(CANCER RESEARCH 46, 1889-1893, April 1986J

mine structure-function correlations among presently availablecompounds. Our initial findings are reported here, togetherwith a summary of unpublished observations in plant tissues.

MATERIALS AND METhODS

Materials.All derivativesof DPP werepreparedas describedpreviously (1). GTP, vinblastine, colchicine, and MES were obtained fromSigma podophyllotoxin was from Aldrich; (8-'4CJGTP was from Moravek Biochemicals and (3HJcolchicineand (3HJvinblastinewere fromAmersham. Maytansine was provided by the Natural Products Branch,National Cancer Institute, and [3Hjmaytansinewas a generous gift ofDr. D. G. Johns (9). GTP and [8-'4CJGTPwere repurifiedby ionexchange gradient chromatography. Electrophoretically homogeneouscalf brain tubulin and MAPs purified by DEAE-cellulose chromatography were preparedas described previously (10). All drugs weredissolved in dimethyl sulfoxide, and control reaction mixtures alwayscontained an equivalent amount of the solvent.

Methods. Measurement ofGTP hydrolysisand colchicine binding totubulin, turbidimetric analysis of microtubule assembly, and electronmicroscopy of negatively stained specimens were performed as described elsewhere (10, 11). Measurement of the binding of radiolabeledvinblastineandmaytansineto tubulinwasperformedbythecentrifugalgel filtrationmethodof Penefsky(12) as describedin detailpreviously(13). Analysis of drug effects on the mitotic index and on cytotoxicityin cell culturewas performedwith murine L1210 leukemiacells, asdescribed by Wolpert-DeFilippes et a!. (14).

RESULTS

The basic structure of the DPP derivatives described here ispresented in Fig. 1, with structural details presented in Table1. A variety of substituents at position 2 of the pyridazinonering and on the two phenyl rings have been observed in activecompounds (in all cases the substituents on the phenyl ringshave been identical). Virtually all active agents in both plantand mammalian systems have had a nitrile group at position 4.

Summary of Findings in Plant Tissues. The observation ofsignificant herbicidal activity in the DPP derivative NSC362448 led to efforts to define its mechanism ofaction. Studieswith root tip tissue demonstrated defective cell division, withcoalescence of condensed chromosomes in the cell center, andthe disappearance of microtubules. Unusual new structureswere also observed: macrotubules about 75 nm in diameter, 3times as wide as normal microtubules. A large number of DPPderivatives were also observed to inhibit flagellar regrowth inChiamydomonascells (cf Refs. 15 and 16). In this study theprimary comparison was between a group of derivatives withvaried substituents at position 2 of the pyridazinone ring, anitrile group at position 4, and unsubstituted phenyl rings.Although some of the compounds inhibited flagellar regrowthby 50% at drug doses as low as 0.4 gsM,none of the agents mostactive in inhibiting flagellar regrowth were among the drugsmost active in inhibiting bovine tubulin polymerization in vitro.

Demonstration That Tubulin Is the Probable Target of DPPDerivatives in Mammalian Tissues. A group of the DPP denyatives was submitted to the National Cancer Institute for screen

ABSTRACT

A series of derivatives of 5,6-diphenylpyridazin-3-one (DPP) wasexamined for interactions with calf brain tubulin followingthe demonstration that many members ofthe class caused significant mitotic effectsinintactanimals,whileothershadactivityagainstmarineP388leukemia.In L1210cells severalDPP derivativescauseda rise in the mitoticindexwhich correlated well with the cytotoxicity of the drugs. Active DPPderivatives markedly stimulated tubulin-dependent guanosine triphosphatehydrolysisandinhibitedtubulinpolymerizationor inducedtubulinoligomerformation,dependingonspecificreactionconditions.Thesenewagents, however,did not interfere with the bindingto tubulin of radiolabeled colchicine, vinblastine, maytansine, or guanosine triphosphate.They thus appearto bindat a previouslyUndescribedsite on the tubulinmolecule. Some DPP derivativeshave significantherbicidalactivity,causing mitotic disruption and a rise in the mitotic index in seedlingroottissues. Although the DPP derivatives most toxic to plant tissues differfromthose most active in inhibitingcalf braintubulinpolymerization,virtually all active compoundsbear a nitrile substituent at position 4 ofthe pyridazinonering. Most activederivativesalso bearsubstituentsofvarying structure at position 2 ofthis ring, but no clear structure-functionpattern is apparent at this position. The phenyl rings in the most activeherbicidal DPP derivatives either are unsubstituted or bear fluorineatoms. Derivativeswith chlorinesubstituentshave no detectableherbicidal activity. In contrast, interactions with calf brain tubulin are substantiallyenhancedif the phenylringsbearchlorinesubstituents.

INTRODUCTION

The observation of antihypertensive activity for a DPP2 derivative led to the synthesis of several analogues to explorestructure-activity relationships in this novel class of compounds(1, 2) (see Fig. 1 and Table 1). Although several of these agentswere active in reducing blood pressure in a spontaneouslyhypertensive rat model (1, 2), some DPP derivatives displayedsignificant herbicidai activity (3). Preliminary toxicologicalstudies of the herbicidal compounds included the observationof bizarre mitotic figures in renal tissue (4, 5). A number of theagents were therefore submitted to the National Cancer Institute for screening for antineoplastic activity, and a significantincrease in survival of mice given simultaneous injections ofP388 leukemia cells and selected DPP derivatives was observed.Since we have been screening antineoplastic drugs of uncertainmechanism ofaction for their effects on tubulin-dependent GTPhydrolysis (6—8)to identify new antimitotic agents, we exammed these compounds. They were found to stimulate GTPhydrolysis dramatically, and as a consequence we have exammed in detail the interactions ofthis class ofdrugs with tubulin.In particular, we wanted to establish that they were antimitoticagents in mammalian cells, determine their effects on tubulinpolymerization, locate their binding site on tubulin, and deter

Received 8/2/85; revised I 1/1/85; accepted 12/23/85.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I To whom requests for reprints should be addressed, at Building 37, Room

6D28, NIH, Bethesda, MD 20892.2 The abbreviations used are: DPP, 5,6-diphenylpyridazin-3-one; MES, 2-(N-

morpholino)ethanesulfonate;MAPs, microtubule-associatedproteins.

1889

Derivatives of 5,6-Diphenylpyridazin-3-one: Synthetic Antimitotic Agents WhichInteract with Plant and Mammalian Tubulin at a New Drug-bindingSite

Janendra K. Batra, L. J. Powers, F. Dana Hess, and Ernest Hamel'

Laboratory ofPharmo.cology and Experimental Therapeutics,Developmental TherapeuticsProgram, Division ofCancer Treatment, National Cancer Institute, NIH,Bethesda,Maryland 20892 fJ. K. B., E. H.]; AgriculturalChemicaLsSection,SDS BiotechCorporation,Painesvile,Ohio 44077 fL. I. P.1;and Departmentof Botanyand Plant Pathology,PurdueUniversity,WestLafayette,Indiana 47907fF. D. H.]

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Table 2 Stimulation oftubulin-dependent GTP hydrolysis by DPP derivativesEach 50-jd reaction mixture contained tubulin (1 mg/mI), 0.1 mr@i(8-―C@GTP,

the indicated drug at 0.1 mr@i,1%dimethyl sulfoxide (v/v), 0.1 M MES (pH 6.4),and 0.5 mM MgCI2. Incubation was for 20 mm at 37'C. Data are expressed asnmol of (8-'4CJGDP formed per ml of reaction. This experiment was performedtwice with comparable results. In addition, stimulation of GTP hydrolysis byDPP derivatives was observed in six experiments in 1 M glutamate and in twoexperiments with heat-treated MAPs (cf Ref.8).Drug

added nmol GDPformedNone

1.7Colchicine 8.9NSC 362453 44.8

5,6-DIPHENYLPYRIDAZIN-3-ONE DERIVATIVES

Fig. I . Structural formula of the DPP derivatives.

Table I Inhibition ofmicrotubule assembly by DPP derivativesID,@ values were established in 0.25-ml reaction mixtures containing tubulin

(1.0 mg/mI), MAPS purified by DEAE-cellulose chromatography (0.5 mg/mI)(10), 0.1 M MES (pH 6.9), 0.5 mM MgCl2, 1.0 mr@iGTP, 2% (v/v) dimethylsulfoxide, and varying concentrations of the indicated drugs. ID,o values werebasedon a minimum of two independentdeterminations.

Drug (NSC no.) Xe Rb

362458362460601680601683602747603334

a ID,@,, concentration of drug resulting in a 50% reduction in the turbidityreading after a 20-mm incubation at 37T.

a i@ocationsof X and R in the DPP derivatives is as indicated in Fig. 1.

0

z00

362449362453362455602746

351477351478351482602744

HCI at 4'Cl at 3' and 4'CI at 3' and 4'

Cl at 4'Cl at 4'Cl at 4'Cl at 4'

HCl at 4'CH,at4'Br at 4'CI at 4'Cl at 4'

HCl at 2'Fat 4'Fat 4'Cl at 2'

HHH

OCH, at 4'HCl at 4'CI at 4'

0.1 1.0 10 100

@LMDRUG

Fig. 2. Cytotoxic and antimitotic effects of DPP derivatives on murine L1210leukemia cells. These studies were performed as described by Wolpert-DeFilippesetal. (14). Dimethyl sulfoxideconcentrationwas 1%(v/v). 0, 0, @,mitotic index(control, 2.8% mitoses); •,U, A, cytotoxic effects of the drugs (control, 3.9 x10' cells/mI). Details of drug structures are presented in Table 1. NSC 362453(0, •);NSC 351478 (Lx,A); NSC 602746 (0, U). This study was performed inits entirety a single time. Similar results were obtained with NSC 362453 in asecond experiment. In addition, a high mitotic index was obtained two additionaltimes in a cell culture treated with 100 @MNSC 351478.

Fig. 3. Inhibition of microtubule assembly by NSC 362449. Each 0.25-mireaction mixture contained tubulin (0.75 mg/mI), MAPs purified by DEAEcellulose chromatography (0.38 mg/mI), 1.0 mi.i GTP, 0.1 M MES (pH 6.9), 0.5mM MgCl2, 2% (v/v)dimethyl sulfoxide, and NSC 362449 (see Table 1 for detailsof drug structure) at the following concentrations: Curve 1, none; curve 2, 5 @;Curve3, 7.5 aiM;Curve4, 10 @LM.At zero time the electronic temperaturecontroller(see Ref. 10) was set at 37'C; at the time indicated by the arrow on the abscissa,the temperature controller was set at 0C. Similar results were obtained in at leastfour experiments.

1D,@,5-10 @LAf

CH2COOH(CH2)@-O-CO(CH2)2COOH(CH2)20HH

ID,,@,10-15 pM

H(CH2)20H(CH2),COOH(CH2),COOH

ID,@,15-25 pM

CH3(CH2)2-O-COCH2NH2.HBrHHCH2CO-OCH,CH2COCH,

ID,@,25—50pM

CH@CO-O-CH2CH,(CH2)20H(CH2)20HHH

JD,4,50-100

(CH2)20HH(CH2)2COOH

ID,@,> 100 pM

(CH2)20H(CH2hCO-O(CH2)2OCH,(CH2),CO-OCH2CH,(CH2hCI

362448362450362454602745602748

362444362445362451

362447601681601682601684

0;

MINUTES

ing for antineoplastic activity. Several compounds proved activeagainst murine P388 leukemia, but these differed from the most

. active herbicides. (The most promising drugs at this point

appear to be NSC 351478 and NSC 362454.) Nevertheless,since tubulin appeared to be a likely target for this class ofagents, we examined the effect of DPP derivatives on tubulindependent GTP hydrolysis (6—8).Many of these drugs stimulated the reaction under three different conditions (in 1 Mglutamate and in 0. 1 M MES both with and without heattreated MAPs). Table 2 compares the potent stimulation ofGTP hydrolysis by NSC 362453 in 0.1 M MES without MAPs

to the much less marked effect of colchicine, the first drugreported to stimulate tubulin-dependent GTP hydrolysis (17).

The GTPase studies and the observations in plant tissuespredict that the DPP derivatives should cause mitotic arrest inmammalian cells in culture. This was confirmed with threedrugs studied in murine L1210 leukemia cells (Fig. 2). The risein mitotic index correlated well with cytotoxic effects of the 3compounds.

DPP Derivatives Inhibit Microtubule Assembly and/or InduceTubulin Oligomer Formation. Generally antimitotic agents inhibit microtubule assembly in vitro. This was readily demonstrated with many DPP derivatives (Fig. 3; also see Table 1).

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5,6-DIPHENYLPYRIDAZIN-3-ONE DERIVATIVES

@1 f@.@ :*c@

Fig. 4. Morphology of tubulin oligomersinduced with NSC 362453 (see Table 1 fordetails of drug structure) and vinblastine. Aand B, oligomers formed with NSC 362453.The 0.25-mi reaction mixture contained tubuun (1.0 mg/mI), 0.1 mM GTP, 1.0 M monosodium glutamate (pH 6.6), 1.0 mM MgCI2,4% (v/v) dimethyl sulfoxide, and 0.1 mM NSC362453. After 20 mm at 37'C, a negativelystained specimen was prepared and examinedin the electron microscope. C, oligomersformed with vinbIastine. Reaction mixture asabove,exceptthat the drugwas 10pMvinbiastine instead of NSC 362453. A, x 7,900 BandC, x 52,700.

4 44

One of the most inhibitory members of this class of drugs wasNSC 362449. In a system consisting of purified tubulin andMAPs (Fig. 3), progressive inhibition ofnucleation, elongation,and extent of polymerization was observed as the drug concentration was raised, with complete inhibition occurring at 10 tiM.In a second series of experiments we demonstrated that glycerol-induced polymerization of purified tubulin (18) was inhibited by NSC 362449 (data not presented), confirming that thetarget of the DPP derivatives is tubulin and not the MAPs (aspredicted by the GTPase experiments without MAPs).

In 1.0 Mglutamate several of the DPP derivatives were foundto induce cold-irreversible turbidity development rather thansimply inhibit cold-reversible polymerization, as is the case withmost other antimitotic drugs (6—8).Electron microscopic examination of reaction mixtures at 37°Cdemonstrated the for

mation ofwell-defined helical oligomers. Fig. 4A presents a lowmagnification (x7,900) view of these structures; Fig. 4B showsa high magnification (x52,700) view. There is a clearly visiblefilamentous substructure, perhaps representing protofilaments(2 and occasionally 3 filaments can be distinguished). Thehelical oligomers are relatively short, and the repeat distancein the helix is about 90 nm. Neither turbidity development noroligomer formation required GTP, and GTP had little effecton this reaction; nor is there turbidity development or oligomerformation at low ionic strength without MAPs. With somepreparations of MAPs, DPP derivative-induced turbidity development also occurs, but the structures formed are not asordered as those in glutamate. We have not yet been able tocorrelate DPP derivative-induced turbidity development with aspecific MAP component (cf Ref. 19).

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I@ ‘

C

•.‘%@4..

‘..

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addedCoichicine°VinblastinebMaytansinebNone100100100NSC

362449106130103NSC362453100107100Podophyilotoxin1.311399Maytansine1036.9Vinblastine10145

5,6-DIPHENYLPYRIDAZIN-3-ONE DERIVATIVES

Table 3 Failure ofDPP derivatives to inhibit the binding of radiolabeleddrugsto tubulin

% of control after followingradiolabeled drug added

equivalent to a bromine at that position and that a compoundwith a chlorine substituent at position 4' is about 3—4timesmore active than one with a chlorine at position 2', which is inturn 2—3 times more inhibitory than an unsubstituted corn

pound.Finally, Fig. 5 presents the structures of three inactive corn

pounds. The two phenyl rings seem to be essential for inhibitionof microtubule assembly, and they must have at least somedegree of mobility (alternatively, the fused structure presentedin Fig. 5 may represent an unfavorable conformation for an

interaction with tubulin). All compounds with no substituentor a large substituent at position 4 of the pynidazinone ringhave been inactive.

DISCUSSION

The DPP derivatives are a new rnultiagent class of antimitoticdrugs, many ofwhich interact directly with bovine brain tubulin.Although no direct interaction with plant tubulin has beendocumented, it is likely that their effects in inhibiting flagellarregrowth and inducing mitotic disruption derive from an analogous interaction with plant tubulin. It must be emphasizedthat specific DPP derivatives have maximum inhibitory activityonly with one class of organisms. Those most active in planttissues interact only weakly with bovine tubulin and vice versa.In structure-function studies on herbicidal activity, compoundswith unsubstituted phenyl rings were highly active, as werethose with fluorine substituents. Chlorine groups on the twophenyl rings eliminated herbicidal activity.3 The opposite is thecase for interactions of DPP derivatives with bovine tubulin.

The interactions of the DPP derivatives with tubulin arereminiscent of colchicine and many colchicine analogues instimulating tubulin-dependent GTP hydrolysis even as theyinhibit the microtubule assembly reaction associated with GTPbreakdown (6—8,17). However, these drugs do not inhibit thebinding of colchicine to tubulin.

The DPP derivatives are also reminiscent of the Vinca alkabids in that they inhibit microtubule assembly and inducetubulin oligomer formation. In addition, distinct new structures(macrotubules) were observed in DPP derivative-treated seedling roots. Although it is not yet known whether these macrotubules contain tubulin, they may bear some relationship to theoligomeric structures formed from purified bovine tubulin inglutamate. Perhaps analogously, the tight spirals induced byVinca alkaloids in vitro (19, 20) differ greatly morphologicallyfrom the tubulin paracrystals observed in cells treated withVincaalkaloids.Nevertheless,the DPP derivative-inducedoilgomer differs morphologically from the Vinca alkaloid-inducedoligomer, and DPP derivatives are unable to inhibit the bindingof either vinbiastine or maytansine to tubulin (9). Moreover,both vinblastine and maytansine inhibit rather than stimulatetubulin-dependent GTP hydrolysis (7, 8, 17).

The DPP derivatives thus seem to bind to a previouslyundescribed site on tubulin, for they also do not inhibit thebinding of guanine nucleotides at the exchangeable GTP site.Definitive proofofthis hypothesis requires radiolabeled agents,and we are attempting their synthesis.

Some ofthe DPP derivatives (particularly NSC 351478) wereeffective in the treatment of P388 leukemia in mice. Thestructure-activity studies suggest promising leads for the synthesis of additional compounds. While the substituents at position 2 of the pyridazinone ring do not fall into a clear pattern,

3 L. J. Powers, unpublished data.

a Each 0.1-mi reaction mixture contained tubuiin (0.1 mg/mI), 5 pM (3HJ

colchicine, the competing drug at 0.5 m@.i,and 5% (v/v) dimethyl sulfoxide.Further reaction components and further experimental details are as describedpreviously (10). Comparable results were obtained in three different experiments.

a Each 0.4-mi reaction mixture contained tubulin (1.0 mg/mi), 0.1 M MES(pH 6.9), 0.5 mM MgC12,0.1 mr.iGTP, 2.5% (v/v) dimethyl sulfoxide, and either25 pM (3Hlvinbiastine with the competing drugsat I mM or 10 pM (3Hjmaytansinewith the competing drugs at 0.25 mM. Incubation was at room temperature for20 mm. Comparable results were obtained in three different experiments withvinblastine. The maytansine experiment was performed once.

cH3-tk@,.AO @)H2 CH2OH

@ CH2 OH

c@.N

CH2OH

Fig. 5. Formulas of compounds which are structurally related to the DPPderivatives and which do not inhibit in vitro microtubule assembly.

The Vinca alkaloids have long been known to induce formation of aberrant tubulin structures iii vitro and in vivo, includingformation of oligomers of purified tubulin in 1.0 M glutamate.To determine whether there was any relationship between theDPP-induced oligomers and those formed with vinblastine, weexamined the morphology of the vinblastine-induced structures(Fig. 4C). Although these were also helical, they were coiledmuch more tightly and appeared to be considerably larger thanthose formed with the DPP derivatives. The vinblastine-inducedstructures formed in 1.0 M glutamate are morphologically similar to those described by other workers (19, 20) using differentreaction conditions.

Effects of DPP Derivatives on Drug and Nucleotide Bindingto Tubulin. To determine whether the DPP derivatives boundat a previously described site on tubulin, we examined the effectof these drugs on the binding of radiolabeled colchicine, yinblastine, and maytansine (Table 3). No inhibitory effect wasobserved. These new agents also do not affect the binding ofGTP to tubulin (data not presented), as predicted by their briskstimulation of GTP hydrolysis requiring turnover of GTP atthe exchangeable site.

Structure-Activity Relationships. The availability of a largenumber of DPP derivatives permitted a relatively extensiveanalysis of the structural requirements for interaction of thisclass of drug with tubulin in vitro. Our most important findingsare summarized in Table 1, in which the drug concentrationrequired to inhibit the extent of microtubule assembly by 50%after 20 minutes at 37°Cwas determined. The drug concentrations required to inhibit reaction rate by 50% were considerablylower, but relative activities of the drugs were unaltered.

No clear pattern ofactivity related to substituents at position2 of the pynidazinone ring emerged from these experiments. Infact, two ofthe most active inhibitors ofassembly(NSC 351477and NSC 602746) were unsubstituted at this position. A clearpattern is observed, however, when the symmetrical substituentson the phenyl rings are considered. Activity is progressivelyenhanced with a fluorine atom at position 4', a bromine at 4',a chlorine at 4', and two chlonines at 3' and 4'. Availablecompounds also indicate that a methyl group at position 4' is

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5,6-DIPHENYLPYRIDAZIN-3-ONE DERIVATIVES

alterations at this position can have major effects on the activityof the resulting compounds. More specific was the effect ofchlorinating the phenyl rings. Position 4' seems most important, but a second chlorine at position 3' further enhanced invitro inhibition of microtubule assembly.

Position 4 of the pyridazinone ring is also of major importance. Virtually all compounds which inhibit polymerization ofbovine tubulin or which have herbicidal activity bear a nitrilegroup at this position. A large number of inactive compoundshave been examined bearing a variety of substituents (or noneat all; see Fig. 5). A single compound of this group (analogousto NSC 362449, but bearing a chlorine instead of a nitrile atposition 4) has had activity. This compound was about onethird as effective as NSC 362449 in inhibiting microtubuleassembly. Thus, while the nitrile group cannot be consideredessential for inhibitory activity in the DPP derivative series, itis nonetheless ofmajor importance. It appears that a substituentis required at position 4 and that it must be relatively small.

ACKNOWLEDGMENTS

The authors would like to thank Dr. K. Paull for suggesting thiscollaboration and T. Januszewski for performing the electron microscopy.

REFERENCES

I. Buchman, R., Scozzie, J. A., Ariyan, Z. S., Heilman, R. D., Rippin, D. J.,Pyne, W. J., and Powers, L. J. Antihypertensive 5,6-diarylpyridazin-3-ones..J. Med. Chem., 23: 1398—1405,1980.

2. Fogt. S. W., Scozzie, J. A., Heilman, R. D., and Powers, L. J. Lipophilicand hydrophilic esters of 4-acetyi-2-(2-hydroxyethyl)-5,6-bis(4-chlorophenyl)-2H-pyridazin-3-one as hypertensive agents. J. Med. Chem., 23:1445—1448,1980.

3. Pyne, W. J., Lowbridge, J., Chang, I. K., Knotz, F., and Powers, L. J.European Patent Application 81303611.8.

4. Mizens, M., Brown, W. R., Laveglia, J., Killeen, J. C., Jr., and Ignatoski, J.A. Combined 30-day feeding study and one-generation reproduction study in

rats with ethyl 5-cyano-3,4-diphenyl-6-oxo-1@6HJ-pyridazineacetate (DS29399). Toxicologist, 4: 17, 1984.

5. Mizens, M., Brown, W. R., Killeen, J. C., Jr., and Ignatoski, J. A. Subchronicfeeding study in rats with five pyridazinone compounds structurally relatedto DS-29399. Toxicologist, 5: 60, 1985.

6. Hamel, E., and Lin, C. M. Interactions of combretastatin, a new plantderived antimitotic agent, with tubulin. Biochem. Pharmacol., 32: 3864—3867, 1983.

7. Hamel, E. Antimitotic drugs and tubulin-nucleotide interactions. In.@R. I.Glazer (ed.), Developments in Cancer Chemotherapy, pp. 131—164.BocaRaton, FL: CRC Press, 1984.

8. Batra, J.K., Jurd, L., and Hamel, E. Structure-function studies with derivatives or6-benzyl-l,3-benzodioxole, a new class of synthetic compounds whichinhibit tubulin polymerization and mitosis. Mol. Pharmacol, 27: 94-102,1985.

9. Mandeibaum-Shavit, F., Wolpert-fleFilippes, M. K., and Johns, D. G. Binding of maytansine to rat brain tubulin. Biochem. Biophys. Res. Commun.,72:47—54,1976.

10. Hamel, E., and Lin, C. M. Separation of active tubulin and microtubuleassociated proteins by ultracentrifugation and isolation of a componentcausing the formation ofmicrotubuIe bundles. Biochemistry, 23:4173—4184,1984.

II. Gaskin, F., Cantor, C. R., and Shelanski, M. L. Turbidimetnc studies of thein vitroassemblyanddisassemblyofporcine neurotubuies.J. MoL BioL,89:737—758,1974.

12. Penefsky, H. S. Reversible binding of P@by beef heart mitochondrial adenosine triphosphatase. J. BiOLChem., 252: 2891-2899, 1977.

13. Hamel, E., and Lin, C. M. Guanosine 5'-O-(3-thiotriphosphate), a potentnucleotide inhibitor of microtubule assembly. J. Biol. Chem., 259: 11060—11069, 1984.

14. Wolpert-DeFilippes, M. K., Bono, V. H., Jr., Dion, R. L, and Johns, D. G.Initial studies on maytansine-inducedmetaphase arrest in L1210 murineleukemia cells. Biochem. Pharmacol., 24: 1735—1738,1975.

15. Hess, F. D. The influence ofthe herbicide trifluralin on flagellar regenerationin Chiamydomonas. Exp. Cell Res., 119: 99—109,1979.

16. Quader, H., and Filner, P. The action of antimitotic herbicides on flageilarregeneration in Chiamydomonas reinhardtii: a comparison with the action ofcolchicine. Eur. J. Cell BioL, 21: 301—304,1980.

17. David-Pfeuty, T., Erickson, H. P., and Pantaloni, D. Guanosinetriphosphatase activity of tubulin associated with microtubule assembly. Proc. NatI.Aced. Sci. USA, 74: 5372—5376,1977.

18. Lee, J. C., and Timasheff, S. N. The reconstitution of microtubules frompurified calfbrain tubulin. Biochemistry, 14: 5183—5187,1975.

19. Luduena, R. F., Fellous, A., McManus, L, Jordan, M. A., and Nunez, J.Contrasting roles of tau and microtubule-associated protein 2 in the vinblastine-induced aggregation ofbrain tubulin. J. Biol. Chem., 259: 12890—12898,1984.

20. Donoso, J. A., Haskins, K. M., and Himes, R. H. Effect of microtubuleassociated proteins on the interaction of vincristine with microtubules andtubulin.Cancer Res., 39: 1604—1610,1979.

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1986;46:1889-1893. Cancer Res Janendra K. Batra, L. J. Powers, F. Dana Hess, et al. Tubulin at a New Drug-binding SiteAntimitotic Agents Which Interact with Plant and Mammalian Derivatives of 5,6-Diphenylpyridazin-3-one: Synthetic

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