Synthesis, design, and structure–activity relationship of the pyrimidone derivatives as novel selective inhibitors of plasmodium falciparum dihydroorotate dehydrogenase

Article abstract of DOI:10.3390/molecules23061254

The inhibition of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) potentially represents a new treatment option for malaria, as P. falciparum relies entirely on a de novo pyrimidine biosynthetic pathway for survival. Herein, we report a series of pyrimidone derivatives as novel inhibitors of PfDHODH. The most potent compound, 26, showed high inhibition activity against PfDHODH (IC₅₀ = 23 nM), with >400-fold species selectivity over human dihydroorotate dehydrogenase (hDHODH). The brand-new inhibitor scaffold targeting PfDHODH reported in this work may lead to the discovery of new antimalarial agents.

Full text of DOI:10.3390/molecules23061254

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molecules
Article
Synthesis, Design, and Structure–Activity
Relationship of the Pyrimidone Derivatives as Novel
Selective Inhibitors of Plasmodium falciparum
Dihydroorotate Dehydrogenase
Le Xu 1,†, Wenjie Li 1,†, Yanyan Diao 1,†, Hongxia Sun 1, Honglin Li 1, Lili Zhu 1,*,
Hongchang Zhou 2,* and Zhenjiang Zhao 1,
*
1
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and
Technology, Shanghai 200237, China; 13162212770@163.com (L.X.); wjli12345@126.com (W.L.);
diaoyan1025@126.com (Y.D.); shx7982@163.com (H.S.); hlli@mail.shcnc.ac.cn (H.L.)
Department of Microbiology, Medical School of Huzhou Teachers College, Huzhou 313000, China
Correspondence: zhulfl@ecust.edu.cn (L.Z.); zhouhc529@zjhu.edu.cn (H.Z.); zhjzhao@ecust.edu.cn (Z.Z.);
Tel.: +86-21-6425-3379 (L.Z.); +86-21-6425-3962 (Z.Z.); Fax.: +86-21-64250213 (L.Z. & Z.Z.)
These authors contributed equally to this work.
2
*
†
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 2 May 2018; Accepted: 22 May 2018; Published: 24 May 2018
Abstract: The inhibition of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH)
potentially represents a new treatment option for malaria, as P. falciparum relies entirely on a de novo
pyrimidine biosynthetic pathway for survival. Herein, we report a series of pyrimidone derivatives
as novel inhibitors of PfDHODH. The most potent compound, 26, showed high inhibition activity
against PfDHODH (IC50 = 23 nM), with >400-fold species selectivity over human dihydroorotate
dehydrogenase (hDHODH). The brand-new inhibitor scaffold targeting PfDHODH reported in this
work may lead to the discovery of new antimalarial agents.
Keywords: P. falciparum; PfDHODH; pyrimidone; antimalarial agents
1. Introduction
Malaria is a mosquito-transmitted disease caused by protozoan parasites of the Plasmodium
species [
episodes of malaria and 445,000 deaths from malaria globally in 2016 [
therapies (ACTs) are the current first-line treatment for the deadliest form of malaria. However,
the reduced efficacy of first-line treatments using artemisinin (compound ) [ ] and its
derivatives (associated with Kelch 13 propeller protein mutations 7-95430) is now prevalent at the
Cambodia–Thailand border and poses a serious threat to malaria control programs globally [ ].
Chloroquine (compound ) [ ] and pyrimethamine (compound ) [10], used as the mainstay of
antimalarial chemotherapy, have now compromised the development of resistance [11]. To tackle
the problem of drug resistance, various strategies have been developed to treat malaria [12 13]. For
1,
2]. The World Health Organization (WHO) estimated that there were nearly 216 million
3]. Artemisinin combination
1
4
5–8
2
9
3
,
instance, Gilbert’s group discovered that DDD107498 exhibits a novel spectrum of antimalarial activity
against multiple life-cycle stages of the Plasmodium parasite [14].
Dihydroorotate dehydrogenase (DHODH) is a rate-limiting enzyme that is required for the
fourth step of de novo pyrimidine biosynthesis, converting dihydroorotate (DHO) to orotate (ORO)
with the participation of the cofactors flavin mononucleotide (FMN) and ubiquinone (CoQ) [15–17].
Pyrimidine-based biosynthesis represents a basic biological and physiological process that is crucial for
RNA and DNA production and cell proliferation. The mammalian cells generate pyrimidines through
Molecules 2018, 23, 1254; doi:10.3390/molecules23061254
www.mdpi.com/journal/molecules

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Molecules 2018, 23, 1254
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both de novo and salvage pathways for survival, while plasmodium parasites lack the necessary genes
for the former, resulting in de novo pyrimidine synthesis as the vital pathway for the parasite [18].
Therefore, PfDHODH has been considered as the prospective target for the conquest of malaria [19–21].
DHODHs are divided into two families in the light of different amino acid sequences and coenzyme
dependence. Human dihydroorotate dehydrogenase (hDHODH) and PfDHODH belong to family II,
located on the inner mitochondrial membrane, which utilizes ubiquinone as the terminal oxidant [22].
Many hDHODH inhibitors have been corroborated to be effective against a number of diseases,
such as cancer, rheumatoid arthritis (RA), psoriasis, and lupus erythematosus [23
in identifying PfDHODH inhibitors on the basis of the modification of reported hDHODH inhibitors
(e.g., compound ) are well known [24]. Although sharing a highly conserved sequence in their
–25]. Some successes
4
C-terminal domains, PfDHODH and hDHODH possess huge variation in terms of the amino acid
sequence in the N-terminal domain, which facilitates further discovery of specific inhibitors of
PfDHODH [22]. In addition, many different kinds of selective and promising PfDHODH inhibitors
have been developed through target-based high-throughput screening (e.g., compounds
in Figure 1) [26 28]. What is more, X-ray structures of PfDHODH in complex with some representative
compounds were used to develop more potent, non-cross-resistant selective antimalarial agents,
leading to the identification of compound (DSM 256), which has progressed in phase II clinical
trials [29]. Furthermore, compound (DSM 421) is a preclinical development candidate for the
5–7) (as shown
–
8
9
treatment of malaria, which has improved solubility, lower intrinsic clearance, and increased plasma
exposure after oral dosing compared with DSM 265 [30].
In our previous work, we reported the identification of compound 10 as a PfDHODH
inhibitor by the structure-based virtual screening strategy integrating molecular Glide docking with
Prime/MM-GBSA re-scoring on the basis of the complex crystal structure of compound
7 (DSM 1)
with PfDHODH (PDB code 3I65) [31]. Compound 10 selectively inhibited PfDHODH (IC50 = 6 nM,
with >14,000-fold species selectivity over hDHODH) and parasite growth in vitro (IC50 of 15 and
18 nM against 3D7 and Dd2 cells, respectively), but it was less effective in vivo as a result of its
poor metabolic properties [31]. Recently, several pyrimidone derivatives based on compound 10 as a
lead compound were obtained in this work and showed high selectivity over hDHODH, suggesting
potential applications as novel antimalarial agents.
Figure 1. Structures of reported antimalarial agents
compound 10 in this study.
1–3, PfDHODH inhibitors 4–9, and the lead
2. Results and Discussion
2.1. Proposed Binding Pose of Lead Compound 10 in the Ubiquinone Binding Pocket of PfDHODH
To thoroughly explore the binding mode of compound 10 in the ubiquinone binding pocket of
PfDHODH, flexible induced-fit docking [32] was implemented in Maestro v10.1 on the basis of the

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crystal structure of PfDHODH in complex with DSM1 (PDB code 3I65). As shown in Figure 2A,
the entrance of the ubiquinone binding channel is relatively closed in PfDHODH; thus compound
10 is totally buried inside the protein. The naphthyl group sits in the region close to the entrance
and fits the pocket well through hydrophobic interactions with adjacent residues such as Met536,
Leu197, Leu240, Ile237, and Phe227. The ester group is located almost in the same plane of the
dihydrofuranone moiety, with its carbonyl group pointing in the same direction as that on the
dihydrofuranone ring. Both the carbonyl groups could form favorable hydrogen bond interactions
with Arg265. Additionally, the binding pocket near Arg265 has a strongly positive electrostatic surface
potential, which is complementary to the two carbonyl groups with negative electrostatic potential
(the minimum ESP values of the two oxygen atoms are both
−
58.53 kcal/mol). An additional hydrogen
bond is presumably formed between the bridging nitrogen atom and His185, which is considered to
be essential for the activities and selectivities of PfDHODH inhibitors. The ethyl group extends to the
cavity near the FMN binding site, forming beneficial hydrophobic effects with residues Ile263 and
Ile272 that could help to enhance the potency of compound 10.
2.2. The Structural Optimization Strategy using the Lead Compound 10
According to rational drug design and the proposed binding mode described above, the desired
PfDHODH inhibitor should embrace two functional parts, a hydrogen bond forming “head” linked
via a nitrogen to a hydrophobic “tail”. In the hydrophilic pocket composed by subsites 2 and 3,
the functional groups (e.g., carbonyl groups) could form hydrogen bonds with the guanidine group
of Arg265, which is indispensable for maintaining the inhibitory activity against PfDHODH. As for
the hydrophobic pocket of subsite 1, the naphthyl ring is more suitable compared with the reported
PfDHODH inhibitors in terms of the size that the cavity can accommodate [31]. Consequently, with
the attempt to discover more potent PfDHODH inhibitors for further pharmacological study, as well
as to verify the structure–activity relationship, an optimization strategy using the lead compound
10 was carried out through three modifications: (1) hydrophobic modification on the aromatic ring,
(2) hydrophilic group modification on the dihydrofuranone ring, and (3) cyclization forming a new
scaffold to enhance the stability (as shown in Figure 3).
Figure 2.
PfDHODH. Key residues around the binding pocket are shown as white lines and ligand is shown as
green sticks. The hydrogen bonds are labeled as black dashes. ( ) Electrostatic complementarity
between the dihydrofuranone moiety of compound 10 and the binding site near Arg265. The
electrostatic potential surface of PfDHODH shown between 20 (red) and +20 kT/e (blue) was
calculated using APBS tool [33 35]. The ESP of the dihydrofuranone fragment was calculated by Jaguar
with DFT at B3LYP/cc-pVTZ(-f) level.
(A) Proposed binding pose of lead compound 10 in the ubiquinone binding pocket of
B
−
–

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Figure 3. Diagram for the structural optimization strategy.
2.3. Chemistry
The lead compound 10, with a dihydrofuranone core, was regarded as the well-designed structural
foundation in our initial optimization. First, we replaced the easily metabolized ester group with an
amide group. The preparation of the amide derivatives started with compound 11, which served as an
important intermediate (Scheme 1). To avoid byproducts during ester hydrolysis, the temperature must
be controlled accurately [36]. With the purpose to further study the structure–activity relationships
and improve the pharmacological properties, a series of dihydrofuranone derivatives were designed
and synthesized (Scheme 1) [37]. Amides were synthesized in the presence of HOBt, EDCI, and DIPEA.
Compound 20 was obtained by replacing alkylamines with 80% hydrazine hydrate and using N,
N'-carbonyldiimidazole (CDI) in the routes depicted in Scheme 2.
Scheme 1. Reagents and conditions: (
THF, at rt for 1 h, at 40–45 C for 5h, and under reflux for 18 h; (
for 12 h.
a
) sodium hydride, diethyl malonate, 2-chloroacetyl chloride, and
◦
◦
b
) LiOH–H2O, MeOH–H2O (55–60 C)
O
O
Ar
N
H
CONHR1
12-19
c)
d)
O
O
Ar
R1 = Me, Et, n-Pr, n-Bu, cyclo-Pr
and cyclopropylmethyl
N
H
COOH
O
O
Ar
N
H
CONH-NH2
20
Scheme 2. Reagents and conditions: (
80% hydrazine hydrate and N,N -carbonyldiimidazole (CDI), overnight at rt.
c
) RNH2, HOBt, EDC, DIPEA, and CH2Cl2, overnight at rt; (
d
)
0
According to the predicted binding pose of the lead compound 10, two carbonyl groups that
orient in the same direction are necessary for the potent activity. Therefore, we used paraformaldehyde
to form the rigid six-membered ring that could fix the two carbonyl group orientations well in space.
For this reason, compounds 13
derivatives 21 26 by cyclization (Scheme 3) [38]. All structures of the final products were determined
by spectroscopic techniques.
–16,18 and 20 were subsequently transformed to the pyrimidone
–