Synthesis and Structure-Activity Relationships of New 2-Phenoxybenzamides with Antiplasmodial Activity

The 2-phenoxybenzamide 1 from the Medicines for Malaria Venture Malaria Box Project has shown promising multi-stage activity against different strains of P. falciparum. It was successfully synthesized via a retrosynthetic approach. Subsequently, twenty-one new derivatives were prepared and tested for their in vitro activity against blood stages of the NF54 strain of P. falciparum. Several insights into structure-activity relationships were revealed. The antiplasmodial activity and cytotoxicity of compounds strongly depended on the substitution pattern of the anilino partial structure as well as on the size of substituents. The diaryl ether partial structure had further impacts on the activity. Additionally, several physicochemical and pharmacokinetic parameters were calculated (log P, log D7.4 and ligand efficiency) or determined experimentally (passive permeability and CYP3A4 inhibition). The tert-butyl-4-{4-[2-(4-fluorophenoxy)-3-(trifluoromethyl)benzamido]phenyl}piperazine-1-carboxylate possesses high antiplasmodial activity against P. falciparum NF54 (PfNF54 IC50 = 0.2690 µM) and very low cytotoxicity (L-6 cells IC50 = 124.0 µM) resulting in an excellent selectivity index of 460. Compared to the lead structure 1 the antiplasmodial activity was improved as well as the physicochemical and some pharmacokinetic parameters.


Introduction
Over a half of the world's population is at risk of an infection with malaria, especially children and pregnant women in developing countries like Africa. In 2019 more than 229 million cases and over 400,000 deaths were reported [1]. Malaria is caused by singlecelled, eukaryotic protozoans of the species Plasmodium. Five of them are human pathogenic with Plasmodium falciparum causing the most deadly and dangerous Malaria tropica [2]. The current gold standard for malaria treatment are artemisinin-based combination therapies (ACTs). They are combinations of short-acting artemisinins with drugs with longer half-life and different mode of action. Progressive resistance development to ACTs in the Southeast Asian region as well as first reports of artemisinin resistances from Africa in 2020, however, present a tremendous threat to previous accomplishments in the fight against malaria [3][4][5][6]. The last chance to at least temporarily prevent resistance development are triple artemisininbased combination therapies, so called TACTs [7,8]. Vaccine development for malaria is also rather challenging due to the complex life cycle and multiple possible targets. The most advanced candidate in vaccines for P. falciparum malaria, RTS,S/AS01, neither provides long term protection nor significant protection against severe malaria [9][10][11]. Therefore, for now, orally administered drugs remain the most important field of research to successfully fight the malaria parasite.
In 2016 the foundation Medicines for Malaria Venture (MMV) has published results of a huge screening project, the so called "Malaria Box" [12]. It consists of 400 compounds with different activity against various strains of P. falciparum and serves as starting point for further research. Based on the Malaria Box project, a complex study on resistance development and cross resistances on 50 diverse chemically structured compounds discovered by phenotypic screening was performed [13][14][15]. By comparing the data set of this study, compound 1 was picked as promising lead structure. This 2-phenoxybenzamide is one of few structures exhibiting multi-stage activity against sexual, asexual and liver-stages of P. falciparum. Furthermore, in long-term in vitro studies with sub-lethal doses of compound 1 parasites did neither acquire resistances, nor cross-resistances, which is of great significance.
Within a subsequent study, targets of the 2-phenoxybenzamide were identified [16,17]. Synchronized P. falciparum 3D7-A10 parasites with an erythrocytic cycle of 40 h were exposed to different drug concentrations to determine stage specific activity. Compound 1 showed peak activity in sub-micromolar concentrations on late stage trophozoites. Furthermore, dihydroorotate and N-carbamoyl-L-aspartate, characteristic metabolic products that indicate disturbance of the mitochondrial electron transport chain, were detected. Consequently, the dihydroorotate-dehydrogenase as well as the cytochrome bc 1 complex are potential targets of 1. Bloated digestive vacuoles indicate an additional influence on the hemoglobin catabolism [18,19].
The aim of this study was to synthesize new derivatives of the 2-phenoxybenzamide 1 to gain first insights in structure-activity relationships (SAR) and increase antiplasmodial activity. All newly synthesized compounds were tested for their activity against the NF54 strain of P. falciparum. To reveal pharmacokinetic parameters essential for orally administered drugs, compounds were analyzed for passive permeability and CYP3A4 inhibition.

Chemistry
The lead structure 1 was prepared in a multi-step synthesis starting from 3-(trifluoromethyl)anthranilic acid. By this time, no synthetic route to obtain compound 1 has been published. Therefore, a retrosynthetic approach was elaborated to prepare the 2-phenoxy scaffold 2 as well as the 2-substituted derivative of aniline 3. These partial structures were subsequently coupled to obtain the desired carboxamide 1 (Figure 1).
Treatment of 3-(trifluoromethyl)anthranilic acid with sodium nitrite under acidic conditions yielded the diazonium salt. In the course of a Sandmeyer-like reaction with potassium iodide, the diazonium group was substituted with iodine giving the 2-iodo-3-(trifluoromethyl)benzoic acid 4 in high yields [20]. The iodobenzoic acid 4 was afterwards converted into the diaryl ether 2 by means of a copper-catalyzed Ullmann-like ether synthesis [21]. Thereby, it was coupled with 4-fluorophenol to obtain the 2-phenoxy scaffold 2. The nucleophilic aromatic substitution of 1-fluoro-2-nitrobenzene with N-Bocpiperazine and potassium carbonate in dimethyl sulfoxide (DMSO) gave the 1-Boc-4-(2nitrophenyl)piperazine 5 in high yields [22]. To obtain the 2-substituted derivative of aniline 3, the nitro group of compound 5 was subsequently reduced with palladium in an atmosphere of hydrogen at a Parr-apparatus [23]. The desired 2-phenoxybenzamide 1 was synthesized by coupling the carboxylic acid 2 with the anilino derivative 3. Various combinations of DCC, Oxyma Pure, Potassium Oxyma B, EDC x HCl, COMU, CDI and Mukaiyama reagent were used for amide formation. The highest yield was obtained with a combination of 2-chloro-N-methylpyridinium iodide (Mukaiyama reagent) and diisopropylethylamine (DIPEA) [24]. This reaction pathway was used for the majority of new compounds in this paper. The successful amide bond formation was detected by significant changes in the NMR spectra. In the 1 H-NMR spectrum the signal of the aromatic amino protons disappeared and a new signal of the amide hydrogen appeared at much higher frequencies. In the 2D HMBC spectrum we observed a cross-peak from this hydrogen atom to the carbonyl group. To obtain first insights into structure-activity relationships, several series of derivatives were prepared. At first, the 2-(4-fluorophenoxy) substituent was replaced by different functional groups to investigate their influence on the antiplasmodial activity. Compounds 6, 7 and 8 were synthesized as shown in Figure 2.
The carboxylic acids 9, 10 and 11 were prepared from 2-iodo-3-(trifluoromethyl)benzoic acid 4.The latter was treated with the corresponding phenoles to obtain the diaryl ethers 9 and 11 as well as the 3-(trifluoromethyl)benzoic acid 10. Subsequent treatment of the carboxylic acids with the anilino derivative 3, the Mukaiyama reagent and DIPEA in dichloromethane (CH 2 Cl 2 ) yielded the amides 6, 7 and 8.
In order to determine the importance of the N-Boc-piperazinyl group for the antiplasmodial activity, the 2-phenoxybenzoic acid 2 was coupled with different primary aromatic amines giving compounds 12, 13 and 14. The substituted aniline 15 was prepared from 2-nitroaniline. The latter was treated with di-tert-butyldicarbonate giving the tert-butyl-N-(2-nitrophenyl)carbamate 16 [25]. Selective reduction of the nitro group yielded the desired anilino derivative 15. Reaction of the carboxylic acid 2 with aniline or compound 15 gave benzanilides 12 and 13, respectively. Carbamate groups are usually cleaved with trifluoroacetic acid in dichloromethane [26]. Such treatment of compound 13 afforded the N-(2-aminophenyl)benzamide 14 ( Figure 3). In another series, the influence of the tert-butyloxycarbonyl substituent of the 2piperazinylphenyl moiety of compound 1 on the antiplasmodial activity was investigated. Its replacement by diverse substituents yielded compounds 17-22. The corresponding primary aromatic amines 23-28 for the synthesis of the benzamides 17-22 were prepared in multi-stage syntheses from 1-fluoro-2-nitrobenzene ( Figure 4).  The N-Boc-group of carbamate 5 was eliminated using trifluoroacetic acid in CH 2 Cl 2 yielding the 4-unsubstituted piperazinyl derivative 29. Afterwards, the terminal piperazinyl hydrogen was substituted with different functional groups. Compound 29 was treated with acetyl chloride and pivaloyl chloride, respectively, to obtain the acylated derivatives 30 and 31 [27]. In the course of a Reimer-Tiemann reaction the formylpiperazinyl analogue 32 was prepared from 29 with sodium ethanolate and chloroform [28]. Reaction of 29 with potassium cyanate in a mildly acidic environment yielded the carboxamide 33. Its N,N-dimethyl analogue 34 was prepared by reaction of 29 with 1,1carbonyldiimidazole and dimethylamine hydrochloride [29,30]. The nitro group of compounds 30-34 was reduced with palladium in an atmosphere of hydrogen to obtain the desired 2-substituted anilino derivatives 23-27. Their tert-butyl-piperazinyl analogue 28 was prepared by reduction of the nitro group of compound 35, which was obtained from the reaction of 1-fluoro-2-nitrobenzene with tert-butyl-piperazine. The N-[2-(piperazin-1yl)phenyl derivatives 23-28 were subsequently coupled with the carboxylic acid 2 to yield the amides 17-22 (Figure 4).
In order to evaluate the influence of the ortho position of the piperazinyl substituent of 1, we prepared its 3-substituted and 4-substituted analogues 36 and 37. Furthermore, the N-Boc group was replaced by N-pivaloyl groups yielding compounds 38 and 39.
Their syntheses started from the corresponding fluoronitrobenzenes, which reacted in alkaline medium with N-Boc-piperazine giving compounds 40 and 41. Their N-pivaloyl analogues 42 and 43 were obtained in 2 steps from 40 and 41. At first the N-Boc group was cleaved with trifluoroacetic acid affording N-unsubstituted derivatives 44 and 45. Subsequent reaction with pivaloyl chloride and triethylamine in CH 2 Cl 2 gave compounds 42 and 43 [27]. The nitro groups of compounds 40-43 were reduced with palladium in an atmosphere of hydrogen to obtain their anilino derivatives 46-49. Finally, they were coupled with the carboxylic acid 2 yielding benzamides 36-39 ( Figure 5). The positive influence of the piperazinyl substituent per se was examined via its replacement by a primary amino group. Compounds 50 and 51 were prepared by amide synthesis of the carboxylic acid 2 with the corresponding aniline giving the N-(nitrophenyl) benzamides 52 and 53 which were afterwards reduced with palladium in an atmosphere of hydrogen at the Parr-apparatus yielding the amides 50 and 51 ( Figure 6).
In the next series of compounds the piperazinyl ring in position 2 was retained, but its N-Boc group was replaced by diverse substituents. The N-formyl and the N-carbamoyl  27), indicating that the 4-fluorophenoxy substituent has generally an advantageous effect. The para substituted N-Boc and N-pivaloylpiperazinyl derivatives are more active than their ortho substituted analogues. As already demonstrated for ortho substituted derivatives a remarkable decrease of activity was observed when the piperazinyl moieties was replaced by amino groups. The 3-amino and 4-amino analogues 50 and 51 were the least active of all tested compounds (Pf NF54 IC 50 = 51.49-51.85 µM).

Physicochemical and Pharmacokinetic Properties
In addition to antiplasmodial activity and cytotoxicity of compounds 1, 6-8, 12-14, 17-22, 36-39, 50, 51 and 54-56, some physicochemical parameters like log P and log D 7 . 4 were calculated. Furthermore, ligand efficiency (LE) was determined (Table 2) [31]. The log P and log D 7 . 4 values of compounds range between 4.43-6.60. Among the compounds with considerable antiplasmodial activity the N-[4-(4-pivaloylpiperazinyl)phenyl] benzamide 39 (log P = 5.56) exhibits the lowest log P and log D 7 . 4 values, which is compared to compound 1 (log P = 6.44) a remarkable improvement. Ligand efficiency is an important parameter in early drug development. It becomes more apparent, that large molecules often have disadvantageous molecular properties when it comes to oral bioavailability. Ligand efficiency is defined by the free binding energy for a compound divided by its number of heavy atoms (HA). The calculated values ranged from 0.183-0.255 kcal/mol/HA. From the group of more active compounds it was again 39 (LE = 0.236 kcal/mol/HA) that showed the highest value, which is a minor enhancement compared to 1 (LE = 0.214 kcal/mol/HA).
In addition, ADME assays to determine pharmacokinetic parameters were performed (Table 3). Passive permeability and inhibition of Cytochrom P450 3A4 were determined. Permeability of compounds through a semipermeable membrane was detectable for all compounds except 22 due to insufficient solubility in the used solvents. The most active compounds 19, 37, 39 and 56 showed quite low permeability (P e = 0.09-0.24 × 10 −6 cm/s). In the group of compounds with quite good activity the 2-phenyl and the 2-(4-acetamidophenyl) derivative 6 and 8 (P e = 4.06-3.00 × 10 −6 cm/s) possessed improved passive permeability compared to their 2-(4-fluorophenyl) analogue 1 (P e = 2.37 × 10 −6 cm/s). In general, compounds with permeabilities higher than 1.5 × 10 −6 cm/s are considered to be highly permeable.
Inhibition of the phase I liver enzyme Cytochrom P450 3A4 that plays a crucial role in drug metabolism was determined for compounds with the highest antiplasmodial activities. CYP3A4 inhibition of compounds could result in increased bioavailability of simultaneously applied drugs. The lead structure 1 exhibits high enzyme interaction (87%) that is however surpassed by most tested compounds. Only compounds 18 (82%), 54 (80%) and 55 (60%) show less inhibition of CYP3A4.

Instrumentation and Chemicals
Melting points were obtained on an Electrothermal IA 9200 melting point apparatus. IR-spectra were acquired by a Bruker Alpha Platinum ATR FTIR spectrometer (KBr discs), the frequencies are reported in cm −1 . The structures of all newly synthesized compounds were determined by one-and two-dimensional NMR spectroscopy. NMR spectra: Varian UnityInova 400 MHz and Bruker Avance Neo 400 MHz, 5 mm tubes, TMS as internal standard. Shifts in 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra 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 1 (Figures S1-S40).

Syntheses
2-Iodo-3-(trifluoromethyl)benzoic acid (4): 3-(Trifluoromethyl)anthranilic acid (2.11 g (10.33 mmol)) was dissolved in dimethylsulfoxide (17 mL) and the solution was ice-cooled. Sulfuric acid 30 percent (17 mL) was added and the reaction mixture was stirred at 0 • C for 5 min. After that, NaNO 2 (1.54 g (22.35 mmol)) was added, the ice bath was removed and the reaction mixture was stirred at room temperature for 2 h. KI (3.02 g (18.21 mmol)) was dissolved in water (10 mL) and added dropwise with a syringe through a septum. The reaction mixture was stirred at ambient temperature for 1 h. After that, the second portion of KI (1.71 g (10.33 mmol)) dissolved in water (7 mL) was added and the reaction mixture was stirred for another hour at room temperature. Then, ethyl acetate (50 mL) were added. The aqueous and organic phases were separated. The organic phase was washed with water and brine, dried over anhydrous sodium sulfate and filtered. The solvent was evaporated in vacuo. The residue was recrystallized from water, giving compound 4 as brownish solid (2.97 g (91%)). m.P. 134 • C. NMR data were in accordance with literature data [32].
3.2.1. General Procedure for the Synthesis of Compounds 2, 9, 10 and 11 The corresponding iodobenzoic acid derivative (4.00 mmol) was dissolved in dry dimethylformamide. Phenol (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 at 160 • C for 2-48 h. Then, the mixture was acidified with 2N HCl to a pH of 1. Ice and dichloromethane were added. The aqueous and organic phases were separated. The aqueous phase was extracted three times with dichloromethane. The combined organic phases were washed with water and brine, dried over anhydrous sodium sulfate and filtered. The solvent was evaporated in vacuo yielding the raw diaryl ether, which was purified by column chromatography.

General Procedure for the Synthesis of Compounds 14, 29, 44 and 45
To an ice-cooled solution of the corresponding N-Boc derivative (1.00 mmol) in dry CH 2 Cl 2 (10 mL) a solution of trifluoroacetic acid (30 mmol) in dry CH 2 Cl 2 (3 mL) was added dropwise via a dropping funnel. The ice-bath was removed and the reaction mixture was stirred at room temperature for 24 h. After that, the solvent and excess trifluoroacetic acid were evaporated in vacuo. The residue was suspended in a solution of potassium carbonate (6.00 mmol) in water (12 mL). The aqueous phase was extracted five times with CH 2 Cl 2 /isopropyl alcohol (3:1). The organic phases were combined, dried over anhydrous sodium sulfate and filtered. The solvent was evaporated in vacuo yielding the amino or piperazine derivative, which was either purified by column chromatography or used without further purification. 1-(2-Nitrophenyl)piperazine (29): Reaction of compound 5 (2.24 g (7.30 mmol)) with trifluoroacetic acid (5.00 g (43.80 mmol)) in dichloromethane (95 mL) gave the protonated form of 29. Work-up with a solution of potassium carbonate (6.06 g (43.80 mmol)) in water (88 mL) yielded compound 29 as orange oil (1.36 g (90%)), which was used without further purification. NMR data were in accordance with literature data [37].
4-(2-Nitrophenyl)piperazine-1-carbaldehyde (32): Sodium (498 mg (21.70 mmol)) was added in portions to dry ethanol (12 mL). After that, a solution of compound 29 (746 mg (3.60 mmol)) in dry ethanol (5 mL) was added. The reaction mixture was stirred at 50 • C for 15 min. Dry chloroform (1.55 g (13.02 mmol)) was added dropwise with a syringe through a septum. The reaction mixture was stirred for another 45 min at 50 • C. Then, the mixture was quenched with water (30 mL). The aqueous and organic phases were separated, and the aqueous phase was extracted with CH 2 Cl 2 . The combined organic phases were washed with 1N HCl, dried over anhydrous sodium sulfate and filtered. The solvent was evaporated in vacuo yielding compound 32 as brown oil (661 mg (78%)), which was used without further purification. NMR data are in accordance with literature data [39].
4-(2-Nitrophenyl)piperazine-1-carboxamide (33): To a solution of 29 (559 mg (2.73 mmol)) in 1N HCl (3.5 mL), 2N KOH was added dropwise up to a pH of 3. After that, potassium cyanate (292 mg (3.60 mmol)) was added, and the reaction mixture was stirred at room temperature for 2 h. The precipitate was filtered and washed with water. It was dissolved in CH 2 Cl 2 . The organic phase was washed with 2N NaOH, dried over anhydrous sodium sulfate and filtered. The solvent was evaporated in vacuo yielding compound 33 as orange solid (396 mg (58%)), which was used without further purification.  13 g (10.00 mmol)) was added. The reaction mixture was stirred at room temperature for 30 min. After that, dimethylamine hydrochloride (726 mg (8.00 mmol)) was added. The reaction mixture was stirred at 80 • C for 6 h. The solvent was evaporated in vacuo and the residue was mixed with water (5 mL). The aqueous phase was extracted three times with ethyl acetate. The combined organic phases were washed with 8% aq NaHCO 3 and brine, dried over anhydrous sodium sulfate and filtered. The solvent was evaporated in vacuo giving the raw product. Purification by column chromatography (silica gel, EtAc) yielded compound 34 as yellow solid (395 mg (71%)). IR = 3441, 2843, 1645, 1605, 1520, 1488, 1451, 1385, 1349, 1287, 1232, 1210, 1173, 1044 To an ice-cooled solution of the corresponding piperazine (2.00 mmol) in dry CH 2 Cl 2 (8 mL), dry triethylamine (3.00 mmol) was added. After that, pivaloyl chloride (2.10 mmol) was added dropwise with a syringe through a septum. The ice-bath was removed and the reaction mixture was stirred at room temperature for 24 h. Then, the reaction was quenched with water (30 mL). The aqueous and organic phases were separated, and the organic phase was washed with 2N NaOH, 8% aq NaHCO 3 and brine. It was dried over anhydrous sodium sulfate, filtered and the residue was evaporated in vacuo yielding the pivaloyl-piperazine derivative, which was either purified by column chromatography or used without further purification. 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. The reduction of the nitro group was performed in an atmosphere of 50 psi hydrogen at the Parr-apparatus at room temperature for 24 h. After that, the reaction mixture was filtered and the solvent was evaporated in vacuo yielding the corresponding amino compound, which was either purified by column chromatography or used without further purification.

In Vitro Cytotoxicity with L-6 Cells
In vitro cytotoxicity was determined using a primary cell line of rat skeletal myofibroblasts. The assay was performed in 96-well microtiter plates, each well containing 0.1 mL of RPMI 1640 medium, supplemented with 0.1% L-glutamine (200 mM) and 10% fetal bovine serum, as well as 4000 L-6 cells [46,47]. Serial drug dilutions of 11 3-fold dilution steps (covering a range from 100-0.002 µg/mL) were prepared. After 70 h of incubation time, the plates were inspected under an inverted microscope to ensure sterile conditions and growth of the controls. Then, 0.01 mL of Alamar Blue was added to each well and the plates were incubated for another 2 h. After that, the plates were read with a SpectraMax Gemini XS microplate fluorometer (Molecular Devices Corporation, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and an emission wavelength of 588 nm. IC 50 values were determined by linear regression from the sigmoidal dose inhibition curves using SoftmaxPro software (Molecular Devices Corporation, Sunnyvale, CA, USA) [45]. Podophyllotoxin (Sigma P4405) was used as control.

Parallel Artificial Membrane Permeability Assay
The PAMPA was performed using a Corning Gentest Pre-coated PAMPA Plate System at a pH of 7.4. It consists of 96-well polystyrene plates, whereas the donor-plate (bottom plate) is a conventional 96-well microtiter plate. The base of the acceptor-plate (top plate) consists of a porous membrane. The pores are lined with a lipid-oil-lipid trilayer. Stock solutions (10 mM) of each test compound were prepared in DMSO or MeOH and then further diluted to a final concentration of 200 µM with phosphate-buffered saline (PBS) at a pH of 7.4. Compound solutions were then added to the wells of the donor plate and pure PBS was added to each well of the acceptor plate. Compounds and negative control (pure PBS) were tested in quadruplicates. Donor and acceptor plate were coupled and incubated at ambient temperature for 5 h. After that, the plates were separated and solutions from each well of both plates were transferred onto 96-well UV-Star Microplates (Greiner Bio-One). The UV-absorption was measured at different wavelengths (between 200 and 300 nm) by a SpectraMax M3 UV plate reader (Molecular Devices). The concentrations were received from a calibration curve for each substance. The plates were analyzed at a wavelength were the R 2 value of the calibration curve was higher than 0.99 [48]. Hydrochlorothiazide (P e = 0.90) and caffeine (P e = 80.00) were used as standards. The effective permeability (P e ) was calculated as shown in the following Equations (1)- (3): where: P e -effective permeability; S-filter area (0.3 cm 2 ); V D -donor well volume; V A -acceptor well volume; t-incubation time (18,000 s); c A (t)-compound concentration in acceptor well at time t; c equ -equilibrium concentration. where: c D (t)-compound concentration in donor well 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 (%); c A (t)-compound concentration in acceptor well at time t; c D (t)-compound concentration in donor well at time t; c 0 -initial compound concentration in donor well; V D -donor well volume; V A -acceptor well volume.

Cytochrom P450 3A4 Inhibition Assay
The CYP3A4 inhibition assay was performed using 96-well White Plates (Greiner Bio-One) at a pH of 7.4. Stock solutions (4 mM) of test compounds were prepared in DMSO, stock solution of the standard ketoconazole (5 mM) was prepared in acetonitrile. Stock solutions were further diluted to a final concentration of 20 µM using water (HPLC grade). The luciferin IPA stock solution (3 mM) was diluted to a final concentration of 0.3 mM using water (HPLC grade). The CYP3A4 reaction mixture was prepared by mixing water (HPLC grade) with potassium phosphate buffer (1 M), luciferin IPA (0.3 mM) and CYP3A4 membrane (1 pmol/µL). The control reaction mixture was prepared using water (HPLC grade), potassium phosphate buffer (1 M), luciferin IPA (0.3 mM) and membrane without CYP activity (1 pmol/µL). Solutions A and B of the NADPH regeneration system were mixed and HPLC grade water was added. The reconstituted luciferin detection reagent was prepared by mixing the reconstituted buffer with esterase with the luciferin detection reagent. Then, solutions of test compounds and standard were added to the wells of the 96-well White Plate, each was tested in triplicate. The CYP3A4 reaction mixture was added to each well and the plate was incubated for 10 min at room temperature. After that, the NADPH regeneration system was added, inducing the reaction followed by an incubation time of 10 min at ambient temperature. By adding the reconstituted luciferin detection reagent, the reaction was terminated, and a luminescent signal was formed. Luminescence was measured by a SpectraMax M3 UV plate reader (Molecular Devices). The relative light units (RLU) were received from a calibration curve with beetle luciferin. Ketoconazole (100% enzyme inhibition) was used as standard [49]. The CYP3A4 inhibition (%) was calculated from the RLU.

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, antiplasmodial activities and first insights into structure-activity relationships of a series of new 2-phenoxybenzamides. The lead compound MMV030666 from MMV's Malaria Box Project is a 2-(4-fluorophenoxy)benzanilide with a 4-(N-Boc)piperazinyl substituent in ortho position of the anilide nitrogen. It is of particular interest as it exhibits multi-stage activity against different strains of P. falciparum.
Moreover, development of resistant parasites could not be observed in long-term in vitro studies with sub-lethal doses. We aimed on synthesizing derivatives to gain first insights into structure-activity relationships. Our main focus was put on derivatization of the anilino moiety.
Modifications in Figure 8 showed the great importance of the phenoxy substituent as well as the beneficial effect of its 4-fluoro substitution. Replacement of the piperazinyl moiety by a hydrogen atom or an amino group led to inactive compounds. Substitution of the N-Boc group usually caused a considerable decrease in activity, but compounds with a Npivaloyl group showed comparable activity. Their enhanced stability in acidic environment could be of advantage. Moreover, the ring position of the piperazinyl substituent was of particular importance. Meta substituted derivatives were only moderately active. The most active compounds had a 4-(N-Boc)piperazinyl or a 4-(N-pivaloyl)piperazinyl substituent in position 2 or 4 of the anilide nitrogen (Table 4).
Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available in this article.