molecules Article Chemical Derivatization and Characterization of Novel Antitrypanosomals for African Trypanosomiasis Aboagye Kwarteng Dofuor 1,2, Temitayo Samson Ademolue 1,3 , Cynthia Mmalebna Amisigo 1,3 , Kwaku Kyeremeh 4 and Theresa Manful Gwira 1,3,* 1 West African Center for Cell Biology of Infectious Pathogens, University of Ghana, Legon, Accra P.O. Box LG 54, Ghana; akdofuor@uesd.edu.gh (A.K.D.); tsademolue@gmail.com (T.S.A.); amismacyndyy@yahoo.com (C.M.A.) 2 Department of Biological, Physical and Mathematical Sciences, University of Environment and Sustainable Development, PMB, Somanya, Ghana 3 Department of Biochemistry, Cell and Molecular Biology, University of Ghana, Legon, Accra P.O. Box LG 54, Ghana 4 Department of Chemistry, University of Ghana, Legon, Accra P.O. Box LG 56, Ghana; kkyeremeh@ug.edu.gh * Correspondence: tmanful@ug.edu.gh Abstract: The search for novel antitrypanosomals and the investigation into their mode of action remain crucial due to the toxicity and resistance of commercially available antitrypanosomal drugs. In this study, two novel antitrypanosomals, tortodofuordioxamide (compound 2) and tortodofuorpyra- mide (compound 3), were chemically derived from the natural N-alkylamide tortozanthoxylamide (compound 1) through structural modification. The chemical structures of these compounds were  confirmed through spectrometric and spectroscopic analysis, and their in vitro efficacy and possible  mechanisms of action were, subsequently, investigated in Trypanosoma brucei (T. brucei), one of the Citation: Dofuor, A.K.; Ademolue, causative species of African trypanosomiasis (AT). The novel compounds 2 and 3 displayed sig- T.S.; Amisigo, C.M.; Kyeremeh, K.; nificant antitrypanosomal potencies in terms of half-maximal effective concentrations (EC50) and Gwira, T.M. Chemical Derivatization selectivity indices (SI) (compound 1, EC50 = 7.3 µM, SI = 29.5; compound 2, EC50 = 3.2 µM, SI = 91.3; and Characterization of Novel compound 3, EC50 = 4.5 µM, SI = 69.9). Microscopic analysis indicated that at the EC50 values, the Antitrypanosomals for African compounds resulted in the coiling and clumping of parasite subpopulations without significantly Trypanosomiasis. Molecules 2021, 26, affecting the normal ratio of nuclei to kinetoplasts. In contrast to the animal antitrypanosomal drug 4488. https://doi.org/10.3390/ molecules26154488 diminazene, compounds 1, 2 and 3 exhibited antioxidant absorbance properties comparable to the standard antioxidant Trolox (Trolox, 0.11 A; diminazene, 0.50 A; compound 1, 0.10 A; compound 2, Academic Editor: Baoan Song 0.09 A; compound 3, 0.11 A). The analysis of growth kinetics suggested that the compounds exhibited a relatively gradual but consistent growth inhibition of T. brucei at different concentrations. The Received: 23 June 2021 results suggest that further pharmacological optimization of compounds 2 and 3 may facilitate their Accepted: 22 July 2021 development into novel AT chemotherapy. Published: 25 July 2021 Keywords: Trypanosoma brucei; tortodofuordioxamide; tortodofuorpyramide; tortozanthoxylamide; Publisher’s Note: MDPI stays neutral Z. zanthoxyloides; antitrypanosomal with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction A major neglected tropical disease that is of significant health and economic con- cern to humans and livestock of Sub-Saharan Africa is African trypanosomiasis (AT), a Copyright: © 2021 by the authors. tsetse-transmitted disease of humans and livestock caused by protozoan parasites of the Licensee MDPI, Basel, Switzerland. Trypanosoma genus [1,2]. Even though the recent advances in the treatment of human AT This article is an open access article are encouraging [3], the impact of animal AT on livestock productivity is still of great distributed under the terms and economic concern. Chemotherapy coupled with effective community screening remains conditions of the Creative Commons the main mode of parasite control due to the absence of vaccines. However, the resistance Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ and toxicity of commercially available drugs pose serious challenges to chemotherapy. 4.0/). Thus, there is the need to search for alternative sources of AT chemotherapy. Molecules 2021, 26, 4488. https://doi.org/10.3390/molecules26154488 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 4488 2 of 10 The antitrypanosomal activities of several plants have been reported in different parts of the world [4–7]. We previously reported the antitrypanosomal activities of the Ghanaian plant species Zanthoxylum zanthoxyloides (Lam.) Zepern and Timler (Z. zanthoxyloides) in Trypanosoma brucei (T. brucei) [8]. The main secondary metabolites responsible for the antitrypanosomal properties of Z. zanthoxyloides were, subsequently, identified and characterized using various methods of spectrometry and spectroscopy [9,10]. Collectively, these are strong indications that natural plant products could serve as alternative sources of antitrypanosomal chemotherapy. Despite their valuable medicinal values, natural products are usually beset with chemotherapeutic limitations that hinder their further development into drugs. These limi- tations include aspects of the potency, selectivity, toxicity, solubility, stability and bioavail- ability of compounds [11]. For instance, low chemical stability might have interfered with an efficient spectroscopic confirmation of lanyuamide, as well as the antitrypanosomal sensitivity of 9-oxo-10,12-octadecadienoic acid (9-oxo-ODA) upon their identification in Z. zanthoxyloides, as previously reported [9,10]. Moreover, the oxidant capacities of skimmi- anine and 9-oxo-ODA in T. brucei may serve as future limitations to their antitrypanosomal chemotherapeutic capacities as far as safety issues are concerned [10]. Thus, natural prod- ucts usually require various forms of pharmacological optimization before their successful development into drugs can be achieved. N-alkylamides are a diverse group of bioactive natural plant products with reported pharmacological, nutritional, medicinal and cosmeceutical properties [12]. They consist of polyunsaturated fatty acids and relatively shorter aliphatic chains as well as a cen- tral amide bond at varying levels of cyclic and heteromolecular systems to the aliphatic moieties [12]. The potentially lipophilic properties of N-alkylamides could interfere with the identification of their targets due to the tendency to clump together instead of freely interacting with key metabolic proteins [12]. The aliphatic nature might also facilitate easy and uncontrolled access to the central nervous system, thereby ultimately maximizing the potentially toxic effects in the brain and spinal cord [13]. Thus, as with most natural products, N-alkylamides may require pharmacological improvements to be considered as promising chemotherapeutic agents. We previously isolated and characterized a new antitrypanosomal N-alkylamide tortozanthoxylamide from Z. zanthoxyloides [9]. In order to improve the general pharmaco- logical properties of tortozanthoxylamide, the present study sought to chemically derive and characterize two novel antitrypanosomals from the compound through structural modification. These derivatives were, subsequently, investigated in T. brucei for their potential in vitro efficacies and mechanisms of action. The results provide key insights into the antitrypanosomal capacity of the compounds for their development of chemotherapy for AT. 2. Results 2.1. Derivatization and Characterization of Compounds We previously isolated and characterized the novel N-alkylamide antitrypanosomal N-(isobutyl)-3,4-methylenedioxy cinnamoyl amide (tortozanthoxylamide, compound 1) from the root of the Ghanaian plant species Z. zanthoxyloides through bioactivity-guided chromatography, spectrometry and spectroscopy [9]. In order to improve the antitrypanoso- mal activities of compound 1, we designed putative chemical derivatives of compound 1 based on the structural analysis of the available functionalities. The labile bond cleav- age analysis of the mass fragmentation suggested the importance of the benzodioxole functionality in the base structure of compound 1 [9]. Pyrrolidine, a structure found in many natural alkaloids and synthetic drugs [14–16], was identified as a compact moi- ety for compound 1. Coupled with the goal of reducing the aliphaticity of compound 1, the 2-methylpropan-1-amine moiety or its 2-methylpropyl substructure was replaced by a pyrrolidine or benzodioxole functionality, respectively (Table 1). This led to the derivatization of 2-propenamide, N,3-bis(1,3-benzodioxol-5-yl)-(2E) (compound 2) and Molecules 2021, 26, x FOR PEER REVIEW 3 of 11 M olecules 2021, 26, x FOR PEER REVIEW 3 of 11 Molecules 2021, 26, x FOR PEER REVIEW 3 of 11 Molecules 2021, 26, x FOR PEER REVIEW 3 of 11 Molecules 2021, 26, x FOR PEER REVIEW 3 of 11 methylpropan-1-amine moiety or its 2-methylpropyl substructure was replaced by a pyr- mroelitdhiynlep roorp baenn-1z-oadmioinxoe lme fouientcyt ioorn iatlsi t2y-,m reesthpyelcptirvoeplyy l (sTuabbslter u1c).t uTrhei sw laesd r teoplaced by a pyr- rmoelitdhiynlep roorp baenn-z1o-admioixnoel em fouinetcyti oorn aitlsi t2y-,m reesthpyeclptirvoeplyy l( sTuabbsletr u1)c.t uTrhei sw laesd rteop ltahcee dd ebryiv aa tpizyar-- trmiooelnitdh oiynfl epropan-1-amine moiety or imethylp2 -ropropena ide, N,3-bis (1, t3s- b2e-mnzeothdyiolpxoropyl substructure was rep tlahcee dde bryiv aa tpizyar-- trioolnid oinf e2 -opo rrp obaenn-1z-odr b am ioixnoel me fouientcyt iorn iatlsi t2y-,m resthpyelcptirvole-p5ly-y l( lsT)u-a(b2sElte)r u1(cc).ot umTrhepi osw ulaensdd r te2op) ltaahncede d 2 eb-rpyivr aoa tpiezynar--- Molecules 2021, 26, 4488 1trio-oolnind eoi,nf3 e-2 (-o1p,r3r ob-b peenennamide, N,3-bis (1, 3-benzopenzzoaodmdioiidoxxeo,ol eNl- 5f,3u-y-nblc)i-st1i o(-1(n1,a -3lpi-tybyre,rn orzeloisd di dpiien ocyxoxtli ol-5- o)v-le(-25lyE y -y )( l l T ) )( -ca(2E) (compoun-(o2bmEle)p 1(oc)u.o nTmdhp is led d t2o) tahned d 2e-rpivroaptiezna-- 1ti-oonn eo,f3 -2(-1p,3ro-bpeenennzzoaodmdioiidoxxeo,ol eNl- 5f,3u-y-nblc)i-st1i o(-1(n1,a -3pli-tybyre,rn orzeloisddpiienocyxtlio)v-le(-25lyE-y )(l T)(-ca(o2bmEle)p 1(oc)u.o nTmdhp i 3so )ul efnrddo m t2o) ttahneed pd 32aeo-rrfpei1vnr0oat ptniezanat--- u1ti-rooannl eoc,fo3 -2m(-1p,3roo-ubpneedn za1om.d iiTdohexe,o sNle-5, 3-dy-eblr)i-siv1 (-a1(t1,i -v3pe-ybs errwnozeloirdei inocyxolom)-l-(m52-Eey)rl c)(i-ca(o2lly synthe 3os)iu zfnerdom 2b) y tah neAd pu 2ar-ropernraot mpound 3) from the paren p neant-- u1-roanl ec,o3-m(1p,3o-ubnedn z1o.d iTohxeosle-5 -dyelr)-iv1-a(t1iv-peys rrwoelirdei ncyolm)-(m2Eer) c(icaollmEy) p s(ocyuonnmthdpe 3os)iuz fnerddo m 2b) y tah neAd pu 2aro-rperarno t p Fneiante-- C1u-rhoaem Fine Chenl ec, i3oc-ma(1lps, 3oL-ubimnednitz e1od. , TGh t nat- ural diox reaoszle-,5 A-dyuels)r-it1vri-aa(t1 i(-vcpeoysmr rwpooleiurdeni ndcy o2lm), -C(m2hEee)rm c(iciacolamlyl pAsobyusnnttrdhae c3st)si z fSereodrm vbi ycthe eA( CpuAarorSer)na r te Fngiainste-- tCurryha eln m ucimocmablesp roL =uim n1d0it5 e61d6. ,3 TG9h-r8ae8sz-e,3 A;d cueosrmtivripaaot i(uvcnoesdm 3wp,o eCurAen dSc o2=m, 2C6mh1e9er1mc3ii-ac1lal9yl- 3 As)b.y snttrhaecstsiz Seedr vbicye (CAS) regis- try nm ucimocmablepsr oL =ui mn1d0ited, Graz, Austria (compound 2, Chemical Aurora Fine Chemicals Limit5 e61d6. ,3 TG9h-r8ea8sz-e,3 A;d cueorsmitvrpiaaot i(uvnesd 3w, eCrAe Sc o=m 26m1e9r1c3i-a1l9ly-3 As).by snttrhaecstsiz Seedr vbiyce A(CuAroSr)a r eFginise- 2-proCtprhye nen-u1m-obneer,3 =- (110,35-6b6e3n9z-8o8d-i3o;x coolm-5p-yolu) c-no1dm-( 13p-,o pCuyAnrrdSo =2li ,d2 C6inh1y9el1m)3-i(-c21aE9l-) 3A()c.b osmtrapcotsu nSderv3i)cfer o(CmAtSh)e regis- parenTttraynb anlmetu 1imrc. aaCbllhesc eroL =mim i1cpa0iolt5 eus6dtn6r,ud3 Gc91t-ru.8ar8Tze-hs,3 Aea; sncuedos mdtrepioraoli ev(uccaunotlidmav re3p ws,o CewuiAgnehdrSet s=2 co ,o2 fCm6 ch1om9e1mep3rio-cc1uia9anl-l dl3Ays) .bas snytdnr atmhcteossi eiSztieerdsv. ibcye A(CuAroSr)a regis- Fine TCtrayhb enlmeu 1im.c CablheserLm =i im1ca0il5t es6td6ru,3cG9t-ur8ar8e-s3 a; ncdo mpoTable 1. Chemical structurezs, aAnuds mtroia loeuc(cunldar 3 w, Ceilecuolamr pwoeu AgnhSdt s= 2o 2,f 6Cc1o9m1p3o-1uights of cohmempoiuc 9na-dl3sA) .a bnsdt rmacotiseties. nds and moietSieesr. vice (CAS) registTable 1. CTraybnleu 1m. Cbe hemical structures and olecMuloalrecular hrem=Ni1ca0aml5 s6et6r u39ct-u8r8e-s3 ;ancodm mpooleuWecM nuigl doahlr 3e w,cCueilAgahrS ts= o2f 6c1o9m1p3o-1u9n-d3Cs) h.aenmd moieties. Name Molte w (ge/igmhot ical Structure Weight c(gu/lmaro sl )o f compoundCs haenmd micoaile Stitersu. cture Name Molecular l) Chemical Structure Table 1. Chemical stNruacmtuere s and moleculaWr weMiegioghlhet tc(sguo/lfmacro ml) pounds and moieties.Weight (g/mol) Chemical Structure Name Weight (g/mol) Chemical S Otructure ToNrtaomzaenthoxylamide O Molecular Weight (g/mol) ChemiHcal Structure Tort(ozanthoxylamide 247.29 H O N O NH Tor(t Cozoamnpound 1) O HCompthoouxnydla 1m) ide 247.29 O HTor(tCozoamnpthoouxnydla 1m) ide 247.29 O H H N H H HN TortoTzaonrt(thCoozoxamynlapthmoouidxnyedla 1m) ide 247.29 247.29 O NHO H H (Comp(Couonmdp1o)und 1) 247.29 O H O H O O O O Tortodofuordioxamide O O H Tortod N OO Tort(oCdo omfupoorduinodx a2m) ide 311.29 H OO NH OO Tort(oCdo omfupoorudniodx a2m) ide 311.29 H HO H Tortodofu(oCrdoimofuxpaoomrudidnioedx a2m) ide 311.29 O H O N O Tortodofuordioxamide 311.29 O H NH OO H H H O (Comp(C(Cou o on mdpmp2 o)und 2) 331111.2.299 OO Nound 2) H H O O H O O Tortodofuorpyramide H O Tort(oCdoomfuorpyramide 245.27 H O N Tort(oCdoomfu poorupnydpoundr a 3 3m ) ) ide 245.27 O H O N TortoTdoofrutoordpoyfruaomide O rpyramide 245.27 O H H N (CToomrtp(oCoduoonmfdup3oo)rupnydra 3m) ide 22445(Compound 3) 5 .2.277 H H NO H N (Compound 3) 245.27 OO H O H O O O Benzodioxole 122.12 O BenzoBdeionxzooledioxole 11222.12 OBenzodioxole 1222.1.122 O Benzodioxole 122.12 O Benzodioxole 122.12 OO PyrrolPidyirnreolidine 71.12 OHN Pyrrolidine 7711.1.122 HN Pyrrolidine 71.12 HN CompoCuonmd poundP1 was uP s1 y wrraosl iudsiende eydrraos lthide itneme a psl athe tem 71.12 HN te for thepdlaetseig fnora7 nt1hd.e1s y2dn ethsiegsnis abnasde dsyontshtreuscitsu bralsiendsHi goNhnt sstinruf uctnuctriaoln ianlistiegshts in and laCfbuiolnemcbtpoiononduanclldieta i1ev saw gaeansad nu alsaleybdsiil sae. sb tohned t ecmleapvlaatgee f oarn athlyes dise. sign and synthesis based on stru ctural insights in fCuonmctpioonuanlidti 1es w aansd u lsaebdil ea sb tohned t ecmleapvlaatgee f aonr athlyes idse. sign and synthesis based on stru ctural insights in C Cfu o on m mct p pio onuanldit i1e sw aansd u lsaebdil ea sb tohned t ecmleapvlaatgee f oanr athlyes dise. sign and synthesis based on structural insights in CfuonmctpiCoooonuuamnnldipdt i1oes suw2 anaandsnd sud lsa2e3b diawl eane sdb rtoe hn3esd utw ecbmljerapcevlt aeastdgeue bft oaojnerG cathtlayeessd dic seh.t sorio gGmn aaasnt odcg hsryranoptmhheiacst-iosm gbarasasspedshp iocen-cm strtarousmsct eustrpraiecl ci(ntGrsoiCgm-hetst riinc MS) a(fuGnnaCclyt-iCMosionsSma.)l Tiaptihnoeeasu laniynndstdis la2Compoundess r. 2pT b rhiaeleent a dbitno iotn3e dnrw pcolrefeerametva aatsgisuoesbn asj enpocafet lcmeytdsraia sst. swo saGpseas chromatographic-mass spec and 3 were subjected to Gacsot rncadh wruocamtse acdotoungsdriunacpgtheaidcr -oumbsuianssgst das aprteoacb turaostmetrictrome deattraic- of th(ebG(GaNs C-M Cea- tCMoifo S S nmt ) a )ha apel n n oIN al anulas ynsytdi itossun . t2aTe lh aoIenf disSnsi . The int ti t3ateteu n r rwCompounds 2 and 3 wt pderpreea eorrtedfa Stsaituonanbnd jdeoTcafe trmecddha anstnso spectra was conducted using a robust data- base of the National Institute eortefa Stsituobnj eocft meda sts d l so GpTgeaeycsct (hrcNanh oIwrSlooTamgs) .yacT to(onhNgderIuSampTctah)e.si dcsT -hufmresai nmgsgms spectrometric (GC-MS) analysis. The interpretati o Gas chromatographic-mass a asses pnr eoftracbatturigosmntm deenatttraaic-- pattert(biGnaossCneo - pfMoacftS ott)meh arepn Nosa uloaynfts idcioson.m 2aTlpha Ineon duisnt3itdteiusnrt pd2er iaecontafa tdStei d3 oa nnain dodafai rcmpdap ataresondsxd sai pmnTe eaccttpehrpanmr owo/logy (NIST). The mastoan doaf rmd aasns ds pTeeccthran owlxzo aisgom yfcao 3(tn1Ne1d mIuSa/cTnzteas conductd) eo. d d2fT u 43uh51e s,1i rng asinm eagsna pad se r 2coft4rbiagss fr5avu,ge sr m mltye s edep n nae t tt aac---- (Suppttbitiila ovnoese nmel y p peo a an(f tSt tterns of compou robust data- base of tttaue h hrpr eynp National Ine NsFl eioamgftu iceoronemtsaalpSr Iy2on uFa sntingidtdusr tS2ee4 s a)o .Snf2 TdS hat3ane nidncd hdSari4rco)da.m tTaeahndtedo a g cnThr eaarcpophmhpniracootlexoligumrytaai op(tNenh imIocS f/Tezcl) uoo. ftmTi 3ohp1neo1 om uafnn acddsos 245, ressntidtust 2e aonf dS t3a nin fragme ddaircda taendd a nT eacphpnrolxoigmya (tNe ImS/Tz) o. fT 3h1e1 m anasds m 2fi4nrpa5otg, hurmeensed p pns e et i ca-cn-- GC-MtttithiiS vove enea l lG n y ypa C(S a(lSt-ytM up uesi plementary prSsnp osal cenocmafu lceyronrsemtidsa proayo nta- tion patterns of compoct Fi uFcrnuie g gdtr uersenres S2 anur 2deti s ao aSnt2 drt aie3mnt ei d den s d St 4). The chromSio4iocf)na.3 tT1teih.dm8e 6a ecn(shc aroopfm mp3pr1 ao.t8oux6ignm rd(aatogrcao p2ptme) h aihimp c cn e o/edzu l uo2nt1fdi .o38 1n201) o ( acafo ncmdo m p2241op5.uo8,n 0urd ne(scdposem icn-- 3) (Sutpthipovepue lGnyedCm ( S-3eMu)n pSt(apS aruleynpmapFleyliegns und muitsar ereonysct FcaSuir1gyrur se 2 lution aFrndeid gs aa nuStr2 d3er ) ae3s. nt Se idTnn1 h d tSiae4 ioc)naI. R tedndT tiShms3ep a ) e.ce n shc taTorhro pfa mpe3 o r1aof.t8xIRo b6 i sgo m ate mpr(tecahocptmchroaipcm o /ezpul o of uonbt fudi 3on 11ot2hnd) o aaf compounds in the GC-MS analysis occurred at retention times of 31.86 (compound 2)s ocafod nn cdidos m22l14pa.5y8o,0 eur en(scdposme icn-- absorpttbihovuelnyd ( S3u) p(pSulepmpelenmtaernyt Fairgyu Freigsu Sr2e sa nSd1 Sa4n).d T Sh3e) .c hTrhoem IaRto sgpreacpthraic oefl ubtoiothn of n cmdopcomp m2 o1pundou.o8−n0u1d n( scd ds odsm i ii s sn - p - - ppth l lo aaeuny GnceeddCi n-a3Mtb)e sSn(oS saruibtnpieaslywsisth oinccaurarendg eaot frwetaevne GC-MS anaapnlylcesemi sine ontectcanursyirtr ieFedisg wautri etrhse itnSe n1a e t ainronaunndm g tbiSeme3 or)es.f s sTw uohafge vg3 eeI1Rns.8tu i6svm pe(ebcocoeftmrrNsa p- sHouufg (ngb1d6eo5s t2th0i)– v c1aeo5n om8d0f pN2com1.80 )(,comtion times of 31.86 (compound 2) and 21-u.H8n0 d ((1sc 6od5mi0– - C-C (1p15ayed a−b1 sorba−n1ce intens s- lo5a8u0y00ne c–dm1 4a30b)) 0s,( oCScrum-bCpap n(1lc)e5eam0 ni0end–nt1etC4an0-rsNy0 it ciF(em1si −witiesg2 w5 1u)0r iat–ehns1id 0nS2 10a acrnmand−g 1Se)3 ob).fe Tnwdhaesv aeIRnnud −sm1pstberceetrrcsah seousfg, gbaesostethviv icedo eomnf tpNion-uHtnh d(e1s6 d50is– -- arom1ppa5lota8iucy0n eacddnm da3−1b)a) ,( CSu-Cp p(1le5m00e–n1t4a0r0y cFmig−1u) r iaetnhsd inS C 1a - aNrna (dn1g 2Se53 0o)–.f 1Tw0h2ae0v ecIRmnu smp) bebceetnrrsda sso uafgn gbdeo sstthtri evcteco hmoef psN, as evident −1 in the aro C-N (1250–1020 cm ) bends and stretches, o a-usHn e dv(1sid 6d5ei0ns–t- i1pn5l a8tyh0ee cd ma ra−o1b) ms,m oCarit-dbiCcae n(ag1nc5redo0 iu0an–pmt1es4indo0sef0it cgcieomrsmo −uw1p)p ioatsnuhinndsorbance intensities with odinf C caso- Nm2ra ap(n1nog2due5 n03od–f( 1sSw 02u2a pa0vpn ecdlne um3m (eSbnuetparsrp ylsuFgi guersetisvmatic and a range of wavemnu−1m) benrsd s eu amgngedne sttatrirveyt Se 2oaf nNd-HS4 ()1.650– The U15V8–0V cImS −s1)p, eCc-tCra (1w5e0 e c Fhoiefg suN, ra-eHss e Sv(12id6 a5en0nd–t S1in45 )8t.h0 T ech maer− o1U)m,V Ca–t-ViCcI (aS1n 5sd0p r 0 aeae–c m1trre4iads0 te0wr igcecmrtroee−ud 1r)p eatsson tordafi bc Cctsoe-oNdmr p pt(o1toi 2oua5nbn0sd–bo1sar 0n2p2 and 3−1 (Supplementary Figures S2 and −1 aromSa4t)ic. Tahned Ua V–idVeISm sopi0ee–ctmi1ter4isad0 (e0w2 cg5emr0roe–u 3r)5pe a0ssn tnordifm c Cct)eo-Nd(mS tup(o1po 2aup5bnl0esd–mo1sr0 ep22n dt0aiso ncmcon)s bisetnendts wanidth streatncshietiso, nas eovfident in the aromatic and amide groups of compounds 2 t0aito nac ndm b3−1a )(n Sbduesnp dpconsistent nry bFaingdsr sle amnedn sttarreyt wc Fhiiteghsu ,t rareass ne Ssvi2itd iaoenndst oSinf4 )at.hr Toe mhaera otUmicV aa–tnVicdI aSan msdpi deacmet rmiad oewi egetrrioeeus r p(e2s5 to0rif–c 3ctoe5d0m nptomo ua) bn(Ssdousrp p2p tailoemn d nd eb 3na (3 (tn SadupSurysp epcFolSiegn5mus).ireseCnt eSthna5etr)m y.w C iFichitaghelu mtsrrhieacisnaf tSsls i2sth iaoinfnconsistent with transitio td ss fromofS1rf4Ho )am.r aTo n1mhHdea 1tUa3incCV da–N nV1d3M aRmsipdeec mtroairetifleesc (t2iv50e–o3f5s0h nifmti)n (gSuefpfpecletsmdeunetatroy c pFaliregbmuoreneny Stla5(r)1y. HC F:hi1ge0umpriepcsam Sl )2sh ainftdss and afSorr4fo )am. rTo a1hmtHieca aUtsincuVd ba– nsV1t3d CI Sa NmspMidecRet rmsap owIS spectra weiec ettrrieas r r(ee2sf5lte0ric–ct3tiev5d0e ntoomf as)h b(iSsfoutirpnppgtil oeemnff eebcnattnsa drysu cFeo itgnous istenere restricted to absorption bands consircseat reSbn5o t nwyilt h(1 Htr:a1n0s iptipomns) of aromatic andiCt ua NmtioMidnRes m(s1poHeice: tt6rie.a5s –r (e72f.5le0cp–t3piv5m0e ;no1mf3 Cs)h :(iS1fut2ipn0p–g1l ee3mf0feepcntptsam dry)u weF ietgorue rcea vrSbi5dot ) ) en . .w n C Cytil h ht hi( e en1 m Htrbi:acal smic1ona0tlsh pist h hpio i im fntftss) compoforfou amnr 1 13 do smH(aS tauinpc dpa nledmC a eNmntMiadrRey mFspectra reflective of shifting ff cts due to carbonyl ( 1H:10 ppm) from 1H and 13C NMR spiogeiuecrtteireass S r(6e2f5ale0nc–dt3iS5v70e) n.oTmf hs)e h(Si1fDutipnapgnl deemf2fDeecntNtsa MrdyuR Fed igtaout arceaa rrSeb5os)u.n Cmylhm e(1maHrii:zc1ae0ld pshpimfts) in Sufprpolmem 1Hen taanrdy T13aCb lNesMSR1 asnpdecStr2a. reflective of shifting effects due to carbonyl (1H:10 ppm) 2.2. Antitrypanosomal Potency Analysis The antitrypanosomal activities of compounds in T. brucei were determined in a 48-h alamar blue cell viability analysis. As shown in their dose–response curves, compounds 2 and 3 displayed potent antitrypanosomal activities in terms of their EC50 values (compound 1, EC50 = 7.3 µM; compound 2, EC50 = 3.2 µM; compound 3, EC50 = 4.5 µM) (Table 2; Molecules 2021, 26, 4488 4 of 10 Supplementary Figure S8). In the presence of normal mouse macrophages (RAW 264.7 cell lines), compounds 2 and 3 were more selective to T. brucei than compound 1, as evidenced by their selectivity indices (SI) (compound 1, SI = 29.5; compound 2, SI = 91.3; compound 3, SI = 69.9) (Table 2). Table 2. Antitrypanosomal potencies and selectivity indices of compounds. COMPOUNDS Mean EC50 ± SEM (µM) SI T. brucei RAW 264.7 Compound 1 7.3 ± 0.08 215.6 ± 0.9 29.5 Compound 2 3.2 ± 0.09 292.1 ± 1.5 91.3 Compound 3 4.5 ± 0.05 314.6 ± 1.9 69.9 DA 1.7 ± 0.07 138.7 ± 2.0 81.6 SI (selectivity index) was calculated as the ratio of the EC50 value in RAW 264.7 cell lines to that in T. brucei. DA = diminazene aceturate; SEM = standard error of the mean. 2.3. Antioxidant Capacity of Compounds in T. brucei The antioxidant properties of the compounds in T. brucei were determined by em- ploying the reducing properties of ABTS (2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid). For a standard antioxidant such as the water-soluble analog of vitamin E (Trolox), a dose-dependent reduction in the absorbance of the ABTS radical at 405 nm is expected [10]. Thus, a dose-dependent increase in the absorbance for a compound in T. brucei is an in- dication of oxidant activity. As was shown previously [10], the animal antitrypanosomal drug diminazene exhibited a strong oxidant potential (Figure 1). At the maximum tested concentration of 100 µg/mL, compounds 1, 2 and 3 exhibited absorbance intensities that Molecules 2021, 26, x FOR PEER REVIwEWer e not significantly different from Trolox, thereby suggesting antioxidant prope 5r otfi e1s1 comparable to Trolox (Trolox, 0.11 A; compound 1, 0.10 A; compound 2, 0.09 A; compound 3, 0.11 A) (Figure 1). 0.6 TL (0.11 A) 0.4 DA (0.50 A) compound 1 (0.10 A) compound 2 (0.09 A) 0.2 compound 3 (0.11 A) 0.0 0 50 100 Concentration of compounds (μg/mL) FFiigguurree 11.. AAnnttiiooxxiiddaanntt aaccttiivviittiieess ooff ccoommppoouunnddss iinn TT.. bbrruucceeii:: AAbbssoorrbbaannccee rreeaaddiinnggss wweerree rreeccoorrddeedd wwiitthhiinn aa ccoonncceennttrraattiioonn rraannggee ooff 33..112255 ttoo 110000 μµgg//mmLL. .AAnnttiiooxxiiddaanntt ccaappaacciittiieess wweerree eessttiimmaatteedd ffrroomm dduupplliiccaattee aabbssoorrbbaannccee rreeccoorrddiinnggss aatt tthhee mmaaxxiimmuumm oobbsseerrvveedd ccoonncceennttrraattiioonn ooff 110000 μµgg//mmLL usuisnign gthteh eTrTorloolxo xcucruvrev aesa tshteh setasntadnadrdar adnatinotxioidxaidnat.n Dt.AD =A D=imDiinmazineanzee anceetaucreattuer;a TteL; T= LTr=oTlorxo;l oAx=; Aab=soarbbsaonrcbea nucneitus naitt s10a0t 1μ0g0/mµgL/. mL. 22..44.. Annttiittrryyppaannoossoomaall SSeennssiittiivviittyy Annaallyyssiiss ooff Coomppoouunnddss TThhee ccuumuullaattiivvee ggrroowtthh ooff ppaarraassiitteess wwaassm moonnitiotorreeddf oforr9 9d adyasysa taEt CE5C05a0 nadnd2 2×x EECC5500 vvaalluueess ooff ccoomppoouunnddss.. AAs sexepxpecetcetde,d t,htehreer ewwasa snon oeffeefcfet cotno ntheth ceucmuumlautliavteiv gerogwrotwh tohf oTf. brucei in the untreated cells (Figure 2). However, all the compounds resulted in a con- sistent growth inhibition of parasites with respect to time and dose (Figure 2). At the EC50 values, compounds 1, 2 and 3 resulted in the complete eradication of parasites after 96, 192 and 216 h, respectively (Figure 2A). At 2 × EC50 values, both the diminazene- and com- pound 1-treated cells were exterminated within 24 h, while all the compound 2- and 3- treated parasites were eliminated at a relatively slow rate, within 120 and 144 h, respec- tively (Figure 2B). Figure 2. Compound sensitivity analysis in T. brucei: The growth kinetics of T. brucei was investigated after treatment with diminazene (DA), compounds 1, 2 and 3 for 9 days at EC50 and 2 × EC50. NC = negative control. Absorbance at 405 nm Molecules 2021, 26, x FOR PEER REVIEW 5 of 11 0.6 TL (0.11 A) 0.4 DA (0.50 A) compound 1 (0.10 A) compound 2 (0.09 A) 0.2 compound 3 (0.11 A) 0.0 0 50 100 Concentration of compounds (μg/mL) Figure 1. Antioxidant activities of compounds in T. brucei: Absorbance readings were recorded within a concentration range of 3.125 to 100 μg/mL. Antioxidant capacities were estimated from duplicate absorbance recordings at the maximum observed concentration of 100 μg/mL using the Trolox curve as the standard antioxidant. DA = Diminazene aceturate; TL = Trolox; A= absorbance units at 100 μg/mL. Molecules 2021, 26, 4488 2.4. Antitrypanosomal Sensitivity Analysis of Compounds 5 of 10 The cumulative growth of parasites was monitored for 9 days at EC50 and 2x EC50 values of compounds. As expected, there was no effect on the cumulative growth of T. Tbr.ubcreui cieni itnhet huenturneatrteeadt ecdellcse l(lFsig(Fuirgeu 2r)e. H2)o. wHeovwere, vaellr ,thaell ctohme pcoomunpdosu rnedssulrteesdu ilnte da cinona- sciosntesnistt egnrotwgrtohw inthhiibnihtiiobnit oiofn poarfapsaitreass iwteisthw rietshpreecstp teoc ttimtoet iamnde adnodsed (oFsigeu(Freig 2u).r eA2t )t.hAe tEtCh5e0 EvaCluevs,a cluoems,pcooumnpdos u1n, d2 sa1n,d2 3a nrdes3ulrteesdu litne dthien cthome cpolmetep leertaedeircaadtiiocnat ioofn poafras50 pairteass iatefstearf t9e6r, 91962, 1a9n2d a2n1d6 h2,1 r6eshp,ercetsivpeelcyt i(vFeiglyur(eF i2gAu)r.e A2tA 2) ×. AECt 520 v×alEuCes, bvoathlu tehse, dbiomthintahzeendei-m a50 innda zceonme-- apnoduncdo m1-ptroeuantedd1 c-terlelsa twedercee lelxstweremreineaxtteedrm wiintahtiend 2w4 ihth, iwnh2i4leh a, lwl thhiele caolml tphoeucnodm 2p-o aunndd 32-- atrnedate3d-t rpeaarteadsitpeas rwaseirtee selwimerienaetleimd iant aat erdelaattivaerleyl astliovwel yrastelo, wwirtahtien, 1w2i0t hainnd1 12404a hn,d re1s4p4ehc-, rtievsepleyc (tFivigeluyre(F 2igBu).r e 2B). FFiigguurree 22.. CCoommppoouunndd sseennssiittiivviittyy aannaallyyssiiss iinn TT.. bbrruucceeii:: TThhee ggrroowwtthh kkiinneettiiccss ooff TT.. bbrruucceeii wwaass iinnvveessttiiggaatteedd aafftteerr ttrreeaattmmeenntt wwiitthh ddiimmiinnaazzeennee ((DDAA)),, ccoommppoouunnddss 11,, 22 aanndd 33 ffoorr 99 ddaayyss aatt EECC5500 aanndd 22 ×× EECC505.0 N. NCC = =nengeagtaivtiev ecocnotnrtorlo. l. 2.5. Effects of Compounds on the Structure and Distribution of T. brucei The effects of compounds on the morphology and distribution of T. brucei were investigated using fluorescence microscopy. The elongated slender shape of T. brucei, helical flagella and normal ratio of nuclei to kinetoplasts under untreated conditions (1N1K, 1N2K and 2N2K) were observed in the negative control (Figure 3A,B; Table S3). At the EC50 values, diminazene caused the loss of kinetoplasts in about 70% of the cells, in contrast to the approximately 3, 4 and 3% of cells for compounds 1, 2 and 3, respectively (Figure 3A; Table S3). However, amongst selected subpopulations, the compounds induced significant cell clumping and coiling of the normal spiral shape of parasites at the tested EC50 values (Figure 3B). Absorbance at 405 nm Molecules 2021, 26, x FOR PEER REVIEW 6 of 11 2.5. Effects of Compounds on the Structure and Distribution of T. brucei The effects of compounds on the morphology and distribution of T. brucei were in- vestigated using fluorescence microscopy. The elongated slender shape of T. brucei, helical flagella and normal ratio of nuclei to kinetoplasts under untreated conditions (1N1K, 1N2K and 2N2K) were observed in the negative control (Figure 3A,B; Table S3). At the EC50 values, diminazene caused the loss of kinetoplasts in about 70% of the cells, in con- trast to the approximately 3, 4 and 3% of cells for compounds 1, 2 and 3, respectively (Figure 3A; Table S3). However, amongst selected subpopulations, the compounds in- Molecules 2021, 26, 4488 duced significant cell clumping and coiling of the normal spiral shape of parasites at 6thofe1 0 tested EC50 values (Figure 3B). Figure 3. Effects of compounds on structure and distribution of T. brucei: T. brucei cells were treated at the EC50 values of Fciogmurpeo3u.nEdfsf.e (cAts) oFof rc oemacpho cuonmdpsoounndst, rpuecrtcuernetaagned cdelils tproibpuutliaotnioonf wT.asb rcuacleciu: lTat.ebdr ufcroeimc ealnls awveerraegter ecaotuednt aotft 2h4e0E cCel5l0s vina l1u0e s omf cicormospcoopuincd fsie. l(dAs). (FBo)r Reeadch acrroomwp =o uDnAd-,tpreeartceedn tcaeglles;c gerlleepno parurloawtio =n cwomaspcoaulcnudl a1t-etrdeafrtoedm saunbpaovperualagteiocno;u pnutropfle2 4a0rrcoewlls =i n 1c0ompicoruonscdo p2-itcrefiaetledds .su(Bbp) oRpeudlaatriroonw; o=raDngAe- tarreraotwed =c ecollms;pgorueennd a3r-rtorewate=dc osumbppooupnudlat1i-otnre; aKte =d ksiunbeptooppluaslat;t iNon =; pnuucrlpeluesa; r- rPoHwA=ScEo m= Pphouasned c2o-ntrterastet;d DsAubPpI o=p 4u′,l6a-tdioianm; oidrainog-e2a-prrhoewny=licnodmopleo; uDnAd =3 -Dtriematiendazseunbep aocpeutulartaioten;; NKC= =k Ninegtoaptilvaes tc;oNnt=ronlu. cleus; PHASE = Phase contrast; DAPI = 4′,6-diamidino-2-phenylindole; DA = Diminazene aceturate; NC = Negative control. 3. Discussion 3. DiTshcue snsaiotunral products of plants are endowed with several pharmacological and me- dicinTalh pernoapteurrtaielsp. rHodowucetvseorf, tphlaeyn tasraer ueseunadlloyw beedsewt withithse pvheraarlmpahcaorlmogaicoallo ligmiciatal tainodnsm theadti c- hininadl perr othpeirrt iuetsi.lizHaotiwone voerr ,suthbesyeqaurenut sdueavlelylobpemsent tw inittho pdhruargms. aWcoiltohg tihcea lgloimal iotaf tdioenaslinthga t whiinthd ethr itsh cehiar luletnilgizea, thioen anotritsruybpsaenqousoenmtadl etovretloozpamntehnotxyinlatomdidreu (gcso.mWpoituhntdh e1) gwoasl cohfedme-al- iicnagllyw mithodthifiisedc hinatlole tnogrteo,dtohfeuaonrdtiitorxyapmanidoes o(cmomalptourtnodz a2)n athnodx tyolratomdiodfeuo(cropmyrpaomuidned (1co) mw-as pchoeumndic 3a)ll. yTmheo sduibfiseedqiunetnot tcohretomdiocfaul oarnddi oaxnatmitriydpea(ncosmompoaul nchda2r)acatnedriztoartitond odfeumoropnysrtraamteisd e h(coowm pinosuignhdts3 )i.nTtoh ethseu bsstreuqcuteunrat lc haenmd ifcuanl catniodnanl tpitrroyppearntioeso omf apllcahnat rancteitrriyzpaatinoonsodmemalosn - strates how insights into the structural and functional properties of plant antitrypanosomals could facilitate their modification into novel compounds for the purpose of drug discovery. The chemical derivatization increased the period of time required for the complete eradication of parasites, despite a relative increase in selectivity and potency. This slow rate of action may also account for the insignificant impact on the ratio of nuclei to kinetoplasts, despite the corresponding deformation of parasite morphology at EC50 values. Even though this property may not be temporally advantageous in terms of the time-dependent action of the compounds, it could have spatial benefits with regard to the effects on a selected spectrum of parasite populations as well as a minimization of the potential toxicity on host cells. Future studies that would provide insights into reaction kinetics at longer time periods are, therefore, encouraged. Molecules 2021, 26, 4488 7 of 10 The excessive production of reactive oxygen species may serve as a source of damage to cells. The mode of action of nifurtimox, a human African antitrypanosomal drug, is pro- posed to involve the release of free radical and non-radical forms of reactive oxygen species that could damage proteins, DNA and lipids [17]. The animal African antitrypanosomal drug, diminazene, is known to cause damage to the kidney, liver and brain [18], of which a high oxidant activity could play an essential role [10]. Furthermore, despite the promising efficacies of other natural antitrypanosomals similarly isolated from Z. zanthoxyloides, there is the possibility that their significant oxidant activities may have damaging effects on host cells [10]. Thus, the antioxidant properties of the compounds, exhibited through the possible inhibition of reactive oxygen species in the parasites, can be advantageous in the context of the tendency to reduce the overall toxic effects that arise from oxidative stress. Moreover, a combined effect of antitrypanosomal and antioxidant properties could have pharmacological advantages. In one study, derivatives of 4-hydroxycoumarins were shown to be good antioxidants and moderate antitrypanosomals [19]. A selected series of synthetic hydroxy-3-arylcoumarins were also reported to exhibit varying levels of antioxi- dant and trypanocidal activities potentially beneficial to the control of Chagas disease [20]. In light of their reducing properties, several natural antioxidants have also been proposed as adjuvants or supplementary therapy for the treatment of Chagas disease [21]. This is due to the potential induction of T. cruzi-mediated oxidative stress in host cells, which aids the progression of Chagas disease [21]. Thus, the inhibition of oxidative stress in the parasites could be of importance as far as the mechanisms of action of compounds 1, 2 and 3 are concerned. However, despite these chemotherapeutic advantages, natural antioxidants may also aid the establishment of trypanosomes, which calls for careful moderation in their usage [22]. Collectively, this study paves the way for a further pharmacological evaluation of tortodofuordioxamide and tortodofuorpyramide to facilitate their development into com- mercially available antitrypanosomal drugs. The advantageous chemotherapeutic proper- ties of tortodofuordioxamide and tortodofuorpyramide against T. brucei included improved parasite selectivity indices, the preservation of antioxidant properties and consistency in growth inhibition. Future studies may seek to investigate ways of increasing the rate of growth inhibition while retaining or improving other beneficial properties. Future studies should also focus on identifying targets of the compounds in T. brucei to shed light on the mechanisms of antitrypanosomal sensitivities. 4. Materials and Methods 4.1. Culture of Parasites and Mammalian Cell Lines Blood stream forms of the subspecies T. brucei brucei (T. b. brucei) (GUTat 3.1 strains) were cultured in vitro to the logarithm phase using Hirumi’s Modified Iscove’s Medium (HMI9, Thermo Fisher Scientific, Oxford, UK) with 10% fetal bovine serum (Thermo Fisher Scientific) at 5% CO2 and 37 ◦C. Mouse macrophages (RAW 264.7 cell lines, Sigma-Aldrich, Kent, 91062702) were cultivated in vitro to the logarithm phase using Dulbecco‘s Modified Eagle Media (DMEM, Thermo Fisher Scientific, Oxford, UK) with 10% fetal bovine serum at 5% CO2 and 37 ◦C. 4.2. Derivatization, Spectrometric and Spectroscopic Analysis The natural compound 1 previously isolated from Z. zanthoxyloides [9] was used as the template for the design of compounds 2 and 3. We designed putative chemical deriva- tives of compound 1 based on the structural analysis of available functionalities. Labile bond cleavage analysis suggested the importance of benzodioxole functionality in the base structure of compound 1. Pyrrolidine was also employed as another compact moiety in the design process. The 2-methylpropan-1-amine moiety or its 2-methylpropyl substructure was replaced by a pyrrolidine or benzodioxole functionality. This led to the derivatiza- tion of 2-propenamide, N,3-bis(1,3-benzodioxol-5-yl)-(2E) (compound 2) and 2-propen-1- one,3-(1,3-benzodioxol-5-yl)-1-(1-pyrrolidinyl)-(2E) (compound 3) from the parent natural Molecules 2021, 26, 4488 8 of 10 compound 1. These structural designs were submitted to Aurora Fine Chemicals Limited, Graz, Austria, for custom synthesis. Compounds 2 and 3 are commercially available at Aurora Fine Chemicals Limited with the following Chemical Abstracts Service (CAS) reg- istry numbers (unique registered chemical identifiers): compound 2, CAS = 1056639-88-3; compound 3, CAS = 261913-19-3. The experimental protocols employed in the synthesis of compounds 2 and 3 are copyrighted to Aurora Fine Chemicals Limited, Graz, Austria. GC-MS analysis of compounds 2 and 3 was performed using a PerkinElmer GC Clarus 580 Gas Chromatograph interfaced to a Mass Spectrometer PerkinElmer (Clarus SQ 8S) equipped with Elite-5MS (5% diphenyl/95% dimethyl polysiloxane) fused to a capillary column (L × I.D. 30 m × 0.25 mm, df 0.25 µm). The oven temperature was programmed from 40 ◦C with a 3 ◦C/min increase to 90 ◦C, then 10 ◦C/min to 240 ◦C and holding for 15 min at 240 ◦C. For GC-MS detection, an electron ionization system was operated in electron impact mode with ionization energy of 70 eV. Helium gas (99.999%) was used as a carrier gas at a constant flow rate of 1 mL/min and injection volume of 1 µL. The injector temperature and ion-source temperature were 250 and 150 ◦C, respectively. Mass spectra were taken at 70 eV with a scan interval of 0.1 s and fragments from 45 to 450 Da. The solvent delay was 0 to 2 min with a total GC-MS running time of about 42 min. The mass-detector used in this analysis was a PerkinElmer TurboMass (Software = TurboMass version 6.1.0.). Interpretation of mass spectra was conducted using the database of the National Institute of Standard and Technology (NIST). Mid-infrared (IR) spectroscopy was performed using the Attenuated Total Reflectance (ATR) spectrometer with the following specifications: instrument model = BRUKER ALPHA FT-IR platinum ATR; software ver- sion = OPUS-7.2.139.1294; number of scans = 24. UV–VIS spectroscopy was performed with the SPECORD 200 PLUS-223E1451 designation using the following specifications: lamp change = 320 nm; measurement mode = spectral scan; range = 200–700 nm; delta lambda = 1 nm; speed = 50 nm/s. All NMR spectra were acquired with a Bruker FT-NMR Avance 500 spectrometer (Ettlingen, Germany) at 300 K. All solvents used were of the HPLC grade. 4.3. Analysis of Cell Viability and Cytotoxicity The subspecies T. b. brucei were seeded at a density of 1.5 × 105 cells/mL in 96- well plates in a two-fold dilution of compounds and incubated for 24 h. Normal mouse macrophages (RAW 264.7) were initially seeded at a density of 1.5 × 105 cells/mL for 48 h to allow for sufficient adherence to plates before treatment with the compounds in a two-fold dilution and subsequent incubation for another 24 h. Resazurin (10% v/v) was added to wells and incubated for another 24 h. Experiments were run in quadruplicates. Spectrophotometric absorbance was recorded at a wavelength of 570 nm. Diminazene aceturate (Sigma-Aldrich, Kent, UK), a known antitrypanosomal drug, was used as a positive control. 4.4. Antioxidant Analysis of Antitrypanosomals The ABTS (2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) antioxidant assay kit (Sigma-Aldrich, Kent, UK) was used for the investigation of the antioxidant capacity of compounds by following the manufacturer’s protocols. We followed the same procedures as elaborated previously with minor modifications [10]. Briefly, T. b. brucei cells were seeded at a density of 1.5 × 105 cells/mL on 96-well plates in a two-fold dilution of compounds. Myoglobin was added to each well and incubated for 24 h. ABTS was added to each well and incubated for approximately 5 min at room temperature. After inactivating the reaction by adding a stop solution, absorbance was read at 405 nm. The final volume of cells and reagents in each well was 200 µL. Trolox ((±)-6-hydroxy-2,5,7,8-tetramethylchromane-2- carboxylic acid) was used as the positive control antioxidant. Experiments were performed in duplicates. Molecules 2021, 26, 4488 9 of 10 4.5. Growth Kinetics Analysis T. b. brucei cells were grown to a density of 1 × 106 cells/mL and split into fresh media with the antitrypanosomal compounds at an initial cell density of 1 × 105 cells/mL (day 0). Cells were then monitored and subcultured every 24 h by treating with the compounds at 1 × 105 cells/mL for the next 8 days. 4.6. Fluorescence Microscopy T. b. brucei cells were treated with compounds at the EC50 values for 24 h and cen- trifuged at 27,000 rpm for 10 min. Cells were resuspended in 1 mL of FBS-free HMI9 media and incubated for 30 min. Cells were pelleted and resuspended in 1 mL of FBS-free HMI9 media and incubated for another 30 min. Fixation was performed by incubating cells at 4 ◦C for 1 h in 1 mL of 8% paraformaldehyde in Voorheis modified PBS. Washing of cells was carried out by pelleting and resuspending in PBS, after which 10–20 µL of cell suspension was spread on poly-L-lysine-coated microscope slides, sprayed and wiped clean with 70% ethanol. The slides were allowed to air-dry for 15 min in a humid chamber and placed in a container with methanol at −20 ◦C for 30 min. The slides were rinsed in PBS, after which 0.1 µg/mL DAPI was added to the cells. Slides were rinsed again in PBS and 30 µL of mounting media was applied, along with coverslips and sealed with nail varnish for observation with the Zeiss Axio Vert.A1 inverted microscope. Data were analyzed with Image J version 2.1.0/1.53c. 4.7. Statistical Analysis Data from the cell viability, cytotoxicity, antitrypanosomal sensitivity and antioxidant activity assays were analyzed with GraphPad Prism version 5 (Graph Pad Software, San Diego, CA, USA). The half-maximal effective concentration (EC50) was calculated as the concentration that caused a 50% reduction in cell viability. EC50 values were calculated from a non-linear regression model using the Hill function. p-values < 0.05 were considered to be significant. Supplementary Materials: The following are available online, Figure S1: GC-MS total ion chro- matogram for compound 2. Figure S2: Mass fragmentation and ATR-IR spectra for compound 2. Figure S3: GC-MS total ion chromatogram for compound 3. Figure S4: Mass fragmentation and ATR-IR spectra for compound 3. Figure S5: UV–VIS spectra for compounds 2 and 3. Figure S6: 1H and 13C NMR spectra for compound 2 in C2D6OS. Figure S7: 1H and 13C NMR spectra for compound 3 in CDCl3. Figure S8: Dose–response curves of compounds in T. brucei. Table S1: 1D and 2D NMR data for compound 2 in C2D6OS. Table S2: 1D and 2D NMR data for compound 3 in CDCl3. Table S3: Effect of compounds on ratio of nuclei and kinetoplasts in T. brucei. Author Contributions: Conceptualization, A.K.D., K.K. and T.M.G.; formal analysis, A.K.D., K.K. and T.M.G.; investigation, A.K.D., T.S.A. and C.M.A.; methodology, A.K.D., T.S.A. and C.M.A.; mentorship, K.K. and T.M.G.; validation, A.K.D.; visualization, A.K.D. and T.M.G.; writing—original draft, A.K.D. and T.M.G.; writing—review and editing, A.K.D. and T.M.G. All authors have read and agreed to the published version of the manuscript. Funding: Aboagye Kwarteng Dofuor was supported by a World Bank African Centres of Excellence grant (ACE02-WACCBIP: Awandare). The work was supported by funds from a World Bank African Centres of Excellence grant (ACE02-WACCBIP: Awandare) and a DELTAS Africa grant (DEL-15-007: Awandare). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article or supplementary material. 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