prevent angiogenesis.

Furthermore, it was found that MTN and the derived MTN derivatives of the general formula 2 have pronounced antitumor properties. According to the invention, compounds of the general formula 2 are:

wherein R ^{1 is} either H, an unsubstituted, monosubstituted or polysubstituted Cig-alkyl, where the alkyl may be straight, branched or cyclic, a monosubstituted or polysubstituted, straight, branched or cyclic - s-alkenyl or an acyl group such as formyl , Acetyl, trichloroacetyl, fumaryl, maleyl, succinyl, benzoyl, branched or heteroatom or aryl-substituted acyl groups, and R ² to R ⁶ independently of one another are either H, an unsubstituted, monosubstituted or polysubstituted C 1 -C ₁₈ -alkyl, the alkyl may be straight, branched or cyclic, alkenyl, an unsubstituted, monosubstituted or polysubstituted aryl or heteroaryl radical, an unsubstituted, monosubstituted or polysubstituted benzyl group, an acyl group, such as formyl, acetyl, trichloroacetyl, succyl or benzyl, maleyl, maleyl branched or heteroatom or aryl substituted acyl group, an alkoxy substituent, such as eg -OMe, -OEt, -O «Pr, - / Pr, -OnBu, -O Bu, -OsecBu, - OtBu, whose alkyl group is branched, unbranched or cyclic, an alkyl group bonded via a sulfur atom, such as, for example, -SMe, -SEt, or a Sulfonyl group, such as e.g. B. -SO ₃ H, -SO ₂ Me, -SO ₂ CF ₃ , -SO ₂ C ₆ H CH ₃ or SO ₂ C ₆ H ₄ CH ₂ Br, or a nitrogen substituent such as -NH ₂ , -NHR, -NRR '(with R, R' = alkyl, aryl, heteraryl, alkenyl.), -NC or -NO ₂ , or fluorine, chlorine, bromine, iodine, -CN or a hetero substituent.

For the compound 2-methylthio-l, 4-naphthoquinone (MTN) described here for the first time as a natural product, R ¹ = CH ₃ and R ² -R ⁶ = H. The synthetic derivative 2,3-dimethylthio-l, 4-naphthoquinone ( DMTN) has the substitution pattern R ¹ = CH ₃ , R ² = SCH ₃ and R ³ -R ⁶ = H.

Furthermore, it was found that MTN and the MTN derivatives have a pronounced and unpredictable anti-tumor and anti-angiogenic property. Because of this property and based on the finding that MTN and the MTN derivatives are largely non-toxic for mammals (example mouse), the substances described here for the treatment of tumors in general and especially of tumors whose growth depends on vascularization, suitable. In addition, the use for the treatment / prevention of restenosis processes is indicated. It is recommended to use these substances either in the present form or in the form of a depot substance or as a precursor together with a suitable, pharmaceutically acceptable dilution solution or carrier substance.

The above-mentioned anti-tumor or anti-angiogenic properties were also found in the derivatives of claims 4 and 5 in the same manner and effectiveness as in MTΝ.

These compounds according to the invention can be processed with the customary solvents, auxiliaries and carriers to give tablets, dragées, capsules, drip solutions, suppositories, injection and infusion preparations in order to find therapeutic use for oral, rectal or parenteral administration.

The present invention further relates to a method for producing an above-mentioned compound, the MTΝ, which is characterized in that the substance is preferably isolated from a marine organism, such as, for example, a marine bacterium which is associated with the sponge Dysidea avara.

As used herein, in the context of the present invention, a “derivative” is to be understood as meaning a compound derived from the general formula (2), which is substituted, for example, by various of the residual groups specified above for R ^] -R ⁶ , and mixtures of various of these compounds that are being processed in the coordinated with each disease to be treated and / or the patient based on diagnostic data, or data on the treatment success or progress message to a "personalized" medicament ^"may. a derivative is also a compound of the class of the 2 -Methylthio-l, 4-napthoquinones understood, which can be isolated from other (eg) marine organisms than those mentioned here (by way of example).

In the context of the present invention, a “precursor” of a substance is to be understood on the one hand as a substance that is changed in the course of its administration for treatment by the conditions in the body (for example pH in the stomach or the like) or by the body Metabolism is metabolized in such a way that the compound according to the invention or its derivatives form as active substance. Secondly, derivatives of 2-methylthio-1,4-napthoquinone isolated from organisms are understood which already have the properties of 2-methylthio- 1,4-naphthoquinone.

3. Formulations Another aspect of the present invention relates to the use of at least one of the above-mentioned compounds for the treatment of diseases, such as e.g. of tumor diseases. This use can be, for example, in the form of a depot substance or as

Precursors are made together with a suitable, pharmaceutically acceptable diluent or carrier. In the case of the treatment of

Tumors can be treated in a concentration range between 0.1 and 10 mg / kg body weight. Another aspect of the present invention relates to a pharmaceutical

A composition comprising a compound of the invention together with suitable additives or auxiliaries. This pharmaceutical composition can be characterized in that the compound in the form of a depot substance or as a precursor together with. a suitable, pharmaceutically acceptable diluent or vehicle. Particularly preferred are pharmaceutical compositions in which the compound according to the invention is present in an amount or in which the compound according to the invention is added in such an amount that a concentration range between 0.3 and 3.0 μg / ml (blood level) during the treatment in vivo is present. Preferred is a pharmaceutical composition ^" according to the present invention, which contains further chemotherapeutic agents. These chemotherapeutic agents can comprise all chemotherapeutic agents commonly used for a person skilled in the art in the context of cancer therapy (eg taxol or anti-angiogenic compounds such as Bayl2-9566). According to the invention, the above-mentioned pharmaceutical Compositions in the form of tablets, dragees, capsules, drip solutions, suppositories, injection or infusion preparations for oral, rectal or parenteral use are available, such dosage forms and their preparation are known to the person skilled in the art. According to the invention, the compounds can be introduced into organic matrices, for example as a coating for The substances used according to the invention are derivatized with the aim of attaching a linker to the bioactive substances, via which the naphthoquinones are attached to an organi linked polymer. A long-chain carboxylic acid can be used as the linker, which is linked via an ester bond to the 4-OH group of the active ingredient reduced to the hydroquinone. The release of the hydroquinone is caused by the body's own lipases. The hydroquinone is then spontaneously oxidized to bioactive naphthoquinone. As a further application for producing a coating of stents with the compounds used according to the invention, these are enclosed non-covalently in a likewise aromatic matrix using the aromatic property of the substance. The physico-chemical reaction that leads to this reaction is state of the art. Brief description of the figures:

Figure 1 shows a general formula 2 of the compounds according to the present invention.

Figure 2 shows the formula for 2-methylthio-1,4-naphthoquinone.

Figure 3 shows the formula for 2,3-dimethylthio-1,4-naphthoquinone.

FIG. 4 shows the results of a chorioallantoic membrane test (CAM test), FIG. 4a showing the capillary pattern of embryos which were incubated with an agar block which did not contain any test substance, while FIG. 4b and FIG. 4c the capillary pattern after treatment with 0.25 ng of 2-methylthio-l, 4-naphthoquinone or 1 ng of 2-methylthio-l, 4-naphthoquinone.

The invention will now be illustrated by the following examples, which are presented for purposes of illustration, not limitation. Examples

Example 1: Representation of the compounds according to the invention as a natural product.

The bacterial strain was isolated or cultured as follows. A 5 mm ² sample was punched out of the central tissue of the Dysidea avara sponge. Under sterile conditions, the tissue sample was cleaned three to four times of sterile sea water from scaffold structures. The sample was then crushed between two sterile glass slides. The extract obtained was diluted to a dilution factor of 10 ^"1 to 10 ^{" 5} . An agar culture was set up from each dilution stage; the culture medium was composed of the following components: 0.25% peptone, 0.15% yeast extract, 0.15% glycerol and 1.6% agar (dissolved in 100% seawater). The plates were incubated at 30 ° C for 24 to 72 hours. The bacterial colonies were separated using dilution series and thus represented purely. In the proliferation inhibition assay emerged among the eight isolated bacterial strains (Dl - D8) the strain designated as Dl as the most active, specifically in the experimental approach aimed at inhibiting angiogenesis. The characterization of the bacterium has just been carried out. Sequence comparisons of the rDNA showed that the bacterial strain D1 has a 99 percent identity to the alpha-proteobacterium MBIC3368. The bacterial strain Dl was extracted after cultivation with n-butanol using the Elyakov method (Elyakov GB, Kutznetsova TA, Mikhailov VV: From chemistry of marine natural products to marine technologies: research at pacific institute of bioorganic chemistry. Mar Tech Soc J 30: 21-28, 1996). After the incubation, 150 ml of ri-butanol was added to the bacterial culture (in 500 ml aliquots). The suspension was shaken at 40 ° C for 24 hours and then stirred on a magnetic stirrer for 20 minutes. The n-butanol phase obtained after centrifugation was drawn off and brought to dryness with a rotary evaporator under medium vacuum. The dry residue (100-150 mg) was stored at 5 ° C. For gel filtration purification, the extract was applied to a column (23 x 3 cm) filled with Sephadex LH-20. Methanol was used as the eluent; the fraction size was 18 ml. Fractions 14 and 15 of the fractions obtained were combined after activity determination and evaporated to dryness. This was followed by a preparative "high-performance" purification by liquid chromatography (HPLC separation). Xterra 19 x 300 mm (Waters) was used as the column; the mixture was water and acetonitrile; the components were mixed with 0.05% trifluoroacetic acid. The gradient was adjusted as follows: start (0 min) with 10% acetonitrile, 100% acetonitrile was used after 30 min, the flow rate was 12 ml / min, the compound was eluted after 23 min and, after drying, gave 0.5 mg, the The substance obtained was identified by NMR analysis and on the basis of the MS spectra as 2-methylthio-1,4-naphthoquinone (MTN), the melting point being 176-178 ° C. The corresponding literature data for the Melting point is 165-166 ° C (Kametani T, Nemotό H, Takeuchi M, Takeshita M, Fukumoto K: A novel alkylation in the 4-position of isoquinoline derivatives. J Chem Soc Perkin Trans 1: 386-390, 1977) or 185-186 ° C (C oll G, Morey J, Costa A, Saa JM: Direct lithiation of hydroxyaromatics. J Org Chem 53: 5345-5348, 1988). Example 2: Biological activity: inhibition of the proliferation of tumor cells

Test approach: The anti-tumor activity of the 2-thiomethylnaphthoquinones was tested on a number of tumor-transformed cells such as L5178y mouse lymphoma cells (ATCC CRL 1722). As described (Müller WEG, Zahn RK: Metabolism of 1-ß-D-arabinofuranosyluracil in mouse L5178y. Cancer Res 39: 1102-1107, 1979) the cells were cultivated in RPMI medium to which 10% fetal calf serum had been added , 10,000 lines / ml were chosen as the inoculum concentration. At the start, the selected substance was added and the culture was incubated for 72 hours. The number of living cells was then determined using the colorimetric XTT approach and evaluated using an ELISA reader (see: Scudiero DA, Shoemaker RH, Pauli KD, Monks A, Tierney S, Nofziger TH, Currens MJ, Seniff D, Boyd MR : Cancer Res 48: 4827-4833, 1988; Daum Th, Engels J, Mag M, Muth J, Lücking S, Schröder HC, Matthes E, Müller WEG: Antisense oligonucleotides: inhibitors of splicing of mRNA of human immunodeficiency virus. Intervirology 33 : 65-75, 1992). The ED ₅ o concentrations of some selected MTN derivatives for the above tumor cell lines were Durcliführung by the colorimetric XTT assays by linear regression determined (L. Sachs, Angewandte Statistik. Springer, Berlin, 1984, 1-168). The respective mean values with the associated standard deviation were determined from 10 independent experiments.

Result:

The experimental data are summarized in the following table. It becomes clear that the MTN derivatives significantly reduce cell proliferation after 72 hours even at low concentrations down to 0.07 μg / ml. MTN and DMTN with an activity (ED ₅₀ - concentration) of 0.07 (or 0.09) μg / ml in the L5178y cell system are particularly inhibitory - and highly selective. The other derivatives also have a pronounced effect on cell proliferation. The ED ₅₀ concentrations of the MTN derivatives in cell culture batches of PC-12 cells and of HeLa-S3 cells are in the comparable range lower. By way of example, the derivative MTN can be mentioned that at L5178Y cells an ED ₅ o-concentration of 0.07 µg / ml; MTN is less effective for batches with HeLa S3 cells (1.0 μg / ml) or PC-12 cells (2.5 μg / ml).

Actionable conclusion:

It is therefore concluded that the antitumor spectrum of both MTN and MTN derivatives is wide. The compounds have a particularly strong proliferation-inhibiting effect on L5178y cells. Comparative inhibition tests show that MTN and DMTN have a significantly higher inhibitory activity compared to unsubstituted 1,4-naphthoquinone. Similar results were obtained when using the derivatives of claims 4 and 5.

Example 3: Biological activity: anti-angiogenic effect

Test Batch:

The method used here, the chorioallantoic membrane (CAM) test, is based on a published method (Crum R, Sazbo S, Fokeman J: A new class of steroids inhibit angiogenesis in presence of heparin and heparin fragments. Science 231: 1375- 1378, 1985). The test substance MTN was introduced in at least four different doses from 0.25 ng to 1 ng in agar blocks with a diameter of 4 mm. The agar concentration was 2.5%. After the substance had penetrated the agar, the block was placed on the chorioallantoic membrane applied. Fertilized 5 day old eggs were sterilized externally with 70% alcohol. After locating the CAM, a 1 mm x 1 mm window was opened in the shell. The agar block was placed 2-3 mm from the central blood vessel on the region with the proliferating capillaries. The dish window was then closed again with parafilm and the eggs were incubated at 38 ° C. for a further 48 hours. The potential antiangiogenic effect was then determined. As a positive control, hydrocortisone (60 μg / disk) was used together with heparin (100 μg / disk). At least 15 fertilized eggs were used per test substance.

Result:

The Chorioallantoic Membrane (CAM) test is considered a very reliable method for determining potentially anti-angiogenic effects of substances (see: Auerbach R, Lewis R, Shinners B, Kuba, L, Akhtar N: Angiogenesis assays: a critical overview. Clinical Chem 49: 32-40, 2003).

Figure (see also Fieur 4): Effect of MTN on vascularization in chicken embryos in the CAM assay. FIG. 4 A shows the capillary pattern of embryos which were incubated with an agar block which did not contain any test substance for 48 hours. FIG. 4B shows the capillary pattern after treatment with 0.25 ng MTN per disk and FIG. 4 C shows a corresponding approach with 1 ng MTN per disk. A very pronounced disorder of the capillary organization can be seen where the agar disks impregnated with MTN ( Arrowheads and marked with "d") were applied. Magnification: x 10.

In the / 72-v vo series of investigations carried out it was found that

MTN is a very effective angiogenic inhibitor. At a dose of 1 ng / disk, 100% elimination of the capillaries is measured. As shown in Figure 4 (partial image C), there is a complete suppression of capillary formation in the region in which the agar block was applied. in the In comparison, the physiological pattern of capillary formation is shown in partial image A. At the lower dose of 0.25 ng per disk, a misorganization of the capillary pattern can also be seen (partial picture B). A quantitative analysis showed that at a dose of 0.25 ng per disk 20% of the embryos had a significant anti-angiogenic effect (number of embryos examined: n = 20), while at a slightly higher dose between 0, 50 ng and 1 ng (per disk) MTN produces a 100% anti-angiogenic effect.

Feasible conclusion: The results presented here show that MTN shows a pronounced anti-angiogenic effect at very low doses of 0.25 ng to 1 ng per disk. It is therefore to be expected that MTN will show this effect on vascularization not only in chicken embryos but also in other vertebrates and in human tumors at comparable low doses. Similar results were also obtained for DMTN and the derivatives of claims 4 and 5.

Example 4: Biological activity: antibacterial effect

The following two were used to test for antibacterial activity

Bacterial strains used: Escherichia coli (DH5alpha), a gram-negative

Bacterium, and Bacillus subtilis (168), a gram-positive bacterium. The

Investigations were carried out with MTN as well as with DMTN and naphthoquinone.

naphthoquinone

Test Batch:

A bacterial lawn was grown after overnight culture and subsequent streaking on solid agar. LB agar (10 g peptones, 5 g yeast extract, 5 g NaCl and 15 g agar ad 1 liter) was used as the culture medium. The antibacterial test was carried out according to the established methods. Serially diluted concentrations of MTN were applied to paper discs (6 mm in diameter). After the solvent had evaporated, the disks were placed on the bacterial turf and the cultures were incubated at 30 ° C. for 24 hours. The diameter of the zone in which no bacteria were visible was then determined (inhibitory zone).

Result: The investigations showed that naphthoquinone and its derivatives produce a measurable antibacterial effect in Bacillus subtilis (gram-positive) at a dose of 10 μg per disk; the derivatives tested are active accordingly. The effect on the gram-negative bacterium Escherichia coli is low.

The activity is indicated with: weak <2 mm inhibitory zone; strong> 2 mm inhibition zone. Actionable conclusion: It becomes clear that the naphthoquinone derivatives have an anti-bacterial effect on gram-positive bacteria, here the bacterium Bacillus subtilis.

Example 5: Biological activity: fungicidal activity To test the potentially fungicidal activity of the substances to be registered (MTN, DMTN and naphthoquinone and derivatives thereof) Saccharomyces cerevisiae selected.

Test Batch:

After overnight culture, the yeast strain was sown as a lawn on YPD agar (20 g peptones, 10 g yeast extract, 20 g glucose, 15 g agar ad 1 liter). Serially diluted concentrations of MTN or its derivatives were applied to paper disks (diameter 6 mm). After evaporation of the

These disks were placed on the yeast lawn and the cultures 24

Incubated at 30 ° C for hours. The diameter of the zone in which no yeast could be recognized was then determined (inhibition zone).

Result:

The investigations showed that naphthoquinone at a dose of 10 μg per disk does not produce a strong antifungal effect. The inhibition zone is <2 mm.

Example 6: Biological activity: Effect in vivo

To determine the toxicity, the test substance MTN was tested on mice.

Both the survival rate and the change in the weight of the animals were as

Parameters were used for a potentially toxic effect. Male (outbred) NMRI mice (32-36 g; age: 8-9

Months).

Test Batch:

The test substance MTN was dissolved in methyl cellulose and the animals i.p. injected. The animals were given a dose of 30 mg / kg (per day) and 3 mg / kg (per day) for five days. The weight of the animals was determined daily. 5 animals were treated per group. The survival rate and the mean weight of the collective were determined after 5 and after 11 days.

Result:

During this time, the weight of the MTN-treated animals at a dose of 3 mg / kg did not change significantly compared to that of the controls. However, the animals treated with 30 mg / kg experienced a significant weight loss; also died in the animals treated with MTN at 30 mg / kg a total of two animals after the 3rd and 4th day. In the control group or in the animal group treated with 3 mg / kg MTN, none of the experimental animals died.

8.3. Feasible conclusion: Result: It is concluded from these data that the subacute toxicity of MTN after a five-day ip treatment is »3 mg / kg.

The features of the invention disclosed in the above description, the claims and the drawings can be essential both individually and in any combination for realizing the invention in its various embodiments.

Claims

Expectations

1. Compound of the general formula 2:

in which

R ^{1 is} either H, an unsubstituted, monosubstituted or polysubstituted C 1 -C ₁₈ alkyl, where the alkyl can be straight, branched or cyclic, a monosubstituted or polysubstituted, straight, branched or cyclic C 1 -C ₁₈ alkenyl or an acyl group, such as eg formyl, acetyl, trichloroacetyl, fumaryl, maleyl, succinyl, benzoyl, branched or heteroatom or aryl-substituted acyl groups, and R ² to R ⁶ independently of one another are either H, an unsubstituted, monosubstituted or polysubstituted C 1 -C ₁₈ -alkyl, where the alkyl may be straight, branched or cyclic, alkenyl, an unsubstituted, monosubstituted or polysubstituted aryl or heteroaryl radical, an unsubstituted, monosubstituted or polysubstituted benzyl group, an acyl group, such as formyl, acetyl, trichloroacetyl, fumarylylyl, fumaryyl Benzoyl, or a branched or heteroatom or aryl substituted acyl group, an alkoxy substituent, such as B -OMe, -OEt, -O «Pr, -z ^' Pr, -OnBu, -Oz ^' Bu, -OsecBu, -OtBu, whose alkyl group is branched, unbranched or cyclic, an alkyl group bonded via a sulfur atom, such as - SMe, -SEt, or a sulfonyl group, such as. B. -SO ₃ H, -SO ₂ Me, -SO ₂ CF ₃ , - SO ₂ C ₆ H ₄ CH ₃ or SO ₂ C ₆ H CH ₂ Br, or a nitrogen substituent such as -NH ₂ , -NHR , - NRR '(with R, R' = alkyl, aryl, heteroaryl, alkenyl), -NC or -NO ₂ , or fluorine, chlorine, bromine, iodine, -CN or a hetero substituent, and pharmaceutically acceptable salts or solvates of Connection.

2. Compound according to claim 1, characterized in that R ¹ = CH ₃ and R ² -R ⁶ = H.

3. A compound according to claim 1, characterized in that R ¹ = CH ₃ , R ² = SCH ₃ and R ³ -R ⁶ = H.

4. A compound according to claim 1, characterized in that R 1 _ = -CH ₂ -CH (CH ₃ ) ₂ and R ² -R ⁶ = H.

5. A compound according to claim 1, characterized in that R ¹ = -C (CH ₃ ) ₃ and R ² -R ⁶ = H.. ^{"" •}

6.. A method of making a compound according to any of claims 1-5, comprising culturing a bacterium in culture medium, said bacterium being related to the alpha proteobacterium MBIC3368, having 99% sequence identity with respect to the rDNA to the alpha proteobacterium MBIC3368 and the marine one Sponge Dysidea avara occurs associated, and isolating at least one compound according to any one of claims 1-3 from the culture medium and / or the bacterial biomass, the isolation comprising at least one of the following steps: a) extraction with an organic solvent, preferably a Ci - C ₆ - alcohol, more preferred n-butanol, b) gel filtration.

7. The method of claim 6, further comprising a subsequent synthetic derivatization of the isolated compound.

A compound according to any one of claims 1-5 for use as a medicament.

9. A pharmaceutical composition comprising a compound according to any one of claims 1-5 together with suitable additives or adjuvants.

10. Pharmaceutical composition according to claim 9, characterized in that the compound is in the form of a depot substance or as a precursor together with a suitable, pharmaceutically acceptable diluent or carrier substance.

11. Pharmaceutical composition according to claim 9 or 10, characterized in that the composition is in the form of a dosage unit, a dosage unit containing the compound according to any one of claims 1-5 in an amount which is in the range from 5 μg to 50 μg, preferably from 10 ug to 40 ug, more preferably from 15 ug to 30 ug, most preferably about 20 ug.

12. A pharmaceutical composition according to any one of claims 9-11, characterized in that the compound is present in the composition in an amount sufficient to achieve a blood concentration in the range 0.3 to 3.0 when administered to a mammal μg / ml.

13. Pharmaceutical composition according to one of claims 9 to 12, characterized in that it contains further chemotherapeutic agents.

14. Pharmaceutical composition according to one of claims 9 to 13 in the form of tablets, dragees, capsules, drip solutions, suppositories, injection or infusion preparations for peroral, rectal or parenteral use.

15. Use of a compound according to any one of claims 1-5 for the manufacture of a medicament for the treatment of tumor diseases or infections by Gram-negative bacteria.

16. Use of a compound according to any one of claims 1-5 for the manufacture of a medicament for preventing or inhibiting angiogenesis.

17. Use according to claim 16 for the manufacture of a medicament for preventing or inhibiting angiogenesis in tumor diseases.

18. Use of a compound according to any one of claims 1-5 for the manufacture of a medicament for preventing or inhibiting neointimal proliferation.

19. Use according to claim 18, wherein the connection is applied to a stent, preferably as a coating.

20. Use according to any one of claims 15-18, wherein the compound is in the form of a depot or as a precursor, together with a suitable, pharmaceutically acceptable diluent or carrier.

21. Use according to any one of claims 15-18 and 20, wherein the compound is used in an amount which is most preferred in the range from 5 ug to 50 ug, preferably from 10 ug to 40 ug, preferably from 15 ug to 30 ug is approximately 20 μg.

22. Use according to any one of claims 15-18 and 20, wherein the compound is used in an amount sufficient to achieve a blood concentration in the range of 0.3 to 3.0 ug / ml when administered to a mammal lies.