Novel selective and potent inhibitors of malaria parasite dihydroorotate dehydrogenase: Discovery and optimization of dihydrothiophenone derivatives

Article abstract of DOI:10.1021/jm400938g

Taking the emergence of drug resistance and lack of effective antimalarial vaccines into consideration, it is of significant importance to develop novel antimalarial agents for the treatment of malaria. Herein, we elucidated the discovery and structure-activity relationships of a series of dihydrothiophenone derivatives as novel specific inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH). The most promising compound, 50, selectively inhibited PfDHODH (IC₅₀ = 6 nM, with >14 000-fold species-selectivity over hDHODH) and parasite growth in vitro (IC₅₀ = 15 and 18 nM against 3D7 and Dd2 cells, respectively). Moreover, an oral bioavailability of 40% for compound 50 was determined from in vivo pharmacokinetic studies. These results further indicate that PfDHODH is an effective target for antimalarial chemotherapy, and the novel scaffolds reported in this work might lead to the discovery of new antimalarial agents.

Full text of DOI:10.1021/jm400938g

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Article
Novel Selective and Potent Inhibitors of Malaria
Parasite Dihydroorotate Dehydrogenase: Discovery
and Optimization of Dihydrothiophenone Derivatives
Minghao Xu, Junsheng Zhu, Yanyan Diao, Hongchang Zhou, Xiaoli Ren, Deheng
Sun, Jin Huang, Dongmei Han, zhenjiang zhao, Lili Zhu, Yufang Xu, and Honglin Li
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 27 Sep 2013
Downloaded from http://pubs.acs.org on September 29, 2013
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Novel Selective and Potent Inhibitors of Malaria Parasite
Dihydroorotate Dehydrogenase: Discovery and Optimization of
Dihydrothiophenone Derivatives
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,2,†
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Minghao Xu , Junsheng Zhu , Yanyan Diao , Hongchang Zhou , Xiaoli Ren ,
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1,*
1,2,*
Deheng Sun , Jin Huang , Dongmei Han , Zhenjiang Zhao , Lili Zhu , Yufang Xu
,
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Honglin Li
1
Shanghai Key Laboratory of New Drug Design, State Key Laboratory of Bioreactor
Engineering, School of Pharmacy, East China University of Science and Technology,
Shanghai 200237, China.
2
Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China
University of Science and Technology, Shanghai 200237, China.
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Department of Microbiology, Medical School of Huzhou Teachers College, Huzhou
313000, China.
4
Instrumental Analysis Center, Shanghai Jiao Tong University, Shanghai 200240,
China
†
Authors contributed equally to this work
*
To whom correspondence should be addressed. Email: zhulfl@ecust.edu.cn,
yfxu@ecust.edu.cn, hlli@ecust.edu.cn
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Please address correspondence and requests for reprints to:
Prof. Honglin Li
School of Pharmacy, East China University of Science and Technology
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Phone/Fax: +86ꢀ21ꢀ64250213
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Abstract. Taking the emergence of drug resistance and lack of effective antiꢀmalarial
vaccines into consideration, it’s of significant importance to develop novel
antimalarial agents for the treatment of malaria. Herein, we elucidated the discovery
and structureꢀactivity relationships of a series of dihydrothiophenone derivatives as
novel specific inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase
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(
PfDHODH). The most promising compound 50 selectively inhibited PfDHODH
IC50 = 6 nM, with >14000ꢀfold speciesꢀselectivity over hDHODH) and parasite
(
growth in vitro (IC50s = 15 nM and 18 nM against 3D7 cells and Dd2 cells,
respectively). Moreover, an oral bioavailability of 40% for compound 50 was
determined from in vivo pharmacokinetic studies. These results further indicate that
PfDHODH is an effective target for antimalarial chemotherapy and the novel
scaffolds reported in this work might lead to the discovery of new antimalarial agents.
Keywords: Malaria, Plasmodium falciparum dihydroorotate dehydrogenase
(PfDHODH), structureꢀactivity relationship (SAR), virtual screening
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Introduction
Malaria is a global parasitic infectious disease caused by Plasmodium parasites,
among which Plasmodium falciparum is the most dangerous one with the highest
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rates of complications and mortality. It has been estimated that there were 216
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million episodes of malaria and 655,000 people died from this disease in 2011,
mostly African children under the age of five. On one hand, treatment of malaria only
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, 3
relies on chemotherapy owing to the absence of effective vaccines, on the other
hand, the widespread drugꢀresistance renders many traditional antiꢀmalarial drugs
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ꢀ6
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ineffective. For instance, chloroquine (compound 1), previously one of the most
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commonly used antiꢀmalarial drugs, has been withdrawn in many regions as resistant
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parasite strains have evolved. Moreover, the occurrence of “artemisinin (compound 2)
resistant” Plasmodium falciparum malaria in western Cambodia and Thailand is a real
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threat to artemisininꢀbased therapies.
Other antiꢀmalarial agent such as
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pyrimethamine (compound 3), a typical malarial dihydrofolate reductaseꢀthymidylate
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4
synthase (DHFRꢀTS) inhibitor, has been nagged by the problem of gene mutation
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and inefficacy for a long time. Under the circumstance of a current lack of efficient
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antiꢀmalarial drugs, there is an emergency to identify novel parasites biochemical
process to fill the research pipeline and develop new, nonꢀcrossꢀresistant, antiꢀmalarial
17, 18, 19
agents.
Pyrimidine bases biosynthesis represents a basic biological and physiological
20, 21
process which is crucial for RNA and DNA production and cell proliferation.
In
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most organisms, dihydroorotate dehydrogenase (DHODH) catalyzes the fourth and
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rateꢀlimiting step in the de novo pyrimidine biosynthesis, and it converts
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dihydroorotate (DHO) to orotate (ORO).
DHODHs are divided into two families
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based on different amino acid sequence and coenzyme dependence. Both human and P.
falciparum DHODHs belong to family II that are located on the inner mitochondrial
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membrane and use ubiquinone as the terminal oxidant. Unlike human cells which
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could acquire pyrimidine bases from salvage pathway, Plasmodium falciparum relies
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entirely on its de novo pyrimidine biosynthetic pathway for survival . Therefore, P.
falciparum dihydroorotate dehydrogenase (PfDHDOH) has been regarded as an
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attractive target for the treatment of malaria.
Although PfDHODH and hDHODH share highly conserved sequence in their
Cꢀterminal domains, there is huge variation of the amino acid sequence in the putative
ubiquinoneꢀbinding site formed by two αꢀhelices in the Nꢀterminal domain, which
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contributes to further identification of speciesꢀspecific DHODH inhibitors. Many
hDHODH inhibitors have been developed and studied for their use against a number
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of antiꢀproliferative and antiꢀinflammatory diseases such as rheumatoid arthritis,
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psoriasis, and lupus erythematosus. However, in the past decade, some success in
identifying PfDHODH inhibitors has been reported based on the modification of
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known human DHODH inhibitors (e.g. compound 4). Most notably, in the past
decade, a number of potent and selective PfDHODH inhibitors from different
chemical scaffolds have been discovered through enzymeꢀbased highꢀthroughput
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screening (e.g. compounds 5, 6 and 7 (DSM1)).
Coꢀcrystal structures of
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PfDHODH in complex with representative compounds from these series, 7
and 8
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(
Genzꢀ667348) were subsequently solved by Phillips et al. Lead optimization of
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the triazolopyrimidine series led to the identification of 9 (DSM190), which was
shown to have good efficacy in a P. berghei mouse model and finally to facilitate the
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discovery of 10 (DSM265), which is currently in clinical development (MMV.org).
(Figure 1)
Figure 1
In our previous work, we reported the discovery of diverse hDHODH inhibitors by
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structureꢀbased virtual screening method. To find novel PfDHODH inhibitors, the
same virtual screening strategy integrating molecular Glide docking with
Prime/MMꢀGBSA rescoring was implemented based on the coꢀcrystal structure of
PfDHODH complexed with compound 7 (PDB code 3I65), and then the lead
compound 11 (IC50 = 1.11 ꢁM) was identified from SPECS database (ID:
AGꢀ690/40639878). After subsequent structural optimization,
a
series of
dihydrothiophenone derivatives with more potent inhibitory effects against
PfDHODH were obtained in this study, which showed an overall selectivity over
hDHODH. Additionally, most of the hits, which had been demonstrated in vitro
potency against the 3D7 and Dd2 strains of P. falciparum, were highly correlated with
enzyme inhibitory activity, suggesting their potential applications as novel
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antiꢀmalarial agents.
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Binding Modes Predicted by Molecular Docking and Optimization Strategy of
Lead Compound 11：
In the predicted binding pose of the lead compound 11 (Figure 2), the
chlorineꢀsubstituted phenyl group binds deeply into the hydrophobic pocket of which
is close to the entrance of the channelꢀlike ubiquinoneꢀbinding site, making favorable
hydrophobic effects and van der Waals (VDW) interactions with residues Leu197,
Ile237, Leu240, and Met536. An edgeꢀtoꢀface πꢀπ interaction is probably present
between Phe227 and the phenyl ring of 11, which could help to enhance the potency
of the lead compound. The dihydrothiophenone moiety occupies the hydrophilic
section of the ubiquinoneꢀbinding site, and could form hydrogen bonds with residues
Arg265 and the water molecule (W15) reserved from crystal structure in the
molecular docking process, and the bridging nitrogen atom forms an additional
hydrogen bond with His185. Moreover, it is noteworthy that the Hꢀbonding between
NH and His 185 was a common feature for the binding and contributed to the
3
8
selectivity of PfDHODH inhibitors over hDHODH.
Figure 2
Based on the proposed binding mode described above, our lead compound could be
divided into two functional parts, the aromatic section as a “Hydrophobic Group” and
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the dihydrothiophenone section as a hydrogen bondꢀforming “Hydrophilic Group”. In
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the hydrophobic pocket of subsite 1, there is still some space between the chlorine
atom and the residues around to accommodate larger substitution. In the hydrophilic
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pocket composed by subsites 2 and 3, the functional groups (carbonyl groups) which
could form hydrogen bonds with polar residues or resident waters should be reserved
to maintain the inhibitory activity against PfDHODH. Accordingly, to identify more
potent PfDHODH inhibitors for further pharmacological study as well as verify the
structureꢀactivity relationship in the absence of crystal structure, the lead optimization
was carried out through three modifications: 1) hydrophobic modification on the
aromatic ring to improve hydrophobic effects, 2) hydrophilic modification on the
dihydrothiophenone ring and 3) scaffold hopping of the dihydrothiophenone core (as
shown in Figure 3).
Figure 3
Chemistry:
Aromatic amine Mimics
Scheme 1
Firstly, to discover some proper hydrophobic moieties, the target inhibitors 11ꢀ28
were obtained via a threeꢀstep procedure (Scheme 1). In our design, the preparation of
dihydrothiophenone analogues started with commercially available amines and aryl
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isothiocyanates served as the key intermediates. Triphosgene was used instead to
avoid poisonous and environmentally hazardous reagents such as thiophosgene and
phosgene. After several attempts, electronꢀwithdrawing aryl isothiocyanates which
were hard to acquire were finally synthesized and then involved in the condensation
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with ethyl 4ꢀchloroacetoacetate to afford desired products.
Formate Mimics
Scheme 2
Next we focused on synthesizing various formates. Extensive efforts had been
made to control the hydrolysis of these substrates to corresponding acids (30ꢀ31)
o
(Scheme 2). To avoid byꢀproducts, temperature must be kept below 60 C and the
concentration of the base must be controlled accurately. Moreover, we synthesized 29
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to explore the role of the ethoxycarbonyl group in the scaffold. At last, after a
purification by chromatography over silica gel, the desired products (32ꢀ35, 42ꢀ44)
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were reꢀcrystallized to ensure >95% purity.
Formamide Mimics
Scheme 3
To further study the structureꢀactivity relationships of dihydrothiophenone
derivatives and improve their pharmacological properties, target amides (36ꢀ41, 45ꢀ49)
were designed and prepared. The intermediates (30ꢀ31) were made using the same
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method described above and the amides were synthesized in the presence of HOBt,
EDC and DIPEA which served as condensating agents and base (Scheme 3).
Scaffold Hopping
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Scheme 4
The general synthetic scheme for preparation of dihydrofuranone derivatives (50ꢀ56)
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is shown in Scheme 4. In these cases, the treatment of diethyl malonate with
2ꢀchloroacetyl
ꢀethoxyꢀ4ꢀoxoꢀ4,5ꢀdihydrofuranꢀ3ꢀcarboxylate which could further react with
aromatic amines to generate target molecules. All structures of the final products were
chloride
provided
the
intermediate
ethyl
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determined by H NMR, C NMR and HRMS (ESI).
Results and Discussion：
Table 1
Hydrophobic Modification on Aromatic Moiety
In the first stage of optimization, we focused on the modification of the aromatic
hydrophobic moiety with different substituents on the phenyl ring (Table 1). As
exemplified by compound 12, the removal of chlorine from the phenyl ring resulted in
a complete loss of activity, which implied that the larger the group occupying the
hydrophobic cavity, the better its potency is, i.e. the inhibition potency against
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PfDHODH could be achieved by substitution with larger hydrophobic groups to
improve hydrophobic effects. A similar improvement in potency was observed for
those with large alkyl group at paraꢀposition of the phenyl ring (compounds 13-15),
compound 15 was the most active one against PfDHODH with an IC50 value of 0.23
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ꢁ
M. For these compounds containing paraꢀsubstituted groups on the phenyl ring, the
improved activity correlated well with increased hydrophobicity (ꢀC(CH > ꢀCF
Cl), verifying our previous optimization strategy.
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)
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3
>
ꢀ
Compared with compound 11, 16 with the introduction of trifluoromethyl group,
exhibited slightly improved activity. Compound 17, which was similar to 16, retained
comparative potency against PfDHODH. Compound 18 containing
metaꢀtrifluomethyl substituted phenyl moiety showed huge loss of activity (13 vs 18),
which may result from the absent effects of hydrophobicity in the paraꢀposition. For
compound 19, which had two chlorine atoms in the metaꢀpositions of the phenyl ring,
strong PfDHODH inhibition was observed with an IC50 value of 1.19 ꢁM.
Substitution with halogen or methyl at the metaꢀ and paraꢀpositions yielded more
potent compounds 20 and 21, which displayed 4ꢀ and 2ꢀfold better potency against
PfDHODH compared to lead compound 11, respectively. Some small substituents,
such as methyl, fluorine or trifluoromethyl, at the metaꢀposition of the phenyl ring
might have some influence on potency improvement. However, no activity against
PfDHODH was observed for compound 22, in which methyl was alternatively
introduced into the orthoꢀposition. The results summarized above indicated that the
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size and position of substituents on the phenyl ring significantly affected the in vitro
inhibitory potency against PfDHODH: a) a hydrophobic substituent at paraꢀposition
is preferential and a larger alkyl group is well tolerated, b) more attention should be
paid to the size of metaꢀsubstituent since halogens or small alkyl groups are suitable, c)
substituents at orthoꢀposition should be avoided.
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Since the PfDHODH inhibition potency was particularly sensitive to substituent
pattern of the phenyl ring, an alternative optimization strategy was carried out by the
introduction of cyclic systems with strong hydrophobicity and conformational
flexibility as the “hydrophobic group”. Thus, we next developed compounds 23ꢀ28.
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Consistent with the hydrophobic nature of the interactions in this area, the
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ꢀnaphthyl containing compound 25 showed greater fold improvement in potency
against PfDHODH with the IC50 value of 0.02 ꢁM, which turned out to be the most
potent inhibitor of this series with up to 56ꢀfold improvement in potency relative to
the lead compound 11. A demonstration that hydrophilic group/polar functionality was
relatively less acceptable at the entrance of the binding site was evidenced by the
significantly reduced activity of 24 compared to 25 (IC50 = 0.35 vs 0.02 ꢁM). Notably,
all these compounds still retained the inhibitory activity against PfDHODH and were
inactive against hDHODH except for 23. It was also noteworthy that the replacement
of 5,6,7,8ꢀtetrahydroꢀ2ꢀnaphthyl (compound 23) with similar but smaller 5ꢀindanyl
(compound 26) would reduce the activity by nearly 10ꢀfold. This significant variety
25
might be caused by the slight difference of the size and shape of the substituent.
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Larger substituents such as anthracene (27, IC50 = 0.20 ꢁM) and carbazole (28, IC50
=
0
.39 ꢁM) were also well tolerated.
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Hydrophilic Group Modification on the Dihydrothiophenone Scaffold with Acids,
Esters and Amides
1
On the basis of previously established proper hydrophobic groups (R
=
3,4ꢀdiꢀCH
3
ꢀPh and 2ꢀnaphthyl), compounds 29ꢀ49 were achieved to explore SAR in
the hydrophilic region. As illustrated in the predicted binding mode of 11, the ester
group (ꢀCOOC H ) was pointed toward a small deep hydrophobic cavity formed by
2
5
residues Ile263 and Ile272, and featured hydrogen bonds formed with Tyr528 at the
same time. Therefore, the ethoxycarbonyl moiety could interact with PfDHODH via
hydrogen bond and hydrophobic contacts simultaneously. In this section, emphasis
was laid on the influence of structural variation of the ethoxycarbonyl group on the
inhibitory activity against PfDHODH.
The inhibitor without ethoxycarbonyl group on the dihydrothiophenone scaffold
(29) displayed poor enzyme inhibitory activity in comparison with 20, which could be
concluded that the derivatives without any substituent at 3ꢀposition of the scaffold
would be strongly disfavored. The addition of carboxyl group to the 3ꢀposition on the
dihydrothiophenone ring did not make much improvement, as no activity against
PfDHODH was observed for compound 30. Although compound 31 showed an IC50
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value of 1.24 ꢁM, the inhibitory activity against PfDHODH was 6ꢀfold lower
compared to that of ethoxycarbonylꢀsubstituted derivative 25. The greatly reduced
activity was possibly due to the absence of hydrophobic interactions with the small
pocket formed by residues Ile263 and Ile272. Next some derivatives with different
alkoxycarbonyl groups (32-34 and 42ꢀ44) were synthesized to identify the optimal
alkyl chain which could fit the small cavity. Notably, this series of derivatives did not
display improved potency compared to the corresponding ethoxycarbonyl analogues
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0 and 25. On one hand, the decrease of PfDHODH IC50 values observed for 32 and
2 (IC50 = 1.49 ꢁM and 0.033 ꢁM, respectively) should arise from the reduced
4
hydrophobic interaction caused by the shorter length of methoxycarbonyl group. On
the other hand, the reduced binding affinity of 33, 34, 43 and 44 (IC50 = 4.03 ꢁM, 2.35
ꢁ
M, 0.95 ꢁM and 35.57 ꢁM, respectively) was probably resulted from the size
constraint of the cavity when increasing chain length. Likewise, the bulkier such as 35
IC = 4.16 ꢁM), could not be well accommodated by the hydrophobic cavity neither.
(
5
0
Formamide derivatives 36ꢀ41 and 45ꢀ49 were also evaluated. Interestingly, this
series of compounds were collectively much less potent in the PfDHODH enzyme
activity assay than corresponding ester derivatives. Two representative compounds, 37
and 45, replaced the ester group (20 and 25) with amide group, displayed up to 160ꢀ
and 600ꢀfold decrease in potency against PfDHODH respectively. The pronounced
difference in activity against PfDHODH probably resulted from the intramolecular
hydrogenꢀbond in these amide derivatives, the carbonyl group engaging in
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intramolecular NHꢀC=O interactions with adjacent amide group, which formed
additional hydrogen bond with Arg265 of PfDHODH. Meanwhile, the change of
35, 38
substituents led to some selectivity changes against hDHODH (38ꢀ41).
Overall,
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50
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these amide derivatives seemed not to be appropriate PfDHODH inhibitors.
Scaffold Hopping and Replacement of Sulfur with Oxygen on the
Dihydrothiophenone Skeleton
After discussing the SAR through hydrophobic group modification and hydrophilic
group modification, the role of sulfur on the skeleton still remained ambiguous even
43
by the aid of molecular docking simulation. Replacement of sulfur with oxygen on
the dihydrothiophenone skeleton gave compound 50, which had an IC50 value as low
as 6 nM, 3ꢀfold more active than matched sulfur derivative 25 and 185ꢀfold than the
lead compound 11. To further verify the significance of oxygen supplement, more
dihydrofuranone derivatives possessing different hydrophobic groups were
2
synthesized with R fixed to ethoxycarbonyl group. As displayed in Table 1, an
increased trend in activities against PfDHODH was found for compounds 51ꢀ56
relative to corresponding dihydrothiophenone analogues. Notably, the
dihydrofuranone scaffold made compound 54 approximately 13ꢀfold more potent than
its matched dihydrothiophenone derivative 26. Meanwhile, this series of derivatives
remained the high speciesꢀselectivity over hDHODH, which indicated that our
scaffold hopping was quite successful and the novel dihydrofuranone scaffold, could
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be further investigated to discovery more PfDHODH inhibitors.
In Vitro Evaluation of Compound Activity against Pf3D7 and PfDd2 cells
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To further evaluate their antiplasmodial activities, these dihydrothiophenone
derivatives were tested against CQꢀsensitive Pf3D7 and CQꢀresistant PfDd2 strains in
vitro assay. It was found that the inhibition potencies of these compounds against
PfDHODH were maintained in Pf3D7 and PfDd2 cells (Table 1). For hydrophobic
group modification, previous conclusion that paraꢀsubstituted large hydrophobic
group on the phenyl ring is preferential could also be evidenced by the comparison of
1
3 and 18 in the cell line. “Naphthylꢀlike” derivatives such as 23 and 25 showed good
potency against Pf3D7 strains and PfDd2 strains as well (23, Pf3D7 IC50 = 0.17 ꢁM,
PfDd2 IC50 = 0.31 ꢁM; 25, Pf3D7 IC50 = 0.077 ꢁM, PfDd2 IC50 = 0.057 ꢁM).
Moreover, compounds with hydrophilic group modification were still less effective in
Pf3D7 cells and PfDd2 cells and these results also proved that the ethoxycarbonyl
group is essential for the inhibitory activity against PfDHODH. The trend of cell
inhibitory activity of 42, 43 and 44 was consistent with the enzyme inhibition activity
(Figure 4). Amide derivatives such as 37ꢀ41, 45ꢀ49 also showed IC50 values in
micromolar range. To our satisfaction, scaffold hopping was successful which could
be evidenced by the great cell potency demonstrated by 50 and 53 in two kinds of Pf
cells. Other dihydrofuranone derivatives such as 52, 54, 55, 56 with attractive activity
could also be optimized to find more effective PfDHODH inhibitors.
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Figure 4
The IC50 values of these compounds in both cell lines (Table 1) and linear regession
analysis (Figure 4) showed an excellent correlation between inhibitory activity against
PfDHODH and antiꢀmalarial potency in Pf3D7 cells and PfDd2 cells (For Pf3D7 cells,
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2
2
slope = 1.256 and r = 0.750; For PfDd2 cells, slope = 1.347 and r = 0.746). Novel
inhibitors described in Table 1 displayed promising activity in both cells with IC50
values in micromolar range either. Meanwhile, consistent with the previous inhibitory
activity against PfDHODH (Table 1), compounds which showed poor activity on
PfDHODH also exhibited less inhibitory potency against Pf3D7 cells and PfDd2 cells.
On the basis of above data and analysis, it could be demonstrated that cell killing by
these inhibitors results from the inhibition of PfDHODH, which confirmed
PfDHODH was an effective target for antimalarial chemotherapy.
Table 2
In Vivo Pharmacokinetic Studies of compounds 25 and 50 in Sprague-Dawley
Rats
Pharmacokinetic studies with potent and selected PfDHODH inhibitors (25 and 50)
were carried out in rats at intravenous dose of 1 mg/kg and oral dose of 10 mg/kg
(
Table 2). After IV dosing, 25 exhibited high clearance of 129 mL/min/kg and short
halfꢀlife (0.3 h), whereas 50 showed a little lower clearance of 84 mL/min/kg and
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halfꢀlife of 0.26 h. Neither compounds had high volume of distribution (3.35 and 1.91
L/kg, respectively).
After oral administration, 25 showed very low exposure (AUC0ꢀlast) of 39 ng/mL*hr
and maximum plasma concentration (Cmax) of 25 ng/mL, giving rise to an oral
bioavailability of only 3%. However, 50 displayed pharmacokinetic properties with
systemic exposure (AUC0ꢀlast) of 790 ng/mL*hr and maximum plasma concentration
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(Cmax) of 608 ng/mL, which lead to an oral bioavailability of 40%. The halfꢀlives (T1/2)
and the time to reach the maximum concentration (Tmax) for both inhibitors were short.
The obvious individual difference in overall exposure and pharmacokinetic
parameters of 25 and 50 indicated that these compounds might be instable. In contrast
with 25, 50 showed higher bioavailability and better pharmacokinetic properties
(AUC, Cmax, Clearance) which strongly suggested that sulfur atom in the core was a
soft spot and scaffold hopping on the dihydrothiophenone skeleton could improve the
stability of the compound.
Conclusion
In summary, a novel series of dihydrothiophenone derivatives as Plasmodium
falciparum dihydroorotate dehydrogenase inhibitors were identified through virtual
screening, and subsequent structural optimization and SAR analysis led to dozens of
novel PfDHODHꢀspecific inhibitors that achieved excellent inhibitory potency.
Compounds in this series with three preferential structural fragments were regarded as
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potent PfDHODH inhibitors: 1) paraꢀsubstituted larger alkyl group on the phenyl ring
or bicyclic systems such as “naphthylꢀlike” substituents as the hydrophobic group, 2)
ethoxycarbonyl group at 3ꢀposition on the dihydrothiophenone ring or
dihydrofuranone ring as the hydrophilic group, 3) dihydrofuranone ring was more
potent skeleton than dihydrothiophenone ring.
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50
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The potency of compounds described in this study was particularly encouraging for
the development of antiꢀmalarial agents to conquer the widespread drugꢀresistance.
The most potent inhibitors 25, 50 and 53 showed excellent inhibitory activity against
PfDHODH (with IC50 values 20 nM, 6 nM, 18 nM) and high speciesꢀselectivity over
hDHODH. Moreover, the high inhibition potencies of these PfDHODH inhibitors
were maintained in the low doubleꢀdigit nanomolar range against Pf3D7 and PfDd2
cells strains in vitro assay. Furthermore, excellent correlation between inhibitory
activity against PfDHODH and antiꢀmalarial potency in Pf3D7 cells and PfDd2 cells
were observed for the dihydrothiophenone derivatives and dihydrofuranone
derivatives. These results further suggested that PfDHODH is an effective target for
antimalarial chemotherapy and novel scaffolds found in this work might lead to the
discovery of new antimalarial agents. Study on the crystal structure of the PfDHODH
inhibitors complex and efforts to improve pharmacokinetic properties are underway to
further understand SAR and make them “druggable”.
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Experimental Section
Materials:
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A771726 and chloroquine (CQ) were obtained from J&K and SigmaꢀAldrich.
Albumax II, SYBR Green I and 1640 incomplete medium were acquired from
Invitrogen. DSM1 was synthesized inꢀhouse and at least 97% pure. Materials for
enzyme and cells assay which were not listed out were purchased from Sigma.
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DHODH inhibition assays. Plasmid construction, protein expression and purification
of PfDHODH (Phe158ꢀSer569) and hDHODH (Met30ꢀArg396) were performed
44
35
following the protocols of Liu Shengpin et al and Xiaoyi Deng et al respectively.
The enzyme was diluted into a final concentration of 10 nM with assay buffer
containing 50 mM HEPES (pH 8.0), 150 mM KCl, 0.1% Triton Xꢀ100 supplemented
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60
0
with 100 ꢁM UQ , 120 ꢁM DCIP. The mixture was transferred into a 96ꢀwell plate
and incubated for 5 min at room temperature, and then the dihydroorotate was added
to a final concentration of 500 ꢁM to initiate the reaction. The reaction was monitored
by measuring the decrease of DCIP in the absorption at 600 nm for each 30 s over a
period of 6 min. Inhibition studies were performed in this assay with additional
various amounts of inhibitors. A771726 and DSM1 were measured as the positive
control for hDHODH and PfDHODH respectively. Percent inhibition relative to no
i 0
inhibitor control was calculated from (1ꢀV /V ) ×100. For the determination of the
IC50 value of the inhibitor, 8ꢀ9 different concentrations were applied. Each inhibitor
concentration point was tested in triplicate and the IC50 values were calculated using
the sigmoidal fitting option of the program Origin 8.0.
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In Vitro Plasmodium falciparum whole cell assays. CQꢀsensitive strain 3D7 and
CQꢀresistant strain Dd2 of Plasmodium falciparum were used in vitro blood stage
culture to test the antimalarial efficacy. The cultures were maintained according to the
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method described by Trager and Jensen in a medium containing 0.5% Albumax II
instead of human serum. Stock solution of CQ was prepared in water (milliꢀQ grade)
o
with the concentration of 50 ꢁg/mL and stored at ꢀ20 C. The test compounds stock
solution was prepared in DMSO with the concentration of 10 ꢁmol/mL and stored at
room temperature. All of the stock solution was diluted with 1640 incomplete medium
to achieve the required concentration during the experiment. (In all cases except CQ,
the highest final concentration of DMSO was 0.2%, which was found to be nonꢀtoxic
to the parasite). The antiplasmodial activity of the compounds was determined by a
fluorometric method using SYBR Green I. In brief, asynchronous parasites were
plated in triplicate at 2% hematocrit and 1% parasitemia in 100 ꢁL with the absence or
presence of a series of concentration ranging from 0.15625 to 20 ꢁM of each
compound by serial dilution. A series of concentration of CQ were used as the positive
control and 0.2% DMSO was used as the negative control. Plates were incubated at 37
o
C for 72 h. After that, the supernate was removed and 100 ꢁL of SYBR Green I in
4 3
lysis buffer (8.26 g/L NH Cl, 1 g/L KHCO , 0.037 g/L EDTA and 5×SYBR Green I)
was added to each well. Plates were covered and incubated in the dark at room
temperature for 1 h. Plates were read at 485/520 nm on a Synergy MX, Biotek
fluorescent plate reader. This experiment was repeated twice. Data analysis was
46
processed as described by Michael J. B et al with a little modification. Briefly,
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fluorescent counts from untreated parasites represent the maximum amount of DNA in
viable parasites while those from uninfected erythrocytes represent background
fluorescence. The mean fluorescent counts in test or control wells were adjusted by
deducting the background. The growth inhibition rate at each drug concentration was
obtained as described below.
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Mean fluorescence counts in test wells
%ꢀGrowth Inhibitionꢀ=ꢀ ꢁ 1ꢀ
ꢂ ×100%ꢀ
Mean fluorescence counts in control wells
IC50 values were determined using a Growth/Sigmoidal option of Origin 8.0.
In Vivo Pharmacokinetic Assay. Single dose pharmacokinetic studies on the most
active compounds were performed in male SpragueꢀDawley Rats in compliance with
the Guide for the Care and Use of Laboratory Animals. Blood samples were collected
with heparin sodium as antiꢀcoagulate at specified intervals (5 min, 15 min, 30 min, 1
h, 2 h, 4 h, 8 h and 12 h). PK parameters were calculated by nonꢀcompartment model
using WinNonlin5.2 based on the LCꢀMS/MS quantitation data.
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Chemistry: Reagents and Methods. All chemical reagents and solvents were
obtained from commercial sources and used without further purification. Thinꢀlayer
chromatography (TLC) was carried out to monitor the process of reactions.
Purification of compounds was achieved by column chromatography with silica gel
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(
HaiYang, Qingdao) 200ꢀ300 mesh. H NMR and C NMR spectra were recorded on
a Bruker AMꢀ400 spectrometer with chemical shifts expressed as ppm (in CDCl or
DMSOꢀd , Me Si as internal standard). Melting points were recorded on a
3
6
4
WRSꢀ1Bꢀdigital melting point apparatus. The mass spectra were measured at The
Institute of Fine Chemistry of ECUST. Analytical HPLC was performed on a
HewlettꢀPackard 1100 chromatography system equipped with photodiode array
detector and a Zorbax XDBꢀC18 or BonusꢀRP (compounds 30ꢀ35, 42ꢀ44) column
(250 mm×4.6 mm) was used to determine the purity of the products. The mobile
phase A was 10 mM NH OAc in water (pH 6.0) and mobile phase B was acetonitrile.
4
A gradient of 10ꢀ100% B over 20 minutes was run at a flow rate of 1.0 mL/min.
Compounds synthesized in our laboratory were generally varied from 95% to 99% in
purity, and the biological experiments were employed on compounds whose purity is
at least 95%.
General procedure for aryl isothiocyanate:
A mixture of 1,4ꢀdiazabicyclo[2.2.2]octane (9 mmol), aniline (3 mmol), carbon
disulfide (5 mL) in acetone (3 mL) was stirred overnight at room temperature. The
precipitated solid was filtered, washed with hexane and dried under vacuum. To a
stirred solution of the solid in chloroform (10 mL), a solution of triphosgene (1 mmol)
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in chloroform (10 mL) was added dropwise over 30 min. The stirring was continued
for 10 h at room temperature. After resulting mixture was filtered, the filtrate was
concentrated under reduced pressure and purified by column chromatography (100%
PE) with 45%ꢀ70% yield as white solid or colorless oil.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
General procedure for compounds 11-28:
Ethyl 4ꢀchloroacetoacetate (1 mmol) was added to a solution of sodium hydride (2
mmol, 60%) in THF (5 mL) , and the mixture was stirred for 1 h at room temperature
before a solution of aryl isothiocyanate (1 mmol) in THF (5 mL) was added dropwise
over 10 min. After the mixture was stirred overnight under argon, 5% HCl solution
(20 mL) was added and the resulting oil was extracted with EtOAc (2×15 mL). The
organic layer was washed with brine (2×15 mL), dried (Na SO ), concentrated under
2
4
reduced pressure and purified by column chromatography (PE : EtOAc = 2 : 1, v/v)
with 40ꢀ45% yield as white solid.
General procedure for compounds 30 and 31:
LiOHꢀH
2
O (5 mmol) was slowly added to a solution of 20 or 25 (1 mmol) in
o
MeOHꢀH
2
O (3 mL, MeOH : H
2
O = 5:1, v/v) at 0 C over 15 min. The reaction
o
mixture was allowed to warm to 55ꢀ60 C for 12 h with stirring. After MeOH was
evaporated off, the aqueous residual was acidified to pH 1ꢀ2 with 1N HCl and
precipitated solid was filtered, washed with water and dried under vacuum with 70ꢀ80%
yield as yellow solid.
General procedure for compound 29:
After a solution of 30 (1 mmol) in pyridine (2 mL) had been heated for 2 h, the
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2
2
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3
3
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3
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4
4
4
4
4
4
4
4
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5
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5
5
5
5
6
reaction mixture was washed by 5% aqueous HCl (2×10 mL) and extracted with
EtOAc (2×10 mL). The organic layer was washed with brine (2×15 mL), dried
2 4
(Na SO ) and concentrated under reduced pressure with purification by column
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
chromatography (PE : EtOAc = 4 : 1, v/v) with 45% yield as yellow solid.
General procedure for compounds 32-35 and 42-44:
Diethyl azodicarboxylate (DEAD, 1.1 mmol) was slowly added to a solution of 30 (or
3
1) (1 mmol), triphenyl phosphine (1.1 mmol), and alcohol (1.2 mmol) in toluene (5
mL) with a pressure equalized dropping funnel under an argon atmosphere. The
o
mixture was stirred overnight at 45 C, then extracted with EtOAc (2×15 mL), washed
2 4
with brine (2×15 mL), dried (Na SO ) and concentrated under reduced pressure with
purification by column chromatography (PE : EtOAc = 3 : 1, v/v) with 20ꢀ25% yield
as white solid.
General procedure for compounds 36-41 and 45-49:
HOBt (1.1 mmol), EDC (1.1 mmol) and DIPEA (1 mmol) were added to a solution of
o
amine (1 mmol) and 30 (or 31) (1 mmol) in dry CH
2
Cl
2
(5 mL) at 0 C were added.
The reaction mixture was stirred overnight at room temperature, then washed with 5%
aqueous HCl (2×15 mL), 5% aqueous NaHCO (2×15 mL), brine (2×15 mL), dried
Na SO ) and concentrated under reduced pressure with purification by column
3
(
2
4
chromatography (PE : EtOAc = 6 : 1, v/v) with 20%ꢀ25% yield as white solid.
General procedure for compounds 50-56:
Diethyl malonate (2 mmol) was added to a solution of sodium hydride (2 mmol, 60%)
o
in dry THF (20 mL) and the mixture was stirred for 30 min at 0ꢀ10 C. Then the
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reaction mixture was stirred at room temperature for 10 min and 2ꢀchloroacetyl
chloride (1 mmol) in dry THF (5 mL) was added. The solution was kept at room
o
temperature for 1 h, at 40ꢀ45 C for another 1 h and aniline (1 mmol) in dry THF (100
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
mL) was added dropwise over 20 min. The reaction mixture was stirred at room
temperature overnight, heated under reflux for 4 h. After excess THF was evaporated
off, the resulting residue was extracted with EtOAc (2×15 mL) and washed with brine
(
2×15 mL). The organic layer was dried (Na
pressure and purified by column chromatography (PE : EtOAc = 2.5 : 1, v/v) with
0ꢀ35% yield as white solid.
Ethyl 2-(4-chlorophenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate (11). mp
2 4
SO ), concentrated under reduced
3
o
1
213.2ꢀ213.7 C. H NMR (400 MHz, DMSOꢀd
6
) δ 11.12 (s, 1H), 7.55 (d, J = 8.8 Hz,
2
H), 7.47 (d, J = 8.4 Hz, 2H), 4.22 (q, J = 7.2 Hz, 2H), 3.69 (s, 2H), 1.25 (t, J = 7.2
13
Hz, 3H). C NMR (100 MHz, DMSOꢀd
6
) δ 191.2, 183.2, 165.3, 136.9, 132.5, 129.9,
+
1
27.8, 97.7, 59.8, 38.5, 14.9. HRMS (ESI) calcd for C H ClNO S [M+H] 298.0305,
13
12
3
R
found 298.0307. Purity: 99.59% (t 13.95 min).
Ethyl
146.8ꢀ147.5 C. H NMR (400 MHz, CDCl
.37 (d, J = 8.0 Hz, 2H), 7.35 (s, 1H), 4.39 (q, J = 7.2 Hz, 2H), 3.67 (s, 2H), 1.42 (t, J
2-(phenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate
(12).
mp
o
1
3
) δ 11.51 (s, 1H), 7.47 (t, J = 8.0 Hz, 2H),
7
13
= 7.2 Hz, 3H). C NMR (100MHz, DMSOꢀd ) δ 191.0, 183.3, 165.5, 137.8, 130.0,
6
+
1
28.3, 125.6, 97.4, 59.7, 38.4, 14.9. HRMS (ESI) calcd for C13
3
H13NO S [M+H]
264.0694, found 264.0694. Purity: 99.89% (t 12.21 min).
R
Ethyl 2-(4-(trifluoromethyl)phenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxyl
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1
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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
o
1
ate (13). mp 180.1ꢀ181.0 C. H NMR (400 MHz, DMSOꢀd
6
) δ 11.34 (s, 1H), 7.86 (d,
J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 4.23 (q, J = 7.2 Hz, 2H), 3.74 (s, 2H), 1.26
13
(
t, J = 7.2 Hz, 3H). C NMR (100 MHz, DMSOꢀd
6
) δ 191.1, 182.7, 165.4, 141.4,
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
27.1, 127.1, 125.9, 98.4, 59.9, 38.5, 14.8. HRMS (ESI) calcd for C H F NO S
1
4
12
3
3
+
[
M+H] 332.0568, found 332.0559. Purity: 99.42% (t
R
14.83 min).
Ethyl 2-(4-pentafluorosulfenylphenyl)-4-oxo-4,5-dihydrothiophene-3-carboxylate
o
1
(
14). mp 177.3ꢀ178.1 C. H NMR (400 MHz, DMSOꢀd
6
) δ 11.38 (s, 1H), 8.03 (d, J =
9
.2 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H), 4.25 (q, J = 6.8 Hz, 2H), 3.76 (s, 2H), 1.26 (t, J
13
=
6.8 Hz, 3H). C NMR (100 MHz, DMSOꢀd ) δ 191.1, 182.6, 165.4, 141.1, 127.74,
6
1
12 5 3 2
27.7, 127.7, 125.7, 98.7, 59.9, 38.6, 14.8. HRMS (ESI) calcd for C13H F NO S
+
[M+H] 390.0257, found 390.0262. Purity: 99.49% (t
R
15.69 min).
Ethyl 2-(4-tert-butylphenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate (15).
o
1
mp 167.7ꢀ168.2 C. H NMR (400 MHz, CDCl
3
): δ 11.44 (s, 1H), 7.47 (d, J = 8.4 Hz,
2
H), 7.30 (d, J = 9.6 Hz, 2H), 4.40 (q, J = 6.8 Hz, 2H), 3.66 (s, 2H), 1.43 (t, J = 6.8
13
Hz 3H), 1.36 (s, 9H). C NMR (100 MHz, CDCl
3
): δ 191.2, 183.2, 166.6, 151.0,
1
34.30, 126.6, 123.6, 97.7, 60.4, 38.0, 34.7, 31.3, 14.5. HRMS (ESI) calcd for
+
C
17 3 R
H21NO S [M+H] 320.1320, found 320.1318. Purity: 99.49% (t 17.37 min).
Ethyl 2-(4-chloro-3-(trifluoromethyl)phenylamino)-4-oxo-4,5-dihydrothiophene-3-
o
1
carboxylate (16). mp 211.0ꢀ211.6 C. H NMR (400 MHz, CDCl ) δ 11.67 (s, 1H),
3
7
.81 (d, J = 8.4 Hz, 1H), 7.73 (s, 1H), 7.45 (d, J = 8.4 Hz, 1H), 4.39 (q, J = 6.8 Hz,
1
3
2H), 3.71 (s, 2H), 1.42 (t, J = 6.8 Hz, 3H). C NMR (100 MHz, DMSOꢀd
6
) δ 191.1,
1
83.0, 165.1, 137.8, 136.5, 131.5, 129.8, 129.4, 125.9, 125.9, 124.3, 121.6, 118.0,
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7
8
9
+
9
8.5, 59.8, 38.6, 14.8. HRMS (ESI) calcd for C14
found 366.0177. Purity: 95.77% (t 15.64 min).
Ethyl 2-(4-bromo-3-(trifluoromethyl)phenylamino)-4-oxo-4,5-dihydrothiophene-3-
3 3
H11ClF NO S [M+H] 366.0179,
R
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
o
1
carboxylate (17). mp 223.8ꢀ224.5 C. H NMR (400 MHz, CDCl ): δ 11.66 (s, 1H),
3
7
2
1
3
4
.73 (s, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 4.40 (q, J = 7.2 Hz,
1
3
6
H), 3.70 (s, 2H), 1.42 (t, J = 7.2 Hz, 3H). C NMR (100 MHz, DMSOꢀd ) δ 191.2,
83.1, 165.1, 137.5, 133.1, 131.6, 129.9, 127.9, 127.6, 125.9, 125.8, 125.7, 98.4, 59.8,
+
8.6, 14.9. HRMS (ESI) calcd for C14
09.9675. Purity: 95.72% (t 15.87 min).
H
11BrF
3
NO
3
S [M+H] 409.9673, found
R
Ethyl 2-(3-(trifluoromethyl)phenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxyl
o
1
ate (18). mp 190.2ꢀ191.2 C. H NMR (400 MHz, DMSOꢀd
6
): δ 11.23 (s, 1H), 7.86 (s,
1
H), 7.78ꢀ7.72 (m, 3H), 4.24 (q, J = 6.8 Hz, 2H), 3.71 (s, 2H), 1.26 (t, J = 6.8 Hz, 3H).
1
3
C NMR (100 MHz, DMSOꢀd
6
): δ 191.1, 183.3, 165.2, 138.7, 131.1, 130.1, 124.9,
1
24.8, 122.9, 122.8, 98.1, 59.8, 38.5, 14.8. HRMS (ESI) calcd for C H F NO S
1
4
12
3
3
+
[M+H] 332.0568, found 332.0572. Purity: 99.04% (t
R
14.52 min).
Ethyl 2-(3,5-dichlorophenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate (19).
o
1
mp 188.1ꢀ188.9 C. H NMR (400 MHz, DMSOꢀd
6
) δ 11.12 (s, 1H), 7.66 (s, 1H),
13
7
.61 (s, 2H), 4.22 (q, J = 7.2 Hz, 2H), 3.71 (s, 2H), 1.25 (t, J = 7.2 Hz, 3H). C NMR
(100 MHz, DMSOꢀd ) δ 191.2, 183.2, 165.1, 140.2, 134.8, 127.8, 125.0, 98.5, 59.9,
6
+
3
8.7, 14.8. HRMS (ESI) calcd for C13
H11Cl
2
NO
3
S [M+H] 331.9915, found 331.9918.
Purity: 99.89% (t 16.34 min).
R
Ethyl 2-(3,4-dimethylphenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate (20).
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