Synthesis of Novel Artemisinin, Ciprofloxacin, and Norfloxacin Hybrids with Potent Antiplasmodial Activity

The synthesis and antiplasmodial evaluation of new hybrids combining the pharmacophore structures of artemisinin, ciprofloxacin or norfloxacin, and 7-chloroquinoline are reported in this study. The first step for all of the syntheses is the obtainment of key piperazine esters intermediates bearing the drugs ciprofloxacin and norfloxacin. Using these platforms, 18 final compounds were synthesized through a multistep procedure with overall yields ranging between 8 and 20%. All compounds were screened for their antiplasmodial activity against the chloroquine-resistant Plasmodium falciparum FcB1 strain. Compounds 20, 21, 22, and 28, bearing an artesunate fragment with ciprofloxacin, exhibited IC50 values in the range of 3.5–5.4 nM and excellent selectivity indices. Among the compounds bearing the artesunate moiety on the norfloxacin, two of them, 23 and 24, afforded IC50 values of 1.5 nM and 1.9 nM, respectively. They also showed excellent selectivity indices. The most potent compounds were also evaluated against the CQ-resistant Dd2 strain of Plasmodium falciparum, demonstrating that those compounds incorporating the artesunate fragment were the most potent. Finally, the combination of artesunate with either ciprofloxacin or norfloxacin moieties in a single molecular entity proved to substantially enhance the activity and selectivity when compared to the administration of the unconjugated counterparts artesunate/ciprofloxacin and artesunate/norfloxacin.


Introduction
Malaria is a parasitic disease that is caused by Plasmodium.It affects millions of people every year and can lead to death, mainly caused by Plasmodium falciparum.There is no efficacious malaria vaccine, and, currently, the WHO recommends as a treatment the use of artemisinin-based combination therapies (ACTs), which involve the combination of an artemisinin (ART) derivative, such as artemether or sodium artesunate (AS), with a partner drug, mostly a quinoline-based drug, with a different mechanism of action [1].Unfortunately, the parasites have developed resistance to the known antimalarial drugs, and the current frontline antimalarial ART is now under threat.Several research groups have focused on a newer strategy for the discovery of the next generation of antimalarial drugs.More specifically, the combination of two antimalarial drugs in a single hybrid molecule could overcome these limitations, allowing each pharmacophore to act simultaneously on multiple targets [2,3].Furthermore, these hybrids may improve efficiency, due to their increased cellular uptake in comparison with the cellular uptake of the corresponding parent drugs and act as prodrugs, where the hydrolysis of the ester moiety provides the individual pharmacological activity.Thus, various quinoline- [4,5] and ART-based [6] hybrids have been synthesized in recent years and tested for their activity against different strains of the P. falciparum.
In brief, an ART-acridine hybrid 1 [7] was found to be sevenfold more active than chloroquine (CQ) against the P. falciparum NF54 strain, whereas an ART-estrogen hybrid 2 [8] exhibited higher antiplasmodial activity than standard drugs against the 3D7 strain.Tsogoeva et al. [9] prepared five ART-quinazoline hybrids and evaluated them against the 3D7 strain.All of them provided very good EC 50 (half maximal effective concentration), with hybrid 3 being even more active than the clinical drugs dihydroartemisinin (DHA) and CQ, with EC 50 values of 1.4 ± 0.4 nM, 2.4 ± 0.4 nM, and 9.8 ± 2.8 nM, respectively.Furthermore, another work from the same group [10] provided a small library of ART-CQ and ART-isoCQ hybrids via a click chemistry approach to enhance the potency against CQ resistance and against multidrug-resistant Plasmodium falciparum strains.In fact, all these compounds were active with EC 50 values ranging from 780 pM to 27.5 nM.The ART-CQ hybrid 4 demonstrated significant efficacy (EC 50 = 1.7-4.5 nM) against all three P. falciparum parasite strains (3D7, Dd2, and K1) in comparison to AS (EC 50 = 5.2-14.4nM) and was therefore comparable to ACTs.
Recent studies from our group have demonstrated that the combination of ART with a phytohormone endoperoxide G factor analogue (GMeP) and/or polyamines, such as spermidine and homospermidine, led to the discovery of three candidates more potent than ART and CQ [11].The ART-GMeP hybrid 5 and compounds 6 and 7, with two units of ART and one of GMeP conjugated through a polyamine linker, exhibited antiplasmodial activities at nM concentrations (IC 50 values between 2.6 and 10.6 nM) against the CQresistant P. falciparum strain FcB1, with hybrid 5 being 21 times more active than ART (IC 50 = 55 ± 13.6 nM).Furthermore, an ART core was also combined with the antibiotic fosmidomycin (FSM), which is also known for its antimalarial activity as it targets the nonmevalonate isoprenoid synthesis pathway that is essential for the malaria parasite [12,13].The ART-FSM conjugates 8 and 9 exhibited antiplasmodial activity against the FcB1 strain 41.5 and 23.1 times more potent than FSM, respectively (Figure 1).
In this work, we report the synthesis and the biological evaluation of 18 novel hybrids that combine the pharmacophore structures of ART, CPX or NRX, and 7-chloroquinoline (7-CQ).Structure-activity relationship studies (SARS) of this suitably designed library will allow us to study the following: (a) which one of the drugs CPX or NRX provides higher antiplasmodial activity; (b) how the esterification of the fluorquinolone moiety free carboxylic group, such as ethyl or butyl ester, affects the activity of hybrids; (c) the difference in the antiplasmodial activity between the 10-carba-ART-CPX and 10-carba-ART-NRX hybrids and the corresponding artesunate hybrids, and (d) how the introduction of a third pharmacophore through an amide bond between the fluoroquinolone and piperazine-(7-CQ) modifies the activity of the compounds (Figure 2).In this work, we report the synthesis and the biological evaluation of 18 novel hybrids that combine the pharmacophore structures of ART, CPX or NRX, and 7-chloroquinoline (7-CQ).Structure-activity relationship studies (SARS) of this suitably designed library will allow us to study the following: (a) which one of the drugs CPX or NRX provides higher antiplasmodial activity; (b) how the esterification of the fluorquinolone moiety free carboxylic group, such as ethyl or butyl ester, affects the activity of hybrids; (c) the difference in the antiplasmodial activity between the 10-carba-ART-CPX and 10-carba-ART-NRX hybrids and the corresponding artesunate hybrids, and d) how the introduction of a third pharmacophore through an amide bond between the fluoroquinolone and piperazine-(7-CQ) modifies the activity of the compounds (Figure 2).

Synthesis of the Key Intermediates 38-43
The first step in the synthesis of the key intermediates of drugs CPX and NRX is the protection of the secondary amine of the piperazine fragment after the temporary silylation of the free carboxylic group in a one-pot reaction.Subsequently, the corresponding N-trityl-protected analogues 30 and 31 (Scheme 1) were subjected either to a Steglich-type esterification with commercially available alcohols using 1-ethyl-3-(3-dimehtylaminopropyl)carbodiimide hydrochloride (EDC•HCl) in the presence of a catalytic amount of 4dimehtylaminopyridine (DMAP), affording compounds 32-35, or to an amide bond formation with 7-chloro-4-(piperazin-1-yl)quinoline (47) using O-[(Ethoxycarbonyl)cyanomethylenamino]-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TOTU) as the coupling reagent in the presence of DIPEA, thereby providing the conjugates 36 and 37 in a 61% yield after purification through flash column chromatography (FCC).Compound 47 was obtained from the commercially available 4,7-dichloro-quinoline via the nucleophilic aromatic substitution of the latter with piperazine according to a published procedure [34].Finally, the treatment of conjugates 32-37 with TFA/DCM/anisole for 1 h at room temperature led to the key intermediates 38-42 (Scheme 1).

Synthesis of the Key Intermediates 38-43
The first step in the synthesis of the key intermediates of drugs CPX and NRX is the protection of the secondary amine of the piperazine fragment after the temporary silylation of the free carboxylic group in a one-pot reaction.Subsequently, the corresponding N-trityl-protected analogues 30 and 31 (Scheme 1) were subjected either to a Steglich-type esterification with commercially available alcohols using 1-ethyl-3-(3dimehtylaminopropyl)carbodiimide hydrochloride (EDC•HCl) in the presence of a catalytic amount of 4-dimehtylaminopyridine (DMAP), affording compounds 32-35, or to an amide bond formation with 7-chloro-4-(piperazin-1-yl)quinoline (47) using O-[(Ethoxycarbonyl) cyanomethylenamino]-N,N,N ′ ,N ′ -tetramethyluronium tetrafluoroborate (TOTU) as the coupling reagent in the presence of DIPEA, thereby providing the conjugates 36 and 37 in a 61% yield after purification through flash column chromatography (FCC).Compound 47 was obtained from the commercially available 4,7-dichloro-quinoline via the nucleophilic aromatic substitution of the latter with piperazine according to a published procedure [34].Finally, the treatment of conjugates 32-37 with TFA/DCM/anisole for 1 h at room temperature led to the key intermediates 38-42 (Scheme 1).

Synthesis of the ART-and AS-CPX or NRX Hybrids 14-19 and 21-24
The TFA-piperazine salts of esters 38-41 were then conjugated through an amide bond either with the ART-derived carboxylic acids 44 and 45 (Scheme 2) or with the artesunate fragment 46 using BOP or TOTU as the coupling reagent in the presence of DI-PEA.The desirable hybrid compounds 14-19 and 21-24 were obtained in good to excellent yields ranging from 56% to 88% after flash column purification.Regarding the synthesis of the ART derivatives 44-46, they were prepared starting from ART according to published procedures [11,35].

Synthesis of the ART-and AS-CPX or NRX Hybrids 14-19 and 21-24
The TFA-piperazine salts of esters 38-41 were then conjugated through an amide bond either with the ART-derived carboxylic acids 44 and 45 (Scheme 2) or with the artesunate fragment 46 using BOP or TOTU as the coupling reagent in the presence of DIPEA.The desirable hybrid compounds 14-19 and 21-24 were obtained in good to excellent yields ranging from 56% to 88% after flash column purification.Regarding the synthesis of the ART derivatives 44-46, they were prepared starting from ART according to published procedures [11,35].

Synthesis of the ART-Drug-(7-CQ) Hybrids 25-29
Compounds 42 and 43, bearing two pharmacophores (a fluoroquinolone and 7-CQ) already tethered through an amide bond, were further combined with one of the three artemisinin derivatives 44, 45, and 46.The reactions were performed in DMF using the TOTU/DIPEA system, which resulted in the final hybrids 25-29 after flash column purification in 50-75% yields (Scheme 3).

Synthesis of the ART-Drug-(7-CQ) Hybrids 25-29
Compounds 42 and 43, bearing two pharmacophores (a fluoroquinolone and 7-CQ) already tethered through an amide bond, were further combined with one of the three artemisinin derivatives 44, 45, and 46.The reactions were performed in DMF using the TOTU/DIPEA system, which resulted in the final hybrids 25-29 after flash column purification in 50-75% yields (Scheme 3).

Synthesis of the ART-CPX and AS-CPX Hybrids 12, 13, and 20
Finally, for the sake of comparison, condensation occurred between the piperazine ring of the commercially available CPX with the C-10-carba-artemisinin fragments 44 and 45 and the artesunate (46), which led to the ART-CPX conjugates 12, 13, and 20 (Scheme 4).All reactions were conducted in DCM under peptide coupling conditions using BOP as the activating reagent and DIPEA as a base.

Biological Investigation
The synthesized compounds were screened for their antiplasmodial activity against the CQ-resistant P. falciparum FcB1 strain, using AS, CPX, and NRX as control drugs (Figure 1).Moreover, their cytotoxicity was measured upon the primary human fibroblast cell line AB943, which allowed the calculation of their selectivity index (SI).The IC50 and SI (the ratio between the IC50 of cytotoxicity and the IC50 of antiplasmodial activity) values for the hybrids bearing the CRX or the NRX fragment and the drug controls are reported Finally, for the sake of comparison, condensation occurred between the piperazine ring of the commercially available CPX with the C-10-carba-artemisinin fragments 44 and 45 and the artesunate (46), which led to the ART-CPX conjugates 12, 13, and 20 (Scheme 4).All reactions were conducted in DCM under peptide coupling conditions using BOP as the activating reagent and DIPEA as a base.

Synthesis of the ART-CPX and AS-CPX Hybrids 12, 13, and 20
Finally, for the sake of comparison, condensation occurred between the piperazine ring of the commercially available CPX with the C-10-carba-artemisinin fragments 44 and 45 and the artesunate (46), which led to the ART-CPX conjugates 12, 13, and 20 (Scheme 4).All reactions were conducted in DCM under peptide coupling conditions using BOP as the activating reagent and DIPEA as a base.

Biological Investigation
The synthesized compounds were screened for their antiplasmodial activity against the CQ-resistant P. falciparum FcB1 strain, using AS, CPX, and NRX as control drugs (Figure 1).Moreover, their cytotoxicity was measured upon the primary human fibroblast cell line AB943, which allowed the calculation of their selectivity index (SI).The IC50 and SI (the ratio between the IC50 of cytotoxicity and the IC50 of antiplasmodial activity) values for the hybrids bearing the CRX or the NRX fragment and the drug controls are reported

Biological Investigation
The synthesized compounds were screened for their antiplasmodial activity against the CQ-resistant P. falciparum FcB1 strain, using AS, CPX, and NRX as control drugs (Figure 1).Moreover, their cytotoxicity was measured upon the primary human fibroblast cell line AB943, which allowed the calculation of their selectivity index (SI).The IC 50 and SI (the ratio between the IC 50 of cytotoxicity and the IC 50 of antiplasmodial activity) values for the hybrids bearing the CRX or the NRX fragment and the drug controls are reported in Tables 1 and 2, respectively.In addition, for the most interesting compounds, the IC 50 values were also evaluated against the CQ-resistant Dd2 strain of P. falciparum, and, consequently, their SIs were also evaluated and are reported in Table 3 (with the CPX fragment present) and Table 4 (with the NRX fragment present).
Concerning the artemisinin-derived carboxylic acids 44 and 45, they presented IC 50 values of 401 and 264 nM.When 7-CQ was present in addition to ART and CPX moieties (Table 1, entries 11 and 12), compounds 25 and 26 presented comparable IC 50 values (7.5 nM and 13.7 nM, respectively).In the absence of 7-chloroquinoline, it is important to note that the n-Bu esters were more potent again than the corresponding ethyl esters.In fact, compound 16 was sixteen times more potent than compound 14 (Table 1, entries 6 and 4), and compound 17 was four times more potent than compound 15 (Table 1, entries 7 and 5).Concerning the acids 12 and 13 (Table 1, entries 2 and 3), we observed a large variation between the IC 50 values, with compound 12 being around 200 times more potent than compound 13.Finally, concerning the selectivity indices of this subgroup, we observed very good values for compounds 12, 16, and 28 (SI = 4453, 2000, and 4046, respectively), although these were lower in comparison with the best-performing artesunate-CPX derivatives.
For compounds bearing only the NRX fragment, the IC 50 values were quite poor, ranging from 14 to 84 µM.Nevertheless, we again observed a slightly better activity for the n-Bu esters than for ethyl esters (Table 2, entries 8 vs. 9 and 11 vs. 12).For compounds bearing the artemisinin moiety on the NRX ester derivatives, we observed very potent activities (Table 1, entries 2-5).Here again, coupling with the artesunate moiety afforded tenfold better IC 50 values than coupling with artemisinin-derived carboxylic acids 44 and 45.Thus, compounds 23 and 24 (Table 2, entries 4 and 5) presented IC 50 values of 1.5 nM and 1.9 nM, respectively, with the former having an excellent and much higher SI value (28,382 vs. 16,047).The 7-chloroquinoline moiety, when coupled with NRX alone or with the ART-NRX derivatives, did not contribute to better results, neither for antiplasmodial activity nor for the SIs (Table 2, entries 6, 7, 10, and 13).The most potent compounds in both the CPX and NRX series were also evaluated against the CQ-resistant Dd2 strain of P. falciparum; the results are presented in Tables 3 and 4. AS (46) was taken as the reference compound.We can generally observe the same trends in the antiplasmodial activities, and the IC 50 values are in the same range for the FCB1 and Dd2 strains.The same is also true for the SI values when calculated using the Dd2 IC 50 values, although they are less high than those for the FcB1 strain.When examining all the evaluated compounds, we can notice that those bearing the artesunate fragment are the most potent.In order to clarify the importance of the covalent conjugation of the pharmacophores, for three of them (22, 23, and 24) we also examined the antiplasmodial activity measured against the FcB1 strain when there is no covalent bond between the artesunate and the CPX or NRX fragment.In this respect, we determined the IC 50 values of the CPX and NRX fragments alone (compounds 39, 40, and 41) and mixed with AS (46) at an equimolar ratio and compared them to the IC 50 values of the corresponding hybrid compounds (22, 23, and 24) and AS (the results are shown in Table 5).The CPX and NRX fragments (compounds 39, 40, and 41) have very poor antiplasmodial activity in the micromolar range.When mixed with artesunate at an equimolar ratio, the IC 50 values (expressed as the equivalent AS concentration present in the mixture) were similar to the IC 50 value of AS alone, indicating no deleterious action of the CPX or NRX fragment by itself on the AS antiplasmodial activity.Most gratifyingly, not only are the excellent IC 50 values, which are even better than artesunate, maintained in the hybrid compounds 22, 23, and 24 but also the cytotoxicity of each of them measured against the human primary fibroblasts AB943 and, consequently, the selectivity indices are much better for the best hybrid compounds than for the parent AS (46).

General Methods
All solvents were dried and purified according to the standard procedures prior to use.When required, the reactions were carried out under a dry argon atmosphere in preflamed glassware.Anhydrous Na 2 SO 4 was used for drying the solutions, and the solvents were then routinely removed at ca. 40 • C under reduced pressure using a rotary vacuum evaporator.All reagents employed in this present work were commercially available and used without further purification.Flash column chromatography (FCC) was performed on silica gel (70-230 and 230-400 mesh, Merck, Darmstadt, Germany) and analytical thin layer chromatography (TLC) on silica 60gel-F254 precoated aluminum foils (0.2 mm film, Merck, Germany).The spots on the TLC plates were visualized with UV light at 254 nm and ninhydrin solution or charring agents. 1 H NMR spectra were obtained at 600.13 MHz, 13 C NMR spectra at 150.90 MHz, and 19 F NMR spectra at 564.63 MHz on a Bruker AVANCEIII HD spectrometer in CHCl 3 .Chemical shifts (δ) are indicated in parts per million (ppm) downfield from TMS and referenced to residual undeuterated solvents (7.26 for 1 H NMR and 77.16 for 13 C NMR).Copies of the 1 H and 13 C spectra of all the final compounds are reported in the Supplementary Material (Figures S1-S36).The referencing of the 19 F NMR spectra was calculated with the instrument using the default methods.Coupling constants (J) are reported in hertz.Electrospray ionization (ESI) mass spectra were recorded at 30V on a Waters Micromass Platform LC spectrometer (Waters, Wilmslow, UK) using MeOH as the solvent.Melting points were determined with a Buchi SMP-20 apparatus (Buchi, Flawil, Switzerland) and are uncorrected.

Synthesis of the N-Trt-Protected CPX 30 and NRX 31
To a stirred suspension of commercially available CPX or NRX (6.0 mmol) in CHCl 3 /MeCN (5:1, 9 mL), TMSCl (0.84 mL, 5.8 mmol) was added at room temperature, and the reaction mixture was heated to reflux for 1 h.It was subsequently cooled to 0 • C. The addition of Et 3 N (1.84 mL, 13.2 mmol) and of four equal portions of TrtCl (1.74 g, 6.2 mmol) every 15 min was undertaken, and the reaction mixture was stirred at 0 • C for another 3 h.Upon completion of the reaction, MeOH (1.2 mL, 30 mmol) was added, and the precipitate was filtered under vacuo with CHCl 3 .The filtrate was concentrated to approximately onefifth of its original volume under a reduced pressure, diluted with CHCl 3 , and acidified with 5% aqueous citric acid.The organic layer was washed with water and brine, dried over anhydrous Na 2 SO 4 , and concentrated to dryness under vacuum.The residues thus obtained were subjected to FCC, affording the corresponding Trt-protected analogs 30 and 31.

Deprotection of Compounds 32-37
To an ice-cold solution of the Trt-protected analogue or conjugate (0.05 mmol) in DCM (0.23 mL), anisole (10 µL, 0.09 mmol) and TFA (23 µL, 0.3 mmol) were added, and the reaction mixture was stirred for 15 min at 0 • C and then for 45 min at room temperature.Then, a mixture of Et 2 O and n-hexane was added dropwise until full precipitation of the corresponding tris-trifluoroacetate salt, which was collected upon filtration under vacuum and used in the next step without further purification.

General Procedure for the Synthesis of the Hybrids 12-14, 20, and 21
To a stirred solution of 44, 45, or 46 (0.25 mmol) in anhydrous DCM (6.1 mL), BOP (84 mg, 0.19 mmol) was added, the mixture was cooled to 0 • C and the addition of DIPEA (80 µL, 0.46 mmol) was undertaken.After 40 min, CPX or CPX analogue (0.25 mmol) was added, and the reaction mixture was left under overnight stirring at room temperature.Upon completion of the reaction, monitored with the TLC, the mixture was diluted with DCM, and the organic layer was washed sequentially with cold 5% aqueous citric acid, water, and brine.After being dried over anhydrous Na 2 SO 4 , the organic extracts were filtered and evaporated to dryness under reduced pressure.The obtained residues were subjected to FCC to provide the pure hybrids as yellow solids.

Antibiotics 2024 , 23 Figure 1 .
Figure 1.Structures of various hybrids (1-11) with antimalarial activity and of clinical drugs related to this work.

10 Figure 1 .
Figure 1.Structures of various hybrids (1-11) with antimalarial activity and of clinical drugs related to this work.

3. 3 . 2 .
In Vitro Growth Inhibition of P. falciparum The chloroquine-resistant FcB1/Colombia and Dd2/Indochina strains of Plasmodium falciparum were obtained from the Protist Collection of the National Museum of Natural History, Paris, France (n • MNHN-CEU-2016-224-PfFCB1 and MNHN-CEU-2016-222-PfDD2).The parasites were maintained in vitro on human erythrocytes in RPMI 1640 medium

Table 1 .
Antiplasmodial activity of compounds bearing the CPX fragment against the CQ-resistant P. falciparum FcB1 strain, cytotoxicity upon human primary fibroblasts AB943, and selectivity index.

IC 50 upon Fibroblasts AB943 (µM) a Selectivity Index (IC 50 AB943/IC 50 FcB1)
n.d.: not determined; a : mean +/− standard deviations of the IC 50 values determined from at least three independent experiments; b : IC 50 value determined from the compound tested at the concentrations of 50, 5, and 0.5 µg/mL and 50 and 5 ng/mL in triplicate; and c : mean +/− standard deviations of the IC50 values determined from at least four independent experiments.

Table 2 .
Antiplasmodial activity of compounds bearing the NRX fragment against the CQ-resistant P. falciparum FcB1 strain, cytotoxicity upon human primary fibroblasts AB943, and selectivity index.

IC 50 upon Fibroblast AB943 (µM) a Selectivity Index (IC 50 AB943/IC 50 FcB1
: IC 50 value determined from compound tested at the concentrations of 50, 5, and 0.5 µg/mL and 50 and 5 ng/mL in triplicate; and c : mean +/− standard deviations of the IC 50 values determined from at least four independent experiments.
n.d.: not determined; a : mean +/− standard deviations of the IC50 values determined from at least three independent experiments; b

Table 3 .
Comparison of the antiplasmodial activity of compounds bearing the CPX fragment against the CQ-resistant P. falciparum FcB1 and Dd2 strains.: mean +/− standard deviations of the IC 50 values of at least three independent experiments. a

Table 4 .
Comparison of the antiplasmodial activity of compounds bearing the NRX fragment against the CQ-resistant P. falciparum FcB1 and Dd2 strains.
n.d.: not determined; and a : mean +/− standard deviations of the IC 50 values of at least three independent experiments.