Ameliorated or Acquired Cytostatic/Cytotoxic Properties of Nucleosides by Lipophilization

A series of nucleolipids, containing one of the β‐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.     

Cytostatic/Cytotoxic Effects of 5‐Fluorouridine Nucleolipids on Colon,Hepatocellular, and Renal Carcinoma Cells: in vitro Identification of a Potential Cytotoxic Multi‐Anticancer Drug

The insufficient penetration through the cell membranes is one of the major drawbacks of chemotherapeutics such as 5‐fluorouracil (5‐FU; 1 ). To improve the penetration, a useful strategy is the attachment of lipophilic moieties. Thus, we have synthesized a series of nucleolipid derivatives of 5‐fluorouridine (5‐FUrd; 2a ), carrying lipophilic moieties at N(3) and/or at the 2′,3′‐O position, i.e., 3a, 3b, 4 – 7 , and tested their cytostatic/cytotoxic activities towards three carcinoma cell lines (colon (HT‐29), hepatocellular (HepG2), and renal (RENCA)) in comparison with 5‐FU ( 1 ) and 5‐FUrd ( 2a ). After 48 h of incubation, four derivatives, 3a, 3b, 5 , and 7 , showed inhibitory effects on the survival of HT‐29, HepG2, and RENCA cells. Additionally, to differentiate between anticancer and side‐effects, we tested the cytotoxicity of the derivatives in human macrophages. Interestingly, the derivatives 4, 5 , and 6 did not exhibit any effects on survival of THP‐1 macrophages. Furthermore, we investigated the apoptosis induction of compound 1 and 2a , and the above‐mentioned derivatives in HT‐29 cells. Derivative 5 showed the highest significant (p<0.05; p<0.01) increase of the apoptosis at 80 μM after 2‐h or 4‐h treatment, as well as after 6‐h incubation at 40 μM (p<0.05). Real‐time PCR revealed that 40‐μM derivative 5 showed a 1.8‐fold increase of the pro‐apoptotic caspase‐3 gene and a twofold significant increase (p<0.01 and p<0.05 vs. control and 1 , resp.) of the tumor suppressor TP53 gene, whereas the other compounds did not show any effect. We demonstrated that some 5‐FUrd derivatives such as compound 5 are more effective than 5‐FU or 5‐FUrd concerning a cytotoxic (vs. cytostatic (5‐FU, 5‐FUrd)) effect on different cancer cell lines, but without cytotoxic side‐effects on differentiated macrophages. Thus, compound 5 is suggested as a novel potent cytotoxic multi‐anti‐cancer drug.     

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.     

Mitsunobu Alkylation of Cancerostatic 5‐Fluorouridine with (2E)‐10‐Hydroxydec‐2‐enoic Acid,a Fatty Acid from Royal Jelly with Multiple Biological Activities

5‐Fluorouridine ( 1 ) – a nucleoside antimetabolite with strong cancerostatic properties – was protected i) at the 2′‐ and 3′‐OH groups with a heptan‐4‐ylidene residue and ii) at the 5′‐OH group with a (4‐methoxyphenyl)(diphenyl)methyl residue. This fully protected compound, 3 , was submitted to a Mitsunobu reaction with the N‐hydroxysuccinimide (NHS) ester, 5 , of (2E)‐10‐hydroxydec‐2‐enoic acid ( 4 ) which gave nucleolipid 6 . The latter was detritylated with Cl₂CHCOOH to yield the co‐drug 7 as NHS ester.     

Nucleolipids of the Cancerostatic 5‐Fluorouridine: Synthesis,Adherence to Oligonucleotides,and Incorporation in Artificial Lipid Bilayers

5‐Fluorouridine ( 1a ) was converted to its N(3)‐farnesylated nucleoterpene derivative 8 by direct alkylation with farnesyl bromide ( 4 ). Reaction of the cancerostatic 1a with either acetone, heptan‐4‐one, nonadecan‐10‐one, or hentriacontan‐16‐one afforded the 2′,3′‐O‐ketals 2a – 2d . Compound 2b was then first farnesylated (→ 5 ) and subsequently phosphitylated to give the phosphoramidite 6 . The ketal 2c was directly 5′‐phosphitylated without farnesylation of the base to give the phosphoramidite 7 . Moreover, the recently prepared cyclic 2′,3′‐O‐ketal 11 was 5′‐phosphitylated to yield the phosphoramidite 12 . The 2′,3′‐O‐isopropylidene derivative 2a proved to be too labile to be converted to a phosphoramidite. All novel derivatives of 1a were unequivocally characterized by NMR and UV spectroscopy and ESI mass spectrometry, as well as by elemental analyses. The lipophilicity of the phosphoramidite precursors were characterized by both their retention times in RP‐18 HPLC and by calculated log P values. The phosphoramidites 6, 7 , and 12 were exemplarily used for the preparation of four terminally lipophilized oligodeoxynucleotides carrying a cyanine‐3 or a cyanine‐5 residue at the 5′‐(n–1) position (i.e., 14 – 17 ). Their incorporation in an artificial lipid bilayer was studied by single‐molecule fluorescence spectroscopy and fluorescence microscopy.     

Synthesis of 5‐Fluorouridine Nucleolipid Derivatives and Their Cytostatic/Cytotoxic Activities on Human HT‐29 Colon Carcinoma Cells

One of the major drawbacks of chemotherapeutics is their insufficient penetration through cell membranes due to a high hydrophobicity. Thus, we have synthesized a series of selected nucleolipid derivatives of 5‐fluorouridine (5‐FUrd; 2a ), carrying lipophilic moieties at N(3) and/or in the 2′,3′‐O‐position (i.e., 3a – 7a and 3c ), and tested their cytostatic/cytotoxic activities using HT‐29 human colon carcinoma cells, in comparison with, e.g., 5‐FU ( 1 ) and 5‐FUrd ( 2a ). Incorporation and intracellular localization of the substances under test were performed after conjugation with the fluorochrome Atto 425. We showed that all 5′‐O‐labelled Atto 425 derivatives were incorporated by the human HT‐29 cells and accumulated in their cytoplasm. Moreover, after 24‐h treatment of HT‐29 human colon carcinoma cells, 1 or 2a (10, 20, 40, or 80 μM ) revealed a significant (14–23 or 33–45%, resp.) decrease of the viability in comparison with the (negative) control. Interestingly, derivatives 3a and 3c (40 and 80 μM ) led to a significant (77–95 or 89–96%, resp.) inhibition of survival of human HT29 cells, i.e., these two substances were ca. 63–72% or ca. 75%, respectively more effective than 5‐FU ( 1 ; positive control). Furthermore, derivative 5a showed a significant, i.e., 30 and 86%, inhibition of the survival at 40 and 80 μM , respectively in comparison with the (negative) control. Some synthesized 5‐FUrd derivatives turned out to be more effective than 5‐FU ( 1 ) or 5‐FUrd ( 2a ).     

Mitsunobu Reactions of 5‐Fluorouridine with the Terpenols Phytol and Nerol: DNA Building Blocks for a Biomimetic Lipophilization of Nucleic Acids

The cancerostatic 5‐fluorouridine (5‐FUrd; 1 ) was sequentially sugar‐protected by introduction of a 2′,3′‐O‐heptylidene ketal group (→ 2 ), followed by 5′‐O‐monomethoxytritylation (→ 3 ). This fully protected derivative was submitted to Mitsunobu reactions with either phytol ((Z and E)‐isomer) or nerol ((Z)‐isomer) to yield the nucleoterpenes 4a and 4b . Both were 5′‐O‐deprotected with 2% Cl₂CHCOOH in CH₂Cl₂ to yield compounds 5a and 5b , respectively. These were converted to the 5′‐O‐cyanoethyl phosphoramidites 6a and 6b , respectively. Moreover, the 2′,3′‐O‐(1‐nonyldecylidene) derivative, 7a , of 5‐fluorouridine was resynthesized and labelled at C(5′) with an Eterneon‐480 fluorophor^® (→ 7b ). The resulting nucleolipid was studied with respect to its incorporation in an artificial bilayer, as well as to its aggregate formation. Additionally, two oligonucleotides carrying terminal phytol‐alkylated 5‐fluorouridine tags were prepared, one of which was studied concerning its incorporation in an artificial lipid bilayer.