Synthesis and biological evaluation of 4 arylcoumarin analogues as tubulin-targeting antitumor agents

Accepted Manuscript Synthesis and biological evaluation of 4 arylcoumarin analogues as tubulintargeting antitumor agents Peggoty Mutai, Gilles Breuzard, Alessandra Pagano, Diane Allegro, Vincent Peyrot, Kelly Chibale PII: DOI: Reference:

S0968-0896(16)31073-2 http://dx.doi.org/10.1016/j.bmc.2017.01.035 BMC 13514

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

27 October 2016 13 January 2017 19 January 2017

Please cite this article as: Mutai, P., Breuzard, G., Pagano, A., Allegro, D., Peyrot, V., Chibale, K., Synthesis and biological evaluation of 4 arylcoumarin analogues as tubulin-targeting antitumor agents, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.01.035

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Synthesis and biological evaluation of 4 arylcoumarin analogues as tubulin-targeting antitumor agents. Peggoty Mutai

[a, b] *

, Gilles Breuzard

[c] *

, Alessandra Pagano

[c]

, Diane Allegro

[c]

, Vincent

Peyrot [c] ¥ §, Kelly Chibale [a, d, e] ¥ §

[a]

Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa.

[b]

Department of Pharmacology & Pharmacognosy, College of Health Sciences, University of

Nairobi, PO Box 19676-00202, Nairobi Kenya. [c]

Aix-Marseille Université, INSERM UMR_S 911 CRO2, Faculté de Pharmacie, 27 bd Jean-

Moulin, 13385, Marseille, France. [d]

South African Medical Research Council Drug Discovery and Development Research Unit,

University of Cape Town, Rondebosch 7701, South Africa. [e]

Institute of Infectious Disease and Molecular Medicine, University of Cape Town,

Rondebosch 7701, South Africa.

* These authors contributed equally to this work. §

These authors co-managed equally this work.

¥

Corresponding authors: [email protected]; [email protected]

1

Abstract The synthesis of twenty-six 4-arylcoumarin analogues of combretastatin A-4 (CA-4) led to the identification of two new compounds (25 and 26) with strong cytotoxic activity. Both compounds had a high cytotoxic effect on a CA-4-resistant colon adenocarcinoma cell line (HT29D4). The compounds affected cell cycle progression characterized by a mitotic block. The activity of these compounds against microtubules both in vitro and in cells was examined and both compounds were found to potently inhibit in vitro microtubule formation via a substoichiometric mode like CA-4. By immunofluorescence, it was observed that both compounds induced strong microtubule network disruption. Our results provide a strong experimental basis to develop new potent anti-tubulin molecules targeting CA-4-resistant cancer cells. Graphical Abstract

Induce cell cycle arrest

4-arylcoumarin

Inhibit microtubule formation

2

INTRODUCTION The microtubule cytoskeleton is an essential functional network in the eukaryotic cell that is involved in many biological processes such as cell division, the maintenance and alteration of cell morphology, vesicle trafficking and cell migration. Microtubule is a heterodimer assembly of α and β tubulin. The microtubule network is constantly reworking and each microtubule is the seat of assembly and disassembly cycles, also called dynamic instability 1, which is tightly regulated by associated proteins such as MAP1A-B, tau or stathmin

2–5

. This mechanism is the

basis for development of anticancer therapeutic agents that target the microtubule cytoskeleton such as colchicine, taxane and vinca-alkaloids

6–8

. Despite their effect on the microtubule

network to promote cell death, the administration of these molecules remains ineffective in the treatment of certain cancers (such as colorectal cancer

9,10

), especially due to the poor

accessibility of drug to tumor cells within a healthy tissue and to the development of cell chemoresistance. In view of this, the development of new antimitotic drugs has the potential to lead to new cancer chemotherapy approaches and to a better knowledge of microtubule pharmacology and biochemistry. Considering the success of the anti-tubulin pharmacological class

11

, numerous studies

are now focused on the discovery and clinical trial development of new derivatives. The 4arylcoumarins, also known as neoflavones, are a small class of naturally occurring compounds reported to have various pharmacological properties such as antitumor, antiprotozoal, antiplasmodial, antifungal and antidiabetic activities 12–15.

3

With regard to antitumor activity, 4-arylcoumarins have been reported to share the colchicine binding site with combretastatin A-4 (CA-4) (Figure 1A), a natural antimitotic and anti-angiogenic agent from the African willow tree Combretum caffrum

16

. Structurally, the 4-

arylcoumarins and CA-4 have two phenyl rings (A and B) but have different chemical linkers between them. Combretastatin A-4 has an olefinic group while the 4-arylcoumarins have a benzopyranone group. Previous structure-activity relationship studies of 4-arylcoumarin derivatives revealed that the trimethoxy substituents on ring A are essential for antitumor activity 17,18

. However, we previously reported that even the unsubstituted ring A analogues (A) (Figure

1) possessed significant cytotoxic activity against cancer cells 18,19 with the highest activity being in the mono-substituted ring A analogues (B). It is noteworthy that all the compounds tested had either hydroxyl or methoxy substituents on the ring B moiety, which are the most commonly occurring substituents in nature.

4

Figure 1: (A) Structures of 4-arylcoumarins and combretastatin A-4 (CA-4); (B) Structures of the two most cytotoxic analogues of 4-arylcoumarin investigated by Combes et al. 19: concentrations half inhibiting HBL-100 cell proliferation (IC50) are 39 nM (A) and 84 nM (B). Prompted by previously reported antitumor activities of 4-arylcoumarin analogues

19–21

,

we synthesized twenty six 4-arylcoumarin derivatives by substituting the hydroxyl or methoxy groups on rings A and B. The resulting compounds were evaluated for their cytotoxic effect on colon adenocarcinoma HT29D4 cells 22 a clone made in our lab and derived from CA-4 resistant HT29 cells

23

and CA-sensitive non-small lung cancer A549 cell lines. We then tested them for

their microtubule-depolymerizing activities in quantifying the cellular microtubule network by immunofluorescence imaging24 and testing in vitro microtubule assembly by turbidity time course25

5

METHODOLOGY Chemistry. Modifications were carried out on rings A and B of the 4-arylcoumarin skeleton using previously reported synthetic routes

26,27

. A common synthetic scheme was used for both ring

modifications. The synthetic sequence (Scheme 1) began with the commercially available substituted 2’-hydroxyacetophenones, which were cyclized to form 4-hydroxycoumarins. Further treatment of the 4-hydroxycoumarins with trifluoromethanesulfonic anhydride provided triflate precursors which were then coupled with various boronic acids (R2) via the Suzuki crosscoupling reaction to yield the target compounds 1-26 in good to moderate yields.

Scheme 1. Synthesis of 4-arylcoumarin derivatives 1-29. Reagents and conditions: (i) NaH, DEC, Toluene, 120°C, 12h (ii) Triflic anhydride, CH2Cl2, 0°C, 1h (iii) Boronic acid, dioxane, Pd(PPh3)2Cl2, 70°C, reflux, 14h. Modifications on ring A Eight analogues were synthesized with variation of substituents at position 6 of ring A. For the first five compounds (1-5) (Figure 2), the 4-methoxy-3-hydroxyphenyl ring B present in the natural compound, CA-4 was retained at position B, while for the next three compounds (6-

6

8), this ring was replaced by a 4-methoxypyridyl ring in order to eliminate the hydroxyl metabolic hotspot present in compounds 1-5.

Modifications on ring B Previous work reported that the 4-methoxy-3-hydroxy substituted phenyl ring B found in the naturally occurring compound CA-4 was essential for antitumor activity

17,18

. Studies

exploring the structure activity relationships of combretastatin A-4 have demonstrated that this substitution pattern is not essential for antitumor activity 28,29. Most reported substitution patterns mainly involved the methoxy and hydroxyl groups, which are widespread in nature. In this paper, various substituents including those that are not typically found in nature, as well as various heterocyclic rings were introduced in place of the phenyl ring B (Figure 2). For compounds 9-20, ring A has a methoxy substituent; for compounds 21-24 ring A is unsubstituted and for compounds 25 and 26 ring A has a chloro substituent at position 6.

7

Figure 2: Structures of 4-arylcoumarin analogues synthesized.

Biology. A tetrazolium-based assay to determine the drug concentration required to inhibit cell growth by 50% after incubation in the culture media for 72h was performed. The values of calculated IC50 for all compounds were compared to those obtained after treatment with CA-4 of the colon adenocarcinoma HT29D4 cells22 and the lung adenocarcinoma A549 cells (Tables 1 and 2). Next, the microtubule-depolymerizing activities of CA-4 and compounds 25 and 26 were quantified using immunofluorescence of the cellular microtubule network in A549 cells (Figures 3 – 5), as well as with a turbidity time course of in vitro microtubule assembly (Figure 6). RESULTS AND DISCUSSION 8

Cytotoxic effects of Ring A substitution Eight 4-arylcoumarin derivatives (1-8) were tested for their cytotoxicity on HT29D4 and A549 cell lines in comparison with CA-4 treatment (Table 1). CA-4 exhibited poor antitumor activity in HT29D4 cells with an IC50 value of 12.5 ± 1.4 µM and good activity in A549 cells with an IC50 value of 0.010 ± 0.001 µM. It has been previously reported that the parental HT29 cells are highly resistant to CA-4

23,30

. We confirm that also clonal derived HT29D4 cells are

resistant to this drug. In the series with a 3’-hydroxy-4’-methoxyphenyl ring B (1-5), the most cytotoxic compound against HT29D4 cells was 2 (IC50 = 4.8 ± 0.6 µM), which has a chloro substituent in position 6. Table 1. Cytotoxic effects of CA-4 and compounds 1-8 towards A549 and HT29D4 cell lines.

IC50 b (µM) Compounds

R1

Solubility a (µM) A549

HT29D4

CA4

-

n.d.

0.010 ± 0.001

12.5 ± 1.4

1

OCH3

40

˃40

10.0 ± 1.1

2

Cl

40

0.4 ± 0.1

4.8 ± 0.6

9

(a)

3

F

80

1.5 ± 0.2

10.5 ± 1.2

4

CH3

40

0.2 ± 0.1

13.4 ± 1.5

5

H

160

1.7 ± 0.2

8.3 ± 1.0

6

OCH3

80

38.5 ± 4.3

˃80

7

F

80

˃80

˃80

8

H

80

41.0 ± 4.6

˃80

Solubility obtained in PBS;

(b)

Drug concentration that inhibits cell line growth by 50 % after

incubation in culture media for 72 h. Data are the mean ± SD of three independent experiments. The order of activity starting with the most active to the least cytotoxic was Cl>H>OCH3>F>CH3. When the same series was tested using A549 cells, the cytotoxic order changed to CH3>Cl>F>H>OCH3. Our data show that introduction of the electron withdrawing chloro group (2) at position 6 of ring A results in a highly pronounced cytotoxic effect with a 2.6-fold increase in cytotoxicity in the HT29D4 compared to CA-4. A significant difference in activity was observed between the chloro- (2) and corresponding fluoro- (3) substituted analogues with the former substituted compound being up to 4 fold more active. In the series with a methoxypyridyl ring B (6-8) all compounds displayed no activity against the HT29D4 cell line at their solubility limits. When these compounds were tested against the A549 cell line, the only active compounds were the methoxy substituted (6) and the unsubstituted compounds (8). Compound 7 did not elicit a cytotoxic effect on A549 cells. Cytotoxic effects of Ring B substitution

10

Various substituents were introduced to the phenyl ring B as well as different ring systems that are bio-isosteric to the benzene ring to yield compounds 9-26. The cytotoxic effects of compounds are reported in Table 2.

Table 2. Cytotoxic effects of compounds 9-26 towards A549 and HT29D4 cell lines.

IC50 b (µM)

Solubility a Compounds

R1

R2 (µM)

A549

HT29D4

OCH3

80

˃80

˃80

OCH3

40

0.2 ± 0.1

0.5 ± 0.1

11

OCH3

40

13.0 ± 1.5

˃40

12

OCH3

80

˃80

˃80

13

OCH3

40

13.0 ± 1.6

˃40

14

OCH3

120

˃120

˃120

9

10

11

15

OCH3

120

34 ± 4

˃120

16

OCH3

20

˃20

˃20

17

OCH3

80

2.5 ± 0.5

1.0 ± 0.2

18

OCH3

80

˃80

˃80

19

OCH3

80

˃80

˃80

20

OCH3

40

˃40

˃40

21

H

200

35 ± 4

˃200

22

H

80

˃80

˃80

23

H

80

˃80

˃80

24

H

80

˃80

˃80

25

Cl

5-10

0.11 ± 0.02

0.10 ± 0.02

12

26

(a)

Cl

Solubility obtained in PBS;

10-20

(b)

0.11 ± 0.04

0.44 ± 0.05

Drug concentration that inhibits cell line growth by 50 % after

incubation in culture media for 72 h. Data are the mean ± SD of three independent experiments. In the series where R1 is a methoxyl group (9-20), the most active compound on both cell lines was 10 (0.2 ± 0.1 µM and 0.5 ± 0.1 µM in A549 and HT29D4 cells, respectively) which has a N-methylaminophenyl ring B. The next active compound was 17 with a meta-fluoro-paramethoxyphenyl ring B. When the para-methoxyl substituent on 17 was replaced by a paratrifluoromethyl group as in compound 11, activity was reduced from IC50 = 2.5 ± 0.3 µM to IC50 = 13.0 ± 1.5 µM in the A549 cell line and lost in the HT29D4 cell line (IC50 = 1.0 ± 0.2 µM to no activity). When the fluoro substituent as in 17 was replaced by a hydroxyl group as in 1, activity was lost (IC50 = 2.5 ± 0.3 µM to no activity) on the A549 cell line and decreased (IC50 = 1.0 ± 0.2 µM to IC50 = 10.0 ± 1.1 µM) in the HT29D4 cell line. From these observations, activity may be related to the electronic and hydrophobic properties of the compound, with the hydrophobic substituents at the meta position and electron donating substituents at the para position being favorable for activity. Based on the results from the ring modifications described above, the two analogues 25 and 26 were designed to be the most active ring A and ring B substituents. The two compounds displayed the highest cytotoxic activity against both cell lines with IC50 values in the submicromolar range (Table 2). The cytotoxic effects on the A549 cell line were moderate (25; IC50 0.11 ± 0.02 µM and 26; IC50 0.11 ± 0.04 µM) compared to CA-4 (IC50 0.01 ± 0.001 µM) while both compounds were 124- and 28-fold more potent against HT29D4 cells (IC50 = 0.10 ±

13

0.02 µM for 25 and 0.44 ± 0.05 µM for 26) than CA-4 (IC50 = 12.44 ± 1.37 µM). These two compounds were selected for further biological evaluation within the context of understanding their mechanisms of action.

Effects of CA-4 and compounds 25 and 26 on cell cycle Using flow cytometry, modifications in the cell cycle progression of HT29D4 and A549 cell lines after 24h of treatment with CA-4, 25 and 26 were compared (Figure 3A – B). For this purpose, 0.5 µM CA-4, 1 µM compound 25 and 1 µM compound 26 were used.

14

B

A549 n.s

60

CTRL

counts

240

120

0 n.s

*

60 40

* n.s

20

n.s

n.s

CTRL CA-4 (25) (26) CTRL CA-4 (25) (26) CTRL CA-4 (25) (26) CTRL CA-4 (25) (26)

0

sub-G1

G1

S

80

0 90 80 70 60 50 40 30 20 10 0 100

0

G2/M

zoom

counts

enlarged

200 150

100 50 0

(25)

60 40

CTRL

20

CA-4

counts

(26)

0

50

100 150 200 250

80 70 60 50 40 30 20 10 0 90 80 70 60 50 40 30 20 10 0

(25)

(26)

0

50

100 150 200 250

(25)

fluorescence intensity (a.u)

(26)

HT29D4

counts

0 80 70 60 50 40 30 20 10 0

CA-4

250

80

Phases

C

CA-4

counts

HT29D4

160

60

counts

80

CTRL

320

180

*

20

400

240

counts

Percentage of cells

*

*

40

HT29D4

A549

300

counts

A 80

15

Figure 3: Compounds 25 and 26 block cells in G2/M phase during the cycle progression. (A) Cell cycle distribution in A549 and HT29D4 cells treated for 24 h with 0.5 µM CA-4 (black bars), 1 µM (25) (dark grey bars) and 1 µM (26) (light grey bars); cells were analyzed with the FACScan flow cytometer; all reported values (mean ± SD of three independent experiments) correspond to percentages of cells in sub-G1, G1, S and G2/M phases; significant statistical differences were calculated using a non-parametric Mann-Whitney test with (*) P < 0.01 from three independent experiments. (B) Results show a typical experiment. (C) Confocal LaserScanning microscopy (CLSM) reveals abnormal mitosis with dysfunctional and multipolar spindles (white arrows on image insets) in HT29D4 cells; scale bars: 15 µm.

When HT29D4 cells were treated with CA-4, the percentages of cells in phases G1 (20.5 ± 1.2 %), S (51.4 ± 4.1 %) and G2/M (13.0 ± 1.3 %) were similar to those of untreated cells (24.8 ± 3.1 %, 63.3 ± 6.5% and 11.1 ± 3.7 % for phases G1, S and G2/M, respectively), indicating that CA-4 had no effect at this concentration. This was true even when cells were treated at a 10 times higher concentration (data not shown). On the other hand, when A549 cells were treated with CA-4 at the same concentration, 33.1 ± 7.6 % of cells were arrested in the G2/M phase of the cell cycle. These results confirm the very low anti-proliferative activity of CA-4 on HT29D4 cells compared to A549 cells. Compounds 25 and 26 caused a significant accumulation of HT29D4 cells in the G2/M phase (38.3 ± 2.3 % and 29.9 ± 1.1 % respectively in Figure 3A) while a significant decrease of cells in the G1 phase (10.4 ± 1.9% and 12.8 ± 5.4 % for 25 and 26, respectively) was noted, indicating that these compounds induced an increase in cells blocked in the mitotic phase. Similarly, in the A549 cells treated with the same concentration of compounds 25 and 26, 32.5 ± 3.2 % and 33.5 ± 7.7 % of cells were arrested in G2/M, and 10.1 ±

16

4.7 % and 9.1 ± 6.6 % of cells were in G1 phase, , respectively. The percentages of the two cell lines in the S phase were similar in untreated and drug-treated cells. For A549 cells, an apoptosis induction could be implied after all drug treatment, as strongly suggested by the enhanced accumulation of sub-G1 cells (1.3 ± 0.4 % for untreated cells vs. 6.6 ± 4.5 %, 6.5 ± 4.6 % and 7.5 ± 4.2 % of cells treated with CA-4, compound 25 and 26, respectively; Figure 3A). In HT29D4 cells, no significant difference in sub-G1 cells was observed for all treatments compared to untreated cells. As a complementary approach, HT29D4 cells were pre-incubated or not with CA-4 (0.5 µM), compounds 25 and 26 (1 µM) for 24 h before to be co-labelled for -tubulin and nuclei and then cells were observed by confocal fluorescence microscopy (Figure 3C). Following incubation with 25 and 26, we clearly identified many dysfunctional and multipolar spindles (white arrows in image ‘zoom’ of Figure 3C) which led to polyploid cells and mis-segregation of chromatids. Our observations strongly suggest that disturbance of cell cycle progression could have originated from abnormal mitosis. Moreover, mitosis indexes were calculated in A549 and HT29D4 cells treated or not with compounds CA-4, 25 and 26. The quantification of nucleilabelled cells showed that for A549 cells, 2.7 ± 0.8 % of untreated cells, 11.6 ± 1.8 % of cells treated with CA-4, 9.7 ± 0.9 % and 10.3 ± 2.4 % of cells treated with compounds 25 and 26, respectively, were in mitosis. For HT29D4 cells, 3.0 ± 0.6 % of untreated cells and 4.4 ± 0.4 % of cells treated with 0.5 µM CA-4 were found in mitosis, whereas 28.9 ± 3.8 % and 29.9 ± 3.8 % was observed with compound 25 and 26, respectively. All these results confirm the higher efficacy of compounds 25 and 26 on CA-4 resistant colon cancer cells.

17

Compounds 25 and 26 modified both the A549 and HT29D4 cell cycle progression and triggered cell death. The parent compound, CA-4, was inactive in HT29D4 cells, providing evidence of the cytotoxic potential of compound 25 and 26 against colon adenocarcinoma. In addition, for both compounds, the modification of cell cycle progression led to numerous anomalous mitosis as observed by fluorescence microscopy. However, the two compounds displayed a lower pro-apoptotic activity compared to other 4-arylcoumarin derivatives

19

. Using

the HBL100 cell line, we previously determined cell cycle effects of compounds A (IC50 = 39 nM) and B (IC50 = 84 nM) which have either OCH3 or H on R1 of ring A and a 4-methoxy-3hydroxyphenyl ring B. In addition to the G2/M block, we also observed apoptosis concomitantly19. In the present study, following treatment with compounds 25 and 26, the accumulation of cells in the G2/M and sub-G1 phases was rather sequential as previously described

31

. This may imply activity on two different targets and/or the activation of different

cell death pathways. On the basis of the structural similarity between the two compounds and CA-4, the next step of our program focused on exploring the effects on tubulin assembly in vitro and in cells.

Effects of compounds 25 and 26 on cytoplasmic microtubule network and tubulin assembly. Since CA-4 is a well-known microtubule-depolymerizing agent, we investigated whether the cytotoxic effect of compounds 25 and 26 was associated with microtubule disorganization. For this purpose, three experimental conditions were designed to examine (1) the effect of drug concentration, (2) the effect of drug incubation time and (3) the effect of washing on the reassembly of the microtubules after removal of the drug. For this purpose, we performed a direct immunofluorescence staining of the microtubule network in untreated or drug-treated 18

A549 cells. From images collected by CLSM, the state of microtubule disassembly was calculated as being the ratio of the surface of microtubule network to the total surface of cells (for details, see the “Material and method” section). To investigate the effect of drug concentration, the microtubule network was visualized after a 4 h-treatment with 0.01 µM, 0.05 µM and 0.50 µM of CA-4 and similar concentrations of compounds 25 and 26 (Figure 4A-B).

Ratio of MT on cell surfaces (%)

A

B

* 40 35

* * n.s

n.s

CTRL

CA-4

(25)

(26)

n.s

30 25 20

15 10 5 0

CA-4

(25)

(26)

Figure 4: Disassembly of cytoplasmic microtubule networks of A549 cells by compounds 25 and 26. Cells were incubated with 0.01 – to – 0.50 µM drugs for 4 h at 37°C, fixed and then labelled by a direct immunofluorescence of -tubulin. The microtubule networks are visualized by CLSM. (A) Ratios of microtubules on total cell surfaces (in %) are calculated for cells untreated (‘CTRL’, white bar) and treated with 0.5 % DMSO (hatched bar), CA-4 (black bars), 25 (dark grey bars) and 26 (light grey bars); all reported values correspond to means ± SD of 60 cells; significant statistical differences are calculated using Student’s t test with *P < 0.001 between the value obtained for conditions ‘CTRL’ and the others; n.s: non-significant. (B) is 19

representative of the results obtained with CTRL cells, cells treated with 0.50 µM CA-4, 0.50 µM compound 25, 0.50 µM compound 26; scale bar: 20 µm.

In untreated cells, it was determined that 31 ± 4 % of the cell surface was occupied by the fluorescent microtubule network. All these values come close to the range of 31-41% of tubulin in microtubules in tissue cultured cells previously determined by Ostlund et al.

32

and our team24.

For CA-4, the disassembly of microtubules was evident after treatment with a concentration of 0.01 µM (25 ± 2 %), and was more apparent at 0.05 µM (3 ± 1 %) and total disassembly occurred at 0.50 µM. When cells were treated with compounds 25 and 26, no effect was observed at a concentration of 0.01 µM and a partial disassembly effect was observed (29 ± 3 % for the two compounds) at a concentration of 0.05 µM. When the compounds were tested at a concentration of 0.50 µM, the microtubule network was reduced 2.6 times related to untreated cells (13 ± 2 % and 12 ± 2 % for compound 25 and 26, respectively) (Figure 4A). As shown in Figure 4B, the set of representative images illustrate the complete microtubule network disassembly with 0.05 µM CA-4. At these concentrations, only the microtubule organizing centers (MTOC) were still visible for CA-4, while compounds 25 and 26 at a 0.5 µM exhibited a reduced ability to disrupt microtubule networks. This highlights the lower anti-proliferative activity between the two analogues and CA-4 in A549 cells. The effect of incubation time with CA-4 and compounds 25 and 26 on microtubule network was also investigated. For this, cells were incubated with 0.05 µM of CA-4 and 0.50 µM of compounds 25 and 26 for 1-8 h at 37°C (Figure 5). With CA-4, the microtubule disassembly (5 ± 2 %) was effective after 1 h of incubation (Figure 5A). On the other hand, compounds 25

20

and 26 induced a maximal disruption of the microtubule network after 4 h (13 ± 6 % and 16 ± 7 % for compound 25 and compound 26, respectively; Figure 5B – C). Our results indicate that compounds 25 and 26 are less active than CA-4, which correlates well with the lower cytotoxic activity of these two compounds compared to CA-4 in A549 cells.

Figure 5: Effect of the incubation time with compounds 25 and 26 on cytoplasmic microtubule network. The A549 cells were incubated with 0.05 µM CA-4 (A), 0.50 µM 25 or 0.50 µM 26 for 1 – to – 8 h at 37°C, fixed and then labelled by a direct immunofluorescence of -tubulin. The microtubule networks are visualized by CLSM. Ratios of microtubules on total cell surfaces (in %) are calculated in all conditions; all reported values correspond to means ± SD of 60 cells; significant statistical differences are calculated using Student’s t test with *P < 0.001 between the value obtained for incubation time ‘0 h’ and the others.

Next, the reversibility of inhibition of cellular microtubules by the drugs was investigated (Figure 6). Cells were treated with 0.05 µM CA-4 and 0.50 µM compounds 25 and 26 for 4 h, which corresponds to the maximal effect of drugs on microtubule disassembly. Drugs were 21

washed out from cells and the state of microtubule reassembly monitored. At time 0 min, it was established that in cells treated with CA-4 and compounds 25 and 26, 2.8 ± 1.5 %, 14.5 ± 5.4 % and 14.7 ± 7.0 %, respectively of the cytoplasm was occupied by the fluorescent microtubule network. These data are consistent with previous results (Figures 4 and 5). Moreover, in the case of CA-4 (Figure 6A), we observed a complete microtubule repolymerization after 120 min, while with compounds 25 and 26 microtubule repolymerization was observed after 30 min and 60 min, respectively (Figure 6B – C). After removing drugs from the medium, the time recovery of microtubules formation was quicker for compounds 25 and 26 than for CA-4.

Figure 6: Reversibility of the inhibition of cytoplasmic microtubule networks by compounds 25 and 26. The A549 cells were incubated with 0.05 µM CA-4 (A), 0.50 µM 25 or 0.50 µM 26 for 4 h at 37°C, then washed in complete medium without compounds during 0 – 120 min at 37°C. Cells are fixed and a direct immunofluorescence of -tubulin is performed. The microtubule networks are visualized by CLSM. Ratios of microtubules on total cell surfaces (in %) are calculated in all conditions; all reported values correspond to means ± SD of 60 cells; significant

22

statistical differences are calculated using Student’s t test with *P < 0.001 between the value obtained for incubation time ‘0 min’ and the others.

The fast recovery of cellular microtubule assembly could be related to faster binding dissociation equilibrium constants for compounds 25 and 26. In a previous study, we showed that the cytoplasmic microtubules of PtK2 cells were strongly disrupted in a concentration and timedependent manner by MDL 27048

25

. Moreover, maximal depolymerization took placed with

2.10-6 M MDL 27048 in 3 h. When the inhibitor was washed off from the cells, fast microtubule assembly ( 8 min) and complete reorganization of the cytoplasmic microtubule network (15-30 min) were observed. Our data are in agreement with this previous study and could indicate a good plasma membrane permeability of the drugs. To confirm that the activity of these compounds is due to a direct interaction with tubulin, turbidity time courses of in vitro microtubule assembly were performed with CA-4, as well as compounds 25 and 26 (Figure 7 A – C). For the three drugs, the rate of assembly as well as the final amount of microtubules was lower in the presence of drugs (curves (b – f), in panels A – C) than in the control experiment (curve (a) corresponding to 15 µM tubulin without ligand). Our data suggests an efficient inhibition of microtubule assembly by drugs at very low concentration. Moreover, as reported in Figure 7 D – F, the extent of inhibition increased linearly with the ratio of the total ligand concentration to total tubulin concentration until to a plateau value corresponding to a full inhibition of the microtubule formation. As a result, 0.06 mole of CA-4 per mole of tubulin was necessary to halve the microtubule formation, which is consistent with previously reported values 20. In the presence of compounds 25 and 26, 50% inhibition occurred

23

at a mole ratio of 0.12 mole of drugs per mole of tubulin. Our data indicate that as with CA-4, compounds 25 and 26 have a sub-stoichiometric mode of inhibition. This mode means there is blocking of microtubule assembly by the binding of either a tubulin-drug complex or a drug molecule to the growing polymer. Moreover, compounds 25 and 26 were two times less potent in inhibiting the microtubule formation compared to CA-4, which could be due to lower binding affinity constants to tubulin.

24

0.04

a b c

0.03

d

0.02

0.06

f 4

12 8 Time (min)

16

(25)

0.05

a b c d

0.04 0.03

e

0.02

f 0.01 0.00 0

4

0.05

(26)

8 12 Time (min)

16

20

CA-4

100 80 60 40 20 0

0.00

0.10 0.20 [drug]total / [tubulin]total

0.30

120

(25)

100 80 60 40 20 0

0.00

0.10 0.20 [drug]total / [tubulin]total

0.30

F

C  Absorbance 350 nm

120

E % polymerization inhibition

 Absorbance 350 nm

e

0.01 0.00 0

B

CA-4

a b c d e

0.04 0.03 0.02

f

0.01 0.00 0

4

12 8 Time (min)

16

% polymerization inhibition

 Absorbance 350 nm

0.05

% polymerization inhibition

D

A

120

(26)

100 80 60 40 20 0

0.00

0.10 0.20 [drug]total / [tubulin]total

0.30

25

Figure 7: Inhibitory effects of compounds 25 and 26 on the turbidity time course of in vitro microtubule assembly. The reaction was started by warming the solution at 37°C. Panels A – C show (a) tubulin at 15 µM and (b – f) aliquots of the same solution with 0.05 µM (b), 0.1 µM (c), 0.5 µM (d), 1 µM (e), 2 µM (f) CA-4 (panel A), compound 25 (panel B) and compound 26 (panel C). Panels D – F show the percentages of polymerization inhibition as a function of the ratio of the total drug concentration to total tubulin concentration; CA-4 (panel D), compound 25 (panel E), compound 26 (panel F).

Overall, we observed that compounds 25 and 26 induced the disassembly of the cytoplasmic microtubule network of A549 cells. Moreover, turbidity time courses of in vitro microtubule assembly in the presence of compounds 25 and 26 revealed a sub-stoichiometric mode of inhibition as CA-4. All these data indicate that the cytotoxic activity of these compounds is mainly due to a binding to tubulin. Moreover, it is likely that these two compounds exert their biological effect through partial disruption and/or suppressed dynamics of microtubules, as colchicine or Vinca-alkaloids 33–36. Despite their strong effects on the cell cycle, compounds 25 and 26 displayed a weaker effect than CA-4 on the microtubule disassembly at similar concentrations (Figure 4B). In addition, we found that inhibition of cell proliferation and mitotic arrest occur at concentrations of drugs much lower than those needed for a full disassembly of cytoplasmic microtubule. This indicates that the biological effects of compounds 25 and 26, as well as that of CA-4, could be due to perturbation of the spindle microtubule dynamics and not due to depolymerization of the cytoplasmic microtubules, as demonstrated by Jordan et al. for HeLa cells and Vinca-alkaloids 34 . Otherwise, compounds could exert their cell growth inhibitory effect through additional interactions with other molecular targets besides 26

tubulin. The planar nature and charges of compounds 25 and 26 may result in disruption of replication and/or repair mechanisms of DNA such as other classes of drugs (e.g. anthracyclines, cisplatin) 37–40. All this work outlines the higher efficacy of the compounds 25 and 26 on CA-4-resistant colon cancer cells. The causes of innate resistance of cells remain undefined, even if several mechanisms could be proposed. Some studies have suggested that this resistance is associated with a higher expression of the multidrug resistance proteins MRP-1 in these cells 41,42. However, treatment with the MRP-1 inhibitor MK-571 weakly increased efficacy of CA-4, indicating that this mechanism cannot fully explain HT29 chemoresistance 41 It has also been suggested that in HT29 cells a protective mechanism of autophagy may contribute to their resistance, however the inhibition of the autophagy pathway with the reagent BAF-A1 in these cells enhanced the efficacy of CA-432 derivative but not of CA-4 43. In another publication it has been reported that some oxazole-bridged CA-4 analogues are more efficient than CA-4 in inducing HT29 cell death by increasing the expression of p21cip1/waf1 at the RNA and protein level

44

. This molecule is a

cell cycle regulator by inhibiting it at G1 and S phase when DNA damage is present 45. Compounds 25 and 26 induced toxicity on HT29D4 cells by clearly causing cell cycle arrest in G2-M phase, as described for CA-4 and all anti-microtubules agents. Even if these two compounds inhibit the polymerization of tubulin in vitro almost as effectively as CA-4, we cannot exclude that in HT29D4 cells these compounds may have other targets such topoisomerase activity, as already shown for other CA-4 analogues18.

27

CONCLUSIONS In conclusion, 26 analogues of 4-arylcoumarins were synthesized and structure activity relationships derived. The two most active compounds, 6-chloro-4-(3-fluoro-4-methoxyphenyl)2H-chromen-2-one (25) and 6-chloro-4-(4-(methyl-amino) phenyl)-2H-chromen-2-one (26) show that the presence of a chloro substituent at position 6 of Ring A enhance the activity of the 4 arylcoumarins. However, the two compounds displayed a lower pro-apoptotic activity compared to other 4-arylcoumarin derivatives that have previously been reported by other studies, though the cell lines used in those studies were different.

18,19

. The two compounds (25

and 26) had poor solubility profiles, which may pose a problem in in vivo studies. Compounds 25 and 26 led to an accumulation of the A549 and HT29D4 cells in G2/M phase which correlates well with a disassembly of the microtubule network. We showed in vitro that compounds 25 and 26 have a sub-stoichiometric anti-tubulin activity and two-fold lower than that of CA-4. Our results provide a strong experimental basis to develop new potent anti-tubulin molecules for targeting CA-4-resistant cancer cells. Further work with these compounds may include testing them for other biological activities, given that the coumarin skeleton is a priviledged structure. Further work may also explore efforts to improve the solubilities of these compounds.

28

EXPERIMENTAL SECTION Chemistry. General equipment, reagents, solvents and materials. Melting points were determined using a Reichert-Jung Thermovar hot stage microscope apparatus and are uncorrected. 1H NMR and

13

C NMR data for synthetic compounds were

obtained using a Bruker (400 MHz) or a Varian (300 MHz) spectrometer. Chemical shifts (δ) are expressed in parts per million (ppm) relative to tetramethylsilane (TMS). Peak multiplicities are described as singlet (s), broad singlet (br s), doublet (d), doublet of a doublet (dd), doublet of a doublet of a doublet (ddd), triplet (t) and multiplet (m). Coupling constants are reported in Hertz (Hz). Low resolution electron impact mass spectra were obtained using a JEOL GC Mate II single magnetic sector mass spectrometer. Hexane, ethyl acetate and dichloromethane were general purpose reagents and were distilled before use. Anhydrous dioxane and toluene were obtained commercially from SigmaAldrich Chemical Company. Ultra-filtered water was purified using a Millipore Synergy water purification system. Unless specified, all solvents and chemicals were purchased from SigmaAldrich, Merck or Kimix, South Africa. Boronic acids were purchased from Combi-Blocks Inc., USA. Column chromatography was carried out on normal phase silica gel 60 from Sigma Aldrich. Flash chromatography was carried out using Biotage SNAP cartridges on a Biotage Isolera Flash Purification system. Thin layer chromatography was carried out on normal phase silica GF254 (250 µm) plates visualized under UV light (254 and 366 nm) and stained using

29

iodine vapour. Preparative TLC was carried out using normal phase silica GF 254 (1000 µm) plates. General procedure for the synthesis of 4-hydroxycoumarin intermediates46 Sodium hydride (6 equiv.) was stirred in anhydrous toluene under nitrogen gas. The appropriate 2’-hydroxyacetophenone (1 equiv.) in anhydrous toluene was added dropwise with stirring. After evolution of hydrogen had ceased, diethylcarbonate (2 equiv.) in dry toluene was added dropwise. The reaction mixture was refluxed at 110°C for 6 h then cooled to room temperature. The flask was then put in an ice bath and distilled water (30 mL) added slowly. HCl (2 M) was added dropwise to form a thick precipitate which was filtered under suction to yield the desired 4-hydroxycoumarin intermediates. General procedure for triflate formation47 Tri-fluoromethane sulfonic anhydride (1.3 equiv.) was added dropwise to a mixture of the appropriate 4-hydroxycoumarins (1 equiv.)

and tri-ethylamine (1.3 equiv.)

in dry

dichloromethane at 0°C under nitrogen. The reaction was stirred at 0°C for 1 hour, and then diluted with 50 % ether-petroleum (40°C - 60°C). The reaction mixture was then passed through a pad of celite and the solvent evaporated in vacuo. The triflates were then crystallized from dichloromethane and used without further purification. General procedure for the synthesis of target 4-arylcoumarins using Suzuki coupling48 The appropriate triflates (1 equiv.) were dissolved in anhydrous dioxane under nitrogen and boronic acids (1.1 equiv.) were added. The reaction mixture was degassed by bubbling nitrogen gas for about 20 min. Bis(triphenyphosphine)palladium (II) dichloride (0.05 equiv.) was added under nitrogen gas, followed by aqueous K2CO3 (3 equiv.) The reaction mixture was then 30

refluxed at 85°C for 14h. For work up, the reaction mixture was poured into distilled water and partitioned three times with ethyl acetate. The organic fractions were combined and the solvent evaporated under pressure. The 4-arylcoumarins were isolated using column or flash chromatography on a Biotage machine. Some compounds were purified using preparative HPLC. All compounds were obtained in moderate to good yields of 35 to 90 %. 4-(3-hydroxy-4-methoxyphenyl)-6-methoxy-2H-chromen-2-one (1) Yellow solid (29 mg, 52 %) m.p. 165°C (Lit 167°C 19) 1H NMR (400 MHz, CD3OD) δ 7.36 (d, J = 9.1 Hz, 1H, H-8), 7.22 (dd, J = 9.0, 2.6 Hz, 1H, H-7), 7.09 (m, 2H, Ar-H), 7.00 (d, J = 2.6 Hz, 1H, H-5), 5.47 (br, s, 1H, OH) 6.98 (d, J = 1.9 Hz, 1H, H-2’), 6.31 (s, 1H, H-3), 3.94 (s, 3H, OCH3), 3.75 (s, 3H, OCH3). 13C NMR (101 MHz, MeOD) δ 160.9, 156.4, 156.1, 148.4, 148.4, 146.1, 127.8, 120.0, 119.5, 119.2, 117.8, 115.1, 113.8, 111.6, 109.6, 55.1, 54.9 m/z (LRMS, EI) Found 298 (M+) C17H14O5 requires M, 298.08. HPLC 98 % (Method A described in supporting information, t R 13.30 min). 6-chloro-4-(3-hydroxy-4-methoxyphenyl)-2H-chromen-2-one (2) Yellow crystalline solid (37 mg, 60 %) m.p. 175°C , 1H NMR (400 MHz, CDCl3) δ 7.57 (d, J = 2.5 Hz, 1H, H-5), 7.51 (dd, J = 8.8, 2.5 Hz, 1H, H-7), 7.36 (d, J = 8.8 Hz, 1H, H-8), 7.05 (d, J = 2.1 Hz, 1H,H-2’), 7.03 (d, J = 8.3 Hz, 1H, H-5’), 6.97 (dd, J = 8.3, 2.1 Hz, 1H, H-6’), 6.40 (s, 1H, H-3), 5.81 (br, s, 1H, OH), 4.02 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3) δ 160.2, 154.3, 152.5, 148.3, 146.2, 132.0, 129.4, 127.9, 126.5, 120.8, 120.3, 118.5, 115.7, 114.6, 110.8, 56.0 m/z (LRMS, EI) Found 302 (M+) C16H11ClO4 requires M, 302.03. HPLC 98 % (Method A, t R 14.32 min). 6-fluoro-4-(3-hydroxy-4-methoxyphenyl)-2H-chromen-2-one (3) 31

White solid, (31 mg, 47 %) m.p. 154°C, 1H NMR (400 MHz, CDCl3) δ 7.36 (d, J = 8.9 Hz, 1H, H-8), 7.29 – 7.22 (m, 2H, H-5, H-7), 7.03 (d, J = 2.1 Hz, 1H, H-2’), 7.00 (d, J = 8.3 Hz, 1H, H5’), 6.95 (dd, J = 8.3, 2.1 Hz, 1H, H-6’), 6.39 (s, 1H, H-3), 5.82 (br, s, 1H, OH), 3.99 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3) δ 160.5, 159.9, 157.5, 154.4, 150.3, 148.0, 146.1, 127.9, 120.6, 119.2 (d, 2JCF = 24.5 Hz, C-7), 118.8 (d, 3JCF = 8.3 Hz, C-8), 115.62, 114.63, 112.69 (d, 2

JCF = 25.2 Hz, C-5). 111.00, 56.15. m/z (LRMS, EI) Found 286 (M+) C16H11FO4 requires M,

286.06. HPLC 98 % (Method A, t R 13.35 min). 4-(3-hydroxy-4-methoxyphenyl)-6-methyl-2H-chromen-2-one (4) Colourless crystalline solid (52 mg, 73 %) m.p. 164°C, 1H NMR (400 MHz, CDCl3) δ 7.35 – 7.31 (m, 2H, H-5, H-8), 7.29 – 7.25 (m, 1H, H-7), 7.04 (d, J = 2.0 Hz, 1H, H-2’), 6.99 (d, J = 8.3 Hz, 1H, H-5’), 6.95 (dd, J = 8.3, 2.0 Hz, 1H, H-6’), 6.31 (s, 1H, H-3), 3.98 (s, 3H, OCH3), 2.34 (s, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ 160.8, 155.3, 152.1, 148.0, 145.9, 133.8, 132.8, 128.6, 126.8, 120.6, 118.8, 117.0, 114.9, 114.7, 110.9, 56.1, 20.9 m/z (LRMS, EI) Found 282 (M+) C17H14O4 requires M, 282.09. HPLC 97 % (Method A, tR 13.92 min). 4-(3-hydroxy-4-methoxyphenyl)-2H-chromen-2-one (5) White solid (56 mg, 82 %) m.p. 165°C, 1H NMR (400 MHz, CD3OD) δ 7.67 – 7.58 (m, 2H, ArH), 7.41 (dd, J = 8.3, 1.2 Hz, 1H, H-8), 7.32 (d, J = 70.5 Hz, 1H, H-5’), 7.12 – 7.07 (m, 1H, ArH), 6.99 (dd, J = 7.5, 2.2 Hz, 1H, H-6’), 6.97 (d, J = 2.2 Hz, 1H, H-2’), 6.32 (s, 1H, H-3), 3.94 (s, 3H, OCH3).13C NMR (101 MHz, MeOD) δ 161.5, 156.3, 153.9, 149.2, 146.6, 131.9, 127.6, 127.0, 124.2, 120.1, 118.9, 116.8, 115.3, 113.5, 111.6, 55.1 m/z (LRMS, EI) Found 286 (M+) C16H12O4 requires M, 286.07. HPLC 98 % (Method A, t R 12.78 min). 6-methoxy-4-(6-methoxypyridin-3-yl)-2H-chromen-2-one (6) 32

Yellow crystalline powder, (70 mg, 78 %), m.p. 135- 139°C, 1H NMR (400 MHz, CDCl3) δ 8.30 (d, J = 2.5 Hz, 1H, H-2’), 7.68 (dd, J = 8.6, 2.5 Hz, 1H, H-4’), 7.35 (d, J = 9.0 Hz, 1H, H-8), 7.14 (dd, J = 9.0, 2.7 Hz, 1H, H-7), 6.91 (d, J = 2.7 Hz, 1H, H-5), 6.91 (d, J = 8.6 Hz, 1H, H-5’), 6.36 (s, 1H, H-3), 4.02 (s, 3H, OCH3), 3.76 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3) δ 165.1, 160.6, 156.1, 152.0, 148.6, 146.5, 138.5, 124.3, 119.2, 118.4, 118.4, 115.8, 111.2, 109.5, 55.9, 53.9. m/z (LRMS, EI) Found 283 (M+) C16H13NO4 requires M, 283.08. HPLC 98 % (Method A, tR 13.91 min). 6-fluoro-4-(6-methoxypyridin-3-yl)-2H-chromen-2-one (7) White needle-like crystals (61 mg, 46 %) m.p. 150- 153°C, 1H NMR (300 MHz, CDCl3) δ 8.23 (d, J = 2.5 Hz, 1H, H-2’), 7.61 (dd, J = 8.6, 2.5 Hz, 1H, H-4’), 7.34 (dd, J = 9.2, 4.6 Hz, 1H, H8), 7.21 (m, 1H, H-7), 7.10 (dd, J = 8.9, 2.9 Hz, 1H, H-5), 6.86 (d, J = 8.6 Hz, 1H, H-5’), 6.35 (s, 1H, H-3), 3.97 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3) δ 165.3, 160.0, 157.5, 151.5, 150.4, 146.5, 138.4, 123.7, 119.6 (d, 2JC-F = 24.5 Hz, C-7), 119.1 (d, 3JC-F = 8.3 Hz, C-8), 116.2, 116.2 112.1 (d, 2JC-F = 25.2 Hz, C-5), 111.4, 53.9. m/z (LRMS, EI) Found 271 (M+) C15H10FNO3 requires M, 271.06. HPLC 98 % (Method A, t R 13.87 min). 4-(6-methoxypyridin-3-yl)-2H-chromen-2-one (8) White amorphous powder, (69 mg, 76 %,) m.p. 132-134°C, (Lit- 125°C)21, 1H NMR (400 MHz, CDCl3) δ 8.28 (d, J = 2.5 Hz, 1H, H-2’), 7.66 (dd, J = 8.6, 2.5 Hz, 1H, H-4’), 7.57 – 7.52 (m, 1H, H-7), 7.46 (dd, J, 8.3, 1.2 Hz, 1H, H-8), 7.39 (dd, J = 8.2, 1.5 Hz, 1H, H-5), 7.24 (ddd, J = 8.2, 6.4, 1.2 Hz, 1H, H-6), 6.89 (d, J = 8.6 Hz, 1H, H 5’), 6.35 (1H, s, H-3), 4.00 (3H, s, OCH3). 13C NMR (101 MHz, CDCl3) δ 165.1, 160.4, 154.3, 152.3, 146.6, 138.6, 132.1, 126.5, 124.3, 124.2,

33

118.8, 117.5, 115.3, 111.2, 53.8 m/z (LRMS, EI) Found 253 (M+) C15H11NO3 requires M, 253.07. HPLC 98 % (Method A, t R 13.72 min). 4-(3,4-dimethoxyphenyl)-6-methoxy-2H-chromen-2-one (9) Colorless crystalline solid (50 mg, 61 %) m.p. 190-191°C (Lit 178°C14, 148-150°C 47), 1H NMR (300 MHz, CDCl3) δ 7.34 (d, J = 9.0 Hz, 1H, H-8), 7.12 (dd, J = 9.0, 2.2 Hz, 1H, H-7), 7.01 – 7.08 (m, 3H, H-5/H-2’, H-5’, H-6’), 6.97 (d, J = 2.2 Hz, 1H, H-2’/H-5), 6.37 (s, 1H, H-3), 3.96 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 3.75 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3) δ 161.0, 155.9, 155.1, 150.4, 149.3, 148.7, 127.8, 121.3, 119.6, 119.1, 118.3, 115.2, 111.5, 111.4, 109.9, 56.1, 56.1, 55.8. m/z (LRMS, EI) Found 312 (M+) C18H16O5 requires M, 312.10. HPLC 99 % (Method B as described in supporting information, t R 10.09 min). 6-methoxy-4-(4-(methylamino)phenyl)-2H-chromen-2-one (10) Yellow crystalline solid (73 mg, 67 %) m.p. 156-160°C, 1H NMR (400 MHz, CDCl3) δ 7.28 – 7.36 (m, 3H, Ar-H), 7.11 (m, 2H, Ar-H), 6.71 (d, J = 7.6 Hz, 2H, H-3’, H-5’), 6.32 (s, 1H, H-3), 4.10 (br, s, 1H, N-H), 3.76 (s, 3H, OCH3), 2.92 (s, 3H, N-CH3),13C NMR (101 MHz, CDCl3) δ 161.4, 155.8, 155.5, 150.6, 148.8, 129.8, 129.8, 123.6, 119.8, 118.8, 118.2, 114.0, 112.2, 112.2, 110.2, 55.8, 30.4 m/z (LRMS, EI) Found 281 (M+) C17H15NO3 requires M, 281.10. . HPLC 98 % (Method A, tR 14.23 min). 4-(3-fluoro-4-(trifluoromethyl)phenyl)-6-methoxy-2H-chromen-2-one (11) White crystals (28 mg, 30 %) m.p. 158-160°C, 1H NMR (300 MHz, CDCl3) δ 7.78 (d, J = 7.6 Hz, 1H, H-5’), 7.32 – 7.37 (m, 3H, H-8, H-2’, H-6’), 7.15 (dd, J = 9.1, 2.9 Hz, 1H, H-7), 6.75 (d, J = 2.9 Hz, 1H, H-5), 6.36 (s, 1H, H-3).

13

C NMR (101 MHz, CDCl3) δ 160.1, 157.5, 156.2,

34

152.4, 148.5, 141.3, 128.2, 128.1, 124.2, 119.4, 118.6, 117.2, 117.0, 116.5, 109.5, 109.5, 55.9 m/z (LRMS, EI) Found 338 (M+) C17H10F4O3 requires M, 338.06. HPLC 95 % (Method B, t R 11.51 min). 6 methoxy-4-(6’-(triflouromethyl)pyridine-3-yl)-2H-chromen-2-one (12) Colorless crystalline solid (50 mg, 43 %) m.p. 154-156°C, 1H NMR (400 MHz, CDCl3) δ 8.85 (d, J = 2.1 Hz, 1H, H-2’), 8.01 (dd, J = 8.1, 2.1 Hz, 1H, H-4’), 7.89 (d, J = 8.1 Hz, 1H, H-5’), 7.38 (d, J = 9.1 Hz, 1H, H-8), 7.18 (dd, J = 9.1, 2.9 Hz, 1H, H-7), 6.71 (d, J = 2.9 Hz, 1H, H-5), 6.42 (s, 1H, H-3), 3.75 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3) δ 159.9, 156.4, 150.5, 149.0, 148.5, 138.6, 137.4, 134.1, 122.9, 120.6, 119.7, 118.7, 118.5, 117.2, 109.2, 55.9 m/z (LRMS, EI) Found 321 (M+) C16H10F3NO3 requires M, 321.06. HPLC 99 % (Method B, t R 10.49 min). 3-(6-methoxy-2-oxo-2H-chromen-4-yl) benzonitrile (13) White solid, (44 mg, 58 %) m.p.181-183°C, 1H NMR (400 MHz, CDCl3) δ 7.83 (ddd, J = 6.6, 2.3, 1.6 Hz, 1H, H-4’), 7.76 (d, J = 1.6, 1H, H-2’), 7.67-7.70 (m 2H, H-5’, H-6’), 7.37 (d J = 9.1Hz, 1H, H-8), 7.17 (dd, J = 9.1, 2.9 Hz, 1H, H-7), 6.73 (d, J = 2.9 Hz, 1H, H-5), 6.37 (s, 1H, H-3), 3.75 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3) δ 160.0, 156.2, 152.7, 148.6, 136.6, 133.2, 132.6, 131.7, 130.0, 119.2, 118.7, 118.6, 117.8, 116.6, 113.6, 109.6, 55.9 m/z (LRMS, EI) Found 277 (M+) C17H11NO3 requires M, 277.07 HPLC 97 % (Method B, t R 10.24 min). 6-methoxy-4-(6’-(methylsulfonyl)pyridin-3-yl)-2H-chromen-2-one (14) Yellow solid, (55 mg, 43 %) 143-145°C, 1H NMR (400 MHz, CDCl3) δ 8.80 (d, J = 2.1 Hz, 1H, H-2’), 8.24 (d, J = 8.0 Hz, 1H, H-5’), 8.09 (dd, J = 8.0, 2.1 Hz, 1H, H-4’), 7.33 (d, J = 9.1 Hz, 1H, H-8), 7.15 (dd, J = 9.1, 2.8 Hz, 1H, H-7), 6.67 (d, J = 2.8 Hz, 1H, H-5), 6.38 (s, 1H, H-3),

35

3.72 (s, 3H, OCH3), 3.28 (s, 3H, SO2CH3) 13C NMR (101 MHz, CDCl3) δ 159.6, 159.2, 156.4, 150.0, 149.0, 148.5, 138.2, 135.1, 121.1, 119.8, 118.8, 118.3, 117.5, 109.1, 56.0, 40.0. m/z (LRMS, EI) Found 331 (M+) C16H13NO5S requires M, 331.05. HPLC 96 % (Method B, t R 9.08 min). 3-(6-methoxy-2-oxo-2H-chromen-4-yl)benzamide (15) White needle-like crystals (26 mg, 28 %) m.p. 199- 202°C, 1H NMR (300 MHz, DMSO) δ 8.06 (m, 3H, H-2’, NH2), 7.70 (m, 3H, H-4’, H-5’, H-6’), 7.48 (d, J = 9.1 Hz, 1H, H-8), 7.30 (dd, J = 9.1, 2.9 Hz, 1H, H-7), 6.84 (d, J = 2.9 Hz, 1H, H-5), 6.52 (s, 1H, H-3), 3.71 (s, 3H, OCH3). 13C NMR (101 MHz, DMSO) δ 167.4, 160.1, 155.7, 154.4, 148.1, 135.3, 135.0, 131.4, 129.3, 129.2, 127.8, 119.6, 119.1, 118.5, 115.9, 110.0, 56.0 m/z (LRMS, EI) Found 295 (M+) C17H13NO4 requires M, 295.08. HPLC 98 % (Method A, t R 12.15 min).

4-(4-chlorophenyl)-6-methoxy-2H-chromen-2-one (16) White crystals, (79 mg, 61 %) m.p. 148- 150°C, 1H NMR (300 MHz, CDCl3) δ 7.52 (d, J = 8.1 Hz, 2H, H-3’, H-5’), 7.40 (d, J = 8.1 Hz, 2H, H-2’, H-6’), 7.35 (d, J = 9.0 Hz, 1H, H-8), 7.14 (dd, J = 9.0, 2.4 Hz, 1H, H-7), 6.86 (d, J = 2.4 Hz, 1H, H-5), 6.36 (s, 1H, H-3), 3.75 (s, 3H, OCH3).13C NMR (100 MHz, CDCl3) δ 160.5, 155.9, 154.0, 148.5, 135.9, 133.6, 129.6, 129.6, 129.2, 129.2, 119.1, 119.0, 118.2, 115.7, 109.6, 55.7. m/z (LRMS, EI) Found 286 (M+) C16H11ClO3 requires M, 286.04. HPLC 98 % (Method A, t R 15.33 min). 4-(3-fluoro-4-methoxyphenyl)-6-methoxy-2H-chromen-2-one (17) White solid (63 mg, 68 %) m.p. 134-136°C, 1H NMR (400 MHz, CDCl3) δ 7.34 (d, J = 9.0 Hz, 1H, H-8), 7.19 – 7.24 (m, 2H, H-2’, H-6’), 7.14 (dd, J = 9.0, 3.0 Hz, 1H, H-7), 7.12 (d, J = 8.2 36

Hz, 1H, H-5’), 6.95 (d, J = 3.0 Hz, 1H, H-5), 6.34 (s, 1H, H-3), 3.98 (s, 3H, OCH3), 3.76 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3) δ 160.7, 156.0, 153.8, 153.5, 151.0, 148.5, 127.9, 124.7 (d, 4

JC-F = 3.4 Hz, C-5’), 119.2, 119.1, 118.3, 116.4 (d, 2JC-F = 19.7 Hz, C-2’).115.6, 113.8, 109.8,

56.4, 55.8 m/z (LRMS, EI) Found 300 (M+) C17H13FO4 requires M, 300.8. HPLC 99 % (Method B, tR 10.72 min). 4-(4-fluorophenyl)-6-methoxy-2H-chromen-2-one (18) Light yellow crystals (56 mg, 64 %) m.p. 127-129°C, 1H NMR (400 MHz, CDCl3) δ 7.42 – 7.47 (m, 2H, H-3’, H-5’), 7.33 (d, J = 9.0 Hz, 1H, H-8), 7.19 – 7.26 (m, 2H, H-2’, H-6’), 7.12 (dd, J = 9.0, 3.0 Hz, 1H, H-7), 6.87 (d, J = 3.0 Hz, 1H, H-5), 6.34 (s, 1H, H-3), 3.74 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3) δ 162.3, 160.7, 156.0, 154.2, 148.8, 131.3, 130.3 (d, 3JC-F = 8.3 Hz, C2’, C-6’), 119.4, 119.1, 118.3, 116.2 (d, 2JC-F = 21.8 Hz, C-3’, C-5’), 115.8, 109.9, 55.8 m/z (LRMS, EI) Found 270 (M+) C16H11FO3 requires M, 270.07. HPLC 95 % (Method B, t R 10.88 min). 4-(benzo[d][1,3]dioxol-5-yl)-6-methoxy-2H-chromen-2-one (19) White solid (78 mg, 76 %) m.p. 191-193°C (Lit 188-190°C

47 1

), H NMR (400 MHz, CDCl3) δ

7.36 (d, J = 9.0 Hz, 1H, H-8), 7.15 (dd, J = 9.0, 3.0 Hz, 1H, H-7), 7.03 (d, J = 3.0 Hz, 1H, H-5), 2H), 6.95 – 6.98 (m, 3H, H-2’, H-5’, H-6’), 6.37 (s, 1H, H-3), 6.10 (s, 2H, H-2’’), 3.79 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3) δ 160.9, 155.9, 154.8, 149.0, 148.6, 148.2, 129.0, 122.5, 119.5, 118.9, 118.3, 115.4, 110.1, 108.8, 108.8, 101.7, 55.7 m/z (LRMS, EI) Found 296 (M+) C17H12O5 requires M, 296.07. HPLC 98% (Method A, t R 14.39 min). 6-methoxy-4’-phenyl-2H-chromen-2-one (20)

37

White solid (63 mg, 80 %) m.p. 144-146°C (Lit 148-149°C

49

), 1H NMR (400 MHz, CDCl3) δ

7.55 (m, 3H, Ar-H), 7.48 (m, 2H, Ar-H), 7.36 (d, J = 9.0 Hz, 1H, H-8), 7.15 (dd, J = 9.0, 3.0 Hz, 1H, H-7), 6.95 (d, J = 3.0 Hz, 1H, H-5), 6.39 (s, 1H, H-3), 3.76 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3) δ 160.9, 155.9, 155.3, 148.6, 135.3, 129.7, 128.9, 128.9, 128.3, 128.3, 119.5, 119.0, 118.2, 115.7, 110.1 55.8 m/z (LRMS, EI) Found 252 (M+) C16H12O3 requires M, 252.08. HPLC 98 % (Method A, t R 14.53 min). 4-phenyl-2H-chromene-2-one (21) White solid (46 mg, 84 %) m.p. 95°C (Lit 97-98 °C

49

), 1H NMR (400 MHz, CDCl3) δ 7.59 –

7.38 (m, 7H, Ar-H), 7.27 – 7.19 (m, 2H, Ar-H), 6.38 (s, 1H, H-3). 13C NMR (101 MHz, CDCl3) δ 160.7, 155.7, 154.3, 135.3, 131.9, 129.7, 128.9, 128.9, 128.5, 128.5, 127.0, 124.2, 119.1, 117.4, 115.3 m/z (LRMS, EI) Found 222 (M+) C15H10O2 requires M, 222.07. HPLC 98 % (Method A, t R 14.16 min). 4-(3-chlorophenyl)-2H-chrome-2-one13 (22) White powder (40 mg, 53%) m.p 129-130°C (Lit- 122-124°C)50), 1H NMR (300 MHz, CDCl3) δ 7.53 – 7.57 (m, 1H, Ar-H), 7.47 – 7.52 (m, 1H, Ar-H), 7.43 – 7.47 (m, 1H, Ar-H), 7.38 – 7.43 (m, 2H, Ar-H) 7.31 (dd, J = 7.8, 1.7 Hz, 1H, Ar-H), 7.23 – 7.15 (m, 1H, Ar-H), 7.09 (d, J = 7.8 Hz, 1H, Ar-H), 6.36 (s, 1H, H-3).13C NMR (101 MHz, CDCl3) δ 160.4, 153.7, 153.3, 134.0, 132.5,131.9, 130.6, 130.1, 127.1, 126.7, 126.6, 124.2, 118.7, 117.1, 116.5. m/z (LRMS, EI) Found 256 (M+) C15H9ClO2 requires M, 256.03. HPLC 98% (Method A, t R 15.35 min). 4-(2-chlorophenyl)-2H-chrome-2-one13 (23)

38

White powder (40 mg, 48 %) m.p. 104°C, 1H NMR (300 MHz, CDCl3) δ 7.53 – 7.57 (m, 1H, ArH), 7.47 – 7.52 (m, 1H, Ar-H), 7.43 – 7.47 (m, 1H, Ar-H), 7.40 (dd, J = 9.1, 1.7 Hz, 2H, Ar-H), 7.31 (dd, J = 7.8, 1.7 Hz, 1H, Ar-H), 7.23 – 7.15 (m, 1H, Ar-H), 7.09 (d, J = 7.8 Hz, 1H, Ar-H), 6.36 (s, 1H, H-3).13C NMR (101 MHz, CDCl3) δ 160.4, 153.7, 153.3, 134.0, 132.5,131.9, 130.6, 130.1, 127.1, 126.7, 126.5, 124.2, 118.7, 117.1, 116.5. m/z (LRMS, EI) Found 256 (M+) C15H9ClO2 requires M, 256.03. HPLC 98 % (Method A, t R 14.65 min). 4-(4-chlorophenyl)-2H-chromen-2-one (24) Light yellow crystalline powder (43 mg; 50 %) m.p. 176- 179°C (Lit- 164-166°C)50, 1H NMR (400 MHz, CDCl3) δ 7.49 – 7.59 (m, 3H, Ar-H), 7.38 – 7.45 (m, 4H, Ar-H), 7.27 – 7.21 (m, 1H, Ar-H), 6.36 (s, 1H, H-3).

13

C NMR (100 MHz, CDCl3) δ 160.2, 154.2, 154.1, 135.9, 133.5,

132.0, 129.7, 129.7, 129.1, 129.1, 126.5, 124.1, 118.7, 117.3, 115.3 m/z (LRMS, EI) Found 256 (M+) C15H9ClO2 requires M, 256.03. HPLC 98 % (Method A, t R 15.27 min). 6-chloro-4-(3-fluoro-4-methoxyphenyl)-2H-chromen-2-one (25) Colorless crystalline solid (23 mg, 46 %) m.p. 160°C, 1H NMR (400 MHz, CDCl3) δ 7.51 (dd, J = 8.7, 2.5 Hz, 1H, H-7), 7.47 (d, J = 2.5 Hz, 1H, H-5), 7.35 (d, J = 8.7 Hz, 1H, H-8), 7.22 – 7.20 (m, 1H, H-6’), 7.18 (dd, J = 4.6, 2.0 Hz, 1H, H-2’), 7.13 (dd, J = 6.6, 3.4 Hz, 1H, H-5’), 6.37 (s, 1H, H-3), 3.99 (s, 3H, OCH3) 13C NMR (101 MHz, CDCl3) δ 160.0, 153.6, 153.1, 152.6, 151.2, 132.0, 129.8, 127.1, 126.1, 124.7 (d, 3JC-F = 3.5 Hz, C-5’), 120.0, 118.9, 116.4 (d, 2JC-F = 19.8 Hz, C-2’), 116.1, 113.9, 56.4 m/z (LRMS, EI) Found 304 (M+) C16H10ClFO3 requires M, 304.03. HPLC 98 % (Method A, t R 15.71 min) 6-chloro-4-(4-(methylamino)phenyl)-2H-chromen-2-one (26)

39

Brown solid (21 mg, 57 %) m.p. 155°C, 1H NMR (400 MHz, CDCl3) δ 7.63 (d, J = 2.5 Hz, 1H, H-5), 7.47 (dd, J = 8.8, 2.5 Hz, 1H, H-7), 7.34 – 7.30 (m, 3H, H-8, H-2’, H-6’), 6.74 (dd, J = 8.7, 2.1 Hz, 2H, H-3’, H-5’), 6.35 (s, 1H, H-3), 4.00 (br, s, 1H, N-H), 2.93 (s, 3H, N-CH3). 13C NMR (101 MHz, CDCl3) δ 160.6, 154.8, 152.8, 150.6, 131.5, 129.9, 129.4, 129.4, 126.6, 123.0, 120.6, 118.7, 114.4, 112.5, 112.5, 30.5 m/z (LRMS, EI) Found 285 (M+) C16H12ClNO2 requires M, 285.06. HPLC 97 % (Method A, t R 15.30 min).

Solubility testing51 All the compounds were dissolved in DMSO to make a 10 mM stock solution. The stock solution was pipetted into a 96 well pre-dilution plate to make serial dilutions ranging from concentrations of 10 mM to 0.25 mM, pipetting each sample in triplicate. From the pre-dilution plate, the samples were pipetted into 96 well plates containing either DMSO or PBS buffer at pH 7.4. The samples were pipetted so as to end up with a final concentration of 200 µM to 5 µM in DMSO and in PBS buffer. The plates were then incubated on the bench at room temperature for 2 hours then absorbance read at 620 nm using a SpectraMax® 340 PC384 Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Microsoft Excel program was then used to generate graphs and the solubility limit was interpreted as the concentration at which an inflection was observed on the graph.

Biology Cell culture.

40

Cells from human non-small lung carcinoma (clone A549; ATCC n°: CCL2, MD, USA) and human colonic adenocarcinoma (HT-29 D4 cells sub-cloned from the HT-29 line

22

) were

routinely grown at 37°C in a humidified atmosphere of 5 % CO2. Cells were maintained in a standard medium composed of RPMI 1640 (Lonza, France) for A549 and with DMEM for HT29 D4 line, supplemented with 10 % fetal bovine serum (FBS), 2mM L-glutamine and 1 % penicillin and streptomycin (Invitrogen). Cells were free from mycoplasma as determined by mycoalert tests (Lonza).

Cytotoxicity Assays. For cell viability assay, cells harvested from sub-confluent monolayers were seeded at 2.5104 cells per mL in 96-well microtiter plate (Cambridge Technology, France) and cultured 24 h under standard conditions. Standard medium was then replaced by fresh medium containing no drugs or compounds at different concentrations. The surviving cells were quantified after 72 h by the MTT assay, according to the manufacturer’s instructions. Briefly, 20 µL of 3-(4,5diMethylThiazol-2-yl)-2,5diphenyl Tetrazolium bromide (MTT) solution at 5 mg.mL -1 were added to cells for 2 h at 37°C. The supernatant was discarded and replaced by 200 µL of dimethyl sulfoxide to dissolve formazan crystals. The absorbance was then read at 540 nm by spectrophotometry. For all concentrations of compound, cell viability was expressed as the percentage of the ratio between the mean absorbance of treated cells and the mean absorbance of untreated cells. Three independent experiments were performed, and the IC50 values (i.e., concentration half inhibiting cell proliferation) were graphically determined.

Fluorescence microscopy 41

For immunofluorescence microscopy of the microtubule network, 106 cells were plated on cover-glass and incubated with the different drugs. Cells were then fixed in 3.7 % formaldehyde (in PBS pH 7.4) for 20 min at room temperature, permeabilized with PBS-Triton X-100 0.5 % for 10 min at room temperature. Direct immunostaining was carried out for 2 h at room temperature with a primary FITC-conjugated anti--tubulin antibody (dilution 1:400 in PBS-BSA 1 % from a 1 mg.mL-1 solution; monoclonal antibody, clone DM1A, Sigma-Aldrich, France). Next, cells were washed in PBS and cover-glasses were mounted with a drop of ProLong® anti-fade solution (Invitrogen). The cytoskeleton was imaged by a confocal laser scanning microscope (CLSM) Leica SP5 with a Leica inverted microscope, equipped with a Plan-Apochromat 63 oil immersion objective (NA = 1.4). Each image was recorded with the CLSM’s spectral mode selecting specific domains of the emission spectrum. The FITC fluorophore was excited at 488 nm with an argon laser and its fluorescence emission was collected between 496 nm and 535 nm. The public-domain ImageJ software was used for image analysis 52. Here, 60 cells immuno-labelled for -tubulin were examined from three independent experiments to quantify the ratio of microtubule on cell surfaces.

Cell cycle analysis and mitosis index For flow cytometric analysis of DNA content, 4.10 5 cells in exponential growth were treated with 0.5 µM CA-4, 1 µM compound (25) and (26) for 24 h. The supernatant and trypsinated cells were harvested then centrifuged (1200 rpm, 5 min, at 4°C). The cell pellet was re-suspended in 1 mL of cold 70 % ethanol for 30 min at -20°C. Cells were centrifuged (2000 rpm, 5 min, at 4°C) to remove ethanol then the cell pellet was re-suspended in the staining mix containing 50 µg.mL-1 propidium iodide (Molecular Probes, France) and 100 µg.mL-1 RNAse A 42

(Sigma, France) in PBS for 20 min at room-temperature in darkness. Samples were analyzed on a Becton Dickinson FACScan flow cytometer using the CellQuest software. Propidium iodide was excited at 488 nm and fluorescence was analyzed at 620 nm on channel Fl-2. For the analysis, there is considerable overlap between early S phase and G1 and between late S phase and G2/M due to broadening of the distribution caused by variability in the staining of the cells and also instrumental variability. Therefore, the percentages of cells in the sub-G1, G1, S and G2/M phases of the cell cycle were calculated from the modified ‘pragmatic approach’ described by Watson et al53: a gate to surround the sub-G1 population was dragged on DNA histograms from zero to 30 in fluorescence intensity (the value of 30 was considered as the beginning of G1 phase); for G1 and G2/M phases, the step was repeated for creating interval gates from 30 to 45 and from 95 to 110 in fluorescence intensity which corresponding to G1 and G2/M populations, respectively; for S phases, an interval gate was dragged from 45 to 95 in fluorescence intensity in order to estimate at better cells in early and late S phases. The number of cells in all phases were divided by the total number of analyzed cells and reported in percentage. For scoring mitotic indexes, 105 cells were plated on cover-glass day 1. Day 2, cells were incubated with 0.5 µM CA-4, 1 µM compounds 25 and 26 for 24 h. Day 3, cells were washed in PBS, then fixed with 3.7 % formaldehyde in PBS for 20 min at room-temperature. After washing in PBS, nuclei were labelled with 1 µM Draq5 (Biostatus, GB) then mounted on glass. Cells were imaged with the CLSM’s meta mode selecting specific domains of the emission spectrum, i.e. Draq5 was excited at 633 nm with a He/Ne laser and its fluorescence emission was collected between 670 and 745 nm. The mitotic index was calculated by dividing the mitotic count determined in 20 fluorescence images by the total cell number in these fields, and expressed as the number of mitotic figures per 100 cells.

43

Analysis of microtubule network in cells The analysis of microtubule network was performed as we previously reported24. Briefly, series of 8-bit images were transferred to ImageJ software and all images were corrected from contribution of fluorescence intensity of the cell background. Next, the surface of 60 single cells was measured by considering the cell shape as the boundary of labelling of cytosolic -tubulin. Then, the surface area of the microtubule network in individual cells was measured by systematically executing Otsu’s method via the plugin ‘Otsu threshold’ of ImageJ. This algorithm is an implementation of the Otsu thresholding technique

54,55

. The histogram of pixel

intensities is divided into two classes and the inter-class variance is minimized. This plugin outputs a binary image of MT and the ratios of MT on ROI surfaces are then calculated as percentages.

Tubulin Purification and microtubule assembly Lamb brain tubulin was purified from brain soluble extract by modified Weisenberg procedure consisting in ammonium sulfate fractionation and ion-exchange chromatography 56,57. Pure protein was stored in liquid nitrogen and prepared as described for used in Barbier et al 58. Protein concentrations were determined spectrophotometrically at 275 nm with a Perkin-Elmer spectrophotometer Lambda 800 with an extinction coefficient of 1.07 L.g-1.cm-1 in 0.5 % SDS in neutral aqueous buffer or with an extinction coefficient of 1.09 L.g-1.cm-1 in 6 M guanidine hydrochloride. Microtubule assembly was performed in PG buffer (20 mM sodium phosphate buffer, 1 mM EGTA, 10 mM MgCl2, 3.4 M glycerol and 0.1 mM GTP, pH 6.7). The reaction was started by

44

warming the samples at 37°C and the mass of polymer was monitored by turbidimetry at 350 nm with a POLARstar BMG Labtech spectrophotometer using 96-well plate. Samples containing compounds and their controls had less than 2 % residual DMSO. For all concentrations of compounds (from 0 to 10 µM), the percentage of polymerization inhibition was calculated as following: % polymerization inhibition = 100 – [A350 nm{microtubules with drug}  100 / A350 nm{microtubules

without drug}]. Three independent experiments were performed. Graphs in

Figure 6 D – F show all collected values until 100% polymerization inhibition. The values of mole ratio of 50% inhibition of tubulin assembly was determined from the equation of linear regression lines.

Statistical analysis Data are presented as mean ± SD. For immunofluorescence microscopy of the microtubule network and calculations of mitosis indexes, the statistical significances of data was analyzed using a parametric Student’s t test. Reported p-values are two-sided and *P < 0.001 was considered statistically significant. For cell cycle analyses, the statistical significances of data was analyzed using a non-parametric Mann-Whitney test. Asterisks indicate significant level vs control *P < 0.05. All statistical analyses were performed using Excel Microsoft software.

45

ASSOCIATED CONTENT Supporting Information available: The synthesis and the characterization of all tested compounds. AUTHOR INFORMATION Corresponding

Authors:

[email protected]

(for

the

biological

part);

[email protected] (for the chemical part). ACKNOWLEDGEMENTS PM is grateful to the University Science, Humanities and Engineering Partnerships in Africa Fellowship for funding. The University of Cape Town, South African Medical Research Council, and South African Research Chairs initiative of the Department of Science and Technology administered through the South African National Research Foundation are gratefully acknowledged for support (KC). We thank Charles Prévot (INSERM UMR_S 911 CRO2), Stephane Robert (AMUTICYT), engineers of the flow cytometry and cell sorting platform, for measurements of cell cycle progression. We also acknowledge the financial support of AixMarseille Université, INCa, INSERM and DGOS (SIRIC label). The authors declare that they have no conflict of interest. NON-STANDARD ABBREVIATIONS A549: adenocarcinome human alveolar basal epithelial cells; FBS: fetal bovine serum; FITC: fluorescein

isothiocyanate; HT29D4:

human colonic

adenocarcinoma;

MTT:

3-(4,5-

diMethylThiazol-2-yl)-2,5diphenyl Tetrazolium bromide; NA: numerical aperture; SD: standard deviation.

46

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Potent anti-proliferative activities against different cancer cells

4-arylcoumarin

Inhibits microtubule formation

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