Beiträge zur Chemie übergangsmetallkoordinierter P-Cp*- substituierter Heterocyclen und P,C-Käfigverbindungen

Im Rahmen dieser Arbeit werden umfangreiche Studien zur Synthese übergangsmetall-koordinierter P,C- und O,P,C-Käfigliganden (Käfigkomplexe) vorgestellt. Zur besseren Verständlichkeit beim Vergleich der verschiedenen Polycyclen wird im Sinne einer Kurznomenklatur die allgemeine Formel n[ExPyCz](Rp(R) * verwendet. [*: n: Anzahl kondensierter Ringe, E: Heteroelement (O, N), x, y, z: Anzahl der jeweiligen Elemente im Käfiggerüst; Rp: Ringgrößen: Phosphorheterocyclen (aufsteigende Reihenfolge); (R): Ringgrößen: verbleibende kondensierte Ringe]. Im ersten Teil (Kapitel II) wird das Potential eines P-Cp*-substituierten 2H-Azaphosphirenkomplexes an Wolfram- bzw. Molybdänpentacarbonyl als Precursor in der Synthese von P,C-Käfigkomplexen weiterführend untersucht und auch erste Reaktionen an ausgewählten polycyclischen Phosphirankomplexen studiert. So konnte gezeigt werden, dass die erhaltenen Produkte durch geeignete Wahl der Substituenten und Synthesebedingungen gesteuert werden können. Erhalten werden können im Falle des Wolframkomplexe entweder 1H-Phosphirenkomplexe oder 4[P1C7]3,4,4(5)-Käfigkomplexe; im Falle des Molybdänkomplexes konnte zusätzlich ein 4[P2C7]3,5,5(5)-Käfigkomplex als Produkt einer Konkurrenzreaktion identifiziert werden. Ein interessanter Seitenaspekt ist hierbei, dass auch ein Einfluss des Metalls auf die Stabilität eines 1H-Phosphirenkomplexes nachgewiesen werden kann. Erste Folgestudien zur Chemie der P,C-Käfigkomplexe am Beispiel der 4[P1C7]3,4,4(5)-Wolframkomplexe zeigten, dass die Freisetzung des P,C-Käfigliganden durch Erhitzen in Toluol in Gegenwart von DPPE oder durch Erhitzen in Pyridin möglich ist. Die basenkatalysierte Hydrolyse der C-Ester-substituierten P,C-Käfigkomplexe mit Lithiumhydroxid-Monohydrat führte unerwarteterweise zur partiellen P,C-Käfig-Gerüstöffnung bzw. zur Umwandlung des P,C-Käfigsystems unter mehrfachem Bindungsbruch und Bindungsneubildung. Als Vergleichsverbindungen wurden monocyclische P-Cp*-substituierte Phosphirankomplexe synthetisiert, wobei festgestellt wurde, dass deren Bildung weniger begünstigt verläuft als die der polycyclischen Derivate. Wird jedoch dieser Reaktionskanal durchlaufen, werden die Komplexe als Mischung zweier Diastereomere erhalten. Weiterhin wurde im Falle der Umsetzung mit Acrylsäureethylester auch die Konkurrenzreaktion unter Bildung von 3,4-Dihydro-2H-1,2-azaphospholkomplexen aufgeklärt sowie deren Tautomerisierung zu dem entsprechenden 2,3-Dihydro-1H-1,2-azaphospholkomplex festgestellt. Im zweiten Teil der Arbeit wird schwerpunktmäßig die Synthese von P-Cp*-substituierten Oxaphosphirankomplexen untersucht (Kapitel III). Hierbei zeigt sich, dass P-Cp*-substituierte 2H-Azaphosphirenkomplexe nicht als Precursor geeignet sind. Die vorgestellten Reaktionen mit Aryl-substituierter Aldehydderivate unter thermischen Bedingungen lieferten stattdessen selektiv Zugang zu den 1,3,4-Dioxaphospholankomplexen. Dennoch gelang es im Rahmen dieser Arbeit ein neues Synthesekonzept für P-Cp*-substituierte Oxaphosphirankomplexe zu entwickeln, welches auf der Verwendung eines Li/Cl-Phospinidenoidkomplexes basiert. Dieser konnte auf zwei Wegen selektiv bei tiefer Temperatur in Gegenwart von 12-Krone-4 aus einem P-Cp*-substituierten Monochlorphosphanwolframkomplex mittels Deprotonierung mit Lithiumdiisopropylamid (LDA) (Methode A) oder durch Chlor-/Lithiumaustausch an einem P-Cp*-substituierten Dichlorphosphanwolframkomplex mittels t-Butyllithium (Methode B) erzeugt und NMR-spektroskopisch charakterisiert werden. Hierbei liegt der Vorteil von Methode A in der höheren (thermischen) Stabilität, während sich Methode B durch die leichtere Zugänglichkeit der Edukte auszeichnet. Die Folgechemie des Li/Cl-Phospinidenoidkomplexes erwies sich als sehr ergiebig, da beim Erwärmen der Lösung auf Raumtemperatur, selektiv ein weiterer neuer 3[P2C5]3,4(5)-Käfigkomplex gebildet wurde. Außerdem gelang unter Verwendung der jeweiligen Precursorsysteme und äquimolarer Mengen eines Aldehydderivates problemlos die Synthese der entsprechenden P-Cp*-substituierten Oxaphosphiranwolframkomplexe und so erstmals auch die Einführung einer funktionellen Gruppe am Phosphorzentrum. Die Synthesen nach Methode A verliefen in guten bis sehr guten Ausbeuten. Im Falle der Methode B hingegen bildeten sich geringe Mengen an Nebenprodukten, sodass die Oxaphosphirankomplexe mittels Tieftemperatursäulenchromatographie gereinigt werden mussten, was die Ausbeute maßgeblich erniedrigte. Durch die Reaktion der analogen Monochlorphosphanmolybdän- bzw. -chromkomplexe mit LDA und Acetaldehyd in Gegenwart von 12-Krone-4 gelang auch die Darstellung der ersten Oxaphosphiranmolydän- und -chromkomplexe. Am Beispiel des P-Cp*-C-Ph-substituierten Oxaphosphiranwolframkomplexes wurden erste Untersuchungen zur thermischen Stabilität durchgeführt und eine erste Verwendung in der Synthese neuer O,P,C-Käfigverbindungen aufgezeigt. Die Reaktionen unter thermischer Belastung in Gegenwart von Benz- bzw. Acetaldehyd lieferten O,P,C-Käfigkomplexen die unterschiedliche Gerüststrukturen aufweisen (3[O2P1C7]5,6(5) bzw. 3[O2P1C7]6,6(5)).Contributions to the chemistry of transition metal coordinated P-Cp*-substituted heterocycles and P,C-cage compounds In the framework of this thesis diverse studies on the synthesis of transition metal coordinated P,C- and O,P,C-cage ligands (cage complexes) are presented. As miscellaneous polycycles (cages) will be discussed this general formula n[ExPyCz](Rp(R) is introduced in terms of a short nomenclature; where n is the number of condensed rings in the frame, E a heteroelement (O, N), x, y, z are the numbers of atoms of the elements within the cages frame; Rp are the ring sizes of contained phosphorus heterocycles and (R) the ring sizes of the remaining condensed rings (in increasing order). In the first part (Chapter II) the potential of a P-Cp*-substituted 2H-azaphosphirene complex at tungsten- and molybdenum pentacarbonyl respectively as a precursor in P,C-cage complex synthesis is further studied and first reactions of selected polycyclic phosphirane complexes are presented. In the reaction of P-Cp*-substituted 2H-azaphosphirene complexes with alkynes the obtained products depended strongly on substituents and reaction conditions. In the reactions of the tungsten complex either 1H-phosphirene complexes or 4[P1C7]3,4,4(5)-cage complexes were obtained while in case of the molybdenum complex a 4[P2C7]3,5,5(5)-cage complex was identified as the product of a competing reaction pathway. An interesting aspect from this context is that the metal had an influence on the stability of the 1H-phosphirene complex. First studies on the chemistry of P,C-cage complexes using the example of the 4[P1C7]3,4,4(5)-tungsten complexes showed that decomplexation of the P,C-cage ligand can be achieved by thermolysis of the complex either in toluene in the presence of DPPE or in pyridine. The base catalyzed hydrolysis of the C-ester-substituted P,C-cage complexes with lithium hydroxide monohydrate surprisingly lead to partial P,C-cage opening and rearrangement through various bond ruptures and formations respectively. As both types of P,C-cage compounds contained a phosphirane ring in their framework monocyclic P-Cp*-substituted phosphirane complexes were synthesized for comparison. In the course of the study it became apparent that their formation is less favored than that of the polycyclic derivatives. However if this reaction pathway is followed then the complexes are obtained as a diastereomeric mixture. In Addition for the reaction with ethyl acrylate the competing pathway was studied and a diastereomeric mixture of a 3,4-dihydro-2H-1,2-azaphosphole complex was obtained and the tautomerization to the corresponding 2,3-dihydro-1H-1,2-azaphosphole complex was observed. In the second part the focus was placed on the synthesis of P-Cp*-substituted oxaphosphirane complexes (Chapter III). It is shown that P-Cp*-substituted 2H-azaphosphirene complexes are not suitable as precursors. Thermolysis reactions with aryl-substituted aldehydes gave access to 1,3,4-dioxaphospholane complexes selectively instead. Nevertheless a new concept for the synthesis for P-Cp*-substituted oxaphosphirane complexes was developed based on a Li/Cl-phosphinidenoid complex. This complex could be generated selectively at low temperature and in the presence of 12-crown-4 either by deprotonation of a P-Cp*-substituted monochlorophosphine tungsten complex with lithium diisopropylamide (LDA) (method A) or by chloro-/lithium-exchange at a P-Cp*-substituted dichlorophosphine tungsten complex with t-butyl lithium (method B). The advantage of method A lies in the higher (thermal) stability, whereas method B stands out due to the easily accessible starting materials; NMR-spectroscopic characterization of this complex is also presented. Chemistry of the Li/Cl-phosphinidenoid complex proved to be fruitful as warming of the reaction solution to room temperature gave access to another new 3[P2C5]3,4(5)-cage complex. Furthermore was the synthesis of diverse P-Cp*-substituted oxaphosphirane tungsten complexes successful, when either of the two precursor systems was reacted with equimolar amounts of an aldehyde and so for the first time a functional group was introduced at phosphorus. Through analogous reactions of monochlorophosphine molybdenum- and -chromium complexes respectively with LDA und acetaldehyde in the presence of 12-crown-4 the first oxaphosphirane molybdenum- and -chromium complexes could also be obtained. First studies on the thermal stability were carried out at the example of the P-Cp*-C-Ph-substituted oxaphosphirane tungsten complex and a first application in the synthesis of new O,P,C-cage compounds was found. In thermally induced reactions with benz- and acetaldehyde respectively two different O,P,C-cage complexes were obtained (3[O2P1C7]5,6(5) and 3[O2P1C7]6,6(5))

Worse prognosis of osteosarcoma patients expressing IGF-1 on a tissue microarray

BACKGROUND It is hardly possible to define osteosarcoma (OS) patients at greatest risk for non-response to chemotherapy, metastasis and short survival times. Our goal was the investigation of local expression of insulin-like growth factor (IGF-1) with regard to survival time of OS patients using a tissue microarray (TMA). MATERIALS AND METHODS Tumor tissue specimens from surgical primary tumor resections were collected from patients with OS. A TMA was composed, sections were stained with rabbit anti-IGF-1 and grading was performed. Statistics involved Kaplan-Meier curves and the log-rank test. RESULTS We analyzed immunohistochemical expression of local IGF-1 on a TMA based on surgical primary tumor resections of 67 OS patients. The mean clinical follow-up time was 98 months. Twenty-two (33%) OS patients stained negatively and 44 (66%) OS patients stained positively for IGF-1. Significantly shorter survival was detected with expression of IGF-1 (p=0.007). The 5-year survival rate for patients expressing IGF-1 was 63% compared to 92% in patients without expression of IGF-1. Non-responders to chemotherapy and patients with metastasis, who also stained positively for IGF-1 manifested a significantly (p=0.002 and p<0.0001, respectively) shorter survival. CONCLUSION Expression of local IGF-1 in primary tumor tissue appears to significantly affect the aggressiveness of OS, may predict survival time and, above all, may discriminate patients with non-response to chemotherapy and metastasis. This represents the basis for successful patient selection with regard to the decision process for or against chemotherapy and the choice of the most effective therapeutic drug. It may be a more important marker of tumor progression and indicator of prognosis than serum IGF-1. Novel tumor markers and therapeutic agents targeting the local IGF-1 pathway may increase the likelihood of therapeutic success

Studien zum Metabolismus von Pestiziden und Xenobiotika durch humane Cytochrom-P450-Monooxygenasen in transgenen Tabakzellkulturen

Detailed data on the metabolism of pesticides in plants is required for registration, therefore several in vitro systems have been developed to generate and identify a broad spectrum of possible metabolites. Therefore transgenic plant cell cultures with the human isoenzymes CYP1A1 and CYP1A2 were established for an oxidative metabolic profiling of pesticides. The cDNA of human P450 (CYP1A1 and CYP1A2) was introduced into tobacco cells (N. tabacum L.) by Agrobacterium-mediated transformation. For the screening of the transgenic calli an in vivo 7?Ethoxycoumarin O-de-ethylation (ECOD)-assay was established, which was performed with intact plant cells. The ECOD activity was measured as the concentration of non-metabolised 7?ethoxycoumarin remaining in the media. The developing transgenic calli or transgenic suspension cultures were characterised regarding their ECOD-activity. Expression of the P450 genes was further examined by PCR and western blotting. For metabolism studies in the transgenic tobacco cell cultures 14C-labelled atrazine, metamitron and dimethoat were used as model substances. The herbicide atrazine was metabolised by oxidative N-Dealkylation. The conversion rate was considerably higher in the transformed cell cultures (CYP1A1 and CYP1A2) than in non-transformed cell culture. However, CYP1A2 exhibited a higher conversion rate than CYP1A1. In a time-course study the enzyme CYP1A2 catalysed predominately N-deethylation whereas the enzyme CYP1A1 catalysed predominately the N-deisopropylation. In experiments with a constant amount of CYP1A2 plant cell culture the originally used concentration of atrazine was raised up to a 20fold quantity. In these large-scale experiments the high activity of the transgenic clones was demonstrated. Metabolism studies with the herbicide metamitron demonstrated that the arylhydroxylation and the previous desamination both can be catalysed by the isoenzymes CYP1A1 and CYP1A2. The conversion rate was considerably higher in the transformed cell cultures (CYP1A1 and CYP1A2) than in non-transformed cell culture. However, CYP1A2 exhibited a higher conversion rate than CYP1A1. In a time-course study using a CYP1A2 cell culture the transformation of metamitron could be demonstrated, starting with the formation of the metabolite desaminometamitron, followed by 4?hydroxydesaminometamitron and ended with the glucosid of the later. In experiments with a constant amount of CYP1A2 plant cell culture the originally used concentration of metamitron was raised up to a 20fold quantity. In these large-scale experiments the relative metabolic profile had changed slightly. This was due to the slower glucosylation compared to P450-oxidation. Therefore a high P450 activity of the transgenic clones was demonstrated. The third substance used was the insecticide dimethoate with three potential P450 reaction centres present in this molecule. The three reactions are the desulfuration, N-demethylation and O-demethylation. Nevertheless, there was no evidence that dimethoat was a suitable substrate for the two P450 isoenzymes. Only a slight increase in the conversion rate of dimethoate could be found by the CYP1A2 enzyme which was attributed to the O-demethylation. Finally the metabolism of dimethoate was tested in liver microsomes of rats, which were prior induced with Aroclor 1254 to increase the P450 enzyme activity. On the basis of the obtained results it is obvious, that dimethoate is not metabolised by P450-isoenzymes in rats. Plant cell cultures in which the appropriate P450 cDNAs are introduced, appear to be suitable in vitro systems in order to produce large quantities of primary oxidised pesticide metabolites

Studien zum Metabolismus von Pestiziden und Xenobiotika durch humane Cytochrom-P450-Monooxygenasen in transgenen Tabakzellkulturen

Detailed data on the metabolism of pesticides in plants is required for registration, therefore several in vitro systems have been developed to generate and identify a broad spectrum of possible metabolites. Therefore transgenic plant cell cultures with the human isoenzymes CYP1A1 and CYP1A2 were established for an oxidative metabolic profiling of pesticides. The cDNA of human P450 (CYP1A1 and CYP1A2) was introduced into tobacco cells (N. tabacum L.) by Agrobacterium-mediated transformation. For the screening of the transgenic calli an in vivo 7?Ethoxycoumarin O-de-ethylation (ECOD)-assay was established, which was performed with intact plant cells. The ECOD activity was measured as the concentration of non-metabolised 7?ethoxycoumarin remaining in the media. The developing transgenic calli or transgenic suspension cultures were characterised regarding their ECOD-activity. Expression of the P450 genes was further examined by PCR and western blotting. For metabolism studies in the transgenic tobacco cell cultures 14C-labelled atrazine, metamitron and dimethoat were used as model substances. The herbicide atrazine was metabolised by oxidative N-Dealkylation. The conversion rate was considerably higher in the transformed cell cultures (CYP1A1 and CYP1A2) than in non-transformed cell culture. However, CYP1A2 exhibited a higher conversion rate than CYP1A1. In a time-course study the enzyme CYP1A2 catalysed predominately N-deethylation whereas the enzyme CYP1A1 catalysed predominately the N-deisopropylation. In experiments with a constant amount of CYP1A2 plant cell culture the originally used concentration of atrazine was raised up to a 20fold quantity. In these large-scale experiments the high activity of the transgenic clones was demonstrated. Metabolism studies with the herbicide metamitron demonstrated that the arylhydroxylation and the previous desamination both can be catalysed by the isoenzymes CYP1A1 and CYP1A2. The conversion rate was considerably higher in the transformed cell cultures (CYP1A1 and CYP1A2) than in non-transformed cell culture. However, CYP1A2 exhibited a higher conversion rate than CYP1A1. In a time-course study using a CYP1A2 cell culture the transformation of metamitron could be demonstrated, starting with the formation of the metabolite desaminometamitron, followed by 4?hydroxydesaminometamitron and ended with the glucosid of the later. In experiments with a constant amount of CYP1A2 plant cell culture the originally used concentration of metamitron was raised up to a 20fold quantity. In these large-scale experiments the relative metabolic profile had changed slightly. This was due to the slower glucosylation compared to P450-oxidation. Therefore a high P450 activity of the transgenic clones was demonstrated. The third substance used was the insecticide dimethoate with three potential P450 reaction centres present in this molecule. The three reactions are the desulfuration, N-demethylation and O-demethylation. Nevertheless, there was no evidence that dimethoat was a suitable substrate for the two P450 isoenzymes. Only a slight increase in the conversion rate of dimethoate could be found by the CYP1A2 enzyme which was attributed to the O-demethylation. Finally the metabolism of dimethoate was tested in liver microsomes of rats, which were prior induced with Aroclor 1254 to increase the P450 enzyme activity. On the basis of the obtained results it is obvious, that dimethoate is not metabolised by P450-isoenzymes in rats. Plant cell cultures in which the appropriate P450 cDNAs are introduced, appear to be suitable in vitro systems in order to produce large quantities of primary oxidised pesticide metabolites

Expression of MSH2 and MSH6 on a Tissue Microarray in Patients with Osteosarcoma

BACKGROUND/AIM: Reliable prognostic factors for the outcome of patients with osteosarcoma (OS) remain elusive. We analyzed the relationship between immunohistochemical expression of deoxyribonucleic acid (DNA) mismatch repair (MMR) proteins, MutS protein homolog 2 (MSH2) and MSH6 using a tissue microarray (TMA) with respect to OS patient demographics and survival time. MATERIALS AND METHODS: We retrieved tumor tissue specimens from bone tissue originating from surgical primary tumor specimens of OS patients to generate a TMA and stained sections with antibodies against MSH2 and MSH6. RESULTS: Tumor resections of 67 patients with a mean follow-up of 98 months were evaluated. MSH2 was expressed in nine (13%), MSH6 in ten (15%) and combined MSH2 and MSH6 (MSH2/6) in six (9%) patients. Significantly shorter survival times were associated with expression of MSH6, MSH2/6, as well as simultaneous non-response to chemotherapy and presence of metastasis. CONCLUSION: The survival time of patients with OS may be predicted by local expression of MSH6 and MSH2/6 in surgical primary tumor resections. This study shows the prognostic value of the local expression of DNA MMR proteins, as markers for poor prognosis of OS patients

Insights into the complex biosynthesis of the leupyrrins in Sorangium cellulosum So ce690.

The anti-fungal leupyrrins are secondary metabolites produced by several strains of the myxobacterium Sorangium cellulosum. These intriguing compounds incorporate an atypically substituted γ-butyrolactone ring, as well as pyrrole and oxazolinone functionalities, which are located within an unusual asymmetrical macrodiolide. Previous feeding studies revealed that this novel structure arises from the homologation of four distinct structural units, nonribosomally-derived peptide, polyketide, isoprenoid and a dicarboxylic acid, coupled with modification of the various building blocks. Here we have attempted to reconcile the biosynthetic pathway proposed on the basis of the feeding studies with the underlying enzymatic machinery in the S. cellulosum strain So ce690. Gene products can be assigned to many of the suggested steps, but inspection of the gene set provokes the reconsideration of several key transformations. We support our analysis by the reconstitution in vitro of the biosynthesis of the pyrrole carboxylic starter unit along with gene inactivation. In addition, this study reveals that a significant proportion of the genes for leupyrrin biosynthesis are located outside the core cluster, a 'split' organization which is increasingly characteristic of the myxobacteria. Finally, we report the generation of four novel deshydroxy leupyrrin analogues by genetic engineering of the pathway