Coumarin derivatives as potential antitumor agents: Growth inhibition, apoptosis induction and multidrug resistance reverting activity

Accepted Manuscript Coumarin derivatives as potential antitumor agents: Growth inhibition, apoptosis induction and multidrug resistance reverting activity Alessandra Bisi, Concettina Cappadone, Angela Rampa, Giovanna Farruggia, Azzurra Sargenti, Federica Belluti, Rita M.C. Di Martino, Emil Malucelli, Alessia Meluzzi, Stefano Iotti, Silvia Gobbi PII:

S0223-5234(17)30028-4

DOI:

10.1016/j.ejmech.2017.01.020

Reference:

EJMECH 9171

To appear in:

European Journal of Medicinal Chemistry

Received Date: 9 November 2016 Revised Date:

11 January 2017

Accepted Date: 12 January 2017

Please cite this article as: A. Bisi, C. Cappadone, A. Rampa, G. Farruggia, A. Sargenti, F. Belluti, R.M.C. Di Martino, E. Malucelli, A. Meluzzi, S. Iotti, S. Gobbi, Coumarin derivatives as potential antitumor agents: Growth inhibition, apoptosis induction and multidrug resistance reverting activity, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.01.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Graphical abstract

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Previously reported series

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Coumarin Derivatives as Potential Antitumor Agents: Growth Inhibition, Apoptosis Induction and Multidrug Resistance Reverting Activity Alessandra Bisi,*a Concettina Cappadone,*b Angela Rampa,a Giovanna Farruggia,b,c Azzurra

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Sargenti,b Federica Belluti,a Rita M.C. Di Martino,a,1 Emil Malucelli,b Alessia Meluzzi,b Stefano Iotti,b,c Silvia Gobbia

Department of Pharmacy and Biotechnology, Alma Mater Studiorum University of Bologna aVia

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Belmeloro, 6, 40126 and bVia S. Donato, 19/2, 40127, Bologna, Italy; cNational Institute of

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Biostructures and Biosystems, Via delle Medaglie D’oro, 305, 00136, Roma, Italy.

*Corresponding authors:

*Phone: +39-051-2099710. Fax: +39-051-2099734. E-mail: [email protected] *Phone: +39-051-2095626. Fax: +39-051-2095626. E-mail: [email protected] 1

Rita M. C. Di Martino present address: Istituto Italiano di Tecnologia (IIT), Via Morego, 30,

16163 Genova, Italy

ACCEPTED MANUSCRIPT Abstract A small library of coumarins, carrying butynyl-amino chains, was synthesized continuing our studies in the field of MDR reverting agents and in order to obtain multipotent agents to combat malignancies. In particular, the reported anticancer and chemopreventive natural product 7-

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isopentenyloxycoumarin was linked to different terminal amines, selected on the basis of our previously reported results. The anticancer behavior and the MDR reverting ability of the new compounds were evaluated on human colon cancer cells, particularly prone to develop the MDR

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phenotype. Some of the new derivatives showed promising effects, directly acting as cytotoxic

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compounds and/or counteracting MDR phenomenon. Compound 1e emerged as the most interesting of this series, showing a multipotent biological profile and suggesting that conjugation of an appropriate coumarin core with a properly selected butynyl-amino chain allows to obtain novel

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hybrid molecules endowed with improved in vitro antitumor activity.

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Keywords: P-glycoprotein; anticancer; coumarin; MDR modulators; chemosensitizers

Abbreviations used

ABC, ATP binding cassette; P-gp, P-glycoprotein; MDR, Multidrug resistance; MRP-1, Multidrug resistance associated protein-1; BCRP, Breast cancer resistance protein; CS, Collateral sensitivity; 7-IP, 7-isopenthenyloxy coumarin; DEAD, Diethyl azodicarboxylate; Dx, Doxorubicine

ACCEPTED MANUSCRIPT 1. Introduction Oxygenated heterocyclic compounds represent an important class of natural products endowed with several biological activities [1]. Among them, coumarins (2H-1-benzopiran-2-one derivatives) are a large class of compounds found as naturally produced secondary metabolites in several plant

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families, mainly Rutaceae and Umbelliferae. Beside the natural occurring coumarins, thousands of synthetic derivatives have been reported [2], mainly due to easily affordable synthetic procedures and straightforward elucidation of their chemical structures. The interest of medicinal chemists in

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this class of compounds is closely related to the wide array of biological activities that they display

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[3]. In particular, coumarin itself, but mostly its metabolite 7-hydroxycoumarin and derivatives, exhibit antitumor activity against several human tumor cell lines and hold promise as potential inhibitors of cellular proliferation [4]. In addition, both natural and synthetic coumarins have also been studied as inhibitors of P-glycoprotein (P-gp), the best known member of ATP-binding cassette (ABC) transporters involved in cancer multi-drug resistance (MDR), albeit in this respect

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mixed results have been observed [5].

MDR represents a challenging clinical problem for cancer chemotherapy and several mechanisms

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contribute to its onset, among which the increased ATP-dependent efflux of hydrophobic antitumor drugs from cells is the most investigated. The overexpression of membrane efflux pumps is indeed a

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well characterized mechanism contributing to MDR, being P-gp, multidrug resistance associated protein-1 (MRP) and breast cancer resistance protein (BCRP) the most extensively characterized MDR transporters [6,7]. In humans, these ABC pumps are constitutively expressed in most barrier organs, such as intestine, blood brain barrier, liver, kidneys, placenta, where they play a crucial role in detoxifying cells from both normally produced metabolites and exogenous toxic agents, including structurally unrelated chemotherapy drugs [8,9]. The co-administration of the anti-cancer drug with an agent capable of inhibiting its efflux from the cell, through the inhibition of these efflux pumps (MDR reverting agent), currently represents the most investigated strategy to

ACCEPTED MANUSCRIPT overcome ABC-mediated drug resistance. At present, three generations of reverting agents have been reported [10], among which Tariquidar (XR9576) [11] and Zosuquidar (LY335979) [12] (Figure 1) entered clinical trials, unfortunately without much success. Indeed, this approach often produces unpredictable or undesirable pharmacokinetics interactions and lacks effectiveness due to

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the complexity of the MDR phenomenon. An alternative strategy to reverse clinical MDR could be the identification of small molecules able to selectively destroy the MDR cells with respect to the non-resistant parental cells from which they are derived. This particular behaviour has been

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described as “collateral sensitivity” (CS) and compounds endowed with this ability could be useful in resensitizing MDR tumors towards antitumor drugs, as well as in preventing MDR through co-

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administration in standard chemotherapeutic treatments [13].

In previous papers [14,15] we described the design, synthesis and in vitro antitumor profile of phenothiazine and polycyclic derivatives bearing a common but-2-ynyl amino side chain (Figure 2). These studies revealed for some of these compounds a broad range of cellular activities, in

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particular the ability to induce antiproliferative effects and apoptosis in resistant tumor cell lines, to exert an appreciable MDR reverting effect and to be endowed with CS. These cellular properties were related to the presence of the nitrogen-containing butinyl side chain, and in particular the

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terminal amine seemed to be a key feature in promoting specific antitumor activity. In continuing

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our studies in this field, in order to broaden the biological profile of our compounds and obtain hybrid multipotent agents, able to reverse MDR and endowed with intrinsic cytotoxicity, a series of butinyl-amino derivatives bearing a 7-isopenthenyloxycoumarin (7-IP) core was designed and synthesized (Figure 2). 7-IP, first isolated in 1966 from the fruit of Libanotis Intermedia and widely found in edible vegetables and fruits [16], was selected due to its reported anticancer and chemopreventive activity [17,18], and as a potential coumarin-based additional MDR reverting moiety. The different terminal amines inserted on the rigid side chain were selected on the basis of previously obtained results. In particular, the N-methylhomoveratrilamine moiety (compound 1a, Table 1), the same amine found in the structure of verapamil, a first generation chemosensitizer still

ACCEPTED MANUSCRIPT used as reference standard in most biological assays, was maintained, as it gave the most interesting results in previous series of compounds. 6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline (1b), 1(piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one (1c) and 1-(bis(4-fluorophenyl)methyl)piperazine (1d) were also introduced as functional groups of the well-known chemosensitizers tariquidar,

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pimozide [19] and flunarizine [20], respectively (Figure 1). In this framework, compounds bearing 1-(9H-fluoren-9-yl)piperazine (1e) and benzyl piperazine (1f) could be viewed, in turn, as a rigid analogue and a simpler flexible derivative of 1d. Further structural simplifications of the benzyl

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piperazine amino moiety of 1f led to derivatives 1g, carrying a phenyl piperazine substituent, which also gave stimulating results in the phenothiazine series [14], and 1h, bearing a plain and less

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lipophilic piperidine moiety. Notably, the new compounds possess the limited common features reported as structural requirements for P-gp ligands [10], namely a protonable nitrogen, aromatic rings, overall high lipophilicity, but also groups able to establish H-bonds and weak polar interactions. Moreover, the compounds were prepared via Mannich reaction, and Mannich bases as

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anticancer and cytotoxic agents have been recently reviewed [21]. The antitumor profile and the MDR reverting ability of the new compounds was then evaluated on human colon cancer cells, being this tumor particularly prone to develop the MDR phenotype. In this respect, in order to

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definitively assess the role of the side chain purposely introduced in these new derivatives, the

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synthetic intermediate 2 (Scheme 1), without the Mannich base, and 7-IP itself were also tested.

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Figure 1. Representative MDR reversal agents.

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2. Chemistry

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Figure 2. Design strategy for compounds 1a-h.

All the synthesized compounds, collected in Table 1, were prepared starting from (E)-7-((3-

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methylpent-2-en-4-yn-1-yl)oxy)-2H-chromen-2-one (2, Scheme 1), that was obtained applying the Mitsunobu reaction to 7-hydroxycoumarin and (E) 3-methylpent-2-en-4-yn-1-ol, as previously

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described [22]. According to Scheme 1, the final compounds 1a-h were synthesized in parallel by using a Carousel work-station via a readily accessible Mannich reaction, refluxing 2 with the selected amine, formaldehyde and CuSO4 in ethanol/water.

ACCEPTED MANUSCRIPT Scheme 1. Synthesis of Compounds 1a-ha

Reagents and conditions: (i) (E) 3-methylpent-2-en-4-yn-1-ol, PPh3, DEAD; (ii) selected amine,

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formaldehyde, CuSO4, EtOH/H2O, reflux.

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Table 1. Structures and Antiproliferative Effects Against LoVo and LoVo/Dx Cells. for the Studied

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Compounds.

1a-h

2

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LoVo/Dx

IC50 (µM)a

IC50 (µM)a

40 ± 8.3

15.4 ± 4

10.7 ± 1.1

10.6 ± 0.6

2.5 ± 0.1

5 ± 0.19

1d

47 ± 4.2

60 ± 18

1e

3.5 ± 1.1

1.8 ± 0.42

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Compound

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1a

1b

1c

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NH

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67.6 ± 16.8

19.6 ± 7

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41 ± 7.3

12 ± 4.08

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62 ± 2.8

33 ± 2.7

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48 ± 16.8

71.5 ± 15

7-IP

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30 ± 5.3

50.3±16

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The IC50 was determined from the dose-response curve by MTT assay. Each value

3. Results and discussion

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represents the mean from triplicate determination ± SD.

3.1 Effect of growth inhibition of colon cancer cells

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The antiproliferative effects of coumarin derivatives were evaluated on LoVo colon cancer cell line and on its multidrug resistant subline LoVo/Dx, obtained by prolonged exposure to doxorubicin [23]. It is well known that the diminished sensitivity of cells to the original drug leads to the onset

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of a cross-resistance to other unrelated drugs, falling in the MDR complex phenomenon. Moreover,

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differences in energy metabolism between sensitive and resistant cells have been described. In particular, the increase of energy-yielding pathways in LoVo/Dx correlates with higher levels of ATP with respect to LoVo cells [24,25] and this feature can be ascribed to a higher activity of ABC pumps, whose overexpression characterizes MDR cells. The resistance to doxorubicin has been verified by MTT assay: the IC50 was 50 ± 15 µM for LoVo/Dx versus 1 ± 0.2 µM for LoVo cells. The in vitro IC50 growth inhibitory concentration was determined for each coumarin-based derivative, incubating the cells with increasing concentrations of the compounds (0.01-100 µM) for 24 hours. The obtained results show that 7-IP, the synthetic intermediate 2 and some of the new compounds exhibit a moderate activity in sensitive cells (Table 1). On the other hand, three

ACCEPTED MANUSCRIPT derivatives were able to reduce the number of viable cells at very low concentrations (≤ 10 µM), being 1c the most active (IC50 = 2.5 µM), followed by 1e (IC50 = 3.5 µM) and 1b (IC50 = 10.7 µM). In LoVo/Dx cells, all the new compounds proved to be more effective than 2 and 7-IP, with the sole exception of 1d. Interestingly, 1b, 1c and 1e were again the most potent derivatives. Moreover, a

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general increase in inhibitory activity with respect to sensitive cells was observed for most of the new derivatives, which proved to be endowed with CS. The ability of these agents to selectively hit resistant cells could be seen as an additional feature to overcome MDR in cancer cells. This

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behaviour is of particular interest for compound 1e, that proved to be twice as active (IC50 = 1.8 µM) when compared to sensitive cells, and the most cytostatic of the whole series in LoVo/Dx. On

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the contrary, 1b maintained the same activity (IC50 = 10.6 µM), suggesting a mechanism of action independent from the overexpression of efflux pumps. Finally, 1c acted at double concentration (IC50= 5 µM) on resistant cells.

It is to note that compound 1a, bearing the dimethoxyphenylethyl-N-methylamino side chain of

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verapamil, did not show appreciable cytotoxicity, in contrast to what was observed in previously reported series [14,15]. This could indicate an interaction between the coumarin core and the side

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chain in modulating cytotoxic effects in this series of compounds. 3.2 Effects on cell cycle

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The interest in coumarins as anticancer agents arose from reports that these compounds had achieved objective responses in some patients with advanced malignancies [26]. To further elucidate the antitumor properties of our most active coumarin derivatives on colon cancer, their effects on cell cycle progression were evaluated by flow cytometry. LoVo and LoVo/Dx cells were treated with 1b, 1c and 1e at IC50 concentration and its double, to better appreciate any cytotoxic effect.

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A

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Figure 3. Effects of 1b, 1c, and 1e on cell cycle distribution in LoVo (A) and Lovo/Dx cells (B).

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The cells were treated with the indicated concentration of the compounds for 24 h.

As shown in Figure 3, coumarin derivatives induced significant changes on cell percentage distribution in the different phases of the cell cycle. In particular, in LoVo sensitive cells (Figure 3A), 1b determined G0/G1 arrest, barely visible at the IC50 dose. The antiproliferative activity of 1c

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was accompanied by a block in G0/G1, which switched to a G2/M arrest at higher concentration. Finally, 1e always induced a consistent increase of cells in G2/M phase.

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Cell cycle analysis of treated LoVo/Dx cells, at the correspondent IC50s and double concentrations, are depicted in Figure 3B. At both tested concentrations, 1b induced a significant G0/G1 arrest

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while 1c caused a block in S phase. Antiproliferative effects of compound 1e were accompanied by an arrest in G0/G1 phase at the lower concentration and by a G2/M block at the higher concentration.

Considering these results, it could be noticed that 1b induced a dose-dependent increase of cell population in G0/G1 both in sensitive and in resistant cells. This finding is in line with the growth inhibition observed via MTT test, suggesting that the activity of this compound is not affected by the MDR phenotype. A completely different behaviour was observed for 1c, for which a significant difference could be seen between the cell types. While this compound acted as doxorubicine and

ACCEPTED MANUSCRIPT other antitumor drugs, having a dose-dependent effect on cell cycle in sensitive cells [15,27], its effect on resistant cells is a dose-independent S phase accumulation. On the other hand, compound 1e induced a massive and dose independent block in G2/M on LoVo cells, which is maintained only

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at higher concentration on LoVo/Dx cells.

3.3 Effects on cell death

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To discriminate whether the growth inhibitory activity of a given compound was due to cytostatic or cytotoxic effects, we performed a morphological analysis by using phase contrast microscopy.

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The cytostatic effect is defined as growth delay without observing cell death; on the contrary, cytotoxicity is characterized by massive cell death [28].

+ 1b

+ 1e

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+ 1c

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LoVo/Dx

LoVo

CTRL

Figure 4. Morphological changes induced by compounds 1b, 1c and 1e on the growth of LoVo (upper panel) and LoVo/Dx cells (lower panel).

The treatment of both sensitive and resistant LoVo cells with the most effective compounds 1b, 1c and 1e induced a reduction of cell number in all samples (cytostatic effects). Moreover, in both cell types, cytotoxic effects were observed: the presence of several floating cells at 24 hours of

ACCEPTED MANUSCRIPT treatment was more pronounced for 1e, quite visible for 1c and just significant for 1b treated cells (Figure 4). To determine whether coumarin derivatives caused apoptotic cell death, the activity of caspase 3 was assayed by a fluorimetric method. In cells treated with the compounds at cytotoxic

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concentration, the activity of effector caspases was significantly increased with respect to the controls (Figure 5). In particular, an increase of 39% for derivative 1b, 19% for 1c and 72% for 1e,

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respectively, were observed in LoVo sensitive cells.

Figure 5. Activation of caspase protease. Caspase acting on the peptide sequence DEVD

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(DEVDase activity) was measured in LoVo (black bars) and LoVo/Dx (gray bars) cells after 24 h of treatment with 1b, 1c, and 1e. Bars indicate the percentage increase in activity of treated cells compared to the control arbitrarily taken as 100%. Data are reported as mean ± SD determined from at least three independent experiments.

The three tested coumarins were able to induce caspase activation also in the resistant phenotype, but in this case 1b became less active than 1c, whereas 1e appeared very effective, with a three-fold increase in caspase activity with respect to LoVo sensitive cells. Remarkably, compound 1e,

ACCEPTED MANUSCRIPT endowed with CS, caused a strikingly higher increase in caspase activity in resistant cells with respect to sensitive ones.

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3.4 MDR reverting activity The reverting activity of the synthesized coumarins was evaluated in P-gp over-expressing human colon cancer cells LoVo/Dx. A flow cytometry test based on the measurement of intracellular

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rhodamine 123 (fluorescent substrate analogue for P-gp) accumulation was performed.

The results, collected in Figure 6, showed a wide range of reverting activities for different

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derivatives. Notably, all compounds, with the only exception of the synthetic intermediate 2, lacking the Manich base, proved to be more active than verapamil at 2.5 µM, confirming our working hypothesis. In particular, the 7-IP fragment, starting point of our hybrid molecules, while being as active as verapamil when tested at 2.5 µM, proved to be significantly less potent than most

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of the new derivatives. The mean fluorescence of the cell population was higher after treatment with 1a, 1b, 1c, 1d and 1e at 2.5 µM, compared to a positive control represented by cells treated with verapamil, a well-known P-gp substrate. This revertant effect was significantly increased at 10 µM,

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with the only exception of 1e, for which no substantial difference was observed. This could be explained by an early activation of the apoptotic pathway: although rhodamine efflux was evaluated

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at a very short time (75 minutes), it is to note that a concentration of 10 µM is almost three-fold higher than the cytotoxic one for compound 1e. An efflux of rhodamine comparable to that of the positive control verapamil was observed after treatment with 1f, 1g and 1h.

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Figure 6. Effect of coumarin derivatives on reversal of multidrug resistance in LoVo/Dx. Bars indicate the fluorescence intensity of treated cells with respect to the negative control taken arbitrarily as unit. Data are reported as mean ± SD determined from at least three independent

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experiments. * denotes significant statistical difference (p<0.05) with respect to verapamil treated cells.

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These data once again assess the crucial role of the amino side chain of verapamil, inserted on

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appropriately selected cores, in conferring MDR reverting abilities, as reported in our previous papers for molecules bearing different scaffolds [14,15]. Even if unfortunately this subset of molecules do not allow to draw clear structure activity relationships, some interesting information could be gained. 1a proved to be the most active compound of the series already at the lower tested dose, with a ten-fold increase with respect to verapamil. Generally speaking, the insertion of bulky amino groups, as in 1b, 1c, 1d and 1e, seemed to be favorable for MDR reverting effects. Indeed, going from 1d to 1f, lacking one of the aromatic rings, an appreciable decrease in activity could be seen, whereas a reduction in flexibility as in compound 1g did not lead to significant variations with respect to 1f.

ACCEPTED MANUSCRIPT Compounds that interact with P-gp are classified into three groups: substrates, modulators and inhibitors. Substrates are actively transported by the pump, while modulators interact at the binding sites reducing substrate binding through a negative allosteric interaction. Specifically, modulators alter substrate binding in a noncompetitive manner, reducing the maximal receptor density for

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substrate binding, without affecting the dissociation equilibrium constant (Kd). Inhibitors interfere with the substrates or ATP binding steps, blocking P-gp activity and therefore determining drug release [29]. In order to define whether the revertant compounds are substrates or

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modulators/inhibitors, we investigated the consumption of ATP after treatment. The obtained data (Figure 7) demonstrated that the revertant agents 1a and 1b are not substrates of efflux pumps, as

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they did not induce ATP consumption. On the contrary, compounds 1c, 1d and 1e certainly bind to

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substrate site, like verapamil, as after treatment the cellular ATP content is lower than the control.

Figure 7. ATP content of LoVo/Dx after treatment with10 µM coumarin derivatives for 2 h.

4. Conclusions

ACCEPTED MANUSCRIPT A series of 7-IP-based butynyl-amino derivatives was synthetized and tested to evaluate antitumor and MDR reverting activity. Generally speaking, the presented data demonstrated that the 7-IP coumarin core conferred to all newly synthesized molecules a very high capacity to reverse induced MDR in colon carcinoma cells. A potent MDR reverting agent with no appreciable cytotoxicity was

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obtained (1a), possessing the same side chain seen in the reference compound verapamil, but showing a ten-fold increase in activity. This could suggest a possible role of 7-IP as a tool for (hemi)synthesis of novel and more effective MDR inhibitors. In view of the identification of

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multipotent agents for resistant cancers treatment, some of the new compounds were found to be endowed with promising effects, directly acting as cytotoxic compounds and counteracting MDR

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phenomenon. In particular, compounds 1b and 1c showed significant cytotoxicity in both sensitive and resistant cell lines, as well as a remarkable MDR reverting profile. Finally, compound 1e emerged as the most interesting of this series, being able to act at different levels as potential anticancer agent. Indeed, it proved to be highly cytotoxic and to affect cell viability with an

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appreciable selectivity toward the resistant cell line, causing a striking increase in caspase activity with respect to sensitive cells, proving to be at the same time a potent MDR modulating agent at the lower tested dose. The absence of a dose-dependent increase in the MDR effect, seen with other

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dose.

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derivatives, could be explained by an early activation of the apoptotic pathway at the higher tested

Altogether, the biological profile of the tested compounds indicates that conjugation of an appropriate coumarin core with different butinyl-amino chains yields novel hybrid molecules endowed with improved in vitro antitumor profile and collateral sensitivity. Indeed, molecular hybridization again proves to be an attractive strategy to develop novel therapeutics that can be used for the treatment of complex multifactorial diseases such as cancer.

5. Experimental part

ACCEPTED MANUSCRIPT 5.1 Chemistry. Starting materials, unless otherwise specified, were used as high grade commercial products. Solvents were of analytical grade. Reaction progress was followed by thin layer chromatography (TLC) on precoated silica gel plates (Merck Silica Gel 60 F254) and then visualized with a UV254

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lamplight. Chromatographic separations were performed on silica gel columns by flash method (Kieselgel 40, 0.040-0.063 mm, Merck). Melting points were determined in open glass capillaries, using a Büchi apparatus and are uncorrected. 1H NMR and

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C NMR spectra were recorded on a

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Varian Gemini spectrometer 400 MHz in CDCl3 solutions unless otherwise indicated, and chemical shifts (δ) were reported as parts per million (ppm) values relative to tetramethylsilane (TMS) as

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internal standard; coupling constants (J) are reported in Hertz (Hz). Standard abbreviations indicating spin multiplicities are given as follow: s (singlet), d (doublet), t (triplet), br (broad), q (quartet) or m (multiplet). Mass spectra were recorded on Waters ZQ 4000 apparatus operating in electrospray mode (ES). Chemical purities of tested compounds were determined by elemental

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analysis (C, H, N) and were within ± 0.4 % of the theoretical values.

Compounds were named relying on the naming algorithm developed by CambridgeSoft

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Corporation and used in Chem-BioDraw Ultra 15.0.

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5.1.1. General parallel procedure for the synthesis of the Mannich bases (1a-h) A suspension of formaldehyde (0.21 mL, 2 mmol), the selected amine (2 mmol), and CuSO4 (0.05 g) was added to distinct reactors containing a water/ethanol 1:1 solution (10 mL) of (E)-7-((3methylpent-2-en-4-yn-1-yl)oxy)-2H-chromen-2-one (2) (0.48 g, 2 mmol), prepared as previously described [22], and the mixture was heated to reflux for 24 h. After cooling, ammonium hydroxide solution (15 mL) was added and the mixture was extracted with diethylether (3 x 20 mL), the organic layers were dried over anhydrous Na2SO4 and evaporated to give an oily crude, which was purified by flash column chromatography on silica gel using a suitable eluent and/or by

ACCEPTED MANUSCRIPT crystallization. The compounds were obtained with yields ranging from 30 to 65%. Where indicated, the oxalate salt was prepared dissolving the compound in ethanol and adding an equimolar amount of oxalic acid. The salts crystallized from ethanol and were collected by filtration. (E)-7-((6-((3,4-dimethoxyphenethyl)(methyl)amino)-3-methylhex-2-en-4-yn-1-yl)oxy)-2H-

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5.1.1.1.

chromen-2-one (1a). Purified by flash chromatography, eluent toluene/acetone 4:1. Mp 135-137 °C (oxalate) 1H NMR δ: 1.94 (s, 3H, CH3), 2.41 (s, 3H, N-CH3), 2.70-2.77 (m, 4H, N-CH2, CH2-Ph),

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3.55 (s, 2H, CH2-N), 3.86 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.67 (d, J = 6.8 Hz, 2H, OCH2-), 6.04 (t, J = 0.8 Hz, 1H, CH=), 6.27 (d, J = 9.6 Hz, 1H, CH= cum), 6.75-6.86 (m, 5H, arom), 7.38 (d, J = 13

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8.0 Hz, 1H, arom), 7.64 (d, J = 9.6 Hz, 1H, CH=cum).

C NMR δ: 18.2, 33.6, 41.8, 46.1, 55.8,

55.9, 57.6, 64.8, 74.0, 101.5, 111.2, 111.3, 112.0, 112.7, 113.1, 113.3, 120.5, 128.8, 130.1, 143.3, 148.2, 155.8, 161.1, 161.6, 163.5. ES-MS: m/z 448 (M + H). Anal. Calc. (C27H29NO5) C, 72.46; H,

5.1.1.2.

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6.53; N, 3.13. Found C, 72.49; H, 6.51; N, 3.14.

(E)-7-((6-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-3-methylhex-2-en-4-yn-1-

yl)oxy)-2H-chromen-2-one (1b). Purified by flash chromatography, eluent toluene/acetone 4:1. Mp

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159-162 °C (oxalate) 1H NMR δ: 1.93 (s, 3H, CH3), 2.83-2.87 (m, 4H, N-CH2 + CH2-Ph), 3.62 (s, 2H, CH2-N), 3.71 (s, 2H, N-CH2), 3.84 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 4.66 (d, J = 6.4 Hz, 2H,

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OCH2-), 6.04 (t, J = 0.8 Hz, 1H, CH=), 6.26 (d, J = 9.2 Hz, 1H, CH= cum), 6.55 (s, 1H, arom), 6.60 (s, 1H, arom), 6.80-6.85 (m, 2H, arom), 7.37 (d, J = 8.4 Hz, 1H, arom), 7.63 (d, J = 9.8 Hz, 1H, CH= cum). 13C NMR δ: 18.2, 29.7, 47.4, 49.9, 54.0, 55.9, 64.8, 83.7, 87.0, 101.5, 109.4, 111.3, 112.7, 113.1, 113.2, 123.0, 125.6, 126.2, 128.8, 130.1, 143.4, 147.2, 147.5, 155.8, 161.2, 161.6. ESMS: m/z 446 (M + H) Anal. Calc. (C27H27NO5) C, 72.79; H, 6.11; N, 3.14. Found C, 73.01; H, 6.13; N, 3.15 5.1.1.3. (E)-1-(1-(4-methyl-6-((2-oxo-2H-chromen-7-yl)oxy)hex-4-en-2-yn-1-yl)piperidin-4-yl)-1,3dihydro-2H-benzo[d]imidazol-2-one (1c). Purified by flash chromatography, eluent toluene/ethyl

ACCEPTED MANUSCRIPT acetate 7:3. Mp 134-137 °C (oxalate) 1H NMR δ: 1.86-1.89 (m, 2H, pip), 1.96 (s, 3H, CH3), 2.452.51 (m, 4H, pip), 3.07-3.10 (m, 2H, pip), 3.50 (s, 2H, CH2-N), 4.37-4.40 (m, 1H, CH pip-N), 4.69 (d, J = 6.4 Hz, 2H, OCH2-), 6.06 (t, J = 0.8 Hz, 1H, CH=), 6.27 (d, J = 9.6 Hz, 1H, CH= cum), 6.82-6.88 (m, 2H, arom), 7.04-7.10 (m, 3H, arom), 7.29-7.31 (m, 1H, arom), 7.38 (d, J = 8.8 Hz, 13

C NMR δ: 18.2, 29.13, 47.7,

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1H, arom), 7.64 (d, J = 9.6 Hz, 1H, CH= cum), 8.97 (s, 1H, NH).

50.3, 52.1, 64.8, 83.6, 87.1, 101.6, 109.5, 109.8, 112.7, 113.1, 113.2, 121.1, 122.9, 127.7, 128.8, 129.1, 130.3, 143.3, 154.6, 155.8, 161.1, 161.7. ES-MS: m/z 470 (M + H), 492 (M + Na). Anal.

(E)-7-((6-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-3-methylhex-2-en-4-yn-1-yl)oxy)-

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5.1.1.3.

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Calc. (C28H27N3O4) C, 71.62; H, 5.80; N, 8.95. Found C, 71.38; H, 5.79; N, 8.92.

2H-chromen-2-one (1d). Purified by flash chromatography, eluent toluene/ethyl acetate 4:1. Mp 160-161 °C (oxalate) 1H NMR δ: 1.92 (s, 3H, CH3), 2.15-2.21 (m, 4H, pip), 2.45-2.51 (m, 4H, pip), 3.15 (s, 2H, CH2-N), 3.94 (s, 1H, CH), 4.45 (d, J = 6.4 Hz, 2H, OCH2), 5.84 (t, J = 0.8 Hz, 1H, CH=), 6.17 (d, J = 9.6 Hz, 1H, CH= cum), 6.82-6.88 (m, 2H, arom), 7.02-7.10 (m, 3H, arom),

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7.19-7.21 (m, 1H, arom), 7.38-7.45 (m, 5H, arom), 7.63 (d, J = 9.6 Hz, 1H, CH= cum). 13C NMR δ: 18.15, 47.63, 48.65, 52.87, 64.86, 69.89, 84.39, 86.68, 101.61, 112.69, 113.05, 113.23, 116.03, 116.92, 119.62, 123.11, 129.01, 129.75, 129.94, 140.28, 140.96, 155.81, 160.40, 161.09, 161.64

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ES-MS: m/z 541 (M + H), 563 (M + Na). Anal. Calc. (C33H30 F2N2O3) C, 73.32; H, 5.59; N, 5.18.

5.1.1.4.

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Found C, 73.47; H, 5.61; N, 5.16

(E)-7-((6-(4-(9H-fluoren-9-yl)piperazin-1-yl)-3-methylhex-2-en-4-yn-1-yl)oxy)-2H-

chromen-2-one (1e). Purified by flash chromatography, eluent toluene/ethyl acetate 7:3. Mp 144145 °C (oxalate) 1H NMR δ: 1.92 (s, 3H, CH3), 2.55-2.61 (m, 4H, pip), 2.70-2.73 (m, 4H, pip), 3.38 (s, 2H, CH2-N), 4.66 (d, J = 6.8 Hz, 2H, OCH2), 4.86 (s, 1H, CH), 6.02 (t, J = 0.8 Hz, 1H, CH=), 6.26 (d, J = 9.2 Hz, 1H, CH= cum), 6.80-6.86 (m, 2H, arom), 7.25-7.29 (m, 2H, arom), 7.35-7.38 (m, 3H, arom), 7.62-7.70 (m, 5H, arom + CH= cum). 13C NMR δ: 18.2, 47.6, 48.6, 52.9, 64.8, 69.8, 84.1, 86.7, 101.6, 112.7, 113.1, 113.2, 119.6, 123.1, 126.0, 126.9, 128.0, 128.8, 129.9, 141.0, 143.3,

ACCEPTED MANUSCRIPT 144.0, 155.8, 161.1, 161.6. ES-MS: m/z 503 (M + H). Anal. Calc. (C33H30N2O3) C, 78.86; H, 6.02; N, 5.57. Found C, 78.68; H, 6.04; N, 5.56. 5.1.1.5.

(E)-7-((6-(4-benzylpiperazin-1-yl)-3-methylhex-2-en-4-yn-1-yl)oxy)-2H-chromen-2-one

(1f). Purified by flash chromatography, eluent toluene/ethyl acetate 7:3. Mp 210-211 °C (oxalate) H NMR δ: 1.94 (s, 3H, CH3), 2.55-2.62 (m, 4H, pip), 2.69-2.75 (m, 4H, pip), 3.34 (s, 2H, CH2-Ph),

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3.58 (s, 2H, CH2-N), 4.67 (d, J = 6.8 Hz, 2H, OCH2), 6.03 (t, J = 0.8 Hz, 1H, CH=), 6.24 (d, J = 9.2 Hz, 1H, CH= cum), 6.78-6.84 (m, 2H, arom), 7.25-7.38 (m, 6H, arom), 7.64 (d, J = 9.6 Hz, 1H, 13

C NMR δ: 18.2, 47.6, 52.9, 64.4, 64.8, 73.0, 86.7, 101.6, 112.7, 113.1, 113.2, 119.6,

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CH= cum).

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126.3, 126.9, 127.1, 128.5, 130.1, 143.3, 144.0, 155.8, 161.1, 161.6. ES-MS: m/z 429 (M + H). Anal. Calc. (C27H28N2O3) C, 75.68; H, 6.59; N, 6.54. Found C, 75.91; H, 6.61; N, 6.55. 5.1.1.6.

(E)-7-((3-methyl-6-(4-phenylpiperazin-1-yl)hex-2-en-4-yn-1-yl)oxy)-2H-chromen-2-one

(1g). Purified by flash chromatography, eluent toluene/ethyl acetate 7:3. Mp 155-156 °C (oxalate) H NMR δ: 1.95 (s, 3H, CH3), 2.56-2.63 (m, 4H, pip), 2.70-2.75 (m, 4H, pip), 3.37 (s, 2H, CH2-N),

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4.65 (d, J = 6.8 Hz, 2H, OCH2), 6.01 (t, J = 0.8 Hz, 1H, CH=), 6.22 (d, J = 9.2 Hz, 1H, CH= cum), 6.68-6.85 (m, 4H, arom), 7.18-7.21 (m, 3H, arom), 7.37 (d, J = 8.8 Hz, 1H, arom), 7.66 (d, J = 9.6

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Hz, 1H, CH= cum). 13C NMR δ: 18.2, 47.6, 48.6, 52.9, 64.8, 70.0, 86.7, 101.6, 112.7, 113.1, 113.2, 119.6, 126.0, 126.9, 128.0, 128.8, 129.9, 143.3, 144.0, 156.0, 161.1, 161.6. ES-MS: m/z 415 (M +

5.1.1.7.

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H). Anal. Calc. (C26H26N2O3) C, 75.34; H, 6.32; N, 6.76. Found C, 75.48; H, 6.34; N, 6.74. (E)-7-((3-methyl-6-(piperidin-1-yl)hex-2-en-4-yn-1-yl)oxy)-2H-chromen-2-one

(1h).

Purified by flash chromatography, eluent toluene/acetone 4:1. Mp 122-124 °C (oxalate) 1H NMR δ: 1.62-1.66 (m, 6H, pip), 1.93 (s, 3H, CH3), 2.48-2.57 (m, 4H, pip), 3.38 (s, 2H, CH2-N), 4.67 (d, J = 6.8 Hz, 2H, OCH2), 6.02 (t, J = 0.8 Hz, 1H, CH=), 6.27 (d, J = 9.6 Hz, 1H, CH= cum), 6.80-6.86 (m, 2H, arom), 7.37 (d, J = 8.0 Hz, 1H, arom), 7.64 (d, J = 9.6 Hz, 1H, CH= cum).

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C NMR δ:

18.2, 23.8, 25.8, 48.2, 53.3, 64.8, 84.2, 86.7, 101.6, 112.7, 113.0, 113.1, 123.1, 128.8, 129.8, 143.3,

ACCEPTED MANUSCRIPT 155.8, 161.1, 161.6. ES-MS: m/z 338 (M + H). Anal. Calc. (C21H23NO3) C, 74.75; H, 6.87; N, 4.15. Found C, 74.52; H, 6.89; N, 4.14

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5.2. Biological Evaluation Methods 5.2.1. Cell culture and treatment

Human colorectal adenocarcinoma cell line LoVo and its resistant subline LoVo/Dx were used.

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L-glutamine, in humidified air at 37 °C with 5% CO2.

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Cells were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum and 2 mM

Compounds were dissolved in DMSO at 10 mmol/L and diluted with medium to obtain the desired concentration. The cells were plated at 2x104 cells/cm2 and treated for 24 h in triplicate. In control

5.2.2. MTT assay

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cells, only DMSO was added to the culture medium.

To evaluate coumarin derivatives activity, the cells were treated for 24 h without (control) or with

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concentrations between 0.01-100 µM of the test samples. To test the doxorubicin sensitivity, the

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cells were treated for 72 hours with doxorubicin concentrations between 0.01-250 µM. The culture medium was removed and cells were further incubated for 4 h with 0.2 mg/mL MTT in PBS. After removal of the medium, the cells were lysed with 0.1 mL of DMSO. The absorbance at 540 nm of the solubilized formazan pellet, (which reflects the relative viable cell number), was determined by microplate reader (Victor). From the dose-response curve, the 50% growth inhibition concentration (IC50) was determined by using Sigma Plot 10.0 software.

5.2.3. Flow cytometric analyses

ACCEPTED MANUSCRIPT 5.2.3.1 Cell cycle. To determine cell cycle distribution at the end of incubation LoVo cells were stained according to Andreani et al [30]. Briefly, 1x106 cells were pelleted and resuspended in trisodium citrate 0.1%, RNAse 0.1 mg/L, Igepal 0.01%, Propidium Iodide (PI) 50 µg/L. After 30 min at 37 °C in the dark, cells were analyzed on a Coulter Epics Elite flow cytometer (Beckman

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Coulter) equipped with a 15 mW argon ion laser tuned at 488 nm. PI fluorescence was collected on a linear scale at 600 nm and the DNA distribution, obtained after doublet discrimination, was

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analyzed by the Modifit 5.0 software.

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5.2.3.2. Efflux assay. The efflux of rhodamine 123 was performed according to Wieslowa et al [31]. In brief, the LoVo/Dx cells were resuspended at the concentrations of 3 x 105 cells/mL in the culture medium without serum and Phenol Red, and treated at room temperature with the different molecules for 15 min at two concentrations: 2.5 and 10 µM. Then, 2 µM of rhodamine 123 was

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added and the cells were further incubated at 37 °C in the dark for 1 h. After washing of the cells in PBS, the fluorescence of the cell population was measured on cytometer. Analysis were performed selecting the excitation band centered at 488 nm and the emission of probe was acquired on a

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logarithmic scale at 530 nm. Negative control cells were stained with rhodamine 123 without any pretreatment with the modulator, while positive control cells were pretreated with verapamil.

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Data were analyzed by the Winmdi software and the mean value of control cells fluorescence distribution was subtracted to the mean value of the distribution of the treated samples.

5.2.4. Caspase activity The enzymatic activity of caspases hydrolysing the peptide sequence Asp-Glu-Val-Asp (DEVD) is indicated as DEVDase activity, which was measured in cell extracts by a fluorimetric assay [32].

ACCEPTED MANUSCRIPT 5.2.5. Bioluminescence ATP assay These experiments were performed as reported in the technical sheet of the ATPlite 1-step Kit for luminescence ATP detection based on firefly (Photinus pyralis) luciferase (PerkinElmer Life

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Sciences) [33]. The ATPlite assay is based on the production of light caused by the reaction of ATP with added luciferase and d-luciferin (substrate solution), and the amount of light emitted is proportional to the

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ATP concentration. LoVo and LoVo/Dx cells were seeded into black CulturePlate 96-well plates in 100 µL of complete medium at a density of 2x104 cells per well. Plates were incubated for 24 h at

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37 °C. The medium was removed, and 100 µL of complete medium was added, in the presence or absence of test compounds (10 µM). Plates were incubated for 1 h at 37 °C. Mammalian cell lysis solution (50 µL) was then added to all wells, and the plates were agitated for 5 min on an orbital shaker. Substrate solution (50 µL) was added to all wells, and the plates were stirred for another 5

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min on an orbital shaker. Plates were dark adapted for 10 min, and luminescence was measured on a microplate reader Victor 2 (PerkinElmer Life Sciences).

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5.2.6. Statistical analysis

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All experiments were performed in triplicate. Data are described as means±SD and analyzed by the Student’s test. P-Values below 0.05 were considered statistically significant. 6. Supplementary data

Representative NMR spectra (compound 1e).

7. References

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Highlights

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►A small series of coumarin derivatives were designed and synthesized ►Antitumor profile and MDR reverting ability of the new compounds were evaluated ►Some derivatives act as cytotoxic compounds and counteract MDR phenomenon ►Compound 1e emerged as the most interesting, showing a multipotent biological profile ►Molecular hybridization appears an attractive strategy for multifactorial diseases