Synthesis and biological evaluation of B-ring modified colchicine and isocolchicine analogs

A series of modified colchicine and isocolchicine analogs (C-7 substituent) were synthesized and evaluated in vitro against a PC3 cancer cell line and for inhibition of microtubule polymerization. The colchicine analogs all displayed strong inhibition of tubulin polymerization, while compounds 6 and 20 also possessed an increased cytotoxic activity as compared to colchicine. More importantly, isocolchicine analogs 7, 15, and 17 showed inhibition of microtubule polymerization with IC(50) values ranging from 58 to 68muM. In addition, 7 displayed strong cytotoxic activity with an IC(50)=93nM which was more potent than colchicine analog 12.     

Phenylimino-10H-anthracen-9-ones as novel antimicrotubule agents-synthesis, antiproliferative activity and inhibition of tubulin polymerization

A novel series of phenylimino-10H-anthracen-9-ones and 9-(phenylhydrazone)-9,10-anthracenediones were synthesized and evaluated for interaction with tubulin and for cytotoxicity against a panel of human tumor cell lines. The 10-(3-hydroxy-4-methoxy-phenylimino)-10H-anthracen-9-one 15h and its dichloro analog 16b were identified as potent inhibitors of tumor cell growth (16b, IC(50) K562 0.11 μM), including multidrug resistant phenotypes. Compound 15h had excellent activity as an inhibitor of tubulin polymerization. Concentration-dependent cell cycle analyzes by flow cytometry confirmed that KB/HeLa cells treated by 15h and 16b were arrested in the G2/M phases of the cell cycle. In competition experiments, 15h strongly displaced radiolabeled colchicine from its binding site on tubulin, showing IC(50) values similar to that of colchicine. The results obtained demonstrate that the antiproliferative activity is related to the inhibition of tubulin polymerization.     

Antitumor agents. Part 215: antitubulin effects of cytotoxic B-ring modified allocolchicinoids

N-Acetylcolchinol methyl ether 1 served as the starting material to prepare the chloroacetamide (3) and epoxide (5) analogues. Both 3 and 5 were potent inhibitors of tubulin polymerization in vitro. Compound 3 was also 4-fold more cytotoxic than colchicine against the 1A9 tumor cell line and showed a unique cross-resistance profile.     

Triethylated chromones with substituted naphthalenes as tubulin inhibitors

Previously synthesized 2-(benzo[b]thiophene-3′-yl)-6,8,8-triethyldesmosdumotin B (1, TEDB-TB) and 2-(naphth-1′-yl)-6,8,8-triethyldesmosdumotin B (2) showed potent activity against multiple human tumor cell lines, including a multidrug-resistant (MDR) subline, by targeting spindle formation and/or the microtubule network. Consequently, ester analogues of hydroxylated naphthyl substituted TEBDs (3–5) were prepared and evaluated for their effects on tumor cell proliferation and on tubulin assembly. Among all new compounds, compound 6, a 4′-acetoxynaphthalen-1′-yl derivative, displayed the most potent antiproliferative activity (IC₅₀ 0.2–5.7 μM). Selected analogues were confirmed to be tubulin assembly inhibitors in cell-free and cell-based assays using MDR tumor cells. The new analogues partially inhibited colchicine binding to tubulin, suggesting their binding mode would be different from that of colchicine. This observation was supported by computational docking model analyses. Thus, the newly synthesized triethylated chromones with esterified naphthalene groups have good potential for development as a new class of mitotic inhibitors that target tubulin.     

Oxadiazole derivatives as a novel class of antimitotic agents: Synthesis, inhibition of tubulin polymerization, and activity in tumor cell lines

Oxadiazole derivatives were synthesized and evaluated for their ability to inhibit tubulin polymerization and to cause mitotic arrest in tumor cells. The most potent compounds inhibited tubulin polymerization at concentrations below 1 microM. Lead analogs caused mitotic arrest of A431 human epidermoid cells and cells derived from multi-drug resistant tumors (10, EC(50)=7.8 nM). Competition for the colchicine binding site and pharmacokinetic properties of selected potent compounds were also investigated and are reported herein, along with structure-activity relationships for this novel series of antimitotic agents.     

Synthesis and biological evaluation of aryl azide derivatives of combretastatin A-4 as molecular probes for tubulin

Two new aryl azides, (Z)-1-(3'-azido-4'-methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)ethene 9 and (Z)-1-(4'-azido-3'-methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)ethene 5, modeled after the potent antitumor, antimitotic agent combretastatin A-4 (CA-4), have been prepared by chemical synthesis as potentially useful photoaffinity labeling reagents for the colchicine site on beta-tubulin. Aryl azide 9, in which the 3'-hydroxyl group of CA-4 is replaced by an azido moiety, demonstrates excellent in vitro cytotoxicity against human cancer cell lines (NCI 60 cell line panel, average GI50 = 4.07 x 10(-8) M) and potent inhibition of tubulin polymerization (IC50 = 1.4+/-0.1 microM). The 4'-azido analogue 5 has lower activity (NCI 60 cell line panel, average GI50 = 2.28 x 10(-6) M, and IC50 = 5.2+/-0.2 microM for inhibition of tubulin polymerization), suggesting the importance of the 4'-methoxy moiety for interaction with the colchicine binding site on tubulin. These CA-4 aryl azide analogues also inhibit binding of colchicine to tubulin, as does the parent CA-4, and therefore these compounds are excellent candidates for photoaffinity labeling studies.     

Synthesis, biological evaluation, and molecular docking studies of 2,6-dinitro-4-(trifluoromethyl)phenoxysalicylaldoxime derivatives as novel antitubulin agents

A series of 2,6-dinitro-4-(trifluoromethyl)phenoxysalicylaldoxime derivatives (1h-20h) have been designed and synthesized, and their biological activities were also evaluated as potential antiproliferation and tubulin polymerization inhibitors. Among all the compounds, 2h showed the most potent activity in vitro, which inhibited the growth of MCF-7, Hep-G2 and A549 cell lines with IC(50) values of 0.70 ± 0.05, 0.68 ± 0.02 and 0.86 ± 0.05 μM, respectively. Compound 2h also exhibited significant tubulin polymerization inhibitory activity (IC(50)=3.06 ± 0.05 μM). The result of flow cytometry (FCM) demonstrated that compound 2h induced cell apoptosis. Docking simulation was performed to insert compound 2h into the crystal structure of tubulin at colchicine binding site to determine the probable binding model. Based on the preliminary results, compound 2h with potent inhibitory activity in tumor growth may be a potential anticancer agent.     

Chloroacetates of 2- and 3-demethylthiocolchicine: specific covalent interactions with tubulin with preferential labeling of the beta-subunit.

We synthesized two chemically reactive A ring modified analogs of colchicine, 2-chloroacetyl-2-demethylthiocolchicine (2-CTC) and 3-chloroacetyl-3-demethylthiocolchicine (3-CTC). Both are similar to colchicine as inhibitors of tubulin polymerization and act as competitive inhibitors of colchicine binding (apparent Ki values, 3 microM). [14C]-labeled 2-CTC and 3-CTC bound to tubulin at 37 degrees C but not at 0 degree C, and bound drug formed covalent bond(s) with tubulin. The binding and covalent reactions were inhibited by podophyllotoxin. About 60% of the bound 3-CTC rapidly formed a covalent bond with tubulin. With 2-CTC the covalent reaction was slower than the binding reaction, and only one-third of the bound 2-CTC reacted covalently with tubulin. The ratio of radiolabel in beta-tubulin to that in alpha-tubulin was about 4:1 with both 2-CTC and 3-CTC.     

New Combretastatin Analogs as Anticancer Agents: Design,Synthesis, Microtubules Polymerization Inhibition,and Molecular Docking Studies

A new series of 4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole-3-thiol derivatives were synthesized as analogs for the anticancer drug combretastatin A-4 ( CA-4 ) and characterized using FT-IR, ¹H-NMR, ¹³CNMR, and HR-MS techniques. The new CA-4 analogs were designed to meet the structural requirements of the highest expected anticancer activity of CA-4 analogs by maintaining ring A 3,4,5-trimethoxyphenyl moiety, and at the same time varying the substituents effect of the triazole moiety (ring B ). In silico analysis indicated that compound 3 has higher total energy and dipole moment than colchicine and the other analogs, and it has excellent distribution of electron density and is more stable, resulting in an increased binding affinity during tubulin inhibition. Additionally, compound 3 was found to interact with three apoptotic markers, namely p53, Bcl-2, and caspase 3. Compound 3 showed strong similarity to colchicine , and it has excellent pharmacokinetics properties and a good dynamic profile. The in vitro anti-proliferation studies showed that compound 3 is the most cytotoxic CA-4 analog against cancer cells (IC₅₀ of 6.35 μM against Hep G2 hepatocarcinoma cells), and based on its selectivity index (4.7), compound 3 is a cancer cytotoxic-selective agent. As expected and similar to colchicine , compound 3 -treated Hep G2 hepatocarcinoma cells were arrested at the G2/M phase resulting in induction of apoptosis. Compound 3 tubulin polymerization IC₅₀ (9.50 μM) and effect on V_max of tubulin polymerization was comparable to that of colchicine (5.49 μM). Taken together, the findings of the current study suggest that compound 3 , through its binding to the colchicine-binding site at β-tubulin, is a promising microtubule-disrupting agent with excellent potential to be used as cancer therapeutic agent.     

Synthesis and evaluation of diaryl sulfides and diaryl selenide compounds for antitubulin and cytotoxic activity

We have devised a procedure for the synthesis of analogs of combretastatin A-4 (CA-4) containing sulfur and selenium atoms as spacer groups between the aromatic rings. CA-4 is well known for its potent activity as an inhibitor of tubulin polymerization, and its prodrugs combretastatin A-4 phosphate (CA-4P) and combretastatin A-1 phosphate (CA-1P) are being investigated as antitumor agents that cause tumor vascular collapse in addition to their activity as cytotoxic compounds. Here we report the preparation of two sulfur analogs and one selenium analog of CA-4. All synthesized compounds, as well as several synthetic intermediates, were evaluated for inhibition of tubulin polymerization and for cytotoxic activity in human cancer cells. Compounds 3 and 4 were active at nM concentration against MCF-7 breast cancer cells. As inhibitors of tubulin polymerization, both 3 and 4 were more active than CA-4 itself. In addition, 4 was the most active of these agents against 786, HT-29 and PC-3 cancer cells. Molecular modeling binding studies are also reported for compounds 1, 3, 4 and CA-4 to tubulin within the colchicine site.     

Synthesis, biological evaluation and molecular modeling of 1,2,3-triazole analogs of combretastatin A-1

The synthesis, cytotoxicity, inhibition of tubulin polymerization data and anti-angiogenetic effects of seven 1,5-disubstituted 1,2,3-triazole analogs and two 1,4-disubstituted 1,2,3-triazole analogs of combretastatin A-1 (1) are reported herein. The biological studies revealed that the 1,5-disubstituted 1,2,3-triazoles 3-methoxy-6-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-5-yl)benzene-1,2-diol (6), 3-methoxy-6-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-5-yl)benzene-1,2-diamine (8) and 5-(2,3-difluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (9) were the three most active compounds regarding inhibition of both tubulin polymerization and angiogenesis. Molecular modeling studies revealed that combretastatins 1 and 2 and analogs 5-11 could be successfully docked into the colchicine binding site of α,β-tubulin.     

Induction of Tumor Cell Death through Targeting Tubulin and Evoking Dysregulation of Cell Cycle Regulatory Proteins by Multifunctional Cinnamaldehydes

Multifunctional trans-cinnamaldehyde (CA) and its analogs display anti-cancer properties, with 2-benzoyloxycinnamaldehyde (BCA) and 5-fluoro-2-hydroxycinnamaldehyde (FHCA) being identified as the ortho-substituted analogs that possess potent anti-tumor activities. In this study, BCA, FHCA and a novel analog 5-fluoro-2-benzoyloxycinnamaldehyde (FBCA), were demonstrated to decrease growth and colony formation of human colon-derived HCT 116 and mammary-derived MCF-7 carcinoma cells under non-adhesive conditions. The 2-benzoyloxy and 5-fluoro substituents rendered FBCA more potent than BCA and equipotent to FHCA. The cellular events by which these cinnamaldehydes caused G₂/M phase arrest and halted proliferation of HCT 116 cells were thereby investigated. Lack of significant accumulation of mitosis marker phospho-histone H3 in cinnamaldehyde-treated cells indicated that the analogs arrested cells in G₂ phase. G₂ arrest was brought about partly by cinnamaldehyde-mediated depletion of cell cycle proteins involved in regulating G₂ to M transition and spindle assembly, namely cdk1, cdc25C, mad2, cdc20 and survivin. Cyclin B1 levels were found to be increased, which in the absence of active cdk1, would fail to drive cells into M phase. Concentrations of cinnamaldehydes that brought about dysregulation of levels of cell cycle proteins also caused tubulin aggregation, as evident from immunodetection of dose-dependent tubulin accumulation in the insoluble cell lysate fractions. In a cell-free system, reduced biotin-conjugated iodoacetamide (BIAM) labeling of tubulin protein pretreated with cinnamaldehydes was indicative of drug interaction with the sulfhydryl groups in tubulin. In conclusion, cinnamaldehydes treatment at proapoptotic concentrations caused tubulin aggregation and dysegulation of cell cycle regulatory proteins cdk1 and cdc25C that contributed at least in part to arresting cells at G₂ phase, resulting in apoptotic cell death characterized by emergence of cleaved forms of caspase 3 and poly (ADP-ribose) polymerase (PARP). Results presented in this study have thus provided further insights into the intricate network of cellular events by which cinnamaldehydes induce tumor cell death.     

Separation of tubulin-binding and anti-inflammatory activity in colchicine analogs and congeners

The effects of colchicine and its analogs on the carrageenin-induced footpad edema in rats were investigated. The anti-inflammatory effects of colchicine analogs were measured at 3 and 5 hr after the carrageenin injection. Colchicine, 1-demethylcolchicine and 3-demethylcolchicine markedly inhibited the carrageenin edema whereas 2-demethylcolchicine was much less active. Thiocolchicinoids, having a thiomethyl group at C-10 instead of a methoxy group, were considerably less potent. These results suggest that the presence of methoxy groups at C-2 and C-10 in colchicine is necessary to maintain anti-inflammatory activity. Inactivity of deacetylcolchicine indicates that substitution of the amino group at C-7 with electron withdrawing groups is also important. Significant inhibition of carrageenin edema and strong binding to tubulin in vitro were manifested by colchicine, 3-demethylcolchicine, N-butyryldeacetylcolchicine and colchifoline. On the other hand, N-carbethoxydeacetylcolchicine which did bind well to tubulin, did not show much effect on the carrageenin edema. These results suggest that the anti-inflammatory action of colchicinoids may not be regulated through the microtubule system.     

Synthesis and preliminary biological evaluation of new anti-tubulin agents containing different benzoheterocycles

A new series of compounds, in which the 2-amino-4-methoxyphenyl ring of phenstatin analogue 5 was replaced with 2- or 3-amino-benzoheterocycles, was synthesized and evaluated for antiproliferative activity and inhibition of colchicine binding. The lack of activity of 3',4'-dimethoxy- and 4'-methoxy-benzoyl derivatives (8 and 9, respectively) indicates that the 3',4',5'-trimethoxybenzoyl moiety is critical for the activity. Two compounds, 7 and 11, displayed potent antiproliferative activity, with IC50 values ranging from 25 to 100 nM against a variety of cancer cell lines. Derivative 11 was more active than CA-4 as an inhibitor of tubulin polymerization. The results demonstrated that the antiproliferative activity was correlated with inhibition of tubulin polymerization.     

Identification of diaryl 5-amino-1,2,4-oxadiazoles as tubulin inhibitors: The special case of 3-(2-fluorophenyl)-5-(4-methoxyphenyl)amino-1,2,4-oxadiazole

The combination of experimental (inhibition of colchicine binding) and computational (COMPARE, docking studies) data unequivocally identified diaryl 5-amino-1,2,4-oxadiazoles as potent tubulin inhibitors. Good correlation was observed between tubulin binding and cytostatic properties for all tested compounds with the notable exception of the lead candidate, 3-(3-methoxyphenyl)-5-(4-methoxyphenyl)amino-1,2,4-oxadiazole (DCP 10500078). This compound was found to be substantially more active in our in vitro experiments than the monofluorinated title compound, 3-(2-fluorophenyl)-5-(4-methoxyphenyl)amino-1,2,4-oxadiazole (DCP 10500067/NSC 757486), which in turn demonstrated slightly better tubulin binding activity. Comparative SAR analysis of 25 diaryl 5-amino-1,2,4-oxadiazoles with other known tubulin inhibitors, such as combretastatin A-4 (CA-4) and colchicine, provides further insight into the specifics of their binding as well as a plausible mechanism of action.     

Biosynthesis of radiolabeled curacin A and its rapid and apparently irreversible binding to the colchicine site of tubulin.

Curacin A is a potent competitive inhibitor of colchicine binding to tubulin, and it inhibits the growth of tumor cells. We prepared [(14)C]curacin A biosynthetically to investigate its interaction with tubulin. Binding was rapid, even at 0 degrees C, with a minimum k(f) of 4.4 x 10(3) M(-1) s(-1). We were unable to demonstrate any dissociation of the [(14)C]curacin A from tubulin. Consistent with these observations, the K(a) value was so high that an accurate determination by Scatchard analysis was not possible. The [(14)C]curacin A was released from tubulin following urea treatment, indicating that covalent bond formation does not occur. We concluded that curacin A binds more tightly to tubulin than does colchicine. Besides high-affinity binding to the colchicine site, we observed significant superstoichiometric amounts of the [(14)C]curacin A bound to tubulin, and Scatchard analysis confirmed the presence of two binding sites of relatively low affinity with a K(a) of 3.2 x 10(-5) M(-1).     

The Novel Microtubule-Destabilizing Drug BAL27862 Binds to the Colchicine Site of Tubulin with Distinct Effects on Microtubule Organization

Microtubule-targeting agents are widely used for the treatment of cancer and as tool compounds to study the microtubule cytoskeleton. BAL27862 is a novel microtubule-destabilizing drug that is currently undergoing phase I clinical evaluation as the prodrug BAL101553. The drug is a potent inhibitor of tumor cell growth and shows a promising activity profile in a panel of human cancer models resistant to clinically relevant microtubule-targeting agents. Here, we evaluated the molecular mechanism of the tubulin–BAL27862 interaction using a combination of cell biology, biochemistry and structural biology methods. Tubulin-binding assays revealed that BAL27862 potently inhibited tubulin assembly at 37 °C with an IC₅₀ of 1.4 μM and bound to unassembled tubulin with a stoichiometry of 1 mol/mol tubulin and a dissociation constant of 244 ± 30 nM. BAL27862 bound to tubulin independently of vinblastine, without the formation of tubulin oligomers. The kinetics of BAL27862 binding to tubulin were distinct from those of colchicine, with evidence of competition between BAL27862 and colchicine for binding. Determination of the tubulin–BAL27862 structure by X-ray crystallography demonstrated that BAL27862 binds to the same site as colchicine at the intradimer interface. Comparison of crystal structures of tubulin–BAL27862 and tubulin–colchicine complexes shows that the binding mode of BAL27862 to tubulin is similar to that of colchicine. However, comparative analyses of the effects of BAL27862 and colchicine on the microtubule mitotic spindle and in tubulin protease-protection experiments suggest different outcomes of tubulin binding. Taken together, our data define BAL27862 as a potent, colchicine site-binding, microtubule-destabilizing agent with distinct effects on microtubule organization.     

Synthesis,cytotoxic evaluation and molecular docking study of 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazoles as tubulin polymerization inhibitors

A series of cis-restricted 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazole analogues of combretastatin A-4 were synthesized and investigated for inhibition of cell proliferation against three cancer cell lines, HT-29, MCF-7, and AGS, and a normal mouse fibroblastic cell line, NIH-3T3, using an MTT assay. The biological study showed that 2-(methylthio) substituted compounds showed little cytotoxic activity against the four cell lines. In contrast, the presence of the 2-(benzylthio) group on the thiazole ring resulted in a significant improvement in cytotoxic activity relative to the 2-(methylthio) substituted derivatives. Furthermore, the inhibition of tubulin polymerization by some potent compounds was evaluated. All the compounds studied were moderate tubulin polymerization inhibitors. The flow cytometry analysis confirmed that the synthesized compounds led to cell cycle arrest at the G₂/M phase. Docking simulation was performed to insert these compounds into the crystal structure of tubulin at the colchicine binding site to determine a probable binding model.     

Antimitotic natural products combretastatin A-4 and combretastatin A-2: studies on the mechanism of their inhibition of the binding of colchicine to tubulin

Combretastatin A-4 (CS-A4), 3,4,5-trimethoxy-3'-hydroxy-4'-methoxy-(Z)-stilbene, and combretastatin A-2 (CS-A2), 3,4-(methylenedioxy)-5-methoxy-3'-hydroxy-4'-methoxy-(Z)-stilbene, are structurally simple natural products isolated from the South African tree Combretum caffrum. They inhibit mitosis and microtubule assembly and are competitive inhibitors of the binding of colchicine to tubulin [Lin et al. (1988) Mol. Pharmacol. 34, 200-208]. In contrast to colchicine, drug effects on tubulin were not enhanced by preincubating CS-A4 or CS-A2 with the protein. The mechanism of their binding to tubulin was examined indirectly by evaluating their effects on the binding of radiolabeled colchicine to the protein. These studies demonstrated rapid binding of both compounds to tubulin even at 0 degrees C (binding was complete at the earliest times examined), in contrast to the relatively slow and temperature-dependent binding of colchicine. Although the binding of the C. caffrum compounds to tubulin was quite tight, permitting ready isolation of near-stoichiometric amounts of drug-tubulin complex even in the absence of free drug, both CS-A4 and CS-A2 dissociated rapidly from tubulin in the presence of high concentrations of radiolabeled colchicine. Apparent rate constants for drug dissociation from tubulin at 37 degrees C were 3.2 x 10(-3) s-1 for CS-A4, 4.8 x 10(-3) s-1 for CS-A2, and 2.9 x 10(-5) s-1 for colchicine (half-lives of 3.6, 2.4, and 405 min, respectively). Thus, the effectiveness of the C. caffrum compounds as antimitotic agents appears to derive primarily from the rapidity of their binding to tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-acetylcolchinol O-methyl ether and thiocolchicine, potent analogs of colchicine modified in the C ring. Evaluation of the mechanistic basis for their enhanced biological properties

Two colchicine analogs with modifications only in the C ring are better inhibitors than colchicine of cell growth and tubulin polymerization. Radiolabeled thiocolchicine (with a thiomethyl instead of a methoxy group at position C-10) and N-acetylcolchinol O-methyl ether (NCME) (with a methoxy-substituted benzenoid instead of the methoxy-substituted tropone C ring) were prepared for comparison with colchicine. Scatchard analysis indicated a single binding site with KD values of 1.0-2.3 microM. Thiocolchicine was bound 2-4 times as rapidly as colchicine, but the activation energies of the reactions were nearly identical (18 kcal/mol for colchicine, 20 kcal/mol for thiocolchicine). NCME bound to tubulin in a biphasic reaction. The faster phase was 60 times as fast as colchicine binding at 37 degrees C, and a substantial reaction occurred at 0 degrees C. The rate of the faster phase of NCME binding changed relatively little as a function of temperature, so the activation energy was only 7.0 kcal/mol. Dissociation reactions were also evaluated, and at 37 degrees C the half-lives of the tubulin-drug complexes were 11 min for NCME, 24 h for thiocolchicine, and 27 h for colchicine. Relative dissociation rates as a function of temperature varied little among the drug complexes. Activation energies for the dissociation reactions were 30 kcal/mol for thiocolchicine, 27 kcal/mol for NCME, and 24 kcal/mol for colchicine. Comparison of the activation energies of association and dissociation yielded free energies for the binding reactions of -20 kcal/mol for NCME, -10 kcal/mol for thiocolchicine, and -6 kcal/mol for colchicine. The greater effectiveness of NCME and thiocolchicine as compared with colchicine in biological assays probably derives from their more rapid binding to tubulin and the lower free energies of their binding reactions.