Pd-catalyzed C–C and C–N cross-coupling reactions in 2-aminothieno[3,2-d]pyrimidin-4(3H)-one series for antiplasmodial pharmacomodulation

In 2015, we identified gamhepathiopine (M1), a 2-tert-butylaminothieno[3,2-d]pyrimidin-4(3H)-one antiplasmodial hit targeting all development stages of the human malarial parasite P. falciparum. However, this hit compound suffers from sensitivity to hepatic oxidative metabolism. Herein, we describe the synthesis of 33 new compounds in the 2-aminothieno[3,2-d]pyrimidin-4(3H)-one series modulated at position 6 of this scaffold. The modulations were performed using three palladium-catalyzed cross coupling reactions, namely Suzuki–Miyaura, Sonogashira, and Buchwald–Hartwig. For the latter, we developed the reaction conditions. Then, we evaluated the synthesized compounds for their antiplasmodial activity on the K1P. falciparum strain and their cytotoxicity on the human HepG2 cell line. Although we did not obtain a compound better than M1 in terms of the antiplasmodial activity, we identified compound 1g bearing a piperidine at position 6 of the thieno[3,2-d]pyrimidin-4(3H)-one ring with an improved cytotoxicity and metabolic stability. 1g is an interesting new starting point for further pharmacomodulation studies. This study also provides valuable antiplasmodial SAR data regarding the nature of the ring at position 6, the possible substituent on this ring, and the introduction of a spacer between this ring and the thienopyrimidinone moiety.


Introduction
Malaria is a heavy burden for endemic countries, mainly in Africa, with an estimated 241 million cases and 627 000 deaths in 2020, according to the World Health Organization. 1 Seventy seven percent of the victims were children under 5 years old.The disease is caused by parasites of the Plasmodium genus transmitted by the bite of an infected female mosquito belonging to the Anopheles genus.Of the ve species causing malaria in humans, P. falciparum is responsible for the most deaths.Along with the spread of long-lasting insecticide-treated bed nets, the introduction of artemisinin-based combined therapies (ACT) as rst line treatments against severe P. falciparum infections were key to the ght against malaria. 2 However, the emergence of artemisinin-resistant P. falciparum in South-East Asia rst reported in 2009, 3 mediated by Pfkelch13 mutations, 4 and its spread in the greater Mekong subregion is currently limiting the clinical effectiveness of ACT in this area. 5While the combination of ganaplacide (Fig. 1) and lumefantrine is the most promising drug combination currently being investigated by Medicines for Malaria Venture (MMV), 6 the recent withdrawal of MMV048, P218, and DSM265 in clinical trials (for teratogenicity, low half-time in vivo, and high selection of resistances, respectively; Fig. 1) 7,8 increases the urgent need for new antimalarial compounds with novel mechanisms of action.
In 2015, we described the synthesis and activity of a new molecule called gamhepathiopine (also M1, Scheme 1) that is active on all stages of P. falciparum, including quiescent parasites of the artemisinin-resistant strain F32-ART5, with an unknown mechanism of action. 9We later discovered that the in vivo activity of M1 was limited by its sensitivity to mouse hepatic metabolism.Indeed, the in vivo activity in P. berghei infected mice was only achieved with the addition of 1-aminobenzotriazole, a pan-CYP450 inhibitor. 10Therefore, the synthesis of new thieno [3,2-d]pyrimidines to afford a new hit compound with improved metabolic stability and similar activity prole was initiated.We also expected to obtain new antiplasmodial SAR data on the thieno [3,2-d]pyrimidine series.Indeed, we observed important modications to the biological values with minor modications on the molecular scaffold during the rst round of pharmacomodulations, leading to compound M1. 9 Previously explored substituents on the phenyl ring at position 6 of the thienopyrimidinone scaffold were simple, i.e., halogen atoms or methoxy group, but some of them maintained good antiplasmodial potencies.Moreover, on M1, position 6 (and position 2) is sensitive to oxidative metabolism because of the methyl group. 10Thus, we decided to explore more diverse substituents at this position with the help of palladium-catalyzed cross-coupling reactions.Herein, we describe the synthesis of 2-aminothieno [3,2-d]pyrimidin4(3H)ones, structurally modulated on the west part of the scaffold at position 6 (Scheme 1) by Buchwald-Hartwig, Sonogashira, or Suzuki-Miyaura cross-coupling reactions, along with their antiplasmodial and cytotoxicity evaluations.
First, we used the conditions described in the literature involving a thiophene ring (entry 1, 11 entries 2-3, 12 and entries 4-5, 13 Table 1).We did not observe any reaction with these conditions.From the conditions of entry 5, we then decided to study the inuence of the phosphine ligand using SPhos instead of RuPhos (entry 6, Table 1).While we observed 1a for the rst time, the main product of the reaction resulted from the dehalogenation of the starting material.Also, from entry 5, we decided to improve the solubility of starting material 1 by changing the reaction solvent from toluene to dioxane (entry 6, Table 1).We observed the formation of 1a in an estimated 4% yield.By combining the conditions of entries 6 and 7, the estimated yield increased to 46% (entry 8, Table 1).Using the closely related XPhos phosphine, instead of SPhos, the same estimated yield was maintained (entry 9, Table 1).However, tBuXPhos, a more hindered version of XPhos, did not allow the formation of 1a.From entry 8, we tried to explore the changes in the base and the solvent using LiHMDS instead of NaOtBu (entry 11, Table 1) and using DME instead of dioxane (entry 12, Table 1).The use of LiHMDS was unsuccessful in affording 1a, while DME caused a signicant dehalogenation of 1. From entries 8 and 9, we decided to increase the load of p-toluidine in the reaction (entries 13 and 14, Table 1).We observed an increase in the estimated yield with full conversion in 1 h when XPhos was used.From these conditions, we reduced the load of p-toluidine or the phosphine/palladium duo (entries 15 and 16, Table 1), and we observed only an increase in the reaction time from 1 to 2 h.We nally decided to use the conditions of entry 16 thereaer since amines are cheap starting materials compared to palladium and phosphine.
The Ullman reaction, a possible alternative to the Buchwald-Hartwig coupling reaction, was also tried on compound 1 or its precursor 2. In both cases, no reaction was observed aer 24 h at 100 C with the following tested conditions: para-toluidine (3 eq.), dimethylglycine (20 mol%), CuI (10 mol%), and Cs 2 CO 3 (3 eq.).
Therefore, the conditions of entry 16 (Table 1) were then applied on various substrates to explore the scope of the operating procedure and obtain a new series of functionalized 2tertbutylaminothieno[3,2-d]pyrimidin-4(3H)-one (Table 2).
Purications, using ash chromatography, were challenging for the whole series, thus leading to a moderate isolated yield of 55% for compound 1a.The isolated yields for the other compounds were decreased by two additional factors-incomplete conversion and dehalogenation of 1.When moving the methyl substituent from the para position to the ortho (1c) position, the conversion was not complete aer 24 h.Conversion was also incomplete aer 24 h of heating when the methyl group was replaced by an electron-donating (1d) or electronwithdrawing (1e) group.When performing the reaction with morpholine (1f) or piperidine (1g), we also observed moderate dehalogenation of the starting material in addition to incomplete conversion aer 24 h, both reasons being responsible for the low reaction yields.For N-methylpiperazine (1h), we observed a complete and rapid (less than 2 h) dehalogenation of the starting material.The same result was observed with alkyl amines such as cyclopropylamine (1l) and ethanolamine (1m).This dehalogenation could be caused by the choice of our catalyst ligand. 14Finally, heteroaromatic amines, such as pyrazole, imidazole, and 3-aminopyridine, did not react using the same conditions.Although our conditions seemed to work for a simple substrate such as para-toluidine, they are strongly impacted by the modication of the para-substituent and the nature of the amine used.
We applied these Buchwald-Hartwig conditions to compound 2, the synthetic precursor of compound 1, to possibly overcome the dehalogenation problem encountered with aliphatic amines and N-methylpiperazine (Table 3).While our conditions properly functionalized position 5 of the thiophene aminoester, we also observed a partial saponication of   the methyl ester group.The corresponding carboxylic acid derivative could not be isolated because of its high polarity.We hypothesized that sodium tertbutoxyde could saponify the ester at position 2 of thiophene.We decided to explore other bases to observe the behavior of the ester; caesium carbonate promoted the homo-coupling of compound 2, while LiHMDS led to the formation of a cyclopropylamide derivative.The replacement of the phosphine ligand with caesium carbonate did not cause any change and the homo-coupling product was still formed.

Sonogashira coupling
From the reaction conditions found in the literature, 15 we performed a conclusive test on compound 2 (Scheme 2).We then used these reaction conditions on compound 1 and its isopropylamine analog 3, leading to seven 2-aminothieno[3,2-d] pyrimidin-4(3H)-ones bearing an alkyne substituent at position 6 in yields ranging from 23 to 85% (Table 4).The poor yields are due either to purication difficulties (compound 2c and 2g) or the volatile behavior of some alkynes (compound 2e).Depending on the boiling point of the alkyne, we tried to improve the low yields by increasing the load of the alkyne in the reaction, but still observed low yields.Compound 2f was obtained aer the protodesilylation of the trimethylsilyl alkyne using potassium carbonate in a mixture of methanol and dichloromethane.

Suzuki-Miyaura coupling
When Suzuki-Miyaura coupling was performed on compound 1 or 3, applying the reaction conditions used for the synthesis of  M1 metabolites, 10 we observed the dehalogenation of the starting material, which complicated the purication.Therefore, we decided to perform Suzuki-Miyaura coupling on 2 with conditions from the literature and previously used in our lab. 16leven thiophene aminoesters functionalized at position 5 were synthesized in yields above 50%, except for compound 3l (Table 5).In this specic case, we removed water from the reaction medium to avoid the substitution of the chlorine atom contained in the chloroimine moiety.The chlorine atom of compound 3l was then engaged in a S N Ar reaction with morpholine or N-methylpiperazine (Scheme 3).The functionalized thiophene aminoesters (3a-3k, 3m, and 3n) were then engaged in a cyclization reaction to form the thieno [3,2-d]pyrimidin-4(3H)-one core.This three step one-pot reaction afforded 15 compounds, 13 including a tertbutylamine group and 2 bearing an isopropyl group at position 2 (Table 6).The 4-nitrophenyl-containing compounds 4b and 4c were then engaged in a reduction reaction to afford the 4-aminophenyl compounds 4p and 4q in poor yields (Scheme 4).We initially performed this reduction at 120 C for 30 min, but this led to the formation of compound 4r, resulting from the acylation of compound 4p, which was isolated.Compound 4j was used to synthesize the sulfoximine derivative 4s (Scheme 4) using a one-step reaction with ammonium carbamate and (diacetoxyiodo)benzene (PIDA).

Biological evaluation
The synthesized compounds were evaluated for their antiplasmodial activity (EC 50 ) on the multi-resistant K 1 strain of P. falciparum (resistant to chloroquine, sulfadoxine, and pyrimethamine) and their cytotoxicity (CC 50 ) on the human HepG2 cell-line in order to determine their selectivity index (SI, EC 50 / CC 50 ) and to compare them to reference drugs (chloroquine, atovaquone, and doxycycline for activity and doxorubicine for cytotoxicity; Table 7).Of the 33 tested compounds, 16 displayed aqueous solubility problems in the test medium, limiting the determination of precise values for EC 50 , CC 50 , or both.Such behavior for the thieno [3,2-d]pyrimidin-4(3H)-one series was already seen during the pharmacomodulation, leading to compound M1. 9 The thienopyrimidinone scaffold is found in cytotoxic compounds, 17 but apart from compound 4r (CC 50 ¼ 0.16 mM, more toxic than doxorubicine), the CC 50 of the compounds ranged widely from 2.1 to 89.1 mM, while the EC 50 ranged from 0.77 to 43.2; 18 compounds showed an antiplasmodial activity below 5 mM.Compound 1g was the best among all the synthesized compounds with submicromolar antiplasmodial activity (0.77 mM), which is still far from the M1 potency (0.045 mM) but with improved cytotoxicity (33.9 mM) compared to M1 (24 mM). 9 These results provided valuable data regarding the SAR of the thieno [3,2-d]pyrimidin-4(3H)-one core.
The introduction of an amine spacer between the thienopyrimidinone core and the C6-substituent (1a) signicantly reduced the antiplasmodial activity.The cytotoxicity value was worsened when the methyl was moved on the phenyl ring (para 1a < ortho 1c < meta 1b).The replacement of the methyl group by an electron-donating (1d) or withdrawing (1e) group moderately   improved the antiplasmodial activity.The introduction of an alkyne spacer in the M1 structure (2b) reduced the antiplasmodial activity and aqueous solubility in the test medium of the compound.Among the different alkyne substituents, only cyclopropylalkyne 2e showed a decent combination of the biological values.Therefore, the conformational changes of the C6-Scheme 3 S N Ar reaction on compound 3l.sidechain induced by the amine spacer or sidechain elongation following the introduction of an alkyne are not effective strategies to obtain an antiplasmodial hit compound in the thienopyrimidinone series.
We also investigated the para-substituent on the phenyl ring at position 6 of the thienopyrimidinone core.The replacement of the methyl substituent by a more polar group to potentially improve the aqueous solubility (methyl ester 4a, phenol 4d, anilines 4p and 4q and acetamide 4r, and sulphur-containing functions 4j-4m and 4s) was favorable for neither the antiplasmodial activity nor the cytotoxicity, while the replacement of the methyl by a uorine atom was interesting in our previous work; 9 the addition of a chlorine atom at position 3 of the 4-uorophenyl cycle induced a loss of activity and aqueous solubility in the test medium (4e and 4f) compared to M1.We also replaced the methyl substituent by different saturated heterocycles (4g, 4h and 4i), but this was associated with an increased cytotoxicity and reduced antiplasmodial activity.The cytotoxicity values were improved when the phenyl ring was replaced by a pyrimidine (4n and 4o).
Finally, Buchwald-Hartwig cross-couplings gave us the opportunity to replace the phenyl ring at position 6 by saturated heterocycles.When the phenyl ring is replaced by a piperidine (1g), we observed a moderate loss of activity and improved cytotoxicity.The addition of an oxygen atom in the cyclic amine, leading to morpholine 1f, signicantly decreased the antiplasmodial activity.
With this observation and the biological results of compounds bearing a polar substituent (4a, 4d, 4g, 4h, and 4k-4s), we concluded that a polar head (linked to c log D 7.4 decrease) at this position is detrimental to the antiplasmodial activity (Fig. 2).We also concluded that a ring-type structure (aromatic or saturated) is required at position 6 since compounds lacking one of these structures (1, 3, 2d, and 2f) displayed a lack of antiplasmodial activity.
Considering 1g gave good biological results, it was evaluated in vitro for its mouse microsomal stability.We observed a more than 3-fold improvement in the half-time between the tolyl moiety (M 1 , t 1/2 ¼ 11 min, Cl int ¼ 240 mLmin À1 mg) and the piperidine moiety (1g, t 1/2 ¼ 40 min, Cl int ¼ 42 mLmin À1 mg).This important improvement shows that the piperidine moiety is stable.

Conclusion
We synthesized 33 original compounds in the thieno [3,2-d]  pyrimidin-4(3H)-one series using three different palladiumcatalyzed cross-coupling reactions, namely Buchwald-Hartwig, Sonogashira, and Suzuki-Miyaura.For the rst time, we developed reaction conditions to directly functionalize the position 6 of the thienopyrimidinone core with anilines and saturated heterocycles.However, additional work on this reaction is required to extend the scope of useable substrates and to improve the reaction yields.Sonogashira coupling provided compounds with the side chains of various natures.However, the introduction of the alkyne spacer that reduced the antiplasmodial activity led us to stop the further synthesis of 6alkynyl-thieno[3,2-d]pyrimidin-4(3H)-ones.Seventeen compounds were obtained using Suzuki-Miyaura coupling with various substituents on the phenyl ring, and there are still a lot of possibilities to explore with this coupling.The synthesized compounds were tested on the blood-stage of PfK 1 and the human HepG2 cell-line to measure their antiplasmodial activity and cytotoxicity.This work led us to the discovery of compound 1g, which has submicromolar antiplasmodial activity that is associated with a better cytotoxicity value than compound M1 and an improved in vitro metabolic stability compared to M1.
Moreover, this work provided valuable RSA data on the thienopyrimidinone series (Fig. 2).Considering the microsomal stability of the piperidine moiety, 1g is an interesting starting point to synthesize new thieno [3,2-d]pyrimidines bearing, at position 6, a substituted piperidine.The substitution on this piperidine could provide us compounds with biological values at par with the M1 antiplasmodial prole and further improve the metabolic stability of the compounds..50 pm for 1 H and 39.52 ppm for 13 C. Data for 1 H NMR are reported as follows: chemical shis (d) in parts per million (ppm), multiplicity (described as follows: s, singlet; bs, broad singlet; d, doublet; t, triplet; q, quadruplet; dd, doublet of doublet; ddd, doublet of doublet of doublet; m, multiplet), coupling constants (J) in Hertz (Hz), and integration.Data for 13 C NMR are reported as follows: chemical shis (d) in parts per million (ppm).Melting points were determined on a Köer melting point apparatus (Wagner & MunzGmbH, München, Germany) and are uncorrected.HRMS spectra (ESI) were recorded on a SYNAPT G2 HDMS (Waters) and were performed at the "Faculté des Sciences" of Marseille (St Jérôme campus).Silica Gel 60 (Merk 70-230) was used for column chromatography.Flash chromatography was performed on a puriFlash® 5.020 apparatus (Interchim, Montluçon, France).TLC was performed on aluminium plates coated with silica gel 60F-254 (Merck) using an appropriate eluent.Visualization was carried out with ultraviolet light (254 or 365 nm).Reactions using microwave heating were performed with a Biotage® Initiator or Initiator + apparatus (Biotage, Uppsala, Sweden).
If a precipitate was formed, this precipitate was ltered, washed with water, and dried in vacuo.If needed, the precipitate was then puried with the appropriate method.
If no precipitate was formed, the mixture was extracted with ethyl acetate.The organic layer was washed with brine, dried over sodium sulphate, and the excess solvent was removed in vacuo.Then, the obtained crude was puried with the appropriate method.
If a precipitate was formed, this precipitate was ltered, washed with water, and dried in vacuo.If needed, the precipitate was then puried with the appropriate method.
If no precipitate was obtained, the mixture was extracted with ethyl acetate.The organic layer was washed with brine, dried over sodium sulphate, and the excess solvent was removed in vacuo.Then, the obtained crude was puried with the appropriate method.

Biology
4.2.1.Antiplasmodial evaluation.In this study, the K 1 culture-adapted P. falciparum strain resistant to chloroquine, pyrimethamine, and proguanil was used in an in vitro culture.It was maintained in continuous culture, as described previously by Trager and Jensen. 18Cultures were maintained in fresh A + human erythrocytes at 2.5% hematocrit in complete medium (RPMI 1640 with 25 mM HEPES, 25 mM NaHCO 3 , 10% of A + human serum) at 37 C under reduced O 2 atmosphere (gas mixture 10% O 2 , 5% CO 2 , and 85% N 2 ).Parasitemia was maintained daily between 1 and 3%.The P. falciparum drug susceptibility test was carried out by comparing quantities of DNA in treated and control cultures of parasite in human erythrocytes according to a SYBR Green I uorescence-based method 19 using a 96-well uorescence plate reader.Compounds previously dissolved in DMSO (nal concentration less than 0.5% v/v) were incubated in a total assay volume of 200 mL (RPMI, 2% hematocrit and 0.4% parasitemia) for 72 h in a humidied atmosphere (10% O 2 and 5% CO 2 ) at 37 C in 96well at bottom plates.Duplicate assays were performed for each sample.Aer incubation, plates were frozen at 20 C for 24 h.Then, the frozen plates were thawed for 1 h at 37 C. Fieen mL of each sample were transferred to 96-well at bottom non-sterile black plates (Greiner Bio-one, Kremsmünster, Austria), already containing 15 mL of the SYBR Green I lysis buffer (2X SYBR Green I, 20 mM Tris base pH 7.5, 20 mM EDTA, 0.008% w/v saponin, 0.08% w/v Triton X-100).Negative control treated by solvents (DMSO or H 2 O) and positive controls (chloroquine and doxycycline) were added to each set of experiments.Plates were incubated for 15 min at 37 C and then read on a TECAN Innite F-200 spectrophotometer with excitation and emission wavelengths at 485 and 535 nm, respectively.The concentrations of compounds required to induce a 50% decrease in parasite growth (EC 50 K 1 ) were calculated from three independent experiments.4.2.2.Cytotoxic evaluation.The HepG2 cell line was maintained at 37 C, 5% CO 2 , at 90% humidity in MEM supplemented with 10% fetal bovine serum, 1% L-glutamine (200 mM), and penicillin (100 U per mL)/streptomycin (100 mg mL À1 ) (complete RPMI medium).The cytotoxicity of the tested molecules on the HepG2 (hepatocarcinoma cell line purchased from ATCC, ref HB-8065) cell line was assessed according to the method of Mosmann 20 with slight modications.Briey, 5 Â 10 3 cells in 100 mL of complete medium were inoculated into each well of 96-well plates and incubated at 37 C in humidied 5% CO 2 .Aer 24 h incubation, 100 mL of medium with various product concentrations dissolved in DMSO (nal concentration less than 0.5% v/v) were added and the plates were incubated for 72 h at 37 C. Triplicate assays were performed for each sample.Each plate was then microscopically examined for possible precipitate formation before the medium was aspirated from the wells.Next, 100 mL of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide) solution (0.5 mg mL À1 in medium without FBS) was added to each well.Cells were incubated for 2 h at 37 C. Aer this time, the MTT solution was removed and DMSO (100 mL) was added to dissolve the resulting blue formazan crystals.Plates were shaken vigorously (700 rpm) for 10 min.The absorbance was measured at 570 nm with 630 nm as a reference wavelength using a BIO-TEK ELx808 Absorbance Microplate Reader (LabX, Midland, ON, Canada).DMSO was used as a blank and doxorubicin (purchased from Sigma Aldrich) as a positive control.Cell viability was calculated as a percentage of control (cells incubated without the compound).The 50% cytotoxic concentration (CC 50 ) was determined from the dose-response curve using TableCurve soware 2D v.5.0.CC 50 values to represent the mean value calculated from three independent experiments.4.2.3.Microsomal stability.The tested product and propranolol, used as a reference, were incubated in duplicate (reaction mixture volume of 0.5 mL) with female mouse microsomes (CD-1, 20 mg mL; BD Gentest) at 37 C in a 50 mM phosphate buffer, pH 7.4, in the presence of MgCl 2 (5 mM), NADP (1 mM), glucose-6-phosphate dehydrogenase (G6PD) (0.4 U per mL), and glucose-6-phosphate (5 mM).For the estimation of the intrinsic clearance, 50 mL aliquots were collected at 0, 5, 10, 20, 30, and 40 min and the reaction was stopped with 4 volumes of acetonitrile (ACN) containing the internal standard.Aer centrifugation at 10 000Âg for 10 min at 4 C, the supernatants were conserved at 4 C for immediate analysis or placed at À80 C in case of postponement of the analysis.Controls (time zero and nal time point) in triplicate were prepared by the incubation of the internal standard with microsomes denatured by acetonitrile.The LC-MS system used for this study was a Waters Acquity I-Class/Xevo TQD equipped with a Waters Acquity BEH C 18 column, 50 by 2.1 mm, 1.7 mm.The mobile phases were 10 mM ammonium acetate (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B).The injection volume was 1 mL, and the ow rate was 600 mL min À1 .Chromatographic analysis, with a total duration of 4 min, was made with the following gradient: 0 < t < 0.2 min, 2% mobile phase B; 0.2 < t < 2 min, linear increase to 98% mobile phase B; 2 < t < 2.5 min, 98% mobile phase B; 2.5 < t < 2.6 min, linear decrease to 2% mobile phase B; 2.6 < t < 4 min, 2% mobile phase B. The quantication of each compound was obtained by converting the average of the ratios of the analyte/internal standard surfaces to the percentage of consumed product.The ratio of the control at t0 corresponded to 0% of the product consumed.The calculation of the half-life (t 1/2 ) of each compound in the presence of microsomes was carried out according to the equation t 1/2 ¼ (ln 2 )/k, where k is the rst-order degradation constant (the slope of the logarithm of the compound concentration versus incubation time).The intrinsic clearance in vitro (Cl int , expressed in mlmin À1 mg) was calculated according to the equation Cl int ¼ (dose/AUC N )/ [microsomes], where dose is the initial concentration of the product in the sample, AUC N is the area under the concentration-time curve extrapolated to innity, and [microsomes] is the microsome concentration expressed in mg mL À1 .

Fig. 1 Scheme 1
Fig. 1 Chemical structures of antimalarial compounds in clinical trial or recently withdrawn from them.

4. 1 .
Chemistry 4.1.1.Materials and methods.Starting materials were purchased from Sigma-Aldrich (Saint Louis, MO, USA) or Fluorochem (Derbyshire, UK).NMR spectra were recorded on a Bruker Avance 250 MHz or a Bruker Avance NEO 400 MHz NanoBay spectrometer at the "Faculté de Pharmacie" of Marseille.The residual proton signal of the deuterated solvent was used as an internal reference: CDCl 3 d ¼ 7.26 ppm for 1 H and 77.16 for 13 C, and DMSO-d 6 d ¼ 2

Fig. 2
Fig. 2 RSA data obtained from the biological results.

Table
Optimization of the Buchwald-Hartwig cross-coupling reaction between 1 and p-toluidine a Using LCMS.b Pd(dba) 2 .c Dehalogenation of the starting material mainly observed.

Table 2
Scope of the Buchwald-Hartwig cross-coupling reaction on compound 1 a Conversion rate (determined by LCMS) indicated between brackets if the conversion was not complete.

Table 3
Optimization attempts for Buchwald-Hartwig cross-coupling reaction on compound 2 a from LCMS observations.

Table 4 Synthesis
a Two steps.