Identification, Characterization, and Optimization of 2,8-Disubstituted-1,5-naphthyridines as Novel Plasmodium falciparum Phosphatidylinositol-4-kinase Inhibitors with in Vivo Efficacy in a Humanized Mouse Model of Malaria

Article abstract of DOI:10.1021/acs.jmedchem.8b00648

A novel 2,8-disubstituted-1,5-naphthyridine hit compound stemming from the open access Medicines for Malaria Venture Pathogen Box formed a basis for a hit-to-lead medicinal chemistry program. Structure-activity relationship investigations resulted in compounds with potent antiplasmodial activity against both chloroquine sensitive (NF54) and multidrug resistant (K1) strains of the human malaria parasite Plasmodium falciparum. In the humanized P. falciparum mouse efficacy model, one of the frontrunner compounds showed in vivo efficacy at an oral dose of 4 × 50 mg·kg^-1. In vitro mode-of-action studies revealed Plasmodium falciparum phosphatidylinositol-4-kinase as the target.

Full text of DOI:10.1021/acs.jmedchem.8b00648

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Article
Identification, Characterization and Optimization of 2,8-Disubstituted-1,5-
naphthyridines as Novel Plasmodium falciparum Phosphatidylinositol-4-
kinase Inhibitors with in Vivo Efficacy in a Humanized Mouse Model of Malaria
Nishanth Kandepedu, Diego Gonzalez Cabrera, Srinivas Eedubilli, Dale Taylor, Christel
Brunschwig, Liezl Gibhard, Mathew Njoroge, Nina Lawrence, Tanya Paquet, Charles J
Eyermann, Thomas Spangenberg, Gregory S. Basarab, Leslie J. Street, and Kelly Chibale
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00648 • Publication Date (Web): 11 Jun 2018
Downloaded from http://pubs.acs.org on June 12, 2018
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Identification, Characterization and Optimization of
2,8ꢀDisubstitutedꢀ1,5ꢀnaphthyridines
as
Novel
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Plasmodium falciparum Phosphatidylinositolꢀ4ꢀkinase
Inhibitors with in Vivo Efficacy in a Humanized Mouse
Model of Malaria
†
†
†
∞
Nishanth Kandepedu , Diego Gonzàlez Cabrera , Srinivas Eedubilli , Dale Taylor , Christel
∞
∞
∞
∞
†
Brunschwig , Liezl Gibhard , Mathew Njoroge , Nina Lawrence , Tanya Paquet , Charles J.
†
¥
†
†
*,† ,#
Eyermann , Thomas Spangenberg , Gregory S. Basarab , Leslie J. Street and Kelly Chibale
†
Drug Discovery and Development Centre (H3D), University of Cape Town, Rondebosch 7701,
∞
South Africa; Drug Discovery and Development Centre (H3D), DMPK/Pharmacology, University of
¥
Cape Town, Observatory, 7925, South Africa; Merck Global Health Institute, Ares Trading S.A., a
#
subsidiary of Merck KGaA (Darmstadt, Germany), Coinsins 1267, Switzerland; South African
Medical Research Council, Drug Discovery and Development Research Unit, Department of
Chemistry and Institute of Infectious Disease and Molecular Medicine, University of Cape Town,
Rondebosch 7701, South Africa.
ABSTRACT
A novel 2,8ꢀdisubstitutedꢀ1,5ꢀnaphthyridine hit compound stemming from the open access
Medicines for Malaria Venture Pathogen Box, formed a basis for a hitꢀtoꢀlead medicinal
chemistry program. Structureꢀactivity relationship investigations resulted in compounds with
potent antiplasmodial activity against both chloroquine sensitive (NF54) and multiꢀdrug
resistant (K1) strains of the human malaria parasite Plasmodium falciparum. In the
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humanized P. falciparum mouse efficacy model, one of the frontrunner compounds showed
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in vivo efficacy at an oral dose of 4 × 50 mg.kg . In vitro modeꢀofꢀaction studies revealed
Plasmodium falciparum phosphatidylinositolꢀ4ꢀkinase as the target.
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INTRODUCTION
Malaria represents a major global health burden with an estimated 216 million new cases and
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nearly 445000 deaths in 2016, mostly affecting young children and pregnant women. It is a
vectorꢀborne infectious disease caused by the hematoprotozoan parasite of genus
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Plasmodium. According to the recent data from the World Health Organization (WHO),
Plasmodium falciparum was responsible for 99% of the malaria related morbidity and
mortality in subꢀSaharan Africa, whereas, Plasmodium vivax caused 36% of malarial
infections in the rest of the world.
Currently, the WHO recommended artemisininꢀbased combination therapy (ACT) and
1,3
vector control measures are key players in relieving the malarial burden. However, recent
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reports of emerging resistance towards ACTs, and the availability of a limited number of
validated drug targets exemplified by dihydrofolate reductase, cytochrome cꢀoxidoreductases
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and hemozoin formation, emphasises the need to expand chemical matter towards more
efficacious drugs with novel modes of action and multiꢀstage antiparasitic activity. Within
this context, Plasmodium kinases are attractive targets for new generation antimalarials as
both protein and lipid kinases are involved in key signalling pathways at various stages of the
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parasite lifecycle and have had some level of genetic or phenotypic validation. Even though,
to our knowledge, there has yet not been any inhibitor(s) of Plasmodium protein kinases
(PKs) in human clinical trials, substantial knowledge pertaining to the validity of PKs as drug
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targets has been obtained using genoproteomic approaches. On the other hand, lipid kinases
are important in all stages of the Plasmodium lifecycle; this includes phosphatidylinositolꢀ4ꢀ
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kinase (PI4K) that catalyses the conversion of phosphatidylinositol (PI) to
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phosphatidylinositolꢀ4ꢀphosphate (PI4P). Plasmodium PI4K is therefore important for
signal transduction and membrane trafficking and has been shown to be a validated drug
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target for prevention, treatment, and elimination of malaria. The 2ꢀaminopyridine
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MMV390048 was recently reported as a Plasmodium PI4K inhibitor and is currently in
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Phase IIa clinical trials, and several other published antimalarials including BQR695,
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KDU691, BRD73842 also target Plasmodium PI4K (Figure 1). The series presented herein
also inhibits PI4K, thus offering a new chemotype to interrogate this important new
antimalarial target. However, none of the known PI4K inhibitors have yet cleared the clinical
phase of development and are available to patients.
Medicines for Malaria Venture (MMV) has assembled a set of small molecules
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termed the Pathogen Box, containing 400 openꢀsource drugꢀlike compounds that are active
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against neglected diseases
from which the previously unexplored 1,5ꢀnaphthyridine
MMV024101 (8, Figure 2), active on P. falciparum with a described Pf3D7 IC of 600 nM
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was selected as a starting point for our medicinal chemistry campaign. When resynthesized
for validation, 8 showed submicromolar potency against P. falciparum NF54 (IC50 = 543
nM), low aqueous solubility (<5 µM) and rapid clearance by mouse liver microsomes with
only 2% of parent compound remaining after 30 minutes of incubation. In order to address
these shortcomings, we explored substitutions at both the 2ꢀ and 8ꢀpositions of the 1,5ꢀ
naphthyridine scaffold to identify a drugꢀlike candidate with an overall improved Absorption,
Distribution, Metabolism, and Excretion (ADME) profile. Herein, we report the synthesis, in
vitro antiplasmodial activity, and ADME profiles of a selection of 1,5ꢀnaphthyridines. A
representative compound 55, with low nanomolar in vitro antiplasmodial activity, good
ADME and oral pharmacokinetic (PK) properties, was tested for in vivo efficacy in the
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NODꢀscid IL2Rγ (NSG) murine malaria disease model of P. falciparum infection, in the
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recently established PfSCID platform at the University of Cape Town. In addition, three
compounds (21, 26, and 30) were used to determine the modeꢀofꢀaction of the 1,5ꢀ
naphthyridine series and were contextualized with other P. falciparum PI4K inhibitors.
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RESULTS AND DISCUSSION
Chemistry. Target compounds 8ꢀ55 were synthesized using a linear synthetic route starting
from commercially available 6ꢀmethoxypyridinꢀ3ꢀamine (Scheme 1). The key intermediate 4
17
was synthesized using a previously reported procedure. Briefly, the first step involves
condensation of 1 with Meldrum’s acid and trimethoxymethane. The domino sequence
includes an additionꢀelimination reaction to form the enamine intermediate 2 that, in the
subsequent step, undergoes thermal cyclization with decarboxylation to form 3. A facile
bromination of 3 at 0 °C resulted in 8ꢀbromoꢀ2ꢀmethoxyꢀ1,5ꢀnaphthyridine 4, a key
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intermediate that enabled investigation of the structural changes at R (position 8 of the 1,5ꢀ
naphthyridine ring). This intermediate was either subjected to a Suzuki cross coupling
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reaction or nucleophilic aromatic substitution using commercially available boronates or
amines, respectively, to give compound 5. The intermediate 5 was subjected to demethylation
to form 6, which upon a second bromination gave 7. Intermediate 7 was then subjected either
to a second Suzuki or an amination reaction using boronates or amines respectively, to furnish
target compounds 8ꢀ55 (Tables 1ꢀ4). Commercially available aryl boronates were used in
almost all the Suzuki reactions. When unavailable, boronates were synthesized using classical
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Miyaura borylation from the corresponding halogenated substrates.
In vitro antiplasmodial activity. All synthesized compounds were tested for in vitro growth
inhibition activity against a drug sensitive strain of P. falciparum, NF54, and selected
compounds were tested for activity against a multiꢀdrug resistant strain, K1, with chloroquine
and artesunate as positive controls. Compounds with good antiplasmodial activity (IC ’s
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<200 nM) were also evaluated for in vitro mammalian cytotoxicity in the Chinese Hamster
Ovary (CHO) cell line, using emetine as a positive control. The preliminary structureꢀactivity
relationship (SAR) investigation was focused on identifying substituents at the 2ꢀ and 8ꢀ
positions of the 1,5ꢀnaphthyridine ring that gave potent antiplasmodial activity (Table 1). At
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R , the general SAR trend showed that pyridine analogues (15ꢀ19) displayed improved
activity compared to other heterocyclic substituents like pyrazole (8, 9), phenyl (10ꢀ14) and
piperazine (20ꢀ22). Both 3ꢀ and 4ꢀlinked pyridines (16, 17) were equally potent and small
substitutions on the pyridine rings (18, 19) were tolerated.
For optimization of antiplasmodial activity, by making changes at the 2ꢀposition of
the 1,5ꢀnaphthyridine ring, the analogue with the 4ꢀpyridyl group at position 8 of the scaffold
was synthesized first and maintained for matched pair analyses (Table 2). This work was
facilitated by the synthetic accessibility of multiꢀgram quantities of the required pyridine
precursor, intermediate 5. As can be seen in Tables 1 and 2, replacing the pyridine
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sulfonamide at the 2ꢀposition of the naphthyridine ring (R ), e.g. compound 8 (NF54 IC =
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543 nM) and compound 15 (NF54 IC50 = 377 nM), with phenyl sulfonamide in the metaꢀ
position as in compound 9 (NF54 IC50 = 277 nM) and 16 (NF54 IC50 = 122 nM), improved
the NF54 activity 2ꢀ to 3ꢀfold. Orthoꢀ and paraꢀ phenyl sulfonamides diminished NF54
activity for compounds 23, 24 and 31 (IC50’s >5000 nM). The carboxamide, as in compound
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7, was not as wellꢀtolerated (NF54 IC = 745 nM) compared to the sulfonamide matched
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pair, compound 16. Overall, aryl carboxamides 37ꢀ41 and amines 27, 43ꢀ45 at R displayed
diminished antiplasmodial activity.
As shown in Table 1 and 2, crossꢀresistance against K1 was not observed for the
compounds tested. Compounds 16, 26, 28, 29, 30, 35 were also assayed for cytotoxicity using
CHO cells. The CHO CC /NF54 IC selectivity indices (SI) were greater than 100ꢀfold,
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except in the case of compounds 16 and 29, where the SI values were 16ꢀfold and 85ꢀfold
respectively. In vitro cytotoxicity indications were thus compound specific and not an
intrinsic issue for the series.
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Some of the noteworthy analogues identified from the SAR investigations include
compounds 26 (NF54/K1 IC50 = 87/110 nM), 30 (NF54/K1 IC50 = 22/19 nM) and 35
(NF54/K1 IC50 = 31/65 nM). In addition, 30 showed good aqueous solubility at pH 6.5 (115
µM). However, incubation of the compound with human, mouse and rat liver microsomes
revealed moderate clearance (Table 2), which needed improving.
Compounds 21 and 26 were screened for offꢀtarget inhibitory activity against a panel
of 140 human protein kinases at a concentration of 10 µM. The 11% hit rate (<30% residual
activity at 10 µM), suggested that these analogues have low promiscuity for human kinases.
(Supporting Information Table S3) and supported continued progression of the series.
SAR exploration around compound 30. Phenyl sulfonamide analogues at the 2ꢀposition of
the 1,5ꢀnaphthyridine ring showed that antiplasmodial activity could be ameliorated with
larger substituents as in compounds 30 and 35. Further SAR studies were focused at the 2ꢀ
position towards improving drug metabolism and pharmacokinetic (DMPK) properties of 30
(Table 3). With the exception of the alphaꢀmethyl analogue 46, all the analogues showed
good potency against NF54 with IC50’s less than or equal to 200 nM. Compounds 48, 51, 52,
and 53 displayed higher potency (NF54 IC ’s ≤50 nM) and the analogues containing a
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piperazine ring (51), a morpholine ring (52) or a pyrrolidine ring (53) showed higher aqueous
solubility (>150 µM) with only 53 showing comparable metabolic stability to 30.
Metabolite identification studies on 30 (Supporting information Section 8) supported
the fact that the sulfonamide moiety was the major metabolic soft spot in mice and mouse
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liver microsomes, with Nꢀdealkylation producing the unsubstituted sulfonamide 16. To
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mitigate the risk of CYP inhibition due to the presence of the pyridine ring, and in order to
explore SAR at the 8ꢀposition of the naphthyridine ring, compounds 54 and 55, in which a
trifluoromethyl group was incorporated at the 2ꢀposition of the pyridine ring, were
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synthesized. Although compound 54 showed good potency (NF54/K1 IC = 20/46 nM) and
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aqueous solubility (145 µM), it displayed poor microsomal stability, suggesting that the
substituted sulfonamide group remained a metabolic liability. The unsubstituted sulfonamide
55, showed good antiplasmodial activity (NF54/K1 IC50 = 63/102 nM) and good microsomal
stability in human, mouse, and rat liver microsomes (Table 4). Metabolic soft spots were
blocked as confirmed by metabolite identification studies in mice, where only a minor
oxidation metabolite of 55 occurring on the naphthyridine core or, on the pyridine
substituent, was detected (Supporting Information Section 8).
The partition coefficient, log D7.4’s of selected compounds ranged from moderate to
high (1.8 to 4.2) (Supporting Information Table S1). During the course of the program,
several compounds in the series were evaluated for inhibition of CYP450’s and for activity
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against the hERG K channel. Compound 30 showed no inhibition of several CYP450’s at 20
µ
M (Table 5). Four compounds 19, 30, 35, and 55 were profiled for potential cardiotoxicity
hERG) liabilities (Table 6). With the exception of 35, all compounds were clean against
hERG (IC >10 µM), suggesting that a potential cardiotoxicity risk is not associated with the
(
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series. This is especially gratifying since the cell line used for the assay overexpresses the
hERG channel relative to cardiomyocytes. Based on the aggregate of antiplasmodial activity,
microsomal stability and in vitro safety, compounds 30 and 55 were chosen for further
profiling towards demonstrating in vivo proofꢀofꢀconcept (PoC).
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Gametocytocidal activity. The potential of the 1,5ꢀnaphthyridine class to behave as dualꢀ
active antimalarials was evaluated by testing 55 against gametocyte stages of the malaria
parasite. A single concentration of 1 µM of 55 displayed 42% inhibition of early stage and
51% inhibition of late stage gametocytes as demonstrated by decreased activity of
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Plasmodium lactate dehydrogenase. The gametocytocidal activity of compound 55 suggests
that the 1,5ꢀnaphthyridine class of compounds are more potent against asexual blood stage
parasites compared to sexual gametocyte stage parasites. This observation is consistent with
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what has been noted for other PI4K inhibitors.
Pharmacokinetic studies. When dosed intravenously, 30 was cleared quickly from blood
ꢀ1
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(90 mL.min .kg ). Tissue distribution was high (25.7 L.kg ) and halfꢀlife was moderate (3.3
h) (Table 7). A comparative oral pharmacokinetic study of 30 between mice preꢀtreated with
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ꢀaminobenzotriazole (ABT, a nonꢀselective CYP inhibitor), and untreated mice, was
carried out to ascertain whether or not the oral exposure of the compound could be improved
and metabolism of 30 into the less active metabolite 16 minimized. Without ABT, 30 was
absorbed quickly (Tmax of 0.5 h), but, the oral exposure was low with an oral bioavailability
of 8%. Exposure of the metabolite 16 was 10ꢀfold the exposure of the parent 30, but with a
short halfꢀlife (1.6 h). ABT did partially inhibit the biotransformation of 30 into 16, with a 2ꢀ
fold increase and 2ꢀfold decrease of the oral exposure of the parent and metabolite,
respectively. As a result, the oral bioavailability of 30 did not improve significantly (15%). In
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fact, ABT is known to inhibit CYP enzymatic activity but not completely. These results
suggested that metabolism was not the only limiting factor with respect to oral exposure of 30
and that other clearance mechanisms were involved. As 16 was about 6ꢀfold less active than
30, it was not expected to contribute significantly to the pharmacodynamic profile of 30, and
the latter was not progressed to efficacy studies.
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When dosed intravenously, 55 was cleared slowly from systemic circulation (11
ꢀ1
ꢀ1
ꢀ1
mL.min .kg ) and tissue distribution was moderate (7 L.kg ), resulting in a long halfꢀlife
(
8.5 h). When dosed orally, 55 was absorbed quickly (Tmax of 1 h) with a relatively good
bioavailability (39%) (Table 7) (Supporting Information Figures S1 & S2). Oral absorption
was likely limited by low aqueous solubility (5 µM) and moderate permeability (efflux ratio
of 2.9 in Cacoꢀ2 cells) (Supporting Information Table S2). A 10% free fraction was
determined for human plasma protein binding and was used as surrogate for mouse plasma
protein binding towards determining free drug levels in vivo. (Supporting Information
Figures S3). As compound 55 showed potent in vitro antiplasmodial activity and good
DMPK properties, it was progressed to efficacy studies.
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In vivo efficacy studies. Compound 55 was assessed for in vivo efficacy using the NSG
mouse model of P. falciparum infection (Pf3D7 IC50 = 142 nM). NSG mice are genetically
immunodeficient and thereby able to support engraftment by human red blood cells and
2
4
infection by the human specific P. falciparum parasite. A quadrupleꢀdose regimen of 50
ꢀ
1
mg.kg of 55 for four consecutive days showed 80% reduction in parasitemia compared to
untreated mice (Figure 3). The pharmacokinetics of 55 from the study showed good exposure
and a doseꢀdependent correlation with the reduction in parasitemia (Table 8 and Figure 4).
However, 55 had relatively slow killing kinetics (see below) and was cleared quickly. This
likely accounts for parasitemia remaining flat throughout the course of the experiment.
Mechanism of action studies. The original hit compound 8 was disclosed in an oncology
directed patent for compounds that act through inhibition of phosphatidylinositolꢀ3ꢀkinase
25
(
PI3K), and assessment against Plasmodium lipid kinases showed it to be an inhibitor of
PI4K from P. vivax with an IC50 of 1.9 M. To support the inhibition of PI4K as the modeꢀofꢀ
µ
action of the series, structurally diverse compounds, 21 and 26 were assessed against a panel
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of six mechanistically distinct laboratory generated drug resistant strains of P. falciparum:
10,26ꢀ29
PfeEF2, Pfdxr, Pfpi4k, Pfdhodh, Pfcarl, PfcytB.
Reduced susceptibility (2ꢀ and 3ꢀfold,
respectively) was only seen with the Pfpi4k mutant strain in line with the 5ꢀfold reduced
susceptibility of the strain for MMV390048 (Supporting Information Table S6) and in line
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with the PI4K modeꢀofꢀaction.
Plasmodium vivax PI4K enzyme assay. Compounds 21 and 26 showed potent inhibition (IC50
of 5 and 15 nM, respectively, Figure 5) of PvPI4K, the only Plasmodium PI4K that has been
9
,10
isolated for enzymology work.
The catalytic domain of PI4KIIIβ in P. falciparum and P.
vivax are well conserved and share 97% similarity; hence, the PvPI4k is thought to be an
adequate surrogate for expression of activity against P. falciparum. The clinical PfPI4K
inhibitor MMV390048 showed an IC50 of 3.4 nM in the PvPI4K assay and 28 nM against
10
PfNF54. This data supports inhibition of PfPI4K as the antimalarial modeꢀofꢀaction for the
series though there is not a direct correlation between inhibitory potencies and antiplasmodial
activity. Compound permeability through the host red blood cell and the parasite necessarily
30
plays a role for expression of wholeꢀcell activity.
Parasite reduction ratio assay. Clearing the parasite quickly from the host system is deemed
to be ideal to overcome mutations within the parasite and to mitigate resistance development.
The IC50 of 30 against Pf3D7, the strain used in the parasite reduction ratio (PRR) assay, was
46 nM correlating closely to the value of 22 nM against NF54. The PRR experiment was run
over 48 h showing a log PRR of 30 = 0.8 at a concentration of 10ꢀfold over the IC50
,
categorising the compound as also slow acting (Figure 6). PfPI4K inhibitors have been
previously reported to be moderately slow acting in the in vitro PRR assay and similar to
1
0
atovaquone. Hence the data is consistent in that the PI4K modeꢀofꢀaction is associated with
slower killing kinetics in the in vitro PRR assay.
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Stageꢀspecificity assay. The stageꢀspecificity assay was performed using synchronous
cultures of the drug sensitive NF54 strain to assess concentrationꢀdependant growth response
3
1
of rings and schizonts in the presence of compounds. Compound 30 acted mostly on the
later intraꢀerythrocytic schizonts stage of the parasite and showed little effect on the early
ring stage. This correlates with the slow acting profile in the PRR assay and is comparable to
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10
the profile of MMV390048 (Figure 7).
Computer Aided Drug Design (CADD) studies
Since compounds 21 and 26 showed potent PI4K inhibition, we investigated the putative
receptorꢀligand interactions in a homology model of PfPI4K. The crystal structure of any
plasmodium PI4K is unavailable at present, and so the Xꢀray crystal structure of human
32
PI4KIIIβ (PDBID:4D0L) was used as a template and the PfPI4K homology model was built
33
using Schrodinger’s PRIME protein modelling tool. Docking studies revealed that 26 sits
quite deep in the binding pocket (Figure 8) and delineated five interactions including four
hydrogen bond interactions and one hydrophobic πꢀπ stacking interaction. The naphthyridine
5ꢀposition nitrogen displayed a hydrogen bond with the backbone NꢀH of hinge residue
Val1357 (Figure 9). The pyridine nitrogen of 26 formed a hydrogen bond with catalytic
Lys1308. This interaction can account for the improvement in activity of 16 (NF54 IC50
=
122 nM) compared to the phenyl matched pair 10 (NF54 IC50 = 719 nM). The oxygen atoms
in the sulfonamide group plausibly interacted with Ser1362 and Lys825. The residues
highlighted in Figure 9, and others in the binding site, are strictly conserved in P. vivax, the
isozyme which was used for enzyme inhibition studies. To support this model, the activities
of 16 and 37 could be compared, where, replacement of sulfonamide in 16 with carboxamide
in 37 (NF54 IC50 = 745 nM) resulted in a decrease in antiplasmodial activity. A faceꢀtoꢀface
πꢀπ stacking of the aryl ring at the naphthyridine 2ꢀposition with Phe827 was also observed.
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As per the SAR interpretation, this interaction seems quite significant, as removal of the
phenyl ring from 26 (NF54 IC50 = 87 nM) to afford 27 (NF54 IC50 >5000 nM) resulted in
complete loss in activity. In summary, potential interactions envisaged using the PfPI4K
homology model appear to be consistent with the SAR Both the methylsulfonyl and pyridyl
groups of 26 extends towards solvent by this model suggesting further room for analogue
modification, which might address physicochemical properties, pharmacokinetics and target
potency.
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CONCLUSION
Starting from an open source hit compound, MMV024101 (compound 8) from the MMV
Pathogen Box, a series of 2,8ꢀdisubstitutedꢀ1,5ꢀnaphthyridine analogues was synthesized and
evaluated for in vitro antiplasmodial activity. From a total of 48 analogues made for SAR
investigation, 26 showed improved blood stage activity compared to the original hit. Several
analogues exhibited low nanomolar activity (NF54 IC50’s <100 nM) and were equipotent
against both NF54 and K1. Two compounds, 21 and 26, were crossꢀresistant with a Pfpi4k
laboratory mutant strain pointing to PI4K as the modeꢀofꢀaction. The compounds were
confirmed as potent inhibitors of PvPI4K using a biochemical assay. The realization that the
compounds operate on PI4K is significant in that the importance of the target is magnified by
MMV390048 now in Phase IIa clinical trials. Major routes of metabolism of the 2ꢀ and 8ꢀ
substituents of the naphthyridine ring were blocked en route to delivering the lead compound
55. Compound 55 showed improved bioavailability in the mouse, and was chosen for in vivo
efficacy studies based on the pharmacokinetics and antiplasmodial activity. In the PfSCID
ꢀ1
mouse model of infection, 55 resulted in 80% reduction in parasitemia at 4 × 50 mg.kg . At
this juncture it is noteworthy that this compound meets the MMV Late Lead criteria with
respect to CYP inhibition (IC >20 µM), oral bioavailability (>30%) and hERG (>10 µM)
5
0
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but does not meet the in vitro antiplasmodial potency (IC50 <10 nM) criteria. By contrast,
MMV390048 shows much higher efficacy with a similar reduction in parasitemia at 4 × 1
ꢀ1
mg.kg and achieved 100% reduction in parasitemia at higher doses. This is a consequence
of the much better pharmacokinetic profile and higher asexual blood stage activity of
1
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MMV390048. Future optimization studies will aim to further improve pharmacokinetics
and in vivo potency to enhance efficacy and to achieve the complete clearance of the malaria
parasite that is comparable to other PfPI4K inhibitors in development.
EXPERIMENTAL SECTION
All commercially available chemicals were purchased from either SigmaꢀAldrich or Combiꢀ
1
Blocks. Unless otherwise stated, all solvents used were anhydrous. H NMR spectra were
13
recorded on a Bruker Spectrometer at 300 MHz. C NMR were recorded either on a Bruker
1
13
1
spectrometer at 400 MHz ( H 400.2 MHz and C 100.6 MHz) or Brukerꢀ600 ( H 600.3
13
MHz, C 150.9 MHz). Analytical thinꢀlayer chromatography (TLC) was performed on
aluminumꢀbacked silicaꢀgel 60 F254 (70ꢀ230 mesh) plates. Column chromatography was
performed with Merck silicaꢀgel 60 (70ꢀ230 mesh). Chemical shifts (δ) are given in parts per
million (ppm) downfield from TMS as the internal standard. Coupling constants, J, are
recorded in Hertz (Hz). Standard acronyms representing multiplicity are used as follows: br s
=
broad singlet, s = singlet, d = doublet, t = triplet, m = multiplet. Purity was determined by
Agilent 1260 Infinity binary pump, Agilent 1260 Infinity diode array detector (DAD), Agilent
290 Infinity column compartment, Agilent 1260 Infinity standard autosampler, and Agilent
1
6120 quadrupole (single) mass spectrometer, equipped with APCI and ESI multimode
ionization source and all compounds tested for biological activity were confirmed to have
≥
95% purity. The HPLC method used is described in the Supporting Information.
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6
Synthesis of 5ꢀ(((6ꢀmethoxypyridinꢀ3ꢀyl)amino)methylene)ꢀ2,2ꢀdimethylꢀ1,3ꢀdioxaneꢀ
4,6ꢀdione (2). A mixture of 2,2ꢀdimethylꢀ1,3ꢀdioxaneꢀ4,6ꢀdione (29 g, 0.2 mol) and
trimethoxymethane (198 mL, 1.8 mol) in ethanol (60 mL) was heated to 105 °C for 2 hours.
6ꢀmethoxypyridinꢀ3ꢀamine 1 (25 g, 0.2 mol) was then added and the resulting solution stirred
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0
at 105 °C for an additional 12 hours. The reaction mixture was allowed to cool to room
temperature and diluted with hexane (200 mL). The precipitate was filtered and washed with
hexane to afford 5ꢀ(((6ꢀmethoxypyridinꢀ3ꢀyl)amino)methylene)ꢀ2,2ꢀdimethylꢀ1,3ꢀdioxaneꢀ
1
4,6ꢀdione in 80% yield as a pale yellow solid. H NMR (300 MHz, CDCl
3
) δ = 11.18 (d, 1H,
J = 13.8 Hz), 8.49 (d, 1H, J = 13.8 Hz), 8.13 (d, 1H, J = 2.8 Hz), 7.53 (dd, 1H, J = 8.9 Hz &
.9 Hz), 6.83 (d, 1H, J = 8.9 Hz), 1.76 (s, 6 H), 3.97 (s, 3 H); Anal. RPꢀHPLC t = molecular
2
R
ion peak not observed.
Synthesis of 6ꢀmethoxyꢀ1,5ꢀnaphthyridinꢀ4(1H)ꢀone (3). 5ꢀ(((6ꢀmethoxypyridinꢀ3ꢀ
yl)amino)methylene)ꢀ2,2ꢀdimethylꢀ1,3ꢀdioxaneꢀ4,6ꢀdione 2 (7.4 g, 26.6 mmol) was added
portion wise to a preheated solution of Dowtherm A at 220 °C. After bubbling stopped, the
mixture was allowed to cool to room temperature. The mixture was diluted with hexane, the
precipitate was isolated, washed with hexane and dried to afford 6ꢀmethoxyꢀ1,5ꢀ
1
naphthyridinꢀ4(1H)ꢀone 3, in 64% yield as a brown solid. H NMR (300 MHz, DMSOꢀd
6
) δ
=
11.82 (br s, 1H), 7.95 (d, J = 8.3 Hz, 2H), 7.16 (d, J = 9.0 Hz, 1H), 6.23 (s, 1H), 3.93 (s,
3
H); Anal. RPꢀHPLC t = 0.804 min (method 1, purity 99%); LCꢀMS APCI: m/z 177.1
R
+
+
2
[M+H] (anal. calcd for C H N O : m/z = 176.1).
9
8
2
Synthesis of 8ꢀbromoꢀ2ꢀmethoxyꢀ1,5ꢀnaphthyridine (4). Phosphorus tribromide (5 mL,
3.1 mmol) was added to a solution of 6ꢀmethoxyꢀ1,5ꢀnaphthyridinꢀ4(1ꢀH)ꢀone 3 (8.5 g, 48.2
5
mmol) in N,Nꢀdimethylformamide (10 mL) at 0 °C. The mixture was continued to stir at 0 °C
for 1 hour. The reaction mixture was diluted with deionized water (500 mL), and 6N NaOH
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(1.3 mL) was added. The light brown precipitate was filtered off and thoroughly washed with
deionized water. The resulting solid was taken up in ethyl acetate, and the solution was
2
filtered through a pad of SiO to afford 8ꢀbromoꢀ2ꢀmethoxyꢀ1,5ꢀnaphthyridine 4, in 87%
1
yield as a pale brown solid. H NMR (300 MHz, DMSOꢀd ) δ = 8.36 (d, 1H, J = 4.8 Hz),
6
1
1
1
1
1
1
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8.06 (d, 1H, J = 9.0 Hz), 7.83 (d, 1H, J = 4.8 Hz), 7.09 (d, 1H, J = 9.0 Hz), 3.83 (s, 3H);
+
Anal. RPꢀHPLC t
R
= 4.471 min (method 1, purity 98%); LCꢀMS APCI: m/z 239.0 [M+H]
+
(anal. calcd for C
9
H
7
BrN
2
O : m/z = 238.0).
General procedure 1 for synthesis of intermediate 6 (GP1).
Synthesis of 8ꢀ(pyridinꢀ4ꢀyl)ꢀ1,5ꢀnaphthyridinꢀ2ꢀol. A 25 mL round bottom flask was
loaded with 2ꢀmethoxyꢀ8ꢀ(pyridinꢀ4ꢀyl)ꢀ1,5ꢀnaphthyridine (0.91 g, 3.84 mmol) followed by
dropwise addition of HBr (4.17 mL, 77 mmol) at room temperature. The resulting reaction
mixture was heated at 85 °C for 12 hours. Excess of hydrogen bromide was evaporated from
the reaction mixture in vacuo. The resulting residue was dissolved in 20 mL of
dichloromethane/methanol 9:1 and loaded with Amberlyst 21. The resulting suspension was
left to stir for 1 hour and filtered. The filtrate was evaporated in vacuo to afford 8ꢀ(pyridinꢀ4ꢀ
1
yl)ꢀ1,5ꢀnaphthyridinꢀ2ꢀol in 94 % yield as white solid. H NMR (300 MHz, DMSOꢀd
6
) δ =
10.99 (br s, 1H), 8.79 – 8.72 (m, 2H), 8.58 (d, J = 4.7 Hz, 1H), 8.03 (d, J = 9.7 Hz, 1H), 7.65
–
7.57 (m, 2H), 7.47 (d, J = 4.7 Hz, 1H), 6.83 (d, J = 9.7 Hz, 1H); Anal. RPꢀHPLC t
R
= 1.475
+
+
min (method 1, purity 99%); LCꢀMS APCI: m/z 224.1 [M+H] (anal. calcd for C H N O :
13
9
3
m/z = 223.1).
General procedure 2 for synthesis of intermediate 7 (GP2).
Synthesis of 2ꢀbromoꢀ8ꢀ(pyridinꢀ4ꢀyl)ꢀ1,5ꢀnaphthyridine. To a dry 25 ml round bottom
flask, 8ꢀ(pyridinꢀ4ꢀyl)ꢀ1,5ꢀnaphthyridinꢀ2ꢀol (0.3 g, 1.34 mmol) and phosphorus oxybromide
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5
6
(1.16 g, 4.03 mmol) were added and stirred under inert atmosphere at 60 °C for 10 minutes.
When the resulting reaction mixture liquefied, the temperature was increased to 120 °C and
continued to stir for 2 hours. The resulting residue was dissolved in ethyl acetate (30 mL) and
washed with deionized water (3 x 5 mL), dried over MgSO and concentrated in vacuo to
4
0
1
2
3
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8
9
0
1
2
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1
give 2ꢀbromoꢀ8ꢀ(pyridinꢀ4ꢀyl)ꢀ1,5ꢀnaphthyridine in 80% yield as a light brown powder. H
4
NMR (300 MHz, MeODꢀd ) δ = 9.08 (d, 1H, J = 4.5 Hz), 8.74ꢀ8.67 (m, 2H), 8.35 (d, 1H, J =
8.8 Hz), 7.96ꢀ7.89 (m, 2H), 7.87ꢀ7.83 (m, 2H); Anal. RPꢀHPLC t
R
= 3.927 min (method 1,
+
+
3
purity 98%); LCꢀMS APCI: m/z 286.0 [M+H] (anal. calcd for C13
H
8
BrN
: m/z = 285.0).
General procedure 3 for nucleophilic substitution reaction (GP3).
Synthesis of Nꢀ(8ꢀ(pyridinꢀ4ꢀyl)ꢀ1,5ꢀnaphthyridinꢀ2ꢀyl)methanesulfonamide (27). To a
solution of 2ꢀbromoꢀ8ꢀ(pyridinꢀ4ꢀyl)ꢀ1,5ꢀnaphthyridine (0.05 g, 0.18 mmol) in N,Nꢀ
dimethylformamide (1.5 mL), methanesulfonamide (0.066 g, 0.699 mmol) and caesium
carbonate (0.17 g, 0.52 mmol) were added into a 5 mL sealed tube. The resulting reaction
mixture was heated at 110 °C and stirred for 12 hours. N,Nꢀdimethylformamide was
azeotropically evaporated from the reaction mixture in vacuo with the help of toluene. The
residue was subjected to column chromatography on silica gel using hexane/ethyl acetate 5:5
v/v ratio initially and slowly increased to ethyl acetate/methanol 9:1 v/v ratio to elute Nꢀ(8ꢀ
(pyridinꢀ4ꢀyl)ꢀ1,5ꢀnaphthyridinꢀ2ꢀyl)methanesulfonamide 27, in 30% yield as a yellow solid.
1
H NMR (300 MHz, MeODꢀd ) δ = 8.92 (d, J = 4.6 Hz, 1H), 8.81 – 8.64 (m, 2H), 8.39 (d, J =
4
9.1 Hz, 1H), 8.07 – 7.97 (m, 2H), 7.88 (d, J = 4.6 Hz, 1H), 7.35 (d, J = 9.1 Hz, 1H), 3.17 (s,
13
3H). C NMR (151 MHz, DMSOꢀd
6
) δ = 152.07, 149.83, 149.14, 144.06, 142.26, 141.88,
1
40.54, 139.23, 125.54, 124.81, 117.28, 41.81; Anal. RPꢀHPLC t
R
= 2.309 min (method 1,
+
+
purity 99%); LCꢀMS APCI: m/z = 301.1 [M+H] (anal. calcd for C14
H
12
N
4
O
2
S : m/z =
300.1).
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General procedure 4 for BuchwaldꢀHartwig crossꢀcoupling reaction (GP4).
Synthesis of Nꢀ(3ꢀ(methylsulfonyl)phenyl)ꢀ8ꢀ(pyridinꢀ4ꢀyl)ꢀ1,5ꢀnaphthyridinꢀ2ꢀamine
(28). Pd
2 3
(dba) (7.68 mg, 8.39 µmol) and dicyclohexyl(2',4',6'ꢀtriisopropylꢀ3,6ꢀdimethoxyꢀ
1
1
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9
0
[
1,1'ꢀbiphenyl]ꢀ2ꢀyl)phosphane (6.75 mg, 0.01 mmol) were dissolved in tertꢀbutanol (1 mL)
and toluene (1 mL) and stirred for 5 minutes. At which time 3ꢀ(methylsulfonyl)aniline (52.3
mg, 0.25 mmol) and caesium carbonate (96 mg, 0.29 mmol) were added. The reaction
mixture was then heated at 115 °C for 12 hours. After concentration, the residue was
subjected to column chromatography on silica gel using ethyl acetate to afford Nꢀ(3ꢀ
(methylsulfonyl)phenyl)ꢀ8ꢀ(pyridinꢀ4ꢀyl)ꢀ1,5ꢀnaphthyridinꢀ2ꢀamine 28, in 34% yield as pale
1
yellow solid. H NMR (300 MHz, DMSOꢀd ) δ = 10.11 (br s, 1H), 8.78ꢀ8.75 (m, 3H), 8.51ꢀ
6
8
1
1
.47 (m, 1H), 8.52 (d, 1H, J = 9.1 Hz), 7.84 (t, 1H, J = 1.9 Hz), 7.77ꢀ7.75 (m, 2H), 7.68 (d,
13
H, J = 4.5 Hz), 7.49ꢀ7.34 (m, 3H), 3.12 (s, 3H). C NMR (101 MHz, DMSOꢀd
6
) δ =
54.07, 149.88 (x 2), 147.20, 145.50, 142.24, 141.69, 141.61, 140.08, 139.16, 130.12,
125.25, 124.63, 123.36, 120.02, 118.47, 116.87, 44.08; Anal. RPꢀHPLC t = 0.312 min
R
+
+
(method 2, purity 98%); LCꢀMS APCI: m/z 377.1 [M+H] (anal. calcd for C H N O S : m/z
20
16
4
2
=
376.1).
General procedure 5 for microwave mediated Suzuki crossꢀcoupling reaction (GP5).
Synthesis
of
Nꢀ(2ꢀhydroxyethyl)ꢀ3ꢀ(8ꢀ(pyridinꢀ4ꢀyl)ꢀ1,5ꢀnaphthyridinꢀ2ꢀ
yl)benzenesulfonamide (30). To a solution of 2ꢀbromoꢀ8ꢀ(pyridinꢀ4ꢀyl)ꢀ1,5ꢀnaphthyridine
(0.200 g, 0.699 mmol) in Dioxane (5.5 mL) in 25mL microwave vial, (3ꢀ(Nꢀ(2ꢀ
hydroxyethyl)sulfamoyl)phenyl)boronic acid (0.206 g, 0.839 mmol), Pd
.070 mmol), tricyclohexylphosphane (0.047 g, 0.168 mmol) and Potassium phosphate
17
2 3
(dba) (0.064 g,
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6
2
tribasic (0.445 g, 2.097 mmol) were added and degassed using N for 5 minutes. Deionized
water (1.38 mL) was then added and the reaction mixture was stirred for an additional 5
minutes. The mixture was microwaved in dynamic mode at 125 °C, 250 watts, 17.5 bar for
15 minutes. 1,4ꢀDioxane was removed under reduced pressure and the residue purified by
0
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column chromatography, hexane/ethyl acetate 5:5 v/v ratio initially and slowly increased to
ethyl acetate/methanol 9.5:0.5 v/v ratio to elute Nꢀ(2ꢀhydroxyethyl)ꢀ3ꢀ(8ꢀ(pyridinꢀ4ꢀyl)ꢀ1,5ꢀ
1
naphthyridinꢀ2ꢀyl)benzenesulfonamide 30, in 37% yield as yellow amorphous solid. H
NMR (300 MHz, DMSOꢀd
6
) δ = 9.17 (d, 1H, J = 4.4 Hz), 8.89 (d, 2H, J = 5.3 Hz), 8.72ꢀ8.65
(m, 2H), 8.57 (d, 1H, J = 8.9 Hz), 8.51ꢀ8.44 (m, 1H), 8.13 (d, 2H, J = 5.3 Hz), 8.03 (d, 1H, J
=
=
4.5 Hz), 7.93 (ddd, 1H, J = 7.8 Hz, 1.9 Hz & 1.1 Hz), 7.79 (t, 1H, J = 7.7 Hz), 7.69 (t, 1H, J
13
5.9 Hz), 3.40 (t, 2H, J = 6.2 Hz), 2.86 (q, 2H, J = 6.1 Hz). C NMR (151 MHz, DMSOꢀd
6
)
δ = 155.40, 152.12, 149.88, 144.57, 144.05, 143.73, 142.13, 140.42, 139.39, 139.17, 131.34,
R
130.53, 128.18, 125.85, 125.80, 125.13, 122.59, 60.40, 45.66; Anal. RPꢀHPLC t = 3.416
+
min (method 1, purity 99%); LCꢀMS APCI: m/z = 407.1 [M+H] (anal. calcd for
+
C H N O S : m/z = 406.1).
21
18
4
3
General procedure 6 for Suzuki crossꢀcoupling reaction (GP6).
Synthesis
of
3ꢀ(8ꢀ(2ꢀ(trifluoromethyl)pyridinꢀ4ꢀyl)ꢀ1,5ꢀnaphthyridinꢀ2ꢀ
yl)benzenesulfonamide (55). To a solution of 2ꢀbromoꢀ8ꢀ(2ꢀ(trifluoromethyl)pyridinꢀ4ꢀyl)ꢀ
1
1
,5ꢀnaphthyridine (0.300 g, 0.847 mmol) in 1,4ꢀDioxane (12.5 mL), 3ꢀ(4,4,5,5ꢀtetramethylꢀ
,3,2ꢀdioxaborolanꢀ2ꢀyl)benzenesulfonamide (0.264 g, 0.932 mmol), PdCl (dppf) (0.062 g,
2
0.085 mmol) and caesium carbonate (0.828 g, 2.54 mmol) were added, followed by deionized
water (3.13 mL) in a 25 mL round bottom flask. The resulting reaction mixture was heated at
95 °C for 12 hours. 1,4ꢀDioxane was evaporated from the reaction mixture in vacuo. The
resulting residue was dissolved in ethyl acetate (50 mL) and washed with water (3 x 15 mL),
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dried over MgSO
4
and concentrated in vacuo to give a light brown solid. The residue was
subjected to column chromatography on silica gel using hexane/ethyl acetate 8:2 v/v ratio
initially and slowly increased to ethyl acetate/methanol 9:1 v/v ratio to elute 3ꢀ(8ꢀ(2ꢀ
(trifluoromethyl)pyridinꢀ4ꢀyl)ꢀ1,5ꢀnaphthyridinꢀ2ꢀyl)benzenesulfonamide 55, in 49% yield as
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
white amorphous solid. H NMR (300 MHz, MeODꢀd ) δ = 9.09 (d, 1H, J = 4.5 Hz), 8.94 (dt,
4
1H, J = 5.0 Hz & 0.7 Hz), 8.73 (td, 1H, J = 1.8 Hz & 0.5 Hz), 8.61 (d, 1H, J = 8.9 Hz), 8.46
(d, 1H, J = 8.9 Hz), 8.43ꢀ8.36 (m, 2H), 8.22 (dd, 1H, J = 5.1 Hz & 1.6 Hz), 8.04 (ddd, 1H, J
= 7.9 Hz, 1.9 Hz & 1.1 Hz, 1H), 7.99 (d, 1H, J = 4.5 Hz), 7.69 (td, 1H, J = 7.8 Hz & 0.5 Hz).
1
3
C NMR (101 MHz, DMSOꢀd ) δ = 155.78, 152.13, 150.50, 147.20, 146.22, 145.59, 143.74,
6
1
1
4
43.04, 140.18, 139.54, 138.89, 130.81, 130.23, 129.36, 127.57, 125.46, 124.94, 123.60,
22.71, 120.88; Anal. RPꢀHPLC t
R
= 2.370 min (method 1, purity 98%); LCꢀMS APCI: m/z
+
+
31.0 [M+H] (anal. calcd for C20
H
13
F
3
N
4
O
2
S : m/z = 430.1).
ASSOCIATED CONTENT
Supporting Information
Additional details of the characterization of selected compounds and the procedures used for
the in vitro and in vivo antimalarial studies as well as PK and metabolism studies
SMILES nomenclature, NF54 and K1 IC50 values, biochemical and biological data (CSV)
AUTHOR INFORMATION
Corresponding Author
*Phone: +27ꢀ21ꢀ6502553. Fax: +27ꢀ21ꢀ6505195. Eꢀmail: Kelly.Chibale@uct.ac.za
ORCID
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Kelly Chibale: 0000ꢀ0002ꢀ1327ꢀ4727
Notes
The authors declare no competing financial interest.
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ACKNOWLEGEMENTS
We thank the Merck Global Health Institute for financial support for this research. The
University of Cape Town, South African Medical Research Council, and South African
Research Chairs initiative of the Department of Science and Technology, administered
through the South African National Research Foundation, are gratefully acknowledged for
support (K.C.). We thank Paul. A. Willis and James Duffy of MMV for assistance with the
Pathogen Box. We thank Laura Maria Sanz Alonso of GlaxoSmithKline for support with the
PRR assay. We thank Sergio Wittlin and Christian Scheurer for support with the crossꢀ
resistance and stageꢀspecificity assays. We thank John E. Burke of University of Victoria for
the PvPI4K enzyme assay as well as Anjo Theron and Dalu Mancama of the Council for
Scientific and Industrial Research (South Africa) for gametocytocidal activity assays. We
extend our thanks to Virgil Verhoog and Sumaya Salie for assistance in performing the
antiplasmodial assays and Nesia Barnes, Warren Olifant and Duane Knowles for their
assistance with the ADME assays. TS thanks his colleagues from Merck KGaA Darmstadt
Germany for logistical support and for generating hERG, Cacoꢀ2, and LogD data. We also
thank the South African Western Province Blood Transfusion Services for donating human
erythrocytes used in the study.
ABBREVIATIONS USED
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WHO, world health organization; ACT, artemisininꢀbased combination therapy; PI4K,
phosphatidylinositolꢀ4ꢀkinase; PI, phosphatidylinositol; PI4P, phosphatidylinositolꢀ4ꢀ
phosphate; MMV, medicines for malaria venture; PK, pharmacokinetics; ADME, absorption,
distribution, metabolism, and excretion; SAR, structureꢀactivity relationships; DMPK; drug
metabolism and pharmacokinetics; ABT, 1ꢀaminobenzotriazole; PRR, parasite reduction
ratio; NMR, nuclear magnetic resonance; TMS, tetramethylsilane; TLC, thinꢀlayer
chromatography; HPLC, high pressure liquid chromatography; LCꢀMS, liquid
chromatographyꢀmass spectrometry
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ClinicalTrials.gov. (2018). MMV390048 POC in Patients With P. vivax and P.
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https://clinicaltrials.gov/ct2/show/NCT02880241 [Accessed 26 Mar. 2018].
Kato, N.; Comer, E.; SakataꢀKato, T.; Sharma, A.; Sharma, M.; Maetani, M.; Bastien,
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G. L.; Duffy, S.; Eckley, S.; Itoe, M. A.; Koolen, K. M. J.; Lewis, T. A.; Lui, P. S.;
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TABLE OF CONTENTS GRAPHIC
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F3C
N
NH2
O
N
NH
F
O
N
NH
N
O S O
OH
O
MMV390048
BRD73842
PvPI4K - 3.4 nM
PvPI4K - 21 nM
N
N
O
O
O
N
N
H
N
N
N
N
O
H
Cl
NH
O
KDU691
BQR695
PvPI4K - 1.5 nM
PvPI4K - 3.5 nM
Figure 1: Antiplasmodial agents targeting PfPI4ꢀkinases.
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Figure 2: Structure of MMV024101
a
Scheme 1: Synthesis of 2,8ꢀdisubstituted 1,5ꢀnaphthyridines
a
Reagents and conditions: (i) 2,2ꢀdimethylꢀ1,3ꢀdioxaneꢀ4,6ꢀdione, trimethoxymethane, ethanol, 105 ºC, 12 h,
1
80%; (ii) Dowtherm A, 220 ºC, 64%; (iii) PBr
3
, DMF, 0 ºC to rt, 87%; (iv) R ꢀB(OH)
2
, PdCl
2
(dppf), Cs
2
CO
3
,
1
dioxane, 95 ºC, 41%ꢀ91%; or R ꢀappropriate amine, Cs CO , DMF, 110 ºC, 12 h, 45%ꢀ71%; (v) HBr, 85 ºC,
2
3
2
8
3 2 2 2 3
9%ꢀ95%; (vi) POBr , 60 ºC to 120 ºC, 55%ꢀ91%; (vii) R ꢀB(OH) , PdCl (dppf), Cs CO , dioxane, 95 ºC, 8ꢀ
2
2
6
4%; or R ꢀB(OH) , Pd (dba) , PCy K PO , dioxane, 125 ºC, 17ꢀ23%; or R ꢀappropriate amine, Cs CO , DMF,
2
2
3
3,
3
4
2
3
2
110 ºC, 12 h, 30ꢀ41%; or R ꢀappropriate aromatic amine, Pd
2
(dba)
3
, BrettPhos, Cs
2
CO
3
, toluene, tertꢀbutanol,
115 ºC, 34%.
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