tubulin as a target for anticancer drugs: agents which interact with the mitotic spindle

Tubulin as a Target forAnticancer Drugs:Agents WhichInteract with the Mitotic Spindle

Allan Jordan,1,2 John A. Hadfield,2 Nicholas J. Lawrence,1 Alan T. McGown2

1Department of Chemistry, University of Manchester Institute of Science and Technology, PO Box 88, Manchester, M60 1QD, UK

2Cancer Research Campaign Department of Drug Development and Imaging, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester, M20 4BX, UK

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Abstract: Tubulin is the biochemical target for several clinically used anticancer drugs, includingpaclitaxel and the vinca alkaloids vincristine and vinblastine. This review describes both the natu-ral and synthetic agents which are known to interact with tubulin. Syntheses of the more complexagents are referenced and the potential clinical use of the compounds is discussed. This review de-scribes the biochemistry of tubulin, microtubules, and the mitotic spindle. The agents are discussedin relation to the type of binding site on the protein with which they interact. These are thecolchicine, vinca alkaloid, rhizoxin/maytansine, and tubulin sulfhydryl binding sites. Also in-cluded are the agents which either bind at other sites or unknown sites on tubulin. The literatureis reviewed up to October 1997. © 1998 John Wiley & Sons, Inc. Med. Res. Rev., 18, No. 4, 259–296, 1998.

Key words: tubulin; cancer; binding site; antimitotic

1. I N T R O D U C T I O N

Following heart disease, cancer is the biggest cause of death in the West. Cancer is a generic termfor over 200 diseases, which share a number of characteristics including uncontrolled cellular pro-liferation. This uncontrolled growth can impinge on surrounding organs, causing disruption of nor-mal bodily functioning which in turn can lead to death. Another feature of cancer is the ability of tu-mor cells to migrate to other sites in the body. This process (metastasis) also increases the difficultyin treating these diseases as these secondary tumors can also disrupt bodily functions. Under theseconditions the removal of tumors by surgery becomes less practicable and other methods of treat-ment are needed. Chemotherapy (the use of drugs) therefore becomes the therapy of choice underthese circumstances.

Cancer chemotherapy is designed to exploit differences between normal and malignant cells.Thus the ultimate goal of this therapy is to produce a drug which will specifically destroy, or other-wise render benign, cancer cells without having a significant effect on normal cells. Clearly cancercells differ from their normal counterparts in a number of biochemical processes, particularly in the

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© 1998 John Wiley & Sons, Inc. CCC 0198-6325/98/040259-38

Correspondence to: A. T. McGown

control of cell growth and division. However, it is clear that these differences are a result of altera-tions or damage to a minute proportion of normal cellular processes. Thus the design and synthesisof a truly selective anticancer drug has yet to be accomplished.

A cancer can be viewed as a condition where the delicate balance between cell production andcell death is lost, resulting in an overproduction of cells. This increase in cell growth and divisionpresents an attractive and achievable target for drug design. Mitosis is the stage in the process of celldivision where segregation of chromosomes occurs prior to cell replication. This process also occursin normal cells undergoing division and thus does not represent a truly specific target. However se-lectivity toward the more frequently dividing cancer cell can be demonstrated, and this has been con-firmed by the clinical results obtained with the agents such as the vinca alkaloid drugs. These drugshave been shown to be effective in a number of neoplastic conditions.

2. T H E M I T O T I C S P I N D L E

Though the basic sequences leading to cell division are well established, many specific aspects ofmitosis and the ordering of replicated DNA are little understood by molecular biologists. However,it is known that the involvement of a dynamic pipe-like protein fiber, known as a microtubule, is es-sential for the processes to occur. Disturbance of this organelle causes major structural changes with-in the cell. These effects have led to a great deal of interest in the molecular biology of microtubules,and much has been discovered in recent years.

A. Tubulin and Microtubules

Within every nucleated cell in the human body exist two similar spherical proteins a and b tubulin,each with a molecular weight of about 50 kDa. The two proteins are found in virtually all other nu-cleated cells. There is considerable homology between the tubulins of different species.

Through a series of events still not fully understood, these two proteins come together to forman a–b heterodimer. Bound to these heterodimers are two molecules of energy-rich guanosinetriphosphate (GTP). One of these GTP molecules is tightly bound, and cannot be removed withoutdenaturing the heterodimer, while the other GTP molecule is freely exchangeable with unbound GTP.It is widely thought that this exchangeable GTP molecule is intimately involved in the regulation oftubulin function. These heterodimers, in the presence of additional GTP and at 378C, can combinein a head-to-tail arrangement to give a long protein fiber composed of alternating a- and b-tubulin,known as a protofilament. After an induction period, typically several minutes, the protofilamentsgroup together to form a C-shaped protein sheet, which then curls around to give a pipe-like struc-ture known as a microtubule. These microtubules typically consist of 12 or 13 protofilaments, withan external diameter of around 24 nm and an internal bore of around 15 nm, as shown in Figure 1.Associated with these microtubules are a number of proteins, known as Microtubule Associated Pro-teins (MAPs), each with a mass of about 200 kD. The exact purpose of these MAPs is unclear, how-ever microtubules form faster in their presence and the MAPs also appear to protect the microtubulefrom agents which induce depolymerization, namely low temperature and Ca2+ ions.

Also associated with the microtubules are Microtubule Organizing Centers (MTOCs). TheseMTOCs form a focus for microtubule growth, and all microtubules initially begin to grow from oneof these centers. In most cells, there is one major type of MTOC known as the cell center, or cen-trozome, which contains two microtubular structures known as centrioles. It appears that this orga-nization of microtubule growth at the MTOC involves the presence of a third type of tubulin protein,known as g-tubulin. Similar to both a- and b-tubulin, the presence of g-tubulin is essential for mi-crotubule growth in vivo.1 It is thought that an aggregation of g-tubulin occurs on the surface of theMTOC, perhaps forming a ring or short cylinder and that this aggregate acts as the site of nucleationfor incoming a–b tubulin heterodimers.

260 • JORDAN ET AL.

Once formed, these complex protein tubes are not static. They exist in an equilibrium, withdimers constantly adding to one end of the microtubule [known as the “plus” (1) end], and leavingat the other [the “minus” (2) end]. This finely balanced equilibrium, and the resulting control of thelength of the microtubules, is vital for a number of their functions within the cell.

But why does the body, and the biological kingdom at large, have these complicated, dynamicproteins present in all its cells? Amongst the known functions of the microtubules is that of cell sup-port, with the microtubule acting as a form of internal scaffold, giving the cell both shape and an or-ganized structure. Also, the microtubules seem to be used for cellular transport, moving the cellaround its environment and transporting organelles around the cellular interior. However, arguablythe most important role of the microtubules is the formation of the mitotic spindle, which is inti-mately involved in cell replication.

B. Microtubules, Cell Replication, and the Cell Cycle2

Cell division is arguably one of the most complex and demanding processes undertaken by the body.During cell division, the cell must completely duplicate its internal components, including the wholeof its DNA, such that it can form two identical daughter cells. Once duplication of the internal com-ponents has been completed, the cell must then order its DNA into two identical sets of chromosomesand separate them into two distinct parcels at opposite ends of the cell, ready to form the two nucleiin the daughter cells. Once these new nuclei have fully separated, the cell is then ready to split intothe two new daughter cells. This ordering and relocation of the genetic material, which takes aboutan hour, is known as mitosis, and falls into five distinct phases, as depicted in Figure 2.

During the first phase, the prophase, the DNA in the nucleus is replicated and the two sets of ge-netic material organized into two identical daughter sets of chromosomes. Towards the end ofprophase, the microtubules required for cell division begin to form and grow toward the newlyformed chromosomes. This bundle of microtubules is the structure known as the mitotic spindle. Thisspindle grows concurrently from two MTOCs, which begin to separate and migrate toward oppositeends of the cell.

In the next stage, prometaphase, the nuclear envelope rapidly disintegrates and the microtubulesattach themselves to the center of the chromosomes at a point known as the kinetochore. The cell

TUBULIN AS TARGET FOR ANTICANCER DRUGS • 261

Figure 1. Microtubule structure.

then enters metaphase, where the chromosomes gradually become arranged in the plane between thetwo centrozomes. Once these chromosomes are accurately arranged, the cell abruptly entersanaphase, triggered by specific cellular signals. The daughter chromosomes then begin to separateslowly as the microtubules decay, slowly drawing and guiding the daughter chromosomes apart toopposite ends of the cell.

During the final phase, telophase, the chromosomes reach the opposite ends of the cell and newnuclear envelopes form around them. This marks the end of mitosis, and it only remains for the cy-toplasm surrounding the nuclei to begin to divide, in a process known as cytokinesis. The nuclei thusbecome partitioned, eventually dividing to give two new daughter cells. Microtubules, therefore, areintimately involved with the replication of cells. If the microtubules in a tumor cell can be prevent-ed from forming or decaying, the chromosomes cannot separate, the cell cannot reproduce and hencethe tumor cannot grow.

Thus, agents which interfere with the dynamics of tubulin may also act as inhibitors of cell di-vision. Indeed, a number of these agents have been shown to act as clinically useful anticancer agents.This review discusses the major classes of antimicrotubular drugs, commonly known as spindle poi-sons, grouped according to their site of action on tubulin.

C. Classes of Spindle Poisons

Drugs which bind to tubulin can be subdivided into separate classes. The class into which a particu-lar drug fits is dependent upon the effect which that drug exerts on the binding of five well-charac-terized tubulin agents to tubulin. These agents are colchicine, the two vinca alkaloids (vincristine and


Figure 2. Stages of Mitosis.

vinblastine), and the two macrocyclic natural products rhizoxin and maytansine. The drugs are cat-egorized dependent upon whether they prevent the binding of colchicine, the binding of the two vin-ca alkaloids or the binding of rhizoxin and maytansine to tubulin. However, some drugs do not haveany effects of the binding of these drugs, having an affinity instead for a separate, distinct region oftubulin. Some of these may bind covalently to certain reactive groups on the protein, particularly thetubulin sulfhydryl groups. The mode of action of others is unknown.

Thus, the spindle poisons can be defined as being associated with the colchicine binding site,the vinca alkaloid binding site, the rhizoxin/maytansine binding site, the tubulin sulfhydryl groupsor with a separate, uncharacterized binding sites.

3. T H E C O L C H I C I N E B I N D I N G S I T E

A. Colchicine

Colchicine 1 has long been associated with microtubules, and is the classic tubulin-binding agent.3

Indeed, tubulin was once known as “colchicine binding protein,” and radio-labeled colchicine wasutilized in the first preparations of purified tubulin. Isolated from the meadow saffron, Colchicumautumnale, colchicine is a highly soluble alkaloid,4 long known to be an effective treatment for gout,and still employed in this role today. Its high toxicity, however, prevented its use in other therapies.

Being one of the first antimitotic drugs to be investigated, the study of colchicine 1 has led tomuch of our present understanding of antimicrotubular drug action. The drug 1 induces various ef-fects on tubulin, the major one being a change in secondary structure of the protein caused by bind-ing to a high affinity site on the tubulin heterodimer. This binding, which is both temperature de-pendent and irreversible, induces an alteration in dimer structure which hinders tubulin assembly,5

inducing partial unfolding of the secondary structure of b-tubulin at the carboxy terminal. It has beensuggested that it is this unfolding which disrupts the protein regions necessary for microtubule for-mation.6 The drug 1 can additionally bind to a second, lower affinity site on tubulin in a reversiblemanner.7


Much effort has been directed toward simplified analogues of colchicine, as exemplified byMTC 2. Synthesized as part of a study of bifunctional analogues, MTC binds to the colchicine siteof tubulin and prevents microtubule assembly at concentrations comparable to that of colchicine it-self.8

B. Ampethinile

Ampethinile 3 aroused interest when it was shown to terminate pregnancy in rats,9 and further stud-ies suggested termination was caused by cytotoxic effects. Studies with HeLa cells added further evi-

dence to this proposition, and it was later shown that ampethinile 3 became bound to tubulin, bind-ing competitively to the colchicine site.10 Interest in this synthetic antimitotic agent 3 increased asinitial in vitro results showed great promise and these results enabled amphethinile 3 to enter PhaseI clinical trials in the mid-1980s.

C. Aromatic Carbamates

In an effort to prepare analogues of the known anticancer agent methotrexate [an inhibitor of the en-zyme Dihydrofolate Reductase (DHFR)] a number of dihydropyridopyrazines 4 were synthesized asprecursors of the proposed inhibitors and routinely tested for anticancer activity. These intermedi-ates prevent cell growth, but not by DHFR inhibition.11 The carbamates 4 were later shown to bindto tubulin at nanomolar concentrations, preventing the formation of microtubules and also competi-tively inhibiting the binding of colchicine.12 The carbamates 4 additionally showed activities in anumber of multidrug resistant cell lines.13


Many of the derivatives synthesized possessed planar geometries, but the more active com-pounds were shown by molecular modeling to be puckered, giving a slight twist between the phenyland pyrazine rings. This increase in activity with the puckered molecules is in accordance with pre-vious work suggesting a relationship between the shapes of ampethinile 3, colchicine 1 and com-bretastatin A-4 16 and their abilities to bind to tubulin.14 Indeed, one of these puckered compounds,the 2-methyl derivative 4, was recommended for clinical evaluation,15 and is now in phase II clini-cal trials.16

The related diphenyl quinazolone 5 is also a potent antimitotic agent, and is highly active invivo.17 The compound is highly insoluble, though incorporation of the compound into liposomes(several concentric layers of lipid bilayers) imparts water solubility to the drug, allowing transportinto the cell. Once inside the cell, the liposome shell dissipates, leaving the drug free to exert its ef-fects.18 Further investigations into these compounds are ongoing.19 In a second related series, a num-

ber of imidazo [4,5-c] pyridines (exemplified by 6 and 7) were produced, replacing the pyridine ringwith an imidazole ring system, and these also showed activity against the formation of microtubulesin vitro, and some activity in vivo. However, these compounds were much less potent than the dihy-dropyridopyrazines.20 The activities of the dihydropyridopyrazines promoted the study of a numberof related pyridine derivatives, analogues where the pyrazine has ring-opened, as exemplified by 8.These molecules also showed some antitubulin activity in vivo.


In a similar vein, the bis-carbamate 9, also possesses tubulin inhibitory properties in vitro, andis active at micromolar concentrations.21 These compounds, along with their related cyclic andacyclic analogues, all contain the aromatic carbamate functionality, that is, Ar-NHCO2R, and in thisrespect are also related to nocodazole 28 and tubulazole-C 10.

Developed as a treatment for Leishmania protoza parasitic infections, tubulazole-C 10 wasfound to be cytotoxic to mammalian cells as well as to the parasitic organisms themselves.22 Inves-tigation of this toxicity demonstrated23 that the drug was an effective microtubule destabilizingagent, being more effective than both colchicine 1 and nocodazole 28. Solid tumors appeared to beparticularly susceptible to treatment. However, tubulazole-C 10 showed poor water solubility whichled to the development of the more soluble derivative, erbulazole 11. Showing a ten-fold increase inactivity, erbulazole 11 is now in phase I clinical trials.24

D. Centauriedin

Isolated from Polymnia fruticosa, centauriedin 12 is a natural flavone which prevents the binding ofcolchicine 1 to tubulin and prevents the formation of microtubules with an IC50 of 3 mM.25 This com-

pound 12 was the first flavone found to cause mitotic arrest in cells by destabilizing the mitotic spin-dle. Since then, the flavone 13 has been isolated from both Zieridium pseudobtusifolium and Polan-sia dodecandra,26,27 and inhibits tubulin formation at a concentration of 0.8 mM.

Further work in this area has led to the synthesis of a series of 2-phenyl-4-quinolones, exem-plified by 14, which are potent inhibitors of tubulin polymerization,28 and can prevent cell growthin a variety of human tumor lines.29


E. The Chalcones

A series of chalcones, typified by 15, have been synthesized and found to be potent cytotoxic agents,with IC50 values of around 4 nM in HeLa cells.30 Further tests also showed promising activity againstL1210 leukaemia and B16 melanoma cell lines. The chalcones also bind to tubulin and induce mi-totic arrest in cell cultures. Furthermore, studies in animal tumor models indicated that these drugswere up to 300 times more potent at halting cell division than colchicine 1. The chalcones are dis-placed from their binding site on tubulin by podophyllotoxin 31, inferring that it binds wholly or par-tially to the colchicine site on tubulin.31

F. Combretastatin

Isolated from the South African Willow, Combretum caffrum,32 combretastatin A-4 16 is one of thesimpler compounds to show antimitotic effects by interaction with the colchicine binding site of tubu-lin, and is one of the most potent inhibitors of colchicine binding presently known.33 Not only is itsspectrum of activity impressive, but combretastatin A-4 16 is not recognized by the multidrug resis-tance (MDR) pump, a cellular pump which rapidly ejects foreign molecules, including many anti-cancer drugs, from the cell. Additionally, it has recently been reported that combretastatin A-4 16can inhibit angiogenesis (the growth and development of blood vessels), a process essential for tu-mor growth.34 Owing to these factors, combretastatin A-4 16 was considered for clinical trials un-

der the supervision of the Cancer Research Campaign, though the progress of these has been hin-dered by the low water solubility of the drug. There is currently a great deal of interest in this area,trying to form new, potent combretastatin analogues with enhanced solubility.

Among these are a series of benzylaniline hydrochlorides, typified by compound 17.35 Thesehighly water-soluble compounds bind to tubulin in the same manner as combretastatin A-4 16, withIC50 values in the region of 3.5 mM in tubulin binding assays.


More recently, the combretastatin core has been coupled to a small number of sugar moieties,in an attempt to produce water-soluble analogues.36 The most active of these compounds was thecombretastatin glucuronide 18, which though highly water-soluble, was about 100-fold less potentthan the parent phenol 16.

A second class of analogues produced in an attempt to mimic combretastatin are the diphenylse-lenides and diphenylsulfides, exemplified by compounds 19 and 20. These compounds 19, 20, how-ever, do not appear to inhibit microtubule formation, but disrupt the cell cycle and cause mitotic ar-rest by interaction with microtubules.37 These agents 19, 20 bind strongly to tubulin, but, unlikecombretastatin A-4 16, they seem to promote microtubule formation, and may not bind to thecolchicine site on the tubulin dimer. It is thought that the binding of the compounds 19, 20 to thedimer may somehow alter the structure of the tubulin protein, encouraging polymerization to occur.

G. Cornigerine

A natural product isolated from Colchicum cornigerum, cornigerine 21 resembles a structural hybridof colchicine 1, podophyllotoxin 31, and steganacin 38. As would perhaps be expected from its close

structural similarity, cornigerine 21 binds to the colchicine site on tubulin and elicits similar effectswith approximately a three-fold increase in potency.38

H. Curacin A

Isolated from the blue–green cyanobacterium, Lyngbya majuscula, curacin A 22 is one of a familyof related natural lipids found to inhibit microtubule formation and the binding of colchicine to tubu-lin dimers.39–41 Isolated in large yields (8–10% w/w) from the crude bacterial extract, curacin A 22is the most potent of this family, and has an IC50 of 1.8 pM in Chinese hamster Aux B1 cells. Stud-ies have indicated that curacin A 22 competes with colchicine 1 in tubulin binding assays, but notwith the vinca alkaloids, establishing its mode of action as a member of the colchicine class of mi-crotubule assembly inhibitors. The activity of this compound 22 has resulted in a great deal of in-terest in its synthesis A and a number of groups have recently reported total syntheses.42–46


I. Diethylstilbestrol

Developed in the 1940s as a synthetic oestrogen, and used in the treatment of threatened abortion,diethylstilbestrol 23 was found to induce cervical and vaginal tumors in the daughters of somewomen treated with the drug. This neoplastic effect was caused by depolymerization of microtubulesand binding to the colchicine site on tubulin.47

J. E7010

The most active of a series of sulfonamide antimitotic agents, E7010 24 has recently been shown toinhibit microtubule formation by binding at the colchicine site.48 In vivo tests showed good oral ac-tivity for the drug 24 across a wide range of tumor types and good activity against vinca alkaloid re-sistant solid tumors. Results from animal studies have indicated activity against gastric, colorectal,breast, and lung cancer tissues, and the clinical viability of the drug is currently being investigatedin Phase I clinical trials.49

K. 2-Methoxyestradiol

The naturally occurring metabolite of the mammalian hormone oestradiol, 2-methoxyestradiol 25,is formed in the body by oxidation in the liver, followed by O-methylation.50 The diol 25 is cyto-toxic to several tumor cell lines, binds to the colchicine site of tubulin, inducing the formation of ab-normal microtubules.51 Additionally, compound 25 inhibits angiogenesis.52 Interestingly,methoxyestradiol 25 does not appear to cause destabilization of microtubules at concentrations suf-ficient for tubulin binding and mitotic block and causes little noticeable change in the morphologyof assembled microtubules. It is thought that methoxyestradiol 25 exerts its effects by altering thedynamics of tubulin polymerization.53


L. Nocodazole

Discovered54 as part of an effort to find effective agents against the helminthic parasite Ascaris lum-bricoides, (which infects about a quarter of the world’s population and is responsible for protein-calorific malnutrition and malabsorbtion of vitamin A) nocodazole 28 is the most active member ofa family of benzimidazole derivatives 26–29. These compounds 26–29 are competitive inhibitors ofthe binding of colchicine to tubulin. Although the drugs 26–29 do not have a trimethoxyphenyl ring,common to many colchicine-site binders, it is thought that the phenyl group may act in the same role,and bind to the same site as the trimethoxyphenyl ring of colchicine 1.

Further work in this area led to 1069C 30, which was developed in an effort to generate a se-lective microtubule destabilizing agent.55 Compound 30 is effective in nanomolar concentrations andin a standard assay against brain tubulin, was about five times more active as a microtubule desta-bilizer than was colchicine 1. This activity is comparable to that of the vinca alkaloids.56 The com-pound 30 also exhibited useful toxicity against a variety of vinca alkaloid and adriamycin resistanttumor cell lines.

M. Podophyllotoxin, Etoposide, and Teniposide

The major component of a resinous mixture known as podophyllin, isolated from the dried roots ofthe American mandrake Podophyllum peltatum, podophyllotoxin 31 has been used as a medical treat-ment for many hundreds of years for conditions ranging from sclerosis of the liver, through to con-stipation, rheumatism, and cancer. More recently, podophyllotoxin 31 has been shown to bind quick-ly and reversibly to at least part of the colchicine binding site of tubulin, and binds as a competitiveinhibitor.57 This ease of reversibility and the low temperature dependence of binding, however, in-dicate that the binding does not occur at a completely identical site to that of colchicine 1.


Along with its other medical uses, podophyllotoxin 31 can be applied topically, for example inthe treatment of venereal warts, but it is too toxic to be of clinical use for cancer chemotherapy. Toovercome this, two less toxic semisynthetic analogues, etoposide 32 and teniposide 33, have beendeveloped.58

Etoposide 32, or VP-16, is currently used in the treatment of small-cell lung, testicular, and ma-lignant lymphoid cancers, among others.59 However, poor water solubility restricts the effectivenessof the drug, and it has to be administered intravenously in a complex formulation. Despite these prob-lems, etoposide 32 is clinically a very useful antimitotic agent, and often used in combinationchemotherapy. Teniposide 33 appears to be a less useful clinical agent, though it is undergoing tri-als in combination therapy for the treatment of metastatic brain tumors.60

Though these agents 32, 33 bind to tubulin, it is now thought that they function predominantlyby inhibition of DNA topoisomerase II, an enzyme involved in the folding and unfolding of DNAduring cell replication, rather than by microtubular interactions.61

N. Rotenone

Extracted from Clitoria macrophyllia, 6-deoxyclitoriacetal, or rotenone 34 is used in Thailand as atraditional remedy for pest control and skin diseases.62 However, rotenone 34 is also a potent an-timitotic agent, interacting with the colchicine binding site. Clinically it is used in combination withvinblastine 40.


O. Sanguinarine, Chelidonine, and Chelerythrine

Isolated as a trace component from the seeds of the Opium poppy, Papaver somniferum, sanguinar-ine 35 is a natural benzophenanthridine inhibitor of the binding of both colchicine 1 and podophyl-lotoxin 31 to tubulin, binding in an acid-reversible manner.63 The compound 35 also inhibits pacli-taxel-mediated polymerisation of microtubules at micromolar concentrations. Sanguarine 35 and therelated alkaloid chelidonine 36, isolated from Chelidonium majus, had both been mentioned as an-timitotic drugs before tubulin was discovered.64 Chelidonine 36, unlike sanguinarine 35, does notinhibit the binding of podophyllotoxin 31 to tubulin.

A further structurally similar alkaloid, chelerythrine 37, also inhibits both colchicine 1 andpodophyllotoxin 31 from binding to tubulin, though greater concentrations are required. This inhi-bition is about 20% weaker than with sanguinarine 35, which is in turn about 10-fold less potent thanchelidonine 36.As well as inhibition of tubulin assembly by a mechanism similar to that of colchicine1, the alkaloids 35, 36 additionally appear to have the potential to react reversibly with tubulinsulfhydryl groups, also preventing tubulin assembly (see Sec. 6). The syntheses of these three alka-loids 35–7 has been accomplished.65,66

P. Steganacin

Isolated in 1973 as a major component from the stem bark of Steganotaenia araliacea,67 steganacin38 binds to tubulin with similar affinity16 to that of colchicine 1. Indeed compound 38 is a competi-tive inhibitor of colchicine 1. Despite the potency of binding, and much early interest, steganacin 38has yet to find a medical application.


Q. TN-16

The synthetic agent TN-16 39 inhibits microtubule formation, and competition assays have demon-strated that this effect is mediated by binding to the colchicine site on tubulin.68 The drug also pre-vents the stabilization of microtubules in cells dosed with paclitaxel (taxol) 104.

4. T H E V I N C A A L K A L O I D B I N D I N G S I T E

A. Vincristine, Vinblastine, and Vindesine

Isolated from the periwinkle Catharanthus roseus, the vinca alkaloids vinblastine 40 and vincristine41 have both been widely used as clinical antitumor agents for the treatment of leukemias, lym-phomas, and some solid tumors.69 Both these agents 40, 41, induce the destabilization of polymer-ized tubulin, by binding to a site recently localized on b-tubulin,70 and both have a high affinity forthe protein.71 It has been reported that this destabilizing effect results from the stoichiometric end-wise poisoning of the tubulin heterodimer, presumably preventing polymerization from occurring byblocking the region involved in heterodimer attachment.1

As the microtubule is a dynamic protein, constantly polymerizing and depolymerizing, vinca al-kaloid poisoned dimers could easily be incorporated into the microtubule polymer, preventing fur-ther growth. The incorporation of the vinca alkaloids onto the heterodimer is rapidly reversible, andappears to occur at two sites per tubulin dimer. At higher concentrations of drug, microtubular crys-tals are formed, consisting of two intertwined helices of tubulin. In addition to the two natural com-pounds 40, 41, a third effective alkaloid, vindesine 42, has been produced by functional group trans-formation, and appears to work by the same mechanism.

B. Halichondrin B

First isolated from Halichondria okadai,72 and later from the unrelated sponges Axinella carteri73

and Phankella carteri,74 halichondrin B 43 is a complex polyether macrolide which has recently beensynthesized75 and arrests cell growth at subnanomolar concentrations. In cell line screenings, thecompound 43 showed a similar pattern of activity to other antitubulin drugs, suggesting its possiblemode of action as an antimitotic agent, and this prediction was later confirmed experimentally. Hali-chondrin B 43 and the related halistatins are noncompetitive inhibitors of the binding of both vin-cristine 41 and vinblastine 40 to tubulin, suggesting the drugs bind to the vinca binding site, or a sitenearby. The isolation of halichondrin B 43 from two unrelated genera of sponge, however, has ledto speculation that the compound 43 is in reality a microbial, rather than sponge metabolite, assponges are known to support a wide range of microbes. If this is the case, fermentation technolo-gies could provide a useful supply of the drug 43.76


C. Hemiasterlins

Isolated77 from the marine sponge, Cymbastela sp the hemiasterlins 45 and 46 show potent activityagainst the P388 cell line (IC50 0.4 ng/ml) and, by binding to the vinca alkaloid site on tubulin, in-hibit cell division.78 Indeed, these agents 45, 46, which have recently been synthesized,79 exhibitstronger antiproliferative activities than both the vinca alkaloids 40, 41 and paclitaxel 104.

D. Spongistatin

Isolated from Spirastrella spinispirulifera, little is known about the spongistatins 47–55, other thantheir relative stereochemistry and their extraordinary potency against human cancer cell line screens.This family of complex cyclic compounds are among the most potent compounds tested to date, dis-


playing cytotoxicities80 with IC50 of 10 fg/ml. Similarly to halichondrin B 43, the spectrum of ac-tivity of the spongistatins was so similar to other known anti-mitotic agents that it was proposed thatthe compounds interacted with tubulin. Later studies vindicated this hypothesis, and showed that thespongistatins were inhibitors of the binding of vincristine 41 to tubulin.81 However, recent researchhas also shown that the spongistatins are inhibitors of dolastatins 58, 59 binding to tubulin, and it hasbeen proposed that the spongistatins (and possibly the halichondrins) possess their own unique bind-ing site, distinct from both the vinca alkaloid site and the rhizoxin/maytansine site (where the dola-statins 58, 59 bind), but sufficiently close to disrupt the binding of both classes drugs to their re-spective binding sites.82


5. T H E R H I Z O X I N / M A Y T A N S I N E ( R Z X / M A Y )B I N D I N G S I T E

A. Rhizoxin

Isolated from the fungus causing rice seedling blight, Rhizopus chinensis, rhizoxin 56 is an antifun-gal agent which also acts as an antimitotic agent. As with most of the drugs binding to the RZX/MAYsite, rhizoxin 56, was originally thought to bind to the vinca alkaloid site, but the drug has since beenshown to possess its own, distinct binding site, also referred to as the peptide site. Though in a sep-arate region of tubulin, this binding site may overlap slightly with the vinca alkaloid site and rhi-zoxin can still affect the binding of the vinca alkaloids to the protein.83 Effective against many hu-man cancer cell lines, and some vinca alkaloid resistant tumors, rhizoxin 56 is also a competitiveinhibitor of maytansine binding and exhibits many similar effects.84 The drug 56 is currently in theearly stages of clinical evaluation in human patients,85 but results so far have proved to be disap-pointing.86

B. Cryptophycin 1

Isolated from the blue–green algae Nostoc sp. GSV 224, the cryptophycins are a family of relateddepsipeptides showing87 highly potent cytotoxic activity (IC50 < 2 pM). Cryptophycin 1 57, whichhas been synthesized,88 was originally developed as a fungicide, but was too toxic for clinical use inthis role.89 Later work highlighted the role of cryptophycin 1 57 as a microtubule poison, prevent-ing the formation of the mitotic spindle. Competition binding assays indicated that this effect ap-peared to be mediated by binding to tubulin at the vinca alkaloid site,90 though this result was laterrevised in light of data indicating binding occurred at the RZX/MAY site.91 This binding is substoi-chiometric, and it has been proposed that depsipeptide 57 exerts its effects by “end-poisoning” theassembling microtubule, preventing the addition of further a–b heterodimers.92 Interest in the com-pound has been further heightened by the discovery that cryptophycin 1 57 shows reduced suscep-tibility to the multidrug resistance pump, and shows no reduction of activity in a number of drug-re-sistant cell lines.93

C. The Dolastatins

Isolated from the sea hare Dolabella auricularia, a small sea mollusc, and thought to be the sourceof poison used to murder the son of Emperor Claudius of Rome in 55 A.D., putting Nero on thethrone, the dolastatins 10 58 and 15 59 are novel pentapeptides. These compounds 58, 59 exhibitpowerful antimitotic properties,94 and are cytotoxic in a number of cell lines at subnanomolar con-centrations. Originally thought to bind at the vinca alkaloid site, these peptides 58, 59 completelyand noncompetitively inhibit the binding of vincristine 41 to tubulin, but have been shown to bindto the RZX/MAY region on tubulin.95 Like the vinca alkaloids 40 and 41, however, the dolastatinsalso enhance and stabilize the binding of colchicine 1 to tubulin.96 The total synthesis of dolastatin10 58 has been accomplished.97

D. Maytansine

Derived from various plants in the species the Maytenus, maytansine 60 is the most potent memberof a family of highly cytotoxic macrolides.98 Despite this potency, so far no useful clinical applica-tions have been found for this agent 60. However, the attachment of tumor specific antibodies tomaytansine derivatives, in order to increase target specificity, is under investigation.99 Maytansine60 binds to tubulin in a reversible manner, and can competitively inhibit100 the binding of vinblas-tine 40 and vincristine 41; however, colchicine 1 binding is only marginally affected by the drug.101

Upon binding to assembled tubulin, maytansine 60 causes extensive disassembly of the microtubule,and totally prevents tubulin spiralization.102


E. Phomopsin A

Phomopsin A 61 is produced by the fungus Phomopsin leptostromiformis, the mold responsible forlupin poisoning, or lupinosis, a severe liver disease which affects grazing animals.103 The damagedliver cells are arrested in mitosis and phomopsin A 61 was shown to be the active agent. Later pho-mopsin A 61 was shown to cause disassembly of assembled microtubules, and to prevent furtherpolymerization.104 Though phomopsin A 61 binds to the RZX/MAY site, it also appears to have anaffinity for an additional, distinct binding site on tubulin.105


F. The Ustiloxins

The ustiloxins 62–65, produced by the rice plant pathogen Ustilaginoidea virens, are a family of fourcyclic peptides structurally related to phomopsin A 61 and have been found to inhibit the formationof microtubules. This binding is competitive with respect to rhizoxin 56, and ustiloxin A 62, the mostpotent member of the family, shows similar biological activity to rhizoxin 56, stabilizing the bind-ing of colchicine 1 to tubulin, and preventing mitosis at concentrations below 1 mg/ml.106

G. Arenastatin A

Arenastatin A 66, isolated107 from the Okinawan marine sponge Dysidea arenaria, is a potently cy-totoxic (IC50 5 8.7 pM, KB cells). The depsipeptide 66, which has recently been synthesized,108 in-hibits109 the assembly of tubulin (IC50 5 2.3 mM) and binds to the RZX/MAY site on tubulin. Are-nastatin A 66 binding is inhibited by vinblastine 40 in a partially competitive manner while havingno effect of the binding of colchicine to tubulin.107


6. D R U G S W H I C H B I N D T O T U B U L I NS U L F H Y D R Y L S

Unlike the binding sites discussed previously, the tubulin sulfhydryls are not a true binding site assuch. They are composed not of a cleft or pocket, but of a reactive function on a protein; specifical-ly a thiol group on a cysteine residue. The drugs which bind to these residues often possess Michaelacceptor functionalities which react with the nucleophilic thiols. Though not fully understood, thiscovalent interaction appears to prevent the formation of stable microtubules, and thus prevents celldivision. It appears that these sulfhydryl groups have a regulatory role in the formation of the mitot-ic spindle, perhaps involving the formation and breakage of a disulfide linkage. This alteration of thelinkage may induce a structural modification in the tertiary structure of the protein, either allowingpolymerization to occur, or causing the microtubule to decay. Regardless of the true molecular causeof these effects, the sulfhydryl-directed reagents effectively prevent microtubule formation, and thusrepresent a major class of spindle poisons.

A. Calvatic Acid

Calvatic acid 67 is a known antibiotic and cytostatic agent isolated from Calvatica liacina.110,111

These cytostatic properties arise from the prevention of microtubule assembly. Cysteine prevents thisinhibition of assembly which indicated that the tubulin sulfhydryl groups were the biological targetfor the compound. Structure–activity relationship studies determined that the chloro derivative 68 tobe the most active agent in the class, and that the compound 68 also prevented the binding ofcolchicine 1 to tubulin.112 However, this binding is believed to occur by a structural alteration of theprotein structure, rather than by direct competitive binding of the calvatic acid derivatives to thecolchicine binding site.

B. Cystamine

Cystamine 69 is a known physiological compound, which regulates the action of some enzymes byinteraction with their sulfhydryl groups. Cystamine 69 can interact with tubulin in a similar manner,inhibiting the assembly of microtubules, and causing abnormal tubulin structures to form at higherconcentrations.113


C. Cytochalasin A

A fungal metabolite, cytochalasin A 70 inhibits the assembly of microtubules in vitro, and preventsthe binding of colchicine 1 to tubulin. Later studies showed that the alkaloid 70 acted by forming acovalent adduct with the sulfhydryl groups of tubulin, and in doing so not only prevented the as-sembly of microtubules, but also destroyed the colchicine binding site.114 Further research has shownthat ethyl acetylacrylate 71, an analogue of the reactive portion (Michael acceptor) of cytochalasinA 70, also reacts with tubulin sulfhydryls in a manner akin to the parent compound 70.115

D. 2,4-Dichlorobenzyl Thiocyanate

Shown to cause mitotic arrest and disruption of microtubules,116 many studies of 2,4-dichloroben-zyl thiocyanate (DCBT) 72 failed to show any marked effects on polymerization or assembly of mi-crotubules. Further studies identified the compound 72 as an alkylating agent, which forms a disul-fide bond with the sulfur-containing amino acids of tubulin, particularly cysteine.117

E. Disulfiram

Disulfiram (“Antabuse”) 73, commonly used for the treatment of alcohol dependency, was found tocause neurotoxicity in some patients undergoing long-term treatment.118 Investigation of this toxic-ity indicated that disulfiram 73 bound to tubulin and prevented its assembly into microtubules, thisinhibition being caused by reaction with free tubulin sulfhydryl groups. However, though binding ofthe drug is instantaneous, there exists a lag time before inhibition of polymerization occurs. It hasbeen proposed that, after the drug binds, a slower conformational change occurs within the tubulinprotein and this change causes the inhibition of microtubule formation. Disulfide 73 also inhibits the

binding of the vinca alkaloids to tubulin, perhaps by alteration of the binding site, but has no effecton the binding of colchicine 1.


G. N,N’-Ethylenebis(iodoacetamide)

N,N’-Ethylenebis(iodoacetamide), or EBI 75, is a bifunctional compound which reacts with tubulin,forming cross-links between sulfhydryl groups, probably via nucleophilic displacement of iodide bythiol. This cross linking prevents assembly of tubulin into microtubules.121 EBI 75 is a commonsulfhydryl-directed reagent, and has been used in a large number of experiments to ascertain the na-ture and function of the tubulin sulfhydryl groups.

H. 4-Hydroxynon-2-enal

A product of lipid peroxidation in the body, 4-hydroxynon-2-enal 76, inhibits cell proliferation in anumber of cell lines by preventing DNA replication. However, enal 76 also prevents mitosis by re-action with tubulin sulfhydryls, alkylating tubulin at neutral pH.122 The aldehyde 76 is rapidly tak-en up by cells, efficiently prevents microtubule assembly and causes the destruction of those micro-tubules already formed.

F. Ethacrynic Acid

Developed as an inhibitor of glutathione-S-transferase during studies of muscle relaxants,119

ethacrynic acid 74 may also have a role in the treatment of glaucoma. Although ethacrynic acid 74induces structural changes in microtubules,120 little has been published on its antimitotic properties.These changes appear to be a result of interaction with the sulfhydryl groups of b-tubulin, prevent-ing the normal assembly of the tubulin polymer.

I. MPMAP

During a study of b-amino ketone derivatives, which possess many biological properties, MPMAP77 was shown to exhibit antitubulin properties. A strong antimitotic agent,123 though about three-fold weaker than colchicine 1 in its effects, the ketone 77 represented a novel class of inhibitor whichwere both easy to synthesize and water-soluble. MPMAP 77 covalently binds to the sulfhydryl

groups of tubulin, and thus is irreversibly bound. Slight interference with the binding of colchicine1 has been observed, though it is not known whether MPMAP interacts with sulfhydryls located inthe colchicine site itself or a related nearby region.


J. PCMPS

Similarly to EBI 75, or p-chloromercuribenzenesulfonate, is a sulfhydryl-directed reagent often usedin experimental work to determine the properties of the tubulin sulfhydryl groups.124 Again, as withEBI 75, treatment of tubulin with PCMPS 78 leads to a disruption of the polymerization of tubulin.

K. Stypoldione

A marine natural product, secreted by the brown algae Stypopoldium zonale as a defensive measureagainst reef dwelling herbivorous fish,125 stypoldione 79 inhibits cell division in a number of mam-malian cell cultures. More recently, the o-quinone 79 has been shown to react covalently with thesulfhydryl groups of a number of small biomolecules and proteins, including tubulin.126 This dis-ruption of tubulin thus halts cell division, and stypoldione 79 is active at concentrations less than 10mM. The synthesis of stypoldione 79 has been accomplished.127,128

7. D R U G S W I T H O T H E R O R U N K N O W N B I N D I N G S I T E S

A. Avarol

Isolated129 from the abundant sponge Dysidea avara, avarol 80 is a potent cytostatic agent, presentin high quantities within the sponge. Studies of the quinol 80 shows potent antileukemic activity bothin vitro and in vivo,130 and acts by preventing microtubule formation. The total synthesis of avarol80 has been achieved.131

B. Azatoxin

Rationally designed as an analogue of podophyllotoxin 31 to inhibit the action of DNA topoisom-erase, the enzyme responsible for DNA coiling and supercoiling after replication, azatoxin 81 wasshown to be a potent antimitotic agent in vitro. It acts by inhibition of tubulin polymerization, witha potency comparable132 to that of etoposide 32.


C. Bis-ANS

Bis-ANS 82 was discovered in 1984 during research on fluorescent probes for locating hydrophilicregions on proteins.133 It binds strongly to apolar regions of tubulin, and inhibits in vitro polymer-ization at micromolar concentrations. Binding to tubulin increases the fluorescence of the molecule,indicating that, even if no therapeutic use can be found for the compound 82, it may serve as a use-ful probe for investigating microtubule assembly and the action of antimitotic drugs. Primary bind-ing to tubulin is noncovalent, and occurs at a single binding site. However, at higher concentrations,when its initial binding site has become saturated, Bis-ANS 82 additionally binds to six low-affini-ty binding sites. This binding increases the rate of alkylation of the tubulin sulfhydryls, indicatingthat a conformational change may be occurring upon binding, making this reactive functionalitymore accessible to alkylating agents.134

D. CIPC

Originally developed as carbamate herbicides, CIPC 83, and its less active parent compound IPC 84,were found to be potent microtubule disrupting agents in higher plants, and also in animal cells. Un-like most other spindle poisons, these carbamates 83, 84 do not bind to tubulin or to microtubules,but to the microtubule organizing centers (MTOCs), disrupting the organized growth of functionalmicrotubules.135

E. Cyclocreatine

Cyclocreatine 85 is a substrate-analogue inhibitor of the enzyme creatine kinase, first synthesized136

in the 1970s. This enzyme has been implicated in tumorigenesis and cyclocreatine 85 prevents thegrowth of a number of solid tumors.137 This cytostatic effect has been attributed to the formation of

stable microtubules which resist depolymerization by microtubule destabilizing agents.138 Further-more, the drug acts synergistically when administered with paclitaxel 104, highlighting a possiblecombination chemotherapeutic use. This, and the differing effects of cyclocreatine 85 and paclitax-el 104 on cells, indicates that these drugs perhaps do not bind to the same region of tubulin. Alsonoteworthy creatine kinase is over-expressed in tumor cells, indicating that cyclocreatine 85 maypreferentially target mutant cells, and thus exhibit lesser side effects in vivo.139


F. Diazepam

Diazepam 86 is a known hypnotic, and is able to halt cell division. Diazepam 86 binds to the ben-zodiazepine receptor, but this binding is not responsible for the observed inhibition of cell replica-tion.140 Diazepam 86 appears to induce shrunken mitotic spindles, and seems to weaken the con-nections between the microtubules and the MTOCs.141 Thus, it halts cell division by acting as aspindle poison, but in a way that is distinct from the majority of antimitotic agents. Lactam 86 is alsoless potent than most agents, with the microtubules recovering rapidly after drug treatment, where-as recovery from most spindle poisons is a gradual process.

G. 5,6-Diphenylpyridazin-3-ones

Originally synthesized142 as candidates for the treatment of hypertension, the diphenylpyridazi-nones, for example 87 and 88, were found to be effective herbicides. This herbicidal action wascaused by interaction with microtubules. The effects of the compounds 87, 88 are similar to thoseseen with colchicine 1, though they do not inhibit the binding of colchicine 1 to tubulin, nor do theyaffect the binding of the vinca alkaloids. Attempts are currently underway to produce radiolabeleddrugs, in an effort to localize and identify the binding site on tubulin.

H. Discodermolide

Isolated from the Caribbean sponge Discodermia dissoluta, discodermolide 89 is a potent immuno-suppressive lactone, which also inhibits microtubule function via the formation of abnormally sta-

ble microtubules.143 Competitive binding studies have shown that the binding of discodermolide 89and paclitaxel 104 are mutually exclusive, indicating that the compounds 89 and 104 bind to the sameor overlapping binding sites, though discodermolide 89 binds with a much higher affinity than doespaclitaxel 104.144 The microtubule stabilizing effects are seen at concentrations of around 10 nM,whereas similar effects with paclitaxel 104 require concentrations about 100-fold greater. The syn-thesis of discodermolide 89 has been accomplished.145


I. Ecteinascidin

Ecteinascidin 90, isolated146 from the tunicate Ecteinascidia turbinata, causes disorganization of mi-crotubules. However, the drug 90 does not appear to interfere directly with microtubules. Rather thandisrupting polymerization and depolymerization, the drug seems to prevent the attachment of as-sembled microtubules to the centrozome.147 These detached, disorganized fibers then aggregatearound the outside of the cell nucleus. This lack of organization prevents the microtubules from form-ing a mitotic spindle, and thus mitosis is halted. The synthesis of ecteinascidin 90 has beenachieved.148

J. Epothilones

A family of 16-membered macrolides isolated from the myxobacterium Sorangium cellulosum theepothilones (e.g., 91 and 92) exhibit both antifungal and cytotoxic properties.149 These epothilonesare competitive inhibitors of the binding of paclitaxel 104 to tubulin, exhibiting activity at similarconcentrations.150 This finding has led to the speculation that the epothilones and paclitaxel 104adopt similar conformations in vivo, and a common pharmacophore has recently been proposed.151

However, the epothilones are around 30 times more water-soluble than paclitaxel 104 and more read-

ily available, being easily obtained by fermentation of the parent myxobacterium.152 Additionally,the epothilones have been successfully prepared by total synthesis.153,154 Interestingly, theseepothilones also appear not to be recognized by multidrug resistant mechanisms and therefore showmuch higher potency than paclitaxel 104 in multidrug resistant cell lines.

K. Estramustine Phosphate

Estramustine phosphate 93 was first synthesized in the 1960s as a potential treatment for breast can-cer, shows considerable target selectivity. It was originally thought that phosphate 93, an oestradiolattached to a nitrogen mustard, would liberate the mustard moiety after uptake in cells.155 It washoped that the steroidal nature of the drug would increase its uptake in estrogen-receptor positive tu-mors. However, the drug 93 acts efficiently in cells lacking this estrogen receptor, and later workdemonstrated that the cytotoxic effects of the drug arise from microtubular interactions, and not fromthe effects of liberated nitrogen mustard.156 It was later elucidated157 that the drug binds to the mi-crotubule-associated proteins, specifically MAP-2, and 2 molecules of estramustine bind to each ofthe three tubulin binding domains of MAP-2.


L. Geiparvarin

Isolated158 from the leaves of Geijera parviflora, geiparvarin 94 does not to affect the rate of poly-merization of microtubules at doses below 500 mM, but effectively reverses159 the stabilizing effectsof paclitaxel 104 at a concentration of 10 mM. This coumarin 94 shows no effect on the binding ofcolchicine 1 or the exchangeable GTP nucleoside, and competitive inhibition of the paclitaxel bind-ing site may explain its mode of action.

M. Griseofulvin

Originally developed as a Penicillium derived antifungal agent for plants, but deemed too expensivefor agricultural use, griseofulvin 95 was found to have useful activity against fungal infections in hu-mans, and is a common prescription drug for these complaints. However, griseofulvin 95 inducesmajor conformational changes to the structure of tubulin, and acts as a weak destabilizing and de-polymerizing agent.160 These effects appear to occur in a concentration dependent manner,161 andaffect both a- and b-tubulin equally. The binding site of griseofulvin 95 has been subject to somecontroversy, with reports of it binding to the tubulin protein itself, or to the MAPs.162 Recent re-ports163 suggest that the binding is localized to tubulin itself, but that this binding appears to differ

from that of colchicine 1, vinblastine 40 and paclitaxel 104. Further analysis of the binding indicat-ed the presence of only a single binding site, but the nature of this site is still unclear. Owing to theseproperties, griseofulvin 95 is now under investigation as a possible antimitotic agent.


N. 2,5-Hexanedione

Prolonged exposure to the solvent n-hexane causes many adverse effects in humans, in particular se-vere neuropathy, and it is believed that the ultimate in vivo metabolite, 2,5 hexanedione 96 is respon-sible for these toxic effects.164 The dione 96 can cause the formation of extensive inter- and in-tramolecular cross-links across adjacent lysine residues of tubulin heterodimers.165 These cross-linkedtubulin dimers form microtubules at a greater rate than untreated tubulin, and appear to be more cold-stable than their noncross-linked counterparts. 2,5-Hexanedione 96 forms pyrrole derivatives with ly-sine,166 and this process may be partially involved with the above cross-linking action.

O. HO-221

The synthetic benzoylphenylurea derivative HO-221 97 is not only of interest because of its abilityto prevent the formation of the mitotic spindle, but also because of its low toxicity. In animal exper-iments, pyrimidine 97 was found to have a potency similar to that of colchicine 1, but these resultsindicated that the drug causes little suppression of white blood cell production and also may not causesystemic toxicity, thus reducing its side effects.167

P. IKP-104

The synthetic pyridinone IKP-104 98 induces mitotic arrest168 in cultured cells and causes break-down of the mitotic spindle, leaving abnormal chromosomes scattered throughout the cell. These ef-

fects are similar to those induced by the vinca alkaloids, though no evidence exists as yet to suggestthat the drug 98 binds to the vinca alkaloid site on tubulin.

Q. Lupeol

Isolated169 as a major component of the leaves of Lxora coccinea, lupeol 99 shows activity as ananti-inflammatory agent, and additionally inhibits cell division. Studies indicate that this mitoticblock may arise by a combination of three modes of action. Firstly, the compound 99 may inhibitDNA replication. Secondly, it may induce depolymerization of existing DNA, destroying the dou-ble helix, and thirdly, it appears to induce the breakdown of microtubules, preventing chromosomeseparation.


R. Lidocaine

A local anesthetic, commonly used in sunburn remedies, lidocaine 100, and to a lesser extent its sis-ter compound procaine 101, affect the polymerization of tubulin into microtubules.170 It appears thatthe amide 100 binds to tubulin and prevents further polymerization, rather than causing the depoly-merization of formed microtubules. The compound 100 is much less potent than colchicine 1, andthe effects are only observed at levels far in excess of those required to give a local anesthetic effect.Similar effects have also been observed with inhalation anesthetics, such as halothane and methoxy-fluorane, which cause rapid and reversible dispersion of microtubules in cell culture.171

S. Melatonin

Melatonin 102, (N-acetyl-5-methoxytryptamine) is a natural pineal gland hormone which inducesthe aggregation of a number of granular structures within cells. This granulation effect inhibits theeffect of colchicine 1 on microtubules,172 but the indole 102 has since been shown to cause micro-tubule inhibition in its own right.173

T. Nostodione A

Isolated174 from the blue–green algae Nostoc. commune, Nostodione A 103 disturbs mitosis in seaurchin eggs. This effect was caused by microtubular disruption, in a manner almost identical to thatof colchicine 1.

U. Paclitaxel (Taxol)

Isolated175 in 1962 as part of the American National Cancer Institute (N.C.I.) natural products screen,which screened over 35,000 plant extracts in 22 years, paclitaxel 104 was largely ignored as an an-timitotic agent owing to a number of seemingly insurmountable problems (low availability, poor wa-ter solubility). However, when it was discovered176 that paclitaxel 104 was a microtubule stabilizer,rather than a destabilizer (like every antitubulin compound then known) the level of research in-creased dramatically, resulting in paclitaxel 104 becoming one of the most popular areas of phar-maceutical interest for many years. Paclitaxel 104 is now clinically used to treat ovarian and breastcancer. Four groups have succeeded177,178 in the total synthesis of paclitaxel 104.


V. Taxotere (Docetaxel)

Discovered as a late-stage intermediate in a synthetic strategy toward paclitaxel 104, taxotere 105shows a similar spectrum of action to paclitaxel 104, but with a four-fold increase in potency and im-proved water solubility.179 However the improved water solubility of taxotere 105 reduces the com-plexity and side effects of administration180,181 in comparison to paclitaxel 104. Clinical trials to es-tablish the scope of the drug’s usefulness are currently in progress and taxotere 105 has already beenlicensed for the treatment of breast and ovarian cancers.

Produced semisynthetically179 from a precursor readily obtained from the needles of the yewtree, taxotere 105 is less encumbered by the problem of supply which troubled the early develop-ment and use of paclitaxel 104. This improved availability, allied with its increased efficacy, wide

range of clinical activity and reduced side effect almost guarantee the future liability and success oftaxotere 105 as a clinically useful agent.

W. Pyrimidine Nucleoside Analogues

The exchangeable GTP nucleoside, bound to b-tubulin is often implicated as an important part ofthe regulation of microtubule dynamics. Recently, two GTP analogues (106, 107) have been shownto bind very weakly to this region of b-tubulin. Though these analogues 106, 107 barely show anybinding, assembly of polymerized microtubules was found to occur at much lower concentrationsthan with the normal GTP nucleoside, and these microtubules also exhibited much greater stabilityto cold and calcium ions.182


Similarly, the natural product tubercidin 108, extracted183,184 from the cyanobacterium Plec-tonema Radiosum and the bacteria Streptomyces tubercidius, protects polymerized microtubulesfrom destabilizing agents such as the vinca alkaloids. However, it is thought that tubercidin 108 bindsnot to the GTP site on b-tubulin, but to an ATP binding site on a-tubulin.

X. Rhazinilam

First isolated185 from Rhazya stricta, and probably derived from auto-oxidation of the parent alka-loid dehydroasidospermine, (2)-rhazinilam 109 acts as an inhibitor of tubulin polymerization, func-tioning in a manner similar to that of the vinca alkaloids.186 It is interesting to note that this activityis absent both from the parent alkaloid and from its optical isomer (1)-rhazinilam, indicating thatthe activity is an artifact of the spatial arrangement of the aromatic system.

Y. Spatol

Though there is no direct evidence for the binding of spatol 110 to tubulin, the epoxide 110 (isolat-ed from the brown seaweed Spatoglossum schmitti and recently synthesized187) was highly active intests against sea urchin eggs.188 This assay is highly selective for antitubulin agents, and thus it hasbeen inferred that spatol 110 exerts its cytostatic effects by interaction with tubulin and microtubules.

Z. Welwistatin

A member of a family of alkaloids isolated from the blue–green algae Hapalosiphon welwitchii, wel-wistatin 111 caused depolymerization of microtubules in vitro.189 This effect is reversible upon re-moval of the drug 111, and competitive assays show no inhibition of colchicine or vinca alkaloidbinding, nor interference with the binding of GTP. However, welwistatin 111 is resistant to elimina-tion by the multidrug resistance pump, and appears to weakly reverse its effects.190


AA. Eleutherobin

Eleutherobin 112 a diterpene glycoside, isolated from an unknown species of Eleutherobia, is a po-tent cancer cell inhibitor with an IC50 range of 10–15 nM in vitro in a diverse panel of tumor tissuecell lines.191 The glycoside 112 is particularly potent toward breast, renal, ovarian, and lung cancercell lines. Like paclitaxel 104, eleutherobin 112 stabilizes microtubules by competing for the pacli-taxel binding site on the microtubule polymer.191

BB. Protoanemonin

Isolated originally from Ranunculus bulbosus for its antimicrobial properties,192 protoanemonin 113is a simple lactone which exhibits a wide spectrum of activity against various nucleated organisms.Though little was known about the mode of action of the compound 113, its spectrum of activity sug-gested a site of action common in many biological systems. It has now been shown193 that lactone113 primarily targets intracellular microtubules.

8. C O N C L U S I O N

This review clearly highlights the importance of tubulin as a target for anticancer drugs. Tubulin caninteract with a range of structurally diverse natural and synthetic agents. Compounds can react orbind at various sites on tubulin or with sulfhydryl groups on tubulin. Also several antimitotic agents(paclitaxel, the vinca alkaloids) are clinically effective.

The recent successes with paclitaxel has led to renewed interest in natural product chemistry.The discovery of agents such as the epothilones and eleutherobin, which act in a similar way to pac-litaxel, has stimulated great activity among chemists, biologists, and clinicians. It is evident thatmany new drugs for the treatment of cancer will act by targeting tubulin.

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Allan Jordan was born in Stockport, England in 1972, and received his B.Sc. in Chemistry from UMIST in1993. After a semester as a Graduate Teaching Assistant at Arizona State University, he returned to the U.K. tocommence his postgraduate studies under the direction of Dr. Nicholas Lawrence and Dr. Alan McGown. Thesestudies focused upon the synthesis and mode of action of anticancer agents derived from taxane natural prod-ucts. He was awarded his Ph.D. in July 1997, and he is currently a Postdoctoral Fellow at the University ofReading, investigating prodrug treatment strategies for malignant melanoma, with Dr. Helen Osborn.

John Hadfield graduated from Nottingham University, U.K., in 1979 with a B.Sc. in Chemistry. In 1984 he wasawarded a Ph.D. at Nottingham Trent University for his work on isoquinoline chemistry. Between 1984 and1986 he worked as a Postdoctoral Research Fellow in the Department of Chemistry at the University of War-wick studying the biosynthesis of riboflavin. In 1987 he was appointed as a Postdoctoral Fellow at the Pater-son Institute for Cancer Research, Manchester, U.K. where he has studied a range of topics including alkylat-ing agents, protein tyrosine kinase inhibitors, and anti-mitotic agents. At present he leads a team which isdeveloping anti-vascular agents.

Nicholas Lawrence graduated in Natural Sciences from Cambridge University in 1985. He remained at Cam-bridge to work with Dr. Ian Fleming, FRS, gaining his Ph.D. in 1989, having investigated synthetic organosili-con chemistry and the synthesis of the pancreatic lipase inhibitor tetrahydrolipstatin (orlistat). This was fol-lowed by postdoctoral research at Leicester University, U.K., with Dr. Paul Jenkins, involving synthetic routestowards paclitaxel. He was appointed as a lecturer at UMIST in 1991 and Senior Lecturer in organic chemistryin 1997. His research interests include the development of new synthetic methods and the synthesis, design, andisolation (from medicinal herbs) of potential anticancer drugs.

Alan McGown was awarded a B.Sc. in Chemistry from the University of Newcastle-upon-Tyne, U.K., in 1975.In 1979 he gained Ph.D. from the University of Newcastle-upon-Tyne for his work on Radiation Chemistry. Hewas appointed to a Postdoctoral position at the Paterson Institute for Cancer Research, Manchester U.K. in1979. In 1993 he was appointed as Head of Experimental Chemotherapy at the Paterson Institute and as anHonorary Lecturer at the University of Manchester. In 1997 he was selected as Section Head of Drug Devel-opment and Imaging at the Paterson Institute. He is Liaison and Information Officer for the Cancer ResearchCampaign.


tubulin as a target for anticancer drugs: agents which interact with the mitotic spindle

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