Antimalarials with benzothiophene moieties as aminoquinoline partners

Article abstract of DOI:10.3390/molecules22030343

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 x 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.

Full text of DOI:10.3390/molecules22030343

molecules
Article
Antimalarials with Benzothiophene Moieties as
Aminoquinoline Partners
1
2
3
3
Jelena Konstantinovic´ , Milica Videnovic´ , Jelena Srbljanovic´ , Olgica Djurkovic´-Djakovic´ ,
Katarina Bogojevic´ , Richard Sciotti 4 and Bogdan Šolaja 1,
*
1
1
Faculty of Chemistry, University of Belgrade, Studentski trg 16, P.O. Box 51, 11158 Belgrade, Serbia;
jelena_konstantinovic@chem.bg.ac.rs (J.K.); bogojevickatarina@gmail.com (K.B.)
Innovation Center of the Faculty of Chemistry, Studentski trg 12-16, 11158 Belgrade, Serbia;
milica_videnovic@chem.bg.ac.rs
Institute for Medical Research, University of Belgrade, Dr. Subotic´a 4, 11129 Belgrade, Serbia;
jelena.srbljanovic@imi.bg.ac.rs (J.S.); olgicadj@imi.bg.ac.rs (O.D.-D.)
Experimental Therapeutics Branch, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA;
richard.j.sciotti.civ@mail.mil
2
3
4
*
Correspondence: bsolaja@chem.bg.ac.rs; Tel.: +381-11-263-86-06
Academic Editor: Diego Muñoz-Torrero
Received: 9 January 2017; Accepted: 16 February 2017; Published: 24 February 2017
Abstract: 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.
Keywords: antimalarials; thiophene; benzothiophene; aminoquinoline
1. 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 [
has spread, posing a threat to malaria control [ ], in order to prevent progression to life-threatening
malaria artemisinin-based combination therapies (ACTs) have been recommended by the WHO [ ].
A vaccine has yet to be discovered [ ] 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
2]. As artemisinin resistance
3
4
5
Molecules 2017, 22, 343; doi:10.3390/molecules22030343
www.mdpi.com/journal/molecules

Molecules 2017, 22, 343
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for malaria. Widespread chloroquine (CQ) resistance is largely attributable to mutations in the vacuole
transmembrane protein (PfCRT) protein [
hemoglobin digestion takes place.
6,7], found in the membrane of the digestive vacuole where
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 [ 10], fatty acid
synthase (PfFAS) [11], N-myristoyltransferase (NMT) [12 13], hexose transporter (PfHT1) [14 16],
8–
,
–
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.
N
R
R
Cl
R1
H
N
()n
N
H
N
R
R2
()n
N
H
X
N
S
S
1: R = H, n = 3, IC50(D6) = 8 nM [27]
2: R = F, n = 3, IC50 (D6) = 10 nM [27]
3: R = CN, n = 2, IC50 (D6) = 5 nM [27]
R1, R2 = H, F, CN; R = H, Me
n = 1,2,4
X = H, Cl
23-32
this work
R
H
N
S
R
N
R
S
()n
NH
N
()n
NH
N
Cl
X
4: R = CN, n = 2, IC50(D6) = 2 nM [28]
R1 = CN, C CH; R = H, Me
n = 1,2,3,4,5,7
X = H, Cl
5: R = OCH3, n = 2, IC50 (D6) = 7 nM [28]
6: R = CH3, n = 1, IC50 (D6) = 5 nM [28]
37-46, 52
Figure 1. Benzothiophene and thiophene based antimalarials 23–32, 37–46 and 52.

Molecules 2017, 22, 343
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2. Results
2.1. Chemistry
The syntheses of the aminoquinoline antimalarials with a benzothiophene carrier were executed
using procedures we developed earlier. Derivatives 12 and 13 were obtained by reductive
8, 9,
amination, starting from the corresponding aldehydes (Scheme 1).
i)
NH
NH
8:
n=2 (59%)
9: n=3 (58%)
()n
CHO
CHO
S
S
N
7
R1
R1
R2
i)
12: n=2, R1=H, R2=F (50%)
13: n=3, R1=F, R2=H (59%)
R2
NH
()n
NH
S
N
S
10: R1=F, R2=H [27]
11: R1=H, R2=F [27]
i) 1) aminoquinoline, AcOH glac, MeOH/CH2Cl2, r.t., 2 h 2) NaBH4, r.t., 12 h
Scheme 1. Synthesis of benzothiophene derivatives 8, 9, 12 and 13.
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
21 and 22. These fragments are used in the key
acid via Suzuki reactions to give the aldehydes 20
,
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.
CHO
R3
N
Br
R1
R1
R1
R2
i)
R1
R2
ii)
iii)
N
()n NH
R2
R2
R4
S
S
X
S
S
14: R1=F, R2=H [27]
17: R1=F, R2=H (84%)
18: R1=CN, R2=H (87%)
19: R1=H, R2=CN (81%)
20: R1=F, R2=H (88%)
21: R1=CN, R2=H (37%)
22: R1=H, R2=CN (76%)
23: n=1, R1=F, R2,R3,R4=H, X=H (59%)
24: n=4, R1=F, R2,R3,R4=H, X=H (63%)
15: R1=CN, R2=H [27]
16: R1=H, R2=CN [27]
25: n=2, R1=F, R2,R4=H, R3=Me, X=Cl (66%)
26: n=2, R1=F, R2,R4=H, R3=Me, X=H (38%)
27: n=2, R1=F, R2=H, R3,R4=Me, X=H (60%)
28: n=2, R1=CN, R2,R3,R4=H, X=H (20%)
29: n=2, R1=CN, R2,R4=H, R3=Me, X=H (21%)
30: n=4, R1=CN, R2,R3,R4=H, X=H (19%)
31: n=2, R2=CN, R1,R3,R4=H, X=H (52%)
32: n=4, R2=CN, R1,R3,R4=H, X=H (33%)
iv)
i) Br2, C2H4Cl2, 0 oC to r.t., 2 h; ii) 4-formylphenylboronic acid, Pd(OAc)2, SPhos, DME, Na2CO3, EtOH, 100 oC, 2 h, MW; iii) 1) aminoquinoline, AcOH glac,
MeOH/CH2Cl2, r.t., 2 h 2) NaBH4, r.t., 12 h; iv) HCHO, ZnCl2, NaBH3CN, MeOH, r.t., 4 h
Scheme 2. Synthesis of benzothiophene derivatives 23
2-Dicyclohexylphosphino-2 ,6 -dimethoxybiphenyl; DME = 1,2-dimetoxyethane.
–
32 with the phenylene linker. SPhos =
0
0
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

Molecules 2017, 22, 343
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additional Suzuki reaction with 4-formyphenylboronic acid afforded 36 in excellent yield (92%).
After submitting 36 to reductive amination reactions with different aminoquinolines, the target
aminoquinoline derivatives 37–44 were obtained in 26%–59% isolated yield. The compound 45
was further methylated providing derivative 46 with a tertiary amino group (Scheme 3).
Br
i)
ii)
i)
NC
S
S
S
Br
S
O
NC
NC
33
34: 82%
35: 91%
36: 92%
iii)
37: n = 1, R=H, X = Cl (32%)
38: n = 2, R=H, X = Cl (59%)
39: n = 3, R=H, X = Cl (38%)
40: n = 4, R=H, X = Cl (40%)
41: n = 5, R=H, X = Cl (39%)
42: n = 7, R=H, X = Cl (36%)
43: n = 3, R=H, X = H (45%)
44: n = 5, R=H, X = H (26%)
45: n = 7, R=H, X = H (35%)
46: n = 7, R=Me, X = H (45%)
NH
S
N
R
X
N
NC
iv)
i) ArB(OH)2, PdO x 1.4H2O, K2CO3, EtOH/H2O, 60 oC; ii) Br2, C2H4Cl2, 0 oC to r.t.; iii) 1) aminoquinoline, AcOHglac, MeOH/CH2Cl2,
r.t., 2 h 2) NaBH4, r.t; iv) HCHO, ZnCl2, NaBH3CN, MeOH, r.t.
Scheme 3. Synthesis of thiophene derivatives 37–46.
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.
i)
ii)
i)
Br
Br
S
S
S
S
Br
O
O
O
33
47: 87%
48: 80%
49: 56%
iii)
NH
HN
S
( )7
iv)
TMS
S
R
O
N
51: R=TMS (25%)
52: R=H (99%)
v)
50: 41%
i) ArB(OH)2, Pd(OAc)2, PPh3, Na2CO3, MeOH/PhMe, 110 oC; ii) NBS, THF, r.t.; iii) ethynyltrimethylsilane, PdCl2(PPh3)2, PPh3, CuI,
Et2NH, DMF, MW; iv) 1) AQ10, AcOH glac, MeOH/CH2Cl2, r.t., 2 h 2) NaBH4, r.t.; v) K2CO3, MeOH, r.t.
Scheme 4. Synthesis of acetylene derivative 52. NBS = N-bromosuccinimide; THF = tetrahydrofuran;
DMF = N,N-dimethylformamide; MW = microwave irradiation; AQ10 = N-(quinolin-4-yl)octane-
1,8-diamine.
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.

Molecules 2017, 22, 343
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2.2. 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).
Table 1. In vitro antiplasmodial activity of benzothiophene derivatives.
In Vitro Antimalarial Activity
SI g
HepG2/D6
RI h
W2/D6
HepG2 f
(IC50, nM)
(P. falciparum, IC50, nM) a,b
Structure
D6 c
C235 e
W2 d
NH
8
9
NH
S
20
104
107
8867
4867
4477
440
339
211
5.2
13.6
8.8
N
N
14
21
191
185
205
191
NH
S
NH
N
F
NH
NH
12
S
F
NH
N
NH
13
23
24
25
16
28
60
71
201
144
277
12
177
111
242
121
2726
3825
2233
2087
172
136
37
12.6
5.1
S
N
F
NH
NH
S
F
NH
NH
N
4.6
S
F
NH
NH
N
29
0.2
S
Cl
F
NH
NH
N
26
27
28
36
29
6
55
45
2345
4998
2575
65
1.5
4.3
S
F
NH
N
N
126
188
126
129
173
401
S
NC
NH
NH
N
31.3
S
NC
NH
NH
29
13
44
46
2413
192
3.4
N
S

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Table 1. Cont.
In Vitro Antimalarial Activity
SI g
HepG2/D6
RI h
W2/D6
HepG2 f
(IC50, nM)
(P. falciparum, IC50, nM) a,b
Structure
D6 c
C235 e
W2 d
NC
NH
NH
N
30
31
32
12
81
51
1976
3577
2049
162
276
37
6.8
S
NH
NH
NC
N
13
55
197
102
93
29
15.2
1.8
S
NH
NH
NC
N
S
N
HN
N
CQ i
15 (6)
23 (6)
595 (5)
7 (5)
206 (5)
55 (5)
39.7
0.3
Cl
HN
HO
MFQ i
F3
C
N
CF3
a
Antiplasmodial IC50 values (nM) (Malaria SYBR Green Fluorescence Assay) for isolates and clones of P. falciparum;
b
c
All in vitro experiments were done as technical quadruplicates, mean values are given; Chloroquine (CQ)
d
susceptible P. falciparum African D6 clone;
Chloroquine (CQ) resistant P. falciparum Indochina W2 clone;
P. falciparum CQ, MFQ and pyrimethamine resistant TM91C235 strain (Thailand); f Hepatocellular carcinoma;
Selectivity index; h Resistance index; i Control drugs: CQ as diphosphate; MFQ as HCl salt (number of replicates
e
g
given in parentheses).
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.

Molecules 2017, 22, 343
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Table 2. In vitro antiplasmodial activity of thiophene derivatives.
In Vitro Antimalarial Activity
SI g
HepG2/D6
HepG2 f
(IC50, nM)
RI h
W2/D6
(P. falciparum, IC50, nM) a,b
Structure
D6 c
C235 e
W2 d
N
S
37
49
26
60
> 30605
624
30
0.5
NC
NH
NH
Cl
NH
S
NH
N
NC
38
84
20
43
2564
0.2
Cl
N
39
59
80
46
41
52
54
> 5792
6702
98
84
0.8
0.5
NH
S
NH
Cl
NC
Cl
NH
S
40
NC
NH
N
N
NH
41
27
55
4
13
12
20
30
34
2415
2092
8153
89
38
0.5
0.2
26
S
NH
Cl
NC
N
S
42
43
NC
NH
NH
Cl
N
S
106
2038
NH
NH
NC
N
44
46
52
101
7
74
5
54
13
4085
2570
9230
40
367
52
0.7
0.7
1.5
NH
S
NH
NC
N
S
NC
NH
N
N
177
259
274
S
NH
NH
N
HN
N
CQ i
15 (6)
23 (6)
595 (5)
7 (5)
206 (5)
55 (5)
39.7
0.3
Cl
HN
HO
MFQ i
F3
C
N
CF3
a
Antiplasmodial IC50 values (nM) (Malaria SYBR Green Fluorescence Assay) for isolates and clones of P. falciparum;
All in vitro experiments were done as technical quadruplicates, mean values are given; CQ susceptible
b
c
P. falciparum African D6 clone; d CQ resistant P. falciparum Indochina W2 clone; e P. falciparum multidrug resistant
TM91C235 strain (Thailand). Hepatocellular carcinoma; g Selectivity index; h Resistance index; i Control drugs:
f
CQ as diphosphate; MFQ as HCl salt (number of replicates given in parentheses).
3. 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
×
106 P. berghei

Molecules 2017, 22, 343
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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
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
.
However, rather low selectivity indices were calculated for all derivatives
(
×
×
×
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
examined for its antimalarial activity in vivo, despite a relatively low SI (282). Surprisingly, compound
cured 5/5 mice using a modified Thompson test (Figure 3, Table 3) and cleared all parasites on day 7
1 [27] (Figure 2), which was
1
(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
was no detectable parasitemia up to day 11 at a dose as low as 40 mg/kg/day
occurred between day 11 (D11) and day 14 (D14). In vivo activity results for
Figure 3. On the other hand, compound 53 27] with a propylene linker, which showed much more
1
did not clear the parasites (Table 3). There
however, recrudescence
are summarized on
;
1
[
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; IC50(W2) = 31 nM; IC50(D6) = 8 nM; SI(HepG2/D6) = 282 [27]
53: n=1; IC50(W2) = 24 nM; IC50(D6) = 7 nM; SI(HepG2/D6) = 1111 [27]
N
this work
()n
NH
NH
1: n=2
Toxicity 5/5 survived @ 160 mg/kg/day
S
Cl
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
Figure 2. Antimalarial activity of compounds 1 and 53. RBC—red blood cell.
Figure 3. Effect of treatment with compound
1 at four dosage regimens (20, 40, 80, 160 mg/kg) on the
survival of mice infected with P. berghei Antwerpen-Kasapa (ANKA) strain.

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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).
1000
950
900
850
800
750
700
650
600
550
500
450
400
350
300
250
200
150
100
50
0
-50
-100
-150
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
Cycle
Threshold:
208 (Noiseband)
Baseline settings: automatic, Drift correction OFF
Figure 4. Real time (quantitative) PCR (qPCR) analysis for compound 1. Amplification curves for
blood and tissues of five mice. Horizontal line represents threshold for amplification. All values >40 Ct
and amplification curves below threshold indicate absence of target DNA. The three amplification
curves above threshold correspond to positive controls.
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.

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Table 3. In Vivo Antimalarial Activity a,b
.
Mean Survival Time
(MST, Days)
Comp.
mg/kg/Day
Parasitemia (Day: # Mice, %Parasitemia)
Mice Dead/Day Died
Mice Alive on Day 31
D3: 5 mice positive, 0.6%–2.4%;
D7: 2 mice negative, 3 mice positive, 0.1%–0.2%;
D11: 3 mice positive, 0.8%–1.9%;
D14: 2 mice positive, 1.6%–3.7%;
D18: 1 mouse positive, 6%;
D21: 1 mouse positive, 27%;
D25: 1 mouse positive, 63%;
2/11
1/14
20 c
0/5
16.8
19.4
1/18
1/30
D28: 1 mouse positive, 66%
D3: 5 mice positive, 0.4%–0.9%;
D7: 5 mice negative;
D11: 5 mice negative;
D14: 5 mice positive, 0.6%–2.4%;
D18: 1 mouse positive, 2.2%;
D21: 1 mouse positive, 7.3%;
D25: 1 mouse positive, 37%;
D28: 1 mouse positive, 53%
1/15
1/16
1
40 c
0/5
2/18
1/30
D3: 4 mice positive, 0.7%–1.1%;
D7: 4 mice negative;
D11: 4 mice negative;
D14: 4 mice positive, 0.1%–0.5%;
D18: 1 mouse positive, 2.3%
3/18
1/19
80 c
160
0/4
5/5
18.2
>31
D3: 5 mice positive, 0.3%–0.9%;
D7–D31: 5 mice negative
D3: 4 mice positive, 0.4%–0.6%;
D7: 4 mice positive, 0.2%–0.4%;
D10: 4 mice positive, 0.3%–0.6%;
D14: 4 mice positive, 0.5%–0.8%;
D17: 2 mouse positive, 1.5%–3.7%;
D21: 1 mouse positive, 2.7%;
D24: 1 mouse positive, 52%;
D28: 1 mouse positive, 72%;
D31: 1 mouse positive, 79%
2/17
1/18
1/4
20.8
25
160

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Table 3. Cont.
Mean Survival Time
(MST, Days)
Comp.
42
mg/kg/Day
Parasitemia (Day: # Mice, %Parasitemia)
Mice Dead/Day Died
Mice Alive on Day 31
D3: 5 mice positive, 0.2%–0.6%;
D7: 5 mice positive, 0.2%–10%;
D10: 4 mice positive, 9.2%–15%;
D14: 3 mice positive, 12.8%–47%;
D17: 3 mice positive, 50%–60%;
D21: 2 mice positive, 64%–70%;
D24: 1 mouse positive, 75%
1/9
1/13
1/20
1/23
1/28
18.6
15.8
0/5
160
D3: 5 mice positive, 0.6%–2.2%;
D7: 1 mouse negative, 4 mice positive, 0.2%–0.4%;
D10: 4 mice positive, 1.6%–4.9%;
D14: 2 mice positive, 8.1%–12%;
D18: 2 mice positive, 16%–20%;
D21: 2 mice positive, 22%–41%;
D25: 1 mouse positive, 43%
1/10
2/11
40 c
0/5
1/22
1/25
46
D3: 5 mice positive, 0.8%–2.3%;
D7: 2 mice negative, 2 mice positive, 0.1%–0.2%;
D10: 4 mice positive, 0.2%–6.6%;
D14: 1 mouse positive, 4.7%;
D18: 1 mouse positive, 12%;
D21: 1 mouse positive, 35%;
1/7
1/12
1/13
80 c
14.4
>31
0/5
5/5
1/14
1/26
D25: 1 mouse positive, 41%
D3: 5 mice positive, 0.3%–0.8%;
D6-D31: 5 mice negative
CQ
160
0
Infected controls
All mice died on day 7–8
a Groups of five P. berghei (ANKA strain) infected mice were treated per os (p.o.) once per day on days 3–5 postinfection with aminoquinolines suspended in 0.5% hydroxyethylcellulose-0.1%
Tween 80. Mice alive on day 31 with no parasites in a blood film are considered cured; b All compounds were tested in separate experiments (mice groups) for toxicity. None exerted any
toxic effect when dosed orally at 160 mg/kg/day
with aminoquinolines suspended in 0.5% hydroxyethylcellulose-0.1% Tween 80. Mice alive on day 31 with no parasites in a blood film are considered cured.
×
3 days; c Groups of five P. berghei (ANKA strain) infected mice were treated per os (p.o.) once per day on days on 3–6 postinfection

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4. Materials and Methods
4.1. Chemistry
4.1.1. General Experimental Procedures
Melting points were determined on a Boetius PHMK apparatus (Carl Zeiss, Germany) and
were not corrected. IR spectra were recorded on a Nicolet 6700 Fourier-transformed infrared (FT-IR)
1
diamond crystal spectrophotometer (Thermo-Scientific, Waltham, MA, USA). H- and 13C-NMR
spectra were recorded on a Ultrashield Advance III spectrometer (at 500 and 125 MHz, respectively,
Bruker, Billerica, MA, USA) and Gemini-200 spectrometer (at 50 MHz for 13C-NMR spectra, Varian,
Palo Alto, CA, USA), in the indicated solvents (vide infra) using tetramethylsilane as the internal
standard. Chemical shifts are expressed in ppm (δ) values and coupling constants (J) in Hz.
High-resolution electrospray ionization mass spectrometry (ESI–MS, HRMS) spectra of the synthesized
compounds were acquired on a 1200 Series instrument (Agilent Technologies, Santa Clara, CA, USA)
equipped with a Zorbax Eclipse Plus C18 (100
(DAD, 190–450 nm) in combination with a 6210 Time-of-Flight LC/MS instrument in positive ion
×
2.1 mm i.d. 1.8 µm) column and diode array detector
mode. The samples were dissolved in MeOH (HPLC grade). The selected values were as follows:
◦
capillary voltage 4 kV; gas temperature 350 C; drying gas 12 L
·
min-1; nebulizer pressure 45 psig;
fragmentator voltage 70 V. Gas chromatography tandem mass spectrometry (GC-MS) analyses were
performed on an Agilent 7890A GC (Agilent) system equipped with a 5975C inert XL EI/CI MSD
and a flame ionization detector (FID) connected by capillary flow technology through a 2-way splitter
with make-up gas. An HP-5 MS capillary column (Agilent Technologies, 25 mm i.d., 30 m length,
0.25
(Biotage AB, Uppsala, Sweden) equipped with UV detector and FLASH 12+, FLASH 25+ or FLASH 40+
columns charged with KP-SIL (40–63 m, pore diameter 60 Å), KP-C18-HS (40–63 m, pore diameter
90 Å) or KP-NH (40–63 µm, pore diameter 100 Å) as an adsorbent.
µm film thickness) was used. The flash chromatography was performed on a Biotage SP1 system
µ
µ
4.1.2. Method A—General Procedure for Reductive Amination for Compounds 8, 9, 12, 13, 23–26,
28–32, 37–45, 51
Amine (1.5 eq.) and appropriate aldehyde (1 eq.) were dissolved in MeOH/CH2Cl2 mixture
(2:1 v:v), AcOH glac (1.5 eq.) was added, and the mixture was stirred under an Ar atmosphere at r.t.
After 3 h, NaBH4 (6 eq.) was added, and stirring was continued for another 18 h. Solvent was removed
under reduced pressure, and the residue was dissolved in CH2Cl2. The organic layer was washed with
2M NH4OH, water and then extracted with CH2Cl2. The combined organic layers were washed with
brine and dried over anhydrous Na2SO4. Finally, the solvent was evaporated under reduced pressure.
4.1.3. Method B—General Experimental Procedure for Bromination for Compounds 17–19, 35
Substituted benzothiophene (1 eq.) was dissolved in 1,2-dichloroethane. A solution of bromine
◦
(1.12 eq.) in 1,2-dichloroethane (CH2Cl2 for 17) was slowly added to the reaction mixture at 0 C, then
warmed to r.t., and stirred for 2 h. Reaction progress was monitored by thin-layer chromatography
(TLC, reverse phase silica gel, MeOH). Aqueous Na2S2O3 solution was added, and the desired product
was extracted with CH2Cl2. Combined organic layers were washed with brine, and dried over
anhydrous Na2SO4. After filtration, the solvent was removed under reduced pressure. The product
was purified using column chromatography. [34]
4.1.4. Method C—General Procedure for Suzuki Coupling for Compounds 20–22
A suspension of Pd(OAc)2 (5.00 mol %) and SPhos (20.0 mol %) in DME was purged with Ar and
stirred at r.t. for 10 mins. Brominated benzothiophene (1 eq.) in DME and 2 M Na2CO3 were then
added. After 5 min, 4-formylphenylboronic acid (1.2 eq.) in EtOH was added. The mixture is once
more purged with Ar and heated in a sealed vessel in microwave reactor at 100 ◦C for 2 h. Reaction

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mixture was filtered through Celite and transferred to a separation funnel. Water was added and
extracted with CH2Cl2. Combined organic layers were washed with brine and dried over anhydrous
Na2SO4. After filtration, the solvent was removed under reduced pressure. The product was purified
using column chromatography.
4.1.5. Method D—General Procedure for Synthesis of Aminoquinolines AQ88, AQ9 and AQ10
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 CH2Cl2. Combined organic layers were dried
over anhydrous Na2SO4. After filtration, the solvent was removed under reduced pressure. The crude
product was subjected to silica-gel column chromatography using CH2Cl2/MeOH (NH3 satd.) as
eluent to afford the final compound.
4.1.6. Method E—General Procedure for N-Methylation of Aminoquinolines for Compounds 27
and 46
To a stirred solution of aminoquinolines (1 eq.) in MeOH containing 37% aqueous formaldehyde
(2 eq.) was added mixture of ZnCl2 (2 eq.) and NaHB3CN (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 CH2Cl2, the combined
extracts were washed with water and brine and dried over anhydrous Na2SO4. The solvent was
evaporated under reduced pressure. [35]
4.1.7. Method F—General Procedure for the Suzuki Coupling Reaction Using PdO × 1.4 H2O for
Compounds 34, 36
An appropriate aryl-bromide (1 eq.) was added to the mixture of arylboronic acid (1.2 eq.), catalyst
PdO
×
1.4 H2O (0.01–0.05 eq.), K2CO3 (1.2 eq.) and ethanol/H2O (3:1 v/v). The mixture was stirred at
60 ◦C for 5 h, then diluted with water and extracted with CH2Cl2. The combined organic layers were
washed with brine and dried over anhydrous Na2SO4. After filtration, the solvent was removed under
reduced pressure. The product was purified using silica gel flash chromatography [29].
4.1.8. Method G—General Procedure for the Suzuki Coupling in MeOH/Toluene for
Compounds 47, 49
A solution of Pd(OAc)2 (0.05 eq.) and PPh3 (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. Na2CO3 (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.
N-(7-Chloroquinolin-4-yl)ethane-1,2-diamine (AQ2), N-(7-chloroquinolin-4-yl)propane-1,3-diamine
(AQ3), N-(7-chloroquinolin-4-yl)butane-1,4-diamine (AQ4), N-(7-chloroquinolin-4-yl)pentane-1,5-
diamine (AQ5), N-(7-chloroquinolin-4-yl)hexane-1,6-diamine (AQ6), N-(quinolin-4-yl)propane-
1,3-diamine (AQ7), N-(quinolin-4-yl)butane-1,4-diamine (AQ8), were prepared according to known
procedures [36–39].
Full details are given in the Supplementary Materials.
4.2. 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

Molecules 2017, 22, 343
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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].
4.3. 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).
4.4. Genomic DNA Extraction and qPCR Analysis
4.4.1. 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
gDNA extraction.
µL of sample was used per each spin column for
4.4.2. 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 20 L 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 L template gDNA. The following PCR conditions
:
µ
×
µ
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 (H2O) were included in each run [32].
5. 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 IC50 = 6 nM, and cured 5/5

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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.
Supplementary Materials: Supplementary Material—I (Chemistry); Supplementary Material—II (NMR spectra
of synthesized compounds, HPLC analyses for purity) are available online.
Acknowledgments: This research was supported by The Ministry of Science and Technological Development of
Serbia (grant #: 172008 and 41019) and the Serbian Academy of Sciences and Arts.
Author Contributions: Bogdan Šolaja and Jelena Konstantinovic´ designed thiophene and benzothiophene
antiplasmodials; Jelena Konstantinovic´, Milica Videnovic´ and Katarina Bogojevic´ synthesized and characterized
the compounds; Richard Sciotti coordinated research at WRAIR; Jelena Srbljanovic´, Jelena Konstantinovic´, and
Olgica Djurkovic´-Djakovic´ performed in vivo antimalarial screening and toxicity tests; Bogdan Šolaja was the
head research advisor, directed research activities and edited the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 8, 9, 12, 13, 23–32, 37–44, 46, 52 are available from the authors.
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