Discovery of Antimalarial Azetidine-2-carbonitriles That Inhibit P. falciparum Dihydroorotate Dehydrogenase

Article abstract of DOI:10.1021/acsmedchemlett.7b00030

Dihydroorotate dehydrogenase (DHODH) is an enzyme necessary for pyrimidine biosynthesis in protozoan parasites of the genus Plasmodium, the causative agents of malaria. We recently reported the identification of novel compounds derived from diversity-oriented synthesis with activity in multiple stages of the malaria parasite life cycle. Here, we report the optimization of a potent series of antimalarial inhibitors consisting of azetidine-2-carbonitriles, which we had previously shown to target P. falciparum DHODH in a biochemical assay. Optimized compound BRD9185 (27) has in vitro activity against multidrug-resistant blood-stage parasites (EC₅₀ = 0.016 μM) and is curative after just three doses in a P. berghei mouse model. BRD9185 has a long half-life (15 h) and low clearance in mice and represents a new structural class of DHODH inhibitors with potential as antimalarial drugs.

Full text of DOI:10.1021/acsmedchemlett.7b00030

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Letter
Discovery of antimalarial azetidine-2-carbonitriles that
inhibit P. falciparum dihydroorotate dehydrogenase
Micah Maetani, Nobutaka Kato, Valquiria A.P. Jabor, Felipe Antunes
Calil, Maria Cristina Nonato, Christina A. Scherer, and Stuart L. Schreiber
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00030 • Publication Date (Web): 27 Feb 2017
Downloaded from http://pubs.acs.org on February 27, 2017
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Discovery of antimalarial azetidine-2-carbonitriles that inhibit P. fal-
ciparum dihydroorotate dehydrogenase
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Micah Maetani,†,‡ Nobutaka Kato,‡ Valquiria A.P. Jabor,§ Felipe A. Calil,§ Maria Cristina Nonato,§
Christina A. Scherer,‡ Stuart L. Schreiber*,†,‡,║
†Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States
‡Broad Institute, Cambridge, Massachusetts 02142, United States
§School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo 14040ꢀ903, Brazil
║Howard Hughes Medical Institute, Cambridge, Massachusetts 02138, United States
KEYWORDS: BRD7539, BRD9185, DHODH, malaria, diversity-oriented synthesis, Plasmodium falciparum
ABSTRACT: Dihydroorotate dehydrogenase (DHODH) is an enzyme necessary for pyrimidine biosynthesis in protozoan parasites
of the genus Plasmodium, the causative agents of malaria. We recently reported the identification of novel compounds derived from
diversityꢀoriented synthesis with activity in multiple stages of the malaria parasite life cycle. Here, we report the optimization of a
potent series of antimalarial inhibitors consisting of azetidineꢀ2ꢀcarbonitriles, which we had previously shown to target P. falcipa-
rum DHODH in a biochemical assay. Optimized compound BRD9185 (27) has in vitro activity against multidrugꢀresistant bloodꢀ
stage parasites (EC50 = 0.016 µM) and is curative after just three doses in a P. berghei mouse model. BRD9185 has a long halfꢀlife
(15 h) and low clearance in mice and represents a new structural class of DHODH inhibitors with potential as antimalarial drugs.
Malaria is a global health concern with nearly 200 million
cases annually, many of which occur in subꢀSaharan Africa.
The disease is caused by parasitic protozoans of the genus
Plasmodium and transmitted by female Anopheles mosquitos.1
Malaria is treatable using chemotherapy, but reduced efficacy
of firstꢀline treatments artemisinin and its derivatives at the
CambodiaꢀThailand border underscores the need for new, safe,
and effective antimalarial therapies.2ꢀ5 Moreover, while most
current antimalarial drugs target asexual bloodꢀstage parasites,
nextꢀgeneration antimalarials should ideally also target the
liverꢀ and/or sexual bloodꢀstage parasites to impede parasite
replication and transmission from hostꢀtoꢀvector, respectively.6
New antimalarial candidates have entered clinical trials in this
regard, including one that targets dihydroorotate dehydrogenꢀ
ase (DHODH).
organisms use both de novo and salvage pathways to generate
pyrimidines, Plasmodium parasites lack the necessary genes
Scheme 1. Elaboration of the azetidineꢀ2ꢀcarbonitrile scafꢀ
folda
Chart 1. Structures of PfDHODH inhibitors in clinical or preꢀ
clinical development.
aReagents
and
conditions:
(a)
Pd(PPh3)4,
1,3ꢀ
°
dimethylbarbituric acid, 2:1 EtOH/DCM, 40 C, 16 h, 92%;
(b) propylisocyanate, DIPEA, DCM, 23 C, 1 h, 96%; (c)
trifluoroacetic acid, Et3SiH, DCM, 23 C, 1 h, 87%; (d) 1ꢀ
°
DHODH catalyzes the flavin mononucleotide (FMN)ꢀ
°
dependent oxidation of
fourth step in de novo pyrimidine biosynthesis. While most
Lꢀdihydroorotate to orotate as the
o
ethynylꢀ3ꢀfluorobenzene, XPhos PdꢀG3, Et3N, MeCN, 70 C,
6 h, 91%.
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Table 1. Activity of BRD7539 analogs at R1 position against Dd2 parasitesa
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7
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9
Cmpd
EC50 (ꢀM)
0.010
0.249
0.083
0.013
0.016
5.640
0.427
0.020
0.016
0.035
0.046
0.019
0.051
0.106
0.019
M/Hb
Cmpd
17
18
19
20
21
22
23
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25
26
27
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29
30
31
EC50 (ꢀM)
8.375
0.005
0.010
12.630
0.139
5.541
4.390
0.097
0.012
0.039
0.016
0.352
0.015
0.015
0.041
M/Hb
ꢁ
R1
R1
F
BRD7539
86/99
H
R1
Br
O
3
4
ꢁ
8/51
34/74
ꢁ
CN
F
N
HO
O
N
N
85/85
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HN
H
N
5
F
100/100
F
O
6
ꢁ
ꢁ
N
7
ꢁ
ꢁ
N
CN
O
S
Me
O
N
8
ꢁ
91/90
0/16
39/96
81/91
95/90
ꢁ
N
HO
O
CF3
Cl
HN
9
85/96
MeO
F3C
BRD7539
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16
45/94
F
CF3
F3C
56/87
F3C
F3C
F
0/14
CN
F3C
OMe
N
ꢁ
ꢁ
ꢁ
ꢁ
HO
O
MeO
HN
F
0/74
0/7
27 (BRD9185)
F
MeO
F
50/66
aEC50 values are the mean of at least two independent experiments. bMouse (M) and human (H) microsomal stability (% remaining after 1
hour). Data were obtained from a single experiment performed in duplicate and calculated from a 6ꢀpoint curve over 1 hour.
for the latter, making de novo pyrimidine synthesis an essenꢀ
tial pathway for the parasite.7 One compound in the antimalarꢀ
ial pipeline, DSM265, has progressed to phaseꢀII clinical trials
and has activity against both asexual bloodꢀstage and liverꢀ
stage parasites.8 DSM265 and secondary candidate DSM421
(Chart 1) comprise a class of selective and potent antimalarial
DHODH inhibitors.9ꢀ15 These triazolopyrimidines remain the
most wellꢀstudied and clinically relevant antimalarial
DHODHꢀ inhibitors to date, but 5ꢀbenzimidazolylꢀthiopheneꢀ
2ꢀcarboxamides16 and 7ꢀarylaminopyrazolo[1,5ꢀα]pyrimidines
have also been reported.17
couple–pair strategy.19 The success of this strategy in revealꢀ
ing novel therapeutic targets is illustrated by the discovery of
smallꢀmolecule antimalarial inhibitors of phenylalanylꢀtRNA
synthetase18, PI4K18, and cytochrome bc1 Qi,20ꢀ21 as well as
others with asꢀyet unidentified targets.22 While our initial efꢀ
forts focused on smallꢀmolecule inhibitors of phenylalanineꢀ
tRNA synthetase, we became intrigued in BRD7539, which
we had previously shown to target PfDHODH (IC50 = 0.033
µM) selectively over human (Hs) DHODH (IC50 > 50 µM).
BRD7539 was reported to have potent activity against both
multidrugꢀresistant asexual bloodꢀstage (P. falciparum, Dd2
strain, EC50 = 0.010 µM) and liverꢀstage (P. berghei, EC50
=
We recently identified numerous compounds with multiꢀ
stage activity by growthꢀinhibition phenotypic screening of
100,000 compounds prepared in advance using diversityꢀ
oriented synthesis (DOS).18 The DOS collection was syntheꢀ
sized using modern asymmetric organic chemistry to impart
threeꢀdimensional topographical features using the build–
0.015 µM) parasites but no activity against sexual bloodꢀstage
(P. falciparum, stages IVꢀV, EC50 > 20 µM) parasites.
BRD7539 is an azetidine carbonitrile with three contiguous
stereocenters (2S,3S,4S), and stereochemistryꢀbased structure–
activity relationships (SSAR) showed that only two of eight
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possible stereoisomers are active.18 The clinical relevance of
late mouse and human microsomal stability while maintaining
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PfDHODH inhibitors and the selectivity and potency of
BRD7539 arising directly from a highꢀthroughput screen enꢀ
couraged us to pursue this series further. Here, we report our
efforts to optimize this compound and to evaluate this series in
vivo.
activity. Improved stability should correlate with favorable
pharmacokinetic (PK) properties. Analogs bearing a 4ꢀpyridyl
(22) and 4ꢀmethanesulfonyl (23) distal aryl were synthesized
in an effort to improve solubility but were inactive in vitro.
CF3ꢀsubstitution (24-26) was found to impart greater in vitro
microsomal stability than methoxy substituents (28-30). Ultiꢀ
mately, we found that the addition of two –CF3 groups on the
distal phenyl ring (BRD9185, 27) to be comparable in activity
and microsomal stability to BRD7539.
To confirm the biological activity of BRD7539 and to exꢀ
plore structure–activity relationships (SAR) of the scaffold, we
resynthesized core structure 1 (Scheme 1) as reported.23 Dealꢀ
lylation of the protected azetidine core and sequential capping
of the nitrogen with propyl isocyanate gave urea 2. Trityl
deprotection followed by a Heck alkynylation or Suzuki reacꢀ
tion diversified the paraꢀBr position and served as a route to
most analogs. Biological activity of BRD7539 was confirmed
in 20ꢀpoint dose (N ≥ 2 independent experiments in triplicate)
against a multidrugꢀresistant strain (P. falciparum, Dd2 strain)
using a phenotypic bloodꢀstage growth inhibition assay that
models a human bloodꢀstage infection. Additionally, in vitro
stability against mice and human microsomes for 1 h was used
as a guide to identify analogs with potential in vivo stability.
9
We briefly sought to evaluate the role of the primary alcohol
(R2) and secondary nitrile (R3) on activity (Table 2). Analogs
were synthesized from BRD7539 in 1ꢀ3 steps or from comꢀ
mercially available starting materials (Scheme S2-S7). Modiꢀ
fying the nitrile to a methyl ester (32) or alcohol (33) abolꢀ
ished activity. This is unsurprising given our previous result
that the 2S,3S,4R diastereocenter – which only differs from
BRD7539 at the nitrileꢀbearing stereocenter – is inactive, hintꢀ
ing at the importance of this functional group. This also illusꢀ
trates the subtle but significant role stereochemistry can have
on small molecule–protein interactions and highlights the
strength of diversityꢀoriented synthesis in identifying these
key interactions. Any modification of the primary alcohol,
including methylation (34) or conversion to a primary amine
(36), resulted in large loss in activity.
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Our initial SAR focused primarily on the acetylene (R1) reꢀ
gion of BRD7539 as this was the most facile point of diversiꢀ
fication to explore and a possible toxicity concern (Table 1).24
Activity was assessed using the phenotypic bloodꢀstage
growth inhibition assay. The activity of BRD7539 was reconꢀ
firmed in dose, and in vitro activity was maintained with a
wide variety of hydrophobic acetylenes (4-6, 9-10). Heteroarꢀ
omatic 2ꢀ and 3ꢀpyridyl analogs (7-8) showed significant loss
in activity compared to aromatic analogs. Interestingly, cisꢀ
alkene (11) and alkane (12) derivatives of BRD7539 showed
only a slight loss in activity, suggesting that the acetylene was
not necessary. Indeed, unsubstituted biaryl 13 is essentially
equipotent to BRD7539 while removing the distal ring (17,
Scheme S1) abolished activity, indicating the need for a large
hydrophobic region in the scaffold. Having removed the acetyꢀ
lenic toxicity concern, we decided to use this region to moduꢀ
To confirm that our lead compound 27 inhibits PfDHODH
despite removal of the acetylene motif, we performed bioꢀ
chemical assays against both recombinant P. falciparum and
Table 3. Key properties of lead compound 27
a
in vitro enzyme inhibition, IC50
PfDHODH (µM)
HsDHODH (µM)
plasma protein bindingb
mouse (%)
0.012
>50
Table 2. Activity of BRD7539 analogs at R2 and R3 positions
99.3
against Dd2 parasitesa
human (%)
>99.0
mouse PKc
t1/2 (h)
15.2
4.9
C0 (µM)
Cmax (µM)
16.9
>54.3
0.37
0.40
94
DNAUC0ꢀinf (µM·h)
Vss (L/kg)
CL (mL/min/kg)
F (%)
b
aMean of a single experiment in triplicate. Single experiment,
calculated from a 6ꢀpoint curve over 1 hour. c t1/2, terminal halfꢀ
life; C0, initial serum concentration at t = 0; Cmax, peak serum
concentration; DNAUC0ꢀinf, doseꢀnormalized area under the
plasma concentration vs. time curve following PO dosing; Vss,
volume of distribution at steady state; CL, plasma clearance;
F%, bioavailability. IV dosing in 5% DMSO/10% cremoꢀ
phor/85% H2O at 0.25 mg/mL (1 mg/kg). PO dosing in 70%
PEG300/30% (5% dextrose in H2O) at 0.50 mg/mL (5 mg/kg).
n = 3 mice in both IV and PO groups.
aEC50 values are the mean of at least two independent experiꢀ
ments.
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human DHODH enzyme (Table 3, Figure S1). Similar to hit vivo P. berghei model after just three doses. Additional efforts
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compound BRD7539, 27 is a potent inhibitor of PfDHODH
(IC50 = 0.012 µM) but not HsDHODH (IC50 > 50 µM), sugꢀ
gesting that this class of DHODH inhibitors provides selectiviꢀ
ty between orthologs. The IC50 of selected analogs against
PfDHODH was also shown to track well with Dd2 EC50 (Table
S1).To assess the suitability of BRD9185 further for in vivo
use, we measured plasma protein binding and obtained mouse
PK data. Lead compound 27 is highly protein bound in both
mouse and human plasma (>99%) and is a highly bioavailable
(94%), long halfꢀlife (15 h) compound in mice (PO 5 mg/kg;
IV 1 mg/kg) with low clearance (0.40 mL/min/kg). Notably,
the DNAUC0ꢀinf of 27 is >54.3 µM, higher than the EC50 in
vitro.
assessing the inhibition of azetidineꢀ2ꢀcarbonitriles against
DHODH from other Plasmodium species and evaluating effiꢀ
cacy of this series in the humanized NSG mouse P. falciparum
model are underway.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: XXXXXXXXXXXXXX.
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Supplementary figures, experimental details, compound characterꢀ
ization, and abbreviation, (PDF)
Based on the promising PK, we were interested in evaluatꢀ
ing the efficacy of 27 in vivo (Figure 1). We used a bloodꢀ
stage model with the rodent malaria parasite P. berghei that
expresses luciferase and treated infected CDꢀ1 mice for three
days with 66.6 mg/kg of 27 or vehicle (70% PEG300, 30%
solution of 5% dextrose in H2O). Bioluminescence intensity
was used to measure parasite growth, and artesunate was used
as a positive control. No parasites were detected after 30 days
in mice treated with 27, suggesting that the analog achieved a
sterile cure for P. berghei. These results are particularly interꢀ
esting in light of the properties of DSM265 and the triazolopyꢀ
rimidine series, which are not effective in the P. berghei (Pb)
model due to poor binding to the PbDHODH enzyme.9,14 This
raises the possibility that 27 has a different mechanism of acꢀ
tion from DSM265 on PfDHODH. However, 27 binds comꢀ
petitively with decylubiquinone (Figure S2), the same proꢀ
posed binding site as DSM265.8 Xꢀray crystallography studies
are underway to gain insights into binding features of these
compounds.
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: stuart_schreiber@harvard.edu.
Funding Sources
This work was supported in part by the Bill & Melinda Gates
Foundation (grant OPP1032518 awarded to S.L.S.). S.L.S. is an
Investigator at the Howard Hughes Medical Institute. M.M. was
supported by a fellowship from the National Science Foundation
(DGE1144152) and from Harvard University’s Graduate Prize
Fellowship. Biochemical and mechanistic studies for DHODH
were supported by Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP), grant number 2012/25075ꢀ0 (M.C.N.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Leslie N. Aldrich, Jennifer A. Beaudoin, Christopher J.
Gerry, and Jingqiang Wei (Broad Institute) for advice regarding
compound synthesis; Eamon Comer and Marshall Morningstar
(Broad Institute) for helpful discussions; and Broad Institute
Compound Management and analytical teams for assistance with
compound access and characterization.
These data collectively show that azetidineꢀ2ꢀcarbonitriles
comprise a promising, potent, and selective new class of inhibꢀ
itors of PfDHODH. In contrast to other antimalarial DHODH
inhibitors to date, compound 27 exhibits a sterile cure in an in
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