Nucleolipids of the Nucleoside Antibiotics Formycins A and B: Synthesis and Biomedical Characterization Particularly Using Glioblastoma Cells

Two lipophilic derivatives of formycin A ( 1 ) and formycin B ( 5 ) carrying an O‐2′,3′‐(ethyl levulinate) ketal group have been prepared. These were base‐alkylated at N(1) (for 1 ) and N(1) and N(6) (for 5 ) with both isopentenyl and all‐trans‐farnesyl residues. Upon the prenylation, side reactions were observed, resulting in the formation of nucleolipids with a novel tricyclic nucleobase (→ 4a , 4b ). In the case of formycin B, O‐2′,3′‐(ethyl levulinate) ( 6 ) farnesylation gave the double prenylated nucleolipid 7 . All new compounds were characterized by ¹H‐, ¹³C‐, UV/VIS and fluorescence spectroscopy, by ESI‐MS spectrometry and/or by elemental analysis. Log P determinations between water and octanol as well as water and cyclohexane of a selection of compounds allowed qualitative conclusions concerning their potential blood‐brain barrier passage efficiency. All compounds were investigated in vitro with respect to their cytotoxic activity toward rat malignant neuroectodermal BT4Ca as well as against a series of human glioblastoma cell lines (GOS 3, U‐87 MG and GBM 2014/42). In order to differentiate between anticancer and side effects of the novel nucleolipids, we also studied their activity on PMA‐differentiated human THP‐1 macrophages. Here, we show that particularly the formycin A derivative 3b possesses promising antitumor properties in several cancer cell lines with profound cytotoxic effects partly on human glioblastoma cells, with a higher efficacy than the chemotherapeutic drug 5‐fluorouridine.     

Synthesis of New Potential Lipophilic Co‐Drugs of 2‐Chloro‐2′‐deoxyadenosine (Cladribine, 2‐CdA,Mavenclad®, Leustatin®) and 6‐Azauridine (z6U) with Valproic Acid

2‐Chloro‐2′‐deoxyadenosine (cladribine, 1 ) was acylated with valproic acid ( 2 ) under various reaction conditions yielding 2‐chloro‐2′‐deoxy‐3′,5′‐O‐divalproyladenosine ( 3 ) as well as the 3′‐O‐ and 5′‐O‐monovalproylated derivatives, 2‐chloro‐2′‐deoxy‐3′‐O‐valproyladenosine ( 4 ) and 2‐chloro‐2′‐deoxy‐5′‐O‐valproyladenosine ( 5 ), as new co‐drugs. In addition, 6‐azauridine‐2′,3′‐O‐(ethyl levulinate) ( 8 ) was valproylated at the 5′‐OH group (→ 9 ). All products were characterized by ¹H‐ and ¹³C‐NMR spectroscopy and ESI mass spectrometry. The structure of the by‐product 6 (N‐cyclohexyl‐N‐(cyclohexylcarbamoyl)‐2‐propylpentanamide), formed upon valproylation of cladribine in the presence of N,N‐dimethylaminopyridine and dicyclohexylcarbodiimide, was analyzed by X‐ray crystallography. Cladribine as well as its valproylated co‐drugs were tested upon their cancerostatic/cancerotoxic activity in human astrocytoma/oligodendroglioma GOS‐3 cells, in rat malignant neuro ectodermal BT4Ca cells, as well as in phorbol‐12‐myristate 13‐acetate (PMA)‐differentiated human THP‐1 macrophages. The most important result of these experiments is the finding that only the 3′‐O‐valproylated derivative 4 exhibits a significant antitumor activity while the 5′‐O‐ as well as the 3′,5′‐O‐divalproylated cladribine derivatives 3 and 5 proved to be inactive.     

Novel Nucleolipids of Pyrimidine β‐D‐Ribonucleosides: Combinatorial Synthesis,Spectroscopic Characterization,and Cytostatic/Cytotoxic Activities

Four series of nucleolipids with either uridine, 5‐methyluridine, 5‐fluorouridine, and 6‐azauridine as β‐D ‐ribonucleoside component have been prepared in a combinatorial (not parallel!) manner (see Formulae). All compounds have been characterized by elemental analyses, ESI mass spectrometry as well as by ¹H‐, and ¹³C‐NMR, and UV spectroscopy. A selection of eight nucleolipids with different lipophilizing moieties, based on earlier findings, as well as of 5‐fluorouridine as control were first tested on their cytotoxic effect towards PMA‐differentiated human THP‐1 macrophages. Those compounds which did not exhibit a significant inhibitory effect on the survival of the macrophages were next tested on their cytostatic/cytotoxic effect towards the human astrocytoma/oligodendroglioma GOS‐3 cells as well as against the rat malignant neuroectodermal BT4Ca cell line. Additionally, induction of apoptosis of the cell lines was evaluated. It turned out that particularly a combined lipophilization of the nucleosides by an 2′,3′‐O‐ethyl levulinate residue plus a farnesyl moiety at N(3) of the pyrimidine moiety of the corresponding nucleolipids leads to an active compound with the highest probability.     

Deep Bisulfite Sequencing of Aberrantly Methylated Loci in a Patient with Multiple Methylation Defects

NLRP7 is a maternal effect gene as maternal mutations in this gene cause recurrent hydatidiform moles, spontaneous abortions and stillbirths, whereas live births are very rare. We have studied a patient with multiple anomalies born to a mother with a heterozygous NLRP7 mutation. By array-based CpG methylation analysis of blood DNA from the patient, his parents and 18 normal controls on Illumina Infinium HumanMethylation27 BeadChips we found that the patient had methylation changes (delta ß ≥ 0.3) at many imprinted loci as well as at 87 CpGs associated with 85 genes of unknown imprinting status. Using a pseudoproband (permutation) approach, we found methylation changes at only 7-24 CpGs (mean 15; standard deviation 4.84) in the controls. Thus, the number of abberantly methylated CpGs in the patient is more than 14 standard deviations higher. In order to identify novel imprinted genes among the 85 conspicuous genes in the patient, we selected 19 (mainly hypomethylated) genes for deep bisulfite amplicon sequencing on the ROCHE/454 Genome Sequencer in the patient and at least two additional controls. These controls had not been included in the array analysis and were heterozygous for a single nucleotide polymorphism at the test locus, so that allele-specific DNA methylation patterns could be determined. Apart from FAM50B, which we proved to be imprinted in blood, we did not observe allele-specific DNA methylation at the other 18 loci. We conclude that the patient does not only have methylation defects at imprinted loci but (at least in blood) also an excess of methylation changes at apparently non-imprinted loci.     

Synthese der pentasaccharid-kette des forssman-antigens

The pentasaccharide chain of the Forssman antigen, O-(2-acetamido-2-deoxy-α-d-galactopyranosyl)-(1→3)-O-(2-acetamido-2-deoxy-β-d-galactopyranosyl)-(1→3)-O-α-d- galactopyranosyl-(1→4)-O-β-d-galactopyranosyl-(1→4)-d-glucopyranose (46) was synthesized by a block synthesis in which an α-d-glycoside linkage between two d-galactose residues was formed. The trisaccharide O-(6-O-acetyl-2-azido-3,4-di-O-benzoyl-2-deoxy-α-d-galactopyranosyl)- (1→3)-O-(6-O-acetyl-4-O-benzyl-2-deoxy-2-phthalimido-β-d-galactopyranosyl)-(1→3)-6-O-acetyl-2,4-di-O-benzyl- α-d-galactopyranosyl bromide (40) (this was obtained through acetolysis of O-(6-O-acetyl-2-azido-3,4-di-O-benzoyl-2-deoxy-α-d-galactopyranosyl)- (1→3)-O-(6-O-acetyl-4-O-benzyl-2-deoxy-2-phthalimido-β-d-galactopyranosyl)-(1→3)-1,6-anhydro-2,4-di-O-benzyl-β-d- galactopyranose to the acetyl derivative, followed by reaction with titanium tetrabromide under anhydrous conditions) was condensed with benzyl-4-O-(6-O-benzoyl-2,3-di-O-benzyl-β-d-galactopyranosyl)-2,3,6- tri-O-benzyl-β-d-glucopyranoside were in the presence of silver carbonate and perchlorate. The resulting pentasaccharide was deprotected to give 46.     

DNA-methylation profiling of fetal tissues reveals marked epigenetic differences between chorionic and amniotic samples

Epigenetic mechanisms including DNA methylation are supposed to play a key role in fetal development. Here we have investigated fetal DNA-methylation levels of 27,578 CpG loci in 47 chorionic villi (CVS) and 16 amniotic cell (AC) samples. Methylation levels differed significantly between karyotypically normal AC and CVS for 2,014 genes. AC showed more extreme DNA-methylation levels of these genes than CVS and the differentially methylated genes are significantly enriched for processes characteristic for the different cell types sampled. Furthermore, we identified 404 genes differentially methylated in CVS with trisomy 21. These genes were significantly enriched for high CG dinucleotid (CpG) content and developmental processes associated with Down syndrome. Our study points to major tissue-specific differences of fetal DNA-methylation and gives rise to the hypothesis that part of the Down syndrome phenotype is epigenetically programmed in the first trimester of pregnancy.