Novel Chrysin-De-Allyl PAC-1 Hybrid Analogues As Anticancer Compounds: Design, Synthesis, and Biological Evaluation

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Article Novel Chrysin-De-Allyl PAC-1 Hybrid Analogues as Anticancer Compounds: Design, Synthesis, and Biological Evaluation

1, 2, 2 3 Buthina A. Al-Oudat †, Hariteja Ramapuram †, Saloni Malla , Suaad A. Audat , Noor Hussein 2, Jenna M. Len 2, Shikha Kumari 4, Mel F. Bedi 5 , Charles R. Ashby, Jr. 6 and Amit K. Tiwari 2,*

1 Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan; [email protected] 2 Department of Pharmacology and Experimental Therapeutics, College of Pharmacy & Pharmaceutical Sciences, University of Toledo, Toledo, OH 43614, USA; [email protected] (H.R.); [email protected] (S.M.); [email protected] (N.H.); [email protected] (J.M.L.) 3 Department of Chemistry, College of Science and Arts, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan; [email protected] 4 Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198, USA; [email protected] 5 Department of Medicinal and Biological Chemistry, College of Pharmacy & Pharmaceutical Sciences, University of Toledo, Toledo, OH 43614, USA; [email protected] 6 Department of Pharmaceutical Sciences, College of Pharmacy & Pharmaceutical Sciences, St. John’s University, Queens, NY 11439, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-419-383-1913; Fax: +1-419-383-1909 These authors contributed equally to this work. † Academic Editor: Qiao-Hong Chen Received: 2 June 2020; Accepted: 30 June 2020; Published: 4 July 2020

Abstract: New chrysin-De-allyl-Pac-1 hybrid analogues, tethered with variable heterocyclic systems (4a–4o), were rationally designed and synthesized. The target compounds were screened for in vitro antiproliferative eﬃcacy in the triple-negative breast cancer (TNBC) cell line, MDA-MB-231, and normal human mammary epithelial cells (HMECs). Two compounds, 4g and 4i, had the highest eﬃcacy and selectivity towards MDA-MB-231 cells, and thus, were further evaluated by mechanistic experiments. The results indicated that both compounds 4g and 4i induced apoptosis by (1) inducing cell cycle arrest at the G2 phase in MDA-MB-231 cells, and (2) activating the intrinsic apoptotic pathways in a concentration-dependent manner. Physicochemical characterizations of these compounds suggested that they can be further optimized as potential anticancer compounds for TNBC cells. Overall, our results suggest that 4g and 4i could be suitable leads for developing novel compounds to treat TNBC.

Keywords: triple-negative breast cancer; cytotoxicity; chrysin analogues; ﬂavonoid; anticancer compounds

1. Introduction Breast cancer is the second leading cause of death among all cancers aﬀecting women [1]. Triple-negative breast cancer (TNBC) lacks the expression of hormone receptors (estrogen (ER) or progesterone (PR)) and/or human epidermal growth factor receptor 2 (HER2), which are more amenable to targeted therapy [2]. TNBC often presents as a high-grade invasive ductal carcinoma (IDC) in

Molecules 2020, 25, 3063; doi:10.3390/molecules25133063 www.mdpi.com/journal/molecules Molecules 2020, 25, 3063x FOR PEER REVIEW 2 of 21 20

(IDC) in patients, accounting for one-fourth of all breast cancer deaths. Consequently, there is an patients,urgent need accounting for the development for one-fourth of ofcompounds all breast cancerthat are deaths. efficacious Consequently, and safe for there the treatment is an urgent of needbreast for cancer. the development Flavonoids are of compoundsubiquitous thatnatura arelly e ffioccurringcaciousand polyphenolic safe for the compounds treatment ofthat breast are cancer.commonly Flavonoids found in are fruits ubiquitous and vegetables naturally [3]. occurring Flavonoids polyphenolic comprise several compounds classes that of low are commonlymolecular foundweight incompounds, fruits and vegetables including [ 3].flavanones, Flavonoids anthoc compriseyanidins, several flavonols, classes of flavanols, low molecular isoflavones, weight compounds,dihydroflavonols, including and flavanones,flavones [4–6]. anthocyanidins, Chrysin (5, 7- flavonols,dihydroxyflavone, flavanols, isoflavones,Figure 1) is dihydroflavonols,a natural flavone andfound flavones in many [4 plant–6]. Chrysinextracts, (5,such 7-dihydroxyflavone, as the blue passionflower, Figure1 )as is well a natural as in honey flavone and found propolis in many [7,8]. plantChrysin extracts, has been such reported as the blue to passionflower,have properties, as wellsuch as as in antioxidant honey and propolis[9], antihypertensive [7,8]. Chrysin [10], has beenantibacterial reported [11], to haveanti-inflammatory properties, such [12], as antiviral antioxidant [13], [antiallergic9], antihypertensive [14], antidiabetic [10], antibacterial [15], anxiolytic [11], anti-inflammatory[16], and anticancer [12 efficacy], antiviral [17,18]. [13], The antiallergic activation [14 of], antidiabeticapoptosis plays [15], a anxiolytic major role [16 in], andproducing anticancer the eanticancerfficacy [17 efficacy,18]. The of activation chrysin [19–21]. of apoptosis Mechanistically, plays a major apoptosis role in producing involves a the cascade anticancer of initiator efficacy and of chrysineffector [caspases19–21]. Mechanistically,[22]. Among these apoptosis caspases, involves caspase-3 a cascade and caspase-7 of initiator are anddownstream effector caspases executioner [22]. Amongcaspases thesethat play caspases, an essential caspase-3 role andin inducing caspase-7 apoptosis are downstream by cleaving executioner a variety of caspasescellular substrates that play an[23]. essential Therefore, role caspase-dependent in inducing apoptosis apoptosis by cleaving pathways a variety represent of cellular targets substrates for the development [23]. Therefore, of caspase-dependentefficacious anticancer apoptosis drugs. pathways Recently, represent a number targets of forstudies the development have been ofdone effi caciousto augment anticancer the drugs.pharmacological Recently, activity a number of chrysin of studies by producing have been synthetic done to analogues augment the[24–29]. pharmacological Moreover, there activity are ofstudies chrysin indicating by producing that chrysin-based synthetic analogues compounds [24 have–29]. in Moreover, vitro efficacy there inare brea studiesst cancer indicating cells [30–32]. that chrysin-basedThe compound, compounds de-allyl procaspase-activating have in vitro efficacy compound in breast cancer 1 (PAC-1), cells [30 induces–32]. The apoptosis compound, in different de-allyl procaspase-activatingtypes of cancer cells by compound activating 1caspase-3 (PAC-1), and/or induces caspase-7 apoptosis [33–36]. in diff erentPreviously, types ofwe cancer reported cells that by activatingmolecular caspase-3hybridization and /betweenor caspase-7 chrysin [33– 36and]. Previously,de-allyl PAC-1 we reportedcan be used that to molecular produce hybridizationnovel hybrid betweenmolecules chrysin with cytotoxic and de-allyl efficacy PAC-1 [29]. can Molecular be used to producehybridization novel is hybrid a process molecules that withcomprises cytotoxic the eamalgamationfficacy [29]. Molecular of two or hybridizationmore pharmacophoric is a process moieties that comprises of different the bioactive amalgamation molecules of twointo ora single more pharmacophoricmolecular framework moieties [37,38]. of di fferent bioactive molecules into a single molecular framework [37,38].

Figure 1. ChemicalChemical structures structures of of chrysin, chrysin, de-allyl de-allyl PAC-1, PAC-1, and a representative designed hybrid molecule 4a4a..

In this study, newnew chrysin-de-allylchrysin-de-allyl PAC-1 hybridhybrid analogues,analogues, substituted with variable aromatic heterocyclic cores, were synthesized and evaluated to identify a more potent bioactive hybrid against breast cancercancer cellscells (Figure(Figure1 ).1). The The in in silico silico parameters parameters of of the the synthesized synthesized compounds compounds were were calculated calculated to predictto predict their their pharmacokinetic pharmacokinetic proﬁle profile and and drug-likeness drug-likeness using using SwissSimilarity SwissSimilarity ADME, ADME, a web a web tool [tool39]. Subsequently,[39]. Subsequently, the target the target compounds compounds were screenedwere scre forened antiproliferative for antiproliferative eﬃcacy efficacy in the humanin the human breast cancerbreast cellcancer line MDA-MB-231,cell line MDA-MB-231, using the MTT 3-(4,5-using dimethylthiazol-2-yl)-2,5-Diphenyltetrazoliumthe MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5- Diphenyltetrazolium Bromide) colorimetric assay. Finally, we determined the effects of the most potent compounds on the cell cycle.

Molecules 2020, 25, 3063 3 of 20

Bromide) colorimetric assay. Finally, we determined the eﬀects of the most potent compounds on the cell cycle.

2. Results and Discussions

2.1. Chemistry Molecules 2020, 25, x FOR PEER REVIEW 3 of 21 The target compounds (4a–4o) were prepared according to Scheme1, starting from the 2. Results and Discussions commercially available chrysin, using previously published synthetic procedures [29]. Briefly, chrysin was added2.1. to Chemistry methyl 2-bromoacetate at a low temperature in the presence of K2CO3, yielding the desired alkylationThe target product compounds2. The conversion(4a–4o) were ofprepared ester 2 accordinginto hydrazide to Scheme3 was 1, starting accomplished from the using 80% hydrazinecommercially hydrate and available a few dropschrysin, of using hydrochloric previously acidpublished at low synthetic temperature. procedures1H [29]. and Briefly,13C NMR data confirmedchrysin the formation was added to of methyl hydrazide 2-bromoacetate3 as apure at a low single temperature product. in the Target presence compounds of K2CO3, yielding (4a– 4o) were the desired alkylation product 2. The conversion of ester 2 into hydrazide 3 was accomplished using obtained by80% adding hydrazine hydrazide hydrate and3 toa few an drops appropriate of hydrochloric aldehyde acid at in low the temperature. presence of1H aand catalytic 13C NMR amount of hydrochloricdata acid confirmed at room the temperature.formation of hydrazide All analogues 3 as a pure gave single adequate product.analytical Target compounds and spectroscopic (4a–4o) data, which werewere in fullobtained accordance by adding with hydrazide their structures. 3 to an appropriate1H and aldehyde13C NMR in spectrathe presence showed of a catalytic that compounds 4a–4o existedamount as geometricalof hydrochloric isomers acid at (Eroom/Z isomers).temperature. The All E:Z analogues ratio for gave each adequate compound analytical was and determined 1 13 1 spectroscopic data, which were in full accordance with their structures. H and C NMR spectra from H NMRshowed spectra that compounds utilizing 4a– the4o existed integration as geometrical of the isomers neat methylene(E/Z isomers). groupThe E:Z peaksratio for that each appeared as two separatedcompound singlets was determined for each from isomer. 1H NMR Target spectra compounds utilizing the integration were analyzed of the neat using methylene SwissSimilarity (Swiss Institutegroup peaks of Bioinformatics, that appeared as Lausanne, two separated Switzerland). singlets for each Compounds isomer. Target4a compounds–4j and 4n were–4o were the most structurallyanalyzed similarusing toSwissSimilarity apigenin, a flavonoid(Swiss Institute similar of toBioinformatics, chrysin. The Lausanne, physicochemical Switzerland). properties of Compounds 4a–4j and 4n–4o were the most structurally similar to apigenin, a flavonoid similar to the compoundschrysin. ( 4aThe– 4ophysicochemical) are shown properties in Table 1of. the compounds (4a–4o) are shown in Table 1.

Scheme 1.SchemeReagents 1. Reagents and conditions: and conditions: (a) ( methyla) methyl 2-bromoacetate, 2-bromoacetate, K2CO K23CO, DMF,3, DMF, 0 °C, 3 0h;◦ (C,b) hydrazine 3 h; (b) hydrazine hydrate 80%, EtOH, 0 °C, HCl (cat.), 1 h; (c) different substituted aldehydes, CH3OH, HCl (cat), rt, 1 hydrate 80%, EtOH, 0 ◦C, HCl (cat.), 1 h; (c) diﬀerent substituted aldehydes, CH3OH, HCl (cat), rt, 1 h. h. Table 1. Physiochemical properties of the compounds 4a–4o collected from Swiss ADME.

Table 1. Cont.

Solubility Pharmacokinetics Drug-Likeness Medicinal Chemistry Lipophilicity Compound MW GI BBB Lipinski Lead-likeness PAINS ESOL Class Log P Absorption Permeant Violations Violations Alerts Moderately 4g 446.41 Low No 0 2 2 2.71 soluble Moderately 4h 474.46 High No 0 3 0 3.65 soluble Moderately 4i 462.41 Low No 0 2 3 2.38 soluble Moderately 4j 462.41 Low No 0 2 2 2.43 soluble Moderately 4k 404.37 High No 0 2 0 2.81 soluble Moderately 4l 415.4 High No 0 2 0 2.66 soluble Moderately 4m 415.4 High No 0 2 0 2.72 soluble Poorly 4n 480.47 Low No 0 2 1 4.04 soluble Molecules 2020, 25, x FOR PEER REVIEW 1 of 21 Moderately 4o 475.41 Low No 1 3 1 2.52 Molecules 2020, 25, x FOR PEER REVIEWsoluble 1 of 21 Molecules 2020, 25, x FOR PEER REVIEW 1 of 21 2.2. 3-(4,5-DimethylthiESOL Class: Estimatedazol-2-yl)-2,5-Diphenyltetrazolium Solubility Class [40]. GI Absorption: Bromide-Based Gastrointestinal Cytotoxicity Absorption. Assay BBB Permeant: Blood Molecules 2020Brain, 25,Barrier x FOR PEER Permeant. REVIEW Lipinski Violations examines orally active compounds to determine ranges for1 highof 21 2.2. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide-Based Cytotoxicity Assay Molecules 2020probability, 25, x FOR to PEER be an oralREVIEW drug. Lead-likeness Violations examines compounds’ likeliness to become a lead based1 of on21 2.2. 3-(4,5-DimethylthiThe synthesizeda rule-based method azol-2-yl)-2,5-Diphenyltetrazoliumchrysin [41]. derivatives PAINS Alerts: were compounds evaluate that Br givedomide-Based for false cytotoxic positives Cytotoxicity whereefficacy the compoundin Assay the human nonspecifically breast cancer2.2. 3-(4,5-DimethylthiThe cell synthesizedbinds line, to numerous MDA-MB-231, azol-2-yl)-2,5-Diphenyltetrazoliumchrysin biological derivatives targets,using instead werethe ofevaluate3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium one desired Brdomide-Based for target cytotoxic [42]. Cytotoxicity Log efficacy P: partition in Assay coetheffi humancient measuring breast The synthesizedlipophilicity [41 chrysin]. derivatives were evaluated for cytotoxic efficacy in the human breast 2.2.bromidecancer 3-(4,5-Dimethylthi cell (MTT) line, colorimetric MDA-MB-231,azol-2-yl)-2,5-Diphenyltetrazolium assay andusing doxorubicin the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium was Br usedomide-Based as a reference Cytotoxicity anticancer Assay drug. The IC50 The synthesized chrysin derivatives were evaluated for cytotoxic efficacy in the human breast valuescancerbromide (concentrationcell (MTT) line, colorimetric MDA-MB-231, of compound assay and inusing µM doxorubicin requiredthe 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium towas reduce used 50% as a ofreference cell viability) anticancer are shown drug. inThe Table IC50 2.cancerbromidevalues 2.2.The (concentrationcell 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium(MTT)synthesized line, colorimetric MDA-MB-231, chrysinof compound assay derivatives and inusing µM doxorubicin wererequiredthe evaluate3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium towas reduce dused for Bromide-Based 50%ascytotoxic a referenceof cell efficacy viability) Cytotoxicity anticancer in are the Assayshown humandrug. Thein breast Table IC50 cancerbromide cell (MTT) line, colorimetric MDA-MB-231, assay andusing doxorubicin the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium was used as a reference anticancer drug. The IC50 values2. (concentrationThe synthesized of compound chrysin derivatives in µM required were to evaluated reduce 50% for of cytotoxic cell viability) efficacy are inshown the human in Table breast bromidevalues (concentration (MTT) colorimetric of compound assay and in µM doxorubicin required towas reduce used 50%as a referenceof cell viability) anticancer are shown drug. Thein Table IC50 2. cancerTable 2. cell The line, effects MDA-MB-231, of chrysin derivatives using the (4a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium–4o) on the survival of the MDA-MB-231 cancer cell line. bromide values2. Table (concentration 2. The effects of of compound chrysin derivatives in µM required(4a–4o) on to the reduce survival 50% of ofthe cell MDA-MB-231 viability) are cancer shown cell line. in Table (MTT) colorimetric assay and doxorubicin was used as a reference anticancer drug. The IC50 values 2. TableCompound 2. The effects of chrysin derivatives (4a–4o) Structureon the survival of the MDA-MB-231 cancerIC cell50 line.(µM) (concentration of compound in µM required to reduce 50% of cell viability) are shown in Table2. TableCompound 2. The effects of chrysin derivatives (4a–4o) Structureon the survival of the MDA-MB-231 cancerIC cell50 line.(µM) Compound Structure IC50 (µM) Table 2. The effects of chrysin derivatives (4a–4o) on the survival of the MDA-MB-231 cancer cell line. CompoundTable4a 2. The effects of chrysin derivatives (4aStructure–4o) on the survival of the MDA-MB-231 cancerIC6.850 ± (µM) cell2.76 line. Compound4a Structure IC6.850 ± (µM) 2.76 Compound Structure IC50 (µM) 4a 6.8 ± 2.76 4a 6.8 ± 2.76

4a 4a 6.8 2.766.8 ± 2.76 ± 4b 9.4 ± 3.38

4b 9.4 ± 3.38 4b 9.4 ± 3.38 4b 4b 9.4 3.389.4 ± 3.38 4b ± 9.4 ± 3.38

4c 23.3 ± 6.98

4c 23.3 ± 6.98 4c 4c 23.3 6.9823.3 ± 6.98 4c ± 23.3 ± 6.98 4c 23.3 ± 6.98

4d >100

4d 4d 100 >100 4d >100 4d >100 4d >100

4e 54.8a ± 5.7 a 4e 54.8 5.7 4e ± 54.8 ± 5.7 a 4e 54.8 ± 5.7 a 4e 54.8 ± 5.7 a 4e 54.8 ± 5.7 a

4f 11.9 ± 3.5 a

4f 11.9 ± 3.5 a 4f 11.9 ± 3.5 a 4f 11.9 ± 3.5 a 4f 11.9 ± 3.5 a

4g 5.98 ± 2.25

4g 5.98 ± 2.25 4g 5.98 ± 2.25 4g 5.98 ± 2.25 4g 5.98 ± 2.25

4h >100

4h >100 4h >100 4h >100 4h >100

Molecules 2020, 25, x FOR PEER REVIEW 1 of 21 Molecules 2020, 25, x FOR PEER REVIEW 1 of 21 Molecules 2020, 25, x FOR PEER REVIEW 1 of 21 2.2. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide-Based Cytotoxicity Assay 2.2. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide-Based Cytotoxicity Assay 2.2. 3-(4,5-DimethylthiThe synthesizedazol-2-yl)-2,5-Diphenyltetrazolium chrysin derivatives were evaluate Brdomide-Based for cytotoxic Cytotoxicity efficacy inAssay the human breast cancerThe cell synthesized line, MDA-MB-231, chrysin derivatives using werethe evaluate3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumd for cytotoxic efficacy in the human breast cancerThe cell synthesized line, MDA-MB-231, chrysin derivatives using werethe evaluate3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumd for cytotoxic efficacy in the human breast bromide (MTT) colorimetric assay and doxorubicin was used as a reference anticancer drug. The IC50 cancer cell line, MDA-MB-231, using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromidevalues (concentration (MTT) colorimetric of compound assay and in µM doxorubicin required towas reduce used 50%as a referenceof cell viability) anticancer are shown drug. Thein Table IC50 bromide (MTT) colorimetric assay and doxorubicin was used as a reference anticancer drug. The IC50 values2. (concentration of compound in µM required to reduce 50% of cell viability) are shown in Table values2. (concentration of compound in µM required to reduce 50% of cell viability) are shown in Table 2. Table 2. The effects of chrysin derivatives (4a–4o) on the survival of the MDA-MB-231 cancer cell line. Table 2. The effects of chrysin derivatives (4a–4o) on the survival of the MDA-MB-231 cancer cell line. TableCompound 2. The effects of chrysin derivatives (4a–4o) onStructure the survival of the MDA-MB-231 cancer ICcell50 line. (µM) Compound Structure IC50 (µM) Compound Structure IC50 (µM) 4a 6.8 ± 2.76 4a 6.8 ± 2.76 4a 6.8 ± 2.76

4b 9.4 ± 3.38 4b 9.4 ± 3.38 4b 9.4 ± 3.38

4c 23.3 ± 6.98 4c 23.3 ± 6.98 4c 23.3 ± 6.98

4d >100 4d >100 4d >100

Molecules4e2020 , 25, 3063 54.8 ± 5.7 a 5 of 20 4e 54.8 ± 5.7 a 4e 54.8 ± 5.7 a Table 2. Cont.

Compound Structure IC50 (µM) 4f 11.9 ± 3.5 a 4f 11.9 ± 3.5 a a 4f 4f 11.9 3.511.9a ± 3.5 ±

4g 5.98 ± 2.25 Molecules 20204g, 25 , x FOR PEER4g REVIEW 5.98 2.255.98 ± 2.252 of 21 Molecules 20204g, 25 , x FOR PEER REVIEW ± 5.98 ± 2.252 of 21 Molecules 2020, 25, x FOR PEER REVIEW 2 of 21 Molecules 2020, 25, x FOR PEER REVIEW 2 of 21 Molecules 2020, 25, x FOR PEER REVIEW 2 of 21 Molecules 20204i, 25 , x FOR PEER REVIEW 9.40 ± 1.452 of 21 4h 4h 100 >100 Molecules 20204h4i, 25 , x FOR PEER REVIEW 9.40>100 ± 1.45 2 of 21 4h4i 9.40>100 ± 1.45 4i 9.40 ± 1.45 4i 9.40 ± 1.45 4i 9.40 ± 1.45 4i 4i 9.40 1.459.40 ± 1.45 4j ± >100

4j >100 4j >100 4j >100 4j >100 4j >100 4j 4j 100 >100

4k 66.65 ± 11.10 4k 66.65 ± 11.10 4k 66.65 ± 11.10 4k 66.65 ± 11.10

4k 4k 66.65 66.6511.10 ± 11.10 4k ±66.65 ± 11.10 4k 66.65 ± 11.10

4l >100 4l >100 4l >100

4l 4l 100 >100 4l >100 4l >100 4l >100

4m >100 4m >100 4m >100 4m 4m 100 >100 4m >100 4m >100 4m >100

4n >100

4n >100 4n 4n 100 >100 4n >100 4n >100 4n >100 4n >100

4o 7.90 ± 1.82 4o 7.90 1.82 4o ± 7.90 ± 1.82 4o 7.90 ± 1.82 4o 7.90 ± 1.82 4o 7.90 ± 1.82 Doxorubicin4o Doxorubicin NANA 0.2 0.17.90 0.2 ±± 0.11.82 4o ± 7.90 ± 1.82 CellDoxorubicin survivalCell survival was was determined determined by by MTT MTT assay as as described describedNA in materialsin materials and methods. and methods. The IC50 Thevalues IC50 are0.2 values represented± 0.1 Doxorubicin NA a 0.2 ± 0.1 as mean SD of two independent experiments performed in triplicate. The data was takena from [29]. areCellDoxorubicin represented survival ±was as determinedmean ± SD of by two MTT indepe assayndent as described experimentsNA in materials performed and in methods. triplicate. The The IC data50 0.2 values was± 0.1 Cell survival was determined by MTT assay as described in materials and methods. The IC50 values takenareDoxorubicin represented from [43]. as mean ± SD of two independent experimentsNA performed in triplicate. a The data 0.2 was± 0.1 Cell survival was determined by MTT assay as described in materials and methods. Thea IC50 values takenareDoxorubicin represented from [43]. as mean ± SD of two independent experimentsNA performed in triplicate. The data 0.2 ±was 0.1 Cell survival was determined by MTT assay as described in materials and methods. Thea IC50 values aretakenDoxorubicin represented from [43]. as mean ± SD of two independent experimentsNA performed in triplicate. The data 0.2 was± 0.1 AsCellare shown represented survival in wasTable as determined mean 2, the ± SDstructure–activity ofby two MTT indepe assayndent as relati described experimentsonship in (SAR)materials performed results and in methods. indicated triplicate. The athat The IC thedata50 values cytotoxic was takenCell survival from [43]. was determined by MTT assay as described in materials and methods. The IC50 values efficacyAsare shownofrepresented the synthesized in Table as mean 2, the ±compou SD structure–activity of twonds indepe was ndentaffected relati experiments onshipby the (SAR)modifications performed results in indicatedtriplicate. on the benzene a that The datathe ring cytotoxic was (ring Astakenare shownrepresented from in[43]. Table as mean 2, the ± SD structure–activity of two independent relati experimentsonship (SAR) performed results in indicatedtriplicate. a that The datathe cytotoxic was D)efficacy oftaken the of parent fromthe synthesized [43]. compound compou 4a. Compoundnds was affected4a, possessing by the amodifications hydroxyl group on theat C-2benzene of ring ring D, (ring had efficacyAstaken shownof fromthe synthesized in[43]. Table 2, the compou structure–activitynds was affected relationship by the (SAR)modifications results indicated on the benzene that the ring cytotoxic (ring cytotoxicD) ofAs the shown efficacy,parent in compound Tablewith an2, the IC 4a50 structure–activity value. Compound of 6.8 µM. 4a, relati Topossessing determineonship a(SAR) hydroxylthe importance results group indicated of at the C-2 that hydroxyl of thering cytotoxic D, group had efficacyD) of the of parent the synthesized compound compou 4a. Compoundnds was affected4a, possessing by the amodifications hydroxyl group on the at C-2benzene of ring ring D, (ring had cytotoxicAs shown efficacy, in Table with 2,an the IC 50structure–activity value of 6.8 µM. relati To determineonship (SAR) the importance results indicated of the that hydroxyl the cytotoxic group atD)efficacy the ofAs C-2the shownof position,parent the synthesized in compound Table compound 2, the compou 4a structure–activity 4b. Compound wasnds synthesized, was affected4a ,relati possessing and onshipby its the efficacy a(SAR)modifications hydroxyl resultswas similargroup indicated on the toat compoundC-2benzene that of thering ring cytotoxic 4aD, .(ring Thehad atefficacycytotoxic the C-2 of efficacy,position, the synthesized with compound an ICcompou50 4bvalue wasnds of synthesized, was6.8 µM. affected To anddetermine by itsthe efficacy modifications the importance was similar on theof to the compoundbenzene hydroxyl ring 4a group .(ring The methylationcytotoxicD)efficacy of the of efficacy,parent the of synthesizedthe compound withhydroxyl an IC compou group4a50 value. Compound innds of 4a 6.8was yielded µM. affected4a, Topossessing compound determine by the amodifications 4c hydroxylthe, which importance hadgroup on a lowerofthe at the C-2benzene efficacyhydroxyl of ring ring than D, group (ring had 4a D)at theof theC-2 parentposition, compound compound 4a 4b. Compound was synthesized, 4a, possessing and its efficacy a hydroxyl was similargroup atto C-2compound of ring 4aD,. hadThe andmethylationcytotoxic 4b, whereas efficacy, of the ethylation withhydroxyl an IC group50of value the in hydroxof 4a 6.8 yielded µM.yl group,To compound determine which 4c theyielded, which importance hadcompound a lowerof the 4d, efficacyhydroxyl produced than group 4aa atmethylationD) the of C-2the parentposition, of the compound hydroxylcompound 4agroup 4b. Compound was in synthesized,4a yielded 4a, possessing compound and its efficacy a 4chydroxyl, which was similarhadgroup a lower atto C-2compound efficacy of ring than4aD,. Thehad 4a significantandcytotoxic 4b, whereasefficacy, decrease withethylation in cytotoxic an IC 50of value efficacy,the ofhydrox 6.8 compared µM.yl group,To determine to compoundwhich theyielded importance 4a. Previously,compound of the we 4d,hydroxyl reported produced group that a methylationatcytotoxic the C-2 efficacy,position, of the with hydroxylcompound an IC group50 4bvalue was in of synthesized,4a 6.8 yielded µM. To compound determineand its efficacy 4c the, which importance was similarhad a lowerof to the compound efficacyhydroxyl than4a group. The 4a significantatand the 4b C-2, whereas position, decrease ethylationcompound in cytotoxic of 4b efficacy, thewas hydrox synthesized, comparedyl group, and to compounditswhich efficacy yielded was4a. Previously,similarcompound to compound we4d, reported produced 4a. thatThe a theandmethylationat the shifting 4b C-2, whereas position, ofof thethe ethylation hydroxylcompound groupgroupof 4b the was in fromhydrox synthesized,4a yieldedC-2yl togroup, C-4compound and ( 4e whichits) efficacyproduced 4c yielded, which was a similarsignificanthadcompound a lower to compound reduction 4d,efficacy produced than4ain. Thethe 4aa themethylationsignificant shifting decrease of the hydroxylhydroxyl in cytotoxic groupgroup efficacy, infrom 4a yielded comparedC-2 to compoundC-4 to ( 4ecompound) produced 4c, which 4a .a Previously,hadsignificant a lower wereduction efficacy reported than in thatthe 4a significantandmethylation 4b, whereas decrease of the ethylationhydroxyl in cytotoxic groupof efficacy,the in hydrox 4a yieldedcomparedyl group, compound to compoundwhich 4c yielded, which 4a. Previously, hadcompound a lower we 4d,efficacy reported produced than that 4aa andthe shifting4b, whereas of the ethylation hydroxyl groupof the fromhydrox C-2yl togroup, C-4 (4ewhich) produced yielded a compoundsignificant reduction4d, produced in the a thesignificantand shifting4b, whereas decrease of the ethylationhydroxyl in cytotoxic groupof efficacy,the fromhydrox comparedC-2yl togroup, C-4 to ( 4ecompoundwhich) produced yielded 4a .a Previously, compoundsignificant wereduction4d, reported produced in thatthe a significant decrease in cytotoxic efficacy, compared to compound 4a. Previously, we reported that thesignificant shifting decrease of the hydroxyl in cytotoxic group efficacy, from comparedC-2 to C-4 to ( 4ecompound) produced 4a .a Previously, significant wereduction reported in thatthe the shifting of the hydroxyl group from C-2 to C-4 (4e) produced a significant reduction in the the shifting of the hydroxyl group from C-2 to C-4 (4e) produced a significant reduction in the

Molecules 2020, 25, 3063 6 of 20

As shown in Table2, the structure–activity relationship (SAR) results indicated that the cytotoxic efficacy of the synthesized compounds was affected by the modifications on the benzene ring (ring D) of the parent compound 4a. Compound 4a, possessing a hydroxyl group at C-2 of ring D, had cytotoxic efficacy, with an IC50 value of 6.8 µM. To determine the importance of the hydroxyl group at the C-2 position, compound 4b was synthesized, and its efficacy was similar to compound 4a. The methylation of the hydroxyl group in 4a yielded compound 4c, which had a lower efficacy than 4a and 4b, whereas ethylation of the hydroxyl group, which yielded compound 4d, produced a significant decrease in cytotoxic efficacy, compared to compound 4a. Previously, we reported that the shifting of the hydroxyl group from C-2 to C-4 (4e) produced a significant reduction in the cytotoxic efficacy (IC > 50 µM). However, methylation of the hydroxyl group in 4e yielded compound 4f, which 50 Molecules 2020, 25, x FOR PEER REVIEW 3 of 21 was ~5 times more efficacious than 4e [29]. The addition of a second hydroxyl group at the C-4 position in 4acytotoxicresulted efficacy in compound (IC50˃50 µM).4g (ICHowever,50 = 5.98 methylationµM), which of the had hydroxyl cytotoxic group effi incacy 4e similaryielded to compound 4acompound. Interestingly, 4f, which the was methylation ~5 times more of efficacious the two hydroxylthan 4e [29]. groups The addition in 4g ,of yielding a second compoundhydroxyl 4h, significantlygroup decreasedat the C-4 position the cytotoxic in 4a resulted effi cacy.in compound The addition 4g (IC50 = 5.98 of a µM), third which hydroxyl had cytotoxic group efficacy to 4g at similar to compound 4a. Interestingly, the methylation of the two hydroxyl groups in 4g, yielding position 3 of ring D, yielding compound 4i, did not significantly alter the antiproliferative efficacy compound 4h, significantly decreased the cytotoxic efficacy. The addition of a third hydroxyl group of the compound,to 4g at whereasposition 3 addingof ring D, a yielding third hydroxyl compound group 4i, didat not position significantly 6 of alter ring the D antiproliferative yielded a totally inactive compound,efficacy of 4jthe. compound, Next, we whereas determined adding the a third effect hydroxyl of ring group D on at theposition cytotoxic 6 of ring e ffiD cacyyielded of a the synthesizedtotally compounds. inactive Therefore,compound, 4j compounds. Next, we determined4k–4m were the effect synthesized, of ring D whereon the cytotoxic ring D was efficacy replaced of with aromaticthe heterocyclicsynthesized compounds. moieties. CompoundsTherefore, compounds4k, 4l, and4k–4m4m were, containing synthesized, furan, where 3-pyridine, ring D was and replaced with aromatic heterocyclic moieties. Compounds 4k, 4l, and 4m, containing furan, 3- 2-pyridine moieties, respectively, did not have significant cytotoxic efficacy. Furthermore, adding pyridine, and 2-pyridine moieties, respectively, did not have significant cytotoxic efficacy. another fusedFurthermore, benzene ring adding to ringanother D, whilefused benzene keeping ring the to C-2 ring hydroxyl D, while keeping group(2-hydroxynaphthalene), the C-2 hydroxyl group yielded the inactive(2-hydroxynaphthalene), compound, 4n yielded. The the addition inactive ofcompound, a nitro group 4n. The at addition the C5-position of a nitro group of ring at the D C5- in 4a yielded compoundposition 4oof ring, which D in had4a yielded an effi compoundcacy similar 4o, which to compound had an efficacy4a (IC similar50 = 7.9 to compoundµM). 4a (IC50 = Next, the7.9 cytotoxicityµM). and selectivity of the most active compounds, 4g and 4i, were determined in a panel of cellNext, lines, the including cytotoxicity the and normal selectivity cell lines,of the most HMECs, active and compounds, cancer cell4g and lines, 4i, were BT-20, determined U-251, and in a panel of cell lines, including the normal cell lines, HMECs, and cancer cell lines, BT-20, U-251, HCT116, as shown in Figure2 and Table3. Compound 4g had antiproliferative efficacy in the BT-20, and HCT116, as shown in Figure 2 and Table 3. Compound 4g had antiproliferative efficacy in the 4i U-251, and HCT116,BT-20, U-251, with and IC50 HCT116,values ofwith 5.32, IC 7.64,50 values and 2.68,of 5.32, respectively. 7.64, and 2.68, In contrast, respectively. compound In contrast,had IC50 values rangingcompound from 4i had 10–25 IC50 valuesµM. The ranging results from indicated 10–25 µM. thatThe resu althoughlts indicated compound that although4g is compound more potent than compound4g is 4imorein thepotent three than cancer compound cell lines,4i in the compound three cancer4i cellis more lines, selectivecompound for 4i is the more cancer selective cells for than the normal cells.the cancer cells than the normal cells. A

Figure 2. Cont.

Molecules 2020Molecules, 25, 3063 2020, 25, x FOR PEER REVIEW 4 of 21 7 of 20

Figure 2. The efficacy and selectivity of 4g and 4i in MDA-MB-231, BT-20, U-251, and HCT116, and compared to the normal cell line, HMEC (A) the viability curves for cancer cells (MDA-MB-231, BT-20, U-251,Figure HCT116), 2. The efficacy and and the selectivity normal of cell4g and line, 4i in HMEC; MDA-MB-231, (B) the BT-20, IC50 U-251,values and ofHCT116,4g and and4i for these cellcompared lines are to also the normal compared cell line, to HMEC the normal (A) the viability cell line. curves Cell for survivalcancer cells was (MDA-MB-231, determined BT- using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium20, U-251, HCT116), and the normal cell line, HMEC; (B) bromidethe IC50 values (MTT) of 4g assay. and 4i for The these IC50 cellvalues lines are representedare as also the meanscompared SDto ofthe three normal independent cell line. experimentsCell survival performedwas determined in triplicate using the with 3-(4,5- * p < 0.05, ± **** p < 0.0001dimethylthiazol-2yl)-2,5-diphenyltetrazolium vs. control group. bromide (MTT) assay. The IC50 values are represented as the means ± SD of three independent experiments performed in triplicate with * p < 0.05, **** p < Table 3. The0.0001 eff ectsvs. control of the group. chrysin-like compounds (4a–4o) on the survival of cancer cell lines (breast, glioblastoma, and colon) and a normal non-cancerous cell line (normal human mammary epithelial cells). Table 3. The effects of the chrysin-like compounds (4a–4o) on the survival of cancer cell lines (breast, Compoundglioblastoma, Breast and Cancercolon) and a normal Brain non-cancer Cancerous cell line Colon (normal Cancer human mammary Normal epithelial Cells cells). BT-20 U-251 HCT116 HMEC Compound Breast Cancer Brain Cancer Colon Cancer Normal Cells 4a 4.41 0.99 8.63 4.86 2.88 0.09 7.12 4.55 ± BT-20 ± U-251 HCT116± HMEC ± 4g 5.32 0.72 7.64 0.11 2.68 0.15 8.00 1.33 4a ± 4.41 ± 0.99 8.63± ± 4.86 2.88 ±± 0.09 7.12 ± 4.55 ± 4i 10.43 0.20 25.36 7.53 20.09 0.13 >100 4g ± 5.32 ± 0.72 7.64± ± 0.11 2.68 ± ±0.15 8.00 ± 1.33 4o 7.25 1.82 11.27 2.84 3.85 0.34 7.17 4.10 4i ± 10.43 ± 0.20 25.36± ± 7.53 20.09 ±± 0.13 >100 ± Cell survival was determined4o by MTT7.25 ± assay 1.82 as described 11.27 in± 2.84 the materials 3.85 and ± methods.0.34 The 7.17 IC50 ± values4.10 (µM) are represented as the mean SEM of three independent experiments performed in triplicate. The compounds were screened onCell breast survival (MDA-MB-231, was± determined BT-20), by brain MTT (U-251), assay as and described colon (HCT116) in the materials cancer celland linesmethods. and normalThe IC50 human mammary epithelialvalues (µM) cells are (HMECs). represented as the mean ± SEM of three independent experiments performed in triplicate. The compounds were screened on breast (MDA-MB-231, BT-20), brain (U-251), and colon (HCT116) cancer cell lines and normal human mammary epithelial cells (HMECs). 2.3. 4g and 4i Induce Apoptosis and G2 Cell Cycle Arrest in a Triple-Negative Breast Cancer Cell Line Apoptosis,2.3. 4g and a 4i type Induce of Apoptosis programmed and G2 Cell cell Cycle death, Arrest isin a one Triple-Negative of the major Breast Cancer mechanisms Cell Line by which chemotherapeuticApoptosis, drugs a type produce of programmed their therapeutic cell death, e ffiis cacyone of [43 the]. major Morphologically, mechanisms by apoptosis which is characterizedchemotherapeutic by cellular shrinkage,drugs produce which their is accompaniedtherapeutic efficacy with [44]. nuclear Morphologically, chromatin condensation apoptosis is and fragmentationcharacterized followed by cellular by blebbing shrinkage, of thewhich plasma is accompanied membrane. with nuclear This leads chromatin to the condensation formation and of small fragmentation followed by blebbing of the plasma membrane. This leads to the formation of small apoptoticapoptotic bodies that bodies have that an have intact an cellular intact cellular membrane membrane and unalteredand unaltered organelle organelle integrity. integrity. These These bodies are then releasedbodies are in then the released extracellular in the extracellular environment envi andronment removed and removed by the by process the process of phagocytosis of phagocytosis [44 ,45]. Apoptosis[45,46]. can occur Apoptosis by two can pathways: occur by two The pathways: extrinsic The pathway extrinsic pathway and intrinsic and intrinsic pathway. pathway. In either In either pathway, when the cellpathway, is exposed when the to certaincell is exposed extrinsic to certain or intrinsic extrinsic stimuli, or intrinsic the integrity stimuli, the of integrity the inner of mitochondrialthe inner membranemitochondrial of the cell ismembrane compromised, of the resultingcell is compromi in thesed, loss resulting of the mitochondrial in the loss of the membrane mitochondrial potential, and causing the release of several apoptotic factors, including cytochrome c [46,47]. Numerous studies indicate that during apoptosis, phosphatidylserine (PS), in the cytoplasmic side of the plasma membrane, is translocated to the extracellular cell surface [48]. The flipped anionic PS binds to the Ca2+-dependent phospholipid-binding protein, annexin V [49]. We discovered and reported several potent apoptosis-inducing compounds [50–57]. In this study, the result of our morphological studies in MDA-MB-231 cells after incubation with our lead compounds, 4g and 4i, indicated that apoptosis was Molecules 2020, 25, x FOR PEER REVIEW 5 of 21

membrane potential, and causing the release of several apoptotic factors, including cytochrome c [47,48]. Numerous studies indicate that during apoptosis, phosphatidylserine (PS), in the cytoplasmic side of the plasma membrane, is translocated to the extracellular cell surface [49]. The flipped anionic PS binds to the Ca2+-dependent phospholipid-binding protein, annexin V [50]. We discovered and Molecules 2020reported, 25, 3063 several potent apoptosis-inducing compounds [51–58]. In this study, the result8 of 20 of our morphological studies in MDA-MB-231 cells after incubation with our lead compounds, 4g and 4i, indicated that apoptosis was occurring (Figure 3A). At a concentration of 20 µM, both compounds occurring (Figure3A). At a concentration of 20 µM, both compounds decreased the number of adherent decreased the number of adherent MDA-MB-231 cells and induced cellular shrinkage. The cells were MDA-MB-231 cells and induced cellular shrinkage. The cells were rounded and loosely attached, rounded and loosely attached, and apoptotic bodies were present. Similarly, the incubation of MDA- and apoptotic bodies were present. Similarly, the incubation of MDA-MB-231 cells with compounds 4g MB-231 cells with compounds 4g or 4i for 24 h produced a significant loss of the mitochondrial or 4i for 24 h produced a significant loss of the mitochondrial membrane potential. For compound 4g, membrane potential. For compound 4g, the population of cells undergoing apoptosis increased from the population of cells undergoing apoptosis increased from 14.30% at 0 µM to 26.56% and 58.05% at 5 14.30% at 0 µM to 26.56% and 58.05% at 5 and 10 µM, respectively (p value < 0.0001; Figure 3B,C). and 10 µM, respectively (p value < 0.0001; Figure3B,C). Compound 4i also produced a significant shift Compound 4i also produced a significant shift in the apoptotic cell population in quadrant II, from in the apoptotic cell population in quadrant II, from 14.78% at 0 µM to 32.25% and 42.56% at 5 and 14.78% at 0µM to 32.25% and 42.56 % at 5 and 10 µM, respectively (p value < 0.0001, Figure 3B,C). 10 µM, respectively (p value < 0.0001, Figure3B,C). Cell cycle analysis indicated that compounds 4g and Cell cycle analysis indicated that compounds 4g and 4i produced a significant disruption in the cell 4i produced a significant disruption in the cell cycle of MDA-MB-231 cells (Figure3D). Data obtained cycle of MDA-MB-231 cells (Figure 3D). Data obtained using flow cytometry indicated that, when using flow cytometry indicated that, when incubated with vehicle alone, MDA-MB-231 cells had a incubated with vehicle alone, MDA-MB-231 cells had a normal cell cycle (5.43%, 83.4 %, 4.63%, and normal cell cycle (5.43%, 83.4%, 4.63%, and 4.46% in the subG1, G1, S, and G2 phases, respectively). 4.46% in the subG1, G1, S, and G2 phases, respectively). However, incubation of MDA-MB-231 cells However, incubation of MDA-MB-231 cells with compound 4g resulted in a significant shift towards with compound 4g resulted in a significant shift towards the G2 phase (59.12% and 54.13% for 5 and the G2 phase (59.12% and 54.13% for 5 and 10 µM, respectively (p value < 0.0001, Figure3E). 10 µM, respectively (p value < 0.0001, Figure 3E). 0 hr 24 hrs 48 hrs 72 hrs A

Control

4g(5μM)

4g(20μM)

4i(5μM)

4i(20μM)

Figure 3. Cont.

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B 4g(µM) 05 10 5 Q1 Q2 5 Q1 Q2 5 Q1 Q2 10 10 10 85.12% 14.30% 70.09% 26.56% 39.90% 58.05% 3 3 3 10 10 10

1 1 1 10 10 10 Q3 Q3 1 Q4 1 Q3 Q4 1 Q4 -10 -10 0.58% 0.00% -10 3.36% 0.00% 2.05% 0.00% 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 4i(µM) 05 10 5 Q1 Q2 5 Q1 Q2 5 Q1 Q2 10 83.16% 14.78% 10 63.94% 32.25% 10 53.08% 42.56%

3 3 3 10 10 10

1 1 1 10 10 10 MitoTracker Fluorescence Red Q3 Q3 Q4 Q3 Q4 Q4 1 1 1 -10 2.06% 0.00% -10 3.81% 0.00% -10 4.36% 0.00% 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Alexa Fluor 488 Dye Fluorescence C

Figure 3. Cont.

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D 4g(µM) 05 10 85 85 G2:59.12% 85 G1:24.68% G1:28.43% G1:83.40%

SubG1: 64 SubG1: 64 SubG1: 64 5.43% S:4.63% 0.82% 0.39% G2:54.13%

43 43 43 G2:4.46% S:12.35% S:17.67%

21 21 21

0 0 0 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 PE A 4i(µM)

Cell count 05 10 85 85 85 G1:83.40% G1:33.41% G1:34.95% G2:48.15% G2:43.44%

64 SubG1: 64 SubG1: 64 SubG1: 5.43% S:4.63% 1.33% 1.56%

43 43 43 G2:4.46% S:19.88% S:16.76%

21 21 21

0 0 0 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 PE A FL2-H (PI) E

Figure 3. Effects of 4g and 4i on cellular morphology, mitochondrial membrane potential, and cell cycle Figure( A3.) Effects Morphological of 4g and observations 4i on cellular of MDA-MB-231morphology, cellsmitochondrial incubated membrane with 0, 5, and potential, 20 µM concentrationsand cell cycle (A) Morphologicalof 4g and observations4i at different of time MDA-MB-231 intervals of cells 0, 24, incu 48,bated and 72 with h, respectively; 0, 5, and 20 µM (B) Theconcentrations MDA-MB-231 of 4g cells and in 4i at differentcomplete time intervals medium of were0, 24, incubated48, and 72 with h, respectively;4g and 4i at (B 0,) 5,The or MDA-MB-231 10 µM for 24 h. cells Cells in werecomplete then medium incubated were incubatedwith with the 4g reagents and 4i at of 0, the 5, or MitoTracker 10 µM for 24 Red h. Cells and were Alexa then Fluor incubated 488 annexin with the V kitsreagents for flow of the cytometry. MitoTracker Red andRepresentative Alexa Fluor 488 results annexin of MDA-MB-231 V kits for flow cells cytometry. from two Representative independent experiments, results of MDA-MB-231 each performed cells in from two independenttriplicate, are experiments, shown; (C) Histograms each performed quantitatively in triplicate, summarize are theshown; results (C following) Histograms incubation quantitatively with summarize4g and the4i results, respectively; following (D incubation) The induction with 4g of and cell 4i cycle, respectively; arrest in MDA-MB-231(D) The induction cells of by cell4g cycleand arrest4i is in MDA-MB-231shown. cells The MDA-MB-231by 4g and 4i is cellsshown. were The incubated MDA-MB-231 with di cellsfferent were concentrations incubated with (0, different 5, and 10 concentrationsµM) of 4g (0, 5, andand 104i µM)for of 24 4g h and 4i were for 24 subjected h and were to cell subjected cycle analysis to cell cycle by flow analysis cytometry by flow of cytometry PI (X axis) of/cell PI (X counts axis)/cell counts (Y(Y axis);axis); ((EE)) AA histogramhistogram quantitativelyquantitatively summarizingsummarizing the change in % of cells in each phase of the cell cycle duecell to cycle incubation due to incubation with 4g and with 4i.4g Theand data4i. represents The data represents means ± SEM means of threeSEM independent of three independent experiments ± performedexperiments in triplicate performed with **** in p triplicate < 0.0001 vs. with control **** p

2.4. Compounds 4g and 4i Activate Apoptosis by Activating the Intrinsic Apoptotic Pathway

Molecules 2020, 25, 3063 11 of 20

Molecules 2020, 25, x FOR PEER REVIEW 8 of 21 2.4. Compounds 4g and 4i Activate Apoptosis by Activating the Intrinsic Apoptotic Pathway ApoptosisApoptosis can be induced can be by induced the activation by the ofactivation two major of two pathways: major pathways: The intrinsic The and intrinsic extrinsic and extrinsic apoptotic pathwaysapoptotic [pathways22]. The activation [22]. The activation of the intrinsic of the apoptotic intrinsic pathwayapoptotic induces pathway the induces activation the activation of of proapoptoticproapoptotic proteins, such proteins, as apoptosis such as regulatorapoptosis Bakregulator (Bcl-2 Bak homologous (Bcl-2 homologous antagonist antagonist/killer)/killer) and Bax and Bax (Bcl-2-associated(Bcl-2-associated X protein) X [58 protein)]. The activated 59]. The theactivated Bax and the Bak Bax proteins and Bak subsequently proteins subsequently permeabilize permeabilize the mitochondrialthe mitochondrial outer membrane outer membrane by forming by pores forming on itspore outers on surface its outer [58 surface–60]. Consequently, [59–61]. Consequently, cytochromecytochrome c (Cyt C) is c released (Cyt C) intois released the cytosol, into the where cytosol, it combines where it with combines the adaptor with the protein adaptor (Apaf-1) protein (Apaf- to form an1) apoptosome to form an [apoptosome22]. The initiator [22]. The caspases initiator (i.e., caspases caspase-2, (i.e., caspase-8, caspase-2, caspase-9, caspase-8, or caspase-9, caspase-10) or caspase- are activated10) andare activated recruited and to large recruited protein to large complexes, protein resultingcomplexes, in resulting the cleavage in the of cleavage the executioner of the executioner caspases, caspase-3caspases, orcaspase-3 caspase-7 or [caspase-761]. [62]. Since MDA-MB-231Since MDA-MB-231 cells incubated cells with in compoundscubated with4g andcompounds4i had a decrease 4g and in the4i mitochondrialhad a decrease in the membranemitochondrial potential, which membrane is an early potential, event of which intrinsic is apoptosis,an early event i.e., altered of intrinsic permeability apoptosis, of the i.e., altered inner mitochondrialpermeability membrane, of the inner we mitochondrial conducted experiments membrane, to determinewe conducted if these experiments compounds to determine altered if these the expressioncompounds of key apoptotic altered the proteins, expression including of key cytochrome apoptotic c,proteins, in MDA-MB-231 including cells cytochrome using Western c, in MDA-MB- blotting analysis.231 cells Ourusing results Western indicated blotting that analysis. compounds Our results4g at 5 indicatedµM, and compoundthat compounds4i at 104g µatM 5 µM, and produced acompound significant 4i increase at 10 µM in produced the expression a significant of cytochrome increase c, in compared the expression to cells of incubated cytochrome with c, compared vehicle (Figureto cells4A,B). incubated This may with be duevehicle to an (Figure increase 4A,B). in the This expression may be ofdue Bak to following an increase incubation in the expression with of Bak 4g and 4i (Figurefollowing4A,B), incubation compared with to 4g cells and incubated 4i (Figure in 4A,B), the absence compared of lead to cells compounds. incubated In in addition, the absence of lead both compoundscompounds. (4g at In 5 µaddition,M and 4i bothat 10 compoundsµM) produced (4g at significant 5 µM and cleavage 4i at 10 µM) of the produced initiator significant caspase, cleavage caspase 9, inof MDA-MB-231the initiator caspase, cells, compared caspase to9, cellsin MDA-MB-231 incubated with cells, vehicle compared (Figure to4A,B). cells Theseincubated events with vehicle activated caspase(Figure 74A,B). in breast These cancer events cells incubatedactivated withcaspase both 7 concentrationsin breast cancer of 4g cellsand 10incubatedµM of 4i ,with both comparedconcentrations to cells incubated of 4g with and vehicle 10 µM (Figure of 4i,4 A,B).compared In contrast, to cells there incubated was no with significant vehicle change (Figure 4A,B). In in the mammaliancontrast, targetthere was of rapamycin no significant (mTOR) change expression, in the mammalian indicating target that cell of rapamycin death induced (mTOR) by 4g expression, and 4i in MDA-MB-231indicating that cells cell death is not induced due to autophagy by 4g and 4i (Figure in MDA-MB-2314A,B). Thus, cells our is results not due suggest to autophagy that (Figure cytochrome4A,B). c release Thus, induces our results intracellular suggest initiator that cytochrome caspase activation, c release followedinduces byintracellular the activation initiator of caspase executioneractivation, caspases, followed thus activating by the apoptotic activation cell of death executio machineryner caspases, through thus intrinsic activating the pathway.apoptotic cell death machinery through intrinsic the pathway. A

Figure 4. Cont.

MoleculesMolecules 20202020, ,2525, ,x 3063 FOR PEER REVIEW 129 of of 21 20

FigureFigure 4. 4. TheThe effect effect on on apoptosis apoptosis induction induction at at different different concentrations concentrations of of 4g4g andand 4i4i inin MDA-MB-231 MDA-MB-231 cells.cells. (A (A) )Western Western blots blots for for the the proteins proteins caspase caspase 3, 3, casp caspasease 7, 7, cleaved cleaved caspase caspase 7, 7, caspase caspase 9, 9, cleaved cleaved µ caspasecaspase 9, 9, Bak, Bak, cyt C and mTORmTOR followingfollowing incubation incubation with with4g 4gand and4i 4iat at 0, 0, 5, 5, or or 10 10M. µM. The The values values of theof β theproteins proteins were were normalized normalized to to-actin β-actin levels. levels. (B) (B Histograms) Histograms summarizing summarizing the the levels levels of eachof each protein protein are also shown. All the data are presented as the means SEM of three independent studies with * p < 0.05, are also shown. All the data are presented as the means± ± SEM of three independent studies with * p ** p < 0.01, *** p < 0.001 vs. control group. Cleaved is abbreviated as Clvd. < 0.05, ** p < 0.01, *** p < 0.001 vs. control group. Cleaved is abbreviated as Clvd. 3. Materials and Method 3. Materials and Method 3.1. Chemistry 3.1. Chemistry All chemicals and solvents were procured from commercial sources (reagent grade) and were used withoutAll chemicals further purification. and solvents The were reaction procured progress from was commercial monitored bysources thin layer (reagent chromatography grade) and were (TLC) usedusing precoatedwithout further TLC plates purification. of silica gel 60The F254. reacti1H-NMRon progress and 13C-NMR was spectramonitored were recordedby thin usinglayer a chromatography400 MHz Bruker Avance(TLC) using Ultrashield precoated spectrometer. TLC plates The of spectra silica weregel 60 obtained F254. 1H-NMR in ppm using and 13 automaticC-NMR 1 spectracalibration were to recorded the residual using proton a 400 peak MHz of Bruker the solvent, Avance dimethyl-sulphoxide Ultrashield spectrometer. (DMSO-d The6). spectra The H were NMR obtaineddata are presentedin ppm using as follows: automatic Chemical calibration shift ( δtoppm), the residual multiplicity proton (s = peaksinglet, of the d = solvent,doublet, dimethyl- t = triplet, sulphoxideq = quartet, (DMSO-d m = multiplet),6). The coupling1H NMR constants data are (Hz), presented and integration. as follows: The Chemical13C NMR shift analyses (δ ppm), were multiplicityreported in terms(s = singlet, of the d chemical = doublet, shift. t = Thetriplet,1H andq = quartet,13C NMR m spectra= multiplet), for all coupling compounds constants are included (Hz), andin the integration. supporting The information 13C NMR analyses (Figure were S1). HRMSreported data in terms were acquiredof the chemical using shift. a Thermo The 1H QExactive and 13C NMRPlus massspectra spectrometer for all compounds equipped are with included an electrospray in the supporting ionization information source (Thermo (Figure S1). Fisher HRMS Scientific, data wereGreensboro, acquired NC, using USA). a Thermo QExactive Plus mass spectrometer equipped with an electrospray ionization source (Thermo Fisher Scientific, Greensboro, NC, USA). 3.1.1. Synthesis of methyl 2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (2) 3.1.1. Synthesis of methyl 2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (2) The title compound was synthesized as previously described [62]. The title compound was synthesized as previously described [63]. 3.1.2. Synthesis of 2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetohydrazide (3) 3.1.2. TheSynthesis title compound of 2-((5-Hydroxy-4-oxo-2-phenyl-4 was synthesized accordingH-chromen-7-yl)oxy)acetohydrazide to our previously published procedure (3) [29]. The title compound was synthesized according to our previously published procedure [29]. 3.1.3. General Procedure for the Synthesis of N’-arylidene-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen- 7-yl)oxy)acetohydrazide (4a–4o) 3.1.3. General procedure for the synthesis of N’-arylidene-2-((5-Hydroxy-4-oxo-2-phenyl-4H- chromen-7-yl)oxy)acetohydrazideTo a stirred suspension of hydrazide(4a–4o) 3 (1.0 g, 3.0 mmol) in anhydrous methanol (60 mL), the appropriate aldehyde (3.0 mmol) was added, along with a few drops of concentrated hydrochloric To a stirred suspension of hydrazide 3 (1.0 g, 3.0 mmol) in anhydrous methanol (60 mL), the acid. After one hour, the reaction was completed and the formed precipitate was separated by filtration, appropriate aldehyde (3.0 mmol) was added, along with a few drops of concentrated hydrochloric washed with methanol, and dried in air to give pure compounds in good yields. acid. After one hour, the reaction was completed and the formed precipitate was separated by filtration, washed with methanol, and dried in air to give pure compounds in good yields.

(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-(2- hydroxybenzylidene)acetohydrazide (4a)

Molecules 2020, 25, 3063 13 of 20

(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-(2-hydroxybenzylidene)acetohydrazide (4a)

1 The product was obtained as a pale-yellow powder. Yield (64%). H NMR (DMSO-d6)(E:Z = 1:1): δ 12.84–12.81 (m, 2H), 11.84 (s, 1H), 11.62 (s, 1H), 10.99 (s, 1H), 10.05 (s, 1H), 8.57 (s, 1H), 8.33 (s, 1H), 8.12–8.10 (d, J = 8 Hz, 4H), 7.77–7.55 (m, 8H), 7.31–7.24 (m, 2H), 7.07–6.85 (m, 8H), 6.51–6.42 (m, 2H), 13 5.31 (s, 2H), 4.87 (s, 2H). C NMR (DMSO-d6): δ 182.11, 182.06, 167.94, 164.42, 163.65, 163.61, 163.48, 163.37, 161.17, 161.07, 157.36, 157.22, 156.45, 148.27, 141.63, 132.18, 132.12, 131.59, 131.29, 130.57, 130.54, 129.18, 129.15, 129.11, 126.56, 126.47, 120.01, 119.40, 118.65, 116.39, 116.14, 105.44, 105.39, 105.31, 105.09, + 98.81, 98.72, 93.62, 93.46, 66.57, 65.36. HRMS (ESI, m/z): calculated for C24H19N2O6 [M + H] 431.1237; found 431.1238.

(E,Z)-N’-Benzylidene-2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetohydrazide (4b) The synthesis and full characterization of the title compound have been previously reported [29].

(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-(2-methoxybenzylidene)acetohydrazide (4c)

1 The product was obtained as a yellow powder. Yield (55%). H NMR (DMSO-d6)(E:Z = 1:0.5): δ 12.84–12.80 (m, 1.5H), 11.64 (s, 1.5H), 8.68 (s, 0.5H), 8.37 (s, 1H), 8.11–8.09 (d, J = 8 Hz, 3H), 7.91–7.89 (d, J = 8 Hz, 1H), 7.82–7.80 (d, J = 8 Hz, 0.5H), 7.63–7.55 (m, 4.5H), 7.44–7.4 (m, 1.5H), 7.12–7.0 (m, 4.5H), 13 6.88–6.84 (m, 1.5H), 6.5–6.42 (m, 1.5H), 5.32 (s, 2H), 4.82 (s, 1H), 3.86 (s, 4.5H). C NMR (DMSO-d6): δ182.09, 182.03, 168.09, 164.43, 163.71, 163.60, 163.48, 163.22, 161.13, 161.05, 157.80, 157.64, 157.21, 143.56, 139.73, 132.15, 132.10, 131.72, 131.48, 130.55, 129.09, 126.45, 125.67, 125.53, 121.94, 120.72, 120.66, 111.84, 111.77, 105.42, 105.31, 105.06, 98.77, 98.70, 93.62, 93.45, 66.65, 65.36, 55.68, 54.87. HRMS (ESI, + m/z): calculated for C25H21N2O6 [M + H] 445.1394; found 445.1392.

(E,Z)-N’-(2-Ethoxybenzylidene)-2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetohydrazide (4d)

1 The product was obtained as a light-brown powder. Yield (80%). H NMR (DMSO-d6)(E:Z = 1:0.5): δ 12.84–12.80 (m, 1.5H), 11.68–11.62 (m, 1.5H), 8.66 (s, 0.5H), 8.40 (s, 1H), 8.11–8.10 (d, J = 4 Hz, 3H), 7.90–7.80 (m, 1.5H), 7.60–7.57 (m, 4.5H), 7.41–7.37 (m, 1.5H), 7.10–6.84 (m, 6H), 6.5–6.42 (m, 1.5H), 5.31 13 (s, 2H), 4.82 (s, 1H), 4.14–4.09 (q, J = 8 Hz, 3H), 1.39–1.35 (t, J = 8 Hz, 4.5H). C NMR (DMSO-d6): δ 182.08, 182.03, 168.03, 164.42, 163.77, 163.57, 163.46, 163.23, 161.14, 161.04, 157.20, 157.13, 156.98, 143.46, 140.01, 132.10, 131.67, 131.42, 130.54, 129.09, 126.45, 125.76, 125.58, 122.14, 122.09, 120.61, 112.75, 105.41, 105.29, 105.05, 98.71, 93.63, 93.45, 66.62, 65.36, 63.80,14.63. HRMS (ESI, m/z): calculated for + C26H23N2O6 [M + H] 459.1550; found 459.1548.

(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-(4-hydroxybenzylidene)acetohydrazide (3e) The synthesis and full characterization of the title compound were previously published [29].

(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-(4-methoxybenzylidene)acetohydrazide (3f) The synthesis and full characterization of the title compound were previously published [29].

(E,Z)-N’-(2,4-Dihydroxybenzylidene)-2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7- yl)oxy)acetohydrazide (4g)

1 The product was obtained as an oﬀ-white powder. Yield (85%). H NMR (DMSO-d6)(E:Z = 1:0.8): δ 12.84–12.80 (m, 1.8H), 11.67 (s, 1H), 11.43 (s, 0.8H), 11.15 (s, 1H), 9.98 (s, 1.8H), 9.83 (s, 0.8H), 8.42 (s, 1H), 8.20 (s, 0.8H), 8.12–8.10 (d, J = 8 Hz, 3.6H), 7.63–7.53 (m, 6.2H), 7.33–7.31 (d, J = 8 Hz, 1H), 7.08–7.06 (d, J = 8 Hz, 1.8H), 6.89 (d, J = 2 Hz, 1H), 6.8 (d, J = 2.4 Hz, 0.8H), 6.5 (d, J = 2 Hz, 1H), 6.4 (d, 13 J = 2 Hz, 0.8H), 6.36–6.30 (m, 3.6H), 5.26 (s, 1.6H), 4.83 (s, 2H). C NMR (DMSO-d6): δ182.10, 182.05, 167.42, 164.44, 163.68, 163.64, 163.50, 162.86, 161.14, 161.04, 160.86, 160.53, 159.35, 158.07, 157.23, 149.28, 142.68, 132.18, 132.12, 131.10, 130.55, 129.15, 129.12, 128.27, 126.47, 111.49, 110.37, 107.84, 107.77, 105.45, Molecules 2020, 25, 3063 14 of 20

105.37, 105.32, 105.07, 102.60, 102.37, 98.81, 98.72, 93.63, 93.45, 66.59, 65.33. HRMS (ESI, m/z): calculated + for C24H19N2O7 [M + H] 447.1186; found 447.1187.

(E,Z)-N’-(2,4-Dimethoxybenzylidene)-2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7- yl)oxy)acetohydrazide (4h)

1 The product was obtained as a yellow powder. Yield (96%). H NMR (DMSO-d6)(E:Z = 1:0.5): δ 12.81 (s, 1.5H), 11.49 (s, 1.5H), 8.58 (s, 0.5H), 8.27 (s, 1H), 8.11–8.10 (m, 3H), 7.84–7.57 (m, 6H), 7.05 (s, 1.5H), 6.88–6.84 (m, 1.5H), 6.63–6.41 (m, 4.5H), 5.29 (s, 2H), 4.79 (s, 1H), 3.86–3.82 (m, 9H). 13C NMR (DMSO-d6): δ182.04, 167.79, 164.43, 163.69, 163.55, 163.44, 162.91, 162.55, 162.33, 161.09, 161.02, 159.19, 159.02, 157.18, 143.62, 139.82, 132.10, 130.53, 129.09, 126.78, 126.43, 114.76, 106.41, 105.38, 105.28, 105.02, 98.68, 98.24, 98.07, 93.59, 93.42, 66.66, 65.35, 55.74, 55.40.HRMS (ESI, m/z): calculated for C26H23N2O7 [M + H]+ 475.1499; found 475.1499.

(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-(2,3,4- trihydroxybenzylidene)acetohydrazide (4i)

1 The product was obtained as a yellow powder. Yield (85%). H NMR (DMSO-d6)(E:Z = 1:0.5): δ 12.84–12.80 (m, 1.5H), 11.71 (s, 1H), 11.47 (s, 0.5H), 11.18 (s, 1H), 9.57–9.51 (m, 1.5H), 9.37 (s, 0.5H), 8.54–8.50 (m, 1.5H), 8.38 (s, 1H), 8.18–8.10 (m, 3.5H), 7.61–7.59 (m, 4.5H), 7.07–7.0 (m, 2H), 6.90–6.78 (m, 13 2.5H), 6.51–6.38 (m, 3H), 5.26 (s, 1H), 4.85 (s, 2H). C NMR (DMSO-d6): δ 182.12, 182.07, 167.34, 164.42, 163.68, 163.54, 162.93, 161.16, 161.07, 157.25, 150.45, 148.91, 148.42, 147.47, 146.65, 144.18, 132.76, 132.71, 132.21, 132.16, 130.56, 129.18, 126.49, 121.11, 118.36, 112.11, 110.70, 107.84, 107.73, 105.46, 105.40, 105.34, + 105.10, 98.83, 98.75, 93.65, 93.48, 66.59, 65.32. HRMS (ESI, m/z): calculated for C24H19N2O8 [M + H] 463.1134; found 463.1133.

(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-(2,4,6- trihydroxybenzylidene)acetohydrazide (4j)

1 The product was obtained as a brown powder. Yield (76%). H NMR (DMSO-d6)(E:Z = 1:0.2): δ 12.83–12.79 (m, 1.2H), 11.65 (s, 1H), 11.48 (s, 0.2H), 10.99 (s, 2H), 10.32 (s, 0.4H), 9.85 (s, 1.2H), 8.72 (s, 1H), 8.41 (s, 0.2H), 8.09–8.08 (m, 2.4H), 7.59–7.58 (m, 3.6H), 7.04 (m, 1.2H), 6.88–6.84 (m, 1.2H), 6.5–6.42 13 (m, 1.2H), 5.86–5.83 (m, 2.4H), 5.2 (s, 0.4H), 4.82 (s, 2H). C NMR (DMSO-d6): δ 182.08, 166.56, 164.28, 163.61, 163.51, 162.55, 161.74, 161.46, 161.12, 161.04, 159.73, 159.24, 157.20, 147.32, 144.33, 132.15, 130.54, 129.13, 126.45, 105.43, 105.37, 105.11, 98.85, 98.79, 94.37, 93.61, 93.50, 66.58, 65.26. HRMS (ESI, m/z): + calculated for C24H19N2O8 [M + H] 463.1134; found 463.1134.

(E,Z)-N’-(Furan-2-ylmethylene)-2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetohydrazide(4k)

1 The product was obtained as a yellow powder. Yield (87%). H NMR (DMSO-d6)(E:Z = 1:0.5): δ 12.83–12.79 (m, 1.5H), 11.63–11.57 (m, 1.5H), 8.23 (s, 0.5H), 8.11–8.09 (d, J = 8 Hz, 3H), 7.92 (s, 1H), 7.85 (s, 1.5H), 7.63–7.55 (m, 4.5H), 7.06–7.05 (m, 1.5H), 6.95–6.83 (m, 3H), 6.64 (m, 1.5H), 6.49–6.39 (m, 13 1.5H), 5.24 (s, 2H), 4.83 (s, 1H). C NMR (DMSO-d6): δ182.03, 167.97, 164.34, 163.66, 163.59, 163.46, 163.35, 161.13, 161.02, 157.21, 149.12, 148.94, 145.34, 145.10, 137.86, 134.20, 132.11, 130.54, 129.09, 126.45, 113.95, 113.78, 112.16, 105.42, 105.29, 105.08, 98.70, 93.60, 93.38, 66.65, 65.14.HRMS (ESI, m/z): calculated + for C22H17N2O6 [M + H] 405.1081; found 405.1077.

(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-(pyridin-3-ylmethylene)acetohydrazide (4l)

1 The product was obtained as a yellow powder. Yield (86%). H NMR (DMSO-d6)(E:Z = 1:0.5): δ 12.82(bs, 1.5H), 11.98 (bs, 1.5H), 8.43 (s, 0.5H), 8.30–8.28 (d, J = 8 Hz, 3H), 8.13–7.96 (m, 7H), 7.62–7.55 (m, 4.5H), 7.07–7.06 (m, 1.5H), 6.88 (s, 1.5H), 6.5–6.45 (m, 1.5H), 5.39 (s, 2H), 4.9 (s, 1H). 13C NMR (DMSO-d6): δ 182.12, 168.50, 164.42, 163.67, 163.55, 161.08, 157.27, 150.51, 148.52, 145.40, 141.22, 133.75, 132.20, 130.59, 129.96, 129.16, 126.51, 123.94, 105.36, 105.12, 98.76, 93.55, 66.61, 65.40. HRMS (ESI, m/z): + calculated for C23H18N3O5 [M + H] 416.1241; found 416.1240. Molecules 2020, 25, 3063 15 of 20

(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-(pyridin-2- ylmethylene)acetohydrazide (4m)

1 The product was obtained as a brown powder. Yield (52%). H NMR (DMSO-d6)(E:Z = 1:0.5): δ 12.84–12.81 (m, 1.5H), 11.89–11.87 (m, 1.5H), 8.61 (m, 1.5H), 8.35 (s, 0.5H), 8.11–8.03 (m, 5H), 7.94–7.87 (m, 2H), 7.62–7.55 (m, 4.5H), 7.44–7.41 (m, 1.5H), 7.07–7.06 (m, 1.5H), 6.88 (m, 1.5H), 6.51–6.45 (m, 1.5H), 13 5.37 (s, 2H), 4.88 (s, 1H). C NMR (DMSO-d6): δ 182.02, 168.45, 164.34, 163.75, 163.65, 163.59, 163.48, 161.13, 161.05, 157.21, 152.93, 152.78, 149.51, 149.44, 148.26, 144.45, 136.86, 136.75, 132.09, 130.53, 129.07, 126.44, 124.52, 124.34, 119.98, 119.90, 105.42, 105.30, 105.09, 98.70, 93.63, 93.48, 66.61, 65.30. HRMS (ESI, + m/z): calculated for C23H18N3O5 [M + H] 416.1241; found 416.1238.

(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-((2-hydroxynaphthalen-1- yl)methylene)acetohydrazide (4n)

1 The product was obtained as a yellow powder. Yield (54%). H NMR (DMSO-d6)(E:Z = 1:0.5): δ 12.86–12.81 (m, 1.5H), 12.46 (s, 1H), 11.91 (s, 1H), 11.68 (s, 0.5H), 10.77 (s, 0.5H), 9.40 (s, 1H), 8.89 (s, 0.5H), 8.77–8.75 (d, J = 8 Hz, 0.5H), 8.30–8.28 (d, J = 8 Hz, 1H), 8.12–8.09 (m, 3H), 7.94–7.84 (m, 3H), 7.63–7.58 (m, 6H), 7.42–7.36 (m, 1.5H), 7.24–7.21 (m, 1.5H), 7.08–7.06 (m, 1.5H), 6.94–6.88 (m, 1.5H), 13 6.55–6.46 (m, 1.5H), 5.38 (s, 1H), 4.94 (s, 2H). C NMR (DMSO-d6): δ182.09, 182.03, 167.52, 164.38, 163.64, 163.54, 163.49, 163.17, 161.17, 161.06, 157.95, 157.23, 156.88, 147.37, 143.29, 132.91, 132.52, 132.16, 132.10, 131.59, 131.25, 130.53, 129.13, 128.89, 128.68, 128.13, 127.80, 127.75, 126.46, 123.54, 123.40, 120.97, 118.75, 118.13, 110.08, 108.45, 105.45, 105.33, 105.10, 98.87, 98.73, 93.68, 93.46, 66.72, 65.59.HRMS (ESI, + m/z): calculated for C28H21N2O6 [M + H] 481.1394; found 481.1394.

(E,Z)-2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)-N’-((2-hydroxy-5- nitrobenzylidene)acetohydrazide (4o)

1 The product was obtained as a yellow powder. Yield (91%). H NMR (DMSO-d6)(E:Z = 1:0.75): δ 12.85–12.80 (m, 1.75H), 11.97 (bs, 2H), 11.78 (s, 1.5H), 8.64–8.54 (m, 2.75H), 8.33 (s, 1H), 8.15–8.10 (m, 5.25H), 7.64–7.55 (m, 5.25H), 7.08–7.04 (m, 3.25H), 6.90–6.86 (m, 1.75H), 6.52–6.44 (m, 1.75H), 5.39 (s, 13 2H), 4.89 (s, 1.5H). C NMR (DMSO-d6): δ 182.08, 182.03, 168.28, 164.42, 163.71, 163.61, 163.46, 162.04, 161.14, 161.04, 157.20, 144.30, 139.91, 138.65, 132.16, 132.10, 130.55, 129.12, 129.08, 126.73, 126.45, 123.37, 121.68, 120.98, 119.95, 117.10, 116.72, 105.43, 105.37, 105.30, 105.08, 98.78, 98.73, 93.62, 93.46, 66.53, 65.42. + HRMS (ESI, m/z): calculated for C24H18N3O8 [M + H] 476.1088; found 476.1089.

3.2. Biological Studies

3.2.1. Cell Lines and Cell Culture A panel of cancer cell lines, including breast (MDA-MB-231, BT20), brain (U251), and colon (HCT116), as well as a normal cell line (human mammary epithelial cells: HMECs), were grown as adherent monolayers in flasks with Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin in a humidified incubator with 5% CO2 at 37 ◦C.

3.2.2. MTT Assay The (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay was used to determine the cytotoxicity of the 15 chrysin derivatives in the above-mentioned cell lines. Briefly, cells were harvested with 0.05% trypsin, 2.21 mM ethylenediaminetetraacetic acid (EDTA), 1 from × Corning (Corning, NY, USA), and suspended at a final density of 5 103 cells/well. Cells were seeded × (180 µL/well) into 96-well plates. Initially, four different concentrations of each compound were added to find the compounds with the greatest antiproliferative efficacy in the MDA-MB-231 cell line (0, 1, 10, and 100 µM). Subsequently, eight different concentrations (0.1, 0.3, 1, 3, 10, 30, and 100 µM) of each compound were added to the remaining cell lines mentioned above. After 68 h of incubation, Molecules 2020, 25, 3063 16 of 20

20 µL of the MTT solution (4 mg/mL) were added to each well, and the plates were incubated for 4 h. This allowed viable cells to biotransform the yellow-colored MTT into dark-blue formazan crystals. Subsequently, the medium was discarded, and 150 µL of DMSO were added to each well to dissolve the formazan crystals. The absorbance was determined at 590 nm using a DTX 880 multimode detector (Beckman Coulter life sciences, IN, USA). The IC SD concentrations were calculated from three 50 ± experiments performed in triplicate/duplicate. The IC50 values were calculated from the cell survival percentages obtained for each compound tested at diﬀerent concentrations. Similarly, the cytotoxicity of the test compounds was compared to the normal cell line (HMEC).

3.2.3. Cell Cycle, Apoptosis, and Mitochondrial Membrane Potential Analysis MDA-MB-231 cells were plated into 6-well plates at 2.5 105 cells/well. The cells were incubated × with 0, 5, or 10 µM of compounds 4g or 4i and incubated for 12 h. Next, the cells were trypsinized with 0.05% trypsin, 2.21 mM EDTA, 1 , washed, counted, and resuspended in 1 mL of ice-cold PBS. The cells × then were stained with propidium iodide (PI) dye and incubated for at least 15 min. The distribution of the cells in each cell cycle phase for the different concentrations was measured using a BD Accuri™ C6 flow cytometer from BD Biosciences (Becton-Dickinson, San Jose, CA, USA) and analyzed using FCS Express 5 plus De Novo software (Glendale, CA, USA). MitoTracker Red and Alexa Fluor 488 annexin V kits for flow cytometry (Molecular Probes Inc., Invitrogen, Eugene, OR) were used to measure the mitochondrial membrane potential and apoptosis in MDA-MB-231 cells. Briefly, cells were seeded into 6-well plates and incubated with 0, 5, or 10 µM of compounds 4g and 4i for 12 h. The cells were then lysed using 0.05% trypsin, 2.21 mM EDTA, 1 , counted, and 4 µL of 10 µM of the MitoTracker Red working solution were added to 1 mL of the × harvested cells. The cells were incubated at 37 ◦C with 5% CO2 for 30 min. The cells were washed once with PBS and resuspended in 100 µL of the annexin binding buffer. The cell suspensions were incubated with 5 µL of Alexa Fluor 488 annexin V for 15 min. This was followed by the addition of 400 µL of the annexin-binding buffer. Finally, flow cytometry was used to detect the fluorescence of stained cells at the following excitation/emission maxima: Alexa Fluor® 488 annexin V: 499/521 nm; MitoTracker® Red: 579/599 nm with the BD Accuri™ C6 flow cytometer from BD Biosciences (Becton-Dickinson, San Jose, CA, USA) and analyzed using FCS express 5 plus De Novo software (Glendale, CA, USA).

3.2.4. Protein Expression Analysis Using Western Blot To measure the expression of Bak, cytochrome c, caspase-7, caspase-9, and mTOR, Western blotting was performed by lysing MDA-MB-231 cells using a lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1% Triton, 0.1% SDS) containing a protease inhibitor cocktail that consisted of Aprotinin, Bestatin, E-64, Leupeptin, and Pepstatin A (Sigma-Aldrich Life Science, St. Louis, MO, USA). The bicinchoninic acid (BCA) quantification assay was used to determine the protein levels in the cell extracts (G-BIOSCIENCES, St. Louis, MO, USA). The extracted proteins were loaded onto a 10–20% tris-glycine gel. After separation, the proteins were transferred from the gel onto a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked using 5% milk in Tris-buffered saline Tween 20 for 30 min and incubated overnight with primary antibodies against Bak (1:1000), cytochrome C (1:1000), caspase-3 (1:1000), caspase 7 (1:1000), caspase 9 (1:1000), mTOR (1:1000), or B-actin (1:2000) in 5% BSA (bovine serum albumin) at 4 ◦C. The next day, membranes were washed and incubated with horseradish peroxidase-labelled (HRP) anti-rabbit secondary antibody (1:5000 dilutions). The membrane was incubated with the antibody for an additional 1 h. Subsequently, the membranes were washed and developed by Clarity Western ECL substrate (Bio-Rad; Hercules, CA, USA). Protein was detected using a ChemiDoc Imaging System (Bio-Rad). Densitometry analyses of the blots for the detected protein were quantified using the ImageJ software. Data was calculated as ratios of protein/β-actin. Molecules 2020, 25, 3063 17 of 20

4. Conclusions In conclusion, a series of novel chrysin derivatives were designed, synthesized, and characterized. Upon screening these compounds for their antiproliferative eﬃcacy in the TNBC cell line, MDA-MB-231, and normal breast HMEC cells, two compounds, 4g and 4i, had the highest eﬃcacy and selectivity towards MDA-MB-231 cells. Upon investigating the mechanism by which these compounds produce cytotoxicity, it was determined that 4g and 4i cause the death of MDA-MB-231 cells by inducing apoptosis, producing cell cycle arrest at the G2 phase, and activating the intrinsic apoptotic pathway. Physicochemical characterizations of these compounds suggested that they can be further optimized as potential anticancer compounds for TNBC cells. The compounds were determined to have some solubility issues that need to be overcome in the future design of additional compounds. Overall, our results suggest that 4g and 4i could be suitable leads for developing novel compounds to treat TNBC.

Supplementary Materials: The following are available online, Figure S1: Chemical characterization of compounds 4a–4o. Author Contributions: Conceptualization, B.A.A.-O., S.A.A. and A.K.T.; methodology, B.A.A.-O., S.A.A., H.R., S.M., N.H. and J.M.L.; software, S.M. and N.H.; validation, S.M. and N.H.; formal analysis, B.A.A.-O.; investigation, H.R., S.M., N.H., J.M.L.; resources, B.A.A.-O. and A.K.T.; data curation, B.A.A.-O., H.R., S.M., N.H. and J.M.L.; writing—original draft preparation, B.A.A. and S.M.; writing—review and editing, B.A.A.-O., S.A.A., S.M., S.K., M.F.B., C.R.A.J. and A.K.T.; visualization, A.K.T.; supervision, A.K.T.; project administration, A.K.T.; funding acquisition, B.A.A.-O., and A.K.T. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the Deanship of Scientific Research at Jordan University of Science and Technology (grant # 20190360 to BAA). This manuscript has been supported, in part, by University of Toledo startup grants (F110760 to AKT) and Susan G. Komen Breast Cancer Foundation (CCR18548498 to AKT). Acknowledgments: The authors acknowledge the financial support given by the Deanship of Research at the Jordan University of Science and Technology (grant # 20190360 to BAA). The high-resolution mass spectrometry data were acquired from the Triad Mass Spectrometry Laboratory at the University of North Carolina at Greensboro, Greensboro, NC, USA. This manuscript has been supported, in part, by the University of Toledo startup grants (F110760 to AKT) and Susan G. Komen Breast Cancer Foundation (CCR18548498 to AKT). Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 4a, 4g, 4i and 4o are available from the authors on request.

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