molecules

Review Exploiting the Indole Scaffold to Design Compounds † Binding to Different Pharmacological Targets

Sabrina Taliani 1,* , Federico Da Settimo 1, Claudia Martini 1 , Sonia Laneri 2, Ettore Novellino 2 and Giovanni Greco 2,* 1 Department of Pharmacy, University of Pisa, Via Bonanno Pisano, 6, 56126 Pisa, Italy; [email protected] (F.D.S.); [email protected] (C.M.) 2 Department of Pharmacy, University of Naples “Federico II”, Via D. Montesano, 49, 80131 Naples, Italy; [email protected] (S.L.); [email protected] (E.N.) * Correspondence: [email protected] (S.T.); [email protected] (G.G.); Tel.: +39-050-2219547 (S.T.); +39-081-678645 (G.G.) The authors wish to dedicate this review to the memory of Professor Barbara Cosimelli, our dear friend and † colleague, who was a key participant in the research discussed herein.



Academic Editors: Marco Catto and Cosimo Damiano Altomare
 Received: 20 April 2020; Accepted: 15 May 2020; Published: 16 May 2020

Abstract: Several indole derivatives have been disclosed by our research groups that have been collaborating for nearly 25 years. The results of our investigations led to a variety of molecules binding selectively to different pharmacological targets, specifically the type A γ-aminobutyric acid (GABAA) chloride channel, the translocator protein (TSPO), the murine double minute 2 (MDM2) protein, the A2B adenosine receptor (A2B AR) and the Kelch-like ECH-associated protein 1 (Keap1). Herein, we describe how these works were conceived and carried out thanks to the versatility of indole nucleus to be exploited in the design and synthesis of drug-like molecules.

Keywords: type A γ-aminobutyric acid (GABAA) chloride channel; translocator protein (TSPO); murine double Minute 2 (MDM2) protein; A2B adenosine receptor (A2B AR); Kelch-like ECH-associated protein 1 (Keap1)

1. Introduction During the last 25 years our research groups have been engaged in preparing and testing several indole derivatives as ligands for some pharmacologically relevant targets: namely, the type A γ-aminobutyric acid chloride channel, the translocator protein, the murine double minute 2 protein, the A2B adenosine receptor and the Kelch-like ECH-associated protein 1. We chose indole as a privileged scaffold owing to its recognized ability of being exploited to obtain drug-like molecules [1]. In virtue of its chemical reactivity, this heterocycle is amenable to be readily modified in order to introduce multiple decorations, so to obtain a multitude of indole-based compounds. Indole is widely distributed among biologically active molecules, either natural (many alkaloids, tryptophan, plant hormones) and synthetic, acting on a huge number of therapeutic targets [1]. The present review summarizes our studies taking indole as the polar star of our medicinal chemistry strategies.

2. Indole Derivatives as Ligands of the Benzodiazepine Receptor Many drugs structurally related to diazepam bind to a site known for a long time as the benzodiazepine receptor (BzR). Although this term was changed in 1998 to “benzodiazepine binding site” [2] the acronym BzR has been widely used for decades and it is still usually employed by the scientific community. This binding site is located within the transmembrane type A γ-aminobutyric acid (GABAA) chloride channel, one of the most important members of the family of pentameric

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acid (GABAA) chloride channel, one of the most important members of the family of pentameric ligand-gatedligand-gated ionion channels channels [2]. When[2]. When this channel this ischannel activated is byactivated interaction by with interaction the neurotransmitter with the GABA,neurotransmitter the flow of GABA, chloride the into flow the of cell chloride increases into and the produces cell increases hyperpolarisation. and produces Mammals hyperpolarisation. express a numberMammals of GABAexpressA aisoforms number localized of GABA inAthe isoforms CNS which localized are composed in the CNS by which five subunits: are composed two β3 ,by one fiveγ2 andsubunits: two α twoamong β3,six one types γ2 and (α1, αtwo2, α 3α, αamong4, α5, α 6six). Such types a pentameric(α1, α2, α3, organization α4, α5, α6). (FigureSuch a1 )pentameric originates theorganization following (Figure six GABA 1) Aorigsubtypes:inates theα1 followingβ3γ2, α2β 3sixγ2 ,GABAα3β3γA2, subtypes:α4β3γ2, α 5αβ1β3γ3γ22,and α2β3αγ62β, α3γ3β23[γ32–, 5α].4β The3γ2, aboveα5β3γ2 subtypesand α6β3γ can2 [3–5]. be also The named above bysubtypes specifying can thebe alsoα subunit named which by specifying imparts specific the α subunit physiological which andimparts pharmacological specific physiological properties and to thepharmacological GABAA complex properties [6–10]. to The theα 1GABAsubtype,A complex the dominant [6–10]. one,The isα1 presentsubtype, in the both dominant the cortex one, and is present cerebellum in both and the mediates cortex and sedation; cerebellum the α and2 and mediatesα3 subtypes sedation; are foundthe α2 mainlyand α3 subtypes in the cortex are andfound the mainly hippocampus in the cortex andare and involved the hippocampus in anxiolytic and and are myorelaxant involved in eanxiolyticffects; the andα5 subtypemyorelaxant is largely effects; expressed the α5 subtype in the hippocampusis largely expressed and is in associated the hippocampus with cognition and is processesassociated likewith learning cognition and processes memorising; like thelearningα4 and andα6 memorising;subtypes, less the investigated, α4 and α6 subtypes, are known less as benzodiazepine-insensitiveinvestigated, are known as binding benzodiazepine-insensi sites because theytive do binding not bind sites diazepam because but they recognize do not several bind non-benzodiazepinediazepam but recognize ligands. several non-benzodiazepine ligands.

β 3 γ α 2 GABA site n Cl- BzR β 3 αn

Figure 1.1. SchematicSchematic representation representation of of the the organization organization of theof fivethe five protein protein subunits subunits composing composing the major the GABAmajor AGABAcomplexA complex isoforms. isoforms. There areThere two areβ3 subunits,two β3 subunits, one γ2 subunitsone γ2 subunits and two and among two six amongα subunits six α (wheresubunits n varies(where from n varies 1 to 6).from The 1 circleto 6). in The bold circle corresponds in bold corresponds to the chloride to channel.the chloride The channel. binding sitesThe ofbinding the neurotransmitter sites of the neurotransmitter GABA and of theGABA BzR and ligands of the are BzR evidenced ligands by are filled evidenced circles and, by filled respectively, circles aand, square. respectively, a square.

Following the introduction in the clinical usage of chlordiazepoxide in 1960, a huge number of chemically heterogeneous classes of compounds have been reported in literature as BzR ligands [11]. [11]. This bindingbinding site site is is located located at theat theα/γ αsubunits’/γ subunits’ interface, interface, distinct distinct from thefrom GABA the bindingGABA binding sites situated sites atsituatedα/β interfaces at α/β interfaces (Figure1 ).(Figure 1). The pharmacologicalpharmacological actions actions of BzRof BzR ligands ligands range range from fullfrom agonism full agonism (associated (associated with anxiolytic, with anticonvulsant,anxiolytic, anticonvulsant, sedative-hypnotic, sedative-hypnotic, and myorelaxant and myorelaxant effects) to effects) antagonism to antagonism (implying (implying the ability the to reverseability to sedation reverse caused sedation by caused agonists) by and agonists) to inverse and agonismto inverse (characterized agonism (characterized by anxiogenic, by anxiogenic, somnolytic andsomnolytic proconvulsant and proconvulsant effects) [12,13 effects)]. [12,13]. In 19871987 SchofieldSchofield andand coworkerscoworkers reportedreported thethe sequencesequence and the functionalfunctional expression of the GABAA channel [14]. [14]. Subsequently Subsequently to to this pioneeri pioneeringng work, further knowledge about the subunitssubunits composing the GABAGABAAA isoformsisoforms was was achieved [15–17]. [15–17]. Since 2000, molecular geneticsgenetics experimentsexperiments and measurements of binding and eefficacyfficacy ofof BzRBzR ligandsligands atat eacheach ofof thethe GABAGABAAA isoformsisoforms [[18–21]18–21] paved thethe wayway forfor the the search search of of compounds compounds provided provided by by affi affinity-nity- and and/or/or effi efficacy-basedcacy-based selectivity selectivity for αfor1, α12,/ α32/,αα35, αBzR5 BzR subtypes subtypes [22 [22].]. Currently, thethe manymany BzR ligands available as drugs are agonists endowed with the above- mentioned depressantdepressant e ffeffectsects (ascribed (ascribed to their to their action action on the αon1 subtype)the α1 subtype) and the antagonist and the flumazenil,antagonist employedflumazenil, as employed antidote as to antidote treat benzodiazepine to treat benzod overdose.iazepine overdose. Given the Given therapeutic the therapeutic potential potential of BzR ligands,of BzR ligands, this class this of compoundsclass of compounds has been has one been of the one most of intriguingthe most intriguing and challenging and challenging field of medicinal field of chemistrymedicinal research.chemistry research. Our studies of indole derivatives as BzR ligands began in 1985 with the synthesissynthesis and the evaluation of the binding affinities affinities of racemic N-(indol-3-ylglyoxylyl)amino acid acid derivatives derivatives ( (11))[ [23].23]. Compounds of series 1 were designed taking the 3-ethoxycarbonyl-β-carboline (2), reported to bind with nanomolar affinity to the BzR [24], as a reference structure.

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Molecules 2020, 25, x FOR PEER REVIEW 3 of 26 CompoundsMolecules 2020, 25 of, x series FOR PEER1 were REVIEW designed taking the 3-ethoxycarbonyl-β-carboline (2), reported to3 bindof 26 with nanomolar affinity to the BzR [24], as a reference structure.

In series 1, the groups at the 5 position of the indole nucleus (R) were H, Cl, Br, OCH3, NO2, In series 1, the groups at the 5 position of the indole nucleus (R) were H, Cl, Br, OCH3, NO2, whileIn the series R’ substituents1, the groups of at the the aminoacid 5 position ofmoiety the indole were nucleushydrogen, (R) werealkyl, H, benzyl Cl, Br, and OCH indolylmethyl,, NO , while while the R’ substituents of the aminoacid moiety were hydrogen, alkyl, benzyl and indolylmethyl,3 2 somethe R’ of substituents which bearing of the small aminoacid groups moiety on their were benz hydrogen,ene rings. alkyl, R’’ benzylwas a andhydrogen indolylmethyl, in the subset some of some of which bearing small groups on their benzene rings. R’’ was a hydrogen in the subset of whichcarboxylic bearing acids small and a groups methyl on or theiran ethyl benzene in the rings. subset R” of wasesters. a hydrogen in the subset of carboxylic carboxylic acids and a methyl or an ethyl in the subset of esters. acidsAt and that a methyltime, the or endpoint an ethyl inin theour subset biological of esters. experiments was the ability of the tested compound At that time, the endpoint in our biological experiments was the ability of the tested compound to displaceAt that a time, radioligand the endpoint (generally in our [ biological3H]flunitrazepam) experiments from was neuronal the ability membranes of the tested obtained compound by the to 3 to displace a radioligand (generally3 [ H]flunitrazepam) from neuronal membranes obtained by the cortexdisplace of a bovine radioligand brain. (generally In the light [ H]flunitrazepam) of the knowledge from about neuronal the GABA membranesA isoforms obtained acquired by the several cortex cortex of bovine brain. In the light of the knowledge about the GABAA isoforms acquired several yearsof bovine after brain. our initial In the studies, light of such the knowledge binding data about correlated the GABA mainlyA isoforms with the acquired α1 BzR severalsubtype. years after years after our initial studies, such binding data correlated mainly with the α1 BzR subtype. our initialMost studies,of the compounds such binding of dataseries correlated 1 exhibited mainly affinity with values the α inBzR the subtype. micromolar-submillimolar Most of the compounds of series 1 exhibited affinity values1 in the micromolar-submillimolar range.Most The ofesters the were compounds more potent of series than1 theexhibited corresponding affinity acids. values In in the the ester micromolar-submillimolar subset two compounds range. The esters were more potent than the corresponding acids. In the ester subset two compounds wererange. endowed The esters by were submicromolar more potent than affinity, the corresponding both bearing acids. a methyl In the estergroup subset as R’, two while compounds the R were endowed by submicromolar affinity, both bearing a methyl group as R’, while the R weresubstituent endowed was by a chlori submicromolarne or a nitro affi group.nity, both bearing a methyl group as R’, while the R substituent substituent was a chlorine or a nitro group. was aIn chlorine a subsequent or a nitro paper group. [25], we described the synthesis and the binding affinity for BzR of In a subsequent paper [25], we described the synthesis and the binding affinity for BzR of opticallyIn a subsequentactive forms paper of some [25], N we-(indol-3-ylglyoxylyl)aminoacids described the synthesis and the bindingof series a ffi1.nity Expectedly, for BzR of the optically esters optically active forms of some N-(indol-3-ylglyoxylyl)aminoacids of series 1. Expectedly, the esters performedactive forms better of some thanN -(indol-3-ylglyoxylyl)aminoacidsthe corresponding acids. The two of seriescompounds1. Expectedly, provided the with esters the performed highest performed better than the corresponding acids. The two compounds provided with the highest affinitybetter than as theracemic corresponding mixtures acids.in the The previous two compounds work [23] provided did not withshow the appreciable highest affi nitydifferences as racemic in affinity as racemic mixtures in the previous work [23] did not show appreciable differences in affinitymixtures when in the tested previous as pure work optica [23] didl isomers, not show thus appreciable suggesting di ffthaterences the α in-methyl affinity side when chain tested does as pure not affinity when tested as pure optical isomers, thus suggesting that the α-methyl side chain does not occupiesoptical isomers, a sterically thus hindered suggesting cleft that within the α the-methyl BzR. side chain does not occupies a sterically hindered occupies a sterically hindered cleft within the BzR. cleftIn within 1992 the we BzR. reported a series of N-(indol-3-ylglyoxylyl)-β-arylethylamines (3) as BzR ligands In 1992 we reported a series of N-(indol-3-ylglyoxylyl)-β-arylethylamines (3) as BzR ligands [26]. InIn 1992this series, we reported R was a H, series Cl, Br, of N NO-(indol-3-ylglyoxylyl)-2; R’ was H or CH3;β the-arylethylamines β-arylethyl side (3) chain as BzR derived ligands from [26]. [26]. In this series, R was H, Cl, Br, NO2; R’ was H or CH3; the β-arylethyl side chain derived from tryptamine,In this series, tyramine, R was H, Cl, dopamine, Br, NO ; R’α-phenylethlylamins was H or CH ; the β -arylethylbearing variou side chains substituents derived from on tryptamine,the phenyl tryptamine, tyramine, dopamine,2 α-phenylethlylamins3 bearing various substituents on the phenyl ring.tyramine, dopamine, α-phenylethlylamins bearing various substituents on the phenyl ring. ring. O H R O NH Ar R N Ar O O N N R' R' 3 3 All the 1-methyl derivatives (R’ = CH3) were inactive, suggesting that the NH of the indole All the 1-methyl derivatives (R’ = CH3) were inactive, suggesting that the NH of the indole nucleusAll is the engaged 1-methyl in derivativesa H-bond with (R’ =theCH BzR3) wereor, alternatively, inactive, suggesting is sterically that repelled the NH by of the the binding indole nucleus isis engagedengaged inin aa H-bond H-bond with with the the BzR BzR or, or, alternatively, alternatively, is stericallyis sterically repelled repelled by theby bindingthe binding site. site. The best ligands of the new series showed Ki values of 85 nM (R = H, R’ = H, Ar = site. The best ligands of the new series showed Ki values of 85 nM (R = H, R’ = H, Ar = Them-methoxyphenyl) best ligands of theand new 90 seriesnM (R showed = H, R’ Ki values= H, Ar of = 85 p nM-methoxyphenyl). (R = H, R’ = H, Surprisingly, Ar = m-methoxyphenyl) when the m-methoxyphenyl) and 90 nM (R = H, R’ = H, Ar = p-methoxyphenyl). Surprisingly, when the hydrogenand 90 nM at (R the= H, 5-position R’ = H, Ar of= thep-methoxyphenyl). indole scaffold of Surprisingly, the two above when compounds the hydrogen was at replaced the 5-position by a ofhydrogen the indole at scatheff old5-position of the two of the above indole compounds scaffold was of the replaced two above by a nitro compounds group affi wasnity replaced decreased by to a nitro group affinity decreased to a considerable exent (Ki > 10 μM). The remaining compounds considerablenitro group exentaffinity (K decrease> 10 µM).d Theto a remainingconsiderable compounds exent (K showedi > 10 μ KM).values The inremaining the micromolar compounds range. showed Ki values in thei micromolar range. i showed Ki values in the micromolar range. In orderorder to to highlight highlight the the role role of theof indolethe indole NH fragmentNH fragment in the interactionin the interaction with the with BzR, the we preparedBzR, we In order to highlight the role of the indole NH fragment in the interaction with the BzR, we prepareda number ofa number benzofurane of benzofurane and benzothiophene and benz derivativesothiophene of derivatives general formula of general4 and, respectively,formula 4 and,5 in prepared a number of benzofurane and benzothiophene derivatives of general formula 4 and, whichrespectively, the above 5 in fragment which the was above replaced fragment by an was oxygen replaced or, respectively, by an oxygen a sulphur or, respectively, [27]. a sulphur respectively, 5 in which the above fragment was replaced by an oxygen or, respectively, a sulphur [27]. [27].

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The new compounds were much worse in terms of potency compared with the corresponding TheThe new new compounds compounds werewere muchmuch worseworse inin termsterms of potency compared with with the the corresponding corresponding isostericThe newindoles compounds reported werein our much previous worse papersin terms [23,26], of potency clearly compared indicating with that the thecorresponding indole NH isostericisosteric indoles indoles reported reported in ourin our previous previous papers papers [23,26 [23,26],], clearly clearly indicating indicating that the that indole the NHindole donates NH isostericdonates aindoles H-bond reported to an acceptor in our heteroatom previous papers of the BzR.[23,26], clearly indicating that the indole NH adonates H-bond a to H-bond an acceptor to an heteroatomacceptor heteroatom of the BzR. of the BzR. donatesWith a H-bondthe aim toof anfinding acceptor indole heteroatom derivatives of the with BzR. improved affinity for the BzR, three subsets of WithWith the the aim aim of of finding finding indoleindole derivativesderivatives with improved affinity affinity for for the the BzR, BzR, three three subsets subsets of of indolylglyoxylamidesWith the aim of finding (general indole formulae derivatives 6, 7 and with 8) were improved shortly affinity after synthesized for the BzR, and three tested subsets [28]. ofIn indolylglyoxylamidesindolylglyoxylamides (general (general formulaeformulae 66,, 77 andand 88)) werewere shortlyshortly after synthesized and and tested tested [28]. [28]. In In indolylglyoxylamidesthe three subsets R = H, (general Cl, NO formulae2 while R’ 6 =, 7H, and p-F, 8 )p were-Cl, p -OCHshortly3, afterm,p-(OCH synthesized3)2. and tested [28]. In thethe three three subsets subsets R R= = H,H, Cl,Cl, NONO22 while R’ = H,H, pp-F,-F, pp-Cl,-Cl, pp-OCH-OCH3,3 ,mm,p,p-(OCH-(OCH3)32.) 2. the three subsets R = H, Cl, NO2 while R’ = H, p-F, p-Cl, p-OCH3, m,p-(OCH3)2.

WhileWhile thethe anilidesanilides 77 andand thethe γ-phenylpropylamides 88 showedshowed poorpoor affinity,affinity, severalseveral While thethe anilides anilides7 and 7 theandγ -phenylpropylamidesthe γ-phenylpropylamides8 showed 8 poorshowed affinity, poor several affinity, benzylamides several benzylamidesbenzylamides 66 displayeddisplayed nanomolarnanomolar potency. The scarce performanceperformance ofof indolesindoles 77 andand 88 waswas benzylamides6 displayed nanomolar 6 displayed potency. nanomolar The scarce potency. performance The scarce of performance indoles 7 and of8 wasindoles ascribed 7 and to 8 steric was ascribedascribed to to steric steric repulsiverepulsive interactionsinteractions taking place between theirtheir sideside chainchain phenylphenyl rings rings and and the the repulsive interactions taking place between their side chain phenyl rings and the boundaries of the ascribedboundariesboundaries to steric ofof thethe repulsive BzR.BzR. TheThe interactions excellentexcellent binding taking placedata betweenof some benzylamidestheirbenzylamides side chain 66 phenylsuggestedsuggested rings thatthat and their their the BzR. The excellent binding data of some benzylamides 6 suggested that their phenyl ring was correctly boundariesphenylphenyl ringring of waswas the correctly correctlyBzR. The orientedoriented excellent to bindingfind room data into of asome hydrophobic benzylamides regionregion 6 ofofsuggested thethe BzR.BzR. that TheThe besttheir best phenyloriented ring to findwas room correctly into aoriented hydrophobic to find region room ofinto the a BzR. hydrophobic The best performingregion of the ligand BzR. of The series best6 performingperforming ligaligandnd ofof seriesseries 66 (K(Kii 1111 nM)nM) had R = NO22 andand R’R’ == mm,,pp-(OCH-(OCH33))22. . TheThe structure-affinitystructure-affinity i m p 2 3 2 6 performing(Krelationshipsrelationshipsi 11 nM) hadliga in in Rndseries series= ofNO series 662 werewereand 6 R’characterizedcharacterized(K= 11 ,nM)-(OCH had by3 ) R2interdependent. = The NO structure-a and R’ ee=ffectsffects ffimnity,p-(OCH ofof relationships thethe R )R .and andThe R’ R’structure-affinity substituents insubstituents series were on on relationshipscharacterized in by series interdependent 6 were characterized effects of the by R interdependent and R’ substituents effects on of potency. the R and Particularly, R’ substituents affinity on of potency.potency. Particularly,Particularly, affinityaffinity ofof thethe 5-Cl/NO2 derivatives waswas improvedimproved byby hydroxyl/methoxyhydroxyl/methoxy potency.thesubstituentssubstituents 5-Cl/ NOParticularly,2 onderivativeson thethe sideside affinity chain waschain improved ofphenylphenyl the 5-Cl/NOring, by hydroxyl while2 derivatives affinity/methoxy ofof wasthethe substituents 5-H5-Himproved derivativesderivatives on by the hydroxyl/methoxy was sidewas increased chainincreased phenyl by by substituentsring,halogenshalogens while on aon ffion thenitythe the same ofsame side the 5-Hchainphenylphenyl derivatives phenyl riring.ng. ring,Actually, was while increased such affinity bya halogenspattern of the 5-Hofof on interdependent interdependentthederivatives same phenyl was effectseffects ring.increased Actually, ofof the theby halogenssuchsubstituentssubstituents a pattern on at atthe ofthe the interdependentsame 5-position5-position phenyl ofof thethe eriffng.ects indoleindole Actually, of thescaffold substituents such had aalready pattern at the beenbeen 5-position of observedobservedinterdependent of thein in series indoleseries effects 3 3. sca . ff oldof hadthe substituentsalreadyInIn order beenorder at observedto to the identify identify 5-position in indoleindole series of derivatives derivatives3the. indole scaffoldas BzR li hadgands already characterized been observed byby good good in water waterseries solubility, solubility,3. we we preparedpreparedIn order order a a numbernumber to identify identify ofof N Nindole indole’-phenylindol-3-ylglyoxylhydrazides’-phenylindol-3-ylglyoxylhydrazides derivatives derivatives as as BzR BzR li ligandsgands characterized characterized ((99)) [29].[29]. InIn by by thisthis good good seriesseries water water we we solubility, solubility,inserted inserted the we the preparedfollowingfollowing a substituents substituents number of N N onon’-phenylindol-3-ylglyoxylhydrazides’-phenylindol-3-ylglyoxylhydrazides thethe parentparent structure:structure: R = H, Cl,Cl, NONO ( (2929 and)and)[ [29].29 R’].R’ In In== H,this thisH, p p-F,series series-F, pp-Cl,-Cl, we we p p -NOinserted -NO2,2 ,m m- -the or or followingpp-CH-CH3,3 ,p p-OH,-OH, substituents p p-OCH-OCH33.. on the parent structure:structure: R = H, Cl, NO2 andand R’ R’ == H,H, pp-F,-F, p-Cl, p-NO2,, mm-- or p-CH3,, pp-OH,-OH, pp-OCH-OCH33. .

R'R' O H RR N R' O HN N R N NH O H N O H NH H 9 9 9 AffinityAffinity ofof compoundscompounds 99 waswas restrictedrestricted to thethe 5-H5-H derivatives,derivatives, whereaswhereas thethe 5-Cl/NO5-Cl/NO2 2 derivativesAffinity wereof compounds all devoid of9 wasaffinity. restricted Again, tothe the structure-affinity 5-H derivatives, relationships whereas ofthe these 5-Cl/NO two 2 derivativesAffinity were of compounds all devoid9 wasof affinity. restricted Again, to the 5-Hthe derivatives,structure-affinity whereas relationships the 5-Cl/NO of derivativesthese two derivativessubsets of werecompounds all devoid were of divergent. affinity. Again,In an athettempt structure-affinity to explain the relationships reasons underlying of2 these such two weresubsets all devoidof compounds of affinity. were Again, divergent. the structure-a In anffi nityattempt relationships to explain of these the tworeasons subsets underlying of compounds such subsetsfindings, of wecompounds searched werethe Cambridge divergent. StructuralIn an attempt Database to explain [30] looking the reasons for differences underlying in suchthe werefindings, divergent. we searched In an the attempt Cambridge to explain Structural the reasons Database underlying [30] looking such findings,for differences we searched in the findings,conformational we searched properties the of Cambridge N’-arylhydrazides Structural and DatabaseN-arylamides. [30] Welooking realized for that differences benzylamides in the 6 theconformational Cambridge properties Structural of Database N’-arylhydrazides [30] looking and for N di-arylamides.fferences in We the realized conformational that benzylamides properties 6 conformationalcancan adopt adopt aa transoidtransoid properties conformationconformation of N’-arylhydrazides aboutabout the C-N-C-Ar and N-arylamides. torsiontorsion angleangle We realized (values(values comprisedthatcomprised benzylamides between between 6 can adopt a transoid conformation about the C-N-C-Ar torsion angle (values comprised between

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Molecules 2020, 25, x FOR PEER REVIEW 5 of 26 of N’-arylhydrazides and N-arylamides. We realized that benzylamides 6 can adopt a transoid conformation about the C-N-C-Ar torsion angle (values comprised between 150 and +150 ). Such a -150° and +150°). Such a staggered conformation is forbidden to hydrazides− 9 which◦ are forced◦ into a staggeredgauche disposition conformation about isthe forbidden corresponding to hydrazides C-N-N-Ar9 which torsion are angle forced (values into a gauchecompriseddisposition between about -60° the corresponding C-N-N-Ar torsion angle (values comprised between 60 and 120 or between and -120° or between +60° and +120°). − ◦ − ◦ +60◦ Theand above+120◦ ).data led us to hypothesize that our indole derivatives might bind to BzR by adopting one Theout aboveof two data conformations led us to hypothesize and orientations that our indolewithin derivatives the binding might cavity bind todepending BzR by adopting on the onesubstituent out of two at conformationsthe 5-position andof the orientations indole nucleu withins. theOn binding the basis cavity of this depending conjecture, on the we substituent began to atconsider the 5-position the 5-Cl/NO of the2 and indole the nucleus.5-H indole On derivatives the basis ofasthis different conjecture, “families” we began of BzR to ligands, consider each the 5-Cldisplaying/NO2 and peculiar the 5-H structure-affi indole derivativesnity relationships. as different “families” In our ofspeculations BzR ligands, we each were displaying aided peculiarby the structure-apharmacophore/topologicalffinity relationships. model In our proposed speculations by C weook were and aided coworkers by the pharmacophore[31] defined by/topological the same modelauthors proposed as “comprehensive” by Cook and coworkersbecause it [31holds] defined for agonists, by the same antagonists authors asand “comprehensive” inverse agonists. because This itmodel holds includes for agonists, the following antagonists interaction and inverse sites: agonists. (i) a H-bond This model acceptor includes (A2), (ii) the a following H-bond donor interaction (H1), sites:(iii) a (i)bifunctional a H-bond acceptorH-bond (Adonor/acceptor2), (ii) a H-bond (H donor2/A3), and (H1 ),(iv) (iii) four a bifunctional lipophilic H-bondpockets donor(L1, L2/,acceptor L3, and (HLDi2)./ AThe3), andboundaries (iv) four of lipophilic the binding pockets site are (L1 ,Ldefi2,Lned3, andin terms LDi). of The sterically boundaries forbidden of the sites binding (S1, siteS2, and are definedS3). Regarding in terms the of efficacy sterically profile, forbidden the only sites safe (S1 ,Sstatement2, and S 3was). Regarding that filling the of ethefficacy L3 pocket profile, (occupied the only safeby the statement diazepam was pendant that filling phenyl of the ring) L3 pocket was (occupiedmandatory by for the agonism. diazepam The pendant two putative phenyl ring) binding was mandatorymodes (named for agonism. A and B) The of twoour putativeindole derivatives binding modes are depicted (named Ain and Figure B) of 2 ourin the indole framework derivatives of areCook’s depicted model. in The Figure binding2 in themode framework A of the 5-Cl/NO of Cook’s2 indoles model. requires The binding a staggered mode conformation A of the 5-Cl of/NO the2 indolesside chain requires and gives a staggered rise to conformationthe following ofinteractions: the side chain (i) the and indole gives riseNH tois theH-bond following to the interactions: A2 site; (ii) (i)the the C=O1 indole and NH C=O2 is H-bondare H-bound to the to A 2thesite; H2 (ii) and the H C1 sites,=O1 andrespectively; C=O2 are (iii) H-bound the CH to2, the the phenyl H2 and and H1 sites,the fused respectively; benzene (iii)ring the fillCH the2 ,L the1, L2 phenyl, and L andDi pockets, the fused respectively. benzene ring The fill binding the L1 ,Lmode2, and B is LDi accessiblepockets, respectively.only to 5-H indoles The binding because mode the Bsterically is accessible forbidden only to S2 5-H site, indoles closely becausefacing the the 5-position sterically forbiddenof the indole S2 site,nucleus, closely cannot facing host the substituents 5-position of larger the indole than nucleus, a hydrogen. cannot Such host a substituents binding mode, larger compatible than a hydrogen. with a Suchfolded a bindingconformation mode, of compatible the side chain, with a is folded charac conformationterized by the of thefollowing side chain, interactions: is characterized (i) C=O1 by and the followingC=O2 are H-bound interactions: to the (i) CH=2 O1and and H1 sites, C=O2 respectively; are H-bound (ii) to the the lipophilic H2 and H L1 1sites, and L respectively;2 pockets are (ii) filled the lipophilicby the pyrrole L1 and and, L2 pockets respectively, are filled the by benzene the pyrrole moieties and, respectively, of indole; (iii) the benzenethe indole moieties NH donates of indole; a (iii)H-bond the indole to a heteroatom NH donates belonging a H-bond to to the a heteroatom S1 site. It is belongingworth noticing to the that S1 site. each It of is worththe two noticing postulated that eachbinding of the modes two postulatedbenefits from binding three modes H-bonds benefits with from the threeBzR, H-bondsconsistently with with the BzR, the consistentlysimilar affinities with thedisplayed similar by affi thenities best displayed performing by the5-Cl/NO best performing2 and 5-H indoles 5-Cl/NO derivatives2 and 5-H [29]. indoles derivatives [29].

L2 L2 S2 S2 X H2 H2 O2N X O L O L1 1 HN N LDi LDi HN H O O S1 Z N S1

H H1 H1

A A2 2 binding mode A binding mode B

Figure 2. The binding modes A and B hypothesized for the 5-Cl5-Cl/NO/NO22 indoles and, respectively of the 5-H indoles oriented in the frameworkframework of thethe Cook’sCook’s pharmacophorepharmacophore/topological/topological model [[31].31]. Z is a CH2 inin benzylamides benzylamides 6 or a NH in hydrazides 9.

Some years later, wewe preparedprepared andand testedtested severalseveral NN-(indol-3-ylglyoxyl)arylalkylamides-(indol-3-ylglyoxyl)arylalkylamides 10–15 characterized byby conformationallyconformationally oror geometricallygeometrically constrained constrained side side chains, chains, most most of of which which featured featured a chirala chiral center center [32 [32].]. We We reasoned reasoned that that such such properties properties of the of new the side new chains side mightchains increasemight increase the chances the ofchances conferring of conferring affinity-based affinity-based selectivity se forlectivity any of for the any BzR of subtypes.the BzR subtypes.

Molecules 2020,, 25, 2331x FOR PEER REVIEW 66 of of 2726 Molecules 2020, 25, x FOR PEER REVIEW 6 of 26

H O R R' O H R' R O HN R' R O HN S R' R N R R N S O CH3 O CH3 O CH3 O CH3 N N NH NH H H 10 11 10 11

O H O R O HN R O N R N R N O O O N O N NH H NH 13 H 12 13 12

O H O H S R O HN R R O HN R N R R N S O O O O N N NH NH H H 14 15 14 15 In series 10–15 the R substituents were H, Cl or NO2. In series 10 and 11 the R’ substituents on In series 1010––1515 thethe R R substituents substituents were were H, H, Cl Cl or or NO NO2. 2In. Inseries series 10 10andand 11 11thethe R’ substituents R’ substituents on the phenyl ring were H, p-CH3, p-OCH3, m,p-(OCH3)2 and p-NO2. All of the S isomers (11 and 15) onthe thephenyl phenyl ring ring were were H, H,p-CHp-CH3, p3-OCH, p-OCH3, m3,,pm-(OCH,p-(OCH3)2 and3)2 andp-NOp-NO2. All2. of All the of S the isomersS isomers (11 and (11 and 15) lacked affinity, likewise the optically inactive compounds 13. Some of the R isomers displayed Ki 15) lacked affinity, likewise the optically inactive compounds 13. Some of the R isomers displayedi lackedvalues affinity,in the micromolar/nanom likewise the opticallyolar inactiverange. The compounds structure-af 13.finity Some relationships of the R isomers of the displayed R isomers K Kvaluesi values in inthe the micromolar/nanom micromolar/nanomolarolar range. range. The The structure-af structure-afinityffinity relationships relationships of of the the R isomersisomers confirmed our hypothesis about the different binding modes of the 5-Cl/NO2 derivatives and of the confirmedconfirmed our hypothesis about the difffferenterent bindingbinding modes of thethe 5-Cl5-Cl/NO/NO22 derivatives and of the 5-H derivatives. Specifically, in series 10 and 14 the 5-Cl/NO2 were significantly more potent than the 5-H derivatives. Specifically, Specifically, in series 10 and 14 the 5-Cl5-Cl/NO/NO22 were significantly significantly more potent than the 5-H derivatives. Conversely, in series 12 affinity was appreciable (Ki 123 nM) only if R = H. The most 5-H derivatives. Conversely, inin seriesseries 1212 aaffinityffinity waswas appreciableappreciable (K(Kii 123123 nM)nM) onlyonly ifif RR == H. The most potent compound described in this study belonged to series 10 (R = NO2, R’ = H, Ki 17 nM). An 10 2 i potentoverlay compoundcompound of molecular described described models in ofin this a th few studyis studyindole belonged belonged derivatives to series to representativeseries(R 10= NO(R 2=, of R’NO the=,H, fourR’ K =i 17seriesH, nM). K investigated17 An nM). overlay An ofoverlay molecular of molecular models ofmodels a few of indole a few derivatives indole derivatives representative representative of the four of the series four investigated series investigated showed showed that the inactive ones projected portions of their side chains into the sterically forbidden S1 thatsubsiteshowed the inactiveofthat Cook’s the onesinactive model. projected ones A projected portionssubset of ofportions theircompound side of their chainss were side into chainstested the sterically intofor thetheir forbiddensterically ability forbidden Sto1 subsitedisplace ofS1 Cook’s model. A subset of compounds were tested for their ability to displace [3H]flumazenil from [subsite3H]flumazenil of Cook’s from model. recombinant A subset rat α 1,of α 3 compoundand α5 BzRs subtypes.were tested All offor them their displayed ability highto displace affinity recombinant3 rat α , α and α BzR subtypes.1, 3 All of5 them displayed high affinity for α β γ receptors for[ H]flumazenil α1β3γ2 receptors from1 and recombinant3 moderate5 rat to goodα α selectivityand α BzR for subtypes. α3 and α All5 subtypes. of them displayed 1 3 high2 affinity 1 3 2 α α 3 5 andfor α moderateAβ numberγ receptors to of good N and-(heteroarylmethyl)indol-3-ylglyoxylamides selectivity moderate for to good3 and selectivity5 subtypes. for α and α 16 subtypes.were subsequently investigated A numbernumber ofofN N-(heteroarylmethyl)indol-3-ylglyoxylamides-(heteroarylmethyl)indol-3-ylglyoxylamides16 16were were subsequently subsequently investigated investigated [33] [33] to probe the H-bonding properties previously ascribed to the S1 subsite of the BzR [29] (see to[33] probe to probe the H-bonding the H-bonding properties properties previously previously ascribed ascribed to the Sto1 subsitethe S1 subsite of the BzR of the [29 ]BzR (see [29] binding (see binding mode B in Figure 2). The new compounds had R = H or NO2 at the 5-position of the indole modemoietybinding B and inmode Figure several B 2in). Figureheterocycles The new 2). compounds The on new the compoundsside had chain R = H(Het). had or NO R =2 atH theor NO 5-position2 at the of5-position the indole of moiety the indole and severalmoiety and heterocycles several heterocycles on the side chainon the (Het). side chain (Het). N N X X X N X NH Het = X = O, S, NH, N-CH3 H Het = X = O, S, NH, N-CH3 N NH N H N N N N N The 5-NO2 derivatives bearing a 2-pyrrolyl, a 2-furyl, a 4-methyl-2-furyl, a 3-pyrrolyl or a The 5-NO2 derivatives bearing a 2-pyrrolyl, a 2-furyl, a 4-methyl-2-furyl, a 3-pyrrolyl or a 3-furyl ring in the side chain exhibited affinities in the nanomolar range (Ki values comprised 3-furyl ring in the side chain exhibited affinities in the nanomolar range (Ki values comprised

Molecules 2020, 25, 2331 7 of 27

The 5-NO derivatives bearing a 2-pyrrolyl, a 2-furyl, a 4-methyl-2-furyl, a 3-pyrrolyl or a 3-furyl Molecules 2020, 25,2 x FOR PEER REVIEW 7 of 26 ring in the side chain exhibited affinities in the nanomolar range (Ki values comprised between 13 nM andbetween 33 nM) 13 comparablenM and 33 nM) with comparable the most active with compoundsthe most active in the compounds benzylamide in the series benzylamide6. All the 5-NO series2 derivatives6. All the 5-NO bearing2 derivatives in the side bearing chain in a the 2-thienyl, side chain a 1-methyl-2-pyrrolyl, a 2-thienyl, a 1-methyl-2-pyrrolyl, a 2-indolyl or a a 2-imidazolyl 2-indolyl or werea 2-imidazolyl scarcely potent were (Kscarcelyi values potent in the micromolar(Ki values in range) the micromolar or practically rang devoide) or ofpractically affinity. Thesedevoid data of wereaffinity. explained These data as follows: were explained the side chains as follow of thes: mostthe side potent chains 5-NO of2 theindoles most make potent H-bonds 5-NO2 atindoles the S1 sitemake (Figure H-bonds3); the at lessthe potentS1 site (Figure or inactive 3); the NO less2 indoles potent cannot or inactive make NO the2 same indoles H-bonds cannot if make the heterocycle the same lacksH-bonds a H-bond if the donor heterocycle fragment lacks (2-thienyl, a H-bond 1-methy-2-pyrrolyl), donor fragment is (2-thienyl, exceedingly 1-methy-2-pyrrolyl), bulky (2-indolyl) or is hydrophilicexceedingly (2-imidazolyl).bulky (2-indolyl) or is hydrophilic (2-imidazolyl).

L2 L2 S2 S2

H2 H2 N H O2N H O2N H O L1 N O L1 HN H O HN O

O O S N S1 N 1 H H1 H H1

A2 A2 L2 L2 S2 S2

H2 H2 O O2N O2N H O L1 O O L1 HN H O HN O

O O N S1 N S1 H H1 H H1 A A 2 2 Figure 3. Putative binding mode A of some of the most potent ligands of series 16 hypothesized to interact with a hydroxy groupgroup locatedlocated atat thethe SS11 subsite of the BzR.

All the 5-H derivatives of series 16 showedshowed significantsignificant lower aaffinitiesffinities (Kii in the submicromolar-micromolar range), range), probably probably because because they they are are not not able able to engage to engage H-bonds H-bonds with their with theirside chains side chains in the in binding the binding mode mode B. B. Four of the most potent compounds of series 16 were tested for their ability to displace 3 [3H]flumazenilH]flumazenil from recombinant rat αα11, α2 and α5 GABAA/BzR/BzR pure pure isoforms. All All of them showed binding selectivity for the α1 subtype over the α2 andand αα55 subtypes.subtypes. In continuingcontinuing our our research research of indoleof indole derivatives derivatives endowed endowed with selectivewith selective affinity foraffinity BzR subtypes,for BzR wesubtypes, came back we tocame indol-3-ylglyoxylamides back to indol-3-ylglyoxylamides of series 6 by of inserting series 6 lipophilic by inserting substituents lipophilic at substituents the 40-position at ofthe the 4’-position side phenyl of ring,the side some phenyl of which ring, were some characterized of which were by considerable characterized steric by considerable bulk (i.e., Br, CHsteric3,C bulk2H5, C(i.e.,CH, Br,C CHC-CH3, C2H3,C5, CCSi(CH≡CH, C3≡)C-CH3 and3 C, CC-CH≡CSi(CH2Si(CH3)3 and3)3)[ C34≡C-CH]. In addition2Si(CH3)3 to) [34]. the novelIn addition benzylamide to the ≡ ≡ ≡ ≡ derivatives,novel benzylamide in the same derivatives, paper we in alsothe same disclosed paper indole we also derivatives disclosed in indole which derivatives the benzyl in moiety which was the replacedbenzyl moiety by alkyl groupswas replaced (i.e., (CH 2by)3CH alkyl3, (CH groups2)4CH3 , CH(CH(i.e., (CH3)2,2 CH(CH)3CH3, 3)CH(CH22)CH4CH3,3, C(CH CH(CH3)3 and3)2, CHCH(CH2CH(CH3)CH32)CH2). Unfortunately,3, C(CH3)3 and theCH above2CH(CH structural3)2). Unfortunately, changes did the not above improve structural affinity forchanges the BzR. didOne not ofimprove the new affinity compounds for the (R =BzR.NO 2Oneand of R’ the= p -CHnew3) compounds exhibited nanomolar (R = NO potency2 and R’ at = the p-CH rat recombinant3) exhibited αnanomolar1 BzR subtype potency (Ki 31at the nM) rat with recombinant no appreciable α1 BzR a ffisubtypenity for (K thei 31α nM)2 and with the noα5 appreciablesubtypes, displaying affinity for a fullthe agonistα2 and e ffithecacy α5 profile subtypes, and adisplaying zolpidem-like a full sedative-hypnotic agonist efficacy activity profile in vivo. and a zolpidem-like sedative-hypnotic activity in vivo. According to the Cook’s research group, the shapes of the BzR subtypes are very similar, with the exception of α1 and α5 subtypes that seem to be slightly larger at the lipophilic pockets, called LDi and L2 regions, respectively [35]. The above steric differences have been exploited to obtain ligands

Molecules 2020, 25, 2331 8 of 27

According to the Cook’s research group, the shapes of the BzR subtypes are very similar, with the exception of α1 and α5 subtypes that seem to be slightly larger at the lipophilic pockets, called LDi and L2 regions, respectively [35]. The above steric differences have been exploited to obtain ligands that bind selectively to either α1 or α5 subtypes [22,36], but have also hampered the identification of affinity-based α2 and/or α3 selective ligands [37]. Investigators realized that a more fruitful strategy to obtain non-sedating anxiolytic agents or non-sedating cognition enhancers would be identifying compounds binding to all four subtypes, but preferentially activating only the targeted subtypes. The search of efficacy-based α2 and/or α3 selective ligands has indeed yielded better results [38]. Following this approach, we selected some of our indole derivatives of series 6, 10 and 14 and tested their affinity and efficacy for the rat recombinant α1, α2 and α5 subtypes [39]. Efficacy was evaluated by measuring 36 the Cl− uptake in transfected human embryonic kidney cells stably expressing the three rat subtypes upon treatment with the tested compound. The results of this work led to the identification of two N-(indol-3-ylglyoxyl)benzylamides showing α2 selective efficacy in vitro and anxioselective effects in vivo according to the mouse light/dark box test [40], namely one from series 6 (R = H and R’ = p-F) and one from series 10 (R = NO2 and R’ = p-CH3). Docking calculations of the two anxioselective indole derivatives, using a homology-built model of α1 BzR provided by Cromer Bet al. [41], were carried out by means of 10 ns molecular dynamics. Although homology building approaches should regard with cautions, our in silico simulations showed that the selected 5-NO2 and the 5-H indole derivatives adopt two different orientations and conformations within the BzR, corresponding to mode A and, respectively, to mode B conjectured in our previous papers [29,32–34]. Most of the interactions (H-bonds, lipophilic pockets, sterically forbidden regions) observed in the docking complexes were in a reasonable agreement with those hypothesized using the Cook’s pharmacophore/topological model (Figure2).

3. Indole Derivatives as Ligands of the Translocator Protein In 1977 Braestrup and Squires discovered an alternative binding site for diazepam in rat kidneys and called it peripheral benzodiazepine receptor (PBR) to distinguish it from the BzR of the GABAA ion channel [42]. Subsequently, wide studies on this protein allowed a deeper knowledge about its structure, tissue distribution and subcellular localization as well as physio-pathological functions. In virtue of these new findings, in 2006 Papadopoulos and collaborators proposed a new name for PBR, that is translocator protein (TSPO) [43]. TSPO is a transmembrane protein distributed in many tissues, mainly in those involved in steroid biosynthesis, including kidney, testis, liver and lung; in the CNS, it is mainly expressed in glial cells and, at lower levels, in neurons [43]. TSPO is located at the contact site between the inner and outer mitochondrial membrane, in strict association with other proteins that make up the mitochondrial permeability transition pore (MPTP): the voltage dependent anion channel, the adenine nucleotide transporter and the steroidogenesis regulatory protein. TSPO is involved in many biological processes, including mitochondrial respiration, cell proliferation and differentiation, induction of apoptosis. Moreover, TSPO plays a crucial role in steroid biosynthesis [44–46], specifically in the translocation of cholesterol from cytoplasm to inner mitochondria, which represents the rate-limiting step of steroidogenesis. As next step, cholesterol is converted by cytochrome P450 side chain cleavage (P450scc) into pregnenolone, the precursor of all steroid hormones [44–46]. The basal expression of TSPO is altered in different pathological conditions: an up-regulation occurs in brain injury and pathologies involving neuroinflammation (e.g., neurodegenerative diseases, gliomas) and in certain tumors; a down-regulation is often observed in correlation with anxiety disorders and post-traumatic stress [47–49]. In addition to cholesterol [50], TSPO binds with high affinity to structurally different synthetic compounds [51], such as Ro5 4864 (17a)[52] and PK11195 − (17b)[53], both widely employed as reference ligands. Molecules 2020, 25, x FOR PEER REVIEW 9 of 26 Molecules 2020, 25, x FOR PEER REVIEW 9 of 26 Molecules 2020,, 25,, 2331x FOR PEER REVIEW 99 of of 2726

H3C H3C O H3C O O CH3 N O O CH3 N O CH3 N CH3 N CH3 N 3 Cl N N CH3 Cl N N CH3 Cl N N CH3 Cl Cl

Cl PK11195 (17b) Cl Ro5-4864Cl (17a) PK11195 (17b) Ro5-4864 (17a) Ro5-4864 (17a) TSPO ligands may act as positive allosteric modulators of their target protein. Consequently, TSPO ligands may act as positive allosteric modulators of their target protein. Consequently, such TSPOligands ligands promote may pregnenoloneact as positive formation allosteric modulatorsand increase of thetheir levels target of protein. endogenous Consequently, steroids such TSPOligands ligands promote may actpregnenolone as positive allosteric formation modulators and increase of their the target levels protein. of endogenous Consequently, steroids such which,such ligands in turn, promote produce pregnenoloneseveral physiological formation effects and and increase result beneficialthe levels in of pathological endogenous conditions steroids ligandswhich, in promote turn, produce pregnenolone several formationphysiological and effects increase and the result levels beneficial of endogenous in pathological steroids conditions which, in ofwhich, the CNS in turn, including produce inflammatory, several physiological neurological effects and and psychiatric result beneficial disorders in [48,54]. pathological In the conditionslast years, turn,of the produce CNS including several physiologicalinflammatory, e ffneurologicalects and result and beneficial psychiatric in pathologicaldisorders [48,54]. conditions In the of last the years, CNS TSPOof the CNSligands including are emerging inflammatory, as promising neurological anxiolytics and psychiatric with favorable disorders safety [48,54]. and Intolerability the last years, and includingTSPO ligands inflammatory, are emerging neurological as promising and psychiatric anxiolytics disorders with favorable [48,54]. In safety the last and years, tolerability TSPO ligands and limitedTSPO ligands unwanted are emergingeffects, aslike promising sedation, anxiolytics with respect with favorableto benzodiazepines safety and tolerability[55–57]. Such and arelimited emerging unwanted as promising effects, anxiolytics like sedation, with favorable with respect safety andto tolerabilitybenzodiazepines and limited [55–57]. unwanted Such pharmacologicallimited unwanted properties effects, are like ascribed sedation, to their with ability respect to stimulate to benzodiazepines the synthesis of neurosteroids[55–57]. Such in epharmacologicalffects, like sedation, properties with respect are ascribed to benzodiazepines to their ability [ 55to –stimulate57]. Such the pharmacological synthesis of neurosteroids properties are in thepharmacological CNS, such as propertiespregnenolone are andascribed allopregnanolone to their ability, whichto stimulate act as positivethe synthesis allosteric of neurosteroids modulators ofin ascribedthe CNS, to such their as ability pregnenolone to stimulate and the allopregnanolone synthesis of neurosteroids, which act in as the positive CNS, such allosteric as pregnenolone modulators and of thethe GABACNS, suchA chloride as pregnenolone channel [55–57]. and allopregnanolone Examples of these, which non-sedating act as positive anxiolytic allosteric agents modulators are emapunil of allopregnanolone,the GABAA chloride which channel act as[55–57]. positive Examples allosteric of modulators these non-sedating of the GABA anxiolyticA chloride agents channel are emapunil [55–57]. (the17c GABA) [58] andA chloride etifoxine channel (17d) [55–57].[59], the Exampleslatter dually of thesebinding non-sedating to TSPO and anxiolytic to a site agents on the are β-subunit emapunil of Examples(17c) [58] and of these etifoxine non-sedating (17d) [59], anxiolytic the latter agents dually are binding emapunil to TSPO (17c)[ and58] to and a site etifoxine on the (17d β-subunit)[59], the of the(17c GABA) [58] andA channel. etifoxine (17d) [59], the latter dually binding to TSPO and to a site on the β-subunit of latterthe GABA duallyA channel. binding to TSPO and to a site on the β-subunit of the GABAA channel. the GABAA channel.

O H3C O H3C H O H3C H H C N NH CH3 H3C N N N N N CH3 H3C N N N N N CH3 3 N N N O O Cl O O Cl O N O Cl CH3 N CH3 N N CH3 N

etifoxine (17d)

emapunil (17c) etifoxine (17d) emapunil (17c) emapunil (17c) In 2004 and 2008 our research groups described the synthesis and the biological evaluation of a In 2004 and 2008 our research groups described the synthesis and the biological evaluation of a seriesIn 20042004of and Nand,N 2008 -dialkyl-2-phenylindol-3-y2008 our our research research groups groups described describedlglyoxylamides the the synthesis synthesis (PIGAs, and and the biological th18e) biological[60,61] evaluation evaluationdesigned of a series ofas a series of N,N-dialkyl-2-phenylindol-3-ylglyoxylamides (PIGAs, 18) [60,61] designed as conformationallyofseriesN,N -dialkyl-2-phenylindol-3-ylglyoxylamidesof N,N-dialkyl-2-phenylindol-3-y constrained analogues of 2-arylindoleacetamideslglyoxylamides (PIGAs, 18)[(PIGAs,60,61 19] designedreported18) [60,61] by as Kozikowsky conformationally designed et al.as conformationally constrained analogues of 2-arylindoleacetamides 19 reported by Kozikowsky et al. asconstrainedconformationally TSPO ligands analogues [62].constrained ofIn 2-arylindoleacetamides the generalanalogues formula of 2-arylindoleacetamides 1819, reportedR1 and R by2 were Kozikowsky 19symmetric reported et al. byor as Kozikowskyasymmetric TSPO ligands alkylet [62 al.]. as TSPO ligands [62]. In the general formula 18, R1 and R2 were symmetric or asymmetric alkyl as TSPO ligands [62]. In the general formula 18, R1 and R2 were symmetric or asymmetric alkyl (linearIn the generaland branched) formula or18 benzyl,R1 and chains, R2 were R3 = symmetricH, CH3, F, Cl, or asymmetricNO2, CF3; R4alkyl = H, OCH (linear3, F, and Cl,branched) NO2; R5 = H, or (linear and branched) or benzyl chains, R3 = H, CH3, F, Cl, NO2, CF3; R4 = H, OCH3, F, Cl, NO2; R5 = H, (linear and branched) or benzyl chains, R3 = H, CH3, F, Cl, NO2, CF3; R4 = H, OCH3, F, Cl, NO2; R5 = H, CHbenzyl3, Cl. chains, R3 = H, CH3, F, Cl, NO2, CF3;R4 = H, OCH3, F, Cl, NO2;R5 = H, CH3, Cl. CH3, Cl. CH3, Cl.

Most of the PIGAs showed affinity values for TSPO in the low nanomolar/subnanomolar range Most ofof thethe PIGAs PIGAs showed showed a ffiaffinitynity values values for for TSPO TSPO in thein the low low nanomolar nanomolar/subnanomolar/subnanomolar range range and and fullMost selectivity of the PIGAs over showed the BzR. affinity Noticeably, values the for mostTSPO performant in the low nanomolar/subnanomolarcompounds exhibited a gain range in fulland selectivityfull selectivity over theover BzR. the Noticeably, BzR. Noticeably, the most the performant most performant compounds compounds exhibited exhibited a gain in aaffi gainnity ofin affinityand full ofselectivity one order over of the magnitude BzR. Noticeably, with re spectthe most to theperformant indoleacetamide compounds counterparts exhibited a19 gain. The in oneaffinity order of of one magnitude order of with magnitude respect towith the indoleacetamiderespect to the indoleacetamide counterparts 19 .counterparts The structure-a 19ffi. nityThe structure-affinityaffinity of one order relationships of magnitude within with thisrespect class to thewere indoleacetamide rationalized counterpartsby means 19of. Thea structure-affinity relationships within this class were rationalized by means of a

Molecules 2020, 25, 2331 10 of 27

Molecules 2020, 25, x FOR PEER REVIEW 10 of 26 relationships within this class were rationalized by means of a pharmacophore/topological model made pharmacophore/topologicalby three lipophilic pockets (L1, model L3 and made L4, by hosting three the lipophilic 2-susbtituted pockets phenyl (L1, L3 group and and, L4, respectively,hosting the 2-susbtitutedthe substituents phenyl R1, and group R2 on and, the respectively, amide nitrogen) the substituents and a H-bond R1 donor, and R group2 on the H1 amide interacting nitrogen) with and the aamide H-bond carbonyl donor oxygengroup H1 (Figure interacting4). with the amide carbonyl oxygen (Figure 4).

R4 R4 4 R 4 R R5 R5 H H1 H H1 N 1 N 1 O O Ar N R L4 N R L4 O N R1 O N R1 R L1 R L1 R R2 L1 R2 L1 R3

L3 L3

Figure 4. Pharmacophore/topologicalPharmacophore/topological model of interaction between PIGAs and TSPO.

PIGA ligands were further characterized for their efficacy efficacy measured as ability to improve the synthesis ofof pregnenolone pregnenolone in ratin C6rat gliomaC6 glioma cells. Ace considerablells. A considerable number ofnumber them resulted of them stimulators resulted stimulatorsof steroid biosynthesis of steroid biosynthesis more active more than PK11195active than and PK11195 Ro5-4864 and [60 Ro5-4864,61]. [60,61]. The best-performing PIGAs, in terms of affinity affinity for TSPO and pregnenolone production, were evaluated in rats for their anxiolytic properties by means of the elevated plus-maze testtest [[61,63,64].61,63,64]. In this assay,assay, compounds compounds18a ,b18aexhibited,b exhibited non-sedating non-sedating anxiolytic anxiol properties.ytic properties. Results from Results investigations from investigationsabout the mechanism about ofthe their mechanism anxiolytic activityof their indicated anxiolytic that activity it involves indicated the stimulation that itof involves endogenous the stimulationneurosteroid of production, endogenous which neurosteroid in turn determines production, a positive which modulation in turn ofdetermines the GABA aA chloridepositive modulationchannel permeability of the GABA [63].A chloride channel permeability [63].

H C H3C

CH O CH3 O N

O N H CH H CH3 18b 18b

More recently, we investigated two novel series of PIGAs 18 featuring polar R3 groups (OH, More recently, we investigated two novel series of PIGAs 18 featuring polar R3 groups (OH, NH2, COOH) on the 2-phenyl moiety or different 2-aryl substituents (Ar = 3-thienyl, p-biphenyl, NH2,, COOH) COOH) on on the the 2-phenyl moiety or didifferentfferent 2-aryl2-aryl substituentssubstituents (Ar == 3-thienyl, p-biphenyl, 2-naphthyl)2 [65]. The 2-naphthyl derivatives exhibited the highest affinity values, thus confirming 2-naphthyl) [[65].65]. The 2-naphthyl derivatives exhibi exhibitedted the the highest affinity affinity values, thus confirmingconfirming the crucial role of the ligand-receptor interaction involving the L1 pocket. In the same paper we the crucial rolerole ofof thethe ligand-receptorligand-receptor interactioninteraction involvinginvolving thethe L1L1 pocket.pocket. In the same paper we reported a docking model of interaction between TSPO and three selected PIGAs based on a 3D reported a docking model of interaction between TSPO and three selected PIGAs based on a 3D structure of the target protein complexed to PK11195 [66,67]. This model was fairly consistent with structure ofof thethe targettarget protein protein complexed complexed to to PK11195 PK11195 [66 [66,67].,67]. This This model model was was fairly fairly consistent consistent with with the the pharmacophore/topological scheme depicted in Figure 4, with the exception of the H1 site. pharmacophorethe pharmacophore/topological/topological scheme scheme depicted depicted in Figure in Figure4, with 4, with the exception the exception of the of H1 the site. H1 site. In two subsequent studies, a set of highly steroidogenic PIGA ligands demonstrated promising In two subsequent studies, a set of highly steroidogenic PIGA ligands demonstrated promising pharmacological activities [68,69]. Particularly, a number of these compounds were found to pharmacological activities [[68,69].68,69]. Particularly Particularly,, a a number number of of these these compounds compounds were were found to promote the oxidative metabolism of human astrocytes and prevent the oxidative damage and the promote the oxidative oxidative metabolism metabolism of of human human astroc astrocytesytes and and prevent prevent the the oxidative oxidative damage damage and andthe inflammatory response in C6 glioma cells. The observed effects were completely counteracted by the theinflammatory inflammatory response response in C6 in glioma C6 glioma cells. cells. The Theobse observedrved effects effects were were completely completely counteracted counteracted by the by co-treatment with D,L-aminoglutethimide, an inhibitor of P450scc involved in steroid biosynthesis, theco-treatment co-treatment with with D,L D,L-aminoglutethimide,-aminoglutethimide, an aninhibitor inhibitor of ofP450scc P450scc involved involved in in steroid steroid biosynthesis, biosynthesis, supporting the hypothesis that the PIGA-mediated protective mechanisms are mainly related to supporting thethe hypothesis hypothesis that that the the PIGA-mediated PIGA-mediated protective protective mechanisms mechanisms are mainly are mainly related related to steroid to steroid production [68,69]. According to these results, PIGAs can be regarded as potential new productionsteroid production [68,69]. According[68,69]. Accord to theseing results,to these PIGAs results, can PIGAs be regarded can be as regarded potential as new potential therapeutic new therapeutic tools for the treatment of inflammatory-based neurodegenerative diseases characterized by astrocyte loss [68,69].

Molecules 2020, 25, 2331 11 of 27

toolsMolecules for 2020 the, 25 treatment, x FOR PEER of inflammatory-basedREVIEW neurodegenerative diseases characterized by astrocyte11 of 26 Molecules 2020, 25, x FOR PEER REVIEW 11 of 26 loss [68,69]. TSPO has been reported as a marker to reveal the onset of diseases related to its expression TSPO has has been been reported reported as a as marker a marker to reveal to revealthe onset the of onset diseases of related diseases to relatedits expression to its [47,49]. In virtue of the high affinity and selectivity of PIGAs for TSPO, we exploited the [47,49].expression In [virtue47,49]. of In the virtue high of theaffinity high and affinity selectivity and selectivity of PIGAs of PIGAsfor TSPO, for TSPO, we exploited we exploited the 2-phenylindol-3-ylglyoxylamide scaffold to develop specific TSPO molecular probes. In detail, 2-phenylindol-3-ylglyoxylamidethe 2-phenylindol-3-ylglyoxylamide scaffold scaff oldto develop to develop specific specific TSPO TSPO molecular molecular probes. probes. In In detail, reversible and irreversible fluorescent probes featuring the 7-nitrobenz-2-oxa-1,3-diazol-4-yl group reversible and irreversible fluorescentfluorescent probes featuringfeaturing thethe 7-nitrobenz-2-oxa-1,3-diazol-4-yl7-nitrobenz-2-oxa-1,3-diazol-4-yl group (18c,d) were synthesized, characterized for their optical properties and tested in spectroscopy (18c,d) were synthesized,synthesized, characterized for their op opticaltical properties and tested in spectroscopy experiments to evaluate their ability to specifically label the mitochondrial localization of TSPO in experiments to to evaluate evaluate their their abi abilitylity to to specifically specifically label label the the mitochondrial mitochondrial localization localization of ofTSPO TSPO in Drosophila S2, rat C6 and human U87MG glioma cells [70–72]. These molecular probes emerged as Drosophilain Drosophila S2,S2, ratrat C6C6 and and human human U87MG U87MG glioma glioma cells cells [70–72]. [70–72 These]. These molecular molecular probes probes emerged emerged as useful tools to study the physiological role and the expressions levels of TSPO, especially the usefulas useful tools tools to tostudy study the the physiological physiological role role and and the the expressions expressions levels levels of of TSPO, TSPO, especially the irreversible probes, whose lasting signal is maintained even after multiple washes, allowing a irreversible probes,probes, whose whose lasting lasting signal signal is maintained is maintained even aftereven multiple after multiple washes, washes, allowing allowing a detection a detection that is less affected by unspecific signal [71,72]. detectionthat is less that affected is less by affected unspecific by unspecific signal [71 ,72signal]. [71,72].

11 Shortly after, we synthesized N,N-di-n-propyl-(N1-[1111C]methyl-2-(4′-nitrophenyl)indol-3-yl) Shortly after,after, wewe synthesizedsynthesized NN,N,N-di--di-nn-propyl-(N1-[-propyl-(N1-[ C]methyl-2-(4C]methyl-2-(40′-nitrophenyl)indol-3-yl) glyoxylamide (18e) as a high affinity radiolabelled probe of TSPO [73]. The corresponding unlabeled glyoxylamide (18e) as a high affinity affinity radiolabelled probe of TSPO [[73].73]. The The corresponding corresponding unlabeled compound was selected from a small library of PIGAs thanks to its optimal combination of high compound waswas selectedselected from from a smalla small library library of PIGAsof PIGAs thanks thanks to its to optimal its optimal combination combination of high of TSPOhigh TSPO binding affinity and moderate lipophilicity (calculated logP = 3.9) to ensure adequate brain TSPObinding binding affinity affinity and moderate and moderate lipophilicity lipophilicity (calculated (calculated logP = 3.9) logP to ensure= 3.9) to adequate ensure brainadequate entry brain and entry and low non-specific binding [73]. Compound 18e was evaluated with positron emission entrylow non-specific and low non-specific binding [73 binding]. Compound [73]. Compound18e was evaluated 18e was with evaluated positron with emission positron tomography emission tomography in monkey after being administered by intravenous injection. This probe readily tomographyin monkey after in monkey being administered after being byadministered intravenous by injection. intravenous This probeinjection. readily This entered probe monkeyreadily entered monkey brain and gave a high proportion of specific and reversible TSPO binding, auguring enteredbrain and monkey gave a brain high proportionand gave a ofhigh specific proportion and reversible of specific TSPO and binding, reversible auguring TSPO binding, well for auguring its future well for its future application in humans [73]. wellapplication for its future in humans application [73]. in humans [73].

CH3 CH3 CH3 O N CH3 O N O O N N 11 NO2 11CH3 NO2 CH3 18e 18e In the last years, our attention has focused on the lack of correlation between binding affinity In the last years, our attention has focused on the lack of correlation between binding affinity and Insteroidogenic the last years, efficacy our attention of TSPO has focusedligands on[74]. the This lack ofrepresents correlation a betweenproblem binding affecting affi nitythe and steroidogenic efficacy of TSPO ligands [74]. This represents a problem affecting the andidentification steroidogenic of effective efficacy lead of TSPO compounds ligands [by74 ].a traditional This represents affinity-based a problem drug affecting discovery the identification strategy as identification of effective lead compounds by a traditional affinity-based drug discovery strategy as ofwell eff ectiveas the leadinterpretation compounds of bypharmacological a traditional a ffidanity-basedta [75]. Our drug efforts discovery took advantage strategyas from well studies as the well as the interpretation of pharmacological data [75]. Our efforts took advantage from studies interpretationshowing that ofthe pharmacological biological effectiveness data [75]. of Our a certain efforts tookmolecule advantage cannot from directly studies be showing deduced that by theits showing that the biological effectiveness of a certain molecule cannot directly be deduced by its biologicalaffinity for e fftheectiveness target, but of ait certainmay rather molecule be related cannot to directlythe period be deducedfor which by it itsinteracts affinity with for theits target, affinity for the target, but it may rather be related to the period for which it interacts with its target, butdefined it may as rather“residence be related time” to (RT) the period[75]. This for kineti whichc itparameter interacts withcorresponds its target, to defined the reciprocal as “residence of the defined as “residence time” (RT) [75]. This kinetic parameter corresponds to the reciprocal of the time”dissociation (RT) [ 75rate]. This (Koff kinetic) of the parameter ligand-target corresponds complex. to the Base reciprocald on these of the findings, dissociation we raterecently (Koff) dissociation rate (Koff) of the ligand-target complex. Based on these findings, we recently ofinvestigated the ligand-target whether complex. RT could Based be employed on these to findings, estimate wethe recentlysteroidogenic investigated efficacy whether of a TSPO RT ligand. could investigated whether RT could be employed to estimate the steroidogenic efficacy of a TSPO ligand. beFor employed this purpose, to estimate we selected the steroidogenica set of representa efficacytive ofPIGAs a TSPO showing ligand. different For this combinations purpose, we of selected TSPO For this purpose, we selected a set of representative PIGAs showing different combinations of TSPO aaffinity set of and representative steroidogenic PIGAs properties showing in vitro different [76]. combinationsThen, a kinetic of radioligand TSPO affinity binding and steroidogenicassay was set affinity and steroidogenic properties in vitro [76]. Then, a kinetic radioligand binding assay was set propertiesup with ratin kidney vitro [76 membrane]. Then, a kinetic homogenates radioligand and binding validated assay for was determination set up with rat of kidney RT values membrane using up with rat kidney membrane homogenates and validated for determination of RT values using [3H]PK11195. Our experiments showed a direct correlation between the efficacy of TSPO ligands and [3H]PK11195. Our experiments showed a direct correlation between the efficacy of TSPO ligands and their RT values [76,77]. These findings were further supported by two studies in which we their RT values [76,77]. These findings were further supported by two studies in which we

Molecules 2020, 25, 2331 12 of 27 homogenates and validated for determination of RT values using [3H]PK11195. Our experiments showed a direct correlation between the efficacy of TSPO ligands and their RT values [76,77]. These findings were further supported by two studies in which we retrospectively assessed the relationship between RT and the steroidogenic activities of emapunil and etifoxine [78,79]. Subsequently, computational studies were performed to get insights into the different kinetics of PIGAs. Specifically, the unbinding paths of three representative compounds were studied by enhanced-sampling molecular dynamics simulations, revealing that subtle structural differences between PIGAs produce relevant effects on the unbinding energetics, involving mainly the LP1, TM2, and TM5 domains of TSPO. This study accounted for the molecular basis of the efficacy of TSPO ligands [80].

4. Indole Derivatives as Dual Ligands of the Translocator Protein and the Murine Double Minute 2 Protein The pathogenesis of malignant gliomas involves the aberration of several signaling pathways, and a number of targets have been identified for therapeutic approaches, including growth factor ligands, receptors and intracellular downstream effectors [81]. As these deregulated intracellular signaling pathways are points of convergence from different stimuli, the concept of multi-target therapy is currently considered a rational approach to develop innovative and more efficient therapies [82–84]. In glioblastoma multiforme (GBM), a particularly aggressive form of brain malignancy, the p53 protein and TSPO, both acting as apoptosis inducers, represent two attractive intracellular targets [85]. Indeed, the loss of the ability of cells to undergo programmed cell death is a common step in cancer. A crucial step in the regulation of apoptosis is an increase of mitochondrial outer membrane permeability (mediated by the opening of the MPTP) and the release of specific transcription factors [86,87]. As reported in the previous chapter, TSPO is an important constitutive protein of the MPTP, holding a major regulatory significance in apoptosis [88]. Actually, a ligand from the class of PIGAs (18, R1 = R2 = n-butyl, R = Cl, R4 = Cl) had shown moderate antiproliferative and pro-apoptotic activity by enhancing the MPTP opening in rat C6 glioma cells [89]. The tumour suppressor protein p53 promotes apoptosis by interacting with members of the protective Bcl2-family proteins which in turn mediate the release of cytochrome c. P53 is a transcription factor that controls cellular response to stress by inducing cell cycle arrest or apoptosis [90,91]. The murine double minute 2 protein (MDM2) downregulates p53 activity by binding to the transactivation domain of p53. In response to stress, phosphorylation of p53 decreases the affinity of this protein for MDM2. A number of human tumors are associated with inhibition of the p53 pathway and consequent uncontrolled cell proliferation. Disruption of the p53-MDM2 interaction is therefore a therapeutic goal for the treatment of cancer [92]. The MDM2-p53 complex is stabilized mainly by a strong hydrophobic interaction between a region of MDM2 and the Phe19, Trp23, and Leu26 residues of p53. A synthetic molecule displaying three lipophilic groups in an orientation that mimics the presentation of the side chains of the above aminoacids can occupy the MDM2 cleft and thereby inhibit the p53-MDM2 interaction [92]. Based on these findings, computational methods were applied on our in-house library of indole-based TSPO ligands to identify those suitable to undergo appropriate decorations in order to inhibit the p53-MDM2 interaction and to maintain TSPO affinity. Following this approach, we synthesized a series of 2-phenylindol-3-yl-glyoxylamides (20), bearing on the glyoxylyl bridge a number of dipeptide moieties (L-Leu-L-Phe, L-Phe-L-Leu, L-Val-L-Leu, L-Leu-L-Val, L-Ile-L-Val, L-Ile-L-Ile and L-Val-L-Ile) capped as methyl or ethyl esters [93,94]. An immune enzymatic assay on − native human MDM2/p53 complex was performed to determine the ability (expressed as IC50 values) of the new compounds to bind MDM2 and disrupt the MDM2/p53 complex; affinity to TSPO was 3 evaluated by competition binding assays employing the radioligand [ H]-PK11195 and expressed as Ki values. The strategy resulted successful as all the new compounds revealed to disrupt the p53-MDM2 complex and bind to TSPO at nanomolar concentrations. Molecules 2020, 25, 2331 13 of 27 Molecules 2020, 25, x FOR PEER REVIEW 13 of 26 Molecules 2020, 25, x FOR PEER REVIEW 13 of 26

R2 O O R2 O O NH NH O R3 R1 O R R1 3 O NH O NH O O N HN H 20 20

The compound from series 20 (R1 = CH2C6H5, R2 = CH2CH(CH3)2, R3 = CH3), showing the highest The compound from series 20 (R1 = CH2C6H5, R2 = CH2CH(CH3)2, R3 = CH3), showing the highest abilityThe to compounddissociate the from p53-MDM2 series 20 complex(R1 = CH (IC2C506 H= 4.35,R nM)2 = CHand2 CH(CHthe highest3)2,R affinity3 = CH for3), TSPO showing (Ki = the 87 ability to dissociate the p53-MDM2 complex (IC50 = 4.3 nM) and the highest affinity for TSPO (Ki = 87 highestnM), was ability selected to dissociate for further the biological p53-MDM2 studies, complex giving (IC50 the= 4.3 following nM) and results: the highest (i) reactivation affinity for of TSPO the nM), was selected for further biological studies, giving the following results: (i) reactivation of the (Kp53i = function87 nM), and was inhibition selected for of furtherthe GBM biological cell growth, studies, triggering giving subsequent the following apop results:tosis; (i)(ii) reactivation no efficacy p53 function and inhibition of the GBM cell growth, triggering subsequent apoptosis; (ii) no efficacy ofon thea GBM p53 function cell line and expressing inhibition mutant of the GBMp53, supporting cell growth, the triggering involvement subsequent of this apoptosis; protein in (ii) the no on a GBM cell line expressing mutant p53, supporting the involvement of this protein in the eobservedfficacy on effect; a GBM (iii) cell reduction line expressing of viability mutant of glioma p53, supporting cancer stem the cells involvement (CSCs), which of this areprotein less sensitive in the observed effect; (iii) reduction of viability of glioma cancer stem cells (CSCs), which are less sensitive observedto anticancer effect; agents (iii) reduction and responsible of viability for of GBM glioma re cancercurrence stem [95]. cells These (CSCs), effects which were are lesssignificantly sensitive to anticancer agents and responsible for GBM recurrence [95]. These effects were significantly tostronger anticancer than agents those andelicited responsible by the p53 for activator GBM recurrence nutlin-3 [95 and]. Thesethe TSPO effects ligand were PK11195 significantly [91], stronger thanks stronger than those elicited by the p53 activator nutlin-3 and the TSPO ligand PK11195 [91], thanks thanto the those synergism elicited resulting by the p53 from activator the simultaneous nutlin-3 and ac thetivation TSPO of ligand both PK11195targets [93,94]. [91], thanks Finally, to cell the to the synergism resulting from the simultaneous activation of both targets [93,94]. Finally, cell synergismviability assays resulting performed from the on simultaneous non-tumor activationhuman mesenchymal of both targets stem [93, 94cells]. Finally, (MSCs) cell showed viability that assays the viability assays performed on non-tumor human mesenchymal stem cells (MSCs) showed that the performedantiproliferative on non-tumor effect of humanthe selected mesenchymal indole derivative stem cells was (MSCs) preferentially showed that directed the antiproliferative toward tumor antiproliferative effect of the selected indole derivative was preferentially directed toward tumor ecells.ffect All of the these selected findings indole confirmed derivative that was dual preferentially targeting MDM2-p53 directed toward and TSPO tumor is cells. a valuable All these anticancer findings cells. All these findings confirmed that dual targeting MDM2-p53 and TSPO is a valuable anticancer confirmedstrategy against that dual GBM, targeting where the MDM2-p53 downstream and p53 TSPO signaling is a valuable is not mutated. anticancer strategy against GBM, strategy against GBM, where the downstream p53 signaling is not mutated. whereAnticancer the downstream drugs binding p53 signaling reversibly is not to mutated.their targets may have several limitations in sustaining a Anticancer drugs binding reversibly to their targets may have several limitations in sustaining a therapeuticAnticancer effect drugs over binding time, thereby reversibly favoring to their the targets activation may haveof alternative several limitations signaling inpathways sustaining that a therapeutic effect over time, thereby favoring the activation of alternative signaling pathways that therapeuticescape drug eactionffect over and time,cause thereby resistance. favoring Research the in activation the field ofof alternativeoncology has signaling recently pathways been focused that escape drug action and cause resistance. Research in the field of oncology has recently been focused escapeon the synthesis drug action and and development cause resistance. of new Research irreversible in the and field long-lasting of oncology drugs has recently [96]. As been a continuation focused on on the synthesis and development of new irreversible and long-lasting drugs [96]. As a continuation theof synthesisour studies and on development 2-phenylindol-3-ylglyoxylyldipeptides of new irreversible and long-lasting 20, we drugs synthesized [96]. As a continuation21, bearing ofa of our studies on 2-phenylindol-3-ylglyoxylyldipeptides 20, we synthesized 21, bearing a ourchemo-reactive studies on 2-phenylindol-3-ylglyoxylyldipeptides isothiocyanate group at the 5-position20 of, wethe synthesizedindole nucleus.21, bearing This compound, a chemo-reactive thanks chemo-reactive isothiocyanate group at the 5-position of the indole nucleus. This compound, thanks isothiocyanateto its ability to group form atcovalent the 5-position bonds ofwith the indoleelectrophilic nucleus. groups, This compound, displayed thanksa potent to long-lasting its ability to to its ability to form covalent bonds with electrophilic groups, displayed a potent long-lasting formbinding covalent affinity bonds for TSPO with and electrophilic a prolonged groups, inhibiti displayedon of the a MDM2-p53 potent long-lasting complex binding [97]. Furthermore, affinity for binding affinity for TSPO and a prolonged inhibition of the MDM2-p53 complex [97]. Furthermore, TSPO21 caused and aGBM prolonged cell death inhibition by arresting of the MDM2-p53 the cell cy complexcle and inducing [97]. Furthermore, apoptosis;21 bothcaused effects GBM were cell 21 caused GBM cell death by arresting the cell cycle and inducing apoptosis; both effects were deathgreater by and arresting more thelong-lasting cell cycle and than inducing those of apoptosis; the reversible both eff analoguesects were greater of series and 20 more. The long-lasting observed greater and more long-lasting than those of the reversible analogues of series 20. The observed thanapoptotic those effects of the reversiblewere irreversible analogues so that of series the cells20. Thewere observed not able apoptoticto regain eproliferativeffects were irreversible activity after so apoptotic effects were irreversible so that the cells were not able to regain proliferative activity after thatdrug the wash-out cells were [97]. not able to regain proliferative activity after drug wash-out [97]. drug wash-out [97].

Compound 21 has been very recently employed in a study aimed to highlight the role played by Compound 21 has been been very very recently recently employed employed in in a astudy study aimed aimed to tohighlight highlight the the role role played played by the p53-MDM2 complex in osteoblast generation from MSCs [98]. The long-lasting MDM2-p53 bythe thep53-MDM2 p53-MDM2 complex complex in inosteoblast osteoblast generation generation from from MSCs MSCs [98]. [98]. The The long-lasting MDM2-p53MDM2-p53 dissociation determined by 21 enhances the MSC differentiation into osteoblasts through a pathway dissociation determined by 21 enhances the MSC differentiation into osteoblasts through a pathway involving the G protein-coupled receptors kinase 2 and the A2B adenosine receptor. involving the G protein-coupled receptors kinase 2 and the A2B adenosine receptor.

Molecules 2020, 25, 2331 14 of 27

Molecules 2020, 25, x FOR PEER REVIEW 14 of 26 Molecules 2020, 25, x FOR PEER REVIEW 14 of 26 dissociation determined by 21 enhances the MSC differentiation into osteoblasts through a pathway 5. Indole Derivatives as Allosteric Modulators of the Human Adenosine A2B Receptor involving5. Indole Derivatives the G protein-coupled as Allosteric receptors Modulators kinase of 2 the and Human the A2B Adenosineadenosine receptor.A2B Receptor Adenosine plays a key role in a variety of physiological and pathological processes by 5. Indole Derivatives as Allosteric Modulators of the Human Adenosine A2B Receptor interactinginteracting withwith specificspecific receptors.receptors. FourFour differentdifferent subclassessubclasses ofof adenosineadenosine receptorsreceptors (ARs)(ARs) havehave beenbeen identifiedAdenosine to date, plays A1 a, keyA2A role, A2B in, and a variety A3, all of physiologicalbelonging toand thepathological superfamily processes of G-protein-coupled by interacting identified to date, A1, A2A, A2B, and A3, all belonging to the superfamily of G-protein-coupled withreceptorsreceptors specific [99,100].[99,100]. receptors. ActivationActivation Four di ffofoferent ARsARs subclasses byby adenosadenos ofine adenosineine oror aa syntheticsynthetic receptors agonistagonist (ARs) have determinesdetermines been identified differentdifferent to date,intracellular A ,A events,A , andstarting A , all with belonging inhibition to the(A1 superfamily and A3) or ofstimulation G-protein-coupled (A2A and receptorsA2B) of adenylate [99,100]. intracellular1 2A events2B starting3 with inhibition (A1 and A3) or stimulation (A2A and A2B) of adenylate Activationcyclase.cyclase. AdditionalAdditional of ARs by molecularmolecular adenosine mechanismsmechanisms or a synthetic coupledcoupled agonist toto determines occupationoccupation di ofoffferent ARsARs by intracellularby agonistsagonists areare events stimulationstimulation starting withof phospholipase inhibition (A Cand (A1 A, A)2B or, and stimulation A3), activation (A and of Apotassium) of adenylate channels, cyclase. and Additionalinhibition of molecular calcium of phospholipase 1C (A1, A3 2B, and A3), activation2A of potassium2B channels, and inhibition of calcium mechanismschannels (A1) coupled [101]. to occupation of ARs by agonists are stimulation of phospholipase C (A ,A , channels (A1) [101]. 1 2B and ABeing3), activation ubiquitously of potassium distributed channels, in tissues and inhibition and organs of calcium of mammalians, channels (A1)[ ARs101]. have been consideredconsideredBeing ubiquitously attractiveattractive targetstargets distributed forfor thethe indevelopmentdevelopment tissues and of organsof agonist-agonist- of mammalians, andand antagonist-basedantagonist-based ARs have therapiestherapies been considered againstagainst attractivea wide range targets of pathologies, for the development including of CNS agonist- disorders, and antagonist-based cardiac arrhythmia, therapies ischemic against injuries, a wide asthma, range ofrenalrenal pathologies, failurefailure andand including inflammatoryinflammatory CNS disorders, diseasesdiseases [102].[102]. cardiac arrhythmia, ischemic injuries, asthma, renal failure and inflammatoryInIn thethe coursecourse of diseasesof ourour researchesresearches [102]. onon BzRBzR ligandsligands (discussed(discussed inin thethe secondsecond chapter),chapter), wewe preparedprepared and testedIn the coursea number of ourof [1,2,4]triazino researches on [4,3- BzRaa ligands]benzimidazoles]benzimidazoles (discussed (TBI,(TBI, in the 22)) second asas geometricallygeometrically chapter), we constrainedconstrained prepared andanalogues tested of a numberindole derivatives of [1,2,4]triazino[4,3- (23)) [103,104].[103,104].a]benzimidazoles (TBI, 22) as geometrically constrained analogues of indole derivatives (23)[103,104].

O O R R' R N N O N N N H H 22 23

The most potent TBIs (R = C6H5, C6H4-pp-OCH3, 2-furyl, 2-thienyl) displayed Ki values at the BzR TheThe mostmost potentpotent TBIsTBIs (R(R= = CC66H55,,C C6HH44--p-OCH-OCH33, ,2-furyl, 2-furyl, 2-thienyl) 2-thienyl) displayed displayed Ki values at the BzR (obtained(obtained fromfromfrom bovinebovinebovine cerebralcerebralcerebral cortex)cortex)cortex) rangingrangingranging fromfromfrom 13 1313 nM nMnM to toto 56 5656 nM. nM.nM. ItIt isis worthworth notingnoting thatthat thethe structuresstructures ofof certaincertcertain BzRBzR ligandsligands areare similarsimilar toto thosethose ofof severalseveral antagonists of the A1 AR. Compare, as an example, the BzR agonist CGS-9895 (24) [105] with the antagonistsantagonists of thethe AA11 AR. Compare, as an example, the BzR agonist CGS-9895 (24)[) [105]105] withwith thethe triazoloquinazoline derivative CGS-15943 (25) identified as the first non-xantine antagonist of the A1, triazoloquinazolinetriazoloquinazoline derivativederivative CGS-15943CGS-15943 ((25)) identifiedidentified asas thethe firstfirst non-xantinenon-xantine antagonistantagonist ofof thethe AA11, A2A and A3 ARs [106]. AA2A2A and A 3 ARsARs [106]. [106]. OCH OCH33

O N N N N N N N Cl N O N

N NH2 N 2 H 25

24

In the light of the above consideration, we prepared some novel TBIs (26) purposely designed InIn thethe lightlight ofof the the above above consideration, consideration, we we prepared prepared some some novel novel TBIs TBIs (26 ()26 purposely) purposely designed designed as as potential A1 AR antagonists through insertion of substituents not only at position 3 (R) but also at potentialas potential A1 AAR1 AR antagonists antagonists through through insertion insertion of of substituents substituents not not only only at at position position3 3 (R)(R) butbut alsoalso atat position 10 (R’) of the tricyclic system [107]. positionposition 1010 (R’)(R’) ofof thethe tricyclictricyclic systemsystem [[107].107].

Molecules 2020, 25, 2331 15 of 27 Molecules 2020, 25, x FOR PEER REVIEW 15 of 26 Molecules 2020, 25, x FOR PEER REVIEW 15 of 26 Molecules 2020, 25, x FOR PEER REVIEW 15 of 26 O R O R N N N N N N N N N R'N R' R'26

26

Among the new TBI derivatives, the most potent (Ki 83 nM) and selective one at the human A1 AR Among the the new new TBI TBI derivatives, derivatives, the the most most potent potent (K (Ki 83i 83 nM) nM) and and selective selective one one at the at thehuman human A1 AR A1 had RAmong = R’ = phenyl the new [108]. TBI derivatives, the most potent (Ki 83 nM) and selective one at the human A1 AR ARhad hadR = R’ R == phenylR’ = phenyl [108]. [ 108]. had RIn = 2012R’ = phenyl we disclosed [108]. a TBI derivative 26a provided with high potency (IC50 of 3 nM) and In 20122012 we we disclosed disclosed a TBIa TBI derivative derivative26a provided26a provided with highwith potency high potency (IC50 of 3(IC nM)50 of and 3 selectivitynM) and selectivityIn 2012 for we the disclosed human A a2B TBIAR derivative[109]. For a26a long provided time, this with receptor high potency has been (IC less50 of characterized 3 nM) and selectivityfor the human for the A2B humanAR [109 A].2B For AR a long[109]. time, For thisa long receptor time, hasthis been receptor less characterized has been less compared characterized with comparedselectivity withfor the the humanother AR A 2Bsubtypes, AR [109]. partly For dua elong to the time, scarcity this ofreceptor specific has ligands been [110]. less characterized the other AR subtypes, partly due to the scarcity of specific ligands [110]. compared with the other AR subtypes, partly due to the scarcity of specific ligands [110].

O Cl O Cl N N N N N N N N N N

26a 26a 26a The therapeutic potential of agonists and antagonists of the A2B AR is remarkable. Particularly, The therapeutic potential of agonists and antagonists of the A2B AR is remarkable. Particularly, selectiveThe agonists therapeutic of this potential receptor of have agonists been and reported antagonistsantagonists to reduce of thethe in flammation AA2B2B AR is remarkable.remarkable. after ventilator-induced Particularly, selective agonists of this receptor have been reported to reduce inflammation after ventilator-induced selectivelung injury agonists [111] of and this to receptor modulate have myocardial been reported adaptation toto reducereduce inflammationtoin flammationischemia [112]. afterafter ventilator-inducedventilator-inducedSelective A2B AR lung injury [111] and to modulate myocardial adaptation to ischemia [112]. Selective A2B AR lungantagonists injuryinjury have [[111]111 ]been andand regarded toto modulatemodulate as candidates myocardialmyocardial for adaptationtheadaptation treatment toto ischemiaofischemia cancer [[113,114],112[112].]. SelectiveSelective colitis [115,116] AA2B2B AR antagonists have been regarded as candidates for the treatment of cancer [113,114], colitis [115,116] antagonistsand asthma [117–119].have been regarded as candidates for the treatmenttreatment of cancercancer [[113,114],113,114], colitiscolitis [[115,116]115,116] and asthma [117–119]. and asthmaContinuing [117–119]. [117 –our119 ].searches of novel lead compounds binding to ARs, we synthesized five indole Continuing our searches of novel lead compounds binding to ARs, we synthesized five indole derivativesContinuing 27a–c our, 28a searches,b featuring of novel a diketo lead moiety compoundscompounds as a linker binding designed to ARs, as open wewe synthesizedsynthesized chain analogues fivefive indole indoleof the derivatives 27a–c, 28a,b featuring a diketo moiety as a linker designed as open chain analogues of the derivativesTBI 26a. Additionally, 27a27a––cc,, 28a28a, b,bwe featuringfeaturing purchased a adiketo diketo two moietyindole moiety derivativesas as a linker a linker de29a designedsigned,b characterized as asopen open chain chainby analoguesan analoguesamide linkerof the of TBI 26a. Additionally, we purchased two indole derivatives 29a,b characterized by an amide linker the[120].TBI TBI26a . 26aAdditionally,. Additionally, we purchased we purchased two indole two indole derivatives derivatives 29a,b 29acharacterized,b characterized by an byamide an amidelinker [120]. linker[120]. [120].

O O O O O O O O O O S O S O O O O O O O N N O N N N N N N

27b 27c 27a 27b 27c 27a 27b 27c

Molecules 2020,, 25,, x 2331 FOR PEER REVIEW 1616 of of 26 27

O H N

O N

28a

O N H

N

29b

The affinity of indoles 27–29 for the human A1, A2A, and A3 AR expressed in CHO cells was The affinity of indoles 27–29 for the human A1,A2A, and A3 AR expressed in CHO cells was determined by by measuring their their abilit abilityy to to displace specific specific radiolig radioligandsands from from the the above above receptors. receptors. Compounds 27a and 27b exhibited submicromolar affinities for the A1 AR (Ki values of 161 nM and, Compounds 27a and 27b exhibited submicromolar affinities for the A1 AR (Ki values of 161 nM and, respectively,respectively, 343 nM), whereas the remaining five five indo indolesles showed no appreciable affinity affinity for the three AR subtypes. Functional experiments showed that 27a and 27b behaved at the A1 AR as antagonists. AR subtypes. Functional experiments showed that 27a and 27b behaved at the A1 AR as antagonists. To evaluate the pharmacological effect effect resulting from interaction between 27–2929 and the human A2B AR, we measured to what extent our compounds modified the levels of cAMP in CHO expressing A2B AR, we measured to what extent our compounds modified the levels of cAMP in CHO expressing only this receptor. None None of of the the compounds compounds increa increasedsed the the cAMP cAMP levels levels at at the the concentration concentration of of 10 10 μµM,M, clearly showing a lack of A2B AR agonist activity. However, when the experiments were repeated in clearly showing a lack of A2B AR agonist activity. However, when the experiments were repeated in the presence of 5′-(N-ethylcarboxamido)adenosine (NECA), which acts as unspecific agonist of the the presence of 50-(N-ethylcarboxamido)adenosine (NECA), which acts as unspecific agonist of the ARs, 27a27a andand 28a28a,b,b potentiatedpotentiated its its agonist agonist effe ects,ffects, suggesting suggesting that that these these three three compounds compounds interact interact with the A2B AR as positive modulators. Conversely, 27b,c and 29a,b potently counteracted the with the A2B AR as positive modulators. Conversely, 27b,c and 29a,b potently counteracted the NECA-mediated increase in cAMP, indicating that they act as negative modulators of the A2B AR. NECA-mediated increase in cAMP, indicating that they act as negative modulators of the A2B AR. We were very satisfied by these unexpected preliminary results (a typical case of serendipity) as We were very satisfied by these unexpected preliminary results (a typical case of serendipity) as our indole derivatives 27–29 are so far the only A2B AR allosteric modulators reported in literature. our indole derivatives 27–29 are so far the only A2B AR allosteric modulators reported in literature. In a subsequent paper, the pharmacology of the new indole derivatives at the A2B AR was In a subsequent paper, the pharmacology of the new indole derivatives at the A2B AR was characterized in more detail [121]. The potencies of compounds 27–29 in modulating the activity of A2B characterized in more detail [121]. The potencies of compounds 27–29 in modulating the activity AR agonists were determined by assessing the effects of different concentrations of each of them on of A2B AR agonists were determined by assessing the effects of different concentrations of each of cAMP accumulation induced by an EC50 concentration of NECA (100 nM). The resulting them on cAMP accumulation induced by an EC50 concentration of NECA (100 nM). The resulting concentration-response curves indicated that 27a and 28a,b exhibit similar submicromolar potencies at concentration-response curves indicated that 27a and 28a,b exhibit similar submicromolar potencies at A2B AR, with EC50 values between 250 nM and 446 nM. A2B AR, with EC50 values between 250 nM and 446 nM. The concentration-response curves of 27b and 29a,b, obtained under the same conditions, fitted a The concentration-response curves of 27b and 29a,b, obtained under the same conditions, fitted two-site equation model, suggesting that these compounds recognize two sites of the A2B AR with a two-site equation model, suggesting that these compounds recognize two sites of the A2B AR different affinities. The potency values obtained for the high and low affinity states of the receptor with different affinities. The potency values obtained for the high and low affinity states of the were in the subnanomolar/nanomolar and micromolar range, respectively. Conversely, the receptor were in the subnanomolar/nanomolar and micromolar range, respectively. Conversely, the concentration-response curve of 27c fitted a one-site equation model, revealing that this compound concentration-response curve of 27c fitted a one-site equation model, revealing that this compound recognizes a unique site of the A2B AR with nanomolar affinity. recognizes a unique site of the A2B AR with nanomolar affinity. Concentration-response curves in which the cAMP was measured by varying the concentration of Concentration-response curves in which the cAMP was measured by varying the concentration of the tested compound as well as the concentration of NECA gave us further information about their the tested compound as well as the concentration of NECA gave us further information about their mechanisms of action. Particularly, from these curves we could infer that 27a and 28a,b enhance the mechanisms of action. Particularly, from these curves we could infer that 27a and 28a,b enhance the efficacy of the agonist without affecting its potency, while 27b,c and 29a,b decrease either the efficacy

Molecules 2020, 25, 2331 17 of 27 efficacy of the agonist without affecting its potency, while 27b,c and 29a,b decrease either the efficacy and potency of the agonist. Several studies report that agonist efficacy and potency are not necessarily both modified by allosteric modulators [122,123]. A plausible hypothesis is that 27a and 28a,b affects specific conformational states of A2B AR so as to improve the functional coupling to the intracellular signaling system without altering the conformation of the orthosteric site. Probably, 27b,c and 29a,b affects the agonist potency by shifting the receptor conformational states toward the resting ones; at the same time, they decrease the agonist affinity by deforming the conformation of the orthosteric site. In virtue of their indirect mechanism of receptor modulation, allosteric modulators of G-protein-coupled receptors offer therapeutic advantages compared to agonists and antagonists. Particularly, they tune pharmacological responses only when and where the endogenous agonist is present in the specific tissue. Given the role played by A2B AR in several physiological and pathological processes, discussed previously in this chapter, the positive and negative allosteric modulators of this receptor represent promising tools to identify novel druggable compounds. Shortly after we reported the therapeutic potential of the indole derivatives acting as allosteric enhancers of A2B AR agonists in the treatment of bone-related diseases (e.g., osteoporosis, rheumatoid arthritis, osteogenesis imperfecta, multiple myeloma, fracture mal-union) [124]. Particularly, we demonstrated that compound 28b potentiates the effects of either adenosine and synthetic A2B AR agonists in mediating osteoblast differentiation in vitro. In detail, by treating the MCSs with 28b we observed an increase in the expression of osteoblast-related genes (runx2 and osterix) and osteoblast marker proteins (phosphatase alkaline and osteocalcin) associated with a stimulation of osteoblast mineralization.

6. Indole Derivatives as Ligands of the Kelch-like ECH-Associated Protein 1 Very recently, we have published a paper [125] disclosing novel indole derivatives binding to a pharmacological target playing a key role in cellular oxidative stress, namely the Kelch-like ECH-associated protein 1 (Keap1) [126]. Oxidative stress [127] is associated with an excess of reactive oxygen species (ROS), such as superoxide anion (O ), hydrogen peroxide (H O ), hydroxyl radical 2−· 2 2 (OH ). ROS are potentially cytotoxic as they damage DNA, RNA, enzymes and cellular membranes; they · are generated from molecular oxygen during physiological processes (e.g., oxidative phosphorylation) or pathological events (e.g., inflammatory responses that protect our body from foreign pathogens). The cells reduce oxidative stress through radical scavengers (those best known are vitamins C, E and K) and antioxidant enzymes, both inactivating the ROS. The antioxidant enzymes include superoxide dismutase, catalase, heme oxygenase-1, glutathione S-transferase, NADPH:quinone oxidoreductase 1 and transketolase [128–130]. The expression of the above enzymes is regulated by the so called Keap1-Nrf2-ARE system [131], whose mechanism can be briefly schematized as follows. Under physiological conditions, the activity of the nuclear factor erythroid 2-related factor 2 (Nrf2) [132], a transcription factor, is inhibited by a strong interaction with Keap1. When the ROS exceed a safety threshold concentration, they disrupt the Keap1-Nrf2 complex by oxidizing a group of cysteine residues belonging to a specific domain of Keap1. This event triggers the release of Nrf2, allowing it to act as a transcriptional activator of genes that contain an enhancer sequence known as antioxidant response element (ARE) [133]. Inhibitors of the Keap1-Nrf2 interaction (KNI) are considered a promising new class of anti-inflammatory agents to treat diseases involving chronic oxidative stress, such as diabetes, cancer and neurodegenerative disorders [134]. The Keap1 binding cavity hosting the KNI inhibitors can be divided into six subpockets (P1-P6) [135]. P1 and P2 contain protonated arginine residues (Arg483, Arg415, Arg380), which give rise to strong electrostatic interactions with electron-rich parts of their ligands: salt bridges with carboxylate groups, H-bonds with nitro oxygens or azole nitrogens, cation-π contacts with aromatic rings. In the above mentioned six subpockets there are also lipophilic aminoacids. Molecules 2020, 25, 2331 18 of 27

Molecules 2020, 25, x FOR PEER REVIEW 18 of 26 Most of the KNI inhibitors reported in literature are chemically heterogeneous small molecules featuring a planar or quasi-planar scaffold which bear at least one aromatic ring and/or a weak acidic group involved in the abovementioned electrostatic interactions [136]. Our experience with indole as scaffold, led us to believe that it would be feasible to design indole derivatives acting as KNI inhibitors. The design of the compounds to be tested was mainly guided by the 3D structures of some KNI inhibitors co-crystallized with Keap1 [135–137]. With the help of molecular modelling and docking approaches, we selected nine indole derivatives 30a–i, among which 30a–d were synthesized, whereas 30e–i were purchased.

O O

HN HN O N N OCH3 O

30a 30b

H H H N N N

OCH3 OCH3 N N N OCH3 OCH3 OCH3 S S S COOH COOH COOH

30e 30f 30f NH NH

OCH3 N OCH3 N

COOH COOH 30i 30h Based on their acid-basic properties, these compounds can be divided into three groups: a) non-ionizableBased on ( their30a–d acid-basic); b) acidic properties, (30e,f); ampholytic these compounds (30g–i). The can presence be divided of methoxy into three group(s) groups: or (a) a methylendioxynon-ionizable ( 30amoiety–d); (b)in the acidic structures (30e,f); of ampholytic 30a–i was ( 30gregarded–i). The aspresence a chance of for methoxy our compounds group(s) to or strengtha methylendioxy potential moiety cation- inπ theinteractions structures and/or of 30a– iengagewas regarded H-bonds as awith chance the for target ourcompounds protein. The to thiophenestrength potential ring featured cation- πbyinteractions 30e–g confers and/ orconformational engage H-bonds rigidity with theand target represents protein. an The electron-rich thiophene ring potentially able to interact with arginine residues. Compounds 30a–i were evaluated for their ability to inhibit the Keap1-Nrf2 interaction through a cell-based luciferase reporter assay [138]. Nearly all of them were tested at the concentration of 10 μM; 30g was tested at the concentration of 5 Molecules 2020, 25, 2331 19 of 27 ring featured by 30e–g confers conformational rigidity and represents an electron-rich ring potentially able to interact with arginine residues. Compounds 30a–i were evaluated for their ability to inhibit the Keap1-Nrf2 interaction through a cell-based luciferase reporter assay [138]. Nearly all of them were tested at the concentration of 10 µM; 30g was tested at the concentration of 5 µM owing to its limited solubility in phosphate buffer. t-Butylhydroxyquinone (t-BHQ) was employed as a positive control [139] at the concentration of 50 µM, a value which gave in our experiments the maximum luciferase activity. Compounds 30e,f and 30g, characterized by a 5-carboxythien-2-yl substituent, increased luciferase activity by 152%, 263% and 486%, respectively; their activities were higher by 3.2, 5.5 and 10-fold, respectively, than that exhibited by t-BHQ (48% increase). The remaining compounds displayed activities below 50%. A western blot analysis confirmed that 30e,f and 30g increase the expression of Nrf2 and of two enzymes encoded by its downstream target ARE genes, namely NADPH:quinone oxidoreductase 1 and transketolase. The same three best performing compounds showed to be non-cytotoxic when tested on human peripheral blood lymphocytes. Docking simulations of the interaction between 30g and Keap1, using available 3D structures of this protein [140], allowed us to explain the outstanding activity of this thiophene-contaning compound. The carboxylate group of 30g makes a salt bridge with the Arg483 protonated side chain and a charge-reinforced H-bond with the Ser508 hydroxy group; the thiophene ring establishes a cation-π interaction with the Arg415 positively charged side chain. Such a cation-π interaction cannot be established by the less active compounds 30h,i which bear a carboxylic group but lack an aromatic ring attached to the indole nitrogen. Furthermore, 30h,i are much more flexible than 30e,f and 30g. The (m-methoxy)benzylaminomethyl substituent of 30g establishes hydrophobic interactions with Val512 and Leu472 side chains and a H-bond between the m-methoxy oxygen and the Leu472 backbone NH. The protonated nitrogen of 30g is not involved in any type of electrostatic interaction. This suggests that the higher activity of 30g with respect to those of 30e,f may be ascribed to the different length of the linker between the indole nucleus and the m-methoxy moiety. The indole ring of 30g contributes to the binding affinity through weak hydrophobic interactions with the Ala556 methyl group and the Arg415 dimethylene fragment.

7. Conclusions The works described in this review confirm how useful and versatile indole can be as a molecular scaffold in designing drug-like molecules.

Author Contributions: All authors have read and agreed to the published version of the manuscript. Funding: This paper was supported by University of Pisa and University of Naples “Federico II”. Conflicts of Interest: The authors declare no conflict of interest

References and Notes

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