Seleno-Analogs of Scaffolds Resembling Natural Products a Novel Warhead toward Dual Compounds

Nowadays, oxidative cell damage is one of the common features of cancer and Alzheimer’s disease (AD), and Se-containing molecules, such as ebselen, which has demonstrated strong antioxidant activity, have demonstrated well-established preventive effects against both diseases. In this study, a total of 39 Se-derivatives were synthesized, purified, and spectroscopically characterized by NMR. Antioxidant ability was tested using the DPPH assay, while antiproliferative activity was screened in breast, lung, prostate, and colorectal cancer cell lines. In addition, as a first approach to evaluate their potential anti-Alzheimer activity, the in vitro acetylcholinesterase inhibition (AChEI) was tested. Regarding antioxidant properties, compound 13a showed concentration- and time-dependent radical scavenging activity. Additionally, compounds 14a and 17a showed high activity in the melanoma and ovarian cancer cell lines, with LD50 values below 9.2 µM. Interestingly, in the AChEI test, compound 14a showed almost identical inhibitory activity to galantamine along with a 3-fold higher in vitro BBB permeation (Pe = 36.92 × 10−6 cm/s). Molecular dynamics simulations of the aspirin derivatives (14a and 14b) confirm the importance of the allylic group instead of the propargyl one. Altogether, it is concluded that some of these newly synthesized Se-derivatives, such as 14a, might become very promising candidates to treat both cancer and AD.


Introduction
Cancer and Alzheimer's disease (AD) entail a socio-economic burden for society, accounting for almost 10.0 million deaths, presenting 19.3 million new cases diagnosed in 2020 for cancer [1] and at least 50 million people living with AD or other dementias worldwide [2]. Although treatment options for both illnesses have increased over the past few years, all of them present important drawbacks such as toxicities and resistance. Thus, the development of novel and more effective agents for both diseases is an urgent need.
Overall, considering all the above mentioned, herein we decided to combine several of the DDTs to design "pseudo-natural products" with the potential to become dual agents towards cancer and AD. Thus, the designed compounds gather in the same structure a natural occurring functionality modified with the selenium (Se) atom following a fragment-based design. After an extensive review of the literature, we rule to choose allylic and propargylic fragments present in active molecules of natural products [13,14] (see Figure 1). Molecules containing allylic fragments are present in garlic, nutmeg, parsley, or mustard [15] and active molecules containing propargylic fragments have been identified in cyanobacteria and marine mollusks [16]. Numerous extensive literature review papers have compiled many of the pharmacological effects of garlic extracts and some of its allyl sulfides [17][18][19][20][21][22][23], including preclinical, in vivo, and clinical trials. Two of the most characterized allylsulfides are allicin and Sallylcysteine (depicted in Figure 1). The first one possesses anticancer activity through the STAT3 signaling pathway [24,25] along with its anti-amyloidogenic property that prevents the progression of AD [26,27]. Notwithstanding, S-allylcysteine has suppressed ovarian cancer proliferation [28]. On the other hand, the propargyl group has been broadly exploited since its privileged structural feature for targeting a wide range of therapeutic target proteins, including MAO or tyrosine kinases. Moreover, propargyl compounds have attracted great interest due to their wide application in organic synthesis as well as the development of active propargyl molecules such as erlotinib and noreynodrel [29,30]. A common feature among these garlic active principles, depicted in Figure 1, is Numerous extensive literature review papers have compiled many of the pharmacological effects of garlic extracts and some of its allyl sulfides [17][18][19][20][21][22][23], including preclinical, in vivo, and clinical trials. Two of the most characterized allylsulfides are allicin and Sallylcysteine (depicted in Figure 1). The first one possesses anticancer activity through the STAT3 signaling pathway [24,25] along with its anti-amyloidogenic property that prevents the progression of AD [26,27]. Notwithstanding, S-allylcysteine has suppressed ovarian cancer proliferation [28]. On the other hand, the propargyl group has been broadly exploited since its privileged structural feature for targeting a wide range of therapeutic target proteins, including MAO or tyrosine kinases. Moreover, propargyl compounds have attracted great interest due to their wide application in organic synthesis as well as the development of active propargyl molecules such as erlotinib and noreynodrel [29,30]. A common feature among these garlic active principles, depicted in Figure 1, is the presence carried out to evaluate the effect of NSAIDs in AD and have demonstrated their capacity to inhibit Aβ aggregation, such as indomethacin via a α2-macroglobulin-activating lrp1dependent mechanism [69][70][71]. Thus, the development of novel compounds that include NSAIDs in their structure seems to be a promising approach in the search for dual drugs against cancer and AD.
Based on the above facts, the current study presents the synthesis and in vitro evaluation of 38 Se-containing compounds as dual anticancer and anti-Alzheimer agents (see Figure 2). These Se derivatives were designed based on a fragment-based approach, gathering, in the same molecule, two active fragments and the Se atom, in the form of selenoester. In the acyl moiety, we envisioned the introduction of different small carbo-and heterocycles, as well as NSAIDs. In the opposite location of the molecule, allyl (series a) or propargyl (series b) fragments were included with the aim of mimicking the active ingredients of natural products. To date, the library of Se-containing compounds is extensive due to its high chemical versatility, and it is greatly increasing yearly [72]. There are molecules with acylselenourea and selenourea groups in their structures that have been found to be excellent radical scavenger agents. Within these groups of Se derivatives, molecules with dual in vitro antioxidant and antiproliferative activities have been also reported [36,73]. Moreover, selenoester derivatives have been observed as potent cytotoxic agents, albeit not all of them exhibit antioxidant activity [74,75]. The most studied Se-containing compound is ebselen, with a benzoisoselenazolone ring, which has exhibited anti-inflammatory, antioxidant, and anticancer activities [43,[76][77][78]. However, no molecules have been reported so far that combine in their structure NSAIDs and an active fragment present in garlic-derived natural products through a selenoester group. All the synthesized Se derivatives were evaluated as dual agents towards breast, colorectal, lung, and prostate cancer and AD using the MTT assay and Ellman's method, respectively. Moreover, DPPH assay was performed to evaluate the antioxidant capacity of the synthesized compounds. The two most active compounds in the screening against cancer cell lines were submitted to the Developmental Therapeutics Program (DTP) of the National Cancer Institute (NCI). Furthermore, molecular dynamics simulations were carried out to elucidate the mode of interaction between compound 14a and the active site of AChE.
ioxidants 2023, 12, x FOR PEER REVIEW 4 of 31 [69][70][71]. Thus, the development of novel compounds that include NSAIDs in their structure seems to be a promising approach in the search for dual drugs against cancer and AD. Based on the above facts, the current study presents the synthesis and in vitro evaluation of 38 Se-containing compounds as dual anticancer and anti-Alzheimer agents (see Figure 2). These Se derivatives were designed based on a fragment-based approach, gathering, in the same molecule, two active fragments and the Se atom, in the form of selenoester. In the acyl moiety, we envisioned the introduction of different small carbo-and hetero-cycles, as well as NSAIDs. In the opposite location of the molecule, allyl (series a) or propargyl (series b) fragments were included with the aim of mimicking the active ingredients of natural products. To date, the library of Se-containing compounds is extensive due to its high chemical versatility, and it is greatly increasing yearly [72]. There are molecules with acylselenourea and selenourea groups in their structures that have been found to be excellent radical scavenger agents. Within these groups of Se derivatives, molecules with dual in vitro antioxidant and antiproliferative activities have been also reported [36,73]. Moreover, selenoester derivatives have been observed as potent cytotoxic agents, albeit not all of them exhibit antioxidant activity [74,75]. The most studied Se-containing compound is ebselen, with a benzoisoselenazolone ring, which has exhibited anti-inflammatory, antioxidant, and anticancer activities [43,[76][77][78]. However, no molecules have been reported so far that combine in their structure NSAIDs and an active fragment present in garlic-derived natural products through a selenoester group. All the synthesized Se derivatives were evaluated as dual agents towards breast, colorectal, lung, and prostate cancer and AD using the MTT assay and Ellman's method, respectively. Moreover, DPPH assay was performed to evaluate the antioxidant capacity of the synthesized compounds. The two most active compounds in the screening against cancer cell lines were submitted to the Developmental Therapeutics Program (DTP) of the National Cancer Institute (NCI). Furthermore, molecular dynamics simulations were carried out to elucidate the mode of interaction between compound 14a and the active site of AChE.   , Acros Organics (Janssen Pharmaceuticalaan 3a, 2440 Geel, Belgium) and Lancaster (Bischheim-Strasbourg, France). Melting points (mp) were determined with a Mettler FP82+FP80 apparatus (Greifensee, Switzerland). All the compounds are >95% pure by quantitative NMR ( 1 H q-NMR) using dimethyl sulfone as reference. The results are expressed as the percentage of purity and were calculated tracking the signal of the first alkene hydrogen, which appears around 5.9 ppm, for series a, and the signal of CH 2 which appears around 3.7 ppm, for series b.
2.1.2. General Procedure for the Synthesis of the Compounds (1a-19a and 1b-19b) NaBH 4 (0.9 g, 11.39 mmol) was added to a mixture of elemental Se (0.9 g, 11.39 mmol) in water (30 mL). The mixture was stirred at room temperature for 20 min. The corresponding acyl chloride (11.39 mmol) was added in situ and stirred for 60 min. Allyl bromide (0.9 mL, 11.39 mmol) for series a, and propargyl bromide (0.8 mL, 11.39 mmol) for series b, and tetrahydrofuran (10 mL) were added in situ and stirred at room temperature for 90 min. The product was isolated by extraction with methylene chloride (3 × 50 mL) and the organic phases were dried using sodium sulfate anhydrous. After that, the organic phases were filtered, and the methylene chloride was removed by rotatory evaporation under vacuum. The final product was purified by column chromatography. A gradient of hexane/ethyl acetate, ranging between 0% and 50% of ethyl acetate, was used as eluent. The mobile phase of the TLCs was hexane/ethyl acetate with a ratio of 9:1. The Rf range was from 0.18 to 0.75.
The acyl chlorides of compounds 1, 4, 10, 7-8, and 15-19 were formed from the reaction of the corresponding carboxylic acids with oxalyl chloride in methylene chloride using drops of N,N-DMF as a catalyst. The mixture was stirred at room temperature for twelve hours and the reaction media was removed by rotatory evaporation under the vacuum. Tables 1 and 2 provide the chemical name, starting reagent, yield, appearance, 1 H, 13 C, and 77 Se NMR spectra, and purity data for the compounds of series a and series b, respectively.
Orange oily liquid            The antioxidant activity was determined by the colorimetric assay of DPPH, described by Svinyarov [79], in which the capacity of the compounds for scavenging radicals in vitro is measured. It is based on the reduction of a stable free radical, DPPH, with the presence of antioxidants, by the donation of a hydrogen atom. Then, this reduction of DPPH causes the decrease in its absorbance at 517 nm, and the corresponding DPPH radical-scavenging activity can be determined. The measurements were recorded on a BioTeck PowerWave XS spectrophotometer (BioTeck Instruments, Winooski, VT, USA) and the data were collected using KCJunior v.1.41. software (BioTeck Instruments, Winooski, VT, USA).
Each compound was dissolved in absolute methanol at the concentration of 1 mg/mL, and then, serial dilutions were prepared. Ascorbic acid and trolox were used as positive controls because of their well-known and potent antioxidant capacity. A methanolic solution (0.04 mg/mL) of DPPH (Aldrich) was prepared daily and was protected from light. The blank of colorless sample was absolute methanol. Moreover, 100 µL of each sample were dissolved in 100 µL of DPPH solution, and the control was prepared dissolving 100 µL of absolute methanol in 100 µL of the DPPH solution. The decolorization of the purple radical to the yellow reduced form was followed at 517 nm and the absorbances were read after different times intervals. All the measurements were carried out in triplicate. Results are expressed as the percentage of the radical scavenger, calculated using the following formula:

Cell Culture Conditions
The cell lines were obtained from the American Type Culture Collection (ATCC). Five tumor cell lines (MDA-MB-231, MCF-7, HCT116, HTB-54, and DU-145) and two nontumorigenic cells (184B5 and BEAS-2B) were grown in RPMI 1640 medium (Gibco), and the HT-29 tumor cell line was grown in McCoy's medium. Both mediums were supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% antibiotics (10.00 units/mL penicillin and 10.00 µg/mL streptomycin; Gibco). Cells were preserved in tissue culture flasks at 37 o C and 5% CO 2 . The culture medium was replaced every three days.

Cell Viability Assay
The effect of each compound on cell viability was tested using the MTT assay [80].

NCI-60 Analysis
Compounds 14a and 17a were submitted to the Developmental Therapeutics Program (DTP) of National Cancer Institute (NCI). Cytotoxicity activity was evaluated by performing One-Dose screening (10 −5 M) against a panel of 60 human tumor cell lines. As both compounds demonstrated effective cytotoxic activity, they were selected for the Five-Dose (0.01 µM-100 µM) assay against the same cell panel comprising different leukemia, nonsmall cell lung, colon, central nervous system (CNS), melanoma, ovarian, renal, prostate and breast tumor cell lines. Cells grew in the RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine and were inoculated into 96-well microtiter plates in 100 µL at plating densities ranging from 5.000 to 40.000 cells/well. The microtiter plates inoculated with cells were incubated at 37 • C, in 5% humidified CO 2 for 24 h prior to addition of experimental drugs. Then, experimental drugs were added, and the microtiter plates were incubated for 48 h. The protocol is available on https://dtp.cancer.gov/discovery_ development/nci-60/methodology.htm accessed on 31 December 2022.

AChE Inhibition
The AChE inhibitory capacity of the synthesized compounds, 1a-19a and 1b-19b, was assessed spectrophotometrically by Ellman's method [81] with minor modifications [82]. Frontal cortex tissue obtained from male Wistar rats was homogenized in 39 volumes of 75 mM sodium phosphate buffer (pH 7.4). A mixture of 260 µL containing the compounds assessed, acetylthiocholine iodide and 5 µL tissue homogenate was incubated for 8 min.
The reaction was then terminated by adding 50 µL 3% (w/v) sodium dodecyl sulphate followed by 50 µL 0.2% (w/v) 5,5 -dithio-bis(2-nitrobenzoic) acid to produce the yellow anion of 5-thio-2-nitro-benzoic-acid. The extent of the color production was measured spectrophotometrically at 405 nm using Multiskan Ex (Thermo Electron Corporation). Compounds were assessed at a screening concentration of 10 −6 M. IC 50 values were calculated as the concentration of compound that produces 50% enzyme activity inhibition with the OriginPro 9.0.0 software. Results are expressed as the mean ± SD and each experiment was repeated three times.

In Vitro Brain-Blood Barrier Permeation Study
The parallel artificial membrane permeability assay (PAMPA) described by Di et al. was used to evaluate the brain penetration of the compounds 10a, 13a, 14a, and galantamine (used as reference drug) [83]. Franz diffusion cells (Microette 8910130, Hanson Research) were employed to perform this assay. The compounds were dissolved in DMSO at 5 mg/mL and diluted to 500 µg/mL with phosphate-buffered saline (PBS)/EtOH (7:3) to make a stock solution. A total of 10 µL of porcine brain lipid (PBL) diluted in dodecane solution (20 mg/mL) was spread onto a PVDF membrane that was placed between the donor and acceptor compartments, forming a sandwich structure. Then, 4.5 mL of PBS/EtOH (7:3) was added to the acceptor compartment and 700 µL of the stock solution was added to the donor cell. After maintaining this structure for 20 h at 25 • C, the donor cell was carefully removed, and the concentrations of the tested compounds in the acceptor and donor cells were measured as UV-visible (λ = 272 nm for compound 10a, and λ = 290 nm for compound 14a and galantamine) (8453 UV-Visible Agilent Technologies). The concentration of the compound was calculated from the standard curve and expressed as permeability (Pe) by the following formula [84]: Every sample was analyzed at least in duplicate and the data were reported as mean ± SD.

Statistical Analysis
Data were expressed as the mean ± SD (standard deviation) and experiments were performed three times in triplicates unless otherwise specified. Non-linear curve regression analysis calculated by OriginPro 9.0.0 software (OriginLab Corporation; Northampton, MA, USA) was used to assess the IC 50 . Data were analyzed using GraphPad Prism version 8.0.2 (GraphPad Software; San Diego, CA, USA).

Molecular Dynamics Simulations
The following crystal structures deposited in the Protein Data Bank (PDB) under the ID 4BDT [85] and 4EY6 [86] were used to prepare the starting structure to run the Molecular Dynamics Simulations (MD). The ligand (galantamine) of the 4BDT structure was replaced by the different selenium derivatives before running the MD simulations. Both the system preparation and the simulations were performed in the AMBER 18 suite software. The protocol for the system preparation and the MD simulations is detailed as follows. Firstly, the system is neutralized by adding sodium ions and later immersed in a cubic box of 10 Å length, in each direction from the end of the protein, using TIP3P water parameters. The force fields used to obtain topography and coordinates files were ff14SB [87] and GAFF [88]. The first step of the simulation protocol followed to run the MD simulations is a minimization of the solvent molecules position only, keeping the solute atom positions restrained, and the second stage minimizes all the atoms in the simulation cell. Heating the system is the third step, which gradually raises the temperature 0 to 300 K under a constant volume (ntp = 0) and periodic boundary conditions. In addition, Harmonic restraints of 10 kcal·mol −1 were applied to the solute, and the Berendsen temperature coupling scheme [89] was used to control and equalize the temperature. The time step was kept at 2 fs during the heating phase. Long-range electrostatic effects were modelled using the particle-mesh-Ewald method [90]. The Lennard-Jones interactions cut-off was set at 8 Å. An equilibration step for 100 ps with a 2 fs time step at a constant pressure and temperature of 300 K was performed prior to the production stage. The trajectory production stage kept the equilibration step conditions and was prolonged for 500 ns longer at the 1 fs time step. In addition, the selenium derivatives required a previous preparation step where the parameters and charges were generated by using the antechamber module of AMBER, using the GAFF force field and AM1-BCC method for charges [91].

Synthesis of Target Compounds
We hypothesized that small molecules containing Se-allyl or Se-propargyl functionalities, mimicking active ingredients of natural products, would yield potent antitumor and anti-Alzheimer agents. Thus, a total of 38 new selenocompounds, grouped in two series, were obtained following the synthetic procedure depicted in Scheme 1. Series a comprises Se-allyl and series b consists of Se-propargyl, all of which are decorated with cynnamoyl, hydrocynnamoyl, adamantyl, phenyl, small heterocycles, and NSAID derivatives (see Figure 2). substitution over the allyl/propargyl bromide. Allyl/propargyl bromide are organic reagents with poor aqueous solubility, making the addition of tetrahydrofuran necessary to achieve good yields. The synthetic procedure was carried out in one-pot and yields ranging from 10 to 68 % were achieved. The structures of all the synthetic compounds were confirmed using spectroscopic methods ( 1 H NMR, 13  Compounds were synthesized following three reaction steps represented in Scheme 1. This synthesis procedure was previously reported with slight modifications [92]. First, the starting reagent sodium selenide (NaSeH), which was common to all the target compounds, was synthesized in good yields. For that, elemental Se was reduced by NaBH 4 in water. Once the selenating agent was synthesized, the corresponding acid chloride was added to the reaction mixture to form the corresponding sodium selenoate by a nucleophilic acyl substitution. The yield of this reaction step depends on the acid chloride aqueous solubility. Finally, the target compounds were obtained through a nucleophilic substitution over the allyl/propargyl bromide. Allyl/propargyl bromide are organic reagents with poor aqueous solubility, making the addition of tetrahydrofuran necessary to achieve good yields. The synthetic procedure was carried out in one-pot and yields ranging from 10 to 68% were achieved. The structures of all the synthetic compounds were confirmed using spectroscopic methods ( 1 H NMR, 13 C NMR, and 77 Se NMR).

Antioxidant Activity
One of the common features of cancer and AD is oxidative damage. Therefore, it is feasible to believe that the development of a molecule with antioxidant capacity could be beneficial for the prevention and/or treatment of both diseases. Taking this objective into account, radical scavenging ability of the synthesized compounds were evaluated using the DPPH assay. Firstly, determinations were performed at 0.03 mg/mL in the time range (0, 5, 15, 30, 60 and 90 min). Ascorbic acid and trolox were used as the positive controls. The data are expressed as a percentage of DPPH scavenging activity in at least three independent experiments performed in triplicates (Table S2). Compound 13a stood out as the only active compound showing a DPPH scavenging activity of 22.38% at 90 min. Thus, further determinations of compound 13a were performed at five different concentrations ranging from 3.00 × 10 −4 to 0.30 mg/mL and were recorded at different time points (15,30,60,90,120,150, and 180 min). The data are expressed as the percentage of cell growth ± SD in at least three independent experiments performed in triplicates (Table S3).
Compound 13a was able to scavenge the DPPH activity, with values that reached 76% of inhibition at 0.3 mg/mL (see Figure 3). It showed concentration-and time-dependent radical scavenging activity in vitro.

Antioxidant Activity
One of the common features of cancer and AD is oxidative damage. Therefore, it is feasible to believe that the development of a molecule with antioxidant capacity could be beneficial for the prevention and/or treatment of both diseases. Taking this objective into account, radical scavenging ability of the synthesized compounds were evaluated using the DPPH assay. Firstly, determinations were performed at 0.03 mg/mL in the time range (0, 5, 15, 30, 60 and 90 min). Ascorbic acid and trolox were used as the positive controls. The data are expressed as a percentage of DPPH scavenging activity in at least three independent experiments performed in triplicates (Table S2). Compound 13a stood out as the only active compound showing a DPPH scavenging activity of 22.38 % at 90 min. Thus, further determinations of compound 13a were performed at five different concentrations ranging from 3.00 × 10 −4 to 0.30 mg/mL and were recorded at different time points (15,30,60,90,120,150, and 180 min). The data are expressed as the percentage of cell growth ± SD in at least three independent experiments performed in triplicates (Table S3).
Compound 13a was able to scavenge the DPPH activity, with values that reached 76 % of inhibition at 0.3 mg/mL (see Figure 3). It showed concentration-and time-dependent radical scavenging activity in vitro.

Antiproliferative Activity
As a first approach, all the compounds were tested in vitro against lung (HTB-54), prostatic (DU-145), colon (HT-29), and triple-negative breast (MDA-MB-231) cancer cell lines at two concentrations (50 and 10 µ M) for 48 h. Then, the MTT colorimetric assay [67] was used to evaluate cell proliferation after the treatment. The data are expressed as the percentage of cell growth ± SD in at least three independent experiments performed in quadruplicates (Table S1). Results at 10 µ M concentration are summarized in Figure 4.

Antiproliferative Activity
As a first approach, all the compounds were tested in vitro against lung (HTB-54), prostatic (DU-145), colon (HT-29), and triple-negative breast (MDA-MB-231) cancer cell lines at two concentrations (50 and 10 µM) for 48 h. Then, the MTT colorimetric assay [67] was used to evaluate cell proliferation after the treatment. The data are expressed as the percentage of cell growth ± SD in at least three independent experiments performed in quadruplicates (Table S1). Results at 10 µM concentration are summarized in Figure 4.
As a first approach, all the compounds were tested in vitro against lung (HTB-54), prostatic (DU-145), colon (HT-29), and triple-negative breast (MDA-MB-231) cancer cell lines at two concentrations (50 and 10 µ M) for 48 h. Then, the MTT colorimetric assay [67] was used to evaluate cell proliferation after the treatment. The data are expressed as the percentage of cell growth ± SD in at least three independent experiments performed in quadruplicates (Table S1). Results at 10 µ M concentration are summarized in Figure 4.  As shown in Figure 4, some compounds were active under our experimental conditions. These results allowed us to determine some preliminary structure-activity relationships, since: • The presence of the propargyl group leads to a lower antiproliferative effect (series b) compared to compounds of series a, that showed more activity against the four cancer cell lines.

•
Among the compounds of series a: • The presence of the adamantyl ring (compound 1a) instead of a benzene ring (compound 2a) failed to increase the antiproliferative activity.

•
Among the different substituents of the phenyl ring, the incorporation of the chlorine atom in the "2" position of the ring (compound 3a) seems to not be important for the inhibition of cell viability, as no significant differences were observed with the unsubstituted phenyl ring compound (compound 2a). Nevertheless, the presence of the benzodioxol ring (compound 4a), which is a benzene derivative containing the methylenedioxyl functional group, appears to lose cytotoxic effect in HTB-54, DU-145, and MDA-MD-231 cancer cell lines, whereas it seems to be active in the HT-29 cancer cell line compared to the unsubstituted phenyl ring compound (compound 2a).

•
The unsaturation of the 2-carbon linker (compound 5a) between the phenyl ring and the carbonyl group seems to lose the cell inhibitory effect, since the saturated linker leads to a higher antiproliferative activity (compound 6a). Moreover, no significant differences were observed with the phenyl ring compound directly bonded to the carbonyl group (compound 2a).

•
The incorporation of a chlorine atom in position "2" of the pyridyl ring seems not to be important for the inhibition of the cell viability, since no significant As shown in Figure 4, some compounds were active under our experimental conditions. These results allowed us to determine some preliminary structure-activity relationships, since:

•
The presence of the propargyl group leads to a lower antiproliferative effect (series b) compared to compounds of series a, that showed more activity against the four cancer cell lines. • Among the compounds of series a: • The presence of the adamantyl ring (compound 1a) instead of a benzene ring (compound 2a) failed to increase the antiproliferative activity.

•
Among the different substituents of the phenyl ring, the incorporation of the chlorine atom in the "2" position of the ring (compound 3a) seems to not be important for the inhibition of cell viability, as no significant differences were observed with the unsubstituted phenyl ring compound (compound 2a). Nevertheless, the presence of the benzodioxol ring (compound 4a), which is a benzene derivative containing the methylenedioxyl functional group, appears to lose cytotoxic effect in HTB-54, DU-145, and MDA-MD-231 cancer cell lines, whereas it seems to be active in the HT-29 cancer cell line compared to the unsubstituted phenyl ring compound (compound 2a).

•
The unsaturation of the 2-carbon linker (compound 5a) between the phenyl ring and the carbonyl group seems to lose the cell inhibitory effect, since the saturated linker leads to a higher antiproliferative activity (compound 6a). Moreover, no significant differences were observed with the phenyl ring compound directly bonded to the carbonyl group (compound 2a).

•
The incorporation of a chlorine atom in position "2" of the pyridyl ring seems not to be important for the inhibition of the cell viability, since no significant differences were observed between the chlorine-substituted derivative and the unsubstituted derivative (compounds 8a and 7a, respectively). • Nevertheless, the incorporation of a chlorine atom in position "3" of the thiophenyl ring (compound 10a) does appear to increase the inhibition of cell viability compared to the unsubstituted ring (compound 9a).

•
The presence of a furyl ring instead of a thiophenyl ring leads to increased cytotoxic activity (compounds 9a and 11a, respectively). In contrast, the incorporation of the isoxazolyl ring in place of the furyl ring results in less inhibition of cell viability (compounds 11a and 12a, respectively).

•
NSAIDs derivatives appear to exert greater anti-proliferative activity than carboand hetero-cycle derivatives (compounds 13a-19a). Aspirin, ibuprofen, naproxen, and indomethacin derivatives (compounds 14a, 15a, 16a, and 17a, respectively) stand out by demonstrating cell viability of less than 55% at 10 µM against the panel of the four cancer cell lines. The mefenamic acid derivative (compound 18a) showed selectivity against the lung cancer HTB-54 cell line. The flumenamic acid derivative (compound 19a), however, showed potent anti-proliferative activity against three tumor lines (HTB-54, DU-145, and HT-29) with no activity against the MDA-MD-231 tumor line.
As shown in Figure 4, compounds 14a-17a were found to be the most active, as ones with a reduction of the cell growth greater than 45%, after 48h of treatment at 10 µM in at least 3 of the 4 tumor cell lines. Thus, they were selected to further investigate the cytotoxicity at seven concentrations between 0.1 and 100 µM against HTB-54 (lung), HT-29 (colon), HCT116 (colon), and MCF-7 (breast) cancer cell lines. Moreover, these compounds were also evaluated against mammary gland (184B5) and bronchial epithelium (BEAS-2B) nonmalignant cell lines, and the selectivity indexes (SI) were determined as the ratio of IC 50 values obtained for nonmalignant cells and the homolog cancer cells. IC 50 and SI values are shown in Table 3. The selected cancer cell lines displayed sensitivity to the action of these Se-derivatives. In this context, compounds 14a and 17a exhibited potent antiproliferative activity with IC 50 values below 10 µM in all tested cancer cell lines. Interestingly, these Se-NSAID derivatives showed greater antiproliferative activity than their parent drug (Table 3). Thus, the introduction of the selenoester moiety along with the allyl chain in the structure of NSAIDs led to far more potent analogs. However, these compounds exhibited SI values below 1.5 in breast and lung cancer. It is known that high SI values are desirable since they reflect efficacy with less toxicity. Remarkably, these NSAID derivatives that present the allyl chain in their structure, demonstrated slightly better cytotoxic activity than other Se-NSAID derivatives recently published in relation to MCF-7 cells [93]. Table 3. IC 50 values (in µM) for compounds, 14a-17a, and the parent NSAIDs in HTB-54, HT-29, HCT116, MCF-7, BEAS-2B and 184B5 cell lines, and selectivity indexes.

NCI-60 Analysis of the Compounds 14a and 17a
Compounds 14a and 17a showed the lowest IC 50 values against the tested cancer cell lines. Therefore, they were further submitted to the Developmental Therapeutics Program (DTP) of National Cancer Institute (NCI). Both compounds presented promising results in the One Dose (10 µM) assay against a panel of 60 cancer cell lines (Figures S1 and S2).  [94]. In addition, these compounds were highlighted for the potent antiproliferative activity against the most resistant cancer cell lines of the panel [95]. Compound 14a showed GI 50 values of 0.34 µM, 0.49 µM, 1.9 µM, 1.9 µM, and 1.5 µM against NCI-H322M (non-small cell lung), SNB-19 (CNS), SK-MEL-5 (melanoma), OVCAR-3 (ovarian), and OVCAR-8 (ovarian) cancer cell lines, respectively. Likewise, compound 17a demonstrated GI 50 values of 0.42 µM, 0.66 µM, 1.7 µM, 1.8 µM, and 1.7 µM against the same cancer cell lines, respectively. These outstanding results emphasize the successful design of these hybrid molecules combining NSAIDs and allylic fragments derived from natural products, to achieve molecules with high antiproliferative activity.

(CNS)
, SK-MEL-5 (melanoma), OVCAR-3 (ovarian), and OVCAR-8 (ovarian) cancer cell lines, respectively. Likewise, compound 17a demonstrated GI50 values of 0.42 µ M, 0.66 µ M, 1.7 µ M, 1.8 µ M, and 1.7 µ M against the same cancer cell lines, respectively. These outstanding results emphasize the successful design of these hybrid molecules combining NSAIDs and allylic fragments derived from natural products, to achieve molecules with high antiproliferative activity. C o l o n C a n c e r C N S C a n c e r M e l a n o m a O v a r i a n C a n c e r R e n a l C a n c e r P r o s t a t e C a n c e r B r e a s t C a n c e r O v a r i a n C a n c e r R e n a l C a n c e r P r o s t a t e C a n c e r B r e a s t C a n c e r

AChE Inhibition
With the aim of developing dual compounds for the treatment of cancer and AD, all compounds were evaluated as inhibitors of AChE enzyme. AChE enzyme has played a major role in AD, since clinical data have demonstrated that the brain of patients with AD

AChE Inhibition
With the aim of developing dual compounds for the treatment of cancer and AD, all compounds were evaluated as inhibitors of AChE enzyme. AChE enzyme has played a major role in AD, since clinical data have demonstrated that the brain of patients with AD have significant neurodegeneration, reduced cholinergic neurons, and a severe deficiency of acetylcholine (ACh) [68]. Therefore, inhibitors of this enzyme have been developed for the treatment of this disease, such as galantamine, donepezil, rivastigmine, and tacrine.
All the synthetic derivatives were tested and commercially available galantamine was used as a reference standard. In the screening at 1 µM (see Table 4), most of the compounds showed a moderate AChE inhibition, with values ranging from 15 to 30%, and no significant differences were observed between allyl and propargyl derivatives. However, compounds 10a, 13a and 14a demonstrated inhibitory activity similar to galantamine, with inhibition values of 42.46%, 51.80%, and 43.46%, respectively. Therefore, their dose-response curves were determined, and they showed IC 50 values in the low micromolar range (Table 4). Compound 10a, presenting both the 2-(3-chloro)thiophenyl and allyl fragments, exhibited an IC 50 value of 2.4 µM, which is comparable to galantamine activity. Compound 13a, the salicyl and allyl derivative, showed an IC 50 value greater than galantamine, and compound 14a (the aspirin and allyl derivative), demonstrated an IC 50 of 0.9 µM lower than the IC 50 value of galantamine. Thus, compound 14a has exhibited similar or even slightly greater AChE inhibitory activity than galantamine. Figure 6 depicts the dose-inhibition curve of compound 14a and galantamine.

In Vitro Blood-Brain Barrier Permeation Assay
Good penetration through the blood-brain barrier (BBB) is a necessary condit drugs designed for the treatment of AD. Therefore, the BBB penetration of the comp that were found to be the most potent in vitro AChE inhibitors (10a, 13a, and 14a) to with galantamine were tested using the PAMPA-BBB assay [83]. This assay measu passive diffusion of a compound across a membrane coated with PBL. It is know compounds with a Pe value greater than 4 × 10 −6 cm s −1 can easily cross the BBB and the CNS, whereas compounds with a Pe value below 2 × 10 −6 cm s −1 cannot pass it pound 13a was tested but signs of degradation were shown through the experiment pounds 10a and 14a showed 3-fold higher Pe values (38.63 × 10 −6 and 36.92 × 10 −6 respectively) compared to galantamine (12.27 × 10 −6 cm s −1 ). Moreover, the simulatio formed with the preADMET predictor was confirmed by the experimentally ob data, as both studies suggest higher BBB penetration for compounds 10a and 14 pared to galantamine (reference drug). Results are depicted in Table 5.

In Vitro Blood-Brain Barrier Permeation Assay
Good penetration through the blood-brain barrier (BBB) is a necessary condition for drugs designed for the treatment of AD. Therefore, the BBB penetration of the compounds that were found to be the most potent in vitro AChE inhibitors (10a, 13a, and 14a) together with galantamine were tested using the PAMPA-BBB assay [83]. This assay measures the passive diffusion of a compound across a membrane coated with PBL. It is known that compounds with a Pe value greater than 4 × 10 −6 cm s −1 can easily cross the BBB and reach the CNS, whereas compounds with a Pe value below 2 × 10 −6 cm s −1 cannot pass it. Compound 13a was tested but signs of degradation were shown through the experiment. Compounds 10a and 14a showed 3-fold higher Pe values (38.63 × 10 −6 and 36.92 × 10 −6 cm s −1 , respectively) compared to galantamine (12.27 × 10 −6 cm s −1 ). Moreover, the simulation performed with the preADMET predictor was confirmed by the experimentally obtained data, as both studies suggest higher BBB penetration for compounds 10a and 14a compared to galantamine (reference drug). Results are depicted in Table 5.

Molecular Dynamics Simulations
The structure of AChE has been extensively studied since it was first characterized in 1991 [97]. The active site of AChE is not on the surface of the protein but is inside a 20 Å deep gorge lined with many aromatic residues. At the entrance to the gorge is the peripheral anionic site (PAS), where aromatic residues (predominantly tryptophan) interact with cationic ligands. This interaction can also be observed at the catalytic anionic site (CAS), which is located at the base of the gorge. PAS plays a key role in the binding and orientation of acetylcholine into the gorge. Acetylcholine transiently forms a π-cation interaction with tryptophan and the carbonyl of the acetyl group forms a weak hydrogen bridge with tyrosine further down the gorge. These interactions position the acetylcholine towards the active site. At the base of the gorge, a second tryptophan plays a key role in the CAS. Again, acetylcholine forms a π-cation interaction between its quaternary amine and the tryptophan ring. The acetyl group is bound in the acyl pocket, formed from further aromatic residues (Phe295, Phe297, and Trp236) lining the base of the gorge. This binding of acetylcholine to the CAS and the acyl pocket places it in the active site of the enzyme, where three amino acids, glutamate, histidine, and serine, known as the catalytic triad, are located [98,99].
To further investigate the interaction mode of compound 14a with recombinant human AChE (PDB code: 4BDT), we performed a series of molecular dynamics simulations. The ligand from the crystal was replaced by the allyl derivative prior to the molecular dynamic simulations. As shown in Figure 7, compound 14a establishes interactions in the PAS of the enzyme, at the entrance to the gorge. Tyr124 in grey color forms two hydrogen bonds with the oxygens from the two carbonyl functional groups (acetyl and selenoester groups) and Trp286 interacts with the aromatic ring through a CH-π interaction. Likewise, Tyr341 interacts with the same aromatic ring from the ligand through a π-π stacking interaction, and the allylic chain lies between Phe338 and Phe297 from the acyl binding pocket.
Given the results obtained in the in vitro inhibition of AChE (Table 4), we decided to perform molecular dynamics studies with compound 14b as well. Compounds 14a and 14b differ only in the presence of a triple (14b) or a double (14a) terminal bond in the alkyl chain; however, compound 14a showed higher in vitro AChE inhibition activity, similar to that of galantamine. Therefore, molecular dynamics studies were performed to analyze the interaction of compound 14b with the enzyme binding site and compare it with compound 14a. In this study, it was observed that with the majority conformation of compound 14b, the interactions with the protein were much weaker than in the case of compound 14a. As can be shown in Figure 7C, hydrogen bonds between the carbonyl groups and Tyr124 of PAS was not found, and only very weak CH-π or π-π stacking interactions were observed with Tyr124, Tyr341, and Trp286 (green) of PAS and the aromatic ring of the ligand. It was also revealed that the acyl binding site remains free (purple) for interaction with acetylcholine, which would explain why compound 14b is not a good inhibitor of AChE.
All these data seem to suggest that compound 14a prevents the interaction of acetylcholine with the aromatic residues of the PAS, which is critical for the molecule to settle into the active site. In addition, it appears that the allylic chain plays a key role, as it would hinder the interaction of the acyl binding site residues with acetylcholine, preventing its proper placement in the CAS and the action of the catalytic triad.
All these data seem to suggest that compound 14a prevents the interaction of acetylcholine with the aromatic residues of the PAS, which is critical for the molecule to settle into the active site. In addition, it appears that the allylic chain plays a key role, as it would hinder the interaction of the acyl binding site residues with acetylcholine, preventing its proper placement in the CAS and the action of the catalytic triad.

Conclusions
Overall, 14a was identified as a dual anticancer and AChE inhibitory agent, showing a concentration-dependent inhibition. For example, to exert its AChE inhibitory capacity, it exhibited an IC50 value of 0.9 μM and for this concentration it showed no antiproliferative activity. In addition, it has been observed that the introduction of the allyl fragment, which is present in natural products such as garlic, as opposed to the propargyl fragment, provides antiproliferative activity, AChE inhibitory activity, and a slight radical scavenging ability. Compound 14a is the first Se-containing agent with dual in vitro antiproliferative and AChE inhibitory activities reported so far. The introduction of Se into molecules with potential AChE inhibitory activity is a novel strategy currently under development. Thereby, scientific references are scarce. Ebselen has been demonstrated to inhibit AChE

Conclusions
Overall, 14a was identified as a dual anticancer and AChE inhibitory agent, showing a concentration-dependent inhibition. For example, to exert its AChE inhibitory capacity, it exhibited an IC 50 value of 0.9 µM and for this concentration it showed no antiproliferative activity. In addition, it has been observed that the introduction of the allyl fragment, which is present in natural products such as garlic, as opposed to the propargyl fragment, provides antiproliferative activity, AChE inhibitory activity, and a slight radical scavenging ability. Compound 14a is the first Se-containing agent with dual in vitro antiproliferative and AChE inhibitory activities reported so far. The introduction of Se into molecules with potential AChE inhibitory activity is a novel strategy currently under development. Thereby, scientific references are scarce. Ebselen has been demonstrated to inhibit AChE activity with an IC 50 value of 29 µM, which is 32-fold higher than the IC 50 presented by compound 14a [100]. However, the compound selenepezil, a derivative of the fusion of donepezil and ebselen, exhibited potent AChE inhibition with an IC 50 value of 0.097 µM (9-fold lower than the IC 50 value of 14a). Additionally, selenepezil mimicks the activity of GPx and exhibits scavenging activity towards radical species generated by hydrogen peroxide in vitro [100].
On the other hand, no relation was found between radical scavenging ability and AChE inhibitory activity or antiproliferative activity. For anticancer activity, this was to be expected, since it has been observed that molecules with antioxidant capacity do not provide any benefit during the treatment of the disease, but rather worsen it by protecting the tumor cells from oxidative damage and, hence, promoting their progression. However, molecules with antioxidant activity might be favorable in the prevention of the disease. Previously, our research group has reported the synthesis of Se-molecules with antioxidant and cytotoxic activity in vitro, highlighting the acylselenourea and selenourea derivatives [36,73]. Therefore, a new approach could be to substitute the selenoester group of the synthesized molecules by the acylselenourea or selenourea group and study their antioxidant and antiproliferative capacity. On the other hand, ebselen, which acts as a glutathione peroxidase mimic, is widely known because of its potent antioxidant capacity [43].
Altogether, the work presented herein warrants future studies to further assess the in vivo efficacy, toxicity, and characterization of the mechanism of action of compound 14a in both cancer and AD models.   3-(4,5-dimethylthiazol-2-yl)-2, 5-