Endowing Indole-Based Tubulin Inhibitors with an Anchor for

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Endowing Indole-Based Tubulin Inhibitors with an Anchor for Derivatization: Highly Potent 3-Substituted Indole Phenstatins and Isocombretastatins. Raquel Álvarez, Mª Pilar Puebla, Jose Fernando Díaz, Ana C. Bento, Rósula García-Navas, Janis de la Iglesia-Vicente, Faustino Mollinedo, Jose Manuel Andreu, Manuel Medarde, and Rafael Pelaez J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm3015603 • Publication Date (Web): 07 Mar 2013 Downloaded from http://pubs.acs.org on March 11, 2013

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Figure 1. Structures of combretastatin analogues undergoing clinical trials. 165x39mm (300 x 300 DPI)

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Figure 5. Effects of compounds 10, 11, 14, 22, 24, 29 and 32 on the microtubule network of HeLa cells. Cells were incubated in the absence (Control) or in the presence of 10-8 M of compounds 10, 11, 14, 22, 24, 29 and 32 for 18 h and then fixed and processed to study the immunofluorescence of microtubules (red fluorescence) and nuclei (blue fluorescence), as described in Experimental Section. Bar: 25 µm. The photomicrographs shown are representative of at least three independent experiments performed. 120x127mm (150 x 150 DPI)


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Figure 6. Caspase-3 activation following treatment of HeLa cells with compounds 14, 24, 29 and 32. Cells were treated with 50 nM of compounds 14, 24, 29 and 32 for the indicated times and analyzed by immunoblotting with anti-activated caspase-3 (SDS-12% polyacrylamide gels) and anti-PARP antibodies (SDS-8% polyacrylamide gels). Untreated control cells (C) were run in parallel. The migration positions of activated caspase-3, as well as of full-length PARP and its cleavage product p85, are indicated. Data shown are representative of three experiments performed. 188x40mm (150 x 150 DPI)



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Figure 8. Molecular models of carboxylate 17 and amide 29 bound at the colchicine site (upper and middle rows respectively, two different views) and superimpositions of the former with the electron diffraction structure of paclitaxel-stabilized tubulin sheets 1JFF.pdb (lower left) and with the tubulin - colchicine complex 1SA0.pdb (lower right). The carboxylate hydrogen-bonds to the sidechain of Asnβ350 and to a water molecule and forms a salt bridge with the sidechain ammonium group of Lysβ352. The amide hydrogen-bonds to the backbone carbonyl of Proβ358. The superimposition with 1JFF.pdb shows that the ligand occupies the space taken by Lysβ352 in 1JFF.pdb (shown in light green) and displaces the sheets underneath downwards. The superimposition with the colchicine complex (shown in blue) shows a high resemblance between the binding modes of the two ligands. 184x123mm (72 x 72 DPI)



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Table of Contents Graphic 231x51mm (72 x 72 DPI)


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Endowing Indole-Based Tubulin Inhibitors with an Anchor for Derivatization: Highly Potent 3-Substituted Indole Phenstatins and Isocombretastatins. Raquel Álvarez,† Pilar Puebla,† J. Fernando Díaz,‡ Ana C. Bento,# Rósula García-Navas,# Janis de la Iglesia-Vicente,# Faustino Mollinedo,# José Manuel Andreu,‡ Manuel Medarde† and Rafael Peláez*,† †

Laboratorio de Química Orgánica y Farmacéutica, CIETUS and IBSAL, Facultad de Farmacia,

Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain, ‡Centro de Investigaciones Biológicas, CSIC, Madrid 28040, Spain, and # Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain.

RECEIVED DATE

*

To whom correspondence should be addressed. Telephone: 34 923 294528. Fax: 34 923 294515. E-mail adress: [email protected]

†

Laboratorio de Química Orgánica y Farmacéutica, CIETUS and IBSAL

‡

Centro de Investigaciones Biológicas

#

Instituto de Biología Molecular y Celular del Cáncer


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ABSTRACT. Colchicine site ligands with indole B rings are potent tubulin polymerization inhibitors. Structural modifications at the indole 3-position of 1-methyl-5-indolyl based isocombretastatins (1,1diarylethenes) and phenstatins endowed them with anchors for further derivatization and resulted in highly potent compounds. The substituted derivatives displayed potent cytotoxicity against several human cancer cell lines due to tubulin inhibition, as shown by cell cycle analysis, confocal microscopy and tubulin polymerization inhibitory activity studies and promoted cell killing mediated by caspase-3 activation. Binding at the colchicine site was confirmed by means of fluorescence measurements of MTC displacement. Molecular modeling suggests that the tropolone-binding region of the colchicine site of tubulin can adapt to hosting small polar substituents. Isocombretastatins accepted substitutions better than phenstatins, and the highest potencies were achieved for the cyano and hydroxyiminomethyl substituents, with TPI values in the submicromolar range and cytotoxicities in the subnanomolar range. A 3,4,5-trimethoxyphenyl ring usually afforded more potent derivatives than a 2,3,4-trimethoxyphenyl ring.

INTRODUCTION

The eukaryotic microtubule system serves a variety of architectural functions in cells and provides a dynamic scaffold for cell shape maintenance, flagellar and ciliary movement, and the intracellular transport of vesicles and other cellular components. It also supplies the conveyor belt of the mitotic spindle for chromosomes during mitosis, thus allowing them to become properly positioned at the metaphase plate and after which the sister chromatids are pulled apart towards the cell poles in anaphase.1 The important role of microtubule dynamics makes them important targets in anticancer chemotherapy. Most drugs interfering with microtubule dynamics act directly on tubulin, such as the stabilizing taxanes, and the destabilizing vinca alkaloids and colchicine site ligands.2


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Among the colchicine site ligands, combretastatins have become structural models for the design of new analogues as a result of their cytotoxic effects on tumor cells and their ability to collapse the tumor vasculature, in the shape of different salts of the phosphate prodrug of combretastatin A-4 (fosbretabulin), the diphosphate prodrug of combretastatin A-1 (OXi4503) and the combretastatin analogue AVE 8062, which are currently undergoing phase I and II clinical trials (Fig. 1).3 However, combretastatins show low aqueous solubility due to the hydrophobic nature of the colchicine site, chemical instability brought about by their isomerization to the inactive but thermodynamically more stable trans isomers,4 and insufficient cytotoxicity against cells in the tumor rim, resulting in tumor regrowth.5 Several approaches have been undertaken to solve each of these disadvantages, with different degrees of success.

Figure 1. Structures of combretastatin analogues undergoing clinical trials.

We have recently prepared a new family of macrocyclic combretastatin analogues that are unable to become converted to the trans isomers but they display reduced potency, probably owing to steric reasons.6 With the same aims, but following a different strategy, we have also recently described a new family of combretastatin isomers, which we have called isocombretastatins, and structurally related ketones and oximes, which are potent inhibitors of tubulin polymerization and cytotoxic compounds.7 The most potent analogues of these families carry a 1-methyl-5-indolyl B ring, but these compounds suffer from low water solubility. Other groups have also found that N-methyl-5-indolyl rings are good scaffolds for colchicine site ligands in terms of potency but are associated with poor physicochemical properties.8 In the same vein, we have also found that N-methyl-5-indolyl-substituted combretastatins ACS Paragon Plus Environment

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are poor cytotoxic compounds despite their potent inhibition of tubulin polymerization.9 Liou et al have explored the possibility of incorporating acidic substituents at the indole nitrogen of 5-aroylindoles in order to improve their physical chemical properties, but these result in much lower potencies.10 The most successful strategy applied to increase water solubility in combretastatins is the formation of phosphate prodrugs of phenol groups already present in the parent compounds, but 2- or 3hydroxyindoles are not stable and therefore this approach cannot be easily applied to 5-indolyl analogues.11 In this work, we set out to study the possibility of incorporating polar groups at the position three of the indole of 5-benzoylindoles that might serve as an anchor for further derivatization in ways similar to what is seen for phenolic groups in combretastatins. The phenolic group of combretastatin A-4 can be replaced by a hydrogen atom or with amines with only small changes in potency, but this position has proved to be important as a solubilizing group and as anchors for derivatization purposes.12 A similar group is thus needed to extend the possibilities of the development of 5-indolyl-containing tubulin polymerization inhibitors. Following the pharmacophore model of Nguyen13 for colchicine site ligands and our docking studies of 5-indolylisocombretastatins, phenstatins and related compounds7 it could be suggested that the 3 position of the indole ring might accommodate hydrogen bond donors (such as amino substituents), which might hydrogen-bond to the carbonyl oxygen atom of Thrβ177 (corresponding to pharmacophore point D1, such as the phenolic group of combretastatin A-4) but also hydrogen bond acceptors to bind the amide nitrogen of Valβ179 (corresponding to pharmacophore point A2, such as the carbonyl oxygen of the tropolone ring of colchicine), as has been discussed for CA-4 and related 4-arylcoumarins.14 To date few substitutions on the indole ring have been described, but potent combretastatin, sulfonamide and phenstatin analogues carrying carbazole rings suggested to us that an expanded tropolone site might be exploited for improved potency and/or pharmacokinetic modulation.9,15 In order to explore new possibilities for the indole-based interfacial inhibitors, and continuing our


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previous work studying the effect of different nitrogen substituents or other modifications at the phenstatin bridge and the effect of a 2,3,4-trimethoxyphenyl as A-ring, here we prepared and assayed a set of compounds characterized by the presence of substituents at the 3-position of the 5-indolyl unit. Considering that the introduction of the more sterically demanding indoles might affect the overall disposition of the ligands in the colchicine site, the effect of a 2,3,4-trimethoxyphenyl ring was also explored. Additionally, the replacement of the indole by benzimidazole and benzoxazole systems was addressed. These modifications are very interesting in terms of interactions with the protein, because new possibilities for binding can be assessed, as well as for activity modulation, solubilization improvement and derivatization (Figure 2). Y

Z

Y

MeO

Z

R

MeO N

MeO

Z = O, CH2, H/CH3 N

MeO

X

X

Described indolephenstatins

X, Y = MeO/H

New indolephenstatins

R= CHO, CN, CH2OH, CHNOH, COOH, CONH2, NH2 , CH2NH2 protein binding, solubilization and derivatisation

Figure 2. Structures of the new 3-substituted indole analogues.

RESULTS AND DISCUSSION Chemical synthesis The synthesis of the phenstatin-like scaffolds was planned and carried out according to our previously described methodologies for the synthesis of N-substituted indole-phenstatins, and their derivatives modified on the bridge, such as isocombretastatins (Scheme 1). The introduction of the 3-substituents was attempted at two different stages: on the monoarylic starting materials and on the preassembled non-substituted diarylic precursors. Electrophilic reactions on the 5-substituted-N-methyl-1H-indoles proved to be problematic, easily evolving into complex red mixtures. Therefore, substitutions were performed at the diarylic stage. Several attempts to introduce amino groups at the three positions of the indole rings of phenstatins and isocombretastatins by means of a nitration - reduction sequence failed


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due to chemical instability of the amines.16 Instead, Vilsmeier-Haack formylation was selected and we prepared the other derivatives from the aldehydes. Starting from commercial 5-bromo-1H-indole, N-methylation and coupling with 2,3,4- or 3,4,5trimethoxybenzaldehydes yielded benzhydrols 2 and 3, which upon PDC oxidation to phenstatins 4 and 5 followed by Wittig methylenations yielded isocombretastatins 6 and 7. The corresponding dihydroderivatives 14 and 15 were obtained by hydrogenation (Scheme 1). Alternatively, transmetalation of 5-bromo-N-methyl-1H-indole and coupling of the organolytic compound with the sodium salts of the trimethoxybenzoic acids yielded the benzophenones directly in similar overall yields. From these already known compounds, the preparation of the new derivatives carrying the substitution at the indole 3-position can be completed by formylation of the highly reactive indole nucleus. Under Vilsmeier-Haack conditions, aldehydes 8 and 9 were readily produced, whereas for isocombretastatins mixtures of the monoformylated (10 or 11) and diformylated (12 or 13) products were formed by sequential reaction with the indole and with the electron-rich olefin under more forcing conditions. As shown by NMR, 12 and 13 were obtained as Z+E mixtures.

Scheme 1: Synthesis of 3-formylated indole-phenstatins and indole-isocombretastatins.a

a. Reagents and conditions: (a) (i) NaOH (10%), nBu4NHSO4, CH2Cl2, 1 h, rt; (ii) IMe, 24 h. (b) (i) ACS Paragon Plus Environment

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BuLi (1.6M), THF, -40ºC, 1h; (ii) 3,4,5- or 2,3,4-trimethoxybenzaldehyde, THF, -40ºC→rt, 24 h. (c) PDC, CH2Cl2, 0ºC, 4Å ms, 4 - 24 h. (d) (i) Ph3PMeI, THF, -40ºC, BuLi (1.6M), 1 h; (ii) 4 or 5, THF, 40 ºC → rt, 24 h. (e) (i) POCl3, DMF, 0ºC, 1 h; (ii) 4 or 5 or 6 or 7, 60ºC, 2 h. (f) Pd/C (10%), EtOH, H2 atmosphere, 24 h.

With keto-aldehydes 8 and 9 at hand, the first modification performed was the preparation of the carboxylic acids in an attempt to improve water solubility and as a means of synthesizing further derivatives. Direct oxidation of the isocombretastatins was complicated by the double bond, and phenstatin 16 was produced after the oxidation of aldehyde 10. This problem was circumvented by previous oxidation of aldehyde 8 to carboxylic acid 16, followed by its methylenation to 17 (Scheme 2). Unexpectedly, acid 16 was extremely insoluble in many solvents (including THF, water, methanol, CH2Cl2 and DMSO), which made it very difficult to handle and led to very low reaction yields in all reactions attempted. The presence of minor amounts of non-oxidized ketoaldehyde 8 accounts for the isolation of diolefin 18 as a by-product of the olefination. Although the yield of this process was very low, attempts to prepare the carboxylic acid by alternative procedures (trifluoroacetylation-hydrolysis, ethoxycarbonylation-hydrolysis) failed or resulted in the degradation of the reacting material.

Scheme 2: Synthesis of isocombretastatin indole-3-carboxylic acid derivativesa

a. Reagents and conditions: (a) 8 or 10 in tBuOH, 2-methyl-2-butene, THF; add NaClO2, NaH2PO4,


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H2O, rt, 12 h. (b) (i) Ph3PMeI, THF, -78ºC, BuLi (1.6M), 1 h; (ii) 16, THF, -78ºC→rt, 24 h.

Hydrogenation of olefin-aldehyde 10 reduced both the double bond and the carbaldehyde to the corresponding alkanes (19). Several transformations aimed at increasing solubility by means of additional nitrogen or oxygen atoms and/or enabling further modifications that could in some cases also regenerate the simple carbonyl group by hydrolysis were also carried on the carbaldehydes (Scheme 3). Thus, hydrazones 20 and 21 and oximes 22 and 23 were prepared as stereoisomeric Z+E mixtures, because isomerization of the isomers isolated was observed. In the case of 21, simultaneous hydrazone formation and bridge reduction occurred under the conditions assayed. The oximes can be used to easily prepare other derivatives at the 3-position: the acetyloximes and the nitriles. By treatment with acetic anhydride in pyridine, compounds 24-27 were produced by acetylation and acetylation-elimination of the oximes, as well as a minor amount of keto-nitrile 28. Compound 24 was also used to prepare amide 29. Finally, aldehydes 10 and 11 were modified by reduction to the 3-hydroxymethyl derivatives 30 and 31. In view of the high potency displayed by the nitrile derivative 24, the synthesis of the dinitrile derivative 32 was tackled from dialdehyde 12 (Scheme 4) following the same procedure.

Scheme 3: Preparation of isocombretastatin indole-3-carbaldehyde derivatives.a


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a. Reagents and conditions: (a) Pd/C (10%), EtOH, H2 atmosphere, 24 h. (b) NH2NH2·H2O, MeOH, HOAc (2 droplets), rt, 24 h. (c) NH2OH·HCl, MeOH, pyridine (2 droplets), rt, 24 h. (d) Ac2O, pyridine, rt, 4 h. (e) NaOH (10%), MeOH, rt, 16 h. (f) NaBH4, MeOH, rt, 30 min.

Scheme 4: Preparation of dinitrile 32.a

a. Reagents and conditions: (a) (i) NH2OH·HCl, MeOH, pyridine (2 droplets), rt, 24 h; (ii) Ac2O,


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pyridine, rt, 4 h.

Simultaneously

to

the

synthesis

of

indole

phenstatins,

isocombretastatins

and

dihydroisocombretastatins, which in general display high potency in the inhibition of tubulin polymerization and cytotoxicity assays (see below), we decided to replace the indole nucleus by related bicyclic systems, such as benzimidazole and benzoxazole. Accordingly, 5(6)-bromo-1-methyl-1Hbenzimidazoles were obtained as a mixture of regioisomers by treatment of 4-bromo-o-phenylene diamine with methyl orthoformate followed by methylation of the 5-bromo-1H-benzimidazole thus obtained. The mixture of bromides was treated with n-butyl-lithium and reacted with 3,4,5trimethoxybenzaldehyde, yielding products 33 and 34, arising from the metalation at the 2-position instead of those derived from the metal-halogen exchange. Oxidation of this reaction mixture produced the keto-imidazole derivatives 35 and 36. Under identical conditions, 5-bromobenzoxazole (obtained from 4-bromo-2-nitrophenol by reduction to 4-bromo-2-aminophenol and treatment with methyl orthoformate) yielded alcohol 37, which was oxidized to the benzoxazole-phenstatin 38. Owing to the low tubulin polymerization inhibitory activity and cytotoxicity displayed by the latter compound, the synthesis of the 5-(3,4,5-trimethoxybenzoyl)-1-methyl-1H-benzimidazole was not further pursued.

Scheme 5: Synthesis of benzimidazole and benzoxazole derivatives 33-38.a


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a. Reagents and conditions: (a) (i) BuLi (1.6M), THF, -78ºC, 1 h; (ii) 3,4,5-trimethoxybenzaldehyde, THF, -78ºC→rt, 24 h. (b) PDC, CH2Cl2, 0ºC, 4Å ms, 24h. (c) KMnO4, nBuNSO4, H2O, CH2Cl2, rt, 24 h.

The 1H-indole derivatives without substitution at 1-position were synthesized according to Scheme 6, using the N-benzenesulphonyl-protected starting material to prepare benzhydrol 39, oxidation to phenstatin 40, olefination to isocombretastatin 41 and deprotection to 42. The latter was formylated to dihydroisocombretastatin 44 , through previous hydrogenation to 43, and directly to the dicarbaldehyde derivative 45 (Z+E).

Scheme 6: Preparation of 1H-indole isocombretastatin and dihydroisocombretastatin derivatives.a

a. Reagents and conditions: (a) (i) 5-bromo-1,2,3-trimethoxybenzene, BuLi (1.6M), THF, -60ºC, 1.5 h; (ii) 1-(phenylsulfonyl)-1H-indole-5-carbaldehyde, THF, -60ºC→rt, 24 h. (b) PDC, CH2Cl2, 0ºC→rt, 4Å ms, 12 h. (c) (i) Ph3PMeI, THF, -40ºC, BuLi (1.6M), 1.5 h; (ii) 40, THF, -40ºC→rt, 12 h. (d) NaOH (5M), EtOH, reflux, 20 h. (e) Pd/C (10%), EtOH, H2 atmosphere, 24 h. (f) (i) POCl3, DMF, 0ºC, 30 min;


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(ii) 42 or 43, 60ºC, 2 h.

Following these methodologies a large number of analogues can be prepared by standard procedures, but these compounds are sufficiently representative to accomplish assays aimed at elucidating the effects of the modification at the 3-position at the indole nucleus of this class of antimitotics on tubulin and on cytotoxicity.

Biology The compounds synthesized were assayed by the XTT method for growth inhibition against four types of human cancer cell lines: A-549 human lung carcinoma, HeLa human cervix epithelioid carcinoma, HL-60 human acute myeloid leukemia, and HT-29 human colon adenocarcinoma. The results are summarized in Table 1. Some of these compounds inhibited the proliferation of the indicated human cancer cell lines at very low drug concentrations, with IC50 values in the 10-9 M and 10-10 M range. These IC50 values are up to 10-100 times lower than those shown by doxorubicin (IC50 at a range of 10-8 M) (Fig. 3).


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Figure 3. Effects of compound 14 and doxorubicin on the cell proliferation of HeLa cells. Cells were incubated with compound 14 and doxorubicin at the indicated concentrations. After 3 days, cell proliferation was determined by the XTT assay and plotted as a percentage of untreated control cells. Results are mean values ± SD of a representative experiment carried out in triplicate, out of three performed. Most of the compounds assayed were cytotoxic at micromolar concentrations or lower (Table 1), thus indicating that the common 5-(trimethoxybenzyl)indole is a very favorable scaffold for cytotoxicity. The benzophenones (phenstatins) with 3-substituted indoles (e.g. 8, 9, and 28) displayed potencies in the hundreds of nanomolar concentrations and were less potent than the corresponding isocombretastatins


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(1,1-diarylethenes, e.g. 24, 26, and 29), the most potent of them being cytotoxic in the nanomolar range. This result is in agreement with our previous findings showing that indoleisocombretastatins are more potent than indolephenstatins7 and suggests that, contrary to what was previously considered,13 the carbonyl oxygen of phenstatins is not an essential pharmacophoric anchor point. Binding at the colchicine site of tubulin is usually considered to involve a geometric arrangement with two noncoplanar aromatic rings13 that must disrupt the conjugation of the carbonyl group with the aromatic rings and impose a more severe conformational penalty on binding for the carbonyls than for the olefins. Irrespective of the bridge and the nature of the indole substitution, the 2,3,4-trimethoxyphenyl analogues were less potent than the 3,4,5-trimethoxyphenyl ones (e.g. compare 8 to 9, 10 to 11, and 24 to 26), similar to what has been reported previously for the unsubstituted indoles.7 This is probably a consequence of the scaffold geometry, with the one-carbon bridge placing the two aromatic moieties when the compound is in a complex with tubulin similar to the aromatic rings of podophyllotoxin, and the cleft for the A ring hosting the methoxy groups at the 3, 4 and 5 positions better. Allocating a 2,3,4trimethoxyphenyl ring at this site would require tilting of the indole ring, which does not seem to be possible in sight of the lower potency observed, thus suggesting a scarce adaptability of the B ring pocket in the colchicine site. The introduction of substituents at the indole three position increases the bulk and likely aggravates this difficulty for the 2,3,4-trimethoxyphenyl analogues. For these reasons, we selected the complex between tubulin and 1sa1.pdb for the molecular docking studies (see below) and below we mainly discuss the results for the more potent 3,4,5-trimethoxyphenyl analogues. Overall, the introduction of substituents at the indole three position results in a decrease in cytotoxic potency, in good agreement with the low expandability of the B ring pocket at the colchicine site. Consistent with the previously discussed effect of the bridge, this decrease was more pronounced in the indolephenstatins (comparing unsubstituted 4 with aldehyde 8, with acid 16, or with nitrile 28, revealed 10- to 100-fold potency decreases) than in the isocombretastatins with the same substituents (comparing unsubstituted 6 with aldehyde 10 or with acid 17 caused a 10- to 100-fold potency decrease, but potency


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remained similar for nitrile 24). Further exploration of the isocombretastatins series led to amide 29, which is an improvement over the nanomolar potencies of the parent indoles. The presence of hydrogenbond donors and acceptors in 29 seemed to improve the cytotoxic potency with respect to substituents with only acceptors (e.g. aldehyde 10 or nitrile 24) or with only hydrogen-bond donors (e.g. hydrazones 20), although in this latter case the presence of two stereoisomers might also contribute to the loss of potency. This might also be the case for oximes 22, which despite their similarity to the amide arrangement of hydrogen-bond donors and acceptors showed a lower cytotoxic potency, although they represented an improvement with respect to their parent aldehyde 10, with only a hydrogen-bond acceptor. This observation is interesting, since if they behaved as prodrugs releasing the aldehyde, one would expect at most a potency equal to that of aldehyde 10 (which is in fact a more potent inhibitor of tubulin polymerization, see Table 1 and below). Therefore, if they behave as prodrugs of the aldehyde they must have other advantageous properties, such as for instance better solubility or cell uptake, therefore attracting more interest and suggesting a starting point for potential double prodrug derivatization of the aldehydes. The charged carboxylate resulted in a lower cytotoxic potency, which might reflect a combination of poor solubility (despite of its charge), cell uptake, and tubulin polymerization inhibitory activity (even if the carboxylate is close to the ammonium group of lysine 350β, see later). Reduction of aldehydes 10 and 11 to methanols 30 and 31 respectively, produced a sharp decrease in potency, thus confirming the importance of a hydrogen-bond acceptor moiety placed in the same plane as the indole ring. The more potent 3-substituted indoleisocombretastatins, such as 10, 24 and 29, provide anchor points for further derivatization and provide interesting opportunities for increases in solubility or drug targeting.17 Taking into account the apparent importance of the overall indole bulk on cytotoxic potency, we explored the effect of removing the methyl group on the indole nitrogen and also replaced the Nmethylindole by a benzoxazole, which also incorporates a less bulky hydrogen-bond acceptor in the ring plane. However, the methyl group seemed to be important for the activity of the substituted derivatives,


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since the compounds with a free indole amino group (compare 6 to 42, or 14 to 43) showed reduced potencies, as did benzoxazole 38. Considering the previously discussed potentially deleterious effect on cytotoxicity of the conjugation of the aromatic rings with the sp2 bridges we decided to explore the effect of reducing the olefin bridge to ethyl analogues. The unsubstituted hydrogenated derivative 14 proved to be very potent, but substitution of the indole caused a sharp decrease in potency (e.g. 19) and therefore was not further pursued. The introduction of other modifications on the olefinic bridge of the isocombretastatins did not result in a consistent improvement with respect to the unsubstituted olefins, although the dinitrile derivative 32 displayed consistent nanomolar potencies in all four cell lines studied. In order to interpret the results on cytotoxicity, the effect of the synthesized compounds 2 - 45 at a concentration of 20 µM on in vitro tubulin assembly was studied with bovine brain tubulin. For compounds showing more than 50 % inhibition at this concentration the IC50 values of tubulin polymerization inhibition (TPI) were calculated in order to compare potencies. Overall, there was good agreement between the TPI and cytotoxic potencies, mainly for HeLa cells (Supplementary material: Fig. S1). However, slightly different effects against A-549, HL-60 and HT-29 were observed for some highly potent inhibitors of tubulin polymerization, such as with 22. The TPI results show that aldehyde, oxime, nitrile and amides at position three of the indole are acceptable for tubulin binding, regardless of the differences they cause in cytotoxic potencies against different cell lines. These results suggest that both hydrogen-bond donors and acceptors are suitable modifications to obtain indole-based tubulin inhibitors, in agreement with previous results with colchicine site analogues incorporating both types of functionalities, as already mentioned.14 The binding site and the association constant of the most cytotoxic and potent inhibitors of tubulin assembly were obtained using the MTC18 displacement assay.19 MTC is a bona-fide fluorescent probe of the colchicine site,17 which largely increases its fluorescence upon binding. For phenstatins and isocombretastatins, displacement of MTC from its binding site, which can be observed by the decay of


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the fluorescence of the MTC upon ligand addition, was used. The observed displacement confirms binding at or close to the colchicine binding site. Consistent with the TPI activities, the strongest association constant was for the cyano-substituted indole isocombretastatin 24 (1.8±0.8 107 M-1), whereas the unsubstituted indolic phenstatin 4, isocombretastatin 6 and formylated isocombretastatin 10 displayed association constants in the range of 106 M-1. In addition, a positive correlation was observed between the cytotoxicity of the compounds studied in HL-60 cells and their affinity for the colchicinebinding site of tubulin (Supplementary material: Fig. S2). The affinity range achieved with this ligand series spanned almost three orders of magnitude. In order to ascertain that the cytotoxic effects of the assayed compounds were due to tubulin inhibition, we analyzed the effect of the different compounds on cell cycle analysis by flow cytometry. We used different drug concentrations (5, 10, 50 and 100 nM) and the HeLa cancer cell line in timecourse experiments (18, 24, 48 and 72 h incubation times). Compound 24, as one of the most active compounds, totally arrested the HeLa cell cycle at the G2/M phase (> 83%) when used at 10-8 M for 18 h (Fig. 4). Similarly, compounds 14, 29 and 32, which together with compound 24 were the most active compounds, arrested practically all the cells at G2/M phase of the cell cycle (> 85%) when used at 10-8 M after 18 h incubation (data not shown).


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Figure 4. Effects of compound 24 on the cell cycle in HeLa cells. Cells were incubated with 10-8 M compound 24 for 18 h and stained with propidium iodide (PI). Their DNA content was analyzed by fluorescence flow cytometry. Control untreated cells were run in parallel. The positions of the G0/G1 and G2/M peaks are indicated by arrows. Data shown are representative of three experiments performed.

This cell cycle arrest was followed by the onset of apoptosis at later incubation times, as detected by the appearance of cells with DNA contents at the sub-G0/G1 region of cell cycle analysis, a significant percentage of apoptotic cells being reached at 72 h incubation (e.g.: 20% apoptosis following incubation of HeLa cells with 10-8 M compound 24). Compounds that interfere with the microtubule system typically cause mitotic arrest at the G2/M phase before the induction of an apoptotic cell death,20 consistent with our results. Furthermore, confocal microscopy studies showed severe disruption of the microtubule system of HeLa cells treated with 10 nM of the compounds (Fig. 5), further confirming that the observed effects were due to interaction with tubulin, resulting in a prolonged mitotic arrest that


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ultimately leads to cell death.21

Figure 5. Effects of compounds 10, 11, 14, 22, 24, 29 and 32 on the microtubule network of HeLa cells. Cells were incubated in the absence (Control) or in the presence of 10-8 M of compounds 10, 11, 14, 22, 24, 29 and 32 for 18 h and then fixed and processed to study the immunofluorescence of microtubules (red fluorescence) and nuclei (blue fluorescence), as described in Experimental Section. Bar: 25 µm. The photomicrographs shown are representative of at least three independent experiments performed.


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The type of cell killing induced by the most potent inhibitors of tubulin polymerization and cytotoxic compounds was investigated by Western Blot, measuring the activation of caspase-3 (Fig. 6) in HeLa cells after incubation with the compounds. Following 24-h of incubation, caspase-3 activation was detected and after 72 h cells started to accumulate in the sub-G0/G1 region, indicating the induction of apoptosis. We found that the most active compounds, namely 14, 24, 29 and 32, induced caspase-3 activation in HeLa cells (Fig. 6) as assessed by the processing of procaspase-3 into the p19/p17 active forms, and the cleavage of the typical caspase-3 substrate PARP, using a polyclonal antihuman caspase3 antibody that recognized the p19/p17 forms of the active caspase-3, and the anti-PARP C2.10 monoclonal antibody that detected both the 116-kDa intact form and the 85-kDa cleaved form of the caspase-3 substrate PARP. A similar type of behavior was detected with the other active dugs assayed in this work (data not shown). In addition, the incubation of HeLa cells with the cell permeable pancaspase inhibitor z-VAD-fmk (50 µM) prevented the induction of apoptosis triggered by compound 24 (19.5% vs. 5% apoptosis in 24-treated HeLa cells incubated in the absence or presence of z-VAD-fmk for 72 h), further supporting the involvement of apoptosis in the cell killing process.

Figure 6. Caspase-3 activation following treatment of HeLa cells with compounds 14, 24, 29 and 32. Cells were treated with 50 nM of compounds 14, 24, 29 and 32 for the indicated times and analyzed by immunoblotting with anti-activated caspase-3 (SDS-12% polyacrylamide gels) and anti-PARP antibodies (SDS-8% polyacrylamide gels). Untreated control cells (C) were run in parallel. The migration positions of activated caspase-3, as well as of full-length PARP and its cleavage product p85,


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are indicated. Data shown are representative of three experiments performed.

Table 1. Tubulin polymerization inhibitory activity, antineoplastic cytotoxic activity against human tumor cell lines and binding parameters. TPI (%)

TPI

IC50 (nM)a

IC50 (nM)a

IC50 (nM)a

IC50 (nM)a

HL-60

A-549

HeLa

HT-29

Kass

Compound at 20µ µM IC50 (µ µM)

2.16 ± 0.3 x 106

4

98

7.9

27 ± 1

190 ± 30

25 ± 6

27 ± 4

5

100

0.4

240 ± 10

380 ± 10

30 ± 1

240 ± 20

6

96

0.7

2.8 ± 0.4

28 ± 1

2.7 ± 0.2

3.1 ± 0.1

3.8 ± 2 x 106

7

97

4.7

280 ± 10

730 ± 60

32 ± 1

310 ± 10

7.6 ± 1 x 106

8

61

16.9

310 ± 10

> 104

320 ± 10

3000 ± 200

3.1 ± 2 x 105

9

73

780 ± 120

> 104

780 ± 110

5300 ± 900

2.6 ± 1 x 105

10

100

0.9

28 ± 2

3000 ± 100

20 ± 3

62 ± 8

3.6 ± 3 x 105

11

98

4.0

32 ± 1

1800 ± 200

32 ± 1

230 ± 20

104

> 104

7000 ± 1600

> 104

< 104

17

91

1100 ± 200

1100 ± 500

880 ± 50

550 ± 140

19

90

280 ± 10

320 ± 10

310 ± 10

310 ± 10

20b

12

180 ± 20

3200 ± 100

38 ± 1

300 ± 10

5.4 ± 4 x 104

21

0

1800 ± 100

>104

430 ± 20

3200 ± 100

< 104

22b

93

2.6

21 ± 4

30 ± 1

6.2 ± 0.5

33 ± 1

8.6 ± 4 x 106

23b

95

4.0

270 ± 10

290 ± 10

34 ± 1

320 ± 10

5.4 ± 3 x 105

24

93

1.2

1.3 ± 0.4

8.4 ± 0.2

2.9 ± 0.1

3.9 ± 0.2

1.8 ± 0.8 x 107

26

89

2.2

27 ± 3

360 ± 10

32 ± 1

32 ±1

4.0 ± 3 x 106

28

40

290 ± 10

2000 ± 400

270 ± 10

310 ± 20

29

93

0.11 ± 0.01

1.8 ± 0.4

0.25 ± 0.05

0.51 ± 0.07

30

32

250 ± 40

3200 ± 100

310 ± 10

2000 ± 100

≈5

0.9


5.3 ± 2 x 104

104

>104

>104

>104

34

22

>104

>104

>104

>104

35

37

29 ± 3

400 ± 30

190 ± 10

340 ± 20

36

37

2400 ± 100

~104

2300 ± 200

2700 ± 300

38

0

470 ± 40

~104

340 ± 30

2800 ± 200

42

36

3600 ± 500

9000 ± 1100

2100 ± 500

5100 ± 700

43

5

3400 ± 300

4900 ± 800

2300 ± 600

3300 ± 600

44

0

5400 ± 400

9100 ± 200

5800 ± 700

5800 ± 300

4.7

1.0 ± 0.6 x 106

Values are derived from concentration-response curves using the XTT assay as described in the

Experimental section. Data are shown as the main values ± S.D. of three experiments performed in triplicate. bAssayed as Z+E mixtures.

Theoretical calculations were performed in order to explain the relative biological activity of the different compounds. Figure 7 shows the superimpositions of the DFT optimized indolephenstatin 4 and indoleisocombretastatin 6 onto podophyllotoxin (carbon atoms colored in cyan), revealing an extensive overlapping of the aromatic rings. This is in good agreement with the pharmacophoric models for the colchicine site, which assign an essential role to the spatial arrangement of the two aromatic rings. However, the higher potency of isocombretastatins when compared to the phenstatins, despite these latter having one more potential hydrogen-bond acceptor point due to the carbonyl oxygen atom, could be explained in terms of the greater preference of the carbonyl to remain conjugated to the phenyl rings. This preference results in a less favorable disposition of the phenstatins when bound to tubulin.


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B)

A)

Figure 7. Superimposition of the most stable DFT conformers of 3-unsubstituted compounds (A) 4 and 6, and formylated analogues (B) 8 and 10 onto podophyllotoxin (carbons in cyan). Only those conformations which place the phenyl rings close to those of podophyllotoxin are shown.

When DFT-minimized 3-substituted indolephenstatin (8, R=CHO) and indoleisocombretastatin (10, R=CHO) were superimposed onto podophyllotoxin (carbon atoms colored in cyan), there was a similar overlapping of the aromatic rings (figure 7) as that described for the unsubstituted analogues. The substituents protrude from the podophyllotoxin pocket towards its walls. For the phenstatins, the tilted aromatic ring directs the substituents towards the upper and lower walls, while in the case of the isocombretastatins they are in the podophyllotoxin benzodioxole plane, which might allow for their better interaction with tubulin. Attempts to dock the ligands into the rigid unmodified podophyllotoxin site of 1SA1.pdb22 resulted in poses which did not place the trimethoxyphenyl rings in close proximity to those of podophyllotoxin and colchicine, although the unsubstituted indoles did. These results pointed to the new substituents being responsible. We therefore attempted to take site flexibility into account by allowing the sidechains of the surrounding residues to rotate. This resulted in a more diffuse binding pattern for all the ligands studied,

including

podophyllotoxin,

colchicine,

the

unsubstituted

indolic

phenstatins

and

isocombretastatins, and other ligands previously studied by us. Despite these unsuccessful results, which suggested a different binding mode extending to additional regions of the site, as has also been suggested for other families, but considering that all the available tubulin X-ray structures with colchicine site ligands present ligands with very similarly sized B rings and that the structure – activity ACS Paragon Plus Environment

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relationships of ours did not deviate much from the unsubstituted analogues, we set out to run molecular dynamics simulations in an attempt to further explore the flexibility of the B-ring site of colchicine site ligands. We started off with the unsubstituted isocombretastatins docked at the podophyllotoxin site and we later introduced a nitrile, formyl or carboxy group. After initial relaxation, molecular dynamics simulations showed that the site can easily adapt to the expanded ligands, and that the ammonium sidechain of Lysβ252 can form an ionic interaction with the ligand carboxylates, or even hydrogen bond with suitable acceptors on the ligands (figure 8). This observation is also consistent with the X-ray structure of tubulin in complex with taxol™ (1JFF.pdb), where the colchicine site is occupied by this residue, thus suggesting an important structural role for Lysβ252 in tubulin structural transitions (Figure 8). Finally, docking attempts with models arising from the molecular dynamics simulations successfully resulted in similar binding modes for the substituted ligands, as well as for podophyllotoxin, colchicine and other unsubstituted indole analogues.


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Figure 8. Molecular models of carboxylate 17 and amide 29 bound at the colchicine site (upper and middle rows respectively, two different views) and superimpositions of the former with the electron diffraction structure of paclitaxel-stabilized tubulin sheets 1JFF.pdb (lower left) and with the tubulin colchicine complex 1SA0.pdb (lower right). The carboxylate hydrogen-bonds to the sidechain of Asnβ350 and to a water molecule and forms a salt bridge with the sidechain ammonium group of Lysβ352. The amide hydrogen-bonds to the backbone carbonyl of Proβ358. The superimposition with 1JFF.pdb shows that the ligand occupies the space taken by Lysβ352 in 1JFF.pdb (shown in light green) and displaces the sheets underneath downwards. The superimposition with the colchicine complex (shown in blue) shows a high resemblance between the binding modes of the two ligands.

CONCLUSION ACS Paragon Plus Environment

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We have synthesized a new family of potent indole-based colchicine site inhibitors with modifications on the indole ring that might serve in the future as anchors for derivatization, in the same way as the amino and phenolic groups serve for prodrug establishment in combretastatins under clinical development. We have shown that some of the substituents do not interfere with tubulin binding at the colchicine site and result in potent cytotoxic activities against several human cancer cell lines. The most active compounds lead cancer cells to accumulate in the G2/M phase of the cell cycle and eventually induce apoptotic death, which is dependent upon caspase-3 activity. Finally, we propose a binding mode for the substituted ligands based on molecular dynamics simulations that is consistent with the observed structure-activity relationships for this class of compounds. Comparison of the molecular dynamics results, the diffraction structures of tubulin dimers in complexes with colchicine site ligands and the paclitaxel tubulin complexes highlights the importance of Lysβ352 in the binding of colchicine site ligands and in filling the colchicine pocket when tubulin becomes arranged in microtubules, as represented by the paclitaxel-tubulin sheets.

EXPERIMENTAL SECTION1 General chemical techniques. Reagents were used as purchased without further purification. Solvents (THF, DMF, CH2Cl2, toluene) were dried and freshly distilled before use according to procedures described in the literature. Chromatographic separations were performed on silica gel columns by flash (Kieselgel 40, 0.040-0.063;

1

Abbreviations used. bs: broad singlet; EGTA: Ethyleneglycol-bis(2-aminoethyl)-N,N,N’,N’-tetraacetic

acid; kDa: kilodalton; MES: 2-(N-Morpholino)ethanesulfonic acid hydrate; MTC: 2-methoxy-5-(2',3',4'trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one; TPI: tubulin polymerization inhibition; XTT: 2,3-Bis(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt; β−ΜΕ: β-mercaptoethanol.


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Merck) or gravity (Kieselgel 60, 0.063-0.200 mm; Merck) chromatography. TLC was performed on precoated silica gel polyester plates (0.25 mm thickness) with a UV fluorescence indicator 254 (Polychrom SI F254). Melting points were determined on a Buchi 510 apparatus and are uncorrected. 1

H NMR and 13C NMR spectra were recorded in CDCl3 on a Bruker WP 200-SY spectrometer at 200/50

MHz or on a Bruker SY spectrometer at 400/100 MHz. Chemical shifts (δ) are given in ppm downfield from tetramethylsilane as internal standard and coupling constants (J values) are in Hertz. IR spectra were run on a Nicolet Impact 410 Spectrophotometer. For FABHRMS analyses, a VG-TS250 apparatus (70 eV) was used. HPLCs were run on at least three different columns (5 µm, 4.6x150 mm): Waters XTerra® MS C8, Waters X-Terra® MS C18 and Waters X-Terra® MS CF on an Agilent HP series 1100 with at least two different solvent gradients (typically acetonitrile/water or methanol/water). Elemental analyses were run on a Perkin Elmer 2400 CHN apparatus. All the compounds described here were obtained with at least 95% of purity by quantitative HPLC and/or elemental analysis, unless otherwise stated.

Chemical syntheses of key compounds

1-Methyl -5-[1-(3,4,5-trimethoxyphenyl)vinyl]-1H-indole-3-carbaldehyde (10). A solution of POCl3 (0.48 mL, 5.2 mmol) in DMF (5.5 mL) was stirred at 0ºC for 1 h, after which 1methyl-5-(1-(3,4,5-trimethoxyphenyl)vinyl)-1H-indole (6) (1.6 g, 4.9 mmol) was added and heated at 60 ºC for 2 h. Then, the reaction mixture was poured onto ice with sodium acetate, water (500 mL) was added and the mixture was kept at 4 ºC. After 24 h the obtained precipitate was dissolved in CH2Cl2, dried over anhydrous Na2SO4, filtered, and concentrated under a vacuum to obtain 1-methyl-5-[1-(3,4,5trimethoxyphenyl)vinyl]-1H-indole-3-carbaldehyde (10) (230 mg, 13%), a mixture of the Z,E isomers of 1-methyl-5-(3-oxo-1-(3,4,5-trimethoxyphenyl)prop-1-enyl)-1H-indole-3-carbaldehyde (12Z+E) (306 mg, 16%), (Z)-1-methyl-5-(3-oxo-1-(3,4,5-trimethoxyphenyl)prop-1-enyl)-1H-indole-3-carbaldehyde


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(12Z) (87 mg, 4.6%) and a mixture of 10+12Z+12E (851 mg, 47%). 1-Methyl-5-[1-(3,4,5trimethoxyphenyl)vinyl]-1H-indole-3-carbaldehyde (10). M.p.: 156-158 ºC. (CH2Cl2/Hex). IR (film): 1656, 1457, 1124, 785 cm-1. 1H NMR (400 MHz, CDCl3): δ 3.75 (6H, s), 3.82 (3H, s), 3.86 (3H, s), 5.42 (1H, s), 5.45 (1H, s), 6.54 (2H, s), 7.26 (2H, s), 7.66 (1H, s), 8.34 (1H, s), 9.91 (1H, s). 13C NMR (100 MHz, CDCl3): δ 33.8 (NCH3), 56.1 (2) (OCH3), 60.9 (OCH3), 105.6 (2) (CH), 109.6 (CH), 114.1 (CH2), 118.3 (C), 121.7 (CH), 125.1 (CH), 125.3 (C), 136.7 (2) (C), 137.7 (C), 137.8 (C), 140.1 (CH), 150.4 (C), 152.9 (2) (C), 184.5 (CH). HRMS (C21H21NO4+Na): calcd. 374.1368 (M++Na), found 374.1365. HPLC: C18 tR: 14.25 min. C8 tR: 13.61 min. Phenylic tR: 14.16 min. 1-Methyl-5-(1-(3,4,5-trimethoxyphenyl)ethyl)-1H-indole (14). Pd/C (8 mg, 0.08 mmol of active Pd) was added to a solution of 1-methyl-5-(1-(3,4,5trimethoxyphenyl)vinyl)-1H-indole (26 mg, 0.08 mmol) in absolute EtOH (8 mL) under an H2 atmosphere. After 24 h stirring at room temperature, the suspension was filtered through Celite and the solvent was evaporated in vacuo. The crude product was purified by column chromatography over silica gel to yield 1-methyl-5-(1-(3,4,5-trimethoxyphenyl)ethyl)-1H-indole (14) (20 mg, 78%). IR (film): 1588, 1457, 1126 cm-1. 1H NMR (400 MHz, CDCl3): δ 1.68 (3H, d, J = 7.2), 3.74 (3H, s), 3.81 (6H, s), 3.82 (3H, s), 4.20 (1H, c, J = 7.2), 6.44 (1H, d, J = 3.0), 6.50 (2H, s), 7.02 (1H, d, J = 3.0), 7.09 (1H, dd, J = 8.4 and 1.6), 7.24 (1H, d, J = 8.4), 7.49 (1H, d, J = 1.6). 13C NMR (100 MHz, CDCl3): δ 22.6 (CH3), 32.9 (NCH3), 45.1 (CH), 56.1 (2) (OCH3), 60.9 (OCH3), 100.8 (CH), 104.7 (2) (CH), 109.1 (CH), 119.0 (CH), 121.9 (CH), 128.7 (CH), 132.1 (C), 135.5 (C), 136.0 (C), 137.3 (C), 143.2 (C), 153.0 (2) (C). HRMS (C20H23NO3+Na): calcd. 348.1576 (M++Na), found 348.1580. HPLC: C18 tR: 17.31 min. C8 tR: 15.76 min. Phenylic tR: 16.02 min. 1-Methyl-5-(1-(3,4,5-trimethoxyphenyl)vinyl)-1H-indole-3-carboxylic acid (17) Methyltriphenylphosphonium iodide (1.71 g, 4.25 mmol) was suspended in dry THF (50 mL) and cooled to -78 ºC under Argon atmosphere. Then, nButyl lithium (1.6 M in hexane, 2.5 mL, 4.00 mmol) was added dropwise, and the resulting yellow solution was stirred for 1 h. Following this, a solution of


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1-methyl-5-(3,4,5-trimethoxybenzoyl)-1H-indole-3-carboxylic acid (16) (315 mg, 0.85 mmol) in THF was added and the mixture was warmed to room temperature. Once completed, the reaction mixture was cooled to 0ºC, treated with a saturated NH4Cl solution, and extracted with CH2Cl2. The organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under a vacuum. The residue was purified by flash chromatography to yield 1-methyl-5-(1-(3,4,5-trimethoxyphenyl)vinyl)1H-indole-3-carboxylic acid (17) (13 mg, 4%) and 1-methyl-5-(1-(3,4,5-trimethoxyphenyl)vinyl)-3vinyl-1H-indole (18) (72 mg, 24%). 1-Methyl-5-(1-(3,4,5-trimethoxyphenyl)vinyl)-1H-indole-3carboxylic acid (17): M.p.: 318-319 ºC. (CH2Cl2/Hex). 1H NMR (400 MHz, CDCl3): δ 3.80 (6H, s), 3.88 (3H, s), 3.89 (3H, s), 5.47 (1H, d, J = 1.4), 5.49 (1H, d, J = 1.4), 6.60 (2H, s), 7.23 (1H, dd, J = 8.6 and 1.6), 7.31 (1H, d, J = 8.6), 7.90 (1H, s), 8.31 (1H, d, J = 1.6). 13C NMR (100 MHz, CDCl3): δ 33.8 (NCH3), 56.2 (2) (OCH3), 61.0 (OCH3), 105.7 (2) (CH), 106.6 (C), 109.5 (CH), 113.9 (CH2), 121.5 (CH), 124.2 (CH), 126.9 (C), 136.0 (C), 137.0 (CH), 137.2 (C), 137.8 (C), 138.0 (C), 150.6 (C), 152.9 (2) (C), 170.4 (C). HRMS (C21H21NO5+Na): calcd. 390.1312 (M++Na), found 390.1303. HPLC: C18 tR: 20.31 min. 1-Methyl-5-[1-(3,4,5-trimethoxyphenyl)vinyl]-1H-indole-3-carbaldehyde oxime (22). NH2OH·HCl (311 mg, 4.5 mmol) and 2 droplets of pyridine were added to a stirred solution of 1methyl-5-[1-(3,4,5-trimethoxyphenyl)vinyl]-1H-indole-3-carbaldehyde (157 mg, 0.45 mmol) in MeOH (10 mL). After 23 h, the solvent was evaporated off, and the residue was re-dissolved in CH2Cl2, washed with brine, and the organic layers were dried, filtered, and evaporated to obtain a 1:1 mixture (Z+E) 1-methyl-5-[1-(3,4,5-trimethoxyphenyl)vinyl]-1H-indole-3-carbaldehyde oxime (22Z+E) (145 mg, 88%). By column chromatography one isomer (34 mg, 21%) and another one (53 mg, 32%) were isolated, but both pure compounds isomerized to regenerate the original 1:1 mixture. (Z or E)-1Methyl-5-[1-(3,4,5-trimethoxyphenyl)vinyl]-1H-indole-3-carbaldehyde oxime. M.p.: 162-164 ºC. (CH2Cl2/Hex). IR (film): 3430, 1576, 1100 cm-1. 1H NMR (400 MHz, CDCl3): δ 3.80 (9H, s), 3.89 (3H, s), 5.41 (1H, s), 5.48 (1H, s), 6.60 (2H, s), 7.25 (2H, s), 7.27 (1H, s), 8.15 (1H, s), 8.32 (1H, s).13C NMR


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(100 MHz, CDCl3): δ 33.3 (NCH3), 56.2 (2) (OCH3), 61.0 (OCH3), 105.8 (2) (CH), 109.1 (CH), 113.4 (CH2), 118.2 (C), 121.8 (CH), 124.0 (CH), 125.2 (2) (C), 131.8 (CH), 134.6 (C), 137.5 (C), 138.1 (C), 145.5 (CH), 150.8 (C), 152.8 (2) (C). HRMS (C21H22N2O4+Na): calcd. 389.1477 (M++Na), found 389.1478. (Z or E)-1-Methyl-5-[1-(3,4,5-trimethoxyphenyl)vinyl]-1H-indole-3-carbaldehyde oxime. M.p.: 162-164 ºC. (CH2Cl2/Hex). IR (film): 3450, 1579, 1093 cm-1. 1H NMR (400 MHz, CDCl3): δ 3.80 (6H, s), 3.85 (3H, s), 3.90 (3H, s), 5.44 (1H, s), 5.46 (1H, s), 6.60 (2H, s), 7.31 (2H, s), 7.77 (1H, s), 7.80 (1H, s), 8.27 (1H, s). 13C NMR (100 MHz, CDCl3): δ 33.4 (NCH3), 56.2 (2) (OCH3), 61.0 (OCH3), 105.7 (2) (CH), 109.4 (CH), 113.3 (CH2), 118.2 (CH), 123.5 (CH), 127.2 (2) (C), 134.4 (C), 135.7 (CH), 135.7 (C), 137.7 (C), 138.0 (CH), 139.5 (C), 150.8 (C), 152.9 (2) (C). HRMS (C21H22N2O4+Na): calcd. 389.1477 (M++Na), found 389.1478. HPLC (original mixture or pure isomers after standing in solution): C18 tR: 14.92 and 15.50 min. (1:1). C8 tR: 13.67 and 13.97 min. (1:1). Phenylic tR: 14.41 and 14.69 min. 1-Methyl -5-[1-(3,4,5-trimethoxyphenyl)vinyl]-1H-indole-3-carbonitrile (24). Acetic

anhydride

(0.4

mL)

was

added

to

a

solution

of

(Z+E)-1-methyl-5-[1-(3,4,5-

trimethoxyphenyl)vinyl]-1H-indole-3-carbaldehyde oxime (85 mg, 0.23 mmol) in pyridine (0.4 mL). After 4 h stirring, the mixture was poured onto EtOAc, washed with 2 N HCl, 5% NaOH and brine, and the organic layers were dried over anhydrous Na2SO4, filtered, and evaporated under a vacuum. After column chromatography of the crude, 1-methyl-5-[1-(3,4,5-trimethoxyphenyl)vinyl]-1H-indole-3carbonitrile (24) (27 mg, 34%) and (Z+E)-1-methyl-5-[1-(3,4,5-trimethoxyphenyl)vinyl]-1H-indol-3carbaldehyde oxime acetate (25Z+E) (20 mg, 21%) were obtained. 1-Methyl-5-[1-(3,4,5trimethoxyphenyl)vinyl]-1H-indole-3-carbonitrile (24): IR (film): 2216, 1580, 1125 cm-1. 1H NMR (400 MHz, CDCl3): δ 3.80 (6H, s), 3.87 (3H, s), 3.89 (3H, s), 5.46 (2H, s), 6.55 (2H, s), 7.34 (2H, s), 7.54 (1H, s), 7.77 (1H, s). 13C NMR (100 MHz, CDCl3): δ 33.8 (NCH3), 56.1 (2) (OCH3), 61.0 (OCH3), 85.8 (C), 105.8 (2) (CH), 110.0 (CH), 114.2 (CH2), 115.9 (C), 119.6 (CH), 124.9 (CH), 127.9 (C), 135.8 (C), 135.9 (CH), 136.1 (C), 137.6 (C), 138.2 (C), 150.2 (C), 153.0 (2) (C). HRMS (C21H20N2O3+Na):


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calcd. 371.1372 (M++Na), found 371.1368. HPLC: C18 tR: 19.61 min. 1-Methyl-5-(1-(3,4,5-trimethoxyphenyl)vinyl)-1H-indole-3-carboxamide (29) NaOH (10 M, 1 mL) was added to a solution of 1-methyl-5-(1-(3,4,5-trimethoxyphenyl)vinyl)-1Hindole-3-carbonitrile (90 mg, 0.30 mmol) in methanol (3.0 mL). After 16 h stirring, the mixture was poured onto ice, neutralized with 2N HCl, extracted with EtOAc and washed with brine to obtain 1methyl-5-(1-(3,4,5-trimethoxyphenyl)vinyl)-1H-indole-3-carboxamide (29) (89 mg, 81%). The crude was purified by crystallization in CH2Cl2/Hex. M.p.: 206-207 ºC. (CH2Cl2/Hex). 1H NMR (400 MHz, CDCl3): δ 3.79 (6H, s), 3.84 (3H, s), 3.88 (3H, s), 5.44 (1H, s), 5.45 (1H, s), 6.58 (2H, s), 7.25 (1H, dd, J = 8.6 and 1.4), 7.30 (1H, d, J = 8.6), 7.74 (1H, s), 8.01 (1H, bs). 13C NMR (100 MHz, CDCl3): δ 33.4 (NCH3), 56.1 (2) (OCH3), 60.9 (OCH3), 105.7 (2) (CH), 109.6 (CH), 109.9 (C), 113.7 (CH2), 120.1 (CH), 123.8 (CH), 125.5 (C), 133.7 (CH), 135.4 (C), 137.1 (C), 137.7 (C), 137.8 (C), 150.6 (C), 152.8 (2) (C), 167.0 (C). HRMS (C21H22N2O4+Na): calcd. 389.1477 (M++Na), found 389.1477. HPLC: C18 tR: 16.58 min. 5-(2-Cyano-1-(3,4,5-trimethoxyphenyl)vinyl)-1-methyl-1H-indole-3-carbonitrile (32) Hydroxylamine hydrochloride (186 mg, 2.69 mmol) and two drops of pyridine were added to a solution of aldehyde 12 (102 mg, 0.27 mmol) in MeOH (6 mL). After 24 at reflux, the solvent was evaporated off, the residue obtained was solved in CH2Cl2 and extracted with H2O. The combined organic layers were dried over Na2SO4 and evaporated off to give a residue that was dissolved in pyridine (0.5 mL). Then, acetic anhydride (0.5 mL) was added. After 9 h at room temperature, the reaction was treated with 2N HCl, extracted with CH2Cl2, washed with 2% NaOH, dried over anhydrous Na2SO4 and the solvent evaporated off. The residue was purified by flash chromatography with hexane/EtOAc (1:1) yielding a mixture of compounds 32Z+32E, which could not be separated (36.2 mg, 36%). IR (film): 2339, 2216, 1737, 1650 cm-1. Major isomer: 1H NMR (400 MHz, CDCl3): δ 3.83 (6H, s), 3.89 (3H, s), 3.90 (3H, s), 5.71 (1H, s), 6.67 (2H, s), 7.29 (1H, d, J = 8.8), 7.39 (1H, d, J = 8.8), 7.65 (1H, s), 7.76 (1H, s). 13C NMR (100 MHz, CDCl3): δ 33.8 (NCH3), 56.2 (2) (OCH3), 60.9 (OCH3),


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77.6 (C), 86.6 (C), 94.1 (CH), 106.1 (CH), 107.2 (2) (CH), 115.1 (C), 118.1 (C), 120.4 (CH), 124.6 (CH), 127.7 (C), 132.4 (C), 133.4 (C), 136.8 (CH), 137.0 (C), 153.2 (2) (C), 163.3 (C). Minor isomer: 1

H NMR (400 MHz, CDCl3): δ 3.78 (6H, s), 3.94 (6H, s), 5.72 (1H, s), 6.49 (2H, s), 7.49 (1H, d, J =

8.8), 7.54 (1H, d, J = 8.8), 7.75 (1H, s), 7.90 (1H, s). 13C NMR (100 MHz, CDCl3): δ 33.8 (NCH3), 56.2 (2) (OCH3), 60.9 (OCH3), 77.6 (C), 86.6 (C), 94.4 (CH), 103.7 (CH), 110.5 (2) (CH), 115.1 (C), 117.2 (C), 121.6 (CH), 125.5 (CH), 127.6 (C), 131.2 (C), 134.7 (C), 136.6 (CH), 139.7 (C), 153.1 (2) (C), 163.1 (C). HRMS (C22H19N3O3+Na): calcd. 396.1319 (M++Na), found 396.1323. HPLC: C18 tR: 15.38 and 15.88 min.

Cell cultures HL-60 (human acute myeloid leukemia) cell line was cultured in RPMI-1640 culture medium containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin at 37ºC in humidified 95% air and 5% CO2. A549 (human lung carcinoma), HeLa (human cervical), and HT-29 (human colon carcinoma) cell lines were cultured in DMEM culture medium containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), 2 mM Lglutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37ºC in humidified 95% air and 5% CO2. Cells were periodically tested for Mycoplasma infection and found to be negative.

Cell growth inhibition assays The effect of the different compounds on the proliferation of human tumor cell lines (cytostatic activity)

was

determined

as

previously

described23

using

the

XTT

(sodium

3’-[1-

(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)-benzenesulfonic acid hydrate) cell proliferation kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions. Cells (1.5 x 103 - A549, HeLa, HT-29- and 3 x 103 -HL-60- in 100 µl) were incubated in culture medium containing 10% heat-inactivated FBS in the absence and in the presence of the indicated ACS Paragon Plus Environment

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compounds at different concentration ranges from 10-5 to 10-13 M, in 96-well flat-bottomed microtiter plates, and following 72 h of incubation at 37 ºC in a humidified atmosphere of air/CO2 (19/1) the XTT assay was performed. Measurements were done in triplicate, and each experiment was repeated three times. The IC50 (50% inhibitory concentration) value, defined as the drug concentration required to cause a 50% inhibition in cellular proliferation with respect to the untreated controls, was determined for each compound. Non-linear curves fitting the experimental data were carried out for each compound.

Cell cycle analyses For cell cycle analyses, untreated and drug-treated HeLa cells (2-4 x 105) were centrifuged and fixed overnight in 70% ethanol at 4ºC. Then, cells were washed three times with PBS, incubated for 1 h with 1 mg/mL RNAse A and 20 µg/mL propidium iodide at room temperature, and analyzed with a Becton Dickinson Fluorescence activated cell sorter (FACSCalibur) flow cytometer (San Jose, CA) as described previously.24 Quantification of apoptotic cells was calculated as the percentage of cells in the sub-G0/G1 peak in cell cycle analysis.24

Confocal microscopy HeLa cells were grown on poly-L-lysine-coated coverslips, and after drug treatment the coverslips were washed three times with HPEM buffer (25 mM HEPES, 60 mM PIPES, 10 mM EGTA, 3 mM MgCl2, pH 6.6), fixed with 4% paraformaldehyde in HPEM buffer for 15 min, and permeabilized with 0.5% Triton X-100 as previously described.9 Coverslips were incubated with a specific Ab-1 anti-alphatubulin mouse monoclonal antibody (diluted 1:150 in PBS) (Calbiochem, san Diego, CA) for 1 h, washed four times with PBS, and then incubated with CY3-conjugated sheep anti-mouse IgG (diluted 1:100 in PBS) (Jackson ImmunoResearch, West Grove, PA) for 1 h at 4ºC. After washing four times with PBS, cell nuclei were stained with DAPI (Sigma, St. Louis, MO) for 5-10 min, washed with PBS,


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and then the samples were analyzed by confocal microscopy using a ZeissLSM 310 laser scan confocal microscope. A drop of SlowFade light antifading reagent (Molecular Probes, Eugene, OR) was added to preserve fluorescence. Negative controls, lacking the primary antibody or using an irrelevant antibody, showed no staining.

Western blot About 5 X 106 cells were pelleted by centrifugation, washed with PBS, lysed, and subjected to Western blot analysis as described previously.24b Proteins were separated through SDS polyacrylamide gels under reducing conditions, transferred to nitrocellulose filters, blocked with 5% defatted milk powder, and incubated overnight with anti–activated caspase-3 (1:1000 dilution) rabbit monoclonal antibody, which detects the the p19/p17 forms of cleaved caspase-3 (BD Biosciences Pharmingen, Franklin Lakes, NJ), or C2.10 anti-116 kDa poly(ADP-ribose) polymerase (PARP) (1:1000 dilution) mouse monoclonal antibody that also detects the p85 subunit (BD Biosciences Pharmingen). Signals were developed using an enhanced chemiluminescence detection kit (Amersham Biosciences, Aylesbury, UK). Immunoblotting with AC-15 anti-42 kDa β-actin (1:5000 dilution) mouse monoclonal antibody (Sigma) was used as an internal loading control, revealing equivalent amounts of protein in each lane of the gel. Prestained protein molecular mass standards (Bio-Rad) were run in parallel.

Inhibition of tubulin polymerization Bovine brain tubulin was isolated as previously described.9 The assays were carried out at pH 6.7 with 1.5 mg/mL protein and the measured ligand concentration in 0.1 M MES buffer, 1 mM EGTA, 1 mM MgCl2, 1 mM β-ME, 1.5 mM GTP. The samples were incubated at 20 °C for 30 min and subsequently cooled on ice for 10 min. Tubulin polymerization was monitored by measuring the absorbance increase in the UV at 450 nm caused by a temperature shift from 4ºC to 38ºC. After reaching a stable plateau, temperature was switched back to 0ºC and return to the initial absorption values was ascertained, in


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order to confirm the reversible nature of the monitored process. The difference in amplitude between the stable plateau and the initial baseline of the curves was taken as the degree of tubulin assembly for each experiment. Comparison with control curves with identical conditions but without ligands yielded tubulin polymerization inhibition as a percentage value. Initially, all compounds were assayed at a concentration of 5 µM in at least two independent measurements, and those displaying a TPI higher than 40% were selected for further studies. The tubulin polymerization inhibitory activity of the selected compounds was measured at different ligand concentrations and, as expected, the extent of inhibition by all compounds increased monotonically with the mole ratio of the total ligand to total tubulin in the solution. The obtained values were fitted to monoexponential curves and the IC50 values of tubulin polymerization inhibition were calculated from the best fitting curves.

Interaction with tubulin Tubulin was prepared from calf brain by the modified Weisenberg procedure, stored in liquid nitrogen, and equilibrated in the desired buffer before use. Tubulin concentrations were determined spectrophotometrically by employing an extinction coefficient of 107000 M-1 cm-1 at 275 nm in 10 mM phosphate buffer containing 1% SDS, pH 7, and employing a Hitachi U2000 spectrophotometer.25 The binding constants of the ligands to the colchicine site of tubulin were measured by displacement of MTC from its binding site. The displacement isotherm of each ligand was measured at least three times. A mixture of 10 µM tubulin and 10 µM MTC in a phosphate buffer was placed in a cuvette. Increasing concentrations of the ligands (dissolved in DMSO) were added progressively and the decrease in fluorescence was recorded using a Fluoromax 4 fluorimeter from Horiba Jobin-Yvon (excitation wavelength: 350 nm; emission wavelength: 422 nm). A sample with 10 µM of MTC was employed as a blank and a sample with 10 µM tubulin and 10 µM MTC and increasing amounts of DMSO was used as standard. Knowing MTC binding constant (4.7*105 M-1), the fractional saturation values at different concentrations of the competitor, and assuming unitary stoichiometry, the binding


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constants were calculated using the Equigra v5 program.26

Chemical structure Calculations were performed at the molecular mechanics level (MMFF94s), semiempirical level (AM1), and HF and B3LYP 6-31+G* DFT levels using the Spartan’08 and Gaussian G03 software packages. Conformational analyses with the MMFF94s forcefield were performed by systematically rotating in 18º steps the two rotatable bonds of the bridges of indolephenstatin 4 and indoleisocombretastatin 6. The trimethoxyphenyl rings of the conformations obtained were superimposed over one another and over the trimethoxyphenyl ring of podophyllotoxin extracted from the X-ray crystal structure with tubulin (pdb-1SA1) and the conformations for which the indole rings covered a region close to the benzodioxole ring of podophyllotoxin were subjected to further unrestrained energy minimization steps at the semiempirical (AM1), 6-31G** HF and, then, B3LYP/631+G* DFT levels of theory. The conformations were then superimposed again onto podophyllotoxin by means of the Spartan 08 pharmacophore option, by selecting as pharmacophoric references the two aromatic rings of podophyllotoxin (hydrophobic), the two external oxygens of the trimethoxyphenyl moiety (hydrogen bond acceptors) and the excluded volume, as calculated from the complex of podophyllotoxin with tubulin.

Docking experiments The pdb of tubulin in complex with podophyllotoxin (1SA1.pdb)22 was retrieved from the protein data bank,27 chains C, D and E were removed by hand, and the models were energy-minimized and subjected to molecular dynamics simulations at 300 K, initially with a restrained backbone and then an unrestrained one.28 The ligands were built with Spartan´08, prepared with AutodockTools and docked with AutoDock 4.2,29 by running the Lamarckian genetic algorithm (LGA) 100-300 times with a maximum of 2.5x106 energy evaluations, 150 individuals in the population and a maximum of 27000


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generations. Different docking runs were performed with either rigid protein or with several combinations of the VALα165, GLUβ200, TYRβ202, LEUβ255, METβ257, LYSβ350 and/or THRβ312 flexible sidechains. The results were analyzed with AutoDockTools,30 with Marvin31 and with an in-house developed java based software tool.32

Molecular Dynamics Simulations The initial structure for the molecular dynamics simulations was based on our previous docking results of unsubstituted indole isocombretastatins at the colchicine site of tubulin. All MD simulations were carried out with the AMBER1133 program using the ff10 force-field parameters for the protein,34 the gaff for the ligand,35 a set of parameters developed for a better thermodynamic description of guanine nucleotides and Mg2+ ions,36 the Joung and Cheatham parameters for non-polarizable spherical ions,37 and TIP3P for water molecules.38 The initial conformation was simulated with periodic boundary conditions in a truncated octahedron box of roughly 30000 TIP3P water molecules surrounding at least 10 Å between the solute and the faces and with the solute charges compensated by the addition of 38 Na+ ions. The system was energy-minimized with harmonic restraints on the solute atom positions followed by unrestricted energy minimization, heated to 300 K and then subjected to molecular dynamics simulations for 10 ns using an isothermal−isobaric ensemble and a 2 fs time-step. The trajectories were analyzed by means of the ptraj program within AMBER and visualized with vmd.39

Acknowledgments This work was supported by Spanish MEC (Ref CTQ2004-00369/BQU), the EU (Structural Funds), Spanish MICINN (SAF 2008-04242), the Consejería de Educación de la Junta de Castilla y León (SA067A09) and the AECID (PCI-Mediterráneo A1/037364/11). Different parts of the work were performed thanks to financial support from the Consejería de Educación de la Junta de Castilla y León


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(Programa de Apoyo a Proyectos de Investigación, EDU/940/ 2009, CSI052A11-2, and CSI221A12-2), Spanish Ministerio de Economía y Competitividad (BIO2010-16351, SAF2008-02251, SAF201130518), Comunidad de Madrid (S2010/BMD-2457 BIPEDD2 to JFD), Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, cofunded by the Fondo Europeo de Desarrollo Regional of the European Union (RD06/0020/1037 and RD12/0036/0065) and the European Community’s Seventh Framework Programme FP7-2007-2013 (grant HEALTH-F2-2011-256986, PANACREAS). R. A. thanks the Spanish MEC for FPU and the Fundación Ramón Areces for a predoctoral grant. We thank Beatriz G. Sierra for her excellent technical assistance during the early stages of this research, Nicholas Skinner for carefully proofreading the manuscript, Mariano Redondo Horcajo for his tubulin purification, and the people at Asocarsa S.A. slaughterhouse and Matadero Municipal Vicente de Lucas de Segovia for providing us with the calf brains. Compound 38 was synthesized during a pre-doctoral stay of R.A. in the laboratory of Prof. Westwell (University of Cardiff), whose collaboration is acknowledged.

Supporting Information Available. Full synthetic data and NMR spectra for compounds 1 - 45 and graphs (Fig. S1-2) of the correlation between the cytotoxicity of the compounds and their tubulin polymerization inhibitory activity or their affinity for the colchicine-binding site of tubulin. This material is available free of charge via the Internet at http://pubs.acs.org.


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Table of contents graphic


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Microtubules: A New Molecular Perspective Med. Res. Rev. 2011, 31, 443-481 (b) Dumontet, C.; Jordan, M. A. Microtubule-binding agents: a dynamic field of cancer therapeutics. Nat. Rev. Drug Discovery 2010, 9, 790-803. (c) April L. Risinger, A. L.; Giles, F. J.; Mooberry S. L. Microtubule dynamics as a target in oncology. Cancer Treat. Rev. 2009, 35, 255-261. 3

(a) Chaudhary, A.; Pandeya, S. N.; Kumar, P.; Sharma, P. P.; Gupta, S.; Soni, N.; Verma, K. K.;

Bhardwaj, G. Combretastatin A-4 analogs as anticancer agents. Mini-Rev. Med. Chem. 2007, 7, 11861205. (b) Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazzani, A. A. Medicinal chemistry of combretastatin A4: Present and future directions. J. Med. Chem. 2006, 49, 3033-3044. 4

(a) Pettit, G. R.; Rhodes, M. R.; Herald, D. L.; Chaplin, D. J.; Stratford, M. R.; Hamel, E.; Pettit, R.

K.; Chapuis, J. C.; Oliva, D. Antineoplastic agents 393. Synthesis of the Trans-Isomer of Combretastatin A4 Prodrug. Anti-Cancer Drug Des. 1998, 13, 981–993. (b) Pettit, G. R.; Toki, B. E.; Herald, D. L.; Boyd, M. R.; Hamel, E.; Pettit, R. K.; Chapuis, J. C. Antineoplastic agents. 410. Asymmetric hydroxylation of trans-combretastatin A-4. J. Med. Chem. 1999, 42, 1459-1565. 5

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disrupting agents: combating treatment resistance. Br. J. Radiol. 2008, 81, S12-S20. (b) Tozer, G. M.; Kanthou, C.; Baguley, B. C. Disrupting tumour blood vessels. Nat. Rev. Cancer 2005, 5, 423-435.


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(a) Mateo, C.; Pérez-Melero, C.; Peláez, R.; Medarde, M. Stilbenophane Analogues of

Deoxycombretastatin A-4. J. Org. Chem. 2005, 70, 6544-6547. (b) Mateo, C.; Álvarez, R.; PérezMelero, C.; Peláez, R.; Medarde, M. Conformationally restricted macrocyclic analogues of combretastatins. Bioorg. Med. Chem. Lett. 2007, 17, 6316-6320. (c) Mateo, C.; López, V.; Medarde, M.; Peláez, R. Synthesis and Conformational Analysis of Macrocyclic Dihydroxystilbenes linked up between the para-para positions. Chem. --Eur. J. 2007, 13, 7246-7256. (d) Álvarez, R.; López, V.; Mateo, C.; Medarde, M.; Peláez, R. New para–para Stilbenophanes: Synthesis by McMurry Coupling, Conformational Analysis and Inhibition of Tubulin Polymerisation. Chem. --Eur. J. 2011, 17, 34063419. 7

(a) Álvarez, R.; Álvarez, C.; Mollinedo, F.; Sierra, B. G.; Medarde, M.; Peláez, R.

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(a) Brancale, A.; Silvestri, R. Indole, a Core Nucleus for Potent Inhibitors of Tubulin

Polymerization. Med. Res. Rev. 2007, 27, 209-238. (b) Wang, L.; Woods, K. W.; Li, Q.; Barr, K. J.; McCroskey, R. W.; Hannick, S. M.; Gherke, L.; Credo, R. B.; Hui, Y.; Marsh, K.; Warner, R.; Lee, J. Y.; Zielinski-Mozng, N.; Frost, D.; Rosenberg, S. H.; Sham, H. L. Potent, Orally Active HeterocycleBased Combretastatin A-4 Analogues: Synthesis, Structure-Activity Relationship, Pharmacokinetics,


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and In Vivo Antitumor Activity Evaluation. J. Med. Chem. 2002, 45, 1697-1711. (c) Durrant, D. E.; Richards, J.; Tripathi, A.; Kellogg, G. E.; Marchetti, P.; Eleopra, M.; Grisolia, G.; Simoni, D.; Lee, R. M. Development of water soluble derivatives of cis-3, 4′, 5-trimethoxy-3′-aminostilbene for optimization and use in cancer therapy. Invest. New Drugs 2009, 27, 41-52. 9

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We have successfully nitrated the indolephenstatins but attempts to prepare the amines by reduction

or the isocombretastatins by Wittig reaction failed. García, R.; Álvarez, R.; Medarde, M.; Peláez, R. unpublished. 17

a) Examples of soluble prodrugs for aldehydes can be found in Adkison, K. K.; Barrett, D. G.;

Deaton, D. N.; Gampe, R. T.; Hassell, A. M.; Long, S. T.; McFadyen, R. B.; Miller, A. B.; Miller, L. J.; Payne, A.; Shewchuk, L. M.; Wells-Knecht, K. J.; Willard, D. H.; Wright, L. L. Semicarbazone-based inhibitors of cathepsin K, are they prodrugs for aldehyde inhibitors? Bioorg. Med. Chem. Lett. 2006, 16, 978-983. b) Examples of soluble amide prodrugs can be found in Almansa, C.; Bartrolí, J.; Belloc, J.; Cavalcanti, F. L.; Ferrando, R.; Gómez, L. A.; Ramis, I.; Carceller, E.; Merlos, M.; García-Rafanell, J. New Water-Soluble Sulfonylphosphoramidic Acid Derivatives of the COX-2 Selective Inhibitor Cimicoxib. A Novel Approach to Sulfonamide Prodrugs. J. Med. Chem. 2004, 47, 5579-5582.


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Very similar results to those described here were obtained by following the same protocol and using

alternative pdb structures such as 3HKC.pdb, 3HKD.pdb, 3HKE.pdb, 3N2G.pdb, 3N2K.pdb and 3UT5.pdb. 23

David-Cordonnier, M. H.; Gajate, C.; Olmea, O.; Laine, W.; de la Iglesia-Vicente, J.; Pérez, C.;

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by

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reversible

microtubule-disrupting

agent

2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-

cycloheptatrien-1 -one: protection by Bcl-2 and Bcl-X(L) and cell cycle arrest. Cancer Res. 2000, 60, 2651-2659. (b) Gajate, C.; Santos-Beneit, A. M.; Macho, A.; Lázaro, M.; Hernández-de Rojas, A.;


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http://www.wwpdb.org/

28

Discover program from http://www.accelrys.com/ was used.

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Endowing Indole-Based Tubulin Inhibitors with an Anchor for

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