Synthesis of New Potential Lipophilic Co-Drugs of 2-Chloro-2'-deoxyadenosine (Cladribine, 2-CdA, Mavenclad®, Leustatin®) and 6-Azauridine (z6 U) 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 1 H- and 13 C-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.

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 1 H-, 13 C-, 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.

Guanosine Nucleolipids: Synthesis, Characterization, Aggregation and X-Ray Crystallographic Identification of Electricity-Conducting G-Ribbons.

The lipophilization of ß-d-riboguanosine (1) with various symmetric as well as asymmetric ketones is described (→3a-3f). The formation of the corresponding O-2',3'-ketals is accompanied by the appearance of various fluorescent by-products which were isolated chromatographically as mixtures and tentatively analyzed by ESI-MS spectrometry. The mainly formed guanosine nucleolipids were isolated and characterized by elemental analyses, 1 H-, 13 C-NMR and UV spectroscopy. For a drug profiling, static topological polar surface areas as well as 10 logPOW values were calculated by an increment-based method as well as experimentally for the systems 1-octanol-H2 O and cyclohexane-H2 O. The guanosine-O-2',3'-ketal derivatives 3b and 3a could be crystallized in (D6 )DMSO - the latter after one year of standing at ambient temperature. X-ray analysis revealed the formation of self-assembled ribbons consisting of two structurally similar 3b nucleolipid conformers as well as integrated (D6 )DMSO molecules. In the case of 3a ⋅ DMSO, the ribbon is formed by a single type of guanosine nucleolipid molecules. The crystalline material 3b ⋅ DMSO was further analyzed by differential scanning calorimetry (DSC) and temperature-dependent polarization microscopy. Crystallization was also performed on interdigitated electrodes (Au, distance, 5 µm) and visualized by scanning electron microscopy. Resistance and amperage measurements clearly demonstrate that the electrode-bridging 3b crystals are electrically conducting. All O-2',3'-guanosine ketals were tested on their cytostatic/cytotoxic activity towards phorbol 12-myristate 13-acetate (PMA)-differentiated human THP-1 macrophages as well as against human astrocytoma/oligodendroglioma GOS-3 cells and against rat malignant neuroectodermal BT4Ca cells.

Combinatorial Synthesis of New Pyrimidine- and Purine-ß-d-Ribonucleoside Nucleolipids: Their Distribution Between Aqueous and Organic Phases and Their In Vitro Activity Against Human- and Rat Glioblastoma Cells In Vitro.

Two series of nucleolipids, O-2',3'-heptanylidene- as well as O-2',3'-undecanylidene ketals of six ß-d-ribonucleosides (type A) and partly N-farnesyl derivatives thereof (type B) were prepared in a combinatorial manner. All novel compounds were characterized by elemental analysis and/or ESI mass spectrometry and by UV-, 1 H-, and 13 C-NMR spectroscopy. Conformational parameters of the nucleosides and nucleolipids were calculated from various 3 J(H,H), 3 J(1 H,13 C), and 5 J(F,H) coupling constants. For a drug profiling, the parent nucleosides and their lipophilic derivatives were studied with respect to their distribution (log P) between water and n-octanol as well as water and cyclohexane. From these data, qualitative conclusions were drawn concerning their possible blood-brain barrier passage efficiency. Moreover, nucleolipids were characterized by their molecular descriptor amphiphilic ratio (a.r.), which describes the balance between the hydrophilicity of the nucleoside headgroup and the lipophilicity of the lipid tail. All compounds were investigated in vitro with respect to their cytostatic/cytotoxic activity toward human glioblastoma (GOS 3) as well as rat malignant neuroectodermal BT4Ca cell lines in vitro. In order to differentiate between anticancer and side-effects of the novel nucleolipids, they were also studied on their activity on differentiated human THP-1 macrophages.

Novel Nucleolipids of Pyrimidine ß-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 ß-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 (1) H-, and (13) C-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.

Ameliorated or Acquired Cytostatic/Cytotoxic Properties of Nucleosides by Lipophilization.

A series of nucleolipids, containing one of the ß-D-ribonucleosides 5-fluorouridine, 6-azauridine, uridine, or 5-methyluridine were lipophilized, either at the O-2',3'-position and/or at N(3) of the nucleobase with a large variety of hydrophobic residues. The resulting nucleolipids as well as the parent nucleosides and the lipid precursors were investigated in vitro with respect to their antitumor activity towards i) ten human tumor cell lines from the NCI 60 panel and ii) partly against three further tumor cell lines, namely a) human astrocytoma/oligodendro glioma GOs-3, b) rat malignantneuroectodermal BT4Ca, and c) differentiated human THP-1 macrophages. Inspection of the doseresponse curves allows two main conclusions concerning lipid determinants lending the corresponding nucleoside an ameliorated or an acquired antitumor activity: i) introduction of either a symmetrical O-2',3'-nonadecylidene ketal group or introduction of an O-2',3'-ethyl levulinate moiety plus an N(3)-farnesyl group leads often to nucleolipids with significant cytostatic/cytotoxic properties; ii) for the two canonical and non-toxic nucleosides uridine and 5-methyluridine, the condensation with also non-toxic lipids gives nucleolipids with a pronounced antitumor activity.

Nucleolipids of Canonical Purine ß-d-Ribo-Nucleosides: Synthesis and Cytostatic/Cytotoxic Activities Toward Human and Rat Glioblastoma Cells.

We report on the synthesis of two series of canonical purine ß-d-ribonucleoside nucleolipids derived from inosine and adenosine, which have been characterized by elemental analyses, electrospray ionization mass spectrometry (ESI MS) as well as by (1)H and (13)C NMR, and pH-dependent UV/Vis spectroscopy. A selection of the novel nucleolipids with different lipophilic moieties were first tested on their cytotoxic effect toward human macrophages. Compounds without a significant inhibitory effect on the viability of the macrophages were tested on their cytostatic/cytotoxic effect toward human astrocytoma/oligodendroglioma GOS-3 cells as well as against the rat malignant neuroectodermal BT4Ca cell line. In order to additionally investigate the potential molecular mechanisms involved in the cytotoxic effects of the derivatives on GOS-3 or BT4Ca cells, we evaluated the induction of apoptosis and observed the particular activity of the nucleolipid ethyl 3-{4-hydroxymethyl-2-methyl-6-[6-oxo-1-(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-1,6-dihydro-purin-9-yl]-tetrahydro-furo[3,4-d][1,3]dioxol-2-yl}propionate (8 c) toward both human and rat glioblastoma cell lines in vitro.