TKK130 is a 3-Hydroxy-Propanamidine (HPA) with Potent Antimalarial In Vivo Activity and a High Barrier to Resistance

Abstract

Malaria continues to pose a significant burden on populations in endemic areas and requires innovative treatment options. Here, we report the synthesis and preclinical evaluation of the novel 3-hydroxypropanamidine (HPA) 2 (TKK130), which shows excellent antiplasmodial in vitro activity against drug-sensitive and -resistant Plasmodium falciparum strains. Moreover, in various human cell lines, the compound shows no cytotoxicity and excellent parasite selectivity. The compound inhibits synthetic hemozoin (β-hematin) formation, with IC₅₀ values lower than chloroquine (CQ), and its in vitro rate of activity is comparable with the fast-acting antimalarial drug dihydroartemisinin. Furthermore, selection studies reveal a very low propensity for resistance development. Based on initial in vivo pharmacokinetic snapshot data, 2 (TKK130) has a long-lasting, linear pharmacokinetic profile. In vivo, this novel HPA exhibits curative activity in the Plasmodium bergheimouse model and potent activity in theP. falciparum SCID mouse model after oral administration.

Graphical Abstract

INTRODUCTION

Malaria is a widespread parasitic disease with global prevalence, caused by protozoa belonging to the genus Plasmodium. According to WHO estimates in 2023, Plasmodium falciparum (Pf) infected about 263 million people and caused more than 597,000 deaths worldwide.¹ After nearly two decades of progress in the fight against malaria, the current trend unfortunately indicates a stagnation, if not a deterioration. Although the first malaria vaccine Mosquirix is approved,^2,3 and a similar vaccine, R21/Matrix-M is prequalified by the WHO,⁴ the current vaccines show only moderate efficacy and their long-term impact is difficult to assess.² Thus, treatment and control will continue to depend primarily on effective antimalarial drugs. However, declining clinical efficacy and resistance are affecting almost all currently used antimalarial drug classes, including first-line artemisinin-based combination therapies.^5,6 Parasites displaying artemisinin partial resistance have recently been identified in several countries in Africa, including Rwanda, Uganda, Ethiopia, and Eritrea.⁷ To combat the development of resistance and to address the lack of an efficient vaccine for all population groups, intensive efforts have been made in recent years to develop new antimalarial drugs with novel modes of action. However, antimalarial drug development is a major challenge, and over the past 30 years, very few new antimalarials have received regulatory approval.

Previously, we reported the development of 3-hydroxypropanamidines (HPAs) as a novel chemotype exhibiting potent in vivo antimalarial activity and a high barrier to resistance. The most potent compound, referred to as 1 (TKK129) (compound 22),⁸ displays structural similarities with halofantrine (Hf) and lumefantrine (LUM) (Figure 1). Herein, we describe the structural optimization of 1 (TKK129), achieved through a bioisosteric replacement. In particular, the 4-methoxy group of the benzamidine moiety was replaced by a fluorine substituent to improve antiplasmodial properties and reduce cytotoxicity toward host cells. Furthermore, this bioisosteric replacement is known to improve the metabolic stability of drug candidates. Our data show that the new fluorinated 3-hydroxypropanamidine 2 (TKK130) exhibits an improved antiplasmodial profile and reduced cytotoxicity toward human cells.

Figure 1.

Structural optimization of 2 (TKK130) from 1 (TKK129) and comparison with halofantrine (Hf) and lumefantrine (LUM).

RESULTS

Synthesis of 2 (TKK130).

The synthesis of 2 (TKK130) followed the procedure recently reported by Knaab et al.⁸ To accomplish the bioisosteric optimization, the essential amidine moiety in 2 (TKK130) was introduced in the last reaction step by reacting the 3-hydroxypropanenitrile 3 with 4-fluoroaniline in the presence of trimethylaluminum (Scheme 1). The key intermediate 3 was synthesized according to a method outlined by Leven et al.⁹

Scheme 1.

Synthesis of 2 (TKK130). (i) 1. 4-Fluoroaniline, Al(CH₃)₃, Toluene/THF, rt to 60 °C, 16 h. 2. Ether/HCl, rt, 5 min⁸

Chemical Stability of 2 (TKK130).

The stability of 2 (TKK130) in an aqueous solution (phosphate buffer, pH 7.4) was determined to guarantee chemical stability during in vitro and in vivo studies. 2 (TKK130) is stable (>90%, n = 3) over 24 h at room temperature when dissolved in an aqueous solution with phosphate buffer at pH 7.4 (SI).

Antiplasmodial In Vitro Evaluation and Cytotoxicity Toward Human Cells.

The initial antiplasmodial in vitro assessment demonstrated that the exchange of the methoxy substituent with a fluorine atom led to a significantly improved activity profile against both chloroquine-sensitive and chloroquine-resistant P. falciparum strains (IC₅₀: 0.002–0.012 μM) (Table 1). In addition, 2 (TKK130) exhibited very potent activity against Plasmodium knowlesi parasites, a zoonotic malaria parasite that has gained increasing interest in malaria drug research, because infections are potentially fatal if not treated early enough. Simultaneously, cytotoxicity to human cells decreased, resulting in excellent parasite selectivity. To assess and compare cytotoxicity, three human cell lines (HepG2, HEK293, and HeLa cells; Table 2) were used, and the selectivity indices (SI) were calculated. 2 (TKK130) showed decreased cytotoxicity in all human cell lines resulting in excellent SI values e.g., in HepG2 cells SI 1 (TKK129) = 860; SI 2 (TKK130) = 10,000 (Table 2).

Table 1.

Initial Antiplasmodial In Vitro Evaluation of 2 (TKK130)

compound	Pf3D7a IC50 ± SD [μM]c	Pf Dd2a IC50 ± SD [μM]c	Pf K1b IC50 ± SD [μM]c	Pf NF54b IC50 ± SD [μM]c	P. knowlesi A1-H.1 IC50 ± SD [μM]c
CQ d	0.003 ± 0.002	0.24 ± 0.13	0.28 ± 0.02	0.012 ± 0.001	0.016 ± 0.006
LUM e,f	0.006 ± 0.003	0.002 ± 0.0005	0.001 ± 0.0002	0.005 ± 0.001	n.d.
1 (TKK129) f	0.005 ± 0.001	0.012 ± 0.006	0.006 ± 0.001	0.016 ± 0.0004	n.d.
2 (TKK130)	0.002 ± 0.001	0.003 ± 0.002	0.003 ± 0.001	0.011 ± 0.003	0.001 ± 0.0006

^aValues show the mean ± standard deviation (SD) of at least three independent experiments conducted in duplicate (11-points, serial dilution).

^bTested at Swiss TPH using the 72 h [³H] hypoxanthine incorporation assay.

^cIC₅₀: half-maximal inhibitory concentration, values show the mean ± standard deviation (SD) of three independent experiments conducted in duplicate (7-points, serial dilution).

^dChloroquine.

^eLUM: Lumefantrine.

^fPreviously published by Knaab et al.⁸

n.d.: not determined.

Table 2.

Cytotoxicity of 2 (TKK130) Against Human Cells

compound	HepG2a IC50 [μM]	SIb (HepG2/Pf 3D7)	HeLac IC50 (n1; n2) [μM]	SIb (HeLa/Pf 3D7)	HEK293c IC50 (n1; n2) [μM]	SIb (HEK293/Pf 3D7)
CQ d,f	168.5 ± 15.0	56,000	>1000	>1000	>1000	>1000
LUM e,f	>20	>20	n.d.	n.d.	n.d.	n.d.
1 (TKK129) f	4.2 ± 0.3	860	46.1 (46.3; 45.8)	9220	36.8 (40.4; 33.2)	7360
2 (TKK130)	20 ± 2	10,000	89.3 (89.4; 89.1)	44,650	47.8 (51.6; 43.9)	23,900

^aResults from 3 different assays in duplicate.

^bSelectivity Index (SI).

^cValues show the mean plus single values (n₁;n₂) of two independent experiments.

^dChloroquine.

^eLUM: Lumefantrine.

^fPreviously published by Knaab et al.⁸

n.d. not determined.

In Vitro Parasite Reduction Ratio (PRR) of 2 (TKK130).

The parasite reduction ratio assay was performed to assess the speed of action of 1 (TKK129) and 2 (TKK130) in comparison to standard antimalarial drugs with known killing speed: dihydroartemisinin (fast-acting), pyrimethamine (medium-acting) and atovaquone (slow-acting) (Figure 2). All drugs were used at 10-fold IC₅₀, as determined in a standard growth inhibition assay. Both compounds 1 (TKK129) and 2 (TKK130) exhibited a faster speed of activity than the fast-acting antimalarial drug dihydroartemisinin, reducing the number of viable parasites to baseline within 24 h of exposure. In contrast, the slower-acting antimalarials pyrimethamine and atovaquone reduced the parasitemia to baseline only after 72 and 120 h of drug incubation, respectively.

Figure 2.

Parasite killing time course profiles in response to compounds 1 (TKK129) and 2 (TKK130), and comparative antimalarial drugs. Parasites were treated with the compounds at their respective 10-fold IC₅₀ for different lengths of time and allowed to recover in a drug-free medium for 28 days. Subsequently, parasite growth was measured by HRP2 ELISA. Error bars are the standard error of the mean (SEM) of at least three independent experiments in duplicate.

In Vitro Resistance Studies with 2 (TKK130).

To evaluate the propensity of resistance development to 2 (TKK130), single-step selection assays were carried out with a mutator P. falciparum strain (Dd2 Polδ). An initial inoculum of 2 × 10⁷ parasites per flask was used, and as no resistant parasites were generated, the single-step assay was repeated with a higher starting inoculum (2 × 10⁹). Parasite cultures treated with 2 (TKK130) (9 nM) were clear on day 4 for both starting inocula and no subsequent parasite growth could be observed in any of the cultures treated with 2 (TKK130) after 60 days of recovery time. The former antimalarial candidate DSM265 was used as a positive control for the assay at the lower inoculum of 2 × 10⁷ per flask and recrudescence of parasites was detected on day 16 in all three replicate culture flasks. Resistance development against DSM265 was confirmed in recrudescent parasites by a growth inhibition assay. DSM265 - treated parasites had a 23-fold shift in their mean IC₅₀ values (IC₅₀ (Parent Dd2 Polδ) = 10.2 ± 3.1 nM; IC₅₀ (Resistant Dd2 Polδ) = 231.5 ± 50.3 nM).

Ex Vivo Susceptibilities of Clinical Isolates to 2 (TKK130).

Of the 86P. falciparum isolates from participants with uncomplicated malaria (22 children and 11 adults) included between January and August 2024 in Gabon (Lambaréné and surroundings), 33 successfully grew and yielded drug susceptibility data. Isolates were susceptible with IC₅₀ values in the low nM range to compound 2 (TKK130) (median IC₅₀: 7.5 nM, min, max: 1.4, 25.1 nM). The median age of participants was 11 years, 48% were male, and median parasitemia was 20,757 (min, max: 1102, 439,401) p/μL.

In Vitro Studies with Multidrug-Resistant P. falciparum Parasites.

Assessing the risk of resistance at an early stage of the drug discovery process is critical when developing novel antimalarial therapeutic agents, to avoid investing in further development for compounds with a major resistance liability. Therefore, 2 (TKK130) was tested against various laboratory-adapted P. falciparum strains from various geographical origins and with varying resistance profiles. It was found to potently inhibit growth of all strains tested—irrespective of those conditions—as compared to the drug-sensitive NF54 strain (Table 3). In addition, 2 (TKK130) showed no cross-resistance when tested against a panel of laboratory strains with mutations adapted in recently identified antimalarial targets from compounds currently in clinical development (Table 4).^10,11

Table 3.

In Vitro Antiplasmodial Activity of 2 (TKK130) Against Field Isolates (CQ as Control)

laboratory strain	mutated loci	2 (TKK130) IC50 (μM)a	CQ IC50 (μM)a	fold shift IC50 of 2 (TKK130) relative to NF54
NF54 b	wtc	0.012	0.011	1.00
Dd2	pfcrt, pfmdr1, pfdhfr, pfdhps	0.015	0.172	1.23
K1	pfcrt, pfmdr1, pfdhfr, pfdhps	0.003	0.259	0.29
7G8	pfcrt, pfmdr1, pfdhfr, pfdhps	0.003	0.763	0.21
TM90C2b	pfcrt, pfmdr1, pfdhfr, pfdhps, pfcytb Q0	0.013	0.126	1.09
RF12 (PH-1263-C)	pfcrt, pfmdr1, pfdhfr, pfdhps, pf K13, plasmepsin II	0.004	0.201	0.33

^a72 h [³H]Hypoxanthine incorporation assay, mean values from 2 independent biological replicates. The majority of the individual values varied no more than 2×.

^bSensitive strain.

^cWild type.

Table 4.

In Vitro Antiplasmodial Activity of 2 (TKK130) Against Drug-Resistant Mutant Strains (CQ as Control)

strain generateda	mutated locus	2 (TKK130) IC50 (μM)b	CQ IC50 (μM)a	fold shift IC50 of 2 (TKK130) relative to Dd2	references
Dd2	wt	0.015	0.172	1.00
Dd2 DDD107498	PfeEF2	0.016	0.241	1.08	12
Dd2 GNF156	Pfcarl	0.007	0.166	0.47	13
Dd2 ELQ300	PfcytB	0.014	0.131	0.92	14
Dd2 SJ557733	Pfatp4	0.011	0.246	0.76	15
Dd2MMV183	PfACoAS	0.013	0.172	0.85

^aMutant P. falciparum strains. Mutations were introduced into the Dd2 parental strain via gene editing or drug selection.

^b72 h [³H]Hypoxanthine incorporation assay, with mean values derived from 2 independent biological replicates. The majority of the individual values varied no more than 2x.

Initial Mode of Action Studies.

Given the structural similarity of 2 (TKK130) to Hf and LUM, we speculated that this compound may also target the heme detoxification pathway. The NP-40 detergent assay was used to determine synthetic hemozoin (β-hematin) inhibition activity.^16,17 Results indicate that 1 (TKK129) and 2 (TKK130) both inhibit β-hematin formation with IC₅₀ values lower than CQ (Table 5). To explore how they may inhibit this biocrystallization process, we performed docking calculations to determine each compound’s adsorption energy to the fastest-growing (001) crystal face.¹⁸ The docking scores indicate that 1 (TKK129) and 2 (TKK130) have superior adsorption capabilities compared to standards. By further considering the compounds’ size-independent ligand efficiency (SILE),¹⁹ which takes into account the number of non-hydrogen atoms in each compound, we confirmed that the observed trend is not just a consequence of the larger size of 2 (TKK130) compared to CQ.

Table 5.

Detergent-Mediated NP-40 Assay to Determine Inhibition of β-Hematin Formation

compound	IC50 (μM)a	docking scoreb (kcal/mol)
1 (TKK129)	8.4 ± 0.4	−12.2
2 (TKK130)	9.4 ± 0.3	−12.1
CQ	21.0 ± 0.4	−8.2
Hf	13.4 ± 0.4	−10.4

^aValues show the mean ± standard deviation (SD) of one independent experiment conducted in technical triplicate.

^bValue is from the top-scoring of five repeat docking poses.

In Vitro Pharmacokinetic Evaluation of 2 (TKK130).

Building on the subnanomolar antiplasmodial in vitro activity and excellent parasite selectivity of 2 (TKK130), we assessed its in vitro pharmacokinetics (Figure 3A–D) using several relatively rapid and informative in vitro assays as surrogates and indicators to predict the fate of 2 (TKK130) in vivo, before actual dosing in animals. This pharmacokinetic evaluation not only contributes to the planning and implementation of preclinical studies, but also provides initial insights into potential behavior in humans, as in vivo pharmacokinetic data in animals cannot always be reliably transferred to humans.

Figure 3.

Compilation of in vitro pharmacokinetics of 2 (TKK130). (A) Plasma stability at 37 °C for 3 days sampled in triplicate (concentration 25 ng/mL 2 (TKK130)). (B) Blood-to-plasma ratio at three concentration levels (1, 5, and 25 ng/mL 2 (TKK130)); carvedilol served as the assay reference. (C) Microsomal stability in human liver microsomes (pooled from 150 donors) over 1 h (concentration 1 μM 2 (TKK130)); propranolol served as the assay reference. (D) Plasma protein binding (concentration 25 ng/mL 2 (TKK130)) was obtained by standard equilibrium dialysis assay and dilution method; itraconazole served as the assay reference. SD = Standard deviation, ‡ = Acceptance criteria according to international bioanalytical guidelines. K (B/P) = Blood-to-plasma ratio. ref = Reference.

Plasma Stability of 2 (TKK130).

To monitor degradation and modification by enzymes in human plasma, 2 (TKK130) stability in plasma was observed for 48 h. The compound showed no decrease over 6 h in human plasma (37 °C). However, in long-term monitoring over 24 and 48 h, the concentration of 2 (TKK130) decreased to 76 and 59%, respectively. The elimination half-life of TKK130 is 67 h. Further details are compiled in Figure 3A.

Blood-to-Plasma Ratio of 2 (TKK130).

The mean blood-to-plasma Ratio (K (B/P)) of 2 (TKK130) was 1.12 ± 0.04 (mean ± SD) (Figure 3B), indicating that the compound was slightly higher distributed in the red blood cell (RBC) fraction than in the plasma fraction. 2 (TKK130) showed similar ratios in its distribution at different concentrations (range: 1–25 ng/mL).

Microsomal Stability of 2 (TKK130).

Investigations of liver metabolism showed low degradation (intrinsic clearance (Cl_int) = 1.8 μL/min/mg) for 2 (TKK130) using pooled human liver microsomes (HLMs) of 150 donors. These findings indicate low metabolic fate (Figure 3C) due to slow or very limited biotransformation by hepatic enzymes, such as Cytochrome P450. Microsomal binding of 2 (TKK130) was observed by equilibrium dialysis and was found to be 0.974 (97.4%), resulting in an unbound fraction of 0.026 (2.6%) available for metabolization by the enzymes in HLMs.

Plasma Protein Binding (PPB) of 2 (TKK130).

High protein binding of 2 (TKK130) was observed using the standard equilibrium dialysis assay (>99%, Figure 3D). Considering uncertainties in protein binding measurements due to high binding, the dilution method was additionally applied to accurately determine the fraction unbound.²⁵ Two different dilution approaches (1:10 and 1:20) resulted in similar values of 99.6% PPB of 2 (TKK130). The PPB of the reference itraconazole was 99.4% at both dilutions.

Calculation of Hepatic Clearance and Hepatic Extraction Ratio of 2 (TKK130).

The hepatic clearance was calculated as 0.25 L/min resulting in a hepatic extraction ratio of 0.17. Therefore, 2 (TKK130) can be classified as a low extraction compound.

Table 6 summarizes the in vitro pharmacokinetic characteristics of 2 (TKK130) in comparison to the approved antimalarial drugs lumefantrine (LUM) and halofantrine (Hf) as well as 1 (TKK129). The optimized amidine 2 (TKK130) showed increased microsomal metabolic stability. Additionally, both TKK compounds also had a higher K(B/P) than LUM and Hf, indicating a more pronounced binding to erythrocytes. PPB was high in all tested compounds (>99%).

Table 6.

In Vitro Pharmacokinetic Parameters of 2 (TKK130)

compound	plasma stability	blood-to-plasma ratioya	plasma protein binding (%)	microsomal stability (60 min)
LUM	17 h at RTb	0.48 ± 0.09d	>99d	Clint = <7 μL/min/mgd
Hf	n.d.c	0.68 ± 0.06d	>99d	Clint = 26.9 μL/min/mgd
1 (TKK129)	24 h at 37 °C	1.14 ± 0.07	>99	Clint = 13.6 μL/min/mg
2 (TKK130)	6 h at 37 °C	1.12 ± 0.04	99.6	Clint = 1.8 μL/min/mg

^aMean ± SD.

^bAccording to Pingale et al.²⁶

^cNo published plasma stability data.

^dAccording to Charman et al.,²⁷

Cl_int = intrinsic clearance.

hERG Potassium Channel Inhibition.

To assess potential cardiotoxicity of 2 (TKK130) in vitro, we tested for inhibition of the hERG potassium channel inhibition (Table 7), which is linked to various cardiotoxic effects and is a recognized concern for certain arylamino alcohol antimalarials.^28,29 A fluorescence polarization assay was conducted to compare the hERG potassium channel inhibition of compound 2 (TKK130) with the lead compound 1 (TKK129) and reference compounds Hf and LUM (Table 7). 2 (TKK130) exhibited weaker hERG potassium channel inhibition compared to 1 (TKK129) and Hf. Notably, the hERG potassium channel inhibition of 2 (TKK130) was in the same range as LUM, which exhibits no cardiotoxic effects in clinical use.

Table 7.

hERG Potassium Channel Inhibition of 2 (TKK130)

compound	IC50 [μM]a
Hf b	0.16
LUM c	0.38
1 (TKK129) d	0.19
2 (TKK130)	0.30

^ahERG fluorescence polarization assay, IC₅₀ values were derived from one experiment conducted with 10-point titrations (each time point conducted in duplicate) with 3-fold serial dilutions.

^bHalofantrine (Hf).

^cLumefantrine (LUM).

^dPreviously published by Knaab et al.⁸

In Vivo Studies.

Antiplasmodial In Vivo Evaluation of 2 (TKK130).

Due to its excellent antiplasmodial in vitro profile and remarkable parasite selectivity, 2 (TKK130) was studied in vivo in Plasmodium berghei-infected mice. In a standard 4-day Peters test, groups of n = 3 mice were orally treated with 4 × 50/30/10 or 3 mg of compound 2 (TKK130)/kg body weight, respectively, (Table 8). Compound 2 (TKK130) exhibited excellent activity (99.8%) with curative in vivo efficacy at doses of 50 and 30 mg/kg (100% cure rates, mean survival days (MSD) > 30). At 10 mg/kg, the in vivo activity remained high (99.9% activity, MSD = 28, 33% cure rate). Although the in vivo efficacy decreased at 3 mg/kg, it was still significant (99.9% activity, MSD = 20, 0% cure rate). In addition, 2 (TKK130) showed no obvious signs of toxicity during the in vivo studies. Potent in vivo activity (99.7%) was also observed in the P. falciparum SCID mouse model on day 7 after a 4 × 50 mg/kg per oral administration to n = 2 mice (data see SI).

Table 8.

In Vivo Evaluation of Compound 2 (TKK130)

compound	oral dosea	activity [%]b	MSD [d]c	curedd
CQ	4 × 30	99.9	21	0/10
AS e	4 × 30	99	9	0/10
LUM f	4 × 30	99.8	>30	3/3
1 (TKK129) g	4 × 50	99.8	27	2/3
1 (TKK129) g	4 × 30	99.9	28	1/3
1 (TKK129) g	4 × 10	99.9	17	0/3
1 (TKK129) g	4 × 3	0	euthanized	0/3
2 (TKK130)	4 × 50	99.8	30	3/3
2 (TKK130)	4 × 30	99.9	30	3/3
2 (TKK130)	4 × 10	99.9	28	1/3
2 (TKK130)	4 × 3	99.9	20	0/3
control		0	4

^aStandard four-day Peters test using daily per oral doses of 3, 10, 30, and 50 mg/kg body weight. Experimental groups (n = 3 mice) were treated four times (at 4, 24, 48, and 72 h) postinfection.

^bBlood for parasitemia determination by fluorescence-activated cell sorting (FACS) analysis was collected on day 4 (96 h after infection). Activity was calculated as the difference between the mean parasitemia for the control and treated groups expressed relative to the control group.

^cMean survival time in days (MSD). Control mice and other mice for which the antimalarial activity was <40% were euthanized on day 4 postinfection. The survival of the animals was monitored by microscopy for up to 30 days. A compound was considered curative if the animal survived to day 30 postinfection with no detectable parasites (detection limit of 1 parasite in 10,000 erythrocytes).

^dNumber of parasite-free mice on day 30.

^eArtemisinin (AS).

^fLumefantrine (LUM).

^gPreviously published by Knaab et al.⁸

Snapshot Pharmacokinetics.

First, in vivo pharmacokinetics of 2 (TKK130) were investigated in P. berghei mice after a single oral administration of three different doses (3, 10, and 30 mg/kg). The concentration–time profiles indicate linear pharmacokinetics (Figure 4). Maximum concentration was reached at 4 h after administration with slow elimination over time.

Figure 4.

In vivo snapshot pharmacokinetics in P. berghei mice treated with single oral administration of 2 (TKK130) (3 mg/kg [square, Purple box solid], 10 mg/kg [triangle, Sky Blue triangle up solid], and 30 mg/kg [circle, Blue circle solid] (each n = 2)). Concentrations over time are shown in mean ± standard deviation. A: Linear y-axis. B: Logarithmic y-axis. c_max = maximum concentration. t_max = time to maximum concentration. AUC_0–t = total exposure from 0 to 24 h.

DISCUSSION

Malaria drug development comes with substantial challenges, as seen by the very few new antimalarial drugs that have been approved in the last decades. Currently, the core dependency on artemisinin-based combination therapies is a major concern, as they are encountering declining efficacy and parasite resistance in several endemic areas. Beneficial antiplasmodial characteristics of the new lead compound 2 (TKK130) are a fast-killing rate, a very high barrier to resistance, potent activity against clinical isolates, and curative efficacy in vivo without obvious signs of toxicity.³⁰

On the other hand, inhibition of the hERG potassium channel represents a significant weakness of 1 (TKK129) and 2 (TKK130), which is possibly attributed to the phenanthrene moiety that is also present in Hf. Therefore, further SAR data are required to better assess the structural cause of hERG potassium channel inhibition. If concluded to be due to the phenanthrene moiety, future chemical modifications will focus on its replacement by phenanthridines, phenanthrolines and building blocks that are present in approved antimalarial drugs.

Furthermore, more detailed in vivo pharmacokinetic data are needed to develop improved derivates with longer half-life as suitable combination partners for synergistically acting antimalarials. To optimize the reduced plasma stability of 2 (TKK130) compared to 1 (TKK129), we will focus on the modification of the benzamidine structure. In addition to optimization studies regarding plasma stability, imaging studies on drug distribution and metabolism in specific organs (liver, heart, and brain) are planned.

Finally, to explore whether additional plasmodial targets are affected by 2 (TKK130), intensive mode of action studies are required. However, this represents a major challenge because the mode of action of arylamino alcohol-based antimalarials is not yet fully understood. All of these planned studies into the inhibition of the hERG potassium channels, increasing the half-life and plasma stability, organ-specific metabolism, and the mode of action of the HPA 2 (TKK130) could provide insight for future antimalarial optimization.

CONCLUSIONS

In this work, we describe the development of the HPA 2 (TKK130) obtained by a bioisosteric replacement of the 4-methoxy group of the benzamidine moiety in the predecessor molecule 1 (TKK129) by a fluorine substituent. The new fluorinated 3-hydroxypropanamidine 2 (TKK130) exhibits an improved antiplasmodial activity profile and reduced cytotoxicity toward human cells. Further beneficial characteristics are a fast-killing rate with rapid in vitro parasite clearance and a very low tendency for resistance development in vitro. The hERG potassium channel inhibition is reduced and comparable with LUM, which exhibits no cardiotoxic effects in clinical use, but to minimize risk, new chemical modifications will aim to replace the phenanthrene moiety. Based on initial snapshot pharmacokinetic (PK) data, 2 (TKK130) exhibits a long-lasting, linear pharmacokinetic profile. The compound demonstrates curative in vivo activity in the P. berghei mouse model at 30 mg/kg and no obvious signs of in vivo toxicity in mice. At 3 mg/kg, the antimalarial activity was still significant and potent in vivo activity was confirmed in the P. falciparum SCID mouse model after oral administration. The NP-40 detergent assay for β-hematin inhibition provides evidence that the compound inhibits the formation of synthetic hemozoin (β-hematin), with IC₅₀ values lower than chloroquine (CQ). However, further studies on the mode of action are necessary to better understand the potential of the substance class. Overall, 2 (TKK130) exhibits antiplasmodial properties that position the compound as a new promising lead for further structural optimizations.

EXPERIMENTAL SECTION

Biological Evaluation.

Antiplasmodial In Vitro Evaluation.

All parasite strains were kept in complete culture medium (RPMI 1640 medium [Sigma], 2 mM l-glutamine [Gibco], 12 mL of 1 M HEPES [Gibco], 50 μg/mL gentamicin [Gibco], and 0.5% [wt/vol] AlbuMax II) at 37 °C in 5% CO₂/5% O₂ at 2.5% hematocrit, with a change of medium every other day and with regular parasite dilution with fresh erythrocytes.³¹ P. knowlesi cultures the medium was additionally supplemented with 10% human AB serum (kindly provided by Robert Moon of LSHTM, London, UK under a MTA by the Francis Crick Institute).

In vitro activity of the compounds against asexual stages of the P. falciparum was evaluated with a histidine-rich protein 2 (HRP2) enzyme-linked immunosorbent assay (ELISA) at the Institute of Tropical Medicine, Tübingen as previously reported.^32,33 The antiplasmodial in vitro activity of 2 (TKK130) against field isolates and laboratory-generated strains was tested at Swiss TPH in the [³H]-hypoxanthine incorporation assay, as previously reported.^34–36

The antiplasmodial activity of 2 (TKK130) against P. knowlesi was evaluated with Sybr Green as previously described.³⁷

Sample Collection and Processing.

P. falciparum isolates were collected from individuals aged 2 years or older during January–August 2024. Asymptomatic and symptomatic individuals were screened for P. falciparum infection using a rapid diagnostic test (RDT); those with positive RDT results had a Giemsa-stained thick blood smear evaluated to confirm the diagnosis before enrollment. The study protocol and all related documents were approved by the Comité d’Ethique de Centre de Recherche Médicales de Lambaréné, Gabon (CEI-012/2023).

Individuals with P. falciparum monoinfection ≥1000 parasites/μL determined by thick blood smear and willing to participate were enrolled in the study. Venous blood was collected in citrate vacutainer tubes. Informed consent was provided by adult participants and parents or guardians of children and adolescents under 18 years of age. Adolescents between 12 and 18 years provided written assent. Patients reporting use of antimalarial drugs in the previous 30 days were excluded.

All samples were processed at the same day as collection. Blood was centrifuged at 2000 rpm for 10 min at room temperature, plasma, and buffy coat were removed, and the erythrocyte pellet was washed three times with RPMI 1640 media (Thermo Fisher Scientific). Ex vivo growth inhibition assay was performed as described above for the laboratory P. falciparum strains.

Cytotoxicity Assay.

Cytotoxicity of the compounds against HepG2, HeLa and HEK cells was evaluated based on a neutral red assay as described previously.³⁸ HepG2, HeLa and HEK293 cells (obtained from ATCC) were maintained in DMEM medium (Sigma-Aldrich) supplemented with 10% of inactivated fetal bovine serum (Sigma-Aldrich), 200 mM l-glutamine (Gibco), 12 mL of 1 M HEPES buffer (Gibco), and 50 μg/mL penicillin/streptomycin solution (Gibco). Trypsin (Gibco) was used to detach the cells when they reached a semiconfluent layer. Briefly, 300,000 cells (for HepG2) or 200,000 cells (for HeLa and HEK293) were seeded in supplemented medium as described above to 96-well plates. After 24 h, the cells were incubated with a 2-fold serial dilution of the respective drug diluted in supplemented DMEM medium for another 24 h. Thereafter, the drug-containing medium was replaced by a supplemented medium with 1.5% Neutral Red, and the cells were incubated for an additional 3 h at 37 °C. Cells were then washed with phosphate-buffered saline (pH 7.2) and 100 μL of freshly prepared lysis buffer (50% methanol, 49% distilled water, and 1% acetic acid) were added to the plates. The cells were then shaken for 10 min and the absorption was measured at a wavelength of 540 nm using CLARIOstar (BMG Labtech).

In Vivo Antimalarial Efficacy.

In vivo efficacy on P. berghei was determined as previously described.^39,40 Female mice (Charles River Laboratories) were infected with a GFP-expressing P. berghei ANKA strain (donated by A.P. Waters and C.J. Janse, Leiden University, The Netherlands), and parasitemia was determined using standard flow cytometry techniques. The detection limit was 0.1%, i.e., One parasite in 1000 erythrocytes. Activity was calculated as the difference between the mean parasitemia for the control and treated groups (n = 3, each) expressed relative to the control group. 2 (TKK130) was suspended (50 mg/kg dose) or dissolved (30, 10, and 3 mg/kg) in 70/30 Tween 80/ethanol, diluted 10-fold with water, and administered orally as four consecutive daily doses (4, 24, 48, and 72 h after infection). In vivo efficacy against P. falciparum was conducted according to the standard assay previously described.^41,42 All animal experiments adhered to local and national regulations of laboratory animal welfare in Switzerland (awarded permission no. 1731 and 2303). Protocols are regularly reviewed and revised following approval by the local authority (Veterinäramt Basel Stadt).

Parasite Reduction Ratio Assay.

The parasite reduction ratio assay was performed as previously described.⁴³ Briefly, ring-stage parasites (strain 3D7) were set up at 0.5% parasitemia and 2% hematocrit and treated daily with compound for 5 consecutive days. The speed of action of 2 (TKK130) (30 nM) and 1 (TKK129) (50 nM) was assessed in comparison to the antimalarial drugs dihydroartemisinin (100 nM), pyrimethamine (940 nM), and atovaquone (10 nM), which were used as fast-, medium-, and slow-acting control drugs, respectively. The concentrations used herein correspond to 10-fold the IC₅₀ as previously determined in 72 h-growth inhibition assays. Aliquots of the treated parasites were transferred from the cell culture flasks to 96-well plates daily and allowed to regrow for 28 days in order to detect viable parasites.

In Vitro Selection of 2 (TKK130)-Resistant P. falciparum.

P. falciparum Dd2-Polδ ring-stage parasites were kept under constant 2 (TKK130) pressure using a previously described method for single-step drug selection assay.⁴⁴ Briefly, single-step selections with 2 × 10⁷ and 2 × 10⁹ parasites were set up at 2% parasitemia and 5% hematocrit using 3-fold IC₅₀ of 2 (TKK130) (9 nM, as determined previously in 72 h-growth inhibition assays) in cell culture flasks in triplicate. Drug-containing media was replaced and the cultures were checked by blood smears daily up to day 7, and thereafter, every second day for up to 60 days to detect parasite recrudescence. Cultures were passaged once a week by replacing 40% of the culture with fresh media, and erythrocytes. DSM265 was used as a positive control for parasite recrudescence at 12 nM (3-fold IC₅₀ as determined previously in the 72 h-growth inhibition assay). Recrudescent parasites were subjected to growth inhibition assays as described above to assess IC₅₀ shifts after resistance selection. The Dd2-Polδ strain, which has a 10–30 fold increased mutation rate compared with untreated Dd2,⁴⁵ was provided by David Fidock, Columbia University.

β-Hematin Inhibition.

The Nonidet P-40 (NP-40) detergent-mediated assay,¹⁶ adapted for high-throughput screening in 96-well plates by Sandlin et al.,¹⁷ was used to determine β-hematin inhibition activity. Following a period of 4–5 h incubation at 37 °C, the formation of a bis-pyridyl heme complex was measured at 405 nm to quantify the free heme component that does not convert to β-hematin.⁴⁶ Measurements were made using a Thermo Scientific Multiskan GO plate reader. Sigmoidal dose–response curves were plotted in GraphPad Prism to determine (IC₅₀) values.

Docking Calculations.

The method reported by Amod et al. was followed.⁴⁷ Briefly, energy-minimized 3D ligand conformations were prepared on the Schrödinger 2021–4 Maestro interface using the LigPrep tool at pH 5.0 ± 0.5 with the OPLS4 force field.⁴⁸ Optimized structures were exported from Schrödinger and imported into the Autodock Vina PyRx interface (Python Prescription Virtual Screening Tool),⁴⁹ from where the docking calculations were performed.

Data Analysis.

All assays were performed at least three times in duplicate. Individual IC₅₀ values were determined by nonlinear regression analysis of log concentration–response curves, using the drc v3.0–1 package of R v4.1.2 (R Core Team, 2014).⁵⁰ Mean IC₅₀ values and standard deviations (SDs) were calculated for each growth inhibition assay using Excel. The graphical presentations were done with GraphPad Prism v8.

General Procedure for Pharmacokinetic Evaluation Assays.

For DMPK (Drug, Metabolism & Pharmacokinetics) determination, a tailored LC–MS/MS method was established. The instrument consisted of an Agilent 1200 series HPLC system (Agilent, Waldbronn, Germany) coupled to a TSQ Quantum Ultra triple quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) with an electrospray ionization (ESI) interface. Chromatographic separation was achieved using a Luna 3u C18 100 × 2.00 mm HPLC column with guard column AJ0–4286 C18 4 × 2.00 mm (Phenomenex Inc., Torrance, California, USA). Under gradient conditions (mobile phase: 0.1% formic acid in water and 0.1% formic acid in methanol) the flow rate was set to 400 μL/min. The mass-to-charge ratio of 463.00–309.97 m/z (collision energy 27 V) for compound 2 (TKK130) was monitored by multiple reaction monitoring and positive ionization mode. In the ESI interface, a spray voltage of 4000 V, a vaporizer temperature of 197 °C, an aux gas pressure of 5, a sheath gas pressure of 50, and an ion sweep gas pressure of 1 were set. In addition, a tube lens of 148 V and a capillary temperature of 345 °C were used.

Plasma Stability.

Freshly collected plasma (3K EDTA) was centrifuged at 2000 rpm for 10 min. The plasma was warmed to 37 °C and spiked with 2 (TKK130) solution (5 μg/mL in methanol) to obtain a concentration of 25 ng/mL (final methanolic concentration 0.5%). The spiked plasma was divided into three fractions and incubated at 37 °C with gentle shaking at 400 rpm. At the time points 0, 2, 4, 6, 24, and 48 h, 100 μL was drawn from the sample and precipitated with 300 μL ice-cold acetonitrile (containing 5 ng/mL of a structurally related compound as internal standard), immediately vortexed and shaken for 30 min at 1000 rpm on a ThermoMixer FP (Eppendorf SE, Hamburg, Germany). The samples were centrifuged for 10 min at 14,000 rpm (centrifuge 5427 R, Eppendorf SE, Hamburg, Germany). For each sample, 307 μL of the supernatant was transferred into reaction tubes, evaporated to dryness at 37 °C under a gentle nitrogen stream, and stored in the refrigerator at 4 °C until reconstitution in 115 μL acetonitrile/water 50/50 (v/v). The recoveries were calculated by dividing the area ratio (area 2 (TKK130) divided by area internal standard) of the respective time point by the area ratio of the 0 h time point. Each time point was analyzed in triplicate.

Blood-to-Plasma Ratio.

The blood-to-plasma ratio K(B/P) was obtained to determine the distribution of compound 2 (TKK130) between the plasma and the RBC fraction. Fresh whole blood was spiked with compound 2 (TKK130) at concentrations of 1, 5, and 25 ng/mL. After 30 min of incubation at 37 °C, the whole blood was divided into the plasma and RBC fraction by centrifugation at 2000 rpm for 10 min. To obtain references, additive plasma and RBC fractions were obtained from fresh whole blood and were spiked with 2 (TKK130) at concentrations of 1, 5, and 25 ng/mL. The RBC fractions of samples and references were vortexed to achieve lysis of the red blood cells. 100 μL of both the plasma and RBC fractions (37 °C) were purified by precipitation with 300 μL ice-cold acetonitrile (containing 5 ng/mL internal standard), and immediately vortexed and shaken for 30 min at 1000 rpm on a ThermoMixer FP. The samples were centrifuged for 10 min at 14,000 rpm. Per sample, 307 μL of the supernatant was transferred into a 96-well plate, evaporated to dryness at 37 °C under a gentle nitrogen stream, and the residue was reconstituted finally in 115 μL acetonitrile/water 50/50 (v/v). All samples were analyzed in triplicate. The hematocrit was determined volumetrically. Carvedilol served as reference and its K(B/P) was comparable to ratios reported in the literature.^20–22

The K(B/P) was calculated by eq 1, where H is the hematocrit and RBC fraction is the red blood cell fraction.

$$K(\text{B}/\text{P})=\frac{(\text{area}\text{ratio}\text{RBC}\text{fraction}/\text{area}\text{ratio}\text{RBC}\text{reference})}{(\text{area}\text{ratio}\text{plasma}\text{fraction}/\text{area}\text{ratio}\text{plasma}\text{reference})}\times H+(1-H)$$ (1)

Plasma Protein Binding.

Standard Equilibrium Dialysis Assay.

Equilibrium dialysis was used to determine plasma protein binding. For this purpose, a regenerated cellulose dialysis membrane (limit 6 kDa, Reichelt Chemietechnik GmbH + Co, Heidelberg, Germany) previously soaked in 20% ethanol in water was clamped in a 96-well Teflon plate, creating two chambers in each well. The donor chamber was spiked with 25 ng/mL 2 (TKK130) in plasma, while the acceptor chamber contained 0.9% saline. The covered 96-well plate was incubated at 37 °C. After 24 h, samples from both chambers were sampled. 100 μL plasma (37 °C) was precipitated with 300 μL ice-cold acetonitrile (containing 5 ng/mL internal standard), immediately vortexed and shaken for 30 min at 1000 rpm on the ThermoMixer FP. The samples were centrifuged for 10 min at 14,000 rpm. 307 μL of the supernatant of each sample was transferred into a 96-well plate and was evaporated to dryness at 37 °C under a gentle nitrogen stream. The residue was reconstituted in 115 μL 50/50 acetonitrile/water (v/v). Methanol (containing internal standard) was added to the acceptor side to dissolve 2 (TKK130), resulting in a 40% methanolic solution. Quantification was performed by individual calibration curves in the respective medium.

Plasma protein binding was calculated by eq 2, where fb (p) is the fraction bound in plasma, DTe is the total plasma concentration at equilibrium, DF is the free concentration of the acceptor side, Vi is the initial plasma volume and Ve is the equilibrium plasma volume. Plasma protein binding was analyzed in triplicate. As an internal reference, the PPB of itraconazole was determined.

$$\text{fb}(\text{p})[\%]=\frac{(\text{DTe}-\text{DF})\times (\text{Ve}/\text{Vi})}{[(\text{DTe}-\text{DF})\times (\text{Ve}/\text{Vi})]+\text{DF}}\times 100$$ (2)

Dilution Method.

As a modification of the standard equilibrium dialysis method, we applied a dilution approach.⁵¹ Instead of undiluted plasma as the donor, a 1:10 or 1:20 dilution of plasma in 0.9% saline was used. The diluted plasma was spiked at 25 ng/mL 2 (TKK130) and incubated with 0.9% saline as acceptor for 24 h at 37 °C. Sample purification was performed according to the classical approach. Calibration curves from 1:10 and 1:20 diluted plasma were used to determine the corresponding donor concentrations. PPB was analyzed in triplicate. As reference, the PPB of itraconazole was determined.

PPB was calculated by eqs 3–5, where fu(p) is the fraction unbound in plasma and D is the dilution level of the plasma. The free fraction from the dilution method (fu, d(p)) had to be determined first, and then the free fraction in the nondiluted plasma (fu(p)) had to be back calculated. Fb(p) is the fraction bound in plasma.

$$\text{diluted}\text{fu},d(\text{p})=\frac{\text{acceptor}\text{concentration}}{\text{donor}\text{concentration}}$$ (3)

$$\text{undiluted}\text{fu}(\text{p})=\frac{(1/D)}{[((1/\text{fu},d(\text{p})))-1]+(1/D)}$$ (4)

$$\text{fb}(\text{p})[\%]=(1-\text{fu}(\text{p}))\times 100$$ (5)

Microsomal Stability.

HLMs and the cosolvent method²³ were used to determine microsomal stability. For this purpose, 905 μL of 0.1 M potassium phosphate buffer pH 7.4 and 25 μL of 20 mg/mL pooled (from 150 donors) HLMs (Corning, New York, USA) were added and gently swirled. 10 μL of a 50 μg/mL 2 (TKK130) or a 29.6 μg/mL propranolol–HCl working solution in acetonitrile/DMSO 80/20 (v/v) was added and gently swirled. 47 μL of the mix was pipetted into three reaction tubes per time point. To start the reaction, 3 μL of a freshly prepared mix of 125 μL NADPH Regenerating System Solution A and 25 μL NADPH Regenerating System Solution B (Corning, New York, USA) was added and the tubes were incubated at 37 °C and 450 rpm. The final concentrations of analytes were 1 μM. At time points 0, 15, 30, 45, and 60 min, the reaction was terminated by adding 150 μL of ice-cold acetonitrile (containing 100 ng/mL IS for TKK130 and propranolol-d7 as IS for propranolol) to each preparation, and the reaction tubes were vortexed and placed on ice. Samples were centrifuged at 14,000 rpm for 10 min and the supernatant diluted 1:10 with acetonitrile/water 40/60 (v/v). A negative control (containing everything except cofactors) and a blank (containing everything except the analytes) ran as controls. Samples were analyzed in triplicate. Propranolol served as a control.^23,24

The elimination rate constant (k_e) is the negative slope of the plotted ln test compound peak area ratio versus time. The elimination half-life (t_1/2) and the intrinsic clearance (Cl_int) were calculated by eqs 6 and 7.

$$t_{1/2}=\text{ln}(2)/k_e$$ (6)

$${\text{Cl}}_{\text{int}}[\mu \text{L}/\text{min}/\text{mg}\text{protein}]=\frac{\text{ln}(2)}{t_{1/2}}\times \frac{\text{volume}\text{of}\text{incubation}[\mu \text{L}]}{\text{protein}\text{amount}\text{in}\text{the}\text{incubation}[\text{mg}]}$$ (7)

Microsomal Binding.

To find out what fraction of 2 (TKK130) was freely available for metabolism in HLMs, we determined the nonspecific binding to HLMs by equilibrium dialysis. A regenerated cellulose dialysis membrane (limit 6 kDa, Reichelt Chemietechnik GmbH + Co, Heidelberg, Germany), previously soaked in 20% ethanol in water, was clamped in a 96-well Teflon plate to create two chambers in each well. The donor chamber was spiked with 1 μM 2 (TKK130) in 0.5 mg/mL HLMs in 0.1 M potassium phosphate buffer, and the acceptor chamber contained 0.9% saline. The covered 96-well plate was incubated at 37 °C. Samples were taken from both chambers after 24 h. The donor side was diluted 1:20 with 4% bovine serum albumin (BSA) in potassium phosphate buffer 0.1 M (w/V) and then 100 μL was precipitated with 300 μL of ice-cold acetonitrile (containing 5 ng/mL internal standard), vortexed immediately and shaken at 1000 rpm for 30 min on a ThermoMixer FP. Then, the samples were centrifuged at 14,000 rpm for 10 min. For each sample, 200 μL of the supernatant was transferred to a 96-well plate and evaporated to dryness at 37 °C under a gentle stream of nitrogen. The residue was reconstituted in 100 μL 40/60 acetonitrile/water (v/v). Methanol containing 12.5 ng/mL internal standard was added on the acceptor side to dissolve 2 (TKK130), resulting in a 40% methanolic solution. Quantification was carried out using calibration curves in the respective medium. The microsomal binding was analyzed in triplicate.

Microsomal binding was calculated by eq 8, where fb (mic) is the fraction bound in HLMs, DTe is the total donor concentration at equilibrium, DF is the free concentration of the acceptor side, Vi is the initial donor volume and Ve is the equilibrium donor volume. The unbound fraction in HLMs (fu (mic)) was calculated by eq 9.

The unbound intrinsic clearance (Cl_int,u) was determined by eq 10, where Cl_int is the intrinsic clearance from the microsomal stability assay.

$$\text{fb}(\text{mic})=\frac{(\text{DTe}-\text{DF})\times (\text{Ve}/\text{Vi})}{[(\text{DTe}-\text{DF})\times (\text{Ve}/\text{Vi})]+\text{DF}}$$ (8)

$$\text{fu}(\text{mic})=(1-\text{fb}(\text{mic}))$$ (9)

$${\text{Cl}}_{\text{int},\text{u}}[\mu \text{L}/\text{min}/\text{mg}\text{protein}]=\frac{{\text{Cl}}_{\text{int}}}{\text{fu}(\text{mic})}$$ (10)

Calculation of Hepatic Clearance and Hepatic Extraction Ratio.

By combining the results of the pharmacokinetic in vitro assays, the hepatic clearance (ClH, eq 11) and the hepatic extraction ratio (EH, eq 12) can be calculated.⁵² QH is the hepatic blood flow (set to 1500 mL/min), fu (p) is the fraction unbound in plasma, K(B/P) the blood-to-plasma ratio at 25 ng/mL and Cl_int,u the unbound intrinsic clearance.⁵³

$$\text{ClH}[\text{L}/\text{min}]=\text{QH}\times \frac{(\text{fu}(\text{p})/K(\text{B}/\text{P}))\times {\text{Cl}}_{\text{int},\text{u}}}{\text{QH}+(\text{fu}(\text{p})/K(\text{B}/\text{P}))\times {\text{Cl}}_{\text{int},\text{u}}}$$ (11)

$$\text{EH}=\frac{\text{ClH}}{\text{QH}}$$ (12)

Snapshot Pharmacokinetics in P. berghei Mice (Dried Blood Spots).

Single oral doses of 2 (TKK130) were administered to P. berghei-infected mice. Groups of two mice each received 3, 10, or 30 mg/kg. According to a predefined sampling scheme, mice were sampled after 1, 4, and 24 h postdose. Obtained dried blood spots on filter paper were shipped at room temperature for bioanalysis. The dried blood spots were punched out with a fixed diameter of 5.5 mm and extracted for 30 min at 2000 rpm with 600 μL acetonitrile/water 80/20 (v/v) containing 5 ng/mL internal standard. 450 μL of the supernatant was evaporated to dryness under a gentle nitrogen stream (37 °C) and the residue was reconstituted in 150 μL acetonitrile/water 40/60 (v/v). Samples were diluted 1:5 (3 and 10 mg/kg) and 1:10 (30 mg/kg) before LC–MS analysis. In parallel, a fresh whole blood calibration curve and independently prepared quality control (QC) samples at three different levels (high 600 ng/mL; mid: 400 ng/mL and low 6.25 ng/mL), were pipetted at the day of analysis. 25 μL of each calibration and QC level was spotted and dried on filter paper. Following a 2 h drying process, all calibration samples as well as QCs were treated similarly to unknown samples from mice. The final calibration range was 1.56–800 ng/mL 2 (TKK130). In addition to the three sampling times at 1, 4, and 24 h, one separate “0 h” value was obtained using the reading of blank dried blood spots (triplicate; matrix: human whole blood). Obtained concentrations for the unknown samples were analyzed using PK Solver 2.0 regarding c_max, t_max, and AUC_0–t.

Experimental Data.

General Procedures.

Proton (¹H) and carbon (¹³C) NMR spectra were recorded on a Bruker Avance 500 (500.13 MHz for ¹H and 125.76 MHz for ¹³C) using DMSO-d₆ as solvent. All spectra were performed at room temperature. The coupling constants between two nuclei over n bonds (ⁿJ) are given in Hertz (Hz), and chemical shifts are given in parts per million (ppm). Purity and chemical stability were determined by high performance liquid chromatography (HPLC, Method 1). Instrument: Knauer HPLC system in combination with a Knauer UV Detector K-2600. Column: Vertex Plus (150 × 4 mm with pre column, Eurospher II 100–5 C18). Mobile phase 1: linear gradient (90–0%) of water with 0.1% trifluoroacetic acid. Mobile phase 2: linear gradient (10–100%) of acetonitrile with 0.1% of trifluoroacetic acid. Run time: 20 min, followed by an isocratic elution with 100% acetonitrile for 10 min. Flow rate: 1 mL/min. Detection: 254 nm. The purity of all compounds is >95.0% (determined by HPLC, Method 1).

Synthesis of 2 (TKK130).

Under an argon atmosphere, 4-fluoroaniline (2.0 equiv) was dissolved in anhydrous toluene (0.9 mL) at room temperature. Trimethylaluminum (2.0 equiv, 2 M in toluene) was subsequently added and the solution was stirred at 60 °C for 30 min. Then, a solution of the 3-hydroxypropanenitrile (1.0 equiv) in anhydrous THF (0.9 mL) and anhydrous toluene (0.9 mL) was added dropwise to the reaction mixture and stirred at 60 °C for 16 h. After that, the reaction mixture was poured onto an ice–water mixture (10 mL) and AcOEt (5 mL) was added. Subsequently, the phases were separated. The aqueous layer was extracted with AcOEt (3 × 20 mL); the combined organic phases were washed with saturated sodium chloride solution (3 × 20 mL) and dried over anhydrous Na₂SO₄. After filtration, the solvent was removed under reduced pressure. Hydrogen chloride in anhydrous diethyl ether was added slowly and stirred for 5 min. The precipitant was washed twice with acetonitrile and dried.

(Z)-3-(1,3-Difluoro-6-(trifluoromethyl)phenanthrene-9-yl)-N′-(4-fluorophenyl)-3-hydroxypropanimidamid Hydrochlorid 2 (TKK130).

White solid, yield: 88%. mp: 213.7 °C, ¹H-NMR (500 MHz, DMSO-d₆) δ [ppm] = 11.81 (s, 1H), 9.83 (s, 1H), 9.28 (s, 1H), 9.02 (d, J = 4.1 Hz, 1H), 8.82 (d, J = 10.9 Hz, 1H), 8.70 (s, 1H), 8.41 (s, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.71 (t, J = 9.3 Hz, 1H), 7.40 (d, J = 6.6 Hz, 4H), 6.55 (s, 1H), 6.05 (d, J = 8.5 Hz, 1H), 3.31–3.24 (m, 1H), 3.04–2.96 (m, 1H). ¹³C-NMR (126 MHz, DMSO-d6) δ [ppm] = 165.41, 161.75 (d, ¹J_CF = 245.3 Hz), 161.20 (dd, ¹J_CF = 245.1,12.9 Hz), 159.29 (dd, ¹J_CF = 251.8, 13.9 Hz), 138.83, 132.23 (d, ⁴J_CF = 4.7 Hz), 132.12 (d, ³J_CF = 11.0 Hz), 131.06 (d, ⁴J_CF = 2.3 Hz), 129.27, 128.22 (d, ³J_CF = 8.9 Hz), 127.82 (d, ²J_CF = 32.1 Hz), 127.37, 124.75 (d, ¹J_CF = 272.6 Hz), 123.93–123.79 (m), 122.46–122.27 (m), 118.05 (d, ³J_CF = 14.4 Hz), 117.91 (d, ⁴J_CF = 5.1 Hz), 117.19 (d, ²J_CF = 23.1 Hz), 105.73 (dd, ²J_CF = 22.8, ⁴J_CF = 3.2 Hz), 103.59 (dd, ²J_CF = 27.8, 24.7 Hz), 67.64, 41.49. HPLC (Method 1): t_R = 12.6 min, AUC = 98.1%.

Supplementary Material

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01465.

Chemical stability measurement procedure of 2 (TKK130); SCID mouse model evaluation (Table S1), HPLC-Chromatogram of the purity testing of 2 (TKK130) (Figure S1) (PDF)

Molecular formular strings (CSV)

ABBREVIATIONS USED

ABS

asexual blood stages

AcOE

tethylacetate

AS

artesunate

BW

body weight

Cl_int

intrinsic clearance

CQ

chloroquine

d

douplet

dd

douplet of a douplet

equiv

equivalents

FACS

fluorescence-activated cell sorting

hERG

human Ether-a-Go Go Related Gene

HF

halofantrine

HLM

human liver microsomes

3-HPA

3-hydroxypropanamidine

HPLC

high pressure liquid chromatography

HRMS

high resolution mass spectros-copy

Hz

Hertz

K(B/P)

blood-to-plasma ratio

LUM

lumefantrine

MeOH

methanol

MSD

mean survival time

Na₂SO₄

sodium sulfate

PK

pharmacokinetic

PPB

plasma protein binding

QN

quinine

RBC

red blood cell

ref

reference

rt

room temperature

SCID

severe combined immunodeficient

SD

standard deviation

SEM

standard error of the mean

SI

selectivity index

SILE

size-independent ligand efficacy

THF

tetrahydrofuran

t _R

retention time

wt

wild type