Antitumoral activity of 1,2-diaminocyclohexane derivatives in breast, colon and skin human cancer cells

31/1/2017 Anti-tumoral Activity of 1, 2‐Diaminocyclohexane Derivatives in Breast, Colon and Skin Human Cancer Cells http://powerxeditor.aptaracorp.com/fsg/Portal/Editor/Editor.aspx?assetid=MagMGzL2u%2fs%3d&roleid=TCw3dUKf1OA%3d&editstatus=1LHELv8bezw%3d 1/14 DOI: 10.4155/fmc-2016-0212 [Q uer y-Q 1: Karis hma Bhan( CE) to All(AU)] It is our hous e s ty le to italic iz e genes to differ entiate them from proteins . Can y ou pleas e c hec k and c onfir m that any genes in y our artic le ar e italic iz ed c orr ec tly ? Morales, Ramírez, Morata-Tarifa et al.[Q uer y- Q 2: Karis hma Bhan( CE) to All( AU) ] It is our hous e s ty le to leav e all proteins , genes and growth fac tors in their abbr ev iated form only . Pleas e c ould y ou ens ur e none of thes e hav e been defined ( inc luding the figures and tables of ar tic les )? Antitumoral activity of 1,2‐diaminocyclohexane derivatives in breast, colon & skin human cancer cells S h o r t C o m m u n i c a t i o n Antitumoral activity of 1,2‐diaminocyclohexane derivatives in breast, colon and skin human cancer cells[Q uer y-Q 3: Karis hma Bhan( CE) to All(AU)] Pleas e c hec k all author names , in partic ular y our c o-author’s names , and affiliation details are pres ented c orrec tly (inc luding in the r unning header at the top of the page) . We will not c hange thes e onc e publis hed in mos t c as es . Fátima Morales1, Alberto Ramírez2,3, Cynthia Morata-Tarifa2,4, Saúl A Navarro2,4, Juan A Marchal2,4, Joaquín M Campos‡,1,4 & Ana Conejo-García*,‡,1,4 1Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, Granada, Spain [Q uer y- Q 4: Karis hma Bhan( CE) to All( AU) ] Pleas e pr ov ide the pos tal c odes for affiliations 1,2 and 3. 2Instituto de Biopatología y Medicina Regenerativa (IBIMER), Departamento de Anatomía y Embriología Humana, Facultad de Medicina, Granada, Spain 3Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales y de la Salud, Jaén, Spain 4Instituto Biosanitario de Granada (ibs.GRANADA), Hospitales Universitarios de Granada-Universidad de Granada, 18071, Granada, Spain *Author for correspondence: [email protected] [Q uer y- Q 5: Karis hma Bhan( CE) to All(AU) ] Pleas e pr ov ide the telephone and fax number of the author for correspondence. ‡Authors contributed equally Abstract Aim: Cancer is among the leading causes of death worldwide. Medical interest has focused on macrocyclic polyamines because of their properties as antitumor agents. Results/Methodology: We have designed and synthesized a series of 1,2‐ diaminocyclohexane derivatives with notable in vitro antiproliferative activities against the MCF‐7, HCT‐116 and A375 cancer cell lines. Cell cycle and apoptosis analyses were also carried out. Our results show that all the compounds are potent cytotoxic agents, especially against the A375 cell line. Conclusion: The selective activity of the macrocyclic derivative against A375, via apoptosis, supposes a great advantage for future therapeutic use. This exemplifies the potential of 1,2‐diaminocyclohexane derivatives to qualify as lead structures for future anticancer drug development due to their easy syntheses and noteworthy bioactivity. Graphical abstract 31/1/2017 Anti-tumoral Activity of 1, 2‐Diaminocyclohexane Derivatives in Breast, Colon and Skin Human Cancer Cells http://powerxeditor.aptaracorp.com/fsg/Portal/Editor/Editor.aspx?assetid=MagMGzL2u%2fs%3d&roleid=TCw3dUKf1OA%3d&editstatus=1LHELv8bezw%3d 2/14 Keywords: 1, 2‐diaminocyclohexanes • antitumor • apoptosis • benzenesulfonamides • breast cancer • caspases • colon cancer • macrocycles • melanoma First draft submitted: 11 November 2016; Accepted for publication: 4 January 2017; Published online: TBC Cancer is among the leading causes of death worldwide, being responsible for more deaths than all coronary heart disease or stroke, accounting for 8.2 million deaths in 2012. It is expected that annual cancer cases will rise from 14million in 2012 to 22 within the next two decades. Thus, cancer continues to be a major health problem in developing, as well as undeveloped countries [1,2]. The MCF‐7, HCT‐116 and A375 cell lines are representative for breast, colon and melanoma tumors, respectively. Breast cancer is the main cancer in women both in the developed and the developing countries [3], with an estimated 1.7million new cases and 521,900deaths in 2012, representing 25% of all cancer diagnoses and 15% of all cancer deaths among females. MCF‐7 is a luminal epithelial-like cell line that expresses low levels of the enzyme phosphodiesterase type 5 and its phenotype is conserved in their derived tumors [4,5]. Colorectal cancer is a leading cause of cancer death in developed countries, being the third most commonly diagnosed cancer in men and the second in women, with an estimated 1.4million cases and 693,900deaths in 2012 [6]. Colon cancer is associated with a highfrequency variation of microsatellite repeats as the HCT‐116 cell line shows [7]. A cancer-specific hypermethylation event for the small nucleolar RNAs SNORD123, U70C and ACA59B has recently been found in this cell line [8]. The incidence of both nonmelanoma and melanoma skin cancers has been increasing over the past decades [9], malignant melanoma, being the one that accounts for approximately 75% of all deaths from skin cancer [10]. A375 is a human malignant melanoma cell line derived from skin biopsies that is highly tumorigenic and metastatic in animal models [11,12]. Medical interest has focused on macrocyclic polyamines in the last decades because of their chemical and biological properties as antitumor agents. Their antiproliferative activity is due to their affinity to the DNA [13]. 1,2‐diamines possess a wide range of bioactivities, such as the anticancer and antituberculosis ones [14]. The amine groups are useful for modulating the solubility of the drug, as well as for donating or accepting hydrogen bonds to and from a biological receptor [15]. Diamines (-)-trans-, (+)-trans- and meso‐1 (Figure 1) showed antiproliferative activity against the MCF‐7 cell line [16]. They induce growth inhibition with an IC50 of 0.4, 0.6 and 1.8µM, respectively. The most active compound (-)-trans‐1 shows greater cytotoxic activity toward the MCF‐7 cells than the normal MCF‐10A cells. Real-time RT-PCR analysis 31/1/2017 Anti-tumoral Activity of 1, 2‐Diaminocyclohexane Derivatives in Breast, Colon and Skin Human Cancer Cells http://powerxeditor.aptaracorp.com/fsg/Portal/Editor/Editor.aspx?assetid=MagMGzL2u%2fs%3d&roleid=TCw3dUKf1OA%3d&editstatus=1LHELv8bezw%3d 3/14 demonstrated that (-)-trans‐1 is an extremely efficient regulator of the antiapoptotic genes Bcl-xl, Bcl‐2 and cyclin D1. Later studies of the molecule confirmed that it also regulates p53 [16,17]. Figure 1. Diamines with antiproliferative activity against the MCF‐7 cell line. Data taken from [12]. [Q uery - Q 6: Karis hma Bhan( CE) to All( AU)] Pleas e c hec k and c onfir m that any figur es /tables that appear in the artic le hav e been r edrawn c orr ec tly , and if not, pleas e des c r ibe any c orrec tions that need to be made. Macrocyclic compounds represent a structural class with an exceptional potential for biological activity and specificity [18]. Nowadays, macrocycles are promising chemotypes, with more than 100 marketed macrocycle drugs that included antitumor drugs [19]. Benzenesulfonamides also show anticancer activity through a variety of mechanisms such as cellcycle perturbation on the G1 phase, the disruption of microtubule assembly or angiogenesis inhibition [20]. We have previously designed different molecules that target cancer (Figure 2). Compound 2 is one of the most potent human choline kinase inhibitor reported to date, showing activity in the low micromolar range (IC50=0.3µM) [21], with similar values to others recently published [22-24]. Macrocycle trans-bis(5‐FU O, N-acetal) (3) presents an antiproliferative activity of 5.5µM against the MCF‐7 cell line and provokes a G0/G1 cell cycle arrest in the MCF‐7 breast cancer cells [25]. The benzenesulfonamide-containing compound, named bozepinib (4), is a potent antitumor agent with an IC50 against the MCF‐7 cell line of 0.355µM [26] that induces apoptosis in breast and colon cancer cells. The doublestranded RNA-dependent protein kinase PKR is a target of bozepinib (4). Moreover, the combination with IFN-α potentiates the apoptosis induced by bozepinib (4) and also enhances autophagy and senescence, both processes of great importance in tumor cells that show resistance to conventional chemotherapy [27]. Bozepinib (4) also shows in vivo antitumor and antimetastatic efficacy in xenotransplanted nude mice without presenting subacute toxicity and a selective activity against cancer stem cells [28]. Figure 2. Anticancer agents previously reported by us. In this paper we present the synthesis and biological evaluation of a series of compounds (Figure 3) designed to study the influence of three structural modifications in the 1,2‐diaminocyclohexane derivatives (1): (A) transition of the phenol 31/1/2017 Anti-tumoral Activity of 1, 2‐Diaminocyclohexane Derivatives in Breast, Colon and Skin Human Cancer Cells http://powerxeditor.aptaracorp.com/fsg/Portal/Editor/Editor.aspx?assetid=MagMGzL2u%2fs%3d&roleid=TCw3dUKf1OA%3d&editstatus=1LHELv8bezw%3d 4/14 group to meta (m-) and para (p-) positions (5 and 6, Figure 3), (B) introduction of two p-nitrobenzenesulfonyl groups (7– 9, Figure 3) and (C) its conversion into a 14‐membered macrocycle with an ethylene bridge that links the two phenolic groups of 1 (10, Figure 3). [Q uer y -Q 7: Leigh Nugent(PE) to All(AU) ] What ar e A, B and C r eferr ing to in the pr ec eding s entenc e? Figure 3. Molecules synthesized and analyzed as antitumor agents. Materials & methods Chemistry Melting points were taken in open capillaries on an electrothermal melting point apparatus and are uncorrected. Analytical thin layer chromatography was performed using Merck Kieselgel 60 F254 aluminum sheets, the spots being developed with UV light (λ=254nm). All evaporation was carried out in vacuo with a Büchi rotary evaporator and the pressure controlled by a Vacuubrand CVCII apparatus. For flash chromatography, Merck silica gel 60 with a particle size of 0.040– 0.063mm (230–400 mesh ASTM) was used. [Q uer y -Q 8: Karis hma Bhan( CE) to All( AU)] Pleas e define the following term: ‘AST M.’ Purity of all compounds was determined by NMR studies, mass spectroscopy and elemental analysis. [Q uer y -Q 9: Karis hma Bhan(CE) to All(AU)] Pleas e define the following ter m: ‘NMR.’ Elemental analyses were performed on the Thermo Scientific Flash 2000 analyzer and the measured values indicated by the symbols of the elements or functions were within±0.4% of the theoretical values. Nuclear magnetic resonance spectra have been carried out at the Centro de Instrumentación Científica (CIC)/Universidad de Granada and recorded on a 300MHz 1H and 75MHz 13CNMR Varian Inova-TM spectrometers at ambient temperature. Chemical shifts (δ) are quoted in parts per million (ppm) and are referenced to the residual solvent peak. Signals are designated as follows: s, singlet; bs, broad singlet; d, doublet; dd, double doublet; ddd, double double doublet; t, triplet; m, multiplet. High-resolution nano-assisted laser desorption/ionization or electrospray ionization mass spectra were carried out on a Bruker Autoflex or a Waters LCT 31/1/2017 Anti-tumoral Activity of 1, 2‐Diaminocyclohexane Derivatives in Breast, Colon and Skin Human Cancer Cells http://powerxeditor.aptaracorp.com/fsg/Portal/Editor/Editor.aspx?assetid=MagMGzL2u%2fs%3d&roleid=TCw3dUKf1OA%3d&editstatus=1LHELv8bezw%3d 5/14 Premier Mass Spectrometer, respectively. Anhydrous CH2Cl2 was purchased from the VWR International Eurolab. Anhydrous conditions were performed under argon. All reagents were purchased from Aldrich. Compounds 1, 5–10 have been named according to IUPAC recommendations for phane structures [29,30]. Intermediate 11 was synthesized as previously described [31]. Target compounds meso‐1,5–9, (±)-trans‐1,5–9 and meso‐10 were synthesized according to the previously reported protocols [16,32,33] with the modifications described in the Supplementary Information. Biology Cell culture MCF‐7 (ECACC: 86012803) and HCT‐116 (ECACC: 91091005) provided by the Cell Bank of the University of Granada and A375 cells, a gift from Bosserhoff (Institute of Pathology, Regensburg University, Germany), were grown at 37°C in an atmosphere containing 5% CO2, with Dulbecco’s modified Eagle Medium (Gibco, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 2% L-glutamine, 2.7% sodium bicarbonate, 1% Hepes buffer, 40 mg/l gentamicin and 500mg/l ampicillin [34-36]. Drug treatment Compounds were dissolved in DMSO and stored at -20°C. For each experiment, the stock solutions were further diluted in medium to obtain the desired concentrations. The final solvent concentration in cell culture was≤0.1% v/v of DMSO, a concentration without any effect on cell replication. Parallel cultures of MCF‐7, HCT‐116 and A375 cells in medium with DMSO were used as controls. Biological assays The effect of the compounds on cell viability was assessed using the sulforhodamine-B (SRB) colorimetric assay. The assay procedure is explained in the supplementary information file, as well as the cell-cycle distribution analysis, the apoptosis detection by staining with annexin V-FITC and propidium iodide and the caspase assay. Statistical analyses All the quantitative data in the present study are reported as means±standard derivation from at least three independent experiments. Two-way ANOVA was used for grouped analysis of differences followed by Bonferroni post-tests. Results & discussion Chemistry meso- and (±)-trans‐1,5,6 were synthesized by reductive amination [16]. The yield of meso‐1 was improved to 100% by increasing the reaction time for the Schiff-base condensation from 2 to 48h. Sulfonylation of diamines meso- and (±)- trans‐1,5,6 with p-nitrobenzenesulfonyl chloride gave meso- and (±)-trans‐7,8,9 respectively (Figure 4). [Q uery - Q 10: Leigh Nugent( PE) to All( AU)] Pleas e ignore the s pac e below F igure 5; this will be rec tified in the final lay out. PXE is not r epr es entativ e of the final lay out, pleas e only us e it to rev iew the c ontent. 31/1/2017 Anti-tumoral Activity of 1, 2‐Diaminocyclohexane Derivatives in Breast, Colon and Skin Human Cancer Cells http://powerxeditor.aptaracorp.com/fsg/Portal/Editor/Editor.aspx?assetid=MagMGzL2u%2fs%3d&roleid=TCw3dUKf1OA%3d&editstatus=1LHELv8bezw%3d 6/14 Figure 4. Reagents and conditions. (A) Et3N/EtOH, rt, 48 h; NaBH4, rt, 4h [100% for meso‐1, 41% for (±)-trans‐1, 75% for meso‐5, 74% for (±)-trans‐5, 51% for meso‐6, 57% for (±)-trans‐6]; (B) p-NO2-Ph-SO2Cl, anhydrous CH2Cl2, rt, 4.5h [63% for meso‐7, 26% for (±)-trans‐7, 24% for meso‐8, 24% for (±)-trans‐8, 16% for meso‐9, 9% for (±)-trans‐9]. The first step in the synthesis of meso‐10 (Figure 5) was the reaction of 1,2‐dibromoethane with salicylaldehyde [31]. The reductive amination of 11 yield meso‐10 without further purification [32,33]. Figure 5. Reagents and conditions. (A) NaOH 2%/EtOH, reflux, 72 h (27%); (B) meso‐1,2‐diaminocyclohexane, CH3OH, reflux, 5h; NaBH4, 50°C, 3.5h (38%). Biology Compounds meso‐1, 5–9, (±)-trans‐1, 5–9 and meso‐10 were assayed for their in vitro antiproliferative activity against the MCF‐7, HCT‐116 and A375 cell lines. The antitumor drug oxaliplatin, that contains a diamine in its structure, was used as a control in the assays and their IC50 values are concurrent with the ones in literature [37]. The results are 31/1/2017 Anti-tumoral Activity of 1, 2‐Diaminocyclohexane Derivatives in Breast, Colon and Skin Human Cancer Cells http://powerxeditor.aptaracorp.com/fsg/Portal/Editor/Editor.aspx?assetid=MagMGzL2u%2fs%3d&roleid=TCw3dUKf1OA%3d&editstatus=1LHELv8bezw%3d 7/14 summarized in Table 1. All the compounds are potent antiproliferative agents with the highest potency against the A375 melanoma cancer cell line (submicromolar values of IC50 for compounds 1, 7–10). meso- and (±)-trans‐1 are the best antiproliferative agents in the three cell lines studied (MCF‐7, HCT‐116 and A375 (IC50=58nM for meso‐1 vs 2.5µM for oxaliplatin in MCF‐7 cells, IC50 =66 nM for (±)-trans‐1 vs 3.8µM for oxaliplatin in HCT‐116 cells). [Q uer y -Q 11: Leigh Nugent( PE) to All( AU) ] Wher e do the brac k ets befor e MCF - 7 and IC50 end in the pr ec eding s entenc e? We should highlight that the alteration of the phenol group from ortho (o-) to para (p-) position decreases the antiproliferative activity of compounds 5 (m-) and 6 (p-) substantially (from IC50=0.06–1.2µM for 1 to IC50=10.7–54.6µM for 5 and 6). However, there is not a difference between the molecules with the p-nitrobenzenesulfonyl group (7–9), where the antiproliferative activity is similar between o-, m- and pderivatives (IC50 = 0.94–6.3µM). The best values for antiproliferative activity of the pnitrobenzenesulfonyl derivatives are meso‐9 against HCT‐116 cell line (IC50=1.1µM) and (±)-trans‐9 against A375 cell line (IC50=94nM). meso‐10 shows similar values of antiproliferative activity as oxaliplatin against the MCF‐7 cell line (IC50=2.9, 2.5µM, respectively), being a better agent than oxaliplatin against HCT‐116 and A375 cell lines (IC50=1.5, 1.1µM against 3.8, 3.4µM, respectively). As a result, we should mention that the modification of the phenol groups in compounds 5 and 6, the introduction of the p-nitrobenzenesulfonyl group into meso- and (±)-trans‐1 to give meso- and (±)-trans‐7–9 as well as the macrocyclization to produce meso‐10, do not improve the antiproliferative activity in relation to those of meso- and (±)-trans‐1. Table 1. Antiproliferative activity for meso- and (±)-trans‐1,5–9, meso‐10 and oxaliplatin against the MCF‐7, HCT‐116 and A375 cell lines. Compound MCF‐7 IC50µM HCT‐116 IC50µM A375 IC50µM meso‐10.058±0.014 0.236±0.060 0.099±0.018 (±)-trans‐11.240±0.018 0.066±0.017 0.205±0.015 meso‐542.30±0.012 12.79±1.08 40.30±1.01 (±)-trans‐548,40±0.76 14.84±0.12 35.65±0.92 meso‐654.56±1.16 31.30±0.92 38.70±1.32 (±)-trans‐640.90±0.78 10.65±0.82 32.18±0.69 meso‐77.010±0.044 5.070±0.032 3.413±0.016 (±)-trans‐72.630±0.044 3.680±0.045 2.398±0.020 meso‐86.320±0.011 5.040±0.005 2.040±0.001 (±)-trans‐86.100±0.047 4.390±0.010 2.030±0.001 meso‐94.130±0.014 1.090±0.007 4.390±0.019 (±)-trans‐94.080±0.024 4.330±0.002 0.940±0.003 meso‐10 2.900±0.018 1.460±0.001 1.106±0.064 Oxaliplatin 2.500±0.003 3.770±0.001 3.350±0.002 All experiments were conducted in triplicate and gave similar results. Data are the mean±SD of three independent determinations. µM: Micromolar. The cell cycle is the process by which cells duplicate themselves, grow and prepare to divide again. It regulates cell division in multicellular organisms and it is essential for the replacement of damaged cells. Regulation of cell proliferation 31/1/2017 Anti-tumoral Activity of 1, 2‐Diaminocyclohexane Derivatives in Breast, Colon and Skin Human Cancer Cells http://powerxeditor.aptaracorp.com/fsg/Portal/Editor/Editor.aspx?assetid=MagMGzL2u%2fs%3d&roleid=TCw3dUKf1OA%3d&editstatus=1LHELv8bezw%3d 8/14 is one of the strategies for the treatment of cancer. As a consequence, in order to evaluate whether the antiproliferative effect of these molecules involves changes in cell-cycle distribution, A375 cells were treated with the compounds that present better antiproliferative activity: 1, 7–10 and then analyzed by flow cytometry (see Table 2). We found a cellcycle arrest in the G0/G1 phase induced by meso- and (±)-trans‐7 and 9 and meso‐10. meso‐1 and meso and (±)- trans‐8 did not modify the cell cycle profile and (±)-trans‐1 provoked a G2/M and S-phase cell-cycle arrest at the expense of G0/G1 phase. The antiproliferative properties of several antitumor agents are mediated by the modulation of cell-cycle checkpoint. Palbociclib is an inhibitor of CDK4 and CDK6 that provokes a pronounced G0/G1 arrest, as molecules 7,9 and 10. It is a highly selective and orally active drug, blocking retinoblastoma phosphorylation in a low nanomolar range [38]. Its antitumor activity is associated with reduced Rb phosphorylation and with a decreased expression of Ki‐67 [39], a cell proliferation marker that is associated with ribosomal RNA transcription. It decreased caspase 3/7 activation and CDKN2A and increased levels of RB1 and cyclin D1 [40]. An example of a drug that causes cell-cycle G2/M arrest, as (±)-trans‐1, is panobinostat [41-43]. It is demonstrated that it decreases in vivo tumorigenesis of triple-negative breast cancer cells. Panobinostat is a potent inhibitor with activity against Class I, II and IV HDAC enzymes, with hyperacetylation of histones H3 (Lys9) and H4 (Lys8) [41]. Table 2. Cell cycle analysis in the A375 melanoma cell line upon treatment with meso- and (±)-trans‐ 1,7–9, meso‐10 compounds and oxaliplatin (at 3×IC50 doses, 48h). Compound Cell cycle G0/G1SG2/M Control 38.97±0.22 44.42±0.66 16.61±0.45 meso‐139.04±1.63 42.24±2.98 18.73±1.35 (±)-trans‐119.97±0.13 54.78±1.95 25.26±1.83 meso‐758.01±0.36 30.14±0.24 11.85±0.11 (±)-trans‐762.28±0.14 28.54±1.33 9.19±1.20 meso‐838.95±2.69 46.90±2.62 14.15±1.70 (±)-trans‐836.68±1.91 50.40±0.14 12.93±0.92 meso‐955.63±1.98 36.56±1.20 7.79±0.28 (±)-trans‐942.35±0.99 44.16±0.64 13.49±0.57 meso‐10 66.97±3.59 15.22±1.05 17.82±4.63 Oxaliplatin 53.15±0.71 30.22±0.85 16.62±0.42 Determined by flow cytometry [44]. All experiments were conducted in triplicate and gave similar results. Data are the mean±SD of three independent determinations. Further studies were performed to determinate if the observed growth inhibition was due to apoptosis. Apoptosis is a genetic orderly process in which cells lead to their own death in response to different stimuli and it is an essential process to maintain homeostasis in multicellular organisms. Therefore, apoptosis activation is a fundamental strategy in the improvement of cancer therapy. Apoptosis was determined using an Annexin V-based assay in A375 cells treated with compounds: 1, 7–10 (see Supplementary Table 1). A light apoptotic response was observed by compound (±)-trans‐7 (28.3%) and no significant response by molecules 1-, meso‐7 and 9 (6.35–14.91%). However, we can emphasize that †‡ † ‡ 31/1/2017 Anti-tumoral Activity of 1, 2‐Diaminocyclohexane Derivatives in Breast, Colon and Skin Human Cancer Cells http://powerxeditor.aptaracorp.com/fsg/Portal/Editor/Editor.aspx?assetid=MagMGzL2u%2fs%3d&roleid=TCw3dUKf1OA%3d&editstatus=1LHELv8bezw%3d 9/14 the macrocycle meso‐10, which is the compound that induces more G0/G1 phase accumulation, is also the drug that provokes extremely high levels of apoptosis at 48h in the A375 cancer cells. This compound increases the percentage of apoptotic cells (early and late apoptosis) from 1.05% in DMSO-treated cells to 95.0%. Highly apoptotic drugs have demonstrated efficient antitumor activities in patients. The potent apoptotic properties of the macrocycle meso‐10 in melanoma cells suggest the potential clinical interest of this drug and encourage future deep studies and in vivo experiments. Finally, studies of apoptosis were performed by the activation of caspases to determine which metabolic pathway activated these drugs. Compound meso‐10 (Figure 6) activates the canonical intrinsic caspase‐8/caspase‐3 apoptotic pathway on the MCF‐7 cell line, but also this compound induces slightly caspase‐2 activation (Figure 6A). However, this compound does not significantly activate significantly any caspase in HCT‐116 and A‐375 cell lines (Figures 4B &C). Different cell death modalities exposed to the same molecule in diverse cell lines have been previously described [45]. Indirubin, a CDK inhibitor, induces caspase-independent cell death in human neuroblastoma [46], while other indirubin derivatives induce cell death through the intrinsic caspase‐9 pathway and/or activation of caspase‐8 in breast cancer cells [47].