DOI: 10.1002/cplu.201300036

9-Fluorenone-2-Carboxylic Acid as a Scaffold for TubulinInteracting CompoundsFrancesco Calogero,[a] Stella Borrelli,[a] Gaetano Speciale,[a] Michael S. Christodoulou,[a]

Daniele Cartelli,[c] Dario Ballinari,[d] Francesco Sola,[d] Clara Albanese,[d] Antonella Ciavolella,[d]

Daniele Passarella,*[a] Graziella Cappelletti,[c] Stefano Pieraccini,[b] and Maurizio Sironi*[b]

Introduction

The fluorenone scaffold is widespread both in natural productsand in industrial by-products. Over the last few years its deriva-tives have generated interest because of their use in several di-verse fields ranging from drugs to materials science. The fluo-renone scaffold is found in natural biologically active[1, 2] andsemisynthetic compounds.[3–5] Among these compounds thereare some 2,7-disubstituted fluorenones such as tilorone[6] and2,7-disubstituted amidofluorenones,[7] which show anticancerproperties.

Tubulin is a dimeric protein, whose (a, b) heterodimers self-assemble in a head-to-tail fashion to form protofilaments andmicrotubules (MTs). MTs are key components of the cytoskele-ton, and play a fundamental role in cell division and in manyfundamental processes for cell function such as cell shapemaintenance, cell motility, and morphology.[8] The kinetics anddynamics of MTs polymerization and depolymerization regulatetheir biological functions.[9]

Interfering with this mechanism, either by stabilizing or de-stabilizing MTs,[10] results in cytotoxic effects on duplicating

cells, that are blocked in different phases of mitosis and even-tually undergo apoptosis (programmed cell death). MTs arethus the target of many anticancer drugs that exert their cyto-toxic action during the cell-division process.[11]

In a previous study,[12] we have modeled the network of lon-gitudinal interactions between the a and the b subunit of dif-ferent tubulin heterodimers allowing MTs formation and stabili-ty.

It has been observed that only a subset of the residues lo-cated at the protein-protein interface are responsible for theprotein-protein interaction. In particular, residues Val175, Asp177,and Thr178 on the b subunit were found to strongly interactwith Lys352 and Val353 on the a subunit. This set of residues islocated in the central region of the interface and their mutualinteraction is modified by tubulin targeted agents such as vin-blastine[13] and pironetin.[14] The considered interface regionshows shape and charge complementarity between the a andthe b tubulin subunits, with Lys353 and Asp177 involved ina stable salt bridge. In particular, a large seam-shaped concavi-

The introduction of a hydrophobic group at position 7 of 9-flu-orenone-2-carboxylic acid generates new tubulin binders, thedesign of which is suggested by modeling studies. The synthe-sis is based on the use of 2,7-dibromo-fluorenone as starting

material. The antiproliferative activity on two different celllines, fluorescent microscopy, flow cytometry, and sedimenta-tion assay tests confirmed the supposed mechanism.

[a] F. Calogero, S. Borrelli, Dr. G. Speciale, Dr. M. S. Christodoulou,Prof. Dr. D. PassarellaDipartimento di ChimicaUniversit� degli Studi di MilanoVia Golgi 19, 20133 Milano (Italy)E-mail : daniele.passarella@unimi.it

[b] Dr. S. Pieraccini, Prof. Dr. M. SironiDipartimento di Chimica-INSTM-UdR,and Istituto di Scienze e tecnologie molecolari del CNR (CNR-ISTM)Universit� degli Studi di MilanoVia Golgi 19, 20133 Milano (Italy)E-mail : maurizio.sironi@unimi.it

[c] Dr. D. Cartelli, Prof. Dr. G. CappellettiDepartment of BiosciencesUniversit� degli Studi di MilanoVia Celoria 26, 20133 Milano (Italy)

[d] Dr. D. Ballinari, Dr. F. Sola, Dr. C. Albanese, Dr. A. CiavolellaDepartment of Biology-Nerviano Medical Sciences OncologyVia Pasteur 10, 20014 Nerviano, Milano (Italy)

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ty (Figure S1 in the Supporting Information) is present on a tu-bulin, formed by the residues belonging to the H10 helix, theS9 strand and the H10-S9 loop, which could host 9-fluorenone-2-carboxylic where the carboxylic (or ester) group is able to in-teract with Lys352. The introduction of hydrophobic substituentin position 7 appears to improve the affinity by interactingwith the hydrophobic residues forming the pocket. On thebasis of this observation and pursuing our interest in the syn-thesis of tubulin binders and anticancer compounds,[15] we syn-thesized a series of 2,7-disubstituted fluorenones that werelater tested to verify their ability to inhibit tubulin polymeri-zation and affect cancer cell proliferation in vitro. Docking cal-culations were performed to evaluate the binding geometry ofthe tested molecules in the proposed binding site on a tubu-lin.

Results and Discussion

Biological evaluation of 9-florenone-2-carboxylic acid

We initially considered 9-florenone-2-carboxylic acid (2CF). Togain insight into its potential biological activity we investigat-ed its ability to interfere with MT assembly in vitro (Figure 1).Assembly kinetics recorded with 50 mm tubulin is affected byincreasing concentrations of the compound (Figure 1 A).

As quantitative parameters describing the mechanism of tu-bulin assembly can be deduced from the kinetics with theoret-ical models,[16] we calculated the apparent first-order rate con-stant of elongation and the steady-state extent of assembly.Compound 2CF, already in low concentration (0.279 DA min�1

at 5 mm), inhibited the elongation rate with respect to thevalues of 0.622 DA min�1 that was derived from control kinet-ics. Next, 2CF affected the maximum absorbance at the end ofassembly, which is proportionally related to the mass concen-tration of the tubulin polymer (DA = 0.879 at 5 mm) with re-spect to the value that was derived from control kinetics(DA = 1.437). Differential interference contrast (DIC) microscopy

analysis confirmed that tubulin polymerization into MTs, andnot unspecific aggregation, is responsible for the absorbanceincrease observed in the assay and revealed that increasingconcentrations of 2CF prevented the assembly of conventionalMTs leading to the formation of shorter and fragmented poly-mers (Figure 1 B).

Synthesis of 2,7-disubstituted compounds

In the literature, various approaches have been reported forthe synthesis of fluorenones. Most of them involve the con-struction of the fluorenone moiety using substituted aryl com-pounds as building blocks. These are based on Friedel–Craftsring closures of biarylcarboxylic acids,[17] intramolecular [4+2]cycloaddition,[18] cyclization of o-iodobenzophenones,[19] cyclo-carbonylation of o-halobiaryls,[20] [3+3] cyclization,[21] palladi-um-catalyzed oxidative cyclization,[22] and Suzuki cross-cou-pling.[23] Furthermore, O’Hara and co-workers recently reportedthe introduction of an alkyl chain on the fluorenone scaffoldby exploiting the reactivity of a sodium alkylamidozincate in-termediate.[24] Attracted by the possibility of functionalizingthe fluorenone scaffold, we introduced both alkyl and arylgroups at positions 2 and 7 on the fluorenone skeleton.

The synthesis of compound 4 was accomplished in a three-step synthetic sequence as outlined in Scheme 1. Commerciallyavailable 2,7-dibromofluorenone (1), which can also be synthe-sized by a well-known procedure,[25] was functionalized at posi-

tion 2 by a Suzuki coupling reaction to give compound 2. Sub-sequently, the introduction of the nitrile group was accom-plished by a nucleophilic metal-catalyzed cyanation.[26] Thebasic hydrolysis of cyanide group secured the obtainment ofcarboxylic acid 4.

Preparation of compound 5 is based on the Stille reactionusing dibromo derivative 1 as substrate (Scheme 2). The use ofstannane as the limiting agent, only led to the formation ofthe monosubstituted fluorenone, thus avoiding the formationof 2,7-divinylfluorenone. Catalytic hydrogenation of com-pound 5, provided the desired compound 6. The use ofZn(CN)2 in the presence of [Pd(PPh3)4] permitted the produc-tion of nitrile derivatives 7 and 8, which were respectively con-

Figure 1. Tubulin assembly in vitro. A) Assembly kinetics was recorded asa function of time by measuring the increase in absorbance at 340 nm inthe absence (control) or presence of increasing concentrations of 2CF. B) DICmicroscopy of microtubules assembled in the absence (control) or presenceof 2CF. Scale bar = 10 mm.

Scheme 1. Reactions and conditions: a) PhB(OH)2, [Pd(PPh3)4] , Cs2CO3, H2O,THF; b) Zn(CN)2, [Pd(PPh3)4] , DMF; c) NaOH, H2O, EtOH. DMF = N,N-dimethyl-formamide, THF = tetrahydrofuran.

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verted into the methyl esters 9 and 11 under Pinner’s condi-tions.[27] Basic hydrolysis furnished the corresponding carboxyl-ic acids 10 and 12.

Biological evaluation of the compounds

We evaluated the antiproliferative activity of compounds 2CF,4, and 9–12 on A2780 (human ovarian carcinoma) and U2-OS(human osteosarcoma) cell lines. Compounds 11 and 12showed better performance than the starting reference 2CF(Table 1).

To confirm cellular mechanism of action, compound 12 wasalso tested on U2-OS cells engineered to stably express an a tu-bulin/YFP (yellow fluorescent protein) fusion protein, whichallows direct visualization of tubulin by fluorescent microscopy.The U2-OS a tubulin/YFP cells were treated with 12 at thedoses of 12.5, 25, and 50 mm for 24 hours.

Morphological examination by laser scanning confocal mi-croscopy (LSCM) showed that treated cells tended to possessenlarged nuclei without chromosomal condensation, with in-creased tubulin intensity and without polynuclei formation(Figure 2). These morphological features are typical of cellswhich are unable to transit through early mitosis followingDNA replication, and are consistent with inhibition of tubulinfunction.

However, similar morphologi-cal traits can also result from celldamage following a specific toxi-cological insult. To confirm thatcells treated with 12 are indeedblocked during mitosis, we char-acterized the cell cycle profile ofcontrol and treated cells by flowcytometry after DNA staining.This analysis did indeed confirmthat treatment with 12 dose-de-pendently induces cell cycle

arrest, with an accumulation of cells containing replicatedDNA, again consistent with in vivo interference with tubulinfunction by compound 12 (Figure 3).

Finally, we compared the ability of 2CF to inhibit tubulin as-sembly with respect to its derivatives 9–12 by sedimentationassay (Figure 4). As expected, 2CF induced a significant de-crease of the ratio MTs/dimers being consistent with its abilityto inhibit tubulin polymerization. Among the tested molecules,compounds 11 and 12 heavily affected MT assembly and, al-though their effect does not significantly differ from that ob-served in the presence of 2CF, they seem to inhibit more tubu-lin polymerization.

Table 1. Biological activity (mm).

Compound X R Code Cell linesA2780[a] U2-OS[b]

2CF H H ICARUS1 >100 >1004 Ph H SB018 >119 >1199 C2H3 Me FC001 >100 >10010 C2H3 H SB012 >100 >10011 C2H5 Me FC008 11.93 85.8012 C2H5 H SB019 16.08 29.69Taxol – – – 6–15 nm 9–10 nm

[a] Human ovarian carcinoma. [b] Human osteosarcoma.

Figure 2. LSCM images of U2-OS a tubulin/YFP cells (A) untreated or(B) treated with 50 mm compound 12 for 24 h. a tubulin/YFP is representedin green, while DNA (nuclei) is shown in blue. Scale bar = 10 mm.

Figure 3. Cell cycle profile of (A) untreated U2-OS or cells treated with compound 12 for 24 h at concentrations of(B) 12.5, (C) 25, and (D) 50 mm.

Scheme 2. Reactions and conditions: a) CH2CHSn(Bu)3, [Pd(PPh3)4] , toluene;b) H2, Pd/C, CH2Cl2; c) Zn(CN)2, [Pd(PPh3)4] , DMF; c) PhB(OH)2, [Pd(PPh3)4] ,Cs2CO3, H2O, THF; d) HClgas, MeOH; e) NaOH, CH2Cl2, MeOH.

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Docking studies of 2,7-disubstituted compounds

A simple collection of candidate structures was designed anddocked in the proposed binding site (Table 1). Docking resultswere clustered based on root-mean-square deviation (RMSD)values with a 2 � tolerance. In all cases, the most populatedcluster is the one with the lowest energy. The lowest energystructures of the most populated cluster for each docked mol-ecule are drawn in Figure 5. On the whole, all the compoundsbind in a similar fashion in the binding pocket, with the eitherthe acidic or the ester functional group interacting with Lys352

through a salt bridge or hydrogen bond interactions.The hydrophobic moiety of the molecule, on the other

hand, preferentially interacts with Ile332, Phe351, and the alkylchain of Lys336. Compound 4 and its ester, which bring a bulkyphenyl group in position 7, showed a slightly displaced dockedposition allowing the formation of an additional hydrophobiccontact between Leu248 and the fluorenone moiety. Interesting-ly, the esterification of the carboxylic group does not affect thebinding mode of any of the tested molecules, and does not

significantly modify the binding energy, which is comprisedbetween 6.5 and 7.2 Kcal mol�1 for all the compounds.

Conclusion

The introduction of a hydrophobic group at position 7 of the9-fluorenone-2-carboxylic acid generates compounds thatbetter interact with tubulin. This improvement is suggested bymodeling studies and confirmed by assays that evaluate inter-ference with MT assembly. Assessment of antiproliferative ac-tivity on two different human cancer cell lines indicated mod-erately good activity for the methyl ester 11 and for the car-boxylic acid 12. The similar behavior of compounds 11 and 12could be due to the hydrolysis of the ester group of 11 in thepresence of esterases. Fluorescent microscopy and flow cytom-etry confirm the interference of compound 12 with tubulinfunction. This outcome is a further demonstration of the effica-cy of modeling studies for the design of new tubulin binders.

Experimental Section

Synthesis

2-Bromo-7-phenyl-9H-fluoren-9-one (2): Cs2CO3 (0.385 g,1.18 mmol) was added to a stirred solution of 1 (0.100 g,0.295 mmol), phenylboronic acid (0.036 g, 0.295 mmol), and[Pd(PPh3)4] (0.035 g, 0.029 mmol) in H2O/THF (1:10; 20 mL). The re-action mixture was heated at reflux (70 8C) for 11 h, then cooled toRT and concentrated under vacuum. Then CH2Cl2 (30 mL) wasadded to the crude residue and it was washed with brine (30 mL)and water (30 mL), dried over anhydrous Na2SO4, and concentratedunder vacuum. The crude product was purified by flash columnchromatography on silica gel (n-hexane/CH2Cl2 = 3:1) to give 2 asa yellow powder in 42 % yield. 1H NMR (CDCl3, 400 MHz): d= 7.92(d, J = 4.9 Hz, 1 H), 7.80 (d, J = 1.8 Hz, 1 H), 7.76 (dd, J = 7.7, 1.9 Hz,1 H), 7.62–7.66 (m, 3 H), 7.59 (d, J = 7.7 Hz, 1 H), 7.47–7.51 (m, 2 H),

7.41–7.44 ppm (m, 2 H); 13C NMR(CDCl3, 100 MHz): d= 192.3, 142.9,142.7, 142.4, 139.6, 137.2, 136.1,134.5, 133.5, 129.0 (2C), 128.1,127.6, 126.8 (2C), 123.2, 122.9,121.7, 120.8 ppm.

9-Oxo-7-phenyl-9H-fluorene-2-carbonitrile (3): A solution of 2(0.033 g, 0.098 mmol), Zn(CN)2

(0.052 g, 0.296 mmol), and[Pd(PPh3)4] (0,0116 g, 0.010 mmol)in DMF (4 mL) was stirred for 4 hat 153 8C in a sealed vial underMW irradiation (500 W). After cool-ing, the reaction mixture waswashed with brine (50 mL), andthe aqueous layer was extractedwith AcOEt (3 � 15 mL). The organ-ic layers were dried over anhy-drous Na2SO4 and concentratedunder vacuum. The crude productwas purified by biotage column12 m (n-hexane/CH2Cl2 = 2:1) togive 3 as a yellow powder in 62 %yield. 1H NMR (CDCl3, 300 MHz):

Figure 4. Tubulin assembly in vitro. Ratios of assembled microtubules (MTs)/nonpolymerized tubulin (dimers) obtained in the absence (control) or in thepresence of 10 mm each of 2CF and compounds 9, 10, 11, and 12. Data areexpressed as means�SEM of values of at least three independent experi-ments. *p<0.05, ** p<0.002, vs. control, according to ANOVA, Fisher LSDpost-hoc test.

Figure 5. Binding modes of the docked ligands are depicted. Tubulin residues involved in the protein-ligand inter-action are drawn, as well as the H10 helix and S9 strand forming the proposed binding pocket, outlined in red.

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d= 7.97 (1 H, d, J = 1.6 Hz), 7.91 (1 H, bs), 7.75–7.81 (2 H, m), 7.60–7.68 (4 H, m), 7.37–7.50 ppm (3 H, m).

9-Oxo-7-phenyl-9H-fluorene-2-carboxylic acid (4): NaOH (0.024 g,0.604 mmol) was added to a stirred solution of 3 (0.017 g,0.061 mmol) in EtOH/H2O (1:1; 2 mL). The reaction mixture washeated at reflux (100 8C) for 2 h, then after cooling it was acidifiedto pH 1 with 1 m HCl and extracted with AcOEt (3 � 5 mL). The or-ganic layers were dried and concentrated under vacuum to give 4as a yellow powder in 94 % yield. 1H NMR (CDCl3, 400 MHz): d=8.14–8.17 (m, 2 H), 7.97 (bs, 2 H), 7.94 (d, J = 7.7 Hz, 1 H), 7.90 (bs,1 H), 7.77 (d, J = 7.4 Hz, 2 H), 7.55 (bs, 1 H), 7.51 (t, J = 7.4 Hz, 2 H),7.43 ppm (t, J = 7.4 Hz, 1 H); 13C NMR (CDCl3, 100 MHz): d= 193.4,167.7, 147.2, 143.2, 143.1, 139.9, 136.3, 136.0, 135.9, 134.7, 130.2(2C), 129.3, 127.8 (2C), 123.9, 123.5, 123.2, 122.3 ppm.

2-Bromo-7-vinyl-9H-fluoren-9-one (5): Under anhydrous condi-tions, tributyl(vinyl)tin (0.255 mL, 0.000875 mol) was added drop-wise to a stirred solution of 1 (0.4205 g, 0.00125 mol) and[Pd(PPh3)4] (0.14 g, 0.000125 mol) in anhydrous toluene (40 mL).The reaction mixture was heated at reflux (120 8C) for 13 h, thencooled to RT and quenched with a saturated solution of KF. The or-ganic layer was washed with brine (50 mL) and water (50 mL),dried over anhydrous Na2SO4, and concentrated in vacuum. Thecrude product was purified by column chromatography on silicagel (n-hexane/CH2Cl2 = 4:1) to give 5 as a yellow powder in 41 %yield. 1H NMR (CDCl3, 300 MHz): d= 7.74 (d, J = 1,78 Hz, 1 H), 7.72(bs, 1 H), 7.58 (dd, J = 7,78, 1,84 Hz, 1 H), 7.47 (dd, J = 7.67, 1.76 Hz,1 H), 7.43 (d, J = 7,75 Hz, 1 H), 7.35 (d, J = 7,84 Hz, 1 H), 6.69 (dd, J =16.5, 10.2 Hz, 1 H), 5.80 (d, J = 16.5 Hz, 1 H), 5.32 ppm (d, J =10.2 Hz, 1 H); 13C NMR (CDCl3, 75 MHz): d= 192.2, 142.9, 142.7,139.2, 137.2, 136.1, 135.5, 134.2, 133.1, 127.6, 122.8, 121.7(2C),120.5, 115.4 ppm.

2-Bromo-7-ethyl-9H-fluoren-9-one (6): H2 gas was bubbled intoa solution of 5 (0.135 g, 0.48 mmol) and 10 % Pd on activatedcarbon (0.123 g, 0.09 mmol) in CH2Cl2 (5 mL). After 3 h, all the start-ing material had been consumed, the H2 flow was stopped andthe reaction mixture was filtered through celite and the filter cakewas washed successively with CH2Cl2, AcOEt, and MeOH. The fil-trate was concentrated under vacuum, and the crude residue waspurified by column chromatography on silica gel (n-hexane/CH2Cl2 = 2:1) to give 6 as a yellow powder in 48 % yield. 1H NMR(CDCl3, 300 MHz): d= 7.72 (d, J = 1.73 Hz, 1 H), 7.57 (bd, J = 7.75,2,0 Hz, 1 H), 7.49 (bs, 1 H), 7.39 (d, J = 7.63, 1 H), 7.29–7.35 (m, 2 H),2.66 (q, J = 7,67 Hz, 2 H), 1.243 ppm (t, J = 7,67 Hz, 3 H).

9-Oxo-7-vinyl-9H-fluorene-2-carbonitrile (7): A solution of 5(0.0376 g, 0.126 mmol), Zn(CN)2 (0.0446 g, 0.252 mmol), and[Pd(PPh3)4] (0,0150 g, 0.013 mmol) in DMF (4 mL) was stirred for 5 hat 153 8C in a sealed vial under MW irradiation (500 W). After cool-ing, the reaction mixture was washed with brine (50 mL), and theaqueous layer was extracted with AcOEt (3 � 15 mL). The organiclayers were dried over anhydrous Na2SO4 and concentrated undervacuum. The crude product was purified by biotage column 12 m

(n-hexane/CH2Cl2 = 1:1) to give 7 as a yellow powder in 74 % yield.1H NMR (CDCl3, 300 MHz): d= 7.88 (bs, 1 H), 7.76–7.79 (m, 2 H), 7.61(d, J = 7.8 Hz, 1 H), 7.55 (bs, 2 H), 6.73 (dd, J = 17.5, 10.8 Hz, 1 H),5.86 (d, J = 17.5 Hz, 1 H), 5.38 ppm (d, J = 10.8, 1 H); MS(EI): m/z (%):231 ([M]+).

7-Ethyl-9-oxo-9H-fluorene-2-carbonitrile (8): A solution of 6(0.0566 g, 0.128 mmol), Zn(CN)2 (0.0454 g, 0.256 mmol), and[Pd(PPh3)4] (0,0150 g, 0.013 mmol) in DMF (4 mL) was stirred for 5 h

at 153 8C in a sealed vial under MW irradiation (500 W). After cool-ing, the reaction mixture was washed with brine (50 mL), and theaqueous layer was extracted with AcOEt (3 � 15 mL). The organiclayers were dried over anhydrous Na2SO4 and concentrated undervacuum. The crude product was purified by biotage column 12 m

(n-hexane/CH2Cl2 = 1:1) to give 8 as a yellow powder in 90 % yield.1H NMR (CDCl3, 300 MHz): d= 7.85 (bs, 1 H), 7.75 (dd, J = 7.75,1.55 Hz, 1 H), 7.58 (d, J = 7,64 Hz, 1 H), 7.50 (d, J = 7.68 Hz, 1 H), 7.38(bd, J = 7.70 Hz, 1 H), 2.70 (q, J = 7.59 Hz, 2 H), 1.26 ppm (t, J =7.59 Hz, 3 H); MS(EI): m/z (%): 233 ([M]+).

Methyl 9-oxo-7-vinyl-9H-fluorene-2-carboxylate (9): Dry HCl gaswas bubbled into a solution of 7 (0.0623 g, 0.2694 mmol) in MeOH(5 mL). After 3 h, all the starting material had been consumed, theHCl flow was halted and the solvent was removed under vacuum.The residue was dissolved in DME (5 mL) before 6 N HCl (1 mL) wasadded and the mixture stirred at RT overnight. After complete hy-drolysis, the reaction mixture was neutralized with a solution ofaqueous saturated NaHCO3 and extracted with diethyl ether (3 �15 mL). The organic layers were dried over anhydrous Na2SO4 andconcentrated under vacuum. The crude product was purified bycolumn chromatography on silica gel (CH2Cl2/n-hexane = 5:1) togive 9 as a yellow powder in 58 % yield. 1H NMR (CDCl3, 300 MHz):d= 8.27 (bs, 1 H), 8.19 (bd, J = 7,76 Hz, 1 H), 7.763 (s, 1 H), 7,489–7.598 (m, 3 H), 6.713 (dd, J = 17.5 Hz, 10.8 Hz, 1 H), 5,832 (d, J =17,5 Hz, 1 H), 5.34 (d, J = 10,8 Hz, 1 H), 3.925 ppm (3 H,s) ; 13C NMR(CDCl3, 75 MHz): d= 189.0, 165.7, 147.8, 145.2, 143.1, 136.4, 134.9,133.2, 130.1, 125.4, 122.7, 120.3, 120.1, 115.8, 57.7, 52.4, 29.6,26.2 ppm; MS(EI): m/z (%): 264 ([M]+).

9-Oxo-7-vinyl-9H-fluorene-2-carboxylic acid (10): NaOH (9 mg,0.226 mmol) was added to a stirred solution of 9 (15 mg,0.056 mmol) in CH2Cl2/MeOH (9:1; 1 mL). The reaction mixture wasstirred at RT for 28 h, then concentrated under vacuum, acidifiedwith 1 m HCl, and extracted with CHCl3 (3 � 15 mL). The organiclayers were dried and concentrated under vacuum to give 10 asa yellow powder in quantitative yield. 1H NMR ([D6]DMSO,400 MHz): d= 8.18 (d, J = 7.8 Hz, 1 H), 8.04 (bs, 1 H), 7.92 (d, J =7.7 Hz, 1 H), 7.87 (d, J = 7.7 Hz, 1 H), 7.79 (bs, 1 H), 7.75 (d, J = 7.8 Hz,1 H), 6.83 (dd, J = 17.7, 11.0 Hz, 1 H), 6.01 (d, J = 17.7 Hz, 1 H),5.39 ppm (d, J = 11.0 Hz, 1 H); 13C NMR ([D6]DMSO, 100 MHz): d=192.1, 171.4, 167.3, 148.6, 142.3, 140.3, 137.5, 136.6, 135.9, 135.7,134.3, 125.2, 123.4, 122.5, 122.4, 117.9 ppm; MS(EI): m/z (%): 250([M]+).

Methyl 7-ethyl-9-oxo-9H-fluorene-2-carboxylate (11): Dry HCl gaswas bubbled into a solution of 8 (0.0623 g, 0.2694 mmol) in MeOH(5 mL). After 3 h, all the starting material had been consumed, theHCl flow was halted and the solvent was removed under vacuum.The residue was dissolved in DME (5 mL) before 6 N HCl (1 mL) wasadded and the mixture stirred at RT overnight. After complete hy-drolysis, the reaction mixture was neutralized with a solution ofaqueous saturated NaHCO3 and extracted with diethyl ether (3 �15 mL). The organic layers were dried over anhydrous Na2SO4 andconcentrated under vacuum. The crude product was purified bycolumn chromatography on silica gel (CH2Cl2/n-hexane = 5:1) togive 11 as a yellow powder in 58 % yield. 1H NMR (CDCl3, 300 MHz):d= 8.25 (s, 1 H), 8.17 (d, J = 7.86 Hz, 1 H), 7.54 (bs, 1 H), 7.53 (d, J =7,23 Hz, 1 H), 7.48 (d, J = 7.44 Hz, 1 H), 7.34 (bd, J = 7.79 Hz, 1 H),3.92 (s, 3 H), 2.68 (q, J = 7.68 Hz, 2 H), 1.252 ppm (t, J = 7.68 Hz, 3 H);13C NMR (CDCl3, 75 MHz): d= 192.9, 166.1, 148.6, 147.0, 140.9,136.3, 134.9, 134.4, 130.5, 125.2, 124.0, 121.1, 119.8, 52.3, 29.8, 28.8,15.2 ppm; MS(EI): m/z (%): 266 ([M]+).

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7-Ethyl-9-oxo-9H-fluorene-2-carboxylic acid (12): NaOH (9.2 mg,0.231 mmol) was added to a stirred solution of 11 (15.4 mg,0.0578 mmol) in CH2Cl2/MeOH (9:1; 1 mL). The reaction mixturewas stirred at RT for 28 h, then concentrated under vacuum, acidi-fied with 1 m HCl, and extracted with CHCl3 (3 � 15 mL). The organiclayers were dried and concentrated under vacuum to give 12 asa yellow powder in quantitative yield. 1H NMR (CD3OD, 400 MHz):d= 8.22 (d, J = 7.62 Hz,1 H), 8.16 (s, 1 H), 7.74 (d, J = 7.62 Hz, 1 H),7.67 (d, J = 7,6 Hz, 1 H), 7.54 (s, 1 H), 7.47 (d, J = 7.6 Hz, 1 H), 2.73 (q,J = 7.65 Hz, 2 H), 1.29 ppm (t, J = 7.65 Hz, 3 H); 13C NMR (CD3OD,75 MHz): d= 192.9, 166.1, 147.6, 147.2, 143.8, 138.8, 137.0, 136.82,135.7, 135.3, 124.8, 124.1, 121.9, 120.6, 29.1, 15.0 ppm.

Biological assays

Tubulin assembly assays

Tubulin was purified from bovine brain purchased from a localslaughterhouse as previously described,[12] conserved before use inice-cold phosphate-buffered saline (PBS; 20 mm Na phosphate,150 mm NaCl, pH 7.2) and used as soon as possible. Pure tubulinwas resuspended in BRB80 (80 mm K 1,4-piperazinebis(ethanesul-fonic acid) (PIPES) buffer, pH 6.9, 2 mm ethylene glycol tetraaceticacid (EGTA), 1 mm MgCl2), snap-frozen in aliquots in liquid nitrogenand kept at �80 8C. Protein concentration was determined by Mi-croBCA assay kit (Pierce). Stock solutions of 2CF were prepared bydissolving the powder at a concentration of 5 mm in dimethyl sulf-oxide (DMSO). To assess its effect on tubulin assembly kinetics,bovine tubulin (5 mg mL�1) was mixed with 2CF or an equalvolume of the solvent (final 0.2 % DMSO) in an assembly buffer(80 mm K pipes pH 6.9, 2 mm EGTA, 1 mm MgCl2, 10 % glycerol)containing 1 mm guanidine triphosphate (GTP). Each reaction wasstarted by plating the cuvette at 37 8C in an Ultraspec 300 spectro-photometer (Pharmacia) equipped with a temperature controller.The kinetics of tubulin polymerization were assessed by turbidime-try analysis following the increase in absorbance at 340 nm for30 min. At the end of the assembly assay, microtubules were col-lected by centrifugation at 30,000 g for 30 min at 30 8C, fixed with0.5 % glutaraldehyde in the assembly buffer (80 mm K pipes pH 6.9,2 mm EGTA, 1 mm MgCl2, 10 % glycerol), and put onto coverslips.Image acquisition was performed using a Zeiss Axiovert200 equipped with differential interference contrast (DIC) optics,a 63 � oil objective, and a digital image recording system (AxiocamHRM Rev. 2 camera driven by Axiovision software rel. 4.4, Zeiss).Sedimentation assay was performed as previously described.[15c]

Briefly, reaction mixtures (20 mm tubulin, 10 % glycerol, 1 mm GTPin BRB80 buffer) were incubated for 30 min at 37 8C. At the end ofpolymerization, nonpolymerized and polymerized fractions of tu-bulin were separated by centrifugation at 16500 g for 30 min at25 8C. The collected microtubules were resuspended in sodium do-decyl sulfate-polyacrylamide gels (SDS-PAGE) buffer (2 % w/v SDS,10 % v/v glycerol, 5 % v/v b-mercaptoethanol, 0.001 % w/v bromo-phenol blue, and 62.5 mm tris(hydroxymethyl)aminomethane (Tris)buffer, pH 6.8) and the nonpolymerized tubulin was diluted 3:1with four times SDS-PAGE sample buffer. Equal proportions of eachfraction were resolved by a 7.5 % SDS-gel and stained with Coo-massie blue. Densitometric analysis of stained gels was performedby using ImageJ software (National Institute of Health), and datawere elaborated using STATISTICA (StatSoft Inc. , Tulsa, OK). Signifi-cant differences were assessed by one-way ANOVA with Fisher LSDpost-hoc test. Experiments were done in triplicate and data are ex-pressed as means�SEM.

Cell culture

A2780 human ovarian cancer cells were obtained from ECACC andcultured in monolayer using RPMI 1640 medium (GIBCO), supple-mented with 2 mmL glutamine and 10 % fetal bovine serum. U2-OS human osteosarcoma cancer cells were obtained from ATCCand subcultured using Mc’Coys medium (GIBCO), supplementedwith 2 mmL glutammine and 10 % fetal bovine serum (GIBCO). Celllines were maintained in a humidified 5 % CO2 incubator at 37 8Cand periodically tested for absence of mycoplasma. Identity of thecell lines used was confirmed by DNA fingerprinting using theAmpFlSTR Identifiler Plus PCR Amplification Kit (Applied Biosys-tems) according to the manufacturer’s protocol.

Antiproliferative assays

A2780 and U2-OS cells were seeded in black 384 well plates 24 hbefore the treatment at 1600 cell per well and 3200 cell per well,respectively. Compounds were added in a 24 points dose responsecurve in duplicate. After 72 h of treatment, 25 mL per well of Cell-Titer-Glo (Promega; a luciferase-based assay system that evaluatesthe number of viable cells based on quantification of ATP as indica-tor of metabolic activity) was added to the microplates and thenmixed. After 10 min, the plates were read in a luminometer (Envi-sion). The data were expressed as percentage of viability in sigmoi-dal curves using Symix Assay Explorer (Symix Technologies Inc.)software. The IC50 value was calculated using sigmoidal fitting algo-rithm as average of two independent experiments.

LSCM (Laser Scanning Confocal Microscopy)

U2-OS cells stably transfected with aTubulin fused to the C-termi-nus of yellow fluorescent protein (aTubulin/YFP) were seeded onglass slides. Following incubation and treatment, cells were fixedin 3.7 % formaldehyde, and DNA was stained using DRAQ5. A Con-focal laser mounted on a ZEISS microscope was used (Radiance2000, Biorad) and images were acquired with a 40X objective,using oil immersion.

Flow Cytometry

Twenty-four hours after treatment, U2-OS cells were collected,fixed in 70 % ethanol overnight, washed in PBS and stained for 1 hwith 25 mg mL�1 propidium iodide (Sigma) in 0.1 % Na citrate con-taining 6 mg mL�1 RNase A III (Sigma) and 0.02 % IGEPAL (Sigma,CA-630). Sample acquisition was done on a FACSCalibur instrument(Becton Dickinson) and analysis was performed on at least104 cells, excluding doublets and aggregates. The DNA histogramswere analyzed by Modfit 3.0 (Verity Software House).

Computational details

Fluorenone derivatives were docked on the tubulin a-subunitusing the Autodock 4.2 software.[28] The protein was consideredrigid while ligand flexibility was allowed. The potential grid gener-ated by the Autogrid module was centered on residue Lys352 andhad a 60x64x60 points width with a uniform 0.375 � spacing be-tween grid points. A lamarkian genetic algorithm[29] was employedfor the docking simulation, performing 100 independent runs permolecule. In each run a population of 50 individual evolved along27000 generations and a maximum number of 25 million energyevaluation was performed. The top (best fit, that is, lowest docked

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energy) solutions of the 100 independent runs were stored forsubsequent analysis.

Acknowledgements

This research has been developed under the umbrella of CM1106COST Action “Chemical Approaches for Targeting Drug Resistancein Cancer Stem Cells”. We acknowledge the LISA MODTUB projectand CINECA award N. HP10BRT6OA 2013 for the availability ofhigh performance computing resources.

Keywords: antitumor agents · 2,7-disubstituted fluorenones ·docking · 9-fluorenone-2-carboxylic acid · tubulins

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Received: February 7, 2013Revised: April 8, 2013Published online on May 13, 2013

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