Synthesis and Cytotoxic Evaluation of Combretafurazans - Journal of

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J. Med. Chem. 2005, 48, 3260-3268

Synthesis and Cytotoxic Evaluation of Combretafurazans Gian Cesare Tron, Francesca Pagliai, Erika Del Grosso, Armando A. Genazzani, and Giovanni Sorba* Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universita´ del Piemonte Orientale “A. Avogadro”, Via Bovio 6, 28100 Novara, Italy Received November 9, 2004

Combretastatin A-4 is an antitumoral and antitubulin agent that is active only in its cis configuration. In the present manuscript, we have synthesized cis-locked combretastatins embodying a furazan ring (combretafurazans). To achieve this, we have developed a new strategy that exploits the dehydration of vicinal dioximes using the Mitsunobu reaction. Among the advantages of following such a strategy are the mild conditions used for the construction of the diarylfurazan derivatives, allowing for the presence of highly functionalized substrates and deactivated aromatic rings. Combretafurazans are more potent in vitro cytotoxic compounds compared to combretastatins in neuroblastoma cells, yet maintaining similar structure-activity relationship and pharmacodynamic profiles. Introduction Malignant tumors represent one of the most common human diseases, and the clinical prognosis remains relatively poor. This therefore leaves ample space for the development of new therapeutic strategies for the improvement of drugs that are currently in use. Among the current targets for chemotherapy, alongside DNA, microtubules have a prominent role. Since tubulin-containing structures are crucial in a number of cellular functions, such as chromosome segregation during cell division, intracellular transport, cell motility and the maintenance of cell shape, microtubules have represented a good target for chemotherapy, and a number of drugs, mainly of natural origin, have emerged.1 Microtubules are at a dynamic equilibrium, with monomeric and polymerized tubulin interchanging, and this swaying process is thought to be fundamental for their cellular functions. The ability to disrupt microtubule assembly or disassembly makes tubulin inhibitors preferential for cells that have a high proliferation rate, such as tumors, since it results in cell arrest during mitosis.1 Such a dynamic process is also highly pronounced in the neovasculature of solid tumors, and this represents an additional attraction of tubulin inhibitors in chemotherapy.2 Since inhibition of polymerization or inhibition of the disassembly leads to similar cellular events, drugs that target either process have been developed. For example, taxanes (such as paclitaxel and docetaxel) prevent the disassembly of the microtubule and are drugs of choice for ovarian and breast cancers, while other antimitotic drugs of natural origin (such as vinca alkaloids) prevent the assembly of the complex. Among the most recent additions to this class of antitumoral agents are combretastatins, first identified in the bark of the South African willow tree Combretum caffrum. Combretastatin A-4 (CA-A4) 1 appears to be the most powerful antimitotic agent of this series and is remarkably simple in its chemical structure (Figure 1), while being one of the most potent tubulin inhibitors * To whom correspondence should be addressed. Tel: +39-0321375827. Fax: +39-0321-375821. e-mail: giovanni.sorba@ pharm.unipmn.it.

Figure 1. Known SAR of combretastatin derivatives.

known.3 CA-A4 has been shown to possess a powerful cytotoxic activity against a broad panel of tumoral cell lines4,5 and MDR cells.6 In vivo, CA-A4 displays low or no antitumoral activity7 mainly because of low water solubility, and therefore phosphate pro-drugs have recently entered into clinical trials.8 Because of the interest in these structures, several synthetic analogues of CA-A4 have been developed allowing to sketch structure-activity relationships (Figure 1). The cis configuration of the double bond is fundamental as is the 3,4,5-trimethoxy group on ring A. However, recently Gaukroger questioned the importance of the trimethoxyphenyl group for the antitubulin activity.9 Ring B seems more amenable to isosteric substitutions. For instance, substituting the hydroxyl group with an amine leads to higher cytotoxicity and an increased water solubility through salification.10 By comparing the minimal energy conformations for both colchicine and combretastatin, it has been demonstrated that it is possible to overcome the problem of the isomerization of the active cis double bond to the inactive trans by introducing five-membered heterocycles in place of the olefin group without substantial loss of potency.11 These cis-locked analogues provide three main advantages: (a) prevention of isomerization of combretastatin from cis to trans; (b) increased specificity, since the trans conformation might be recognized by other cellular targets; and (c) the possibility to use heterocyclic systems that might improve the therapeutic potential of these drugs. This is strengthened by computational analysis showing that tubulin, on its colchicine site, can host the compounds with

10.1021/jm049096o CCC: $30.25 © 2005 American Chemical Society Published on Web 04/13/2005

Evaluation of Combretafurazans

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3261

Scheme 1a

a Reagents and conditions: (a) TBDMSiCl, imidazole in CH Cl ; (b) AD mix R (Sharpless asymmetric dihydroxylation reagent), 2 2 methanesulfonamide in H2O/tert-butyl alcohol; (c) NaOCl, KBr, TEMPO in CH2Cl2/H2O; (d) NH2OH‚HCl in pyridine/ethanol at 90 °C; (e) NaOH in 1,2-propanediol at 160 °C; (f) PPh3, DIAD in toluene at 0 °C then reflux.

different spatial orientations and that three different binding domains can be involved in the interaction with these molecules.12 The strategy to improve the efficacy or potency of combretastatin analogues has been widely used and a number of heterocyclic compounds, as new possible bioisosteres of the double bond of the combretastatins, have been prepared.13 These studies have also shown that loss of activity can be ascribed to an incorrect orientation of two phenyl rings into the binding site due to the specific five-membered ring.13 Therefore, a key structural factor for the cytotoxic activity is the presence of the double bond which forces the two aromatic rings to be at an appropriate distance and to have an optimal dihedral angle to maximize interactions with tubulin. In the present manuscript, we have rigidified combretastatin by replacing the olefin group with the furazan system (1,2,5-oxadiazole) maintaining the trimethoxy group on ring A and synthesizing derivatives (e.g. 3-hydroxy-4-methoxy, 4-methoxy, 3-amino-4-methoxy) on phenyl ring B (combretafurazans). To accomplish this task, we have developed a new synthetic procedure which employs the Mitsunobu reaction to close the vicinal dioximes. This has also allowed us to synthesize substituted diphenylfurazans using mild reaction conditions. We hereby show that replacing the olefin group with the furazan moiety increases the cytotoxic potency of

the compounds when tested in neuroblastoma cells, while maintaining similar structure-activity relationship and pharmacodynamic profiles of combretastatin A-4. Chemistry The combretafurazans derivatives were synthesized starting from corresponding stilbene derivatives obtained using the Perkin reaction.14 The furazan analogue of CA-A4 was synthesized according to Scheme 1. Protection of the phenolic group of 1 with tertbutyldimethylsilyl chloride gave a compound which was dihydroxylated using the protocol developed by Pettit15 to obtain the desired diol 2 along with the diketone 3. Our attempt to carry out the dihydroxylation using catalytic osmium tetroxide and morpholine N-oxide failed, as in our hands we were only able to isolate the two aldehydes resulting from the cleavage of the double bond. The diol 2 was oxidized to diketone 3 using TEMPO and sodium hypochlorite, and the following transformation into bis-oxime 4 was performed in an excess of hydroxylamine hydrochloride and pyridine, heated for 3 days at 90 °C. Higher reaction times and temperatures gave only one of four possible isomers of vicinal dioximes, the thermodynamically more stable isomer (E,E bis-oximes or anti form), and this was

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Scheme 2. Proposed Mechanism for the Formation of Nitrile Byproducts

confirmed using the analytical methods based on the complex formations between bis-oximes and nickel(II) salts.16 We then attempted to close the vicinal dioximes to furazan. The traditional synthesis of the furazan ring system starting from vicinal dioximes involves harsh reaction conditions and is performed only on unfunctionalized or poorly functionalized substrates.17 Such difficulty is highlighted by the existence of few such reactions described in chemical databases (SciFinder Scholar and Beilstein CrossFire). The two main methods that have been developed in the literature consist in the dehydration of vicinal dioximes in basic or acidic medium18 or in the deoxygenation of the corresponding furoxans (1,2,5-oxadiazole 2-oxide) with reducing agents such as triethyl phosphite.19 The dehydrating conditions require a molar excess of sodium hydroxide in 1,2-ethanediol or a strong acid, such as p-toluenesulfonic acid at high temperature (ca. 160 °C). Our attempt to carry out this reaction using p-toluenesulfonic acid at reflux led to the formation of a plethora of hardly separable compounds. Dehydration in basic medium successfully formed two main products: the desired compound 5a and a demethylated product 5b. Nonetheless, due to the presence of byproducts, even after column chromatography, it was not possible to obtain 5a with an acceptable purity. Furthermore, since these reactions are unlikely to be tenable with functionalized substrates or deactivated aromatic rings, we abandoned such strategies. We then developed a new protocol that uses the Mitsunobu reaction20 for the synthesis of furazan via vicinal dioximes. It is well-known that the Mitsunobu reaction carried out on 1,2 diols brings epoxide formation deriving from an intramolecular Mitsunobu reaction. Our hypothesis was that the closure of the vicinal dioximes, which mimic the 1,2 diol function (4), via the intramolecular Mitsunobu reaction should directly yield the furazan ring. This new strategy would allow the use of milder reaction conditions which would then be more compatible with functionalized substrates. Usually, the acidic component of the reaction has to possess a pKa not higher than 11 to allow protonation of the triphenylphosphine-DIAD adduct in order for the reaction to

work successfully,21 and therefore the use of diphenyldioximes which have pKa between 8 and 11 should yield 5a.22 The reaction with 4 was performed using toluene as solvent. After the addition of DIAD at 0 °C, the reaction was heated at reflux and was completed in 1 h, giving the desired product 5a. Although the anti configuration of 4 should not favor the ring closure, it has been shown that the isomerization of dioximes can occur easily under various conditions (acid or basic medium, heat, UV light), and this might provide an explanation for the success of the reaction.23 Furthermore, during the course of the reaction, the removal of the protecting group was also observed. Last, the formation of combretafurazan 5a was accompanied by the formation of two nitriles derivatives as byproducts (5c, 24%; 5d 30%) deriving from a Beckmann fragmentation.23 The proposed mechanism for the formation of the two nitriles is outlined in Scheme 2. The ring closing reaction competes with the Beckmann fragmentation, which yields a nitrile derivative and a carbocationic species which is reduced to nitrile via nitrile N-oxide.24 This hypothesis is supported by the knowledge that the Beckmann fragmentation competes with transposition when the carbocation formed is relatively stable.23 This occurs in the reaction described since the trimethoxyphenyl carbocation is stabilized by the three electronreleasing methoxy groups. To prove this hypothesis we carried out the Mitsunobu reaction on diphenyl dioximes displaying in the para position: an electron donating group (OMe, 6), no functional group (7) or an electron-withdrawing group (Br, 8). Such reactions yielded the desired furazan systems 6a (51%), 7a (59%), 8a (68%) in fair yield (Scheme 3). The best yield was obtained, as hypothesized, when the electron-withdrawing group was present (8a), suggesting that the Beckmann fragmentation pathway was depleted.25 The synthesis of combretafurazan bearing only a methoxy group on the B ring is reported in Scheme 4. We chose to prepare the diketone moiety 10 starting from corresponding stilbene 9 using Sharpless protocol.26 The stilbene 9 was prepared starting from the corresponding carboxylic acid 3,4,5,-trimethoxy-R-[(4-



Scheme 3a

Scheme 5a

a Reagents and conditions: (a) PPh , DIAD in toluene at 0 °C 3 then reflux.

methoxyphenyl)methylene]-(E)-benzeneacetic acid.7b After the formation of the dioxime 11, we successfully obtained the corresponding furazan 12a via the Mitsunobu reaction. Parallel execution of this reaction with the dehydration in basic medium gave a significantly lower yield (18% vs 43%). Interestingly, demethylation of the 4-methoxy group to give 12b was, of course, not observed in the Mitsunobu conditions, while it was being predominant (25% yield) in the basic dehydration process. Last, the amine-substituted combretafurazan was prepared starting from the stilbene derivative 13 as outlined in Scheme 5. Decarboxylation of 13 with copper in quinoline gave the desired stilbene 14. After formation of the diketone 15, we were not able to obtain a single isomer of the dioxime, but only a mixture (16).

a Reagents and conditions: (a) Cu, quinoline 180 °C; (b) KMnO 4 in Ac2O; (c) NH2OH‚HCl in pyridine/ethanol 90 °C; (d) NaOH in 1,2-propanediol at 160 °C; (e) PPh3, di-tert-butyl azodicarboxylate in toluene at 0 °C then reflux; (f) H2, Pd/C 5% in EtOH.

Attempts to separate the isomers failed, so we used the mixture without further purification to perform the dehydrating reaction. The basic dehydration applied to electron-withdrawing substituted vicinal dioximes led to a reaction mixture from which no furazan derivative was isolated. The most likely explanation for this is that the nitro group deactivates the aromatic ring and promotes nucleophilic attack both on the phenyl group and on the methoxy group to give unstable phenolic derivatives, which are subsequently oxidized. On the other hand, the desired product (17) was obtained in

Scheme 4a

a Reagents and conditions: (a) KMnO in Ac O; (b) NH OH‚HCl in pyridine/ethanol at 90 °C; (c) PPh , DIAD in toluene at 0 °C then 4 2 2 3 reflux; (d) NaOH in 1,2-propanediol at 160 °C.

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Scheme 6a

a

Reagent and conditions: (a) TBAF sol. 1 M in THF.

good yield (40%) with the Mitsunobu reaction. This strongly suggests that this strategy is an improvement to the published reactions to perform dehydrating reactions to form furazan ring systems from phenyl dioximes bearing a deactivating group. In this last reaction, the Mitsunobu reaction was performed replacing DIAD with di-tert-butyl diazodicarboxylate to allow an easier purification of the furazan from hydrazodicarboxylate. Reduction of the nitro compound with hydrogen and Pd/C 5% gave the final amino derivative 18. Biological Results and Discussion All final products (5a, 5b, 12a, 12b, 17, 18) were tested in cytotoxicity assays. Furthermore, we also decided to test the two main intermediates, the diketone 19 and the bis-oxime 20, after removal of their protecting groups (Scheme 6). Cytotoxicity was evaluated on a neuroblastoma cell line, SH-SY5Y, and cells were treated for 48 h with increasing concentrations of combretastatin A-4 (1) or combretafurazans to obtain an approximate IC50 value. Preliminary data on other experimental time points (i.e. 24 and 72 h) yielded similar results (data not shown). The results (Table 1) show that treatment with combretastatin A-4 was capable of significantly reducing tumoral cell viability at nM concentrations with an approximate IC50 of 5.8 nM. The synthetic intermediates 19 and 20 lost approximately 90- and 40-fold activity compared to 1, although they still remained cytotoxic. In our opinion this signifies that the vicinal dioxime or diketone reduces the affinity of the compound for tubulin, revealing the importance of the correct alignment of the two phenyl rings. Strikingly, compound 5a, where the dioxime was closed via the Mitsunobu reaction, displayed an IC50 approximately 4-fold lower than combretastatin, suggesting that the conformational block of ring A and B elicited by the furazan ring favors tumoral cell cytotoxicity. Compound 5a also displayed a similar Hill slope, vaguely suggesting a similar mechanism of action (Figure 2). When compound 5b was tested, which bears demethylation at the para position of ring A, a significant loss of activity compared to 5a was evident (approximately 12-fold). When compound 12a was tested, which bears meta dehydroxylation of ring B, a similar loss of potency was observed. As might be expected when

Tron et al.

compound 12b was assessed, which displays both the demethylation at the para position of ring A and the dehydroxylation in meta of ring B, a further substantial loss of activity was identifiable compared to 5a (140fold). Finally, the meta position of ring B was either substituted with an electron-withdrawing nitro group (17) or an electron-donating amino group (18). Indeed, compound 17 displayed a 50-fold drop in activity while the potency of compound 18 reflected that of 5a. In conclusion, this preliminary assay shows that the substitution of the double bond with the furazan ring increases the potency of combretastatin. Furthermore, the combretafurazans display a similar structureactivity relationship and relative rank order of potency compared to the original structure.13 For example, demethylations on the A ring generate a loss of potency both in combretafurazans (5b) and combretastatins.10 Similarly, substituting the hydroxyl with the Grimm’s isostere amino group (18) does not result in any significant difference in the activity of either structure,10 while substituting the same position with an electronwithdrawing group (17) causes a loss of potency in both combretastatin and combretafurazan.10 Such similarity would suggest that combretafurazans elicit their cytotoxicity with a similar mechanism of action compared to combretastatin. To establish a qualitative link between combretastatin and these compounds, we performed immunocytochemistry on cells treated with the two most potent combretafurazans, 5a and 18, to establish whether these were able to mimic the reported ability of combretastatin to inhibit microtubule structure in cultured cells.2,13 Indeed, while control cells displayed the typical weblike tubulin architecture (Figure 3a), cells treated with 1, 5a or 18 displayed a disorganized tubulin structure and a change in gross morphology (Figure 3b-d). To confirm this finding, cells were grown in the presence of 1, 5a or 18, and proteins were extracted in the presence of paclitaxel, which prevents further tubulin rearrangements. Western blotting of the pelletable fraction and the soluble fraction then allows distinction between the polymerized and free form of tubulin.27 Indeed, while in control cells tubulin was present in similar amounts in the polymerized (pellet) and free form (supernatant), the equilibrium was significantly shifted toward the free form when cells were treated with 1 or with combretafurazans (Figure 3e). This is yet further evidence that inhibition of tubulin polymerization represents the primary mechanism of action of combretafurazans. Last, the cytotoxic nature of these compounds and the ability to inhibit tubulin might suggest that these drugs might trigger apoptosis in a cell-cycle specific manner,13 causing cell arrest in the G2/M phase. To establish if these assumptions were correct, we aimed at verifying whether treatment of cells with these compounds could activate caspase-3, an effector of apoptosis.28 This enzyme is present in cells as a 33 KDa protein, and is cleaved to an active 17 KDa protein when the apoptotic program is initiated.28 In control cells, while the inactive protein was present, there was no band corresponding to the active fragment. On the contrary, cells treated with combretastatin or combretafuranzans displayed the active fragment suggesting that programmed cell death is responsible for their cytotoxic effects (Figure



Table 1. Cytotoxicity of Combretastatin and Combretafurazans on SH-SY5Y Neuroblastoma Cellsa IC50 (nM) n

1

5a

5b

12a

12b

17

18

19

20

5.8 (16)

1.4 (12)

17.5 (12)

8.9 (12)

196 (12)

72.5 (12)

1.6 (20)

524 (8)

254 (8)

a

IC50 values have been generated by a curve-fitting program (Kaleidograph). Full concentration-response curves for compounds 1, 5a and 18 are illustrated in Figure 2. The number of 8-point concentration-response curves performed for each sample is in parentheses.

Figure 2. Concentration-response curve of combretastatin (1) and of the two most potent combretafurazans synthesized. Values are mean ( SEM of 12-24 determinations in 3-6 separate experimental days. * p < 0.01 if compared to 5a and vs 18 (ANOVA followed by Student’s t-test)

4). These cells also displayed an increased expression of procaspase-3, suggesting that up-regulation of this protein might also take place. Last, cell cycle effects of these compounds were assessed using propidium iodide staining. Indeed, while control cells were mainly in G1 phase, cells treated with combretafurazans or combretastatins resulted in G2/M cell arrest (Figure 5). Such arrest was time-dependent with accumulation in the tetraploid peak observable after 6 h and reaching a plateau after 16 h, after which cell death rendered the experiment difficult to interpret and perform (data not shown). Conclusions In conclusion, we have successfully used a new protocol to synthesize functionalized diphenylfurazan compounds starting from vicinal dioximes using mild reaction conditions. This method represents the first example of using the Mitsunobu reaction for the construction of furazan rings. Replacing the double bond with the furazan moiety leads to slightly more potent compounds as assessed in cytotoxicity assays. Furthermore, in neuroblastoma cells, rigidifying combretastatin with a furazan group does not change the reported structure-activity relationship of this natural product.10 Last, these compounds appear to elicit their tumor cytoxicity in a fashion similar to combretastatin, via inhibition of tubulin polymerization, which then leads to cell cycle arrest in G2/M and subsequent apoptosis. Experimental Section Chemistry. Commercially available reagents and solvents were used without further purification and were purchased from Fluka-Aldrich or Lancaster. Tetrahydrofuran (THF) was distilled immediately before use from Na/benzophenone under a slight positive atmosphere of N2, and toluene was dried by distillation from sodium and stored on activated molecular sieves (4 Å). When needed the reactions were performed in flame- or oven-dried glassware under a positive pressure of dry N2.

Figure 3. Combretastatin and combretafurazans affect tubulin polymerization (a-d). Bright field and immunfluorescence using an anti-tubulin antibody of control cells (a) or cells treated for 24 h with combretastatin (3 nM, b), 5a (3 nM, c), or 18 (3 nM, d). Brightfield images have been obtained with an 20× objective, while immunofluorescence images have been obtained with a 100×/oil immersion objective. (e) Western blot of tubulin extracted in the presence of paclitaxel from neuroblastoma cells treated with the indicated compounds all at 50 nM for 16 h. Results are representative of three separate experiments which yielded comparable results. P represents the pelletable (polymerized) fraction while S represents supernatant (free) fraction of tubulin. Melting points were determined in open glass capillary with a Stuart scientific SMP3 apparatus and are uncorrected. All the compounds were checked by IR (FT-IR THERMO-NICOLET AVATAR); 1H and 13C APT (JEOL ECP 300 MHz) and mass spectrometry (Thermofinningan LCQ-deca XP-PLUS and Thermofinningan TRACE GC-POLARIS Q). Chemical shifts are reported in part per million (ppm). Column chromatography was performed on silica gel (Merck Kieselgel 70-230 mesh ASTM) using the indicated eluants. Thin-layer chromatography (TLC) was carried out on 5 × 20 cm plates with a layer thickness of 0.25 mm (Merck Silica gel 60 F254). When necessary they were developed with KMnO4 or vanillin reagent. Elemental analysis (C, H, N) of the target compounds was performed by REDOX (Monza, Italy) and are within (0.4% of the calculated values unless otherwise noted. Compounds 1,14 6 29, 7 29 and 137b were synthesized as described previously. (1S,2R)-1-(3-{[tert-Butyl(dimethyl)silyl]oxy}-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-1,2-ethanediol (2). AD mix-R (83 g) was added to a biphasic solution of 200 mL of

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Figure 4. Combretastatin and combretafurazans activate caspase-3. Western blot of samples extracted from neuroblastoma cells treated for 24 h with the indicated compounds (all at 3 nM). Results are representative of three separate experiments.

Figure 5. Combretastatin and combretafurazans induce cell cycle arrest. Cell cycle analysis of neuroblastoma cells treated for 16 h with vehicle (a), 1 (b, 10 nM), 5a (c, 10 nM), and 18 (d, 10 nM). Data are representative of three separate experiments. Similar results were obtained in separate experiments when drugs were incubated for 8 or 24 h. The Y-axis represents cell number and the X-axis represents fluorescence on a linear scale. tert-butyl alcohol and 200 mL of water. After its dissolution, methansulfonamide (4.6 g) was added and the contents were cooled to 0 °C. cis-Silyl ether combretastatin A-4 (20 g; 0.046 mol) was added and the reaction mixture stirred vigorously in the dark. After 30 h, sodium sulfite was added (10 g), 30 min later the reaction was diluted with EtOAc, and the layers were separated. The aqueous layer was further washed with EtOAc (× 4), and the combined organic extracts were washed with 2 M NaOH (× 2) and brine (× 1). After drying over sodium sulfate and evaporation of the solvent, the crude product was purified by column chromatography using PE/EtOAc 8:2 to give 5.19 g of corresponding diketone 3 (25%). Finally the eluant was replaced with PE/EtOAc 5:5 to elute 6.2 g of diol 2 as an amorphous gum (29%). 1-(3-{[tert-Butyl(dimethyl)silyl]oxy}-4-methoxyphenyl)2-(3,4,5-trimethoxyphenyl)-1,2-ethanedione (3). To a solution of 2 (1.5 g; 3.23 mmol) in dry CH2Cl2 were added TEMPO (50 mg, 0.323 mmol, 0.1 equiv) and KBr (576 mg, 4.84 mmol, 1.5 equiv). The resulting solution was then cooled at 0 °C, and commercial sodium hypochlorite at 5% was added under vigorous stirring (573.5 mg, 7.75 mmol, 2.4 equiv) to yield a yellow solution. After 10 min, the reaction was worked up by dilution with CH2Cl2 and washed sequentially with water, 2 N HCl and brine. The solution was then evaporated and purified by column chromatography using PE/EtOAc 9:1. Compound 3 was obtained as a yellow powder (1.4 g, 94%). 1-(3-{[tert-Butyl(dimethyl)silyl]oxy}-4-methoxyphenyl)2-(3,4,5-trimethoxyphenyl)-1,2-ethanedione Dioxime (4). To a solution of 3 (5.86 g, 12.75 mmol) in dry pyridine (58 mL) and ethanol (76 mL) was added an excess of hydroxylamine hydrochloride (8.92 g, 127.54 mmol; 10 equiv). The resulting solution was heated at 90 °C for 3 days, worked up by dilution of EtOAc and washed with 2 N HCl (× 3), water (× 1) and brine (× 1). After drying over sodium sulfate and evaporation

Tron et al. of the solvent, the crude vicinal dioxime was purified by column chromatography using PE/EtOAc 4:6 as eluant to obtain 3.3 g of 4 as a colorless gum. (85%). 2-Methoxy-5-[4-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazol-3-yl]phenol (5a) and 4-[4-(3-Hydroxy-4-methoxyphenyl)-1,2,5-oxadiazol-3-yl]-2,6-dimethoxyphenol (5b). Basic Dehydration Method. To a solution of 300 mg of 4 (0.61 mmol) in 1,2-propanediol (4 mL) was added 244 mg of sodium hydroxide (6.1 mmol: 10 equiv). The resulting suspension was heated at 150 °C for 16 h and worked up by diluition with EtOAc and washed sequentially with 2 N HCl and brine. After drying over sodium sulfate and evaporation of the solvent, the residue was purified by column chromatography using PE/ EtOAc 8:2 to elute 5a and 5b. The obtained compounds were then washed with EtOH to give 5a (63 mg, 29%) and 5b (14 mg, 7%) as white powders. Mitsunobu Dehydration Method. To a cooled (0 °C) suspension of 4 (300 mg; 0.61 mmol) in dry toluene (4 mL) was added triphenylphosphine (400 mg, 1.53 mmol, 2.5 equiv). DIAD (301 µL, 309 mg, 1.53 mmol, 2.5 equiv) was then added dropwise, and the resulting solution was heated at reflux for 1 h. The reaction was worked up by evaporation of the solvent, and the residue was purified by column chromatography. Elution with PE/EtOAc 9:1 gave 35 mg of 3,4,5-trimethoxybenzonitrile (5d, 30%). The solvent was then changed to PE/EtOAc 8:2 to elute 42 mg of 3-hydroxy4-methoxybenzonitrile (5c, 24%). Finally, the eluant was changed to PE/EtOAc 7:3 to give 52 mg of 5a as a white powder (24%). 1,2-Bis(4-bromophenyl)-1,2-ethanedione Dioxime (8). To a solution of 1,2-bis(4-bromophenyl)-1,2-ethanedione (2 g, 5.45 mmol) in dry pyridine (20 mL) and ethanol (20 mL) was added an excess of hydroxylamine hydrochloride (3.75 g, 54.5 mmol; 10 equiv). The resulting solution was heated at 90 °C for 2 days and worked up by dilution of EtOAc and washing with 2 N HCl (× 3), water (× 1) and brine (× 1). After drying over sodium sulfate and evaporation of the solvent, the crude product was purified by column chromatography using PE/ EtOAc 8:2 as eluant to obtain 0.76 g of 8 as a colorless gum (35%). 2,3-Dimethoxy-5-[(Z)-2-(4-methoxyphenyl)ethenyl]phenyl Methyl Ether (9). To 3,4,5,-trimethoxy-R-[(4-methoxyphenyl)methylene]-(E)-benzeneacetic acid7b (19 g, 0.055 mol) dissolved in quinoline (200 mL) was added copper powder (16.7 g; 5 equiv). The resulting mixture was heated at 220 °C for 3 h. After cooling, the copper was filtered off through a Celite pad. The filtrate was washed with concentrated hydrochloric acid (20 mL), and the aqueous layer was filtered again through a Celite pad and extracted several times with EtOAc. The combined organic layers were washed with saturated aqueous sodium carbonate and brine. After drying over sodium sulfate and removal of the solvent, the residue was purified by column chromatography using as eluant PE/EtOAc 9:1 to give 9 as an amorphous solid (14.7 g, 89%). 1-(4-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-1,2ethanedione (10). To a cooled (0 °C) and stirred solution of 9 (3 g, 0.02 mol) in acetic anhydride (60 mL) was added portionwise potassium permanganate (12.64 g, 0.08 mol, 4 equiv). During the addition the temperature was increased to 20 °C. After 1 h the reaction was cooled at 0 °C and was worked up by dilution with water. Sodium sulfite was added until all potassium permanganate was reduced. The mixture was extracted with EtOAc, and the organic phases were washed respectively with saturated aqueous NaHCO3 and brine and dried over sodium sulfate. Evaporation of the solvent gave a crude product, which was recrystallized with PE/EtOAc 8:2, obtaining 1.84 g of diketone 10 as a yellow solid (28%). 1-(4-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-1,2ethanedione Dioxime (11). To a solution of 10 (3 g, 9.0 mmol) in dry pyridine (30 mL) and ethanol (40 mL) was added an excess of hydroxylamine hydrochloride (6.25 g, 90 mmol; 10 equiv). The resulting solution was heated at 90 °C for 3 days and worked up by dilution of EtOAc and washing with 2 N sulfuric acid (× 3), water (× 1) and brine (× 1). After drying over sodium sulfate and evaporation of the solvent, the crude



product was purified by column chromatography using PE/ EtOAc 5:5 as eluant to obtain 3 g of 11 as a colorless gum (95%). 3-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5oxadiazole (12a) and 2,6-Dimethoxy-4-[4-(4-methoxyphenyl)-1,2,5-oxadiazol-3-yl]phenol (12b). Basic Dehydration Method. To a solution of 300 mg of 11 (0.833 mmol) in 1,2propanediol (4 mL) was added 333 mg of sodium hydroxide (8.33 mmol; 10 equiv). The resulting suspension was heated at 150 °C for 16 h and worked up by dilution with EtOAc and washed sequentially with 2 N HCl and brine. After drying over sodium sulfate and evaporation of the solvent, the residue was purified by column chromatography (PE/EtOAc 9:1 to elute 12a and PE/EtOAc 8:2 to elute 12b). The compounds obtained were then washed with EtOH to give 12a (52 mg, 18%) and 12b (67 mg, 25%) as white powders. Mitsunobu Dehydration Method. To a cooled (0 °C) suspension of 11 (200 mg; 0.55 mmol) in dry toluene (3 mL) was added triphenylphosphine (216.3 mg, 0.825 mmol, 1.5 equiv). DIAD (162.4 µL, 166.8 mg, 0.825 mmol, 1.5 equiv) was then added dropwise, and the resulting solution was heated at reflux for 1 h. The reaction was worked up by evaporation of the solvent, and the residue was purified by column chromatography. Elution with PE/ EtOAc 95:5 gave 15 mg of 4-methoxybenzonitrile (34%). The solvent was then changed to PE/EtOAc 9:1 to elute 18 mg of 3,4,5-trimethoxybenzonitrile (17%) and 80 mg of 12a as a white powder (43%). 2,3-Dimethoxy-5-[(Z)-2-(4-methoxy-3-nitrophenyl)ethenyl]phenyl Methyl Ether (14). To the carboxylic acid 13 (5 g, 0.013 mol) dissolved in quinoline (50 mL) was added copper powder (4.26 g). The resulting mixture was heated at 180 °C for 2.5 h. After cooling, the copper was filtered off through a Celite pad. The filtrate was washed with concentrated hydrochloric acid (20 mL), and the aqueous layer was filtered again through a Celite pad and extracted several times with EtOAc. The combined organic layers were washed with saturated aqueous sodium carbonate and brine. After drying over sodium sulfate and removal of the solvent, the residue was purified by column chromatography using as eluant PE/EtOAc 7:3 to give 14 as brownish solid (1.83 g, 41%). 1-(4-Methoxy-3-nitrophenyl)-2-(3,4,5-trimethoxyphenyl)1,2-ethanedione (15). To a cooled (0 °C) and stirred solution of 14 (1 g, 2.89 mol) in acetic anhydride (22 mL) was added portionwise potassium permanganate (1.8 g, 11.59 mmol, 4 equiv). During the addition the temperature increased to 4 °C. After 30 min the reaction was cooled at 0 °C and was worked up by dilution with water; solid sodium sulfite was added until all potassium permanganate was reduced. The compound was then extracted twice with EtOAc, and the combined organic extracts were washed with saturated aqueous NaHCO3 (× 2) and brine and dried over sodium sulfate. Evaporation of the solvent gave a crude semisolid residue, to which ethanol was added to decompose excess acetic anhydride. The residue so obtained was then crystallized with MeOH to give 475 mg of diketone 15 as a yellow solid (44%). 1-(4-Methoxy-3-nitrophenyl)-2-(3,4,5-trimethoxyphenyl)1,2-ethanedione Dioxime (16). To a solution of 15 (2 g, 5.33 mmol) in dry pyridine (58 mL) and ethanol (76 mL) was added an excess of hydroxylamine hydrochloride (3.73 g, 53.33 mmol; 10 equiv). The resulting solution was heated at 90 °C for 2 days and worked up by dilution with EtOAc and washed respectively with 2 N HCl (× 3), water (× 1) and brine (× 1). After drying over sodium sulfate and evaporation of the solvent, the crude dioxime was purified by column chromatography using PE/EtOAc 5:5 as eluant to obtain 1.86 g of 22 as a reddish gum (86%). 3-(4-Methoxy-3-nitrophenyl)-4-(3,4,5-trimethoxyphenyl)1,2,5-oxadiazole (17). To a cooled (0 °C) suspension of 16 (1.205 g; 2.97 mmol) in dry toluene (20 mL) were added triphenylphosphine (1.56 g, 5.95 mmol, 2 equiv) and di-tertbutyl azodicarboxylate (1.37 g, 5.95 mmol, 2 equiv). The resulting solution was heated at reflux for 1 h. The reaction was worked up by evaporation of the solvent, and the residue was purified by column chromatography. Elution with PE/

EtOAc 9:1 afforded 3,4,5-trimethoxybenzonitrile along with ditert-butyl hydrazoazodicarboxylate. The eluant was then changed to PE/EtOAc 8:2 and PE/EtOAc 7:3 to give 17 as a yellow solid (453 mg, 40%). The solid was crystallized from ethanol. 2-Methoxy-5-[4-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazol-3-yl]aniline (18). A mixture of 17 (166 mg, 0.428 mmol), 5% palladium on carbon (100 mg) and 20 mL of EtOH was equipped with a balloon of hydrogen gas and stirred at room temperature. After 2 h, the reaction mixture was filtered through a pad of Celite. The filtrate was evaporated and the residue was purified by column chromatography using as eluant PE/EtOAc 7:3 to give 18 as a white powder (123 mg, 80%). 1-(3-Hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-1,2-ethanedione (19). To a solution of 3 (379 mg, 0.824 mmol) in dry THF (3 mL) was added TBAF (1 M solution in THF) (824 µL, 0.824 mmol, 1 equiv). The solution became rapidly red. After 5 min, the reaction was worked up by diluition of EtOAc and washed with saturated aqueous NH4Cl. The water phase was extracted once again with EtOAc. The combined organic phases were then washed with brine and dried over sodium sulfate. After evaporation, the crude dione was purified by column chromatography using as eluant PE/EtOAc 8:2 to give 19 as yellow powder (150 mg, 40%). 1-(3-Hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-1,2-ethanedione Dioxime (20). To a solution of 4 (150 mg, 0.31 mmol) in dry THF (1.5 mL) was added TBAF (1 M in THF) (380 µL, 0.31 mmol, 1 equiv). The solution became rapidly red. After 2 h, the reaction was worked up by dilution with EtOAc and washed with saturated aqueous NH4Cl. The water phase was extracted a second time with EtOAc. The organic phases collected were then washed with brine and dried over sodium sulfate. After evaporation, the crude was purified by column chromatography using as eluant PE/EtOAc 8:2 to give 20 as amorphous solid (108 mg, 93%). Cell Culture and Cytotoxicity Assay. The SH-SY5Y human neuroblastoma cell line was obtained from ATCC (LGC Promochem Teddington, UK) and cultured in 50% DMEM and 50% F-12 supplemented with 10% foetal bovine serum, 2 mM L-glutamine, penicillin (100 µg/mL), and streptomycin (100 µg/ mL). For cytotoxicity assay, cells were plated on 24-well plates and grown for 24, 48 or 72 h in the presence or absence of combretastatin or combretafurazans. On the experimental day, cells were washed twice in Locke’s solution (134 mM NaCl, 5 mM KCl, 4 mM NaHCO3, 10 mM HEPES [pH 7.6], 2.3 mM CaCl2, 1 mM MgCl2, 5 mM sucrose) and incubated for 1 h with MTT (250 µg/mL in Locke’s solution) at 37 °C. Reactions were then stopped and the crystals solubilized in isopropyl alcohol/ HCl before being read at 570 nm in a spectrophotometer. To determine IC50 values, data were plotted and fitted using the Kaleidagraph software (Synergy software, Reading, PA). Immunocytochemistry. SH-SY5Y were plated on glass coverslips and grown to subconfluency in the presence or absence of drugs for 24 h. Cover slips were then fixed in paraformaldehyde (3.7%) for 30 min at 4°C. Cells were then washed once in phosphate buffered saline (PBS) and permeabilized in 0.1% Triton-X100 for 10 min at room temperature. Cells were then washed twice in PBS and blocked with 10% horse serum. Anti-tubulin primary antibody (Sigma-Aldrich; 1:1000) was incubated overnight, and after three further washes, coverslips were incubated with a chicken anti-mouse secondary Texas Red-conjugated antibody (Molecular Probes, 1:250). Slides were then visualized in a Nikon upright fluorescence microscope. Tubulin Polymerization Assay. To measure the degree of tubulin polymerization, we used an adaptation of the methods described by Minotti et al.27 In brief, cells were grown in 75 cm2 flasks in the presence or absence of drugs for 16 h. Cells were then tripsinised and centrifuged twice at 600g for 5 min. Cells were then resuspended in 70 µL of of hypotonic buffer (20 mM Tris-Hcl pH 6.8, 1 mM MgCl2, 2 mM EGTA, protease inhibitors, 0.5% Igepal) containing 4 µg/mL paclitaxel. Lysates were incubated for 10 min at room temperature and

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then vortexed. Lysates were then corrected for protein amounts (Bradford assay, Sigma-Aldrich) and 50 µg (corresponding to 50 µls) was centrifuged at 13000 rpm for 15 min at room temperature. Supernatant and pellet were then resuspended in equal volumes of SDS-loading buffer and run on a 10% SDS-PAGE polyacrylamide gel. After transfer of proteins to nitrocellulose (blocked in 5% milk), tubulin was identified with an anti-tubulin primary antibody (1:1000, Sigma-Aldrich) and anti mouse secondary antibody peroxidase-conjugated (1:8000, Amersham Bioscience) and visualized by chemiluminescence (Supersignal WestPico, Pierce). Assessment of Caspase-3 Activity. Cells were treated for 24 h in the presence or absence of the desired compounds. Cells were then resuspended in 10 mM Tris [pH 7.5], 150 mM NaCl, 5 mM EDTA [pH 8.0], 10 µg/mL of aprotinin, 10 µg/mL of leupeptin and 10 µg/mL of pepstatin A, 1 mM PMSF, 0.2 M sodium orthovanadate, 0.2 M sodium fluoride and 1% Triton X-100 and underwent three freeze-thaw cycles in dry ice. Lysates were the centrifuged at 13 000 rpm for 30 min at 4° C. Supernatants were then collected, and protein was determined. A 25 µg amount of protein was run on a 15% SDSPAGE polyacrylamide gel and transferred onto a nitrocellulose membrane. Procaspase-3 and caspase-3 were identified with an anti-caspase 3 primary antibody (1:1000, BD Pharmingen) and anti rabbit secondary antibody peroxidase-conjugated (1: 10000, Amersham Bioscience) and visualized by chemiluminescence (Supersignal WestPico, Pierce). Flow-Cytometric Analysis of Cell-Cycle Status. SHSY5Y grown in the presence or absence of compounds for 6, 12, 18 or 24 h were washed once in PBS and resuspended in 1 mL of 30:70 ice cold PBS/EtOH and stored at -20 °C. Cells were then washed twice in PBS and resuspended in PBS containing RNAse (100 µg/mL) for 1 h at 37°. DNA was then stained with a PBS solution containing 5 mM EDTA and 100 µg/mL propidium iodide for 30 min at 4 °C in the dark. Cell cycle analysis was determined with a FACSVantage SE DiVa (Becton Dickinson).

Acknowledgment. We dedicate this work, with deep respect, to Professor Alberto Gasco (University of Turin) our “maestro” and pioneer in the pharmacochemistry of 1,2,5-oxadiazole ring. Supporting Information Available: Characterization (mp, IR, MS, and 1H and 13C NMR data) of all new compounds and elemental analyses of all target compounds are available free of charge via the Internet at http://pubs.acs.org.

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