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

Abstract

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).

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

“…The cells were seeded on 96‐well plates ( BD Falcon ™, Becton Dickinson GmbH , Heidelberg, Germany) with a density of 3 × 10 4 for THP‐1, 1.5 × 10 4 for GOS‐3 or 5 × 10 3 for BT4Ca cells per NL_1.4.0.0 ( 3Ab ), NL_2.4.0.0 ( 2Ad ), NL_5.4.0.0 ( 2Ab ), NL_6.4.0.0 ( 2Bb ), NL_5.cycl7.0.0 ( 4 ), as well as NL_1.5.0.0 ( 2Af ), NL_2.5.0.0 ( 2Ag ), NL_5.5.0.0 ( 2Bc ), NL_6.5.0.0 ( 2Bd ), NL_4.5.0.0 ( 2Ah ), and NL_2.4.3.0 ( 3Ac ), NL_4.4.3.0 ( 3Ad ), NL_6.4.3.0 ( 3Ba ), were tested at concentrations of 1.56, 3.12, 6.25, 12.5, 25, or 50 μ m . The survival/viability was detected after 48 h using PrestoBlue ® reagent ( Invitrogen‐Life Technologies GmbH , Darmstadt, Germany) as described earlier . The reagent was added into the culture medium above the cells at a final concentration of 10%, and after 30 min, 1 h, 2 h, or 3 h the optical densitiy ( OD ) at 570 and 600 nm (reference) was measured with a SUNRISE ELISA ‐reader ( Tecan , Salzburg, Austria).…”

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

“…Cell lines and culture conditions : In vitro experiments were performed using the human astrocytoma/oligodendroglioma GOS‐3 cells (DSMZ GmbH, Braunschweig, Germany), the rat malignant neuro‐ectodermal BT4Ca cells (a kind gift from Dr. Nadine John, Hannover Medical School, Hannover, Germany), as well as the human acute monocytic leukemia cell line THP‐1 (DSMZ GmbH, Braunschweig, Germany). The cells were cultured in 90 % RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS), 100 U mL −1 penicillin, and 0.1 mg mL −1 streptomycin, and were maintained at 37 °C in a humidified atmosphere (5 % CO 2 , 95 % air) as described earlier …”

“…After 24 h, the medium was changed and different concentrations of 5‐fluorouridine or its derivatives 5 , 6 , 8 c , were tested at concentrations of 1.56, 3.12, 6.25, 12.5, 25, or 50 μ m . After incubation for 48 h, viability/cytotoxicity was measured using PrestoBlue reagent (Invitrogen‐Life Technologies GmbH, Darmstadt, Germany) as described earlier . PrestoBlue was added to the cells into the culture medium and 30 min, 1 h, 2 h, or 3 h after addition of PrestoBlue ™ , the optical density (OD) was measured at 570 nm and 600 nm (as reference) with a SUNRISE ELISA‐reader (Tecan, Salzburg, Austria).…”

“…Ketal formation of adenosine with nonadecan-10-one gave the nucleolipid 5.B oth O-2',3' ketals (3 and 5)w ere then submittedt oa ne nzymatic deamination using adenosined eaminase (from calf intestine). It could be clearly shown that compound 3 could be deaminated within 72 hy ielding the inosine We report on the synthesis of two series of canonical purine ßd-ribonucleoside nucleolipids derived from inosine and adenosine, which have been characterizedb ye lemental analyses, electrospray ionization mass spectrometry (ESI MS) as well as by 1 Ha nd 13 CNMR, andp H-dependent UV/Vis spectroscopy.A selectionofthe novel nucleolipids with differentlipophilic moieties were first tested on their cytotoxic effect towardh uman macrophages. Compounds without as ignificant inhibitory effect on the viability of the macrophages were tested on their cytostatic/cytotoxic effect toward human astrocytoma/oligo-dendroglioma GOS-3 cells as well as against the rat malignant neuroectodermal BT4Ca cell line.…”

“…In as eries of precedentp ublications we and others have demonstrated that the cancerostatic/cancerotoxic activity of pyrimidine ß-d-ribonucleoside antimetabolites such as 5-fluorouridine and 6-azauridine towards different human tumor cell lines [1,2] as well as neurobiological [3] and antiviral activities [4,5] can be significantly improved by lipophilization. Also, regular, canonical pyrimidine ß-d-ribonucleosides such as uridine and 5-methyluridine acquireasurprisingly high antitumor in vitro activity upon covalenth ydrophobization.…”