Synthesis and antiproliferative potency of 9-beta-D-arabinofuranosyl-2-fluoroadenine phospholipid adducts.

Three novel alkylphospholipid and four novel O-alkylglycerophospholipid derivatives of fludarabine (F-ara-AMP), known as a drug for the clinical treatment of chronic lymphocytic leukemia, were synthesized. The antiproliferative activity was determined in comparison to the parent nucleoside fludarabine in an immortalized but nontumorigenic human mammary epithelial cell line (H 184 A1N4), in two human breast tumor cell lines (MaTu and MCF7), and in two leukemic cell lines (HL 60 and Daudi). Fludarabine inhibited the growth of the leucemic cell lines very effectively. The breast tumor cell lines responded with much less sensitivity. The antiproliferative potency of the new compounds strongly depended on the chemical structure of the lipid component, and derivatives with a high effectiveness against one or both of the breast tumor cell lines were described.

Opposite effects on cytosolic Ca2+ of antitumor phospholipids by induction of calcium influx and activation of endoplasmic reticulum Ca(2+)-ATPase.

The ability of four different antitumor phospholipids, 1-O-hexadecyl-2-chloro-2-deoxyglycero-3-phosphocholine (ET16CIPC), hexadecylphosphocholine (C16OPC), hexadecylphospho-L-serine analogs (C16OPS, C16OPS-N-Ac) and cytidine-5'-hexadecylphosphonophosphate (C16PCMP) to modulate the cytosolic Ca2+ concentration [Ca2+]i was studied in an immortalized human mammary epithelial cell line H184 A1N4. The compounds induced different modes of activity depending on their structure and concentration. ET16CIPC induced between 0.31 and 5 microM a concentration dependent transient increase which was followed by a sustained increase at 10 microM. Studies using LaCl3 and Mn2+ quench of the Fura-2 fluorescence indicated that both effects are the result of an extracellular Ca2+ influx. Low concentrations of C16OPC, C16OPS and C16OPS-N-Ac induced no, or only a small, transient increase, whereas C16PCMP caused a decrease in [Ca2+]i. Thapsigargin and cyclopiazonic acid, specific inhibitors of the endoplasmic reticulum Ca(2+)-ATPase, prolonged the transient [Ca2+]i increase following ET16CIPC concentration dependently, increased markedly the small transient increase following C16OPC and the C16-phosphoserine analogs and converted the decrease in the basal [Ca2+]i level induced by C16PCMP to an increase. The identical effects with thapsigargin and cyclopiazonic acid provide evidence that the [Ca2+]i response observed is an expression of the balance between the ability of an analog to raise [Ca2+]i and to remove Ca2+ by activation of the endoplasmic reticulum Ca(2+)-ATPase. This behaviour might contribute to the antiproliferative effectiveness of antitumor phospholipids.

1-beta-D-Arabinofuranosylcytosine-5'-alkylphosphonophosphates and diphosphates: new orally active derivatives of ara-C.

ara-Cytidine-5'-alkylphosphonophosphates and the corresponding -diphosphates were found to be cytostatically active in vitro against the human mammary epithelial cell line H184 A1N4 and the human mammary tumor cell line MaTu. Our results indicate that the replacement of the diphosphate by the phosphonophosphate group has no influence on antiproliferative activity in this case. The compounds were more active than the corresponding cytidine phospholipid conjugates and related compounds lacking a cytostatically active nucleoside, the ara-C prodrug Cytoros, and were slightly less active than ara-C. The cytostatic effect was prevented by 2'-deoxycytidine indicating their action as prodrugs of ara-C. In contrast to ara-C, they increase [Ca2+]1 in H184 A1N4 cells, pointing to a different mechanism of action in addition to their prodrug effect. In combination with phospholipid analogs, synergistic effects could be observed. Further studies within the disease-oriented in vitro Anticancer Screening Program of the National Cancer Institute show selectivity for certain cancer cell lines. The hexadecyl derivatives revealed a significant antitumor activity in vivo against the murine lymphatic leukemia P 388 cells being equally potent or even superior to ara-C. In contrast to ara-C, they were found to be orally active. Side effects measured as leukopenia and body weight reduction were less pronounced than with the parent drug.

New nucleoside-5'-alkylphosphonophosphates and related compounds containing 2'-deoxycytidine, thymidine and adenosine as nucleoside component. Syntheses and their effects on tumor cell growth in vitro.

Recent studies have shown that phosphono analogs of cytidine-5'-diphosphate diacylglycerol (CDP-DAG) possessing a structurally modified lipid moiety exhibit antiproliferative activity in vitro. As an extension of our previous work we tried to elucidate whether the presence of the cytidine component is necessary for cytostatic activity. In this context we have synthesized similarly structured nucleoside-phospholipid conjugates containing nucleoside components other than cytidine, which also do not exhibit cytostatic properties as such. The compounds include 5'-alkyldiphosphates and 5'-alkylphosphonophosphates of 2'-deoxycytidine, thymidine and adenosine with different alkyl chain length as well as selected 3-hexadecyl-2-chloro-2-deoxyglycero-(1)-diphosphates and -phosphonophosphates of these nucleosides. The chemical structures of the newly synthesized nucleoside-phospholipid conjugates were confirmed by fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry. It was found that these compounds also inhibit the cell growth of different human cell lines, i.e. the presence of the cytidine component is not a necessary prerequisite for the antiproliferative activity of these nucleoside-phospholipid conjugates.

Modulation of the cytosolic Ca++ concentration by alkylphospho-L-serine analogs: relation to their antiproliferative action.

Antiproliferative alkyllysophospholipid (ALP) analogs produced multiple effects on the cytosolic Ca++ concentration ([Ca++]i) in an immortalized human breast epithelial cell line (H 184). The addition of small concentrations resulted in a short transient [Ca++]i response. With higher concentrations the transient rise was followed by a sustained increase. Pretreatment of cells with the ALP analogs for two minutes inhibited the transient [Ca++] response. Increases in [Ca++]i and inhibition of the transient increase were studied in relation to the dose and structure of several ALP analogs. In a series of alkylphospho-L-serine analogs with different lengths of the alkyl chain we found different dependencies of the stimulatory and inhibitory effects on the dose and the structure. The ability to increase [Ca++]i is absent with the C14 and C15 analogs, is low with the C16 and high with the C18 analog. With the exception of the C12 analog, a dose-related inhibition was observed with all derivatives but the effective concentrations differed very strongly and the maximal potency was reached with the C15 and C16 analogs. The antiproliferative action seems to correlate rather with the potency to inhibit the transient [Ca++]i response than with its stimulation.

Synthesis and antiproliferative activity of cytidine-5'-alkylphosphonophosphates and structurally related compounds.

The chemical synthesis of cytidine-5'-alkyl- and cytidine-5'-alkyl (acyl)deoxyglycerophosphonophosphates is reported. The compounds obtained represent a novel class of cytostatically active agents based on phospholipids, which inhibit the growth of various tumor cell lines in vitro. They are phosphono analogs of the cytidine-5'-diphosphate-diacylglycerol (CDP-DAG) possessing a structurally modified lipid moiety and a phospholipase C-resistant P-C bond. The antiproliferative efficacy of the cytidine-5'-alkylphosphonophosphates strongly depends on the alkyl chain length. The cytidine-5'-hexadecylphosphonophosphate was found to be the most effective compound tested in this study. Its cytostatic effect was distinctly higher than that of the alkyl(acyl) deoxyglycero derivatives and of the corresponding diphosphates. The structure of the new compounds were confirmed by fast atom bombardment mass spectrometry (FAB). The FAB fragmentation pattern is discussed.

Cellular signalling as a target in cancer chemotherapy. Phospholipid analogues as inhibitors of mitogenic signal transduction.

Mitogenic signalling mechanisms emerged as novel targets for tumor chemotherapy. Current strategies for pharmacological interventions are briefly discussed. Phospholipid analogues are treated in greater detail. It is shown here that this new class of antitumor agents acts as inhibitors of mitogenic signal transduction. The common target of all phospholipid analogues studied so far is the phosphatidylinositol (PI)-specific phospholipase C (PLC). This results in an attenuated formation of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). The reduction in IP3-levels leads to a depressed release of Ca2+ from internal stores, and the reduced formation of DAG interferes with the growth factor-induced activation of protein-kinase C (PKC). In addition to the effect on PI-specific PLC, most phospholipid analogues inhibit PKC directly by interacting with the regulatory domain of the enzyme. This effect, however, is not observed with all phospholipid analogues. Some potent growth inhibitory representatives from this group like hexadecylphosphoserine or hexadecylphosphonoserine do not affect PKC in cell-free extracts. It is concluded, therefore, that the direct inhibition of PKC is not required for the growth-inhibitory activity of these agents. The ability of phospholipid analogues to interact with PKC was also not found to be correlated the occurrence of unwanted side effects. Phospholipid analogues have also been found to act as inhibitors of phospholipase D (PLD). However, in this case the correlation to the growth inhibitory potency of various phospholipid analogues was less clear, so that the contribution of the PLD inhibition to the growth inhibitory effect of these agents still remains to be established. The inhibition of the thrombin-induced rise in cytosolic free Ca2+ by phospholipid analogues is reversible by washing the cells in phospholipid-free medium. These findings suggest that phospholipid analogues do not cause persistent membrane damage and may act as cytostatic rather than cytotoxic agents.

Interference of new alkylphospholipid analogues with mitogenic signal transduction.

The interference of several new hexadecylphosphocholine analogues with mitogenic signal transduction was investigated in NIH3T3 fibroblasts by studying the effects of these agents on thrombin-induced inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation and the subsequent Ca2+ release, on protein kinase C (PKC) in cell-free extracts, on the PKC-mediated activation of the Na+/H+ antiporter and on c-fos induction. The compounds investigated include hexadecylphosphocholine (HePC), octadecyl-[2-(N-methyl-piperidinio)-ethyl]-phosphate (D20133), octadecyl-(N,N-dimethyl-piperidinio-4-yl)-phosphate (D21266); octadecyl-[2-(trimethyl-arsonio)-ethyl]-phosphate (D21805) and hexadecylphospho-L-serine (HePS). The data indicate that (i) all compounds inhibit the thrombin-induced progression of growth-arrested NIH3T3 cells into S phase with similar IC50 values; (ii) the common denominator of all compounds is a reduction of Ins(1,4,5)P3 formation, resulting in an attenuation of Ca2+ release; (iii) the direct interaction with PKC does not significantly contribute to the antitumor activity of these agents; (iv) the new HePC congeners D21266, D21133 and D21805 affect the same targets as HePC, i.e. PKC and phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC). The lower toxicities of these compounds cannot be explained by a less pronounced inhibition of PKC or PLC, respectively.

Analogs of alkyllysophospholipids: chemistry, effects on the molecular level and their consequences for normal and malignant cells.

In the search for new approaches to cancer therapy, the first alkyllysophospholipid (ALP) analogs were designed and studied about two decades ago, either as potential immunomodulators or as antimetabolites of phospholipid metabolism. In the meantime, it has been demonstrated that they really act in this way. However, their special importance is based on the fact that, in addition, they interfere with key events of signal transduction, such as hormone (or cytokine)-receptor binding or processing, protein kinase C or phospholipase C function and phosphatidylinositol and calcium metabolism. There are no strict structural requirements for their activity. Differences in the cellular uptake or the state of cellular differentiation seem to be mainly responsible for higher or lower sensitivities of cells towards ALP analogs. Consequences of the molecular effects mentioned on the cellular level are cytostasis, induction of differentiation (while in contrast the effects of known inducers of differentiation such as 12-O-tetradecanoylphorbol-13-acetate are inhibited, probably as a consequence of protein kinase C inhibition) and loss of invasive properties. Already in sublytic concentrations, alterations in the membrane structure were observed, and lysis may begin at concentrations not much higher than those causing the other effects described. Few ALP analogs have already entered clinical studies or are in clinical use. ALP analogs are the only antineoplastic agents that do not act directly on the formation and function of the cellular replication machinery. Therefore, their effects are independent of the proliferative state of the target cells. Because of their interference with cellular regulatory events, including those failing in cancer cells, ALP analogs, beyond their clinical importance, are interesting model compounds for the development of new, more selective drugs for cancer therapy.

Effect of ALP analogs on inositol trisphosphate formation in H184 mammary epithelial cells before and after transfection with v-erb B oncogene.

The influence of cytostatically active alkyllysophospholipid analogs with different chemical structure on IP3 (inositol-1,4,5-trisphosphate) formation and intracellular Ca++ concentration was studied in human mammary epithelial cells before and after transfection with v-erb B oncogene DNA. Transformed cells showed an increased IP3 formation compared with normal cells. In the presence of ALP (alkyllysophospholipid) analogs IP3 formation is inhibited more strongly in transformed cells than in normal cells, dependent on the structure of ALP analogs. Furthermore, these compounds increased [Ca++]i (intracellular Ca++ concentration) in transformed cells much more strongly than in normal cells. From these results, obtained in pursuit of an oncogene-related cancer treatment, it follows that ALP analogs may inhibit processes in signal transduction in cells more strongly after transfection with a defined oncogene than before.

Multiple effects of antitumor alkyl-lysophospholipid analogs on the cytosolic free Ca2+ concentration in a normal and a breast cancer cell line.

Synthetic alkyl-lysophospholipids (ALP) are a new class of antitumor agents which interact with the cell membrane and the intracellular signal transduction at several sites. We studied the modulation of the intracellular calcium concentration ([Ca++]i) induced by two alkylglycerophosphocholines as well as hexadecylphosphocholine and hexadecylphosphoserine in a nontumorigenic and in a tumorigenic breast cell line. We found three distinct [Ca++]i-modulating effects: a transient increase, a decrease and a sustained increase. Their relative contribution to the observed response varies with different cell types, with the proliferation state, with the structure and with the concentration of the ALP analogs. The transient as well as the sustained increase in [Ca++]i depend mainly on extracellular Ca++; however, the Ca++ influx-inducing pathways might be different. The multiple [Ca++]i-increasing and decreasing effects induced by ALP analogs are discussed in relation to their influence on numerous Ca(++)-dependent effects, e.g. proliferation, differentiation, apoptosis and cytotoxicity.

Cytostatic effects of various alkyl phospholipid analogues on different cells in vitro.

Phospholipid analogues were studied with regard to their cytostatic activity on different tumour cell lines and on murine bone marrow cells. Compounds compared for their activity were alkylglycero- and alkyl-phosphocholines with the corresponding serines and the alkylphosphocholines and -serines with the corresponding phosphono derivatives. Moreover, compounds containing cytidine 5'-diphosphate instead of the phospho (or phosphono-) choline or serine moiety were studied. rac-2-Chloro-2-deoxy-2-deoxy-1-0-hexadecyl-glycero-3-phosphocholine (cpd. Id), hexadecylphosphocholine (cpd. Ia) as well as hexadecylphosphonocholine (cpd. Ib) inhibited growth of tumour cells in suspension and monolayer culture and their colony and cluster formation in agar culture but not that of bone marrow cells. The exchange of choline for serine in these compounds results in the loss of this type of antitumour specificity. However, dodecylphospho-L-serine (cpd. IIc) is as specific as the choline derivatives Ia, b, d mentioned. Thus, for serine compounds the specificity for tumour cells might depend in a critical way on the length of the alkyl chain. The phosphono compounds Ib, IIb show almost the same activity as the corresponding compounds hexadecylphosphocholine (cpd. Ia) or hexadecylphosphoserine (cpd. IIa). The CDP-derivatives (IIIa, d, e, f) inhibited growth of tumour cells in suspension or monolayer cultures but not the colony and cluster formation in agar (i.e. they do not decrease the plating efficiency) from either tumour or bone marrow cells.

Alteration of DNA topoisomerase II activity during infection of H9 cells by human immunodeficiency virus type 1 in vitro: a target for potential therapeutic agents.

Infection of H9 cells with human immunodeficiency virus type 1 (HIV-1) was found to decrease the phosphorylation of DNA topoisomerase II during the initial phase of infection. Simultaneously, with a later overshoot of phosphorylation and the subsequent activation of DNA topoisomerase II, the production of HIV-1 started. Applying three new protein kinase C inhibitors from the class of O-alkylglycerophospholipids we demonstrated that inhibition of protein kinase C-mediated phosphorylation of DNA topoisomerase II resulted in an inhibition of HIV-1 production. Based on the differential effect of the two protein kinase C activators, phorbol ester and bryostatin, we conclude that phosphorylation of DNA topoisomerase II is mediated by the form alpha and gamma of protein kinase C. These data suggest that agents which inhibit these two forms of protein kinase C are also potential candidates for an anti-HIV therapy.

Halo lipids. 10. Synthesis and cytostatic activity of O-alkylglycerophospho-L-serine analogs.

The synthesis of O-alkylglycerophospho-L-serine analogs is described, which represent a new class of cytostatically active agents based on phospholipids. The new compounds were obtained by conversion of O-alkylglycerophosphoric ester analogs by means of phospholipase D and by condensing O-alkylglycerophosphoric acid analogs with protected L-serine followed by the removal of protective groups of the resulting intermediates. The structure of the O-alkylglycerophospho-L-serines was confirmed by fast atom bombardment mass spectrometry. The compounds were found to inhibit the growth of Ehrlich ascites tumor cells in vitro. Half maximum inhibition was observed at concentrations between 7 and 15 microM. For the 1-O-alkyl-2-methoxy glycerophosphoserine only a higher value (30 microM) was found. With most of the substances tested growth was completely inhibited at a concentration of 40 microM.

Toxic effects of alkyl-lysophospholipids on human bone marrow and de novo leukaemias. A short overview.

Alkyl-lysophospholipids (ALPs) are reported to have an antineoplastic activity against leukaemic cells. We have tested some halogen-containing ALPs from the Central Institute of Molecular Biology (H. Brachwitz) in comparison with racemic 1-ostadecyl-2-methyl-glycero-3-phosphocholine (ET-18-OCH3) (P.G. Munder, Max-Planck-Institut für Immunobiologie, Freiburg, FRG). We found freshly dissolved ALPs to be very toxic both to human bone marrow and to leukaemic cells of patients. ALP-incubation before cryopreservation is more toxic to bone marrow (but not to AML blasts) than after cryopreservation. All experiments to test the selectivity and to establish a purging protocol should be done using 1, remission marrow including a cryopreservation step and 2, blasts of de novo leukaemias instead of cell as sensitive as HL 60 to ALP-incubation. We found direct toxicity of ALPs to be not suitable for purging lines of bone marrow from patients in remission.

[The effect of alkyl-lysophospholipids and membrane potentials, proliferation and migration of isolated calf aorta endothelial cells]. / Zum Einfluss von Alkyllysophospholipiden auf Membranpotential, Teilung und Migration von isolierten Kälberaortenendothelzellen.

Cancerostatical active synthetic alkyl-lysophospholipids were examined with regard to their effects on membrane potential, proliferation and migration of isolated endothelial cells. In sublytic concentrations all alkyl-lysophospholipids tested caused a hyperpolarization of the membrane of endothelial cells. The effect of alkyl-lysophospholipids on proliferation of endothelial cells was dependent on the serum supplement. The migration of endothelial cells was strongly inhibited by 1-O-octadecyl-2-O-methyl-glycero-phosphocholine. Possible mechanisms of action are discussed.

Cytostatic activity of synthetic O-alkylglycerolipids.

A series of halogen-containing alkylglycerolipid analogs has been checked for their cytostatic activity both in vitro and in vivo. The compounds included alkyldeoxyhaloglycerols (I), alkyldeoxyhaloglycerophosphocholines (II), and alkyldeoxyhaloglycerophosphoric acids and alkyl esters (III). While compounds I and III were moderately active, compounds II were found to have a strong inhibitory effect on the proliferation of Ehrlich ascites tumor cells in vitro. Cell growth inhibition of 50% or more was found mainly in the late S- or G2-phase of the cell cycle as revealed by flow cytometry. Alkyl lysophospholipid analogs II and cholesterol form liposomes with high encapsulation efficiency and low permeability for entrapped substances. Compounds II were active against Lewis lung carcinoma in mice when applied in free form or as liposomes.

Halo lipids. VI. Stimulation of human and rabbit blood platelets by racemic halo analogues of O-alkyl-glycerophosphocholine.

Halo analogues of O-alkyl- glycerophosphocholines are shown to stimulate human and rabbit blood platelets. Using 50-500 microM, a concentration dependent platelet aggregation is triggered in human platelet-rich plasma. Distinctly lower concentrations up to 10 microM activate the platelets in rabbit platelet-rich plasma. Moreover, the halo analogues enhance aggregation and release reaction triggered by suboptimal concentrations of ADP. In protein poor mediums the halo lipids in a concentration of 50 microM or higher cause a complete lysis of platelets. These results indicate that the halo lipids tested show the typical behaviour reported on lysophosphatidic acids which has to be taken into account using these compounds for other purposes.