Design, synthesis and biological evaluation of substituted naphthalene diimides as anticancer agents

It has been proved that naphthalene diimide (NDI) derivatives display anticancer properties as intercalators and G-quadruplex-binding ligands, leading to DNA damage, senescence and down-regulation of oncogene expression. This thesis deals with the design and synthesis of disubstituted and tetrasubstituted NDI derivatives endowed with anticancer activity, interacting with DNA together with other targets implicated in cancer development. Disubstituted NDI compounds have been designed with the aim to provide potential multitarget directed ligands (MTDLs), in order to create molecules able to simultaneously interact with some of the different targets involved in this pathology. The most active compound, displayed antiproliferative activity in submicromolar range, especially against colon and prostate cancer cell lines, the ability to bind duplex and quadruplex DNA, to inhibit Taq polymerase and telomerase, to trigger caspase activation by a possible oxidative mechanism, to downregulate ERK 2 protein and to inhibit ERKs phosphorylation, without acting directly on microtubules and tubuline. Tetrasubstituted NDI compounds have been designed as G-quadruplex-binding ligands endowed with anticancer activity. In order to improve the cellular uptake of the lead compound, the N-methylpiperazine moiety have been replaced with different aromatic systems and methoxypropyl groups. The most interesting compound was 1d, which was able to interact with the G-quadruplexes both telomeric and in HSP90 promoter region, and it has been co-crystallized with the human telomeric G-quadruplex, to directly verify its ability to bind this kind of structure, and also to investigate its binding mode. All the morpholino substituted compounds show antiproliferative activity in submicromolar values mainly in pancreatic and lung cancer cell lines, and they show an improved biological profile in comparison with that of the lead compound. In conclusion, both these studies, may represent a promising starting point for the development of new interesting molecules useful for the treatment of cancer, underlining the versatility of the NDI scaffold.

Small Molecules as Modulators of Different Targets Involved in Tumor Progression

Tumor is a lesion that may be formed by an abnormal growth of neoplastic cells. Many factors increase the risk of cancer and different targets are involved in tumor progression. Within this thesis, we have addressed two different biological targets, independently connected with tumor formation, e.g. Hsp90 and androgen receptor. The ATP-dependent chaperone Hsp90 is responsible for the conformational maturation and the renaturation of proteins. “Client” proteins are associated with the cancer hallmarks, as cell proliferation and tumor progression. Consequently, Hsp90 has evolved into promising anticancer target. Over the past decade, radicicol has been identified as potential anticancer agent targeting Hsp90, but it is not active in vivo. With that aim of obtaining radicicol-related derivatives, we developed the design and synthesis of new chalcones analogs. Chalcones, which are abundant in edible plants, own a diverse array of pharmacological activities and are considered a versatile scaffold for drug design. Antiproliferative assays and western blot analysis on the new compounds showed that some of those display an interesting cytotoxic effect and the ability to modulate Hsp90 client proteins expression. Androgen Receptor (AR) hypersensitivity plays crucial role in prostate cancer, which progression is stimulated by androgens. The therapy consists in a combination of surgical or chemical castration, along with antiandrogens treatment. Casodex® (bicalutamide), is the most widespread antiandrogen used in clinic. However, hormonal therapy is time-limited since many patients develop resistance. Commercially available antiandrogens show a common scaffold, e.g. two substituted aromatic rings linked by a linear or a cyclic spacer. With the aim of obtaining novel pure AR antagonists, we developed a new synthetic methodology, which allowed us to introduce, as linker between two suitably chosen aromatic rings, a triazole moiety. Preliminary data suggest that the herein reported new molecules generally decrease PSA expression, thus confirming their potential AR antagonistic activity.

Untersuchung der DNA-bindenden Eigenschaften neuer Nukleobasen-gekoppelter Pyrrolcarboxamide, Combilexine und Hetaren[a]carbazol-Derivate: Validierung und Etablierung von in-vitro- und in-silico-Methoden

Da maligne Neoplasien durch Mutationen in Proto-Onko- und/oder Tumorsuppressorgenen ausgelöst werden, stellt die DNA eines der wichtigsten Targets für die Entwicklung neuer Zytostatika dar. Auch bei den im Arbeitskreis Pindur designten und synthetisier-ten Verbindungen der Nukleobasen-gekoppelten Pyrrolcarboxamid-, der Hetaren[a]carbazol- und der Combilexin-Reihe handelt es sich um DNA-Liganden mit potentiell antitumoraktiven Eigenschaf-ten. Die einen dualen Bindemodus aufweisenden Combilexine bestehen aus einem Interkalator (u. a. Naphthalimid, Acridon), der über einen Linker variabler Kettenlänge mit einer rinnenbin-denden, von Netropsin abgeleiteten Bispyrrol-, oder einer bioisosteren Imidazol-, Thiazol- oder Thiophen-pyrrolcarboxamid-struktur verknüpft ist. Das N-terminale Ende der Combilexine wird von einer N,N-Dimethylaminopropyl- oder -ethyl-Seitenkette gebildet. Die DNA-Affinitäten der Liganden wurden mittels Tm-Wert-Messung-en bestimmt. Diese Denaturierungsexperimente wurden sowohl mit poly(dAdT)2- als auch mit Thymus-DNA (~42% GC-Anteil) durchge-führt, um Aussagen zur Stärke und zur Sequenzselektivität der DNA-Bindung machen zu können. Des Weiteren wurden die Bindekon-stanten einiger ausgewählter Vertreter mit Hilfe des Ethidium-bromid-Verdrängungsassays ermittelt; einige Testverbindungen wurden zudem auf potentiell vorhandene, TOPO I-inhibierende Eigenschaften untersucht. Diese biochemischen und biophysika-lischen Tests wurden durch Molecular Modelling-Studien ergänzt, die die Berechnung von molekularen Eigenschaften, die Durch-führung von Konformerenanalysen und die Simulation von DNA-Ligand-Komplexen (Docking) umfassten. Durch Korrelation der in vitro-Befunde mit den in silico-Daten gelang es, vor allem für die Substanzklasse der Combilexine einige richtungweisende Struktur-Wirkungsbeziehungen aufzustellen. So konnte gezeigt werden, dass die Einführung eines Imidazol-Rings in die rinnen-bindende Hetaren-pyrrolcarboxamid-Struktur der Combilexine aufgrund der H-Brücken-Akzeptor-Funktion des sp2-hybridisierten N-Atoms eine Verschiebung der Sequenzselektivität der DNA-Bindung von AT- zu GC-reichen Arealen der DNA bedingt. Zudem erwies sich ein C3-Linker für die Verknüpfung des Naphthalimids mit dem rinnenbindenden Strukturelement als am besten geeignet, während bei den Acridon-Derivaten die Verbindungen mit einem N-terminalen Buttersäure-Linker die höchste DNA-Affinität aufwiesen. Dies ist sehr wahrscheinlich auf die im Vergleich zum Naphthalimid-Molekül geringere y-Achsen-Ausdehnung (bzgl. eines x/y-Koordinatensystems) des Acridons zurückzuführen. Die ermittelten Struktur-Wirkungsbeziehungen können dazu herangezogen werden, das rationale Design neuer DNA-Liganden mit potentiell stärkerer DNA-Bindung zu optimieren.

Spectroscopic studies on Cyclodextrin and Metal Organic Framework based potential nanovectors for delivery of Anticancer and Antiviral drugs

The aim of this work is to contribute to the development of new multifunctional nanocarriers for improved encapsulation and delivery of anticancer and antiviral drugs. The work focused on water soluble and biocompatible oligosaccharides, the cyclodextrins (CyDs), and a new family of nanostructured, biodegradable carrier materials made of porous metal-organic frameworks (nanoMOFs). The drugs of choice were the anticancer doxorubicin (DOX), azidothymidine (AZT) and its phosphate derivatives and artemisinin (ART). DOX possesses a pharmacological drawback due to its self-aggregation tendency in water. The non covalent binding of DOX to a series of CyD derivatives, such as g-CyD, an epichlorohydrin crosslinked b-CyD polymer (pb-CyD) and a citric acid crosslinked g-CyD polymer (pg-CyD) was studied by UV visible absorption, circular dichroism and fluorescence. Multivariate global analysis of multiwavelength data from spectroscopic titrations allowed identification and characterization of the stable complexes. pg-CyD proved to be the best carrier showing both high association constants and ability to monomerize DOX. AZT is an important antiretroviral drug. The active form is AZT-triphosphate (AZT-TP), formed in metabolic paths of low efficiency. Direct administration of AZT-TP is limited by its poor stability in biological media. So the development of suitable carriers is highly important. In this context we studied the binding of some phosphorilated derivatives to nanoMOFs by spectroscopic methods. The results obtained with iron(III)-trimesate nanoMOFs allowed to prove that the binding of these drugs mainly occurs by strong iono-covalent bonds to iron(III) centers. On the basis of these and other results obtained in partner laboratories, it was possible to propose this highly versatile and “green” carrier system for delivery of phosphorylated nucleoside analogues. The interaction of DOX with nanoMOFs was also studied. Finally the binding of the antimalarial drug, artemisinin (ART) with two cyclodextrin-based carriers,the pb-CyD and a light responsive bis(b-CyD) host, was also studied.

Design and Synthesis of Naphthalene Diimides Based Small Molecules as Anticancer Agents: Targeting the Polyamine Transporter, G-quadruplex Structures and HDAC

Cancer is a multifactorial disease characterized by a very complex etiology. Basing on its complex nature, a promising therapeutic strategy could be based by the “Multi-Target-Directed Ligand” (MTDL) approach, based on the assumption that a single molecule could hit several targets responsible for the pathology. Several agents acting on DNA are clinically used, but the severe deriving side effects limit their therapeutic application. G-quadruplex structures are DNA secondary structures located in key zones of human genome; targeting quadruplex structures could allow obtaining an anticancer therapy more free from side effects. In the last years it has been proved that epigenetic modulation can control the expression of human genes, playing a crucial role in carcinogenesis and, in particular, an abnormal expression of histone deacetylase enzymes are related to tumor onset and progression. This thesis deals with the design and synthesis of new naphthalene diimide (NDI) derivatives endowed with anticancer activity, interacting with DNA together with other targets implicated in cancer development, such as HDACs. NDI-polyamine and NDI-polyamine-hydroxamic acid conjugates have been designed with the aim to provide potential MTDLs, in order to create molecules able simultaneously to interact with different targets involved in this pathology, specifically the G-quadruplex structures and HDAC, and to exploit the polyamine transport system to get selectively into cancer cells. Macrocyclic NDIs have been designed with the aim to improve the quadruplex targeting profile of the disubstituted NDIs. These compounds proved the ability to induce a high and selective stabilization of the quadruplex structures, together with cytotoxic activities in the micromolar range. Finally, trisubstituted NDIs have been developed as G-quadruplex-binders, potentially effective against pancreatic adenocarcinoma. In conclusion, all these studies may represent a promising starting point for the development of new interesting molecules useful for the treatment of cancer, underlining the versatility of the NDI scaffold.

Tissue engineering strategies for biomaterial-based anticancer drug delivery and enhancement of angiogenesis in tumor-resected bone

Patienten, die an Osteosarkom leiden werden derzeit mit intravenös applizierten krebstherapeutischen Mitteln nach Tumorresektion behandelt, was oftmals mit schweren Nebenwirkungen und einem verzögerten Knochenheilungsprozess einhergeht. Darüber hinaus treten vermehrt Rezidive aufgrund von verbleibenden neoplastischen Zellen an der Tumorresektionsstelle auf. Erfolgreiche Knochenregeneration und die Kontrolle von den im Gewebe verbleibenden Krebszellen stellt eine Herausforderung für das Tissue Engineering nach Knochenverlust durch Tumorentfernung dar. In dieser Hinsicht scheint der Einsatz von Hydroxyapatit als Knochenersatzmaterial in Kombination mit Cyclodextrin als Medikamententräger, vielversprechend. Chemotherapeutika können an Biomaterial gebunden und direkt am Tumorbett über einen längeren Zeitraum freigesetzt werden, um verbliebene neoplastische Zellen zu eliminieren. Lokal applizierte Chemotherapie hat diverse Vorteile, einschließlich der direkten zytotoxischen Auswirkung auf lokale Zellen, sowie die Reduzierung schwerer Nebenwirkungen. Diese Studie wurde durchgeführt, um die Funktionsfähigkeit eines solchen Arzneimittelabgabesystems zu bewerten und um Strategien im Bereich des Tissue Engineerings zu entwickeln, die den Knochenheilungsprozess und im speziellen die Vaskularisierung fördern sollen. Die Ergebnisse zeigen, dass nicht nur Krebszellen von der chemotherapeutischen Behandlung betroffen sind. Primäre Endothelzellen wie zum Beispiel HUVEC zeigten eine hohe Sensibilität Cisplatin und Doxorubicin gegenüber. Beide Medikamente lösten in HUVEC ein tumor-unterdrückendes Signal durch die Hochregulation von p53 und p21 aus. Zudem scheint Hypoxie einen krebstherapeutischen Einfluss zu haben, da die Behandlung sensitiver HUVEC mit Hypoxie die Zellen vor Zytotoxizität schützte. Der chemo-protektive Effekt schien deutlich weniger auf Krebszelllinien zu wirken. Diese Resultate könnten eine mögliche chemotherapeutische Strategie darstellen, um den Effekt eines zielgerichteten Medikamenteneinsatzes auf Krebszellen zu verbessern unter gleichzeitiger Schonung gesunder Zellen. Eine erfolgreiche Integration eines Systems, das Arzneimittel abgibt, kombiniert mit einem Biomaterial zur Stabilisierung und Regeneration, könnte gesunden Endothelzellen die Möglichkeit bieten zu proliferieren und Blutgefäße zu bilden, während verbleibende Krebszellen eliminiert werden. Da der Prozess der Knochengeweberemodellierung mit einer starken Beeinträchtigung der Lebensqualität des Patienten einhergeht, ist die Beschleunigung des postoperativen Heilungsprozesses eines der Ziele des Tissue Engineerings. Die Bildung von Blutgefäßen ist unabdingbar für eine erfolgreiche Integration eines Knochentransplantats in das Gewebe. Daher ist ein umfangreich ausgebildetes Blutgefäßsystem für einen verbesserten Heilungsprozess während der klinischen Anwendung wünschenswert. Frühere Experimente zeigen, dass sich die Anwendung von Ko-Kulturen aus humanen primären Osteoblasten (pOB) und humanen outgrowth endothelial cells (OEC) im Hinblick auf die Bildung stabiler gefäßähnlicher Strukturen in vitro, die auch effizient in das mikrovaskuläre System in vivo integriert werden konnten, als erfolgreich erweisen. Dieser Ansatz könnte genutzt werden, um prä-vaskularisierte Konstrukte herzustellen, die den Knochenheilungsprozess nach der Implantation fördern. Zusätzlich repräsentiert das Ko-Kultursystem ein exzellentes in vitro Model, um Faktoren, welche stark in den Prozess der Knochenheilung und Angiogenese eingebunden sind, zu identifizieren und zu analysieren. Es ist bekannt, dass Makrophagen eine maßgebliche Rolle in der inflammatorisch-induzierten Angiogenese spielen. In diesem Zusammenhang hebt diese Studie den positiven Einfluss THP-1 abgeleiteter Makrophagen in Ko-Kultur mit pOB und OEC hervor. Die Ergebnisse zeigten, dass die Anwendung von Makrophagen als inflammatorischer Stimulus im bereits etablierten Ko-Kultursystem zu einer pro-angiogenen Aktivierung der OEC führte, was in einer signifikant erhöhten Bildung blutgefäßähnlicher Strukturen in vitro resultierte. Außerdem zeigte die Analyse von Faktoren, die in der durch Entzündung hervorgerufenen Angiogenese eine wichtige Rolle spielen, eine deutliche Hochregulation von VEGF, inflammatorischer Zytokine und Adhäsionsmoleküle, die letztlich zu einer verstärkten Vaskularisierung beitragen. Diese Resultate werden dem Einfluss von Makrophagen zugeschrieben und könnten zukünftig im Tissue Engineering eingesetzt werden, um den Heilungsprozess zu beschleunigen und damit die klinische Situation von Patienten zu verbessern. Darüber hinaus könnte die Kombination der auf Ko-Kulturen basierenden Ansätze für das Knochen Tissue Engineering mit einem biomaterial-basierenden Arzneimittelabgabesystem zum klinischen Einsatz kommen, der die Eliminierung verbliebener Krebszellen mit der Förderung der Knochenregeneration verbindet.

Synthesis and functionalization of a lactam-pyrazole molecular scaffold as a promising anticancer compound

The importance of pyrazole and lactam-based molecules in medical and pharmaceutical fields is underlined by the multitude of active ingredients on trade, such as Sildenafil or Apixaban, by Pfizer. In this work, a synthesis of an organic molecule with promising anticancer activity has been developed. This molecular scaffold is characterized by a δ-lactam-fused pyrazolic core, with a well-known biological activity and amenable of further functionalization. The synthetic strategy adopted for the obtainment of the core was based on a 1,3-dipolar cycloaddition of a nitrilimine with an α,β-unsaturated δ-lactam. Secondly, in order to give the final compound an elevated pharmacological activity, a functionalization with a double “side chain”, namely molecular fragment able to improve the interaction with particular biological receptors, was achieved. The target compound was thus obtained, with a highly convergent synthesis, and will be tested for antiproliferative activities towards different cellular lines.

GnRH analogs do not protect ovaries from chemotherapy-induced ultrastructural injury in Hodgkin's lymphoma patients

To determine the protective effect of gonadotropin-releasing hormone analogs (GnRHa) using several ultrasound and endocrine markers to detect ultrastructural ovarian damage in Hodgkin's lymphoma patients.

Anti insulin-like growth factor I receptor immunoliposomes: a single formulation combining two anticancer treatments with enhanced therapeutic efficiency

Context: Through overexpression and aberrant activation in many human tumors, the IGF system plays a key role in tumor development and tumor cell proliferation. Different strategies targeting IGF-I receptor (IGFI-R) have been developed, and recent studies demonstrated that combined treatments with cytostatic drugs enhance the potency of anti-IGFI-R therapies. Objective: The objective of the study was to examine the IGFI-R expression status in neuroendocrine tumors of the gastroenteropancreatic system (GEP-NETs) in comparison with healthy tissues and use potential overexpression as a target for novel anti-IGFI-R immunoliposomes. Experimental Design: A human tumor tissue array and samples from different normal tissues were investigated by immunohistochemistry. An IGFI-R antagonistic antibody (1H7) was coupled to the surface of sterically stabilized liposomes loaded with doxorubicin. Cell lines from different tumor entities were investigated for liposomal association studies in vitro. For in vivo experiments, neuroendocrine tumor xenografts were used for evaluation of pharmacokinetic and therapeutic properties of the novel compound. Results: Immunohistochemistry revealed significant IGFI-R overexpression in all investigated GEP-NETs (n = 59; staining index, 229.1 +/- 3.1%) in comparison with normal tissues (115.7 +/- 3.7%). Furthermore, anti-IGFI-R immunoliposomes displayed specific tumor cell association (44.2 +/- 1.6% vs. IgG liposomes, 0.8 +/- 0.3%; P < 0.0001) and internalization in human neuroendocrine tumor cells in vitro and superior antitumor efficacy in vivo (life span 31.5 +/- 2.2 d vs. untreated control, 19 +/- 0.6, P = 0.008). Conclusion: IGFI-R overexpression seems to be a common characteristic of otherwise heterogenous NETs. Novel anti-IGFI-R immunoliposomes have been developed and successfully tested in a preclinical model for human GEP-NETs. Moreover in vitro experiments indicate that usage of this agent could also present a promising approach for other tumor entities.

Radiolabeled bicyclic somatostatin-based analogs: a novel class of potential radiotracers for SPECT/PET of neuroendocrine tumors

A variety of radiolabeled somatostatin analogs have been developed for targeting of somatostatin receptor (sst)-positive tumors. Bicyclic somatostatin-based radiopeptides have not been studied yet. Hypothesizing that the introduction of conformational constraints may lead to receptor subtype selectivity or may help to delineate structural features determining pansomatostatin potency, we developed and evaluated first examples of this new class of potential radiotracers for imaging or therapy of neuroendocrine tumors.

Agonist-biased signaling at the sst2A receptor: the multi-somatostatin analogs KE108 and SOM230 activate and antagonize distinct signaling pathways

Somatostatin analogs that activate the somatostatin subtype 2A (sst2A) receptor are used to treat neuroendocrine cancers because they inhibit tumor secretion and growth. Recently, new analogs capable of activating multiple somatostatin receptor subtypes have been developed to increase tumor responsiveness. We tested two such multi-somatostatin analogs for functional selectivity at the sst2A receptor: SOM230, which activates sst1, sst2, sst3, and sst5 receptors, and KE108, which activates all sst receptor subtypes. Both compounds are reported to act as full agonists at their target sst receptors. In sst2A-expressing HEK293 cells, somatostatin inhibited cAMP production, stimulated intracellular calcium accumulation, and increased ERK phosphorylation. SOM230 and KE108 were also potent inhibitors of cAMP accumulation, as expected. However, they antagonized somatostatin stimulation of intracellular calcium and behaved as partial agonists/antagonists for ERK phosphorylation. In pancreatic AR42J cells, which express sst2A receptors endogenously, SOM230 and KE108 were both full agonists for cAMP inhibition. However, although somatostatin increased intracellular calcium and ERK phosphorylation, SOM230 and KE108 again antagonized these effects. Distinct mechanisms were involved in sst2A receptor signaling in AR42J cells; pertussis toxin pretreatment blocked somatostatin inhibition of cAMP accumulation but not the stimulation of intracellular calcium and ERK phosphorylation. Our results demonstrate that SOM230 and KE108 behave as agonists for inhibition of adenylyl cyclase but antagonize somatostatin's actions on intracellular calcium and ERK phosphorylation. Thus, SOM230 and KE108 are not somatostatin mimics, and their functional selectivity at sst2A receptors must be considered in clinical applications where it may have important consequences for therapy.

The role of autophagy in anticancer therapy: promises and uncertainties

Autophagy (literally self-eating) is a catabolic mechanism involved in the recycling and turnover of cytoplasmic constituents. Although often referred to as type II programmed cell death, autophagy is primarily a survival rather than a cell death mechanism in response to different stress stimuli. Autophagy is a process in which part of the cytoplasm or entire organelles are sequestered into double-membrane vesicles, called autophagosomes, which ultimately fuse with lysosomes to degrade their contents. Studies show that autophagy is associated with a number of pathological conditions, including cancer, infectious diseases, myopathies and neurodegenerative disorders. With respect to cancer, it has been suggested that the early stages of tumourigenesis are associated with downregulation of autophagy-related (ATG) genes. Indeed, several ATG genes display tumour suppressor function, including Beclin1, which is frequently hemizygously deleted in breast cancer cells. Conversely, in advanced stages of tumourigenesis or during anticancer therapy, autophagy may promote survival of tumour cells in adverse environmental conditions. Therefore, a thorough understanding of autophagy in different cancer types and stages is a prerequisite to determine an autophagy-activating or autophagy-inhibiting treatment strategy.

Insights into cardiovascular side-effects of modern anticancer therapeutics

Modern anticancer therapeutics can be associated with significant cardiovascular side-effects. Detection, risk assessment, and treatment of these unwanted effects are an important task for treating physicians. The purpose of this review is to focus on approved novel cancer therapeutics and discuss the most important cardiovascular side-effects, prognosis, and potential treatment. We will contrast these effects to those of conventional cardiotoxic chemotherapeutics.

Synthesis and biological activity of new functionalized epothilones for prodrug design and tumor targeting

Epothilones are potent antiproliferative agents, which have served as successful lead structures for anticancer drug discovery. However, their therapeutic efficacy would benefit greatly from an increase in their selectivity for tumor cells, which may be achieved through conjugation with a tumor-targeting moiety. Three novel epothilone analogs bearing variously functionalized benzimidazole side chains were synthesized using a strategy based on palladium-mediated coupling and macrolactonization. The synthesis of these compounds is described and their in vitro biological activity is discussed with respect to their interactions with the tubulin/microtubule system and the inhibition of human cancer cell proliferation. The additional functional groups may be used to synthesize conjugates of epothilone derivatives with a variety of tumor-targeting moieties.

Anticancer activity of natural cytokinins: a structure-activity relationship study

Cytokinin ribosides (N(6)-substituted adenosine derivatives) have been shown to have anticancer activity both in vitro and in vivo. This study presents the first systematic analysis of the relationship between the chemical structure of cytokinins and their cytotoxic effects against a panel of human cancer cell lines with diverse histopathological origins. The results confirm the cytotoxic activity of N(6)-isopentenyladenosine, kinetin riboside, and N(6)-benzyladenosine and show that the spectrum of cell lines that are sensitive to these compounds and their tissues of origin are wider than previously reported. The first evidence that the hydroxylated aromatic cytokinins (ortho-, meta-, para-topolin riboside) and the isoprenoid cytokinin cis-zeatin riboside have cytotoxic activities is presented. Most cell lines in the panel showed greatest sensitivity to ortho-topolin riboside (IC(50)=0.5-11.6 microM). Cytokinin nucleotides, some synthesized for the first time in this study, were usually active in a similar concentration range to the corresponding ribosides. However, cytokinin free bases, 2-methylthio derivatives and both O- and N-glucosides showed little or no toxicity. Overall the study shows that structural requirements for cytotoxic activity of cytokinins against human cancer cell lines differ from the requirements for their activity in plant bioassays. The potent anticancer activity of ortho-topolin riboside (GI(50)=0.07-84.60 microM, 1st quartile=0.33 microM, median=0.65 microM, 3rd quartile=1.94 microM) was confirmed using NCI(60), a standard panel of 59 cell lines, originating from nine different tissues. Further, the activity pattern of oTR was distinctly different from those of standard anticancer drugs, suggesting that it has a unique mechanism of activity. In comparison with standard drugs, oTR showed exceptional cytotoxic activity against NCI(60) cell lines with a mutated p53 tumour suppressor gene. oTR also exhibited significant anticancer activity against several tumour models in in vivo hollow fibre assays.