Synthesis and Antiplasmodial Evaluation of 4-Carboxamido- and 4-Alkoxy-2-Trichloromethyl Quinazolines

From three previously identified antiplasmodial hit compounds (A–C) and inactive series (D), all based on a 2-trichloromethylquinazoline scaffold, we conducted a structure-activity relationship (SAR) study at position four of the quinazoline ring by synthesizing 42 novel derivatives bearing either a carboxamido- or an alkoxy-group, to identify antiplasmodial compounds and to enrich the knowledge about the 2-trichloromethylquinazoline antiplasmodial pharmacophore. All compounds were evaluated in vitro for their cytotoxicity towards the HepG2 cell line and their activity against the multiresistant K1 P. falciparum strain, using doxorubicin, chloroquine and doxycycline as reference drugs. Four hit-compounds (EC50 K1 P. falciparum ≤ 2 µM and SI ≥ 20) were identified among 4-carboxamido derivatives (2, 9, 16, and 24) and two among 4-alkoxy derivatives (41 and 44). Regarding the two most potent molecules (16 and 41), five derivatives without a 2-CCl3 group were prepared, evaluated, and appeared totally inactive (EC50 > 50 µM), showing that the 2-trichloromethyl group was mandatory for the antiplasmodial activity.


Introduction
Among parasitic diseases, malaria remains the leading cause of death in 2020. According to the World Malaria Report 2019 [1], some 405,000 deaths were reported in 2018 for 228 million cases of malaria worldwide. Children under five years old are the most vulnerable group, accounting for 67% of the deaths. Malaria in humans is caused by five different species of Plasmodium protozoa, among which, P. falciparum is by far the most lethal, mainly in Africa. P. vivax, responsible for relapses, is mainly found in South-East Asia. The parasites are vectorized during blood-meal by infected female mosquitoes belonging to the Anopheles genus. Huge efforts have been made to control and eradicate the disease, leading to a significant reduction in the number of deaths, from 585,000 in 2010 to 405,000 in 2018 [1]. Despite this improvement, the emergence of forms of resistance to the first line treatments recommended by the WHO, the artemisinin-combination therapies (ACT), currently threaten efforts to control the disease. PfKelch13 mutations have been identified as molecular markers of artemisinin resistance [2]. Recognized since 2002-2004, artemisinin resistance was originally located mainly in the Our group, dedicated to the development of a new antiplasmodial [6][7][8], previously described the antiplasmodial activity of 4-aryl-2-trichloromethylquinazoline derivatives [9]. Indeed, 4-(4′fluorophenyl)-2-trichloromethylquinazoline A (Table 1) showed an efficacy concentration 50% (EC50) of 2.5 µM against the chloroquino-resitant K1 strain of P. falciparum and a cytotoxicity concentration 50% (CC50) above 125 µM toward the human HepG2 cell line, compared with chloroquine, doxycycline and doxorubicin used as reference drugs. Indeed, chloroquine and doxycycline were still in use for antimalarial chemoprophylaxis along with the association of atovaquone and proguanil. Introducing an oxygen atom as a linker between position four of the quinazoline moiety and the phenyl group led to a two-fold improvement in antiplasmodial activity for compound B (Table 1) [10]. To study the Structure-Activity Relationships (SAR) related to the nature of the linker, an atom of carbon was added to provide a two-atom linker in 4-benzyloxy-2-trichloromethylquinazoline derivatives [11]. The resulting modification did not improve antiplasmodial activity, as shown with compound C (Table 1). When introducing a sulfonamide linker, affording 4-arylsulfonamido-2trichloromethylquinazoline derivatives [12], the activity against P. falciparum was lost despite a significant decrease of the cytotoxicity (Table 1).  The aim of current study was to explore other 2-trichloromethylquinazolines bearing a two-atom linker in 4-carboxamido series, analogous to the 4-sulfonamide series D, and in 4-alkoxy series analogous to compounds B or C in aryloxy and benzyloxy series, respectively. The syntheses and in vitro biological evaluations are presented and discussed. Our group, dedicated to the development of a new antiplasmodial [6][7][8], previously described the antiplasmodial activity of 4-aryl-2-trichloromethylquinazoline derivatives [9]. Indeed, 4-(4 -fluorophenyl)-2-trichloromethylquinazoline A (Table 1) showed an efficacy concentration 50% (EC 50 ) of 2.5 µM against the chloroquino-resitant K1 strain of P. falciparum and a cytotoxicity concentration 50% (CC 50 ) above 125 µM toward the human HepG2 cell line, compared with chloroquine, doxycycline and doxorubicin used as reference drugs. Indeed, chloroquine and doxycycline were still in use for antimalarial chemoprophylaxis along with the association of atovaquone and proguanil. Introducing an oxygen atom as a linker between position four of the quinazoline moiety and the phenyl group led to a two-fold improvement in antiplasmodial activity for compound B (Table 1) [10]. To study the Structure-Activity Relationships (SAR) related to the nature of the linker, an atom of carbon was added to provide a two-atom linker in 4-benzyloxy-2-trichloromethylquinazoline derivatives [11]. The resulting modification did not improve antiplasmodial activity, as shown with compound C (Table 1). When introducing a sulfonamide linker, affording 4-arylsulfonamido-2-trichloromethylquinazoline derivatives [12], the activity against P. falciparum was lost despite a significant decrease of the cytotoxicity (Table 1). to control the disease. PfKelch13 mutations have been identified as molecular markers of artemisinin resistance [2]. Recognized since 2002-2004, artemisinin resistance was originally located mainly in the Greater Mekong Subarea [3], and failure rates for first-line ACTs were found to be as high as 93% in Thailand [1]. However, the emergence of potential artemisinin-resistance PfKelch13 mutations was reported in African regions, leading to a major concern [4]. Thus, research efforts need to be pursued with a view to discovering new chemical entities with new mechanisms of action against Plasmodium. Among small molecules displaying antiplasmodial activity, febrifugine (Figure 1), extracted from Dichroa febrifuga, is a natural alkaloid containing a quinazoline scaffold [5]. Our group, dedicated to the development of a new antiplasmodial [6][7][8], previously described the antiplasmodial activity of 4-aryl-2-trichloromethylquinazoline derivatives [9]. Indeed, 4-(4′fluorophenyl)-2-trichloromethylquinazoline A (Table 1) showed an efficacy concentration 50% (EC50) of 2.5 µM against the chloroquino-resitant K1 strain of P. falciparum and a cytotoxicity concentration 50% (CC50) above 125 µM toward the human HepG2 cell line, compared with chloroquine, doxycycline and doxorubicin used as reference drugs. Indeed, chloroquine and doxycycline were still in use for antimalarial chemoprophylaxis along with the association of atovaquone and proguanil. Introducing an oxygen atom as a linker between position four of the quinazoline moiety and the phenyl group led to a two-fold improvement in antiplasmodial activity for compound B (Table 1) [10]. To study the Structure-Activity Relationships (SAR) related to the nature of the linker, an atom of carbon was added to provide a two-atom linker in 4-benzyloxy-2-trichloromethylquinazoline derivatives [11]. The resulting modification did not improve antiplasmodial activity, as shown with compound C (Table 1). When introducing a sulfonamide linker, affording 4-arylsulfonamido-2trichloromethylquinazoline derivatives [12], the activity against P. falciparum was lost despite a significant decrease of the cytotoxicity (Table 1). The aim of current study was to explore other 2-trichloromethylquinazolines bearing a two-atom linker in 4-carboxamido series, analogous to the 4-sulfonamide series D, and in 4-alkoxy series analogous to compounds B or C in aryloxy and benzyloxy series, respectively. The syntheses and in vitro biological evaluations are presented and discussed. to control the disease. PfKelch13 mutations have been identified as molecular markers of artemisinin resistance [2]. Recognized since 2002-2004, artemisinin resistance was originally located mainly in the Greater Mekong Subarea [3], and failure rates for first-line ACTs were found to be as high as 93% in Thailand [1]. However, the emergence of potential artemisinin-resistance PfKelch13 mutations was reported in African regions, leading to a major concern [4]. Thus, research efforts need to be pursued with a view to discovering new chemical entities with new mechanisms of action against Plasmodium. Among small molecules displaying antiplasmodial activity, febrifugine (Figure 1), extracted from Dichroa febrifuga, is a natural alkaloid containing a quinazoline scaffold [5]. Our group, dedicated to the development of a new antiplasmodial [6][7][8], previously described the antiplasmodial activity of 4-aryl-2-trichloromethylquinazoline derivatives [9]. Indeed, 4-(4′fluorophenyl)-2-trichloromethylquinazoline A (Table 1) showed an efficacy concentration 50% (EC50) of 2.5 µM against the chloroquino-resitant K1 strain of P. falciparum and a cytotoxicity concentration 50% (CC50) above 125 µM toward the human HepG2 cell line, compared with chloroquine, doxycycline and doxorubicin used as reference drugs. Indeed, chloroquine and doxycycline were still in use for antimalarial chemoprophylaxis along with the association of atovaquone and proguanil. Introducing an oxygen atom as a linker between position four of the quinazoline moiety and the phenyl group led to a two-fold improvement in antiplasmodial activity for compound B (Table 1) [10]. To study the Structure-Activity Relationships (SAR) related to the nature of the linker, an atom of carbon was added to provide a two-atom linker in 4-benzyloxy-2-trichloromethylquinazoline derivatives [11]. The resulting modification did not improve antiplasmodial activity, as shown with compound C (Table 1). When introducing a sulfonamide linker, affording 4-arylsulfonamido-2trichloromethylquinazoline derivatives [12], the activity against P. falciparum was lost despite a significant decrease of the cytotoxicity (Table 1). The aim of current study was to explore other 2-trichloromethylquinazolines bearing a two-atom linker in 4-carboxamido series, analogous to the 4-sulfonamide series D, and in 4-alkoxy series analogous to compounds B or C in aryloxy and benzyloxy series, respectively. The syntheses and in vitro biological evaluations are presented and discussed. to control the disease. PfKelch13 mutations have been identified as molecular markers of artemisinin resistance [2]. Recognized since 2002-2004, artemisinin resistance was originally located mainly in the Greater Mekong Subarea [3], and failure rates for first-line ACTs were found to be as high as 93% in Thailand [1]. However, the emergence of potential artemisinin-resistance PfKelch13 mutations was reported in African regions, leading to a major concern [4]. Thus, research efforts need to be pursued with a view to discovering new chemical entities with new mechanisms of action against Plasmodium. Among small molecules displaying antiplasmodial activity, febrifugine (Figure 1), extracted from Dichroa febrifuga, is a natural alkaloid containing a quinazoline scaffold [5]. Our group, dedicated to the development of a new antiplasmodial [6][7][8], previously described the antiplasmodial activity of 4-aryl-2-trichloromethylquinazoline derivatives [9]. Indeed, 4-(4′fluorophenyl)-2-trichloromethylquinazoline A (Table 1) showed an efficacy concentration 50% (EC50) of 2.5 µM against the chloroquino-resitant K1 strain of P. falciparum and a cytotoxicity concentration 50% (CC50) above 125 µM toward the human HepG2 cell line, compared with chloroquine, doxycycline and doxorubicin used as reference drugs. Indeed, chloroquine and doxycycline were still in use for antimalarial chemoprophylaxis along with the association of atovaquone and proguanil. Introducing an oxygen atom as a linker between position four of the quinazoline moiety and the phenyl group led to a two-fold improvement in antiplasmodial activity for compound B (Table 1) [10]. To study the Structure-Activity Relationships (SAR) related to the nature of the linker, an atom of carbon was added to provide a two-atom linker in 4-benzyloxy-2-trichloromethylquinazoline derivatives [11]. The resulting modification did not improve antiplasmodial activity, as shown with compound C (Table 1). When introducing a sulfonamide linker, affording 4-arylsulfonamido-2trichloromethylquinazoline derivatives [12], the activity against P. falciparum was lost despite a significant decrease of the cytotoxicity (Table 1). The aim of current study was to explore other 2-trichloromethylquinazolines bearing a two-atom linker in 4-carboxamido series, analogous to the 4-sulfonamide series D, and in 4-alkoxy series analogous to compounds B or C in aryloxy and benzyloxy series, respectively. The syntheses and in vitro biological evaluations are presented and discussed. to control the disease. PfKelch13 mutations have been identified as molecular markers of artemisinin resistance [2]. Recognized since 2002-2004, artemisinin resistance was originally located mainly in the Greater Mekong Subarea [3], and failure rates for first-line ACTs were found to be as high as 93% in Thailand [1]. However, the emergence of potential artemisinin-resistance PfKelch13 mutations was reported in African regions, leading to a major concern [4]. Thus, research efforts need to be pursued with a view to discovering new chemical entities with new mechanisms of action against Plasmodium. Among small molecules displaying antiplasmodial activity, febrifugine (Figure 1), extracted from Dichroa febrifuga, is a natural alkaloid containing a quinazoline scaffold [5]. Our group, dedicated to the development of a new antiplasmodial [6][7][8], previously described the antiplasmodial activity of 4-aryl-2-trichloromethylquinazoline derivatives [9]. Indeed, 4-(4′fluorophenyl)-2-trichloromethylquinazoline A (Table 1) showed an efficacy concentration 50% (EC50) of 2.5 µM against the chloroquino-resitant K1 strain of P. falciparum and a cytotoxicity concentration 50% (CC50) above 125 µM toward the human HepG2 cell line, compared with chloroquine, doxycycline and doxorubicin used as reference drugs. Indeed, chloroquine and doxycycline were still in use for antimalarial chemoprophylaxis along with the association of atovaquone and proguanil. Introducing an oxygen atom as a linker between position four of the quinazoline moiety and the phenyl group led to a two-fold improvement in antiplasmodial activity for compound B (Table 1) [10]. To study the Structure-Activity Relationships (SAR) related to the nature of the linker, an atom of carbon was added to provide a two-atom linker in 4-benzyloxy-2-trichloromethylquinazoline derivatives [11]. The resulting modification did not improve antiplasmodial activity, as shown with compound C (Table 1). When introducing a sulfonamide linker, affording 4-arylsulfonamido-2trichloromethylquinazoline derivatives [12], the activity against P. falciparum was lost despite a significant decrease of the cytotoxicity (Table 1).  The aim of current study was to explore other 2-trichloromethylquinazolines bearing a two-atom linker in 4-carboxamido series, analogous to the 4-sulfonamide series D, and in 4-alkoxy series analogous to compounds B or C in aryloxy and benzyloxy series, respectively. The syntheses and in vitro biological evaluations are presented and discussed. The aim of current study was to explore other 2-trichloromethylquinazolines bearing a two-atom linker in 4-carboxamido series, analogous to the 4-sulfonamide series D, and in 4-alkoxy series analogous to compounds B or C in aryloxy and benzyloxy series, respectively. The syntheses and in vitro biological evaluations are presented and discussed.

Synthesis of 4-Carboxamido-2-Trichloromethylquinazoline Series
We first sought to obtain the target compounds using key intermediate 4-amino-2trichloromethylquinazoline (1), previously described as generating the sulfonamide derivatives belonging to the D series [12]. Thus, using 4-chlorobenzoyl chloride in presence of sodium hydride in DMF, we obtained a mixture of starting material (1) and the formation of dibenzamide (3) resulting from a double substitution of the amino group (Table 2, Entry 1). Surprisingly, no formation of target compound (2) was observed. The same conditions were used at 0 • C and did not afford (2) (Entry 2). Increasing both base and acyl chloride only provided a mixture of (1) and dibenzamide (3) (Entries 3-4). Neither switching solvent types from DMF to THF (Entry 5), nor switching base types from NaH to tBuOK (Entry 6) yielded (2). The use of organic bases such as NaHMDS or NEt 3 was also unsuccessful, in particular with NEt 3 with no conversion observed (Entries 7-9). Finally, in order to isolate the dibenzamide (3), the use of 5 equiv. of NaH led to total conversion and led to (3) with 90% yield (Entry 10). We first sought to obtain the target compounds using key intermediate 4-amino-2trichloromethylquinazoline (1), previously described as generating the sulfonamide derivatives belonging to the D series [12]. Thus, using 4-chlorobenzoyl chloride in presence of sodium hydride in DMF, we obtained a mixture of starting material (1) and the formation of dibenzamide (3) resulting from a double substitution of the amino group (Table 2, Entry 1). Surprisingly, no formation of target compound (2) was observed. The same conditions were used at 0 °C and did not afford (2) (Entry 2). Increasing both base and acyl chloride only provided a mixture of (1) and dibenzamide (3) (Entries 3-4). Neither switching solvent types from DMF to THF (Entry 5), nor switching base types from NaH to tBuOK (Entry 6) yielded (2). The use of organic bases such as NaHMDS or NEt3 was also unsuccessful, in particular with NEt3 with no conversion observed (Entries 7-9). Finally, in order to isolate the dibenzamide (3), the use of 5 equiv. of NaH led to total conversion and led to (3) with 90% yield (Entry 10). Table 2. Studied parameters for the reaction of (1) with 4-chlorobenzoyl chloride.  In view of the ineffectiveness of this synthetic route, we changed tactics and started from 4chloro-2-trichloromethylquinazoline (4), previously described [13] and used by our team in SNAr reactions with various nucleophilic reagents [10,14,15] or Suzuki-Miyaura cross-coupling reactions [9]. Thus, commercial benzamide was deprotonated using NaH in DMF and reacted with chlorimine  In view of the ineffectiveness of this synthetic route, we changed tactics and started from 4-chloro-2-trichloromethylquinazoline (4), previously described [13] and used by our team in S N Ar reactions with various nucleophilic reagents [10,14,15] or Suzuki-Miyaura cross-coupling reactions [9]. Thus, commercial benzamide was deprotonated using NaH in DMF and reacted with chlorimine (4) in DMF, leading to the target compound (5) in 60% yield (Scheme 1). As the conversion of the reaction was total and the yield was only impaired by the purification step, the reaction conditions were not modified and various commercially available substituted benzamides, heteroarylcarboxamides, and alkylcarboxamides were reacted ( Table 3). The benzamides did not show a clear relationship between yields obtained (from 54 to 98%) and their electron-donating/-withdrawing behavior, nor between yields and substituents position borne by their phenyl ring (3, 5-16). For pyridine-containing carboxamides (17)(18)(19), poor to good yields were obtained (22-81%) related to purification issues in silica gel chromatography, even though the silica was deactivated by NEt 3 . With alkylcarboxamides, yields were generally lower than with benzamides (31-55%) (20-25).

Biological Evaluations
All synthesized molecules were then evaluated in vitro against the multi-resistant K1 P. falciparum strain, by determining their 50% efficacy concentration (EC50), and compared with two antimalarial drug-compounds: chloroquine and doxycycline. In parallel, these molecules were assessed in vitro on the HepG2 human hepatocyte cell line, by determining their 50% cytotoxic concentrations (CC50) and comparing them to that of doxorubicin, used as a cytotoxic reference drugcompound, in order to calculate their respective selectivity indices (SI = CC50/EC50). The results are presented in Tables 3 and 4.

SAR of 4-Carboxamido-2-Trichloromethylquinazoline Series
For some synthesized compounds, lack of solubility hampered full determination of cytotoxicity (8, 11, 17, 20-21, and 24) but not evaluation of antiplasmodial activity. It should be noted that in series D, there was no issue concerning solubility of sulfonamide derivatives [12].

Biological Evaluations
All synthesized molecules were then evaluated in vitro against the multi-resistant K1 P. falciparum strain, by determining their 50% efficacy concentration (EC50), and compared with two antimalarial drug-compounds: chloroquine and doxycycline. In parallel, these molecules were assessed in vitro on the HepG2 human hepatocyte cell line, by determining their 50% cytotoxic concentrations (CC50) and comparing them to that of doxorubicin, used as a cytotoxic reference drugcompound, in order to calculate their respective selectivity indices (SI = CC50/EC50). The results are presented in Tables 3 and 4.

SAR of 4-Carboxamido-2-Trichloromethylquinazoline Series
For some synthesized compounds, lack of solubility hampered full determination of cytotoxicity (8, 11, 17, 20-21, and 24) but not evaluation of antiplasmodial activity. It should be noted that in series D, there was no issue concerning solubility of sulfonamide derivatives [12].

Biological Evaluations
All synthesized molecules were then evaluated in vitro against the multi-resistant K1 P. falciparum strain, by determining their 50% efficacy concentration (EC50), and compared with two antimalarial drug-compounds: chloroquine and doxycycline. In parallel, these molecules were assessed in vitro on the HepG2 human hepatocyte cell line, by determining their 50% cytotoxic concentrations (CC50) and comparing them to that of doxorubicin, used as a cytotoxic reference drugcompound, in order to calculate their respective selectivity indices (SI = CC50/EC50). The results are presented in Tables 3 and 4.

SAR of 4-Carboxamido-2-Trichloromethylquinazoline Series
For some synthesized compounds, lack of solubility hampered full determination of cytotoxicity (8, 11, 17, 20-21, and 24) but not evaluation of antiplasmodial activity. It should be noted that in series D, there was no issue concerning solubility of sulfonamide derivatives [12]. For sufficiently soluble 20 23

Biological Evaluations
All synthesized molecules were then evaluated in vitro against the multi-resistant K1 P. falciparum strain, by determining their 50% efficacy concentration (EC50), and compared with two antimalarial drug-compounds: chloroquine and doxycycline. In parallel, these molecules were assessed in vitro on the HepG2 human hepatocyte cell line, by determining their 50% cytotoxic concentrations (CC50) and comparing them to that of doxorubicin, used as a cytotoxic reference drugcompound, in order to calculate their respective selectivity indices (SI = CC50/EC50). The results are presented in Tables 3 and 4.

SAR of 4-Carboxamido-2-Trichloromethylquinazoline Series
For some synthesized compounds, lack of solubility hampered full determination of cytotoxicity (8, 11, 17, 20-21, and 24) but not evaluation of antiplasmodial activity. It should be noted that in series D, there was no issue concerning solubility of sulfonamide derivatives [12].

Biological Evaluations
All synthesized molecules were then evaluated in vitro against the multi-resistant K1 P. falciparum strain, by determining their 50% efficacy concentration (EC 50 ), and compared with two antimalarial drug-compounds: chloroquine and doxycycline. In parallel, these molecules were assessed in vitro on the HepG2 human hepatocyte cell line, by determining their 50% cytotoxic concentrations (CC 50 ) and comparing them to that of doxorubicin, used as a cytotoxic reference drug-compound, in order to calculate their respective selectivity indices (SI = CC 50 /EC 50 ). The results are presented in Tables 3  and 4.
Regarding the alkylcarboxamides, acetamide (20) was not active (>10 µM). The addition of carbon atoms to the alkyl chain led to more cluttered analogs that displayed similar activity to benzamides, like analogs (23) or (24) (1.8 and 1.6 µM). Cyclohexylcarboxamide (25) activity was slightly lower than that of terbutylcarboxamide (23). In conclusion, to provide the best antiplasmodial activity with good SI, para-substituted benzamides with chlorine, fluorine, methoxy, or non-cyclic alkylcarboxamide with fatty carbon chain were required.

SAR of 4-Alkoxy-2-Trichloromethylquinazoline Series
For the whole series, the solubility of the compounds was acceptable. Apart from alcohol (34) with a cytotoxicity value of 7.9 µM, the rest of the series was not cytotoxic (23.4-101.5 µM), in comparison with doxorubicin. In this alkoxy series, amino-containing side chain were the most potent derivatives. We previously showed that 2-(trichloromethyl)quinazolin-4-ol (R = H) was not active against W2 P. falciparum [10]. Interestingly, when R = Me (26), a moderate activity was observed (EC 50 = 7.1 µM), increasing with the number of carbons in the alkyl chain, up to 3 carbon atoms. The best activity (3.5 µM) was obtained with a propoxy substituent (28), whereas with butoxy (29) the activity decreased again (5.8 µM), the compound even becoming inactive with the bulkier isopropoxy group (30). Compound (31) resulting from the reaction of propargyl alcohol showed a modest activity, which was lost with substituted propargyl alcohols (32-33). Compound (34) bearing a 2-hydroxyethoxy group was almost inactive (10 µM). Replacing the hydroxyl group of (34) by a methoxy group (35) or a chlorine atom (36) afforded more active derivatives (2.3 and 2.2 µM). The addition of one (37) or two atoms of carbon (38) in the chain of (36) decreased the activity two-fold (4.2 and 4.3 µM). The replacement of the chlorine atom of (36) by a fluorine atom (39) led to a decrease in activity (8.0 µM) while a bromine atom (40) preserved it. Among aminoethoxy derivatives (41-44), diethylamino (41) was the most potent, with a submicromolar antiplasmodial activity (0.9 µM) and a SI of 35.9. Cyclic analogs (43) and (44) were slightly less active than (41) whereas acetamide (42) was clearly less potent. In conclusion, we succeed in obtaining 4-O-substituted-quinazoline from inactive derivative 4-alcohol [10] to submicromolar active compounds in 4-alkoxyamino series.
To confirm the key role played by the 2-trichloromethylquinazoline moiety of the most potent compounds (16) and (41) in each studied series, derivatives without a -CCl 3 group were synthesized for the two hit molecules: dehalogenated analogs (45, 47), 2-trifluromethyl analogs (46, 48) and unsubstituted analog (49) (Figures 2 and 3). All molecules were inactive against P. falciparum, illustrating the key role played by the -CCl 3 group and consistent with our previous results. Thus, activity cliffs (ACs) [16] were observed when the -CCl 3 group was replaced by similar group (-CF 3 , CH 3 ) or atom (H) as a 50-fold drop of antiplasmodial activity.

General
Melting points were determined on a Köfler melting point apparatus (Wagner & Munz GmbH, München, Germany) and are uncorrected. Elemental analyses were carried out at the Spectropole, Faculté des Sciences de Saint-Jêrome (Marseille) with a Thermo Finnigan EA1112 analyzer (Thermo Finnigan, San Jose, CA, USA). NMR spectra were recorded on a Bruker AV (Billerica, MA, USA) 200 or AV 250 spectrometers or a Bruker Avance NEO 400MHz NanoBay spectrometer at the Faculté de Pharmacie of Marseille or on a Bruker Avance III nanobay 400 MHz spectrometer at the Spectropole, Faculté des Sciences de Saint-Jêrome (Marseille). ( 1 H NMR: reference CHCl3 δ = 7.26 ppm, reference DMSO-d6 δ = 2.50 ppm and 13 C NMR: reference CHCl3 δ = 76.9 ppm, reference DMSO-d6 δ = 39.52 ppm). The following adsorbent was used for column chromatography: silica gel 60 (Merck KGaA, Darmstadt, Germany, particle size 0.063-0.200 mm, 70-230 mesh ASTM). TLC was performed on 5 cm × 10 cm aluminum plates coated with silica gel 60F-254 (Merck) in an appropriate eluent. Visualization was performed with ultraviolet light (234 nm). Purity of synthesized compounds was checked by LC/MS analyses, which were realized at the Faculté de Pharmacie of Marseille with a Thermo Scientific Accela High Speed LC System ® (Waltham, MA, USA) coupled using a single quadrupole mass spectrometer Thermo MSQ Plus ® . The RP-HPLC column is a Thermo Hypersil Gold ® 50 × 2.1 mm (C18 bounded), with particles of a diameter of 1.9 mm. The volume of sample injected on the column is 1 µL. Chromatographic analysis, total duration of 8 min, is on the gradient of the following solvents: t = 0 min, methanol/water 50:50; 0 < t < 4 min, linear increase in the proportion of methanol to a methanol/water ratio of 95:5; 4 < t < 6 min, methanol/water 95:5; 6 < t < 7 min, linear decrease in the proportion of methanol to return to a methanol/water ratio of 50:50; 6 < t < 7 min, methanol/water 50:50. The water used was buffered with ammonium acetate 5 mM. The flow rate of the mobile phase was 0.3 mL/min. The retention times (tR) of the molecules analyzed are indicated in min. The microwave reactions were performed using multimode reactors: ETHOS Synth Lab station and MicroSYNTH ® Lab terminal 1024 (Ethos start, MLS GmbH, Leutkirch, Germany.); or monomode reactors: Biotage Initiator ® classic in sealed vials with a power output of 0 to 400 W. 4-Chloro-2-methylquinazoline, 4-chloro-2-trifluoromethylquinazoline, and 4-chloroquinazoline were purchased from Sigma-Aldrich (Saint Louis, MO, USA) or Fluorochem (Derbyshire, UK). The The mechanism of action of the 2-trichloromethylquinazolines is not known, but it was clearly related to the presence of the -CCl 3 group, which could act as a possibly alkylating group as methyl and trifluoromethyl analogues were inactive or the -CCl 3 group could generate sigma-holes [17] with sulphur nucleophiles in cysteine proteases such as Falcipains as potential plasmodial targets [18].

General
Melting points were determined on a Köfler melting point apparatus (Wagner & Munz GmbH, München, Germany) and are uncorrected. Elemental analyses were carried out at the Spectropole, Faculté des Sciences de Saint-Jêrome (Marseille) with a Thermo Finnigan EA1112 analyzer (Thermo Finnigan, San Jose, CA, USA). NMR spectra were recorded on a Bruker AV (Billerica, MA, USA) 200 or AV 250 spectrometers or a Bruker Avance NEO 400MHz NanoBay spectrometer at the Faculté de Pharmacie of Marseille or on a Bruker Avance III nanobay 400 MHz spectrometer at the Spectropole, Faculté des Sciences de Saint-Jêrome (Marseille). ( 1 H NMR: reference CHCl 3 δ = 7.26 ppm, reference DMSO-d 6 δ = 2.50 ppm and 13 C NMR: reference CHCl 3 δ = 76.9 ppm, reference DMSO-d 6 δ = 39.52 ppm). The following adsorbent was used for column chromatography: silica gel 60 (Merck KGaA, Darmstadt, Germany, particle size 0.063-0.200 mm, 70-230 mesh ASTM). TLC was performed on 5 cm × 10 cm aluminum plates coated with silica gel 60F-254 (Merck) in an appropriate eluent. Visualization was performed with ultraviolet light (234 nm). Purity of synthesized compounds was checked by LC/MS analyses, which were realized at the Faculté de Pharmacie of Marseille with a Thermo Scientific Accela High Speed LC System ® (Waltham, MA, USA) coupled using a single quadrupole mass spectrometer Thermo MSQ Plus ® . The RP-HPLC column is a Thermo Hypersil Gold ® 50 × 2.1 mm (C 18 bounded), with particles of a diameter of 1.9 mm. The volume of sample injected on the column is 1 µL. Chromatographic analysis, total duration of 8 min, is on the gradient of the following solvents: t = 0 min, methanol/water 50:50; 0 < t < 4 min, linear increase in the proportion of methanol to a methanol/water ratio of 95:5; 4 < t < 6 min, methanol/water 95:5; 6 < t < 7 min, linear decrease in the proportion of methanol to return to a methanol/water ratio of 50:50; 6 < t < 7 min, methanol/water 50:50. The water used was buffered with ammonium acetate 5 mM. The flow rate of the mobile phase was 0.3 mL/min. The retention times (t R ) of the molecules analyzed are indicated in min. The microwave reactions were performed using multimode reactors: ETHOS Synth Lab station and MicroSYNTH ® Lab terminal 1024 (Ethos start, MLS GmbH, Leutkirch, Germany.); or monomode reactors: Biotage Initiator ® classic in sealed vials with a power output of 0 to 400 W. 4-Chloro-2-methylquinazoline, 4-chloro-2-trifluoromethylquinazoline, and 4-chloroquinazoline were purchased from Sigma-Aldrich (Saint Louis, MO, USA) or Fluorochem (Derbyshire, UK). The following Supplementary Materials are available online: 1H-NMR, 13C-NMR and HRMS data spectra of compounds 2, 9, 16, 24, 41, 44, 45, 46, 48 and 49.

General Procedure for the Preparation of Compounds (2), (5-25)
To a solution of the appropriate carboxamide compound (1.06 mmol, 1.5 equiv.) in dry DMF (3 mL) at 0 • C under N 2 , 60% sodium hydride in oil (25.5 mg, 1.06 mmol, 1.5 equiv) were added portion wise. The resulting mixture were added dropwise to a solution of 4-chloro-2-(trichloromethyl)quinazoline (4) (200 mg, 0.71 mmol, 1.0 equiv.) in dry DMF (2 mL) at 0 • C under N 2 . The reaction was stirred overnight at rt. Then, the excess of NaH was hydrolyzed with ice. The reaction mixture was extracted with EtOAc and washed three times with brine. The organic layer was dried with Na 2 SO 4 , filtered, and evaporated. The crude product was purified by silica gel column chromatography and recrystallized from appropriate solvent to give the desired compound.