Discovery of new antimalarial agents: Second-generation dual inhibitors against FP-2 and PfDHFR via fragments assembely

Article abstract of DOI:10.1016/j.bmc.2017.10.017

Malaria parasites are a leading cause of worldwide mortality from infectious disease. Cysteine protease falcipain-2 (FP-2) and Plasmodium falciparum dihydrofolate reductase (PfDHFR) play vital roles, which are absolutely essential, in the parasite life cycle. In this study, based on the structures of uniform fragments 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 discovery of 24, which showed high potency against FP-2 (IC₅₀ = 10.0 μM), PfDHFR (IC₅₀ = 84.1 nM), P. falciparum 3D7 (IC₅₀ = 53.1 nM), clinical isolated strains Fab9 (IC₅₀ = 14.2 nM) and GB4 (IC₅₀ = 23.4 nM). The in vivo inhibition assays against P. berghei in 10 days indicated 24 had a more beneficial effect on the growth inhibition of P. berghei than artemisinin and an identical effect with pyrimethamine. Additionally, 24 moderately inhibited the proliferation of chloroquine-resistant P. falciparum Dd2 strain. Collectively, these data revealed that 24 could be an excellent lead compound as FP-2 and PfDHFR dual inhibitor for the treatment of malaria.

Full text of DOI:10.1016/j.bmc.2017.10.017

Accepted Manuscript
Discovery of New Antimalarial Agents: Second-Generation Dual Inhibitors
against FP-2 and PfDHFR via Fragments Assembely
Wenhua Chen, Zhenghui Huang, Wanyan Wang, Fei Mao, Longfei Guan, Yun
Tang, Hualiang Jiang, Jian Li, Jin Huang, Lubin Jiang, Jin Zhu
PII:
S0968-0896(17)31607-3
https://doi.org/10.1016/j.bmc.2017.10.017
BMC 14022
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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7 October 2017
16 October 2017
Please cite this article as: Chen, W., Huang, Z., Wang, W., Mao, F., Guan, L., Tang, Y., Jiang, H., Li, J., Huang, J.,
Jiang, L., Zhu, J., Discovery of New Antimalarial Agents: Second-Generation Dual Inhibitors against FP-2 and
PfDHFR via Fragments Assembely, Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.
2017.10.017
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Graphical Abstract
Discovery of New Antimalarial Agents: Second-Generation Dual Inhibitors against
FP-2 and PfDHFR via Fragments Assembely
Wenhua Chena,†, Zhenghui Huangb,†, Wanyan Wanga,†, Fei Maoa, Longfei Guana, Yun Tanga, Hualiang Jianga,
Jian Lia, Jin Huanga,*, Lubin Jiangb,*, Jin Zhua,*
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, 320 Yueyang Road, Shanghai
200031, China

Bioorganic & Medicinal Chemistry
jour nal homepage: w ww.elsevier. com
Discovery of New Antimalarial Agents: Second-Generation Dual Inhibitors against
FP-2 and PfDHFR via Fragments Assembely
Wenhua Chena,†, Zhenghui Huangb,†, Wanyan Wanga,†, Fei Maoa, Longfei Guana, Yun Tanga, Hualiang
Jianga, Jian Lia, Jin Huanga,*, Lubin Jiangb,*, Jin Zhua,*
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, 320 Yueyang Road, Shanghai 200031, China
† These authors contributed equally to this work.
* To whom correspondence should be addressed: jinz@ecust.edu.cn, huangjin@ecust.edu.cn or lbjiang@ips.ac.cn.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Malaria parasites are a leading cause of worldwide mortality from infectious disease.
Cysteine protease falcipain-2 (FP-2) and Plasmodium falciparum dihydrofolate reductase
(PfDHFR) play vital roles, which are absolutely essential, in the parasite life cycle. In this study,
based on the structures of uniform fragments 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 discovery of 24, which
showed high potency against FP-2 (IC50=10.0 µM), PfDHFR (IC50= 84.1 nM), P. falciparum
3D7 (IC50= 53.1 nM), clinical isolated strains Fab9 (IC50= 14.2 nM) and GB4 (IC50= 23.4 nM).
The in vivo inhibition assays against P. berghei in 10 days indicated 24 had a more beneficial
effect on the growth inhibition of P. berghei than artemisinin and an identical effect with
pyrimethamine. Additionally, 24 moderately inhibited the proliferation of chloroquine-resistant
P. falciparum Dd2 strain. Collectively, these data revealed that 24 could be an excellent lead
compound as FP-2 and PfDHFR dual inhibitor for the treatment of malaria.
Available online
Keywords:
Antimalarial drug
Plasmodium falciparum
FP-2
PfDHFR
Dual inhibitor
2017 Elsevier Ltd. All rights reserved.
1. Introduction
antimalarial drugs used clinically. Although antimalarial drugs
have successfully mitigated the epidemics in the past few
Malaria, a mosquito-borne disease caused by infection
decades, the control of malaria has been severely compromised
in recent years, on account of the widespread resistance of P.
falciparum to nearly all frontline therapeutics which were used
for both prophylaxes and treatments [2,13-18]. Consequently, the
requirement for the development and discovery of new
antimalarial drugs, which are structurally distinct from existing
drugs and endowed with novel mechanisms of action, is greatly
exigent. [19-25]
with Plasmodium parasites, is a devastating parasitic disease
causing widespread mortality and morbidity across many parts of
the developing world[1]. Nearly half the world’s population are
exposed in malaria endemic areas, and according to WHO 2016,
malaria is responsible for an estimated 1.2 billion clinical cases
of infection and 429,000 deaths in 2015 globally [2-3]. Children
below five years of age and pregnant women living in poor
countries are most vulnerable, it is considered that 303,000
malaria deaths happened in children aged under 5 years, which
represented 70% of the global total deaths. Malaria remained a
major killer of children, and it is estimated to take one child’s
life every 2 minutes [4-5]. Mammalian infection is initiated by the
bite of Plasmodium-infected female Anopheles mosquitoes.[6-7]
Among the species of plasmodia, P. vivax and P. falciparum are
responsible for the majority of malaria infections and
recrudescent infection via activation of dormant liver-stage
hypnozoites that re-establish the clinical blood-stage of
infection.[8-12] Natural products and their derivatives, including
artemisinin and its derivatives, are identified as first-line
In P. falciparum, various proteases catalyze the
degradation of human hemoglobin, and the amino acids derived
from this process are incorporated into parasite proteins or
utilized for energy metabolism.[26] Cysteine protease falcipain-2
(FP-2) of P. falciparum is an indispensable protease involved in
this metabolic process. FP-2, belonging to the family of cysteine
proteases (papain-like enzymes known as clan CA), is expressed
during the erythrocytic stage of the life cycle of the parasite.[27-28]
In the past several years, various types of FP-2 inhibitors have
been reported to be capable of inactivating the enzyme and a
large number of studies have confirmed that inhibitors of FP-2
could block parasite hemoglobin hydrolysis, halt the

development of culture parasites, and these inhibitors are
effective against murine malaria [29-39]. FP-2 has emerged as a
promising target for the development of novel antimalarial
drugs[40-43]
improve the pharmacological activity. Finally, a potent dual
inhibitor (24) effectively inhibited the proliferation of P.
falciparum, furthermore, the in vivo assays indicated 24 had a
more beneficial effect on the growth inhibition of P. berghei
than artemisinin. Thus, 24 was identified as a unique lead
compound for the development of antimalarial drugs.
P. falciparum dihydrofolate reductase (PfDHFR) has
received considerable attention for the prophylaxes and the
treatments of P. falciparum infection.[44-45] The de novo synthesis
of folate is required for DNA synthesis in Plasmodium species.
The final step of folate synthesis is the enzymatic reduction of
7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate, which is catalyzed
by the enzyme PfDHFR.[46] Furthermore, PfDHFR is an essential
metabolic enzyme that plays a critical role in one-carbon transfer
reactions, including the biosynthetic pathways for
deoxythymidine monophosphate (dTMP), purines, and several
amino acids,[47] which is necessary for the synthesis of DNA.
Consequently, 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
2. Results and discussion
2.1. Chemistry
As outlined in Scheme 1, tert-butyl(4-bromophenethyl)-
carbamate (2) was prepared from commercially available 2-(4-
bromophenyl)ethan-1-amine (1) in the presence of (Boc)2O in
CH2Cl2 at 25 °C for 4 h in an yield of 84%. 2 was stirred with n-
BuLi in THF at -78 °C under N2 for 1 h, then DMF was added
into the solution for another 1 h to give tert-butyl (4-
formylphenethyl)carbamate (3) in a yield of 70%. Further
treatment of 3 with HCl-dioxane at 25 °C for 4 h gave 4-(2-
aminoethyl)benzaldehyde (4) in a yield of 89%. Subsequent
condensation of 4 with appropriate acids RCOOH in the
presence of HOBt, EDCI and DIPEA in dichloromethane
afforded amide 5 in good yields. 6-Nitroquinazoline-2,4-diamine
(7) was prepared by reaction of guanidine carbonate (6) with 2-
chloro-5-nitrobenzonitrile in DMF under reflux overnight in a
yield of 60%. Reduction of 7 in the presence of 10% Pd/C and
H2 in MeOH and CH3COOH for 12 h in a yield of 80% provided
quinazoline-2,4,6-triamine (8). Analogues 9-29 was performed
by the reaction of 5 and 8, via the condition using NaCNBH3 in
MeOH under reflux overnight in 30%-70% yields. All target
compounds were confirmed to be ꢀ 95% purity (Table S1,
Supporting Information).
combination therapy” in a single agent [48]
.
In this study, based on the structures of uniform fragments
of reported PfDHFR inhibitors and the first-generation dual
inhibitors against FP-2 and PfDHFR identified by us previously,
[48]
a novel series of second-generation dual inhibitors against
FP-2 and PfDHFR had been designed through fragments
assembly. In an attempt to gain novel structures with high
potencies against both FP-2 and PfDHFR, we decided to explore
the 2,4-diaminoquinazoline analogues (Structure D, Figure 2) on
the basis of the computational analysis of three compounds
(compounds A-C, Table 2), we exhaustively evaluated whether
the type of substituent (R, Figure 2) at the terminal amide would
Scheme 1. Synthesis of target compounds 9-29.
Reagents and conditions: (a) (Boc)2O, CH2Cl2, 25 °C, 4 h, 84%; (b) n-BuLi, THF, DMF, -78 °C, N2, 2 h, 70%; (c) HCl-dioxane, 25 °C,
4 h, 89%; (d) HOBt, EDCI, DIPEA, RCOOH, 25 °C, overnight; (e) 2-Chloro-5-nitrobenzonitrile, DMF, reflux, overnight, 60%; (f) 10%
Pd/C, H2, MeOH, CH3COOH, 12 h, 80%; (g) NaCNBH3, MeOH, reflux, overnight, 30-70%.
2.2. Lead compound optimization and SAR
the compound 30, which was randomly identified by screening
FP-2 inhibitors in our laboratory, and gained compound 31 (2o )
which exhibited a high enzymatic inhibition and a moderate in
vivo antimalarial efficacy [48] (Figure 1). The SAR of the first-
generation dual inhibitors showed that, (1) the best length of the
middle phenyl linker was 2 methylene; (2) the sulfonamide and
amide were important fragments, which formed hydrogen bonds
All synthesized analogues were evaluated for FP-2
enzymatic inhibitory activity with the cysteine protease inhibitor
(E-64) as the reference and for PfDHFR enzymatic inhibitory
activity with pyrimethamine as the reference. We assessed the
inhibition rate (IR) of all the analogues against FP-2 and
PfDHFR at 10 µM at first. Previously, we reported the first-
generation dual inhibitors against FP-2 and PfDHFR based on
with the surrounding amino acid residues, and (3) the best
[48]
substituents were (hetero)aromatic rings
(Figure 1).

Considering the weak inhibitory activity against PfDHFR of the
first-generation dual inhibitors, we firstly summarized the
reported potent PfDHFR inhibitors (Table 1).
Furthermore, the binding power was predicted by docking
the three copounds (A, B, C) with proteins FP-2 and PfDHFR
(PDB entry: 2OUL and 1J3I, respectively). In the docking
process, standard-precision (SP) and extra-precision (XP)
docking modes were respectively adopted to generate the
minimized poses, the Glide scoring function (G-Score) was
referred to select at most 10 poses for A, B and C upon
visualized observation. As shown in Table 2, the lower predicted
activity indicated the better inhibitory activity, thus comparing
the predicted activity against FP-2 and PfDHFR, compund B was
the best one, which demonstrated the enzymatic activity of the
compound B might be the most potential one. Therefore, we
decided to evaluate whether the type of substituent (R, Structure
D, Figure 2) at the terminal amide would improve the
pharmacological activity and explore the SAR of the 2,4-
diaminoquinazoline analogues (9-29).
With analysis of SARs of the seven potent PfDHFR
[49-52]
inhibitors
, the terminal heteroaromatic rings (2,4-
diaminopteridine, 2,4-diaminoquinazoline, 2,4-diaminopyrimi-
dine, 2,4-diaminotriazine, which were all 2,4-diamino
heterocyclic fragments) were responsible for the inhibitory
activities against PfDHFR and these 2,4-diamino heterocyclic
fragments were the key pharmacophores. In an attempt to
improve the inhibitory activity against PfDHFR, the three potent
PfDHFR fragments were introduced into the left terminal
substituent Ar. Considering the feasibility of the synthesis and
novelty of scaffold, the sulfonamide was displaced with the
secondary amine (green, Figure 1), and the amide position was
interchanged (gray, Figure 1), therefore, three target compounds
A, B, C were planned to synthesis (Figure 2).
Figure 1. Chemical modification strategies for the second-generation dual inhibitors.
Figure 2. The target compounds A-C and structure D.
Table 1. Reported potent PfDHFR inhibitors.
PfDHFR
IC50/nM
Compd
Structure
Pharmacophore
Methotrexate
84
QN254
0.39
58
Pyrimethamine

P218
P65
4.6
229
Cycloguanil
WR99210
1.5
0.57
Table 2. Computational analysis of 31 and compounds (A-C).
Predicted activity
kcal/mola
Compound
FP-2
PfDHFR
-5.01
-5.22
-8.19
-9.59
-7.14
-5.07
-9.47
-8.10
aThe crystal structures of falcipain-2 (FP-2) binding with cystatin and P. falciparum dihydrofolate reductase (PfDHFR) binding with
WRA from Plasmodium falciparum were retrieved from the Protein Data Bank (PDB entry: 2OUL and 1J3I, respectively). Glide v5.5
(Schrodinger, Inc.) was used for molecular docking. The initial 3D structures of compound A-C were generated with the help of
LigPrep v2.3 (Schrodinger, Inc.).
First, a small set of analogues with various electron-
donating groups (EDGs) at the terminal phenyl ring (9-15, Table
3) were examined. Analogue 13 showed better IR against FP-2 at
10 µM than 14 (IR = 52.6% vs 21.2%), moreover, 11, which
introduced an extra 3-methoxyl in 14, slightly increased the IR
against FP-2 (IR = 31.3% vs 21.2%). Interestingly, 12, which
replaced the 3-methoxylphenyl in 13 with 3,5-dimethoxylphenyl,
decreased the inhibitory activity against FP-2 (IR = 41.1% vs
52.6%). Then we next brought in 1,4-benzodioxane (9) and 1,3-
benzodioxole (10) at the terminal amide. Analogue 10 displayed
the similar inhibitory activity against FP-2 as that of 31 (IR =
59.2% vs 64.7%), however analogue 9 showed poor inhibitory
activity (IR = 34.9%). And analogues 9-14 demonstrated similar
inhibitory activities against PfDHFR at 10 µM (IR = 95.1%-
100.0%), which were greater than 31 (IR = 59.2%).
62.4% vs 44.6%). Moreover, analogues 20-21 displayed
obviously different inhibitory activities (IR = 21.5% vs 67.0%).
Likewise, 16-18 and 20-21 showed potent inhibitory activities
against PfDHFR at 10 µM (IR = 94.7%-100.0%), by contrast 19
showed a poor inhibitory activity (IR = 68.2%).
With the intention of extending the R substitution scope at
the terminal amide, the phenyl ring was replaced with other
heteroaryl rings (22-25, Table 3). Adding a pyrid-4-yl group (22)
and a furan-2-yl group (23) (IR = 22.9% and 28.9%) showed
poor inhibitory activities against FP-2 obviously and 25 with 2-
naphthalene represented a moderate inhibitory activity (IR =
46.4%). Nevertheless, 24 with a thiophene-2-yl dramatically
enhanced the inhibitory activity against FP-2 (IR = 67.6%)
compared to 22 and 23. Additionally, 23-25 remained the potent
inhibitory activities against PfDHFR (IR = 97.4%-100.0%) as
well.
To further develop this 2,4-diaminoquinazoline scaffold,
we next explored analogues 16-21, in which the electron-
donating groups in the terminal phenyl were replaced by
electron-withdrawing groups (EWGs) (Table 3). Analogues 16-
18 with fluorophenyl showed the less inhibitory activities against
FP-2 at 10 µM (IR = 52.7%, 49.8%, 44.6% respectively) than 31
(IR = 64.7%) and the rank was 4-F > 3-F > 2-F. However,
analogue 19 which introduced an additional 4-chloro in 18
improved significantly the inhibitory activity against FP-2 (IR =
Then we further assessed the arylmethyl (26), arylethyl
(27) and alkyl groups (28-29) in the terminal amide. 26-29
displayed the similar inhibitory activities against FP-2 as 31 (IR
= 55.2%-66.6% vs 64.7%) and also remained potent inhibitory
activities against PfDHFR (IR = 92.4%-99.9%). Analysis of the
data shown in Table 3 revealed some noteworthy observations
from the SAR study of analogues 9-29: (1) the inhibitory

activities against FP-2 showed the phenyl substituents (R) with
EDGs preferred 3-position (11 vs 13 vs 14) and the phenyl
substituents with EWGs favored 4-position (16 vs 17 vs 18); (2)
in the studied sets of the R substituents, the potency against FP-2
substantially increased in the order of (aryl)alkyl > EWG-aryl >
EDG-aryl; and (3) in the test of inhibitory activities against
PfDHFR, the R substituents could be well tolerated and eighteen
analogues displayed high potencies (IR at 10 µM > 90%).
Table 3. In vitro inhibitory activities against FP-2 and PfDHFR of analogues 9-29
Inhibition @ 10ꢁM
IC50 SDa
Compd
R
-
FP-2 (%)
PfDHFR (%) FP-2 /ꢁM
PfDHFR /nM
64.7
59.2
7.0 2.0
6.3 1.3b
31(2o)
-
-
n.t.c
n.t.c
n.t.c
57.2 8.2d
n.t.c
E-64
Pyrimethamine
n.t.c
n.t.c
25.5 6.7
34.9
100.0
100.0
n.t.c
80.9 12.2
26.2 1.4
9
59.2
5.1 2.6
10
31.3
41.1
100.0
100.0
n.t.c
n.t.c
61.1 3.0
11
12
32.1 0.01
52.6
21.2
96.8
95.1
6.2 1.0
n.t.c
39.6 8.6
13
14
157.2 12.7
37.6
52.7
49.8
51.2
96.7
98.5
n.t.c
n.t.c
15
16
17
6.7 1.3
n.t.c
118.0 6.0
50.6 0.5
44.6
62.4
21.5
100.0
68.2
96.4
n.t.c
180.2 13.9
n.t.c
18
19
20
9.1 2.1
n.t.c
21.3 0.1
67.0
22.9
28.9
94.7
51.5
97.8
10.0 1.1
n.t.c
55.6 13.7
n.t.c
21
22
23
n.t.c
107.4 14.9
67.6
97.4
10.0 0.3
84.1 12.7
24

46.4
65.0
55.2
100.0
92.4
98.6
n.t.c
67.9 0.02
70.1 2.5
25
26
27
7.6 7.0
4.9 0.6
185.4 23.1
66.6
96.0
7.7 5.9
7.4 0.4
225.5 5.0
28
55.8
99.9
289.9 31.2
29
aAverage of more than two experiments. bUnit: ꢁM. cn.t. indicates no test. dUnit: nM.
2.3. Enzymatic Inhibition of Potent Analogues
PfDHFR substantially increased in the order of EDG-aryl >
EWG-aryl > (aryl)alkyl.
Furthermore, ten analogues, i.e., 10, 13, 16, 19, 21, 24 and
26-29, were identified as inhibitors against FP-2 with IR > 50%
at 10 µM and eighteen analogues, i.e., 9-14, 16-18, 20-21, 23-29,
were identified as potent inhibitors against PfDHFR with IR >
90% at 10 µM. Therefore, they were further evaluated for half
maximal inhibitory concentration (IC50) of two targets,
respectively. As shown in Table 3, the ten inhibitors against FP-2
demonstrated similar enzymatic inhibitory activities (IC50 = 4.9-
10.0 µM), 10 and 27 displayed slightly better than that of 31
(IC50 = 5.1 µM, 4.9 µM vs 7.0 µM). The eighteen potent
2.4. In Vitro Inhibitory Against Multi-Drug-Sensitive P.
falciparum 3D7
From the results described above, six analogues, including
10, 13, 16, 21, 24 and 26 were identified as potent dual inhibitors
against both FP-2 and PfDHFR (IC50 = 5.1-10.0 µM against FP-
2, IC50 = 26.2-118.0 nM against PfDHFR). Therefore, these six
analogues were next evaluated in the inhibitory activity against
the blood stage of the multi-drug-sensitive P. falciparum 3D7
strain. As shown in Figure 3, the selected analogues together
with artemisinin (Art) and compound 31 were tested in 3D7
cells. All the six analogues performed nanomolar potencies
against 3D7 parasites (IC50 = 53.1-390.9 nM), while 31
displayed poor potency against 3D7 parasites (Inhibition Rate
[IR] = 7.9% @ 20 µM), demonstrating that the structural
modification strategy employed in this study is efficient and
valuable for improvement of the antimalarial potency. Moreover,
13 and 24 showed the most potent activities against the parasites
(IC50 = 53.1 nM and 60.5 nM), thus the two analogues were
selected for further development.
inhibitors against PfDHFR presented more potent than 31 (IC50
21.3-289.9 nM vs 6.3 µM). Analogue 12 performed a better
inhibitory activity against PfDHFR than 11, 13 and 14 (IC50
=
=
32.1 nM vs 61.1 nM, 39.6 nM and 157.2 nM respectively),
which indicated the EDGs disfavored 4-position substituents.
And 17, 20 and 21 with EWGs displayed more potent activities
against PfDHFR than 16 and 18 (IC50 = 21.3-55.6 nM vs 118.0-
180.2 nM). Analogues 23-29 showed slightly less inhibitory
activities against PfDHFR than 9-14, which were substituted
with EDGs (IC50 = 67.9-289.9 nM vs 26.2-157.2 nM). In the
examination of the R substituents elicited the potency against
Figure 3. In vitro inhibitory activities against multi-drug-sensitive P. falciparum (3D7). (A) The inhibitory activities of analogues 10,
13, 16 and Art against P. falciparum (3D7); (B) the inhibitory activities of analogues 21, 24, 26 and Art against P. falciparum (3D7).
2.5. In Vivo Antimalarial Efficacy
significantly inhibition in parasitaemia of 20 mg/kg (body
weight) after the oral dose, moreover, 24 at 20 mg/kg
demonstrated similar antimalarial efficacy as that of
pyrimethamine, which were more effective than Art at 20 mg/kg.
Meanwhile, 13 at 20 mg/kg dose also significantly inhibited the
growth, although less effective than Art at 20 mg/kg.
To assess the in vivo antimalarial efficacies of 13 and 24,
the BALB/c mouse infected with the rodent parasite P. berghei
were orally dosed daily with 13 and 24 for 4 days (Figure 4) and
we used Art and pyrimethamine as the reference. 24 had a

Figure 4. In vivo antimalarial efficacies against rodent parasite Plasmodium berghei in the BALB/c mouse model.
2.6. In vitro inhibitory against chloroquine-resistant P.
falciparum Dd2 and clinical isolated strains
inhibition against Dd2 (IC50 > 10 µM) and compound 31
displayed similar poor potency against Dd2 (IR = 6.2% @ 20
µM), which indicated analogues 13 and 24 could also inhibit the
growth of resistant P. falciparum. Furthermore, we next
evaluated the inhibitory activities of compound 24 against two
clinical isolated strains, Fab9 and GB4. To our delight, as shown
in Figure 5B, 24 displayed potent inhibitory activities against the
two clinical isolated strains (IC50 = 14.2 nM against Fab9 and
IC50 = 23.4 nM against GB4). These results indicated 24 could
be a promising antimalarial lead compound.
The remarkable antimalarial efficacies of analogues 13
and 24 supported that the two compounds were potent
antimalarial agents, especially for 24. To further evaluate the
antimalarial potential of 13 and 24, we tested the two compounds
against P. falciparum Dd2 strain which carry a phenotype of
resistance to chloroquine [26, 53]. As shown in Figure 5A, 13 and
24 displayed micromolar potency against Dd2 (IC50 = 2.2 µM
and 1.2 µM), while pyrimethamine presented less effective
Figure 5. Inhibitory activities against chloroquine-resistant P. falciparum (Dd2) and two clinical isolated strains. (A)In vitro inhibitory
activities of analogues 13 and 24 against chloroquine-resistant P. falciparum (Dd2); (B) In vitro inhibitory activities of analogue 24 and
Art against two clinical isolated strains, Fab9 and GB4.
2.7. Molecular Docking Studies
occupied by the 2,4-diaminoquinazoline group in both 13 and
24. The 2,4-diaminoquinazoline groups formed a complicated
hydrogen-bond network with Asp54, Ile14, Cys15 and Ile164,
respectively, which contributed significantly to the higher
affinity of 13 and 24. In the both cases of PfDHFR, the amide
groups formed H-bonds with the key residue Arg122. The
binding modes indicated the 2,4-diaminoquinazoline group was
the critical fragment effecting the inhibitory activity against
PfDHFR, meanwhile, the improved PfDHFR inhibitory activities
of the twenty-one analogues also exhibited 2,4-
diaminoquinazoline group was the critical pharmacophore for
the PfDHFR inhibition.
To understand the structural basis for the inhibitory activities
of the inhibitors against FP-2 and PfDHFR, the 3D binding
models of analogues 13 and 24 with FP-2 and PfDHFR were
studied by molecular docking (Figure 6). Figures 6A and 6C
showed the predicted binding poses of 13 and 24 in the catalytic
site of FP-2, respectively. The amide moieties of 13 and 24
directly interacted with Asn156 and Gly66 via H-bonds,
meanwhile, the terminal 2,4-diaminoquinazoline group
interacted with Asp18 and Lys20 via H-bonds, and 13 formed an
additional H-bond with Trp189. For PfDHFR (Figures 6B and
6D), the catalytic subpocket formed around Asp54 were

Figure 6. Molecular docking studies on 13 and 24. Docked poses of 13 (green sticks) (A), and 24 (green sticks) (C) in the active sites of
FP-2. Docked poses of 13 (green sticks) (B), and 24 (green sticks) (D) in the active sites of PfDHFR. Key residues of the binding
pocket are shown as lines. Hydrogen bonds are shown with yellow dash lines.
Melting points were measured in capillary tubes on an SGW X-4
melting point apparatus without correction. Nuclear magnetic
resonance (NMR) spectroscopy was performed on a Bruker
AMX-400 NMR (TMS as internal standard). Chemical shifts
were reported in parts per million (ppm, ꢂ) relative to
tetramethylsilane. Proton coupling patterns were described as
singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and
doublet of doublets (dd). High-resolution mass spectra (HRMS)
were obtained on a Waters GCT Premie or Waters LCT using
electron ionization (EI). HPLC analysis of analogues 9-29 was
performed on an Agilent 1100 system equipped with a quaternary
pump and a diode-array detector (DAD). The peak purity was
verified by UV spectroscopy. All analogues were confirmed to be
ꢀ95% purity.
3. Conclusion
In summary, we have identified a novel series of 2,4-
diaminoquinazoline analogues (9-29) derived from 31 (2o) as FP-
2 and PfDHFR dual inhibitors. On the basis of the structure of
the lead compound 31, twenty-one completely novel 2,4-
diaminoquinazoline analogues have been synthesized and tested
in FP-2 and PfDHFR enzymatic inhibitory activities. Ten
analogues, i.e., 10, 13, 16, 19, 21, 24 and 26-29, showed good
inhibitory activities against FP-2 (IC50 = 4.9-10.0 µM) and
eighteen analogues, i.e., 9-14, 16-18, 20-21, 23-29, displayed
potent potencies against PfDHFR (IC50 = 21.3-289.9 nM). The
most potent analogue (20) demonstrated a PfDHFR inhibitory
capability that was about 295 times higher than 31. Preliminary
SARs were obtained, which showed that the amide and
secondary amine groups of new analogues inherited FP-2
inhibitory activity from 31 and 2,4-diaminoquinazoline groups
contributed to the dramatic enhancement of the inhibitory activity
against PfDHFR. The molecular docking results also validated
that the 2,4-diaminoquinazoline ring formed a complicated
hydrogen-bond network with Asp54, Ile14, Cys15 and Ile164 in
the catalytic subpocket of the PfDHFR enzyme. Additionally, in
the studied sets of the R substituents, the potency against FP-2
substantially increased in the order of (aryl)alkyl > EWG-aryl >
EDG-aryl, however, the potency against PfDHFR increased in
the order of EDG-aryl > EWG-aryl > (aryl)alkyl. The in vitro
inhibitory activity against P. falciparum with multi-drug-
sensitive strain 3D7 further confirmed that the two analogues (13
4.2. Synthesis of tert-butyl (4-bromophenethyl)carbamate (2)
To a suspension of 2-(4-bromophenyl)ethan-1-amine (1)
(3.1 mL, 20 mmol) in CH2Cl2 (30 mL) was added dropwise
(Boc)2O (5.97 mL, 26 mmol) at 25 °C and the solution was
stirred for 4 h. After the reaction completed, the solvent was
removed under reduced pressure. The residue was dissolved in
water, then the aqueous solution was extracted with ethyl acetate
(50 mL × 3). The combined organic layers were washed with
brine, dried over anhydrous sodium sulfate, and concentrated to
give the crude product, which was purified by column
chromatography to give tert-butyl(4-bromophenethyl)-carbamate
(2) (5.0 g, 84% yield) as a white solid.1H NMR (400 MHz,
CDCl3) δ: 7.42 (d, J = 8.2 Hz, 2H), 7.07 (d, J = 8.1 Hz, 2H), 4.52
(s, 1H), 3.35 (s, 2H), 2.75 (t, J = 6.9 Hz, 2H), 1.43 (s, 9H).
and 24) were the most potent inhibitors against parasites (IC50
=
53.1 nM and 60.5 nM). In the BALB/c mouse model, 24 (20
mg/kg) had a similar antimalarial efficacy as pyrimethamine (20
mg/kg), which were more effective than Art (20 mg/kg).
Meanwhile, 13 (20 mg/kg) dose also significantly inhibited the
growth, although less effective than Art (20 mg/kg). To our
delight, 13 and 24 inhibited chloroquine-resistant P. falciparum
strain Dd2 (IC50 = 2.2 µM and 1.2 µM) which were superior to
pyrimethamine (IC50 > 10 µM), and 24 also displayed potent
inhibitory activities against clinical isolated strains Fab9 and
GB4 (IC50 = 14.2 nM and 23.4 nM respectively). Overall, 24 has
the potential as a good antimalarial lead compound with novel
mechanisms of action.
4.3. Synthesis of tert-butyl (4-formylphenethyl)carbamate (3)
To
a
suspension of tert-butyl(4-bromophenethyl)-
carbamate (2) (1.50 g, 5 mmol) in THF (35 mL) was cooled to -
78 °C under N2. Then n-BuLi (4.58 mL, 11 mmol，2.4 M in
THF) was added dropwise at -78 °C and after the mixture was
stirred for 1 h, DMF(0.96 g, 12.5 mmol) was added dropwise at -
78 °C and the mixture was stirred at for another 1 hour. After the
reaction completed, the solvent was removed under reduced
pressure. The residue was dissolved in water, then the aqueous
solution was extracted with ethyl acetate (50 mL × 3). The
combined organic layers were washed with brine, dried over
anhydrous sodium sulfate, and concentrated to give the crude
product, which was purified by column chromatography to give
tert-butyl(4-formylphenethyl)-carbamate (3) (0.87 g, 70% yield)
as a yellow liquid.1H NMR (400 MHz, CDCl3) δ: 9.99 (s, 1H),
7.83 (d, J = 7.9 Hz, 2H), 7.37 (d, J = 7.9 Hz, 2H), 4.58 (s, 1H),
3.39 (m, 2 H), 2.89 (t, J = 7.0 Hz, 2H), 1.43 (s, 9 H).
4. Experimental
4.1. Synthesis General
Reagents and solvents were purchased from Adamas-Beta, J
& K, Energy Chemical, Target Molecule, TCI, Alfa Aesar, and
Acros, and were used without further purification. Analytical
thin-layer chromatography (TLC) was conducted on HSGF 254
plates (150–200 ꢁm thickness; Yantai Huiyou Co., China) and
spots were visualized with UV light. Yields were not optimized.
4.4. Synthesis of 4-(2-aminoethyl)benzaldehyde (4)
To
a
suspension of tert-butyl(4-formylphenethyl)-

4.7.1. N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methyl)phene-
thyl)-2,3-dihydrobenzo[b]-[1,4]dioxine-6-carboxamide (9)
carbamate (3) (0.75 g, 3 mmol) in dioxane (10 mL) was added
HCl-dioxane (1.5 mL, 6 mmol, 4 M in dioxane) in dropwise.
Then the mixture was stirred at 25 °C for 4 h. The solvent was
removed under reduced pressure and the residue was dissolved in
water, the aqueous solution was extracted with ethyl acetate (50
mL × 3). The combined organic layers were washed with brine,
dried over anhydrous sodium sulfate and concentrated to give the
crude product, which was purified by column chromatography to
give 4-(2-aminoethyl)-benzaldehyde (4) (0.40 g, 89% yield) as a
yellow liquid. 1H NMR (400 MHz, CDCl3) δ: 9.96 (s, 1H), 8.04–
7.96 (m, 2H).
Green solid, 32% yield. mp: 118–120 °C; 1H NMR (400
MHz, DMSO-d6)1H NMR (400 MHz, DMSO) δ: 8.75 (d, J =
11.0 Hz, 1H), 8.55 (s, 1H), 8.46 (t, J = 5.2 Hz, 1H), 7.99–7.82
(m, 1H), 7.60–7.31 (m, 6H), 7.30–7.11 (m, 4H), 6.92 (dd, J =
14.4, 7.7 Hz, 1H), 6.54 (t, J = 5.8 Hz, 1H), 4.16 (s, 2H), 3.64–
3.55 (m, 2H), 3.20–3.06 (m, 4H), 2.80 (t, J = 7.4 Hz, 2H). 13C
NMR (100 MHz, DMSO-d6) δ: 165.79, 163.01, 153.68, 146.42,
146.34, 143.30, 138.58, 137.59, 130.71, 129.01, 128.18, 127.99,
124.58, 121.04, 117.16, 116.62, 110.50, 102.06, 64.77, 64.44,
42.03, 41.33, 35.22. HRMS(EI) m/z calcd for C26H26N6O3(M+)
470.2066, found 470.2064.
4.5. Synthesis of 6-nitroquinazoline-2,4-diamine (7)
To a suspension of guanidine carbonate (6) (1.21 g, 10
mmol) and 2-chloro-5-nitrobenzonitrile (1.83 g, 10 mmol) in
DMF (50 mL) was stirred under reflux overnight. Then the
mixture was cooled to room temperature and the mixture was
dissolved in water, then the aqueous solution was extracted with
ethyl acetate (50 mL × 3). The combined organic layers were
washed with brine, dried over anhydrous sodium sulfate and
concentrated to give the crude product, which was purified by
column chromatography to give 6-nitroquinazoline-2,4-diamine
4.7.2. N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methyl)pheneth-
yl)benzo[d][1,3]dioxole-5-carboxamide (10)
Green solid, 35% yield. mp: 288–290 °C; 1H NMR (400
MHz, DMSO-d6) ꢂ 8.85 (t, J = 6.0 Hz, 1H), 8.70–8.41 (m, 3H),
8.09–7.90 (m, 2H), 7.60 (ddd, J = 6.8, 4.8, 1.7 Hz, 1H), 7.34 (d, J
= 8.0 Hz, 4H), 7.21 (dd, J = 7.3, 3.3 Hz, 5H), 6.51 (t, J = 5.9 Hz,
1H), 4.28 (d, J = 5.7 Hz, 2H), 3.52 (dd, J = 14.5, 6.5 Hz, 2H),
3.17 (d, J = 4.8 Hz, 1H), 2.84 (t, J = 7.5 Hz, 2H). 13C NMR (100
MHz, DMSO-d6) δ: 165.70, 162.68, 155.35, 150.03, 147.73,
145.38, 138.53, 137.79, 129.02, 128.19, 124.37, 122.50, 120.54,
110.72, 108.29, 107.66, 102.09, 101.47, 46.89, 41.42, 35.26.
HRMS(EI) m/z calcd for C25H24N6O3(M+) 456.1910, found
456.1911.
1
(7) (1.23 g, 60% yield) as a red solid. H NMR (400 MHz,
DMSO-d6) δ: 9.08 (d, J = 2.4 Hz, 1H), 8.22 (dd, J = 9.3, 2.4 Hz,
1H), 8.11–7.55 (m, 2H), 7.22 (d, J = 9.3 Hz, 1H), 6.95–6.56 (m,
2H).
4.6. Synthesis of quinazoline-2,4,6-triamine (8)
4.7.3. N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methyl)pheneth-
yl)-3,4-dimethoxybenza-mide (11)
To a suspension of 6-nitroquinazoline-2,4-diamine (7)
(1.03 g, 5 mmol) in MeOH (20 mL) and CH3COOH (1 mL) was
added 10% Pd/C (0.21 g) under H2, and the mixture was at 50 °C
stirred for 12 h. Then the reaction mixture was filtered, the
filtrate was removed under reduced pressure and the residue was
dissolved in water, then the aqueous solution was extracted with
ethyl acetate (50 mL × 3). The combined organic layers were
washed with brine, dried over anhydrous sodium sulfate and
concentrated to give the crude product, which was purified by
column chromatography to give quinazoline-2,4,6-triamine (8)
(0.70 g, 80% yield) as a yellow solid.1H NMR (400 MHz,
DMSO-d6) δ: 7.78 (s, 2H), 7.43 (s, 2H), 7.10 (d, J = 8.9 Hz, 2H),
7.07–7.02 (m, 1H), 5.24 (s, 2H).
Green solid, 42% yield. mp: 289–291 °C; 1H NMR (400
MHz, DMSO-d6) δ: 8.50 (t, J = 5.4 Hz, 1H), 7.89 (d, J = 8.4 Hz,
1H), 7.64 (d, J = 8.3 Hz, 1H), 7.41 (ddd, J = 36.7, 19.6, 5.0 Hz,
7H), 7.22 (d, J = 7.7 Hz, 4H), 7.00 (d, J = 8.4 Hz, 1H), 6.53 (t, J
= 5.9 Hz, 1H), 4.29 (d, J = 5.8 Hz, 2H), 3.80 (t, J = 6.4 Hz, 6H),
3.44 (dd, J = 14.3, 6.1 Hz, 2H), 2.87–2.76 (m, 2H). 13C NMR
(100 MHz, DMSO-d6) δ: 165.75, 162.75, 154.60, 147.50, 146.75,
144.43, 138.72, 137.80, 129.89, 128.30, 127.56, 124.40, 120.79,
116.92, 110.66, 108.59, 102.16, 60.22, 59.88, 44.10, 41.60,
35.20. HRMS(EI) m/z calcd for C26H28N6O3(M+) 427.2223, found
427.2222.
4.7. General procedure for Synthesis of N-(4-formylphenethyl)-
amide (5) and N-(4-(((2,4-diamino-quinazolin-6-
yl)amino)methyl)phenethyl)amide (9-29)
4.7.4. N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methyl)pheneth-
yl)-3,5-dimethoxybenza-mide (12)
Green solid, 45% yield. mp: 222–224 °C; 1H NMR (400
MHz, DMSO-d6) δ: 8.53 (s, 1H), 7.36 (d, J = 7.6 Hz, 2H), 7.20
(d, J = 8.4 Hz, 2H), 7.09–6.83 (m, 7H), 6.63 (s, 1H), 5.86 (s, 1H),
5.43 (s, 2H), 4.24 (s, 2H), 3.77 (s, 4H), 3.43 (s, 2H), 2.81 (s, 2H).
13C NMR (100 MHz, DMSO-d6) δ: 166.14, 162.47, 160.75,
158.11, 144.21, 138.36, 137.15, 129.44, 129.15, 128.97, 128.26,
124.78, 121.89, 113.05, 110.65, 105.53, 55.86, 47.16, 41.44,
35.21. HRMS(EI) m/z calcd for C26H28N6O3(M+) 427.2223, found
427.2220.
To a solution of corresponding acids (RCOOH, 0.63
mmol), HOBt (170 mg, 1.26 mmol), EDCI (242 mg, 1.26 mmol),
and DIPEA (0.208 mL, 1.26 mmol) were added to DMA (60
mL), and then 4 (186 mg, 1.26mmol) were added, the mixture
was stirred overnight. The mixture was dissolved in water, then
the aqueous solution was extracted with ethyl acetate (50 mL ×
3). The combined organic layers were washed with brine, dried
over anhydrous sodium sulfate and concentrated to give the crude
product, which was purified by column chromatography to give
N-(4-formylphenethyl)amide (5) in a good yield. Then the
mixture of 5 and 7 was added NaCNBH3 in MeOH and the
solution was stirred under reflux overnight. After reaction
completed, the solvent was removed under reduced pressure and
the residue was dissolved in water, the aqueous solution was
extracted with ethyl acetate (50 mL × 3). The combined organic
layers were washed with brine, dried over anhydrous sodium
sulfate and concentrated to give the crude product, which was
purified by column chromatography to afford the corresponding
compounds (9-29) in good yields.
4.7.5. N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methyl)pheneth-
yl)-3-methoxybenzamide (13)
Green solid, 55% yield. mp: 150–152 °C; 1H NMR (400
MHz, DMSO-d6) δ: 8.55 (t, J = 5.5 Hz, 1H), 8.35 (s, 2H), 7.45–
7.29 (m, 5H), 7.28–6.98 (m, 8H), 6.42 (s, 1H), 4.26 (d, J = 6.0
Hz, 2H), 3.77 (s, 3H), 3.44 (dd, J = 14.0, 6.1 Hz, 2H), 2.80 (t, J =
7.5 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 166.34, 162.05,
159.59, 158.06, 143.74, 138.38, 138.25, 136.50, 129.87, 128.98,
128.26, 124.44, 124.00, 119.79, 117.33, 112.81, 111.11, 101.00,
55.69, 47.18, 41.42, 35.24. HRMS(EI) m/z calcd for
C25H26N6O2(M+) 422.2117, found 422.2115.

Green solid, 44% yield. mp: 178–180 °C; 1H NMR (400
MHz, DMSO-d6) δ: 8.70 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 7.96
(d, J = 2.0 Hz, 2H), 7.87 (d, J = 8.0 Hz, 2H), 7.61–7.54 (m, 2H),
7.41 (d, J = 8.0 Hz, 2H), 7.37–7.20 (m, 6 H), 4.20 (s, 2H), 3.52
(dd, J = 12.7, 6.7 Hz, 2H), 2.91 (t, J = 6.9 Hz, 2H). 13C NMR
(100 MHz, DMSO-d6) δ: 168.68, 163.15, 160.60, 158.84, 143.42,
143.23, 138.52, 136.38, 134.90, 134.01, 129.72, 129.37, 128.90,
127.35, 125.87, 116.30, 110.71, 47.34, 41.52, 35.05. HRMS(EI)
m/z calcd for C24H22ClFN6O(M+) 464.1528, found 464.1530.
4.7.6. N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methyl)pheneth-
yl)-4-methoxybenzamide (14)
Green solid, 50% yield. mp: 279–281 °C; 1H NMR (400
MHz, DMSO-d6) δ: 8.40 (dd, J = 16.8, 11.4 Hz, 3H), 7.77 (d, J =
8.7 Hz, 2H), 7.30 (d, J = 7.8 Hz, 2H), 7.16 (t, J = 7.4 Hz, 7H),
6.94 (d, J = 8.7 Hz, 2H), 6.40 (s, 1H), 4.24 (d, J = 5.7 Hz, 2H),
3.76 (s, 3H), 3.40 (d, J = 5.7 Hz, 2 H), 2.77 (t, J = 7.5 Hz, 2H).
13C NMR (100 MHz, DMSO-d6) δ: 166.07, 162.26, 161.91,
157.25, 144.27, 138.51, 138.07, 129.38, 128.98, 128.23, 127.26,
124.13, 122.96, 113.91, 110.93, 101.15, 55.78, 47.09, 41.35,
35.37. HRMS(EI) m/z calcd for C25H26N6O2(M+) 422.2117, found
422.2119.
4.7.12. N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methyl)phene-
thyl)-4-(trifluoromethyl)be-nzamide (20)
Green solid, 65% yield. mp: 167–169 °C; 1H NMR (400
MHz, DMSO-d6) δ: 8.79 (t, J = 5.5 Hz, 1H), 8.44 (s, 1H), 8.10
(d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.1 Hz, 2H), 7.82 (d, J = 8.4 Hz,
2H), 7.73 (d, J = 8.1 Hz, 1H), 7.36 (s, 2H), 7.32 (d, J = 8.0 Hz,
2H), 7.23–7.16 (m, 3H), 7.11 (dd, J = 14.0, 6.3 Hz, 2H), 6.44 (t, J
= 5.9 Hz, 1H), 4.26 (d, J = 5.7 Hz, 2H), 3.47 (m, 2H), 2.82 (dd, J
= 14.7, 7.1 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 172.60,
165.82, 163.11, 162.89, 158.36, 151.20, 141.23, 140.04, 138.54,
132.01, 131.56, 129.54, 127.69, 123.91, 123.60, 116.35, 111.79,
102.32, 46.64, 41.33, 35.22. HRMS(EI) m/z calcd for
C25H23F3N6O(M+) 480.1885, found 480.1887.
4.7.7. 3-Chloro-N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methy
-l)phenethyl)-4-methoxyb-enzamide (15)
Green solid, 40% yield. mp: 141–143 °C; 1H NMR (400
MHz, DMSO-d6) δ: 7.58 (d, J = 8.2 Hz, 1H), 7.35 (d, J = 7.9 Hz,
2H), 7.27 (d, J = 8.2 Hz, 1H), 7.21 (d, J = 6.9 Hz, 3H), 7.16 (s,
2H), 7.01 (t, J = 7.6 Hz, 3H), 6.95 (s, 2H), 6.90 (s, 2H), 4.24 (d, J
= 6.3 Hz, 2H), 3.92 (s, 3H), 3.17 (s, 2H), 2.67 (s, 2H). 13C NMR
(100 MHz, DMSO-d6) δ: 164.86, 162.53, 157.08, 155.92, 145.02,
138.45, 137.91, 129.16, 129.00, 128.21, 127.97, 124.28, 121.21,
112.74, 110.80, 101.36, 56.86, 46.94, 41.41, 35.22. HRMS(EI)
m/z calcd for C25H25ClN6O2(M+) 476.1728, found 476.1726.
4.7.13. N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methyl)phene-
thyl)-4-nitrobenzamide (21)
4.7.8. N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methyl)pheneth-
yl)-4-fluorobenzamide (16)
Green solid, 70% yield. mp: 148–150 °C; 1H NMR (400
MHz, DMSO-d6) δ: 8.89 (t, J = 5.6 Hz, 1H), 8.50 (s, 2H), 8.27
(d, J = 8.8 Hz, 2H), 8.00 (d, J = 8.7 Hz, 2H), 7.30 (d, J = 7.9 Hz,
4H), 7.17 (t, J = 11.4 Hz, 6H), 6.47 (t, J = 5.9 Hz, 1H), 4.24 (d, J
= 5.7 Hz, 2H), 3.50–3.44 (m, 2H), 2.82 (dd, J = 17.0, 9.7 Hz,
2H). 13C NMR (100 MHz, DMSO-d6) δ: 172.60, 164.96, 163.21,
162.30, 157.26, 149.41, 140.63, 138.34, 137.74, 129.08, 129.04,
128.19, 124.51, 124.00, 115.45, 110.59, 101.72, 46.74, 41.53,
35.02. HRMS(EI) m/z calcd for C24H23N7O3(M+) 457.1862, found
457.1863.
Green solid, 43% yield. mp: 269–271 °C; 1H NMR (400
MHz, DMSO-d6) δ: 8.59 (s, 1H), 7.95–7.84 (m, 2H), 7.38–7.16
(m, 5H), 7.10–6.84 (m, 5H), 5.86 (s, 2H), 5.44 (s, 2H), 4.25 (d, J
= 6.2 Hz, 2H), 3.44 (s, 2H), 2.81 (s, 2H). 13C NMR (100 MHz,
DMSO-d6) δ: 174.59, 165.53, 163.02, 162.36, 157.01, 144.44,
139.74, 138.39, 138.07, 130.24, 130.15, 128.98, 128.24, 124.20,
115.75, 115.53, 110.82, 101.24, 47.04, 41.42, 35.22. HRMS(EI)
m/z calcd for C24H23FN6O(M+) 430.1917, found 430.1918.
4.7.9. N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methyl)phenet-
hyl)-3-fluorobenzamide (17)
4.7.14. N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methyl)phene-
thyl)isonicotinamide (22)
Green solid, 46% yield. mp: 265–267 °C; 1H NMR (400
MHz, DMSO-d6) δ: 8.70 (d, J = 5.7 Hz, 2H), 8.54 (s, 2H), 7.75–
7.65 (m, 1H), 7.61 (d, J = 9.8 Hz, 1H), 7.52 (dd, J = 13.7, 8.0 Hz,
2H), 7.38 (d, J = 7.9 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.26–7.17
(m, 4H), 6.53 (t, J = 6.3 Hz, 1H), 4.28 (d, J = 5.7 Hz, 2H), 3.46
(dd, J = 14.1, 6.5 Hz, 2H), 2.82 (t, J = 7.5 Hz, 2H). 13C NMR
(100 MHz, DMSO-d6) δ: 165.22, 163.03, 161.20, 153.70, 146.42,
138.46, 137.64, 130.89, 130.78, 129.03, 128.21, 124.64, 123.75,
118.17, 118.01, 114.51, 114.29, 110.52, 101.93, 46.71, 41.43,
35.11. HRMS(EI) m/z calcd for C24H23FN6O(M+) 430.1917,
found 430.1915.
Green solid, 40% yield. mp: 270–272 °C; 1H NMR (400
MHz, DMSO-d6) δ: 9.28 (s, 1H), 8.84 (d, J = 6.7 Hz, 2H), 8.69
(d, J = 5.6 Hz, 2H), 8.47 (s, 2H), 8.15 (d, J = 6.6 Hz, 1H), 7.69
(d, J = 4.4 Hz, 2H), 7.22 (m, 8H), 4.35 (m, 2H), 3.41 (m, 2H),
2.71 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 171.50, 164.62,
163.51, 162.29, 159.06, 152.30, 142.13, 141.14, 139.24, 133.61,
131.06, 128.24, 128.09, 125.11, 124.50, 124.32, 110.59, 46.44,
41.53, 35.21. HRMS(EI) m/z calcd for C23H23N7O(M+) 413.1964,
found 413.1965.
4.7.15. N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methyl)phene-
thyl)furan-2-carboxamide (23)
4.7.10. N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methyl)phene-
thyl)-2-fluorobenzamide (18)
Green solid, 41% yield. mp: 156–158 °C; 1H NMR (400
MHz, DMSO-d6) δ: 8.41 (t, J = 5.4 Hz, 1H), 8.05 (s, 2H), 7.78 (s,
1H), 7.30 (d, J = 7.8 Hz, 2H), 7.25–6.97 (m, 6H), 6.77 (s, 2H),
6.57 (s, 1H), 6.29 (s, 1H), 4.23 (d, J = 5.6 Hz, 2H), 3.39 (s, 2H),
2.78–2.70 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 162.99,
158.14, 153.83, 148.43, 146.34, 145.33, 138.40, 137.64, 131.26,
128.99, 128.21, 124.61, 118.27, 113.64, 112.25, 110.54, 101.87,
46.73, 45.85, 35.25. HRMS(EI) m/z calcd for C22H22N6O2(M+)
402.1804, found 402.1803.
Green solid, 40% yield. mp: 108–110 °C; 1H NMR (400
MHz, DMSO-d6) δ: 8.41 (s, 1H), 7.53 (dt, J = 21.1, 6.6 Hz, 2H),
7.36 (d, J = 7.8 Hz, 2H), 7.31–7.17 (m, 4H), 7.02 (q, J = 8.9 Hz,
2H), 6.94 (d, J = 14.8 Hz, 3H), 5.89 (t, J = 5.9 Hz, 1H), 5.47 (s,
2H), 4.25 (d, J = 5.8 Hz, 2H), 3.52–3.43 (m, 2H), 2.80 (t, J = 7.4
Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 164.07, 161.89,
160.77, 158.79, 158.29, 145.54, 143.31, 138.40, 138.19, 130.46,
130.43, 128.98, 128.26, 125.47, 124.92, 124.89, 123.89, 116.65,
116.43, 111.21, 100.90, 47.26, 41.34, 35.11. HRMS(EI) m/z
calcd for C24H23FN6O(M+) 430.1917, found 430.1920.
4.7.16. N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methyl)phenet-
hyl)thiophene-2-carboxa-mide (24)
Green solid, 46% yield. mp: 170–172 °C; 1H NMR (400
MHz, DMSO-d6) δ: 8.58 (s, 1H), 8.25 (s, 2H), 7.69 (d, J = 3.8
4.7.11. 4-Chloro-N-(4-(((2,4-diaminoquinazolin-6-yl)amino)meth
-yl)phenethyl)-2-fluoroben-zamide (19)

Hz, 2H), 7.30 (d, J = 7.6 Hz, 2H), 7.25–6.90 (m, 8H), 6.37 (s,
1H), 4.24 (d, J = 5.0 Hz, 2H), 3.38 (s, 2H), 2.83–2.68 (m, 2H).
13C NMR (100 MHz, DMSO-d6) δ: 162.88, 161.50, 154.38,
146.02, 140.60, 138.42, 137.74, 132.75, 131.02, 129.01, 128.38,
128.22, 124.54, 119.00, 110.62, 101.81, 46.79, 45.86, 35.29.
HRMS(EI) m/z calcd for C22H22N6OS(M+) 418.1576, found
418.1579.
3H), 2.67 (s, 3H), 2.33 (s, 2H), 1.99 (s, 3H), 1.74 (s, 3H), 0.80
(m, 9H). 13C NMR (100 MHz, DMSO-d6) δ:171.61, 162.42,
160.51, 143.00, 139.11, 136.02, 130.09, 127.97, 127.50, 127.06,
126.40, 125.47, 111.77, 101.65, 46.61, 36.65, 35.41, 33.05,
32.28, 31.02, 28.65, 25.90, 25.33. HRMS(EI) m/z calcd for
C28H38N6O(M+) 474.3107, found 474.3105.
4.8. Enzyme inhibition assays against FP-2 and PfDHFR
4.7.17. N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methyl)phene-
thyl)-2-naphthamide (25)
FP-2 (30 nM) was incubated with different concentrations of
tested compounds for 30 min at room temperature in the solution
of 100 mM sodium acetate and 10 mM DTT (pH 5.5).
Compound solutions were prepared from stock in DMSO
(maximum concentration of DMSO in the assay was 1%). After
Green solid, 57% yield. mp: 235–237 °C; 1H NMR (400
MHz, DMSO-d6) δ: 8.75 (s, 1H), 8.42 (s, 1H), 7.98 (d, J = 8.9
Hz, 3H), 7.91 (d, J = 8.7 Hz, 1H), 7.60 (s, 2H), 7.37 (d, J = 7.6
Hz, 2H), 7.23 (d, J = 7.7 Hz, 2H), 7.01 (t, J = 7.1 Hz, 2H), 6.93
(d, J = 21.9 Hz, 3H), 5.87 (s, 1H), 5.44 (s, 2H), 4.26 (s, 2H), 3.51
(s, 2H), 2.87 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 162.76,
161.85, 158.83, 146.45, 143.26, 138.37, 134.53, 132.43, 131.52,
129.28, 128.98, 128.08, 127.78, 127.19, 124.60, 123.87, 119.52,
111.22, 100.87, 46.50, 45.65, 35.30. HRMS(EI) m/z calcd for
C28H26N6O(M+) 462.2168, found 462.2166.
30 min incubation,
the substrate Z-Leu-Arg-AMC
(benzoxycarbonyl-Leu-Arg-7-amino-4-methylcoumarin, Bachem
AG) in the same buffer was added to a final concentration of 25
ꢁM. The increase in fluorescence (excitation at 355 nM and
emission at 460 nM) was monitored for 30 min at room
temperature
with
an
automated
microtiter
plate
spectrofluorimeter (Molecular Devices, Flex station). P.
falciparum DHFR inhibitory activity assays were carried out in
96-well plates in a total assay volume of 200 ꢁL. The
spectrophotometrically assay was based on measuring the
oxidation of NADPH to NADP+ at 340 nm. The purified DHFR
was incubated for 15 min on ice in assay buffer (100 uM
NADPH, 50 mM TES, pH 7.0, 1 mM EDTA, pH 8.0, 75 mM 2-
mercaptoethanol, 1 mg/mL bovine serum albumin), with various
concentrations of compounds to be tested. After 15min
incubation, the reaction was initiated with 100 ꢁM DHF. Half-
maximal inhibitory concentration (IC50) values were calculated
from plots of percentage inhibition of FP-2 and PfDHFR
activities against various compounds concentration using the
GraphPad Prism software with three independent experiments.
4.7.18. N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methyl)phene-
thyl)-2-phenylacetamide (26)
Green solid, 51% yield. mp: 220–223 °C; 1H NMR (400
MHz, DMSO-d6) δ: 8.67 (s, 1H), 8.54 (s, 1H), 8.07 (s, 1H), 7.76–
7.63 (m, 4H), 7.39 (s, 2H), 7.27–7.15 (m, 6H), 7.08 (d, J = 7.8
Hz, 2H), 6.52 (t, J = 5.7 Hz, 1H), 4.26 (d, J = 5.4 Hz, 2H), 4.14
(p, J = 11.1 Hz, 4H), 2.63 (t, J = 7.2 Hz, 2H). 13C NMR (100
MHz, DMSO-d6) δ: 173.07, 162.94, 154.12, 146.17, 141.70,
138.43, 137.61, 129.00, 128.56, 128.07, 126.87, 124.55, 118.53,
110.58, 101.96, 51.66, 46.69, 35.14, 33.12. HRMS(EI) m/z calcd
for C25H26N6O(M+) 426.2168, found 426.2168.
4.7.19. N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methyl)phenet-
hyl)-3-phenylpropanamid-e (27)
4.9. In vitro inhibition assays against P. falciparum
Green solid, 56% yield. mp: 218–221 °C; 1H NMR (400
MHz, DMSO-d6) δ: 7.82 (s, 1H), 7.28 (dd, J = 20.3, 7.7 Hz, 5H),
7.15 (d, J = 7.6 Hz, 3H), 7.09–6.86 (m, 6H), 5.85 (s, 1H), 5.43 (s,
2H), 4.23 (d, J = 5.3 Hz, 2H), 3.14 (d, J = 6.4 Hz, 2H), 2.98 (s,
2H), 2.53 (s, 2H), 2.37–2.22 (m, 2H). 13C NMR (100 MHz,
DMSO-d6) δ: 171.17, 161.86, 158.83, 145.69, 145.24, 143.25,
138.34, 138.28, 128.86, 128.64, 128.19, 127.88, 126.45, 125.56,
123.87, 111.22, 100.89, 47.20, 43.34, 42.23, 35.57, 35.31.
HRMS(EI) m/z calcd for C26H28N6O(M+) 440.2325, found
440.2323.
P. falciparum clones 3D7, Dd2, Fab9 and GB4 were
cultured in human O+ erythrocytes according to standard
procedures [18]. To prepare the > 80% ring stage parasites, the
asynchronous cultures of parasites were pretreated with 5%
sorbital, Plasmodium falciparum strains mentioned in the text at
the mid-ring stage (6-10 h postinvasion) were used to test
antimalarial effects in 96-well plates. Parasites were incubated in
a 96-well plate with 10, 13, 16, 21, 24, 26, Artemisinin, or
pyrimethamine at equal ratio containing 1% parasitemia, 2%
hematocrit for total 200 ꢁL. For 3D7 the compounds were from
the maximum concentration of 200 nM, for Dd2 were from 10
ꢁM as the maximum concentration. The parasites were allowed
to grow for 72 h at 37 °C with 5% CO2, 5% O2, 90% N2. After 72
h, add 100 ꢁL of lysis buffer (0.12 mg/mL Saponin, 0.12% Triton
X-100 30 mM Tris-Cl, 7.5 mM EDTA) consisting 5X SYBR
Green I (Invitrogen; supplied in 10000 × concentration) to each
well of the plate, the plates were then incubated for 2 h in dark
prior to reading the fluorescence signal on instrument at 485 nm
excitation and 535 nm emission.
4.7.20. N-(4-(((2,4-diaminoquinazolin-6-yl)amino)methyl)phene-
thyl)cyclohexanecarboxam-ide (28)
Green solid, 62% yield. mp: 118–120 °C; 1H NMR (400
MHz, DMSO-d6) δ: 11.83 (s, 1H), 8.62 (s, 1H), 8.49 (s, 1H), 7.70
(s, 1H), 7.35 (s, 2H), 7.27 (d, J = 8.0 Hz, 2H), 7.19 (s, 2H), 7.12
(d, J = 8.2 Hz, 2H), 6.49 (s, 1H), 4.24 (d, J = 5.8 Hz, 2H), 3.22–
3.12 (m, 2H), 2.68–2.59 (m, 2H), 1.99 (s, 2H), 1.59 (d, J = 19.2
Hz, 5H), 1.32 – 1.01 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ:
172.01, 161.20, 159.52, 142.20, 138.41, 137.52, 129.89, 128.67,
128.10, 127.01, 125.50, 124.67, 110.67, 100.65, 47.01, 36.75,
35.21, 33.55, 30.02, 25.50, 25.03. HRMS(EI) m/z calcd for
C24H30N6O(M+) 418.2481, found 418.2482.
4.10. In vivo inhibition assays
Three BALB/c mice per treatment group were infected
with 5 × 106 P. berghei ANKA strain parasites. Mice were
treated with 20 mg/kg orally of test compounds or control (Art
and pyrimethamine) each day for 4 days’ treatments after 24
hours. Parasitemia was quantified by Giemsa-stained blood
sample smear every day for a total 10 days’ observation.
4.7.21. 4-(Tert-butyl)-N-(4-(((2,4-diaminoquinazolin-6-yl)am-
ino)methyl)phenethyl)cyclohe-xane-1-carboxamide (29)
1
Green solid, 60% yield. mp: 99–102 °C; H NMR (400
MHz, DMSO-d6) δ: 7.75 (s, 1H), 7.70–7.63 (m, 1H), 7.33 (d, J =
7.7 Hz, 2H), 7.15 (s, 2H), 7.07–6.98 (m, 2H), 6.95 (s, 1H), 6.89
(s, 1H), 5.85 (s, 1H), 5.43 (s, 2H), 4.24 (s, 2H), 3.26–3.15 (m,
4.11. Molecular docking and binding power prediction

11. Gazzinelli, R. T.; Kalantari, P.; Fitzgerald, K. A.; Golenbock, D. T.
The crystal structures of falcipain-2 (FP-2) binding with
cystatin and P. falciparum dihydrofolate reductase (PfDHFR)
binding with WRA from Plasmodium falciparum were retrieved
from the Protein Data Bank (PDB entry: 2OUL and 1J3I,
respectively). All crystallographic water molecules were removed
from the coordinate set. Hydrogen atoms and charges were added
using the Protein Preparation Wizard in Maestro v9.0, and a
restrained partial minimization was terminated when the root-
mean-square deviation (rmsd) reached a maximum value of 0.3 Å
in order to relieve steric clashes. Glide v5.5 (Schrodinger, Inc.)
was used for molecular docking. The grid-enclosing boxes were
centered on the bound ligands and defined so as to enclose
residues located within 14 Å from the ligands in FP-2 and
PfDHFR, respectively. A scaling factor of 1.0 was set to van der
Waals (VDW) radii of those receptor atoms with the partial
atomic charge less than 0.25. The initial 3D structures of 31,
compunds A-C, analogues 13 and 24 were generated with the
help of LigPrep v2.3 (Schrodinger, Inc.). In the docking process,
standard-precision (SP) and extra-precision (XP) docking modes
were respectively adopted to generate the minimized poses, the
Glide scoring function (G-Score) was referred to select at most
10 poses for 13 and 24 upon visualized observation.
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Financial support for this research provided by the
National Natural Science Foundation of China (Grants 21372001
and 21672064), the “Shu Guang” project supported by the
Shanghai Municipal Education Commission and Shanghai
Education Development Foundation (Grant 14SG28), and the
Fundamental Research Funds for the Central Universities are
gratefully acknowledged.
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