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Current Medicinal Chemistry, 2011, 18, 3035-3081

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Biological Potential and Structure-Activity Relationships of Most Recently Developed Vascular Disrupting Agents: An Overview of New Derivatives of Natural Combretastatin A-4 M. Marrelli1, F. Conforti*,1 , G. A. Statti*,1, X. Cachet2, S. Michel2, F. Tillequin2 and F. Menichini1 1

University of Calabria, Department of Pharmaceutical Sciences, I-87036 Rende (CS), Italy

2

Université Paris Descartes, Faculté des Sciences Pharmaceutiques et Biologiques, UMR 8638 CNRS, Laboratoire de Pharmacognosie, 4 Avenue de l’Observatoire, 75006 Paris, France Abstract: Tumor blood vessels are an important emerging target for anti-cancer therapy. The antimitotic agent combretastatin A-4 (CA4), a cis-stilbene natural product isolated from the South African tree Combretum caffrum Kuntze, is the lead compound of a new class of anti-cancer drugs that target tumor vasculature. CA-4 inhibits tubulin polymerization by interacting at the colchicine binding site on tubulin. This alters the morphology of endothelial cells and causes vascular shutdown and regression of tumor vasculature. Some tubulin-binding vascular-disrupting agents (VDAs) are currently in clinical trials for cancer therapy. As a consequence of the potential favorable applications of these compounds, several analogs projected to induce rapid and selective vascular shutdown in tumors have been synthesized during the last few years. Many of these molecules have already been tested for their effects on tubulin polymerization as well as for their antiproliferative activity and other biological properties, and possible mechanisms of action have been investigated. The aim of the present review is to offer an overview of most recently developed combretastatin derivatives, focusing on biological effects exerted by these compounds. The published data about new analogs are presented and compared, and a detailed investigation of structure-activity relationships is described.

Keywords: Combretastatin A-4, combretastatin analogs, vascular disrupting agents, tubulin, structure-activity relationships. INTRODUCTION Nature offers many potential therapeutic resources against cancer. Over 60% of currently used anti-cancer agents are derived from natural sources, including plants, marine organisms and micro-organisms [1]. A number of novel anti-cancer agents have been identified and some of them have entered into various phases of clinical trials, particularly for use in combination chemotherapy [2]. Among these resources, plants have a long history of medicinal use in the treatment of cancer and have been a primary source of effective conventional drugs. The search for new anti-cancer agents from plant sources reportedly begun in 1950s with the discovery and development of vinca alkaloids, vinblastine and vincristine, and the isolation of cytotoxic podophyllotoxins. More recent plantderived anti-cancer agents are the taxanes and camphothecins [1]. Although there has been significant progress in the treatment of cancer during the last few years, there is still no cure for most forms of human cancer. The main reason being that most chemotherapeutic drugs lack specificity toward cancer cells, i.e, while cancer cells are killed, normal tissues are damaged at the same time. Many researchers have considered the possibility that tumor growth might be suppressed by cutting off the supply of nutrients to tumors [3]. Formation of new blood vessels is an essential prerequisite for tumor growth in order to satisfy an increased demand of oxygen and nutrients. When tumor growth rate exceeds the capacity of the local blood supply, growth factors which stimulate angiogenesis are released [4]. The process of angiogenesis is dynamically regulated by both proangiogenic and antiangiogenic factors. A local excess of tumor-induced angiogenic factors, including basic fibroblast growth factor, vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs), over antiangiogenic agents (e.g. angiostatin, endostatin and thrombospondin) promotes an angiogenic response. Tumor-induced angiogenesis involves several processes, including proliferation of endothelial cells, proteolytic *Address correspondence to this author at the Department of Pharmaceutical Sciences, University of Calabria, I-87036 Rende (CS), Italy, Tel: +39 0984 493168, +39 0984 493063, Fax: +39 0984 493298, E-mail: [email protected], [email protected] 0929-8673/11 $58.00+.00

degradation of the extracellular matrix and migration of endothelial cells, culminating in the formation of a functional vessel with a lumen [5-7]. In order to support an increasing number of cells, the process of angiogenesis leads the surrounding mature vessels to sprout new capillaries that grow toward and infiltrate the tumor [4]. Normally, the resulting level of endothelial proliferation is not found in the adult tissues; tumor vessels present an abnormal structure, characterized by temporary occlusions, blind ends, leaky vessels and a reduction in pericytes [8]. Due to profound differences between tumor blood vessels and normal blood vessels, preclinically and clinically tumor vasculature has been intensively investigated as a new potential target for anti-tumor therapeutic strategy, either by anti-angiogenic drugs to prevent formation of new blood vessels or by vascular disrupting drugs to destroy previously existing tumor blood vessels [4]. The ability to target tumor vessels is an exciting new development in anti-cancer treatment, because the destruction of the blood vessels that support the continuing growth of a tumor can lead to rapid tumor cell death from the lack of nutrients and oxygen in areas of tumors resistant to many anti-cancer agents [9]. The vascular disrupting agents (VDAs), also called vascular targeting agents (VTAs), result in rapid shutdown of blood supply to the tumor, thus killing tumor cells by depriving them of oxygen and nutrients. These agents are different from antiangiogenic agents, which prevent formation of new blood vessels [10]. One class of compounds that have proven to cause antivascular effects in tumors is represented by combretastatins. Combretastatin A-4 (CA-4) (1) is a cis-stilbene natural product isolated from the South African tree Combretum caffrum (Eckl. and Zeyh.) Kuntze (Combretaceae) by Pettit and coworkers in 1989 [11]. CA-4 binds at the colchicine binding site on the -tubulin subunit of tubulin and inhibits polymerization of tubulin into microtubules. Inhibition of tubulin/microtubule polymerization dynamics has two key anticancer effects: the inhibition of cancer cell proliferation through disturbance of mitotic spindle function, which leads to cell apoptosis; the disruption of cell signaling pathways involved in regulating and maintaining the cytoskeleton of endothelial cells in tumor vasculature, leading to selective shutdown of blood flow through tumors [12]. © 2011 Bentham Science Publishers Ltd.

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As a result of the potential favorable applications of vascular disrupting agents, several analogs projected to induce rapid and selective vascular shutdown in tumors have been synthesized and some tubulin-binding vascular-disrupting agents (VDAs) are currently in various phases of clinical trials for cancer therapy. The present review provides an overview of most recently developed combretastatin derivatives. The biological effects exerted by these compounds and the observed structure-activity relationships are reported. MICROTUBULES AND ANTIMITOTIC AGENTS Microtubules are cytoskeletal components present in all eukaryotic cells, that play an essential role in cellular functions including motility, division, shape maintenance and intracellular transport [13]. They are cylindrical protein polymers assembled from head-to-tail and lateral associations of and -tubulin subunits, each approximately 50 kDa in size. The and subunits have a high degree of homology to each other and the primary sequence of these subunits has been determined from protists, plants, fungi and eukaryotes [14]. The secondary and tertiary structures have been elucidated by electron crystallography from protofilaments purified in the presence of zinc ions by Nogales and coworkers [15]. Upon binding of GTP, tubulin polymerizes into microtubules which are helical arrays of alternating and subunits with a cross-sectional diameter of approximately 24 nm [14]. Most microtubules occur as single tubes and form cellular structures such as the mitotic spindle and the interphase network. A subset of microtubules exist as fused structures where a complete microtubule is fused with one or two incomplete tubules to comprise ciliary axonemes (doublet microtubules) or centrioles and basal bodies (triplet microtubules) [16]. The formation of microtubules is a dynamic process that involves assembly of the heterodimers formed by tubulin subunits and degradation of the linear polymers. The balance between these phenomena is important for the structure of the cytoskeleton, as well as for mediating intracellular transport. The alignment of sister chromatids at the spindle equator and their segregation into the nascent daughter cells during mitosis are also mediated by dynamic microtubules. The unusual assembly process of microtubules is characterized by extended periods (seconds–minutes) of growth and shortening and is called dynamic instability. This phenomenon is quantitatively characterized by the microtubule ends growing and shortening rates and by the frequencies of switching between these two states. The frequency at which a growing microtubule tip switches to a degradation state is defined ‘catastrophe’ frequency, while the opposite one is called ‘rescue’ frequency [17-19]. Tubulin polymerizes in presence of non-hydrolysable GTP to form stable microtubules because of the high affinity of the tubulin-GTP dimer for the end of the microtubule. The disassembly is determined by GTP hydrolysis, due to instability of the resulting GDP microtubule [20] .

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and with an affinity that is characteristic of the drug and by disrupting the dynamic equilibrium of microtubules. The binding sites of taxol, colchicines and vinblastine in tubulin are well characterized. Taxol and vinblastine bind to the -subunit whereas colchicine binds at the intradimer interface of and subunits of the tubulin heterodimer. Most of the microtubule depolymerizing agents either bind at the colchicine or vinblastine binding site on tubulin [13, 22]. Even if both MDA classes have been shown to operate by binding to tubulin at different binding sites and with different sensitivities, the dose-dependent series of morphological alterations of the mitotic spindle and of chromosomal disposition are nearly identical. Hence, abnormal spindles have been classified in five different classes (I-V) based on increasing degrees of microtubular disorganization [23]. Combretastatin A-4 is a powerful microtubule-destabilizing agent that inhibits microtubule assembly by binding to tubulin at the colchicine binding site. The antivascular effect of this molecule derives from the role tubulin and microtubules play in determining the elongated shape of vascular endothelial cells. The cellular microtubule networks of the cytoskeleton plays a major role in maintaining cell shape, particularly in the case of the neovasculature. As a result of the tubulin depolymerization, endothelial cells round up and very quickly block blood-flow through the vascular network [24, 25]. VASCULAR DISRUPTING AGENTS The importance of vascular networks in the context of establishment and progression of cancer has led to the development of the concept of ‘vascular targeting’ for therapy [26]. VDAs are a relatively new group of ‘vascular targeting’ agents that exhibit selective activity against established tumor vascular networks, causing severe interruption of tumor blood flow and necrosis to the tumor mass [27]. They are different from factors that aim to inhibit the tumorinitiated angiogenic process, such as agents that interfere with the delivery or export of angiogenic stimuli; antibodies that inhibit or inactivate angiogenic factors after their release; inhibitors of receptor activity, tumor invasion, or endothelial cell proliferation. Antivascular agents, instead, are able to preferentially destroy the established tumor vessel network. VDAs differ from antiangiogenic agents not only in their mode of action but also in their therapeutic application. Whereas VDAs are used in intermittent doses, antiangiogenic treatment is administered continually over months or years.

Due to the key role played by tubulin during cellular division, ligands that interrupt the dynamic instability inherent to this system have been developed as antimitotic, anti-cancer drugs [21]. Different types of naturally occurring ligands that inhibit tubulin polymerization have been reported in the literature, and there has been a continuing discovery of new agents with pronounced structural diversity [14].

VDAs can be divided into two categories: biologics and smallmolecule agents. The former category includes antibodies and peptides that deliver toxins and procoagulant and proapoptotic effectors to the tumor endothelium. The latter category includes agents that do not specifically localize to the tumor endothelium but exploit the known differences between tumor and normal endothelium to induce selective vascular dysfunction. Within minutes of exposure to a VDA, affected tumor blood vessels begin to show signs of damage. This initiates a sequence of events that leads to occlusion of blood vessels and a marked reduction in tumor blood flow. Tumor cell death and necrosis occur as a result of prolonged ischemia.

Microtubule-damaging agents (MDAs) can be divided into two groups, namely polymerization inhibitors and polymerization promoters. Several natural and synthetic compounds such as vinca alkaloids, colchicine, estramustine and combretastatins inhibit microtubule polymerization whereas compounds such as taxanes, laulimalides and discodermolides promote microtubule assembly. Both these classes of microtubule-damaging agents have been shown to operate by binding to tubulin at drug specific binding sites

Tumor vasculature may represent an ideal target for ligandbased treatments, because of the accessibility of the endothelium that significantly attenuates the delivery problems. Antigenic determinants that are selectively expressed on or near the tumor neovasculature, such as endoglin, vascular endothelial growth factor (VEGF) receptors, -integrins, the fibronectin EDB domain and prostate-specific membrane antigen, have been identified as targets for ligand-directed approaches involving VDAs [28].

Biological Potential and Structure-Activity Relationships of New CA-4 Derivatives

Current Medicinal Chemistry, 2011 Vol. 18, No. 20

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OCH3 OCH3 H3CO

H3CO

OCH3

A H3CO

H3CO

B OCH3

H3C OCH3

OH

NH

OP(O)(ONa)2 OCH3

OCH3

O

O

O

P

OH

HO Combretastatin A-4 (CA-4) 1

ZD6126 3

CA4P 2

H3CO HOOC H3CO

O OCH3

NH-Ser-H OCH3 O FAA 5

AVE-8062 4 H3C

CH3

H3CO

O O

OP(O)(ONa)2

H3CO

HO

OCH3 O DMXAA 6

OP(O)(ONa)2 OCH3 CA1P 7

Fig. (1). Leading vascular disrupting agents.

There are two main types of small-molecule VDAs: tubulindepolymerizing agents and flavonoids. Colchicines and vinca alkaloids, which were shown to induce vascular shutdown in preclinical tumor models, stimulated the interest in the vasculardamaging properties of tubulin-binding agents. Presently, the leading candidates belonging to this class are combretastatin A-4 disodium phosphate (CA4P) (2), ZD6126 (3) and AVE-8062 (4), which induce antivascular and antitumor effects at doses that are less than one-tenth of the maximum tolerated dose (Fig. 1). Moreover, these compounds selectively induce shutdown of blood flow in tumor, and have limited or nonexistent effects in normal tissue [28, 29]. The effects induced by flavonoids on blood vessels are determined by the localized release of TNF from activated macrophages [30]. As a matter of fact, antibodies against TNF are able to inhibit the vascular collapse induced by the flavones acetic acid (FAA) (5) [31]. Both FAA and the more active compound 5,6dimethylxanthenone-4-acetic acid (DMXAA) (6) selectively target tumor blood vessels in preclinical rodent tumor models, but only DMXAA is able to stimulate both human and mouse macrophages. Therefore, DMXAA is considered the leading candidate within the class of flavonoid VDAs (Fig. 1) [28, 32]. Even if many VDAs are now being tested in clinical trials, their molecular targets and their mechanisms of action are not completely understood [33]. Their capacity to target the cytoskeleton and compromise the integrity of endothelial cell junctions, are thought to be central to their mechanism of action. Although it is not yet firmly established why VDAs are selective for tumors, current views favor the hypothesis that selectivity

relates to the fragile and immature nature of tumor blood vessels [27]. Vascular targeting agents produce a characteristic early necrosis in the center of tumors while leaving a viable rim of cells that may be supplied with oxygen and nutrients by surrounding host vasculature. It is also possible that the remaining viable rim is also partly a consequence of incomplete shutdown of the vasculature in the outer regions of the tumor. Although this viable rim may ultimately limit the activity of vascular targeting compounds administered as single agents, the susceptibility of this cell population to ionizing radiation and chemotherapy could result in a powerful effect of combining vascular targeting with conventional therapy [34]. COMBRETASTATIN A-4 Combretastatin A-4 is a cis-stilbene based natural product isolated from the bark of the South African tree Combretum caffrum (Eckl. and Zeyh.) Kuntze (Combretaceae) by Pettit and coworkers in 1989 [11]. Over a dozen of combretastatins have been isolated and reported from this and other related plants, such as combretastatin A-1 (CA-1, 8) in 1987 (Fig. 2). Of all the combretastatins, CA-4 is reported to have a very potent cytotoxicity against a wide range of human cancer cell lines, including multidrug resistant (MDR) cancer cell lines, with the IC50's values consistently in low nanomolar to subnanomolar range. The mechanism of cytotoxicity of CA-4 as well as many other combretastatins was shown to be the inhibition of tubulin polymerization [35, 36] . This molecule is one


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H3CO OH

H3CO OCH3

O

HO

O

H3CO OCH3

OH

OCH3

OH

OCH3

OCH3

OCH3

Combretastatin A-2 (CA-2) 9

Combretastatin A-1 (CA-1) 8 HO

OH


HO OCH3

H3CO

H3CO OCH3

OCH3 OCH3


OCH3 H3CO Combretastatin A-6 (CA-6) 12

Fig. (2). Selected combretastatins.

of the most powerful inhibitors of tubulin polymerization known to date. Combretastatin A-4 inhibits tubulin polymerization by binding to tubulin at the colchicine binding site [37]. This agent is a competitive inhibitor of the binding of colchicine to tubulin with an apparent Ki value of 0.14 μM in a competitive binding assay. It was also shown that CA-4 is capable of displacing [3H]-colchicine from its binding site but does not displace [3H]-vinblastine from the vinca binding site. In contrast to colchicine, whose binding to tubulin is slow and temperaturedependent, binding of CA-4 to tubulin appears to be rapid and not temperature sensitive, since preincubation does not enhance the inhibitory effects of CA-4 on tubulin polymerization and the CA-4tubulin complex formation occurs readily even on ice. Binding is reversible, as evidenced by displacement of CA-4 from the colchicine binding site with high concentrations of radio-labeled colchicines [35]. Thus, CA-4 specifically binds at the colchicine binding site. Moreover, combretastatin A-4 binds much more avidly than colchicines to the same site on tubulin, but does not show the same pseudo-irreversible binding activity. Differences in tubulin binding kinetics may influence the in vivo activity of these drugs and account for the wide therapeutic window of combretastatin A-4 compared with other tubulin-binding agents [5]. Despite the very potent cytotoxic and antitubulin activities, CA4 did not exhibit potent antitumor effects in in vivo models. This was ascribed to poor bioavailability, related to the limited water solubility of compound. In order to overcome this issue, several water-soluble sugar derivatives of CA-4 were synthesized [38]. However, these sugar combretastatins showed a substantial decrease in cytotoxicity compared to CA-4. Further efforts directed at synthesizing of water-soluble prodrugs of CA-4 led to the disodium phosphate derivative of CA-4 (CA4P, 2) with excellent water-solubility. CA4P is cleaved to CA-4 by nonspecific endogenous phosphatases and is taken up by the cells [35]. Mechanism of Action Combretastatin A-4 inhibits tubulin polymerization by binding to tubulin at the colchicine binding site, resulting in disruption of dynamic equilibrium needed in formation of microtubules from and - tubulin heterodimers, leading to formation of abnormal mitotic spindles. It results in cell cycle arrest in the M-phase, leading to apoptotic cell death. CA-4 inhibits tubulin polymerization with an IC50 value of 1.2 μM [14, 39-42]. The antivascular effect of CA-4 is related to its antitubulin activity. The cellular microtubule network plays a major role in

determining the elongated shape of vascular endothelial cells and as a result of the depolymerization of microtubules, elongated endothelial cells round up, causing disruption of endothelial cell layer surrounding blood vessel and exposing of underlying basement membrane. This leads to blood vessel congestion and loss of blood flow, loss of oxygen and nutrient supply to tumor cells. Therefore, tumor cells undergo necrosis [43, 44]. It has been shown that compared to normal blood vessels tumor vessels are more susceptible to CA-4 [40]. It has been also reported that CA-4 induces cell death primarily through mitotic catastrophe induced by the activation of the cysteine protease caspase-9 [43]. However, it has also been observed that the inhibition of caspase-9 by a specific inhibitor did not inhibit combretastatin A-4 induced cell death. According to this result, apoptosis is a secondary mechanism of death in a small proportion of cells treated with CA-4 [44]. Moreover, Nabha et al. investigated the molecular aspects of the mitotic catastrophe and whether or not it shares the same pathways of apoptosis. The authors studied the effect of CA4P on selected markers of apoptosis and G2-M protein regulators using lymphocytic leukemia cell line WSU-CLL. Their study suggested that CA4P induces mitotic catastrophe and arrest of WSU-CLL cells mostly in the M phase independent of p53 and independent of chk1 and cdc2 phosphorylation pathways. Apoptosis is a secondary mechanism of death in a small proportion of cells through activation of the two markers of apoptosis caspase 9 and poly(ADPribose) polymerase (PARP) cleavage. This finding suggest that the two mechanisms of cell death, i.e., mitotic catastrophe and apoptosis, are independent of each other [45]. CA-4 is highly cytotoxic against many cancer cell lines, including MDR positive cancer cell lines. This superior activity is explained by the fact that CA-4 is not a substrate of the MDR pump, a cellular pump that transports out foreign molecules, including many anticancer drugs [43]. Structure-Activity Relationships of CA-4 Combretastatin A-4 belongs to the class of natural compounds related to biphenyls and contains, as a key structural feature, the cis-stilbene motif. From structure-activity investigations it has been established that the cis-orientation of the two aryl rings is crucial for the activity. As a matter of fact, in vivo CA-4 has been observed to lose its efficacy not only because of its high lipophilicity and low aqueous solubility, but also due to the isomerization of cis-double


bond to the more thermally stable trans-isomer, which is inactive [43]. The structural simplicity of CA-4 combined with its excellent antivascular activity encouraged the synthesis of numerous analogs. These studies demonstrated that the 3,4,5-trimethoxysubstituted A ring and the 4-methoxysubstituted B ring (Fig. 1) are also very important for the cytotoxic activity [36]. As regard the first one, the presence of the trimethoxybenzene moiety is considered to be fundamental to obtain relevant cytotoxic and antitubulin responses. This statement is mainly based on the recurrence of this chemical motif in other antitubulin drugs, on the potency of CA-4 over CA-3 (Fig. 2), where the meta methoxy group is replaced with a hydroxyl, and by the loss of potency caused by the presence of a simple aromatic ring or the deletions of meta or para methoxy groups. Regards to the modifications at the B-ring, they were historically considered very important for the synthesis of potent compounds, and therefore, this ring has received greater attention from medicinal chemists. It was modified by various substitutions with heterocyclic rings, substituted phenyl rings and nonsubstituted aromatic rings. The olefinic component of CA-4 has been differently modified. The olefinic bond allows placement of the aromatic rings in an appropriate way and imparts the molecule the required flexibility to achieve the right dihedral angle to maximize its interaction with the binding site. Consequently, the presence of a double bond in a Z configuration is fundamental to achieve high cytotoxicity and antitubulin activity. The Z stilbenic double bonds can easily isomerize under the influence of heat, light and protic media. In order to avoid the isomerization, many efforts have been realized to modify the double bond or to replace the olefinic bridge with a ring, in order to stabilize the conformation and to increase the biological effects of the compound [37]. Other Biological Applications

Activities

and

Potential

Therapeutic

The beneficial effects of combretastatin A-4 are not limited to tumor vascularization. In noncytotoxic concentrations, this molecule causes a disruption of actin filaments and tubulin microtubules that results in altered endothelial cell morphology and functions in cell cultures. Some authors, for example, have examined the capacity of CA-4 to inhibit retinal vascularization. Retinal neovascularization is a common feature present in a variety of diseases, including diabetic retinopathy, that is the most common cause of blindness in the developed world. It is a microvascular complication of both type 1 and 2 diabetes, that is characterized by vascular changes of the retinal capillary bed and that leads to the development of neovascularization on the inner surface of the retina [46]. In this context, some studies have shown that a number of antiangiogenic agents can inhibit retinal neovascularization in a murine model of ischemia-induced proliferative retinopathy. Griggs and coworkers examined the capacity of CA-4 to inhibit retinal neovascularization in vivo [47]. CA-4 caused a dose-dependent inhibition of neovascularization with no apparent side effects. The absence of vascular abnormalities or disrupted neovessels in retinas of CA-4-treated mice suggested an anti-angiogenic mechanism in this model, in contrast to the anti-vascular effects observed against established tumor vessels. Moreover, histological and immunohistochemical analyses indicated that CA-4 permitted the development of normal retinal vasculature while inhibiting aberrant neovascularization. These data suggest that CA-4 has potential in the treatment of nonneoplastic diseases with an angiogenic component.


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In a study pilot, Kador et al. [46] evaluated the ability of CA4P to modify retinal neovascularization in aphakic long-term galactose-fed beagles, an animal model that develops diabetes-like retinal neovascularization. Based on the observation that CA4P decreases blood flow into tumors within hours of administration, this compound was anticipated to also alter retinal perfusion in neovascular retinal areas and a single dose was anticipated to be sufficient to disrupt neovascular perfusion that should become evident following a 2-, 4-, and 6-week follow-up. However, it was observed a lack of efficacy of CA4P in this animal model. This suggests that microthrombus formation was not enhanced by CA4P in the dog eye and that the rate of neovascularization of the dog’s ocular capillary bed may be too slow to be significantly affected by tubulin depolymerizing agents. The neovascularization rate in dogs more closely reflects the growth rate of clinical retinal angiogenesis in diabetics and for this reason the administration of this type of vascular targeting may not be appropriate for the clinical treatment of proliferative retinopathy. Alternatively, a prolonged chronic administration of CA4P may be required to destroy retinal endothelial cells. Griggs and coworkers also investigated whether CA-4 is a tumor specific antivascular agent using the hyperplastic thyroid as an in vivo model of neovascularization. CA-4 caused pathological changes in normal tissues, such as the induction of intravascular thrombi. These vascular-damaging effects are another demonstration that the action of CA-4 is specific not for tumor vasculature but for rapidly growing endothelium. For this reason CA-4 may have considerable effects if any non-neoplastic vascular proliferation is occurring, such as in inflammation and angioproliferative diseases [48]. Combretastatin A-4 Disodium Phosphate (CA4P) The limited water solubility of CA-4 led to the synthesis of water-soluble prodrugs. Combretastatin A-4 disodium phosphate (CA4P, 2) was synthesized by Pettit and Rhodes in 1998 [49]. The negatively charged phosphate group prevents cellular transport of the drug, but the phosphate group is readily cleaved by endogenous non-specific phosphatases releasing the uncharged active agent [50, 51]. CA4P causes a rapid and extensive vascular shutdown in experimental tumor models. It has been observed to act most effectively against immature tumor vasculature [52, 53]. It has also been described to be cytotoxic towards proliferating but not the quiescent endothelial cells. The cytotoxic effects of CA4P towards endothelial cells were first reported to be mediated by the induction of apoptosis, but later studies suggested that only a small proportion of cells undergo apoptosis and that endothelial cells undergo predominantly mitotic catastrophe [45]. This work also demonstrated that non-cytotoxic concentration of CA4P cause the disruption of actin filaments and tubulin microtubules of human endothelial cell cultures one hour after treatment. In vivo activity of CA4P has been demonstrated towards a wide number of murine tumors, including mouse models of colorectal and lung metastases [9, 54]. However, cardiovascular toxicity and neurotoxicity were dose limiting for CA4P. These significant side effects currently represent the main obstacles to broad clinical application of this drug. For this reason, it is necessary to develop a new analog of CA-4 with more specificity for tumor endothelial cells than normal endothelial cells to avoid cardiac toxicity from endothelial damage [55, 56]. Despite the potent antivascular effects of CA4P, in tumors, CA4P induces little growth retardation when administered in a single dose which is close to or at its maximum tolerated dose. This lack of activity has been attributed to the survival of narrow rim of peripheral tumor cells adjacent to the more normal vasculature in the surrounding tissue. These cells are capable of proliferation and


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contribute to eventual regrowth and revascularization of the necrotic tumor center [57]. Options for improving the therapeutic efficacy of CA4P have centered on the investigation of CA4P in combination with conventional antiproliferative therapies (e.g. radiation, cisplatin), as it targets the central hypoxic parts of tumors, often considered resistant to conventional therapies, and the anti-proliferative approaches target the viable rim. Beneficial interactions with hyperthermia and radioimmunotherapy have also been reported [9]. Presently, CA4P (ZybrestatTM), followed by CA1P (7), is the lead compound among cancer vasculature disrupting drugs. The potential of CA4P in treating eye diseases, such as diabetic retinopathy and retinoblastoma, is also being studied [58]. Many important recent studies have shown that CA4P is antiangiogenic, increases aberrant organization of metaphase chromosomes in nonsmall cell lung cancer cells, inhibits gastric cancer cell metastasis and improves glucose tolerance in diabetic mice, which suggests a potential new approach for treatment of type 2 diabetes [59]. CLINICAL TRIALS Many VDAs, such as CA4P (2), CA1P (7), AVE-8062 (4) and ZD6126 are undergoing clinical trials. Phase I trials of CA4P (ZybrestatTM) established that when used as single agent the drug is well tolerated, with myocardial ischemia, reversible neurological events and tumor pain as the main limiting toxicities [60]. These trials also demonstrated selective reductions in blood flow and their findings were consistent with data obtained from preclinical studies [61]. Phase II/III clinical trials are now being conducted by OXiGENE. CA4P is tested in combination with radiotherapy, carboplatin, paclitaxel and the anti-VEGF antibody bevacizumab (Avastin). Recent reports show that CA4P causes some constant

vascular changes in the presence of bevacizumab and the combination is safe and well tolerated [27]. Combretastatin A-1-P (CA1P or OXi4503, 7), the prodrug of the natural parent CA-1 is an even more potent VDA than CA4P [62]. It is also now being clinically tested as a single agent and early results point to vascular activity at well-tolerated doses [39, 63]. SYNTHESIS OF CA-4 A general synthesis of Combretastatin A-4 and analogs was first developed by Pettit and co-workers, which utilized the Witting reaction to produce both Z- and E-isomers in an average ratio of about 1:1.5 that required a separation step to isolate pure cis-CA-4 [64]. The simple structure of CA-4 has subsequently encouraged the synthetic efforts toward CA-4 and its analogs and several syntheses have been reported. Synthetic routes based on the Wittig reaction, Perkin condensation, Suzuki cross coupling, Sonogashira crosscoupling and Ramberg Backlund reaction have all been reported. Some of these reactions suffer either from poor stereocontrol of the double-bond geometry or low yield and are therefore difficult to carry out on a preparative scale [65]. Another approach to the synthesis of CA-4 was described by Harrowven and coworkers, who described also a small library of analogs that utilize cooperative ortho-effects in Wittig reactions to achieve high Z-selectivity in the olefination [66]. A very useful reaction for the stereoselective synthesis of Z or E stilbenes is the Kumada-Corriu cross-coupling reaction. LaraOchoa and coworker synthesized CA-4 by coupling 3,4,5trimethoxyphenylacetylene with the corresponding iodomethoxyphenol and bromomethoxyaniline, and by hydrolysis of the Ti(II)alkyne complexes [65]. Robinson et al. described a strategy to synthesize combretastatin A-4 by means of a Ramberg–Bäcklund

O B O B

H3CO

O

H3CO PPh3Br

OCH3 14

13

O B

H3CO 15 Fig. (3). Structures of boron containing analogs.

O

O


reaction to form the key (Z)-stilbene unit [67]. Another short and efficient synthesis of combretastatin A-4 was described by Camacho-Davila, that used the iron-catalyzed cross-coupling of a Grignard reagent and a bromostilbene [68]. MORE RECENT CA4-ANALOGS: STRUCTURE-ACTIVITY RELATIONSHIPS The use of combretastatin A-4 as a clinical antitumor agent is limited by its low aqueous solubility and low bioavailability. Moreover, it tends to isomerize to the thermodynamically more stable and inactive trans-isomer. The goal to find even more potent and selective compounds has induced many researchers to design more soluble, stable and active analogs [12, 69, 70]. Accordingly, hundreds of analogs of CA-4 have been described. Many cis-restricted analogs containing a trimethoxyphenyl group have been prepared and evaluated as tubulin polymerization inhibitors [12]. In order to avoid stability problems of CA-4, many analogs containing 1,2-five membered heterocycles such as imidazole, oxazole, pyrazole [71], thiazole, triazole, tetrazole [72], cyclopentenone [73, 74], furanone [74] or oxazolone [75] were also prepared. Many of these molecules have been tested for their effects on tubulin polymerization as well as for their antiproliferative activity and other biological properties, and possible mechanisms of action have been investigated. The structure-activity relationships for CA-4 have been described from these analogs, and during the last few years some other review were dedicated to these kind of compounds [35, 37, 43, 76, 77]. This review will try to offer an overview of most recently developed combretastatin derivatives, focusing on their biological activities and structure-activity relationships. 1. A-Ring and B-Ring Modified Analogs Recently, Das and coworkers [78] developed a procedure to synthesize new boron-containing stilbene derivatives, and used this method to synthesize CA-4 analogs. Stilbene (1,2-diphenylethene) does not occur in nature, but stilbene derivatives such as resveratrol or combretastatin A-4 are active compounds. The authors synthesized the new derivatives using the Wittig reaction of the ylide 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboratophenyl)-methyl triphnenylphosphonium bromide (13) with 3,4,5-trimethoxy benzaldehyde in the presence of tBuONa in DMF. This procedure allowed the synthesis of new analogs 14 and 15, bearing the boronic acid functionality that replaces the OMe group of the natural CA-4 in ring B (Fig. 3). Experiments are currently underway to test the biological activity of these derivatives. The anti-fungal activity of combretastatins has been reported, and Coccetti [79] evaluated the effects of these molecules on the


cell cycle progression of the budding yeast Saccharomyces cerevisiae, that is one of the best analyzed organisms for cell cycle studies. Combretastatin A-1 (8), CA-4 (1) and eight CA-4 derivatives 16(a-d) and 17(a-d) where the hydroxyl group of the natural compound was replaced by fluoro, nitro, amino and boronic acid moieties, either as Z or E isomer, were synthesized (Fig. 4), and their in vivo activity was analyzed by monitoring their ability to inhibit yeast cell growth. Although the compounds 16 (a-d) and 17 (a-d) were previously reported in the literature, the authors developed a clean strategy for their synthesis. Compounds 16(a-d) and 17(a-d) were synthesized by a transfer-phase Wittig coupling of p-methoxybenzaldehyde bearing in position 3 a boronic moiety, a fluoro atom or a nitro group, respectively and triphenyl(3,4,5-trimethoxybenzyl) phosphonium chloride. Compounds 16(b-d), 17(a-d) and the transCA-1 and trans-CA-4 had no effect on yeast growth at all tested concentrations, while cis-CA-1, CA-4 and molecule 16a arrested cell growth in a dose-dependent manner. CA-4 and 16a determined a G1 arrest due to a decreased level of Clb5, the principal S-phase cyclin, describing a new block of yeast cell cycle not previously identified in mammalian cells. Furthermore, in the case of compound CA-4, the G1 arrest coincides with the activation of the stress activated kinase Snf1. Moreover, Fortin [80] realized a series of novel N-phenyl-N’(2-chloroethyl)urea derivatives of combretastatin A-4. These new compounds were called N-phenyl-N’-(2-chloroethyl)ureas (CEU). While combretastatins bind to the colchicines binding site through electrostatic interactions, these new derivatives covalently bind to the site through a nucleophilic substitution involving the N’-(2chloroethyl)urea pharmacophore. The cell cytotoxic activity of CA-4-CEU hybrids 18-23 was assessed on human colon carcinoma (HT-29), skin melanoma (M21), breast carcinoma (MCF-7) and breast hormone-independent adenocarcinoma (MDA-MB-231). The 1,2-diarylethenyl Z-isomers 22 and 23 were more active than the E-isomers 18 and 19, and the saturated 1,2-diarylethanyl derivatives 20 and 21 (Table 1). Unexpectedly, CEU substituted in position 4 showed a better activity than CEU substituted in position 3. Compounds 20-23 caused a significant accumulation of cells M21 in G2/M, suggesting that these CEU, like combretastatin A-4, might inhibit the tubulin polymerization and consequently induce apoptosis. 2. Benzoheterocycle-Based Combretastatins A recently described pharmacophore model indicates for combretastatins a positive electrostatic potential region situated in OMe MeO

OMe R

OMe

MeO

MeO

OMe

MeO R 16

Fig. (4). Combretastatin analogs tested on yeast growth.

17 R=B(OH)2 R=F R=NO2 R=NH2

3041

16a +17a 16b +17b 16c +17c 16d +17d


Table 1.

Marrelli et al.

Cell Growth Inhibition of CEU Analogs Cytotoxicity (GI50 μM)

Compound

O

HT-29a

M21b

MCF-7c

MDA-MB-231 d

OCH3

68.2

22.7

28.2

16.5

OCH3

9.6

4.7

11.6

4.1

9.2

4.0

4.5

4.9

Ref.

OH NH

18

NH Cl O

OH NH

19

NH Cl

20

H N

HN

HO OCH3

Cl

O

H3CO 21

Cl

HN

HO N H

1.18

8.38

14.0

17.4

15.3

16.3

15.9

11.0

2.4

0.97

1.65

0.86

0.089

0.002

0.003

0.002

O

H3CO 22

[80]

Cl

HO

HN N H

O

O NH 23

OCH3

NH Cl

OH

CA-4 a

b

c

d

Human colon carcinoma cell line. Skin melanoma cell line. Breast carcinoma cell line. Breast hormone-independent adenocarcinoma.

the outer zone of the trimethoxy groups in the A-ring favoring the activity in A- and B- rings. This observation suggests that other hydrogen bond acceptors could replace the trimethoxy groups to obtain active compounds. Heteroaromatic rings, containing electron-rich atoms, may increase the activity of novel combretastatins and could also promote the steric interaction with a large hydrophobic pocket at the active site [81]. Simoni and coworkers [55] replaced the A-ring of CA-4 with benzofuran ring (compounds 26, 27), and benzo[b]thiophene ring (25, 29, 30). The authors also described the replacement of the Aring with a 1,2,3,4-tetrahydro-1,1,4,4-tetramethylnaphthalene portion (28), and also another group of compounds bearing substitutions at both A- and B-ring (Table 2). The first ring is replaced by a benzo[b]thiophene, a methylenedioxobenzene ring or a dimethoxy phenyl group, and the B-ring is replaced by a benzofuran or a benzo[b-]thiophene heterocycle (compounds 3135). Cytotoxicity of all synthesized compounds and their activity in both in vitro tubulin polymerization inhibition (TPI) and colchicine tubulin binding competition assay (CTBC) were analyzed. The obtained results evidence that the compounds containing a benzofuran or benzo[b]thiophene heterocycle possess potent biological activity. Compounds bearing the double

benzo[b]thiophene substitution also showed a good cytotoxic activity. Compounds 25-27 showed the highest activity both on BMEC and H-460 cell lines. The two regioisomers 26 and 27 possess the same IC50 value inhibition on BMEC cell line, showing that activity seems not to be influenced by the position of the endocyclic heteroatom. On the other hand, the substitution of the A-ring with the 1,2,3,4-tetrahydro-1,1,4,4-tetramethylnaphthalene (derivative 28) determined the loss of any activity. The replacement of the characteristic isovanillic moiety of compounds 25-27 with a benzoheterocycle also resulted in loss of activity. Regards to their effects on tubulin assembly, the most active compounds, 25-27, potently inhibit tubulin polymerization with an IC50 value similar to or better than that of CA-4 and, quite surprisingly, the binding of compounds 25-27 at the colchicine binding site resulted in five times higher activity than that of natural CA-4. 3. Bridge Modified Analogs Considering that the Z stilbenic double bonds can easily isomerize under the influence of heat, light and protic media, a number of groups have attempted to modify the olefinic bridge to stabilize the conformation to attempt to increase the biological


Table 2.


3043

Cytotoxicity and Tubulin Interactions of Benzoheterocycle-Based Combretastatins Cytotoxicity (IC50 nM ± SD) Compound BMEC

a

HT-29

b

Tubulin polymerization H-460

c

d

(IC50 nM ± SD )

Colchicine tubulin binding competition

Ref.

(IC50 nM ± SD)

O H3CO

CH3 NH

H3CO

24

-

-

-

-

1.7 ± 0.6

CA-4

3.7 ± 0.3

118 ± 33

1.2 ±0.2

4.9 ± 0.2

0.1 ± 0.02

CA-2

48 ± 1.3

>1000

40 ± 0.4

-

-

17 ± 0.2

260 ± 32

13 ± 2.5

2.9 ± 0.09

0.02 ± 0.002

35 ± 1.8

420 ± 57

19 ± 5

5.1 ± 0.07

0.02 ± 0.003

35 ± 0.3

590 ± 39

12 ± 0.8

3.9 ± 0.1

0.05 0.002

>1000

-

-

21.5 ± 1.9

-

20 ± 2.3

21 ± 1.3

19 ± 1.3

3.6 ± 0.28

6.2 ± 0.12

123 ± 33

-

110 ± 8.5

-

-

110 ± 11

-

-

12.3 ± 0.5

3.8 ± 0.6

800 ± 40

-

-

7.1 ± 0.08

4.0 ± 0.09

880 ± 52

-

-

14.2 ± 0.9

4.9 ± 0.07

>3000

-

-

16.6 ± 0.7

7.7 ± 0.1

250 ± 0.5

-

312 ± 53

24.3 ± 0.8

-

CH3O O OCH3

colchicine 1 9 S

25

OCH3 OCH3

OH

O

26

OCH3 OCH3

27

OH

O

OCH3 OCH3

OH

28 OCH3 OH

S

[55]

29

OCH3 OCH3

NH2xHCl

OCH3

H

S

30

OCH3

H3CO

S

31 OCH3

OH

H3CO

O

32 OCH3

OH

O

33

S

O OCH3

OH

O

34

O

O OCH3

OH

S

S

35 OCH3 a

OH b

Bovine microvascular endothelial cells. Colon carcinoma cells. cNon-small-cell lung carcinoma cells. dStandard deviation.


Table 3.

Marrelli et al.

Biological Activities of Benzyl Derivatives of CA-4 Cytotoxicity (IC50 nM) Compound

Tubulin polymerization

HCT116a

K562b

H1299c

MDA-MB231d

3000

-

-

-

-

NAe

-

-

-

-

NA

-

-

-

-

300

-

-

-

21

Ref.

(IC50 μ M)

O H3CO 36

O

H3CO OCH3

NC O H3CO 37

O

H3CO OCH3

CN

O H3CO 38

O

H3CO OCH3

OMe

O H3CO 39

O

H3CO OCH3

[82]

OMe

O H3CO

OMe

40

300

-

-

-

63

40

25

30

40

-

350

-

-

-

-

700

-

-

-

-

O

H3CO OCH3

OMe

O H3CO

OAc

41

O

H3CO OCH3

OMe

O H3CO

NHAc

42

O

H3CO OCH3

OMe

O H3CO

F

43

O

H3CO OCH3



3045

(Table 3). Contd….. Tubulin polymerization

Cytotoxicity (IC50 nM) Compound

HCT116a

K562b

H1299c

MDA-MB231d

2000

-

-

-

-

35

25

30

40

1.5

38

20

50

30

5.0

3000

-

-

-

-

NA

-

-

-

-

NA

-

-

-

-

1.8

3.6

5.0

Ref.

(IC50 μ M)

O H3CO

OH

44

O

H3CO OCH3

OMe

O H3CO

OH

45

O

H3CO OCH3

OMe

O H3CO

NH2

46

O

H3CO OCH3

O 47

H3CO O

H3CO OCH3

O

N

H3CO 48

O

H3CO OCH3

O 49

H3CO

N O

H3CO OCH3 CA-4

1 a

b

c

3.0 d

1.0 e

Human colon carcinoma cell line. Chronic mylogenous leukemia cells. Non-small lung human carcinoma cell line. Human breast cancer cells. Non-active.

effects of the compound by modification of the double bond or replacement of olefinic bridge with a ring. A series of various substituted benzil derivatives have been synthesized through the oxidation of diarylalkynes promoted by PdI2 [82]. The in vitro antiproliferative activity of the synthesized 1,2diketones was first determined against the human colon carcinoma cell line (HCT116). Compound 45, the most similar to combretastatin A-4, (Table 3) shows a high activity at the nanomolar level (35 nM). This result indicates that the introduction of a 1,2-dicarbonyl unit between the two aromatic rings is favorable for a good antiproliferative activity. Compound 41 and compound 46 related to AVE-8062 showed a similar interesting

antiproliferative activity, demonstrating that the amino and hydroxyl groups are bioequivalent at the C-3 position of these analogs. Compounds 41, 45 and 46, that displayed the better cytotoxicity against HCT116, were next evaluated against K562 (human chronic myelogenous leukemia cell line), H1299 (nonsmall lung human carcinoma) and MDA-MB231 (human breast cancer) cell lines, showing a strong cytotoxic activity (about 30 nM). Regards to the ability to inhibit tubulin assembly, compounds 45 and 46, which are most similar to CA-4 and AVE-8062, displayed potent antimitotic activities. The effects of compounds 45 and 46 on the cell cycle of HCT116 and H1299 cell lines were analyzed by flow cytometry, and a G2/M arrest was observed. The ability of compounds 45 and 46 to induce apoptosis was characterized using the standard caspase cleavage assays. It was


Marrelli et al.

observed after 24 h of treatment that 45 and 46 induced apoptosis in HT1299 and HCT116 cell lines. The obtained results show that treatment of cancer cells with compounds 45 and 46 activates caspases leading to cellular apoptosis in the same manner as that of CA-4. More recently, Tanpure et al. [83] synthesized a series of alkene analogs (50-59) based on combinations of the structural components of tamoxifen and CA-4. Tamoxifen (60) is a triarylethylene compound that has been widely used in the treatment of cancer. The mechanism of action of tamoxifen differs from that of CA-4, because it does not have a significant effect on tubulin polymerization (IC50 >40 μM) but is able to inhibit the estrogen receptor. The authors synthesized a series of tri- and tetraarylethylene compounds (50-59) by means of a McMurry coupling reaction, an important method for the synthesis of highlyfunctionalized alkenes already used for the synthesis of tamoxifen and its related compounds. Compounds 50-59, each containing trimethoxyphenyl and p-methoxy-m-hydroxyphenyl rings, mimic the structural core of tamoxifen and incorporate features of CA-4 and colchicines. These new analogs were evaluated for their ability to inhibit tubulin polymerization and for their antiproliferative activity against a panel of three human cancer cell lines (Table 4). Among triarylethylene analogs with R1 = phenyl (50-51-55-56), the Z isomers 55 and 56 showed a better activity in SK-OV-3 and NCIH460 human cancer cell lines. The opposite trend was observed for the triarylethylene analogs (53-54-58-59) in which R2 = phenyl. However, this second group of molecules was collectively more active than the previous one. Among these compounds, the triarylethylene 54 was the most active one and was also more cytotoxic than tamoxifen against DU-145, SK-OV-3 and NCI-H460 cancer cell lines. None of the compounds inhibited tubulin assembly. Ducki and coworkers synthesized and evaluated various chalcone analogs for their cell growth inhibitory properties [84]. Table 4.

Chalcones are known to possess a range of interesting biological activity and some have been investigated for their anticancer properties. The growth inhibitory properties of new chalcones were determined in vitro using the MTT assay. The drugs were screened against the K562 human chronic myelogenous leukemia cell line. Results obtained from the compounds tested revealed that the introduction of 3,4,5-trimethoxy A-ring in the chalcones leads to markedly high cytotoxicity. The most cytotoxic analogs in the series were those chalcones which resembled CA-4 the most (results showed in Table 5). The chalcone 61 bearing both the Aand B-rings of CA4 was the most potent. The analog 62 resembling 3-deoxy-amino CA-4 is also significantly potent. Incorporation of an alkyl group at the -position leads to an increase in cytotoxic activity. The cytotoxic activity of 63 is 10-fold greater than 61. Cell cycle analysis by flow cytometry indicated that these agents are antimitotic. This study demonstrated that chalcones that exhibited the greatest cell growth inhibitory properties, such as 61, block the cell cycle in the G2/M phase most effectively. As regards the effects of the selected chalcones on in vitro assembly of tubulin, the arrangement of 3,4,5-trimethoxy groups on the ring-A and hydroxyl group at 3'-position on the ring-B, as found in compounds 61 and 63, are clearly advantageous in the ability of these chalcones to inhibit tubulin assembly and illustrate their overriding importance in binding to tubulin. In the [3H]colchicine competition assay compounds 61 and 63 strongly displaced colchicine from its binding site on tubulin. More recently, Kong et al. [85] designed and synthesized boronic acid analogs of chalcones in an effort to compare their biological activities with combretastatin A-4. They designed the chalcone analogs 64-67 as carbonyl-expanded analogs of CA-4. Obtained data showed that chalcone 65 was the most potent inhibitor, with activity comparable to that of CA-4 in the tubulin assay (Table 6). However, 65 was less active than CA-4 as an

Biological Activity of Tri- and Tetra-Arylethylene Analogs

R1

R2

Inhibition of tubulin polymerization IC50 (μ M)

50

Ph

Me

51

Ph

52

Ph

53

Me

54

Compound

OCH3 H3CO

R2 n-Bu

H3CO R1

OCH3 R1

H3CO

R2

H3CO OCH3

OH

Citotoxicity IC50 (μM) DU-145a

SK-OV-3b

NCI-H460c

>40

28.0

24.1

23.4

Et

>40

20.9

27.4

34.1

Ph

>40

21.9

13.8

37.2

Ph

>40

4.25

2.72

5.37

Et

Ph

>40

2.58

0.576

3.41

55

Ph

Me

>40

24.3

8.44

6.54

56

Ph

Et

>40

18.8

17.1

13.3

57

Ph

Ph

>40

19.9

18.9

33.0

58

Me

Ph

>40

16.9

4.35

10.0 5.77

59

Et

Ph

>40

13.5

3.79

>40

6.07

6.40

OCH3

N

O 60

a

Tamoxifen

Prostate cancer cell line. bOvarian cancer cell line. cLung carcinoma cell line.

4.48

Ref.

[83]


Table 5.


3047

Biological Activity of Chalcone Analogs 61, 62 and 63

Compound

K562a IC50 (μM)

IC50 (μM)

G0-G1

S

G2/M

% [3H] Colchicine bound

0.0043

0.62

12

12

76

9

0.01

2.0

48

33

19

ndb

0.00021

8

7

83

0.46

8

Flow cytometry

Tubulin

Ref.

O H3CO

OH

H3CO

OCH3

61 OCH3

O H3CO

NH2

62 H3CO

[84]

OCH3 OCH3

O H3CO

CH3

63

OH

H3CO OCH3 a

OCH3 b

Human chronic myelogenous leukemia cell line. nd, not determined.

inhibitor of MCF-7 cell growth and in disrupting the microtubules of A-10 cells (established aortic smooth muscle cultured cell line). Boronic acid chalcone analog 64 was weakly active as inhibitor of colchicine binding and of tubulin polymerization, but nevertheless had significant cytotoxic activity. SAR analysis determined that the placement of the carbonyl adjacent to the A-ring results in more active analogs than its placement adjacent to the B-ring. Boronic acid analog 64 was evaluated at the National Cancer Institute against a panel of 54 human cancer cell lines in order to determine activity against a broad range of human cancer. This molecule showed inhibition towards 16 of these cancer cell lines in the 10200 nM range, and other three cell lines with GI50 values below 10 nM. Furthermore, this compound had significant anti-angiogenic effects demonstrated by HUVEC tube formation and aortic ring assay. This study showed that positional addition of boronic acid eliminated tubulin activity, but produced nanomolar cytotoxicity.

substitution into the double bond site in C position (69) led to a higher inhibition of tubulin polymerization compared to that produced by one substitution in C (71) or in both positions (68). The substitution of a fluorine with a bromine atom (73) determined a loss of activity compared to difluoro analog 68. The introduction of a NO2 group at the 3-position of the B-ring generated compounds 74, 75 and 79, which displayed a dramatic loss of antiproliferative activity compared to previous molecules. The presence of a fluorine atom in the C position of derivative 77 determined an improved effect on tubulin aggregation.

With the aim of understanding the influence of fluorine on the double bond of the cis-stilbene moiety of combretastatin derivatives, Alloatti and coworkers [86] prepared a series of new analogs in which one or both of the olefinic hydrogens were replaced with fluorine.

Regards to compound 81 and its fluorinated derivatives, 82, 84 and 86, once again, the fluorination of the C into the olefin site (84) incremented the inhibition of tubulin polymerization, in comparison with the other fluorinated analogs 82 and 86. On the contrary, the antiproliferative potency of these molecules was similar to that of the reference compound 81. Therefore, according to the observations reported in literature, obtained results showed a nonlinear relationship between proliferation inhibition and the effect on tubulin polymerization by compounds bearing an amino group in position 3.

The introduction of fluorine atoms into molecules proved to be favorable for biological activity. Electron withdrawal by fluorine results in a strong polarization of the C-F bond, and the resultant pronounced electronic effects can help drug-target interactions. Moreover, the presence of fluorine increases the lipophilicity of drugs, enhancing absorption into biological membranes and facilitating the docking of drugs with their receptors.

Some bridge modified analogs of CA-4 were also prepared by Kerr and coworkers [12]. The authors first synthesized a series of aryl- and aroyl-substituted analogs of CA-4, using a one-pot palladium-mediated hydrostannylation-coupling reaction sequence. These molecules were converted to indanones by Nazarov cyclization, and finally oxidated to give the corresponding indenones.

In order to evaluate the influence of a different halogen, the authors also synthesized a monobromo monofluoro analog, some fluorinated analogs of amino and nitro combretastatins, and a new class of derivatives in which the B-ring is replaced with benzofuran and benzothiophene heterocycles. The fluorinated analogs, as well as reference compounds, were screened for their antiproliferative activity on bovine microvascular endothelial cells (BMEC), and all cis-stereoisomers were at least three times more active than their trans-form (Table 7). The cis-isomers were further evaluated for their ability to inhibit tubulin polymerization and for their antiproliferative activity on NCI-H460 non-small cell lung carcinoma and on HT29 colon carcinoma cells. Fluorine

The biological activities of synthesized compounds are summarized in Table 8. Only compound 90 showed an activity similar to that of CA-4 on tubulin polymerization. All the new analogs did not inhibit growth of the MCF-7 cells at the highest concentration tested (1 μM). Results obtained by Kerr showed that among realized compound, those that lacked a five-membered cycle on the bridge, 90 and 91, retained activity as a tubulin polymerization inhibitor, but had no antiproliferative activity. The aim of the researcher is to find molecules that may affect cell proliferation at doses that do not cause the disruption of other cellular cytoskeletal functions. Therefore, compounds such as 90, that inhibited tubulin polymerization at low concentrations, but that


Table 6.

Marrelli et al.

Biological Activity of Boronic Acid Chalcone Analogs Tubulina Compound

[3H]Colchicine binding inhibitionc (% ± SD)

MCF-7b

IC50 (μM ± SD)

IC50 (μ M ± SD)

31 ± 3.4

2.6 ± 0.2

A-10d EC50 (μM)

5 μM

50 μM

0.9 ± 0.18

4.4 ± 8.6

56 ± 8.6

16.5

0.11 ± 0.024

88 ± 6.3

101 ± 5.5

0.98

Ref.

O H3CO

64 H3CO

B(OH)2 OCH3

OCH3

O H3CO 65

H3CO

OH OCH3

OCH3 O

[85]

H3CO

>40

>2.5

-

-

>15

>40

>2.5

-

-

18

2.0 ± 0.1

0.032 ± 0.021

99 ± 2.0

-

200

-

-

NO2

H, H

6.8 ± 0.1

135 ± 1.3

128 ± 0.6

113 ± 1

Z

NO2

F, F

8.2 ± 0.2

749 ± 75

1166 ± 75

1307 ± 26

E

NO2

F, F

-

2300 ± 53

-

-

77

Z

NO2

F, H

2.3 ± 0.3

38.4 ± 2.5

42.6 ± 6

32 ± 6.5

78

E

NO2

F, H

-

2800 ± 72

-

-

79

Z

NO2

H, F

10.9 ± 1.5

329 ± 2.5

379 ± 0.2

202 ±19

80

E

NO2

H, F

-

>3000

-

-

Ref.

[86]



3049

(Table 7). Contd…..

a

Inhibition of tubulin polymerization (IC50 μM)

BMEC

H, H

3.5 ± 0.2

3.1 ± 0.4

4.9 ± 0.1

5 ± 0.5

F, F

17.5 ± 1.4

3.6 ± 0.2

7.3 ± 0.9

4.5 ± 0.1

F, F

-

204 ± 5.4

-

-

F, H

5.5 ± 0.1

2.0 ± 0.8

2.4 ± 0.2

2.9 ± 0.02

Cytotoxicity

Double bond geometry

X

81

Z

NH2

82

Z

NH2

83

E

NH2

84

Z

NH2

85

E

NH2

F, H

-

86 ± 1.3

-

-

86

Z

NH2

H, F

13.7 ± 0.6

2.1 ± 0.2

11.5 ± 0.9

5 ± 0.3

87

E

NH2

H, F

-

111 ± 15

-

-

88

Z

F

H, F

-

23 ± 1.3-

-

-

89

E

F

H, F

-

610 ± 15

-

-

CA-4 (1)

Z

OH

H,H

4.9 ± 0.2

2.6 ± 0.21

1.24 ± 0.2

118 ± 0.03

Compound

R 1, R 2

a

NCI-H460b

HT29c

Ref.

Bovine microvascular endothelium. bLung carcinoma cell line. cColon carcinoma cell line.

Table 8.

Biological Activities of Bridge Modified Analogs Cytotoxicity (IC50 μM ± SD a)

Compound

MCF-7b

Inhibition of tubulin polymerization

Ref.

(IC50 μ M ± SD)c

OCH3 H3CO

OCH3

H3CO

90

2.5 ± 0.1

> 1.0

6.6 ± 3.0

> 1.0

O

H3CO OCH3 H3CO

OCH3

H3CO

91 O

OCH3

H3CO

OCH3

O

[12]

OCH3

H3CO

92

O

H3CO

20 ± 1

ND d

36 ± 1

ND

2.0 ± 0.3

0.026 ± 0.008

OCH3

OCH3

O

OH

H3CO OCH3 HO

93

O H3CO HO

1 a

OCH3

CA-4

Standard deviation. bBreast cancer cell line. cGI50 value, compound concentration at which the increase in cell protein is 50% of the increase in untreated control cells. dNot done.


Table 9.

Marrelli et al.

Antiproliferative Activity Against L1210 Leukemia Cell Line of -Lactam Analogs Cytotoxicity (IC50 μM)

Compound

L1210a

Ref.

O Cl

N

CH3

94

2.8

H3CO

OCH3 OCH3 [3]

O Cl

N 0.91

95

H3CO

OCH3 OCH3

1 a

CA-4

6.5 x 10-4

Mouse leukemia cell line.

did not show antimitotic or cytotoxic effects, may be a potential selective vascular disrupting agents. In order to lock the cis-configuration between the aromatic systems and to avoid the isomerization to E-forms, Yang et al. synthesized a new series of -lactam analogs of combretastatin [3], in which the olefinic bond was replaced by a four-membered lactam ring. Only compound 95 showed a moderate cytotoxicity against L1210 mouse leukemia cell line (Table 9). Compound 94 was three times less active than compound 95. These new analogs did not show anti-tumor activity. This study provided some rational basis for further structural modification, but demonstrated that the presence of a lactam ring did not significantly influence the cytotoxic effects. The -lactam analogs were inactive and independent of ring modification. More recently, Ty and coworkers [87] realized new CA-4 analogs in which the cis-olefinic bridge was replaced by a cyclopropyl-vinyl moiety or by a cyclopropyl-amide moiety. The authors realized a series of configurationally cis-locked derivatives characterized by a (cis,E)-cyclopropyl-vinyl unit and a series of cyclopropyl derivatives for which the E-double bond was replaced by an amide group. These new analogs were evaluated for their ability to inhibit tubulin polymerization and their antiproliferative activities against HCT-16 (colon carcinoma cell line) and MCF-7 (breast carcinoma cell line). Against the MCF-7 cell line, only derivative 96 displayed a modest activity, with an IC50 value of 6.49 μM (Table 10). The molecule 96 was the only cis-cyclopropyl-vinyl compound that exhibited some inhibitory activity against tubulin assembly. The replacement of the E-double bond of compound 96 by an amide group (98) caused the loss of inhibition on tubulin polymerization, but did not affect cytotoxicity. These results demonstrated that the amide moiety is not a good substitute for the trans double bond of vinylogous CA-4 analogs. Probably the electronic nature and not the geometry of the amide linkage is unsuitable for direct tubulin binding. 4. CA-4 Analogs Containing Heterocyclic Rings Combretastatin A-4 is active in its cis conformation. Heat, light and proton media may favor isomerization of cis molecule into

inactive trans form. In order to avoid this phenomenon, many authors synthesized new analogs in which the olefinic bridge was replaced by heterocycle moieties. Some of these new derivatives, maintaining the correct orientation of the two polyoxygenate aromatic rings, showed a biological activity higher than that of the reference compound CA-4. These heterocycles, if properly suited into the binding pocket, might also influence the specificity and the pharmacodynamic properties of compounds [88]. a. Combretafurans Some rigid analogs of combretastatin bearing a furan ring in place of the olefinic bridge were synthesized by Pirali and coworkers [89]. The authors investigated whether furan could mimic the olefinic bridge of CA-4 and synthesized the new combretafurans through an intramolecular aldolic condensation. These molecules were functionalized at 2 and 5 positions through electrophilic aromatic substitution. This new class of analogs showed high biological activity. Combretafuran 104, which is similar to combretastatin except for the absence of the hydroxyl group on ring B, displayed a good antiproliferative activity against the neuroblastoma cell line SH-SY5Y, with an IC50 value of 39 ± 8.9 nM (Table 11). The analog 107, bearing a hydroxyl group, was the most potent (IC50 = 2.9 ± 0.33 nM). Compound 106 possessing an amino group displayed an IC50 value of 5.1 ± 0.7 nM, and the analog with a nitro group (105) showed an IC50 value of 244.54 μM. Compounds bearing a formyl group in position 2 (108) or 5 (109) of the furan ring were also cytotoxic, with IC50 values of 35.8 ± 20 nM and 231 ± 72 nM, respectively. The studies carried out by Pirali and coworkers demonstrated that realized combretafurans were active as cytotoxic agents and tubulin polymerization inhibitors. However, these molecules did not cause the G2/M block determined by combretastatin A-4, thereby suggesting a distinct mechanism of action. b. Combretatriazoles Cafici et al. synthesized a series of cis-locked combretastatins containing a triazole ring (combretatriazoles) [88]. Many of these compounds exerted potent cytotoxic effects on the neuroblastoma cell line SHSY-5Y (Table 12). Three of these analogs, 112, 117 and


Table 10.


3051

Biological Activity of cis-Locked Vinylogous Combretastatin-A4 Analogs

H3CO

Citotoxicity IC50 (μM)

X

R

Inhibition of tubulin polymerization (μM)

HCT116a

MCF-7b

96 (cis,E)

-

-

5.3

0.53

6.49

97 (cis,Z)

-

-

38

nac

na

98 (cis)

CH

H

>100

0.68

na

99 (trans)

CH

H

92

3.35

na

100 (cis)

C-OH

H

>100

na

na

101 (cis)

C-OH

OCH3

82

na

na na

Compound

Ref.

Ph

H3CO OCH3

H N

H3CO

X

O

H3CO

R

OCH3

102 (cis)

N

H

>100

2.37

103 (trans)

N

H

>100

na

na

1

-

-

0.79

0.003

ndd

CA-4 a

[87]

Colon carcinoma cell line. bBreast carcinoma cell line. cNa, not active. dNd, not determined.

Table 11.

Citotoxicity of Combretafuran Analogs on SH-SY5Y Cell Line Cytotoxicity (IC50 nM)

Compound

SH-SY5Ya

Ref.

O H3CO 104

39 ± 8.9

H3CO H

OCH3 OCH3 O H3CO 105

244.54 ± 150

H3CO NO2

OCH3 OCH3

[89]

O H3CO 106

5.1 ± 0.7

H3CO NH2

OCH3 OCH3 O H3CO 107

2.9 ± 0.33

H3CO OH

OCH3 OCH3


Marrelli et al.

(Table 11). Contd….. Cytotoxicity (IC50 nM)

Compound

SH-SY5Ya

OHC

Ref.

O

H3CO 35.8 ± 20

108

H3CO OCH3 OCH3 O CHO

H3CO 109

231 ± 72

H3CO OCH3 OCH3

O O

O

OH

H3CO N

110

NH

1670 ± 783

H3CO OCH3 OCH3 1 a

CA-4

1.6 ± 0.10

Neuroblastoma cell line.

121, displayed IC50 values of 4.7 nM, 1.9 nM and 12.2 nM, respectively. Combretastatin A-4 showed in this experiment an IC50 of 3.0 nM. The tubulin polymerization assays showed that these three analogs also possess the ability to bind tubulin and interfere with polymerization. The mechanism of action of the more active molecules was investigated by performing cell cycle analysis of cells treated with these compounds for 24 h at the IC50 value. Two of the obtained analogs, 117 and 121, did not induce the G2/M arrest typically determined by tubulin inhibitors. Studies realized by means of confocal microscopy showed for these molecules a new mechanism of action that induces cells to appear multinucleated and with a high number of mitotic spindles. A series of analogs with a locked cis-type bridge between the two phenyl rings have been also synthesized by Odlo [36], that realized a series of cis-restricted 1,5-disubstituted 1,2,3-triazole analogs of combretastatin A-4 in order to avoid isomerization. All prepared triazoles had four methoxy groups of the A- and B-rings and a two atom bridge with a cis-similar arrangement between the A- and B-rings. Compounds 125 and 126 revealed the best in vitro antiproliferative effects against K562 leukemia cancer cell line, that was assessed by the MTT test (Table 13). These compounds showed IC50 values of 0.41 μM and 0.57 μM, respectively. The triazoles 127-129 and 131 showed cytotoxic activity against leukemia cancer cell line K562 in the nano- to micromolar range; 127 and 131 being the most active ones with IC50 value = 27 and 11 nM. All prepared molecules were also evaluated for their ability to inhibit tubulin assembly and only 127 and 131, the most cytotoxic ones, showed inhibition of tubulin assembly with an IC50 value = 7.0 and 4.8 μM, respectively. The other compounds showed IC50 values >10 μM. Triazole 131 was tested on several other cancer cell lines and showed a good activity with IC50 values ranging from 3.9 nM (against WM35 cell line) to 5.1 nm (against WM239 cell line). Triazoles 127 and 131 were more active than their regioisomers 122

and 126, demonstrating that the position of the 1,2,3-triazole moiety is important for biological activity. c. Pyrazoline Analogs Pyrazoline analogs are an important class of CA-4 derivatives. These compounds were developed to address some limitations showed by other series of compounds. During the last years many enone-containing chalcones were synthesized, but these compounds can undergo Michael reactions with glutathione and other biological nucleophiles, and this can compromise their biological activities. Other derivatives, such as the pyrazole-containing analogs of CA-4 revealed a drop in potency due to their geometry. These molecules do not have the twisted geometry of CA-4, but are planar because of the aromaticity of the pyrazole. Consequently, the researchers decided to introduce non-aromatic groups, in order to obtain water soluble compounds but also to prevent the necessary twisted structures required to maintain activity. Because of the non-aromatic, polar pyrazoline moiety, the pyrazoline analogs of CA-4 may adopt the correct geometry for activity and biological solubility is also improved. Fourteen pyrazolines with various substituents on the A- and Brings were synthesized and assessed for biological activity by Johnson and coworkers [70]. The authors realized a series of nonacetylated 3,4,5-trimethoxypyrazolines (compounds 134-136), a group of N-acetylated 3,4,5-trimethoxypyrazolines (137-143) and a series of acetylated 2,5-dimethoxypyrazolines (144-146). The pyrazolines were obtained by reaction of the required chalcone with hydrazine hydrate. The acetylated compounds (137-143 and 144146) were synthesized by dissolving chalcones in acetic acid before adding hydrazine hydrate. Compounds bearing B-ring substituents showed the highest antiproliferative activity against L120 cells, being the most potent within both the non-acetylated and acetylated


Table 12.


Antiproliferative Effects of Combretatriazoles Against SHSY-5Y Cell Line Cytotoxicity (IC50 nM ± SD a) SHSY-5Yb

Compound

Ref.

N

N N 111

630 ± 70

OH H3CO

OCH3

N

N N 112

H3CO

4.7 ± 0.7

OH H3CO

OCH3

OCH3

N

N N 113

126 ± 38

OH F3CO

OCH3

N

N N 114

257 ± 250

OH O

O

OCH3 [88]

N N N OH

115

>1

OCH3

O

N N N OH

116

H3CO

192 ± 23

OCH3

OCH3 N

N N 117

H3CO

1.9 ± 2.0

OH OCH3 N

OCH3

N

N 118

72 ± 6

OH H3CO

OH

OCH3

3053


Marrelli et al.

(Table 12). Contd….. Cytotoxicity (IC50 nM ± SD a) SHSY-5Yb

Compound

N

Ref.

N

N 119

366 ± 80

OH H3CO

F

OCH3

N

OH

N N OCH3

120

41 ± 22

N N N 121

12.2 ± 1.2

OH OCH3 CA-4

1 a

3.0 ± 0.3

b

Standard deviation. Neuroblastoma cell line.

Table 13.

Citotoxicity and Inhibition of Tubulin Polymerization by 1,5-disubstituted 1,2,3-triazole Cytotoxicity (IC50 μ M)

Compound

Tubulinh (IC50 μ M)

K562a

MDAMB231b

SK-BR 3c

SKOVd

OVCARe

WM35f

WM239g

4.00

n.di

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

3.60

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

Ref.

N N

MeO 122

N

MeO OMe OMe N N

MeO

123

N

MeO Br

OMe

[36]

OMe N N

MeO

124

N

MeO

>10

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.41

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

14.0

NO2

OMe OMe

N N

MeO

125

N

MeO OH

OMe OMe



3055

(Table 13). Contd….. Cytotoxicity (IC50 μ M)

Compound

Tubulinh (IC50 μ M)

K562a

MDAMB231b

SK-BR 3c

SKOVd

OVCARe

WM35f

WM239g

0.57

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

>20

0.027

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

7.0

0.057

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

>20

0.035

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

12.6

0.46

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

>20

0.011

>10

4.00

4.90

4.70

3.90

5.10

4.8

>10

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

1.30

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

>20

0.010

6.00

5.10

3.30

3.30

2.50

2.80

0.6

Ref.

N N

MeO

126

N

MeO NH2

OMe OMe

N

N MeO 127

N

MeO OMe OMe N MeO

128

N

N

MeO Br

OMe OMe N MeO

129

N

N

MeO OH

OMe OMe N MeO

130

N

N

MeO NO2

OMe OMe

N MeO

131

N

N

MeO NH2

OMe OMe N MeO

N

N

NO2 OMe

132 MeO OMe

N MeO

N

N

133

NH2 OMe

MeO OMe

1 a

CA-4

Leukemia cancer cell line. bBreast carcinoma cell line. cBreast carcinoma cell line. dBreast carcinoma cell line. e Breast carcinoma cell line. fHuman melanoma cell line. gHuman melanoma cell line. hInhibition of tubulin polymerization. iNot determined.


Table 14.

Marrelli et al.

Citotoxicity Data for Non-Acetylated 3,4,5-trimethoxypyrazolines (134-136), Acetylated 3,4,5-Trimethoxypyrazolines (137-143) and Acetylated 2,5-trimethoxypyrazolines (144-146) Cytotoxicity (IC50 μM)

Compound B16

134

NH

N

H3CO

a

L120b

Ref.

OH

OCH3

H3CO

2.1

0.5

44

42

56

24

>100

44

>100

>100

H3CO

135

NH

N

H3CO

OCH3 OCH3

H3CO H3CO N

H3CO 136

NH

OCH3 OCH3

H3CO OCH3

H3CO

COCH3 N

N

H3CO

OCH3

137

H3CO H3CO COCH3 N

H3CO

OCH3

N

138

H3CO

[70]

H3CO

OCH3 COCH3 N

H3CO

N

OCH3

139

>100

43

>100

52

51

42

35

30

H3CO H3CO

COCH3 N

N

H3CO

NO2

140

OCH3

H3CO H3CO

COCH3 H3CO

N

N

141

NO2

H3CO H3CO COCH3 H3CO

N

N

142

Cl

H3CO H3CO



3057

(Table 14). Contd….. Cytotoxicity (IC50 μM)

Compound

B16a

L120b

>100

>100

58

40

62

28

69

33

0.002

0.003

Ref.

COCH3 N

H3CO

N

OH

143

OCH3

H3CO H3CO COCH3 OCH3

N

N NO2

144

OCH3 H3CO COCH3 OCH3

N

N OCH3

145

OCH3 H3CO

COCH3 OCH3

N

N

OCH3

146

H3CO

OCH3

H3CO 1 a

CA-4 b

Murine melanoma cell line. Murine leukemia cell line.

pyrazolines groups (Table 14). The highest activity was showed by the non-acetylated compound 134, with IC50 value of 0.5 μM. This molecule was significantly more active than its acetylated counterpart 143, that revealed a large drop in activity. Considering the biological activity of all other pyrazoline compounds, the obtained results revealed that the acetyl group on the pyrazoline unit did not meet the desired goals. The activity against B16 cell line followed a similar trend. The most potent analog was 134 (IC50 = 2.1 μM) and, once again, the corresponding acetylated compound 143 was not active (IC50 value >100 μM). The N-acetylpyrazoline analogs bearing two methoxy groups at the para position of the Aring showed generally no activity and were comparable to the trimethoxy equivalents (137-143). These results suggested that the methoxy group on the A-ring is not essential for biological activity. In order to better understand the difference in activity, the microtubule disrupting properties of compounds 134 and 143 were examined. The effects of these molecules on microtubules were evaluated in A-10 cells. Compound 134 was very active, with an EC50 value of 7.1 μM. Contrastingly, only a low microtubule disrupting property was observed for compound 143. Results obtained by Johnson demonstrated that pyrazolines are good structural analogs of CA-4, characterized by a good biological activity and aqueous solubility, and they have the advantage of being readily accessible. Moreover, the presence of substituents similar to that of CA-4 on pyrazoline compounds enhances the biological activity, while an acetyl group at N1 of the pyrazoline structure reduces the activity. d. Thiazoles The cis configuration of double bond and the 3,4,5trimethoxyphenyl group are fundamental for the biological activity

of CA-4. The restricted rotation of rings A and B can be maintained by introducing suitable conformationally restricted heterocycle such as thiazole. A series of 4,5-disubstitute-1,2,3-thiadiazole compounds with 1,2,3-thiadiazole instead of the olefin group of CA-4, were synthesized by Wu and coworkers [90]. These new derivatives were evaluated for their antiproliferative activity against HL-60, HCT116 and HMEC-1 cell lines (human myeloid leukemia, human colon adenocarcinoma and human microvascular endothelial cell line, respectively). Obtained results were interesting, because many of tested compounds possessing the 3,4,5-trimethoxyphenyl at 4 position in 1,2,3-thiadiazole (148-151, 154 and 162) had good activities, while only one of the molecules bearing this group at 5 position (163) displayed activity (Table 15). Tubulin is the intracellular target of these new derivatives, since they all inhibited tubulin polymerization with activities similar to that of CA-4. Compound 148 and 150 were investigated for their influence on cell cycle, and realized experiments confirmed that they induced a dose-dependent increase of HMEC-1 cells in G2/M and a simultaneous decrease in S and G1 cells compared to control. These results suggested that this class of molecules arrest the cell cycle at G2/M phase selectively. According to the in vitro results, four analogs (154, 148, 151 and 162) were investigated for their in vivo antitumor effect. These compounds were tested on mice S180 sarcoma transplant model; results of the experimental therapeutic efficacy are presented in Table 16. After daily i.p. administration with different dosage for 5-7 days, tumors were excised and weighted. Tested compounds showed different anti-cancer efficacy, 148 and 151 being the most interesting.


Table 15.

Citotoxicity and Inhibitory Effect on Tubulin Polymerization of 4,5-disubstitute-1,2,3-thiadiazole Cytotoxicity (IC50 μM)

Compound

HL-60a

HCT116b

HMEC-1c


>1000

>1000

>1000

NDd

14.5 ± 6.3

25.0 ± 8.2

24.6 ± 14.9

0.86

50.2 ± 29.5

86.6 ± 2.3

73.9 ± 18.0

ND

60.9 ± 30.8

50.9 ± 7.5

75.8 ± 27.8

0.72

13.4 ± 1.1

30.2 ± 2.0

26.4 ± 18.5

2.13

ND

>1000

>1000

ND

23.6 ± 5.1

23.6 ± 1.7

ND

ND

Ref.

N

N

S

MeO 147

Marrelli et al.

MeO OMe NO2 N

N

S

MeO 148

MeO OMe OMe N N S

MeO

149

MeO OMe

OCH2OOCH2O-

N N S

MeO 150

MeO OMe

OMe OMe

N

S

MeO 151

N

MeO OMe

NO2 OMe

N N S

MeO 152

MeO OMe F N N S

MeO 153

MeO OMe Cl

[90]



3059


Compound

HCT116b

HMEC-1c


1.5 ± 0.1

3.0 ± 1.3

3.9 ± 2.8

0.7

537 ± 116

616 ± 291

>1000

ND

>1000

>1000

>1000

ND

>1000

>1000

>1000

ND

>1000

>1000

>1000

ND

>1000

>1000

>1000

ND

>1000

>1000

>1000

ND

N

N

S

MeO 154

HL-60a

MeO OMe

OH OMe N N

S MeO 155

MeO OMe OMe S MeO

156

N N

MeO OMe

OMe OMe N N

S MeO

157

MeO OCH2

OMe

O-

OCH2O-

N N

S MeO 158

MeO OMe F N N

S MeO 159

MeO OMe Cl S MeO

160

N N

MeO NO2

OMe OMe

Ref.


Marrelli et al.


Compound

HCT116b

HMEC-1c


>1000

>1000

>1000

ND

1.8 ± 1.3

3.7 ± 1.3

2.5 ± 0.1

0.68

40.9 ± 20.8

70.4 ± 10.4

41.0 ± 5.1

0.86

>1000

>1000

>1000

ND

1.9 ± 0.7

3.0 ± 1.2

3.5 ± 0.9

0.81

Ref.

N N

S MeO 161

HL-60a

MeO OMe NO2 N

S

MeO 162

N

MeO OMe

NH2 OMe

S MeO 163

N N

MeO OH

OMe OMe S MeO

N N

MeO NH2

OMe OMe 1 a

CA-4

Human myeloid leukemia cell line. bHuman colon adenocarcinoma cell line. cHuman microvascular endothelial cell line. dND, not determined.

Table 16.

Effects of Compounds 148, 151, 162 and 154 on the In Vivo Growth of Mice S-180 Sarcoma

Group

Dosage (mg/Kg/day)

Mice (n) Initial/end

Body weight (g) Initial/end

Tumor weight (g) x ± SD

Inhibition rate (%)

NS

_

20/20

17.4/26.6

1.79 ± 0.56

_

CA-4 (1)

40 x 4

10/8

17.0/22.6

0.64 ± 0.33

64.2

40 x 5

10/10

17.2/18.3

0.34 ± 0.28

81.0

10 x 7

10/10

17.0/23.6

1.25 ± 0.58

30.2

100 x 7

10/9

17.5/21.8

0.64 ± 0.45

64.2

20 x 1

10/0

17.2/_

_

_

10 x 1

10/0

17.3/_

_

_

20 x 4

10/6

17.1/19.5

0.84 ± 0.59

53.1

10 x 7

10/9

17.3/23.3

1.46 ± 0.66

18.4

Ref.

148 151

[90]

162

154

Liu and coworkers [91] recently synthesized a series of novel 3,4-diarylthiazolimines (165-167) and 3,4-diarylthiazoles (168179). These analogs were tested for their antiproliferative activity against human CEM leukemia cells. The compound with the B-ring possessing 3,4,5-trimethoxy systems and the A-ring with a 4methoxy substitution was more potent than its isomers characterized by an interchange of the substitution pattern (Table 17).

Compound 167 showed a cytotoxic activity nine-fold higher than that of compound 166, suggesting that the presence of the 3,4,5-trimethoxyphenyl group on the five-membered ring near the imine group is useful for biological activity. Additionally, compound 170 showed eight-fold more cytotoxicity than compound 168. This result indicated that the 3,4,5-trimethoxy phenyl near the carbonyl group of thiazol-2(3H)-one is more favorable for cytotoxicity.


Table 17.


3061

Citotoxicity of 3,4-diarylthiazolimines 165-167 and 3,4-diaryltiazolones 168-179 Cytotoxicity (IC50 μM)

Compound

CEMa

Bel-7402b

HepG2b

SMMC-7721b

MCF-7c

SW-1990d

HCT116e

>20

-

-

-

-

-

-

13.2

-

-

-

-

-

-

1.5

-

-

-

-

-

-

14.9

-

-

-

-

-

-

13.8

-

-

-

-

-

-

1.8

-

-

-

-

-

-

>20

-

-

-

-

-

-

Ref.

S NH N

165

H3CO

OCH3 S NH

H3CO

166

N

H3CO OCH3 OCH3

S NH N

167

H3CO OCH3 H3CO OCH3 S O

H3CO

168

N

H3CO OCH3 OCH3

Cl

S O

H3CO

N

169 H3CO OCH3 OCH3 S O N

170

H3CO OCH3 H3CO OCH3 Cl

S O N

171 H3CO OCH3 H3CO OCH3

[91]


Marrelli et al.


Compound

CEMa

Bel-7402b

HepG2b

SMMC-7721b

MCF-7c

SW-1990d

HCT116e

2.7

-

-

-

-

-

-

>20

-

-

-

-

-

-

10.1

-

-

-

-

-

-

16.2

-

-

-

-

-

-

8.2

-

-

-

-

-

-

7.5

-

-

-

-

-

-

0.24

3.3

4.5

7.4

4.4

20.3

3.4

0.12

23.4

96.0

118.2

52.5

56.7

85.1

0.002

1.72

0.006

0.29

0.03

0.25

0.006

S O

H3CO

172

N

H3CO NO2

OCH3 OCH3

Cl

S O

H3CO

N

173 H3CO NO2

OCH3 OCH3

S O

H3CO

174

N

H3CO NH2

OCH3 OCH3

Cl

S O

H3CO

N

175 H3CO NH2

OCH3 OCH3 S O

O2N

176

N

H3CO OCH3 H3CO OCH3

Cl

S O

O2N

N

177 H3CO OCH3 H3CO OCH3 S O

H2N

178

N

H3CO OCH3 H3CO OCH3

Cl

S O

H2N

N

179 H3CO OCH3 H3CO OCH3

1 a

CA-4 b

c

d

e

Human leukemia cells. Human liver cancer cells. Human breast cancer cell line. Human pancreatic cancer cell line. Human colon adenocarcinoma cell line.

Ref.


Table 18.


3063

Citotoxicity of 3-aroyl Indazoles Cytotoxicity (IC50 nM)

Compound

H460a

HT29b

PC3c

HeLa d

1

3.0

1.1

0.9

8

1.0

1.0

0.8

29

nde

nd

nd

14

nd

nd

nd

Ref.

O H3CO

N NH

180

CH3 H3CO OCH3 OCH3 O H3CO

N NH

181

CH2OH H3CO OCH3 OCH3 O H3CO

N NH

182

CHCH3OH (R) H3CO OCH3 OCH3 O H3CO

N NH

183

CHCH3OH (S) H3CO OCH3 OCH3

[92]

O H3CO

N NH

184

C(CH3)2OH H3CO

195

nd

nd

nd

14

nd

nd

nd

8

nd

nd

nd

3

1.7

3.0

2.3

OCH3 OCH3 O H3CO

N NH

185

CH2CH2OH H3CO OCH3 OCH3

O H3CO

N NH

186

CH2(CH2)2OH H3CO OCH3 OCH3

O H3CO

N NH

187

H H3CO OCH3 OCH3


Marrelli et al.

(Table 18). Contd….. Cytotoxicity (IC50 nM)

Compound

H460a

HT29b

PC3c

Helad

247

nd

nd

nd

13

nd

nd

nd

283

nd

nd

nd

16

nd

nd

nd

>1000

nd

nd

nd

10

>1000

3.2

2.7

Ref.

O H3CO

N NH

188

CH2NH2 H3CO OCH3 OCH3

O H3CO

N NH

189

OH

H3CO OCH3 OCH3

O H3CO

N NH

190

H3CO (CH2)3OH

OCH3 OCH3

O H3CO NH

191

H3CO OCH3 OCH3 O H3CO NH

192

CH3 H3CO OCH3 OCH3

1 a

CA-4

Human non-small lung cancer cell line. bHuman colorectal cancer cell line. cHuman prostate cancer cells. dHuman cervical cancer cells. eNot determined.

Compounds 178 and 179, possessing the electron-donating amino group, showed much stronger cytotoxicity compared to those of 170 and 171. This last result demonstrated that the introduction of the electron-donating amino group at the C-3 position of the 4methoxyphenyl ring influenced the activity of compounds bearing the 3,4,5-trimethoxy phenyl near the carbonyl group of thiazol2(3H)-one. On the other hand, the replacement of the 4-H of the five-member thiazol-2(3H)-one ring with a chlorine atom for 170171 and 172-173 resulted in a loss of activity. The analogs 178 and 179 were further evaluated against a panel of solid human cancer cell lines including human liver cancer cells Bel-7402, HepG2, SMMC-7221, human breast cancer cells MCF-7, human pancreatic cancer cells SW-1990 and human colon adenocarcinoma HCT116. However, both compounds showed the best activity against the non-solid human CEM cell line. Flow cytometric analysis was done for the compounds 178 and 179. Treatment of the HepG2 cells with 178 for 20 h induced a dose-dependent increase of cells at the G2/M and a coincident decrease of the S- and G1-phase cells, while compound 179 did not have effect. e. Aroyl Indazoles Among heterocyclic analogs of combretastatin A-4, 3-(3,4,5trimethoxylbenzoyl)indazoles are highly cytotoxic agents and

inhibitors of tubulin polymerization in vitro. A series of 3-aroyl indazoles were synthesized by Duan and coworkers [92] and a highly potent class of compounds has been identified, based on novel acetylene substitutions of an indazole core. These new analogs were first evaluated for their cytotoxicity toward H460 cells, a human non small lung cancer cell line (Table 18). The propyne derivative 180 proved to be the most potent compound (IC50 value = 1 nM). Linear substituted alcohols 181, 185 and 186 were significant active (IC50 values of 8, 14, and 8 nM, respectively). The tertiary alcohol 184 was found to be much less active (IC50 value = 195 nM). Amino substitution was less interesting, with compound 188 being about 15 times less active than the corresponding alcohol analog 181. The cisallyl alcohol derivative 189 had comparable activity to the propargyl alcohol 181 (IC50 values of 13 nM and 8 nM, respectively), while 190, the completely hydrogenated propanol, was much less active (IC50 value = 283 nM). Three of most potent compounds 180, 181 and 187 were also tested against PC-3, HT29 and HeLa cell lines (human prostate cancer cells, human colorectal cancer cells, and human cervical cancer cells, respectively). Compounds showed analogous activity against these lines. These three lead compounds


Table 19.


3065

Biological Activities of Disubstituted 3-aroylindoles

Compound

ITPa (ratio IC50 compound over IC50 colchicine)

Cytotoxicity (IC50 μM) B16b

Morphological activity on endothelial cells (μM)

31

12.18 ± 5.06

17.49

1.3

0.24 ± 0.14

0.047

12

5.07 ± 0.91

8.74

18

3.14 ± 2.17

1.30

15

3.29 ± 1.15

10.86

-

32.42 ± 2.24

28.71

Ref.

OCH3 H3CO

193

O

H3CO HO

NH H3CO OCH3 H3CO O

H3CO

194 NH H3CO

OH

OCH3 MeO

195

O

MeO H3CO

NH HO OMe MeO

196

O

MeO H3CO

N SO2Ph

MsO

OMe MeO

197

O

MeO MsO

N SO2Ph

H3CO OMe MeO

198

MeO

O

H3CO NH MsO

[93]


Marrelli et al.

(Table 19). Contd….. Compound

ITPa (ratio IC50 compound over IC50 colchicine)

Cytotoxicity (IC50 μM) B16b

Morphological activity on endothelial cells (μM)

-

13.67 ± 1.40

14.35

Ref.

OMe MeO O

MeO 199

MsO NH H3CO 24

Colchicine

1.0

0.031 ± 0.003

0.003

1

CA-4

0.7

0.003 ± 0.001

0.008

a Inhibition of tubulin polymerization (ITP) is expressed as the ratio of the IC50 value of a synthesized compound over the IC50 value of colchicine, used as the reference. bMelanoma cell line . cMorphological activity (rounding up) on modified HUVEC cells (EA.hy 926) is expressed as the lowest concentration at which cell rounding up was observed after a 2 h incubation period with the test compound.

were also tested against cell lines possessing the drug resistance efflux pumps MDR-1 (P-glycoprotein, ABCB1) and MRP-1 (ABCC1). All three compounds exhibited potent antiproliferative activities, with the IC50 values ranging from 1.6 nM to 3.9 nM. These results established that 180, 181 and 187 were not substrates for the MDR-1 and MRP-1 pumps. The authors also investigated the ability of compound 181 to compete with [3H]colchicine binding to purified tubulin and demonstrated that this molecule was able to compete with [3H]-colchicine binding to purified tubulin from Hela cells and MCF-7 cells with IC50 values of 46 nM and 240 nM, respectively. The effects of 181 on microtubule formation in vitro were also evaluated, and it was observed that this compound inhibited tubulin polymerization in a concentration-dependent manner with an IC50 value of 6 μM. f. Aroylindole Ty and Dupeyre described the synthesis of several new disubstituted 3-aroylindole along with their biological evaluation as potential antivascular agents [93]. Compound 194, with a hydroxyl group at the 7-position of the indole nucleus that mimics the hydroxyl group at the 3-position of the B-ring of CA-4, showed a high cytotoxic activity against B16 melanoma cells and also proved to be a potent inhibitor of tubulin polymerization (Table 19). In addition, the authors assessed that compound 194 was able to induce marked morphological changes (rounding up) at nanomolar concentrations on endothelial cells (EA.hy 926 cells), effect which is considered indicative of potential antivascular activity. Moreover, in order to better understand the structure-activity relationships for this series of compounds, Ty and coworkers synthesized and evaluated for their biological activities two other positional isomers (194 and 195). The hydroxylsubstituted analog 194 was more active than the 5-hydroxysubstituted analog 193. g. Oxazoles A series of 4,5-diarylisoxazoles related to combretastatin A-4 were synthesized by Sun and coworkers, that utilized isoxazoles ring to mimic the cis double bond in CA-4 [94]. According to previously described structure-activity relationships known for CA4, the authors chose the 3,4,5-trimethoxyphenyl moiety which is essential for strong cytotoxicity, and the 4-methoxyphenyl or 3hydroxy-4-methoxyphenyl as two aromatic rings, and synthesized the two isomers 200 and 202 (Table 20). Assay results showed that these molecules were active against human cervical epithelioid carcinoma (HeLa), human hepatocellular carcinoma (HepG2) and human ovarian adenocarcinoma cell line (OVCAR-3), being 202 the most potent.

The authors also synthesized the isomers 204 and 205, and evaluated these for their biological cytotoxicity. These results underlined that the 3,4,5-trimethoxyphenyl group near the oxygen atom of isoxazole ring is significant for cytotoxicity. The activity of 202 and 205 was greater than that of 200 and 204, respectively, which indicated that the presence of the hydroxyl group is also important. Among the other analogs prepared, 203 displayed excellent antiproliferative activity and showed better cytotoxicity than CA-4 in HeLa and HepG2 cell lines (IC50 value of 0.022 and 0.065 nM, respectively). The authors assessed by means of flow cytometry that compounds 202 and 203 were able to cause significant arrest of the cells at the G2/M phase in HepG2 human cancer cells, consistent with the behavior of tubulin-binding agents. Compound 202 was recently described as a new inhibitor of tubulin polymerization [41]. Recently, three new oxazole-bridged CA-4 derivatives possessing additional functional groups at the B-ring were also synthesized [95]. These new analogs showed antiproliferative activity against HCT-116 cells at submicromolar IC50 values (Table 21). The potency of thioanisole 207 in combretastatin-refractory HT-29 cells appeared to be related to significant upregulation of p21cip1/waf1 and to induced S/G2 cell-cycle arrest. h. Imidazoles Bellina and coworkers described the synthesis of diarylsubstituted five-membered heterocycles, realized as Z-restricted analogs of combretastatin A-4. The authors developed selective and efficient procedures for the synthesis of 1,5- and 1,2-diaryl-1Himidazoles [96]. The cytotoxic activity of these new compounds (209--217) were tested against 60 tumor cell lines in a screening panel at the US NCI. The results are reported in Table 22, in which activities against the entire NCI cell line panel are reported as the mean Log molar drug concentration (MG_MID Log) for all human cancer cell lines. Among the examined compounds, 209 proved to be the most potent imidazole derivative. Compounds 209 and 210 were more cytotoxic than combretastatin A-4. These activities demonstrated that, as previously reported, the 3-fluoro-4methoxyphenyl and the 2-naphthyl moieties may replace ring B in CA-4. Regards to the structure-activity relationships, it could be underlined also that the 1,5-diaryl-1H-imidazoles 210 e and 211 showed cytotoxic activities higher than those of the corresponding 1,2-diaryl-1H-imidazoles 214 and 216, respectively. Moreover, compounds 210 and 214 bearing a 2-naphthyl substituent were more cytotoxic than analogs 211 and 216, possessing a 4methoxyphenyl moiety. The activity of these molecules was tested in vivo on MDMBA-435 breast cancer cells xenotransplanted in immunodeficient mice. These last experiments confirmed that 211


Table 20.


3067

Citotoxicity and Inhibitory Effect on Tubulin Polymerization of 4,5-disubstitute-1,2,3-thiadiazole Cytotoxicity (IC50 nM)

Compound

O

HeLaa

HepG2b

OVCAR-3c

4.69

4.40

10.3

11.6

13.7

9.6

0.90

1.43

0.53

Ref.

N

MeO 200

MeO OMe OMe O

N

MeO

201

MeO NO2

OMe OMe

O

N

MeO

202

MeO OH

OMe OMe O

[94]

N

MeO

203

MeO

0.022

0.065

0.135

299

396

223

29.4

33.9

14.8

2.75

0.14

0.01

NH2

OMe OMe O

204

N

MeO OMe MeO OMe

O

N

HO

205

MeO OMe MeO OMe

1 a

CA-4

Human cervical epitheloid carcinoma. bHuman hepatocellular carcinoma. chuman ovarian adenocarcinoma cell line.

was able to inhibit the tumor growth at a 150 mg/kg/day dose (52% inhibition after 25 days). In order to investigate the structural basis of the biological activity of these new analogs, the authors analyzed the interactions of 1,5- and 1,2-diaryl-1H-imidazoles with the colchicine binding site of -tubulin. This study revealed that some of the imidazoles were characterized by high total interaction energies with the colchicine binding site. For compound 209 this interaction energy was higher than that of CA-4. These experiments also showed a good linear correlation (R2 = 0.95) between calculated interaction energies of imidazoles with the colchicine binding site of tubulin and the biological activity exerted by these new analogs. i. Thioxopyrimidine Analogs Among the heterocyclic derivatives of combretastatin A-4, some 1,2,3,4-tetrahydro-2-thioxopyrimidine analogs were also

recently synthesized [69]. These new derivatives can be divided into two groups: compounds 218-226, bearing a 3,4,5trimethoxyphenyl moiety (type I), and molecules 227-228, containing a 2,5-dimethoxyphenyl group (type II). Compounds were tested against L1210, murine leukemia cell line and B16 murine melanoma cell line. Obtained results showed interesting structure-activity relationships (Table 23). Three of the analogs (219-223 and 227) displayed significant activity against both cell lines and, as a matter of fact, it was reasonable to expect compound 223 of the type I design to be active, since it has a substitution pattern similar to CA-4. Surprisingly, compound 227, a type II molecule which has a completely different substitution pattern from CA-4, was as potent as 223. The analysis of molecular models showed that the conformation of molecule 227 is twisted due to steric hindrance between ortho-methoxy groups on both phenyl groups, explaining


Table 21.

Marrelli et al.

Biological Activity of Boronic acid Chalcone Analogs Cytotoxicity (IC50 μM)

Compound

HCT-116 (wt)a

HCT-116 (P53_/_)b

HT-29c

0.02 ± 0.01

-

>10

0.01 ± 0.003

0.02 ± 0.004

0.63 ± 0.32

0.1 ± 0.001

0.27 ± 0.01

>10

Ref.

OMe OMe N

206

OMe

O

OMe HO OMe OMe N

OMe

207

[95]

O

SMe OMe OMe N

OMe

208 O HO OMe HO a

Wild-type carcinoma HCT-116. bp53-deficient carcinoma HCT-116. c Colon carcinoma cell line.

Table 22.

Citotoxicity of Imidazole 209-212 and 213-217 Against the NCI 60 Human Cell Lines Screening Panel Cytotoxicity (GI50 μM)

Compound

HCT-15

a

NCI-H460

MG_MID Log b

Ref.

GI50

TGI

N F

209

N

H3CO

100

6.0

0.5

>100

>100

Ref.

S HN

218

NH

H3CO

OCH3

H3CO

OCH3 OCH3

OCH3 S

HN

219

NH

OCH3

H3CO H3CO

H3CO

OCH3

OCH3 S HN

220

NH

H3CO

OCH3

H3CO

OCH3 OCH3 S HN

221

NH

OCH3

H3CO H3CO OCH3

OCH3

S HN

222

NH

H3CO

OCH3

H3CO OCH3 S HN

223

NH

H3CO

OH

H3CO

OCH3 OCH3 S HN

224

H3CO

NH NO2

H3CO

OCH3 OCH3

[69]



3071


Compound

B16a

L1210b

>100

>100

55.5

48.3

6.1

0.4

42

2.2

Ref.

S HN

225

NH

H3CO H3CO

NO2 OCH3 S HN

226

NH

H3CO H3CO

Cl OCH3 S

OCH3

HN

NH

OCH3

227 H3CO

OCH3

OCH3

S OCH3

HN

NH NO2

228

OCH3 OCH3 a

Murine melanoma cell line. bMurine leukemia cell line.

A-10 aortic smooth muscle cells. CA-4 was still significantly more potent in disrupting microtubules under similar conditions, with an EC50 value of 0.007 μM. Among tested compounds 223 and 227 showed the best activity, with an EC50 value of 4.4 μM and 2.9 μM, respectively. The EC50 values were comparable to the cytotoxicity IC50 values. This observation suggested that microtubule disruption might be the mechanism of action through which molecules may exert their effects. j. Quinolines Quinolines are another interesting class of heterocyclic compounds acting as microtubule inhibitors. Recently, a new series of aroylquinoline derivatives obtained by coupling the quinoline core with the 3,4,5-trimethoxybenzoyl group were synthesized and evaluated for anticancer activity [97]. Compounds 229-241 were tested against KB (oral epidermoid carcinoma cells), H460 (nonsmall-cell lung carcinoma cells), HT29 (colorectal carcinoma cells) and MKN45 cells (stomach carcinoma). These new analogs were also evaluated against the MDR-positive cancer cell line KB-vin10 (Table 24). Regards to the position effect of the aroyl group in the quinoline system, obtained results showed that the 2-aroylquinoline 229 and the 6-aroylquinoline 233, bearing the 3,4,5trimethoxybenzoyl group at the C-2 and C-6 position on quinoline ring, showed a good activity (mean IC50 values = 172.8 and 24.4 nM against five cancer cell lines, respectively). Shifting of the aroyl group to the C-3, C-4, C-5, or C-8 position decreased activity, while shifting to the C-7 position, as in compound 234, caused a loss of cytotoxicity. The authors also evaluated the effects of the addition of the methoxy group in aroylquinolines. The 6-methoxy-2-

aroylquinoline 236 and the 8-methoxy-4-aroylquinolime 237, bearing a methoxy group at the C-6 and C-8 position, were more active than the 2-aroylquinoline 229 and the 4-aroylquinoline 231. On the contrary, compound 238, with a methoxy group at the C-2 position in the 6-aroylquinoline system, was less active than its parental compound 233. Regards to the inhibition of microtubule assembly, compounds 233, 236, and 239 were active, with IC50 values of 2.9, 3.5 and 1.6 μM, respectively. The last molecule, 239, showed more potent antitubulin activity as compared to combretastatin A-4 (IC50 = 2.1 μM) and colchicine (IC50 = 4.2 μM). The authors also realized a [3H]-colchicine binding assay, and obtained results indicated that the 5-amino-6-methoxy-(3’,4’,5’trimethoxybenzoyl)quinoline (239) was strongly bound to the colchicine-binding domain of tubulin. k. Other Analogs Incorporating a Ring Between the Cis-Double Bond and One of the Aromatic Substituent In view of reducing the conformational flexibility associated with two phenyl rings of CA-4, some other derivatives incorporating another ring between the cis-double bond and one of the aromatic substituent were considered by Arthuis [98], that realized a series of (E)-3-arylmethyleneoxindoles and a series of (E,E)-3-alkylideneoxindoles (compounds 242-247). These new analogs were obtained by a tandem Heck-SuzukiMiyaura reaction from various alkynamides and 3,4,5trimethoxyphenyl boronic acid or the corresponding styryl derivative (Fig. 5). In order to produce a conformational restriction, Mateo and coworkers [99] realized a series of compounds representing a macrocyclic structure, in which the para position of the aromatic


Table 24.

Marrelli et al.

Antiproliferative Activity and Inhibition of Tubulin Polymerization of Compounds 229-241 Cytotoxicity (IC50 nM)

Compound

a

b

c

d

e

Tubulin f IC50 (μM)

KB

H460

173.6 ± 33.1

180 ± 25.5

148 ± 17

245.5 ± 32.7

117.3 ± 23.7

>10

1800 ± 100

1600 ± 380

1000 ± 210

562 ± 42

1100 ± 220

-

3500 ± 700

3800 ± 520

2300 ± 480

920 ± 90

2200 ± 600

-

2900 ± 400

3800 ± 620

2800 ± 310

1200 ± 150

2300 ± 400

-

24 ±6.1

36 ± 5.5

24.6 ± 3

16.3 ± 4.5

21.5 ± 7.7

2.9 ± 0.5

HT29

MKN45

KB-vin10

Ref.

OCH3 OCH3

229 N

OCH3 O

O OCH3

230 N

OCH3 OCH3

OCH3 OCH3 O

OCH3

231

N

OCH3 H3CO

232

O

H3CO

N O H3CO

233 H3CO

N OCH3

[97]

OCH3 H3CO

234 H3CO

>10000

>10000

>10000

>10000

>10000

-

8100 ± 700

5000 ± 920

4500 ± 1100

3200 ± 700

6000 ± 800

-

27.2 ± 9.8

61.5 ± 20.5

77 ± 5.7

150 ± 31.2

21.5 ± 0.6

3.5 ± 0.6

155 ± 15.1

193 ± 35.3

162.5 ± 28.7

147 ± 25.4

165.2 ± 33

-

300.6 ± 64.4

256.5 ± 34.6

205.5 ± 4.9

138.5 ± 54.4

202.3 ± 26.2

>10

N O

N

235

H3CO

O

H3CO OCH3 OCH3 H3CO

OCH3

236 N

OCH3 O

OCH3 OCH3 O

OCH3

237

N OCH3 O H3CO

238 H3CO

N OCH3

OCH3



3073

(Table 24). Contd…..

KBa

H460b

HT29c

MKN45d

KB-vin10 e

Tubulin f IC50 (μM)

0.3 ± 0.1

0.4 ± 0.3

0.4 ± 0.2

0.3 ± 0.2

0.2 ± 0.1

1.6 ± 0.2

829.5±171.5

1100 ± 150

872 ± 86.2

617.3 ± 85

764 ± 111.1

-

>10000

>10000

>10000

>10000

>10000

-

Cytotoxicity (IC50 nM)

Compound NH2

Ref.

OCH3

H3CO

OCH3

239 N

OCH3 O

O H3CO

240 N+

H3CO

O-

OCH3 O H3CO

241 N+

H3CO

CH3(I-)

OCH3

24

Colchicine

12 ± 1.2

20.1 ± 3

13.2 ± 2.3

12.4 ± 2

128 ± 8

4.2 ± 0.6

1

CA-4

2.4 ± 0.2

2.6 ± 0.3

835 ± 54

4.9 ± 0.2

1.9 ± 0.4

2.1 ± 0.3

a

Oral epidermoid carcinoma cell line. bNon-small-cell lung carcinoma cells. cColorectal carcinoma cell line. dStomach carcinoma cell line. eMDR-positive cancer cell line. fInhibition of tubulin polymerization.

CH3

O

O

H3CO

H3CO NH

N

H3CO

CH3

H3CO OCH3 242

OCH3 OMe

243 CH3

O H3CO

O

H3CO N

Bn

NH

H3CO

H3CO OCH3

OCH3

244

245 O

O H3CO

H3CO N

Bn

N

SEM

H3CO

H3CO

OCH3

OCH3 246

247

Fig. (5). (E)-3-arylmethyleneoxindoles and (E,E) -3-alkylideneoxindoles.

moieties have been linked by a 5- or 6-atoms chain. The authors wanted to verify if the conformational restriction imposed on these compounds by the formation of the macrocycle generated by the presence of a 5- or 6-atoms linker between the para heteroatoms of the aromatic systems could make them adopt an optimal binding geometrics, so they could show an increase in tubulin binding potency. Two linkers with different length (5 or 6 atoms) were chosen to afford a different degree of conformational restriction by varying the macrocycle size. Thus, 3-oxapentamethylene or hexamethylene linkers were employed, giving rise to Families I and II. However, the studies demonstrated that such a restriction prevents them from adopting a suitable conformation or produces a detrimental steric hindrance for binding to the protein and elicit a

potent antitubulin activity. All the macrocyclic combretastatins showed a low activity as tubulin polymerization inhibitors (Table 25). The conformational analysis for these analogs revealed a severe conformational restriction compared to the parent combretastatins. The rotation of the rings is greatly hampered by the presence of the linker and the substituents on the rings. Such a restriction is somewhat relaxed for compounds of Family II, in which a longer 6-atoms linker allows more flexibility and thus a larger conformational space for the aromatic rings. This would account for the highest potency of compound 249 (ITP = 60% at 40 μM). None of the analogs with a shorter linker (3oxapentamethylene) showed any relevant activity.


Table 25.

Marrelli et al.

Inhibition of Tubulin Polymerization for Compounds Macrocyclic Analogs of CA-4

Family

X

Y

R

Assayed concentratio n (μM)

Inhibition of tubulin polymerization (%)

248

I

O

H

-

30

0

249

II

(CH2)2

H

-

40

60

250

II

(CH2)2

OH

-

40

0

251

I

O

H

OH (cis)

40

0

252

I

O

H

OH (trans)

40

0

253

I

O

H

OAc (cis)

40

0

254

I

O

H

OAc (trans)

40

0

255

I

O

OH

OH (cis)

40

11

256

I

O

OH

OH (trans)

40

1

257

I

O

OAc

OAc (cis)

40

0

Compound

H3CO

Ref.

O OCH3

Y

X

O

R R

H3CO O OCH3 X

Y 258

I

O

OAc

OAc (trans)

40

0

259

II

(CH2)2

H

OH (trans)

40

4

260

II

(CH2)2

H

OAc (trans)

40

0

261

II

(CH2)2

OAc

OAc (trans)

40

2

1

-

-

-

-

3

50

O

CA-4 a

[99]

Human myeloid leukemia cell line. bHuman colon adenocarcinoma cell line. cHuman microvascular endothelial cell line. dND, not determined.

CONCLUDING REMARKS Radiations and chemotherapy, together with surgery, have been the only resource against cancer for decades. During the last ten years, a new generation of drugs were designed and developed. These molecules specifically target molecular pathways in the malignant cell itself or in cells supporting tumor growth, such as vascular endothelium [100]. The tumor vasculature has emerged as a promising target for cancer therapy. Blood vessels are important for tumor growth and for metastasis formation and, moreover, the target endothelial cells are adjacent to the bloodstream, therefore easily accessible to the drug [101]. Among new approaches, vascular disrupting therapy aims at destroying the previously existing tumor vascular bed. This new strategy differs from the anti-angiogenic therapy, aimed at inhibiting the formation of new vessels from pre-existing ones. Vascular disrupting agents exploit the antigenic and functional differences between blood vessels in tumor and normal tissues, to cause a selective damage of tumor vasculature. Combretastatins are the most studied class of vascular disrupting agents whose effects are related to their ability to affect microtubules [102, 103]. The use of combretastatin A-4 as a clinical antitumor agent is limited by its low aqueous solubility and low bioavailability. Moreover, it tends to isomerize to the thermodynamically more stable and inactive trans-isomer. The goal to find even more potent and selective compounds has induced many researchers to design more soluble, stable and active analogs [12, 69, 70]. Accordingly, hundreds of analogs of CA-4 have been described. Many cisrestricted analogs containing a trimethoxyphenyl group have been prepared and evaluated as tubulin polymerization inhibitors [12]. Many analogs were also prepared using 1,2-five membered heterocycles such as imidazole, oxazole, pyrazole [71], thiazole, triazole, tetrazole [72], cyclopentenone [73, 74], furanone [74] or oxazolone [75] to avoid stability problems of CA-4. Clear structureactivity relationships for CA-4 have been drawn from these

analogs. Many of these molecules have been tested for their effects on tubulin polymerization as well as for their antiproliferative activity and other biological properties, and possible mechanisms of action have been investigated. Many VDAs are now undergoing clinical testing, such as CA4P, AVE-8062 and ZD6126. Clinical studies realized to date are encouraging. Used as monotherapy, these compound have not had a clinically relevant antitumor effect, because of the survival of cancer cells at the periphery of tumor, but clinical studies combining these agents with conventional approaches such as chemotherapy seems to be very promising [102, 103]. This review, focused on most recently developed derivatives of natural combretastatin A-4, aims to show the variety of structural modification realized to ameliorate the bioavailability and the biological activity of CA-4. Some of the newly synthetized analogs showed better in vitro activity, sometimes even higher than that of CA-4. Further studies are needed to assess their in vivo biological potential, and other studies could be useful to complete and better understand the structure-activity relationships of new analogs. ABBREVIATIONS A10

= established aortic smooth muscle cultured cell line

B16

= murine melanoma cell line

Bel-7402

= human liver cancer cells

BMEC

= bovine microvascular endothelial cells

CA-4

= combretastatin A-4

CA4P

= combretastatin A-4 disodium phosphate

CEM

= human leukemia cells

CEU

= N-phenyl-N’-(2-chloroethyl)ureas

CTBC

= colchicine tubulin binding competition assay


DMXAA

= 5,6-dimethylxanthenone-4-acetic acid

FAA

= flavone-8-acetic acid

H1299

= human non-small lung carcinoma cell line

H460

= human non small lung cancer cell line

HCT-15

= human colon cancer cell line

HCT116

= human colon carcinoma cell line

HeLa

= human cervical cancer cells

HepG2


HL-60

= human myeloid leukemia cells

HMEC

= human microvascular endothelial cell line


3075

20 (Z)-1-(2-Chloroethyl)-3-(3-(3-hydroxy-4-methoxystyryl)phenyl)urea 21 (E)-1-(2-Chloroethyl)-3-(4-(3-hydroxy-4methoxystyryl)phenyl)urea 22 1-(2-Chloroethyl)-3-(4-(3-hydroxy-4methoxyphenethyl)phenyl)urea 23 (Z)-1-(2-chloroethyl)-3-(4-(3-hydroxy-4methoxystyryl)phenyl)urea 25 (Z)-2-Methoxy-5-[2-(4-methoxy-benzo[b]thiophen-6-yl)ethenyl]-phenol 26 (Z)-2-Methoxy-5-[2-(4-methoxy-benzofuran-6-yl)-ethenyl]phenol

HT29

= human colon carcinoma cells

K562

= human chronic myelogenous leukemia cell line

KB

= non-small-cell lung carcinoma

L1210

= mouse leukemia cells

28 2-Methoxy-5-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-vinyl]-phenol

MCF-7

= breast cancer cell line

30 4-Methoxy-6-[2-(4-methoxy-phenyl)-vinyl]-benzo[b]thiophene

27 (Z)-2-Methoxy-5-[2-(7-methoxy-benzofuran-5-yl)-ethenyl]phenol

MDA-MB231 = human breast cancer cell line

31 6-[2-(3,5-Dimethoxy-phenyl)-vinyl]-benzo[b]thiophen-4-ol

MDAs

= microtubule-damaging agents

32 6-[2-(3,5-Dimethoxy-phenyl)-vinyl]-benzofuran-4-ol

MDR

= multidrug resistant cancer cell line

33 6-[2-(7-Methoxy-benzo[1,3]dioxol-5-yl)-vinyl]benzo[b]thiophen-4-ol

MKN45

= stomach carcinoma cells

MMPs

= matrix metalloproteinases

NCI-H460

= human non-small cell lung carcinoma

OVCAR-3

= ovarian adenocarcinoma cell line

36 1-Phenyl-2-(3,4,5-trimethoxy-phenyl)-ethane-1,2-dione

PARP

= poly(ADP-ribose) polymerase

37 2-[2-Oxo-2-(3,4,5-trimethoxy-phenyl)-acetyl]-benzonitrile

PC-3

= human prostate cancer cells

38 4-[2-Oxo-2-(3,4,5-trimethoxy-phenyl)-acetyl]-benzonitrile

SAR

= structure-activity relationships

SH-SY5Y

= human neuroblastoma cell line

39 1-(4-Methoxy-phenyl)-2-(3,4,5-trimethoxy-phenyl)-ethane-1,2dione

SMMC-7221


SK-OV-3

= ovarian cancer cell line

SW-1990

= human pancreatic cancer cells

TPI

= tubulin polymerization inhibition assay

VDAs

= vascular disrupting agents

VEGF

= vascular endothelial growth factor

VTAs

= vascular targeting agents

WSU-CLL

= lymphocytic leukemia cell line

44 1-(3-Hydroxy-phenyl)-2-(3,4,5-trimethoxy-phenyl)-ethane-1,2dione

ZD6126

= N-[(5S)-6,7-dihydro-9,10,11-trimethoxy-3(phosphonooxy)-5H-dibenzo[a,c]cyclohepten-5yl]acetamide

45 1-(3-Hydroxy-4-methoxy-phenyl)-2-(3,4,5-trimethoxy-phenyl)ethane-1,2-dione

COMPOUNDS ABBREVIATIONS 14 4,4,5,5-Tetramethyl-2-{4-[2-(3,4,5-trimethoxy-phenyl)-vinyl]phenyl}-[1,3,2]dioxaborolane 15 2-{4-[2-(4-Methoxy-phenyl)-vinyl]-phenyl}-4,4,5,5-tetramethyl[1,3,2]dioxaborolane

34 6-[2-(7-Methoxy-benzo[1,3]dioxol-5-yl)-vinyl]-benzofuran-4-ol 35 (Z)- 6-[2-(4-Methoxy-benzo[b]thiophen-6-yl)-vinyl]benzo[b]thiophen-4-ol

40 1-(3,4-Dimethoxy-phenyl)-2-(3,4,5-trimethoxy-phenyl)-ethane1,2-dione 41 Acetic acid 2-methoxy-5-[2-oxo-2-(3,4,5-trimethoxy-phenyl)acetyl]-phenyl ester 42 N-{2-Methoxy-5-[2-oxo-2-(3,4,5-trimethoxy-phenyl)-acetyl]phenyl}acetamide 43 1-(3-Fluoro-4-methoxy-phenyl)-2-(3,4,5-trimethoxy-phenyl)ethane-1,2-dione

46 1-(3-Amino-4-methoxy-phenyl)-2-(3,4,5-trimethoxy-phenyl)ethane-1,2-dione 47 1-Naphthalen-2-yl-2-(3,4,5-trimethoxy-phenyl)-ethane-1,2dione 48 1-Pyridin-2-yl-2-(3,4,5-trimethoxy-phenyl)-ethane-1,2-dione 49 1-Quinolin-3-yl-2-(3,4,5-trimethoxy-phenyl)-ethane-1,2-dione

16d (Z)-2-Methoxy-5-[2-(3,4,5-trimethoxy-phenyl)-vinyl]phenylamine

50 (E)- 2-{3'-Hydroxy-4'-methoxyphenyl}-1-phenyl-1-(3,4,5trimethoxy phenyl)-prop-1-ene

17d (E)-2-Methoxy-5-[2-(3,4,5-trimethoxy-phenyl)-vinyl]phenylamine

51 (E)- 2-{3'-Hydroxy-4'-methoxyphenyl}-1-phenyl-1-(3,4,5trimethoxy phenyl)-but-1-ene

18 (E)-1-(2-Chloroethyl)-3-(3-(3-hydroxy-4-methoxystyryl)phenyl)urea

52 (E)- 2-{3'-Hydroxy-4'-methoxyphenyl}-1,2-bis-phenyl-1-(3,4,5trimethoxy phenyl)-ethylene

19 1-(2-Chloroethyl)-3-(3-(3-hydroxy-4-methoxyphenethyl)phenyl)urea

53 (E)- 1-{3'-Hydroxy-4'-methoxyphenyl}-1-phenyl-2-(3,4,5trimethoxy phenyl)-prop-1-ene


Marrelli et al.

54 (E)-1-{3'-Hydroxy-4'-methoxyphenyl}-1-phenyl-2-(3,4,5trimethoxy phenyl)-but-1-ene

84 5-[(E)-2-Fluoro-2-(3,4,5-trimethoxy-phenyl)-vinyl]-2methoxyphenylamine

55 (Z)- 2-{3'-Hydroxy-4'-methoxyphenyl}-1-phenyl-1-(3,4,5trimethoxy phenyl)-prop-1-ene

85 5-[(Z)-2-Fluoro-2-(3,4,5-trimethoxy-phenyl)-vinyl]-2methoxyphenylamine

56 (Z)- 2-{3'-Hydroxy-4'-methoxyphenyl}-1-phenyl-1-(3,4,5trimethoxy phenyl)-but-1-ene

86 5-[(E)-1-Fluoro-2-(3,4,5-trimethoxy-phenyl)-vinyl]-2methoxyphenylamine

57 (Z)- 2-{3'-Hydroxy-4'-methoxyphenyl}-1,2-bis-phenyl-1-(3,4,5trimethoxy phenyl)-ethylene

87 5-[(Z)-1-Fluoro-2-(3,4,5-trimethoxy-phenyl)-vinyl]-2methoxyphenylamine

58 (Z)- 1-{3'-Hydroxy-4'-methoxyphenyl}-1-phenyl-2-(3,4,5trimethoxy phenyl)-prop-1-ene

88 5-[(Z)-2-Fluoro-2-(3-fluoro-4-methoxy-phenyl)-vinyl]-1,2,3trimethoxy-benzene

59 (Z)- 1-{3'-Hydroxy-4'-methoxyphenyl}-1-phenyl-2-(3,4,5trimethoxy phenyl)-but-1-ene

89 5-[(E)-2-Fluoro-2-(3-fluoro-4-methoxy-phenyl)-vinyl]-1,2,3trimethoxy-benzene

61 3-(3-Hydroxy-4-methoxy-phenyl)-1-(3,4,5-trimethoxy-phenyl)prop-2-en-1-one

90 (E)-2,3-Bis-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop2-en-1-one

62 3-(3-Amino-4-methoxy-phenyl)-1-(3,4,5-trimethoxy-phenyl)prop-2-en-1-one

91 (Z)-2,3-Bis-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop2-en-1-one

63 3-(3-Hydroxy-4-methoxy-phenyl)-2-methyl-1-(3,4,5trimethoxy-phenyl)- prop-2-en-1-one

92 4,5,6-Trimethoxy-3-(4-methoxy-phenyl)-2-(3,4,5-trimethoxybenzoyl)-inden-1-one

64 (E)-(2-methoxy-4-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en1-yl)phenyl)boronic acid

93 5-Hydroxy-3-(4-hydroxy-3,5-dimethoxy-benzoyl)-2-(3hydroxy-4-methoxy-phenyl)-6-methoxy-inden-1-one

65 (2E)-3-(4-Hydroxy-3-methoxyphenyl)-1-(3,4,5trimethoxyphenyl)prop-2-en-1-one

95 3-Chloro-1-phenyl-4-(3,4,5-trimethoxy-phenyl)-azetidin-2-one

66 (E)-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en1-yl)phenyl)boronic acid 67 (2E)-1-(4-Hydroxy-3-methoxyphenyl)-3-(3,4,5trimethoxyphenyl)prop-2-en-1-one 68 (Z)-1,2-Difluoro-1-(3,4,5-trimethoxyphenyl)-2-(3-hydroxy-4methoxyphenyl)ethene 69 5-[(E)-2-Fluoro-2-(3,4,5-trimethoxy-phenyl)-vinyl]-2-methoxyphenol 70 5-[(Z)-2-Fluoro-2-(3,4,5-trimethoxy-phenyl)-vinyl]-2-methoxyphenol 71 5-[(E)-1-Fluoro-2-(3,4,5-trimethoxy-phenyl)-vinyl]-2methoxyphenol 72 5-[(Z)-1-Fluoro-2-(3,4,5-trimethoxy-phenyl)-vinyl]-2methoxyphenol 73 5-[(Z)-2-Bromo-1-fluoro-2-(3,4,5-trimethoxy-phenyl)-vinyl]-2methoxy-phenol

94 3-Chloro-1-p-tolyl-4-(3,4,5-trimethoxy-phenyl)-azetidin-2-one 96 (cis, E)- 2-(3,4,5-Trimethoxy-phenyl)ciclopropan-1-styrene 97 (cis, Z)- 2-(3,4,5-Trimethoxy-phenyl)ciclopropan-1-styrene 98 (cis)-2-(3,4,5-Trimethoxy-phenyl)-cyclopropanecarboxylic acid phenylamide 99 (trans)-2-(3,4,5-Trimethoxy-phenyl)-cyclopropanecarboxylic acid phenylamide 100 2-(3,4,5-Trimethoxy-phenyl)-cyclopropanecarboxylic acid (3hydroxy-phenyl)-amide 101 2-(3,4,5-Trimethoxy-phenyl)-cyclopropanecarboxylic acid (3hydroxy-4-methoxy-phenyl)-amide 102 (cis)-2-(3,4,5-Trimethoxy-phenyl)-cyclopropanecarboxylic acid pyridin-3-ylamide 103 (trans)-2-(3,4,5-Trimethoxy-phenyl)-cyclopropanecarboxylic acid pyridin-3-ylamide 104 3-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)furan

74 (Z)-2-Methoxy-5-[2-(3,4,5-trimethoxy-phenyl)-vinyl]nitrobenzene

105 3-(4-Methoxy-3-nitro-phenyl)-4-(3,4,5-trimethoxy-phenyl)furan

75 (Z)-1,2-Difluoro-1-(3,4,5-trimethoxyphenyl)-2-(3-nitro-4methoxy-phenyl)ethene

106 2-Methoxy-5-[4-(3,4,5-trimethoxy-phenyl)-furan-3-yl]phenylamine

76 (E)-1,2-Difluoro-1-(3,4,5-trimethoxyphenyl)-2-(3-nitro-4methoxy-phenyl)ethene

107 2-Methoxy-5-[4-(3,4,5-trimethoxy-phenyl)-furan-3-yl]-phenol

77 5-[(Z)-1-Fluoro-2-(4-methoxy-3-nitro-phenyl)-vinyl]-1,2,3trimethoxy-benzene 78 5-[(E)-1-Fluoro-2-(4-methoxy-3-nitro-phenyl)-vinyl]-1,2,3trimethoxy-benzene 79 5-[(Z)-2-Fluoro-2-(4-methoxy-3-nitrophenyl)-vinyl]-1,2,3trimethoxy-benzene 80 5-[(E)-2-Fluoro-2-(4-methoxy-3-nitrophenyl)-vinyl]-1,2,3trimethoxy-benzene

108 4-(4-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-2furaldehyde 109 3-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2furaldehyde 110 5-(2-{-[3-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2furyl]methylidene} hydrazino)-5-oxopentanoic Acid 111 2-Methoxy-5-[1-(4-methoxyphenyl)-1H-1,2,3-triazol-5yl]phenol

81 2-Methoxy-5-[2-(3,4,5-trimethoxy-phenyl)-vinyl]-phenylamine

112 2-Methoxy-5-[1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-5yl]phenol

82 (Z)-1,2-Difluoro-1-(3,4,5-trimethoxyphenyl)-2-(3-amino-4methoxyphenyl)ethene

113 2-Methoxy-5-{1-[4-(trifluoromethoxy)phenyl]-1H-1,2,3triazol-5-yl}phenol

83 (E)-1,2-Difluoro-1-(3,4,5-trimethoxyphenyl)-2-(3-amino-4methoxyphenyl)ethene

114 5-[1-(1,3-Benzodioxol-5-yl)-1H-1,2,3-triazol-5-yl]-2methoxyphenol


115 2-Methoxy-5-[1-(4-phenoxyphenyl)-1H-1,2,3-triazol-5yl]phenol 116 5-[1-(3,4-Dimethoxyphenyl)-1H-1,2,3-triazol-5-yl]-2methoxyphenol 117 5-[1-(3,5-Dimethoxyphenyl)-1H-1,2,3-triazol-5-yl]-2methoxyphenol 118 5-[5-(3-Hydroxy-4-methoxyphenyl)-1H-1,2,3-triazol-1-yl]-2methoxyphenol 119 5-[1-(3-Fluoro-4-methoxyphenyl)-1H-1,2,3-triazol-5-yl]-2methoxyphenol 120 2-Methoxy-5-[1-(1-naphthyl)-1H-1,2,3-triazol-5-yl]phenol 121 2-Methoxy-5-[1-(2-naphthyl)-1H-1,2,3-triazol-5-yl]phenol 122 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,3triazole 123 1-(3-Bromo-4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)1H-1,2,3-triazole 124 1-(4-Methoxy-3-nitrophenyl)-5-(3,4,5-trimethoxyphenyl)-1H1,2,3-triazole 125 2-Methoxy-5-(5-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-1yl)phenol 126 2-Methoxy-5-(5-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-1yl)aniline 127 5-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,3triazole 128 5-(3-Bromo-4-methoxyphenyl)-1-(3,4,5-tri-methoxyphenyl)1H-1,2,3-triazole 129 2-Methoxy-5-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-5yl)phenol 130 5-(4-Methoxy-3-nitrophenyl)-1-(3,4,5-trimethoxyphenyl)-1H1,2,3-triazole 131 2-Methoxy-5-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-5yl)aniline 132 4-(4-Methoxy-3-nitrophenyl)-1-(3,4,5-trimethoxyphenyl)-1H1,2,3-triazole 133 2-Methoxy-5-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-4yl)aniline 134 2-Methoxy-5-[5-(3,4,5-trimethoxy-phenyl)-3,4-dihydro-2Hpyrazol-3-yl]-phenol 135 5-(3,4-Dimethoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-4,5dihydro-1H-pyrazole 136 3,5-Bis-(3,4,5-trimethoxy-phenyl)-4,5-dihydro-1H-pyrazole 137 1-[5-(3-Methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-4,5dihydro-pyrazol-1-yl]-ethanone 138 1-[5-(2,5-Dimethoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-4,5dihydro-pyrazol-1-yl]-ethanone 139 1-[5-(2-Methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-4,5dihydro-pyrazol-1-yl]-ethanone 140 1-[5-(4-Methoxy-3-nitro-phenyl)-3-(3,4,5-trimethoxy-phenyl)4,5-dihydro-pyrazol-1-yl]-ethanone 141 1-[5-(4-Nitro-phenyl)-3-(3,4,5-trimethoxy-phenyl)-4,5dihydro-pyrazol-1-yl]-ethanone 142 1-[5-(4-Chloro-phenyl)-3-(3,4,5-trimethoxy-phenyl)-4,5dihydro-pyrazol-1-yl]-ethanone 143 1-[5-(3-Hydroxy-4-methoxy-phenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-pyrazol-1-yl]-ethanone 144 1-[3-(2,5-Dimethoxy-phenyl)-5-(4-methoxy-3-nitro-phenyl)-


3077

4,5-dihydro-pyrazol-1-yl]-ethanone 145 1-[3-(2,5-Dimethoxy-phenyl)-5-(3,4-dimethoxy-phenyl)-4,5dihydro-pyrazol-1-yl]-ethanone 146 1-[3-(2,5-Dimethoxy-phenyl)-5-(2,4,6-trimethoxy-phenyl)-4,5dihydro-pyrazol-1-yl]-ethanone 147 5-(4-Nitro-phenyl)-4-(3,4,5-trimethoxy-phenyl)[1,2,3]thiadiazole 148 5-(4-Methoxy-phenyl)-4-(3,4,5-trimethoxy-phenyl)[1,2,3]thiadiazole 149 5-(3,4-dioxo-methanal-phenyl)- 4-(3,4,5-trimethoxy-phenyl)[1,2,3]thiadiazole 150 5-(3,4-Dimethoxy-phenyl)-4-(3,4,5-trimethoxy-phenyl)[1,2,3]thiadiazole 151 5-(4-Methoxy-3-nitro-phenyl)-4-(3,4,5-trimethoxy-phenyl)[1,2,3]thiadiazole 152 5-(4-Fluoro-phenyl)-4-(3,4,5-trimethoxy-phenyl)[1,2,3]thiadiazole 153 5-(4-Chloro-phenyl)-4-(3,4,5-trimethoxy-phenyl)[1,2,3]thiadiazole 154 2-Methoxy-5-[4-(3,4,5-trimethoxy-phenyl)-[1,2,3]thiadiazol-5yl]-phenol 155 4-(4-Methoxy-phenyl)-5-(3,4,5-trimethoxy-phenyl)[1,2,3]thiadiazole 156 4-(3,4-Dimethoxy-phenyl)-5-(3,4,5-trimethoxy-phenyl)[1,2,3]thiadiazole 157 4-(3,4-dioxo-methanal-phenyl)-5-(3,4,5-trimethoxy-phenyl)[1,2,3]thiadiazole 158 4-(4-Fluoro-phenyl)-5-(3,4,5-trimethoxy-phenyl)[1,2,3]thiadiazole 159 4-(4-Chloro-phenyl)-5-(3,4,5-trimethoxy-phenyl)[1,2,3]thiadiazole 160 4-(4-Methoxy-3-nitro-phenyl)-5-(3,4,5-trimethoxy-phenyl)[1,2,3]thiadiazole 161 4-(4-Nitro-phenyl)-5-(3,4,5-trimethoxy-phenyl)[1,2,3]thiadiazole 162 2-Methoxy-5-[4-(3,4,5-trimethoxy-phenyl)-[1,2,3]thiadiazol-5yl]-phenylamine 163 2-Methoxy-5-[5-(3,4,5-trimethoxy-phenyl)-[1,2,3]thiadiazol-4yl]-phenol 164 2-Methoxy-5-[5-(3,4,5-trimethoxy-phenyl)-[1,2,3]thiadiazol-4yl]-phenylamine 165 3,4-Bis-(4-methoxy-phenyl)-3H-thiazol-2-ylideneamine 166 3-(4-Methoxy-phenyl)-4-(3,4,5-trimethoxy-phenyl)-3H-thiazol2-ylideneamine 167 4-(4-Methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-3H-thiazol2-ylideneamine 168 3-(4-Methoxy-phenyl)-4-(3,4,5-trimethoxy-phenyl)-3H-thiazol2-one 169 5-Chloro-3-(4-methoxy-phenyl)-4-(3,4,5-trimethoxy-phenyl)3H-thiazol-2-one 170 4-(4-Methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-3H-thiazol2-one 171 5-Chloro-4-(4-methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)3H-thiazol-2-one 172 3-(4-Methoxy-3-nitro-phenyl)-4-(3,4,5-trimethoxy-phenyl)-3Hthiazol-2-one


Marrelli et al.

173 5-Chloro-3-(4-methoxy-3-nitro-phenyl)-4-(3,4,5-trimethoxyphenyl)-3H-thiazol-2-one

202 2-Methoxy-5-[5-(3,4,5-trimethoxy-phenyl)-isoxazol-4-yl]phenol

174 3-(3-Amino-4-methoxy-phenyl)-4-(3,4,5-trimethoxy-phenyl)3H-thiazol-2-one

203 2-Methoxy-5-[5-(3,4,5-trimethoxy-phenyl)-isoxazol-4-yl]phenylamine

175 3-(3-Amino-4-methoxy-phenyl)-5-chloro-4-(3,4,5-trimethoxyphenyl)-3H-thiazol-2-one

204 5-(4-Methoxy-phenyl)-4-(3,4,5-trimethoxy-phenyl)-isoxazole

176 4-(4-Methoxy-3-nitro-phenyl)-3-(3,4,5-trimethoxy-phenyl)-3Hthiazol-2-one 177 5-Chloro-4-(4-methoxy-3-nitro-phenyl)-3-(3,4,5-trimethoxyphenyl)-3H-thiazol-2-one 178 4-(3-Amino-4-methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)3H-thiazol-2-one 179 4-(3-Amino-4-methoxy-phenyl)-5-chloro-3-(3,4,5-trimethoxyphenyl)-3H-thiazol-2-one 180 (6-Methoxy-7-(prop-1-ynyl)-1H-indazol-3-yl)(3,4,5-trimethoxyphenyl)methanone 181 (7-(3-Hydroxyprop-1-ynyl)-6-methoxy-1H-indazol-3-yl)(3,4,5trimethoxyphenyl) methanone 182 R-(7-(3-Hydroxybut-1-ynyl)-6-methoxy-1H-indazol-3yl)(3,4,5-trimethoxyphenyl) methanone

205 2-Methoxy-5-[4-(3,4,5-trimethoxy-phenyl)-isoxazol-5-yl]phenol 206 2-Methoxy-5-[4-(3,4,5-trimethoxy-phenyl)-4,5-dihydrooxazol-5-yl]-phenol 207 5-[4’-(Methylthio)phenyl]-4-(3’,4’,5’trimethoxyphenyl)oxazole 208 5-(2,3-Dihydroxy-4-methoxyphenyl)-4-(3,4,5trimethoxyphenyl)oxazole 209 5-(3-Fluoro-4-methoxy-phenyl)-1-(3,4,5-trimethoxy-phenyl)1H-imidazole 210 1-(1,4-Dihydro-naphthalen-2-yl)-2-(3,4,5-trimethoxy-phenyl)1H-imidazole 211 1-(4-Methoxy-phenyl)-2-(3,4,5-trimethoxy-phenyl)-1Himidazole 212 1,5-Bis-(4-methoxy-phenyl)-1H-imidazole

183 S-(7-(3-Hydroxybut-1-ynyl)-6-methoxy-1H-indazol-3yl)(3,4,5-trimethoxyphenyl) methanone

213 1-(1,4-Dihydro-naphthalen-2-yl)-2-(3,4,5-trimethoxy-phenyl)1H-imidazole

184 (7-(3-Hydroxy-3-methylbut-1-ynyl)-6-methoxy-1H-indazol-3yl)(3,4,5-trimethoxyphenyl)methanone

214 2-(1,4-Dihydro-naphthalen-2-yl)-1-(3,4,5-trimethoxy-phenyl)1H-imidazole

185 (7-(4-Hydroxybut-1-ynyl)-6-methoxy-1H-indazol-3-yl)(3,4,5trimethoxyphenyl) methanone

215 1-(4-Methoxy-phenyl)-2-(3,4,5-trimethoxy-phenyl)-1Himidazole

186 (7-(5-Hydroxypent-1-ynyl)-6-methoxy-1H-indazol-3-yl)(3,4,5trimethoxyphenyl) methanone

216 2-(4-Methoxy-phenyl)-1-(3,4,5-trimethoxy-phenyl)-1Himidazole

187 (7-Ethynyl-6-methoxy-1H-indazol-3-yl)(3,4,5trimethoxyphenyl)methanone

217 1,2-Bis-(4-methoxy-phenyl)-1H-imidazole

188 (7-(3-Aminoprop-1-ynyl)-6-methoxy-1H-indazol-3-yl)(3,4,5trimethoxyphenyl) methanone 189 (Z)-(7-(3-Hydroxyprop-1-enyl)-6-methoxy-1H-indazol-3-yl)(3,4,5-trimethoxyphenyl) methanone 190 (7-(3-Hydroxypropyl)-6-methoxy-1H-indazol-3-yl)(3,4,5trimethoxyphenyl)methanone 191 (6-Methoxy-1H-indol-3-yl)-(3,4,5-trimethoxy-phenyl)methanone 192 (6-Methoxy-7-(prop-1-ynyl)-1H-indol-3-yl)(3,4,5trimethoxyphenyl)methanone 193 (5-Hydroxy-6-methoxy-1H-indol-3-yl)-(3,4,5-trimethoxyphenyl)-methanone 194 (7-Hydroxy-6-methoxy-1H-indol-3-yl)-(3,4,5-trimethoxyphenyl)-methanone 195 (6-Hydroxy-5-methoxy-1H-indol-3-yl)-(3,4,5-trimethoxyphenyl)-methanone 196 Methanesulfonic acid 1-benzenesulfonyl-5-methoxy-3-(3,4,5trimethoxy-benzoyl)-1H-indol-6-yl ester 197 Methanesulfonic acid 1-benzenesulfonyl-6-methoxy-3-(3,4,5trimethoxy-benzoyl)-1H-indol-5-yl ester 198 Methanesulfonic acid 5-methoxy-3-(3,4,5-trimethoxybenzoyl)-1H-indol-6-yl ester 199 Methanesulfonic acid 6-methoxy-3-(3,4,5-trimethoxybenzoyl)-1H-indol-5-yl ester

218 4,6-Bis-(3,4,5-trimethoxy-phenyl)-3,4-dihydro-1H-pyrimidine2-thione 219 4-(2,4,6-Trimethoxy-phenyl)-6-(3,4,5-trimethoxy-phenyl)-3,4dihydro-1H-pyrimidine-2-thione 220 4-(3,4-Dimethoxy-phenyl)-6-(3,4,5-trimethoxy-phenyl)-3,4dihydro-1H-pyrimidine-2-thione 221 4-(2,5-Dimethoxy-phenyl)-6-(3,4,5-trimethoxy-phenyl)-3,4dihydro-1H-pyrimidine-2-thione 222 4-(3-Methoxy-phenyl)-6-(3,4,5-trimethoxy-phenyl)-3,4dihydro-1H-pyrimidine-2-thione 223 4-(3-Hydroxy-4-methoxy-phenyl)-6-(3,4,5-trimethoxy-phenyl)3,4-dihydro-1H-pyrimidine-2-thione 224 4-(4-Methoxy-3-nitro-phenyl)-6-(3,4,5-trimethoxy-phenyl)3,4-dihydro-1H-pyrimidine-2-thione 225 4-(4-Nitro-phenyl)-6-(3,4,5-trimethoxy-phenyl)-3,4-dihydro1H-pyrimidine-2-thione 226 4-(4-Chloro-phenyl)-6-(3,4,5-trimethoxy-phenyl)-3,4-dihydro1H-pyrimidine-2-thione 227 1,4-Dimethoxy-2-[6-(2,4,6-trimethoxy-phenyl)- 2-thioxo1,3,5,6-tetrahydro-pyrimidin-4-yl]-benzene 228 1,4-Dimethoxy-2-[6-(4-methoxy-3-nitro-phenyl)-2-thioxo1,3,5,6-tetrahydro-pyrimidin-4-yl]-benzene 229 Quinolin-2-yl-(3,4,5-trimethoxy-phenyl)-methanone 230 Quinolin-3-yl-(3,4,5-trimethoxy-phenyl)-methanone

200 4-(4-Methoxy-phenyl)-5-(3,4,5-trimethoxy-phenyl)-isoxazole

231 Quinolin-4-yl-(3,4,5-trimethoxy-phenyl)-methanone

201 4-(4-Methoxy-3-nitro-phenyl)-5-(3,4,5-trimethoxy-phenyl)isoxazole

232 Quinolin-5-yl-(3,4,5-trimethoxy-phenyl)-methanone 233 6-(3,4,5-Trimethoxybenzoyl)quinoline





tricyclo[14.2.2.24,7]docosa-1(19),2,4(22),5,7(21),16(20),17heptaen-6-yl ester

236 6-Methoxy-2-(3’,4’,5’-trimethoxybenzoyl)quinoline

REFERENCES

237 (8-Methoxy-quinolin-4-yl)-(3,4,5-trimethoxy-phenyl)methanone

[1]

238 (2-Methoxy-quinolin-6-yl)-(3,4,5-trimethoxy-phenyl)methanone

[2]

239 5-Amino-6-methoxy-2-(3’,4’,5’-trimethoxybenzoyl)quinoline

[3]

240 (1-Oxy-quinolin-6-yl)-(3,4,5-trimethoxy-phenyl)-methanone 241 1-Methyl-6-(3,4,5-trimethoxy-benzoyl)-quinolinium; iodide 242 6-Methoxy-3-[1-(3,4,5-trimethoxy-phenyl)-ethyl]-1,3-dihydroindol-2-one 243 1-Methyl-3-(3,4,5-trimethoxy-benzyl)-1,3-dihydro-indol-2-one

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244 1-Benzyl-3-(3,4,5-trimethoxy-benzyl)-1,3-dihydro-indol-2-one 245 3-[1-Methyl-3-(3,4,5-trimethoxy-phenyl)-allylidene]-1,3dihydro-indol-2-one

[7]

246 1-Benzyl-3-[3-(3,4,5-trimethoxy-phenyl)-allylidene]-1,3dihydro-indol-2-one

[8]

247 3-[3-(3,4,5-Trimethoxy-phenyl)-allylidene]-1-(2trimethylsilanyl-ethoxymethyl)-1,3-dihydro-indol-2-one

[9]

248 6,20-Dimethoxy-8,11,14-trioxa-tricyclo[13.2.2.24,7]heneicosa1(18),2,4(21),5,7(20),15(19),16-heptaene 249 6,21-Dimethoxy-8,15-dioxa-tricyclo[14.2.2.24,7]docosa1(19),2,4(22),5,7(21),16(20),17-heptaene 250 17,20-Dimethoxy-8,15-dioxa-tricyclo[14.2.2.24,7]docosa1(19),2,4(22),5,7(21),16(20),17-heptaen-6-ol 251 (cis)-6,20-Dimethoxy-8,11,14-trioxatricyclo[13.2.2.24,7]heneicosa1(18),2,4(21),5,7(20),15(19),16heptaene-2,3-diol 252 (trans)- 6,20-Dimethoxy-8,11,14-trioxatricyclo[13.2.2.24,7]heneicosa-1(18),2,4(21),5,7(20),15(19),16heptaene-2,3-diol 253 (cis) Acetic acid 3-acetoxy-16,19-dimethoxy-8,11,14-trioxatricyclo[13.2.2.24,7]heneicosa-1(18),2,4(21),5,7(20),15(19),16heptaen-2-yl ester 254 (trans) Acetic acid 3-acetoxy-16,19-dimethoxy-8,11,14-trioxatricyclo[13.2.2.24,7]heneicosa-1(18),2,4(21),5,7(20),15(19),16heptaen-2-yl ester 255 (cis)16,19-Dimethoxy-8,11,14-trioxatricyclo[13.2.2.24,7]heneicosa-1(18),2,4(21),5,7(20),15(19),16heptaene-2,3,6-triol 256 (trans)16,19-Dimethoxy-8,11,14-trioxatricyclo[13.2.2.24,7]heneicosa-1(18),2,4(21),5,7(20),15(19),16heptaene-2,3,6-triol 257 (cis) Acetic acid 3,16-diacetoxy-6,20-dimethoxy-8,11,14trioxa-tricyclo[13.2.2.24,7]heneicosa1(18),2,4(21),5,7(20),15(19),16-heptaen-2-yl ester 258 (trans) Acetic acid 3,16-diacetoxy-6,20-dimethoxy-8,11,14trioxa-tricyclo[13.2.2.24,7]heneicosa1(18),2,4(21),5,7(20),15(19),16-heptaen-2-yl ester 259 (trans) 6,21-Dimethoxy-8,15-dioxatricyclo[14.2.2.24,7]docosa-1(19),2,4(22),5,7(21),16(20),17heptaene-2,3-diol 260 (trans) Acetic acid 3-acetoxy-17,20-dimethoxy-8,15-dioxatricyclo[14.2.2.24,7]docosa-1(19),2,4(22),5,7(21),16(20),17heptaen-2-yl ester 261 (trans) Acetic acid 2,3-diacetoxy-17,20-dimethoxy-8,15-dioxa-

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Received: February 17, 2011

Revised: May 17, 2011

Accepted: May 19, 2011


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