Konstantin Drandarov

Guignardic Acid, a Novel Type of Secondary Metabolite Produced by the Endophytic Fungus Guignardia sp.: Isolation, Structure Elucidation, and Asymmetric Synthesis

A UV-guided fractionation of the AcOEt extract of the fermentation broth of Guignardia sp., an endophytic fungus from the leaves of the tropical tree Spondias mombin, resulted in the identification of the new metabolite (−)-(2S,5Z)-2-(1-methylethyl)-4-oxo-5-(phenylmethylene)-1,3-dioxolane-2-carboxylic acid (1), isolated as NH salt 1a. The metabolite 1 was designated (−)-(S)-guignardic acid. This first member of a new class of natural compounds contains a dioxolanone moiety formed by fusion of 2-oxo-3-phenylpropanoic acid (phenylpyruvic acid) and 3-methyl-2-oxobutanoic acid (dimethylpyruvic acid), products of the oxidative deamination of phenylalanine and valine, respectively. The structure of 1a was deduced from spectral data (UV, IR, MS, 1H- and 13C-NMR) and confirmed by asymmetric synthesis.

Radiolabeling and in vitro /in vivo evaluation of N-(1-adamantyl)-8-methoxy-4-oxo-1-phenyl-1,4-dihydroquinoline-3-carboxamide as a PET probe for imaging cannabinoid type 2 receptor.

The cannabinoid type 2 (CB2) receptor plays an important role in neuroinflammatory and neurodegenerative diseases such as multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimers disease and is therefore a very promising target for therapeutic approaches as well as for imaging. Based on the literature, we identified one 4‐oxoquinoline derivative (designated KD2) as the lead structure. It was synthesized, radiolabeled and evaluated as a potential imaging tracer for CB2. [11C]KD2 was obtained in 99% radiochemical purity. Moderate blood–brain barrier (BBB) passage was predicted for KD2 from an in vitro transport assay with P‐glycoprotein‐transfected Madin Darby canine kidney cells. No efflux of KD2 by P‐glycoprotein was detected. In vitro autoradiography of rat and mouse spleen slices demonstrated that [11C]KD2 exhibits high specific binding towards CB2. High spleen uptake of [11C]KD2 was observed in dynamic positron emission tomography (PET) studies with Wistar rats and its specificity was confirmed by displacement study with a selective CB2 agonist, GW405833. A pilot autoradiography study with post‐mortem spinal cord slices from amyotrophic lateral sclerosis (ALS) patients with [11C]KD2 suggested the presence of CB2 receptors under disease conditions. Specificity of [11C]KD2 binding could also be demonstrated on these human tissues. In conclusion, [11C]KD2 shows good in vitro and in vivo properties as a potential PET tracer for CB2.

Macrocyclic Spermine Alkaloids from Verbascum: The (E/Z)-Isomeric Pairs (−)-(S)-Verbasitrine/(−)-(S)-Isoverbasitrine and (+)-(S)-Verbametrine/(+)-(S)-Isoverbametrine: Isolation, Structure Elucidation, and Synthesis

The isolation and structure elucidation of the 17-membered macrocyclic spermine alkaloids (−)-(S)-verbasitrine (2), (−)-(S)-isoverbasitrine (4), (+)-(S)-verbametrine (6), and (+)-(S)-isoverbametrine (8) is presented. The synthesis of their racemates is described.

A probable dual mode of action for both L- and D-lactate neuroprotection in cerebral ischemia

Lactate has been shown to offer neuroprotection in several pathologic conditions. This beneficial effect has been attributed to its use as an alternative energy substrate. However, recent description of the expression of the HCA1 receptor for lactate in the central nervous system calls for reassessment of the mechanism by which lactate exerts its neuroprotective effects. Here, we show that HCA1 receptor expression is enhanced 24 hours after reperfusion in an middle cerebral artery occlusion stroke model, in the ischemic cortex. Interestingly, intravenous injection of L-lactate at reperfusion led to further enhancement of HCA1 receptor expression in the cortex and striatum. Using an in vitro oxygen-glucose deprivation model, we show that the HCA1 receptor agonist 3,5-dihydroxybenzoic acid reduces cell death. We also observed that D-lactate, a reputedly non-metabolizable substrate but partial HCA1 receptor agonist, also provided neuroprotection in both in vitro and in vivo ischemia models. Quite unexpectedly, we show D-lactate to be partly extracted and oxidized by the rodent brain. Finally, pyruvate offered neuroprotection in vitro whereas acetate was ineffective. Our data suggest that L- and D-lactate offer neuroprotection in ischemia most likely by acting as both an HCA1 receptor agonist for non-astrocytic (most likely neuronal) cells as well as an energy substrate.

Hydroxylated 2-Amino-3H-phenoxazin-3-one Derivatives as Products of 2-Hydroxy-1,4-benzoxazin-3-one (HBOA) Biotransformation by Chaetosphaeria sp., an Endophytic Fungus from Aphelandra tetragona

The biotransformation of the phytoanticipin HBOA and its major degradation metabolites 2-hydroxy-N-(2-hydroxyphenyl)acetamide (7) and N-(2-hydroxyphenyl)acetamide (8) by Chaetosphaeria sp., an endophytic fungus isolated from Aphelandra tetragona, was studied. Three new metabolites could be identified as 2-amino-7-hydroxy-3H-phenoxazin-3-one (12), 2-acetylamino-7-hydroxy-3H-phenoxazin-3-one (13) and 7-hydroxy-2-(2-hydroxyacetyl)- amino-3H-phenoxazin-3-one (14). Structure elucidation of 12 and 13 was performed by MS, 1H, 13C NMR and 2D NMR techniques and confirmed by chemical transformation.

Towards non-invasive imaging of vulnerable atherosclerotic plaques by targeting co-stimulatory molecules

BACKGROUND Myocardial infarction and stroke are the life-threatening consequences after plaque rupture in coronary or carotid arteries. Positron emission tomography employing [(18)F]fluorodeoxyglucose can visualize plaque inflammation; however, the question remains whether this is specific for plaque vulnerability. The pathophysiology of vulnerable plaques suggests several molecular processes. Here, we propose the co-stimulatory molecules CD80 and CD86 as potential new targets for non-invasive imaging. METHODS AND RESULTS Human atherosclerotic segments were obtained from carotid endarterectomy and classified into stable and vulnerable plaques. We identified CD80 and CD86 with significantly higher mRNA levels in vulnerable than stable plaques. CD80+ and CD86+ cells were found in spatial proximity to CD83+ dendritic cells and CD68+ macrophages of atherosclerotic plaques. As a proof of target-expression we labeled a low molecular weight ligand, which has a high affinity for human CD80, with carbon-11 to perform in vitro autoradiography with human plaque slices. We observed 3-fold higher binding to vulnerable than stable plaques, demonstrating a first approach towards discriminating between the two plaque types. Positron emission tomography studies showed accumulation in CD80+ Raji xenografts, low radioactivity in myocardium and rapid clearance from the blood pool in mice. CONCLUSION In human carotid arteries, the co-stimulatory molecules CD80 and CD86 show significantly higher expression levels in vulnerable compared to stable plaques. With the novel CD80-specific radiotracer we are able to discriminate between stable and vulnerable atherosclerotic plaques in vitro. This is an important step towards non-invasive imaging of the life-threatening vulnerable lesions in humans.

Protoverbine, the Parent Member of a Class of Macrocyclic Spermine Alkaloids

The 17-membered macrocyclic spermine alkaloids protoverbine ((8S)-8-phenyl-1,5,9,13-tetraazacycloheptadecan-6-one, 1) and its N(9),N(13)-methylene-bridged derivative protomethine ((2S)-2-phenyl-1,5,9,14-tetraazabicyclo[12.3.1]octadecan-4-one, 2) were isolated from Verbascum pseudonobileStoj.etStef. (Scrophulariaceae) and characterized. The synthesis of their racemates is described. The possible role of (S)-protoverbine (1) and (S)-protomethine (2) as precursors in the biogenesis of the whole class of N(1),N(9)- and/or N(13)-substituted alkaloids, the groups of verbamethine ((S)-3a – f), verbacine ((S)-4a – f), incasine B′/verdoline ((S)-5a – f), verbamedine ((S)-6a – f), and verbascenine ((S)-7a – f), is discussed.

VERBACINE AND VERBALLOCINE, NOVEL MACROCYCLIC SPERMINE ALKALOIDS FROM VERBASCUM PSEUDONOBILE STOJ. ET STEF. (SCROPHULARIACEAE)

Abstract The isolation and structural elucidation of the novel 17-membered lactam alkaloids verbacine ( 1 ) and verballocine ( 2 ) containing spermine, E - (or Z -) cinnamoyl and phenylpropionyl precursory units, is reported. Evidence for the artificial origin of the previously reported verbaskine is presented.

Macrocyclic Spermine Alkaloids fromVerbascum: Isolation, Structure Elucidation, and Syntheses of the (E/Z)-Isomeric Pairs (S)-Verbasikrine/(S)-Isoverbasikrine and (S)-Verbamekrine/(S)-Isoverbamekrine

The isolation and structure elucidation of the 17-membered macrocyclic spermine alkaloids (S)-verbasikrine (3), (S)-isoverbasikrine (6), (S)-verbamekrine (9), and (S)-isoverbamekrine (12) is presented. The syntheses of their racemates are described. The HPLC/APCI-MS analysis of the original total alkaloid extract of Verbascum pseudonobile is presented.

Phenol oxidative coupling in the biogenesis of the macrocyclic spermine alkaloids aphelandrine and orantine in Aphelandra sp.

Abstract. A crucial step in the biosynthesis of the spermine alkaloid aphelandrine and its diastereoisomer orantine is an intramolecular cyclization of the intermediate (S)-dihydroxyverbacine. In order to elucidate this step of the biosynthetic pathway, microsomes from the roots of Aphelandra squarrosa Nees were incubated with unlabeled and (D8)-labeled (S)-dihydroxyverbacine. It was shown that the microsomal fraction catalyzes the intramolecular coupling of (S)-dihydroxyverbacine to aphelandrine. This was proven by microsomal transformation of (D8)-labeled (S)-dihydroxyverbacine to (D8)-labeled aphelandrine. The reaction absolutely requires NAPDH and O2. The underlying reaction mechanism is probably an oxidative phenol coupling catalyzed by an aphelandrine synthase. This enzyme is proposed to be a cytochrome P-450 oxidase. The intramolecular cyclization of (S)-dihydroxyverbacine represents an important point in the biogenesis of the aphelandrine-type alkaloids.