Novel Antimalarial Tetrazoles and Amides Active against the Hemoglobin Degradation Pathway in Plasmodium falciparum

Article abstract of DOI:10.1021/acs.jmedchem.0c02022

Malaria control programs continue to be threatened by drug resistance. To identify new antimalarials, we conducted a phenotypic screen and identified a novel tetrazole-based series that shows fast-kill kinetics and a relatively low propensity to develop h

Full text of DOI:10.1021/acs.jmedchem.0c02022

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Article
Novel Antimalarial Tetrazoles and Amides Active against the
Hemoglobin Degradation Pathway in Plasmodium falciparum
Aloysus Lawong, Suraksha Gahalawat, John Okombo, Josefine Striepen, Tomas Yeo, Sachel Mok,
Ioanna Deni, Jessica L. Bridgford, Hanspeter Niederstrasser, Anwu Zhou, Bruce Posner, Sergio Wittlin,
Francisco Javier Gamo, Benigno Crespo, Alisje Churchyard, Jake Baum, Nimisha Mittal,
Elizabeth Winzeler, Benoît Laleu, Michael J. Palmer, Susan A. Charman, David A. Fidock,
Joseph M. Ready,* and Margaret A. Phillips*
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ABSTRACT: Malaria control programs continue to be threatened by drug
resistance. To identify new antimalarials, we conducted a phenotypic screen
and identified a novel tetrazole-based series that shows fast-kill kinetics and a
relatively low propensity to develop high-level resistance. Preliminary
structure−activity relationships were established including identification of a
subseries of related amides with antiplasmodial activity. Assaying parasites with
resistance to antimalarials led us to test whether the series had a similar
mechanism of action to chloroquine (CQ). Treatment of synchronized
Plasmodium falciparum parasites with active analogues revealed a pattern of
intracellular inhibition of hemozoin (Hz) formation reminiscent of CQ’s
action. Drug selections yielded only modest resistance that was associated with
amplification of the multidrug resistance gene 1 (pfmdr1). Thus, we have
identified a novel chemical series that targets the historically druggable heme
polymerization pathway and that can form the basis of future optimization
efforts to develop a new malaria treatment.
that has been used in malaria treatment programs.4 The
introduction of artemisinin-based combination therapies
(ACTs) as the first-line treatment regimen restored effective
malaria control, and their use has led to substantial reductions in
malaria disease burden in the last two decades.5 However,
increasing ACT treatment failure rates are being reported,
particularly in the Greater Mekong Subregion. P. falciparum
resistance to artemisinin (ART) is associated with mutations in
Kelch13 and with delayed parasite clearance times.6−9 These
reductions in ART potency have paralleled the emergence of
resistance to partner compounds,10 further jeopardizing the
efficacy of first-line antimalarials and foreshadowing a reversal of
the gains made in the last decade.5 New challenges in emerging
resistance to the current frontline therapies make the discovery
of novel antimalarials a critical component to sustaining and
improving malaria control programs.
INTRODUCTION
■
Malaria remains a leading cause of mortality and morbidity
worldwide. Global malaria deaths in 2020 were estimated at
∼0.4 million, with African children under the age of 5
representing the most vulnerable demographic and accounting
for 272,000 (67%) of all malaria deaths.1 An estimated 228
million cases occurred worldwide in the same period. In high-
burden countries, malaria continues to place a significant strain
on healthcare systems, while leading to substantial losses in
economic output, creating a cycle of poverty. The causative
agent of malaria is an obligate intracellular parasite from the
Plasmodium genus, of which there are five human infective
species. Plasmodium falciparum and Plasmodium vivax account
for most cases of malaria, with the former responsible for almost
all malaria deaths.2
A number of drugs with a range of modes of action (MOA)
have been used to treat malaria, including chloroquine (CQ),
which became the mainstay of treatment and prevention
programs in the late 1940s.3 Widespread P. falciparum resistance
to CQ led to its loss of efficacy, a fate shared by sulfadoxine +
pyrimethamine (which inhibit dihydropteroate synthase and
dihydrofolate reductase, respectively) that was introduced in the
late 1950s. Parasite resistance extends to nearly every other drug
Received: November 21, 2020
Published: February 23, 2021
https://dx.doi.org/10.1021/acs.jmedchem.0c02022
© 2021 American Chemical Society
J. Med. Chem. 2021, 64, 2739−2761
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Figure 1. HTS-based identification of compounds that inhibit growth of P. falciparum 3D7 ABS parasites. (A) Range of Z′ values observed for 384-well
plates covering the 8K screening library. The black bar shows the mean standard deviation (std dev). (B) Representative concentration response
curves for validated hits vs 3D7 parasites. Data for each drug concentration were collected in triplicate wells, and error bars represent the std dev. Data
were fitted to the log(I) vs responsefour-parameter variable slope equation in GraphPad Prism. Replicate data were subsequently collected, yielding
the following EC50’s: 1, 0.31 0.10 μM; 2, 0.71 0.075 μM; and 3, 3.5 0.71 μM, where errors are the std dev for at least three independent biological
replicates. (C) BRRoK rate of kill assay. Serial log-fold dilutions of compounds from 30× EC50 to 0.33× EC50 were incubated for 6 h with NF54-luc
parasites, and viability was assessed by bioluminescence at the end of the incubation. Bioluminescence signals normalized to untreated controls are
plotted and represent mean std dev from triplicate wells. EC50 values against 3D7 parasites were determined in standard 72 h SYBR Green assays
prior to the start of the study and were used as indicated for compounds 1−3. Benchmark compounds included artemisinin, ART (fast-kill) (EC50
0.013 μM), chloroquine, CQ (fast-kill) (EC50 = 0.014 μM), atovaquone, ATQ (slow-kill) (EC50 = 0.0013 μM), and DSM265 (slow-kill) (EC50
=
=
0.0050 μM), with EC50 values used to calculate compound concentrations for the study indicated in parenthesis. Similar EC50 values were obtained
versus NF54-luc parasites using bioluminescence as a readout in a 72 h assay (Table S1).
Academic and commercial antimalarial high-throughput
screening (HTS) campaigns have leveraged whole cell-based
approaches to identify novel antimalarial candidates, some
currently in clinical development.2,11,12 Early phenotypic
screening campaigns focused on targeting asexual blood stage
(ABS) parasites, which cause clinical symptoms. This approach
led to the identification of several clinical candidates for the
treatment of malaria, including the spiroindolone ATP4
inhibitor KAE609 (NITD609),13,14 the imidazolopiperazine,
KAF156,15,16 and PI4 kinase inhibitors17,18 including KDU691
and MMV390048. More recent approaches have since allowed
development of screening platforms targeting multiple stages of
the Plasmodium life cycle.19−21 These phenotypic screening
efforts, combined with the use of forward genetics and whole-
genome sequencing (WGS), have proven powerful in
identifying the protein targets of antimalarial compounds,
including for several of these current development candi-
dates.22,23 Target-based screens, including those against
dihydroorotate dehydrogenase that led to the identification of
DSM265,24,25 have also been deployed but so far have led to
fewer development candidates than the phenotypic approach.
Herein, we describe the identification of a novel tetrazole-
based series from a phenotypic screen against P. falciparum ABS
parasites (using the 3D7 strain). This series shows a fast kill rate
in vitro, and life-cycle profiling reveals it to be ABS-specific.
Synthesis and evaluation of additional analogues in the series
have established preliminary structure−activity relationships
(SARs). MOA studies suggest that parasite killing is mediated by
inhibition of hemozoin (Hz) formation and the toxic buildup of
free heme in the parasite digestive vacuole (DV), similar to CQ.
Selections for resistant parasites have yielded only low-level
resistance, and WGS reveals amplification of the multidrug
resistance 1 gene (pfmdr1). We have thus identified a novel class
of antimalarial compounds with a fast-kill mechanism and low
resistance propensity and whose MOA appears to involve
inhibition of heme detoxification, a critical pathway in the
parasite’s intraerythrocytic development and a well-validated
antimalarial target. These compounds also have the advantage of
rapid synthesis from readily available starting materials.
RESULTS
■
Phenotypic Screen to Identify Novel Compounds
Active against P. falciparum ABS Parasites. A single-
point (5 μM) primary screen of an 8K subset of our larger library
was executed in a high-throughput optimized 384-well format
against the pan-susceptible P. falciparum 3D7 strain of ABS
parasites, using a published SYBR Green assay.26,27 The assay
was highly robust, with an average Z′ of 0.76 0.059 (where
>0.5 is considered a robust assay) across all plates (Figure 1A).
We identified a total of 79 hits based on an RZ score cutoff of
<−3. The RZ score represents the number of robust standard
deviations (RSD) from the corrected median of a measurement
for a library compound, as determined after correction for
systematic errors (plate, row, and column effects).28,29
Compounds with structural alerts (e.g., PAINS compounds30)
or poor drug-like properties31 were removed, and a secondary
screen at three concentrations (5, 1.7, and 0.42 μM) was
performed, yielding 38 confirmed hits. We prioritized
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compounds with EC50 values <3 μM against 3D7 parasites and
selected for chemical novelty based on PubChem and
SciFinder(R) searches, while deprioritizing hits that have been
previously reported in other malaria screening programs. From
our initial hits, we selected three chemotypes (1, 2, and 3) for
commercial resupply and evaluated them in 10-point concen-
tration response assays against 3D7 parasites (Figure 1B). These
compounds ranged in potency on 3D7 from 0.31 to 3.5 μM, with
1 showing the best potency [EC50 values of 0.31 μM (1), 0.71
μM (2), and 3.5 μM (3)] (Figure 1 legend). Cytotoxicity was
assessed against HepG2 cells, with all three compounds showing
a CC50 >30 μM in this assay.
Scheme 1. Synthesis of Antimalarial Compounds
Compounds that show fast kill kinetics are being sought
because they provide rapid relief of symptoms, and a more rapid
reduction in parasite numbers may also reduce the propensity
for resistance to develop.32,33 Kill rates also provide information
about the MOA as the kill rate has been shown to be a property
of the target.34 To this end, compounds were profiled to
estimate the rate of kill (Figure 1C) using a rapid bio-
luminescence relative rate of kill (BRRoK) assay.35 This method
follows bioluminescence in a cytocidal 6 h exposure assay over
multiple drug concentrations using a P. falciparum strain
(Pf NF54luc)36 that expresses luciferase. We benchmarked the
assay against the known fast- (ART and CQ) or slow
[DSM26525 and atovaquone (ATQ)]-acting compounds
previously characterized using the standard parasite reduction
rate (PRR) assay, which measures viability after treatment and
regrowth over 28 days.34 In the BRRoK assay, both 1 and 2
showed slow kill kinetics similar to DSM265 and ATQ, while the
profile for 3 was similar to CQ and thus consistent with a fast kill
mechanism. Based on this desirable property, the 3-series was
selected for more in-depth profiling despite lower potency
compared to 1 and 2.
Chemistry. Tetrazoles were synthesized using a one-pot,
four-component Ugi coupling in the racemic form37 (Scheme
1A). An amine, aldehyde, isocyanate, and trimethylsilyl azide
were combined in methanol and stirred for 12−36 h at room
temperature (RT) to provide the corresponding coupling
product in high yields. In several cases, the enantiomers were
separated by preparative high-performance liquid chromatog-
raphy (HPLC) using a chiral stationary phase. Similarly, most
amides were prepared through a three-component Ugi coupling,
featuring phenyl phosphinic acid38 (Scheme 1B). The resulting
secondary amides were alkylated using methyl iodide in the
presence of sodium hydride. Finally, amide 34 was prepared via a
Strecker reaction involving 3-pyridine carboxaldehyde and
amine 35. Nitrile hydrolysis under basic conditions and amide
coupling provided product 34 in high yield (Scheme 1C).
Medicinal Chemistry to Establish SAR of the 3-Series.
The 3-series is chemically attractive, with a basic amine, a stable
heterocycle, and four vectors for optimization. Initial SAR of the
3-series was established through the synthesis and testing of 19
tetrazole analogues against two strains of P. falciparum. These
included the 3D7 strain that is broadly drug-sensitive and the
Dd2 strain that is resistant to chloroquine and partially resistant
to mefloquine (MFQ) and quinine.39,40 Compounds were also
tested against human HepG2 cells to evaluate cytotoxicity. This
work identified compounds with improved potency versus 3D7
of 10- to 20-fold (Table 1).
Replacing the B-ring phenyl with 3-pyridine (3-Py) further
improved activity to ∼1 μM (6), while also improving drug-like
properties. The N-methyl at the R2 position could be replaced
by cyclopropyl (cPr) (7), yielding an additional threefold
improvement in potency against 3D7. The OMe on the A-ring
could be replaced by F (8) or H (9) without loss of activity.
Further modification of the B-ring showed that the 3-Py of 9
could be replaced by a 5-substituted pyrimidine (5-Pyr, 10) with
similar activity against 3D7. Introduction of fluoro on the A-ring
was also well tolerated at the 3 (11), 4 (12), and 6 (13) positions
on the ring. The B-ring could not be replaced with a small
nonaromatic cPr ring (14), and a basic amine appeared
important because the R2 alkyl group could not be replaced
with COCH3 (15). In contrast, the C-ring could be replaced by
either cyclohexyl (17) or cPr (18) with only a two- or threefold
loss of activity against 3D7 compared to compounds with
matched R2 and B rings, 16 or 9, respectively. However, within
the context of the cPr in the C-ring position, the addition of a 2-
Me to the B-ring 3-Py was not tolerated (19). The benzylic
carbon between the A ring and the tertiary amine could be
substituted with a cPr to block a potential site of metabolism.
When combined with a propyl (Pr) at R2, these compounds
yielded the most potent of the tetrazole series (20 and 21) (3D7
EC50’s ∼ 0.3 μM, Table 1), representing a 10-fold improvement
over the initial hit 3.
On the A-ring, the Br could be replaced with CF3 to give 5
with a slight improvement in activity, while removing the Br
decreased activity by a factor of 3 (4). Removing the A-ring
altogether resulted in an inactive analogue (data not shown).
Three analogues were selected for separation and evaluation
of enantiomer pairs. A small two- to threefold difference in
potency was observed between the separated 3-enantiomers
[(+)-3 vs (−)-3], while the enantiomers of 6 [(+)-6 and (−)-6]
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a
Table 1. SAR of the Tetrazole Series
P. falciparum EC50 (μM)
PfDd2
cytotox CC50 (μM)
cmpd
R
R1
Br
Br
Br
H
R2
CH3
B-ring
Ph
Ph
Ph
Ph
C-ring
Pf3D7
HepG2
3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-F
o-tol
o-tol
o-tol
o-tol
o-tol
c-tol
c-tol
o-tol
o-tol
o-tol
o-tol
o-tol
o-tol
o-tol
o-tol
o-tol
o-tol
o-tol
o-tol
o-tol
cHex
cPr
3.5 0.71 (10)
1.5, 1.9
4.6, 4.4
9.7 3.0 (3)
1.7 0.67 (4)
1.3 0.12 (3)
1.6, 1.5
5.4 0.78 (5)
3.0
4.0
10 1.5 (3)
3.7 0.25 (3)
2.3 0.40 (3)
2.5
>30
ND
ND
ND
ND
ND
ND
ND
16
ND
15
37, 33
36
>30
>30
ND
ND
ND
>30
ND
ND
ND
>30
13
(+)-3
(−)-3
4
5
6
(+)-6
(−)-6
7
8
9
10
11
(+)-11
(−)-11
12
13
14
15
16
CH3
CH3
CH3
CH3
CH3
CH3
CH3
cPr
cPr
cPr
cPr
cPr
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
Ph
3-Py
3-Py
3-Py
3-Py
3-Py
3-Py
5-Pyr
5-Pyr
5-Pyr
5-Pyr
5-Pyr
5-Pyr
cPr
5-Pyr
3-Py
3-Py
3-Py
2Me-3Pyr
5-Pyr
3-Py
1.2, 1.2
1.7
0.49 0.10 (3)
0.60 0.17 (3)
0.38 0.071 (5)
0.40 0.070 (4)
0.37 0.041 (4)
0.35
0.48 0.029 (3)
0.67 0.031 (3)
0.54 0.17 (3)
1.8 0.34 (4)
1.2 0.67 (4)
2.5
2.6
*EC50.1; EC50.2 0.68 0.10; 3.6 1.3 (4)
*EC50.1; EC50.2 0.71 0.13; 4.8 1.3 (4)
3.2, 4.2
>30, 21
2.5 0.26 (3)
2.6 0.49 (3)
3.0 0.55 (3)
21, 16
0.52 0.11 (3)
0.41 0.083 (3)
H
H
3-F
3-F
3-F
4-F
6-F
H
H
2-F
2-F
H
H
H
H
cPr
cPr
cPr
cPr
0.42
0.47 0.11 (3)
0.47 0.18 (3)
4.8, 4.7
cPr
COCH3
CH3
CH3
cPr
cPr
nPr
15, 15
1.0 0.14 (3)
1.7 0.56 (3)
1.5 0.29 (3)
>30, 14
0.31 0.056 (3)
0.23 0.025 (3)
17
18
19
20
cPr
o-tol
o-tol
21
nPr
10
a
Concentration response data were collected with technical duplicates or triplicates at each inhibitor concentration. Data were fitted in GraphPad
Prism to the inhibitor vs responsevariable slope (four parameters) equation to determine the EC50, with the exception of *, where biphasic
curves were observed and data were fitted to the biphasic equation with constraints (top = 1.0, bottom >0, and hill coefficients <0) to determine
EC50.1 and EC50.2. Data are the EC50 mean std dev with the number of independent biological replicates shown in parenthesis. Individual values
are shown for studies with less than three biological replicates. Ph, phenyl; Py, pyridyl; Pyr, pyrimidinyl; tol, tolyl. Compounds are racemic, unless
otherwise indicated.
and 11 [(+)-11 and (−)-11] showed equal potency. Taken
together, the data support an absence of enantiomer selectivity
for the series.
shown). The SAR from the two subseries was found to be
similar, consistent with a similar MOA. This includes the finding
that for the B-ring, both 3-Py (22, 24, 26, and 28) and 5-Pyr (23,
25, 27, and 29) showed similar activity against 3D7, while
similar to the tetrazoles 9 and 10, Dd2 activity on the 5-Pyr
tended to be lower than for the 3-Py. The A-ring CF3 could be
replaced with SCF3 (24 and 25) or OCF3 (26 and 27), yielding
similar potency. The methyl on the C-ring could be extended to
ethyl to improve activity approximately twofold (22 vs 28; 23 vs
29), whereas removing the C-ring methyl was associated with an
approximately twofold loss in activity (23 vs 30). The addition
of a 2-Me to the 5-Pyr at the B-ring led to a substantial 20-fold
loss in potency (31 vs 23). As observed with the tetrazole series,
the addition of 2-F to the A ring was well tolerated (32 vs 22;
compared to 16 in the tetrazole series), while a benzylic
cyclopropyl group again improved activity to give 33 (vs 20 and
21 in the tetrazole series), currently our most potent analogue
(EC50 = 0.21−0.33 μM on 3D7 and Dd2, respectively).
Additionally, as with the tetrazoles, replacement of the C-ring
with a saturated ring (piperidine 34) led to a fivefold reduction
While the initial hit 3 showed similar activity on both 3D7 and
Dd2 parasite lines, a number of subsequent compounds in the
series showed reduced activity on Dd2, with differences of up to
5−10-fold observed for the most disparate compounds (e.g., 10,
12, and 13) (Table 1). These differences will be discussed in
greater detail below. Cytotoxicity was evaluated using human
HepG2 cells. While 3 did not show any activity up to the highest
tested concentration of 30 μM, several of the more potent
analogues (9, 20, and 21) displayed cytotoxicity with CC50
values in the 10−15 μM range, yielding a selectivity window of
40−50-fold in these cases (Table 1).
Molecular modeling suggested that a cis-amide could orient
the A, B, and C rings similarly to the tetrazole. A subseries of
amides was synthesized to test this hypothesis (22−34) (Table
2). Consistently, 22 and 23 were equipotent to their tetrazole
congeners 9 and 10. The NH amides were fivefold less active, as
predicted based on conformational considerations (data not
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a
Table 2. SAR of the Amide Subseries
P. falciparum EC50 (μM)
Pf Dd2
cytotox CC50 (μM)
cmpd
R
R1
R2
R3
B-ring
3-Py
5-Pyr
3-Py
5-Pyr
3-Py
5-Pyr
3-Py
5-Pyr
5-Pyr
2-Me, 5-Pyr
3-Py
3-Py
3-Py
Pf 3D7
HepG2
22
23
24
25
26
27
28
29
30
31
32
33
34
H
H
H
H
H
H
H
H
H
H
F
CF3
CF3
cPr
cPr
cPr
cPr
cPr
cPr
cPr
cPr
cPr
cPr
cPr
nPr
cPr
Me
Me
Me
Me
Me
Me
Et
Et
H
Me
Me
Me
C**
0.34 0.044 (3)
0.40 0.015 (3)
0.31 0.078 (3)
0.26 0.046 (3)
0.32 0.035 (3)
0.24 0.044 (3)
0.19 0.025 (3)
0.24 0.042 (3)
0.72 0.021 (3)
8.1, 7.5
0.59 0.067 (3)
0.79 0.19 (3)
0.39 0.045 (3)
1.0 0.55 (5)
0.52 0.055 (3)
0.59 0.17 (5)
0.48 0.084 (3)
0.57 0.14 (3)
*EC50.1; EC50.2 2.0 1.4; 16 5.4 (4)
9.8, 7.7
>30
>30
>30
>30
>30
>30
>20
>25
>30
>30
>30
>15
>20
SCF3
SCF3
OCF3
OCF3
CF3
CF3
CF3
CF3
CF3
0.31 0.026 (3)
0.21 0.021 (3)
1.8 0.15 (3)
0.46 0.046 (3)
0.33 0.035 (3)
4.2 0.84 (3)
H
H
CF3
CF3
a
Concentration response data were collected with technical triplicates at each inhibitor concentration, and data were fitted in GraphPad Prism to
determine the EC50, as described in the Table 1 footnote. Data are the EC50 mean std dev with the number of independent biological replicates
shown in parenthesis. **C-ring replaced with piperidine.
Figure 2. Relative rate of kill of 10 vs control antimalarials. (A) BRRoK rate of kill assay. Conditions used were as described in Figure 1. The mean EC50
for 10 was 0.32 μM, as determined using 72 h SBYR Green assays. DSM265 and ART controls were collected simultaneously to the 10 data. CQ data
were replotted from Figure 1 and are provided again in this plot as a reference. (B) Kill rate determination using two-color FACS analysis. Benchmark
compounds are described above. Pyrimethamine (PYR) was included as a medium rate of the kill compound. Compounds were plated at 10× EC50,
where EC50 values, determined using 48 h hypoxanthine assays, were 10 (1.7 μM), CQ (0.021 μM), ART (0.03 μM), PYR (0.094 μM), and ATQ
(0.001 μM). (C) PRR washout assay. Data show % parasite survival vs h of drug treatment prior to drug washout and replating. PYR data were collected
simultaneously to 10. CQ and ART data are historical control data collected in a separate study34 and provided as a reference. Triplicate data were
collected at each compound concentration, symbols represent the mean, and error bars represent the std dev.
Table 3. Physicochemical Properties and In Vitro Metabolism in Human Liver Microsomes
cmpd MW cLog D pH 7.4 predicted pKa (basic) CLint (μL/min/mg protein) kinetic solubility pH 2.0 (μg/mL) kinetic solubility pH 6.5 (μg/mL)
7
9
495
465
465
454
454
472
418
5.2
5.3
4.6
5.3
4.6
5.4
3.9
4.6, 3.3
5.1, 2.9
4.7
6.0, 3.4
5.5
154
360
261
781
531
548
244
12.5−25
12−23
1.5−2.9
>100
11−23
>47
1.6−3.1
1.5−2.9
1.5−2.9
6.3−12.5
>45
1.5−2.9
25−50
10
22
23
32
34
5.0, 3.4
6.1, 3.4
>100
in activity but showed some promise. Relative to other
compounds in the series, compound 30 showed the greatest
loss of potency against Dd2 parasites, requiring over 20-fold
higher concentrations for complete killing (Table 2). Cytotox-
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icity in the amide series was improved over the tetrazoles, with
no compounds showing any cytotoxic activity up to the highest
tested concentration of 30 μM (Table 2).
against a selection of drug-sensitive and drug-resistant parasite
lines. Initial work focused on comparing the effectiveness against
3D7 and Dd2 parasites. As noted above, while 3 showed similar
activity against these strains (Table 1), a number of analogues in
both the tetrazole and amide subseries showed lower activity
against Dd2 than 3D7 (Tables 1 and 2). The largest differences
(three- to fivefold) were observed for compounds with a 5-Pyr in
the B-ring (10, 26), whereas the matched compounds with 3-Py
in this position showed smaller (1.4−3.0-fold) effects (9 and 27)
based on comparisons of matched pairs that contained only
variations in this position (Figures 3 and S2). Additionally, for
several analogues (12, 13, and 30) containing the 5-Pyr in the B-
ring, we observed biphasic concentration response curves versus
Dd2. In these cases, up to a 20-fold potency difference was
observed between 3D7 and the second of the fitted EC50 values
determined for Dd2, whereas the more potent EC50 values were
similar to that observed for 3D7 (Figure 4 and Tables 1 and 2).
These data suggest that a second target might be responsible for
the reduced Dd2 efficacy at higher concentrations for these
compounds. In contrast, biphasic concentration response curves
were never observed versus 3D7 for any compound.
Encouragingly, our data show that it is possible to identify
compounds with similar potency against 3D7 and Dd2, for
example, by avoiding the 5-Pyr that frequently led to biphasic
concentration response curves. These results highlight the value
of including multiple parasite lines in early-stage compound
evaluation.
To further probe the MOA, 9 and 10 were evaluated against
two panels of resistant parasite lines that were either obtained as
clinical isolates42 or were generated in Dd2 parasites through in
vitro selections with various antimalarials. Data were compared
to the drug-sensitive 3D7 and NF54 strains (Table 5; note that
3D7 is a clone of NF54). No cross resistance was observed for 9
across the two panels. In contrast, 10 showed reduced potency
against the CQ-resistant Dd2 and piperaquine (PPQ)-resistant
RF12 strains, although reduced activity was not observed against
other tested lines with other drug-resistant phenotypes.
Interestingly, both 9 and 10 were more effective against 7G8
parasites than any of the other tested lines, even though this line,
similar to Dd2, RF12, K1, and TM90C2B, contains point
mutations in the P. falciparum CQ resistance transporter
(Pf CRT; Table 6).
To confirm that the 3-series shows fast kill kinetics, we re-
evaluated the kill rate using more potent analogues from both
the tetrazole (10) and amide (23) subseries using the BRRoK
assay cited above. Like 3, both compounds behaved similarly to
CQ in this assay, consistent with fast kill kinetics (Figures 2A
and S1). Finally, 10 was tested in both a flow cytometry (FACS)
assay41 that detects the ability of parasites to invade new RBCs
after incubation with drug and in the PRR-based washout
assay,34 with both assays confirming that 10 exhibited kill
kinetics similar to CQ, although slower than ART (Figure 2).
ADME Assessment of Selected Tetrazole and Amide
Compounds. A preliminary assessment of the physicochemical
properties and metabolic stability was conducted for several
compounds in the tetrazole and amide subseries (Table 3).
Lipophilicity in the series was high, and this property correlated
with the overall poor metabolic stability in human liver
microsomes for all tested compounds. Compound 7 and some
with lower cLog D (10 and 34) performed marginally better
than other compounds in the series, and overall, the tetrazoles
were slightly better than the amides. Solubility in pH 6.5
phosphate buffered saline (PBS) was poor for the tetrazoles but
was substantially better for the amides at both pH 2.0 and 6.5,
with 34 showing the best overall properties in these assays.
Overall, these studies identified a significant metabolic liability
that would need to be addressed before these series could be
expected to show in vivo efficacy.
Life-Cycle Profiling. Representative compounds, one each
from the tetrazole (9) and amide (32) series, were profiled
against liver- and sexual-stage parasites to determine if the series
would have prophylactic or transmission-blocking activity
(Table 4). Liver-stage activity was assessed against Plasmodium
a
Table 4. Life Cycle Profiling
Pb liver-stage
EC50 (μM)
HepG2 CC50
DGFA (male/female)
gametocytes EC50 (μM)
cmpd
(μM)
9
32
7.3, 12
7.0, 16
19, >17
24, 24
>25/>25
∼60% at 25 μM/>25
a
EC50 values are shown for two independent replicates. DGFA, dual
gamete formation assay.
Pf CRT in certain mutant forms mediates CQ efflux from the
DV and is causal for CQ resistance.23,43−45 While each strain
harbors a key set of Pf CRT mutations (including K76T, Table
6) that confer CQ resistance, the specific set of point mutations
differs across strains and mediates differences in sensitivity to
CQ and other 4-aminoquinolines such as PPQ and amodia-
quine.44,46,47 The 7G8 mutational background has also been
reported to increase sensitivity to lumefantrine (LUM).46,47
Thus, each Pf CRT isoform could display different binding
affinities and transport levels of 3-series compounds as well and
thus modulate parasite susceptibility to these compounds
differentially. This hypothesis was investigated by testing gene-
edited isogenic lines that allow comparison between different
pfcrt alleles with varied sensitivity to CQ in the same genetic
background. These lines were generated previously by zinc-
finger nuclease-mediated editing of Dd2 parasites to insert (1)
the wild-type pfcrt allele (Dd2-GC03); (2) the recombinant
Dd2 pfcrt allele (Dd2-Dd2) (differing from wild-type Pf CRT by
eight point mutations Table 6); or (3) the recombinant Dd2
pfcrt allele and the M343L mutation (Dd2-M343L), resulting in
PPQ resistance.44,46−49 We found that either the Dd2 Pf CRT
berghei to determine whether compounds could block liver-stage
development in HepG2 cells infected by sporozoites.19,21
Modest activity was observed in this assay, but the Pb liver-
stage EC50 for both compounds was 20- to 30-fold higher than
for P. falciparum blood stages. The dual gamete formation assay
(DGFA) was used to assess the ability of compounds to block
maturation of both male and female stage V gametocytes,
providing a readout of functional viability. The assay identifies
compounds that either kill stage V gametocytes or interfere with
gamete formation. No activity was observed against female
gametocytes for either compound, while modest activity was
seen for 32 against male gametocytes (∼60% inhibition at 25
μM). Thus, the 3-series compounds do not have significant liver-
stage or transmission-blocking activity, positioning them for use
solely for treatment of ABS infections.
Evaluation of Cross Resistance to the Known Drugs
Suggests a Common Resistance Mechanism with Hz-
Inhibiting Antimalarials. To obtain insight into the potential
MOA of the 3-series compounds, we evaluated compounds
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Figure 3. Reduced efficacy of 3-analogues containing 5-Pyr against P. falciparum Dd2 compared to 3D7 parasites. Comparison of representative
concentration response curves for tetrazoles 9 (A) and 10 (B) and for amides 26 (C) and 27 (D), tested against 3D7 (green circles) and Dd2 (black
squares) parasites. Results showed that compounds containing 5-Pyr have poorer activity against Dd2 than do compounds containing 3-Py in the B-
ring. Compound structures are shown above the graphs. Data were fitted to the [I] vs response, variable slope (four parameter) equation in GraphPad
Prism to determine the EC50. Triplicate data were collected at each concentration, symbols represent the mean, and error bars represent the std dev.
EC50 data are reported in Tables 1 and 2, which contain average data for at least three biological replicates.
Figure 4. Biphasic growth curves were observed for select 5-Pyr 3-series compounds assayed against Dd2 parasites. Comparison of representative
concentration response curves for tetrazoles 12 (A) and 13 (B) and for amide 30 (C) tested against 3D7 (green circles) and Dd2 (black squares)
parasites. Compound structures are displayed above the graphs. Triplicate data were collected at each concentration, symbols represent the mean, and
error bars represent the std dev. EC50 data and curve fitting methods are reported in Tables 1 and 2.
background or the Dd2-M343L background led to a two- to
threefold increased sensitivity to both 9 and 10 (Table 5)
relative to Dd2-GC03. These data suggest that the wild-type
Pf CRT transporter might bind better to 9 and 10 than do the
variant Dd2 or Dd2 + M343L isoforms, leading to lower drug
concentrations in the DV. Interestingly, the addition of M343L
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Table 5. Activity of 9 and 10 Versus Parasite Strains with Resistance to Compounds from Diverse Structural Classes and
Mechanisms of Action
9
9
10
10
a
a
Pf parasite line
resistance profile
EC50 (μM)
fold diff
EC50 (μM)
fold diff
Drug-Resistant Field Isolates42,44,49
3D7
NF54
K1
sensitive
sensitive
0.38
0.48
0.36
1.0
1.3
0.95
0.47
1.2
0.40
0.83
0.46
0.16 0.024 (3)
0.66
1.1
1.8
1.0
2.1
1.2
0.40
1.7
2.8
4.5
CQ, PYR
CQ, PYR
CQ, ATQ, PYR
PPQ, PYR
CQ, PYR
7G8
0.18 0.042 (3)
TM90C2B
RF12
Dd2
0.46
0.61
0.54
1.6
1.4
Pf CRT Gene Edited Isogenic Lines
Dd2-GC03
Dd2-Dd2
Dd2-M343L
sensitive
CQ
PPQ, CQ
0.89 0.12 (3)
0.45 0.13 (3)
0.38 0.10 (3)
1.0
0.53
0.41
4.8
2.4
0.90
1.0
0.50
0.19
Lab-Derived Dd2 Lines Resistant to Development Candidates
PfeEF2
Pfpi4K
PfDHODH
Pfcarl
DDD10749885
MMV39004818
DSM26525
0.92
0.75
0.80
0.65
0.85
1.7
1.4
1.5
1.2
1.6
1.6
2.0
2.0
2.0
2.0
0.89
1.1
1.1
1.1
1.1
KAF15686
PfCytB
ELQ30087
a
Fold differences were calculated relative to 3D7 for field isolates, relative to Dd2-GC03 for the isogenic gene-edited lines, or relative to Dd2 for
lab-selected Dd2 resistant lines. The GC03 line (which contains the pfcrt wild-type allele, also found in 3D7 or NF54) was previously isolated from
the Dd2 × HB3 cross and is CQ-sensitive. The gene-edited lines were constructed using customized zinc-finger nucleases and have been previously
reported.46−48 Dd2-GC03 was derived by introducing the 3D7 pfcrt sequence into Dd2 in place of the endogenous pfcrt Dd2 locus (sequence
shown in Table 6). Dd2-Dd2 was engineered using the same method to replace the endogenous pfcrt locus with the recombinant Dd2 sequence,
and Dd2-M343L replaced the endogenous pfcrt locus with the recombinant Dd2 sequence and the PPQ resistance-conferring M343L44,49
mutation. Concentration response data were collected with technical duplicates at each inhibitor concentration, and reported data represent the
average of two biological replicates, and where additional biological replicates were collected (number in parenthesis), the std dev is provided.
Additional replicate data for NF54 can be found in Table S2A. Development candidates used for selections are listed, and parasite lines are named
based on the protein target of the resistance allele. CQ, chloroquine, ATQ, atovaquone, PYR, pyrimethamine, and PPQ, piperaquine. Data for Dd2
and 3D7 were taken from Table 1.
ential sensitivity to compounds in the 3-series, suggesting that
like CQ and other quinolines, the 3-series compounds targeted a
process in the DV. Additionally, the lack of enantiomer
selectivity of the 3-series supports the hypothesis of a
nonprotein target such as heme polymerization.
Table 6. Documented Point Mutations in PfCRT Across the
Tested Strains
a
To investigate the activity of 3, 9, and 10 against the heme
detoxification pathway, we tested the ability of these compounds
to inhibit conversion of hematin (Fe(III)PPIX) to the synthetic
β-hematin (βH) form of Hz. These experiments used a pyridine-
based detergent-mediated colorimetric assay that simulates the
physiological microenvironment of the DV.47 Standard
antimalarials with diverse MOAs were tested in parallel to
serve as controls. As expected, CQ, amodiaquine, MFQ, and
pyronaridine inhibited βH formation with IC50 ≤100 μM, while
pyrimethamine, dihydroartemisinin, and doxycycline did not
inhibit conversion of hematin to βH (Figure 5). Among the
tested compounds, 9 inhibited βH formation with the same
potency as MFQ, while 10 showed weaker inhibition.
Compound 3 was nearly inactive in the concentration range
that could be studied (Figure 5). The IC50 values for inhibition
of βH formation (Figure 5) of the 3-series compounds
correlated loosely with their EC50 values for inhibiting NF54
proliferation (Tables 5 and S2). 9 was the most potent in both
assays, supporting the hypothesis that the mechanism of cell
killing was related to inhibition of βH formation. The
concentration of the compound required to inhibit βH
formation was ∼1000-fold higher than the EC50 for parasite
survival not only for the 3-series but also for the 4-
aminoquinolines. These differences are thought to reflect the
a
Data were taken from refs 23, 43, 44, 49, and 88. Colors highlight
residue differences between strains: yellow (3D7 and Dd2), green
(3D7 and 7G8), and turquoise (3D7 and RF12). RF12 is also known
in the literature as PH1263-C. 3D7 is a clone of NF54.49
has also been reported to reduce the degree of CQ resistance by
two- to threefold, compared with the Dd2 isoforms.49 The
finding that the EC50 for 10 on Dd2-GC03 is higher than for the
Dd2-Dd2 line shows that the poor activity of 10 on Dd2 relative
to 3D7 is not linked to differences in the pfcrt allele.
Heme Polymerization is Inhibited by 3-Series Com-
pounds. Similarities between CQ and the 3-series compounds
suggested to us the potential for a common MOA. CQ functions
by interrupting heme detoxification via inhibition of the
biomineralization of toxic labile heme into the chemically inert
Hz in the DV.43,50,51 Similarities include the killing kinetics, the
life-cycle profile showing activity only on ABS, and the
observations that CQ-resistant (7G8 and Dd2) and genetically
engineered lines containing Pf CRT mutations showed differ-
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Figure 5. β-Hematin inhibition assay (βHIA) for 3-series compounds in comparison to antimalarial control compounds. (A) Representative titration
data showing inhibition of β-hematin formation vs concentration profiles for 3, 9, and 10 in comparison to known antimalarials, using the β-hematin
inhibition assay (βHIA).89 Positive (pyronaridine, amodiaquine, CQ, and MFQ) and negative controls were assayed, with the latter representing a
diverse set of MOAs (the DHODH inhibitor DSM1,90 artesunate, sulfadoxine, and doxycycline). Data show the mean std dev for duplicate data at
each compound concentration. Data were fitted to the sigmoidal concentration response (variable slope) equation in GraphPad Prism to determine
the IC50. (B) Fitted IC50 values. Data represent the mean std dev for four independent studies.
fact that drug accumulation in the DV is in a function of pH
trapping;52 hence, the extent of accumulation determines the
concentration at the site of action.
Selections for P. falciparum 3D7 Parasites Resistant to
Compound 9. To obtain additional insight into the potential
mode of resistance to 3-series compounds, we performed single-
step selection studies to obtain parasites resistant to 9. Selections
were performed with a starting parasite inocula of 2 × 109 3D7
parasites per flask, and two separate flasks were pressured with 9
at a concentration of 3× EC50. Drug-treated parasites cleared in
both flasks by day 5 and recrudesced on day 25. Bulk cultures
showed EC50 shifts of only 2.4- to 3.4-fold compared to the
parental line. Three clones were then obtained from each flask
by limiting dilution. EC50 and ED90 shifts for these six clones
were found to be a modest 2.8−2.6-fold (Table 7). The high
starting inocula and the modest shifts in EC50 for the selected
mutant lines suggest that it is relatively difficult to evolve
resistance against 9.
WGS performed on two clones per flask found that each clone
had a copy number variation (CNV) on chromosome 5, with
flask 1 (F1) clones showing three- to fourfold amplification and
flask 2 (F2) clones showing fivefold amplification of the region
(Tables S4 and S5 and Figure S3A). Clones from the two flasks
differed in the chromosome 5 segment that was amplified.
Nonetheless, all clones overlapped in the multidrug resistance
gene 1 (pfmdr1, PF3D7_0523000) locus. This gene has
previously been shown to be involved in MFQ, LUM, and
quinine drug resistance and can also modulate CQ potency.54,55
This was the only change observed in all four clones, suggesting
that it represents the major mechanism of resistance to 9 in these
lines.
In addition to the CNV on chromosome 5, F2 clones A10 and
A11 had another set of CNVs on chromosome 12, showing an
approximately twofold amplification (Table S5 and Figure S3B).
It is interesting to note that one of the eight genes in this
amplified locus (Pf3D7_1223700) encodes a proposed vacuolar
iron transporter, which could potentially be involved in
modulating the consequences of disrupting heme polymer-
ization in the DV. Finally, F1 clones F1-B6 and F1-E4 had single
nucleotide polymorphisms (SNPs) in two common genes
(Table S6), an M199I-substitution in PF3D7_0208200 (KRR1
small subunit processome component) and a N353K-
substitution in PF3D7_1316700 (unknown function), while
F1-B6 also contained a D4190N mutation in PF3D7_1343800
(unknown function). The relevance of these SNPs to the
resistance mechanism of 9 is unknown.
Cellular Heme Fractionation. To mechanistically validate
the in vitro heme polymerization results described above, we
applied a heme fractionation assay to measure inhibition of Hz
formation in NF54 parasites. In this assay, “% free heme”, % Hz
(determined as proportions of total heme species extracted),
and levels of “free” heme-Fe and Hz-Fe (absolute amounts of
each species per cell calculated from the total amount of Fe
obtained using a heme standard curve) were quantified from
isolated trophozoites after their treatment with compounds.
This assay tested a range of compound concentrations at or
above their EC50 for antimalarial activity (0.5× to 3× EC50). For
the positive CQ control, both the % “free” heme and the levels of
“free” heme-Fe showed a significant concentration-dependent
increase as the drug concentration was increased above the EC50
compared to the no-drug control (Figure 6A(i)), whereas Hz
and Hz-Fe decreased over the same concentration range (Figure
6A(ii)). Overlaying the parasite survival curve with the “free”
heme-Fe titration curve showed that the concentration of CQ
leading to 50% activity (EC50 for heme-Fe release vs EC50 for
parasite survival) was similar in both assays (Figure 6A(iii)),
consistent with CQ being a bona fide inhibitor of Hz
formation.53 This result contrasts with the data for the
dihydrofolate reductase inhibitor pyrimethamine (Figure 6B),
included as a negative control to illustrate the robustness of this
protocol in discriminating agents that do not inhibit Hz
formation. No significant increase in free heme was observed
over the course of the titration, regardless of drug concentration.
Treatment with 9 and 10 led to a robust buildup of free heme
(calculated as a percent of total heme species) and the level of
heme-Fe, with a corresponding decrease in Hz levels (Figure
6C,D). These compounds led to significantly larger amounts of
free heme release (sixfold increase in free heme-Fe for 9 and 10
vs only twofold for CQ) and a corresponding reduction in Hz
levels at 3× EC50 compared to CQ (2-fold decrease in Hz heme-
Fe for 9 and 10 compared to 1.3-fold for CQ), demonstrating a
higher amount of free heme at the compound concentrations
that disrupt parasite growth. Similar to CQ, overlaying the
parasite survival curve with the “free” heme-Fe titration curve
showed that the concentrations of 9 and 10 at the EC50 of heme-
Fe release versus EC50 for parasite survival were very similar in
both assays. These data suggest that the MOA of the 3-series
compounds is at least in part due to inhibition of Hz formation.
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Figure 6. Cellular heme fractionation and concentration response versus parasite survival. Data are shown for (A) CQ, the positive control, for (B)
PYR, the negative control, and for two 3-analogues, (C) compound 9 and (D) compound 10. Panels (i,ii) Heme species present as “free” heme (panel
i) and Hz Fe (fg) (panel ii) isolated per cell from drug-treated Pf NF54 parasites. Data represent the mean std dev from three biological replicates,
each arising from two technical replicates (see Table S3). Panel (iii) Overlay of parasite survival with the total amount of “free” heme-Fe as a function of
drug concentration. Free heme data represent the mean std dev from three biological replicates (taken from panels i), while representative parasite
survival data from one biological replicate with technical duplicates is displayed (see Table S2A for average EC50’s from three biological replicates).
Data were fitted to the sigmoidal concentration response (variable slope) equation in GraphPad Prism. Calculated EC50 values for the drug
concentration giving 50% response are as follows: CQ, 58 nM (heme) and 15 nM (survival); PYR, >100 nM (heme) and 33 nM (survival); cmpd 9,
620 nM (heme) and 440 nM (survival); and cmpd 10, 1040 nM (heme) and 910 nM (survival). See Table S2A,B for error analysis. Significance was
assessed using a two-tailed Welch’s t-test, where * <0.05, ** <0.01, and *** <0.001.
showing fast kill kinetics, selection of resistant parasites toward
one of the more potent compounds 9 generated only modest
levels of resistance (two- to threefold) and WGS suggested that
resistance was associated with amplification of the pfmdr1 gene.
We showed that 3-series compounds 9 and 10 inhibited heme
polymerization both in vitro and in P. falciparum cellular assays.
Importantly, 9 showed equal efficacy on both CQ-sensitive and
DISCUSSION
■
The ability of P. falciparum to develop resistance to virtually
every drug that has been used clinically exemplifies the
importance of working on this pathogen to further elucidate
its biology and identify new drugs. Herein, we describe the
identification of a novel, fast-acting antimalarial compound
series that has efficacy against ABS parasites. In addition to
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a
Table 7. In Vitro Selections for Pf3D7 Parasites Resistant to 9 Showing EC50/EC90 Shifts of Clonal Lines
parental
9 resistant clonal 3D7 lines
clone
3D7-A10
F1-B6
F1-C1
F1-E4
F2-A10
F2-A11
F2-H12
EC50 μM
fold shift
EC90 μM
fold shift
0.30 0.033
NA
0.59 0.12
NA
0.55 0.058
1.8
1.1 0.16
1.9
0.54 0.061
1.8
1.0 0.17
1.8
0.74 0.12
2.5
1.5 0.21
2.6
0.83 0.11
2.8
1.5 0.10
2.6
0.67 0.029
2.3
1.3 0.041
2.3
0.64 0.055
2.2
1.3 0.078
2.2
a
Data represent the mean standard error of the mean for three independent replicates.
CQ-resistant strains. Thus, the 3-series meets the objective of
identifying fast kill compounds with a relatively low propensity
of resistance for the treatment of malaria, and they provide a new
scaffold that targets heme polymerization, a highly successful
strategy for killing malaria parasites. Based on their lack of liver-
stage or sexual-stage activity, these compounds would be best
positioned in the portfolio for treatment, and their fast kill
kinetics could allow them to be suitable for development in non-
ART-based therapies. Further work to optimize both potency
and drug-like properties would however be needed for this series
to advance to lead optimization and to in vivo activity.
A limited medicinal chemistry effort was undertaken for the 3-
series to define the structural requirements for activity. We
found that parasite killing was not dependent on the
stereochemistry and that both enantiomers were equally active
for those that were tested. The A- and B-rings could be aryl or
heteroaryl, but they could not be removed or replaced with
aliphatic rings. Substantial substitution on the A ring was
tolerated, which allowed us to replace the original Br/OMe
substitution with more drug-like CF3/F groups. Similarly, we
were able to improve the polarity of the series by replacing the B-
ring phenyl group with heteroaryl rings including 3-pyridine (3-
Py) and 5-pyrimidine (5-Pyr). This ring showed the steepest
SAR profile with substantial loss in activity associated with
substitution on various positions or alternative heteroaryl rings.
Activity was improved by replacing the N-methyl group with
larger N-alkyl groups, and the benzylic CH2 could be replaced
with a cyclopropyl group, blocking a potential metabolic liability.
The tetrazole could be replaced with a conformationally
restricted amide bond, and both series showed similar SAR.
Overall, potency improvements of 10−20-fold were obtained
relative to the initial hit 3. The amide subseries generally showed
better selectivity than the tetrazoles. Lipophilicity, indexed by
Log D, was high throughout both subseries and metabolic
stability was generally poor, although we were able to make some
improvements in aqueous solubility.
Profiling of a panel of drug-sensitive or -resistant strains
provided the first insights that the 3-series might share a
common MOA with Hz inhibitors such as CQ. Direct
biochemical evidence that the 3-series compounds inhibit
heme polymerization was obtained both in vitro and in cell-
based assays. We showed that 9 and 10 inhibited heme
biomineralization in the in vitro βH inhibition assay, while in the
cell-based assay, treatment with these compounds caused a CQ-
like signature of an accumulation of free heme with
commensurate decreases in Hz levels. These effects titrated
with the same dose response as parasite killing. Taken together,
these data strongly implicate inhibition of Hz formation as one,
but perhaps not the sole, MOA of this series. Interestingly, the
levels of free heme generated in the presence of 9 and 10 are
three- to fourfold higher than those observed for CQ across the
dose response range, with the larger differences observed at
higher drug concentrations. These results could reflect divergent
interactions of the two compound classes with heme molecules
during heme−drug complex formation. Indeed atomic-force
microscopy studies have shown that the various antimalarial 4-
aminoquinolines affect the biomineralization of heme molecules
in different ways, with CQ and quinine providing the most
efficient blocking by forming interactions across the flat surface
face (“step-pinning”), whereas MFQ and amodiaquine block the
addition of heme only to the step edge (“kink-blocking”).51
Resistance selection studies uncovered resistance mecha-
nisms that overlapped with the CQ-related quinolines such as
MFQ, providing an additional link between the 3-series and
quinoline compounds. First, we observed that the 3-series has a
relatively low propensity to develop resistance as selections with
109 parasites yielded EC50 shifts of only two- to threefold. Our
WGS analysis found that the one genetic change common to all
the resistant clones was three- to fivefold amplification of the
pfmdr1 gene, which encodes the P-glycoprotein transporter
Pgp1. pfmdr1 amplification is a major driver of clinical and
parasite in vitro resistance to MFQ.55,56 The level of resistance
observed for 9 was less than that reported for MFQ selection
studies, where a lower level of amplification of the pfmdr1 locus
(twofold) yielded a fivefold IC50 increase and which could be
selected with a lower starting inocula (∼108 parasites).57
A
greater impact of amplification was also reported for
dihydroorotate dehydrogenase (DHODH) inhibitors, where
threefold amplification of the dhodh locus led to a fivefold EC50
increase from ∼107 parasites58 and five- to sixfold amplification
arising from ∼108 parasites led to a sevenfold EC50 increase.25
Additionally, for DHODH inhibitors, multiple SNPs resulting in
>10 to 30-fold shifts in EC50 have been identified at a frequency
of ∼108 parasites.25,59 Similar findings have also been reported
for other antimalarials in preclinical development where
minimum inoculum of resistance of 106 to 108 parasites,
mediated often by SNPs or CNVs, led to resistance levels that
can range from 2- up to 1000-fold based on the target, the
genetic change, and the compound (e.g., PfATP4 or eEF2
inhibited by SNPs in KAE609 or DDD107498, respectively4). In
this light, our finding of low-level resistance mediated by pfmdr1
amplification is a favorable feature of the 3-series.
The interplay between Pf MDR1 and Pf CRT and the impact
of their allelic variation on drug resistance are complex and
compound-dependent. While CQ resistance is primarily
associated with Pf CRT mutations,23,43−45 low-grade resistance
to other quinolines such as MFQ and the arylaminoalcohol
LUM is associated with Pf MDR1 genetic changes (amplifica-
tions or point mutations).46,47,60 Pf CRT is localized to the DV
membrane and transports compounds out of the DV. Pf MDR1
also appears to be primarily localized to the Plasmodium DV
membrane of the trophozoite,61 where it has been associated
with solute uptake into the vacuole.55,60,62 Low-level expression
of Pf MDR1 at the plasma membrane has also been reported,61
leaving open the possibility that it could be involved in efflux of
compounds out of the parasitized red cell, similar to its
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mechanism of resistance in mammalian cells. Within this
complex milieu, the potency of any given compound is
dependent on the concentrations that can reach the DV,
which could be influenced by binding affinity to the parasite line-
specific transporter isoforms of Pf CRT and Pf MDR1 and by
nonreceptor-mediated partitioning between the various cellular
compartments related to the physical properties of the
compound. Our studies on the 3-series suggests that like
MFQ and LUM, their efficacy can also be modulated by both
transporters but to a lesser extent than for the quinolines.
Analysis of the gene-edited lines showed that 9 and 10 were
more potent on lines harboring Pf CRT mutations that are
observed in CQ-resistant parasites than on parasites expressing
the wild-type allele. These data provide direct evidence that the
pfcrt allele impacts the potency of 3-series compounds and
suggest that the wild-type isoform might bind to and transport
these compounds better than isoforms found in CQ-resistant
parasites. This result is similar to earlier reports for LUM.47
Importantly, these data provide evidence that the 3-series will be
fully effective against CQ-resistant parasites harboring Pf CRT
mutations found in malaria-endemic regions.
An identified exception to the promising activity on CQ-
resistant strains was identified for 3-series analogues that
contained 5-Pyr in the B-ring (e.g., 10). These tended to show
poorer activity against Dd2, while still being effective against
3D7 and 7G8. Some analogues also exhibited biphasic
concentration response curves versus Dd2 parasites. The Dd2
line has a separate Pf CRT isoform compared to the 7G8 line43,44
for which higher potency for both 9 and 10 was observed, but
data on the gene-edited lines that harbored only the Pf CRT
changes associated with Dd2 suggested that the poor activity of
10 on Dd2 is unrelated to Pf CRT. Pf MDR1 SNPs might also
impact the potency in these cases. Dd2 encodes the N86Y
mutation, whereas 7G8 carries the S1034C/N1042D/D1246Y
haplotype. Both Pf MDR1 isoforms sensitize parasites to MFQ
and LUM (when expressed in parasites with a single copy of
pfmdr1) and impact multiple antimalarials that engage with the
heme degradation pathway, either by causing increased
sensitivity or by mediating a degree of resistance.60,63,64 These
data suggest that the N86Y mutation could be associated with
the poor activity of some 3-series analogues against Dd2 and
could support a model whereby the N86Y PfMDR1 mutation
leads to reduced levels of 5-Pyr analogues such as 10 in the DV,
while not impacting localization of the 3-Py analogues such as 9.
The interplay between the two transporters might also
contribute to the biphasic curves observed with the 5-Pyr 3-
series analogues (e.g., 10). Dd2 parasites also express 2 to 3
copies of pfmdr1, contrasting with the single copy of pfmdr1
expressed by 3D7 and 7G8 parasites, which may further
contribute to the observed differences between Dd2 and other
parasite lines. These differences in potency provide a path
forward for future medicinal chemistry as compounds with 5-Pyr
in the B-ring would be disfavored going forward.
within reasonable physiological pH ranges, with the central
tertiary nitrogen present in all molecules and calculated pKa
values around 5−6, suggesting that 3-series compounds may
also interact with heme through this basic amine (Table 3). For
example, the acetamide 15 lacks a basic amine and is essentially
inactive. Similar to the crystal structure of halofantrine, the
structure of MFQ bound to ferriprotoporphyrin IX shows an
alkoxide bound to Fe and a salt bridge between a basic amine
and the porphyrin carboxylate.66 In this context, we note that the
amine-tetrazole or amine-amide distance in the 3-series is similar
to the amine-alcohol distance in MFQ. This observation
prompts the speculation that the tetrazole or amide might
bind Fe, while orienting the basic amine in a position to interact
with the heme carboxylate.67,68
CONCLUSIONS
■
Herein, we have described a novel tetrazole-based series with
antimalarial activity against ABS parasites. The compound class
exhibits a fast kill rate and a relatively low propensity to develop
resistance, both desirable properties for new antimalarial drugs.
The MOA of the series appears to be inhibition of heme
polymerization in the parasite DV, similar to 4-aminoquinolines
such as CQ. The series thus provides a new chemical scaffold
that functions at least in part by this highly effective mechanism
for killing malaria parasites. Novel compounds that function by
blocking heme biomineralization have been sought through
secondary screening of HTS hits from phenotypic ABS screens,
and while some new chemical entities that target this mechanism
have been discovered, none have yet progressed to preclinical
development.50,69,70 Our initial SAR exploration has revealed
areas of the scaffold that are subject to modification to optimize
activity and physicochemical properties and has shown that
substantial improvements are possible. Further efforts will be
required to identify compounds with improved potency and
ADME properties that will support in vivo efficacy and eventual
development. The 3-series also provides new tools for studying
the heme biomineralization mechanism and the impact of
inhibiting this process on the parasite.
EXPERIMENTAL SECTION
■
Human Samples. Human biological samples were sourced
ethically, and their research use was in accordance with the terms of
the informed consents under approved IRB/EC protocols for each site.
P. falciparum Culture. P. falciparum cultures were maintained at
2% hematocrit using human blood type O+ RBCs (Valley Biomedical)
employing a culturing method adapted from Trager and Jensen.71
Culture media contained the RPMI 1640 medium (Gibco) + 25 mM
HEPES and 0.5% Albumax-I, supplemented with 23 mM sodium
bicarbonate and 92 μM σ-hypoxanthine.
Determination of Compound Potency against P. falciparum
In Vitro. Antiplasmodial activity was evaluated using a parasite growth
SYBR Green-based inhibition assay adapted from published
methods.26,27 For the primary screen (plated at 5 μM) and for hit
validation three-point cherry pick concentration response curves
(plated at 5, 1.7, and 0.42 μM), we acoustically dispensed compounds
[30 nL of 5 mM stocks in dimethyl sulfoxide (DMSO)] into wells using
a Labcyte-Echo-555 dispenser. Concentration response curves on
repurchased or synthetized compounds were generated from serial
dilutions prepared in DMSO in triplicate and plated using a Tecan
D300e digital dispenser. The DHODH inhibitor DSM26525 served as a
positive control, and 0.5% DMSO was plated as a negative control, with
all wells normalized to a final concentration of 0.5% DMSO. Culture
suspensions were prepared to a 0.5% starting parasitemia at 2%
hematocrit and were dispensed (60 μL) using a BioTek-MultiFlo
dispenser into 384-well plates containing prespotted library com-
An open question that remains is how the 3-series analogues
interact with heme. The crystal structure of halofantrine−
ferriprotoporphyrin IX shows that the basic amine of
halofantrine (related to quinine) interacts with a carboxylate
from heme, while the hydroxyl coordinates to the heme iron.65
Indeed, a characteristic of nearly all heme-interacting anti-
malarials is the presence of a protonatable amine. These groups
appear to be important for interaction with heme and for
accumulation in the low pH environment of the DV. All of the 3-
series compounds with EC50 <1 μM have predicted pKa values
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pounds. Prepared plates were then incubated for 72 h at 37 °C in 5%
CO2, 80% humidity. Reference thick film smears from select control
wells were made prior to sealing each plate in aluminum foil and storing
at −80 °C for 24 h. Plates were thawed at room temperature (RT), and
all ensuing steps were conducted under reduced light. SYBR Green
stock solution was prepared by mixing σ-SYBR Green I nucleic acid gel
stock (1.2 μL) into 1 mL of lysis buffer (80 mM Tris-HCl pH 7.52, 20
mM EDTA, 0.032% w/v Saponin, 0.32% v/v 100% Triton X-100).
SYBR Green stock solution (15 μL) was dispensed into each well of the
assay plates, and plates were sealed in aluminum foil and stored in the
dark for 4 h at RT. Fluorescence was measured by detecting emission at
535 nm after excitation at 485 nm using a BioTek Synergy H1 hybrid
reader. Data were fitted to the log(inhibitor) versus responsevariable
slope (four parameters) model in Graph Pad Prism to determine the
effective concentration that led to a 50% reduction in parasitemia
(EC50).
Phenotypic Screen Chemical Library. The chemical library used
for the screen is an 8K subset of the UT Southwestern compound
collection assembled with support from computational chemists at
Chemical Diversity Inc (Chem Div) to represent a plate-based diversity
subset of our 200K chemical library. Compounds in this subset are
compliant with a relaxed set of Lipinski’s rules, with 99% having a
molecular weight less than 550. The abovementioned SYBR Green
assay was optimized on our HTS core platforms as a readout for P.
falciparum growth using the 384-well plate format. Z′ was evaluated as a
measure of assay robustness where a Z′ >0.5 represents a robust assay.
Hits were identified based on an RZ score cutoff of <−3, where the RZ
score represents the number of RSDs that a measurement for a library
compound is from the corrected robust median, which is determined
after correction for systematic errors (plate, row, and column effects) as
described.28,29
Pf Drug-Resistant Laboratory Strains and Cross-Resistance
Testing. Drug sensitivity testing was performed using the modified
[3H]-hypoxanthine incorporation assay, as previously reported.72
HepG2 In Vitro Cytotoxicity Evaluation. Cytotoxicity was
evaluated against HepG2 human hepatoma cell suspensions in the σ-
EMEM medium supplemented with 5% heat inactivated fetal bovine
serum (Gibco) and 2 mM glutamine. Compounds were dispensed into
384-well plates using a Labcyte Echo555 acoustic dispenser.
Methotrexate (Sigma) and 0.5% DMSO were used as positive and
negative controls, respectively. All wells were normalized to an upper
limit of 0.5% DMSO. A volume of 60 μL/well was added using a
BioTek-MultiFlo dispenser from a 10,000 cells/mL cell suspension
preparation of HepG2 cells for a final seeding density of 600 cells/well.
Plates were then incubated for 72 h in a humidity chamber at 37 °C, 5%
CO2, and 80% humidity. Cell viability was established by quantifying
intracellular ATP using a luciferase-coupled ATP quantification assay
(Promega-CellTiter Glo) following the manufacturer’s instructions.
Data were fitted to the log(inhibitor) vs responsevariable slope (four
parameters) model in Graph Pad Prism to determine the cytotoxic
concentration that led to 50% reduction in the signal (CC50).
Two-Color Flow Cytometry Kill Rate Assay. Double-colori-
metric FACS analysis was used to quantify invasion of prestained
human RBCs by drug-treated parasites as described.41 Briefly, killing
profiles were estimated by culturing unlabeled RBCs infected with P.
falciparum 3D7 parasites in the presence of compounds at 10× EC50 for
24 or 48 h. The initial % parasitemia was 0.5% with a hematocrit of 2%.
Compound EC50 was determined prior to the start of the study using
the 48 h in vitro 3H-hypoxanthine incorporation assay as described34 for
parasites cultured in standard RPMI 1640 media supplemented with 25
mM HEPES and 0.225% NaHCO3 supplemented with 2% D-sucrose,
0.3% L-glutamine, 0.005 mM hypoxanthine, and 0.5% AlbuMax II.
Culture media for the kill rate study was identical but contained 0.15
mM hypoxanthine. After drug treatment, compounds were removed
and infected RBCs were diluted using fresh RBCs previously labeled
with carboxylfluorescein diacetate succinimidyl ester (10 μM for 30 min
at 37 °C). Following a further 48 h incubation under standard
conditions, the ability of treated parasites to establish infection in fresh
labeled RBCs was detected by two-color flow cytometry after labeling
parasite DNA with Hoechst 33342 solution (2 μM). Parasite viability
was measured based on the percentage of infected CFDA-SE-stained
RBCs in drug-treated samples versus untreated samples of the initial
inocula after 48 h incubations with labeled RBCs.
PRR Kill Rate Assay. PRR was assessed using the standard drug
washout assay that has been previously described.34 Briefly, parasites
were treated with compounds for either 24 or 48 h at 10× EC50
(determined as described above) with the compound renewed daily
over the treatment period. Parasite samples were removed at the
determined time points (0, for the control of the initial number of
parasites, 24, and 48 h), the drug was washed out, and drug-free
parasites were cultured in 96-well plates after the addition of fresh RBCs
and new media in microtiter plates over a threefold serial dilution.
Cultures were maintained for up to 28 days to enable parasites to
recrudesce. Four independent serial dilutions were carried out with
each sample to correct for experimental variation. The number of viable
parasites was back-calculated based on Xn−1 where n is the number of
wells where growth was observed and X is the dilution factor.34
Liver-Stage and Gametocidal Assays. P. berghei liver-stage
assays were performed in the Winzeler lab (UCSD) as described.19,21 P.
berghei-ANKA-GFP-Luc-SMCON (Pb-Luc) sporozoites were used to
infect HepG2-A16-CD81EGFP cells for this assay, which monitors the
ability of compounds to block liver-stage development of luciferase-
expressing parasites. Assay media (DMEM without Phenol Red (Life
Technology, CA) supplemented with 5% fetal bovine serum, 1.45 mg/
mL glutamine, 500 units of penicillin, and 500 μg/mL streptomycin)
was used for the Pbluc and HepG2tox assays.
Gametocyte assays measuring the viability of mature stage V
gametocytes were conducted to determine the ability of compounds to
block formation of male and female gametes in the Baum lab (Imperial
College, London) as described.74−76 The assay identifies compounds
that either kill stage V gametocytes, cause sterilization, or interfere with
the process of gamete formation.
In Vitro Heme Polymerization Assay. Lipid-mediated Hz
formation was quantified by measuring the formation of βH from
hematin, in a detergent-mediated assay that substitutes neutral lipids for
the commercially available lipophilic detergent Nonidet P-40 (NP-
40).77 Unreacted hematin was detected through the formation of the
bis-pyridyl-Fe(III)PPIX complex which absorbs at a wavelength of 405
nm. Compound stocks (20 mM) of 3-derivatives (3, 9, and 10) and
pyrimethamine were prepared in DMSO, while CQ was prepared in
water. These stocks were then diluted to 2 mM with a water/NP40
detergent solution, resulting in a final solution of compound in 61.1
mM NP40/10% DMSO. Solubility of 3, 9, and 10 to 2 mM in this buffer
was confirmed (see in vitro ADME methods section). Serial dilutions
into this same buffer were then made for each compound. A 25 mM
hematin stock solution was prepared by sonicating hemin in DMSO for
1 min and then suspending 179 μL of this stock in a 1 M acetate buffer
(20 mL, pH 4.8). The homogenous suspension was then added to the
wells to give final buffer and hematin concentrations of 0.5 M and 100
mM, respectively. Plates were covered and incubated at 37 °C for 5 h. A
solution of 50% (v/v) pyridine, 30% (v/v) H2O, 20% (v/v) acetone,
P. falciparum Relative Rate of Kill Bioluminescence Assay
(BRRoK). The parasite kill rate was estimated as previously described35
using a transgenic luciferase-expressing P. falciparum strain, NF54-luc36
(BEI Resources) that had the advantage of expressing luciferase
throughout the asexual life cycle. Cultures were maintained in
WR99210 (5 nM), but the drug was removed by washing and replating
in drug-free media for 2 days prior to the kill rate studies. Briefly, NF54-
luc cultures were sequentially synchronized to ring stages using 5% D-
sorbitol as described.73 The assay was then conducted with trophozoite
stage cultures (20−26 h postinvasion) at 2% parasitaemia and 4%
haematocrit. NF54-luc parasites (200 μL/well) were added to 96-well
plates at 2% hematocrit and 2% parasitemia. Triplicate concentrations
(30× EC50 to 0.33× EC50) of benchmark antimalarials and
experimental compounds were added to each well and then incubated
for 6 h at 37 °C, 5% CO2, and 80% humidity. Culture samples (40 μL/
well) were then transferred to a white-clear bottom 96-well plate, and
parasite viability was determined by luciferase quantification using a
luciferase bioluminescence assay (Promega-Luciferase Assay system).
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and 2 M HEPES buffer (pH 7.4) was prepared, and 32 μL of the
solution was added to each well to give a final pyridine concentration of
5% (v/v). Acetone (60 μL) was then added to each well to assist with
hematin dispersion. The UV−vis absorbance of the plate wells was read
on a SpectraMax P340 plate reader. Sigmoidal concentration−response
curves were fitted to the absorbance data using GraphPad Prism version
8 to obtain a 50% inhibitory concentration (IC50) for each compound.
Cellular Heme Polymerization Assay. Inhibition of Hz
formation was measured in cultured ABS Pf NF54 parasites through a
heme fractionation assay,53,78 where both % “free” heme and levels of
“free” heme-Fe were quantified in femtogram per cell (fg/cell) using a
heme standard curve from the mass of each heme-Fe species per
trophozoite. Percent free heme and amounts of free heme-Fe were
quantified over a range of drug concentrations (0.5−3× EC50) and
compared to the vehicle control. Briefly, early ring-stage NF54 parasites
were treated with a combination of successive sorbitol and Percoll
treatments to prepare young rings synchronized to within 3 h
postinvasion.79 Young rings were incubated with the test drugs at
various multiples of their IC50 values with a no-drug control included
(culture conditions and media as described above). After 32 h, late
trophozoites/early schizonts were harvested by lysis of the RBCs with
0.05% saponin followed by multiple washes with 1× PBS (pH 7.5) to
remove traces of the RBC hemoglobin. Pellets were then resuspended
in 1× PBS (pH 7.5). An aliquot of the trophozoite suspension was used
to quantify, using flow cytometry, the total number of trophozoites
isolated. Contents of the remaining trophozoite pellet were then
released by hypotonic lysis and sonication. Following centrifugation,
treatment with HEPES buffer (pH 7.4), sodium dodecyl sulfate,
pyridine, and NaOH, the fractions corresponding to digested
hemoglobin, “free” heme, and Hz were carefully recovered. The UV−
visible spectrum of each heme fraction as an Fe(III)heme−pyridine
complex was measured using a multiwell plate reader (Spectramax
340PC; Molecular Devices). The total amount of each heme species
was quantified using a heme standard curve whereby the mass of each
heme-Fe species per trophozoite (fg/cell) was calculated by dividing
the total amount of each heme species by the corresponding number of
parasites in that fraction as determined by flow cytometry. Statistical
comparisons and analyses for trends were made on GraphPad Prism
version 8 using Students’ t-test (GraphPad Software Inc., La Jolla, CA,
USA).
In Vitro Selection of Drug-Resistant P. falciparum Lines. P.
falciparum 3D7 parasites (clone A10) resistant to 9 were selected using
previously described methods for single-step drug selections.80 Briefly,
the EC50 of 9 versus 3D7-A10 (genetically homogenous clonal line)
ring-stage parasites (0.2% parasitemia and 1% hematocrit, cultured in
RPMI-1640 media supplemented with 0.5% Albumax) was determined
in a 72 h assay for a twofold dilution series (10 points) of 9 (final DMSO
concentration was <0.5%) in duplicate along with DMSO solvent
controls. Parasite survival was assessed by flow cytometry on an Accuri
C6 (BD Biosciences) using SYBR Green and MitoTracker Deep Red
FM (Life Technologies) as nuclear stain and vital dyes, respectively. A
single-step selection with 2 × 109 parasites in duplicate was set up at 2%
parasitemia and 5% hematocrit using 3× EC50 of 9 (0.78 μM). Wells
were monitored daily by blood smears until the culture was cleared of
live parasites. Drug-containing media was replaced daily until cultures
cleared and then every other day thereafter. Cultures were passaged
once a week by replacing a fourth of the culture with media and fresh
RBCs, with monitoring by blood smears twice a week for up to 60 days
to allow for parasite recrudescence.
(version 4.1.6) was used to identify any SNVs in clones. These were
filtered based on quality scores (variant quality as a function of depth
QD > 1.5, mapping quality > 40, and min base quality score > 18) and
read depth (depth of read > 5) to obtain high-quality SNPs that were
annotated using snpEFF. Comparative SNP analysis between the
resistant clones and parent was performed to generate a final list of
SNPs that were present exclusively in the resistant clones. BIC-Seq was
used to discover CNVs in the resistant mutants against the parent, using
the Bayesian statistical model.82 Integrated genome viewer was used to
visually verify the presence of these SNPs and CNVs in the clones.
ADME: Solubility Measurements. Methods used to assess kinetic
solubility in PBS pH 6.5 and pH 2.0 buffers have been previously
described.25,83 Solubility of 3, 9, and 10 in the buffer used for the in vitro
heme biomineralization studies was conducted by spiking a 40 μL
aliquot of test compound stock solution into 80 μL of NP-40 solution
followed by 280 μL of Milli-Q water. The resulting mixtures contained a
final compound concentration of 2 mM in 10% (v/v) DMSO and 61.1
mM NP-40. Samples were maintained in a 37 °C incubator for the
duration of the study. After 1, 2, 4, and 24 h incubations at 37 °C, the
bulk samples were centrifuged (3 min at 10,000 rpm) and a single
aliquot of the supernatant was taken and diluted 20-fold in 50% aqueous
acetonitrile for quantitative analysis by HPLC with detection by UV−
vis absorbance at 244 nm.
ADME: In Vitro Metabolism and Calculation of Physical
Chemical Properties. Compounds (1 μM) were incubated (up to 60
min at 37 °C) with human liver microsomes (Xenotech LLC, Kansas
City, KS) at a protein concentration of 0.4 mg/mL with an NADPH
regenerating buffer system, as described previously.84 Degradation half-
life and in vitro intrinsic clearance were calculated from the apparent
first-order degradation rate constant. Physical−chemical properties
were calculated using the ChemAxon chemistry cartridge via JChem for
Excel software (version16.4.11).
Chemical Methods. General. All tested compounds have a purity
of >95% as judged by HPLC analysis (UV detection at 210 nM).
Chemical shifts δ are in ppm, and spectra were referenced using the
residual solvent peak. The following abbreviations are used: singlet (s),
doublet (d), triplet (t), quartet (q), double doublet (dd), quintet
(quin), multiplet (m), and broad signal (bs). Mass spectra (m/z) were
recorded on an Agilent LC−MS 1290 Infinity using ESI ionization. All
chemicals were used as received unless otherwise noted.
HPLC/MS Analysis. An Agilent 1290 Infinity HPLC system using
an Eclipse XDB-C18 column (4.6 × 150 mm, 5 μm; Agilent) that was
coupled to an Agilent 6130 quadrupole ESI mass spectrometer run in
the positive mode with a scan range of 100−1100 m/z was used. LC was
carried out at a flow rate of 0.5 mL/min at 20 °C with a 5 μL injection
volume, using a gradient elution with aqueous acetonitrile containing
0.1% formic acid, 0 min: 30% acetonitrile/water; 0 → 6 min: gradient to
95% acetonitrile/water; 6 → 12 min 95% acetonitrile/water.
N-(3-Chlorophenyl)-2-(3-methyl-2-oxo-5-phenyl-2,3-dihydro-
1H-benzo[e][1,4]diazepin-1-yl)butanamide (1). tR = 9.0 min. ESI-MS
(m/z): 446.1 [M + H]+.
N-(4-((4-Acetylphenyl)carbamoyl)phenyl)-2,3-dihydrobenzo[b]-
[1,4]dioxine-2-carboxamide (2). tR = 4.24 min. ESI-MS (m/z): 417.0
[M + H]+.
WGS and Analyses of Drug-Resistant P. falciparum Clones.
For WGS, genomic DNA was used to prepare libraries using the
Illumina Nextera DNA Flex library kit with dual indices, as previously
reported.81 The samples were multiplexed and sequenced on an
Illumina MiSeq to obtain 300 bp paired end reads at 19−44× depth of
coverage across the samples. Sequence reads were aligned to the P.
falciparum 3D7 genome (PlasmoDB version 36) using BWA (Burrow−
Wheeler Alignment). PCR duplicates and unmapped reads were filtered
out using Samtools and Picard. The reads were realigned around indels
using GATK RealignerTargetCreator, and base quality scores were
recalibrated using GATK BaseRecalibrator. GATK HaplotypeCaller
General Syntheses of Tetrazole Derivatives.37 Isocyanide (1.0
mmol), aromatic aldehyde (1.0 mmol), N-benzylamine (1.0 mmol),
and trimethylsilyl azide (1.0 mmol) were dissolved in MeOH (2.0 mL, 2
M) under a N2 atmosphere. The resulting solution was stirred at RT for
2−3 days. MeOH was evaporated, and the residue was purified by flash
column chromatography.
N-(5-Bromo-2-methoxybenzyl)-N-methyl-1-phenyl-1-(1-(o-
tolyl)-1H-tetrazol-5-yl)methenamine (3). The target compound was
obtained in 73% yield as a colorless thick liquid. 1H NMR (400 MHz,
CDCl3): δ 7.48−7.42 (m, 2H), 7.33−7.19 (m, 8H), 7.02 (d, J = 8.4 Hz,
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1H), 6.64 (d, J = 8.6 Hz, 1H), 4.94 (s, 1H), 3.69−3.61 (m, 4H), 3.46 (d,
J = 14.8 Hz, 1H), 2.23 (s, 3H), 1.69 (s, 3H). 13C NMR (101 MHz,
CDCl3): δ 156.7, 155.2, 136.0, 135.5, 132.6, 132.1, 131.6, 131.3, 130.6,
129.2, 128.6 (d, J = 20.8 Hz), 127.3, 127.0, 113.0, 112.0, 63.3, 55.5,
51.7, 39.6, 17.1. ESI-MS (m/z): 478.1 [M + H]+. The enantiomers were
separated by chiral HPLC (CHIRALCEL OD-H column, 1%
isopropanol in hexane, 1.0 mL/min, tR = 25, 32 min). Enantiomer 1,
[α]2D5 +7.99 (c 0.15, EtOH); Enantiomer 2, [α]2D5 −7.99 (c 0.15,
EtOH).
N-(2-Methoxybenzyl)-N-methyl-1-phenyl-1-(1-(o-tolyl)-1H-tetra-
zol-5-yl)methanamine (4). The target compound was obtained in 78%
yield as a colorless liquid. 1H NMR (400 MHz, CDCl3): δ 7.45 (t, J =
7.6 Hz, 1H), 7.38−7.25 (m, 8H), 7.20 (t, J = 7.8 Hz, 1H), 7.01 (d, J =
7.9 Hz, 1H), 6.90 (t, J = 7.4 Hz, 1H), 6.80 (d, J = 8.2 Hz, 1H), 4.94 (s,
1H), 3.74−3.66 (m, 4H), 3.50 (d, J = 14.1 Hz, 1H), 2.26 (s, 3H), 1.73
(s, 3H). 13C NMR (101 MHz, CDCl3): δ 157.6, 155.3, 135.9, 135.7,
132.6, 131.5, 131.1, 129.9, 129.2, 128.4 (d, J = 32.0 Hz), 128.1, 127.2,
126.9, 126.5, 120.4, 110.3, 62.9, 55.2, 52.2, 39.2, 17.1. ESI-MS (m/z):
400.2 [M + H]+.
1H), 5.16 (s, 1H), 4.10 (d, J = 14.4 Hz, 1H), 3.93 (d, J = 14.0 Hz, 1H),
2.05−1.98 (m, 1H), 1.85 (s, 3H), 0.31−0.18 (m, 2H), 0.06 to −0.01
(m, 1H), −0.14 to −0.21 (m, 1H). 13C NMR (101 MHz, CDCl3): δ
153.7, 150.7, 149.9, 140.3, 137.2, 135.5, 132.2 (d, J = 20.7 Hz), 131.9,
131.5, 130.9, 130.2 (q, J = 135.0 Hz) 128.7, 127.3, 126.9, 125.6 (q, J =
15.1 Hz), 123.9 (q, J = 14.8 Hz), 123.4, 58.4, 55.6, 35.1, 17.4, 8.9, 7.5.
ESI-MS (m/z): 465.1.1 [M + H]+.
N-(Pyrimidin-5-yl(1-(o-tolyl)-1H-tetrazol-5-yl)methyl)-N-(3-(tri-
fluoromethyl) benzyl)cyclopropanamine (10). The target compound
1
was obtained in 53% yield as a colorless liquid. H NMR (400 MHz,
CDCl3): δ 9.17 (s, 1H), 8.80 (s, 2H), 7.52−7.43 (m, 2H), 7.40−7.27
(m, 4H), 7.21 (d, J = 7.6 Hz, 1H), 6.93 (d, J = 7.2 Hz, 1H), 5.16 (s, 1H),
4.07 (d, J = 14.0 Hz, 1H), 3.91 (d, J = 13.6 Hz, 1H), 2.09−2.01 (m, 1H),
1.90 (s, 3H), 0.39−0.24 (m, 2H), 0.09−0.01 (m, 1H), −0.12 to −0.20
(m, 1H). 13C NMR (101 MHz, CDCl3): δ 158.7, 157.7, 152.5, 139.3,
135.3, 132.2, 132.1, 132.0, 131.8, 130.8 (q, J = 130.0 Hz), 128.9, 128.7,
127.4, 126.8, 125.6 (q, J = 15.2 Hz), 124.3 (q, J = 17.6 Hz), 56.6, 55.8,
35.0, 17.4, 8.9, 7.7. ESI-MS (m/z): 466.1 [M + H]+.
N-(3-Fluoro-5-(trifluoromethyl)benzyl)-N-(pyrimidin-5-yl(1-(o-
tolyl)-1H-tetrazol-5-yl)methyl)cyclopropanamine (11). The target
N-(2-Methoxy-5-(trifluoromethyl)benzyl)-N-methyl-1-phenyl-1-
1
compound was obtained in 51% yield as a colorless liquid. H NMR
(1-(o-tolyl)-1H-tetrazol-5-yl)methanamine (5). The target compound
1
(400 MHz, CDCl3): δ 9.18 (s, 1H), 8.81 (s, 2H), 7.50 (t, J = 7.2 Hz,
1H), 7.40 (d, J = 7.6 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.15 (d, J = 8.0
Hz, 1H), 7.07 (s, 1H), 6.99−6.91 (m, 2H), 5.17 (s, 1H), 4.09 (d, J =
14.4 Hz, 1H), 3.91 (d, J = 14.4 Hz, 1H), 2.07−1.99 (m, 1H), 1.92 (s,
3H), 0.41−0.26 (m, 2H), 0.12−0.04 (m, 1H), −0.05 to −0.15 (m, 1H).
13C NMR (101 MHz, CDCl3): δ 163.6, 161.2, 158.8, 157.7, 152.4,
was obtained in 70% yield as a colorless liquid. H NMR (400 MHz,
CDCl3): δ 7.59 (s, 1H), 7.48−7.39 (m, 2H), 7.20−7.31 (m, 7H), 7.00
(d, J = 7.7 Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H), 4.98 (s, 1H), 3.78−3.70
(m, 4H), 3.52 (d, J = 14.8 Hz, 1H), 2.24 (s, 3H), 1.69 (s, 3H). 13C NMR
(101 MHz, CDCl3): δ 159.9, 155.0, 135.9, 135.5, 132.5, 131.6, 131.2,
129.1, 128.6 (d, J = 16.0 Hz), 127.6, 127.1, 126.9, 126.4 (q, J = 15.0 Hz),
125.4 (q, J = 16.0 Hz), 122.7 (q, J = 131.9 Hz), 109.9, 63.2, 55.5, 51.6,
39.6, 17.1. ESI-MS (m/z): 468.2 [M + H]+.
N-(2-Methoxy-5-(trifluoromethyl)benzyl)-N-methyl-1-(pyridin-3-
yl)-1-(1-(o-tolyl)-1H-tetrazol-5-yl)methenamine (6). The target com-
pound was obtained in 63% yield as a white solid. 1H NMR (400 MHz,
CDCl3): δ 8.56 (dd, J = 4.8, 1.6 Hz, 1H), 8.49 (d, J = 2.4 Hz, 1H), 7.91
(td, J = 8.0, 2.0 Hz, 1H), 7.52−7.44 (m, 3H), 7.38−7.27 (m, 3H), 7.06
(d, J = 8.0 Hz, 1H), 6.85 (d, J = 8.0 Hz, 1H), 5.03 (s, 1H), 3.73 (s, 3H),
3.71 (d, J = 14.8 Hz, 1H), 3.51 (d, J = 14.8 Hz, 1H), 2.24 (s, 3H), 1.80
(s, 3H). 13C NMR (101 MHz, CDCl3): δ 159.9, 154.0, 150.3, 149.8,
137.0, 135.6, 132.3, 131.9, 131.5, 131.1, 127.2, 126.9 (d, J = 8.68 Hz),
126.6 (q, J = 14.4 Hz), 125.8 (q, J = 16.0 Hz), 123.5, 122.6 (q, J = 134.0
Hz), 110.2, 60.5, 55.5, 51.7, 39.1, 17.2. ESI-MS (m/z): 469.3 [M + H]+.
The enantiomers were separated by chiral HPLC (CHIRALCEL OD-
H column, 10% isopropanol in hexane, 1.0 mL/min, tR = 15, 17 min).
Enantiomer 1, [α]2D5 +5.16 (c 0.15, EtOH); Enantiomer 2, [α]2D5 −5.16
(c 0.15, EtOH).
142.6 (d, J = 25.2 Hz), 135.3, 132.2, 131.9, 131.8, 128.6, 127.5, 126.7,
121.3−121.1 (m), 119.2, 118.9, 112.0 (q, J = 18.7 Hz), 111.8 (q, J =
15.2 Hz), 56.7, 55.4, 35.3, 17.5, 8.9, 7.7. ESI-MS (m/z): 484.0 [M +
H]+. The enantiomers were separated by chiral HPLC (CHIRACEL
OD-H column, 10% isopropanol in hexane, 1.0 mL/min, tR = 10, 12
min). Enantiomer 1, [α]2D5 +24.609 (c 0.195, EtOH); Enantiomer 2,
[α]2D5 −21.558 (c 0.195, EtOH).
N-(4-Fluoro-3-(trifluoromethyl)benzyl)-N-(pyrimidin-5-yl(1-(o-
tolyl)-1H-tetrazol-5-yl) methyl)cyclopropanamine (12). The target
1
compound was obtained in 51% yield as a colorless liquid. H NMR
(400 MHz, CDCl3): δ 9.18 (s, 1H), 8.80 (s, 2H), 7.51 (dt, J = 8.0, 1.6
Hz, 1H), 7.40 (d, J = 7.6 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.24−7.18
(m, 2H), 7.03 (t, J = 10 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H), 5.16 (s, 1H),
4.06 (d, J = 14.0 Hz, 1H), 3.85 (d, J = 14.0 Hz, 1H), 2.00−1.88 (m, 4H),
0.39−0.23 (m, 2H), 0.06−0.01 (m, 1H), −0.11 to −0.19 (m, 1H). 13C
NMR (101 MHz, CDCl3): δ 158.8, 157.7, 152.4, 135.3, 134.7, 134.6,
134.2, 134.1, 132.1, 131.9, 131.8, 128.7, 127.5, 126.8, 117.0, 116.8, 56.9,
54.9, 35.1, 17.4, 8.8, 7.8. ESI-MS (m/z): 484.1 [M + H]+.
N-(2-Methoxy-5-(trifluoromethyl)benzyl)-N-(pyridin-3-yl(1-(o-
tolyl)-1H-tetrazol-5-yl)methyl)cyclopropanamine (7). The target
N-(2-Fluoro-3-(trifluoromethyl)benzyl)-N-(pyrimidin-5-yl(1-(o-
1
compound was obtained in 61% yield as a colorless liquid. H NMR
tolyl)-1H-tetrazol-5-yl)methyl)cyclopropanamine (13). The target
1
(400 MHz, CDCl3): δ 8.55 (m, 2H), 8.08 (d, J = 8 Hz, 1H), 7.48−7.20
(m, 6H), 7.00 (d, J = 8.0 Hz, 1H), 6.76 (d, J = 8.4 Hz, 1H), 5.12 (s, 1H),
4.20 (d, J = 12 Hz, 1H), 3.69 (d, J = 13.2 Hz, 1H), 3.64 (s, 3H), 1.89 (s,
4H), 0.24−0.15 (m, 2H), −0.05 to −0.11 (m, 1H), −0.29 to −0.39 (m,
1H). 13C NMR (101 MHz, CDCl3): δ 159.9, 153.8, 150.9, 149.8, 137.4,
135.5, 132.3, 131.7, 131.4, 130.2, 129.1 (q J = 14.6 Hz), 127.6, 127.1 (d,
J = 7.0 Hz), 125.7 (q, J = 15.0 Hz), 123.2, 122.2 (q, J = 130 Hz), 109.9,
58.5, 55.4, 49.5, 35.1, 17.2, 8.1, 7.5. ESI-MS (m/z): 495.1 [M + H]+.
N-(2-Fluoro-5-(trifluoromethyl)benzyl)-N-(pyridin-3-yl(1-(o-
tolyl)-1H-tetrazol-5-yl)methyl)cyclopropanamine (8). The target
compound was obtained in 50% yield as a colorless liquid. H NMR
(400 MHz, CDCl3): δ 9.20 (s, 1H), 8.88 (s, 2H), 7.52−7.44 (m, 2H),
7.39 (d, J = 7.6 Hz, 1H), 7.34−7.27 (m, 2H), 7.11−7.02 (m, 2H), 5.15
(s, 1H), 4.21 (d, J = 13.2 Hz, 1H), 3.79 (d, J = 13.6 Hz, 1H), 1.97−1.86
(m, 4H), 0.36−0.19 (m, 2H), −0.02 to −0.09 (m, 1H), −0.20 to −0.27
(m, 1H). 13C NMR (101 MHz, CDCl3): δ 158.8, 157.7, 152.5, 135.6 (d,
J = 11.5 Hz), 135.4, 132.0 (d, J = 10.1 Hz), 134.6, 131.7, 128.4, 127.4,
126.9, 126.5 (q, J = 11.6 Hz), 123.7 (d, J = 11.6 Hz), 57.0, 48.5, 35.0,
17.4, 8.2, 7.9. ESI-MS (m/z): 484.1 [M + H]+.
N-(Cyclopropyl(1-(o-tolyl)-1H-tetrazol-5-yl)methyl)-N-(3-
1
compound was obtained in 61% yield as a colorless liquid. H NMR
(trifluoromethyl)benzyl) Cyclo Propanamine (14). The target
1
(400 MHz, CDCl3): δ 8.57 (td, J = 4.8, 1.8 Hz, 1H), 8.52 (bs, 1H),
8.01−7.92 (m, 1H), 7.52−7.22 (m, 6H), 7.00 (t, J = 8.8 Hz, 2H), 5.19
(s, 1H), 4.27 (d, J = 13.8 Hz, 1H), 3.87 (d, J = 13.8 Hz, 1H), 1.91 (bs,
4H), 0.35−0.16 (m, 2H), 0.00 to −0.07 (m, 1H), −0.16 to −0.27 (m,
1H). 13C NMR (101 MHz, CDCl3): δ 153.5, 150.7, 150.0, 137.2, 135.5,
132.2, 131.9, 131.5, 130.3, 129.2−129.0 (m), 127.2, 127.0, 123.3, 115.9,
115.7, 58.7, 48.4, 35.2, 17.3, 8.1, 7.7. ESI-MS (m/z): 483.1 [M + H]+.
N-(Pyridin-3-yl(1-(o-tolyl)-1H-tetrazol-5-yl)methyl)-N-(3-
(trifluoromethyl)benzyl) Cyclopropanamine (9). The target com-
pound was obtained in 62% yield as a colorless liquid. 1H NMR (400
MHz, CDCl3): δ 8.54 (d, J = 4.4 Hz, 1H), 8.45 (bs, 1H), 7.83 (d, J = 7.2
Hz, 1H), 7.47−7.37 (m, 2H), 7.35−7.18 (m, 6H), 6.88 (d, J = 7.2 Hz,
compound was obtained in 71% yield as a colorless liquid. H NMR
(400 MHz, CDCl3): δ 7.50−7.42 (m, 2H), 7.40−7.36 (m, 2H), 7.34−
7.27 (m, 3H), 7.08 (d, J = 6.8 Hz, 1H), 4.17 (d, J = 14.4 Hz, 1H), 4.07
(d, J = 14.4 Hz, 1H), 3.18 (d, J = 10.0 Hz, 1H), 2.13−2.00 (m, 4H),
1.65−1.56 (m, 1H), 0.72−0.64 (m, 1H), 0.61−0.53 (m, 1H), 0.41−
0.31 (m, 1H), 0.22−0.09 (m, 2H), −0.03 to −0.12 (m, 1H), −0.21 to
−0.37 (m, 2H). 13C NMR (101 MHz, CDCl3): δ 155.9, 141.0, 135.8,
132.8, 132.3, 131.7, 131.2, 130.3 (q, J = 127.1 Hz), 128.5, 127.2, 127.0,
125.8 (q, J = 14.6 Hz), 123.7 (q, J = 14.9 Hz), 62.0, 55.3, 35.4, 17.5,
11.4, 8.5, 6.8, 5.6, 3.9. ESI-MS (m/z): 428.1 [M + H]+.
N-(Pyrimidin-5-yl(1-(o-tolyl)-1H-tetrazol-5-yl)methyl)-N-(3-
(trifluoromethyl)benzyl)acetamide (15). 1-(Pyrimidin-5-yl)-1-(1-(o-
2753
https://dx.doi.org/10.1021/acs.jmedchem.0c02022
J. Med. Chem. 2021, 64, 2739−2761

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
tolyl)-1H-tetrazol-5-yl)-N-(3-(trifluoromethyl)benzyl)methanamine
was synthesized using (3-(trifluoromethyl)phenyl)methanamine via a
general procedure. To a suspension of the abovementioned amine (1.0
mmol) in CH2Cl2 was added Et3N (2.0 mmol) at 0 °C. Acetyl chloride
(2.0 mmol) was then added dropwise over 5 min. After stirring at RT
for 1 h, the mixture was diluted with CH2Cl2 and washed with water.
The organic layer was dried over anhydrous Na2SO4, and the solvent
was evaporated under vacuum. The residue obtained was purified by
flash column chromatography (EtOAc/hexane) to furnish the target
compound in 51% yield. 1H NMR (400 MHz, CDCl3): δ 8.97 (bs, 1H),
8.55 (bs, 2H), 7.55−7.27 (m, 5H), 7.20−6.81 (m, 3H), 5.02 (s, 2H),
2.14 (s, 3H), 1.95 (s, 3H). 13C NMR (101 MHz, CDCl3): δ 172.2,
159.0, 157.9, 153.2, 137.2, 135.7, 132.2, 131.9, 131.6, 129.7, 129.3−
129.2 (m), 127.5, 127.0, 124.6 (q, J = 12.5 Hz), 122.5 (q, J = 16.3 Hz),
49.5, 47.5, 22.2, 17.4. ESI-MS (m/z): 468.0 [M + H]+.
7.28 (m, 5H), 7.02 (dd, J = 8.0, 4.8 Hz, 1H), 5.92 (s, 1H), 3.18−3.11
(m, 1H), 2.80−2.73 (m, 1H), 2.47 (s, 3H), 1.49−1.41 (m, 1H), 1.37−
1.27 (m, 1H), 1.25−1.16 (m, 2H), 1.06−0.85 (m, 4H), 0.68−0.50 (m,
2H), 0.32−0.23 (m, 1H), −0.24 to −0.33 (m, 1H). 13C NMR (101
MHz, CDCl3): δ 156.4, 156.0, 148.0, 142.1, 139.0, 132.7, 130.7, 130.2
(q, J = 130 Hz), 128.7, 125.4 (q, J = 14.8 Hz), 124.2 (q, J = 15.6 Hz),
121.3, 56.6, 48.8, 30.5, 28.1, 21.7, 16.7, 13.0, 10.5, 6.8, 6.7, 6.6. ESI-MS
(m/z): 455.1 [M + H]+.
N-Propyl-N-(pyrimidin-5-yl(1-(o-tolyl)-1H-tetrazol-5-yl)methyl)-
1-(3-(trifluoromethyl)phenyl)cyclopropan-1-amine (20). The target
1
compound was obtained in 49% yield as a colorless liquid. H NMR
(400 MHz, CDCl3): δ 9.09 (s, 1H), 8.48 (s, 2H), 7.49 (td, J = 7.6, 1.3
Hz, 1H), 7.39 (dd, J = 10.2, 7.8 Hz, 2H), 7.33−7.24 (m, 3H), 7.16 (s,
1H), 6.87 (d, J = 7.9 Hz, 1H), 5.18 (s, 1H), 2.88 (m, 2H), 1.90 (s, 3H),
1.52−1.34 (m, 2H), 1.18−1.08 (m, 1H), 0.95−0.86 (m, 1H), 0.85−
0.78 (m, 1H), 0.71 (t, J = 7.3 Hz, 3H), 0.55−0.48 (m, 1H). 13C NMR
(101 MHz, CDCl3): δ 158.4, 157.2, 154.3, 143.6, 135.1, 132.2, 131.9,
131.8, 131.7, 131.0, 129.0, 127.4, 126.6, 124.3 (q, J = 13.1 Hz), 124.0
(q, J = 14.4 Hz), 55.4, 48.3, 45.8, 23.6, 17.6, 17.2, 11.4. ESI-MS (m/z):
494.2 [M + H]+.
N-(2-Fluoro-5-(trifluoromethyl)benzyl)-N-methyl-1-(pyridin-3-
yl)-1-(1-(o-tolyl)-1H-tetrazol-5-yl)methenamine (16). The target
1
compound was obtained in 61% yield as a colorless liquid. H NMR
(400 MHz, CDCl3): δ 8.57 (dt, J = 4.8, 1.3 Hz, 1H), 8.45 (d, J = 2.3 Hz,
1H), 7.89 (dt, J = 8.1, 2.0 Hz, 1H), 7.55 (dd, J = 6.6, 2.4 Hz, 1H), 7.53−
7.43 (m, 2H), 7.38−7.27 (m, 3H), 7.10−7.00 (m, 2H), 4.98 (s, 1H),
3.74 (d, J = 14.0 Hz, 1H), 3.58 (d, J = 14.1 Hz, 1H), 2.22 (s, 3H), 1.81
(s, 3H). 13C NMR (101 MHz, CDCl3): δ 164.2, 161.6, 153.9, 150.3 (d,
J = 22.7 Hz), 136.9, 135.6, 132.2, 131.9, 131.6, 130.7, 128.1−127.9 (m),
127.3, 126.9, 126.5−126.3 (m), 126.1, 125.2, 125.1, 123.6, 122.4, 116.2,
115.9, 60.5, 50.5 (d, J = 8.8 Hz), 39.0, 17.2. ESI-MS (m/z): 457.1 [M +
H]+.
1-(1-Cyclo hexy l-1H-tet razol-5-yl)-N-(2- fluoro-5-
(trifluoromethyl)benzyl)-N-methyl-1-(pyridin-3-yl)methanamine
(17). The target compound was obtained in 62% yield as a colorless
liquid. 1H NMR (400 MHz, CDCl3): δ 8.69 (d, J = 2.4 Hz, 1H), 8.62
(dd, J = 4.8, 1.2 Hz, 1H), 7.94 (td, J = 8.4, 4.0, 1.6 Hz, 1H), 7.62 (dd, J =
6.4, 2.0 Hz, 1H), 7.57−7.52 (m, 1H), 7.39−7.35 (m, 1H), 7.15 (t, J =
18, 9.6 Hz, 1H), 5.26 (s, 1H), 4.44−4.35 (m, 1H), 3.70 (t, J = 16.0 Hz,
2H), 2.23 (s, 3H), 2.07−1.70 (m, 7H), 1.42−1.23 (m, 3H). 13C NMR
(101 MHz, CDCl3): δ 164.4, 161.9, 152.1, 150.3, 137.2, 130.1, 128.2−
128.0 (m), 126.9−126.7 (m) 126.2, 126.1, 125.1, 123.7, 122.4, 116.5,
116.2, 61.2, 58.4, 51.4 (d, J = 8.0 Hz), 38.8, 33.3, 32.9, 25.3 (d, J = 22.1
Hz), 24.8. ESI-MS (m/z): 449.1 [M + H]+.
N-Propyl-N-(pyridin-3-yl(1-(o-tolyl)-1H-tetrazol-5-yl)methyl)-1-
(3-(trifluoromethyl)phenyl)cyclopropan-1-amine (21). The target
1
compound was obtained in 56% yield as a colorless liquid. H NMR
(400 MHz, CDCl3): δ 8.48 (dd, J = 4.8, 1.6 Hz, 1H), 8.21 (d, J = 2.4 Hz,
1H), 7.52−7.36 (m, 3H), 7.34−7.26 (m, 3H), 7.24−7.14 (m, 3H), 6.77
(d, J = 7.2 Hz, 1H), 5.14 (s, 1H), 2.94−2.88 (m, 2H), 1.83 (s, 3H),
1.50−1.27 (m, 2H), 1.20−1.12 (m, 1H), 0.95−0.87 (m, 1H), 0.84−
0.77 (m, 1H), 0.70−0.60 (m, 4H). 13C NMR (101 MHz, CDCl3): δ
155.4, 150.4, 149.6, 144.4, 136.5, 135.2, 133.1, 132.0, 131.4, 130.4 (q, J
= 128 Hz), 128.8, 127.2, 126.5, 124.1 (q, J = 14.8 Hz), 123.6 (q, J = 15.2
Hz), 123.2, 57.1, 48.3, 45.7, 23.6, 17.5, 17.0, 11.3. ESI-MS (m/z): 493.1
[M + H]+.
General Synthesis of Amide Derivatives.38 Isocyanide (1
mmol), aromatic aldehyde (1 mmol), N-benzylamine (1 mmol), and
N-((1-Cyclopropyl-1H-tetrazol-5-yl)(pyridin-3-yl)methyl)-N-(3-
(trifluoromethyl)benzyl)cyclopropanamine (18). The target com-
pound was obtained in 59% yield as a colorless liquid. 1H NMR (400
MHz, CDCl3): δ 8.44 (d, J = 2.4 Hz, 1H), 8.41 (dd, J = 5.2, 1.6 Hz, 1H),
7.73 (td, J = 7.6, 2.0 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H), 7.23−7.12 (m,
3.5H), 7.07 (s, 0.5H), 5.34 (s, 1H), 3.86 (d, J = 14.0 Hz, 1H), 3.73 (d, J
= 14.0 Hz, 1H), 3.12−3.05 (m, 1H), 2.04−1.97 (m, 1H), 1.15−1.05
(m, 1H), 1.00−0.76 (m, 3H), 0.31−0.20 (m, 2H), 0.04 to −0.09 (m,
2H). 13C NMR (101 MHz, CDCl3): δ 154.3, 150.3, 149.4, 139.9, 138.1,
132.4, 130.7, 128.9, 125.7 (q, J = 14.6 Hz), 124.2 (q, J = 15.2 Hz), 123.6,
58.1, 56.1, 34.7, 28.5, 8.8, 7.7, 7.3, 6.9. ESI-MS (m/z): 415.1 [M + H]+.
N-Cyclopropyl-N-((1-cyclopropyl-1H-tetrazol-5-yl)(2-methylpyri-
din-3-yl)methyl)-1-(3-(trifluoromethyl)phenyl)cyclopropan-1-
amine (19). Step 1: Synthesis of 1-(3-(Trifluoromethyl)phenyl)-
cyclopropan-1-amine. Ethylmagnesium bromide (0.7 mL, 2.2 mmol,
3 M in ether) was added to a solution of 3-trifluoromethyl benzonitrile
(171 mg, 1.0 mmol) and Ti(Oi-Pr)4 (312 mg, 1.1 mmol) in Et2O (5
mL) at −70 °C. The yellow solution was stirred for 10 min. After the
solution was warmed to RT (1 h), BF3 OEt2 (292 mg, 2.2 mmol) was
added. After the mixture was stirred for 1 h, 1 N HCl (3 mL) and ether
(15 mL) were added. NaOH (10% aq, 10 mL) was added to the
resulting two clear phases, and the mixture was extracted with ether.
The combined ether layers were dried over Na2SO4 and concentrated
under reduced pressure. The residue was purified by flash column
chromatography to furnish the target compound in 63% yield as a
colorless oil.
phosphinic acid (0.1 mmol) were dissolved in anhydrous toluene (1
mL) under a N2 atmosphere. The resulting solution was heated at 80 °C
for 12 h. Toluene was evaporated under reduced pressure, and the
residue was purified by flash chromatography.
To a solution of the abovementioned amide (0.5 mmol) in dry
tetrahydrofuran was added NaH (0.6 mmol) at 0 °C in one portion;
then, after 5 min, MeI (1.0 mmol) was added to the reaction mixture at
the same temperature. The resulting mixture was allowed to warm at
RT and stirred for 30 min. After completion of reaction monitored by
LC−MS, the reaction mixture was quenched with cold H2O, extracted
with ethyl acetate, and dried over anhydrous Na2SO4. The organic layer
was then concentrated under reduced pressure and purified by flash
chromatography.
2-(Cyclopropyl(3-(trifluoromethyl)benzyl)amino)-N-methyl-2-
(pyridin-3-yl)-N-(o-tolyl)acetamide (22). The target compound was
obtained in 61% yield as a colorless liquid. 1H NMR (400 MHz, CDCl3,
1:1 mixture of rotamers): δ 8.49−8.44 (m, 2H, mixture of rotamers),
8.26 (d, J = 2.3 Hz, 1H, one rotamer), 8.15 (d, J = 2.4 Hz, 1H, one
rotamer), 7.63 (dt, J = 7.9, 2.0 Hz, 1H, one rotamer), 7.58 (dt, J = 7.9,
2.0 Hz, 1H, one rotamer), 7.45−7.40 (m, 1H, one rotamer), 7.39−7.31
(m, 4H, mixture of rotamers), 7.25−7.13 (m, 8H, mixture of rotamers),
7.09−7.01 (m, 2H, mixture of rotamers), 6.85 (m, 2H, mixture of
rotamers), 6.29 (dd, J = 7.9, 1.3 Hz, 1H, one rotamer), 4.54 (s, 1H, one
rotamer), 4.39 (s, 1H, one rotamer), 4.30 (d, J = 14.9 Hz, 1H, one
rotamer), 4.24 (d, J = 14.6 Hz, 1H, one rotamer), 3.97 (d, J = 14.7 Hz,
1H, one rotamer), 3.95 (d, J = 14.7 Hz, 1H, one rotamer), 3.23 (s, 3H,
one rotamer), 3.19 (s, 3H, one rotamer), 2.36−2.28 (m, 2H, mixture of
rotamers), 2.27 (s, 3H, one rotamer), 1.50 (s, 3H, one rotamer), 0.36−
0.18 (m, 4H, mixture of rotamers), 0.07 to −0.01 (m, 2H, mixture of
rotamers), −0.13 to −0.26 (m, 2H, mixture of rotamers). 13C NMR
Step 2: Synthesis of N-Cyclopropyl-1-(3-(trifluoromethyl)phenyl)-
cyclopropan-1-amine. This was synthesized via a general procedure
used for synthesis of N-benzylamine.
Step 3. The target compound was obtained in 52% yield as a
colorless liquid using the general procedure for synthesis of tetrazoles.
1H NMR (400 MHz, CDCl3): δ 8.39 (dd, J = 4.8, 1.2 Hz, 1H), 7.47−
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Article
(101 MHz, CDCl3, 1:1 mixture of rotamers): δ 171.5, 171.3, 151.2,
151.0, 149.2, 149.1, 142.5, 141.9, 141.2, 141.1, 137.5, 137.1, 135.8,
135.1, 132.7, 132.0, 131.7, 131.6, 131.5, 131.4, 128.7 (d, J = 13.6 Hz),
128.5 (d, J = 12.0 Hz), 128.2, 128.1, 127.2, 126.9, 125.3 (q, J = 14.4 Hz),
125.1 (q, J = 16.0 Hz), 123.5 (q, J = 16.0 Hz), 123.2 (q, J = 14.4 Hz),
123.1, 123.0, 65.0, 63.5, 56.2, 55.5, 36.2, 35.7, 35.0, 17.5, 16.8, 8.9, 8.7,
7.6, 7.3. ESI-MS (m/z): 454.1 [M + H]+.
157.7, 142.4, 141.8, 140.8, 136.4, 136.2, 135.3, 135.2, 135.0, 134.7,
131.7 (d, J = 12.0 Hz), 131.2, 130.9, 130.6, 129.6, 129.4, 129.1 (d, J =
26.0 Hz), 129.0, 128.3, 128.1, 127.3, 127.0, 62.9, 61.6, 56.2, 55.7, 36.3
(d, J = 7.2 Hz), 35.3, 34.5, 17.4, 16.9, 9.1, 8.9, 7.8, 7.5. ESI-MS (m/z):
487.1 [M + H]+.
2-(Cyclopropyl(3-(trifluoromethoxy)benzyl)amino)-N-methyl-2-
(pyridin-3-yl)-N-(o-tolyl)acetamide (26). The target compound was
obtained in 56% yield as a colorless liquid. 1H NMR (400 MHz, CDCl3,
1:1 mixture of rotamers): δ 8.50−8.45 (m, 2H, mixture of rotamers),
8.26 (bs, 1H, one rotamer), 8.15 (bs, 1H, one rotamer), 7.62 (dt, J = 7.9,
2.0 Hz, 1H, one rotamer), 7.56 (dt, J = 7.8, 2.0 Hz, 1H, one rotamer),
7.25−7.22 (m, 2H, mixture of rotamers), 7.22−7.11 (m, 5H, mixture of
rotamers), 7.10−6.98 (m, 5H, mixture of rotamers), 6.98−6.81 (m, 5H,
mixture of rotamers), 6.32−6.25 (m, 1H, one rotamer), 4.54 (s, 1H, one
rotamer), 4.38 (s, 1H, one rotamer), 4.26 (d, J = 15.0 Hz, 1H, one
rotamer), 4.20 (d, J = 14.7 Hz, 1H, one rotamer), 3.96 (d, J = 14.8 Hz,
1H, one rotamer), 3.94 (d, J = 14.8 Hz, 1H, one rotamer), 3.23 (s, 3H,
one rotamer), 3.19 (s, 3H, one rotamer), 2.40−2.29 (m, 2H, mixture of
rotamers), 2.25 (s, 3H, one rotamer), 1.49 (s, 3H, one rotamer), 0.36−
0.17 (m, 4H, mixture of rotamers), 0.08 to −0.00 (m, 2H, mixture of
rotamers), −0.09 to −0.27 (m, 2H, mixture of rotamers). 13C NMR
(151 MHz, CDCl3, 1:1 mixture of rotamers): δ 149.1, 148.9, 140.9,
135.5, 135.0, 131.5, 131.4, 129.3 (m), 129.1, 128.8, 128.3, 128.0 (m),
127.2, 126.6 (m), 123.3 (m), 120.7, 119.5, 118.9, 64.6, 63.1, 56.0, 55.4,
36.2, 36.1, 35.5, 34.8, 17.3, 16.7, 8.8, 8.6, 7.1. ESI-MS (m/z): 470.1 [M
+ H]+.
2-(Cyclopropyl(3-(trifluoromethyl)benzyl)amino)-N-methyl-2-
(pyrimidin-5-yl)-N-(o-tolyl)acetamide (23). The target compound
1
was obtained in 45% yield as a colorless liquid. H NMR (400 MHz,
MeOD, 1:1 mixture of rotamers): δ 9.04−9.00 (m, 2H, mixture of
rotamers), 8.52−8.46 (m, 4H, mixture of rotamers), 7.53−7.22 (m,
11H, mixture of rotamers), 7.18−7.11 (m, 2H, mixture of rotamers),
7.02−6.91 (m, 2H, mixture of rotamers), 6.45 (d, J = 7.8 Hz, 1H, one
rotamer), 4.57 (s, 1H, one rotamer), 4.50 (s, 1H, one rotamer), 4.24 (d,
J = 14.5 Hz, 1H, one rotamer), 4.18 (d, J = 14.5 Hz, 1H, one rotamer),
4.05−3.98 (m, 2H, mixture of rotamers), 3.25 (s, 3H, one rotamer),
3.21 (s, 3H, one rotamer), 2.38−2.25 (m, 5H, mixture of rotamers),
1.61 (s, 3H, one rotamer), 0.41−0.26 (m, 4H, mixture of rotamers),
0.16−0.05 (m, 2H, mixture of rotamers), −0.07 to −0.15 (m, 1H, one
rotamer), −0.16 to −0.24 (m, 1H, one rotamer). 13C NMR (101 MHz,
MeOD, 1:1 mixture of rotamers): δ 171.9, 171.6, 159.1, 158.9, 158.6,
158.5, 143.2, 142.6, 142.1, 136.5 (d, J = 22.0 Hz), 133.5, 133.2, 132.9,
132.8, 132.3, 131.5, 130.3 (d, J = 21.6 Hz), 130.1, 129.8, 129.5, 129.4,
128.6, 128.0, 126.2 (q, J = 15.2 Hz), 125.9 (q, J = 15.2 Hz), 124.8 (q, J =
16.4 Hz), 124.5 (q, J = 16.4 Hz), 64.4, 63.2, 57.4, 56.9, 36.9, 36.8, 36.7,
36.0, 17.7, 17.0, 9.6, 9.3, 8.5, 8.3. ESI-MS (m/z): 455.2 [M + H]+.
2-(Cyclopropyl(3-((trifluoromethyl)thio)benzyl)amino)-N-meth-
yl-2-(pyridin-3-yl)-N-(o-tolyl)acetamide (24). The target compound
2-(Cyclopropyl(3-(trifluoromethoxy)benzyl)amino)-N-methyl-2-
(pyrimidin-5-yl)-N-(o-tolyl)acetamide (27). The target compound
1
was obtained in 43% yield as a colorless liquid. H NMR (400 MHz,
1
was obtained in 59% yield as a colorless liquid. H NMR (400 MHz,
CDCl3, 1:1 mixture of rotamers): δ 9.09 (bs, 2H, mixture of rotamers),
8.48 (s, 2H, one rotamer), 8.47 (s, 2H, one rotamer), 7.30−7.27 (m,
1H, one rotamer), 7.25−7.23 (m, 1H, one rotamer), 7.22−7.14 (m, 3H,
mixture of rotamers), 7.12−6.95 (m, 7H, mixture of rotamers), 6.94−
6.84 (m, 2H, mixture of rotamers), 6.83−6.76 (m, 1H, one rotamer),
6.32 (dd, J = 7.8, 1.3 Hz, 1H, one rotamer), 4.48 (s, 1H, one rotamer),
4.37 (s, 1H, one rotamer), 4.27 (d, J = 15.0 Hz, 1H, one rotamer), 4.20
(d, J = 14.6 Hz, 1H, one rotamer), 3.96 (d, J = 14.8 Hz, 1H, one
rotamer), 3.94 (d, J = 14.8 Hz, 1H, one rotamer), 3.24 (s, 3H, one
rotamer), 3.20 (s, 3H, one rotamer), 2.45−2.39 (m, 1H, one rotamer),
2.37−2.32 (m, 1H, one rotamer), 2.22 (s, 3H, one rotamer), 1.57 (s,
3H, one rotamer), 0.41−0.19 (m, 4H, mixture of rotamers), 0.10 to
−0.01 (m, 2H, mixture of rotamers), −0.15 to −0.19 (m, 1H, one
rotamer), −0.28 to −0.32 (m, 1H, one rotamer). 13C NMR (101 MHz,
CDCl3, 1:1 mixture of rotamers): δ 170.4, 170.1, 158.0 (m), 157.7,
143.2, 142.6, 140.9, 135.3, 135.2, 131.7, 130.7, 129.6 (d), 129.3, 129.1,
129.0, 128.3, 128.1, 127.3, 127.0, 126.9, 126.6, 121.0, 120.7, 119.6,
119.2, 62.8, 61.5, 56.3, 55.7, 36.3, 35.4, 34.5, 17.4, 16.9, 9.3, 9.0, 7.8, 7.5.
ESI-MS (m/z): 471.1 [M + H]+.
2-(Cyclopropyl(3-(trifluoromethyl)benzyl)amino)-N-(2-ethyl-
phenyl)-N-methyl-2-(pyridin-3-yl)acetamide (28). The target com-
pound was obtained in 53% yield as a colorless liquid. 1H NMR (600
MHz, CDCl3, 1:1 mixture of rotamers): δ 8.48 (d, J = 4.8 Hz, 2H,
mixture of rotamers), 8.24 (s, 1H, one rotamer), 8.19 (s, 1H, one
rotamer), 7.73 (s, 1H, one rotamer), 7.63 (d, J = 8.2 Hz, 1H, one
rotamer), 7.48−7.27 (m, 7H, mixture of rotamers), 7.27−7.19 (m, 6H,
mixture of rotamers), 7.16−7.03 (m, 2H, mixture of rotamers), 6.94−
6.81 (m, 2H, mixture of rotamers), 6.28 (d, J = 7.8 Hz, 1H, one
rotamer), 4.54 (s, 1H, one rotamer), 4.40 (s, 1H, one rotamer), 4.34 (d,
J = 14.8 Hz, 1H, one rotamer), 4.25 (d, J = 14.3 Hz, 1H, one rotamer),
3.95 (s, 1H, one rotamer), 3.93 (s, 1H, one rotamer), 3.25 (s, 3H, one
rotamer), 3.22 (s, 3H, one rotamer), 2.69−2.61 (m, 1H, one rotamer),
2.57−2.50 (m, 1H, one rotamer), 2.34−2.24 (m, 2H, mixture of
rotamers), 2.05−1.95 (m, 1H, one rotamer), 1.25−1.21 (m, 4H), 0.82
(t, J = 7.6, 1.5 Hz, 3H, one rotamer), 0.38−0.25 (m, 2H, one rotamer),
0.24−0.21 (m, 2H, one rotamer), 0.01 (bs, 2H, mixture of rotamers),
−0.189 to −0.24 (m, 2H, mixture of rotamers). 13C NMR (151 MHz,
CDCl3): δ 150.1, 150.01, 148.2, 141.4, 140.8, 140.5, 138.5, 138.1,
132.1, 131.7, 129.5, 129.3, 129.1 (d, J = 22.3 Hz), 128.5 (d), 128.3,
128.0, 127.1, 126.7, 125.4, 125.2, 123.4 (d, J = 28.8 Hz), 64.9, 63.3,
CDCl3, 1:1 mixture of rotamers): δ 8.50−8.45 (m, 2H, mixture of
rotamers), 8.27 (d, J = 2.3 Hz, 1H, one rotamer), 8.17 (d, J = 2.4 Hz,
1H, one rotamer), 7.64 (dt, J = 7.9, 2.0 Hz, 1H, one rotamer), 7.57 (dt, J
= 7.9, 2.0 Hz, 1H, one rotamer), 7.50−7.30 (m, 4H, mixture of
rotamers), 7.25−7.22 (m, 4H, mixture of rotamers), 7.20−7.11 (m, 5H,
mixture of rotamers), 7.10−7.00 (m, 2H, mixture of rotamers), 6.93−
6.80 (m, 2H, mixture of rotamers), 6.29 (d, J = 7.8 Hz, 1H, one
rotamer), 4.55 (s, 1H, one rotamer), 4.39 (s, 1H, one rotamer), 4.27 (d,
J = 14.9 Hz, 1H, one rotamer), 4.22 (d, J = 14.5 Hz, 1H, one rotamer),
3.93 (d, J = 14.7 Hz, 1H, one rotamer), 3.91 (d, J = 14.7 Hz, 1H, one
rotamer), 3.24 (s, 3H, one rotamer), 3.20 (s, 3H, one rotamer), 2.36−
2.29 (m, 2H, mixture of rotamers), 2.26 (s, 3H, one rotamer), 1.52 (s,
3H, one rotamer), 0.34−0.25 (m, 2H, one rotamer), 0.24−0.16 (m, 2H,
one rotamer), 0.06 to −0.02 (m, 2H, mixture of rotamer), −0.14 to
−0.28 (m, 2H, mixture of rotamer). 13C NMR (151 MHz, CDCl3, 1:1
mixture of rotamers): δ 171.3, 150.4, 150.2, 148.4, 141.1, 138.3, 137.9,
136.5, 136.3, 135.7, 135.1, 134.8, 134.5, 131.7, 131.5, 131.2, 131.0,
130.8, 129.2, 129.0, 128.8, 128.7, 128.4, 128.1, 127.2, 126.9, 123.9,
123.7, 123.3, 123.2, 64.9, 63.4, 56.0, 55.5, 36.3 (d, J = 26.4 Hz), 35.6,
35.0, 17.5, 16.9, 8.8 (d, J = 55.0 Hz), 7.6, 7.2. ESI-MS (m/z): 486.1 [M
+ H]+.
2-(Cyclopropyl(3-((trifluoromethyl)thio)benzyl)amino)-N-meth-
yl-2-(pyrimidin-5-yl)-N-(o-tolyl)acetamide (25). The target com-
pound was obtained in 46% yield as a colorless liquid. 1H NMR (400
MHz, CDCl3, 1:1 mixture of rotamers): δ 9.11−9.08 (m, 2H, mixture of
rotamers), 8.50 (s, 2H, one rotamer), 8.48 (s, 2H, one rotamer), 7.51
(dt, J = 7.5, 1.8 Hz, 1H, one rotamer), 7.48−7.38 (m, 2H, mixture of
rotamers), 7.36−7.27 (m, 3H, mixture of rotamers), 7.25−7.14 (m, 5H,
mixture of rotamers), 7.09−7.00 (m, 2H, mixture of rotamers), 6.89
(td, J = 7.6, 1.7 Hz, 1H, one rotamer), 6.82 (dd, J = 7.8, 1.4 Hz, 1H, one
rotamer), 6.33 (dd, J = 7.9, 1.3 Hz, 1H, one rotamer), 4.49 (s, 1H, one
rotamer), 4.38 (s, 1H, one rotamer), 4.29 (d, J = 14.8 Hz, 1H, one
rotamer), 4.22 (d, J = 14.3 Hz, 1H, one rotamer), 3.92 (d, J = 14.6 Hz,
1H, one rotamer), 3.90 (d, J = 14.6 Hz, 1H, one rotamer), 3.24 (s, 3H,
one rotamer), 3.21 (s, 3H, one rotamer), 2.39−2.28 (m, 2H, mixture of
rotamers), 2.22 (s, 3H, one rotamer), 1.59 (s, 3H, one rotamer), 0.37−
0.27 (m, 2H, one rotamer), 0.27−0.18 (m, 2H, one rotamer), 0.05 to
−0.03 (m, 2H, mixture of rotamers), 0.19 to −0.26 (m, 1H, one
rotamer), −0.32 to −0.39 (m, 1H, one rotamer). 13C NMR (101 MHz,
CDCl3, 1:1 mixture of rotamers): δ 170.3, 170.1, 158.2−157.8 (m),
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Article
56.1, 55.6, 37.1, 37.0, 35.7, 35.1, 29.8, 23.3, 22.8, 14.4, 14.1, 8.7, 8.5, 7.7,
7.5. ESI-MS (m/z): 468.1 [M + H]+.
rotamer), 6.86 (t, J = 7.6 Hz, 1H, one rotamer), 6.33 (d, J = 8.0 Hz, 1H),
4.56 (s, 1H, one rotamer), 4.45−4.34 (m, 3H, mixture of rotamers),
4.01 (d, J = 15.1 Hz, 1H, one rotamer), 3.92 (d, J = 14.7 Hz, 1H, one
rotamer), 3.24 (d, J = 1.2 Hz, 3H, one rotamer), 3.22 (d, J = 1.2 Hz, 3H,
one rotamer), 2.33−2.18 (m, 5H, mixture of rotamers), 1.59 (s, 3H, one
rotamer), 0.40−0.18 (m, 4H, mixture of rotamers), 0.10 to −0.06 (m,
2H, mixture of rotamers), 0.11 to −0.19 (m, 1H, one rotamer), −0.19
to −0.30 (m, 1H, one rotamer). 13C NMR (101 MHz, CDCl3, 1:1
mixture of rotamers): δ 171.3, 171.0, 151.2, 150.9, 149.3, 149.2, 141.4,
141.2, 137.5, 137.2, 135.8, 135.2, 132.3, 131.7, 131.5, 131.2, 128.8 (d, J
= 6.4 Hz), 128.5, 128.2, 127.3, 126.9, 125.7−125.3 (m), 123.0 (d, J =
21.9 Hz), 115.7, 115.5, 115.5, 115.3, 65.0, 63.8, 48.6 (d, J = 8.0 Hz),
48.2 (d J = 9.6 Hz), 36.3, 36.2, 35.8, 35.4, 17.5, 16.9, 8.3, 8.0, 7.8, 7.3.
ESI-MS (m/z): 472.1 [M + H]+.
N-Methyl-2-(propyl(1-(3-(trifluoromethyl)phenyl)cyclopropyl)-
amino)-2-(pyridin-3-yl)-N-(o-tolyl)acetamide (33). The target com-
pound was obtained in 42% yield as a colorless liquid. 1H NMR (400
MHz, CDCl3, 1:1 mixture of rotamers): δ 8.46−8.42 (m, 2H, mixture of
rotamers), 8.04 (bs, 1H, one rotamer), 8.01 (bs, 1H, one rotamer),
7.51−7.27 (m, 10H, mixture of rotamers), 7.20−6.90 (m, 8H, mixture
of rotamers), 6.75 (t, J = 7.6 Hz, 1H, one rotamer), 6.12 (d, J = 7.8 Hz,
1H, one rotamer), 4.57 (s, 1H, one rotamer), 4.29 (s, 1H, one rotamer),
3.16 (s, 3H, one rotamer), 3.12 (s, 3H, one rotamer), 3.01−2.88 (m,
2H, one rotamer), 2.85−2.68 (m, 2H, one rotamer), 2.22 (s, 3H, one
rotamer), 1.46−1.34 (m, 4H), 1.41−1.12 (m, 7H, mixture of rotamers),
1.11−0.99 (m, 2H, one rotamer), 0.89−0.81 (m, 2H, one rotamer),
0.65−0.56 (m, 6H, mixture of rotamers). 13C NMR (101 MHz, CDCl3,
1:1 mixture of rotamers): δ 173.1, 172.7, 151.1, 150.9, 148.9, 148.8,
146.4, 146.0, 141.2, 140.9, 137.2, 136.9, 135.9, 134.8, 134.2, 133.1,
131.7, 131.4, 130.6 (d, J = 29.6 Hz), 128.83−128.54 (m), 128.1, 127.2,
126.6, 123.6 (m), 123.0−122.8 (m), 64.0, 63.0, 49.0, 45.6, 45.3, 36.5,
36.3, 31.1, 29.8, 23.7 (d, J = 21.2 Hz), 18.2, 17.6, 16.8, 15.4, 11.4 (d, J =
29.2 Hz). ESI-MS (m/z): 482.1 [M + H]+.
2-(Cyclopropyl(3-(trifluoromethyl)benzyl)amino)-N-(2-ethyl-
phenyl)-N-methyl-2-(pyrimidin-5-yl)acetamide (29). The target
1
compound was obtained in 42% yield as a colorless liquid. H NMR
(400 MHz, CDCl3, 1:1 mixture of rotamers): δ 9.08 (bs, 2H, mixture of
rotamers), 8.48 (s, 2H, one rotamer), 8.45 (s, 2H, one rotamer), 7.48
(d, J = 7.3 Hz, 1H, one rotamer), 7.43−7.25 (m, 10H mixture of
rotamers), 7.13 (d, J = 7.7 Hz, 1H, one rotamer), 7.05 (t, J = 7.6, 1H,
one rotamer), 6.88 (t, J = 7.6 Hz, 1H, one rotamer), 6.83 (d, J = 7.9 Hz,
1H, one rotamer), 6.32 (d, J = 7.8 Hz, 1H, one rotamer), 4.46 (s, 1H,
one rotamer), 4.40−4.32 (m, 2H, mixture of rotamers), 4.25 (d, J = 14.4
Hz, 1H, one rotamer), 3.94 (d, J = 14.6 Hz, 1H, one rotamer), 3.92 (d, J
= 14.6 Hz, 1H, one rotamer), 3.25 (s, 3H, one rotamer), 3.22 (s, 3H,
one rotamer), 2.68−2.57 (m, 1H, one rotamer), 2.52−2.43 (m, 1H, one
rotamer), 2.37−2.27 (m, 2H, mixture of rotamers), 2.09−1.99 (m, 1H,
one rotamer), 1.36−1.15 (m, 4H, one rotamer), 0.88 (t, J = 7.6 Hz, 3H,
one rotamer), 0.36−0.20 (m, 4H, mixture of rotamers), 0.09 to −0.01
(m, 2H, mixture of rotamers), −0.20 to −0.27 (m, 1H, one rotamer),
−0.29 to −0.39 (m, 1H, one rotamer). 13C NMR (101 MHz, CDCl3,
1:1 mixture of rotamers): δ 170.4, 170.1, 158.1, 158.0 (d, J = 18.0 Hz),
157.6, 141.7, 141.1 (d, J = 6.8 Hz), 140.9, 140.3, 132.0, 131.7, 130.5,
129.7, 129.5 (d, J = 18.0 Hz), 128.7, 128.5, 128.4, 128.0, 127.20, 126.9,
125.3 (q, J = 14.8 Hz), 125.1 (q, J = 14.8 Hz), 123.8 (q, J = 16.0 Hz),
123.5 (q, J = 17.6 Hz), 62.9, 61.5, 56.3, 55.8, 37.2, 37.1, 35.4, 34.7, 23.3,
22.9, 14.4, 14.3, 9.03, 8.7, 7.9, 7.7. ESI-MS (m/z): 469.1 [M + H]+.
2-(Cyclopropyl(3-(trifluoromethyl)benzyl)amino)-N-methyl-N-
phenyl-2-(pyrimidin-5-yl)acetamide (30). The target compound was
1
obtained in 45% yield as a colorless liquid. H NMR (500 MHz,
CDCl3): δ 9.10 (s, 1H), 8.50 (s, 2H), 7.46 (d, J = 7.5 Hz, 1H), 7.38 (bs,
1H), 7.36−7.29 (m, 2H), 7.26−7.23 (m, 1H), 7.19 (t, J = 8.1 Hz, 2H),
6.72 (s, 2H), 4.53 (s, 1H), 4.16 (d, J = 14.4 Hz, 1H), 3.97 (d, J = 14.5
Hz, 1H), 3.30 (s, 3H), 2.45−2.40 (m, 1H), 0.37−0.31 (m, 1H), 0.28−
0.22 (m, 1H), 0.06−0.0 (m, 1H), −0.24 to −0.30 (m, 1H). 13C NMR
(101 MHz, CDCl3): δ 169.8, 158.1, 157.7, 142.5, 141.1, 132.0, 130.6,
129.8, 128.7, 128.6, 127.2, 125.3 (q, J = 16.8 Hz), 123.8 (q, J = 16.4 Hz),
61.7, 56.5, 37.6, 34.6, 9.6, 7.2. ESI-MS (m/z): 441.1 [M + H]+.
2-(Cyclopropyl(3-(trifluoromethyl)benzyl)amino)-N-methyl-2-(2-
methyl-pyrimidin-5-yl)-N-(o-tolyl)acetamide (31). The target com-
pound was obtained in 41% yield as a colorless liquid. 1H NMR (400
MHz, CDCl3, 1:1 mixture of rotamers): δ 8.37 (s, 2H, one rotamer),
8.36 (s, 2H, one rotamer), 7.48−7.44 (m, 1H, one rotamer), 7.41−7.31
(m, 4H, mixture of rotamers), 7.31−7.15 (m, 6H, mixture of rotamers),
7.11−6.99 (m, 2H, mixture of rotamers), 6.92 (td, J = 7.7, 1.4 Hz, 1H,
one rotamer), 6.84 (d, J = 8.0 Hz, 1H, one rotamer), 6.37 (d, J = 7.8 Hz,
1H, one rotamer), 4.44 (s, 1H, one rotamer), 4.35 (s, 1H, one rotamer),
4.29 (d, J = 14.8 Hz, 1H, one rotamer), 4.23 (d, J = 14.5 Hz, 1H, one
rotamer), 3.96 (d, J = 14.6 Hz, 1H, one rotamer), 3.93 (d, J = 14.6 Hz,
1H, one rotamer), 3.23 (s, 3H, one rotamer), 3.20 (s, 3H, one rotamer),
2.70 (d, 6H, mixture of rotamers), 2.37−2.24 (m, 5H, mixture of
rotamers), 1.60 (s, 3H, one rotamer), 0.40−0.19 (m, 4H, mixture of
rotamers), 0.09 to −0.04 (m, 2H, mixture of rotamers), −0.14 to −0.21
(m, 1H, one rotamer), −0.24 to −0.31 (m, 1H, one rotamer). 13C NMR
(101 MHz, CDCl3, 1:1 mixture of rotamers): δ 170.6, 170.3, 167.6 (d, J
= 14.8 Hz), 158.1, 157.8, 141.9, 141.3, 141.0 (d, J = 14.4 Hz), 135.4,
135.2, 132.0, 131.7 (d, J = 27.2 Hz), 129.0, 128.9, 128.7, 128.4 (d, J =
28.4 Hz), 128.1, 127.3, 127.1 (d, J = 19.2 Hz), 126.1, 125.3 (q, J = 13.6
Hz), 125.1 (q, J = 15.2 Hz), 123.7 (q, J = 15.2 Hz), 123.4 (d, J = 16.4
Hz), 62.7, 61.5, 56.2, 55.7, 36.3 (d, J = 13.2 Hz), 35.5, 34.8, 25.8, 17.4,
17.0, 8.9, 8.7, 7.9, 7.6. ESI-MS (m/z): 469.1 [M + H]+.
2-(Cyclopropyl(3-(trifluoromethyl)benzyl)amino)-1-(piperidin-1-
yl)-2-(pyridin-3-yl)ethan-1-one (34). To a solution of N-(3-
(trifluoromethyl)benzyl)cyclopropanamine (200 mg, 0.92 mmol) in
trifluoroethanol (2 mL) was added nicotinaldehyde (90 μL, 0.92
mmol), and the solution was stirred for 30 min at 60 °C. The solution
was cooled to RT, and TMSCN (0.11 mL, 0.92 mmol) was added. The
mixture was stirred at 60 °C for 30 min. The solvent was evaporated
under reduced pressure, and the residue was purified by silica gel
column chromatography using EtOAc/hexane as the eluent to furnish
2-(cyclopropyl(3-(trifluoromethyl)benzyl)amino)-2-(pyridin-3-yl)-
acetonitrile as a colorless liquid in 82% yield.
To a solution of the abovementioned 2-(cyclopropyl(3-
(trifluoromethyl)benzyl)amino)-2-(pyridin-3-yl)acetonitrile (100 mg,
0.3 mmol) in EtOH/H2O (3:3) was added NaOH (600 mg, 50 mmol),
and the mixture was stirred at 95 °C for 6 h. Progress of the reaction was
monitored by HPLC/MS. After completion of the reaction, 6 N HCl
was added to pH 7, and the aqueous layer was extracted with EtOAc,
dried over anhydrous Na2SO4, and concentrated under reduced
pressure. The compound was used for the next reaction without any
further purification.
To a solution of the abovementioned acid derivative (40 mg, 0.11
mmol) in dry dimethylformamide (2 mL) at 0 °C were added
piperidine (12 mg, 13 μL, 0.13 mmol), HATU (52 mg, 0.13 mmol), and
i-Pr2NEt (39 μL, 0.22 mmol). The reaction mixture was stirred for 15
min at RT, then diluted with ethyl acetate, and washed with water and
brine. The organic layer was dried over anhydrous Na2SO4, and the
solvent was evaporated under vacuum. The residue obtained was
purified by flash column chromatography (MeOH/DCM 1:20) to
furnish the desired product in 73% yield as a colorless thick liquid.
1H NMR (600 MHz, MeOD): δ 8.53 (d, J = 2.2 Hz, 1H), 8.46 (dd, J
= 4.9, 1.6 Hz, 1H), 7.86 (dt, J = 7.9, 1.9 Hz, 1H), 7.48 (m, 1H), 7.45−
2-(Cyclopropyl(2-Fluoro-5-(trifluoromethyl)benzyl)amino)-N-
methyl-2-(pyridin-3-yl)-N-(o-tolyl)acetamide (32). The target com-
pound was obtained in 46% yield as a colorless liquid. 1H NMR (400
MHz, CDCl3, 1:1 mixture of rotamers): δ 8.48 (m, 2H, mixture of
rotamers), 8.32−8.26 (bs, 1H, one rotamer), 8.26−8.21 (bs, 1H, one
rotamer), 7.72−7.68 (m, 1H, one rotamer), 7.61−7.57 (m, 1H, one
rotamer), 7.45−7.38 (m, 2H, mixture of rotamers), 7.36−7.31 (m, 2H,
mixture of rotamers), 7.28−7.26 (m, 1H, one rotamer), 7.24−7.12 (m,
5H, mixture of rotamers), 7.10−7.05 (m, 2H, mixture of rotamers),
7.01 (t, J = 8.8 Hz, 1H, one rotamer), 6.93 (t, J = 8.8 Hz, 1H, one
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7.39 (m, 4H), 5.07 (s, 1H), 4.13 (d, J = 14.5 Hz, 1H), 3.97 (d, J = 14.5
Hz, 1H), 3.69−3.63 (m, 1H), 3.59−3.54 (m, 1H), 3.39−3.32 (m, 1H),
3.24 (m, 1H), 2.37 (m, 1H), 1.70−1.53 (m, 4H), 1.39 (m, 1H), 1.20−
1.11 (m, 1H), 0.44−0.37 (m, 1H), 0.36−0.27 (m, 2H), 0.06 to −0.04
(m, 1H). 13C NMR (151 MHz, MeOD): δ 170.6, 151.5, 149.5, 143.1,
139.7, 134.5, 133.5, 131.2 (q, J = 128 Hz), 129.8, 126.3 (q, J = 15.6 Hz),
125.0, 124.5 (d, J = 13.2 Hz), 65.1, 57.3, 47.8, 44.1, 35.9, 27.2, 26.8,
25.2, 9.2, 7.6. ESI-MS (m/z): 418.1 [M + H]+.
Hanspeter Niederstrasser − Department of Biochemistry,
University of Texas Southwestern Medical Center, Dallas,
Texas 75390, United States
Anwu Zhou − Department of Biochemistry, University of Texas
Southwestern Medical Center, Dallas, Texas 75390, United
States
Bruce Posner − Department of Biochemistry, University of
Texas Southwestern Medical Center, Dallas, Texas 75390,
United States
Sergio Wittlin − Swiss Tropical and Public Health Institute,
4002 Basel, Switzerland; University of Basel, 4002 Basel,
Switzerland
Francisco Javier Gamo − Medicines Development Campus,
GlaxoSmithKline, 28760 Madrid, Spain; orcid.org/0000-
0002-1854-2882
Benigno Crespo − Medicines Development Campus,
GlaxoSmithKline, 28760 Madrid, Spain
Alisje Churchyard − Department of Life Sciences, Imperial
College London, SW7 2AZ South Kensington, U.K.
Jake Baum − Department of Life Sciences, Imperial College
London, SW7 2AZ South Kensington, U.K.
Nimisha Mittal − Division of Host-Microbe Systems and
Therapeutics, Department of Pediatrics, University of
California San Diego, La Jolla, California 92093, United
States
Elizabeth Winzeler − Division of Host-Microbe Systems and
Therapeutics, Department of Pediatrics, University of
California San Diego, La Jolla, California 92093, United
States; orcid.org/0000-0002-4049-2113
Benoît Laleu − Medicines for Malaria Venture, 1215 Geneva,
Switzerland; orcid.org/0000-0002-7530-2113
Michael J. Palmer − Medicines for Malaria Venture, 1215
Geneva, Switzerland
Susan A. Charman − Centre for Drug Candidate Optimisation,
Monash Institute of Pharmaceutical Sciences, Monash
University, Parkville, Victoria 3052, Australia
David A. Fidock − Department of Microbiology and
Immunology and Division of Infectious Diseases, Department
of Medicine, Columbia University Irving Medical Center, New
York 10032, United States
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02022.
EC50 values against NF54-luc P. falciparum parasites;
EC50 values associated with cellular heme fractionation;
free heme and Hz levels in drug-treated NF54 parasites;
whole-genome sequence metrics from Illumina MiSeq
(300 bp paired end reads) for the four 9-resistant 3D7
clones; CNVs identified in 9-resistant 3D7 clones; SNPs
observed in 9-resistant 3D7 resistant clones; relative rate
of kill versus control antimalarials; reduced efficacy of 3-
analogues containing 5-Pyr on Dd2 compared to 3D7;
snapshots from Integrated Genomics Viewer showing
CNVs; and NMR spectra for compounds 3−34 (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
■
Corresponding Authors
Joseph M. Ready − Department of Biochemistry, University of
Texas Southwestern Medical Center, Dallas, Texas 75390,
United States; orcid.org/0000-0003-1305-9581;
Email: joseph.ready@utsouthwestern.edu
Margaret A. Phillips − Department of Biochemistry, University
of Texas Southwestern Medical Center, Dallas, Texas 75390,
United States; orcid.org/0000-0001-5250-5578;
Email: margaret.phillips@utsouthwestern.edu
Authors
Aloysus Lawong − Department of Biochemistry, University of
Texas Southwestern Medical Center, Dallas, Texas 75390,
United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c02022
Suraksha Gahalawat − Department of Biochemistry, University
of Texas Southwestern Medical Center, Dallas, Texas 75390,
United States
John Okombo − Department of Microbiology and Immunology,
Columbia University Irving Medical Center, New York 10032,
United States
Josefine Striepen − Department of Microbiology and
Immunology, Columbia University Irving Medical Center, New
York 10032, United States
Tomas Yeo − Department of Microbiology and Immunology,
Columbia University Irving Medical Center, New York 10032,
United States
Sachel Mok − Department of Microbiology and Immunology,
Columbia University Irving Medical Center, New York 10032,
United States
Ioanna Deni − Department of Microbiology and Immunology,
Columbia University Irving Medical Center, New York 10032,
United States
Jessica L. Bridgford − Department of Microbiology and
Immunology, Columbia University Irving Medical Center, New
York 10032, United States
Author Contributions
A.L. designed, conducted, and analyzed data from the HTS and
performed parasite and cytotox assays on synthesized
compounds and kill rate studies; S.G. performed synthetic
chemistry; J.O. performed heme binding and heme/Hz
quantitation assays in drug-treated parasites; J.S., T.Y., S.M.,
and I.D. performed resistance selections, WGS, or data analysis;
H.N., A.Z., and B.P. contributed to the design and
implementation of the HTS; S.W., F.J.G., B.C., A.C., J.B.,
N.M., and E.W. performed parasite assays related to life-cycle
profiling and/or kill rate; B.L. and M.J.P. contributed to
chemical triage; S.A.C. supervised ADME studies; D.A.F.
supervised heme binding and heme/Hz quantitation assays
and resistance selections; J.M.R. supervised the medicinal
chemistry program; and M.A.P. supervised the HTS and various
biological studies. J.M.R. and M.A.P. were responsible for the
overall coordination of the project, and they wrote the paper
with help from D.A.F. All authors reviewed the manuscript and
verified the integrity of their data.
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(10) van der Pluijm, R. W.; Imwong, M.; Chau, N. H.; Hoa, N. T.;
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Funding
This work was funded in part by funds from the United States
National Institutes of Health grant, R01AI103947 (to M.A.P.),
NIGMS predoctoral training grant GM007062 (to A.L.), R37
AI50234 (to D.A.F.), and by Medicines for Malaria Venture
through their assay support network to D.A.F., J.B. (RD-08-
2800), and E.W. J.B. also holds an Investigator Award from the
Wellcome Trust (100993/Z/13/Z). B.P. acknowledges the
support of the HTS Core facility by U. T. Southwestern Medical
Center and the State of Texas. M.A.P. and J.M.R. acknowledge
the support of the Welch Foundation (I-1257, I-1612). M.A.P.
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Chair in Biochemistry. S.M. is grateful for the support of the
Human Frontier Science Program Long-term Postdoctoral
Fellowship LT000976/2016-L.
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
The authors would like to acknowledge the use of the PlasmoDB
Plasmodium informatics resource as part of the work of this
paper. We thank Christian Scheurer for technical assistance with
the [3H]-hypoxanthine incorporation assay.
ABBREVIATIONS
■
MOA, modes of action; ABS, asexual blood stage parasites;
ACTs, artemisinin-based combination therapy; CQ, chloro-
quine; ART, artemisinin; ATQ, atovaquone; MFQ, mefloquine;
LUM, lumefantrine; PPQ, piperaquine; WGS, whole genome
sequencing; SAR, structure−activity relationships; Hz, hemo-
zoin; βH, β-hematin; DV, digestive vacuole; BRRoK, bio-
luminescence relative rate of kill; DGFA, dual gamete formation
assay; Pf CRT, P. falciparum CQ resistance transporter; CNV,
copy number variations; SNPs, single nucleotide polymor-
phisms
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NOTE ADDED AFTER ASAP PUBLICATION
■
After this paper was published ASAP on February 23, 2021,
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