molecules Article Structure–Activity Relationships and Biological Evaluation of 7-Substituted Harmine Analogs for Human β-Cell Proliferation 1,2, 3, 3 2 2 Kunal Kumar y, Peng Wang y, Ethan A. Swartz , Susmita Khamrui , Cody Secor , Michael B. Lazarus 1,2 , Roberto Sanchez 1,2, Andrew F. Stewart 3 and Robert J. DeVita 1,2,* 1 Drug Discovery Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; [email protected] (K.K.); [email protected] (M.B.L.); [email protected] (R.S.) 2 Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; [email protected] (S.K.); [email protected] (C.S.) 3 Diabetes, Obesity, and Metabolism Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; [email protected] (P.W.); [email protected] (E.A.S.); [email protected] (A.F.S.) * Correspondence: [email protected]; Tel.: +212-659-5542 These authors contributed equally to this work. y Received: 25 March 2020; Accepted: 20 April 2020; Published: 23 April 2020 Abstract: Recently, we have shown that harmine induces β-cell proliferation both in vitro and in vivo, mediated via the DYRK1A-NFAT pathway. We explore structure–activity relationships of the 7-position of harmine for both DYRK1A kinase inhibition and β-cell proliferation based on our related previous structure–activity relationship studies of harmine in the context of diabetes and β-cell specific targeting strategies. 33 harmine analogs of the 7-position substituent were synthesized and evaluated for biological activity. Two novel inhibitors were identified which showed DYRK1A inhibition and human β-cell proliferation capability. The DYRK1A inhibitor, compound 1-2b, induced β-cell proliferation half that of harmine at three times higher concentration. From these studies we can draw the inference that 7-position modification is limited for further harmine optimization focused on β-cell proliferation and cell-specific targeting approach for diabetes therapeutics. Keywords: dual-specificity tyrosine-regulated kinases (DYRKs); harmine; DYRK1A inhibitor; structure–activity relationship study; β-cell proliferation; diabetes 1. Introduction The dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) is a eukaryotic protein kinase that has been shown to play important roles in biological processes related to various diseases [1–4] and more recently, as a major regulator of human insulin-producing pancreatic β-cells [5–8]. Numerous studies have previously reported the development of DYRK1A inhibitor scaffolds for a variety of therapeutic applications including diabetes [2–6,8–33]. Among all the DYRK1A inhibitors, harmine and its analogues (β-carbolines) are the most commonly studied, and remain one of the most potent and orally bioavailable class of inhibitors known to date [2,10]. Recently, our group found that harmine is able to induce human β-cell proliferation and DYRK1A-NFAT pathway as being the major pathway for this cell proliferation [5]. These results have been confirmed in other labs with other DYRK1A inhibitors unrelated to the harmine scaffold, including from our own lab [6,8,34–37]. Since, modification of harmine has not been explored previously in context of β-cell proliferation, we therefore decided to carry out structure–activity relationship studies of harmine for both DYRK1A inhibition and β-cell proliferation. Based on the known binding pose Molecules 2020, 25, 1983; doi:10.3390/molecules25081983 www.mdpi.com/journal/molecules Molecules 2020, 25, 1983 2 of 18 of harmine,Molecules 20 we20, 2 surmised5, x FOR PEER that REVIEW there are three positions, 1-methyl,7-methoxy and 9-N indole,2 of 18 where rational modifications of harmine can be carried out (Figure1) without disrupting key DYRK1A bindingrelationship contacts. studies Previously, of harmine we for reported both DYRK1A the optimization inhibition and of β the-cell 1-position proliferation. of Based harmine on the which led to twoknown compounds binding pose with of harmine, robust human we surmisedβ-cell that proliferation there are three at positions, doses of 1-methyl,7 3–30 µM-methoxy and one and of those, 9-N indole, where rational modifications of harmine can be carried out (Figure 1) without disrupting compound I (R = CH2OH), showing improved kinase selectivity as compared to harmine (Figure1)[ 38]. key DYRK1A binding contacts. Previously, we reported the optimization of the 1-position of harmine Next, we recently reported the optimization of the N-9 position to identify 2-2c, a highly optimized which led to two compounds with robust human β-cell proliferation at doses of 3–30 µM and one of in vivothose,active compound harmine I (R based = CH DYRK1A2OH), showing inhibitor improved (Figure kinase1)[ 39selectivity]. To extend as compared this systematic to harmine medicinal chemistry(Figure strategy, 1) [38]. Next, we last we focusedrecently reported our effort the on optimization the harmine of the 7-position N-9 position to understand to identify 2 the-2c, impacta of modificationshighly optimized on DYRK1A in vivo inhibition active harmine and βbased-cell proliferationDYRK1A inhibitor (Figure (Figure1). An 1) [39]. additional To extend consideration this was tosystematic explore medicinal the potential chemistry for 7-substituents strategy, we last to linkfocused to putativeour effort targeting on the harmine molecules 7-position for cell to specific deliveryunderstand to the β the-cells, impact without of modifications negatively on a DYRK1Affecting DYRK1Ainhibition and ββ-cell-cell proliferation proliferative (Figure biological 1). An activity. additional consideration was to explore the potential for 7-substituents to link to putative targeting One such prototype targeting technology for cells that highly express GLP1-receptor, including β-cells, molecules for cell specific delivery to the β-cells, without negatively affecting DYRK1A and β-cell was reportedproliferative by biological DiMarchi activity. and colleagues One such prototype [40,41]. targeting Herein, technology we report for the cells results that highly of our express 7-position of harmineGLP1 medicinal-receptor, including chemistry β- studies.cells, was Wereported first determinedby DiMarchi and the ecolleaguesffect of these [40,41]. modifications Herein, we report on DYRK1A inhibition,the results measured of our 7 by-positio FRET-basedn of harmine LanthaScreen medicinal chemistry® Eu Kinase studies. Binding We first Assay determined [42]. Sincethe effect the overall goalof these the project modifications is to develop on DYRK1A DYRK1A inhibition, inhibitors measured that by canFRET induce-based LanthaScreenβ-cells to proliferate,® Eu Kinase we also assayedBinding potent Assay 7-substituted [42]. Since the harmine overall goal analogs of the forproject human is to developβ-cell proliferation DYRK1A inhibitors by quantifying that can Ki67 immunolabelinginduce β-cells into betaproliferate, cells [we5,39 also,43 –assayed45]. potent 7-substituted harmine analogs for human β-cell proliferation by quantifying Ki67 immunolabeling in beta cells [5,39,43–45]. FigureFigure 1. 1. OptimizationOptimization of of the the 7-position 7-position of harmine. of harmine. 2. Results2. Results and and Discussion Discussion 2.1. Synthesis2.1. Synthesis Modification of the harmine 7-position was carried out by several standard reaction sequences Modification of the harmine 7-position was carried out by several standard reaction sequences outlined in Scheme 1. Harmine underwent demethylation in the presence of HBr/AcOH to give outlinedharmol in 1 Scheme-1 in 99%1 yield. Harmine [46]. Alkylation underwent of harmol demethylation 1-1 with various in alkyl the presence bromides in of the HBr presence/AcOH of to give harmolcesium1-1 incarbonate 99% yield as base [46 ].generated, Alkylation in 40 of–68% harmol yield,1-1 7-etherwith harmine various analogs alkyl bromides1-2a–1-2n having in the a presence of cesiumterminal carbonate methyl ester, as base t-butyl generated, ester and BOC in 40–68%-protected yield, amino 7-ether group with harmine carbon analogs chain lengths1-2a– of1-2n 1– having a terminal5 carbons methyl [46]. ester,Harminet-butyl analogs ester 1-2a and–1-2e BOC-protected with terminal methyl amino esters group were with refluxed carbon in chainexcess lengths of 1–5methanolic carbons ammonia [46]. Harmine solution to analogsprovide the1-2a corresponding–1-2e with terminal terminal carboxamide methyl esters analogs were 1-3a–1 refluxed-3e in excessin methanolic84–95% yield. ammoniat-Butyl ester solution harmine toanalogs provide 1-2f– the1-2j correspondingwere treated with terminalhydrochloric carboxamide acid at room analogs temperature to give corresponding acids 1-4a–1-4e in excellent yield. Compounds 1-2k–1-2n 1-3a–1-3e in 84–95% yield. t-Butyl ester harmine analogs 1-2f–1-2j were treated with hydrochloric underwent N-BOC deprotection to generate harmine analogs 1-5a–1-5d with terminal amino acidfunctional at room temperaturegroup which then, to giveunderwent corresponding acylation using acids acetic1-4a anhydride–1-4e in to excellent afford harmine yield. 7-alkoxy Compounds 1-2k–acetamide1-2n underwent analogs 1-N-BOC6a–1-6d. Harmoldeprotection (1-1) was to also generate treated with harmine trifluoromethanesulfonic analogs 1-5a–1-5d anhydridewith terminal aminoto functionalform O-trifluoromethanesulfonyl group which then, underwentharmine analog acylation 1-8 which using underwent acetic anhydride palladium to catalyzed afford harmine