Novel Baicalein-Derived Inhibitors of Plasmodium falciparum

Malaria, a life-threatening mosquito-borne disease caused by Plasmodium parasites, continues to pose a significant global health burden. Despite notable progress in combating the disease in recent years, malaria remains prevalent in many regions, particularly in Southeast Asia and most of sub-Saharan Africa, where it claims hundreds of thousands of lives annually. Flavonoids, such as the baicalein class of compounds, are known to have antimalarial properties. In this study, we rationally designed and synthesized a series of baicalein derivatives and identified a lead compound, FNDR-10132, that displayed potent in vitro antimalarial activity against Plasmodium falciparum (P. falciparum), both chloroquine-sensitive (60 nM) and chloroquine-resistant (177 nM) parasites. FNDR-10132 was evaluated for its antimalarial activity in vivo against the chloroquine-resistant strain Plasmodium yoelii N67 in Swiss mice. The oral administration of 100 mg/kg of FNDR-10132 showed 44% parasite suppression on day 4, with a mean survival time of 13.5 ± 2.3 days vs. 8.4 ± 2.3 days of control. Also, FNDR-10132 displayed equivalent activity against the resistant strains of P. falciparum in the 200–300 nM range. This study offers a novel series of antimalarial compounds that could be developed into potent drugs against chloroquine-resistant malarial parasites through further chemistry and DMPK optimization.


Introduction
Baicalein derivatives have emerged as promising candidates for combating malaria [1], a widespread and severe infectious disease caused by Plasmodium parasites [2].In 2021, approximately 247 million malaria cases were reported worldwide, with the majority concentrated in Africa.The COVID-19 pandemic caused disruptions in access to treatment between 2019 and 2021, resulting in an estimated 13.4 million additional cases [3].Nigeria, the Democratic Republic of the Congo, Uganda, and Mozambique accounted for nearly half of all global malaria cases, and the African region alone accounted for approximately 95% of cases, with an estimated 234 million cases in 2021.
However, the malleability exhibited by mosquitoes and the Plasmodium parasite has resulted in a progressive augmentation of resistance to pharmaceutical drugs and insecticides.Notably, cases of resistance to artemisinin-based combination therapies (ACTs) have been reported in specific countries.However, the global spread of these resistant strains highlights a significant risk with severe consequences and a catastrophic impact.In Africa, resistance to multiple insecticides has been detected in approximately twothirds of endemic regions where malaria is prevalent.Moreover, about 80% of infections exhibit no symptoms, while Plasmodium vivax parasites persist in a dormant state for extended periods from months to years after the initial infection [4].The development The reaction involved the condensation of substituted acetophenones with aldehydes using an inorganic base such as KOH in ethanol and resulted in the formation of intermediate-5.Subsequently, these intermediates underwent cyclization with iodine as a catalyst to produce the desired compounds (Scheme 2,Table 2).trimethoxybenzaldehyde with m-CPBA.Next, an acetyl functional group was introduced to the phenyl ring using a Lewis acid, such as BF3•Et2O, which formed the desired intermediate-2.Following this, two different methods were employed to obtain the condensed product, intermediate-3.In step (c), intermediate-2 and an aldehyde condensation were combined using a strong inorganic base such as KOH in ethanol.Notably, in step (d), the compound FNDR-10142 was synthesized by reacting commercially available dodecanal with tri-methoxy substituted 2-hydroxy acetophenone in the presence of lithium diisopropylamide (LDA) in tetrahydrofuran (THF).Subsequently, these intermediates were cyclized with iodine using a catalytic amount of iodine, followed by demethylation with HBr in acetic acid to produce the desired compounds (Table 1).The reaction involved the condensation of substituted acetophenones with aldehydes using an inorganic base such as KOH in ethanol and resulted in the formation of intermediate-5.Subsequently, these intermediates underwent cyclization with iodine as a catalyst to produce the desired compounds (Scheme 2,Table 2).trimethoxybenzaldehyde with m-CPBA.Next, an acetyl functional group was introduced to the phenyl ring using a Lewis acid, such as BF3•Et2O, which formed the desired intermediate-2.Following this, two different methods were employed to obtain the condensed product, intermediate-3.In step (c), intermediate-2 and an aldehyde condensation were combined using a strong inorganic base such as KOH in ethanol.Notably, in step (d), the compound FNDR-10142 was synthesized by reacting commercially available dodecanal with tri-methoxy substituted 2-hydroxy acetophenone in the presence of lithium diisopropylamide (LDA) in tetrahydrofuran (THF).Subsequently, these intermediates were cyclized with iodine using a catalytic amount of iodine, followed by demethylation with HBr in acetic acid to produce the desired compounds (Table 1).The reaction involved the condensation of substituted acetophenones with aldehydes using an inorganic base such as KOH in ethanol and resulted in the formation of intermediate-5.Subsequently, these intermediates underwent cyclization with iodine as a catalyst to produce the desired compounds (Scheme 2,Table 2).trimethoxybenzaldehyde with m-CPBA.Next, an acetyl functional group was introduced to the phenyl ring using a Lewis acid, such as BF3•Et2O, which formed the desired intermediate-2.Following this, two different methods were employed to obtain the condensed product, intermediate-3.In step (c), intermediate-2 and an aldehyde condensation were combined using a strong inorganic base such as KOH in ethanol.Notably, in step (d), the compound FNDR-10142 was synthesized by reacting commercially available dodecanal with tri-methoxy substituted 2-hydroxy acetophenone in the presence of lithium diisopropylamide (LDA) in tetrahydrofuran (THF).Subsequently, these intermediates were cyclized with iodine using a catalytic amount of iodine, followed by demethylation with HBr in acetic acid to produce the desired compounds (Table 1).The reaction involved the condensation of substituted acetophenones with aldehydes using an inorganic base such as KOH in ethanol and resulted in the formation of intermediate-5.Subsequently, these intermediates underwent cyclization with iodine as a catalyst to produce the desired compounds (Scheme 2,Table 2).trimethoxybenzaldehyde with m-CPBA.Next, an acetyl functional group was introduced to the phenyl ring using a Lewis acid, such as BF3•Et2O, which formed the desired intermediate-2.Following this, two different methods were employed to obtain the condensed product, intermediate-3.In step (c), intermediate-2 and an aldehyde condensation were combined using a strong inorganic base such as KOH in ethanol.Notably, in step (d), the compound FNDR-10142 was synthesized by reacting commercially available dodecanal with tri-methoxy substituted 2-hydroxy acetophenone in the presence of lithium diisopropylamide (LDA) in tetrahydrofuran (THF).Subsequently, these intermediates were cyclized with iodine using a catalytic amount of iodine, followed by demethylation with HBr in acetic acid to produce the desired compounds (Table 1).The reaction involved the condensation of substituted acetophenones with aldehydes using an inorganic base such as KOH in ethanol and resulted in the formation of intermediate-5.Subsequently, these intermediates underwent cyclization with iodine as a catalyst to produce the desired compounds (Scheme 2,Table 2).trimethoxybenzaldehyde with m-CPBA.Next, an acetyl functional group was introduced to the phenyl ring using a Lewis acid, such as BF3•Et2O, which formed the desired intermediate-2.Following this, two different methods were employed to obtain the condensed product, intermediate-3.In step (c), intermediate-2 and an aldehyde condensation were combined using a strong inorganic base such as KOH in ethanol.Notably, in step (d), the compound FNDR-10142 was synthesized by reacting commercially available dodecanal with tri-methoxy substituted 2-hydroxy acetophenone in the presence of lithium diisopropylamide (LDA) in tetrahydrofuran (THF).Subsequently, these intermediates were cyclized with iodine using a catalytic amount of iodine, followed by demethylation with HBr in acetic acid to produce the desired compounds (Table 1).The reaction involved the condensation of substituted acetophenones with aldehydes using an inorganic base such as KOH in ethanol and resulted in the formation of intermediate-5.Subsequently, these intermediates underwent cyclization with iodine as a catalyst to produce the desired compounds (Scheme 2,Table 2).trimethoxybenzaldehyde with m-CPBA.Next, an acetyl functional group was introduced to the phenyl ring using a Lewis acid, such as BF3•Et2O, which formed the desired intermediate-2.Following this, two different methods were employed to obtain the condensed product, intermediate-3.In step (c), intermediate-2 and an aldehyde condensation were combined using a strong inorganic base such as KOH in ethanol.Notably, in step (d), the compound FNDR-10142 was synthesized by reacting commercially available dodecanal with tri-methoxy substituted 2-hydroxy acetophenone in the presence of lithium diisopropylamide (LDA) in tetrahydrofuran (THF).Subsequently, these intermediates were cyclized with iodine using a catalytic amount of iodine, followed by demethylation with HBr in acetic acid to produce the desired compounds (Table 1).The reaction involved the condensation of substituted acetophenones with aldehydes using an inorganic base such as KOH in ethanol and resulted in the formation of intermediate-5.Subsequently, these intermediates underwent cyclization with iodine as a catalyst to produce the desired compounds (Scheme 2,Table 2).

0.40
The reaction involved the condensation of substituted acetophenones with aldehydes using an inorganic base such as KOH in ethanol and resulted in the formation of intermediate-5.Subsequently, these intermediates underwent cyclization with iodine as a catalyst to produce the desired compounds (Scheme 2,Table 2).The synthesis of the compounds involved a stepwise process using commercially available substituted benzaldehydes by following the process described in Scheme 2. The compound FNDR-11096 synthesized by following the Scheme 3. Initially the alcohol was converted to a sulfonic ester by reacting it with Tosyl chloride, resulting in intermediate-6.Subsequently, the thioether moiety was oxidized to sulfone as intermediate-7.The next step involved the reaction of intermediate-7 with benzaldehyde using potassium carbonate as a base, which formed intermediate-8.In the condensation step, strong inorganic bases, specifically KOH in ethanol, were employed to facilitate the formation of various intermediate compounds.These intermediates were then subjected to cyclization,  The synthesis of the compounds involved a stepwise process using commercially available substituted benzaldehydes by following the process described in Scheme 2. The compound FNDR-11096 synthesized by following the Scheme 3. Initially the alcohol was converted to a sulfonic ester by reacting it with Tosyl chloride, resulting in intermediate-6.Subsequently, the thioether moiety was oxidized to sulfone as intermediate- The synthesis of the compounds involved a stepwise process using commercially available substituted benzaldehydes by following the process described in Scheme 2. The compound FNDR-11096 synthesized by following the Scheme 3. Initially the alcohol was converted to a sulfonic ester by reacting it with Tosyl chloride, resulting in intermediate-6.Subsequently, the thioether moiety was oxidized to sulfone as intermediate-7.The next step involved the reaction of intermediate-7 with benzaldehyde using potassium carbonate as a base, which formed intermediate-8.In the condensation step, strong inorganic bases, specifically KOH in ethanol, were employed to facilitate the formation of various intermediate compounds.These intermediates were then subjected to cyclization, The synthesis of the compounds involved a stepwise process using commercially available substituted benzaldehydes by following the process described in Scheme 2. The compound FNDR-11096 synthesized by following the Scheme 3. Initially the alcohol was converted to a sulfonic ester by reacting it with Tosyl chloride, resulting in intermediate-6.Subsequently, the thioether moiety was oxidized to sulfone as intermediate-7.The next step involved the reaction of intermediate-7 with benzaldehyde using potassium carbonate as a base, which formed intermediate-8.In the condensation step, strong inorganic bases, specifically KOH in ethanol, were employed to facilitate the formation of various intermediate compounds.These intermediates were then subjected to cyclization, The synthesis of the compounds involved a stepwise process using commercially available substituted benzaldehydes by following the process described in Scheme 2. The compound FNDR-11096 synthesized by following the Scheme 3. Initially the alcohol was converted to a sulfonic ester by reacting it with Tosyl chloride, resulting in intermediate-6.Subsequently, the thioether moiety was oxidized to sulfone as intermediate-7.The next step involved the reaction of intermediate-7 with benzaldehyde using potassium carbonate as a base, which formed intermediate-8.In the condensation step, strong inorganic bases, specifically KOH in ethanol, were employed to facilitate the formation of various intermediate compounds.These intermediates were then subjected to cyclization, The synthesis of the compounds involved a stepwise process using commercially available substituted benzaldehydes by following the process described in Scheme 2. The compound FNDR-11096 synthesized by following the Scheme 3. Initially the alcohol was converted to a sulfonic ester by reacting it with Tosyl chloride, resulting in intermediate-6.Subsequently, the thioether moiety was oxidized to sulfone as intermediate-7.The next step involved the reaction of intermediate-7 with benzaldehyde using potassium carbonate as a base, which formed intermediate-8.In the condensation step, strong inorganic bases, specifically KOH in ethanol, were employed to facilitate the formation of various intermediate compounds.These intermediates were then subjected to cyclization, The synthesis of the compounds involved a stepwise process using commercially available substituted benzaldehydes by following the process described in Scheme 2. The compound FNDR-11096 synthesized by following the Scheme 3. Initially the alcohol was converted to a sulfonic ester by reacting it with Tosyl chloride, resulting in intermediate-6.Subsequently, the thioether moiety was oxidized to sulfone as intermediate-7.The next step involved the reaction of intermediate-7 with benzaldehyde using potassium carbonate as a base, which formed intermediate-8.In the condensation step, strong inorganic bases, specifically KOH in ethanol, were employed to facilitate the formation of various intermediate compounds.These intermediates were then subjected to cyclization, The synthesis of the compounds involved a stepwise process using commercially available substituted benzaldehydes by following the process described in Scheme 2. The compound FNDR-11096 synthesized by following the Scheme 3. Initially the alcohol was converted to a sulfonic ester by reacting it with Tosyl chloride, resulting in intermediate-6.Subsequently, the thioether moiety was oxidized to sulfone as intermediate-7.The next step involved the reaction of intermediate-7 with benzaldehyde using potassium carbonate as a base, which formed intermediate-8.In the condensation step, strong inorganic bases, specifically KOH in ethanol, were employed to facilitate the formation of various intermediate compounds.These intermediates were then subjected to cyclization, The synthesis of the compounds involved a stepwise process using commercially available substituted benzaldehydes by following the process described in Scheme 2. The compound FNDR-11096 synthesized by following the Scheme 3. Initially the alcohol was converted to a sulfonic ester by reacting it with Tosyl chloride, resulting in intermediate-6.Subsequently, the thioether moiety was oxidized to sulfone as intermediate-7.The next step involved the reaction of intermediate-7 with benzaldehyde using potassium carbonate as a base, which formed intermediate-8.In the condensation step, strong inorganic bases, specifically KOH in ethanol, were employed to facilitate the formation of various intermediate compounds.These intermediates were then subjected to cyclization, The synthesis of the compounds involved a stepwise process using commercially available substituted benzaldehydes by following the process described in Scheme 2. The compound FNDR-11096 synthesized by following the Scheme 3. Initially the alcohol was converted to a sulfonic ester by reacting it with Tosyl chloride, resulting in intermediate-6.Subsequently, the thioether moiety was oxidized to sulfone as intermediate-7.The next step involved the reaction of intermediate-7 with benzaldehyde using potassium carbonate as a base, which formed intermediate-8.In the condensation step, strong inorganic bases, specifically KOH in ethanol, were employed to facilitate the formation of various intermediate compounds.These intermediates were then subjected to cyclization, The synthesis of the compounds involved a stepwise process using commercially available substituted benzaldehydes by following the process described in Scheme 2. The compound FNDR-11096 synthesized by following the Scheme 3. Initially the alcohol was converted to a sulfonic ester by reacting it with Tosyl chloride, resulting in intermediate-6.Subsequently, the thioether moiety was oxidized to sulfone as intermediate-7.The next step involved the reaction of intermediate-7 with benzaldehyde using potassium carbonate as a base, which formed intermediate-8.In the condensation step, strong inorganic bases, specifically KOH in ethanol, were employed to facilitate the formation of various The synthesis of the compounds involved a stepwise process using commercially available substituted benzaldehydes by following the process described in Scheme 2. The compound FNDR-11096 synthesized by following the Scheme 3. Initially the alcohol was converted to a sulfonic ester by reacting it with Tosyl chloride, resulting in intermediate-6.Subsequently, the thioether moiety was oxidized to sulfone as intermediate-7.The next step involved the reaction of intermediate-7 with benzaldehyde using potassium carbonate as a base, which formed intermediate-8.In the condensation step, strong inorganic bases, specifically KOH in ethanol, were employed to facilitate the formation of various

>25
The synthesis of the compounds involved a stepwise process using commercially available substituted benzaldehydes by following the process described in Scheme 2. The compound FNDR-11096 synthesized by following the Scheme 3. Initially the alcohol was converted to a sulfonic ester by reacting it with Tosyl chloride, resulting in intermediate-6.Subsequently, the thioether moiety was oxidized to sulfone as intermediate-7.The next step involved the reaction of intermediate-7 with benzaldehyde using potassium carbonate as a base, which formed intermediate-8.In the condensation step, strong inorganic bases, specifically KOH in ethanol, were employed to facilitate the formation of various intermediate compounds.These intermediates were then subjected to cyclization, following a similar approach as described in Scheme 1, in the presence of catalytic iodine.This final cyclization step formed the desired compounds (Table 3).
Pathogens 2023, 12, x FOR PEER REVIEW 5 of 16 following a similar approach as described in Scheme 1, in the presence of catalytic iodine.This final cyclization step formed the desired compounds (Table 3).We have synthesized the amide linker compounds with tri-hydroxy substitution by following the Scheme 4 synthetic route.This involved a reaction between intermediate-2 and diethyl oxalate to generate the dicarbonate derivative.Then, it was cyclized by refluxing with conc.HCl, resulting in the formation of Intermediate-11, an ester compound.Following this, the ester was reduced to alcohol using sodium borohydride and further converted into a mesylate by reacting it with methane sulfonyl chloride, yielding intermediate-13.Mesylated intermediate-13 was subjected to azide formation by using DMF and sodium azide.The azide compound was then reduced to an amine using THF, diethyl  following a similar approach as described in Scheme 1, in the presence of catalytic iodine.This final cyclization step formed the desired compounds (Table 3).We have synthesized the amide linker compounds with tri-hydroxy substitution by following the Scheme 4 synthetic route.This involved a reaction between intermediate-2 and diethyl oxalate to generate the dicarbonate derivative.Then, it was cyclized by refluxing with conc.HCl, resulting in the formation of Intermediate-11, an ester compound.Following this, the ester was reduced to alcohol using sodium borohydride and further converted into a mesylate by reacting it with methane sulfonyl chloride, yielding intermediate-13.Mesylated intermediate-13 was subjected to azide formation by using DMF and following a similar approach as described in Scheme 1, in the presence of catalytic iodine.This final cyclization step formed the desired compounds (Table 3).We have synthesized the amide linker compounds with tri-hydroxy substitution by following the Scheme 4 synthetic route.This involved a reaction between intermediate-2 and diethyl oxalate to generate the dicarbonate derivative.Then, it was cyclized by refluxing with conc.HCl, resulting in the formation of Intermediate-11, an ester compound.Following this, the ester was reduced to alcohol using sodium borohydride and further converted into a mesylate by reacting it with methane sulfonyl chloride, yielding intermediate-13.Mesylated intermediate-13 was subjected to azide formation by using DMF and following a similar approach as described in Scheme 1, in the presence of catalytic iodine.This final cyclization step formed the desired compounds (Table 3).We have synthesized the amide linker compounds with tri-hydroxy substitution by following the Scheme 4 synthetic route.This involved a reaction between intermediate-2 and diethyl oxalate to generate the dicarbonate derivative.Then, it was cyclized by refluxing with conc.HCl, resulting in the formation of Intermediate-11, an ester compound.Following this, the ester was reduced to alcohol using sodium borohydride and further converted into a mesylate by reacting it with methane sulfonyl chloride, yielding interme- following a similar approach as described in Scheme 1, in the presence of catalytic iodine.This final cyclization step formed the desired compounds (Table 3).We have synthesized the amide linker compounds with tri-hydroxy substitution by following the Scheme 4 synthetic route.This involved a reaction between intermediate-2 and diethyl oxalate to generate the dicarbonate derivative.Then, it was cyclized by refluxing with conc.HCl, resulting in the formation of Intermediate-11, an ester compound.Following this, the ester was reduced to alcohol using sodium borohydride and further converted into a mesylate by reacting it with methane sulfonyl chloride, yielding interme- following a similar approach as described in Scheme 1, in the presence of catalytic iodine.This final cyclization step formed the desired compounds (Table 3).We have synthesized the amide linker compounds with tri-hydroxy substitution by following the Scheme 4 synthetic route.This involved a reaction between intermediate-2 and diethyl oxalate to generate the dicarbonate derivative.Then, it was cyclized by refluxing with conc.HCl, resulting in the formation of Intermediate-11, an ester compound.Following this, the ester was reduced to alcohol using sodium borohydride and further converted into a mesylate by reacting it with methane sulfonyl chloride, yielding interme- We have synthesized the amide linker compounds with tri-hydroxy substitution by following the Scheme 4 synthetic route.This involved a reaction between intermediate-2 and diethyl oxalate to generate the dicarbonate derivative.Then, it was cyclized by refluxing with conc.HCl, resulting in the formation of Intermediate-11, an ester compound.Following this, the ester was reduced to alcohol using sodium borohydride and further converted into a mesylate by reacting it with methane sulfonyl chloride, yielding intermediate-13.Mesylated intermediate-13 was subjected to azide formation by using DMF and sodium azide.The azide compound was then reduced to an amine using THF, diethyl ether, and PPh 3 as the reducing agents, forming an amine.Intermediate-15 underwent demethylation using 47% hydrobromic acid to remove the methyl group.Subsequently, amide formation was achieved by coupling with commercially available carboxylic acids using HATU and DIPEA as coupling agents, which produced the desired compounds (Table 4).
Pathogens 2023, 12, x FOR PEER REVIEW 6 of 16 amide formation was achieved by coupling with commercially available carboxylic acids using HATU and DIPEA as coupling agents, which produced the desired compounds (Table 4).1.12 1 H and 13 C NMR spectra confirmed the structures of all synthesized final products.The mass and purity of the compounds were determined by liquid chromatography-mass spectrometry (LC-MS), confirming a purity of at least 95%.
The in vitro metabolic stability was determined in mouse liver microsomes [22].To prepare a mouse liver microsomes (MLM) homogenate, 100 µL of microsomes was resuspended in 100 mM phosphate buffer (0.607 g K 2 HPO 4 and 0.212 g KH 2 PO4 dissolved in 50 mL of de-ionized water), achieving a working concentration of 0.625 mg/mL.Positive control (Imipramine) and test compounds (FNDR-10132, 10136, 10139, and 10999) were spiked individually into the MLM homogenate at a final concentration of 1 µm.To pre-incubate, they were placed in a water bath at 37 • C for 5 min.At least 30 µL was collected in triplicates (0 min samples), and the reaction was quenched using 90 µL of acetonitrile, which contained 500 ng/mL of Carbamazepine as an internal standard.Nicotinamide Adenine Dinucleotide Phosphate (NADPH) in phosphate buffer was added to the remaining MLM at a 1 mM final concentration and further incubated at 37 • C. At the indicated time intervals, 30 µL aliquots were sampled in triplicates, and the reaction was quenched with acetonitrile containing Carbamazepine as the internal standard [23].The samples were centrifugated for 10 min at 13,000 rpm, and the supernatant was separated for further analysis.

Plasma Protein Binding Assay
Plasma protein binding was performed following the RED method, i.e., rapid equilibrium dialysis [24,25].The test compound was prepared to achieve a 5 µM final concentration when spiked in blank plasma.200 µL of the spiked sample was aliquoted into the sample chamber of RED inserts.A total of 350 µL of the buffer was added into another chamber of the inserts.The plate was covered and incubated at 37 • C in an orbital shaker at 100 rpm for at least 4 h.Aliquots of 100 µL were taken from the buffer and plasma chambers into another tube.A total of 100 µL of plasma was added to the buffer sample, and 100 µL of buffer was added to the plasma sample.We added 400 µL of acetonitrile containing the internal standard to each tube.It was vortexed for 2 min and centrifuged at 13,000 rpm for 10 min at 4 • C.Then, the supernatant was placed into the sample vials and loaded for LC-MS/MS analysis.
The analytical method for microsomal stability and protein binding assay is as follows: Chromatographic elution was achieved following isocratic flow on an X-Terra MS C18 50 × 4.6 mm, 2.5 µm, Waters, USA, maintained at 40 • C. The mobile phase contained aqueous 0.1% formic acid in water in solvent A and acetonitrile acidified with 0.1% formic acid in solvent B delivered at 0.300 mL/min.The flow was 15% from solvent A and 85% from solvent B for 4.5 min.A total of 7 µL of the sample was injected for the assay.Carbamazepine was used as an internal standard as it was observed to be compatible with the extraction method and test compounds.Detection was performed through multiple reaction monitoring (MRM).The ion transitions are as follows: Imipramine, FNDR-10132, FNDR-10136, FNDR-10139, FNDR-10999, and IS at m/z 281.1→86.0,355.2→115.1,354.0→298.0,350.0→186.1,349→305.2, and m/z 237.0→194.0,respectively.An optimized cone voltage and collision energy were used to ionize the compounds.The MS parameters follow as follows: cone gas flow 50 (L/h); ion source temperature 150 • C; desolvation gas flow 900 (L/h); desolvation temperature 400 • C; and collision gas flow 0.10 (mL/min).

Calibration Standards and Quality Control Samples
A stock solution of FNDR-10132 was produced by dissolving in DMSO to achieve a 2 mg/mL concentration.The intermediate stock dilutions were prepared with 0.1% formic acid in 20% acetonitrile in water as a diluent.
The internal standard's stock solution (1 mg/mL) was prepared by dissolving Carbamazepine in DMSO, and its working solution was prepared by further diluting the stock solution using 0.1% formic acid in acetonitrile as a diluent to achieve 100 ng/mL as a final concentration.

Sample Preparation
Sample preparation involved a simple protein precipitation technique determining FNDR-10132 in the mice plasma.A 5% spiking protocol was followed to prepare STDs and QCs using intermediate stock dilutions.A total of 20 µL of spiked samples from STDs and QCs and 20 µL from subject samples were aliquoted into another tube.A total of 180 µL of the IS working solution was added to this tube and vortexed for 2 min.These samples were centrifugated at 4 • C for 10 min at 13,000 rpm, and the supernatant was transferred to autosampler vials and analyzed with the LC-MS/MS instrument.

LC-MS/MS Analysis
The sample analysis was performed using UPLC-MS/MS, i.e., Waters TQD mass spectrometry coupled with UPLC.The system operated with ESI positive ion mode, and the data interpretation was achieved using Target Lynx™.
Chromatographic elution was achieved by isocratic elution on a Zorbax SB-C18, 2.1 × 50 mm, Agilent, Santa Clara, CA, USA, maintained at 40 • C. The mobile phase combines solvents A and B with 0.1% formic acid in a 5 mM ammonium acetate buffer and 0.1% formic acid in acetonitrile, respectively.Isocratic flow at 90% from solvent B was delivered at 0.350 mL/min.The analysis was performed by using 7 µL of the sample.Carbamazepine was used as an internal standard as it was observed to be compatible with the extraction method along with FNDR-10132.Detection was achieved through multiple reaction monitoring (MRM) with transitions of FNDR-10132 and IS at m/z 355.2→115.1 and 237.0→194.0,respectively.A desired cone voltage and collision energy were used to ionize the compound.Optimized MS parameters were as follows: ion source temperature 150 • C; collision gas flow 0.10 (mL/min); cone gas flow 50 (L/h); desolvation temperature 400 • C; and desolvation gas flow 900 (L/h).

Preparation of Blood Smear
The parasite maturity and parasitemia were assessed by making thin blood films of Plasmodium culture.To make a thin blood film, 2-5 µL of RBCs from the culture was taken and smeared on the glass slide, air-dried, fixed with 100% methanol, and stained for 30 min using a 30% Giemsa in staining buffer solution.Slides were evaluated microscopically at 1000× magnification using an oil immersion lens.Parasitemia was measured by collecting and counting the number of infected and uninfected red blood cells up to 10,000.

Preparation of Human RBCs for Culture
Human blood was collected aseptically in an ACD (Acid Citrate Dextrose) solution with the approval of the institutional human ethics committee (CDRI/IEC/2019/A8).The blood was centrifuged at 2000 rpm for 10 min; leukocyte aggregates and residual plasma were removed by aspiration and then used in the parasite culture.The remaining erythrocyte pellet was washed thrice with complete RPMI (CRPMI) at 2000 rpm for 10 min to remove white blood cells and then suspended to 50% hematocrit in CRPMI.The cells were stored at 4 • C and could be kept for 15 days.

Parasite Synchronization (D-Sorbitol Synchronization)
A 5% w/v D-sorbitol method was used for parasite synchronization.Briefly, 5% of D-sorbitol (Sigma Aldrich, St. Louis, MO, USA) was prepared in MilliQ and passed through a 0.22 µM syringe filter.The parasite supernatant was removed after centrifugation at 2000 rpm for 5 min.Then, a 1:5 ratio of D-sorbitol (1 part parasite pellet and 5 part dsorbitol) was added to the parasite palette and incubated at 37 • C for 15 min.Then, the tube was centrifuged at 2000 rpm for 5 min, and the resulting pellet was washed thrice with RPMI to remove late-stage parasites by lysis caused by D-sorbitol and then suspended in CRPMI.

Preparation of Stock Solutions for the Compounds
All tested compounds were dissolved in a DMSO solvent and a prepared stock solution of 10 mM.The reference drug, Chloroquine diphosphate, was dissolved in CRPMI.Working solutions were made from stock solution after diluting in a CRPMI medium during the experiment.The maximum concentration of DMSO used in this study was <1%.It had no parasiticidal effect.

In Vivo Study Design
Healthy BALB/c mice weighing 20 to 25 gm were maintained under standard laboratory conditions before the experiments.An accurately weighed amount of FNDR-10132 was suspended in a mixture of 0.5% HPMC and 0.1% tween-80 to prepare an oral suspension formulation, whereas for the IV formulation, the compound was dissolved in a mixture that contained 5% dimethyl acetamide, 10% solutol, and 85% normal saline.The animals were fed before the compound administration.
The compound was orally administered in BALB/c mice at 30 and 100 mg/kg and the IV route at 5 and 30 mg/kg.However, no plasma levels were observed.Hence, an oral PK at 100 mg/kg of the test compound was performed after dosing the animals with Amino benzotriazole (ABT) at 100 mg/kg.Similarly, 200 mg/kg of Probenecid was dosed before the IV dose of the test compound at 30 mg/kg.Probenecid and Amino benzo triazole were dosed 1 h before the dosage of the test compounds.Probenecid was used to delay renal clearance, and ABT was used as a Pan-Cytochrome P450 inhibitor to increase the bioavailability of FNDR-10132.Blood samples (~50 µL) were collected in a K 2 EDTA tube at exact time points.Samples were centrifuged at 4500 rpm for 10 min, after which plasma was separated and taken for sample preparation.The separated plasma samples were collected and stored at −80 • C until taken for analysis.Non-compartmental pharmacokinetic parameters were determined using a PK calculation tool.

Results
Baicalein is a natural flavonoid compound derived from the root of Scutellaria baicalensis, a traditional Chinese herb.Several studies have investigated the effects of baicalein against malaria, both in vitro (in laboratory settings) and in vivo (in animal models).In a study published in the journal Parasitology Research in 2012, baicalein effectively suppressed the growth of P. falciparum, the most prevalent parasite causing malaria in humans, in cultured human red blood cells [27].Baicalein prominently manifests its antiinflammatory properties through the active intervention and inhibition of producing proinflammatory cytokines and enzymes.By reducing inflammation, baicalein may help alleviate some of the symptoms associated with malaria [28].
Our pursuit to discover an antimalarial compound commenced with the notion that compounds designed based on baicalein may be more likely to exhibit effectiveness against malaria.As a first step, we synthesized five baicalein derivatives with a diversified class of groups.From aryl, we picked up simple phenyl (FNDR-10131), n-hexyl, and dodecane groups (FNDR-10132 and FNDR-10142) from aliphatic and morpholine and piperidine from aliphatic heterocyclic compounds (FNDR-10133 and FNDR-10136).These compounds were synthesized by following the procedure described in Scheme 1.
The antimalarial activity of these compounds was measured by targeting the asexual blood-stage parasite P. falciparum (chloroquine-sensitive strain 3D7).Of the five compounds tested, all showed activity with IC50 values of 0.06-1.2µM (Tables 1-4), while baicalein's antimalarial activity was 32 µM.They displayed a significant increase in antimalarial activity by extending the baicalein scaffold further.There was no difference in the antimalarial activity of aryl, aliphatic, or aliphatic heterocycles among the lipophilic groups, which all showed identical antimalarial activity.However, morpholine showed a 4-5-fold reduction in potency, indicating a hydrophobicity preference.
In parallel, we synthesized another set of compounds with carboxylic acid functional groups at different positions to replace the trihydroxy group on the chromone ring.About ten compounds were produced with the carboxylic acid group at the sixth position on the chromone ring.However, these compounds were not very potent, and the activity was significantly reduced.The simple baicalein mimics with the carboxylic acid group, FNDR-10130, showed no activity at the 25 µM tested concentration.The side chains found to be highly active with trihydroxy groups were rendered inactive by the carboxylic acid group on the chromone ring.The compounds FNDR-10137, FNDR-10138, FNDR-10139, and FNDR-11009 exhibited antimalarial activity against asexual blood-stage parasites with IC50 values of 13.16 µM, 9.65 µM, 4.06 µM, and 10.36 µM, respectively.Replacement of the trihydroxy group on the chromone ring with a carboxylic acid group significantly reduced antimalarial activity.
We synthesized the compounds FNDR-10999, which has a fragment of cyclohexane, and FNDR-11001, which has N, N-di ethyl substituents.Both compounds displayed similar IC50 values around 10 µM in the asexual blood-stage assay and did not lead to improvement in the potency.
Later, we produced three compounds (FNDR-11000, FNDR-11011, and FNDR-11096) with aliphatic chains of various lengths introduced to the scaffold's phenolic group.In order to increase the permeability and optimize the physicochemical properties, the molecule FNDR-11096 was synthesized with a sulfone group.However, none of the compounds were active when the carboxylic acid group was used as an alternative to trihydroxy substitutions.
Additionally, in order to better understand the significance of substitution position, a series of four compounds (FNDR-11003, FNDR-11012, FNDR-11013, and FNDR-11014) with a carboxylic acid group at the seventh position were created.The compounds' antimalarial activities were unaffected by the change in position, and they remained inactive.
We explored another new series (compounds FNDR-10143, FNDR-10146, FNDR-10148, FNDR-10149, and FNDR-10150) where the 2-aminomethyl group was introduced at the second position, and the derivatives were generated via amide bond linkage (Results, Table 4).The linker length of carboxylic acids was examined to optimize the suitable linker length and effect on the antimalarial activity.None of these compounds demonstrated better potency than trihydroxy series compounds (Table 4).However, they showed good activity compared to the carboxylic acid series (Table 2).FNDR-10148 exhibited the highest potency from this series with an IC50 of 0.9 µM.Therefore, we selected the compounds from other series and evaluated them for their PK and metabolic stability properties.
Pathogens 2023, 12, x FOR PEER REVIEW (Results, Table 4).The linker length of carboxylic acids was examined to optimize able linker length and effect on the antimalarial activity.None of these com demonstrated better potency than trihydroxy series compounds (Table 4).Howe showed good activity compared to the carboxylic acid series (Table 2).FNDR-1 hibited the highest potency from this series with an IC50 of 0.9 µ M. Therefore, we the compounds from other series and evaluated them for their PK and metabolic properties.
The stability of the positive control and four test compounds, FNDR-10132 10136, FNDR-10139, and FNDR-10999, was assessed in mouse liver microsom presence of ß-NADPH for 60 min at 37 °C and found to be around 56.95 ± 3.49% 1.26%, 29.92 ± 5.46%, and 32.19 ± 0.29%, respectively (Figure 1).While FND showed moderate stability, the other three compounds showed poor stability i liver microsomes.FNDR-10132 was found to have decent metabolic stability and good in vitro against P. falciparum.FNDR-10132 was profiled further for its P. falciparum dua formation assay (PfDGFA), activity against a cross-resistance panel (6 strains) in FNDR-10132 was found to have decent metabolic stability and good in vitro activity against P. falciparum.FNDR-10132 was profiled further for its P. falciparum dual gamete formation assay (PfDGFA), activity against a cross-resistance panel (6 strains) including lab-derived and clinically isolated strains, plasma stability, cytotoxicity in THP1, Vero cells, and PK in mice (Table 5).In the P. falciparum dual gamete formation assay (PfDGFA), FNDR-10132 displayed poor inhibition of 9.5% in female gametocytes and 1.6% in male gametocytes at a 1 µM concentration.However, FNDR-10132 demonstrated excellent antimalarial activity in the 200-300 nM range against all mutants/strains tested (Table 6).Therefore, FNDR-10132 might operate through a unique mechanism of action as it displayed antimalarial activity despite any resistance.

Figure 1 .
Figure 1.Stability of the test compounds in the mouse liver microsomes with Imipramine itive control.

Figure 1 .
Figure 1.Stability of the test compounds in the mouse liver microsomes with Imipramine as a positive control.

Table 1 .
Antimalarial activity of Baicalein derivatives with trihydroxy groups against Plasmodium falciparum 3D7.

Table 1 .
Antimalarial activity of Baicalein derivatives with trihydroxy groups against Plasmodium falciparum 3D7.

Table 1 .
Antimalarial activity of Baicalein derivatives with trihydroxy groups against Plasmodium falciparum 3D7.

Table 1 .
Antimalarial activity of Baicalein derivatives with trihydroxy groups against Plasmodium falciparum 3D7.

Table 1 .
Antimalarial activity of Baicalein derivatives with trihydroxy groups against Plasmodium falciparum 3D7.

Table 1 .
Antimalarial activity of Baicalein derivatives with trihydroxy groups against Plasmodium falciparum 3D7.

Table 1 .
Antimalarial activity of Baicalein derivatives with trihydroxy groups against Plasmodium falciparum 3D7.

Table 1 .
Antimalarial activity of Baicalein derivatives with trihydroxy groups against Plasmodium falciparum 3D7.

Table 2 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group against Plasmodium falciparum 3D7.

Table 2 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group against Plasmodium falciparum 3D7.

Table 2 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group against Plasmodium falciparum 3D7.

Table 2 .
7. The next step involved the reaction of intermediate-7 with benzaldehyde using potassium carbonate as a base, which formed intermediate-8.In the condensation step, strong inorganic bases, specifically KOH in ethanol, were employed to facilitate the formation of various intermediate compounds.These intermediates were then subjected to cyclization, Antimalarial activity of Baicalein derivatives with carboxylic acid group against Plasmodium falciparum 3D7.

Table 2 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group against Plasmodium falciparum 3D7.

Table 2 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group against Plasmodium falciparum 3D7.

Table 2 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group against Plasmodium falciparum 3D7.

Table 2 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group against Plasmodium falciparum 3D7.

Table 2 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group against Plasmodium falciparum 3D7.

Table 2 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group against Plasmodium falciparum 3D7.

Table 2 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group against Plasmodium falciparum 3D7.

Table 2 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group against Plasmodium falciparum 3D7.

Table 2 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group against Plasmodium falciparum 3D7.

Table 3 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group and oxygen linkers against Plasmodium falciparum 3D7.

Table 3 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group and oxygen linkers against Plasmodium falciparum 3D7.

Table 3 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group and oxygen linkers against Plasmodium falciparum 3D7.

Table 3 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group and oxygen linkers against Plasmodium falciparum 3D7.

Table 3 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group and oxygen linkers against Plasmodium falciparum 3D7.

Table 3 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group and oxygen linkers against Plasmodium falciparum 3D7.

Table 3 .
Antimalarial activity of Baicalein derivatives with carboxylic acid group and oxygen linkers against Plasmodium falciparum 3D7.

Table 4 .
Antimalarial activity of Baicalein derivatives with trihydroxy with amide linker against Plasmodium falciparum 3D7.

Table 4 .
Antimalarial activity of Baicalein derivatives with trihydroxy with amide linker against Plasmodium falciparum 3D7.

Table 4 .
Antimalarial activity of Baicalein derivatives with trihydroxy with amide linker against Plasmodium falciparum 3D7.

Table 4 .
Antimalarial activity of Baicalein derivatives with trihydroxy with amide linker against Plasmodium falciparum 3D7.

Table 4 .
Antimalarial activity of Baicalein derivatives with trihydroxy with amide linker against Plasmodium falciparum 3D7.