Chalcone Derivatives: Promising Starting Points for Drug Design

molecules

Review Chalcone Derivatives: Promising Starting Points for Drug Design

Marcelo N. Gomes 1, Eugene N. Muratov 2, Maristela Pereira 3, Josana C. Peixoto 4, Lucimar P. Rosseto 4, Pedro V. L. Cravo 4,5, Carolina H. Andrade 1 and Bruno J. Neves 1,3,4,*

1 Laboratory for Molecular Modeling and Drug Design, Faculty of Pharmacy, Universidade Federal de Goiás, Setor Leste Universitário, Goiânia 74605-510, Brazil; [email protected] (M.N.G.); [email protected] (C.H.A.) 2 Laboratory for Molecular Modeling, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27955-7568, USA; [email protected] 3 Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia 74001-970, Brazil; [email protected] 4 Programa de Pós-Graduação em Sociedade, Tecnologia e Meio Ambiente, Centro Universitário de Anápolis—UniEVANGÉLICA, Anápolis 75083-515, Brazil; [email protected] (J.C.P.); [email protected] (L.P.R.); [email protected] (P.V.L.C.) 5 GHTM/Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, 1349-008 Lisboa, Portugal * Correspondence: [email protected]

Received: 16 June 2017; Accepted: 14 July 2017; Published: 25 July 2017

Abstract: Medicinal chemists continue to be fascinated by chalcone derivatives because of their simple chemistry, ease of hydrogen atom manipulation, straightforward synthesis, and a variety of promising biological activities. However, chalcones have still not garnered deserved attention, especially considering their high potential as chemical sources for designing and developing new effective drugs. In this review, we summarize current methodological developments towards the design and synthesis of new chalcone derivatives and state-of-the-art medicinal chemistry strategies (bioisosterism, molecular hybridization, and pro-drug design). We also highlight the applicability of computer-assisted drug design approaches to chalcones and address how this may contribute to optimizing research outputs and lead to more successful and cost-effective drug discovery endeavors. Lastly, we present successful examples of the use of chalcones and suggest possible solutions to existing limitations.

Keywords: natural products; chalcone derivatives; chalcone synthesis; molecular modiﬁcation strategies; computer-assisted drug design

1. Introduction Chalcones, or 1,3-diphenyl-2-propen-1-ones, are one of the most important classes of flavonoids across the whole plant kingdom [1,2]. Chalcones are open-chain precursors for biosynthesis of flavonoids and isoflavonoids and occur mainly as polyphenolic compounds whose colour changes from yellow to orange [3]. They exist as either trans (E, 1) or cis (Z, 2) isomers having two aromatic rings that are joined by a three-carbon α,β-unsaturated carbonyl system (Figure1). In most cases, the E isomer is more stable from the perspective of thermodynamics, which makes it the predominant configuration among the chalcones. The configuration of the Z isomer is unstable due to the strong steric effects between the carbonyl group and the A-ring [4]. The chemistry of chalcones remains a fascination among researchers in the 21st century due to the large number of replaceable hydrogens that allows a large number of derivatives and a variety of promising biological activities to be generated, e.g., anti-inflammatory [5], anti-gout [6], anti-histaminic [7],

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Importantly, two chalcones have been used in clinical practice (Figure 2). Metochalcone (3) was anti-oxidant [8], anti-obesity [9], anti-protozoal [10], hypnotic [11], anti-spasmodic [12], etc. Importantly, approved as a choleretic drug [2], and sofalcone (4) as an anti-ulcer agent that increases the amount two chalconesMolecules 2017 have, 22, 1210 been used in clinical practice (Figure2). Metochalcone (3) was approved2 of 24 as of mucosal prostaglandin, conferring a gastroprotective effect against Helicobacter pylori [13]. In a choleretic drug [2], and sofalcone (4) as an anti-ulcer agent that increases the amount of mucosal addition,Importantly, clinical twotrials chalcones have shown have beenthat usedhesperidin in clinical methylchalcone practice (Figure (5 2)., tested Metochalcone for chronic (3) was venous prostaglandin, conferring a gastroprotective effect against Helicobacter pylori [13]. In addition, lymphaticapproved insufficiency) as a choleretic [14,15], drug and[2], and hesperidin sofalcone trimethylchalcone (4) as an anti-ulcer agent(6, evaluated that increases for trunk the amount or branch clinical trials have shown that hesperidin methylchalcone (5, tested for chronic venous lymphatic varicosis)of mucosal [16] led prostaglandin, to the relieving conferring of symptoms, a gastroprotective reached reasonable effect against plasma Helicobacter concentrations, pylori [13]. and In were insufﬁciency)addition, [clinical14,15], andtrials hesperidin have shown trimethylchalcone that hesperidin methylchalcone (6, evaluated for (5, trunktested or for branch chronic varicosis) venous [16] well-tolerated. led tolymphatic the relieving insufficiency) of symptoms, [14,15], reached and hesperidin reasonable trimethylchalcone plasma concentrations, (6, evaluated and for weretrunk well-tolerated.or branch varicosis) [16] led to the relieving of symptoms, reached reasonable plasma concentrations, and were well-tolerated.

Figure 1. Structural and numerical representations of chalcone scaffold. Figure 1. Structural and numerical representations of chalcone scaffold. Figure 1. Structural and numerical representations of chalcone scaffold. Despite medicinal applications of chalcones, their wide bioactivity spectrum indicates a potentially promiscuous target proﬁle, which presents a challenge for clinical development [17]. This is largely promiscuousDespite target medicinal profile, applications which presents of chalcones, a challenge their wide for bioactivity clinical development spectrum indicates [17]. a potentiallyThis is largely attributable to the electrophilic nature of the α,β-unsaturated carbonyl system. This moiety is attributablepromiscuous to the target electrophilic profile, which nature presents of the α a, βchallenge-unsaturated for clinical carbonyl development system. This[17]. Thismoiety is largely is capable ofcapable formingattributable of irreversible forming to the irreversible electrophilic bonds with bondsnature biological of with the αmacromolecules, biological,β-unsaturated macromolecules, carbonyl resulting system. in resultinga This number moiety in of is atoxic capable number effects, of suchtoxic asof effects, formingallergenic such irreversible reactions, as allergenic bonds carcinogenicity, reactions, with biological carcinogenicity, and macromolecules, mutagenicity and resulting mutagenicity[18]. On inthe a numberother [18]. hand, Onof toxic the this othereffects, reactivity hand, maythis reactivitybesuch affected as allergenic may both be reactions, by affected the decoration carcinogenicity, both by theof the decorationand arom mutagenicityatic of rings, the [18]. aromatic and On also,the other rings, even hand, andmore this also, effectively, reactivity even more by αeffectively,-X-substitutionmay be by affectedα-X-substitution of the both double by the bond of decoration the of doublethe enone of bondthe syst arom ofem theatic [19]. enone rings, Therefore, systemand also, the [19 even design]. Therefore, more and effectively, synthesis the design byof new and analogssynthesisα-X-substitution are of particularly new analogs of the important double are particularly bond for theof the future enone important development system for [19]. the Therefore, of future clinically developmentthe designuseful and chalcone synthesis of clinically derivatives. of new useful chalconeanalogs derivatives. are particularly important for the future development of clinically useful chalcone derivatives.

FigureFigure 2. Chemical 2. Chemical structures structures of of approvedapproved and and clinically clinically tested tested chalcones. chalcones. Figure 2. Chemical structures of approved and clinically tested chalcones. Here,Here, we aimed we aimed to reviewto review recent recent data data on on computer-assistedcomputer-assisted design design and and synthesis synthesis of new of new chalcone chalcone Here,derivatives we aimed with improvedto review pharmacodynamics,recent data on comput pharer-assistedmacokinetics, design and toxicologicaland synthesis profiles; of new major chalcone derivatives with improved pharmacodynamics, pharmacokinetics, and toxicological proﬁles; major derivativeschallenges with in improvedthe field; and pharmacodynamics, possible solutions to pharexistingmacokinetics, pitfalls. and toxicological profiles; major challenges in the ﬁeld; and possible solutions to existing pitfalls. challenges in the field; and possible solutions to existing pitfalls. 2. Synthesis of Chalcone Scaffolds 2. Synthesis of Chalcone Scaffolds 2. SynthesisChalcones of Chalcone have a Scaffolds simple chemistry which enables a multiplicity of substitutions with easy Chalconessynthesis. Currently, have a simplea variety chemistry of methods which and sche enablesmes are a multiplicityavailable for the of synthesis substitutions of chalcone with easy Chalcones have a simple chemistry which enables a multiplicity of substitutions with easy synthesis.derivatives. Currently, In each a varietyof these ofmethods, methods the and most schemes important are part available is conden forsation the synthesis of two aromatic of chalcone synthesis. Currently, a variety of methods and schemes are available for the synthesis of chalcone derivatives.systems In (with each nucleophilic of these methods, and electrophilic the most important groups) to part yield is condensationthe chalcone scaffold. of two aromatic Despite systemsthe (withderivatives.multiplicity nucleophilic In each of andsubstitutions of electrophilicthese methods, allowed, groups) wethe describe tomost yield important thebelow chalcone the partreaction scaffold. is conden scheme Despitesation using the ofthe multiplicity twostandard aromatic of systemssubstitutionsscaffold (with of allowed, chalconesnucleophilic we (1,3-diphenyl-2-propen-1-one). describe and electrophilic below the reaction groups) scheme to yield using the the chalcone standard scaffold. scaffold ofDespite chalcones the (1,3-diphenyl-2-propen-1-one).multiplicity of substitutions allowed, we describe below the reaction scheme using the standard scaffold2.1. ofClaisen-Schmidt chalcones (1,3-diphenyl-2-propen-1-one). Condensation 2.1. Claisen-SchmidtAmongst all Condensation methods, the Claisen-Schmidt condensation (Scheme 1) is one of the most common. 2.1. Claisen-SchmidtIn this reaction, chalcones Condensation are form ed by condensation of benzaldehyde and acetophenone derivatives in Amongst all methods, the Claisen-Schmidt condensation (Scheme1) is one of the most common. Amongstthe presence all of methods, alkaline orthe acid Claisen-Schmidt catalysts in liquid condensa solvent attion 50–100 (Scheme °C for 1) several is one hoursof the [20–22]. most common. The In this reaction, chalcones are formed by condensation of benzaldehyde and acetophenone derivatives In this reaction, chalcones are formed by condensation of benzaldehyde and acetophenone derivatives in the presence of alkaline or acid catalysts in liquid solvent at 50–100 °C for several hours [20–22]. The

Molecules 2017, 22, 1210 3 of 25 Molecules 2017, 22, 1210 3 of 24 MoleculesMolecules 2017 2017,, ,22 22,, ,12101210 1210 33 of of 24 24 ◦ inconventional the presence Claisen-Schmidt of alkaline or reaction acid catalysts is typically in liquid carried solvent out in at the 50–100 liquidC phase, for several but certain hours reactions [20–22]. conventional Claisen-Schmidt reaction is typically carried out in the liquid phase, but certain reactions Theconventionalcan conventionaltake place Claisen-Schmidt in Claisen-Schmidtthe solid phase reaction (e.g., reaction acetophenoneis typically is typically carriedcarried derivatives out out in in the are the liquid primarily liquid phase, phase, bound but but certain certainto the resinreactions reactions and can take place in the solid phase (e.g., acetophenone derivatives are primarily bound to the resin and canthen take treated place with in the benzaldehyde solid phase (e.g., derivatives) acetophenone [22] or derivatives solvent-free are primarilyphase (e.g., boundbound condensation to the resin in andthe thenthen treatedtreated withwith benzaldehydebenzaldehyde derivatives)derivatives) [22][22] oror solvent-freesolvent-free phasephase (e.g.,(e.g., condensationcondensation inin thethe thenpresence treatedtreated of catalyst with with benzaldehyde benzaldehyde triazabicyclodecene) derivatives) derivatives) [23]. [22 In] or[22]addition, solvent-free or solvent-free the use phase of microwaves (e.g.,phase condensation (e.g., incondensation liquid in theand presencesolvent- in the presence of catalyst triazabicyclodecene) [23]. In addition, the use of microwaves in liquid and solvent- ofpresencefree catalyst Claisen-Schmidt of triazabicyclodecene) catalyst triazabicyclodecene) reactions reduces [23]. Insynthesis [23]. addition, In tiaddition,me the and use yieldsthe of use microwaves good of microwaves amounts in liquidof chalconesin liquid and solvent-freeand [24,25]. solvent- freeClaisen-Schmidtfree Claisen-Schmidt Claisen-Schmidt reactions reactions reactions reduces reduces reduces synthesis synthesis synthesis time ti timeme and and and yields yields yields good good good amounts amounts amounts of of of chalconeschalcones chalcones [[24,25]. 24[24,25].,25].

Scheme 1. The Claisen-Schmidt condensation. SchemeScheme 1. 1. The TheThe Claisen-Schmidt Claisen-Schmidt Claisen-Schmidt condensation. condensation. condensation. 2.2. Carbonylative Heck Coupling Reaction 2.2.2.2. Carbonylative Carbonylative Heck Heck Coupling Coupling Reaction Reaction In the carbonylative Heck coupling reaction (Scheme 2), chalcones are synthesized by carbonylative In the carbonylative Heck coupling reaction (Scheme 2), chalcones are synthesized by carbonylative vinylationIn the the carbonylativeof carbonylative phenyl halide Heck Heckwith coupling styrene coupling reaction in the reaction (Sch presenceeme (Scheme 2), of chalcones carbon2), chalcones monoxideare synthesized areand synthesizedusing by carbonylative palladium by vinylation of phenyl halide with styrene in the presence of carbon monoxide and using palladium carbonylativevinylation(Pd) as catalyst of phenyl vinylation [26]. halide of with phenyl styrene halide in withthe presence styrene inof thecarbon presence monoxide of carbon and using monoxide palladium and (Pd)using(Pd) as as palladium catalyst catalyst [26]. [26]. (Pd) as catalyst [26].

Scheme 2. Carbonylative Heck coupling reaction. SchemeScheme 2. 2. Carbonylative CarbonylativeCarbonylative Heck Heck Heck coupling coupling coupling reaction. reaction. reaction. 2.3. Coupling Reaction 2.3.2.3. Coupling Coupling Reaction Reaction Chalcones also are synthesized by a coupling reaction (Scheme 3) between benzaldehyde and Chalcones also are synthesized by a coupling reaction (Scheme 3) between benzaldehyde and phenylacetyleneChalcones also in the are presence synthesized of HBr by and a couplingcoupling ionic liquids, reactionreaction such (Scheme(Scheme as 1-butyl-3-methyl-13 3)) betweenbetween benzaldehydebenzaldehydeH-imidazolium andand phenylacetylene in the presence of HBr and ionicionic liquids,liquids, suchsuch asas 1-butyl-3-methyl-11-butyl-3-methyl-1H-imidazolium phenylacetylene4-methylbenzenesulfonate in the presence (BmimOTs) ofof HBrHBr for andand 12 ionicionic h at liquids,100liquids, °C [27]. suchsuch asas 1-butyl-3-methyl-11-butyl-3-methyl-1HH-imidazolium-imidazolium 4-methylbenzenesulfonate4-methylbenzenesulfonate (BmimOTs) (BmimOTs) for for 12 12 h h at at 100 100 °C ◦°CC[ [27]. [27].27].

Scheme 3. Coupling reaction. SchemeScheme 3. 3. Coupling CouplingCoupling reaction. reaction. reaction. 2.4. Sonogashira Isomerization Coupling 2.4. Sonogashira Isomerization Coupling 2.4. Sonogashira Isomerization Coupling In the Sonogashira isomerization coupling reaction (Scheme 4), chalcones are synthesized In the Sonogashira isomerization coupling reaction (Scheme 4), chalcones are synthesized throughIn thethe a Sonogashira reactionSonogashira between isomerization isomerization the equimolar coupling coupling concentr reaction reactionation (Scheme (Schemeof electron-deficient4), chalcones 4), chalcones are synthesized phenyl-halide are synthesized through and throughthrough aa reactionreaction betweenbetween thethe equimolarequimolar concentrconcentration of electron-deficient phenyl-halide and athroughpropargyl reaction a between alcoholreaction theemploying between equimolar themicrowave concentration equimolar irradiation concentr of electron-deficient andation using of electron-deficient PdCl phenyl-halide2(PPh3)2 as and catalyst phenyl-halide propargyl and THF alcohol and as propargylpropargyl alcoholalcohol employingemploying microwavemicrowave irradiationirradiation andand usingusing PdClPdCl222(PPh(PPh333))222 asasas catalystcatalystcatalyst andandand THFTHFTHF asasas employingsolvent [28,29]. microwave irradiation and using PdCl2(PPh3)2 as catalyst and THF as solvent [28,29]. solventsolvent [28,29]. [28,29].

Scheme 4. Sonogashira isomerization coupling. EWG: electron withdrawing group. SchemeScheme 4. 4. Sonogashira SonogashiraSonogashira isomerization isomerizationisomerization coupling. coupling.coupling. EWG: EWG:EWG: electron electronelectron withdrawing withdrawingwithdrawing group. group.group.

Molecules 2017, 22, 1210 4 of 25 MoleculesMolecules 20172017,,, 2222,,, 121012101210 44 ofof 2424

2.5.2.5. Continuous-FlowContinuous-Flow Continuous-Flow DeuteractionDeuteraction ReactionReaction InIn thethe continuous-flow continuous-ﬂowcontinuous-flow reactionreaction reaction (S(S (Schemechemecheme 55),5),), ynonesynones areare initiallyinitiallyinitially preparedprepared byby literatureliteratureliterature proceduresproceduresprocedures throughthrough thethe the couplingcoupling coupling ofof of benzoylbenzoyl benzoyl chlorideschlorides chlorides withwith with phphenylacetylenesenylacetylenes phenylacetylenes underunder under SonogashiraSonogashira Sonogashira conditionsconditions conditions (see(see above). Then, deuterations are carried out in an H-Cube®® ®system with replacement of the H2O above).above).(see above). Then,Then, Then, deuterationsdeuterations deuterations areare arecarriedcarried carried outout out inin inanan an H-CubeH-Cube H-Cube systemsystemsystem withwith with replacementreplacement replacement ofof of thethe the H H22OO hydrogen source to D2O deuterium source [30,31]. hydrogenhydrogen sourcesource to to D D22OOO deuteriumdeuterium deuterium sourcesource [30,31].[30,31]. [30,31].

SchemeScheme 5.5. Continuous-flowContinuous-flowContinuous-ﬂow deuteractiondeuteraction reaction.reaction.

2.6.2.6. Suzuki–MiyauraSuzuki–Miyaura Suzuki–Miyaura CouplingCoupling ReactionReaction InIn thethe Suzuki–MiyauraSuzuki–Miyaura couplingcoupling reactionreaction (Scheme(Scheme 66),6),), chalconechalcone synthesissynthesis involvesinvolves thethe couplingcoupling of benzoyl chloride with styrylboronic acid employing Pd(PPh3)4, CsCO3 and anhydrous toluene or ofof benzoyl benzoyl chloridechloride withwith with styrylboronicstyrylboronic styrylboronic acidacid acid employingemploying employing Pd(PPhPd(PPh Pd(PPh33))443,, )CsCO4CsCO, CsCO33 andand3 and anhydrousanhydrous anhydrous toluenetoluene toluene oror coupling of phenylboronic acid with cinnamoyl chloride employing Pd(PPh3)4, CsCO3, and anhydrous couplingcouplingor coupling ofof phenylboronicphenylboronic of phenylboronic acidacid withwith acid cinnamoylcinnamoyl with cinnamoyl chloridechloride chloride employingemploying employing Pd(PPhPd(PPh33))4 Pd(PPh4,, CsCOCsCO3,33, ) and4and, CsCO anhydrousanhydrous3, and toluenetolueneanhydrous [32].[32]. toluene [32].

SchemeScheme 6.6. Suzuki–MiyauraSuzuki–MiyauraSuzuki–Miyaura couplingcoupling reaction.reaction.

2.7.2.7. One-PotOne-Pot One-Pot SynthesisSynthesis TheThe one-potone-pot synthesissynthesis (Scheme(Scheme 77)7)) isis aa simple,simple, ye yetyett efficient efficientefficient greengreen methodmethod whichwhich allowsallows synthesissynthesis ofof chalconeschalcones inin justjust oneone one reactor.reactor. reactor. ThisThis methodmethod provprov providesidesides severalseveral advantages,advantages, suchsuch asas increasedincreased increased reactionreaction efficiency,efficiency,efficiency, and and avoidance avoidance ofof thethe lengthylengthy purificationpurification purification processprocess ofof thethe the intermediateintermediate intermediate chemicalchemical chemical compounds,compounds, compounds, therebythereby savingsaving resourcesresources andand timetime [22]. [[22].22]. TheThe The reactireacti reactiononon consists consists of of aa mixture mixture of of phenylmethanol phenylmethanol andand acetophenone in the presence of the oxidizing agent CrO3. In this reaction, CrO3 plays the vital role acetophenoneacetophenone in in thethe presencepresence of ofof the thethe oxidizing oxidizingoxidizing agent agentagent CrO CrOCrO3.33. In. InIn this thisthis reaction, reaction,reaction, CrO CrOCrO3 plays33 playsplays the thethe vital vitalvital role rolerole of ofofgenerating generatinggenerating the thethe benzaldehyde benzaldehydebenzaldehyde from fromfrom phenylmethanol, phenylmethanphenylmethanol,ol, which whichwhich further furtherfurther reacts reactsreacts with withwith the thethe acetophenone acetophenoneacetophenone to totoproduce produceproduce the thethe desired desireddesired chalcone chalconechalcone [22 [22].[22].].

SchemeScheme 7.7. One-potOne-pot synthesissynthesis ofof chalcones.chalcones.

2.8.2.8. SolidSolid Solid AcidAcid Acid CatalystCatalyst MediatedMediated ReactionReaction ChalconesChalcones havehave have alsoalso also beenbeen been synthesisynthesi synthesizedzedzed byby employingemploying by employing aa heterogeneousheterogeneous a heterogeneous solidsolid acidacid catalystsolidcatalyst acid (Scheme(Scheme catalyst 8).8). TheThe(Scheme reactionreaction8). Theconsistsconsists reaction ofof thethe consists additionaddition of ofof the anan addition equimolarequimolar of quantityquantity an equimolar ofof benzaldehydebenzaldehyde quantity of andand benzaldehyde phenylacetylenephenylacetylene and inin 1,2-dichloroethane1,2-dichloroethane solventsolvent irradiatedirradiated inin aa microwavemicrowave andand employingemploying ion-exchangeion-exchange resinresin amberlyst-15amberlyst-15 asas heterogeneousheterogeneous solidsolid acidacid catalystcatalyst [33].[33].

Molecules 2017, 22, 1210 5 of 25 phenylacetylene in 1,2-dichloroethane solvent irradiated in a microwave and employing ion-exchange Moleculesresin amberlyst-15 2017, 22, 1210 as heterogeneous solid acid catalyst [33]. 5 of 24

SchemeScheme 8. 8. SolidSolid acid acid catalyst catalyst mediated mediated synthesis. synthesis.

3.3. Design Design of of New New Chalcone Chalcone Derivatives Derivatives DuringDuring the the early early steps steps of of drug discovery proj projects,ects, an an intensive intensive search search ensues ensues to to find find pharmacologicallypharmacologically active active small small molecules with acceptable pharmacokinetic and toxicological properties,properties, which which are are candidates candidates for preclinical and c clinicallinical development [34]. [34]. The The activity activity of of a a molecule molecule cancan be be correlated correlated to to its its structure structure in in terms of the lipophilic, electronic, electronic, and and steric steric features features of of its its functionalfunctional groups. groups. Hence Hence by by investigating investigating the the proper proper functional functional group, group, one one ca cann govern govern the the biological biological patternpattern avoiding avoiding the the occurrence of complications in pre-clinical and clinical assays. The The schematic representationrepresentation ofof thethe nature nature of suchof such bioactive bioactive functional functional groups groups along withalong their with interatomic their interatomic distances distancesis known is as known pharmacophore. as pharmacophore. Once such Once a pharmacophore such a pharmacophore is identified, is molecular identified, modification molecular modificationstrategies can strategies be explored can tobe promote explored chemical to promote alterations chemical in altera the chalconetions in structurethe chalcone which structure lead to whichimprovements lead to inimprovements its physicochemical in its properties physicochemi and biologicalcal properties profile. and Among biological molecular profile. modification Among molecularstrategies used,modification bioisosterism, strategies molecular used, bioisosterism, hybridization, molecular and drug latentiationhybridization, are and the mostdrug widelylatentiation used areexamples. the most We widely address used each examples. of these strategies We address below, each highlighting of these strategies their application(s) below, highlighting in the design their of application(s)new chalcone in derivatives. the design Lastly,of new we chalcone want to derivatives. emphasize Lastly, that chalcones, we want asto emphasize perspective that agents chalcones, against ascancer, perspective bacteria, agents etc., wereagainst strongly cancer, underestimated bacteria, etc., were because strongly of the underestimated PAINS filtering because [35]. Recently, of the PAINSseveral filtering studies have[35]. demonstratedRecently, several the inabilitystudies have of PAINS demonstrated alerts to filter-out the inability promiscuous of PAINS compounds alerts to filter-outand their oversensitivity,promiscuous compounds i.e., rejection and of many their of oversensitivity, non-promiscuous i.e., compounds rejection [of36 ,37many]. Removal of non- of promiscuousPAINS filters fromcompounds the pipeline [36,37]. and Remo theirval substitution of PAINS by filters orthogonal from the assays pipeline [37,38 and] will their greatly substitution increase bythe orthogonal attractiveness assays of chalcones[37,38] will as greatly potential increase starting the points attractiveness in drug discovery. of chalcones as potential starting points in drug discovery. 3.1. Bioisosterism 3.1. Bioisosterism The term bioisostere was introduced by Harris Friedman in 1950. Recognizing the usefulness of isosterismThe term to designbioisostere bioactive was introduced molecules, Friedmanby Harris Fr definediedman bioisosteres in 1950. Recognizing as compounds the usefulness which fit the of isosterismbroadest definition to design for bioactive isosteres molecules, and have aFriedman similar type defined of biological bioisosteres activity, as compounds either through which agonist fit the or broadestantagonist definition actions [for39 ,isosteres40]. This and definition have a has similar been type further of biological expended activity, by Alfred either Burger through as compounds agonist or antagonistor groups thatactions possess [39,40]. near-equal This definition molecular has been shapes further and volumes,expended similarby Alfred physicochemical Burger as compounds properties, or groupsand which that possess exhibit near-equal approximately molecular the same shapes distribution and volumes, of similar electrons physicochemical [40,41]. Today, properties, the design and of whichbioisosteres exhibit frequently approximately introduces the structural same distribution changes that, of dependingelectrons [40,41]. on the context, Today, can the be design beneficial of bioisosteresor deleterious. frequently Electronic introduces density, stericstructural effects, change polarizability,s that, depending dipole, Logon theP and context, LogD can, maintenance be beneficial of orintermolecular deleterious. Electronic interactions, density, molecular steric conformation,effects, polarizability, LogS, and dipole, pKa potentiallyLogP and Log playD, key maintenance contributing of intermolecularroles in molecular interactions, recognition molecular and mimicry conformation, [42,43]. LogS, and pKa potentially play key contributing roles inThere molecular may be recognition innumerous and reasons mimicry for the [42,43]. use of bioisosterism to design new chalcone derivatives, includingThere themay necessity be innumerous to improve reasons pharmacological for the use of bi activity,oisosterism gain to selectivity design new for chalcone a particular derivatives, receptor includingor enzyme the with necessity simultaneous to improve reduction pharmacological of certain activity, adverse gain effects, selectivity or even for a forparticular optimizing receptor the orpharmacokinetics enzyme with simultaneous of the lead candidate reduction [39 ].of Forcertain example, adverse substituting effects, or a hydrogen even for atomoptimizing by fluorine the pharmacokineticsrepresents the most of conservativethe lead candidate example [39]. of For bioisosterism example, substituting given the similarities a hydrogen in stericatom by parameters fluorine represents(van der Waal’s the most radii conservative being 1.2 and example 1.35 Å, of respectively), bioisosterism but given there the are similarities circumstances in steric in drugparameters design (vanwhere der this Waal’s change radii can being offer 1.2 a significant and 1.35 Å, advantage respectively), [40]. but The there influence are circumstances of the electron-withdrawing in drug design whereeffect suchthis aschange fluorine can substitution offer a significant (fluorine advantag being thee most[40]. electronegativeThe influence of element the electron-withdrawing in the periodic table) effectis often such the as basis fluorine for the substitution major differences (fluorine in thebein biologicalg the most properties electronegative of compounds. element Fluorinein the periodic affects table) is often the basis for the major differences in the biological properties of compounds. Fluorine affects the bonding interactions and metabolic stabilities of compounds and has an impact on their physical features and selective reactivity [44]. Burmaoglu and colleagues [45] synthesized a series of bioisosteric molecules with anticancer activity, starting from a 2′,4′,6′-trimethoxy-chalcone (7) structure. Potent anticancer effects were reported for some of these bioisosteres. Of particular interest was

Molecules 2017, 22, 1210 6 of 25 the bonding interactions and metabolic stabilities of compounds and has an impact on their physical features and selective reactivity [44]. Burmaoglu and colleagues [45] synthesized a series of 0 0 0 bioisostericMolecules 2017 molecules, 22, 1210 with anticancer activity, starting from a 2 ,4 ,6 -trimethoxy-chalcone (7) structure.6 of 24 Molecules 2017, 22, 1210 6 of 24 Potent anticancer effects were reported for some of these bioisosteres. Of particular interest was derivativederivative 8 (Figure(Figure3 3),), displaying displaying potencies potencies(IC (IC50)) ranging ranging from from 0.030 to 0.120 μµM on a panelpanel ofof derivative 8 (Figure 3), displaying potencies (IC5050) ranging from 0.030 to 0.120 μM on a panel of cervicalcervical cancer cancer (HeLa), (HeLa), lung lung aden adenocarcinomaadenocarcinomaocarcinoma epithelial epithelial (A549), (A549), rena renalrenall cancer cancer (A498), (A498), skin skin malignantmalignant melanomamelanoma (A375), (A375), and and hepatocellularhepatocellular carcinomacarcinoma (HepG2)(HepG2) cellcell lineageslineages [[45].[45].45].

Figure 3. Bioisosterism represented by replacing the hydrogen (red) with a fluorine (blue) [45]. FigureFigure 3. 3. BioisosterismBioisosterism represented represented by by replacing replacing the hy hydrogendrogen (red) with a fluorine ﬂuorine (blue) [[45].45].

Another fine illustration of bioisosteric replacement has been documented for a series of antibacterial Another fine ﬁne illustration illustration of bioisost of bioisostericeric replacement replacement has been has document been documenteded for a series for of antibacterial a series of chalcones designed by Nielsen and colleagues [46]. Authors showed that the 4′-hydroxy group of the chalconesantibacterial designed chalcones by Nielsen designed and by colleagues Nielsen and [46]. colleagues Authors [ 46showed]. Authors that the showed 4′-hydroxy that the group 40-hydroxy of the A-ring in combination with lipophilic substituents in the B-ring was essential for the activity of A-ringgroup ofin thecombination A-ring in combinationwith lipophilic with substituents lipophilic substituentsin the B-ring in was the essential B-ring was for essential the activity for theof chalcones over Staphylococcus aureus. However, the modest antibacterial potency and the low water chalconesactivity of over chalcones Staphylococcus over Staphylococcus aureus. However, aureus. However, the modest the antibact modest antibacterialerial potency potency and the and low the water low solubility have limited the usefulness of these compounds. Considering this, the hydroxyl group of solubilitywater solubility have limited have limited the usefulness the usefulness of these of these compounds. compounds. Considering Considering this, this, the thehydroxyl hydroxyl group group of the 4′-hydroxy-2,4-dichloro-chalcone (9) was substituted by a carboxylic acid group while halogens theof the 4′-hydroxy-2,4-dichloro-chalcone 40-hydroxy-2,4-dichloro-chalcone (9) ( 9was) was substituted substituted by by a acarboxylic carboxylic acid acid group group while while halogens halogens were substituted by another hydrophobic group (Figure 4). The hydroxyl does not resemble were substitutedsubstituted by by another another hydrophobic hydrophobic group group (Figure (Figure4). The 4). hydroxyl The hydroxyl does not resembledoes not carboxylic resemble carboxylic acid in terms of size or as a strong electron-donating group, but total energy of H-bonding carboxylicacid in terms acid of in size terms or as of a size strong or as electron-donating a strong electron-donating group, but totalgroup, energy but total of H-bonding energy of interactionsH-bonding interactions and dramatic modification of the pKa confers to this group greater solubility. The interactionsand dramatic and modiﬁcation dramatic ofmodification the pKa confers of the to thispKa groupconfers greater to this solubility. group greater The substituents solubility. in The the substituents in the B-ring were systematically changed to obtain the most diverse set of analogues. substituentsB-ring were systematicallyin the B-ring were changed systematically to obtain the chan mostged diverse to obtain set ofthe analogues. most diverse The substituentsset of analogues. were The substituents were selected based on their size, lipophilicity, and electronic properties. Following Theselected substituents based on were their selected size, lipophilicity, based on their and size, electronic lipophilicity, properties. and Followingelectronic properties. these conditions, Following two these conditions, two bioisostere chalcones (10 and 11) showed minimum inhibitory concentrations thesebioisostere conditions, chalcones two bioisostere (10 and 11) chalcones showed minimum (10 and 11 inhibitory) showed concentrationsminimum inhibitory (MICs) concentrations of 2 µM, low (MICs) of 2 μM, low cytotoxicity (>100 μM) against mammalian cells, and markedly improved (MICs)cytotoxicity of 2 (>100μM, lowµM) cytotoxicity against mammalian (>100 μM) cells, against and markedlymammalian improved cells, and solubility. markedly Furthermore, improved solubility. Furthermore, the mechanism of action of 10 and 11 was different, as they were bacteriostatic, solubility.the mechanism Furthermore, of action the of mechanism10 and 11 wasof action different, of 10 and as they 11 was were different, bacteriostatic, as they whilewere bacteriostatic, the hydroxy while the hydroxy chalcones were bactericidal. This might be an advantage, as it is believed that whilechalcones the werehydroxy bactericidal. chalcones This were might bactericidal. be an advantage, This might as it be is believedan advantage, that bactericidal as it is believed compounds that bactericidal compounds are far more toxic to mammalian cells than bacteriostatic compound [46]. bactericidalare far more compounds toxic to mammalian are far more cells toxic than to bacteriostatic mammalian compound cells than bacteriostatic [46]. compound [46].

Figure 4. Bioisosterism represented by the replacing the hydroxyl and chloride groups (red) with a Figure 4. BioisosterismBioisosterism represented represented by by the the replacing replacing th thee hydroxyl hydroxyl and and chloride chloride groups groups (red) (red) with with a carboxylic acid and hydrophobic groups (blue) [46]. carboxylica carboxylic acid acid and and hydrophobic hydrophobic groups groups (blue) (blue) [46]. [46 ].

TheThe replacementsreplacements wherewhere cycliccyclic functionalfunctional moietymoiety mimicsmimics aa noncyclicnoncyclic groupgroup stericallysterically oror electronicallyelectronically alsoalso resultresult inin thethe retentionretention ofof biologicalbiological activity.activity. HereHere thethe abilityability ofof thethe structurestructure toto holdhold thethe criticalcritical functionalityfunctionality inin aa spatialspatial configurationconfiguration couldcould bebe essentialessential forfor activityactivity oror selectivity.selectivity. ForFor example,example, RomagnoliRomagnoli andand colleaguescolleagues [47][47] synthesizesynthesizedd aa seriesseries ofof thiophenethiophene diarylchalconesdiarylchalcones withwith anticanceranticancer activity,activity, inin whichwhich thethe doubledouble bondbond ofof thethe enoneenone systemsystem waswas embeddedembedded withinwithin aa thiophenethiophene ringring (see(see FigureFigure 5).5). TheThe synthesizedsynthesized compoundscompounds werewere foundfound toto inhibitinhibit thethe growthgrowth ofof severalseveral cancercancer

Molecules 2017, 22, 1210 7 of 25

The replacements where cyclic functional moiety mimics a noncyclic group sterically or electronically also result in the retention of biological activity. Here the ability of the structure to hold the critical functionality in a spatial conﬁguration could be essential for activity or selectivity. For example, Romagnoli and colleagues [47] synthesized a series of thiophene diarylchalcones with Moleculesanticancer 2017 activity,, 22, 1210 in which the double bond of the enone system was embedded within a thiophene7 of 24 ring (see Figure5). The synthesized compounds were found to inhibit the growth of several cancer cell lines at nanomolar to low micromolar concentrations. The replacement of the double bond with cell lines at nanomolar to low micromolar concentrations. The replacement of the double bond with a thiophene increases anticancer activity and must not significantly alter the relative conformation of a thiophene increases anticancer activity and must not signiﬁcantly alter the relative conformation of the chalcone derivate 12. Of particular interest was derivative 13, showing IC50’s ranging from 0.160 the chalcone derivate 12. Of particular interest was derivative 13, showing IC50’s ranging from 0.160 to 0.510 μM on a panel of HeLa, human T-lymphocyte (Molt-4 and CEM), murine leukemia (L1210), to 0.510 µM on a panel of HeLa, human T-lymphocyte (Molt-4 and CEM), murine leukemia (L1210), and murine mammary carcinoma (FM3A) cell lineages [47]. and murine mammary carcinoma (FM3A) cell lineages [47].

FigureFigure 5. BioisosterismBioisosterism represented represented by by the the replacing replacing th thee double double bond bond of of the the enone enone (blue) (blue) with with a thiophenethiophene (red) (red) [47]. [47].

3.2.3.2. Molecular Molecular Hybridization Hybridization TheThe hybridization hybridization of biologically biologically active active molecules, molecules, based based on on the the combination combination of of complementary complementary pharmacophoric features features of of two or more known bioactive compounds, which, through adequate fusion,fusion, lead lead to to the the design design of of new new hybrid hybrid architectures architectures that that maintain maintain pre-selected pre-selected characteristics characteristics of the of originalthe original templates, templates, is a powerful is a powerful strategy strategy for drug for drugdesign. design. There Thereare two are main two ways main through ways through which hybridwhich hybridmolecules molecules can be can constructed. be constructed. The Thefirst firstmethod method combines combines two two or or more more entire entire chemical structuresstructures together using a a linker linker chain, chain, whilst whilst th thee second second technique technique directly directly merges merges pharmacophoric pharmacophoric moietiesmoieties of different different compounds [48,49]. [48,49]. In In this this cont context,ext, to to improve improve the the efficacy efficacy of some chalcones in in thethe treatment ofof diseases diseases involving involving dual- dual- or or multi-target multi-target approaches, approaches, molecular molecular hybridization hybridization has been has beeninvestigated investigated for the for design the design of new of ligandsnew ligands or prototypes. or prototypes. Mao and and colleagues colleagues [50] [50] designed and synthesized synthesized a series of compounds compounds that have have a a chalcone chalcone derivatederivate ( 14) and and an an NN-aryl-aryl piperazine piperazine moiety moiety (see (see ciprofloxacin ciprofloxacin structure structure ( 15)) in one frame frame and and evaluatedevaluated them for anticancer anticancer activity activity (Figure (Figure 66).). Ciprofloxacin is a well-known inhibitor of human DNA topoisomerasetopoisomerase II II that that exhibits exhibits potent potent inin vitro anticanceranticancer activity against different cell lineages [51–54] [51–54] while chalcones are are known known to to inhibit inhibit several several anticancer anticancer targets, targets, including including thioredoxin thioredoxin reductase reductase [45], [45], andand tubulin polymerization [[55,56].55,56]. Therefore,Therefore, hybridhybrid moleculesmolecules thatthat contain N-aryl piperazine and and chalconechalcone moieties moieties are are expected to hold improved ac activities.tivities. As As a a result, synergic anticancer effects were reported for some of these hybrids. Hybrid 16 displayed the highest activity against cervical cancercancer (Hela) (Hela) and and gastric gastric ca cancerncer (SGC7901) cells with IC5050 ofof 0.190 0.190 μµMM and and 0.410 0.410 μµM,M, respectively, respectively, in comparisoncomparison to 20 μµM and 12 μµM for the positive control cisplatin [[50].50]. A series of hybrids made of 40-chlorochalcone (17), and the antimalarial drug chloroquine (18) were designed and tested for their antimalarial efficacy against chloroquine-resistant strain (K1) of Plasmodium falciparum [57] (see Figure7). Chloroquine interferes with the parasite detoxification process by inhibiting heme transformation into hemozoin. The accumulation of heme causes severe damage to the parasite, leading to its death [58,59]. The antimalarial property of chalcone derivatives is derived from their ability to inhibit the parasitic cysteine proteases, such as falcipain. These proteases catabolize globin into small peptides within the acidic food vacuole of the intra-erythrocytic malaria parasite [60]. In summary, a series of hybrids were synthesized with different substitution patterns on the chalcone phenyl ring B. The hybrids showed low cytotoxicity in epithelial kidney cells (Vero, selectivity index

Figure 6. Molecular hybrid obtained from the combination of chalcones (red) and N-aryl piperazine moiety (blue) [50].

Molecules 2017, 22, 1210 7 of 24

cell lines at nanomolar to low micromolar concentrations. The replacement of the double bond with a thiophene increases anticancer activity and must not significantly alter the relative conformation of the chalcone derivate 12. Of particular interest was derivative 13, showing IC50’s ranging from 0.160 to 0.510 μM on a panel of HeLa, human T-lymphocyte (Molt-4 and CEM), murine leukemia (L1210), and murine mammary carcinoma (FM3A) cell lineages [47].

Figure 5. Bioisosterism represented by the replacing the double bond of the enone (blue) with a thiophene (red) [47].

3.2. Molecular Hybridization The hybridization of biologically active molecules, based on the combination of complementary pharmacophoric features of two or more known bioactive compounds, which, through adequate fusion, lead to the design of new hybrid architectures that maintain pre-selected characteristics of the original templates, is a powerful strategy for drug design. There are two main ways through which hybrid molecules can be constructed. The first method combines two or more entire chemical structures together using a linker chain, whilst the second technique directly merges pharmacophoric moieties of different compounds [48,49]. In this context, to improve the efficacy of some chalcones in the treatment of diseases involving dual- or multi-target approaches, molecular hybridization has been investigated for the design of new ligands or prototypes. Mao and colleagues [50] designed and synthesized a series of compounds that have a chalcone derivate (14) and an N-aryl piperazine moiety (see ciprofloxacin structure (15)) in one frame and evaluated them for anticancer activity (Figure 6). Ciprofloxacin is a well-known inhibitor of human DNA Molecules 2017, 22, 1210 8 of 25 topoisomerase II that exhibits potent in vitro anticancer activity against different cell lineages [51–54] Molecules 2017, 22, 1210 8 of 24 while chalcones are known to inhibit several anticancer targets, including thioredoxin reductase [45], and tubulin polymerization [55,56]. Therefore, hybrid molecules that contain N-aryl piperazine and (SI) >559) andA series enhanced of hybrids antimalarial made of 4′-chlorochalcone activity as compared (17), and to the chloroquine antimalarial (drugIC50 >chloroquine 0.463 µM )(18 over) the werechalcone designed moieties and are tested expected for their to holdantimalarial improved effi accacytivities. against As achloroquine-resistant result, synergic anticancer strain (K1)effects of resistant strain of P. falciparum (IC50 ≤ 0.315 µM). The most effective hybrid 19 possessed IC50 of Plasmodiumwere reported falciparum for some [57] of these(see Figure hybrids. 7). Hybrid Chloroquine 16 displayed interferes the withhighest the activity parasite against detoxification cervical 0.083 µM and selectivity index of 837. The reference antimalarial drug chloroquine showed IC50 higher processcancer (Hela) by inhibiting and gastric heme ca ncertransformation (SGC7901) intocells hewithmozoin. IC50 of The 0.190 accumulation μM and 0.410 of μhemeM, respectively, causes severe in than 0.463 µM[57]. damagecomparison to the to parasite,20 μM and leading 12 μM to for its thedeath positive [58,59]. control The antimalarial cisplatin [50]. property of chalcone derivatives is derived from their ability to inhibit the parasitic cysteine proteases, such as falcipain. These proteases catabolize globin into small peptides within the acidic food vacuole of the intra-erythrocytic malaria parasite [60]. In summary, a series of hybrids were synthesized with different substitution patterns on the chalcone phenyl ring B. The hybrids showed low cytotoxicity in epithelial kidney cells (Vero, selectivity index (SI) >559) and enhanced antimalarial activity as compared to chloroquine (IC50 > 0.463 μM) over the resistant strain of P. falciparum (IC50 ≤ 0.315 μM). The most effective hybrid 19 possessed IC50 of 0.083 μM and selectivity index of 837. The reference antimalarial drug chloroquine showed IC50 higher than 0.463 μM [57]. Dong and colleagues [61] combined nitrendipine (20) or furoxan (21) to the chalcone scaffold (22, see Figure 8). Nitrendipine is a well-known calcium channel blocker [62,63] while furoxan acts as a nitric oxide (NO) donor [64,65]. In addition to traditional vasodilators, the antioxidant properties of chalcones were also demonstrated to be beneficial in the treatment of cardiovascular diseases [66]. Two series of hybrids were made in which the 1,4-dihydropyridyl or furoxan moiety is located at the 2′-position of the chalcone scaffold. Many of the hybrids exhibited moderate to excellent vasorelaxant activities. Amongst the nine hybrids made, compounds 23 (EC50 = 2.9 μM, Emax = 104%) and 24 Figure 6. Molecular hybrid obtained from the combination of chalcones (red) and N-aryl piperazine Figure(EC50 = 6. 8.9Molecular μM, Emax hybrid = 100%) obtained were the from most the active. combination The positive of chalcones control(red) quercetin and N showed-aryl piperazine EC50 of moiety (blue) [50]. moiety244 μM (blue) [61]. [50].

FigureFigure 7. 7.Molecular Molecular hybrid hybrid obtained obtained from a from combinatio a combinationn of 4'-chlorochalcone of 4'-chlorochalcone (red) and chloroquine (red) and chloroquine(blue) [57]. (blue) [57].

DongA and series colleagues of hybrids [61 ]bearing combined an indole nitrendipine ring system (20) orassociated furoxan to (21 chalcone) to the chalconefibrate with scaffold variable substitution patterns were reported by Sashidhara and colleagues [67] as lipid-lowering (22, see Figure8). Nitrendipine is a well-known calcium channel blocker [ 62,63] while furoxan agents (see Figure 9). Indole-based compounds have been a subject of intense investigations for their acts as a nitric oxide (NO) donor [64,65]. In addition to traditional vasodilators, the antioxidant antihyperlipidemic activity [68]. Fluvastatin (25), compound with indole moiety, was the first propertiessynthetic of chalcones 3-hydroxy-3-methylglutaryl were also demonstrated coenzyme toA bereductase beneficial inhibitor in the (statin) treatment and is of used cardiovascular in the diseasesmanagement [66]. Two of series dyslipidemia of hybrids in the were primary made and in which secondary the 1,4-dihydropyridylprevention of cardiovascular or furoxan disease moiety is located[69,70]. at the In 20 -positionaddition, ofa recent the chalcone literature scaffold. survey Manyrevealed of thethat hybrids chalcone exhibited derivatives, moderate such as to 4′ excellent,4- vasorelaxantdichlorochalcone activities. (26 Amongst), exhibited the potential nine hybrids hypolipidemic made, activities compounds [71]. Fibrates23 (EC50 are= selective 2.9 µM, agonists Emax = 104%) of the peroxisome proliferator-activated receptor gamma (PPARγ). Drugs such as fenofibrate (27) and 24 (EC50 = 8.9 µM,Emax = 100%) were the most active. The positive control quercetin showed lower triglyceride levels and increase high-density lipoprotein (HDL) levels in hyperlipidemic EC50 of 244 µM[61]. patients and reduce the risk of coronary heart disease [72,73]. These interesting biological properties led A series of hybrids bearing an indole ring system associated to chalcone fibrate with variable authors to link indole, chalcone, and fibrate moieties with the aim of obtaining potent antidyslipidemic substitution patterns were reported by Sashidhara and colleagues [67] as lipid-lowering agents (see Figure9). Indole-based compounds have been a subject of intense investigations for their antihyperlipidemic activity [68]. Fluvastatin (25), compound with indole moiety, was the first synthetic 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (statin) and is used in the management of Molecules 2017, 22, 1210 9 of 25 dyslipidemia in the primary and secondary prevention of cardiovascular disease [69,70]. In addition, a recent literature survey revealed that chalcone derivatives, such as 40,4-dichlorochalcone (26), exhibited potential hypolipidemic activities [71]. Fibrates are selective agonists of the peroxisome proliferator-activated receptor gamma (PPARγ). Drugs such as fenofibrate (27) lower triglyceride levels and increase high-density lipoprotein (HDL) levels in hyperlipidemic patients and reduce the risk of coronary heart disease [72,73]. These interesting biological properties led authors to link indole, Molecules 2017, 22, 1210 9 of 24 chalcone,Molecules and 2017 fibrate, 22, 1210 moieties with the aim of obtaining potent antidyslipidemic agents.9 of One 24 of the best hybrids,agents.agents. One compoundOne of of the the best best 28hybrids, hybrids,, was showncompound compound to decrease2828, ,was was shownshown the concentrationtoto decreasedecrease the concentrationconcentration of cholesterol, ofof cholesterol, cholesterol, phospholipids, and triglyceridephospholipids,phospholipids, of hyperlipidemicand and triglyceride triglyceride of of rats hyperlipidemic hyperlipidemic by 32%, 33%, rats rats and byby 32%,32%, 30%, 33%, respectively and 30%, respectivelyrespectively [67]. [67]. [67].

Figure 8.FigureMolecular 8. Molecular hybrids hybrids obtained obtained from from combination combination of of well-known well-known vasodilators vasodilators (nitrendipine (nitrendipine or or Figurefuroxan, 8. Molecular highlighted hybrids by purple obtained and blue from colors, combination respectively) of well-known and chalcones vasodilators [61]. (nitrendipine or furoxan,furoxan, highlighted highlighted by purpleby purple and and blue blue colors, colors, respectively) and and chalcones chalcones [61]. [ 61].

Figure 9. Molecular hybrids obtained from a combination of Fluvastatin (purple), fenofibrate (blue), and chalcones (red) [67]. Figure 9. Molecular hybrids obtained from a combination of Fluvastatin (purple), fenofibrate (blue), Figure 9. Molecular hybrids obtained from a combination of Fluvastatin (purple), fenoﬁbrate (blue), and chalcones (red) [67]. and3.3. chalcones Drug Latentiation (red) [67 ]. 3.3. DrugUndesirable Latentiation properties, including poor aqueous solubility, chemical instability, insufficient oral or local absorption, low half-life, fast pre-systemic metabolism, local irritation, and toxicity are typical Undesirable properties, including poor aqueous solubility, chemical instability, insufficient oral problems involved in drug design. For these reasons, drug latentiation presents an effective strategy or local absorption, low half-life, fast pre-systemic metabolism, local irritation, and toxicity are typical to improve these properties. The term latentiation may be defined as the chemical union between a problems involved in drug design. For these reasons, drug latentiation presents an effective strategy to improve these properties. The term latentiation may be defined as the chemical union between a

Molecules 2017, 22, 1210 10 of 25

3.3. Drug Latentiation Undesirable properties, including poor aqueous solubility, chemical instability, insufficient oral or local absorption, low half-life, fast pre-systemic metabolism, local irritation, and toxicity are typical problemsMolecules 2017 involved, 22, 1210 in drug design. For these reasons, drug latentiation presents an effective strategy10 of 24 to improve these properties. The term latentiation may be defined as the chemical union between acompound compound and and a atransporter transporter group group (e.g., (e.g., ester, ester, amide, amide, carbamate, carbonate, ether, imine,imine, andand phosphate),phosphate), normally by means of of a a covalent covalent labile labile bond, bond, giving giving rise rise to to the the classical classical concept concept of of a aprodrug. prodrug. It Itis is a a temporarily temporarily inactive inactive compound compound that that undergoes in vivovivo biotransformationbiotransformation throughthrough chemicalchemical oror enzymaticenzymatic cleavage,cleavage, enabling the delivery of the activeactive moleculemolecule at efficaciousefficacious levels. Consequently,Consequently, itit cancan facilitatefacilitate thethe accumulationaccumulation ofof aa drugdrug atat the site of action and improve safety [74,75]. [74,75]. ForFor example,example, somesome chalcone derivatives, such as compound 29,, prevents inflammatoryinflammatory reactionsreactions throughthrough neutralization of of the the CXC CXC motif motif chemokine chemokine li ligandgand 12 12 (CXCL12, (CXCL12, inhibitory inhibitory constant constant (Ki) (Ki) of of0.053 0.053 μM)µ M)and and prevents prevents it from it fromacting acting on the on CXCR4 the CXCR4 and CXCR7 and CXCR7receptors, receptors, although althoughpoor solubility poor solubilityin water limits in water their limits use [76]. their To use overcome [76]. To this overcome drawback, this Gasparik drawback, and Gasparik colleagues and [77] colleagues synthesized [77] synthesizeda series of chalcone a series prodrugs of chalcone with prodrugs different with functional different groups, functional such groups, as phosphate, such as l-seryl, phosphate, and sulfate. l-seryl, andThe sulfate.chalcone The prodrug chalcone with prodrug a phosphate with asubstituent phosphate ( substituent30, Figure 10) (30 ,showed Figure 10a )greater showed increase a greater in increasesolubility, in to solubility, at least to3000 at leasttimes 3000 greater times solubili greaterty solubilitythan the parent than the chalcone. parent chalcone. The compound The compound behaves behavesas a prodrug, as a prodrug, remaining remaining inactive inactive in in invitroin vitro assays,assays, but butwhen when cleaved cleaved inin vivo vivo intointo 2929,, inhibitsinhibits eosinophileosinophil recruitmentrecruitment in in the the airways airways by by≥ ≥50%50% atat aa dosedose asas lowlow asas 0.0300.030 µμM/kgM/kg and without any signs ofof toxicitytoxicity [[77].77].

Figure 10. Latentiation represented by thethe replacing thethe hydroxylhydroxyl (red)(red) withwith aa phosphatephosphate (blue)(blue) [[77].77].

AminoAmino acidsacids werewere alsoalso used to improve the physicochemical properties of chalcone derivatives. ForFor example, Canela Canela and and colleagues colleagues [78] [78 investigated] investigated the thetubulin-binding, tubulin-binding, vascular vascular targeting, targeting, anti- anti-tumortumor and andanti-metastatic anti-metastatic activities activities of aof new a new series series of chalcone of chalcone derivatives. derivatives. The The best best compound compound of ofthis this series series (31) (inhibited31) inhibited the proliferation the proliferation of endothelial of endothelial (HMEC-1, (HMEC-1, microvascular microvascular endothelial endothelial cell line- cell1; and line-1; BAEC, and bovine BAEC, aortic bovine endothelial aortic endothelial cells) and cells)tumor and (B16-F10.luc2, tumor (B16-F10.luc2, melanoma melanomacells expressing cells expressing firefly luciferase 2; Cem; and HeLa) cell lines with IC concentrations between 0.001 and firefly luciferase 2; Cem; and HeLa) cell lines with IC50 concentrations50 between 0.001 and 0.004 μM, µ 0.004which M,is slightly which isbetter slightly than better the reference than the referencecompound compound colchicine. colchicine. However, However,poor aqueous poor solubility aqueous solubility(0.016 mg/mL) (0.016 mg/mL)precluded precluded in vivo inevaluation. vivo evaluation. Therefore, Therefore, the authors the authors synthesized synthesized prodrugs prodrugs of of compound 31 by conjugation with amino acids (Figure 11). The L-Lysine-L-Proline derivative 32 compound 31 by conjugation with amino acids (Figure 11). The L-Lysine-L-Proline derivative 32 was wasaround around 2000-fold 2000-fold more more soluble soluble than than the parent the parent compound compound and andwas waseffective effective in inhibiting in inhibiting tumor tumor and andendothelial endothelial cell proliferation. cell proliferation. In addition, In addition, incubation incubation of prodrug of prodrug 32 in32 humanin human serum serum or murine or murine liver liverextract extract showed showed an efficient an efficient release release of 31 of.31 The. The prodrug prodrug candidate candidate 3232 alsoalso showed showed potent potent in vivovivo anticanceranticancer activityactivity inin melanomamelanoma (10(10 mg/kg)mg/kg) and breastbreast cancercancer modelsmodels (15(15 mg/kg)mg/kg) by causingcausing rapidrapid intratumoralintratumoral vascularvascular shutdownshutdown andand massivemassive tumortumor necrosisnecrosis [[78].78].

Figure 11. Latentiation represented by the replacing the amine (red) with an L-Lysine-L-Proline (blue) [78].

4. Computer-Assisted Drug Design (CADD) The concept of design, synthesis, and biological evaluation forms a central pillar of the lead discovery process and provides the basis for our understanding of the underlying structure–activity

Molecules 2017, 22, 1210 10 of 24 compound and a transporter group (e.g., ester, amide, carbamate, carbonate, ether, imine, and phosphate), normally by means of a covalent labile bond, giving rise to the classical concept of a prodrug. It is a temporarily inactive compound that undergoes in vivo biotransformation through chemical or enzymatic cleavage, enabling the delivery of the active molecule at efficacious levels. Consequently, it can facilitate the accumulation of a drug at the site of action and improve safety [74,75]. For example, some chalcone derivatives, such as compound 29, prevents inflammatory reactions through neutralization of the CXC motif chemokine ligand 12 (CXCL12, inhibitory constant (Ki) of 0.053 μM) and prevents it from acting on the CXCR4 and CXCR7 receptors, although poor solubility in water limits their use [76]. To overcome this drawback, Gasparik and colleagues [77] synthesized a series of chalcone prodrugs with different functional groups, such as phosphate, l-seryl, and sulfate. The chalcone prodrug with a phosphate substituent (30, Figure 10) showed a greater increase in solubility, to at least 3000 times greater solubility than the parent chalcone. The compound behaves as a prodrug, remaining inactive in in vitro assays, but when cleaved in vivo into 29, inhibits eosinophil recruitment in the airways by ≥50% at a dose as low as 0.030 μM/kg and without any signs of toxicity [77].

Figure 10. Latentiation represented by the replacing the hydroxyl (red) with a phosphate (blue) [77].

Amino acids were also used to improve the physicochemical properties of chalcone derivatives. For example, Canela and colleagues [78] investigated the tubulin-binding, vascular targeting, antitumor and anti-metastatic activities of a new series of chalcone derivatives. The best compound of this series (31) inhibited the proliferation of endothelial (HMEC-1, microvascular endothelial cell line- 1; and BAEC, bovine aortic endothelial cells) and tumor (B16-F10.luc2, melanoma cells expressing firefly luciferase 2; Cem; and HeLa) cell lines with IC50 concentrations between 0.001 and 0.004 μM, which is slightly better than the reference compound colchicine. However, poor aqueous solubility (0.016 mg/mL) precluded in vivo evaluation. Therefore, the authors synthesized prodrugs of compound 31 by conjugation with amino acids (Figure 11). The L-Lysine-L-Proline derivative 32 was around 2000-fold more soluble than the parent compound and was effective in inhibiting tumor and endothelial cell proliferation. In addition, incubation of prodrug 32 in human serum or murine liver extract showed an efficient release of 31. The prodrug candidate 32 also showed potent in vivo anticancer activity in melanoma (10 mg/kg) and breast cancer models (15 mg/kg) by causing rapid Molecules 2017, 22, 1210 11 of 25 intratumoral vascular shutdown and massive tumor necrosis [78].

FigureFigure 11. 11. LatentiationLatentiation represented represented by the by replacing the replacing the amine the (red) amine with (red) an L with-Lysine- anLL-Proline-Lysine- (blue)L-Proline [78]. (blue) [78]. 4. Computer-Assisted Drug Design (CADD) 4. Computer-AssistedThe concept of design, Drug Designsynthesis, (CADD) and biological evaluation forms a central pillar of the lead discoveryThe conceptprocess ofand design, provides synthesis, the basis and for biologicalour understanding evaluation of formsthe underl a centralying structure–activity pillar of the lead discovery process and provides the basis for our understanding of the underlying structure–activity relationships (SAR) [34]. However, synthesis and bioassays are laborious and the chance of getting false positives is high. Although expert biological knowledge is indisputably important for successfully guiding drug discovery projects, CADD approaches, as well as the availability of supercomputers, parallel processing, and the graphics processing unit (GPU), have been playing a pivotal role on prioritizing compounds for synthesis and/or biological evaluation. As no compounds need to be synthesized or tested before computational studies, CADD represents a time-, labor-, and cost-effective strategy to obtain lead compounds in the early stages of drug discovery [79–81]. In the subsequent sections, we review current developments in CADD, highlighting the main strategies and pitfalls that have solid implications in the medicinal chemistry of chalcone derivatives.

4.1. Structure-Based Drug Design (SBDD) In SBDD, 3D structural information of a macromolecule is used to design or evaluate ligands based on their predicted interactions and affinity with protein binding sites. Thus, identification of a valid biochemical target and the acquisition of its structural information are the first vital steps in SBDD. Researchers from computational and structural biology aided in the generation of thousands of structures with the use of homology modeling, X-ray crystallography, cryo-electron microscopy (EM), and nuclear magnetic resonance (NMR) [81–83]. Currently, more than 13,000 3D structures have been experimentally solved and stored in the Protein Data Bank (PDB), leading to attractive opportunities for the application of SBDD strategies [84]. SBDD can be divided into virtual screening (VS) and de novo approaches. In VS large chemical libraries are processed to search for compounds that possess complementarities toward the targets. De novo design exploits information from the 3D biomacromolecule to find small fragments that match well with the binding site. These fragments should be linked, providing a structurally novel ligand that can be synthesized for further in vitro assays [85,86]. However, because of its knowledge-based characteristic, SBDD strongly depends on the amount and the quality of information available about the system under investigation. Regardless of what kind of protein will be employed as a molecular target, important issues, such as the suitable assignment of protonation states [87,88], solvation [89,90], and protein flexibility [91,92], must be considered. Protein-ligand docking, molecular dynamics simulations, and structure-based pharmacophores are the main SBDD tools. Basic principles, advantages, and disadvantages of these tools are discussed below.

4.1.1. Protein-Ligand Docking Protein-ligand docking is the most extensively used SBVS method. It predicts possible binding modes of a compound or fragment in a target binding site and estimates affinity based on its intermolecular interactions in the binding pocket. Docking is often carried out in two parts. In the first part, the ligand is placed inside the binding site in different orientations and conformations using a search algorithm to facilitate the identification of the binding mode. Then, a scoring function ranks Molecules 2017, 22, 1210 12 of 25 the different poses of the ligand that are generated by the search algorithm and orders them by a score. These scores are features that aid in investigating the interactions between the small molecule and the biological target, thereby providing context about biological activity [82,93,94]. The discussion about search algorithms and scoring functions is beyond the scope of this review, but detailed information can be obtained elsewhere [95–99]. Although the concept of docking seems to be precise, it remains a major challenge of this approach. Usually, the scoring functions are found to have limited accuracy in the ranking of compounds and sometimes cannot distinguish between active and inactive molecules. In addition, the insertion of protein flexibility to some active residues can help in identifying new hits with better molecular complementarity, when compared with traditional rigid docking. However, it requires more complex calculations because the proteins of high conformational energy score would result in idealistic binding mode [85]. Consequently, some streamlined strategies have been developed to solve these methodological limitations. To incorporate protein flexibility, ensemble methods make use of multiple input conformations of a target using a set of different 3D structures experimentally determined (X-ray crystallography or NMR) or computationally produced using molecular dynamics simulations (see below). Instead of single rigid structure, carefully chosen 3D conformations of the target can be used to represent conformational changes in the protein backbone and/or side chain [100,101]. On the other hand, averaging the results from two or more scoring functions and forming a consensus has proved to be more beneficial in scoring and ranking compounds [102]. In addition to consensus scoring, post-processing techniques such as the molecular mechanics Poisson–Boltzmann surface area methods can be used to better estimate the free binding energies [103,104].

4.1.2. Molecular Dynamics (MD) Simulations MD allows the prediction of the time-dependent behavior of a molecular system. It has been particularly useful in the structural refinements of post-docking complexes, such that the complementarity between the ligand and the receptor is enhanced in the complex state, allowing better complementarities. The knowledge of MD functionalities of proteins is important in order to understand several key aspects of SBDD, such as ligand–protein interactions, as well as exploring the energy landscapes of proteins and identifying their physiological conformations, which are, in many cases, not even accessible through experimental techniques [105,106]. Methodologically, MD regards atoms as solid spheres and the bonds connecting them as springs. The trajectories of atoms and molecules are determined by numerically solving Newton’s equations of motion for a system of interacting particles, where forces between the particles and their potential energies are calculated using force fields with predefined parameters. A force field is a mathematical expression describing the dependence of the energy of a system on the coordinates of its atoms. This includes the possible nonbonded interactions (Coulomb potentials and van der Waals potentials) and bonded (bonds, angles, and dihedrals) terms between the different atoms in a 3D macromolecule [105,106]. Several force fields are available for MD simulations, such as GROMOS [107], CHARMM [108], and AMBER [109]. Most of these methods have different functional forms to treat MD simulations, which makes it difficult to apply parameters from one force field to another.

4.1.3. Structure-Based Pharmacophores (SBPs) In addition to docking, 3D structural information may be used to derive pharmacophore hypotheses. They are deﬁned by the International Union of Pure and Applied Chemistry (IUPAC) as “an ensemble of steric and electronic features that is necessary to ensure the optimal supramolecular interactions with a speciﬁc biological target and to trigger (or block) its biological response” and can be used to screen a compound database [110]. Here, SBPs describe the spatial arrangement of essential ligand-macromolecule interactions directly from the 3D structural data. This approach determines chemical features based on complementarities between a ligand and its binding site. Nonetheless, Molecules 2017, 22, 1210 13 of 25 the selection of features in SBPs is a more complex effort than in ligand-based, since there are more possibilities of conformational and spatial states [111,112]. Typically, SBPs can be created using ligand-macromolecule complexes (holo) or using ligand-free macromolecules (apo). The generation of models using holo structures allows complete exploration of the ligand interactions with the binding site, and the inclusion of shape and volume information derived directly from the structural data [111,113]. In the absence of a ligand in the binding site, pharmacophores can be obtained using functional groups or small fragments, also referred to as molecular probes, to map possible interaction sites or hot spots within a binding site. Selected hot spots can then be converted into pharmacophoric features to generate a structure-based pharmacophore model [113].

4.2. Ligand-Based Drug Design (LBDD) In some cases, usually when data pertaining to the 3D structure of a target protein are not available, CADD can instead be based on processes using the known ligands of a target protein or known active compounds in phenotypic assays as starting points. This approach is known as LBDD [114,115]. Similarity search, ligand-based pharmacophores, and quantitative structure-activity relationship (QSAR) analysis are the most popular methods in the LBDD process (described below).

4.2.1. Similarity Search The major principle of medicinal chemistry is the assumption that structurally similar compounds exhibit similar biological activities. Based on this premise, similarity search techniques have been applied to represent chemical structures to allow rapid structural comparison in an effort to identify structurally similar compounds or to cluster collections based on structural similarity. Methodologically, both query ligand and the database of compounds could be represented and further compared using molecular fingerprints, a string made up of binary digits that account for the presence (1) or absence (0) of representative fragments or atoms in the chemical structure. These fingerprints vary greatly in length and complexity ranging from simple topological representations to complicated multi-point 3D pharmacophore arrangements [116,117]. Then, bit vector similarity must be expressed in a way that can be quantified. The most used metric to explore similarity is the Tanimoto coefficient, which is equal to the number of common bits set to 1 in both fingerprints divided by the total number of bits set to 1 between both fingerprints [118]. It assumes that two structures can be considered similar if this coefficient is higher than 0.85.

4.2.2. Ligand-Based Pharmacophores (LBPs) The pharmacophore approach aims to identify containing different scaffolds, but with a similar 3D arrangement of key interacting functional groups. LBPs identify key common features (e.g., hydrogen bond donors or acceptors, aromatic rings, partial charges, and hydrophobicity) and the relative orientations of known active ligands not shared by inactives. Its elucidation involves two main stages: (i) the analysis of the training set molecules to identify common pharmacophoric points and (ii) the alignment of the bioactive conformations of these molecules to determine the best overlay of corresponding features [113,119,120]. During this process, molecular features, which are not consistently observed in active compounds should be made optional or removed from the model. Furthermore, spatial constraints can be employed in moieties occupied by inactive compounds and reﬁned to avoid making the model too restrictive. After model reﬁnement, validation studies using statistical metrics must be performed to determine the ability of the model to discriminate between known active and inactive compounds [120].

4.2.3. QSAR QSAR modeling is widely practiced in industry, government institutions, and universities worldwide. Conceptually, QSAR is a mathematical model describing the relationship between chemical structure and respective biological activity of a set of compounds [121–123]. QSAR modeling Molecules 2017, 22, 1210 14 of 25 could be presented as a two-part process. Firstly, chemical structures are converted into a vector of features, which are also known as descriptors and represented generically by the symbol x [124]. Thousands of descriptors, both freely available and commercial, encode a compound as a feature vector. Then, machine learning methods (e.g., Random Forest [125], Deep Learning [126], Support Vector Machine [127], etc.) are used to establish quantitative relationships between descriptors and biological activities (represented by the symbol y). This involves empirically discovering a function that maps between the feature vectors and activities [124]. Nevertheless, the resulting QSAR model is useful if and only if it is predictive. Hence, the quality of the resulting QSAR model is measured by using the appropriate metrics regarding its ability to correctly predict the activities of compounds experimentally determined [128]. The inﬂuence of various factors on QSAR performance decreases in the following row: data quality > molecular descriptors > machine learning approach [129–132]. Once validated, this model can be applied to untested chemical compounds for numerical prediction of biological activity (continuous model) or the discrimination between active and inactive compounds (binary model) [133].

4.2.4. Quantum Mechanical (QM) Methods The QM approaches explain the behavior of molecules and its interactions with energy on the scale of atoms and subatomic particles. The increasing popularity of QM methods in CADD is not just a consequence of ever-growing computing power but is also due to their highest accuracy. Both the fast semi-empirical approaches and time-consuming ab initio methods do not suffer from the limitation inherent to the ball and spring description of molecular mechanical (MM) methods and the fixed-charge approximation used in the force fields (FFs). Currently available computing power is not enough for the direct ab initio QM calculations of 3D macromolecules with accuracy like that of in vitro experiments [134]. However, hybrid procedures such as QM/MM free energy simulations, that combine the strengths of both QM (accuracy) and molecular mechanics (MM) (efficiency) methods, have been widely employed to model chemical reactions and other electronic processes in biomolecular systems [135–137].

4.3. Practical Applications of CADD in Chalcone Field In the current scenario, design or identiﬁcation of chalcone derivatives using CADD tools still remain poorly explored. Despite the small number of studies available in the literature, most of them led to the discovery of several lead candidates. Below, we discuss some successful applications of ligand- and structure-based approaches leading to the discovery of promising chalcone derivatives.

4.3.1. Design of Anti-Tuberculosis Agents Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a major cause of death around the world. According to the World Health Organization, nearly one-third of the population is infected with tuberculosis, an infectious disease that kills nearly 1.5 million people each year. The current treatment for TB takes approximately six to nine months duration, which normally leads to noncompliance and, hence, the emergence of multidrug-resistant bacteria [138]. Therefore, there is an urgent need for new antitubercular agents that are effective against drug-resistant strains and that reduce the duration of treatment. Aiming at discovering new drugs, our group used QSAR-driven approach to design new series of chalcone derivatives. Initially, we retrieved all chalcone compounds with inhibition data against M. tuberculosis H37Rv from the literature. After compilation of the initial dataset and rigorous curation, chalcones were subject to structure-activity relationships (SAR) analysis. Based on these SAR rules, bioisosteric replacements were employed to design new chalcone derivatives with potential anti-TB activity. In parallel, QSAR models were generated using multiple machine learning methods and ﬁngerprints. Using these models, we prioritized 33 compounds for synthesis and biological evaluation [139]. As a result, ﬁve 5-nitro-substituted heteroaryl chalcones (see structures 33–37 in Figure 12) were found to exhibit MICs at nanomolar concentrations against Molecules 2017, 22, 1210 15 of 25 replicating mycobacteria, low micromolar activity against nonreplicating bacteria, and nanomolar activity against rifampin (RMP) and isoniazid (INH) monoresistant strains (mRMP and mINH, respectively). The series also showed low activity against commensal bacteria and very low cytotoxicity against Vero cells (SI = 61–454). Our results suggest that our designed heteroaryl chalcone compounds are promising anti-TB agents, due to their high potency and selectivity [139].

4.3.2. Discovery of New Tubulin Inhibitors Microtubules are multifunctional cytoskeletal proteins composed of α- and β-tubulin heterodimers. They have an essential role in regulating cell architectures and functioning as part of the spindle to ensure proper chromosome segregation and cell division. Recently, the clinical use of some tubulin inhibitors, such as taxanes and vinca alkaloids, has been limited by neurotoxicity and drug resistance [140,141]. Therefore, new small-molecule tubulin-binding inhibitors must be developed. Aiming at discovering new drugs, Niu and colleagues [142] integrate LBPs and docking approaches to identify new lead candidates with anticancer activities. Initially, high correlation quantitative pharmacophore models were generated using the SAR of known tubulin inhibitors. Then, an external set composed of 40 compounds with experimental activity data and 800 decoys was used to evaluate the discriminative ability of the models when distinguishing the active compounds from the inactive compounds.Molecules 2017 After, 22, validation,1210 the more predictive model was used in virtual screening of15 theof 24 Specs database,Molecules leading 2017, 22 to, 1210 the identification of 952 drug-like compounds (fitted on all the pharmacophoric15 of 24 features)screening with potentialof the Specs activity. database, These leading molecules to the identification were subsequently of 952 drug-like subjected compounds to molecular (fitted on docking screening of the Specs database, leading to the identification of 952 drug-like compounds (fitted on studiesall to the refine pharmacophoric the retrieved features) virtual hits.with Finally, potential five activity. chalcones These with molecules diverse substituentswere subsequently and strong subjectedall the pharmacophoric to molecular docking features) studies with to refinepotential the retrievedactivity. virtualThese hits.molecules Finally, werefive chalcones subsequently with molecular interactions with the key amino acids in tubulin binding site were identified. The biological diversesubjected substituents to molecular and docking strong studies molecular to refine interactions the retrieved with thevirtual key hits.amino Finally, acids fivein tubulin chalcones binding with evaluationsitediverse were indicated substituents identified. that The and compoundsbiological strong moevaluationlecular38 and interactionsindicated39 (see that Figurewith compounds the 13 key) showed amino38 and 39acids potent (see in Figure tubulin inhibitory 13) bindingshowed activity site were identified. The biological evaluation indicated that compounds 38 and 39 (see Figure 13) showed againstpotent MCF-7 inhibitory cells (IC activity50 of against 0.028 µMCF-7M and cells 0.054 (IC50µ ofM, 0.028 respectively) μM and 0.054 [142 μM,]. respectively) [142]. potent inhibitory activity against MCF-7 cells (IC50 of 0.028 μM and 0.054 μM, respectively) [142].

FigureFigure 12. Antitubercular 12. Antitubercular 5-nitro-substituted 5-nitro-substituted heteroarylheteroaryl chalcones chalcones prioritize prioritizedd by our by ourQSAR-driven QSAR-driven drugFigure design 12. Antitubercular strategy [139]. 5-nitro-substituted heteroaryl chalcones prioritized by our QSAR-driven drug designdrug design strategy strategy [139 [139].].

Figure 13. Chalcones with anticancer activity identified by integration of ligand-based pharmacophore FigurescreeningFigure 13. Chalcones 13. andChalcones molecular with with anticancer docking anticancer studies activity activity [142].identiﬁed identified by by integration integration of ofligand-based ligand-based pharmacophore pharmacophore screeningscreening and molecular and molecular docking docking studies studies [142 [142].]. 4.3.3. De Novo Design of Histone Deacetylase 2 Inhibitors 4.3.3. De Novo Design of Histone Deacetylase 2 Inhibitors Inhibition of histone deacetylases (HDAC) has emerged as a highly promising strategy for the developmentInhibition of of new histone therapeutics deacetylas againstes (HDAC) cancer has[143,144] emerged and as vari a highlyous other promising human disordersstrategy for [145]. the Althoughdevelopment promising, of new therapeutics it is particularly against challenging cancer [143,144] to achieve and vari selectiveous other inhibitors human disordersof the HDAC2 [145]. isoformAlthough due promising, to its high it (97.8%)is particularly structural challenging similarities to withachieve HDAC1. selective As ainhibitors result, it ofhas the been HDAC2 very difficultisoform dueto develop to its high HDAC2 (97.8%) selective structural inhibitors similarities by employing with HDAC1. conventi As aonal result, CADD it has approaches. been very Guideddifficult byto previouslydevelop HDAC2 characterized selective HDAC inhibitors reaction by employingmechanism conventi[146], Zhouonal andCADD colleagues approaches. [147] developedGuided by depreviously novo reaction characterized mechanism-based HDAC reaction inhibitor mechanism design [146], strategy Zhou considering and colleagues reactivity [147] differencesdeveloped betweende novo HDAC2 reaction and mechanism-based HDAC1. They worked inhibitor with design the hypothesis strategy thatconsidering a desired reactivity inhibitor shoulddifferences be stable between in solution HDAC2 while and HDAC1. it should They react worked intramolecularly with the hypothesis after binding that to a the desired HDAC inhibitor active siteshould and be thus stable mimics in solution the enzymatic while it transitionshould reac sttate. intramolecularly To examine how after designed binding compoundsto the HDAC and active the enzymesite and thusenvironment mimics the would enzymatic modulate transition the reactivitystate. To examineof the intramolecularhow designed compoundsnucleophilic and attack the reaction,enzyme environmenttheoretical calculations would modulate on several the non-enzyme reactivity of(QM) the andintramolecular corresponding nucleophilic enzyme modelsattack (QM/MM)reaction, theoretical were carried calculations out. Theoretical on several reaction non-enzyme barriers indicated (QM) thatand twocorresponding compounds, enzyme β-aminomethyl models chalcone(QM/MM) (40 were) and carried β-hydroxymethyl out. Theoretical chalcone reaction ( 41barriers), would indicated be stable that at two the compounds, non-enzyme β environment,-aminomethyl whilechalcone the ( 40intramolecular) and β-hydroxymethyl nucleophilic chalcone attack (re41action), would would be stable occur at after the non-enzymebinding to the environment, HDAC1/2 activewhile site.the Computationalintramolecular studiesnucleophilic also showed attack good reaction selectivity would of occurcompounds after binding 40 and 41 to against the HDAC1/2 HDAC2 vs.active HDAC1. site. Computational Then, β-substituted studies chalcones also showed were good synthesized selectivity and of compoundstested on a panel40 and of 41 HDACs. against HDAC2In vitro vs. HDAC1. Then, β-substituted chalcones were synthesized and tested on a panel of HDACs. In vitro

Molecules 2017, 22, 1210 16 of 25

4.3.3. De Novo Design of Histone Deacetylase 2 Inhibitors Inhibition of histone deacetylases (HDAC) has emerged as a highly promising strategy for the development of new therapeutics against cancer [143,144] and various other human disorders [145]. Although promising, it is particularly challenging to achieve selective inhibitors of the HDAC2 isoform due to its high (97.8%) structural similarities with HDAC1. As a result, it has been very difficult to develop HDAC2 selective inhibitors by employing conventional CADD approaches. Guided by previously characterized HDAC reaction mechanism [146], Zhou and colleagues [147] developed de novo reaction mechanism-based inhibitor design strategy considering reactivity differences between HDAC2 and HDAC1. They worked with the hypothesis that a desired inhibitor should be stable in solution while it should react intramolecularly after binding to the HDAC active site and thus mimics the enzymatic transition state. To examine how designed compounds and the enzyme environment would modulate the reactivity of the intramolecular nucleophilic attack reaction, theoretical calculations on several non-enzyme (QM) and corresponding enzyme models (QM/MM) were carried out. Theoretical reaction barriers indicated that two compounds, β-aminomethyl chalcone (40) and β-hydroxymethyl chalcone (41), would be stable at the non-enzyme environment, while the intramolecular nucleophilic attack reaction wouldMolecules occur2017, 22 after, 1210 binding to the HDAC1/2 active site. Computational studies also showed16 good of 24 selectivity of compounds 40 and 41 against HDAC2 vs. HDAC1. Then, β-substituted chalcones studies showed that β-substituted chalcones preferentially inhibit HDAC1 and HDAC2 and not were synthesized and tested on a panel of HDACs. In vitro studies showed that β-substituted HDAC3 (Figure 14). Moreover, a distinct time-dependent (~24 h) selective inhibition for the 41 on chalcones preferentially inhibit HDAC1 and HDAC2 and not HDAC3 (Figure 14). Moreover, a distinct HDAC2 (IC50 = 0.170 μM) against the HDAC1 (IC50 = 2.740 μM) and HDAC3 (IC50 ~50 μM) was time-dependent (~24 h) selective inhibition for the 41 on HDAC2 (IC50 = 0.170 µM) against the HDAC1 observed [147]. (IC50 = 2.740 µM) and HDAC3 (IC50 ~50 µM) was observed [147].

Figure 14. Time-dependentTime-dependent histone histone deacetylase deacetylase 2 2 selectiv selectivee inhibitors identified identiﬁed by integration of quantum mechanics and quantum mechanics/molecularmechanics/molecular mechanics mechanics strategies strategies [147]. [147].

4.3.4. Design of Anti-Leishmanial Agents Leishmaniasis is an infectious poverty-associated disease caused by trypanosomatidtrypanosomatid parasites belonging to genus Leishmania.. In In fact, this term includes a wide spectrum of vector-bornevector-borne diseases with great epidemiological and clinical diversity (e.g., cutaneous, cutaneous, mucocutaneous, mucocutaneous, and visceral) visceral) [148], [148], which areare poverty-related poverty-related and and mostly mostly affect affect the populationthe population with the with lowest the income.lowest Currentincome. treatment Current treatmentdepends on depends a limited on number a limited of number drugs (miltefosine of drugs (mil andtefosine aminoglycosides) and aminoglycosides) that have issuesthat have of toxicity, issues oflong-dose toxicity, regimens, long-dose high regimens, cost, and high the cost, need and for parenteralthe need administrationfor parenteral [administration149]. In addition, [149]. many In addition,of these drugs many were of these developed drugs manywere developed years ago, andmany nowadays years ago, various and nowadays resistant strainsvarious exist resistant [150]. Attemptingstrains exist to[150]. explore Attempting new and to moreexplore potent new anti-leishmanialand more potent agents,anti-leishmanial Rashid and agents, colleagues Rashid [ 151and] pursuedcolleagues an [151] in silico-driven pursued designan in strategysilico-driven to synthesize design strategy novel hybrids to synthesize with common novel pharmacophoric hybrids with commonfeatures ofpharmacophoric dihydropyrimidine features and of chalcone. dihydropyrimidine Dihydropyrimidine and chalcone. based compoundsDihydropyrimidine have received based compoundsconsiderable have attention received as potential considerable anti-leishmanial attention agentsas potential due to anti-leishmanial their inhibition activityagents due on pteridine to their inhibitionreductase 1activity (PTR1), on an pteridine enzyme responsiblereductase 1 for(PTR1) the salvage, an enzyme of pterins responsible in Leishmania for theparasites salvage of [152 pterins,153]. inInitially, Leishmania a molecular parasites docking [152,153]. study Initially, was carried a molecular using the docking 3D structure study of was PTR1 carried from L. using major theaiming 3D structureat designing of PTR1 a series from of L. hybrids major aiming to explore at designing speciﬁc interactionsa series of hybrids of chalcone to explore and specific dihydropyrimidine interactions ofmoieties chalcone in and the dihydropyrimidine active site. Based moieties on the binding in the active modes site. and Based scores on the obtained binding from modes the and docking, scores obtainedauthors prioritized from the docking, 13 hybrid authors compounds prioritized for synthesis 13 hybrid and compounds biological evaluation.for synthesis As and aresult, biological low evaluation.micromolar As activities a result, were low reported micromolar for most activities of these were hybrids. reported Hybrid for most42 displayed of these hybrids. the highest Hybrid activity 42 displayed the highest activity against L. major and L. donovani with IC50 of 1.06 μM and 15.6 μM, respectively (Figure 15). Finally, bioisosteric replacement of the α,β-unsaturated carbonyl system with a pyrazoline significantly altered the conformation of hybrid 43 (see Figure 15) in active site of PTR1 and increased anti-leishmanial activity (IC50 of 0.948 μM and 3.03 μM against L. major and L. donovani, respectively) [151].

Figure 15. Anti-leishmanial chalcone-dihydropyrimidine hybrids (red and blue, respectively) prioritized by molecular docking studies [151].

Molecules 2017, 22, 1210 16 of 24

studies showed that β-substituted chalcones preferentially inhibit HDAC1 and HDAC2 and not HDAC3 (Figure 14). Moreover, a distinct time-dependent (~24 h) selective inhibition for the 41 on HDAC2 (IC50 = 0.170 μM) against the HDAC1 (IC50 = 2.740 μM) and HDAC3 (IC50 ~50 μM) was observed [147].

Figure 14. Time-dependent histone deacetylase 2 selective inhibitors identified by integration of quantum mechanics and quantum mechanics/molecular mechanics strategies [147].

4.3.4. Design of Anti-Leishmanial Agents Leishmaniasis is an infectious poverty-associated disease caused by trypanosomatid parasites belonging to genus Leishmania. In fact, this term includes a wide spectrum of vector-borne diseases with great epidemiological and clinical diversity (e.g., cutaneous, mucocutaneous, and visceral) [148], which are poverty-related and mostly affect the population with the lowest income. Current treatment depends on a limited number of drugs (miltefosine and aminoglycosides) that have issues of toxicity, long-dose regimens, high cost, and the need for parenteral administration [149]. In addition, many of these drugs were developed many years ago, and nowadays various resistant strains exist [150]. Attempting to explore new and more potent anti-leishmanial agents, Rashid and colleagues [151] pursued an in silico-driven design strategy to synthesize novel hybrids with common pharmacophoric features of dihydropyrimidine and chalcone. Dihydropyrimidine based compounds have received considerable attention as potential anti-leishmanial agents due to their inhibition activity on pteridine reductase 1 (PTR1), an enzyme responsible for the salvage of pterins in Leishmania parasites [152,153]. Initially, a molecular docking study was carried using the 3D structure of PTR1 from L. major aiming at designing a series of hybrids to explore specific interactions of chalcone and dihydropyrimidine moieties in the active site. Based on the binding modes and scores obtained from the docking, authors prioritized 13 hybrid compounds for synthesis and biological Moleculesevaluation.2017, 22 As, 1210 a result, low micromolar activities were reported for most of these hybrids. Hybrid17 of 25 42 displayed the highest activity against L. major and L. donovani with IC50 of 1.06 μM and 15.6 μM, respectively (Figure 15). Finally, bioisosteric replacement of the α,β-unsaturated carbonyl system against L. major and L. donovani with IC of 1.06 µM and 15.6 µM, respectively (Figure 15). Finally, with a pyrazoline significantly altered 50the conformation of hybrid 43 (see Figure 15) in active site of bioisosteric replacement of the α,β-unsaturated carbonyl system with a pyrazoline signiﬁcantly altered PTR1 and increased anti-leishmanial activity (IC50 of 0.948 μM and 3.03 μM against L. major and the conformation of hybrid 43 (see Figure 15) in active site of PTR1 and increased anti-leishmanial L. donovani, respectively) [151]. activity (IC50 of 0.948 µM and 3.03 µM against L. major and L. donovani, respectively) [151].

FigureFigure 15.15. Anti-leishmanialAnti-leishmanial chalcone-dihyd chalcone-dihydropyrimidineropyrimidine hybrids hybrids (red and (red blue, and respectively) blue, respectively) prioritized Moleculesprioritizedby 2017 molecular, 22, by1210 molecular docking studies docking [151]. studies [151]. 17 of 24

OurOur group group also also employed employed a computer-aided a computer-aided approach approach to investigate to investigate a set of 32 a recently set of 32synthesized recently synthesizedheteroaryl chalcones heteroaryl as chalconesanti-leishmanial as anti-leishmanial agents [154]. agents Initially, [154 we]. Initially,performed we an performed inverse screening an inverse of screeningthe best mapping of the best poses mapping of the poses heteroaryl of the heteroaryl chalcones chalconesagainst all against the pharmacophore all the pharmacophore models models(51.431 (51.431LBPs and LBPs 16.159 and SBPs) 16.159 available SBPs) available on PharmMapper, on PharmMapper, a freely accessed a freely web accessed server web designed server to designed identify topotential identify target potential candidates target candidates[155]. Then, [155all primar]. Then,y sequences all primary of sequencesthe predicted of the targets predicted were aligned targets wereto thealigned L. infantum tothe genome.L. infantum Subsequently,genome. seven Subsequently, potential L. seveninfantum potential targets L.for infantum the chalconetargets derivatives for the chalconewere investigated derivatives using were investigatedthe underlying using assumption the underlying that assumptionproteins sharing that proteins enough sharing similarity enough (or similaritypharmacophore (or pharmacophore features) have features) enhanced have probability enhanced of probability sharing the of same sharing ligands the same [156,157]. ligands Due [156 to,157 the]. Dueabsence to the of absenceX-ray structures of X-ray structureson PDB, a onhomology PDB, a homologymodeling modelingapproach approachwas employed was employedto build the to buildstructures thestructures of the predicted of the targets. predicted These targets. structures These were structures then used were in then docking used studies in docking to screen studies the tomost screen promising the most anti-leishmanial promising anti-leishmanial heteroaryl chalco heteroarylnes based chalcones on their basedenergy on scores. their energyThe results scores. of Thetarget results fishing of targetfollowed ﬁshing by docking followed studies by docking sugge studiesst that suggestheteroaryl that chalcones heteroaryl could chalcones act in couldL. infantum act in L.because infantum of becausetheir interaction of their interaction with cysteine with cysteineproteases. proteases. Lastly, nine Lastly, promising nine promising compounds compounds and three and threepotentially potentially inactive inactive compounds compounds were wereexperimentally experimentally evaluated evaluated against against L. infantumL. infantum amastigotesamastigotes and andmammalian mammalian cells. cells. Two Two compounds, compounds, 44 44andand 45,45 exhibited, exhibited IC IC50 50ofof 6.30 6.30 μµMM and and 9.60 9.60 μµM againstagainst amastigotes,amastigotes, respectivelyrespectively (see (see Figure Figure 16). In16). addition, In addition, these compounds these compounds demonstrated demonstrated low cytotoxicity low towardscytotoxicity macrophages towards macrophages (SI > 5.2) and (SI Vero > 5.2) cells and (SI Vero > 10.4) cells [154 (SI]. > 10.4) [154].

FigureFigure 16.16. Anti-leishmanial 5-nitro-substituted heteroar heteroarylyl chalcones identiﬁedidentified by integrationintegration ofof pharmacophorepharmacophore screeningscreening andand molecularmolecular dockingdocking studiesstudies [[154].154].

5.5. Conclusions InIn summary,summary, the the main main goal goal of this of reviewthis review was to wa emphasizes to emphasize that chalcone that derivativeschalcone derivatives are promising are startingpromising points starting for drugpoints discovery. for drug discovery. Chalcone-containing Chalcone-containing plants have plants been have used been for used a long for time a long in traditionaltime in traditional medical medical practice. practice. These compoundsThese compounds also have also wellhave deﬁned well defined mechanisms mechanisms of action of action and potentand potentin vitro in vitroand in and vivo inactivities vivo activities against against several several pathological pathological conditions. conditions. Computer-aided Computer-aided drug drug design approaches may establish chalcones as a source of inspiration to design and develop novel and more effective drug candidates. Here, we highlighted the significant advances in organic synthesis as well as molecular modification strategies that have contributed to the design of new chalcone derivatives with improved biological activities. Although expert knowledge is indisputably important for successfully guiding drug design, the considerable amount of biological data generated from chalcone derivatives and the public availability of this information has launched the open data era of drug discovery. The availability of these data boost the use of LBDD and SBDD strategies, allowing the generation and validation of computational models for prioritization of chalcone derivatives for synthesis and/or biological evaluation. Despite the small number of examples of in silico-driven design of chalcones available in the literature, most of them have led to the discovery of new lead candidates with potency or affinity values in the low micromolar range. Based on this, we believe that the integration of molecular modification strategies and computational approaches described in this review can accelerate the discovery of new chalcone derivatives that could ultimately enter the market.

Acknowledgments: The authors would like to thank Brazilian funding agencies, CNPq, CAPES, and FAPEG, for financial support and fellowships. Eugene Muratov also thanks the financial support from NIH (grant 1U01CA207160) and CNPq (grant 400760/2014-2). Maristela Pereira, Carolina H. Andrade, and Pedro V. L. Cravo

Molecules 2017, 22, 1210 18 of 25 design approaches may establish chalcones as a source of inspiration to design and develop novel and more effective drug candidates. Here, we highlighted the significant advances in organic synthesis as well as molecular modification strategies that have contributed to the design of new chalcone derivatives with improved biological activities. Although expert knowledge is indisputably important for successfully guiding drug design, the considerable amount of biological data generated from chalcone derivatives and the public availability of this information has launched the open data era of drug discovery. The availability of these data boost the use of LBDD and SBDD strategies, allowing the generation and validation of computational models for prioritization of chalcone derivatives for synthesis and/or biological evaluation. Despite the small number of examples of in silico-driven design of chalcones available in the literature, most of them have led to the discovery of new lead candidates with potency or affinity values in the low micromolar range. Based on this, we believe that the integration of molecular modification strategies and computational approaches described in this review can accelerate the discovery of new chalcone derivatives that could ultimately enter the market.

Acknowledgments: The authors would like to thank Brazilian funding agencies, CNPq, CAPES, and FAPEG, for financial support and fellowships. Eugene Muratov also thanks the financial support from NIH (grant 1U01CA207160) and CNPq (grant 400760/2014-2). Maristela Pereira, Carolina H. Andrade, and Pedro V. L. Cravo are Research Fellows in productivity of CNPq. We are also grateful to OpenEye Scientific Software, Inc. (Santa Fe, NM, USA) and ChemAxon (Budapest, Hungary) for providing us with the academic license for their software. Conflicts of Interest: The authors declare no conflict of interest.

References

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