Artemisinin–(Iso)quinoline Hybrids by C−H Activation and Click Chemistry: Combating Multidrug‐Resistant Malaria

Abstract A substantial challenge worldwide is emergent drug resistance in malaria parasites against approved drugs, such as chloroquine (CQ). To address these unsolved CQ resistance issues, only rare examples of artemisinin (ART)‐based hybrids have been reported. Moreover, protein targets of such hybrids have not been identified yet, and the reason for the superior efficacy of these hybrids is still not known. Herein, we report the synthesis of novel ART–isoquinoline and ART–quinoline hybrids showing highly improved potencies against CQ‐resistant and multidrug‐resistant P. falciparum strains (EC50 (Dd2) down to 1.0 nm; EC50 (K1) down to 0.78 nm) compared to CQ (EC50 (Dd2)=165.3 nm; EC50 (K1)=302.8 nm) and strongly suppressing parasitemia in experimental malaria. These new compounds are easily accessible by step‐economic C−H activation and copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) click reactions. Through chemical proteomics, putatively hybrid‐binding protein targets of the ART‐quinolines were successfully identified in addition to known targets of quinoline and artemisinin alone, suggesting that the hybrids act through multiple modes of action to overcome resistance.


Introduction
Every year malaria parasites cause approximately 200 million infections in humans and almost half amillion deaths according to the World Health Organization. [1] Plasmodium falciparum (P. falciparum), representing the most pathogenic species of this eukaryotic genus,i sm ostly responsible for lethal courses of infection. Currently,a rtemisinin (ART; Figure 1A)a nd quinoline derivatives (e.g.,c hloroquine (CQ);F igure 1B)a re the two main classes of antimalarial drugs. [2] ARTisane nantiopure sesquiterpene lactone,which was first extracted from the Chinese medicinal plant Artemisia annua L. (sweet wormwood) in 1972 by Youyou Tu (Nobel Prize 2015). [3] Isoquinolines and quinolines are further privileged scaffolds,u sed clinically as antimalarial and antiviral drugs,f or example,t hrough the different commercial drugs amodiaquine,p aritaprevir, and tafenoquine (Figure 1B). Inspired by these traditional and pharmacologically well-established compounds,many derivatives of artemisinin and quinoline have been designed, synthesized, and biologically investigated. [4] Thee mergence of parasite resistance against approved drugs has been on the rise over the last decades,and medical concerns have thus increased dramatically.T wo resistant strains Dd2 and K1 carry mutations and an umber of copies for genes [5] encoding for multidrug resistance transporters, resulting in resistances against currently available antimalarial drugs.Notably,Dd2 is resistant to CQ,mefloquine,and the antifolates sulfadoxine,p yrimethamine,a nd cycloguanil, whereas K1 is resistant to CQ and antifolates. [5] In addition, they show different mutations,l eading to an amino acid exchange of at arget enzyme resulting in resistance against antifolates (sulfadoxine,p yrimethamine,c ycloguanil). Compared with chloroquine/drug-sensitive strains (3D7), the fitness and thus the reproduction rate of chloroquine/multidrug-resistant (Dd2 and K1) parasites is slightly impaired by resistance-mediating polymorphisms in putative drug transporters and/or in target enzymes, [6] even without drug pressure.
CQ-resistant P. falciparum has been reported from almost all malaria-endemic countries.M oreover,t he therapeutic achievements based on the natural product ARTand its semisynthetic derivatives (dihydroartemisinin, artesunate,a rtemether;F igure 1A)a re also threatened by emerging drug resistance. [7] Therefore,the WHO discourages mono-therapy, which primarily promotes further development of resistance to ARTi ne ndemic areas,a nd recommends the use of artemisinin-based combination therapies (ACTs) to reduce the risk of drug resistance.A CTs use ARTori ts derivatives together with one or more other antimalarial drugs that act through different mechanisms. [8] Recently,s tructural fusions of artemisinins and quinolines,n amely hybrid molecules (see Figure 1C for an example), have been described as being even more efficient against different biological threats,s uch as malaria parasites and cancer cells. [9] Hybridization appears as apowerful concept [10] to increase the activity or pharmacological efficacy of known drugs or the bioactive constituents of the hybrid molecule [11] and to potentially overcome drug resistance, [12,13] as hybrids can be less susceptible to drug resistance.
Whereas some examples of hybrid molecules of quinoline and ARTe xist, neither systematic SAR (structure-activity relationship) studies with different linkers nor identifications of their protein targets are known yet, while ART-isoquinoline hybrids have not even been reported thus far.T ofill these gaps,w eh erein present the structural design, synthesis,a nd assessment of the in vitro antimalarial activities of 17 new ART-isoquinoline and ART-quinoline hybrid compounds able to combat multidrug-resistant malaria. Thea ntimalarial activities of the resulting hybrids were tested on the P. falciparum drug-sensitive strain 3D7 and the two multidrugresistant strains Dd2 and K1.
Thea pplied new isoquinoline precursors were accessible by versatile cobalt(III)-catalyzed step-economic CÀHactivation methods,while the linkers in the hybrids were generated using CuAAC(copper-catalyzed azide-alkyne cycloaddition) click reactions and an ovel rearrangement of the in situ formed tertiary amides to secondary amides.T he SAR study revealed important aspects of the linker group,which led us to identify even more potent antimalarial hybrid compounds able to overcome drug resistance.O ne reason for the increased activity of the hybrids versus their individual constituents could be the simultaneous cellular uptake of both pharmacophores owing to their covalent linking,w hich is not possible for combination therapy.B esides,apossible synergistic effect of the applied pharmacophores might partially explain the pronounced bioactivity of the hybrids.
We further experimentally demonstrated the efficacyo f selected hybrid compounds against malaria in P. berghei- Figure 1. Selected artemisinin derivatives and pharmaceutically relevant isoquinolines/quinolines. A) Artemisinin (naturally occurring), dihydroartemisinin,a rtesunate, and artemether (semisynthetic derivatives). B) Clinically applied drugs with aq uinolineo risoquinoline core:paritaprevir, tafenoquine, chloroquine, and amodiaquine. C) First example of an artemisinin-quinine hybrid. [9a] infected mice.I mportantly,t he proteomic characteristics of the hit hybrid compounds were assessed to identify putative targets in the Plasmodium proteome.W ehave shown that the hybridization of two individual pharmacophores into an ew bioactive compound has high potential in addressing resistance issues because hybrids can simultaneously target pro-teins of both pharmacophores (artemisinin and quinoline), as well as new hybrid-binding proteins.Overall, our findings are highly relevant towards the future development of effective multi-action drugs against resistant malaria. Figure 2. Novel hybrids 1-17 applied for activity examination against P. falciparum 3D7, Dd2, and K1 strains. Red, blue, and green indicate the parent pharmacophores of the molecules. The black moieties represent the linker groups of the molecules. The 3-hydroxy-desoxydihydroartemisinin unit of hybrid 3 is shown in orange.

Results and Discussion
Chemistry Thes ynthesis of the new hybrids 1-17 ( Figure 2) is discussed in two parts according to the chemical reactions that were applied to form the corresponding linker:1)hybrid synthesis by Cu I -catalyzed click reactions;a nd 2) hybrid synthesis by esterification, amide bond formation, and anovel rearrangement of the in situ formed tertiary amide into asecondary amide.

Hybrid Synthesis by Copper(I)-Catalyzed Click Reactions
We first planned to investigate the variation of bioactivity when the linker bears at riazole entity.T he CuAACc lick reaction has become one of the most important chemical reactions in the field of medicinal chemistry with its highly preferable features.T he resulting products feature af ivemembered triazole ring as the linker group,f ormed by 1,3dipolar cycloaddition (click) reaction. This five-membered ring is ab uilding block very often employed in valuable pharmaceuticals. [14] As illustrated in Schemes 1A and 1B,we thus obtained hybrids 1-12,b earing atriazole linker.
Theartemisinin-isoquinoline hybrid compounds 1-3 were prepared (Scheme 1A)t hrough aC uAACc lick reaction of new artesunic acid based azide 22 with the isoquinolinederived alkynes 20 and 21 in the presence of CuSO 4 ·5 H 2 O and sodium ascorbate.T he corresponding isoquinoline precursors 20 and 21 were synthesized by facile C À Hcobaltation of O-acetyl oximes with internal alkynes, [15] followed by Sonogashira coupling of the obtained 6-bromo-isoquinolines 18 and 19 with trimethylsilylacetylene (Scheme 1A and the Supporting Information). We were also able to isolate compound 3,w hich is as ide product of the formation of hybrid 2.W ef ound that the artemisinin moiety is hydroxylated at the C3 position to ad esoxydihydroartemisinin derivative in the presence of copper(I) species,a sr eported previously. [16] This 3-hydroxy-desoxydihydroartemisinin derivative 3 was also subjected to bioactivity examination.
Hybrid Synthesis by Esterification, Amidation, and aNovel Rearrangement We next enlarged the scope of the hybrids by diversifying the linker to examine the SAR for further medicinal chemistry insight. Then ew hybrid molecules 13-16 were synthesized from artesunic acid 35 with the corresponding 7-chloroquinoline derivatives 33, 34, 36,a nd 37 (Scheme 1C and the Supporting Information). While the quinolinederived precursors 33, 34,a nd 36 were prepared based on previous reports, [17] compound 37 was synthesized for the first time by an ucleophilic aliphatic substitution reaction of 5-chloropentyne with quinoline-based amine 36 (Scheme 1C and the Supporting Information). Theh ybrid compounds 13 and 14 were obtained by esterification of artesunic acid 35 with the 7-chloroquinoline-derivedp rimary alcohols 34 and 33,u sing the coupling reagents DCC and DMAP in CH 2 Cl 2 under nitrogen atmosphere . Primary and secondary amine derivatives of 7-chloroquinoline (36 and 37)w ere used for amide coupling reactions in the presence of EDCI and DMAP in CH 2 Cl 2 to furnish hybrids 15 and 16 (Scheme 1C).
Thea lkyne-tagged hybrid 16 was synthesized to identify its target proteins in P. falciparum by proteomics.T oo btain another alkyne-tagged hybrid compound for proteomics studies,weapplied hybrid 14 for further modification through acylation of the secondary amine with hept-6-ynoyl chloride (Scheme 1D). This experiment did not result in the expected tertiary amide product. Instead, we observed the product of an ovel metal-free intramolecular rearrangement of the in situ formed tertiary amide into the secondary amide hybrid 17,w hich we used for proteomics studies.Apossible mechanism of this novel multistep domino reaction is shown in Scheme 1D.T riethylamine promotes dehydrohalogenation of chloro acetyl chloride to generate the corresponding ketene, [18] which subsequently reacts with 14 to at ertiary amide (step 1), which undergoes as econd acylation reaction (step 2). Thei nsitu formed C1-ammonium enolate intermediate undergoes an intramolecular CÀCb ond formation/ CÀNb ond cleavage sequence (step 3) towards the final product 17.S econdary amine 14 facilitates the rearomatization process via its acylation to atertiary amide (step 3').
While quinolines are well-known for their antimalarial activity,much less is known about the antimalarial activity of isoquinolines and their structural determinants.Inthe following, the novel ART-isoquinolines hybrids 1-3 were initially investigated. TheEC 50 (half-maximal effective concentration) values of hybrids 1 and 2 against 3D7, Dd2, and K1 strains of P. falciparum are 9.2, 10.0, and 4.2 nm and 5.3, 4.8, and 3.3 nm, respectively,i ndicating the potent activity of these novel hybrids compared to parent compounds artesunic acid 35 and isoquinoline precursor 18.Furthermore,weobserved that the antimalarial activity of hybrid 3 is 100 times lower than that of hybrid 2.T he drop in activity of 3-hydroxy-desoxydihydroar-temisinin derivative 3 underlines the importance of the 1,2,4trioxane fragment for high activity.
Next, we examined the influence of the linker length on the activity of the artesunic acid-quinoline hybrid compounds   Figure 2), where the linker length is elongated from one to four carbon atoms.However,among these hybrids 4-7,the elongation of the linker did not change the antimalarial activity of the compounds in as ubstantial manner (  (Table 1). Thus hybridization can be used to access more potent bioactive compounds than their parent precursors for this set of hybrids 4-7. Similar to our previous results (see Table 1, hybrids 4-7), ART-quinoline hybrids 8-12 showed great activity against malaria parasites ranging from 2.0 nm to 27.5 nm,r evealing that these hybrids are also more active against CQ/multidrugresistant parasites than against CQ/drug-sensitive parasites. Hybrids 8, 10, 11,a nd 12 showed almost the same potency when they were tested against three different strains.T he hybrids exhibited EC 50 values of 9.0 nm,8 .8 nm,9 .4 nm,a nd 8.4 nm against 3D7;5.9 nm,4.5 nm,5.1 nm,and 6.1 nm against Dd2;and 4.3 nm,2.0 nm,3.8 nm,and 3.7 nm against K1 strains, respectively (Table 1). Interestingly,weagain did not observe ac orrelation between activity and linker length. Hybrids 8 and 9 are b-and a-anomers because of the stereochemistry at the C10 position of the artemisinin unit. As previously reported for a-a nd b-arteethers, [19] we have also observed that the b-isomer 8 is more active than the a-isomer 9. Nevertheless,w ith EC 50 values of 27.5 nm,1 0.6 nm,a nd 5.9 nm,the a-isomer of this hybrid is still highly active against P. falciparum strains 3D7, Dd2, and K1.

4-7 (
Next, we tested the new 7-chloroquinoline-artesunicacid hybrids 13-15,c ontaining ester and amide linkers,a gainst P. falciparum parasites.Hybrid 14 is more active than hybrid 13.R eplacing the H-bond acceptor oxygen atom at the C4 position of the 7-chloroquinoline unit of hybrid 13 with an Hbond donor amine group resulted in the formation of 4aminoquinoline chemical space (hybrid 14).
As expected, this chemical modification substantially increased the potencya gainst all P. falciparum strains,w hich is explained through the formation of the 4-aminoquinoline, aknown antimalarial core structure.T he activities increased, as seen from decreases in the EC 50 values from 14.8 to 4.5 nm for 3D7, from 10.6 to 2.3 nm for Dd2, and from 5.5 to 1.7 nm for K1 strains (Table 1). Based on these results,w ea ssumed that the composition of the linker affects the activity. Furthermore,wedecided to replace the second oxygen atom by an itrogen atom. This designed compound 15 gave EC 50 values of 2.7 nm,1.0 nm,and 780 pm against 3D7, Dd2, and K1 strains,r espectively (Table 1). Through this systematic replacement of heteroatoms,weexamined requirements on the linker for high antimalarial activity,which led to EC 50 values as low as 780 pm against the K1 strain with hybrid 15.T hese results are consistent with those obtained with previous hybrids (13 and 14)when comparing the activities of the individual constituents of each hybrid against chloroquine/ drug-sensitive (3D7) and chloroquine/multidrug-resistant (Dd2 and K1) parasites,a sh igher efficiencies were always observed against resistant parasites.I ti s impressive that the generated compounds are able to counteract all resistances presented by the Dd2 and K1 strains.Onthe basis of the observed SAR, we hypothesized that these artesunic acid-7-chloroquinoline based hybrids are more potent when they bear an itrogen atom on the linker.M oreover,t hey show excellent antimalarial activity.
Next, compounds 16 and 17 were evaluated as alkynetagged hybrids.T hey were designed for proteomics experiments,w hich require an alkyne unit to tag the molecule to ab iotin affinity tag through ac lick reaction. Theb inding proteins of these hybrids were investigated to understand the mechanism of action of the most active antimalarial hybrid. To the best of our knowledge,n one of the artemisininquinoline hybrids have been previously investigated based on their target profiling.H ybrid 16 gave EC 50 values of 3.5, 1.6, and 1.3 nm against 3D7, Dd2, and K1 strains,r espectively ( Table 1). As an analogue,o ur hybrid 16 can be compared with hybrid 15,and it can be implied that the hydrogen atom on the secondary amide improves antimalarial activity.I n contrast, hybrid 16 is atertiary amide and does not provide an N À Hb ond, unlike hybrid 15,a nd it is thus less active.W hen the amide nitrogen atom is occupied by an alkyne chain, the activity is lower as it is the case for hybrid 14,which features an ester bond and aH-bond acceptor.This finding might also be ac onfirmation that the H-bond donor improves the activity.Asexpected, when we tested the antimalarial activity of the post-modified hybrid 17,t he activity dropped compared to its pre-modified version, that is,h ybrid 14.T he decrease in activity is possible due to depletion of the 4-aminoquinoline core,which is essential for heme binding. [20] This initial set of compounds has shown that artemisinin and 7-chloroquinoline are apromising pair to improve the activity against malaria parasites,a nd that the composition of the linker is astructural determinant for modulating activity.

Parasitemia Suppression in Experimental Malaria
We evaluated the suppressive activity of the hybrids in P. berghei-infected mice over four consecutive days of treatment. [21] Compared with the vehicle group,m ice treated subcutaneously with 105 mmol kg À1 (40 mg kg À1 )ofartesunate (ARE) showed a > 99 %reduction in blood parasitemia, and this dose also conferred 100 %a nimal survival ( Table 2, entry 2). Based on these results,w ec hose ad ose of [a] Values are mean AE S.D. and were taken from 10 dpi. [b] Until 41 dpi.
[d] One of five mice were blood smear positive for parasites 41 dpi. 105 mmol kg À1 given by subcutaneous injection as as tarting point for testing the hybrid compounds.T og ain insight into the SARs of our compounds,weselected the artesunic acid-7chloroquinoline based hybrid 14,c ontaining an ester linker group (the thermal, hydrolytic,and enzymatic stability of 14 is high;see the Supporting Information), as its potencyissimilar to that of alkyne-tagged hybrid 16,p repared for target identification experiments.I na ddition, the artemisinin-7chloroquinoline based hybrid 12,containing atriazole linker, was selected. Theu se of hybrids 12 and 14 enabled us to investigate the importance of the linker group for antimalarial efficacy( Figures S1-S3 in the Supporting Information). At ad ose of 105 mmol kg À1 (65 mg kg À1 ), hybrid 14 reduced parasitemia by > 99 %, while at ad ose of 35 mmol kg À1 ,itreduced parasitemia by 95.2 AE 4.9 %( Table 2, entries 3a nd 5). As ar esult of the strong parasitemia suppression, all mice treated with 105 mmol kg À1 of hybrid 14 remained free of parasites for up to 41 dpi (days postinfection), and they were considered cured. When hybrid 14 was given at ad ose of 35 mmol kg À1 (s.c.), 80 %o fm ice remained free of parasites up to 41 dpi, which is ac ure rate higher than for artesunate treatment at the same dosage and with the experimental model. [21] In contrast to hybrid compound 14,hybrid 12 featuring atriazole linker did not reduce parasitemia when administered at ad ose of 105 mmol kg À1 ( Figure S2).
Having ascertained the efficacy of hybrid 14 upon subcutaneous administration, we tested whether this compound is able to inhibit parasitemia upon oral administration (Table 2, entry 6). Compound 14 displayed strong antipar-asitic activity,r educing parasitemia by 78.6 AE 10 %a nd increasing the median animal survival time compared to the vehicle group.F rom two independent experiments,6 0% of mice remained free of parasites in the group treated orally with hybrid 14 (Table 2, entry 6). In contrast, infected mice orally treated with ARE at adose of 105 mmol kg À1 were not cured. In fact, ARE is curative only when administered subcutaneously [22] or in combination with mefloquine.T he fact that hybrid 14 inhibited parasitemia and provided protection from mortality upon oral uptake indicates that this compound has oral efficacy, which is ah ighly desired property for anew antimalarial drug candidate.
ARTa nd ARE are known to have af ast onset of antimalarial action, which causes ar apid decrease in parasitemia, but they also have as hort half-life. [23] Unlike ARE, hybrid 14 presented am ore long-lasting action. This was inferred when instead of the standard treatment for four consecutive days,m ice were treated at intercalated days at 3 and 48 hp ost-infection ( Figure S4 and Table S1). At day 10 post-infection, both hybrid 14 and artesunate treatment suppressed parasitemia;h owever,a td ay 12 post-infection, parasitemia had increased rapidly (i.e., recrudescence of parasitemia) in four out of five mice,a nd at day 16 postinfection, all mice treated with ARE showed parasite recrudescence.Incontrast, only two of five mice treated with hybrid 14 presented detectable parasites at day 12 postinfection, showing that treatment with the hybrid compound is more efficient in delaying the recrudescence of parasites compared with ARE treatment.

Curative Potential in Experimental Malaria
Thef act that hybrid 14 presented impressive parasitemia suppression led us to determine its curative potential (Thompson test). As expected, hybrid 14 cured 100 %o f mice,w hile artesunate only cured 33 %o fm ice when administered at the same dose (Figure 3and Table S2). Based on the cure ratio,w ee stimated that hybrid 14 is threefold more effective than ARE.
Apart from piperaquine,t he required dose of antimalarials to cure mice in the curative model of Thompson is at the very least two times higher than the required dose to protect mice from mortality in the suppressive model of Peters. [24] Strikingly,weobserved that the sum of dosages of the hybrid compound employed in the suppressive test was enough to provide cure.T his puts hybrid compound 14 in an efficacy range that is only comparable to those of piperaquine and ACTs.  (Table) from P. berghei-infected mice within 24 hof oral treatment. In (B), parasitemia was determined by GFP signal using flow cytometry.I n(C), heme species were quantified from the peripheral blood after mouse euthanasia. The results of two independentexperiments with n = 3g roups for each experimentare shown. Error bars are means and 95 %CI(95 %confidence interval).I C 50 and %reduction values are mean AE S.D. In the table, b-hematin formation was determined 24 hafter drug incubation.

The Underlying Mechanism of Potency Enhancement
To study the origin of potencyenhancement, we analyzed the potential of hybrid 14 in impairing malarial pigment formation in in vitro and in vivo models.A RE lacks inhibitory properties in the in vitro b-hematin formation while hybrid 14 inhibited b-hematin formation with ap otency as high as that of chloroquine (Figure 4and Table S3). Assuming that only the 7-chloroquinoline part of hybrid compound 14 is responsible for the b-hematin inhibitory properties, [20b,c] the observation that compound 14 is as potent as chloroquine is explained by the fact that the 7-chloroquinoline moiety in the hybrid compound can adopt different conformations and orientations,i np art greatly influenced by the linker group. While this kind of potencye nhancement against b-hematin formation has been observed for quinoline derivatives, [20c] it has not been demonstrated in experimental malaria. Here,we introduce the notion that the potencyenhancement of hybrid compound 14 against b-hematin correlates well with its efficacyi ni mpairing parasite hemozoin in experimental malaria. This was confirmed in ad esigned experiment with P. berghei-infected mice by simultaneously monitoring parasitemia and heme species,i ncluding the cellular hemozoin ( Figure 4).
These experiments revealed that 24 hafter administration of as ingle oral dose (105 mmol kg À1 ), compound 14 had reduced parasitemia by 56.1 %. In addition to an impressive suppression of parasitemia, the hybrid was effective in impairing hemozoin formation by 71.9 %i nc omparison to untreated mice.A tt he same dose,A RE was only half as effective as compound 14 in reducing parasitemia but did not significantly reduce hemozoin. In contrast, CQ did not reduce parasitemia but attenuated hemozoin formation, albeit twice less than compound 14.C oncomitant to ad ecrease in the hemozoin level, both CQ and compound 14 increased the levels of free heme,while ARE did not, clearly showing that CQ and compound 14 reduce parasite viability by interrupting the biosynthesis of hemozoin. In terms of efficacy, compound 14 behaved similarly to the group receiving areference inhibitor of hemozoin (CQ-treated mice at ahigh dose of 100 mg kg À1 ,3 10 mmol kg À1 ). Our combination of in vitro and in vivo experiments,u sing pharmacologically relevant drug doses,inaddition to acritical quantification of free heme and hemozoin levels,a llowed us to recognize that hemozoin inhibition is due to ad irect effect of hybrid drug treatment rather than ac onsequence of parasitemia reduction. This also supports the notion that the efficacyofhybrid 14 in P. berghei-infected mice is not only due to the presence of the artemisinin moiety,b ut also highly dependent on the quinoline.
Chemical Proteomics Profiling of the Interactive Protein Targets of Hybrids 16 and 17 in P. falciparum With the alkyne-tagged artemisinin-chloroquinoline hybrids 16 and 17 in hand, we linked the alkyne-tagged analogues to biotin azide through click chemistry (Figure 5A). Then, the biotin-bearing analogues were incubated with avidin beads to prepare the drug affinity beads.T he enriched and purified P. falciparum parasite protein lysates were incubated with the drug-bearing beads for 4h.The drug binding targets were affinity-purified by avidin beads and identified by tandem mass spectrometry.Atotal of 129 and 107 parasite proteins were identified as direct targets of hybrids 16 and 17 (see Table S5), while pull-down of the dimethyl sulfoxide treated control beads did not identify any parasite proteins.T he exponentially modified protein abundance index (emPAI) was used to provide as emi-quantification of protein abundance. [25] Heat map analysis ( Figure 5B)s howed that the target profiles of hybrids 16 and 17 are generally similar to each other as the hybrids possess as imilar artemisinin moiety. Consistent with the higher antimalarial potency, the target labeling potential of hybrid 16 is also slightly higher than that of hybrid 17.A long with the target identification we found an umber of proteins,s uch as heat shock protein 70, highmolecular-weight rhoptry protein 2, and 40S ribosomal protein S15, that were strongly enriched by both probes. Interestingly,w ef ound that both artemisinin hybrid compounds 16 and 17 can interact with PfATP6, aw ell-known artemisinin target.
Gene ontology pathway analysis was performed to map out the critical pathways of the drug targets involved in the antimalarial targets of the hybrid compounds ( Figure 6). A number of metabolic processes are the most significant pathways affected by the two hybrids 16 and 17,i ncluding glycolytic and organonitrogen compound metabolic processes (Figure 7).
Thefindings of the proteomic analysis support the notion that hybrid compounds 16 and 17 affect multiple pathways classically recognized for artemisinin action (such as the PfATP6 enzyme) and for quinolines (40S ribosomal protein machinery), which are important targets for development and survival during blood stage P. falciparum growth. [26] These findings have important future implications for the development of efficient drug candidates based on ART-derived hybrid compounds.

Conclusion
Theb urden of malaria, especially its most fatal species P. falciparum,a nd the emergence of resistances to known antimalarial drugs (e.g., CQ and ART) are two of the biggest concerns for global infectious disease control. Our work provides access by,among others,environmentally benign C À Ha ctivation to novel ART-based hybrid compounds,w hich show outstanding activities against the drug-sensitive 3D7 wild-type strain and against two multidrug-resistant strains (Dd2, K1) of P. falciparum parasites,with EC 50 values ranging from 780 pm to 27.5 nm.T he hybrids are significantly more potent towards resistant parasite strains.T hus these compounds illustrate the high potential of the hybridization concept as an alternative drug discovery approach for efficient treatment and to overcome drug resistance.T he potencye nhancement of hybrids in comparison to their corresponding parent compounds can be explained through Figure 5. Target Identification. A) The alkyne-taggedh ybrids were reacted with biotin azide to generate the affinity probes for interactive target enrichment. Affinity pull-downc oupled mass spectrometry identification was carried out to characterize the hybrids targets. B) Heat map representation of the relative abundanceofindividualt argetso fhybrids 16 and 17.T he emPAI scores of individual proteins were used to generate the heat map with morpheuss oftware. Complete datasets of the drug targets are shown in Table S5. the simultaneous cellular uptake of both pharmacophores covalently bound via al inker,a nd by possible synergistic effects.T hese concepts are supported by the observation that one of the most potent hybrid compounds against P. falciparum,t he ART-quinoline hybrid 14,d emonstrated superior efficacyincomparison to the antimalarial drug artesunate in P. berghei-infected mice,being in its efficacy only comparable to ACTs.T he outstanding efficacyofhybrid 14 might be due to the combination of the pharmacological features of quinolines (potent hemozoin inhibition) and artemisinins (fast parasite killing). Finally,u sing ac hemical proteomics approach, we identified ac omprehensive set of P. falciparum parasite proteins as direct targets of the selected highly active hybrids 16 and 17.The results demonstrate that the hybrid compounds affect targets important for development and survival during bloodstage P. falciparum development, such as the PfATP6 enzyme (responsible for artemisinin action) and the 40S ribosomal protein machinery (classically recognized for quinoline action).
In general, these findings should provide avaluable basis for further molecular investigations of putative target proteins of these artemisinin-based compounds.T his is an important implication for antimalarial drug design, in particular for hybrid drugs that can simultaneously target protein synthesis (as observed by proteomics) and hemozoin (Hz) formation (as observed by b-hematin assay) as well as surmount multidrug resistance (inferred by EC 50 values). Our results should also encourage further mechanistic studies and be as tepping stone towards overcoming multidrug resistance.