Novel inhibitors of Plasmodium falciparum based on 2,5-disubstituted furans

Article abstract of DOI:10.1016/j.ejmech.2016.12.024

Phenotypic HTS campaigns with a blood stage malaria assay have been used to discover novel chemotypes for malaria treatment with potential alternative mechanisms of action compared to existing agents. N¹-(5-(3-Chloro-4-fluorophenyl)furan-2-yl)-N³,N³-dimethylpropane-1,3-diamine, 1 was identified as a modest inhibitor of P. falciparum NF54 (IC₅₀= 875 nM) with an apparent long plasma half-life after high dose oral administration to mice, although the compound later showed poor metabolic stability in liver microsomes through ring- and side chain-oxidation and N-dealkylation. We describe here the synthesis of derivatives of 1, exploring the influence of substitution patterns around the aromatic ring, variations on the alkyl chain and modifications in the core heterocycle, in order to probe potency and metabolic stability, where 4k showed a long half-life in rats.

Full text of DOI:10.1016/j.ejmech.2016.12.024

Accepted Manuscript
Novel inhibitors of Plasmodium falciparum based on 2,5-disubstituted furans
Susann H. Krake, Pablo David G. Martinez, Jenna McLaren, Eileen Ryan, Gong
Chen, Karen White, Susan A. Charman, Simon Campbell, Paul Willis, Luiz Carlos
Dias
PII:
S0223-5234(16)31030-3
10.1016/j.ejmech.2016.12.024
EJMECH 9115
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 October 2016
Revised Date: 8 December 2016
Accepted Date: 9 December 2016
Please cite this article as: S.H. Krake, P.D.G. Martinez, J. McLaren, E. Ryan, G. Chen, K. White,
S.A. Charman, S. Campbell, P. Willis, L.C. Dias, Novel inhibitors of Plasmodium falciparum
based on 2,5-disubstituted furans, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2016.12.024.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
TITLE: Novel Inhibitors of Plasmodium Falciparum based on 2,5-disubstituted Furans.
AUTHOR NAMES. Susann H. Krakea,‡ Pablo David G. Martineza,‡ Jenna McLarenb, Eileen Ryanb,
Gong Chenb, Karen Whiteb, Susan A. Charmanb, Simon Campbellc, Paul Willisc, Luiz Carlos Diasa,*
AUTHOR ADDRESS
a Instituto de Química, Universidade Estadual de Campinas, Barão Geraldo, PO Box 6154, Campinas,
SP 13084-971, Brazil.
b Centre for Drug Candidate Optimisation, Monash University (Parkville Campus), 381 Royal Parade,
Parkville, VIC 3052, Australia
c Medicines for Malaria Venture, ICC, Route de Pré-Bois 20, P.O. Box 1826, 1215 Geneva,
Switzerland
ABSTRACT.
Phenotypic HTS campaigns with a blood stage malaria assay have been used to discover novel
chemotypes for malaria treatment with potential alternative mechanisms of action compared to
existing agents. N1-(5-(3-Chloro-4-fluorophenyl)furan-2-yl)-N3,N3-dimethylpropane-1,3-diamine, 1
was identified as a modest inhibitor of P.falciparum NF54 (IC50 = 875nM) with an apparent long
plasma half-life after high dose oral administration to mice, although the compound later showed poor
metabolic stability in liver microsomes through ring- and side chain-oxidation and N-dealkylation. We
describe here the synthesis of derivatives of 1, exploring the influence of substitution patterns around
the aromatic ring, variations on the alkyl chain and modifications in the core heterocycle, in order to
probe potency and metabolic stability, where 4k showed a long half-life in rats.
GRAPHICAL ABSTRACT.
1

ACCEPTED MANUSCRIPT
KEYWORDS. Malaria, Furan, Diamines, Synthesis, nM Inhibitors
1. Introduction.
The WHO estimated that 198 million cases of malaria occurred in 2013 with a death toll of 584,000
[1]. The disease, social and economic burdens are highest in Africa, where 90% of all malaria deaths
occur and of those, 78% are accounted for by children under the age of 5 years. Globally, 3.3 billion
people are at risk of contracting malaria, with 1.2 billion living with a high risk. While access to vector
control, preventive therapy and drug treatment have improved and mortality has been reduced, the
development of resistant parasites is still a major concern for the future.
To diminish the likelihood of developing resistance with current antimalarials, combination therapies
with artemisinins and a second longer half-life drug have displaced single agents such as chloroquine.
Even so, the continued development of drug candidates with novel chemotypes and mechanisms of
action against resistant strains is still necessary to respond to the significant clinical challenges. In
particular, recent studies show declining rates of parasite clearance with artemisinin treatment in
Southeast-Asia which is alarming [2-4], and as previously observed with chloroquine, resistance could
spread across Asia to Africa and thus threaten global efforts to control and eliminate malaria.
Coinciding with the development of resistance, the pharmaceutical industry started to screen their
compound libraries against P. falciparum [5-7]; a process aided by the development of phenotypic
HTS, and hits have been made publicly available by the Medicines for Malaria Venture (MMV) by a
number of means, including the MMV Malaria Box [8]. In addition, the decoding of the P. falciparum
genome sequence and analysis of new pathways that identify novel biological targets offers an
opportunity to rationally design improved antimalarial scaffolds [9]. For maximum efficiency, new
drugs should adhere to MMV’s objective of single dose treatments which requires compounds with
long duration of action.
1.1. Initial lead. Furans have recently been described with anti malarial activity [10]. A similar
compound, 1 (Fig. 1), was identified from high throughput screening [6] at GlaxoSmithKline and
showed modest activity against Plasmodium falciparum NF54 cell culture (EC50, 875nM). Profiling
2

ACCEPTED MANUSCRIPT
under Malaria Box protocols showed the compound had a long plasma half-life of greater than 6 h
after oral administration (40 mg/kg) to mice, low cytotoxicity in MRC5 cells (EC50, 17µM) and weak
hERG inhibition (48% inhibition at 10 µM) [11]. Unfortunately, later studies revealed poor metabolic
stability of 1 in human, rat and mouse liver microsomes through oxidation, which suggests that this
high dose, long oral half-life in mice probably results from saturation of clearance pathways.
Therefore, our goal was to enhance antiparasitic activity and improve metabolic stability of this lead
structure. Three main structural elements were considered for optimization: the aryl-ring, the central
furan-template and the diamine side-chain. The main problems with metabolic stability were
associated with dehydrogenation and dealkylation of the amine side-chain, and oxidation of the furan
and aryl rings. Herein, we describe the synthesis and systematic modification of all three subunits, and
effects on biological activity and metabolic stability.
Me2N
NH
F
O
reductive
amination
Cl
Suzuki reaction
1
Fig. 1. Lead structure.
2. Results and discussion
2.1. Chemistry. The synthesis of aryl and amine derivatives of 1 are outlined in Scheme 1 where
boronic acid 2 was obtained in two steps from furfural using known conditions [12, 13]. This was then
coupled with a number of aryl-halides via a Suzuki reaction to obtain intermediates of type 3 [14]. The
substitution pattern of the aryl moiety was changed systematically from mono- to tri-substitution with
a variety of combinations. Products were typically obtained with moderate to very good yields, with
the exception of sterically hindered derivatives. Reductive aminations with primary amines were
conducted in two steps, with confirmation of the identity of the imine-intermediates either by 1H-NMR
or IR. For secondary amines, reactions were carried out in one pot with delayed addition of sodium
triacetoxyborohydride, and products 4 and 5 were obtained after purification. In some cases, free
3

ACCEPTED MANUSCRIPT
amine products were converted into solid hydrochloride salts which are moderately hydroscopic in air,
but stable if kept in a closed container.
Scheme 1. Synthesis of 4 and 5; Reagents and conditions: (a) Ar-Hal, PdCl2(PPh3)2 (5mol%), 2M
Na2CO3 (aq), DME: EtOH (3:2), 60-75°C, 1-3h (36-95%); (b) primary amine, CH2Cl2 or Et2O, then
NaBH4, MeOH (41-96%); (c) secondary amine, CH2Cl2, then NaBH(OAc)3 (83-94%).
The majority of compounds where the furan template is replaced by different aryl rings were obtained
from Suzuki coupling of the corresponding bromo-substituted aldehydes 6 with (3-chloro-4-
fluorophenyl)boronic acid (Scheme 2), which was prepared under standard conditions [15]. In the case
of the pyridine derivative 7b, conditions using tetrakis(triphenylphosphine)palladium were applied to
improve the yield significantly [16]. In the Suzuki coupling with the benzofurancarboxaldehyde, the
temperature was increased to 100°C, maintaining the same palladium catalyst as for the furan series.
The previously established reductive amination of intermediate 7 led to the derivatives of type 8.
Three benzofurans (8d, 9a and 9b) were prepared to investigate the potential for the introduction of
fused ring systems.
Scheme 2. Synthesis of 8a-8d and 9a-b; Reagents and conditions: (a) (3-chloro-4-
fluorophenyl)boronic acid, PdCl2(PPh3)2 (5mol%), 2M Na2CO3 aq, DME, EtOH, 60-100°C, 1-3h or
4

ACCEPTED MANUSCRIPT
Pd(PPh3)4, 2M Na2CO3 (aq), toluene, MeOH, 110°C, 7h (75-88%). (b) (CH3)2N(CH2)3NH2, Et2O or
CH2Cl2, then NaBH4, MeOH (39-96%).
Finally, we were interested in replacing the furan ring by other five-membered heterocycles and we
selected the isoxazole system described in Scheme 3. 4-Bromo benzaldehyde was converted into
intermediate 11 through two steps [17], followed by oxidation with pyridinium chlorochromate and
reductive amination to obtain the isoxazole derivative 12.
O
O
HO
N
NH
N
OHC
a, b
c, d
Br
Me2N
Br
Br
10
11
12
Scheme 3. Synthesis of 12; Reagents and conditions: (a) NH2OH.HCl, Na2CO3, MeOH, H2O, rt; (b)
propargyl alcohol, NEt3, NCS, CH2Cl2, reflux (46% over two steps); (c) PCC, CH2Cl2, 4h, rt; (d)
(CH3)2N(CH2)3NH2, Et2O, then NaBH4, MeOH (65% over two steps).
2.2. Structure-Activity Relationships. The biological activities of compounds were determined
against the drug sensitive P. falciparum NF54 strain in vitro (EC50, NF54) [18]. In vitro intrinsic
clearance was assessed by substrate depletion upon incubation with human (HLM) and mouse (MLM)
liver microsomes in the presence of the co-factor, NADPH. Data are summarized in Tables 1-4.
2.2.1. Modification of the aryl moiety. Of utmost importance for antiparasitic activity is para
halogenation of the aryl moiety at the 2-position of the furan template (Table 1, 4d: Cl or 4e: Br), as
removal (4b) or replacement with a smaller fluoro substituent (4c) leads to a dramatic decrease in
potency. Additional meta-chloro substitution (1 and 4k) is tolerated as long as the para-position is also
halogenated. Substitution in both meta-positions (4l) leads to a decrease in activity. Similar results
were observed for the introduction of ortho-substituents as exemplified by comparison of 4d with 4j.
With respect to rapid clearance, only the two methoxy-substituted derivatives (4h and 4n) show low
intrinsic clearance in human liver microsomes (HLM), which is likely to result from very low
cLogD7.4 (-0.5 and -0.7 respectively), but both derivatives showed a complete loss of antiparasitic
activity.
Table 1.
Substitution of the Aryl Moiety.
5

ACCEPTED MANUSCRIPT
In vitro CLint
(µL/min/mg protein) cLogD7.4
Compound
number
NF54
EC50 (nM)
R
H
HLM
MLM
875
93
84
0.5
1
>10000
7206
0.7
4a
4b
42
34
15
19
110
63
-0.2
2622
342
-0.1
0.4
0.6
4c
4d
4e
67
442
84
3599
7224
6311
N/A
24
N/A
200
74
0.0
0.8
4f
4g
4h
< 7
-0.5
1912
19
159
0.6
4i
1465
263
16
28
100
95
1.0
1.0
4j
4k
1190
16
34
89
285
16
1.0
0.1
4l
4m
4n
> 10000
> 10000
< 7
-0.7
6

ACCEPTED MANUSCRIPT
2.2.2. Modification on the amine side chain. An especially tight SAR was observed for modification
on the amine side chain (Table 2). Complete activity loss followed from shortening the chain length
(5a and 5d), cyclisation to a morpholino derivative (5c) and the absence of the terminal tertiary amine
(5e-5i). The only modifications that showed any activity came from replacement of the dimethylamine
(1) with a diethylamine (5b), and incorporation of an N-methylpiperazine (5j), but without significant
influence on the metabolic stability.
Table 2.
Replacement of the Amine Side Chain.
In vitro CLint
(µL/min/mg
protein)
NF54
EC50
(nM)
Compound
number
R
cLogD7.4
HLM
MLM
875
5360
1210
93
61
61
84
0.5
1.5
0.8
1
377
100
5a
5b
9729
31
101
1.3
5c
6238
49
56
29
147
306
122
2.0
3.8
2.6
5d
5e
5f
> 10000
> 10000
> 10000
> 10000
> 10000
72
90
72
655
371
854
2.1
2.6
3.0
5g
5h
5i
N
1868
25
542
2.4
5j
N
Me
7

ACCEPTED MANUSCRIPT
2.2.3. Modification of the furan ring. Various studies covered the structure activity relationship of
heterocycles in relation to therapeutic properties, including anti malaria activity [19-21].
In an attempt to exchange the core heterocycle, several cyclic replacements were investigated.
Exchange of the furan template with meta- or para-substituted phenyl rings or a 2,6-substituted
pyridine was tolerated and resulted in activity comparable to the lead compound 1 (Table 3, 8a-8c).
Interestingly, the 2,5-disubstituted benzofuran 8d also showed good activity and a significant decrease
in the microsomal intrinsic clearance. Moving the amine side chain to the para position of the phenyl
ring (8c) gave an improvement in potency of 546 nM and also resulted in a significant improvement in
metabolic stability in both HLM and MLM. Further testing of benzofuran analogs identified 9a with
only slightly decreased activity, but introducing a 5-bromo substituent (9b) led to a complete loss of
potency (Table 4). Similarly, the isoxazole derivative 12 was inactive.
Table 3.
Replacement of the Furan Ring.
NF54
EC50
(nM)
In vitro CLint
(µL/min/mg protein) cLogD7.4
Compound
number
Ar
HLM
MLM
875
93
84
0.5
0.7
1
475
113
170
50
52
8a
822
546
0.8
0.8
8b
8c
20
13
41
69
421
1.6
8d
Table 4.
Replacement of the Furan Ring.
8

ACCEPTED MANUSCRIPT
Me2N
NH
Ar
In vitro CLint
(µL/min/mg
protein)
NF54
EC50
(nM)
Compound
number
Ar
cLogD7.4
-0.8
HLM
MLM
O
804
5849
N/A
35
N/A
9a
9b
12
O
105
53
0.0
Br
Br
> 5000
24
0.3
O N
2.3. Stability and clearance. Compound 1 was stable in freshly collected rat plasma in vitro and
demonstrated extensive distribution into erythrocytes with a whole blood to plasma concentration ratio
of approximately 5.
On dosing 1 to Sprague Dawley rats (1 mg/kg IV, 20 mg/kg po, Fig. 2A), the compound rapidly and
extensively distributed from plasma (at least partially into erythrocytes) with a long apparent terminal
half-life. Plasma concentration versus time profiles were not well defined, and there was considerable
variability in the IV plasma concentrations at later time points precluding the assessment of
pharmacokinetic parameters (clearance, volume of distribution, half-life and oral bioavailability).
Following administration to mice (Fig. 2B), plasma concentrations again showed rapid distribution
and concentrations were higher than seen in rats even after accounting for the difference in dose. In
vitro studies suggested that intrinsic clearance in rat liver microsomes (>500 µL/min/mg) was
considerably higher than that in HLM or MLM (Table 1) which may at least partially account for these
differences. Overall, plasma concentrations of 1 after oral dosing to mice were considerably below
what was likely to be needed for efficacy given the EC50 of ~0.9 µM.
9

ACCEPTED MANUSCRIPT
Fig. 2. Plasma concentration versus time profiles of 1 in (A) male Sprague Dawley rats (0.9 mg/kg IV
(filled circles) and 19 mg/kg oral (open squares) administration) and (B) male Swiss outbred mice (1.1
mg/kg IV (filled circles) and 48 mg/kg oral (open squares) administration). Data points represent the
mean and range for n=2 rats or n=2 mice at each time point.
Metabolite ID was performed by incubation of 1 with human, mouse and rat liver microsomes over a
period of 60 min. Several metabolites were detected (Table 5) including a mono-oxygenation or furan
ring opened metabolite (M+16) seen in each species, two distinct M-2 metabolites, one present in
RLM (M-2 (I)) and the other in MLM and HLM (M-2 (II)), an M-14 metabolite likely corresponding
to N-demethylation (HLM and MLM only), and two secondary M+32 bis-oxygenation metabolites in
RLM. Even though full structure elucidation was not conducted, these preliminary results suggest
significant species differences in the metabolic pathways and major metabolites formed.
Table 5.
10

ACCEPTED MANUSCRIPT
Metabolite profile in liver microsomes.
Relative Peak Area (as % of Total Metabolite Peak Area)
% Substrate
Consumed
Species
M+16
12
M-2 (I)
ND
M-2 (II)
57
M-14
31
M+32 (I)
M+32 (II)
Human
Rat
88
ND
5
ND
5
85
99
86
5
ND
7
ND
4
Mouse
ND
89
ND
ND
ND = not detected
As 4k showed good activity and slightly better metabolic stability in HLMs, it was also tested in vivo
to determine pharmacokinetic properties (Fig. 3). As with 1, a rapid decline in plasma concentrations
after IV administration was seen, at least partially due to high partitioning into erythrocytes (in vitro
whole blood to plasma partitioning ratio ~ 6.9). Blood clearance (~17 mL/min/kg) was moderate and
volume of distribution (Vss, 16 L/kg) was high. Oral bioavailability for 4k was ~16%, with maximum
plasma concentrations detected 30 min post-dose suggesting rapid absorption. Plasma profiles after
both oral and IV administration exhibited a prolonged terminal phase with half-lives ranging from 9-
25 h (oral) and 10-17 h (IV). Collectively, the pharmacokinetic parameters for 4k represent a moderate
improvement compared to those seen for 1.
11

ACCEPTED MANUSCRIPT
Fig. 3. Plasma concentrations of 4k in male Sprague Dawley rats following IV (1.2 mg/kg, filled
circles) and oral (5.5 mg/kg, open squares) administration. Data points represent the mean and range
for n=2 animals.
2.4. Malaria assays.
The parasite reduction ratio (PRR) quantifies the speed of killing observed in vitro by measuring the
number of viable parasites after treatment of the Pf 3D7A strain at a concentration of 10 times the IC50
of the selected drug [22]. Both 4k and 8d showed a chloroquine-like profile, which is consistent with a
fast kill rate (Fig. 4). As the rate of killing is believed to be related to the mode of action, this suggests
that the furan and benzofuran disrupt a common target. Compound 8d also maintained potency when
tested on the Pf K1 cell line which is resistant to the antimalarial drugs chloroquine, pyrimethamine
and sulfadoxine (EC50, 490 nM vs drug sensitive Pf NF54 strain EC50, 309 nM) [23], but showed no
efficacy in a P. berghei infected mouse efficacy model after 4 oral daily doses at 50 mg/kg [24, 25].
The same derivatives (4k and 8d) showed no in vitro activity against P. berghei liver stage parasites at
a concentration of 10 µM [26, 27], and no cytotoxicity to HepG2 cells at the same concentration.
Speed of Killing
5
4
4k
3
8d
2
Chloroquine
Pyrimethamine
Atovaquone
1
0
0
24
48
hours of treatment
Fig. 4. Speed of killing.
To assess the potential of the series to generate resistance and to give insights into the mode of action,
Dd2 parasites were incubated with sub-lethal concentrations of 1 for extended periods [28]. After 125
days incubation, no resistant parasites were obtained, suggesting that this series has a low propensity
to generate resistance.
12

ACCEPTED MANUSCRIPT
3. Conclusion. A novel chemotype with blood stage antimalarial activity has been discovered with
nanomolar antiparasitic activity, but with tight SAR limitations. Several analogues showed lower in
vitro intrinsic clearance compared to 1 in HLMs and one derivative (4k) demonstrated reasonable
pharmacokinetic properties in vivo in rats. The low propensity to generate resistance suggests further
work is warranted to determine the mode of action and further characterize the series.
4. Experimental section.
4.1. Materials and Methods. Dichloromethane (CH2Cl2) was distilled from CaH2. Flash column
chromatography was conducted using silica gel (230−400 mesh) or aluminum oxide (activated,
neutral, Brockmann I, ~150 mesh). Analytical thin-layer chromatography was performed on silica gel
60 F254 plates, and the visualization was accomplished using UV light and phosphomolybdic acid
followed by heating. 1H and proton-decoupled 13C NMR spectra were recorded in CDCl3 or CD3OD at
250 MHz (1H) and 60 MHz (13C), 400 MHz (1H) and 100 MHz (13C), 500 MHz (1H) and 125 MHz
(13C), or 600 MHz (1H) and 150 MHz (13C). The chemical shifts (δ) are reported in ppm using
tetramethylsilane (TMS) at 0.00 ppm or the solvent signal as an internal standard (CDCl3 at 7.26 ppm
and CD3OD at 3.30 ppm for 1H NMR spectra and CDCl3 at 77.0 ppm and CD3OD at 49.0 ppm for 13C
NMR spectra). Data are reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, bs = broad
singlet, dd = doublet of doublets, ddd = doublet of doublet of doublets, m = multiplet; coupling
constant(s) in Hz; integration. High-resolution mass spectrometry (HRMS) was performed using the
electrospray ionization (ESI) technique. The parent ions ([M + H]+ or [M + Na]+) are cited. The
purity (≥95%) of all compounds evaluated in this work was determined by 1H NMR.
4.2. Chemistry
4.2.1. Method A - Suzuki Reaction: 1.00 mmol Arylhalide, 1.30 mmol furfural-boronic acid and
0.05 mmol Bis(triphenylphosphine)palladium(II) dichloride were treated with 0.30 ml
dimethoxyethane, 0.50 ml ethanol and 0.30 ml aqueous 2M sodium carbonate solution. The reaction
was heated to 65°C for 1h or until the TLC showed no remaining starting material. The mixture was
evaporated and extracted three times with ethyl acetate. The combined organic layers were washed
13

ACCEPTED MANUSCRIPT
with brine, dried over MgSO4, filtered and concentrated. The crude product was purified by column
chromatography using hexanes/ethyl acetate (9:1).
4.2.2. Method B - Reductive amination with primary amines: 1.00 mmol Aldehyde and 1.20 mmol
primary amine were stirred in 3.33 ml dichloromethane or diethyl ether for 20h at room termperature.
Conversion into the imine was confirmed by 1H-NMR or IR. The reaction was dried over MgSO4,
filtered and concentrated. The crude intermediate was dissolved in 2 ml methanol, treated with
2.00 mmol sodium borohydride and stirred over night at room temperature. The solvent was
evaporated and the residue was stirred with 1M aqueous sodium hydroxide solution and
dichloromethane for 20min. Afterwards, the layers were separated and it was extracted 3 times with
dichloromethane. The combined organic layers were washed with brine, dried over MgSO4, filtered
and concentrated. If necessary, the product was purified by column chromatography on neutral
alumnia with methanol in dichloromethane.
In the case of hydrochlorides, the crude product was dissolved in dichloromethane and dropwise added
to a 0.5M solution of hydrochloric acid in diethylether. The precipitate was recrystalized from
methanol with diethylether and dried in vacuum.
4.2.3 Method C - Reductive amination with secondary amines: 0.40 mmol Aldehyde and
0.52 mmol secondary amine were stirred in 2.50 ml dichloromethane for 24h, then 0.45 mmol sodium
triacetoxyborohydride were added and it was stirred additional 24h. The mixture was washed with
saturated aqueous sodium bicarbonate solution and brine, dried over MgSO4, filtered and
concentrated. The crude product was purified by column chromatography on neutral alumnia with
methanol in dichloromethane.
4.2.4. 6-(3-Chloro-4-fluorophenyl)picolinaldehyde (7b): To a mixture of 6-bromo-2-
pyridinecarboxaldehyde (37 mg, 0.20 mmol) in degassed toluene (0.18 mL) and degassed MeOH
(0.07 mL), 3-chloro-4-fluorophenylboronic (49 mg, 0.29 mmol),
Tetrakis(triphenylphosphine)palladium(0) (9 mg, 0.008 mmol) and degassed Na2CO3 2M aqueous
(0.09 mL) were added. The reaction was heated at 110oC for 7h under argon. Then, it was cooled and
stirred with 0.35 mL aqueous 2M Na2CO3 for 5min. The layers were separated and it was extracted 4
times with dichloromethane (2.5 mL). The combined organic layers were washed with brine (0.5 mL),
14

ACCEPTED MANUSCRIPT
dried over MgSO4, filtered and concentrated. The crude product was purified by column
chromatography using hexanes/ethyl acetate (7:1) and then by recrystallization with CH2Cl2/hexanes.
The procedure provided 45 mg of the title compound as a white solid. Yield: 96%. 1H NMR (250
MHz, CDCl3): δ = 10.15 (s, 1H), 8.20 (dd, J = 2.3, 7.1 Hz, 1H), 8.04-7.86 (m, 4H), 7.28 (t, J = 8.6 Hz,
1H). 13C NMR (60 MHz, CDCl3): δ = 193.5, 159.1 (d, J(C,F) = 252,3 Hz), 155.5, 152.8, 138.1, 135.2
(d, J(C,F) = 3.8Hz), 129.4, 126.7 (d, J(C,F) = 7.5 Hz), 124.0, 121.9 (d, J(C,F) = 18.1 Hz), 120.2,
117.0 (d, J(C,F) = 21.4 Hz).
4.2.5. (3-(4-Bromophenyl)isoxazol-5-yl)methanol (11): To a mixture of 4-bromobenzaldehyde (1.85
g, 10 mmol) in of methanol (5 mL) and of water (15 mL), NH2OH.HCl (836 mg, 12 mmol) was added
at room temperature. After 40min, Na2CO3 (634 mg, 6 mmol) was carefully added to the mixture
containing a white precipitate. The reaction mixture was stirred overnight and then filtered. The white
solid was dried under vacuum and resulted in the desired oxime (1817 mg) which was used in the next
step without purification. Yield: 91%. Under argon, N-chlorosuccinimide (480 mg, 3.6 mmol) was
added to a solution of 4-bromobenzaldehyde oxime (600 mg, 3.0 mmol) in dry dichloromethane (4.5
mL) After 50min, propargyl alcohol (0.22 mL, 3.6 mmol) and dry Et3N (0.46 mL, 3.3 mmol) were
added to the green reaction. (CAUTION! The solution fumes). The mixture was refluxed for 4.5h and
then washed with 2 mL of brine. The layers were separated and it was extracted 3 times with
dichloromethane (15 mL). The combined organic layers were dried over MgSO4, filtered and
concentrated. The crude product was purified by column chromatography using hexanes/ethyl acetate
(1:1) and provided of the title compound as a white solid (392 mg). Yield: 51%. 1H NMR (250 MHz,
CDCl3): δ = 7.54-7.72 (m, 4H), 6.54 (s, 1H), 4.83 (d, J = 4.5 Hz, 1H), 2.28 (bs, 1H).
4.2.6. 3-(4-Bromophenyl)isoxazole-5-carbaldehyde: To a mixture of of PCC (32 mg, 0.15 mmol) in
dry dichlorometane (0.5 mL), (3-(4-bromophenyl)isoxazol-5-yl)methanol (25 mg, 0.10 mmol) was
added at room temperature. The dark brown mixture was stirred for 5h and then filtrated in a short
silica pad with diethyl ether (15 mL). The ethereal phase was concentrated, purified by column
chromatography using hexanes/ethyl acetate (7:3) and provided the title compound as a white solid
(43 mg). Yield: 86%. 1H NMR (250 MHz, CDCl3): δ = 10.04 (s, 1H), 7.77-7.69 (m, 2H), 7.69-7.60
15

ACCEPTED MANUSCRIPT
(m, 2H), 7.26 (s, 1H). 13C NMR (60 MHz, CDCl3): δ = 178.3, 166.5, 162.3, 132.4, 128.3, 126.6,
125.3, 106.2.
4.2.7. N1-((3-(4-Bromophenyl)isoxazol-5-yl)methyl)-N3,N3-dimethylpropane-1,3-diamine
hydrochloride (12): Using method C with 3-(4-bromophenyl)isoxazole-5-carbaldehyde (22 mg, 0.09
mmol), the crude product was dissolved in a small amount of dichlorometane and converting it in the
dihydrochloride salt by adding excess of HCl 0.5M in Et2O. The precipitate was filtered and dried
under vacuum to give the title compound as a white solid (25 mg). Yield: 71%. 1H NMR (500 MHz,
MeOD): δ = 7.84-7.79 (m, 2H), 7.71-7.67 (m, 2H), 7.18 (s, 1H), 4.60 (s, 2H), 3.32-3.24 (m, 4H), 2.94
(s, 6H), 2.28-2.19 (m, 2H). 13C NMR (125 MHz, MeOD): δ = 164.8, 163.6, 133.5, 129.6, 128.7,
125.8, 105.7, 55.5, 45.7, 43.6, 42.6, 22.6. HRMS: (M+H+) calc for C15H21BrN3O, 338.08625; found,
338.08566.
ASSOCIATED CONTENT
Supporting Information. Additional details of the synthesis and characterization of the described
compounds, and the procedures used for the metabolism and PK studies, as well as in the in vitro and
in vivo antimalarial studies. This material is available free of charge via the Internet.
AUTHOR INFORMATION
Corresponding Author
*E-mail: ldias@iqm.unicamp.br. Phone + 55 19 35213097
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to
the final version of the manuscript. ‡These authors contributed equally.
Funding Sources
We gratefully acknowledge financial support from Medicines for Malaria Venture. The National
Council for Scientific and Technological Development (CNPq 300810/2010-5) and The State of São
Paulo Research Foundation (FAPESP 2012-02230-0).
16

ACCEPTED MANUSCRIPT
ACKNOWLEDGMENT
We thank Medicines for Malaria Venture (MMV) for financial support of this research. Further
financial support from FAPESP (The State of São Paulo Research Foundation, grant #2012-02230-0)
and CNPq (The National Council for Scientific and Technological Development, grant #300810/2010-
5), Brazil is gratefully acknowledged. We thank the University of Campinas for support and
infrastructure, especially Prof. Marcus Eberlin (Thomson Laboratory) for HRMS analyses. We are
grateful to Sergio Wittlin, Sibylle Sax and the Swiss Tropical and Public Health Institute (Basel,
Switzerland) for conducting the Plasmodium berghei efficacy studies in mice; to GlaxoSmithKline
(Tres Cantos, Spain) for the Plasmodium falciparum parasite reduction ratio studies; and to Elizabeth
Winzeler and Stephan Meister at the University of California, San Diego, for conducting the
Plasmodium berghei liver stage activity and HepG2 cytotoxicity studies.
ABBREVIATIONS
Ar, aromatic ring; DME, dimethoxyethane; HLM, human liver microsomes; HTS, high throughput
screening; MLM, mouse liver microsomes; NCS, N-chlorosuccinimide; PCC, pyridinium
chlorochromate; PK, pharmacokinetics; PRR, parasite reduction ratio.
REFERENCES
[1] WHO, World Malaria Report 2014, in, Geneva, 2014.
[2] O. Miotto, R. Amato, E.A. Ashley, B. MacInnis, J. Almagro-Garcia, C. Amaratunga, P.
Lim, D. Mead, S.O. Oyola, M. Dhorda, M. Imwong, C.J. Woodrow, M. Manske, J. Stalker, E.
Drury, S. Campino, L. Amenga-Etego, T.-N.N. Thanh, H.T. Tran, P. Ringwald, D. Bethell, F.
Nosten, A.P. Phyo, S. Pukrittayakamee, K. Chotivanich, C.M. Chuor, C. Nguon, S. Suon, S.
Sreng, P.N. Newton, M. Mayxay, M. Khanthavong, B. Hongvanthong, Y. Htut, K.T. Han,
M.P. Kyaw, M.A. Faiz, C.I. Fanello, M.A. Onyamboko, O.A. Mokuolu, C.G. Jacob, S.
Takala-Harrison, C.V. Plowe, N.P. Day, A.M. Dondorp, C.C.A. Spencer, G. McVean, R.M.
Fairhurst, N.J. White, D.P. Kwiatkowski, Genetic architecture of artemisinin-resistant
Plasmodium falciparum, Nat. Genet. 47 (2015) 226-234.
[3] J. Straimer, N.F. Gnadig, B. Witkowski, C. Amaratunga, V. Duru, A.P. Ramadani, M.
Dacheux, N. Khim, L. Zhang, S. Lam, P.D. Gregory, F.D. Urnov, O. Mercereau-Puijalon, F.
Benoit-Vical, R.M. Fairhurst, D. Menard, D.A. Fidock, K13-propeller mutations confer
artemisinin resistance in Plasmodium falciparum clinical isolates, Science 347 (2015) 428-
431.
[4] S. Mok, E.A. Ashley, P.E. Ferreira, L. Zhu, Z. Lin, T. Yeo, K. Chotivanich, M. Imwong,
S. Pukrittayakamee, M. Dhorda, C. Nguon, P. Lim, C. Amaratunga, S. Suon, T.T. Hien, Y.
Htut, M.A. Faiz, M.A. Onyamboko, M. Mayxay, P.N. Newton, R. Tripura, C.J. Woodrow, O.
Miotto, D.P. Kwiatkowski, F. Nosten, N.P.J. Day, P.R. Preiser, N.J. White, A.M. Dondorp,
R.M. Fairhurst, Z. Bozdech, Population transcriptomics of human malaria parasites reveals
the mechanism of artemisinin resistance, Science 347 (2015) 431-435.
[5] W.A. Guiguemde, A.A. Shelat, D. Bouck, S. Duffy, G.J. Crowther, P.H. Davis, D.C.
Smithson, M. Connelly, J. Clark, F. Zhu, M.B. Jimenez-Diaz, M.S. Martinez, E.B. Wilson,
17

ACCEPTED MANUSCRIPT
A.K. Tripathi, J. Gut, E.R. Sharlow, I. Bathurst, F.E. Mazouni, J.W. Fowble, I. Forquer, P.L.
McGinley, S. Castro, I. Angulo-Barturen, S. Ferrer, P.J. Rosenthal, J.L. DeRisi, D.J. Sullivan,
J.S. Lazo, D.S. Roos, M.K. Riscoe, M.A. Phillips, P.K. Rathod, W.C. Van Voorhis, V.M.
Avery, R.K. Guy, Chemical genetics of Plasmodium falciparum, Nature 465 (2010) 311-315.
[6] F.-J. Gamo, L.M. Sanz, J. Vidal, C. de Cozar, E. Alvarez, J.-L. Lavandera, D.E.
Vanderwall, D.V.S. Green, V. Kumar, S. Hasan, J.R. Brown, C.E. Peishoff, L.R. Cardon, J.F.
Garcia-Bustos, Thousands of chemical starting points for antimalarial lead identification,
Nature 465 (2010) 305-310.
[7] S. Meister, D.M. Plouffe, K.L. Kuhen, G.M.C. Bonamy, T. Wu, S.W. Barnes, S.E. Bopp,
R. Borboa, A.T. Bright, J. Che, S. Cohen, N.V. Dharia, K. Gagaring, M. Gettayacamin, P.
Gordon, T. Groessl, N. Kato, M.C.S. Lee, C.W. McNamara, D.A. Fidock, A. Nagle, T.-g.
Nam, W. Richmond, J. Roland, M. Rottmann, B. Zhou, P. Froissard, R.J. Glynne, D. Mazier,
J. Sattabongkot, P.G. Schultz, T. Tuntland, J.R. Walker, Y. Zhou, A.K. Chatterjee, T.T.
Diagana, E.A. Winzeler, Imaging of Plasmodium Liver Stages to Drive Next-Generation
Antimalarial Drug Discovery, Science 334 (2011) 1372-1377.
[8] T. Spangenberg, J.N. Burrows, P. Kowalczyk, S. McDonald, T.N.C. Wells, P. Willis, The
Open Access Malaria Box: A Drug Discovery Catalyst for Neglected Diseases, Plos One 8
(2013) e62906.
[9] M.J. Gardner, N. Hall, E. Fung, O. White, M. Berriman, R.W. Hyman, J.M. Carlton, A.
Pain, K.E. Nelson, S. Bowman, I.T. Paulsen, K. James, J.A. Eisen, K. Rutherford, S.L.
Salzberg, A. Craig, S. Kyes, M.-S. Chan, V. Nene, S.J. Shallom, B. Suh, J. Peterson, S.
Angiuoli, M. Pertea, J. Allen, J. Selengut, D. Haft, M.W. Mather, A.B. Vaidya, D.M.A.
Martin, A.H. Fairlamb, M.J. Fraunholz, D.S. Roos, S.A. Ralph, G.I. McFadden, L.M.
Cummings, G.M. Subramanian, C. Mungall, J.C. Venter, D.J. Carucci, S.L. Hoffman, C.
Newbold, R.W. Davis, C.M. Fraser, B. Barrell, Genome sequence of the human malaria
parasite Plasmodium falciparum, Nature 419 (2002) 498-511.
[10] S. Alnabulsi, E. Santina, I. Russo, B. Hussein, M. Kadirvel, A. Chadwick, E.V.
Bichenkova, R.A. Bryce, K. Nolan, C. Demonacos, I.J. Stratford, S. Freeman, Non-
symmetrical furan-amidines as novel leads for the treatment of cancer and malaria, Eur. J.
Med. Chem. 111 (2016) 33-45.
[11] M. Kaiser, L. Maes, L.P. Tadoori, T. Spangenberg, J.-R. Ioset, Repurposing of the Open
Access Malaria Box for Kinetoplastid Diseases Identifies Novel Active Scaffolds against
Trypanosomatids, J. Biomol. Screen. 20 (2015) 634-645.
[12] M. Dubernet, V. Caubert, J. Guillard, M.-C. Viaud-Massuard, Synthesis of substituted
bis(heteroaryl)maleimides, Tetrahedron 61 (2005) 4585-4593.
[13] K. Rossen, M. Latinovic, M. Sarich, P. Gardner, S. Rowell, Method of producing 5-
formyl-2-furylboronic acid, 2004. EP1403271A1.
[14] T. Hosoya, H. Aoyama, T. Ikemoto, Y. Kihara, T. Hiramatsu, M. Endo, M. Suzuki,
Dantrolene analogues revisited: general synthesis and specific functions capable of
discriminating two kinds of Ca2+ release from sarcoplasmic reticulum of mouse skeletal
muscle, Bioorg. Med. Chem. 11 (2003) 663-673.
[15] K.-T. Wong, Y.-Y. Chien, Y.-L. Liao, C.-C. Lin, M.-Y. Chou, M.-k. Leung, Efficient
and Convenient Nonaqueous Workup Procedure for the Preparation of Arylboronic Esters, J.
Org. Chem. 67 (2002) 1041-1044.
[16] M.P. Jensen, S.J. Lange, M.P. Mehn, E.L. Que, L. Que, Biomimetic Aryl Hydroxylation
Derived from Alkyl Hydroperoxide at a Nonheme Iron Center. Evidence for an FeIVO
Oxidant, J. Am. Chem. Soc. 125 (2003) 2113-2128.
[17] D. Zhang, J. Jia, L. Meng, W. Xu, L. Tang, J. Wang, Synthesis and preliminary
antibacterial evaluation of 2-butyl succinate-based hydroxamate derivatives containing
isoxazole rings, Arch. Pharm. Res. 33 (2010) 831-842.
18

ACCEPTED MANUSCRIPT
[18] R.E. Desjardins, C.J. Canfield, J.D. Haynes, J.D. Chulay, Quantitative assessment of
antimalarial activity in vitro by a semiautomated microdilution technique, Antimicrob. Agents
Chemother. 16 (1979) 710-718.
[19] J.A. Pereira, A.M. Pessoa, M.N.D.S. Cordeiro, R. Fernandes, C. Prudêncio, J.P.
Noronha, M. Vieira, Quinoxaline, its derivatives and applications: A State of the Art review,
Eur. J. Med. Chem. 97 (2015) 664-672.
[20] G. Yadav, S. Ganguly, Structure activity relationship (SAR) study of benzimidazole
scaffold for different biological activities: A mini-review, Eur. J. Med. Chem. 97 (2015) 419-
443.
[21] M.F. Khan, M.M. Alam, G. Verma, W. Akhtar, M. Akhter, M. Shaquiquzzaman, The
therapeutic voyage of pyrazole and its analogs: A review, Eur. J. Med. Chem. 120 (2016)
170-201.
[22] L.M. Sanz, B. Crespo, C. De-Cozar, X.C. Ding, J.L. Llergo, J.N. Burrows, J.F. Garcia-
Bustos, F.-J. Gamo, P. falciparum In Vitro Killing Rates Allow to Discriminate between
Different Antimalarial Mode-of-Action, Plos One 7 (2012) e30949.
[23] C. Snyder, J. Chollet, J. Santo-Tomas, C. Scheurer, S. Wittlin, In vitro and in vivo
interaction of synthetic peroxide RBx11160 (OZ277) with piperaquine in Plasmodium
models, Exp. Parasitol. 115 (2007) 296-300.
[24] C. Le Manach, T. Paquet, D. Gonzalez Cabrera, Y. Younis, D. Taylor, L. Wiesner, N.
Lawrence, S. Schwager, D. Waterson, M.J. Witty, S. Wittlin, L.J. Street, K. Chibale,
Medicinal Chemistry Optimization of Antiplasmodial Imidazopyridazine Hits from High
Throughput Screening of a SoftFocus Kinase Library: Part 2, J. Med. Chem. 57 (2014) 8839-
8848.
[25] D. Gonzalez Cabrera, F. Douelle, Y. Younis, T.-S. Feng, C. Le Manach, A.T. Nchinda,
L.J. Street, C. Scheurer, J. Kamber, K.L. White, O.D. Montagnat, E. Ryan, K. Katneni, K.M.
Zabiulla, J.T. Joseph, S. Bashyam, D. Waterson, M.J. Witty, S.A. Charman, S. Wittlin, K.
Chibale, Structure-Activity Relationship Studies of Orally Active Antimalarial 3,5-
Substituted 2-Aminopyridines, J. Med. Chem. 55 (2012) 11022-11030.
[26] S. Yalaoui, S. Zougbede, S. Charrin, O. Silvie, C. Arduise, K. Farhati, C. Boucheix, D.
Mazier, E. Rubinstein, P. Froissard, Hepatocyte Permissiveness to Plasmodium Infection Is
Conveyed by a Short and Structurally Conserved Region of the CD81 Large Extracellular
Domain, PLoS Pathog 4 (2008) e1000010.
[27] O. Silvie, E. Rubinstein, J.F. Franetich, M. Prenant, E. Belnoue, L. Renia, L. Hannoun,
W. Eling, S. Levy, C. Boucheix, D. Mazier, Hepatocyte CD81 is required for Plasmodium
falciparum and Plasmodium yoelii sporozoite infectivity, Nature Medicine 9 (2003) 93-96.
[28] A. Nzila, L. Mwai, In vitro selection of Plasmodium falciparum drug-resistant parasite
lines, J. Antimicrob. Chemother. 65 (2009) 390-398.
19

ACCEPTED MANUSCRIPT
List of caption:
Fig. 1. Lead structure.
Fig. 2. Plasma concentration versus time profiles of 1
Fig. 3. Plasma concentrations of 4k in male Sprague Dawley rats
Fig. 4. Speed of killing.
Scheme 1. Synthesis of 4 and 5
Scheme 2. Synthesis of 8a-8d and 9a-b
Scheme 3. Synthesis of 12
Table 1. Substitution of the Aryl Moiety.
Table 2. Replacement of the Amine Side Chain.
Table 3. Replacement of the Furan Ring.
Table 4. Replacement of the Furan Ring.
Table 5. Metabolite profile in liver microsomes.
20

ACCEPTED MANUSCRIPT
TITLE: Novel Inhibitors of Plasmodium Falciparum based on 2,5-disubstituted Furans
Highlights
•
•
•
Furans as new chemotype for Plasmodium falciparum inhibition
Examples classified as fast killers in parasite reduction ratio assay
No resistance induced in Dd2 parasites