pharmaceuticals Article Quinolizidine-Derived Lucanthone and Amitriptyline Analogues Endowed with Potent Antileishmanial Activity Michele Tonelli 1,* , Anna Sparatore 2,* , Nicoletta Basilico 3,* , Loredana Cavicchini 3, Silvia Parapini 4 , Bruno Tasso 1 , Erik Laurini 5 , Sabrina Pricl 5,6 , Vito Boido 1 and Fabio Sparatore 1 1 Dipartimento di Farmacia, Università degli Studi di Genova, Viale Benedetto XV, 3, 16132 Genova, Italy; [email protected] (B.T.); [email protected] (V.B.); [email protected] (F.S.) 2 Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy 3 Dipartimento di Scienze Biomediche Chirurgiche e Odontoiatriche, Università degli Studi di Milano, Via Pascal 36, 20133 Milano, Italy; [email protected] 4 Dipartimento di Scienze Biomediche per la Salute, Università degli Studi di Milano, Via Mangiagalli 31, 20133 Milano, Italy; [email protected] 5 Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTS), DEA, Piazzale Europa 1, 34127 Trieste, Italy; [email protected] (E.L.); [email protected] (S.P.) 6 Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, 90-236 Lodz, Poland * Correspondence: [email protected] (M.T.); [email protected] (A.S.); [email protected] (N.B.) Received: 4 October 2020; Accepted: 21 October 2020; Published: 25 October 2020 Abstract: Leishmaniases are neglected diseases that are endemic in many tropical and sub-tropical Countries. Therapy is based on different classes of drugs which are burdened by severe side effects, occurrence of resistance and high costs, thereby creating the need for more efficacious, safer and inexpensive drugs. Herein, sixteen 9-thioxanthenone derivatives (lucanthone analogues) and four compounds embodying the diarylethene substructure of amitriptyline (amitriptyline analogues) were tested in vitro for activity against Leishmania tropica and L. infantum promastigotes. All compounds were characterized by the presence of a bulky quinolizidinylalkyl moiety. All compounds displayed activity against both species of Leishmania with IC50 values in the low micromolar range, resulting in several fold more potency than miltefosine, comparable to that of lucanthone, and endowed with substantially lower cytotoxicity to Vero-76 cells, for the best of them. Thus, 4-amino-1-(quinolizidinylethyl)aminothioxanthen-9-one (14) and 9-(quinolizidinylmethylidene)fluorene (17), with selectivity index (SI) in the range 16–24, represent promising leads for the development of improved antileishmanial agents. These two compounds also exhibited comparable activity against intramacrophagic amastigotes of L. infantum. Docking studies have suggested that the inhibition of trypanothione reductase (TryR) may be at the basis (eventually besides other mechanisms) of the observed antileishmanial activity. Therefore, these investigated derivatives may deserve further structural improvements and more in-depth biological studies of their mechanisms of action in order to develop more efficient antiparasitic agents. Keywords: Leishmania tropica and infantum; antileishmanial agents; lucanthone analogues; amitriptyline analogues; quinolizidine-derived compounds; molecular modelling studies Pharmaceuticals 2020, 13, 339; doi:10.3390/ph13110339 www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2020, 13, 339 2 of 17 Pharmaceuticals 2020, 13, x FOR PEER REVIEW 2 of 19 1. Introduction 1. Introduction Leishmaniases are neglected diseases that are endemic in many tropical and sub-tropical countries,Leishmaniases leading annually are neglected to an estimated diseases 700,000–1,000,000 that are endemic new in casesmany and tropical 20,000–30,000 and sub-tropical deaths [1]. Leishmaniasescountries, leading are annually caused byto an more estimated than 20 700,00 species0–1,000,000 of protozoan new cases parasites and 20,000–30,000 belonging to deaths the genus [1]. LeishmaniaLeishmaniases, which are together caused withby more the genusthan 20Trypanosoma, species of belongsprotozoan to theparasites order Trypanosomatidae belonging to the. Recentgenus advancesLeishmania in, which the taxonomy, together genetics, with the molecular genus Trypanosoma, and cellular belongs biology to and the biochemistry order Trypanosomatidae of these organisms. Recent areadvances well illustrated in the taxonomy, in two reviews genetics, [2,3 ].molecular The pathogen and cellular parasites biology (promastigotes) and biochemistry are transmitted of these to humanorganisms and are other well mammalian illustrated hostsin two by reviews the bites [2,3 of infected]. The pathogen female phlebotomine parasites (promastigotes) sandflies. In theare mammaliantransmitted host,to human the parasites and other diff erentiatemammalian into hosts amastigote by the forms bites andof infected affect skin, female mucosa phlebotomine or internal tissuessandflies. and In organs the mammalian to produce host, cutaneous the parasites (CL), muco-cutaneous differentiate into (MC) amastigote and visceral forms (VL) and leishmaniasis. affect skin, Themucosa last or form internal is fatal tissues in absence and organs of treatment. to produce Current cutaneous therapy (CL), is muco-cutaneous based on pentavalent (MC) antimonials;and visceral pentamidine;(VL) leishmaniasis. amphotericin The last B andform its is liposomal fatal in formulationsabsence of treatment. used by parenteral Current route;therapy and is miltefosine, based on aspentavalent the only oral antimonials; agent. These pentamidine; drugs are burdenedamphotericin by heavy B and side its e ffliposomalects, the occurrence formulations of resistance used by andparenteral high costs, route; so and that miltefosine, the need foras the novel, only more oral eagent.fficacious, These safe drugs and are inexpensive burdened by drugs heavy is veryside stringent.effects, the Indeed, occurrence to meet of resistance this need, aand number high costs, of studies so that are the presently need for ongoing, novel, exploringmore efficacious, a very wide safe chemicaland inexpensive space and drugs also is the very possibility stringent. of Indeed, the repositioning to meet this of need, known a number drugs. Theof studies structures are presently of many interestingongoing, exploring examples a very of investigational wide chemical antileishmanial space and also the agents, possibility able to of hit the di repositioningfferent cellular of targets,known aredrugs. illustrated The structures in several of many reviews interesting [4–9]. Interestingly, examples of highly investigational potent antileishmanial antileishmanial chemical agents, classes,able to ashit the different nitroimidazoles, cellular targets, the benzooxaboroles are illustrated and in theseveral aminopyrazoles reviews [4–9]./pyrazolopyrimidines, Interestingly, highly have potent been identified,antileishmanial mainly chemical within the classes, Drugs foras Neglected the nitroimidazoles, Diseases initiative the (DNDi),benzooxaboroles Geneva, Switzerland. and the Orallyaminopyrazoles/pyrazolopyrimidines, active compounds from these series have (such been as id DNDi-0690,entified, mainly DNDi-6148 within and the GSK-3186899) Drugs for Neglected [10–12] areDiseases presently initiative in clinical (DNDi), development Geneva, Switzerland. (Figure1). Orally active compounds from these series (such as DNDi-0690, DNDi-6148 and GSK-3186899) [10–12] are presently in clinical development (Figure 1). F H OH R N B O N N O O O N O 2 N DNDi-6148 DNDi-0690 (exact structure not disclosed) O N H N CF N SO 3 N 2 N N N H H GSK-3186899 Figure 1. Antileishmanial drugs in clinical development. Figure 1. Antileishmanial drugs in clinical development. Additionally, many tricyclic antidepressant and antipsychotic drugs (and structurally related Additionally, many tricyclic antidepressant and antipsychotic drugs (and structurally related compounds) have been shown to display various degrees of activity against different species of compounds) have been shown to display various degrees of activity against different species of Leishmania, and/or to inhibit enzymes (such as trypanothione reductase) playing essential roles in Leishmania, and/or to inhibit enzymes (such as trypanothione reductase) playing essential roles in parasite development and virulence [13–16]. Among these, clomipramine and cyclobenzaprine have parasite development and virulence [13–16]. Among these, clomipramine and cyclobenzaprine have been recently repurposed for the treatment of visceral leishmaniasis [15,16]. All these compounds been recently repurposed for the treatment of visceral leishmaniasis [15,16]. All these compounds are are characterized by different kinds of linear tricyclic systems, such as phenothiazine, iminodibenzyl, characterized by different kinds of linear tricyclic systems, such as phenothiazine, iminodibenzyl, thioxanthene, dibenzocycloheptane and sulphur isosteric analogues, to which an aliphatic basic side thioxanthene, dibenzocycloheptane and sulphur isosteric analogues, to which an aliphatic basic side chain is attached to the central ring (Figure2A). chain is attached to the central ring (Figure 2A). Pharmaceuticals 20202020,, 1313,, 339x FOR PEER REVIEW 3 of 1719 Figure 2. (A) Examples of basic derivatives of tricyclic systems displaying antileishmanial activity Figure 2. (A) Examples of basic derivatives of tricyclic systems displaying antileishmanial activity and inhibition of trypanothione reductase.