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

molecules 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 y, Hariteja Ramapuram y, 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. y 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

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