New Derivatives of the Multi-Stage Active Malaria Box Compound MMV030666 and Their Antiplasmodial Potencies

MMV’s Malaria Box compound MMV030666 shows multi-stage activity against various strains of Plasmodium falciparum and lacks resistance development. To evaluate the importance of its diarylether partial structure, diarylthioethers and diphenylamines with varying substitution patterns were prepared. A number of evident structure-activity relationships were revealed. Physicochemical and pharmacokinetic parameters were determined experimentally (passive permeability) or calculated. Compared to the lead compound a diarylthioether was more active and less cytotoxic resulting in an excellent selectivity index of 850. In addition, pharmacokinetic and physicochemical parameters were improved.


Introduction
With an estimated 241 million cases and 627,000 deaths in 2020 the hard-earned reduction in malaria casualties fell victim to disruptions in prevention and care due to the COVID-19 pandemic. Furthermore, the number of malaria endemic countries rose from 26 in 2000 to 47 in 2020 [1]. Malaria belongs to the infectious diseases and is caused by eukaryotic, single-celled protozoans of the species Plasmodium. Various strains are human pathogens with Plasmodium falciparum being the most dangerous and deadly [2]. Emerging resistance to the gold standard in malaria therapy, the artemisinins and their partner drugs in the WHO African region are a serious cause for concern, as these countries are among those most affected [3][4][5]. To this day, double or triple Artemisinin-based combination therapies show acceptable efficacy, nonetheless alternative treatments and orally applicable drugs with new modes of action are urgently needed to successfully fight the malaria parasite [6][7][8]. Vaccine development is a difficult task to undertake due to the parasite's complex life cycle and multiple possible targets. RTS,S/AS01, the first vaccine against any parasitic diseases, shows promising features and activities, however only partial efficacy [9][10][11].
The Medicines for Malaria Venture (MMV) is one of many foundations that made it their business to find new strategies to combat the increasing risk of untreatable malaria. Therefore, they published results of a huge screening project, the so-called Malaria Box, a collection of 400 drug-and probe-like compounds with activities against various strains of Plasmodia [12][13][14]. One of these compounds, the 2-phenoxybenzamide 1, exhibits multistage activity against sexual, asexual and liver stages of P. falciparum and lacks resistance development in sub-lethal doses. It shows a metabolomic profile resembling atovaquone.

Chemistry
New derivatives of the lead structure 1 were obtained by firstly synthesizing the corresponding carboxylic acid as well as anilino derivatives and subsequently coupling these partial structures to the desired benzamides. Preparation of the substituted benzoic acids started by treating the respective anthranilic acid with sodium nitrite under acidic conditions yielding the diazonium salts. Subsequent Sandmeyer-like reaction with an aqueous potassium iodide solution gave the desired 2-iodo-benzoic acid derivatives 5, 6, 7 and 8 as light brown solids in mostly high yields [18]. In our last study, only 2-phenoxybenzamides were prepared. In order to evaluate the importance of the diarylether partial structure, corresponding diarylthioethers and diphenylamines were prepared. In the

Chemistry
New derivatives of the lead structure 1 were obtained by firstly synthesizing the corresponding carboxylic acid as well as anilino derivatives and subsequently coupling these partial structures to the desired benzamides. Preparation of the substituted benzoic acids started by treating the respective anthranilic acid with sodium nitrite under acidic conditions yielding the diazonium salts. Subsequent Sandmeyer-like reaction with an aqueous potassium iodide solution gave the desired 2-iodo-benzoic acid derivatives 5, 6, 7 and 8 as light brown solids in mostly high yields [18]. In our last study, only 2-phenoxybenzamides were prepared. In order to evaluate the importance of the diarylether partial structure, corresponding diarylthioethers and diphenylamines were prepared. In the course of a copper-catalyzed Ullmann-like ether synthesis, the obtained iodo-benzoic acids 5, 6, 7 and 8 were coupled with phenols or anilines, respectively, giving diarylethers (9)(10)(11)(12), a diarylthioether (13) as well as diphenylamines (14,15) (Figure 2) [19]. Coupling was confirmed by the appearance of additional proton signals in the 1 H NMR spectrum.
course of a copper-catalyzed Ullmann-like ether synthesis, the obtained iodo-benzoic acids 5, 6, 7 and 8 were coupled with phenols or anilines, respectively, giving diarylethers (9)(10)(11)(12), a diarylthioether (13) as well as diphenylamines (14,15) (Figure 2) [19]. Coupling was confirmed by the appearance of additional proton signals in the 1 H NMR spectrum.  In order to obtain the 2-and 4-substituted derivatives of aniline 16, 17, 18 and 19, 1fluoro-2-nitrobenzene and 1-fluoro-4-nitrobenzene were firstly treated with N-Boc-piperazine and potassium carbonate in dry DMSO in the course of a nucleophilic aromatic substitution yielding compounds 20 and 21 in high yields [20]. Their N-Boc-group was cleaved using trifluoroacetic acid in dry dichloromethane giving 1-(2-nitrophenyl)piperazine 22 and 1-(4-nitrophenyl)piperazine 23 [21]. Treatment of these piperazine derivatives with triethylamine and pivaloyl chloride in dry dichloromethane yielded the N-pivaloylpiperazinyl analogs 24 and 25 [22]. Subsequent reduction of the nitro group of compounds 20, 21, 24 and 25 with palladium in an atmosphere of hydrogen at the parr-apparatus gave the desired aromatic amines 16, 17, 18 and 19 ( Figure 3) [23]. Successful reduction of the nitro group was detected in the 1 H NMR spectrum by a shift of the aromatic protons to lower frequencies as well as by the appearance of a NH2-signal. In order to obtain the 2-and 4-substituted derivatives of aniline 16, 17, 18 and 19, 1-fluoro-2-nitrobenzene and 1-fluoro-4-nitrobenzene were firstly treated with N-Boc-piperazine and potassium carbonate in dry DMSO in the course of a nucleophilic aromatic substitution yielding compounds 20 and 21 in high yields [20]. Their N-Boc-group was cleaved using trifluoroacetic acid in dry dichloromethane giving 1-(2-nitrophenyl)piperazine 22 and 1-(4nitrophenyl)piperazine 23 [21]. Treatment of these piperazine derivatives with triethylamine and pivaloyl chloride in dry dichloromethane yielded the N-pivaloylpiperazinyl analogs 24 and 25 [22]. Subsequent reduction of the nitro group of compounds 20, 21, 24 and 25 with palladium in an atmosphere of hydrogen at the parr-apparatus gave the desired aromatic amines 16, 17, 18 and 19 ( Figure 3) [23]. Successful reduction of the nitro group was detected in the 1 H NMR spectrum by a shift of the aromatic protons to lower frequencies as well as by the appearance of a NH 2 -signal.
Amide synthesis of carboxylic acids and anilino derivatives was achieved using a combination of 2-chloro-N-methylpyridinium iodide (Mukaiyama reagent) and diisopropylethylamine (DIPEA) in dry dichloromethane ( Figure 4) [24]. Efficient amide bond formation was detected in the 1 H NMR spectrum. The NH resonance was shifted 6 ppm downfield.
Coupling of the benzoic acid derivatives 9-15 with the 2-substituted anilino derivatives In order to evaluate the influence of an amino-group compared to electron withdrawing trifluoromethyl or nitro groups in ring position 3 of the benzamide, compounds 46 and 47 were prepared. The nitro group of 28 and 38 was reduced with palladium in an atmosphere of hydrogen at the parr-apparatus yielding compounds 46 and 47 ( Figure 5) [23]. The reduction was detected by the appearance of a singlet for two amino protons in the 1 H NMR spectrum. Amide synthesis of carboxylic acids and anilino derivatives was achieved using a combination of 2-chloro-N-methylpyridinium iodide (Mukaiyama reagent) and diisopropylethylamine (DIPEA) in dry dichloromethane ( Figure 4) [24]. Efficient amide bond formation was detected in the 1 H NMR spectrum. The NH resonance was shifted 6 ppm downfield. Compound In order to evaluate the influence of an amino-group compared to electron withdrawing trifluoromethyl or nitro groups in ring position 3 of the benzamide, compounds 46 and 47 were prepared. The nitro group of 28 and 38 was reduced with palladium in an atmosphere of hydrogen at the parr-apparatus yielding compounds 46 and 47 ( Figure 5) [23]. The reduction was detected by the appearance of a singlet for two amino protons in the 1 H NMR spectrum.

Antiplasmodial Activity and Cytotoxicity
All newly prepared compounds were tested for their in vitro activity against the chloroquine sensitive strain NF54 of P. falciparum. Cytotoxicity was determined using L-6 cells (rat skeletal myofibroblasts). Chloroquine and podophyllotoxin were used as standards. Results obtained are summarized in Table 1.

Antiplasmodial Activity and Cytotoxicity
All newly prepared compounds were tested for their in vitro activity against the chloroquine sensitive strain NF54 of P. falciparum. Cytotoxicity was determined using L-6 cells (rat skeletal myofibroblasts). Chloroquine and podophyllotoxin were used as standards. Results obtained are summarized in Table 1. MMVs Malaria Box compound 1 exhibits sub micromolar antiplasmodial activity (Pf NF54 IC 50 = 0.4134 µM) and promising selectivity (S.I. = 316.9). Apart from the lead compound, its para-substituted analog 2 as well as their N-pivaloyl analogs 3 and 4 served as comparisons for the newly synthesized derivatives.
Replacing the 3-trifluoromethyl group of 1 with other electron-withdrawing groups or hydrogen yielded compounds 26-28 with slightly decreased antiplasmodial activities (Pf NF54 IC 50 = 0.5908-0.6496 µM) and selectivity indices (S.I. = 153.9-245.5). However, their amino analog 46 showed reduced activity (Pf NF54 IC 50 = 1.010 µM) and selectivity (S.I. = 126.0). A stark loss of activities was observed in the groups of para-substituted analogs as well as in both N-pivaloyl groups. In summary, highest antiplasmodial activity and selectivity was observed for compounds with a trifluoromethyl group in ring position 3 and a 4-(fluorophenyl)sulfanyl or a 4-fluorophenoxy substituent in ring position 2 of the benzamide. The anilino moiety should be substituted by a 4-bocpiperazinyl group in ring positions 2 or 4.

Physicochemical and Pharmacokinetic Properties
In addition to in vitro activity and cytotoxicity tests, some key pharmacokinetic parameters were calculated (log P, log D, LE) or determined experimentally (Pe). Results obtained are summarized in Table 2. The log P and log D 7.4 values of compounds range from 4.73 up to 7.68. Ligand efficiency (LE) is an important parameter in early drug development. It is defined as the maximum in vitro binding affinity achievable by ligands, that is 1.5 kcal per mole per heavy atom (HA, non-hydrogen atom). The higher the LE value, the higher the binding affinity [25,26]. The calculated values range from 0.200 up to 0.236 kcal/mol/HA. Compounds 29 and 39 with the most promising antiplasmodial activities also exhibit highest ligand efficiencies of 0.230 and 0.234 kcal/mol/HA, respectively.
The parallel artificial membrane permeability assay (PAMPA) is a fast and easy high-throughput assay to determine passive permeability of compounds through semipermeable membranes (for example the blood-brain-barrier) without the influence of efflux pumps or transporter molecules. Permeability is defined using hydrochlorothiazide (Pe = 0.09 × 10 −6 cm/s) and caffeine (Pe = 8.00 × 10 −6 cm/s) as standards. Compounds with a permeability higher than 1.5 × 10 −6 cm/s are considered to be highly permeable. Permeability through a semi-permeable membrane could be detected for all compounds except 26, 27, 33 and 44 due to insufficient solubility in DMSO and methanol. Compounds 29 and 39 with the highest antiplasmodial activity also exhibit very promising permeability of 4.31 and 3.77 × 10 −6 cm/s, respectively. The unsubstituted 2-phenoxybenzamides 30 (Pe = 8.52 × 10 −6 cm/s) and 40 (Pe = 5.57 × 10 −6 cm/s) as well as the N-pivaloylpiperazinyl derivatives 34 (Pe = 6.92 × 10 −6 cm/s) and 45 (Pe = 5.13 × 10 −6 cm/s) exhibit the by far highest permeabilities.

Instrumentation and Chemicals
IR spectra were acquired using a Bruker Alpha Platinum ATR FTIR spectrometer (Bruker, Ettlingen, Germany) (preparation of KBr discs). HRMS: Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) run by Thermo Q Exactive 2.9 (Thermo Fisher Scientific, Waltham, MA, USA) and Thermo Xcalibur TM Software Version 4.4 (Thermo Fisher Scientific, Waltham, MA, USA). The structures of all new compounds were determined by one-and two-dimensional NMR spectroscopy using a Bruker Avance Neo 400 MHz spectrometer, 5 mm tubes and TMS as internal standard. Shifts in 1 H NMR (400 MHz) and 13 C NMR (100 MHz) are reported in ppm; 1 H-and 13 C-resonances were assigned using 1 H, 1 H-and 1 H, 13 C-correlation spectra and are numbered as given in Figure 4. Signal multiplicities are abbreviated as follows: br, broad; d, doublet; dd, doublet of doublets; ddd, doublet of doublet of doublets; dt, doublet of triplets; m, multiplet; q, quartet; t, triplet; td, triplet of doublet; s, singlet. Melting points were determined using an Electrothermal IA 9200 melting point apparatus (Fisher Scientific, Birmingham, UK).
Materials The corresponding anthranilic acid (6.00 mmol) was dissolved in (DMSO) (11 mL) and the solution was ice-cooled. Upon adding 11 mL of 30% aq sulfuric acid (H 2 SO 4 ), the reaction mixture was stirred at 0 • C for 5 min. After that, the ice bath was removed and sodium nitrate (13.28 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. Subsequently, a solution of KI (10.92 mmol) in 5 mL of demineralized water was added dropwise with a syringe via a septum. The mixture was stirred for another hour before adding a second portion of KI (6.00 mmol) dissolved in 3 mL of aqua dest. After stirring for 1 h at ambient temperature, 50 mL of ethyl acetate was added. The aqueous and organic phases were separated. The organic phase was washed with water and brine, dried over anhydrous sodium sulfate, filtered and the solvent was removed in vacuo. The respective residues were purified by recrystallization from water.

General Procedure for the Synthesis of Compounds 9-15
The corresponding 2-iodobenzoic acid derivative (4.00 mmol) was dissolved in dry DMF (32 mL). The respective phenol, thiophenol or aniline (4.20 mmol), catalytic amounts of copper (0.53 mmol) and copper (I) iodide (0.18 mmol), 1,8-diazabicyclo [5.4.0]undec-7-ene (12.00 mmol) and dry pyridine (0.80 mmol) were added. The reaction mixture was refluxed in an oil bath at 160 • C for 2-24 h. Then, it was cooled to ambient temperature and acidified with 2 N HCl to a pH of 1. Ice and dichloromethane were added. Phases were separated. The aqueous phase was extracted twice with dichloromethane. The organic phases were combined, washed with water and brine, dried over anhydrous sodium sulfate and filtered. The solvent was evaporated in vacuo. The crude products were subsequently purified by column chromatography.

General Procedure for the Synthesis of Compounds 20 and 21
N-Boc-piperazine (14.00 mmol) and potassium carbonate (14 mmol) were suspended in dry DMSO (40 mL). The corresponding fluoronitrobenzene (7.00 mmol) was added and the suspension was refluxed at 80 • C for 72 h. The reaction mixture was cooled to ambient temperature and acidified with 2 N HCl to a pH of 1. Phases were separated. The aqueous phase was extracted with diethyl ether. The organic phases were combined, washed with ice water and brine, dried over anhydrous sodium sulfate and filtered. The solvent was evaporated in vacuo yielding the pure products.

General Procedure for the Synthesis of Compounds 22 and 23
The corresponding N-Boc-piperazinyl derivative (1.00 mmol) was dissolved in dry dichloromethane (10 mL) and cooled to 0 • C in an ice bath. A solution of trifluoroacetic acid (30 mmol) in dry dichloromethane (3 mL) was added via a dropping funnel. After that, the ice bath was removed and the reaction mixture was stirred at room temperature for 24 h. Subsequently, the solvent and access trifluoroacetic acid were evaporated in vacuo. The residue was suspended in a solution of potassium carbonate (20 mmol) in water (6 mL). The aqueous suspension was extracted five times with dichloromethane/propan-2-ol 3:1. The combined organic phases were dried over anhydrous sodium sulfate and filtered. The solvent was evaporated in vacuo yielding the desired compounds as pure products.

General Procedure for the Synthesis of Compounds 24 and 25
The corresponding piperazinyl derivative (2.00 mmol) was dissolved in dry dichloromethane (8 mL) and cooled to 0 • C in an ice bath. Dry triethylamine (3.00 mmol) was added dropwise. Pivaloyl chloride (2.10 mmol) was added with a syringe via a septum. The ice bath was removed and the reaction mixture was stirred at ambient temperature for 24 h. After that, water (30 mL) was added. The aqueous and organic phases were separated. The organic phase was washed with 2 N HCl, 8% aq NaHCO 3 and brine, dried over anhydrous sodium sulfate and filtered. The solvent was evaporated in vacuo yielding the pivaloylpiperazine derivatives which were used without further purification.

General Procedure for the Synthesis of Compounds 16-19, 46 and 47
To a solution of 15% (m/m) palladium on activated carbon in dry methanol (100 mL) the corresponding nitro compound (2.00 mmol) was added. Reduction of the nitro group was performed in an atmosphere of hydrogen (50 psi) at the parr apparatus at ambient temperature for 24 h. The reaction mixture was filtered and the solvent was removed in vacuo yielding the desired anilino-derivatives that were either purified by column chromatography or used without further purification.

General Procedure for the Synthesis of Compounds 26-45
The corresponding benzoic acid derivative (1.00 mmol) and the anilino derivative (1.00 mmol) were dissolved in dry dichloromethane (30 mL) and cooled in an ice bath to 0 • C. The Mukaiyama reagent (1.75 mmol) and DIPEA (5.00 mmol) were added. The reaction mixture was stirred at room temperature for 24-48 h. After that, 20% aq ammonium chloride (50 mL) was added. Phases were separated. The aqueous phase was extracted twice with EtAc. The organic phases were combined, washed with 8% aq NaHCO 3 and brine, dried over anhydrous sodium sulfate and filtered. The solvent was evaporated in vacuo yielding the raw products that were subsequently purified. tert

In Vitro Cytotoxicity with L-6 Cells
The cytotoxicity assays were performed using 96-well microtiter plates, each well containing 4000 L-6 cells (a primary cell line derived from rat skeletal myofibroblasts, ATCC CRL-1458 TM ) in 0.1 mL of RPMI 1640 medium supplemented with 1% glutamine (200 mM) and 10% fetal bovine serum [39,40]. Serial drug dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002 µg/mL were prepared. After 70 h of incubation, the plates were inspected under an inverted microscope to assure growth of the controls and sterile conditions. Then, 0.01 mL resazurin solution (resazurin, 12.5 mg in 100 mL double-distilled water) was added to each well and the plates were incubated for another 2 h. The plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and an emission wavelength of 588 nm. IC 50 values were calculated by linear regression from the sigmoidal dose inhibition curves using SoftmaxPro software (Molecular Devices Cooperation, Sunnyvale, CA, USA) [38]. Podophyllotoxin (Sigma P4405) was used as control.

Parallel Artificial Membrane Permeability Assay
With the high-throughput PAMPA the newly synthesized compounds were tested for their passive permeability through cell membranes without the influence of efflux pumps or transporter proteins. The assay was performed using a Corning ® Gentest TM Precoated PAMPA Plate System with 96-well polystyrene plates. The bottom of the acceptor plate consists of a porous membrane, whereby the pores are lined with a lipid-oil-lipid triple layer. Stock solutions of each test compound at 10 mM were prepared in DMSO or methanol and diluted with phosphate-buffered saline (PBS at a pH of 7.4) to a final concentration of 200 µM. Hydrochlorothiazide (Pe = 0.9 nm/s) and caffeine (Pe = 80 nm/s) were used as standards. The donor plate (bottom plate) was filled with the compound solutions, whereby all compounds were tested in quadruplicates. Each well of the acceptor plate (top plate) was filled with PBS buffer. Donor and acceptor plates were combined and incubated at room temperature for 5 h. After that, the plates were separated and 150 µL of each well of both plates were transferred to 96-well UV plates (Greiner Bio-One). Absorption at different wavelengths covering a range from 200 to 300 nm was measured using a SpectraMax M3 UV plate reader. By measuring serial dilutions of five dilution steps covering a range from 200 to 12.5 µM, a calibration curve was prepared. The plates were analyzed at the wavelength where the R 2 value of the calibration curve was higher than 0.99 [41]. Effective permeability Pe of each test compound was calculated using the following Equations (1)- (3): where: Pe-effective permeability; S-filter area (0.3 cm 2 ); V D -donor well volume (0.3 mL); V A -acceptor well volume (0.2 mL); t-incubation time (18,000 s); c A (t)-acceptor well compound concentration at time t; c equ -equilibrium concentration. where: V D -donor well volume (0.3 mL); V A -acceptor well volume (0.2 mL); c A (t)-acceptor well compound concentration at time t; c D (t)-donor well compound concentration at time t.
Recovery of compounds from donor and acceptor wells (mass retention) was calculated as shown in the equation below. Data were only accepted when recovery exceeded 70%. where: R-mass retention (%); V D -donor well volume (0.3 mL); V A -acceptor well volume (0.2 mL); c A (t)-acceptor well compound concentration at time t; c D (t)-donor well compound concentration at time t; c 0 -initial donor well compound concentration (200 µM).

Ligand Efficiency (LE)
Ligand efficiency was calculated as shown in the following Equation (4): where:

LE-ligand efficiency;
HA-number of heavy atoms; pIC 50 -negative logarithm of IC 50 .

Conclusions
This paper deals with the synthesis of derivates of MMV's Malaria Box compound 1, which exhibits multi-stage activity against different strains of P. falciparum and lack of resistance development. It is a 2-(4-fluorophenoxy)-3-(trifluoromethyl)benzanilide with a Nbocpiperazinyl group in ortho position of the anilide nitrogen. The first series focused on the derivatization of the anilino moiety showing the positive influence of a N-bocpiperazinyl group or a 4-pivaloylpiperazinyl group in ortho or para position, which became partial structure of all of the new compounds. The 3-(trifluoromethyl) group was replaced by hydrogen, fluoro, amino or nitrogen substituents, but turned out to be the preferable substitution. The 2-(4-fluorophenoxy) moiety was replaced by an anilino, a 4-fluoroanilino or a (4-fluorophenyl)sulfanyl substituent. The latter was partial structure of 29 which exhibits a N-bocpiperazinyl group in 2 -position of the benzanilide. Compared to 1 it showed improved activity, selectivity and passive permeability ( Figure 6). This paper deals with the synthesis of derivates of MMV's Malaria Box compound 1, which exhibits multi-stage activity against different strains of P. falciparum and lack of resistance development. It is a 2-(4-fluorophenoxy)-3-(trifluoromethyl)benzanilide with a N-bocpiperazinyl group in ortho position of the anilide nitrogen. The first series focused on the derivatization of the anilino moiety showing the positive influence of a N-bocpiperazinyl group or a 4-pivaloylpiperazinyl group in ortho or para position, which became partial structure of all of the new compounds. The 3-(trifluoromethyl) group was replaced by hydrogen, fluoro, amino or nitrogen substituents, but turned out to be the preferable substitution. The 2-(4-fluorophenoxy) moiety was replaced by an anilino, a 4-fluoroanilino or a (4-fluorophenyl)sulfanyl substituent. The latter was partial structure of 29 which exhibits a N-bocpiperazinyl group in 2′-position of the benzanilide. Compared to 1 it showed improved activity, selectivity and passive permeability ( Figure 6).