4-Arylthieno[2,3-b]pyridine-2-carboxamides Are a New Class of Antiplasmodial Agents

Malaria causes hundreds of thousands of deaths every year, making it one of the most dangerous infectious diseases worldwide. Because the pathogens have developed resistance against most of the established anti-malarial drugs, new antiplasmodial agents are urgently needed. In analogy to similar antiplasmodial ketones, 4-arylthieno[2,3-b]pyridine-2-carboxamides were synthesized by Thorpe-Ziegler reactions. In contrast to the related ketones, these carboxamides are only weak inhibitors of the plasmodial enzyme PfGSK-3 but the compounds nevertheless show strong antiparasitic activity. The most potent representatives inhibit the pathogens with IC50 values in the two-digit nanomolar range and exhibit high selectivity indices (>100).


Introduction
Malaria is a tropical infectious disease caused by unicellular parasites of the genus Plasmodium which is transmitted by the bite of the Anopheles mosquito. The majority of malaria cases occur in African regions where Plasmodium falciparum is the most prevalent [1]. This pathogen causes Malaria tropica, the most dangerous form of the infection [2]. In 2018, there were 228 million cases of malaria worldwide, 405,000 of which were fatal. While the number of malaria infections decreased significantly in the years 2000-2015, this development unfortunately came to a standstill in recent years [2][3][4].
Because resistance to all older drugs against malaria occurs worldwide [5], the World Health Organisation (WHO) in 2001 recommended artemisinin combination therapy as the first treatment option for Malaria tropica [6]. As early as 2006, individual reports of therapy failure under artemisinins in Thailand and West Cambodia were published [7][8][9]. Since then there have been more reports of artemisinin resistance [10][11][12][13].
Alternatives to artemisinins must therefore be developed as quickly as possible and with high priority. These new drugs should have a novel structural design and address previously unexploited biological targets in order to be suitable as combination partners for established antimalarial drugs as well as to avoid cross-resistance.
Molecules 2020, 25 In previous studies the antiplasmodial thieno [2,3-b]pyridine 1 was identified as a selective inhibitor of the plasmodial glycogen synthase kinase-3 (Pf GSK-3) (IC 50 Pf GSK-3 = 0.48 µM) [14]. Since Pf GSK-3 is considered an essential enzyme for the asexual proliferation of the parasite, it was also postulated as a relevant target of antiplasmodial activity of 1 [15][16][17]. In subsequent studies it could be shown that the potency against the enzyme as well as against the pathogen can be increased by attachment of a basic structural element such as in compound 2 [18]. In the framework of the structural modifications, the thieno [2,3-b]pyridine-2-carboxamide 3 (Figure 1) was also synthesized, in which the keto group is replaced by a carboxylic acid amide structure [14].
Molecules 2020, 25,x FOR PEER REVIEW 2 of 38 In previous studies the antiplasmodial thieno [2,3-b]pyridine 1 was identified as a selective inhibitor of the plasmodial glycogen synthase kinase-3 (PfGSK-3) (IC50 PfGSK-3 = 0.48 µM) [14]. Since PfGSK-3 is considered an essential enzyme for the asexual proliferation of the parasite, it was also postulated as a relevant target of antiplasmodial activity of 1 [15][16][17]. In subsequent studies it could be shown that the potency against the enzyme as well as against the pathogen can be increased by attachment of a basic structural element such as in compound 2 [18]. In the framework of the structural modifications, the thieno [2,3-b]pyridine-2-carboxamide 3 (Figure 1) was also synthesized, in which the keto group is replaced by a carboxylic acid amide structure [14]. In this study, we show that structure 3 is neither an inhibitor of plasmodial nor human GSK-3 but inhibits the proliferation of erythrocytic parasite forms much more strongly (IC50 = 199 nM) than the PfGSK-3 inhibitor 1. On the one hand, this observation raises doubts as to whether PfGSK-3 inhibition is also responsible for the antiplasmodial activity of 1 and 2 and, on the other hand, justifies structural modifications of carboxamides such as 3 with the aim of optimizing antiplasmodial activity.
Therefore, the thieno [2,3-b]pyridine-2-carboxamides were modified to allow studies of structure-activity relationships in this class of compounds. On the one hand, the substitution pattern at the two phenyl rings of the parent structure 3 was varied. In addition, it was investigated whether the 5-cyano and the 6-amino group on the heterocyclic parent ring system are necessary for antiplasmodial activity or may be replaced by less polar elements. Furthermore, compounds were synthesized in which the phenyl ring of the amide component was replaced by aliphatic structural elements. Within the scope of these structural variations, compounds were identified which clearly exceeded the original structures 1 and 2 in antiplasmodial activity but no longer caused inhibition of the plasmodial enzyme PfGSK-3.

Syntheses
According to established methods, the thioxo-1,2-dihydropyridines 7 were synthesized in a onepot synthesis from an aromatic aldehyde (4), malononitrile (5) and 2-cyanothioacetamide (6) in the presence of catalytic amounts of piperidine [19,20]. 7 tend to decompose by oxidative dimerization and cannot be purified economically neither by chromatography nor crystallization [21,22]. The intermediates 7 were therefore alkylated directly with 2-chloroacetamide derivatives 8 in the presence of potassium hydroxide. By sequential addition of further potassium hydroxide, the desired thieno [2,3-b]pyridine-2-carboxamides 9 were formed from the intermediate thioethers by Thorpe-Ziegler cyclization (Scheme 1, Table 1) [23,24]. In this study, we show that structure 3 is neither an inhibitor of plasmodial nor human GSK-3 but inhibits the proliferation of erythrocytic parasite forms much more strongly (IC 50 = 199 nM) than the Pf GSK-3 inhibitor 1. On the one hand, this observation raises doubts as to whether Pf GSK-3 inhibition is also responsible for the antiplasmodial activity of 1 and 2 and, on the other hand, justifies structural modifications of carboxamides such as 3 with the aim of optimizing antiplasmodial activity.
Therefore, the thieno [2,3-b]pyridine-2-carboxamides were modified to allow studies of structure-activity relationships in this class of compounds. On the one hand, the substitution pattern at the two phenyl rings of the parent structure 3 was varied. In addition, it was investigated whether the 5-cyano and the 6-amino group on the heterocyclic parent ring system are necessary for antiplasmodial activity or may be replaced by less polar elements. Furthermore, compounds were synthesized in which the phenyl ring of the amide component was replaced by aliphatic structural elements. Within the scope of these structural variations, compounds were identified which clearly exceeded the original structures 1 and 2 in antiplasmodial activity but no longer caused inhibition of the plasmodial enzyme Pf GSK-3. Table 1. 3,pyridine-2-carboxamides 9 a .   N-  The aldehydes 4 needed as starting compounds were either commercially available or were synthesized from 2-chloro-4-fluorobenzaldehyde (10). For this purpose, 10 was converted with secondary amines 11 to 4-(N,N-dialkylamino)-2-chlorobenzaldehydes 12a-e according to a method developed by Yongpruska et al. [25] (Scheme 2).

Com
N- N- N- a For position of residues R 1 -R 4 refer to Scheme 1.

Biological Activity
All synthesized thieno [2,3-b]pyridine-2-carboxamides (Tables 1 and 2) were initially tested for their effects on the viability of erythrocytic forms of the pathogen P. falciparum (strain 3D7, at 3 and 0.3 µM) and the initially assumed biological target structure Pf GSK-3 (at 10 µM). Additionally, the cytotoxicity of all compounds was determined on HEK293T cells at 30 µM (Table 3). For selected compounds the inhibition of the human HsGSK-3 orthologue product was also investigated (Table 4) and the IC 50 values for the viability of the parasites were determined (Table 5). Table 3. Biological activity of thieno [2,3-b]pyridine-2-carboxamides.
The prerequisite for good antiplasmodial activity is obviously an aromatic substituent on the nitrogen atom of the carboxylic acid amide structure, the spatial orientation of which is not disturbed by ortho-substitution. The substitution pattern at the 4-aryl substituent of the heterocyclic basic structure, however, had little influence on the antiplasmodial activity. Both compounds with either a meta-substituted 4-phenyl ring (9a-9i) as well as derivatives with para-amino/ortho-chloro substitution (9p-9af) showed significant inhibition of pathogen viability at 3 µM concentration (<10% of the controls).
Also compounds that were linked to alkyl or carbonyl substituents in the 5-and 6-position of the thieno [2,3-b]pyridine scaffold (17a-17l) showed more than 90% inhibition of the plasmodial pathogens at a concentration of 3 µM. Exceptions were the carboxylic acids 17i and 17j, which in contrast to the analogous tert-butyl esters 17g and 17h showed no (17j) or only low (17i) antiplasmodial activity at the concentration investigated.
The antiplasmodial ketones 1 and 2, which are related in structure to the carboxylic acid amides presented here and which served as the starting point for our investigations, had been shown to be potent inhibitors of the plasmodial enzyme Pf GSK-3. Therefore, the inhibition of this enzyme was supposed to be an antiplasmodial mechanism of action of this substance class [14,18]. However, the testing of carboxylic acid amides on Pf GSK-3 showed that at a concentration of 10 µM most compounds showed no or only low inhibitory activity. Exceptions were three substances with aliphatic substituents on the nitrogen atom of the carboxylic acid amide structure 9j, 9m, 9n, which reduced the Pf GSK-3 activity to below 40% of the controls. Inhibition by these compounds was selective for the plasmodial enzyme, as shown by comparative experiments with the orthologue human HsGSK-3 enzyme (Table 5). In contrast to most of the other compounds in this study, which showed strong antiplasmodial activity in the absence of Pf GSK-3 activity, the opposite was true for the three aliphatic carboxamides 9j, 9m, 9n. This finding makes it very doubtful that Pf GSK-3 is the biological target structure responsible for antiplasmodial activity in this class of drugs.
For particularly potent compounds, IC 50 values for the antiplasmodial activity were determined ( Table 5). The results for four representatives were in the two-digit nanomolar range (17c, 17f, 17g, 17l). These carboxylic acid amides, in which the 5-cyano-6-amino substitution pattern is replaced by other elements, exceed the antiplasmodial activity of the analogous ketones such as 1 [14] and 2 [18] by 1-2 orders of magnitude.
It was shown that with few exceptions, the compounds in this study had only a low toxicity for the human HEK cells investigated for comparison. At a concentration of 30 µM the viability of these cells was reduced by only four compounds to less than 50% of the controls (9i, 9o, 17d, 17h). In contrast, the selectivity indices for the particularly strongly antiplasmodic representatives of this study were well over 100 ( Table 5).
The following structure-activity relationships can be derived from these results: The keto function of compounds like 1 and 2 can be replaced by a carboxamide function without loss of antiplasmodial activity. However, the inhibitory activity for Pf GSK-3 largely disappears through this modification, which is why the enzyme is unlikely to represent the relevant biological target for this class of compounds. Furthermore, in the group of these carboxamides there is no need for the presence of an ortho-halogen substituent at the 4-phenyl residue of the heterocyclic parent scaffold, as had been postulated for the analogous ketone derivatives (e.g., 1 and 2) [14,18]. Eventually, the 5-cyano-6-amino substitution pattern on the heterocyclic parent scaffold is not necessary for antiplasmodial activity. Instead, substances such as 17c, 17f, 17g and 17l, in which the 5-cyano-6-amino substituents have been replaced by other elements, are the most potent representatives in this group. The aromatic substituent on the nitrogen atom of the carboxamide structure cannot be replaced by aliphatic elements without a drastic loss of the antiplasmodial activity, although these compounds still cause significant Pf GSK-3 inhibition. This is a further indication that the Pf GSK-3 inhibition does not contribute to the antiplasmodial effect in this substance class.

General Information
Structures of all test compounds are depicted in Figure S1. The starting materials and reagents were purchased from Acros Organics (Geel, Belgium), Alfa Aesar (Karlsruhe, Germany), Sigma-Aldrich (Steinheim, Germany), Fluorochem (Derbyshire, UK) and Enamine (Riga, Latvia). All reagents and solvents were used without further purification unless otherwise stated. Anhydrous dichloromethane was used if indicated and was dried according to published methods. [29] Silica gel (40-63 µm) was used for purification by column chromatography. Reaction monitoring was performed using thin layer chromatography (TLC): Polygram SIL G/UV 254 , 0.2 mm silica gel 60, 40 × 80 mm (Macherey-Nagel, Düren, Germany), visualization by UV light (254 nm, 366 nm). The melting points (m.p.) were detected in open-glass capillaries on an electric variable heater (Electrothermal IA 9200, Bibby Scientific, Stone, UK). The infrared spectra were recorded on a Thermo Nicolet FT-IR 200 spectrometer (Thermo Nicolet, Madison, WI, USA) using KBr pellets or NaCl windows. 1 H-NMR spectra and 13 C-NMR spectra were recorded on Bruker Avance III 400, Bruker Avance II 600 or Bruker Avance III HD 500 spectrometers (Bruker Biospin, Rheinstetten, Germany) (at the NMR laboratories of the Chemical Institutes of the Technische Universität Braunschweig, Germany) in DMSO-d 6 . Chemical shifts are reported as parts per million (ppm) relative to tetramethylsilane as internal standard (δ = 0 ppm). Signals in 13 C-NMR spectra were assigned based on results of 13 C-DEPT135 experiments. Electron ionization (EI) mass spectra were recorded on a Finnigan-MAT 95 (Thermo Finnigan, Bremen, Germany), (EI) MS: ionization energy 70 eV. Accurate measurements were performed according to the peakmatch method using perfluorokerosene (PFK) as an internal mass reference. Electron spray ionization (ESI) mass spectra were recorded on LTQ-Orbitrap Velos ThermoFisher Scientific (Bremen, Deutschland). Tetradecyltrimethylammonium bromide was used as internal standard. Compounds were dissolved in methanol; concentrations were about 50 µg/mL. The flow rate was 1 µg/min, spray voltage: pos. mode 2.3-2.8 kV, neg. mode: 1.7-2.5 kV (experiments were conducted at the department of mass spectrometry of the Chemical Institutes, TU Braunschweig, Germany). The calculated and found mass data are reported. Atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) spectra were determined with an expression L CMS spectrometer (Advion, Ltd., Harlow, UK). The ESI and APCI measurements were performed by dissolution of the compound in methanol and via direct injection. The elemental analyses were performed on a CE Instruments Flash EA ® 1112 Elemental Analyzer (Thermo Quest, San Jose, CA, USA). If the elemental analysis was inconclusive, a HRMS (described above) study was performed. Purity was determined using high performance liquid chromatography (HPLC) methods with isocratic or gradient elution. All compounds tested in biological systems had purity ≥ 95%. The following HPLC devices and settings were used: System 1: Merck Hitachi Elite LaChrom system (Hitachi High Technologies Corporation, Tokyo, Japan); the threshold of the integration method was set to 1000; (diode array detector (DAD): L-2450; pump: L-2130; autosampler: L-2200; organizer box: L-2000); System 3: Merck Hitachi Elite LaChrom system (Hitachi High Technologies Corporation, Tokyo, Japan); the threshold of the integration method was set to 50; detector: L-2400; pump: L-2130; autosampler: L-2200; organizer box: L-2000); flow rate: 1.000 mL/min; detection wavelength: 254 nm and 280 nm (isocratic), 254 nm (gradient); overall run time: 20 min (gradient), 15 min (isocratic) or 25 min (isocratic for 9ae and 9af); AUC, % method; t ms = retention time, t m = dead time related to DMSO. An acetonitrile/water mixture was used for gradient elution (0-2 min: 10% ACN; 2-12 min: 10%-90% ACN (linear) 12-20 min: 90% ACN). For isocratic elution, various acetonitrile/water or acetonitrile/buffer mixtures were used. Absorption maxima (λ max ) were extracted from the UV spectra recorded by the DAD detector in the peak maxima during HPLC runs. For the measurements of the purity of 9n, 9r, 9ac, 9af and 9ag, a triethylamine/triethylammonium sulfate buffer (pH 2.7) was used. For its preparation, triethylamine (20 mL) was dissolved in water (980 mL) and sodium hydroxide (242 mg) was added. The pH was adjusted to 2.7 by adding concentrated H 2 SO 4 dropwise. The column was equilibrated with ACN/buffer (10/90) for 40 min. Subsequently, the desired ACN/buffer ratio (range 10/90-60/40) was adjusted. Preparative high-performance liquid chromatography (HPLC) was performed on LaPrep (Merck, Darmstadt): LaPrep P110 preparative HPLC pump; sample loop (Knauer, Berlin); LaPrep P216 fraction collector; LaPrep P311 spectral photometer. Column tube: Length 125 mm, inner diameter 25 mm, column packed with self-fill level NW25, column material: LiChrospher ® 100 RP-18, 12 µm (Merck, Darmstadt). Sample preparation: approx. 100 mg substance is dissolved in 5 mL DMSO and injected into the sample loop. Eluent: ACN/H 2 O 70:30, flow rate: 40 mL/min, detection at 254 nm. Because phase transmission was observed during m.p. determination, melting points of 9k and 9m were measured by differential scanning calorimetry. The DSC spectra were recorded on DSC1 STAR e System, Mettler Toledo (Columbus, OH, USA). The onset temperatures are given. 2-Chloro-4-fluorobenzaldehyde (10, 1 equivalent) and potassium carbonate (1.6 equivalents) are dissolved in DMF and the corresponding amine (11, 1.5 equivalents) is added. The mixture is heated at 100 • C for 3 h 30 min-8 h. Finally, ice water (20 mL) is added, forming a yellow-brownish precipitate. The precipitate is filtered off and the product is purified by column chromatography or crystallization.

General
Procedure for the Syntheses of 6-Amino-4-aryl-2-thioxo-1,2-dihydropyridine-3,5dicarbonitriles 7 (Procedure B) A mixture of malononitrile (5, 1 equivalent), 2-cyanothioacetamide (6, 1 equivalent) and the corresponding aromatic aldehyde (4 or 12, 1 equivalent) is dissolved in ethanol. After addition of catalytic amounts of piperidine, the mixture is refluxed for 3-6 h. Afterwards, the solvent is evaporated under reduced pressure. The brown oil obtained is mixed successively with water (10 mL), acetic acid (15 drops) and dichloromethane (2 mL) and stored at 4 • C for 15-30 min. The precipitate is filtered off and the product is used directly without further purification for the synthesis of the corresponding thieno [2,3-b]pyridine. If no precipitate is formed, the further work up is performed as indicated in the specific synthesis procedure. The aromatic aldehyde (4 or 12, 1 equivalent) is dissolved in ethanol. Then 2-cyanothioacetamide (6, 1 equivalent) and catalytic amounts of triethylamine (50 µL) are added. The reaction mixture is stirred at 55 • C for 10 min-2 h 30 min. The solvent is evaporated under reduced pressure and the residue is purified by column chromatography if necessary. The corresponding carbonyl compound (1 equivalent) is dissolved in 1,4-dioxane (1 mL) and the mixture is heated to 80 • C. Then catalytic amounts of piperidine (50 µL) and the respective 3-aryl-2-cyanoprop-2-enthioamide (15, 1 equivalent) are added in portions. The reaction is monitored by TLC. Afterwards, the solvent is evaporated under reduced pressure and the residue is purified by crystallization or column chromatography.

General Procedure for the Syntheses of Aliphatic Amides 8 (Procedure E)
The corresponding amine (14, 1 equivalent) is dissolved in dried dichloromethane (8 mL) and mixed with K 2 CO 3 (1.8 equivalents). The reaction vessel is charged with argon. 2-Chloroacetyl chloride (13, 1.5 equivalents) is added in 100 µL steps every 15 min and the mixture is stirred for 1 h at room temperature. Subsequently, the mixture is refluxed for 1 h 10 min. Then water (15 mL) is added and the mixture is stirred for another 15 min at room temperature. The mixture is extracted with dichloromethane (3 × 50 mL). Subsequently, the combined organic layers are dried over sodium sulfate and evaporated under reduced pressure. A clear liquid is obtained, which becomes solid upon scratching with a glass rod.
3.2.6. General Procedure for the Syntheses of 4-Arylthieno [2,3-b]pyridine-2-carboxamides 9 and 17 (Procedure F) The corresponding thioxo-1,2-dihydropyridine (7 or 16, 1 equivalent) is dissolved in DMF and aqueous potassium hydroxide solution (10%, 1 equivalent) is added. The 2-chloroacetamide derivative (8, 1 equivalent) is added and the mixture is stirred for 10-30 min at room temperature. Finally, further aqueous potassium hydroxide solution (10%, 1 equivalent) is added and the mixture is stirred at 100 • C until the reaction is finished. Upon addition of ice water (20 mL) a yellow-brownish precipitate is formed which is filtered off and washed successively with water, ethanol and petroleum ether. If no precipitate is formed, further work up is performed as indicated in the specific synthesis procedure. Column chromatography and/or crystallization are used for purification.

General Method for Cleavage of Boc-protecting Groups (Procedure G)
The Boc-protected thieno [2,3-b]pyridine (9q or 9ae, 0.1 equivalent) is dissolved in dried dichloromethane (8 mL) in an argon atmosphere. Trifluoroacetic acid (3 mL) is added and the mixture is stirred for 30 min-1 h at room temperature. The solvent is evaporated under reduced pressure. The oily brown residue is dissolved in propan-2-ol (3 mL). After addition of a 5-6 M hydrochloric acid solution (0.1 equivalent) in propan-2-ol a precipitate is formed which is filtered off. In the case of 9ae, the mixture is stored at 4 • C for three days until a precipitate is formed.

General Method for the Cleavage of the tert-Butyl esters 17i and 17j (Procedure H)
The corresponding tert-butyl ester 17i or 17j (1 equivalent) is stirred in dried dichloromethane (2 mL) and trifluoroacetic acid (2 mL) in an argon atmosphere at room temperature for 20-24 h. Afterwards, the solvent is evaporated under reduced pressure. The product is extracted with ethyl acetate (50 mL) and washed with water (3 × 50 mL). The combined organic layers are washed with saturated sodium chloride solution (30 mL), dried over sodium sulfate and the solvent is evaporated under reduced pressure. Purification is performed by crystallization from ethanol (70% v/v).

2-Chloro-N-(2-morpholinoethyl)acetamide 8d (KuSaSch133):
According to Procedure E from 2-morpholinoethan-1-amine (522 µL, 4.00 mmol) in dried dichloromethane (8 mL) and K 2 CO 3 (994 mg, 7.19 mmol). 2-Chloroacetyl chloride (13, 478 µL total, 6.00 mmol) was added and the mixture was stirred for 1 h at room temperature. Subsequently the mixture was refluxed for 1 h 10 min. After addition of water (15 mL), stirring was continued for another 15 min at room temperature. Finally, extraction was carried out with dichloromethane (3 × 50 mL). The combined organic layers were dried over sodium sulfate. Evaporation under reduced pressure yielded a clear liquid which became solid when scratched with a glass rod. A colorless solid (967 mg, 117%) was obtained. The raw product was used without purification. Both the NMR-spectrum and the yield of >100% indicated impurities.

Viability Screening of Antiplasmodial Activity
The proliferation of Plasmodium parasites was analyzed by quantifying the DNA content in infected erythrocytes [43]. Since red blood cells contain no DNA, the proliferation of the parasites in culture can be evaluated by staining the parasite DNA with SYBR-gold (Invitrogen, Carlsbad, CA, USA). To this purpose, the parasitemia of a parasite culture was first determined by flow cytometry (ACEA Novo Cyte 1000, ACEA Biosciences, San Diego, CA, USA) as previously described [44]. Subsequently, parasitemia and hematocrit were adjusted to 0.1% and 0.2%, respectively and the culture was distributed on opaque black 96-well plates (lumox ® multiwell 96 cell culture plate, Sarstedt, DE; 200 µL culture per well). To investigate potentially antiplasmodial substances, these were dissolved in DMSO and then added to the culture. The final DMSO concentration was 0.5% at maximum. DMSO without substance was used as negative control. The culture was then run under standard conditions [45] (Parasites in RPMI complete medium (1.587% (m/v) RPMI 1640, 12 mM NaHCO 3 , 6 mM d-glucose, 0.2 mM hypoxanthine, 0.4 mM gentamicin, 0.5% (w/v) Albumax II, sterile-filtered in H 2 O and adjusted to pH 7.2 with NaOH) with 5% human erythrocytes of blood group 0 Rh+ in the presence of CO 2 (5%), O 2 (1%) and N 2 (94%) at 37 • C) for 96 h and gassed daily. The proliferation of the parasites after 96 h was quantified on the basis of the DNA content by a SYBR-gold staining. For this purpose, 100 µL of the culture supernatant were first removed from each well, then 100 µL lysis buffer (20 mM Tris; 5 mM EDTA; 0.008% saponin; 0.08 triton-X-100; 1x SYBR Gold) were added and re-suspended. Staining was performed by incubation for one hour in the dark at room temperature. The DNA content was then measured in an EnVision Multilable Plate Reader (Perki-nElmer, Waltham, MA, USA). To calculate IC 50 values, the measured values were normalized to uninfected erythrocytes and plotted in GraphPad Prism (version 6) (GraphPad Software, San Diego, CA, USA) as % DMSO control. Dose-response curves were generated using nonlinear regression (curve fit > dose-response inhibition > (log) inhibitor vs. normalized response-variable slope.

Conclusions
The 4-arylthieno [2,3-b]pyridine-2-carboxamides derived from analogous ketones are a class of substances with potent antiparasitic activity against erythrocytic forms of the malaria pathogen Plasmodium falciparum. In contrast to the corresponding ketones, these carboxamides are only weak inhibitors of the plasmodial enzyme Pf GSK-3, which probably does not represent the biological target structure of the new compound class. Molecular structure modifications revealed that the substituents in the 5-and 6-positions of the heterocyclic parent scaffold can be exchanged for aliphatic residues, whereas an aromatic substituent on the nitrogen atom at the carboxamide function is essential. The most potent antiplasmodial representatives of the substance class such as 17f inhibit the pathogens with IC 50 values in the two-digit nanomolar range and exhibit very high selectivity indices (>100). These structures should therefore be excellent starting points for the development of new antimalarial agents. Certain substituents on the parent scaffold may be swapped during this development process. It must be kept in mind that such structural modifications can have a major impact on the metabolic and chemical stability of the congeners. The chemical stability aspects in particular are currently the subject of ongoing investigations.
Funding: S.I.S. is grateful for financial support by a stipend of the Evangelisches Studienwerk Villigst. A.A. is grateful for financial support by a fellowship from the Jürgen Manchot-Stiftung. We acknowledge support by the German Research Foundation and the Open Access Publication Funds of the Technische Universität Braunschweig.