Novel dual inhibitors against FP-2 and PfDHFR as potential antimalarial agents: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.cclet.2017.11.041

Resistance to malaria parasites has quickly developed to almost all used antimalarial drugs. Cysteine protease falcipain-2 (FP-2) and Plasmodium falciparum dihydrofolate reductase (PfDHFR) have crucial roles, which are absolutely necessary, in the parasite life cycle. In this study, based on the uniform pharmacophores of reported PfDHFR inhibitors and the first-generation dual inhibitors against FP-2 and PfDHFR, we identified a novel series of dual inhibitors through fragments assembly. Lead optimization led to the identification of 14, which showed potent inhibition against FP-2 and PfDHFR enzyme (IC₅₀ = 6.8 ± 1.8 μmol/L and IC₅₀ = 8.8 ± 0.3 μmol/L) and P. falciparum 3D7 strain (IC₅₀ = 2.9 μmol/L). Additionally, 14 exhibited more potent inhibition to the proliferation of chloroquine-resistant P. falciparum Dd2 strain (IC₅₀ = 1.1 μmol/L) than pyrimethamine (IC₅₀ > 10 μmol/L), and 14 displayed micromolar inhibitory activities against two clinical isolated strains Fab9 (IC₅₀ = 2.6 μmol/L) and GB4 (IC₅₀ = 1.0 μmol/L). Collectively, these data demonstrated that 14 might be a good lead compound for the treatment of malaria.

Full text of DOI:10.1016/j.cclet.2017.11.041

Accepted Manuscript
Title: Novel dual inhibitors against FP-2 and PfDHFR as
potential antimalarial agents: Design, synthesis and biological
evaluation
Authors: Wenhua Chen, Xue Yao, Zhenghui Huang, Fei Mao,
Longfei Guan, Yun Tang, Hualiang Jiang, Jian Li, Jin Huang,
Lubin Jiang, Jin Zhu
PII:
DOI:
Reference:
S1001-8417(17)30510-7
https://doi.org/10.1016/j.cclet.2017.11.041
CCLET 4356
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Chinese Chemical Letters
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Accepted date:
27-9-2017
25-11-2017
30-11-2017
Please cite this article as: Wenhua Chen, Xue Yao, Zhenghui Huang, Fei
Mao, Longfei Guan, Yun Tang, Hualiang Jiang, Jian Li, Jin Huang, Lubin
Jiang, Jin Zhu, Novel dual inhibitors against FP-2 and PfDHFR as potential
antimalarial agents: Design, synthesis and biological evaluation, Chinese Chemical
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Communication
Novel dual inhibitors against FP-2 and PfDHFR as potential antimalarial agents: Design,
synthesis and biological evaluation
Wenhua Chena,1, Xue Yaoa,1, Zhenghui Huangb,1, Fei Maoa, Longfei Guana, Yun Tanga, Hualiang Jianga,
a
a,
b,*
a,*

Jian Li , Jin Huang , Lubin Jiang , Jin Zhu
a Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
b Key Laboratory of Molecular Virology & Immunology, Unit of Human Parasite Molecular and Cell Biology, Institute Pasteur of Shanghai, University of
Chinese Academy of Science, Shanghai 200031, China
Graphical Abstract
A series of novel 2, 4-diaminopyrimidine-modified compounds were designed and synthesized. Compound 14 showed micromolar dual inhibitory effect on both
FP-2 and PfDHFR, and potential inhibition to the proliferation of P. falciparum 3D7 strain and chloroquine-resistant P. falciparum Dd2 strain.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Resistance to malaria parasites has quickly developed to almost all used antimalarial drugs. Cysteine
protease falcipain-2 (FP-2) and Plasmodium falciparum dihydrofolate reductase (PfDHFR) have crucial
roles, which are absolutely necessary, in the parasite life cycle. In this study, based on the uniform
pharmacophores of reported PfDHFR inhibitors and the first-generation dual inhibitors against FP-2 and
PfDHFR, we identified a novel series of dual inhibitors through fragments assembly. Lead optimization
Available online
led to the identification of 14, which showed potent inhibition against FP-2 and PfDHFR enzyme (IC50
=
6.8 ± 1.8 µmol/L and IC50 = 8.8 ± 0.3 µmol/L) and P. falciparum 3D7 strain (IC50 = 2.9 µmol/L).
Additionally, 14 exhibited more potent inhibition to the proliferation of chloroquine-resistant P.
falciparum Dd2 strain (IC50 = 1.1 µmol/L) than pyrimethamine (IC50 > 10 µmol/L), and 14 displayed
micromolar inhibitory activities against two clinical isolated strains Fab9 (IC50 = 2.6 µmol/L) and GB4
(IC50 = 1.0 µmol/L). Collectively, these data demonstrated that 14 might be a good lead compound for
the treatment of malaria.
Keywords:
Antimalarial drug
Plasmodium falciparum
FP-2
PfDHFR
Dual inhibitor
Malaria, a mosquito-borne disease caused by infection with Plasmodium parasites, is the world’s most deadly parasitic infection
[1,2]. Almost half the world’s population live in malaria endemic areas, and an estimated 1.2 billion people are at high risk of
contracting the disease [3-5]. According to WHO 2016, malaria caused 429,000 deaths and there were approximately 212 million
clinical cases of infection globally in 2015. Since the control of malaria has been severely compromised in recent years by the
widespread resistance to nearly all frontline therapeutics which were used for both prophylaxes and treatments [3, 6-8], hence, there is
an urgent need for the development and discovery of new antimalarial drugs, which are structurally distinct from existing drugs and
endowed with novel mechanisms of action [9].
Cysteine protease falcipain-2 (FP-2) of P. falciparum is an essential hemoglobinase of erythrocytic P. falciparum trophozoites
and provides the amino acids for the growth and proliferation of Plasmodium. Many in vitro and in vivo studies have confirmed that
inhibitors of FP-2 could block parasite hemoglobin hydrolysis, halt the development of culture parasites, and are effective against
 Corresponding authors.
E-mail addressese: huangjin@ecust.edu.cn (J. Huang), lbjiang@ips.ac.cn (L. Jiang), jinz@ecust.edu.cn (J. Zhu).
1 These authors contributed equally to this work.

murine malaria [10,11]. P. falciparum dihydrofolate reductase (PfDHFR) has received considerable attention for the prophylaxes and
treatments of P. falciparum infection. PfDHFR is one of the key enzymes in the process of DNA replication, and could catalyze 7,8-
dihydrofolate to transform into tetrahydrofolate [12,13]. Tetrahydrofolate serves as a necessary cofactor in the important one-carbon
transfer reactions in the biosynthetic pathways of pyrimidines, purines, and amino acids [14]. Thus, a more powerful pesticidal effect
could be achieved by inhibiting FP-2 and PfDHFR simultaneously. Such dual inhibitors might show a good synergetic effect, and
overcome the drug-resistance and be capable of providing “a combination therapy” in a single agent [15].
Previously, we reported the first-generation dual inhibitors against FP-2 and PfDHFR based on the compound 27, which was
randomly identified by screening FP-2 inhibitors in our laboratory, and gained compound 2o (28) which exhibited a high enzymatic
inhibition and a moderate in vivo antimalarial efficacy [15] (Fig. 1). Based on the SAR of the first-generation dual inhibitors [15], to
gain new scaffold dual inhibitors with more potencies, a novel series of dual inhibitors were designed, synthesized by displacing 4-
fluorophenyl with the uniform pharmacophores of reported PfDHFR inhibitors, 2,4-diamino heterocyclic fragments (Table S1 in
Supportion information). Considering the feasibility of the synthesis and novelty of scaffold, the sulfonamide was displaced with the
secondary amine (green, Fig. 1), and the amide position was interchanged (gray, Fig. 1). Therefore, 2,4-diaminopyrimidine analogues
with substituent (R, Fig. 1) at the terminal amide (7−26) were synthesized to obtain better dual target inhibitors and explore the SAR.
Fig. 1. Chemical modification strategies for the second-generation dual inhibitors.
First of all, we synthesized all the 2,4-diaminopyrimidine analogues (7−26) as described in Scheme 1. 2,4-Diaminopyrimidine-5-
carbonitrile (2) was prepared by reaction of guanidine carbonate (1) with ethoxymethylenemalononitrile in EtONa and EtOH in a 71%
yield. Further treatment of 2 with Nickel in MeOH under H2 for 24 h gave rise to 2,4-diaminopyrimidine-5-carbaldehyde (3) in an 80%
yield. Subsequent condensation of commercially available 4-nitrophenethylamine hydrochloride (4) with appropriate acids RCOOH in
the presence of HOBt, EDCI and DIPEA in N,N-dimethylacetamide afforded N-(4-nitrophenethyl)amide 5 in good yields. Reduction of
5 in the presence of 10% Pd/C and H2 in MeOH overnight in good yields provided N-(4-aminophenethyl)amide (6). Analogues 7−26
were performed by the reaction of 3 and 6, via the condition using NaCNBH3 in MeOH under reflux overnight in good yields (30%-
1
70%) and were characterized by H NMR and HRMS. The purity was over 95%, as determined by HPLC analysis (Table S2 in
Supportion information). Supplementary data associated with experiment section, reported PfDHFR inhibitors and HPLC analysis data
of compounds 7−26 can be found in Supporting information.
Scheme 1. Reagents and conditions: (a) Ethoxymethylenemalononitrile, EtONa/EtOH, 5 °C, 8 h; (b) Ni/H2, MeOH, 25 °C, 24 h; (c) HOBt, EDCI, DIPEA,
RCOOH, DMA, 25 °C, overnight; (d) 10% Pd/C, H2, MeOH, reflux; (e) NaCNBH3, MeOH, reflux, overnight.
Then the twenty analogues were evaluated for FP-2 enzymatic inhibitory activity and for PfDHFR enzymatic inhibitory activity
using the cysteine protease inhibitor (E-64) and pyrimethamine as the reference standard in the assay, respectively. And we assessed
the inhibition rate (IR) of all the analogues against FP-2 and PfDHFR at 10 µmol/L firstly. A set of analogues with various substituents
(R), including electron-donating substituted phenyl ring (9−13), electron-withdrawing substituted phenyl ring (14−17), hetero-aryl
rings (18−22) and alkyl groups (23−26), were evaluated for IR against FP-2 and PfDHFR, respectively. From the results in Table 1, 3
analogues, i.e., 11, 14 and 15, were identified as inhibitors against FP-2 with IR > 40% at 10 µmol/L and 19 analogues, i.e., 7−24, 26,
were identified as potent inhibitors against PfDHFR with IR > 70% at 10 µmol/L. Therefore, the IC50 value of them for the enzymatic
inhibitory activity were further evaluated.
Analysis of the data shown in Table 1 revealed some noteworthy observations from the SAR study of analogues 7−26: (1) The
inhibitory activities against FP-2 showed the R substituents which were phenyl rings with electron-donating groups (EDGs) preferred
3-position (9 vs. 10 and 11 vs. 12) and the R substituents which were phenyl rings with electron-withdrawing groups (EWGs) favored

4-position (14 vs. 15 vs. 16); (2) In the studied sets of the R substituents, the potency against FP-2 substantially increased in the order
of EWG-aryl > EDG-aryl > (aryl)alkyl; (3) In the test of inhibitory activities against PfDHFR, the R substituents could be well
tolerated and 19 analogues displayed high potencies (IR at 10 µmol/L > 70%); the potency against PfDHFR substantially increased in
the order of EDG-aryl > (aryl)alkyl > EWG-aryl.
From the results described above, analogue 14 was identified as a potent inhibitor against both FP-2 and PfDHFR (IC50 = 6.8
µmol/L against FP-2, IC50 = 8.8 µmol/L against PfDHFR). Therefore, analogue 14 and the lead compound 28 were next evaluated in
the inhibitory activity against the blood stage of the multi-drug-sensitive P. falciparum 3D7 strain and P. falciparum Dd2 strain, which
carry a phenotype of resistance to chloroquine [16,17]. Compound 28 displayed poor potencies against 3D7 strain (IR = 7.9% @ 20
µmol/L) and Dd2 strain (IR = 6.2% @ 20 µmol/L). As shown in Fig. S1 (Supporting information), analogue 14 together with
artemisinin (Art) were tested in 3D7 cells. Analogue 14 performed micromolar potencies against 3D7 parasites (IC50 = 2.9 µmol/L).
Moreover, as shown in Fig. S2 (Supporting information), 14 displayed micromolar potency against Dd2 (IC50 = 1.1 µmol/L) while
pyrimethamine presented less effecitive inhibition against Dd2 (IC50 > 10 µmol/L), which indicated analogue 14 could also inhibit the
growth of resistant P. falciparum. Furthermore, we evaluated the inhibitory activities of compound 14 against two clinical isolated
strains, Fab9 and GB4. To our delight, as shown in Fig. S3 (Supporting information), 14 also displayed micromolar potencies against
both strains (IC50 = 2.6 µmol/L against Fab9 and IC50 = 1.0 µmol/L against GB4).
Table 1
In vitro inhibitory activities against FP-2 and PfDHFR of analogues 7-26.
Compound
R
FP-2
PfDHFR
Inhibition
@ 10 μmol/L (%)
64.7
IC50 ± SD a
(μmol/L)
7.0 ± 2.0
Inhibition
@ 10 μmol/L (%)
59.2
IC50 ± SD a
(μmol/L)
6.3 ± 1.3
28 (2o)
E-64
n.t.b
57.2 ± 8.2 nmol/L
Pyrimethamine
7
25.5 ± 6.7 nmol/L
1.6 ± 0.1
23.3
100.0
8
9
30.0
35.8
89.7
6.3 ± 0.9
1.9±0.3
100.0
10
39.7
100.0
0.7±0.1
11
12
42.0
29.6
55.5 ± 11.0
87.1
81.7
3.1 ± 0.2
4.2 ± 0.3
13
38.4
91.8
1.3 ± 0.1
14
15
74.0
49.7
6.8 ± 1.8
74.5
87.0
8.8 ± 0.3
3.0 ± 0.3
36.8 ± 2.5
16
17
38.5
37.1
100.0
100.0
1.4 ± 0.2
1.5 ± 0.2
18
19
10.9
28.9
81.5
1.7±0.2
100.0
0.5 ± 0.2
20
21
10.2
11.7
82.7
78.8
1.5 ± 0.2
6.1 ± 0.4

22
23
24
25
34.5
5.6
94.6
100.0
91.5
42.8
0.7 ± 0.2
1.5 ± 0.3
1.9 ± 0.2
12.2
25.6
26
24.7
97.9
3.2 ± 0.2
a Average of more than two experiments.
b Indicates no test.
Moreover, to understand the structural basis for the inhibitory activities of the inhibitors against FP-2 and PfDHFR, the 3D
binding models of analogue 14 with FP-2 and PfDHFR were studied by molecular docking (Fig. 2). Fig. 2A shows the predicted
binding poses of 14 in the catalytic site of FP-2. The amide moiety of 14 directly interacted with Asn156 via H-bonds, meanwhile, the
terminal 2,4-diaminopyrimidine group and secondary amine interacted with Asp18 and Lys20 via H-bonds. For PfDHFR (Fig. 2B), the
catalytic subpocket formed around Asp54 were occupied by the 2,4-diaminopyrimidine group in 14. The 2,4-diaminopyrimidine ring
formed a complicated hydrogen-bond network with Asp54, Ile14, and Ile164, respectively, which contributed significantly to the
higher affinity of 14. Moreover, the amide group formed an H-bond with the key residue Arg122.
Fig. 2. Molecular docking studies on 14. Docked poses of 14 (green sticks) in the active sites of FP-2 (A) and PfDHFR (B), respectively. Key residues of the
binding pockets are shown as lines. Hydrogen bonds are shown with yellow dash lines.
In summary, we have identified a novel series of 2,4-diaminopyrimidine analogues (7−26) derived from 28 as FP-2 and PfDHFR
dual inhibitors. On the basis of the structure of the lead compound 28, 20 novel 2,4-diaminopyrimidine analogues have been
synthesized and tested in FP-2 and PfDHFR enzymatic inhibitory activity assays. Molecular docking studies showed that the amide
and secondary amine groups of new analogues could form hydrogen bonds with the surrounding amino acid residues, which were the
same as the first generation dual inhibitors [15], combining with the preliminary SARs, we speculated the amide and secondary amine
groups inherited FP-2 inhibitory activity from 28 and 2,4-diaminopyrimidine groups contributed to the inhibitory activity against
PfDHFR. The in vitro inhibitory activity against P. falciparum assays with multi-drug-sensitive strain 3D7 further confirmed that
analogue 14 was a good parasites inhibitor (IC50 = 2.9 µmol/L). To our delight, the inhibition of 14 against chloroquine-resistant
Plasmodium falciparum strain Dd2 (IC50 = 1.1 µmol/L) was more superior than pyrimethamine (IC50 > 10 µmol/L) and analogue 14
displayed micromolar inhibitory activities against two clinical isolated strains Fab9 (IC50 = 2.6 µmol/L) and GB4 (IC50 = 1.0 µmol/L).
Overall, 14 has the potential to be developed as a lead compound of antimalarial drugs.
Acknowledgement
Financial support for this research provided by the National Natural Science Foundation of China (Nos. 21372001 and
21672064), the “Shu Guang” project supported by the Shanghai Municipal Education Commission and Shanghai Education
Development Foundation (No. 14SG28), and the Fundamental Research Funds for the Central Universities are gratefully
acknowledged.
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