Colchicine Derivatives Research Articles

Quantitative analysis of the effect of tubulin isotype expression on sensitivity of cancer cell lines to a set of novel colchicine derivatives.

BackgroundA maximum entropy approach is proposed to predict the cytotoxic effects of a panel of colchicine derivatives in several human cancer cell lines. Data was obtained from cytotoxicity assays performed with 21 drug molecules from the same family of colchicine compounds and correlate these results with independent tubulin isoform expression measurements for several cancer cell lines. The maximum entropy method is then used in conjunction with computed relative binding energy values for each of the drug molecules against tubulin isotypes to which these compounds bind with different affinities.ResultsWe have found by using our analysis that αβI and αβIII tubulin isoforms are the most important isoforms in establishing predictive response of cancer cell sensitivity to colchicine derivatives. However, since αβI tubulin is widely distributed in the human body, targeting it would lead to severe adverse side effects. Consequently, we have identified tubulin isotype αβIII as the most important molecular target for inhibition of microtubule polymerization and hence cancer cell cytotoxicity. Tubulin isotypes αβI and αβII are concluded to be secondary targets.ConclusionsThe benefit of being able to correlate expression levels of specific tubulin isotypes and the resultant cell death effect is that it will enable us to better understand the origin of drug resistance and hence design optimal structures for the elimination of cancer cells. The conclusion of the study described herein identifies tubulin isotype αβIII as a target for optimized chemotherapy drug design.

Computational Design and Biological Testing of Highly Cytotoxic Colchicine Ring A Modifications

Microtubules are the primary target for many anti-cancer drugs, the majority of which bind specifically to beta-tubulin. The existence of several beta-tubulin isotypes, coupled with their varied expression in normal and cancerous cells provides a platform upon which to construct selective chemotherapeutic agents. We have examined five prevalent human beta-tubulin isotypes and identified the colchicine-binding site as the most promising for drug design based on specificity. Using this binding site as a template, we have designed several colchicine derivatives and computationally probed them for affinity to the beta-tubulin isotypes. These compounds were synthesized and subjected to cytotoxicity assays to determine their effectiveness against several cancerous cell lines. We observed a correlation between computational-binding predictions and experimentally determined IC(50) values, demonstrating the utility of computational screening in the design of more effective colchicine derivatives. The most promising derivative exhibited an IC(50) approximately threefold lower than values previously reported for either colchicine or paclitaxel, demonstrating the utility of computational design and assessment of binding to tubulin.

Computer assisted design of second-generation colchicine derivatives

Cytoskeletal proteins, such as tubulin, are a primary target for many successful anti-cancer drugs. The expression of several beta-tubulin isotypes in normal and cancerous cells provides a platform upon which to construct chemotherapeutic agents capable of differentiating between them. To test this hypothesis, we have previously designed several colchicine derivatives and computationally probed them for affinity to the beta-tubulin isotypes. Subsequent synthesis and cytotoxicity assays produced a small set of promising compounds exhibiting IC(50) values approximately 30 fold lower than values previously reported for colchicine. Here we describe the creation and testing of these first-generation colchicine derivatives and discuss the subsequent design and preliminary computational screening of a novel set of second-generation derivatives using the most promising first-generation derivatives as scaffolds.

Sekundäre Pflanzenstoffe und Krebs

In dieser Übersicht wird die Bedeutung von sekundären Pflanzenstoffen (phytochemicals) bei der Krebsentstehung, -vorbeugung und -bekämpfung erörtert. Sekundäre Pflanzenstoffe sind niedermolekulare Verbindungen, die häufig biologisch aktiv sind. Es kann sich dabei um akut toxische, chronisch toxische oder auch genotoxische Substanzen handeln. Viele haben aber auch protektive oder kurative Eigenschaften. Im Zusammenhang mit Krebs und Krebsentstehung können sie zytostatische, antikanzerogene, antioxidative oder immunmodulierende Wirkungen entfalten. Sekundäre Pflanzenstoffe lassen sich verschiedenen biogenetischen Stoffgruppen zuordnen: Terpenoide, Alkaloide, Produkte des Shikimisäurewegs, Polyketide und schwefelhaltige Verbindungen. Zu den (potenziell) kanzerogenen sekundären Pflanzenstoffen zählen Pyrrolizidinalkaloide, einige Anthranoide und nitroaromatische Verbindungen. Einige Stoffe, besonders Phorbolester, müssen als Tumorpromotoren eingestuft werden. Kanzerogene entstehen auch bei der Verarbeitung oder Lagerung von Lebensmitteln. Zu den nützlichen sekundären Pflanzenstoffen (dietary phytochemicals) zählen Carotinoide und Polyphenole. Während der Einfluss von Phytoestrogenen (PhytoSERMs) bezüglich Krebsentstehung oder -prophylaxe kontrovers diskutiert wird, scheinen Glucosinolate das Risiko von Krebserkrankungen zu senken. Wichtige protektive Stoffe wie die Ballaststoffe und Phytosterole zählen nicht zu den sekundären Pflanzenstoffen im engeren Sinne. Andererseits zählen potente Krebsmedikamente wie die Mitosehemmer (Colchicin-Derivate, Vinca-Alkaloide und Taxane) und einige Hemmstoffe der DNA-Topoisomerasen (Podophyllotoxin-Derivate, Camptothecin-Derivate) zu den sekundären Pflanzenstoffen. Zusammenfassend muss man erkennen, dass es nicht ganz einfach ist, sekundäre Pflanzenstoffe in „gut” oder „schlecht” einzuteilen.

The efficacy of new colchicine derivatives and viability of the T-Lymphoblastoid cells in three-dimensional culture using 19F MRI and HPLC-UV ex vivo

The paper describes ex vivo applications of colchicine derivatives for the treatment of human T-Lymphoblastoid (CEM) cells. Moreover, the role of the substitutions of ring A at C-1 and C-7 side chain of colchicine analogues was probed by the synthesis and examination of their effects on the three-dimensional (3-D) CEM cells’ growth. The CEM cells were cultured in the hollow fiber bioreactor (HFB) device. We used 1H and 19F magnetic resonance imaging (MRI) to monitor changes in 3-D CEM cell culture. 19F MRI was used for visualization of the cellular uptake of new fluorine derivatives. Before and after treatment CEM cells profile was investigated with high performance liquid chromatography (HPLC-UV).

Free Energy Calculations on the Binding of Colchicine and Its Derivatives with the α/β-Tubulin Isoforms

Tubulin is the target for numerous small molecule ligands which alter microtubule dynamics leading to cell cycle arrest and apoptosis. Many of these ligands are currently used clinically for the treatment of several types of cancer, and they bind to one of three distinct binding sites within beta-tubulin (paclitaxel, vinca, and colchicine), all of which have been identified crystallographically. Unfortunately, serious side effects always accompany chemotherapy since these drugs bind to tubulin indiscriminately, leading to the death of both cancerous and healthy cells. However, the existence and distribution of divergent tubulin isoforms provide a platform upon which we may build novel chemotherapeutic drugs that can differentiate between different cell types and therefore reduce undesirable side effects. We report results of computational analysis that aims at predicting differences between the binding energies of a family of colchicine derivatives against 10 human alpha/beta-tubulin isoforms. Free energy perturbation method has been used in our calculations and the results provide a proof of principle by indicating significant differences both among the derivatives and between tubulin isoforms.

Synthesis and characterization of BODIPY-labeled colchicine

Two BODIPY-labeled colchicine derivatives were synthesized and shown to bind to tubulin but only partially inhibit tubulin polymerization in the presence of GTP. Cytotoxicity studies were carried out in HeLa, HepG2, Raji and Vero cells. Apoptosis-inducing properties were determined by caspase 3/7 activity and flow cytometry and interactions between the derivatives and tubulin were verified by fluorescence microscopy of living cells.

CYTOTOXICITY OF COLCHICINE DERIVATIVES IN PRIMARY CULTURES OF HUMAN HEPATOCYTES

Colchicine has been used to treat gout for centuries. However, owing to its toxicity it displays a variety of side effects. The replacement of colchicine by less toxic but still active derivatives would solve this drawback. The aim of this study was to examine the cytotoxicity of 17 colchicine derivatives. Primary cultures of human hepatocytes were the model of choice. Prior to testing, we measured the biochemical parameters of liver donors and the toxicological response of the hepatocytes cultures. For toxicity testing, cells were treated for 24 h with tested compounds in concentrations 1-100 microM. We monitored lactate dehydrogenase (LDH) leakage into the medium, mitochondrial activity (MTT test) and secretion of albumin. Our data show that LDH and MTT were less sensitive parameters compared to albumin secretion for monitoring the toxicity of colchicine derivatives. Compounds with lower antimitotic activity displayed lowered toxicity. Since human hepatocytes in culture are quiescent cells, they are not as susceptible to tropolone alkaloids as proliferating cells. Screening for colchicine derivatives with lowered cytotoxicity revealed that 10-O-demethylated compounds might be the substances of choice.

Distribution of radioactivity and metabolite profiling in tumour and plasma following intravenous administration of a colchicine derivative (14C-ZD6126) to tumour-bearing mice

The main aim of the study was to investigate the distribution of radioactivity in the tissues and tumours using quantitative whole-body autoradiography (QWBA), together with a more detailed investigation of plasma and tumour samples, following administration of a single intravenous dose at 200 mg kg−1 of 14C-ZD6126 to mice bearing subcutaneous Hras5 tumour xenografts. The study also included an assessment of tumour necrosis following administration of a single intravenous dose of non-labelled ZD6126 at 200 mg kg−1. QWBA analysis showed that drug-related material was widely distributed to the tissues and tumour. In the majority of tissues, concentrations of radioactivity were highest at 15 min and declined rapidly thereafter. The tumour-to-plasma ratio was 0.6:1 at 0.25 h and increased to 6:1 at 48 h, indicating that drug-related material persisted in the tumour longer than in plasma. ZD6126, a phosphate ester, is rapidly hydrolysed to ZD6126 phenol, the active metabolite. The major metabolite in plasma (36% of the sample radioactivity) and all tumour samples (58–83% of the sample radioactivity) was confirmed as ZD6126 phenol. Extensive tumour necrosis was noted by 24 h, which was still evident at 48 h, although there was some evidence of tumour regrowth.

99mTc-labeling of colchicine using [ 99mTc(CO) 3(H 2O) 3] + and [ 99mTc [tbnd]N] 2+ core for the preparation of potential tumor-targeting agents

Multidrug resistance (MDR) mediated by over-expression of P-glycoprotein (Pgp) is one of the major causes of failure of chemotherapy in cancer treatment. Colchicine, a naturally occurring alkaloid, is a Pgp substrate and acts as an antimitotic agent by binding to microtubules. Hence, Colchicine and its analogues radiolabeled with 99mTc may have potential for visualization of MDR in tumors. Here we report 99mTc-labeling of colchicine derivatives using [ 99mTc(CO) 3(H 2O) 3] + and [ 99mTc N] 2+ cores. Trimethylcolchicinic acid synthesized from colchicine was used as the precursor to prepare iminodiacetic acid and dithiocarbamate derivatives which were then radiolabeled with [ 99mTc(CO) 3(H 2O) 3] + and [ 99mTc N] 2+ cores, respectively. Radiolabeling yield for both the complexes was >98% as observed by HPLC and TLC patterns. In vitro studies in tumor cell lines showed significant uptake for 99mTc-carbonyl as well as for 99mTc-nitrido colchicine complexes. Biodistribution studies in Swiss mice bearing fibrosarcoma tumor showed 4.1 ± 1.2% ID/g of uptake at 30 min pi for 99mTc(CO) 3-complex as against 0.42 ± 0.24% ID/g for the 99mTcN-complex. 99mTc(CO) 3–colchicine complex exhibited better pharmacokinetics with lower liver accumulation as compared to the 99mTcN-complex. Thus, colchicine radiolabeled with [ 99mTc(CO) 3(H 2O) 3] + core is more promising with respect to in vivo distribution characteristics in tumor model.

Antitumor Agents. Part 236. Synthesis of Water‐Soluble Colchicine Derivatives

AbstractFor Abstract see ChemInform Abstract in Full Text.

New C(4)‐Functionalized Colchicine Derivatives by a Versatile Multicomponent Electrophilic Aromatic Substitution.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Antitumor agents. Part 236: Synthesis of water-soluble colchicine derivatives

Water-soluble colchicine derivatives were synthesized from 2-demethylcolchicine and 2-demethylthiocolchicine and evaluated in vitro against human tumor cell replication and for inhibition of tubulin polymerization. The glycinate esters ( 4, 5) and their tartaric acid salts ( 4a, 5a) showed potent cytotoxic activity in three different tumor cell lines with IC 50 values ranging from 0.02 to 0.88 μg/mL. The thiocolchicine analogs ( 5, 5a) were more potent than the colchicine analogs ( 4, 4a) in the tubulin polymerization assay. In particular, the water-soluble salt 5a merits preclinical development as an antitumor agent.

New C(4)‐Functionalized Colchicine Derivatives by a Versatile Multicomponent Electrophilic Aromatic Substitution

AbstractElectrophilic alkylation of colchicine at C(4) was accomplished by a multicomponent aromatic electrophilic substitution reaction with electrophilic aldehydes and carboxylic acids or amides in H2SO4. A series of new derivatives were obtained and evaluated for their antiproliferative effect towards various tumor cell lines, and their stimulatory effect on the development of polarity in human neutrophils.

Colchicine derivatives inhibit neopterin production in human peripheral blood mononuclear cells (PBMC).

Colchicine is a microtubule disrupting agent, mostly used as treatment in various kinds of inflammatory diseases such as acute familial Mediterranean fever and Behcet's disease, as well as gout. In patients with familial Mediterranean fever treatment with colchicine induces a decline of urinary neopterin concentrations which indicates a decrease of cell-mediated immune activation. In this study, we investigated a potential effect of colchicine on the T cell/macrophage system in vitro. The human myelomonocytic cell line THP-1 and PBMC were treated with colchicine or the colchicine derivative, colcemide, in the presence or absence of 250 U/ml interferon-gamma (IFN-gamma) or 10 microg/ml lipopolysaccharide (LPS) for 48 h or 96 h. Colchicine and colcemide increased neopterin/protein production in unstimulated THP-1 cells, but no such effect was apparent in cells stimulated with IFN-gamma. By contrast, when PBMC were treated with colchicine or colcemide a significant reduction in neopterin formation was evident in cells without and with prestimulation by IFN-gamma or LPS. In parallel, reduced production of IFN-gamma was observed in PBMC. These data suggest that colchicine and colcemide are able to inhibit T cell activation within the cellular immune response.

Occurrence of colchicine derivatives in plants of the genus Androcymbium

Androcymbium gramineum plants were phytochemically investigated for alkaloids. Two major alkaloids, colchicine and demecolcine, were isolated from the whole plant, and 2-demethylcolchicine, 3-demethylcolchicine, N-formyl- N-deacetylcolchicine and colchifoline have also been identified by HPLC analysis. This is the first report of demecolcine in A. gramineum, and of colchifoline in the genus Androcymbium. In addition, seeds of Androcymbium gramineum, Androcymbium hierrense, Androcymbium palaestinum, Androcymbium psammophilum, Androcymbium rechingerii, and Androcymbium wyssianum were also investigated for their alkaloid content for the first time. All were found to contain colchicine and colchicoside.

Synthesis and antimicrotubule activity of combretatropone derivatives

Combretatropone is a hybrid of combretastatin and colchicine in which the o-methoxyphenol of dihydrocombretastatin A-4 is replaced by an α-methoxytropone. Derivatives of combretatropone have been synthesized and evaluated for antimicrotubule activity. All combretatropones were less active than the corresponding colchicine derivatives, supporting the idea that loss of ligand conformational entropy upon tubulin binding results in decreased potency for colchicinoid ligands. The structure–activity relationship of the combretatropone series was different than that of the colchicine series. These data indicate that conformationally mobile and conformationally rigid colchicinoids do not interact with the receptor site in the same manner.

Effect of Deacetylation on Statmhokinetic Activity of Colchicine Derivatives

Screening of colchicine derivatives that was carried out with the tumor culture CaPa in a cytotoxicity test showed that deacetylation decreases the cytotoxicity and increases the mitotic index. Lower toxicity of the deacetylated colchicine derivatives was also noted.

Highly cohesive monolayers of lipid derivatives of colchicine: a dynamics study

Monolayers of lipid derivatives of colchicine spread at the air–water interface reach the thermodynamic equilibrium over an abnormally long period of time. Dynamics of this equilibration and the behavior of the film during compression–decompression cycles are observed by fluorescence microscopy. The thermodynamically disfavored structures observed are unrelated to previously described unusual shapes in the liquid expanded-gas coexistence regions. The relation between the cholesterol-like effect of the colchicinoid moiety, its propensity to dimerize, and the high viscosity of the monolayer are discussed.

The interaction between lipid derivatives of colchicine and tubulin: Consequences of the interaction of the alkaloid with lipid membranes

Colchicine is a potent antimitotic poison which is well known to prevent microtubule assembly by binding tubulin very tightly. Colchicine also possesses anti-inflammatory properties which are not well understood yet. Here we show that colchicine tightly interacts with lipid layers. The physical and biological properties of three different lipid derivatives of colchicine are investigated parallel to those of membrane lipids in the presence of colchicine. Upon insertion in the fatty alkyl chains, colchicine rigidifies the lipid monolayers in a fluid phase and fluidifies rigid monolayers. Similarly X-ray diffraction data show that lecithin–water phases are destabilized by colchicine. In addition, an unexpectedly drastic enhancement of the photoisomerization rate of colchicine into lumicolchicine in the lipid environment is observed and further supports insertion of the alkaloid in membranes. Finally the interaction of colchicine with lipids makes the drug inaccessible to tubulin. The possible in vivo significance of these results is discussed.