4-Substituted Thieno[3,2-d]pyrimidines as Dual-Stage Antiplasmodial Derivatives

Malaria remains one of the major health problems worldwide. The increasing resistance of Plasmodium to approved antimalarial drugs requires the development of novel antiplasmodial agents that can effectively prevent and/or treat this disease. Based on the structure of Gamhepathiopine, a 2-tert-butylaminothieno[3,2-d]pyrimidin-4(3H)-one hit, active on the sexual and asexual stages of the parasite and thanked for the introduction of various substituents at position 4 of the thienopyrimidine core by nucleophilic aromatic substitution and pallado-catalyzed coupling reactions, a series of 4-substituted thieno[3,2-d]pyrimidines were identified as displaying in vitro activities against both the erythrocytic stage of P. falciparum and the hepatic stage of P. berghei. Among the 28 compounds evaluated, the chloro analogue of Gamhepathiopine showed good activity against the erythrocytic stage of P. falciparum, moderate toxicity on HepG2, and better activity against hepatic P. berghei parasites, compared to Gamhepathiopine.


Introduction
Malaria is a dreaded disease, mainly located in the African region [1]. According to the WHO (World Health Organization), this disease infected in 2021 nearly 241 million people worldwide, and killed 627,000 people [2,3]. Despite the prevention policy carried out by the authorities, such as vector control, and the use of preventive antimalarial drugs and the vaccination of children, populations living in regions with moderate to high transmission remain vulnerable to malaria [4][5][6]. To fight against this scourge, the therapeutic arsenal currently proposes the use of artemisinin or its derivatives in combination with other partner drugs in artemisinin-based combination therapies (ACTs) [7][8][9]. ACTs are recommended as the first line of defense against severe Plasmodium falciparum malaria, but their effectiveness is threatened by the steady growth Since 2015, a new malaria control program has been developed by the WHO, with the aim of reducing the mortality rate by 90% by 2030 [13]. To achieve this goal, innovative treatments need to be developed, especially with multistage activities against Plasmodium [14,15]. In this context, our group identified a thienopyrimidine derivative, named Gamhepathiopine (Figure 1), which presented promising antiplasmodial activity on all the different developmental stages of P. falciparum [16][17][18]. A first structure-activity relationship (SAR) study was performed, including the modulation of positions 2 and 6 of the thienopyrimidine scaffold [18]. A tert-butylamine at position 2, as well as a p-tolyl group at position 6, were found to be the appropriate substituents to maintain the antiplasmodial activity in this series (Figure 1). To further these studies, we report herein the synthesis of new 4-substituted thieno [3,2-d]pyrimidine derivatives and their in vitro activities against the blood stage of P. falciparum and the liver stage of a rodent malaria model, P. berghei. To bring chemical diversity in position 4 of the thienopyrimidine core and to study the influence of a heteroatom at this position, we introduced O-alkyl, O-aryl, S-aryl, (alkyl)amino, and hydrazinyl substituents, as well as an alkynyl or (hetero)aryl group. All these derivatives could be prepared from a common halogenated intermediate, using aromatic nucleophilic substitutions (O-, S-, and N-alkyl or N-aryl groups) or Sonogashira and Suzuki-Miyaura coupling reactions (alkyne or (hetero)aryl groups).

Chemistry
Compound 1 was prepared according to the method described by Cohen et al. (Scheme 1) [18]. Briefly, 4-methylacetophenone was treated with phosphoryl trichloride and hydroxylamine hydrochloride in dimethylformamide (DMF) at room temperature to generate compound 2 in an 89% isolated yield. This latter was reacted with methyl thioglycolate in the presence of sodium methylate in methanol to afford methyl 3-amino-5-(ptolyl)thiophene-2-carboxylate 3, which was isolated in an 82% yield. Then, the condensation of 3 with ethoxycarbonyl isothiocyanate in DMF led to a thiourea intermediate, which was then reacted with tert-butylamine, triethylamine, and EDCI·HCl (1.2 eq) as a coupling agent to form a guanidine intermediate. Finally, the heating of the reaction mixture allowed its cyclization and subsequent deprotection to offer compound 1 in an overall threestep yield of 85%. The chlorination of 1 led to 5, which was isolated in a quantitative yield. A halogen exchange was then realized using an excess of sodium iodide in a dioxane reflux. Under these conditions, the conversion was revealed to be slow and incomplete, even after 72 h of reaction. Finally, compound 6 was isolated only in a 12% yield as a formic acid salt, after a preparative HPLC.

Chemistry
Compound 1 was prepared according to the method described by Cohen et al. (Scheme 1) [18]. Briefly, 4-methylacetophenone was treated with phosphoryl trichloride and hydroxylamine hydrochloride in dimethylformamide (DMF) at room temperature to generate compound 2 in an 89% isolated yield. This latter was reacted with methyl thioglycolate in the presence of sodium methylate in methanol to afford methyl 3-amino-5-(p-tolyl)thiophene-2-carboxylate 3, which was isolated in an 82% yield. Then, the condensation of 3 with ethoxycarbonyl isothiocyanate in DMF led to a thiourea intermediate, which was then reacted with tert-butylamine, triethylamine, and EDCI·HCl (1.2 eq) as a coupling agent to form a guanidine intermediate. Finally, the heating of the reaction mixture allowed its cyclization and subsequent deprotection to offer compound 1 in an overall three-step yield of 85%. The chlorination of 1 led to 5, which was isolated in a quantitative yield. A halogen exchange was then realized using an excess of sodium iodide in a dioxane reflux. Under these conditions, the conversion was revealed to be slow and incomplete, even after 72 h of reaction. Finally, compound 6 was isolated only in a 12% yield as a formic acid salt, after a preparative HPLC. Scheme 1. Synthesis of 4-halogenothienopyrimidines 5 and 6. Reagents and conditions: (i) 1 The introduction of chemical diversity was then studied from the 4-chloro derivative 5, as the iodo analog was obtained only in small quantities. First, O-alkylation was studied using sodium ethylate or methanolate at 60 °C, leading to compounds 7a-b, which were isolated after recrystallization, in 86 and 88% yields, respectively (Scheme 2). The introduction of an O-aryl group or an S-aryl was then carried out by nucleophilic substitution with (thio)phenolic compounds, in the presence of potassium carbonate as a base. Whereas substitution with phenol derivatives required high temperature to proceed, substitution with thiophenol derivatives was efficient at room temperature. Compounds 8ab and 9a-b were isolated after purification by chromatography in 54-88% yields. N-Alkylation was then studied by using compound 5 and a set of aliphatic amines or hydrazines (Scheme 3). Linear, ramified, or functionalized primary amines were introduced at position 4 of the thienopyrimidine core in the presence of a base in ethanol reflux. The introduction of chemical diversity was then studied from the 4-chloro derivative 5, as the iodo analog was obtained only in small quantities. First, O-alkylation was studied using sodium ethylate or methanolate at 60 • C, leading to compounds 7a-b, which were isolated after recrystallization, in 86 and 88% yields, respectively (Scheme 2). The introduction of an O-aryl group or an S-aryl was then carried out by nucleophilic substitution with (thio)phenolic compounds, in the presence of potassium carbonate as a base. Whereas substitution with phenol derivatives required high temperature to proceed, substitution with thiophenol derivatives was efficient at room temperature. Compounds 8a-b and 9a-b were isolated after purification by chromatography in 54-88% yields. The introduction of chemical diversity was then studied from the 4-chloro derivative 5, as the iodo analog was obtained only in small quantities. First, O-alkylation was studied using sodium ethylate or methanolate at 60 °C, leading to compounds 7a-b, which were isolated after recrystallization, in 86 and 88% yields, respectively (Scheme 2). The introduction of an O-aryl group or an S-aryl was then carried out by nucleophilic substitution with (thio)phenolic compounds, in the presence of potassium carbonate as a base. Whereas substitution with phenol derivatives required high temperature to proceed, substitution with thiophenol derivatives was efficient at room temperature. Compounds 8ab and 9a-b were isolated after purification by chromatography in 54-88% yields. N-Alkylation was then studied by using compound 5 and a set of aliphatic amines or hydrazines (Scheme 3). Linear, ramified, or functionalized primary amines were introduced at position 4 of the thienopyrimidine core in the presence of a base in ethanol reflux. N-Alkylation was then studied by using compound 5 and a set of aliphatic amines or hydrazines (Scheme 3). Linear, ramified, or functionalized primary amines were introduced at position 4 of the thienopyrimidine core in the presence of a base in ethanol reflux. Compounds 10c-i were isolated in moderate to good yields (51-77%). In the case of a volatile reagent such as ammonia or (methyl)hydrazine, the reaction was carried out in a sealed tube. Under these conditions, compounds 10a, 10b, and 10i were obtained in high yields after chromatographic purification. Surprisingly, the 1 H NMR spectrum of 10i revealed that the N-3 of the pyrimidine ring was protonated, contrary to what was observed with other amines, where the pyrimidine ring retained its aromaticity (Supplementary Materials). Finally, an amino acid (alanine) was introduced using the same strategy. The resulting compound was revealed to be insoluble in DMSO, which did not allow its biological evaluation. To increase its solubility, it was converted into its sodium salt 10j, using sodium ethylate in ethanol. Compounds 10c-i were isolated in moderate to good yields (51-77%). In the case of a volatile reagent such as ammonia or (methyl)hydrazine, the reaction was carried out in a sealed tube. Under these conditions, compounds 10a, 10b, and 10i were obtained in high yields after chromatographic purification. Surprisingly, the 1 H NMR spectrum of 10i revealed that the N-3 of the pyrimidine ring was protonated, contrary to what was observed with other amines, where the pyrimidine ring retained its aromaticity (Supplementary Materials). Finally, an amino acid (alanine) was introduced using the same strategy. The resulting compound was revealed to be insoluble in DMSO, which did not allow its biological evaluation. To increase its solubility, it was converted into its sodium salt 10j, using sodium ethylate in ethanol. The introduction of phenyl groups at position 4 was then studied using a Suzuki-Miyaura reaction (Scheme 4). First, the conditions described by Fernandez-Mato et al. were tested [19], using compound 5 and 4-methylphenylboronic acid as starting materials. In the presence of 1.3 equivalents of anhydrous potassium carbonate and 2.5% of Pd(PPh3)4 in anhydrous toluene at 100 °C for 12 h, only traces of the coupling product were observed in the LC-MS analysis ( Table 1, Entry 1). The use of polar solvents such as DMF or THF led to the same results (Entry 2, 3). Then, the experimental conditions described by Oh et al. to synthesize substituted benzothienopyrimidines were tested [20]. Using a 1M aqueous solution of potassium carbonate and 10% of Pd(PPh3)4 in THF reflux increased the conversion rate of the coupling reaction, and compound 11a was isolated in a 34% yield after chromatography (Entry 4). Finally, increasing the quantity of the base and catalyst led to the full conversion of the starting materials, and compound 11a was isolated in a 67% yield (Entry 5).
The introduction of phenyl groups at position 4 was then studied using a Suzuki-Miyaura reaction (Scheme 4). First, the conditions described by Fernandez-Mato et al. were tested [19], using compound 5 and 4-methylphenylboronic acid as starting materials. In the presence of 1.3 equivalents of anhydrous potassium carbonate and 2.5% of Pd(PPh 3 ) 4 in anhydrous toluene at 100 • C for 12 h, only traces of the coupling product were observed in the LC-MS analysis ( Table 1, Entry 1). The use of polar solvents such as DMF or THF led to the same results (Entry 2, 3). Then, the experimental conditions described by Oh et al. to synthesize substituted benzothienopyrimidines were tested [20]. Using a 1M aqueous solution of potassium carbonate and 10% of Pd(PPh 3 ) 4 in THF reflux increased the conversion rate of the coupling reaction, and compound 11a was isolated in a 34% yield after chromatography (Entry 4). Finally, increasing the quantity of the base and catalyst led to the full conversion of the starting materials, and compound 11a was isolated in a 67% yield (Entry 5). Compounds 10c-i were isolated in moderate to good yields (51-77%). In the case of a volatile reagent such as ammonia or (methyl)hydrazine, the reaction was carried out in a sealed tube. Under these conditions, compounds 10a, 10b, and 10i were obtained in high yields after chromatographic purification. Surprisingly, the 1 H NMR spectrum of 10i revealed that the N-3 of the pyrimidine ring was protonated, contrary to what was observed with other amines, where the pyrimidine ring retained its aromaticity (Supplementary Materials). Finally, an amino acid (alanine) was introduced using the same strategy. The resulting compound was revealed to be insoluble in DMSO, which did not allow its biological evaluation. To increase its solubility, it was converted into its sodium salt 10j, using sodium ethylate in ethanol. The introduction of phenyl groups at position 4 was then studied using a Suzuki-Miyaura reaction (Scheme 4). First, the conditions described by Fernandez-Mato et al. were tested [19], using compound 5 and 4-methylphenylboronic acid as starting materials. In the presence of 1.3 equivalents of anhydrous potassium carbonate and 2.5% of Pd(PPh3)4 in anhydrous toluene at 100 °C for 12 h, only traces of the coupling product were observed in the LC-MS analysis ( Table 1, Entry 1). The use of polar solvents such as DMF or THF led to the same results (Entry 2, 3). Then, the experimental conditions described by Oh et al. to synthesize substituted benzothienopyrimidines were tested [20]. Using a 1M aqueous solution of potassium carbonate and 10% of Pd(PPh3)4 in THF reflux increased the conversion rate of the coupling reaction, and compound 11a was isolated in a 34% yield after chromatography (Entry 4). Finally, increasing the quantity of the base and catalyst led to the full conversion of the starting materials, and compound 11a was isolated in a 67% yield (Entry 5).   Using these conditions, a series of five other boronic acids were used and the corresponding 4-(hetero)arylthienopyrimidines 11b-f were obtained in good yields (66-80%) after purification. Three 4-alkynyl-substituted derivatives were synthesized according to the conditions described by Wang et al. [21]. Compounds 12a-c were obtained after purification in low to moderate yields (7-35%), due to purification difficulties (compounds 12a and 12c). Finally, the 4-unsubstituted derivative 13 was synthesized from 5, using the conditions described by Muraoka et al. (Scheme 5) [22]. After 3 days of reaction at room temperature, compound 5 was fully converted into 13, which was isolated after chromatography in a 71% yield. The structure of compound 13 was confirmed by 1   Using these conditions, a series of five other boronic acids were used and the corresponding 4-(hetero)arylthienopyrimidines 11b-f were obtained in good yields (66-80%) after purification. Three 4-alkynyl-substituted derivatives were synthesized according to the conditions described by Wang et al. [21]. Compounds 12a-c were obtained after purification in low to moderate yields (7-35%), due to purification difficulties (compounds 12a and 12c). Finally, the 4-unsubstituted derivative 13 was synthesized from 5, using the conditions described by Muraoka et al. (Scheme 5) [22]. After 3 days of reaction at room temperature, compound 5 was fully converted into 13, which was isolated after chromatography in a 71% yield. The structure of compound 13 was confirmed by 1 H NMR with the appearance of a new singlet at 8.88 ppm (Supplementary Materials).

In Vitro Antiplasmodial Activity and Cytotoxicity
All synthesized 4-(un)substituted thienopyrimidines were screened for their activity against drug-resistant P. falciparum K1 asexual intra-erythrocytic-stage parasites, and were compared to Gamhepathiopine 1. This strain is resistant to chloroquine, sulfadoxine, and pyrimethamine. The results are summarized in Table 2.

In Vitro Antiplasmodial Activity and Cytotoxicity
All synthesized 4-(un)substituted thienopyrimidines were screened for their activity against drug-resistant P. falciparum K1 asexual intra-erythrocytic-stage parasites, and were compared to Gamhepathiopine 1. This strain is resistant to chloroquine, sulfadoxine, and pyrimethamine. The results are summarized in Table 2.  Using these conditions, a series of five other boronic acids were used and the corresponding 4-(hetero)arylthienopyrimidines 11b-f were obtained in good yields (66-80%) after purification. Three 4-alkynyl-substituted derivatives were synthesized according to the conditions described by Wang et al. [21]. Compounds 12a-c were obtained after purification in low to moderate yields (7-35%), due to purification difficulties (compounds 12a and 12c). Finally, the 4-unsubstituted derivative 13 was synthesized from 5, using the conditions described by Muraoka et al. (Scheme 5) [22]. After 3 days of reaction at room temperature, compound 5 was fully converted into 13, which was isolated after chromatography in a 71% yield. The structure of compound 13 was confirmed by 1

In Vitro Antiplasmodial Activity and Cytotoxicity
All synthesized 4-(un)substituted thienopyrimidines were screened for their activity against drug-resistant P. falciparum K1 asexual intra-erythrocytic-stage parasites, and were compared to Gamhepathiopine 1. This strain is resistant to chloroquine, sulfadoxine, and pyrimethamine. The results are summarized in Table 2.  Using these conditions, a series of five other boronic acids were used and the corresponding 4-(hetero)arylthienopyrimidines 11b-f were obtained in good yields (66-80%) after purification. Three 4-alkynyl-substituted derivatives were synthesized according to the conditions described by Wang et al. [21]. Compounds 12a-c were obtained after purification in low to moderate yields (7-35%), due to purification difficulties (compounds 12a and 12c). Finally, the 4-unsubstituted derivative 13 was synthesized from 5, using the conditions described by Muraoka et al. (Scheme 5) [22]. After 3 days of reaction at room temperature, compound 5 was fully converted into 13, which was isolated after chromatography in a 71% yield. The structure of compound 13 was confirmed by 1

In Vitro Antiplasmodial Activity and Cytotoxicity
All synthesized 4-(un)substituted thienopyrimidines were screened for their activity against drug-resistant P. falciparum K1 asexual intra-erythrocytic-stage parasites, and were compared to Gamhepathiopine 1. This strain is resistant to chloroquine, sulfadoxine, and pyrimethamine. The results are summarized in Table 2.   From compound 1, which possesses a carbonyl group at position 4, various modulations were studied for this position. The introduction of a chlorine or an iodine atom was tolerated. Compounds 5 and 6 showed similar activities to reference 1 on Pf. K1, with an EC 50 in the submicromolar range. These results indicate that the presence of a hydrogen bond acceptor at this position is not crucial. However, the presence of a substituent remains essential, as the removal of the carbonyl group led to an inactive compound (13 vs . 1). Furthermore, the steric hindrance of the iodine atom suggests that this position could tolerate bulky substituents. The introduction of various substituents was then studied. The introduction of an aryl group led to a loss of activity when this group was linked directly to the thienopyrimidine core (compounds 11), or through an ether (compounds 8) or a thioether link (compounds 9). In the case of an amine link, some activity was maintained (compound 10h), suggesting that the presence of an amino group at this position could be tolerated. Thus, several amino-alkyl derivatives were evaluated. Whereas an amino acid residue (compound 10j) led to the loss of activity, the presence of a primary amine (compound 10a), a short linear amino-alkyl (10c and 10d) or a slightly longer chain (10f and 10g) was tolerated at this position and led to compounds with quite similar activities to compound 1 (EC 50 in the micromolar range). Finally, the introduction of a short ramified amino-alkyl (10e) or a hydrazinyl group (compounds 10b and 10i) was also tolerated but led to a slightly lower activity than compound 1 (IC 50 around 2 µM). Finally, a 4-alkynyl substituent at position 4 (compounds 12) led to totally inactive compounds.
The most active compounds against the erythrocytic stage of P. falciparum were then selected for further studies. Seven compounds (namely compounds 5, 6, 10a, 10c-d, and  10f-g), which displayed an EC 50 lower than 2 µM, were then evaluated for their ability to inhibit hepatic infection by the rodent malaria parasite P. berghei. Their cytotoxicity was also evaluated on the human hepatoma cell line HepG2 and on primary simian hepatocytes. The results are summarized in Table 3. Except compound 10a, which was not active against P. berghei, all selected compounds showed similar or higher activities on the hepatic stage than compound 1. The best results were obtained with compounds 10c, 10d, and 10g, with EC 50 around 5 µM, followed by compounds 10f and 5 with an EC 50 around 15 µM. These results suggested that the presence of an alkylamine group at position 4 increased activity, with a shorter alkyl group being preferable. Activity decreased with the alkyl chain increasing (EC 50 of 10c < EC 50 of 10d and 10g < EC 50 of 10f). All compounds displayed a moderate to high toxicity against the HepG2 human cell line, with their CC 50 values ranging from 2.9 to 29 µM. Surprisingly, these compounds did not show similar toxicity against primary simian hepatocytes, except 10g, which was cytotoxic. All compounds, except 10f-g, showed a CC 50 greater than 20 µM. Finally, compound 5 with a chlorine atom at position 4 showed the best compromise, with an EC 50 against P. berghei slightly lower than that of compound 1, with moderate toxicity on HepG2 (CC 50 = 16.4 ± 5.4 µM) and no toxicity on primary simian hepatocytes (CC 50 > 100 µM).

Materials and Methods
Commercial reagents and solvents were used without further purification. The synthesized products were purified by chromatography on silica gel (40-63 µm) or by preparative HPLC on an apparatus equipped with a Delta Pack C18 radial compression column (100 mm × 40 mm, 15 µm, 100 Å) at a wavelength of 214 nm, at a flow rate of 28 mL/min with a variable elution gradient from X % A (H 2 O + 0.1 TFA) to X + 30% B (ACN + 0.1% TFA) in 30 min. The purest fractions are pooled, freeze-dried, and lyophilized to give the final compounds. LC/MS analyses were recorded on a Quattro microTM ESI triple quadrupole mass spectrometer (ESI+ electrospray ionization mode) or on a Micromass ZQ spectrometer (ESI+ electrospray ionization mode), coupled to an Alliance HPLC system (Waters, Milford, CT, USA) equipped with a Chromolith High Resolution RP-18e column (25 × 4.6 mm), with the samples being previously separated using a gradient from 100% (H 2 O + 0.1% HCO 2 H) to 100% (ACN + 0.1% HCO 2 H) in 3 min at a flow rate of 3 mL/min and with UV detection at 214 nm. UPLC/MS analyses were recorded with an Acquity H-Class UPLC system, coupled to a Waters SQDetector-2 mass spectrometer (Waters, Milford, CT, USA). Chromatographic separation was carried out under the same conditions as above using a Waters Acquity UPLC BEC C18 column (100 × 2.1 mm, 1.5 µm). High-resolution mass spectrometry (HRMS) analyses were performed with a time-of-flight (TOF) spectrometer coupled to a positive electrospray ionization (ESI) source. The elemental analysis was realized with a Elementar Vario Micro Cube. The NMR spectra were recorded on a Brüker 400, 500, or 600 spectrometer. Chemical shifts δ are expressed in parts per million (ppm) relative to the residual signal of the deuterated solvent used (CDCl 3 , DMSO-d 6 , MeOH-d 3 , ACN-d 3 ), and the coupling constants J are expressed in Hertz. The multiplicities are designated as singlet (s), broad singlet (bs) doublet (d), doublet of doublet (dd), triplet (t), quadruplet (q), quintuplet (qt), sextuplet (st), or multiplet (m). Compound 1 was synthesized according to the previous reported procedure and its physical characteristics agreed with the published data [18].

Synthesis of N-(Tert-butyl)-4-chloro-6-(p-tolyl)thieno[3,2-d]pyrimidin-2-amine 5
Phosphorus oxychloride (5.5 eq., 8.37 g, 54.59 mmol, 5.1 mL) was slowly added to a cold solution of compound 1 (1 eq, 3.11 g, 9.93 mmol) and N,N-dimethylaniline (0.7 eq, 842 mg, 6.95 mmol) in CH 3 CN (156 mL) for 2 h at 0 • C. The mixture was then heated to 80-85 • C and was stirred for 18 h. The reaction mixture was cooled to 40 • C and then was quenched into water (400 mL) for 2 h. The precipitate was filtered and washed with water (200 mL). The desired product was dried under vacuum at 50 • C for 48 h to afford 5 as a green/yellow solid (3.26 g, qt). 1  Under argon atmosphere, compound 5 (100 mg, 0.3013 mmol) was dissolved in anhydrous dioxane (7 mL/mmol) and sodium iodide was added (6 eq., 338 mg, 1.808 mmol, 0.09 mL). The reaction mixture was stirred under reflux for 72 h. The solvent was removed under reduced pressure. The crude was dissolved in sodium thiosulfate solution (10%, 25 mL). The aqueous layer was washed three times with EtOAc. Organic layers were combined, washed with water and brine, dried with MgSO 4 , and filtered and concentrated under vacuum. Sodium (2.5 eq.) was dissolved in the desired alcohol (10 mL/mmol) under argon at 0 • C. Then, compound 5 (1 eq.) was added slowly and the reaction mixture was stirred at 60 • C under argon. After completion of the reaction, the medium was evaporated to dryness. A total of 50 mL of water was added and then extracted with diethyl ether (3 × 25 mL). The organic layer was washed with brine, dried with MgSO 4 , and filtered and concentrated under reduced pressure. The crude was recrystallized from ACN.

Synthesis of 4-Arylether-thieno[3,2-d]pyrimidines 8
The appropriate phenol (1 eq., 0.753 mmol) and K 2 CO 3 (1.1 eq., 0829 mmol) were dissolved in DMF (5 mL) and the reaction mixture was stirred for 15 min. Then, compound 5 (250 mg, 0.753 mmol) was added and the reaction mixture was heated at 130 • C for 3 h. Once the reaction finished, the reaction mixture returned to room temperature and was poured in water. The aqueous layer was extracted four times with EtOAc. The organic layers were combined, washed with brine, dried with MgSO 4 , and filtered and concentrated under vacuo. The crude product was purified by chromatography on silica gel (eluent: 9/1 Hexane/EtOAc then 8/2) and was recrystallized in ACN. Compound 5 was dissolved in DMF (5mL/mmol). Then, the appropriate thiophenol (10 eq.) and K 2 CO 3 (1 eq.) were added. The reaction mixture was stirred at room temperature for 1 to 1h 30. Once the reaction was completed, the reaction mixture was concentrated under reduced pressure and water was added. The aqueous layer was extracted three times with DCM. Organic layers were washed with brine, dried with MgSO 4 , and filtered and concentrated under vacuo. The crude was purified by chromatography on silica gel (eluent: 8/2 Hexane/EtOAc for compound 9a and 9/1 Hexane/EtOAc then 7/3 for compound 9b). The isolated product was recrystallized in ACN.
N A mixture of 5 (0.5 g, 1.507 mmol) in EtOH/NH 4 OH (35%) in a 1:1 ratio (50 mL) was heated at 120 • C. The reaction mixture was stirred for 14 h in a sealed tube. After completion, the reaction mixture was poured into 50 mL of water and was extracted three times with EtOAc. The combined organic layers were washed three times with brine, dried with MgSO 4 , and filtered and concentrated under reduced pressure. The crude compound was purified by chromatography on silica gel (eluent: 9/1 to 5/5 Hexane/EtOAc) to afford 10a (295 mg, 63%) as an off-white solid. 1

Synthesis of 4-Hydrazino-thieno[3,2-d]pyrimidines 10b and 10i
A mixture of compound 5 (1.0 eq.) and the appropriate hydrazine (10 eq.) was stirred and heated under EtOH reflux (3 mL/mmol) in a sealed tube. The reaction mixture was stirred for 4 to 12 h. After completion, the reaction mixture returned to room temperature and was concentrated under reduced pressure. The crude was dissolved in EtOAc and the organic layer was washed three times with brine. The organic layer was dried with MgSO 4 and filtered and concentrated under reduced pressure. The crude was purified by recrystallization in ACN.
N-(tert-butyl)-4-(1-methylhydrazinyl)-6-(p-tolyl)-3,4-dihydrothieno [3,2- Compound 5 (1 eq.) was dissolved in EtOH (22 mL/mmol). The appropriate amine (3 eq.) and Na 2 CO 3 (2 eq.) were added to the solution. The reaction mixture was heated at 35 • C for compound 10c or under reflux for other amines. If the reaction was not completed after 72 h, another portion of the appropriate amine was added, and the heating was continued until completion of the reaction (1 eq. was added for compound 10h; 15 eq. for compound 10c; and 4.5 eq. for other compounds). Then, the mixture returned to room temperature and was concentrated under reduced pressure. The mixture was dissolved in water. The aqueous layer was extracted three times with EtOAc. Organic layers were washed with brine, dried with MgSO 4 , and filtered and concentrated under vacuo. The crude was purified by the appropriate method.  Compound 5 (200 mg, 0.603 mmol) was dissolved in EtOH (17 mL/mmol). Alanine (3 eq., 161 mg, 1.808 mmol) and Na 2 CO 3 (2 eq., 192 mg, 1.808 mmol) were added to the solution. The reaction mixture was heated under reflux for 8 days. Then, the mixture returned to room temperature and was concentrated under reduce pressure. Water (50 mL) was added, and the precipitate was filtered and dried under vacuum at 50 • C overnight. The desired compound was obtained without further purification as a white powder (129 mg, 56%); 1  Compound 5 (1 eq.) was dissolved in THF (15 mL/mmol) (for compound 12a-d) or 1,4-dioxane (for compounds 12e-f). The mixture was degassed, filled with argon. Then, the appropriate boronic acid (2 eq.) was added and the mixture was heated (30 to 40 • C) to solubilize the starting material. A solution of 1 M potassium carbonate in water (2.6 eq.) and Pd(PPh 3 ) 4 (0.02 eq.) were added. The reaction mixture was stirred and heated under reflux until completion of the reaction. The resulting mixture was partitioned between DCM and water. The organic layer was washed with a saturated aqueous solution of sodium bicarbonate, dried with MgSO 4 , and filtered and concentrated under vacuum. The residue was purified by column chromatography. vigorously (700 rpm) for 10 min. The absorbance was measured at 570 nm, with 630 nm as a reference wavelength using a TECAN Infinite F-200 Microplate Reader. DMSO was used as a blank and doxorubicin (purchased from Sigma Aldrich, St. Louis, MO, USA) was used as a positive control. Cell viability was calculated as a percentage of control (cells incubated without compound). The 50% cytotoxic concentration (CC50) was determined from the dose-response curve by using the TableCurve software 2D v.5.0. CC50 values represent the mean value calculated from three independent experiments.

Hepatic-Stage Antiplasmodial Evaluation
Parasite Strains and Sporozoite Isolation P. berghei sporozoites constitutively expressing green fluorescent protein (PbGFP, ANKA strain) [26] were obtained by the dissection of salivary glands from infected Anopheles stephensi mosquitoes bred and infected in the insectary facilities of UMR-S 1135 (CIMI, Paris, France). Infected salivary glands were removed by hand dissection, crushed in a potter, and filtered through a 40 µm filter for sporozoite isolation (Cell Strainer, BD BioSciences, San Jose, CA, USA). The sporozoites were counted using a disposable plastic microscope slide (KOVA).

Primary Hepatocyte Culture
Primary simian hepatocytes were isolated from liver segments collected from healthy Macaca fascicularis from CEA, Fontenay aux Roses, France. All hepatocytes were obtained using collagenase perfusion, as previously described [27], and were immediately cryopreserved. One day before infection, the cryopreserved hepatocytes were thawed at 37 • C and were seeded into collagen-coated (5 µg/cm 2 rat tail collagen I, Invitrogen, Carlsbad, CA, USA) 96-well plate, at a density of 80,000 cells per well. They were cultured at 37 • C in 5% CO 2 in William's E medium (Gibco, Life Technologies, Saint Aubin, France) supplemented with 10% of Fetal Bovine Serum FCIII, 5 × 10 -5 M hydrocortisone hemisuccinate (Serb Laboratories, Paris, France), 5 µg per ml bovine insulin (Sigma Aldrich, St. Louis, MO, USA), 2 mM L-glutamine, and 0.02 U per ml-0.02 µg per ml penicillinstreptomycin (Life Technologies) until infection with sporozoites.
In Vitro Infection and Drug Assays Sporozoites of P. berghei were re-suspended in the above complete medium used for the hepatocytes culture. Simian hepatocytes were inoculated in 50 µL of complete media with 20,000 P. berghei-sporozoites/well in 96-well plates. The infected culture plates were centrifuged for 10 min at 900× g to allow fast parasite sedimentation onto the target cells and were further incubated with the serial dilution of drugs that were prepared in advance. After 3 h of incubation, cultures were washed and further incubated in a fresh medium containing the appropriate drug concentration, which were changed every 24 h during the study period. Cultured hepatocytes were fixed using 4% paraformaldehyde (PFA) for 15 min at room temperature. Host cell and parasite nuclei were labelled with 4 ,6-diamidino-2-phenylindole (DAPI). Upon fixation and immunostaining, cell culture plates were analyzed in order to determine the number and size of the parasites using a Cell Insight High Content Screening platform equipped with the Studio HCS software (Thermo Fisher Scientific, Waltham, MA, USA). The parasite size reduction was calculated on the average object area using the total surface area of each selected object (µm 2 ). To assess the cell toxicity of drugs for hepatic cultures, fixed plates were further scanned for the DAPI signal representing host nuclei. The analysis was based on counting of total cell nuclei. GraphPad Prism 7 statistical software (GraphPad. Software, San Diego, CA, USA) was used for data analysis and graphing. All values were expressed as means and standard deviations (SD).

Conclusions
In summary, we have prepared a new library of 28 thienopyrimidines, based on the structure of a first antiplasmodial hit named Gamhepathiopine (compound 1), identified to be active notably on all stages of P. falciparum. The introduction of various substituents at position 4 of the thienopyrimidine core was studied using aromatic nucleophilic substitutions or pallado-catalyzed reactions on the common 4-chloro derivative 5. Among the 28 compounds evaluated, seven with a halogen or an alkylamino group displayed an EC 50 lower than 2 µM against the erythrocytic stage of P. falciparum. These compounds were then selected and evaluated against the hepatic stage of P. berghei. Six of the seven tested compounds were identified as displaying a dual-stage antiplasmodial activity and being more active against the hepatic stage of P. berghei than compound 1. Compound 10c was the most active, but some toxicity on HepG2 cells was also observed with several of these compounds. Thus, compound 5 showed the best compromise, with an EC 50 of 0.3 µM against the erythrocytic stage of P. falciparum, with moderate toxicity on HepG2 (selective index of 54.7) while presenting better activity than compound 1 against hepatic P. berghei parasites. Further modulations on this position are currently under investigation to increase the antiplasmodial activity and reduce the cytotoxicity of these compounds.