Antimalarials with Benzothiophene Moieties as Aminoquinoline Partners

Malaria is a severe and life-threatening disease caused by Plasmodium parasites that are spread to humans through bites of infected Anopheles mosquitoes. Here, we report on the efficacy of aminoquinolines coupled to benzothiophene and thiophene rings in inhibiting Plasmodium falciparum parasite growth. Synthesized compounds were evaluated for their antimalarial activity and toxicity, in vitro and in mice. Benzothiophenes presented in this paper showed improved activities against a chloroquine susceptible (CQS) strain, with potencies of IC50 = 6 nM, and cured 5/5 Plasmodium berghei infected mice when dosed orally at 160 mg/kg/day × 3 days. In the benzothiophene series, the examined antiplasmodials were more active against the CQS strain D6, than against strains chloroquine resistant (CQR) W2 and multidrug-resistant (MDR) TM91C235. For the thiophene series, a very interesting feature was revealed: hypersensitivity to the CQR strains, resistance index (RI) of <1. This is in sharp contrast to chloroquine, indicating that further development of the series would provide us with more potent antimalarials against CQR strains.


Introduction
Malaria is a severe and life-threatening disease caused by Plasmodium parasites that are spread to humans through bites of infected Anopheles mosquitoes. Plasmodium sporozoites injected into the bloodstream travel to the liver, where they are transformed into merozoites. They later re-enter the bloodstream, attacking the red blood cells where they begin the asexual replication. A few of these merozoites develop into sexual forms-gametocytes-which are being taken up by mosquitos during blood feeding, thus completing their life cycle [1]. This suggests that numerous stages of the life cycle of Plasmodium parasite could be potential drug targets for development of new antimalarials.
According to the 2015 World Health Organization (WHO) report it is estimated that 214 million cases of malaria occurred globally and the disease led to 438,000 deaths [2]. As artemisinin resistance has spread, posing a threat to malaria control [3], in order to prevent progression to life-threatening malaria artemisinin-based combination therapies (ACTs) have been recommended by the WHO [4]. A vaccine has yet to be discovered [5] and taking into account the constant loss of therapeutic efficacy due to the drug resistant strains, there is an urgent need for the discovery of new targets and treatments vacuole transmembrane protein (PfCRT) protein [6,7], found in the membrane of the digestive vacuole where hemoglobin digestion takes place.
A number of potential drug targets for novel antimalarials are known so far, most of them parasite proteins, such as Plasmodium falciparum enoyl-acyl carrier protein reductase [8][9][10], fatty acid synthase (PfFAS) [11], N-myristoyltransferase (NMT) [12,13], hexose transporter (PfHT1) [14][15][16], serine hydroxymethyltransferase (SHMT) [17], histone deacetylase [18], and many others. It is unclear if inhibition of these enzymes is the only mechanism of action (MOA) through which novel drugs inhibit parasite growth, but they are important in discovering enzyme function and metabolic pathways. Aminoquinolines, such as chloroquine, are well-known antimalarials. One of the hypotheses of their MOA is the accumulation in the parasite food vacuole, leading to inhibition of hemozoin formation. The toxic effect of free hematin containing ferriprotoporphyrin IX system is suppressed by "polymerization" into hemozoin; therefore, drugs that act as inhibitors of hematin sequestration are involved in the death of the parasite [19]. On the other hand, molecules that contain benzothiophene cores have an important role in medicinal chemistry due to their various biological properties, including antibacterial, antifungal and antitubercular activities [20]. They also act as inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK2) [21,22], C17,20-lyase inhibitors [23], liver receptor homolog-1 (LRH-1) antagonists [24], inhibitors of tyrosine phosphatase 1B and antihyperglycemic agents [25]. Regarding antimalarial activity, only one series of benzothiophene derivatives was evaluated and showed IC50 values up to 0.16 μM for P. falciparum 3D7 strain and inhibited parasite growth up to 65% at 50 mg/kg/day × 4 days in P. berghei infected mice [26]. Now, we report on the efficacy of aminoquinolines coupled to benzothiophene and thiophene rings in inhibiting Plasmodium falciparum parasite growth and compare the obtained results with CQ and previously reported related thiophene and benzothiophene-based 4-amino-7-chloroquinoline derivatives [27,28], as well as other antimalarial benzothiophene derivatives.
The new series possesses an additional phenylene linker between aminoquinoline and benzothiophene/thiophene moieties (Figure 1), and the influence of the extra π-system on antiplasmodial activity was analyzed taking into account the influence of different methylene linkers.

Chemistry
The syntheses of the aminoquinoline antimalarials with a benzothiophene carrier were executed using procedures we developed earlier. Derivatives 8, 9, 12 and 13 were obtained by reductive amination, starting from the corresponding aldehydes (Scheme 1).

Chemistry
The syntheses of the aminoquinoline antimalarials with a benzothiophene carrier were executed using procedures we developed earlier. Derivatives 8, 9, 12 and 13 were obtained by reductive amination, starting from the corresponding aldehydes (Scheme 1). ( Substituted benzothiophene cores were synthesized starting from commercially available difluoro-or bromofluorobenzaldehyde. Bromination of benzothiophene core afforded C(3) substituted products 17, 18 and 19 in good yields (>80%, Scheme 2), which were further coupled to 4-formylphenylboronic acid via Suzuki reactions to give the aldehydes 20, 21 and 22. These fragments are used in the key reductive amination reaction to prepare a diverse set of methylene-linked P. falciparum inhibitors. It is interesting to note that aldehydes 20 and 22 were obtained in good yields (88% and 76%, respectively), in contrast to 21 (37%). Finally, the reductive amination afforded target compounds 23-26 and 28-32 in 19%-77% isolated yield after column purification. In addition, compound 26 was methylated and gave 27 in moderate yield. In the thiophene series, the key intermediate, aldehyde 36, was synthesized in few steps starting from commercially available 2-bromothiophene (33) as follows: 4-(thiophen-2-yl)benzonitrile (34) was obtained from 2-bromothiophene by Suzuki coupling with 4-cyanophenylboronic acid using PdO hydrate as a catalyst (Scheme 3) [29]. Subsequent bromination of thiophene 34 at C(5) position and additional Suzuki reaction with 4-formyphenylboronic acid afforded 36 in excellent yield (92%). Substituted benzothiophene cores were synthesized starting from commercially available difluoroor bromofluorobenzaldehyde. Bromination of benzothiophene core afforded C(3) substituted products 17, 18 and 19 in good yields (>80%, Scheme 2), which were further coupled to 4-formylphenylboronic acid via Suzuki reactions to give the aldehydes 20, 21 and 22. These fragments are used in the key reductive amination reaction to prepare a diverse set of methylene-linked P. falciparum inhibitors. It is interesting to note that aldehydes 20 and 22 were obtained in good yields (88% and 76%, respectively), in contrast to 21 (37%). Finally, the reductive amination afforded target compounds 23-26 and 28-32 in 19%-77% isolated yield after column purification. In addition, compound 26 was methylated and gave 27 in moderate yield.

Chemistry
The syntheses of the aminoquinoline antimalarials with a benzothiophene carrier were executed using procedures we developed earlier. Derivatives 8, 9, 12 and 13 were obtained by reductive amination, starting from the corresponding aldehydes (Scheme 1). Substituted benzothiophene cores were synthesized starting from commercially available difluoro-or bromofluorobenzaldehyde. Bromination of benzothiophene core afforded C(3) substituted products 17, 18 and 19 in good yields (>80%, Scheme 2), which were further coupled to 4-formylphenylboronic acid via Suzuki reactions to give the aldehydes 20, 21 and 22. These fragments are used in the key reductive amination reaction to prepare a diverse set of methylene-linked P. falciparum inhibitors. It is interesting to note that aldehydes 20 and 22 were obtained in good yields (88% and 76%, respectively), in contrast to 21 (37%). Finally, the reductive amination afforded target compounds 23-26 and 28-32 in 19%-77% isolated yield after column purification. In addition, compound 26 was methylated and gave 27 in moderate yield. To examine the significance of the cyano group for antiplasmodial potency, the terminal acetylenic derivative 52 was prepared (Scheme 4). 2-Bromothiophene (33) was coupled with 4-formylphenylboronic acid to afford 47. Brominated product 48 obtained by reaction of 47 with NBS in THF was further submitted to Suzuki coupling with the 4-bromophenylboronic acid to afford the bromo aldehyde 49, which was converted in the next step to 50 by Sonogashira coupling with ethynyltrimethylsilane applying microwave irradiation. The final acetylene derivative 52 was obtained after reductive amination of 50 that was followed by removal of trimethylsilyl group under basic conditions.  All tested compounds were fully characterized and their purity was >95% (as determined by High performance liquid chromatography (HPLC)). Full details are given in the Supplementary Materials. To examine the significance of the cyano group for antiplasmodial potency, the terminal acetylenic derivative 52 was prepared (Scheme 4). 2-Bromothiophene (33) was coupled with 4-formylphenylboronic acid to afford 47. Brominated product 48 obtained by reaction of 47 with NBS in THF was further submitted to Suzuki coupling with the 4-bromophenylboronic acid to afford the bromo aldehyde 49, which was converted in the next step to 50 by Sonogashira coupling with ethynyltrimethylsilane applying microwave irradiation. The final acetylene derivative 52 was obtained after reductive amination of 50 that was followed by removal of trimethylsilyl group under basic conditions.  To examine the significance of the cyano group for antiplasmodial potency, the terminal acetylenic derivative 52 was prepared (Scheme 4). 2-Bromothiophene (33) was coupled with 4-formylphenylboronic acid to afford 47. Brominated product 48 obtained by reaction of 47 with NBS in THF was further submitted to Suzuki coupling with the 4-bromophenylboronic acid to afford the bromo aldehyde 49, which was converted in the next step to 50 by Sonogashira coupling with ethynyltrimethylsilane applying microwave irradiation. The final acetylene derivative 52 was obtained after reductive amination of 50 that was followed by removal of trimethylsilyl group under basic conditions. All tested compounds were fully characterized and their purity was >95% (as determined by High performance liquid chromatography (HPLC)). Full details are given in the Supplementary Materials. All tested compounds were fully characterized and their purity was >95% (as determined by High performance liquid chromatography (HPLC)). Full details are given in the Supplementary Materials.

In Vitro Antiplasmodial Activity
The antimalarial candidates were tested in vitro for their antiplasmodial activity against three P. falciparum strains: D6 (CQ susceptible (CQS) strain), W2 (CQ resistant (CQR) strain), and TM91C235 (Thailand, a multidrug-resistant (MDR) strain), using the Malaria SYBR Green Fluorescence Assay, a microtiter drug sensitivity assay that uses the intercalation of SYBR Green into malaria DNA as a measure of blood stage P. falciparum parasite proliferation in the presence of antimalarial compounds. This assay is performed as a dose response (12 two-fold serial dilutions) to obtain a calculated IC 50 determination [30]. CQ and mefloquine (MFQ) were used as positive controls (Tables 1 and 2).

In Vitro Antiplasmodial Activity
The antimalarial candidates were tested in vitro for their antiplasmodial activity against three P. falciparum strains: D6 (CQ susceptible (CQS) strain), W2 (CQ resistant (CQR) strain), and TM91C235 (Thailand, a multidrug-resistant (MDR) strain), using the Malaria SYBR Green Fluorescence Assay, a microtiter drug sensitivity assay that uses the intercalation of SYBR Green into malaria DNA as a measure of blood stage P. falciparum parasite proliferation in the presence of antimalarial compounds. This assay is performed as a dose response (12 two-fold serial dilutions) to obtain a calculated IC50 determination [30]. CQ and mefloquine (MFQ) were used as positive controls (Tables 1 and 2).

In Vitro Antiplasmodial Activity
The antimalarial candidates were tested in vitro for their antiplasmodial activity against three P. falciparum strains: D6 (CQ susceptible (CQS) strain), W2 (CQ resistant (CQR) strain), and TM91C235 (Thailand, a multidrug-resistant (MDR) strain), using the Malaria SYBR Green Fluorescence Assay, a microtiter drug sensitivity assay that uses the intercalation of SYBR Green into malaria DNA as a measure of blood stage P. falciparum parasite proliferation in the presence of antimalarial compounds. This assay is performed as a dose response (12 two-fold serial dilutions) to obtain a calculated IC50 determination [30]. CQ and mefloquine (MFQ) were used as positive controls (Tables 1 and 2).

In Vitro Antiplasmodial Activity
The antimalarial candidates were tested in vitro for their antiplasmodial activity against three P. falciparum strains: D6 (CQ susceptible (CQS) strain), W2 (CQ resistant (CQR) strain), and TM91C235 (Thailand, a multidrug-resistant (MDR) strain), using the Malaria SYBR Green Fluorescence Assay, a microtiter drug sensitivity assay that uses the intercalation of SYBR Green into malaria DNA as a measure of blood stage P. falciparum parasite proliferation in the presence of antimalarial compounds. This assay is performed as a dose response (12 two-fold serial dilutions) to obtain a calculated IC50 determination [30]. CQ and mefloquine (MFQ) were used as positive controls (Tables 1 and 2).

In Vitro Antiplasmodial Activity
The antimalarial candidates were tested in vitro for their antiplasmodial activity against three P. falciparum strains: D6 (CQ susceptible (CQS) strain), W2 (CQ resistant (CQR) strain), and TM91C235 (Thailand, a multidrug-resistant (MDR) strain), using the Malaria SYBR Green Fluorescence Assay, a microtiter drug sensitivity assay that uses the intercalation of SYBR Green into malaria DNA as a measure of blood stage P. falciparum parasite proliferation in the presence of antimalarial compounds. This assay is performed as a dose response (12 two-fold serial dilutions) to obtain a calculated IC50 determination [30]. CQ and mefloquine (MFQ) were used as positive controls (Tables 1 and 2).

In Vitro Antiplasmodial Activity
The antimalarial candidates were tested in vitro for their antiplasmodial activity against three P. falciparum strains: D6 (CQ susceptible (CQS) strain), W2 (CQ resistant (CQR) strain), and TM91C235 (Thailand, a multidrug-resistant (MDR) strain), using the Malaria SYBR Green Fluorescence Assay, a microtiter drug sensitivity assay that uses the intercalation of SYBR Green into malaria DNA as a measure of blood stage P. falciparum parasite proliferation in the presence of antimalarial compounds. This assay is performed as a dose response (12 two-fold serial dilutions) to obtain a calculated IC50 determination [30]. CQ and mefloquine (MFQ) were used as positive controls (Tables 1 and 2).

In Vitro Antiplasmodial Activity
The antimalarial candidates were tested in vitro for their antiplasmodial activity against three P. falciparum strains: D6 (CQ susceptible (CQS) strain), W2 (CQ resistant (CQR) strain), and TM91C235 (Thailand, a multidrug-resistant (MDR) strain), using the Malaria SYBR Green Fluorescence Assay, a microtiter drug sensitivity assay that uses the intercalation of SYBR Green into malaria DNA as a measure of blood stage P. falciparum parasite proliferation in the presence of antimalarial compounds. This assay is performed as a dose response (12 two-fold serial dilutions) to obtain a calculated IC50 determination [30]. CQ and mefloquine (MFQ) were used as positive controls (Tables 1 and 2).

In Vitro Antiplasmodial Activity
The antimalarial candidates were tested in vitro for their antiplasmodial activity against three P. falciparum strains: D6 (CQ susceptible (CQS) strain), W2 (CQ resistant (CQR) strain), and TM91C235 (Thailand, a multidrug-resistant (MDR) strain), using the Malaria SYBR Green Fluorescence Assay, a microtiter drug sensitivity assay that uses the intercalation of SYBR Green into malaria DNA as a measure of blood stage P. falciparum parasite proliferation in the presence of antimalarial compounds. This assay is performed as a dose response (12 two-fold serial dilutions) to obtain a calculated IC50 determination [30]. CQ and mefloquine (MFQ) were used as positive controls (Tables 1 and 2).

In Vitro Antiplasmodial Activity
The antimalarial candidates were tested in vitro for their antiplasmodial activity against three P. falciparum strains: D6 (CQ susceptible (CQS) strain), W2 (CQ resistant (CQR) strain), and TM91C235 (Thailand, a multidrug-resistant (MDR) strain), using the Malaria SYBR Green Fluorescence Assay, a microtiter drug sensitivity assay that uses the intercalation of SYBR Green into malaria DNA as a measure of blood stage P. falciparum parasite proliferation in the presence of antimalarial compounds. This assay is performed as a dose response (12 two-fold serial dilutions) to obtain a calculated IC50 determination [30]. CQ and mefloquine (MFQ) were used as positive controls (Tables 1 and 2).

In Vitro Antiplasmodial Activity
The antimalarial candidates were tested in vitro for their antiplasmodial activity against three P. falciparum strains: D6 (CQ susceptible (CQS) strain), W2 (CQ resistant (CQR) strain), and TM91C235 (Thailand, a multidrug-resistant (MDR) strain), using the Malaria SYBR Green Fluorescence Assay, a microtiter drug sensitivity assay that uses the intercalation of SYBR Green into malaria DNA as a measure of blood stage P. falciparum parasite proliferation in the presence of antimalarial compounds. This assay is performed as a dose response (12 two-fold serial dilutions) to obtain a calculated IC50 determination [30]. CQ and mefloquine (MFQ) were used as positive controls (Tables 1 and 2).

In Vitro Antiplasmodial Activity
The antimalarial candidates were tested in vitro for their antiplasmodial activity against three P. falciparum strains: D6 (CQ susceptible (CQS) strain), W2 (CQ resistant (CQR) strain), and TM91C235 (Thailand, a multidrug-resistant (MDR) strain), using the Malaria SYBR Green Fluorescence Assay, a microtiter drug sensitivity assay that uses the intercalation of SYBR Green into malaria DNA as a measure of blood stage P. falciparum parasite proliferation in the presence of antimalarial compounds. This assay is performed as a dose response (12 two-fold serial dilutions) to obtain a calculated IC50 determination [30]. CQ and mefloquine (MFQ) were used as positive controls (Tables 1 and 2).

In Vitro Antiplasmodial Activity
The antimalarial candidates were tested in vitro for their antiplasmodial activity against three P. falciparum strains: D6 (CQ susceptible (CQS) strain), W2 (CQ resistant (CQR) strain), and TM91C235 (Thailand, a multidrug-resistant (MDR) strain), using the Malaria SYBR Green Fluorescence Assay, a microtiter drug sensitivity assay that uses the intercalation of SYBR Green into malaria DNA as a measure of blood stage P. falciparum parasite proliferation in the presence of antimalarial compounds. This assay is performed as a dose response (12 two-fold serial dilutions) to obtain a calculated IC50 determination [30]. CQ and mefloquine (MFQ) were used as positive controls (Tables 1 and 2). As a general remark, to our content, all synthesized compounds were more active than CQ against the CQR W2 strain. Importantly, compounds 29, 30 and 31 were as active as CQ against the CQS D6 strain (and had higher activity against W2 and C235 strains), while 28 was two times more active than CQ against the D6 strain. Comparison of in vitro antiplasmodial activities of fluoro and cyano analogues 26 with 29, and 24 with 30, clearly indicate that both cyano derivatives show significantly higher activities against CQS D6 strain. To analyze the effect of the cyano group in different positions of the benzothiophene core, we synthesized the pair of homologs 28, 30, and 31, 32, respectively. The compounds with cyano group at C(5) position were more potent than their C(6) isomers; it was also noticed that the shorter linker n = 2, instead of n = 4, was more favorable for antiplasmodial activity against D6 strain (28 vs. 30, 31 vs. 32).
Unlike benzothiophenes, the compounds of the thiophene series were found to generally exhibit higher in vitro activity against CQR W2 than against CQS D6 strain ( Table 2). We found it indicative that all derivatives in this series bearing cyano group were more active against MDR C235 strain in comparison to CQ, among which 41, 42, 44, and 46 showed higher potency than MFQ as well. In addition, all tested C(7) chloro derivatives were more active than the respective des-chloro aminoquinolines against CQR W2 strain, e.g., 41 vs. 44 (13 nM vs. 74 nM). The effect of the cyano group of this series was additionally confirmed by comparison of its antimalarial activity to the activity of the respective acetylene analogue 52, suggesting that cyano substituent may play an important role in the antiplasmodial activity of these derivatives. As a general remark, to our content, all synthesized compounds were more active than CQ against the CQR W2 strain. Importantly, compounds 29, 30 and 31 were as active as CQ against the CQS D6 strain (and had higher activity against W2 and C235 strains), while 28 was two times more active than CQ against the D6 strain. Comparison of in vitro antiplasmodial activities of fluoro and cyano analogues 26 with 29, and 24 with 30, clearly indicate that both cyano derivatives show significantly higher activities against CQS D6 strain. To analyze the effect of the cyano group in different positions of the benzothiophene core, we synthesized the pair of homologs 28, 30, and 31, 32, respectively. The compounds with cyano group at C(5) position were more potent than their C(6) isomers; it was also noticed that the shorter linker n = 2, instead of n = 4, was more favorable for antiplasmodial activity against D6 strain (28 vs. 30, 31 vs. 32).
Unlike benzothiophenes, the compounds of the thiophene series were found to generally exhibit higher in vitro activity against CQR W2 than against CQS D6 strain ( Table 2). We found it indicative that all derivatives in this series bearing cyano group were more active against MDR C235 strain in comparison to CQ, among which 41, 42, 44, and 46 showed higher potency than MFQ as well. In addition, all tested C(7) chloro derivatives were more active than the respective des-chloro aminoquinolines against CQR W2 strain, e.g., 41 vs. 44 (13 nM vs. 74 nM). The effect of the cyano group of this series was additionally confirmed by comparison of its antimalarial activity to the activity of the respective acetylene analogue 52, suggesting that cyano substituent may play an important role in the antiplasmodial activity of these derivatives. As a general remark, to our content, all synthesized compounds were more active than CQ against the CQR W2 strain. Importantly, compounds 29, 30 and 31 were as active as CQ against the CQS D6 strain (and had higher activity against W2 and C235 strains), while 28 was two times more active than CQ against the D6 strain. Comparison of in vitro antiplasmodial activities of fluoro and cyano analogues 26 with 29, and 24 with 30, clearly indicate that both cyano derivatives show significantly higher activities against CQS D6 strain. To analyze the effect of the cyano group in different positions of the benzothiophene core, we synthesized the pair of homologs 28, 30, and 31, 32, respectively. The compounds with cyano group at C(5) position were more potent than their C(6) isomers; it was also noticed that the shorter linker n = 2, instead of n = 4, was more favorable for antiplasmodial activity against D6 strain (28 vs. 30, 31 vs. 32).
Unlike benzothiophenes, the compounds of the thiophene series were found to generally exhibit higher in vitro activity against CQR W2 than against CQS D6 strain ( Table 2). We found it indicative that all derivatives in this series bearing cyano group were more active against MDR C235 strain in comparison to CQ, among which 41, 42, 44, and 46 showed higher potency than MFQ as well. In addition, all tested C(7) chloro derivatives were more active than the respective des-chloro aminoquinolines against CQR W2 strain, e.g., 41 vs. 44 (13 nM vs. 74 nM). The effect of the cyano group of this series was additionally confirmed by comparison of its antimalarial activity to the activity of the respective acetylene analogue 52, suggesting that cyano substituent may play an important role in the antiplasmodial activity of these derivatives. As a general remark, to our content, all synthesized compounds were more active than CQ against the CQR W2 strain. Importantly, compounds 29, 30 and 31 were as active as CQ against the CQS D6 strain (and had higher activity against W2 and C235 strains), while 28 was two times more active than CQ against the D6 strain. Comparison of in vitro antiplasmodial activities of fluoro and cyano analogues 26 with 29, and 24 with 30, clearly indicate that both cyano derivatives show significantly higher activities against CQS D6 strain. To analyze the effect of the cyano group in different positions of the benzothiophene core, we synthesized the pair of homologs 28, 30, and 31, 32, respectively. The compounds with cyano group at C(5) position were more potent than their C(6) isomers; it was also noticed that the shorter linker n = 2, instead of n = 4, was more favorable for antiplasmodial activity against D6 strain (28 vs. 30, 31 vs. 32).
Unlike benzothiophenes, the compounds of the thiophene series were found to generally exhibit higher in vitro activity against CQR W2 than against CQS D6 strain ( Table 2). We found it indicative that all derivatives in this series bearing cyano group were more active against MDR C235 strain in comparison to CQ, among which 41, 42, 44, and 46 showed higher potency than MFQ as well. In addition, all tested C(7) chloro derivatives were more active than the respective des-chloro aminoquinolines against CQR W2 strain, e.g., 41 vs. 44 (13 nM vs. 74 nM). The effect of the cyano group of this series was additionally confirmed by comparison of its antimalarial activity to the activity of the respective acetylene analogue 52, suggesting that cyano substituent may play an important role in the antiplasmodial activity of these derivatives. As a general remark, to our content, all synthesized compounds were more active than CQ against the CQR W2 strain. Importantly, compounds 29, 30 and 31 were as active as CQ against the CQS D6 strain (and had higher activity against W2 and C235 strains), while 28 was two times more active than CQ against the D6 strain. Comparison of in vitro antiplasmodial activities of fluoro and cyano analogues 26 with 29, and 24 with 30, clearly indicate that both cyano derivatives show significantly higher activities against CQS D6 strain. To analyze the effect of the cyano group in different positions of the benzothiophene core, we synthesized the pair of homologs 28, 30, and 31, 32, respectively. The compounds with cyano group at C(5) position were more potent than their C(6) isomers; it was also noticed that the shorter linker n = 2, instead of n = 4, was more favorable for antiplasmodial activity against D6 strain (28 vs. 30, 31 vs. 32).
Unlike benzothiophenes, the compounds of the thiophene series were found to generally exhibit higher in vitro activity against CQR W2 than against CQS D6 strain ( Table 2). We found it indicative that all derivatives in this series bearing cyano group were more active against MDR C235 strain in comparison to CQ, among which 41, 42, 44, and 46 showed higher potency than MFQ as well. In addition, all tested C(7) chloro derivatives were more active than the respective des-chloro aminoquinolines against CQR W2 strain, e.g., 41 vs. 44 (13 nM vs. 74 nM). The effect of the cyano group of this series was additionally confirmed by comparison of its antimalarial activity to the activity of the respective acetylene analogue 52, suggesting that cyano substituent may play an important role in the antiplasmodial activity of these derivatives. As a general remark, to our content, all synthesized compounds were more active than CQ against the CQR W2 strain. Importantly, compounds 29, 30 and 31 were as active as CQ against the CQS D6 strain (and had higher activity against W2 and C235 strains), while 28 was two times more active than CQ against the D6 strain. Comparison of in vitro antiplasmodial activities of fluoro and cyano analogues 26 with 29, and 24 with 30, clearly indicate that both cyano derivatives show significantly higher activities against CQS D6 strain. To analyze the effect of the cyano group in different positions of the benzothiophene core, we synthesized the pair of homologs 28, 30, and 31, 32, respectively. The compounds with cyano group at C(5) position were more potent than their C(6) isomers; it was also noticed that the shorter linker n = 2, instead of n = 4, was more favorable for antiplasmodial activity against D6 strain (28 vs. 30, 31 vs. 32).
Unlike benzothiophenes, the compounds of the thiophene series were found to generally exhibit higher in vitro activity against CQR W2 than against CQS D6 strain ( Table 2). We found it indicative that all derivatives in this series bearing cyano group were more active against MDR C235 strain in comparison to CQ, among which 41, 42, 44, and 46 showed higher potency than MFQ as well. In addition, all tested C(7) chloro derivatives were more active than the respective des-chloro aminoquinolines against CQR W2 strain, e.g., 41 vs. 44 (13 nM vs. 74 nM). The effect of the cyano group of this series was additionally confirmed by comparison of its antimalarial activity to the activity of the respective acetylene analogue 52, suggesting that cyano substituent may play an important role in the antiplasmodial activity of these derivatives.

Discussion
To investigate possible toxicity issues, the synthesized compounds were evaluated in vitro against a human hepatoma cell line (HepG2). The range of their activity falls within 1976-30605 nM, significantly higher in comparison to their respective antimalarial activities (Tables 1 and 2).
Among all examined derivatives, the compounds 25, 42, and 46 have been chosen for in vivo evaluation since they showed significant activities against W2 clone in vitro (Tables 1 and 2, RI values), which is in sharp contrast to CQ. In a separate host toxicity study, benzothiophene 25 was subjected to in vivo toxicity evaluation. At the 160 mg/kg/day × 3 days dose, 25 proved to be non-toxic, as all 5 mice survived 30 days after administration and showed normal appearance and behavior. Yet, using a modified Thompson test model [31], administering 25 at the same concentration of 160 mg/kg/day × 3 days to C57Bl6 female mice infected with 1 × 10 6 P. berghei parasites, unfortunately led to death of 3 out of 4 mice, with only one surviving 31 days but with parasitemia. In this test model, P. berghei infected mice were treated with aminoquinolines suspended in 0.5% hydroxyethylcellulose-0.1% Tween 80 and administered per os (p.o.) once per day on days 3-5 postinfection (Table 3).
Regarding thiophene derivatives, they were all well tolerated by Hep G2 cells, possessing IC 50 > 2000 nM. However, rather low selectivity indices were calculated for all derivatives (SI HepG2/D6 = 30-624) with an exception of derivative 43, SI HepG2/D6 > 2000. In a host toxicity study at the concentration of 160 mg/kg × 3 days, compounds 42 and 46 with the longest methylene linker (eight methylene groups), proved to be non-toxic (all 5 mice survived). The compounds were further subjected to in vivo evaluation, using the modified Thompson test model, at 160 mg/kg/day × 3 days for 42, and at 80 and 40 mg/kg/day × 4 days for 46. Under applied treatment conditions, three sets of 5 infected mice died of malaria. Derivative 46 enabled one mouse survival until day 25 (D25) at both administered doses, while compound 42 provided survival with high level of parasitemia until day 24 (D24) at a higher dose (Table 3).
It is important to be aware that direct correlation of in vitro and in vivo results is not always justified. One of many examples is our previously reported compound 1 [27] (Figure 2), which was examined for its antimalarial activity in vivo, despite a relatively low SI (282). Surprisingly, compound 1 cured 5/5 mice using a modified Thompson test ( Figure 3, Table 3) and cleared all parasites on day 7 (D7) (parasitemia before treatment was 0.3%-0.9%, and at the end of the study all 5 mice were parasite negative). The same compound was assessed in a dose response test in which P. berghei infected mice were treated per os (p.o.) for 4 days, starting with day 3 postinfection. Unfortunately, at all three applied doses (80, 40 and 20 mg/kg/day) benzothiophene 1 did not clear the parasites (Table 3). There was no detectable parasitemia up to day 11 at a dose as low as 40 mg/kg/day; however, recrudescence occurred between day 11 (D11) and day 14 (D14). In vivo activity results for 1 are summarized on Figure 3. On the other hand, compound 53 [27] with a propylene linker, which showed much more promising SI index (1111), proved to be toxic at concentration of 160 mg/kg/day, thus it was not evaluated in vivo ( Figure 2). significantly higher in comparison to their respective antimalarial activities (Tables 1 and 2).
Among all examined derivatives, the compounds 25, 42, and 46 have been chosen for in vivo evaluation since they showed significant activities against W2 clone in vitro (Tables 1 and 2, RI values), which is in sharp contrast to CQ. In a separate host toxicity study, benzothiophene 25 was subjected to in vivo toxicity evaluation. At the 160 mg/kg/day × 3 days dose, 25 proved to be non-toxic, as all 5 mice survived 30 days after administration and showed normal appearance and behavior. Yet, using a modified Thompson test model [31], administering 25 at the same concentration of 160 mg/kg/day × 3 days to C57Bl6 female mice infected with 1 × 10 6 P. berghei parasites, unfortunately led to death of 3 out of 4 mice, with only one surviving 31 days but with parasitemia. In this test model, P. berghei infected mice were treated with aminoquinolines suspended in 0.5% hydroxyethylcellulose-0.1% Tween 80 and administered per os (p.o.) once per day on days 3-5 postinfection (Table 3).
Regarding thiophene derivatives, they were all well tolerated by Hep G2 cells, possessing IC50 > 2000 nM. However, rather low selectivity indices were calculated for all derivatives (SIHepG2/D6 = 30-624) with an exception of derivative 43, SI HepG2/D6 > 2000. In a host toxicity study at the concentration of 160 mg/kg × 3 days, compounds 42 and 46 with the longest methylene linker (eight methylene groups), proved to be non-toxic (all 5 mice survived). The compounds were further subjected to in vivo evaluation, using the modified Thompson test model, at 160 mg/kg/day × 3 days for 42, and at 80 and 40 mg/kg/day × 4 days for 46. Under applied treatment conditions, three sets of 5 infected mice died of malaria. Derivative 46 enabled one mouse survival until day 25 (D25) at both administered doses, while compound 42 provided survival with high level of parasitemia until day 24 (D24) at a higher dose (Table 3).
It is important to be aware that direct correlation of in vitro and in vivo results is not always justified. One of many examples is our previously reported compound 1 [27] (Figure 2), which was examined for its antimalarial activity in vivo, despite a relatively low SI (282). Surprisingly, compound 1 cured 5/5 mice using a modified Thompson test ( Figure 3, Table 3) and cleared all parasites on day 7 (D7) (parasitemia before treatment was 0.3%-0.9%, and at the end of the study all 5 mice were parasite negative). The same compound was assessed in a dose response test in which P. berghei infected mice were treated per os (p.o.) for 4 days, starting with day 3 postinfection. Unfortunately, at all three applied doses (80, 40 and 20 mg/kg/day) benzothiophene 1 did not clear the parasites (Table 3). There was no detectable parasitemia up to day 11 at a dose as low as 40 mg/kg/day; however, recrudescence occurred between day 11 (D11) and day 14 (D14). In vivo activity results for 1 are summarized on Figure 3. On the other hand, compound 53 [27] with a propylene linker, which showed much more promising SI index (1111), proved to be toxic at concentration of 160 mg/kg/day, thus it was not evaluated in vivo ( Figure 2). 1: n=2; IC 50 (W2) = 31 nM; IC 50 (D6) = 8 nM; SI(HepG2/D6) = 282 [27] 53: n=1; IC 50 (W2) = 24 nM; IC 50 (D6) = 7 nM; SI(HepG2/D6) = 1111 [27] this work 1: n=2 Toxicity 5/5 survived @ 160 mg/kg/day RBC (Thompson, 3 days therapy) 5/5 cure @ 160 mg/kg/day RBC (4 days therapy) no cure @ 80 and 40 mg/kg/day (clearance with recrudescence on D11-14) no cure @ 20 mg/kg/day Parasite clearance provided by compound 1 with a 160 mg/kg/day × 3 days dose regimen, was further examined at the molecular level by polymerase chain reaction (PCR) [32]. Briefly, genomic DNA was extracted from the blood and liver of experimental animals using the DNeasy blood and tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The primers and corresponding TaqMan (Applied BioSystems. Foster City, CA, USA) probe amplify and detect a highly Parasite clearance provided by compound 1 with a 160 mg/kg/day × 3 days dose regimen, was further examined at the molecular level by polymerase chain reaction (PCR) [32]. Briefly, genomic DNA was extracted from the blood and liver of experimental animals using the DNeasy blood and tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The primers and corresponding TaqMan (Applied BioSystems. Foster City, CA, USA) probe amplify and detect a highly conserved region of the 18S rRNA gene of the genus Plasmodium. Pure P. berghei gDNA samples were used as positive controls. Both blood and tissue samples of all 5 mice proved to be negative for P. berghei DNA (Figure 4). Parasite clearance provided by compound 1 with a 160 mg/kg/day × 3 days dose regimen, was further examined at the molecular level by polymerase chain reaction (PCR) [32]. Briefly, genomic DNA was extracted from the blood and liver of experimental animals using the DNeasy blood and tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The primers and corresponding TaqMan (Applied BioSystems. Foster City, CA, USA) probe amplify and detect a highly conserved region of the 18S rRNA gene of the genus Plasmodium. Pure P. berghei gDNA samples were used as positive controls. Both blood and tissue samples of all 5 mice proved to be negative for P. berghei DNA (Figure 4). The possible mechanism of action of compound 1 could include inhibition of β-hematin formation, since this compound showed a low IC50 value of 0.34 [33], which is 3.7 times lower than for CQ .   Cycle   45  44  43  42  41  40  39  38  37  36  35  34  33  32  31  30  29  28  27  26  25  24  23  22  21  20  19  18  17  16  15  14  13  12  11  10  9  8  7  6  5  4  3   The possible mechanism of action of compound 1 could include inhibition of β-hematin formation, since this compound showed a low IC 50 value of 0.34 [33], which is 3.7 times lower than for CQ.  A mixture of 4-chloroquinoline or 4,7-dichloroquinoline (1 eq.) and an appropriate diamine (5 eq.) were subjected to microwave irradiation using an Initiator 2.5 apparatus (Biotage) for 15 mins at 80 • C, followed by 30 mins at 95 • C and then 2 h at 140 • C. After cooling to room temperature 0.1 M aqueous NaOH was added and then extracted with CH 2 Cl 2 . Combined organic layers were dried over anhydrous Na 2 SO 4 . After filtration, the solvent was removed under reduced pressure. The crude product was subjected to silica-gel column chromatography using CH 2 Cl 2 /MeOH (NH 3 satd.) as eluent to afford the final compound. To a stirred solution of aminoquinolines (1 eq.) in MeOH containing 37% aqueous formaldehyde (2 eq.) was added mixture of ZnCl 2 (2 eq.) and NaHB 3 CN (4 eq.) in MeOH. After the reaction mixture was stirred at r.t. for 4 h, the solution was taken up in 0.1 M NaOH and most of the MeOH was evaporated under reduced pressure. Aqueous solution was extracted with CH 2 Cl 2 , the combined extracts were washed with water and brine and dried over anhydrous Na 2 SO 4 . The solvent was evaporated under reduced pressure. [ An appropriate aryl-bromide (1 eq.) was added to the mixture of arylboronic acid (1.2 eq.), catalyst PdO × 1.4 H 2 O (0.01-0.05 eq.), K 2 CO 3 (1.2 eq.) and ethanol/H 2 O (3:1 v/v). The mixture was stirred at 60 • C for 5 h, then diluted with water and extracted with CH 2 Cl 2 . The combined organic layers were washed with brine and dried over anhydrous Na 2 SO 4 . After filtration, the solvent was removed under reduced pressure. The product was purified using silica gel flash chromatography [29] A solution of Pd(OAc) 2 (0.05 eq.) and PPh 3 (0.2 eq.) in toluene was stirred at r.t. under an argon atmosphere for 10 mins. After that, the solution of an appropriate aryl-bromide (1 eq.), an appropriate arylboronic acid (1.1 eq.) and 2M aq. Na 2 CO 3 (2 eq.) in MeOH and toluene was added. The mixture was stirred at 110 • C under an argon atmosphere for 3 h. The reaction work up method is provided for each compound.
Full details are given in the Supplementary Materials.

In Vitro Antiplasmodial Activity
Synthesized aminoquinolines were screened in vitro against P. falciparum strains: CQ and MFQ susceptible strain D6 (clone of Sierra Leone/UNC isolate), CQ resistant but MFQ susceptible strain W2 (clone of Indochina isolate), and CQ and MFQ resistant strain TM91C235 (clone of South East Asian isolate) using the Malaria SYBR Green Fluorescence Assay. Full method details are given in Supplementary Materials I. Assessment of compound toxicity in a HepG2 (hepatocellular carcinoma) cell line followed the protocol described in Ref. [40].

In Vivo Antiplasmodial Activity and Toxicity
The P. berghei (ANKA) mouse efficacy tests were conducted using a modified version of the Thompson test. Groups of five mice were inoculated intraperitoneally with erythrocytes infected with P. berghei on day 0. Drugs were suspended in 0.5% hydroxyethylcellulose-0.1% Tween 80 and administered orally once a day beginning on day 3 post infection. Dosings are given in Table 3. All untreated infected (control) mice showed parasitemia on day 3, which reached levels between 11.2% and 30% on day 6 and succumbed to the infection on day 6-8. Therefore, the test was considered valid. Cure was defined as survival (with no parasitemia) until day 31 post-treatment. Parasitemia was determined by thin-blood Giemsa-stained smears prepared from mice tail blood of each animal on days 0, 3, 6, 10, 13, 17, 20, 24, 27, and 31 (postinfection). The slides were examined under a light microscope.
In a separate host toxicity study, groups of five healthy mice were administered 160 mg/kg/day × 3 days of the investigational compounds and individually monitored for behavior and appearance two times a day for 31 days. No toxicity was defined as survival past day 31 with no overt clinical manifestations of toxicity (changes in behavior and appearance).
The study followed the International Guiding Principles for biomedical research involving animals, and was reviewed by a local Ethics Committee and approved by the Veterinary Directorate at the Ministry of Agricultutre and Environmental Protection of Serbia (decision no. 323-07-02444/2014-05/1).

DNA Extraction
Genomic DNA was extracted from the blood and liver of experimental animals using the DNeasy blood and tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Briefly, mice alive past day 31 with complete parasite clearance were sacrificed. Next, between 300 and 500 µL of blood was removed after heart puncture and the organs were removed, rinsed with dPBS and homogenized. The liver was homogenized using mechanical disruption and then subjected to proteinase K digestion. Approximately 100 µL of sample was used per each spin column for gDNA extraction.

Real Time PCR
Briefly, the primers and corresponding TaqMan probe amplify and detect a highly conserved region of the 18S rRNA gene of the genus Plasmodium. The primer and probe sequences were as follows: forward primer Plasmo 1: 5-GTTAAGGGAGTGAAGACGA TCAGA-3; reverse primer Plasmo 2: 5-AACCCAAAGACTTTGATTTC TCATAA-3; TaqMan probe Plasprobe: 5-FAM-ACCGTCGTAA TCTTAACCAT AAACTATGCC GACTAG-TAMRA-3. Each 20µL reaction contained 1× Maxima Probe qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), 200 nM of each primer, 50 nM probe, 1U UNG (Thermo Fisher Scientific) and 3 µL template gDNA. The following PCR conditions were used: one holding step at 50 • C for 2 min, one holding step at 95 • C for 10 min, then 45 cycles of 95 • C for 15 s, 60 • C for 1 min. The PCR was performed in a StepOne Plus instrument (Applied BioSystems). Samples starting with Ct40 were considered negative. A positive (P. berghei gDNA) and negative control (H 2 O) were included in each run [32].

Conclusions
Benzothiophenes presented in this paper, in comparison to compounds discussed in [26] showed improved activities against the CQS strain, with potencies against D6 of IC 50 = 6 nM, and cured 5/5 P. berghei infected mice dosed per os (p.o.) at 160 mg/kg/day × 3 days. However, comparing the two series of benzothiophenes (and thiophenes), with and without the phenyl linker, we found that introducing the phenyl linker did not improve the in vitro activity of the first series of aminoquinolines [27] In both benzothiophene series, all compounds were more active against the CQS strain D6, than against strains CQR W2 and MDR TM91C235, with the sole exception of 25 which was more active against the W2 strain. As for thiophene series-37-42, 44 and 46-a very interesting feature was revealed: hypersensitivity to the resistant strains, with a resistance index of less than 1. This is in sharp contrast to chloroquine, thus indicating that further development of the series could provide us with antimalarials more powerful against CQR strains.