Synthesis of novel coumarin-triazole hybrids and first evaluation of the 4-phenyl substituted hybrid loaded PLGA nanoparticles delivery system to the anticancer activity

Despite the discovery of many chemotherapeutic drugs that prevent uncontrolled cell division processes in the last century, many studies are still being carried out to develop drugs with higher anticancer efficacy and lower level of side effects. Herein, we designed, synthesized, and characterized six novel coumarin-triazole hybrids, and evaluated for anticancer activity of the one with the highest potential against the breast cancer cell line, MCF-7 and human cervical cancer cell line, human cervical adenocarcinoma (HeLa). Compound 21 which was the coumarin derivative including phenyl substituent with the lowest IC50 value displayed the highest cytotoxicity against the studied cancer cell line. Furthermore, the potential use of poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) prepared by the emulsifying solvent evaporation method as a platform for a drug delivery system was studied on a selected coumarin derivative 21. This coumarin derivative-loaded PLGA NPs were produced with an average size of 225.90 ± 2.96 nm, −16.90 ± 0.85 mV zeta potential, and 4.12 ± 0.90% drug loading capacity. The obtained 21-loaded PLGA nanoparticles were analyzed spectroscopically and microscopically with FT-IR, UV–vis, and scanning electron microscopy as well as thermogravimetric analysis, Raman, and x-ray diffraction. The in vitro release of 21 from the nanoparticles exhibited a controlled release profile just over one month following a burst release in the initial six hours and in addition to this a total release ratio of %50 and %85 were obtained at pH 7.4 and 5.5, respectively. 21-loaded PLGA nanoparticles displayed remarkably effective anticancer activity than 21. The IC50 values were determined as IC50 (21-loaded PLGA nanoparticles): 0.42 ± 0.01 mg ml−1 and IC50 (free 21 molecule): 5.74 ± 3.82 mg ml−1 against MCF-7 cells, and as IC50 (21-loaded PLGA nanoparticles): 0.77 ± 0.12 mg ml−1 and IC50 (free 21 molecule): 1.32 ± 0.31 mg ml−1 against HeLa cells after the incubation period of 24 h. Our findings indicated that triazole-substituted coumarins may be used as an anticancer agent by integrating them into a polymeric drug delivery system providing improved drug loading and effective controlled drug release.

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Introduction
Coumarin and its derivatives constitue an important class of heterocyclic compounds with their benzopyran-2-one ring.They are useful compounds in various areas due to their important photophysical and biological activity properties.Especially, their biological activities such as anticoagulant, anticancer, antioxidant, antibacterial, antifungal, antimalarial make them good drug compounds and there are several coumarin derivatives in drug market [1][2][3].These important biological activities are supplied by their active benzopyrone ring system with the ability to make hydrophobic, π-π and electrostatic interactions, hydrogen bonds besides van der Waals forces with receptors and a variety of enzymes [4].
The searches for compounds with specific activities turned the reseachers attention to hybrid molecules.The structure of a hybrid molecule can be created by the combination of two or more pharmacophores with different paths and activities to enhance the drug effect and balance the side effects.The pharmacophores in hybrid molecules may exhibit their own act on target or one of them can counterbalance the side effects of another parts.The hybrid compound may show properties from all pharmacophores with improved activity, affinity and selectivity, and they have the potential to broaden the biology spectrum and reduce side effects [5].Especially, heterocyclic scaffold hybridization of coumarin compounds opens a new door for the novel drug development [6][7][8][9][10][11][12][13].The easy functionalization of coumarin derivatives from different positions of ring carbons provides new hybrid molecules with promising activities.1,2,3-triazole group containing molecules as therapeutic agents are known to show a wide range biological activity, such as antibiotic, antifungal and anticancer [4,14,15].Coumarin and 1,2,3-trizole rings are important pharmacophore systems.Their combination in a hybrid molecule promise important biological activities [16][17][18][19][20]. Therefore, recently there has been a growing interest among researchers in the synthesis an evaluation of novel coumarin-triazole hybrid molecules.
Cancer is the second most deathful disease in the word.Early diagnosis and effective treatment can reduce the death ratio for most cancer types.There are some chemotherapeutic treatment methods using different drugs, but most of them have serious side effects [21].So, the researces have been focused to develop effective drugs and drug systems without side effects.One of the recent approaches is drug delivery systems which are pharmaceutical formulations or devices.Drug delivery systems carry the bioactive molecules to the target organ through various biological barriers and release it in the targeted side of action.The encapsulation of drug molecule in nanoparticles is a promising approach to cancer treatment.
Polymeric nanoparticles with a size between 1 and 1000 nm are biomaterials that have a high drug loading capacity and provide controlled release of the loaded drug.The solvent evaporation method is the first method developed to prepare nanoparticles from a synthetic or natural polymers [22].Many biocompatible polymers are used for production of nanoparticles, from natural polymers such as chitosan and alginate to synthetic polymers such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(lactide-co-glycolide) (PLGA) [23].Since it is biocompatible and biodegradable (degradation to CO 2 and H 2 O in the body) as well as approved by the FDA (Food and Drug Administration), PLGA is one of the most preferred polymers [23][24][25][26][27][28].Also, there are various studies in which coumarin-derived compounds are loaded into PLGA nanoparticles [29,30].
In this context, six novel coumarin-triazole hybrids (16-21, figure 1) were designed and chemically synthesized.The cytotoxic activities of the molecules whose structures were determined by FT-IR, TLC, 1 H and 13 C-NMR, and MS were evaluated by the MTT method and the best biocompatible compound 21 was loaded into the PLGA nanoparticles.The anticancer activity of the obtained 21-loaded PLGA nanoparticles on MCF-7 cells was assessed.
Commercial quality reagents were used for all experiments, and any further purification was not performed for reagent quality solvents.Millipore Milli-Q system (∼18.2MΩ cm) was used to obtain ultra-pure water.Silica gel 60 (40-63 μM) (Merck) was stationary phase for column chromatography and silica gel 60F 254 coated aluminum sheets (Merck) were used for TLC.
The devices used to obtain spectral data were Perkin Elmer, Spectrum One FT-IR spectrometer and Bruker Tensor 27 spectrometer for IR spectra, Bruker Avance III 500 MHz spectrometer for NMR spectra, Agilent G6530B model TOF/ Q-TOF Mass Spectrometer for LC-MS (Q-TOF) spectra.The chemical shifts δ were given in ppm according to TMS internal standard.
The MCF-7 human breast cancer, CCD1072 human fibroblast, HEK-293 (human embryonic kidney) and He-La (Human Cervical Adenocarcinoma) cell lines were provided from the Melisa Türkoğlu Laçin Regenerative Tissue and Cell Culture Laboratory (TURKEY) cell culture stocks.

Chemical synthesis.
The synthesis of compounds 1-9 were carried out according to the literature procedure [31,32].Among the bromoalkyoxy compounds, only 3-(6-bromohexyloxy)−7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one (6) is not identified in the literature.The protocols for the synthesis of 6 and 10-21 according to the well-known methods were given in the following section.Spectroscopic data were used for characterization of all compounds.Original spectra of new compounds were given in the Supporting Information (figures S1-S36).The data of all known compounds were in accordance with literature data [31,33,34].
3-(6-Bromohexyloxy)-7,8,9,10-tetrahydro-6H-benzo[c] chromen-6-one (6) The coumarin derivative 3 (1.0 mmol) and potassium carbonate (3.0 mmol) were stirred for 1 h at 55 °C in dry acetone, and then the dibromohexane (2.0 mmol) was added quite slowly.The mixture was refluxed for 24 h under nitrogen atmosphere.After cooling to room temperature, the solid was filtered and washed with acetone.The filtrate was concentrated under reduced pressure and purified by column chromatography using ethyl acetate/n-hexane as eluent to get the intermediates of General procedure for the synthesis of 7-azidoalkyloxy substituted coumarin derivatives (10)(11)(12)(13)(14)(15) A solution of sodium azide (NaN 3 ) (9 mmol) dissolved in distilled water (2 ml) was added to bromoalkyloxy coumarin derivatives (4-9) (3.0 mmol) dissolved in DMF (8 ml).The mixture was stirred at 80 °C for 24 h.The formed solution was cooled to room temperature then mixed with ice-water followed by extraction with ether.The ether evaportion was done after drying with MgSO 4 .The obtained compounds were pure enough and used for the next step without any purification.
7-((6-Azidohexyl)oxy)-   PLGA nanoparticles were prepared by the single emulsion solvent evaporation process according to our previous study with minor modifications [37].Firstly, 21 and PLGA were dissolved in 750 μl and 2.5 ml DCM, respectively, and slightly vortexed.Then they were mixed each other.While the obtained solution was added dropwise into 4 ml of 3% (w/v) PVA in an ice bath, sonication was applied by using a probe sonicator (power of 80%, Bandelin Sonopuls, Germany) for 90 s.The formed single emulsion system was diluted in 35 ml of 0.1% (w/v) PVA solution.The obtained NPs were collected by centrifugation at 9000 rpm for 30 min (Nuve, NF 800R) and washed three times with ultrapure water using centrifugation at 9000 rpm for 20 min.Then, the NPs were lyophilized and storage −40 °C.The reaction yield (RY) of NPs was calculated using equation (1): RY % Total amount of lyophilized NPs mg Amount of all excipients used in the formulation mg 100.

= Ín
order to calculate encapsulation efficiency (EE), and drug loading (DL) of NPs direct method was applied and equations (2) and (3) were used.For this purpose, 10 mg of solid, powder NPs was dissolved in 3 ml of DCM, vortexed, and shaken in an incubator (ES-20, Orbital Shaker, Biosan) at 180 rpm for one hour.All organic solvent was evaporated and the obtained solid was redispersed with water: ethanol (4:1).Insoluble particles were precipitated by centrifugation at 6000 rpm for 15 min and the supernatant was separated.This process was repeated two times [38].The quantitative analysis was carried out by using a Shimadzu LC-MS (liquid chromatography-mass spectrometry) system (model LC-ESI-MS 2010 EV).The column was a Shim-Pack MRC-ODS-C18 HPLC with column oven temperature of 25 °C.The mobile phase consisted of A (water, 0.1% (v/v) FA) and B (ACN, 0.1% (v/v) FA) was at a flow rate of 0.6 ml min −1 .

( ) ( ) ( ) ( )
EE % Amount of drug determined in the NPs mg Amount of initially added drug mg 100 2 DL % Amount of drug determined in the NPs mg Total amount of lyophilized NPs mg 100. 3 = 5 mg of the powder NPs were dispersed in 2.5 ml PBS at pH 7.4 or MES at pH 5.5 buffers to evaluate the in vitro release profile of the 21 from the NPs.At predetermined time intervals, all supernatant was separated by centrifugation at 9000 rpm for 15 min.The release media was filtered with 0.45 μm regenerated cellulose (RC) filter, and subjected to the analysis described above using the same LC-MS method and an appropriate calibration curve.
2.2.3.Characterization of the PLGA NPs.The obtained NPs were characterized by Zetasizer, ultraviolet-visible spectroscopy (UV-vis), Fourier-transform infrared spectroscopy-attenuated total reflectance (FT-IR-ATR), x-ray diffraction (XRD), Raman spectroscopy, differential thermal analysis (DTA), thermogravimetric analysis (TGA), and scanning electron microscopy (SEM).The mean size distribution, polydispersity index (PDI), and zeta potential of the NPs were determined by using Zetasizer (Zetasizer Nano ZS, Malvern, UK).Samples were freshly prepared before use by adding aliquots of NPs to PBS (pH 7.2) to make a solution with a NPs concentration of 1 mg ml −1 .Samples were diluted to 1:30 for the analysis and injected into a disposable capillary cell DTS1070 (Malvern Instruments, MA).Measurements were performed at 25 °C ± 1 °C with a material refraction index of 1.33, and viscosity of 0.8872 cp.All measurements were done in triplicate.The FT-IR spectra were recorded on Thermo Scientific Nicolet iS10 in ATR mode in ranging from 600 to 4000 cm −1 with resolution of 4 cm −1 and 32 scans were used [40][41][42].The crystalline and/or amorphous structure of the NPs were evaluated by XRD analyes.Powder XRD patterns of the NPs were analyzed at room temperature with PANalytical X'Pert PRO powder diffractometer.The XRD spectra were recorded between 5°and 60°(2θ).The chemical composition of the free 21 molecule, empty and 21-loaded PLGA nanoparticles were studied by Raman spectroscopy (Perkin Elmer Raman Station 400F) using an excitation wavelength of 785 nm at 100 mW.Raman spectra were recorded by averaging three acquisitions of 3 s in the range 250-3200 cm −1 .The thermal features of the free 21 molecule, empty and 21-loaded PLGA nanoparticles were measured using DTA and TGA (SII Nanotechnology-SII6000 Exstar TG/DTA 6300).Shortly, approximately 5 mg of each sample was placed on an aluminum pan under a nitrogen atmosphere.The thermograms were obtained at a temperature range of 30 °C-800 °C with a heating rate of 2 °C min −1 [43,44].
PVA content of the 21-loaded PLGA nanoparticles was calculated by a colorimetric method [45].10 mg of the NPs were dispersed in 2 ml of 0.5 M NaOH and stirred for 15 min at 60 °C.900 μl of 1 N HCl was added to the mixture for the aim of neutralization and the final volume was completed to 5 ml with distilled water.Then, freshly prepared 3 ml of 0.65 M boric acid, 1 ml of I 2 /KI and 1.5 ml of distilled water were added to the environment, respectively.The UV-vis analysis were performed at 690 nm and PVA content of the 21-loaded PLGA nanoparticles was determined by using a calibration curve of the PVA (R 2 > 0.99) given in Supporting Information, figure S38 (UV-vis calibration curve of the PVA).
The surface morphology of the 21-loaded PLGA nanoparticles was evaluated by SEM, Zeiss, EVO-LS 10 as previously described [40].The complex was fixed on metal studs and then coated with gold under vacuum.SEM photomicrograph was taken at an acceleration voltage of 7 kV.
2.2.4.1.Cytotoxicity assay.We utilized the MTT assay to determine the cytotoxicity of molecules 1-3 and 16-21 on human fibroblast L929 (CCD1072-SK) and HEK-293 cell lines by following the approach of the literature [46].Briefly, the cells (1 × 10 5 cells ml −1 ) were implanted on 96-well plates and cultured overnight.Various concentrations ranging from 0.0001 to 1 mg ml −1 (0.1; 0.5; 1; 5; 50; 500 and 1000 μg ml −1 , n = 4) of the molecules 1-3 and 16-21 solved in serum-free DMEM-F12 medium were added onto cells and incubated the cells for 24 h period of time at 37 °C.For investigation of the cell sensitivity against the agent, 10 μl of MTT [3-(4, 5-dimethylthiazol-2-yl)−2, 5-diphenyl tetrazolium bromide] solution (10 mg ml −1 ) was added into all wells.When the conversion of intracellular purple formazan crystals was detected under the microscope as an indicator of viability (4 h at 37 °C), these crystals were isolated from the medium containing MTT and dissolved in 100 μl of DMSO.As a final step, colorimetric density in well plates was measured by means of an ELISA reader (Thermo Scientific Multiskan GO Microplate, USA) at 570 nm for each well [24,47].DMSO which is well known cytotoxic activity agent was used as a positive control and DMEM + 10% FBS culture medium was used as negative control.The percentage of the viable cells was calculated using the equation (4): Anticancer activity assay.The anticancer activities of the compounds 16-21 were evaluated by in vitro method using MTT assay against MCF-7 and HeLa.The MTT assay described before was performed by inoculating cultured MCF-7 and HeLa cells (the number of cells was 10 4 cells per well) [48].The six compounds (16)(17)(18)(19)(20)(21) were added to wells in a concentrations of 1 mg ml −1 in four replicate (n = 4) after 24 h of seeding in DMEM-F12 medium including 10% FBS.The cells were incubated for 24 h and 48 h periods of time.MTT reagent was added to wells and incubated for 4 h at 37 °C until intracellular purple formazan crystals are visible under the microscope.MTT reagent were removed and 100 μl of the DMSO was added to each well and mixed gently on an orbital shaker for one hour at room temperature.The absorbance was measured at 570 nm for each well on an absorbance plate reader and cell viability ratios were calculated.A positive control DMSO was used as a known cytotoxic activity agent [49].
2.2.5.Statistical analysis.Statistical analyzes of the results were carried out using MS Excel 2016 and OriginPro 9.0 programs.A p value less than 0.05 was considered to be significant.All experiments were repeated at the least three times.The obtained data were given as mean ± standard deviation.

Chemistry
At the beginning of synthesis, three coumarin derivatives (1-3) were prepared by Pechmann condensation starting from resorcinol and β-ketoester in TFA under ultrasonic effect.Then, the alkylation of the coumarin derivatives (1-3) from C-7 was carried out with the dihaloalkane derivatives in acetone under nitrogen atmosphere to give compounds 4-9.
In the third part of our study, coumarin azide products (10-15) were prepared by S N 2 reaction of 4-9 and NaN 3 in DMF.In the last part of synthesis, the triazole-coumarin hybrid molecules (16-21) whose structures were given in figure 1 were obtained for the first time in the literature by the azide-alkyne 1, 3-dipolar cycloaddition reaction between azides (10-15) and phenylacetylene.The synthesis of all coumarin derivatives were summarized in scheme 1.The structures of all new compounds and coumarin-triazole derivatives were elucidated by IR, mass, 1 H and 13 C-NMR spectral data.
1,2,3-triazole derivatives have gained increasing popularity in recent years as an excellent building block for the discovery of potential anticancer agents [14,51].In this study, when the common hydroxyl of 1-3 molecules was replaced with 4-phenyl-1H-1,2,3-triazole of 16-21, some differences, although not significant, were observed in the cytotoxicity values of the six molecules, 16-21, compared to starting molecules 1-3.With this result, it was thought that the three nitrogen atoms of the 1,2,3-triazole groups of the 16-21 molecules will may be exert an cytotoxic effect through different mechanisms by interacting with biological targets via hydrogen bonds [52].The difference of substituents (methyl, cyclohexyl and phenyl) which are attached to the C4 carbon of the 17, 19, 21 (with n = 4C linker) and 16, 18, 20 (with n = 6C linker) molecules, may be affect the cytotoxicity levels as presented in the figure 2. The cytotoxicity values of 16-17, 18-19 and 20-21 molecules with the alkyl linker with 6C and 4C atoms in the C7 carbon were also presented in the figure 2.
It was seen that decreasing of the carbon number in linker causes a significant increase in cytotoxicity values.In addition, it was observed that the methyl and cyclohexyl-linked molecules were more cytotoxic than the phenyl-linked ones when compared molecules 16, 18, and 20 substituted with same linker.This result supports that both of the linker and alkyl difference in the coumarin core directly affects the cellular behavior of the molecule.
However, with the decrease of the carbon number, there was observed a noticeable diminish in the solubility of molecule 19 in the experimental process so molecule 19 was considered inapplicable to in vitro analyzes and was excluded from the anticancer studies.Also, inconsistency of cellular analysis data due to high standard deviation for molecule 17 was determined.Considering these mentioned cons, the effect of substitutions at carbon C7 and C4 on the anticancer potential was evaluated with only molecules 16, 18, 20 and 21.As a result of anticancer activity analyzes with MCF-7 cell line, the percent viability values for molecules 16, 18, 20 and 21 at the concentrations of 1 mg ml −1 were calculated as 80.2 ± 11.9%, 85.7 ± 2.6%, 84.3 ± 9.7% and 37.3 ± 2.6% (13.5 ± 0.1% for positive control) respectively (figure 3).It is seen in the graph that 16, 18 and 20 molecules have very close to each other and low cytotoxicity values.In addition, it has been observed that when compared 20 and 21 containing phenyl substituents on C4, 21 with 4C linker was detected to have the highest anticancer effect as the optimum linker.In light of cell viability values obtained from the cell culture studies, molecule 21 was chosen for further studies.Primarily, a purity study was carried out utilizing HPLC for the 21 to make sure that the potential anticancer activity of the designed compound is not caused by possible impurities.The purity of 21 was found as 96% (Supporting Information, figure S39 HPLC chromatogram and purity information of 21).To determine whether the low anti-cancer activities of the other three compounds (16, 18, and 20) were due to possible impurities, HPLC analysis was performed to present their impurities.The purity rates for molecules 16, 18, and 20 were 95% and above (Supporting Information, figures S40-42).Therefore, 21, selected as the highest biocompatible molecule, was loaded into PLGA nanoparticles by a single emulsion solvent evaporation method to convert it into a functional material having the potential to treat breast carcinoma.It was aimed to diminish the existing toxicity and to increase the anticancer activity of the 21 molecule by developing an effective controlled release system.

Preparation of the 21-loaded PLGA nanoparticles
21 derivative which of 6 new coumarin derivatives obtained in this work with the lowest toxicity and the best biocompatible one in the human fibroblast cell line (CCD1072-SK), and the highest anticancer activity against MCF-7 cell line was incorporated into the PLGA nanoparticles by the solvent evaporation method.Thereby, it was aimed to minimize the existing toxicity of 21 and to enhance its anticancer activity with the controlled long release.RY, EE, and DL of the obtained 21-loaded PLGA nanoparticles were found to be 66.35 ± 2.3%, 52.46 ± 3.8%, and 4.12 ± 0.9%, respectively.Our study exhibited not much difference in these data from previous studies [26,53,54].The average particle sizes of empty and 21-loaded PLGA nanoparticles were found to be 195.5 ± 1.411 nm and 225.9 ± 2.957 nm with PDI values of 0.102 ± 0.016 and 0.095 ± 0.023, respectively, which proved the homogenous distribution of the nanoparticles (figure 4).Some increase in average size occurred after the encapsulation process of 21 into PLGA NPs compare to empty NPs.
Furthermore, empty and 21-loaded PLGA nanoparticles exhibited similar negative zeta potential of −16.9 ± 0.850 and −16.0 ± 0.115 mV thanks to the presence of PLGA terminal carboxylic groups and PVA residues on the surface (figure 5).This similarity also suggested to us that there was no significant drug adsorption on the NPs surface.At the same time, sufficiently negative zeta potential values provided prominent information about the stability of the nanoparticles.The zeta potential value of the 21-loaded PLGA nanoparticles stored in lyophilized powder form at +4 °C for approximately 6 months was measured by the ELS technique.The zeta potential value of 21-loaded NPs measured under the same analysis conditions as in the initial measurements was found to be −15.0 ± 5.39 mV.This value proved to us that these nanoparticles were stable in long-term storage as well as there was no significant difference with the initial measurement.
Additionally, for the full characterization of nanoparticles, it is also interesting to determine the ratio of the emulsifying agent used in the manufacturing of them in the produced nanoparticles.In this study, PVA, which is frequently used as an emulsifying agent in the production of PLGA nanoparticles, was preferred.Although excessive washing for removing PVA after the nanoparticles are produced, some of the PVA remains incorporated with the nanoparticles because it forms a mesh structure with the polymer at the interface.The PVA content of the 21-loaded PLGA nanoparticles was found to be 3.87 ± 0.49% according to the colorimetric method and this value is convenient with the literature [55] (Supporting Information, figure S40, UV-vis spectrum of the 21-loaded PLGA nanoparticles after the PVA concentration calculation method was applied).
FT-IR spectrum of the 21 showed some characteristic peaks (figure 6(a)).While 3120 and 3069 cm −1 corresponded to aromatic C-H stretches, 1716 and 1603 cm −1 referred to aromatic C=O and C=C groups, respectively.In addition, bands at 1471 and 1374 cm −1 represented aliphatic in-plane C-H bending, while 1206 cm −1 supported C-N oscillation, and 1114 cm −1 supported C-O tension.For PLGA, some peaks were examined in figure 6(a).The peaks between 2900 and 3000 cm −1 referred to C-H stretching of CH 3 groups, while the peak at 1748 cm −1 remarked carbonyl -C=O stretching and the other peaks between 1090 and 1171 cm −1 displayed the presence of C-O stretching.The spectra of empty and 21-loaded PLGA nanoparticles exhibited completely the same characteristics.In addition, the absence of any specific peak belonging to the free 21 molecule in the spectrum of the 21-loaded PLGA nanoparticles proved that the encapsulation process was carried out successfully and that the molecule did not adhere to the surface.This result was consistent with the results obtained in our previous studies [24,27,56].
The crystallization properties of the nanoparticles were examined by XRD.The XRD patterns of free 21 molecule, empty and 21-loaded PLGA nanoparticles were presented in figure 6(b).There were sharp peaks in the 12-26 theta range, indicating that the 21 molecule is crystalline.In the spectra of empty and 21-loaded PLGA nanoparticles, broadband in the 18.59 theta supporting the amorphous property of the PLGA were observed, similar to each other [57].Thus, it was shown the 21 molecule was not found on the nanoparticle surface and successfully entrapped into the NPs.
The thermal behavior of the free 21 molecule, empty PLGA nanoparticles, and 21-loaded PLGA nanoparticles was investigated by differential thermal analysis (DTA) and thermogravimetric analysis (TGA).Analysis of the free 21 molecule, (figure 7   addition, the findings showed that the decomposition temperature reduces slightly with drug loading (figure 7(c)).The same result is well described in the literature [58].TGA is also used to identify weight loss because of the volatilization of residual solvents in nanoparticles that take place around the boiling point of it [59].Curiously, when both empty and 21loaded PLGA nanoparticles were analyzed by TGA, no considerable weight loss was monitored at the boiling point of DCM at 40 °C approving its absence as residual solvent.
In the Raman spectroscopy analysis, the representative peaks obtained from 21 and empty PLGA NPs were compared with the peaks for 21-loaded PLGA nanoparticles (figure 7(d)).In the spectrum of empty PLGA NPs, peaks at 2752, 2946, and 3182 cm −1 refer to C−H stretching vibrations.One another band at 1770 cm −1 belongs to C=O bond stretching vibration while bands at 1114 cm −1 and 1036 cm −1 correspond to C-O stretching.Additionally, the C-H bend was noticed at 1436 cm −1 .When the Raman spectrum of the 21 was examined, it was determined that C-H bands at 3062, 2932 and 2890 cm −1 , C=O band at 1720 cm −1 , CH 3 band at 1378 cm −1 , C-C band at 1660 cm −1 .Besides the fact that the spectra of empty PLGA nanoparticles and 21-loaded PLGA nanoparticles exhibited similar properties, no peaks of the 21, the main peaks of which were given above, did not appear in  SEM image showing the morphology of the 21-loaded PLGA nanoparticles was presented in figure 8(a).In SEM imaging, parameters such as being able to determine the morphological distribution of the nanoparticles, being able to monitor the structural form of the nanoparticles, resolution of the images, and not deforming the samples during the analysis are important.We observed homogeneously dispersed and spherical nanoparticles supported by a high-resolution SEM image compatible with the Zetasizer measurement results.
Figure 8(b), represented the in vitro release profile of 21 from PLGA nanoparticles in two different buffered solutions at pH 5.5 and 7.4 at 37 °C.In vitro release behavior was evaluated for various time intervals (1, 6, 32 h; 4, 11, 20, 30, and 45 d).In the beginning, a typical initial burst release of 21 from the PLGA nanoparticles was seen for both release patterns and 15%-17% of the 21 was released from the nanoparticles in the first six hours.Burst release usually occurs when a certain amount of the drug encapsulated in a particle system is released into both the in vitro and in vivo release environment within a short period of 24-48 h.Even though this situation is attributed to different physical and chemical conditions, the primary mechanism of burst release in monolithic polymeric systems has not been utterly elucidated [60,61].The release of the active substance from PLGA nanoparticles occurs due to both diffusion and erosion.In addition, since PLGA is a polymer with the capacity to absorb water, polymeric degradation takes place both within the bulk and on the surface of the obtained nanoparticles.Since all these parameters interact with each other during the release of the active substance, their analysis becomes difficult.In vitro cumulative drug release patterns exhibit behavior in four different categories: monophasic, burst biphasic, delayed biphasic, and triphasic.The burst biphasic, termed 'burst release,' is typically characterized by rapid drug release upon initial exposure of PLGA nanoparticles to aqueous media, with the duration of this phase varying from several hours to 1-2 d [62].Subsequently, it was assessed that 21 showed controlled release over a one-month period for both pH values.Following the initial burst release seen in the PLGA nanoparticles, a phase happens with a power law relationship between the cumulative amount of drug released and the time elapsed in this phase.In such a case, the zeroorder release phase is observed, where the drug release rate becomes constant, meaning that the active material is released from the polymer depot via non-Fickian diffusion for a period of several days to weeks [62].This constant release drew a profile that remained stable at the end of 45 d and continued to release at a low sustained rate which was undetectable by LC-MS.A similar kinetic pattern has been determined for other PLGA NPs [63].The release profile of 21 from PLGA nanoparticles appears similar to coumarin C75 release from the same polymeric system found by Fernandes et al.where around 30% of the active ingredient was released until the first 24 h, followed by a sustained release which prolongs up to 7 d.Overall, while there was nearly 85% release of the drug at pH 5.5, the in vitro release is just about 50% at pH 7.4 for the same sample.It was evaluated that the parameters contributing to the in vitro release of 21 from nanoparticles were swelling of the PLGA polymer, pore diffusion, polymer erosion and degradation [57].The fact that the release was notably higher under cancer microenvironment conditions at pH 5.5 can provide more valuable data for the anticancer cell study compared to pH 7.4.

In vitro anticancer activities
The in vitro cytotoxicity levels and anticancer activities of the three groups consisting of molecule 21, 21-loaded PLGA nanoparticles and empty PLGA NPs at five different concentrations were determined according to the survival rates of human fibroblast L929 cells, human embryonic kidney HEK-293 cells, (Supporting Information, figures S44-S45), human breast cancer MCF-7 cells, and HeLa cell lines by MTT analysis as well (figures 9-10).The negative control absorbance value was accepted as 100% viability for both tests.As shown in figures S44 and S45, 21, 21-loaded PLGA nanoparticles and empty PLGA NPs had almost no cytotoxicity against human fibroblasts (CCD1072-SK) and HeLa cervical adenocarcinom cells.The viability of cells ranged between 70%-95% and 71%-94%, respectively.
With the encapsulation of molecule 21, we observe an increase in cell viability (after incubation time of 24 h) against both MCF-7 breast cancer and HeLa cervical adenocarcinoma cells (figures 9-11).While an increase in cytotoxicity values indicating cell viability was observed from IC50 (21-loaded PLGA nanoparticles) : 0.77 ± 0.12 mg ml −1 to IC50 (free 21 molecule) : 4.66 ± 3.36 mg ml −1 for HeLa cells, was observed from IC50 (21-loaded PLGA nanoparticles) : 0.42 ± 0.01 mg ml −1 to IC50 (free 21 molecule) : 5.74 ± 3.82 mg ml −1 for MCF-7 cells (figure 11).This dramatic increase was evaluated as a result consistent with the burst release character resulting from the structure of the polymeric nanoparticle.The fact that about 20% of the total active ingredient release occurs during the first six hours (figure 8(b)) supports both the time-dependent and dose-dependent cytotoxic effects observed after incubation of 24 h (figures 9, 10).However, a series of cellular tests are needed to prove through which pathway the observed cytotoxic effect occurs.According to the results obtained in this study, it is clear that active molecule 21 acts at a sufficient dose and time to inhibit the proliferation of the tested cancer cells.However, when the IC50 graphs of the studied cancer cells are examined, the cytotoxicity values no increasing consistently with concentration and time may suggest that molecule 21 acts as a pro-apoptic agent on cancer cells.The proliferation of cancer cells, which reduce mitochondria permeability as a result of apoptosis, regardless of the applied dose, may result in increased cell viability.For this reason, despite the cumulative release in the second 24 h (figure 8(b)), cell viability may increase compared to the first 24 h.On the other hand, considering the hydrophobic character of the PLGA nanoparticles compared to the free molecule 21, the delivery time and dose of the activated substance into the cell may differ and the duration of the cytotoxic effect may increase due to the increase in the exposure time to the active substance.In fact, as emphasized in the literature regarding coumarin derivatives, molecule 21 can suppress the growth and proliferation of cancer cells even at low doses and may not have a significant toxic effect on normal cells [64,65].This may be further evidence that coumarins are a promising class of anti-tumor drugs with high selectivity.
By the end of 2020, approximately 8 million women worldwide have been diagnosed with breast cancer, making it the most common and leading cause of death in women [66].Different treatment methods and/or drugs that have been researched for many years aim not only to eliminate the disease but also to protect existing healthy tissues.There are many studies in literature on the structure-activity relationship of coumarin derivatives as anticancer drug candidates [11,[67][68][69][70][71].However, the study examining the anticancer effect of synthetic coumarin-7 derivatives both to increase the drug-activity relationship and with biodegradable polymeric controlled release system is quite limited.When the activities of coumarin derivatives integrated with polymeric and other nanocarrier systems on MCF-7 breast cancer line are examined; the determined lowest IC50 value in which different coumarin extract types was calculated as 1.755 mg ml −1 [72].
In a separate study that treated breast cancer cell lines with zinc oxide nanostructures coated with ruthenium(III) complexes of synthetic coumarin derivatives, it was emphasized that ruthenium(III) complexes (IC50 < 10 μg ml −1 ) had a better value than the coated complex coated nanostructures (IC50 < 80 μg ml −1 ) [73].In our study, IC50 values determined by encapsulating to the PLGA carrier system of synthetic coumarin-7 derivatives against MCF-7 breast cancer and HeLa cervical cancer cells were calculated as 0.42 ± 0.01 mg ml −1 and 0.77 ± 0.12 mg ml −1 respectively.Although the effect of polymeric controlled release system on cytotoxicity is already known, many synthetic coumarin molecules with different functional groups are difficult and limiting for in vitro cell studies due to solubility, hydrophobicity and high toxicity problems to healthy cells when  compared to plant extract and protein/peptide structures.For this reason, we believe that our study will contribute significantly to the existing literature in terms of not only the unique molecules it contains, but also its combination with a delivery system and its in vitro evaluation.21-loaded PLGA nanoparticles can be given active targeting properties by conjugating them with peptides that have specific binding properties to the cancer cells of interest, thereby achieving a much higher level of anticancer activity.However, in cancer therapies, therapeutic agents are administered to patients in saline (0.9% NaCl solution).Therefore, evaluation of in vitro analyzes in this solution may strengthen the study.

Conclusion
Six new coumarin-triazole hybrids featuring different substituents at C4 position (methyl, phenyl or cyclohexyl) with two different chain linker lengths (n = 4, 6) were prepared and characterized by using 1 H NMR, 13 C NMR, and mass spectroscopy techniques.Since the relation of anticancer activity with cytotoxicity is known, first of all, a kind of preelimination was performed for all structures as a result of the cytotoxicity study.Even molecules that could be applied to laboratory analysis procedures were eliminated.In this study, 21 was prominent as the most suitable molecule for the highest cytotoxicity against MCF-7.The superior properties of nanoparticles such as aggregation in leaky blood vessels of tumors and destroying tumors, minimizing damage to healthy organs, circulating in the bloodstream for a longer time, and optimizing the effective dose are known.Therefore, despite a certain level of in vitro anticancer activity of molecule 21, the nanoparticle system was preferred for increasing its anticancer efficiency.The release profile, size, and production quality of the produced nanoparticles were evaluated and compared with the active substance itself in terms of its anticancer properties.On the whole, coumarin compounds can inhibit the growth, proliferation and metastasis of various tumor cells through various mechanisms (inhibition of carbonic anhydrase, PI3K/AKT/mTOR signaling pathway, microtubule polymerization, angiogenesis, monocarboxylate transporters, hypoxia inducible factor-1) acting on apoptosis proteins and inhibiting tumor multidrug resistance, ROS regulation, etc Coumarin derivatives have long been proven to be drug candidates acting as antitumor agents, with selective and effective cytotoxicity through various antitumor mechanisms.The failure to comprehensively study coumarin derivatives modified with different functional groups results in the limitation of in vitro and in vivo experiments to explore the relationship between these derivatives and their biological mechanisms and misunderstanding of drug pharmacokinetics and safety profile.The most well-known limitations can be listed as the fact that antitumor mechanisms have not been evaluated comprehensively, in vitro experiments are not supported by in vivo experiments in proving the in vivo antitumor potential and safety profile of newly discovered coumarin compounds, and finally, the newly discovered coumarin compounds are poorly soluble in water.Presented study was designed to overcome the limitation of in vitro studies due to the poor water solubility of coumarin compounds and the problem of low bioavailability.It involves increasing water solubility and bioavailability by modifying the functional groups of coumarin core, as well as increasing the biological performance of the molecule with a carrier system.We aimed to canalize the literature on the modification and delivery systems of coumarins by ensuring high selectivity, lower side effects and higher bioavailability.Due to the 2H-chromen-2-one ring (contributes to aromatic, planar and lipophilic structure), lactone group and heterocyclic conformation in the structure of coumarin enables interaction with different biological target proteins and forms strong polar bonds.Considering the results of cytotoxic activity, 21-loaded PLGA nanoparticles (0.5 mg ml −1 ) were determined as the most effective group with about 45% and 60% or higher survivals in MCF-7 cells and HeLa cells.In conclusion, in the study, the efficiency of molecule 21 selected among the novel six coumarin derivatives as potential anticancer drug candidates against breast and cervical adenocarcinom cells was extended with the integration to the polymeric nanosystem.We hope that this work can hereby chart a new avenue for the future optimization of candidate molecules with stronger anticancer properties.

Scheme 1 .
Scheme 1. Schematic route of the synthesis.

Figure 3 .
Figure 3. Percentage of MCF-7 cells viable following incubation with coumarin-triazole hybrid molecules (16, 18, 20 and 21 at the concentrations of 1 mg ml −1 ) relative to a control of untreated cells.Each bar represents the mean percentage survival and SD (where n = 4, four fields per repeat).
(a)) revealed one TGA step and three DTA peaks.The free 21 molecule had a single step TGA degradation point at 287 °C.The DTA steps were located at 121 °C, 386 °C and 662 °C.While the TGA thermogram of PLGA displayed a single phase of weight loss between 246 and 600 C, DTA revealed one step at 303 °C (figure 7(b)).Similar to empty nanoparticles, 21-loaded PLGA nanoparticles also showed a single point weight loss, indicating that polymer and 21 molecule mixed but did not interact.In

Figure 4 .
Figure 4. Size distribution by intensity of the NPs-1 (21-loaded PLGA nanoparticles) and NPs-2 (empty PLGA nanoparticles).For the measurements, NPs were freshly prepared at a concentration of 1 mg ml −1 and diluted with distilled water at a ratio of 1:30.The solutions were passed through neither 0.45 nor 0.22 μm filters.

Figure 5 .
Figure 5. Zeta potential distribution of the NPs-1 (21-loaded PLGA nanoparticles) and NPs-2 (empty PLGA nanoparticles).For the measurements, NPs were freshly prepared at a concentration of 1 mg ml −1 and diluted with distilled water at a ratio of 1:30.The solutions were passed through neither 0.45 nor 0.22 μm filters.

Figure 6 .
Figure 6.(a) The FT-IR spectra of empty and 21-loaded PLGA nanoparticles comparatively with free 21 molecule (b) the XRD patterns of free 21 molecule, empty PLGA nanoparticles, and 21-loaded PLGA nanoparticles.

7 .
Thermal gravimetry and differential thermal analysis curves of the (a) free 21 molecule, (b) empty PLGA nanoparticles, (c) 21loaded PLGA nanoparticles, (d) Raman spectra of the free 21 molecule, empty PLGA nanoparticles, and 21-loaded PLGA nanoparticles. the spectra of the encapsulated PLGA nanoparticles, and this confirmed the encapsulation of 21 into PLGA nanoparticles with no surface adsorption.

Figure 8 .
Figure 8.(a) SEM photograph of 21-loaded PLGA nanoparticles produced by single emulsion solvent evaporation method (b) in vitro release profile of 21 from PLGA NPs in PBS buffer at pH 7.4 and in MES buffer at pH 5.5.

Figure 10 .
Figure 10.Dose response in MCF-7 cells treated with free 21 molecule, 21-loaded PLGA nanoparticles, and empty NPs by MTT assay after 24 h and 48 h incubation time (all cell viability values of represent the mean ± SD (n = 5), Apsis values; M (mg ml −1 )).