Evaluation of the anti-malarial activity and cytotoxicity of 2,4-diamino-pyrimidine-based kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.08.055

A series of 2,4 diamino-pyrimidines have been identified from an analysis of open access high throughput anti-malarial screening data reported by GlaxoSmithKline at the 3D7 and resistant Dd2 strains. SAR expansion has been performed using structural knowledge of the most plausible parasite target. Seventeen new analogs have been synthesized and tested against the resistant K1 strain of Plasmodium falciparum (Pf). The cytotoxicity of the compounds was assessed in Vero and A549?cells and their selectivity towards human kinases including JAK2 and EGFR were undertaken. We identified compound 5n and 5m as sub-micromolar inhibitors, with equivalent anti-malarial activity to Chloroquine (CQ). Compounds 5d and 5k, μM inhibitors of Pf, displayed improved cytotoxicity with weak inhibition of the human kinases.

Full text of DOI:10.1016/j.ejmech.2016.08.055

Accepted Manuscript
Evaluation of the anti-malarial activity and cytotoxicity of 2,4-diamino-pyrimidine-
based kinase inhibitors
Oraphan Phuangsawai, Paul Beswick, Siriluk Ratanabunyong, Lueacha Tabtimmai,
Praphasri Suphakun, Phongphat Obounchoey, Pimonwan Srisook, Natharinee
Horata, Irina Chuckowree, Supa Hannongbua, Simon E. Ward, Kiattawee
Choowongkomon, M. Paul Gleeson
PII:
S0223-5234(16)30710-3
10.1016/j.ejmech.2016.08.055
EJMECH 8851
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 June 2016
Revised Date: 23 August 2016
Accepted Date: 24 August 2016
Please cite this article as: O. Phuangsawai, P. Beswick, S. Ratanabunyong, L. Tabtimmai, P. Suphakun,
P. Obounchoey, P. Srisook, N. Horata, I. Chuckowree, S. Hannongbua, S.E. Ward, K. Choowongkomon,
M.P. Gleeson, Evaluation of the anti-malarial activity and cytotoxicity of 2,4-diamino-pyrimidine-based
kinase inhibitors, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.08.055.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Evaluation of the anti-malarial activity and cytotoxicity of 2,4-diamino-
pyrimidine-based kinase inhibitors.
Oraphan Phuangsawaia, Paul Beswickb, Siriluk Ratanabunyongc, Lueacha Tabtimmaic, Praphasri
Suphakunc, Phongphat Obounchoeyc, Pimonwan Srisookc, Natharinee Horatad, Irina Chuckowreeb, Supa
Hannongbuaa, Simon E. Wardb,*, Kiattawee Choowongkomonc, and M. Paul Gleesona,
a Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
b School of Life Sciences, University of Sussex, Brighton, BN1 9QJ, United Kingdom
c Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
d Faculty of Medicinal Technology, Huachiew Chalermprakiet University, Samut Prakarn 10540, Thailand
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of 2,4 diamino-pyrimidines have been identified from an analysis of open access high
throughput anti-malarial screening data reported by GlaxoSmithKline at the 3D7 and resistant
Dd2 strains. SAR expansion has been performed using structural knowledge of the most
plausible parasite target. Seventeen new analogs have been synthesized and tested against the
resistant K1 strain of Plasmodium falciparum (Pf). The cytotoxicity of the compounds was
assessed in Vero and A549 cells and their selectivity towards human kinases including JAK2
and EGFR were undertaken. We identified compound 5n and 5m as sub-micromolar inhibitors,
with equivalent anti-malarial activity to Chloroquine (CQ). Compounds 5d and 5k, µM
inhibitors of Pf, displayed improved cytotoxicity with weak inhibition of the human kinases.
Available online
Keywords:
2,4 diaminopyrimidine
Plasmodium falciparum
Antimalarials
Drug design
Tyrosine kinase inhibitors
K1 strain
2009 Elsevier Ltd. All rights reserved
.
JAK2 kinase
1. Introduction
MMV development compound MMV390048 was proposed to be
phosphoinositide 4-kinase (PfPI4K).15 Additional protein kinases
have also been implicated as possible targets.16-18 A number of
studies aimed at improving our understanding of the
approximately 65 Pf specific kinases have been reported, many of
which share considerable homology with human kinases.10,16,19
This has led to kinase screening campaign20 and SAR studies on
specific protein kinases, including PfCDPK1,21,22, PfPK523 and
PfPK7.24 Nevertheless, it is not yet established which parasite
kinase represents the most valid target,10 or whether
polypharmacology approaches could represent a useful approach,
as found for cancer.25
Malaria is tropical infectious disease of humans and other
animals caused by mosquito-borne parasites that include
Plasmodium falciparum (Pf) and Plasmodium vivax (Pv). The
former causes the greatest percentage of deaths due to its more
severe symptoms. The disease is prevalent in tropical climates in
the African and Asian sub-continents, affecting countries with
less developed economies.1,2 In 2015 the World Health
Organization (WHO) reported that approximately 5.9 million of
children under five years of age died from this infectious
disease.3 To reduce the ongoing issue of drug resistance, the
current frontline treatment artemisinin is given as a combination
only, yet there are signs that its efficacy is under threat.
4-6
In 2010, GlaxoSmithKline reported the results of screening of
almost 2 million compounds to identify anti-malarial hits.
Approximately 13,500 compounds with activity at P. falciparum
(Pf) 3D7, multi-drug resistant Dd2 Pf strain and for cytotoxicity
in the HepG2 cell line were released in the public domain.26 In
2016, these hits were re-assessed at five Pf kinases (PfCDPK1,
PfCDPK4, PfPK6, PfPK7 and PfMAPK2) and led to the
identification of twelve series with potential for optimization.20
New
antimalarial drugs that target novel modes of action are therefore
highly desirable to tackle this disease.7-11
The malaria parasite has a complex life cycle in which
multiple kinase enzymes play an important role.12,13 The target of
the artemisinin has recently been suggested to be
phosphoinositide 3-kinase (PfPI3K),14 while the target of the
———
Corresponding authors. Tel.: +66-2-562-5555 extn. 2210; fax: +66-2-562-3955; e-mail: paul.gleeson@ku.ac.th, simon.ward@sussex.ac.uk

Table 1
Table 2
ACCEPTED MANUSCRIPT
Representative 2,4-diamino-pyrimidine hits identified from GSK HTS data.26
Pf 3D7 (IC50, µM) and HepG2 cytotoxicity (% at 10 µM) are reported for:
TCMDC-134115 (1a), TCMDC-134116 (1b), TCMDC-141384 (1c) and
TCMDC-141383 (1d).
Sequence similarity, identity to probable Pf and host kinases.
PDB
ID
% similarity
to PfCDPK1
% identity to
PfCDPK1
Gatekeeper
residue
ID
PfCDPK1
PbCDPK1
hAUR
-
100
91.8
45.1
36.1
33.9
100
87.0
25.3
18.6
15.4
Thr
Thr
Leu
Met
Met
R2
3Q5I
3UOL
4JI9
HN
N
R3
X
hJAK2
R1
N
N
hEGFR
3IKA
NH
3D7
IC50 (µM)
HepG2
(%)
ID
1a
1b
1c
1d
R1
CN
R2
R3
X
In this study we report the design, synthesis and biological
evaluation of new 2,4-diaminopyrimidine compounds. Our goal
is to improve our SAR understanding of this series by screening
additional analogs at the drug-resistant K1 Pf strain in the belief
that molecules which target either individual, or a defined subset
of kinases, offer potential new approaches to kill resistant strains.
We also assess the cytotoxicity of the series using monkey
kidney epithelial cell line (Vero), and the adenocarcinomic
human alveolar basal epithelial cell line (A549). The human
kinase activity of a subset are also evaluated for their potential to
inhibit the human protein kinases: JAK2, JAK3 and EGFR.
2-CONH2
2-CONH2
3-NO2
Me
H
NCH3
NCH3
NCH3
O
0.009
99
43
16
19
CN
0.040
0.138
0.759
CONHCH3
CONHCH3
H
3-OiPr
H
Our separate analysis of the GSK screening data led to the
identification of a cluster of 2,4-diaminopyrimidine compounds
with good activity, efficiency, physical properties and synthetic
tractability (Table 1). The compounds display activity at the
multidrug resistant Dd2 strain, whilst the HepG2 SAR suggests
an anti-malarial vs cytotoxicity window of 50 fold is achievable
(Table 1). 1b is a confirmed inhibitor of PfCDPK1 (0.012 µM),
CDPK4 (0.089 µM) and PfPK6 (0.054 µM). Host kinase
interactions also require consideration as these could either lead
to cytotoxicity or also potentially facilitate parasite
eradication.17,27
2. Results
2.1. Design
The mode of action of compounds 1a-d is unconfirmed,
however PfCDPK1, hAUR, hJAK and EGFR are probable
targets. A focused library of 2,4-diamino-pyrimidines derivatives
were prepared using established synthetic methods and directed
towards improving our understanding the link between Pf
activity, cytotoxicity and activity at key kinases in the hosts.
The most probable human kinases targeted by 1a-d include
Janus kinase 2 (hJAK2),28 Aurora A hAURKA,29,30 Indeed, other
hJAK and hAUR inhibitor classes have shown anti-malarial
activity from repurposing studies.10,31 The 2,4-diaminopyrimidine
chemotype also appears in Epidermal growth factor (hEGFR),32
and c-Jun N-terminal kinase (hJNK)33 publications, as well as in
patents targeting cancer-related pathways via hJAK34 and
hAURK (Figure 1).35 Kinases inhibitors offer great potential as
anti-malarials due to the wealth of chemical, biological and
safety data amassed over many years to treat diseases in human,
potentially reducing the development burden.
Crystal structures of the Plasmodium Berghei (Pb) CDPK1
homolog and the principal human targets were downloaded for
analysis (Table 2). Despite dramatic differences in sequence
identity, the conserved ATP binding site means inhibitors can
have considerably cross reactivity.10 A key difference between
these targets is the moderately sized, non-polar gatekeeper for the
human proteins, compared to the smaller, more polar Thr
sidechain.37
We have undertaken modification to the chemotype at four
different positions as highlighted in Figure 2. Region 1 exploits
difference at the gatekeeper while region 2 (pyrimidine 4
position) targets differences within the kinase backpocket.38
Modifications at the latter position have resulted in dramatic
changes in the hAURA protein conformation though the flipping
of the DFG-loop despite only subtle changes in substitution of
the 4-position aniline.39 Modifications at region 3 will induce
subtle changes in ring conformation, which can benefit
selectivity, while region 4 can be exploited to improve compound
solubility.40 Anti-malarial SAR shows that compounds with R1=
-CN have higher anti-malarial activity, however, this is mirrored
by increases in the % cytotoxicity. We therefore evaluated the
effect of incorporation of -H, -CN, -OMe and CONH2. A range
of substituted anilines at the 2- and 4- positions were selected to
exploit interaction toward the kinase solvent pocket and
backpocket, respectively, and maintain reasonable molecule
properties (Table 3). We attempted to bias the reagent selection
to focus on lower MWT (mean=405 da) and clogP (mean=4.04).
This was with the goal of increasing the likelihood of achieving
better solubility and permeability and solubility, a pre-requisite
for reliable phenotypic testing.
NHMe
SO2NtBu
O
CH3
CH3
HN
N
HN
N
N
NC
N
H3C
N
N
N
N
NH
NH
TCMDC-134116 (1b), PfCDPK1 IC50= 12 nM
TG101209, hJAK2 IC50= 6 nM
O
H2C
NH
NHMe
O
O
O
HN
O
Cl
N
Cl
N
N
N
N
NH
N
NH
OMe
OMe
WZ4002, hEGFR IC50= 16 nM
SID103904601, hAURKA IC50= 4 nM
Fig. 1. 2,4-diamino-pyrimidines kinase inhibitors; TCMDC 134116,20
TG101209,28 SID103904601 (www.pubchem.com) and covalent inhibitor
WZ4002.32,36

ACCEPTED MANUSCRIPT
gatekeeper
1
H
hinge
N
NH
CH 2
N
3
HN
H
3
N
4
protein
solvent
O
Fig. 2. Schematic (left) and docked model of 2,4 diamino-pyrimidines to PbCDPK1. The binding model of 5k (carbons in grey) bound to the X-ray crystal
structure of PbCDPK1 (3Q5I, magenta). The model was constructed using the JAK2- TG101209 complex (4JIA, cyan).41 Hydrogen bonds to the hinge and
structural waters are indicated by dotted lines.
2.2. Synthesis
activity 0.57 µM, Mefloquine 0.07 µM and artemisin at 0.002
µM.
Intermediates were synthesized using the methodology
described in Scheme 1. 2a was produced from the reaction of p-
fluoronitrobenzene and piperazine in DMSO with the assistance
of K2CO3. The amine product was hydrogenated to 3a using Pd/C
and H2. Intermediates 3b & 3c were produced in the same
manner.
Systematic analysis of the results presented in Table 3
suggests that compounds with –H at R1 are preferred for anti-
malarial activity (as exemplified by 5a,r). R2 position shows a
preference for anilines with R2 = 3-Me (5a,r) or 2-CONH2
(5k,m). The R3 position does not display a clear preference
towards the substituents employed. R4 shows
a general
Compound 1a was synthesized following Scheme 2. The Cl at
the 4-position of 5-cyano-2,4-dichloropyrimidine was substituted
by p-aminobenzamide with the assistance of DIPEA giving
intermediate 4a (Scheme 2). Intermediate 4a was reacted at with
3a assisted of DIPEA, leading to substitution at the 2-position.
Compounds 5a-e were synthesized in an analogous manner to 1a.
Compound 5e was oxidized using hydrogen peroxide to yield
compound 5f. Compound 5g, having an -OMe substituent at the
5-position was synthesized using the same protocol as 1a.35
Compounds 5h-r, with H at the 5-position, were synthesized as
follows; nucleophilic substitution at the 4-position of 2,4-
dichloro pyrimidine was achieved using DIPEA base for R2=3-
SO2NH2 and 3-CH3, a HCl promoted reaction for R2=2-CONH2.
Nucleophilic attack at the corresponding 2-position was achieved
using TFA to promote the reaction.
preference towards Me-piperazine, compared to morpholine or
the simpler substituted anilines.
2.3.2. Cytotoxicity
Compounds 1a, 5a-r were evaluated for their ytotoxicity at
10 µM against the Vero cell line (ATCC CCL-81) using the
method described by Hunt and co-workers.44 The compounds
show a range in potency from 25-96%. A degree of correlation
between activity and cytotoxicity, with the most potent
compounds (5n,o) also displaying high (94%) cytotoxicity. This
compares to 50% for chloroquine, a standard anti-malarial
treatment. Compounds 5d, 5k and 5m display µM anti-malarial
activity and low to moderate cytotoxicity at 31, 66 and 35%,
respectively
Nineteen compounds were synthesized (Table 3) with reaction
yields varied from low (<30%) to excellent (>90%). Among the
19 compounds synthesized, compounds 1a and 5j. 1a was
originally reported in the study of Gamo et al26 and in the recent
paper of Crothers et al.20 5j was reported as a JNK2 inhibitor by
Song et al targeted towards anti-cancer therapies.33
Five compounds were taken forward for further assessment;
potent but cytotoxic compounds (5n,o), moderately potent and
cytotoxic compounds (5k), and moderately potent and weakly
cytotoxic compounds (5d,m). EC50s against the Vero (ATCC
CCL-81) and A549 (ATCC CCL-185) cells using an MTT based
method (Table 4).45 The EC50’s are in good agreement with the %
cytotoxicity data, with 5d,k&m demonstrating better activity
than compounds 5n,o. The A549 cytotoxicity data broadly
mirrors those obtained in the Vero cell line. The lower EC50
values would be consistent with the higher expression of certain
kinases in this disease tissue.46-50
2.3. Biological testing
2.3.1. Anti-malarial activity
The titled compounds 1a, 5a-r were evaluated for their in
vitro anti-malarial activity at the Pf K1 strain (EC50) using a
hypoxanthine incorporation assay.42,43 The compounds show a
range in potency from 0.43-8.97 μM. Compound 1a, 0.009 µM
against the 3D7 strain, was found to be 3.9 µM at the resistant K1
strain. Compounds 5n and 5o were 0.65 and 0.43 µM
respectively. In comparison, standards chloroquine displayed an
No clear SAR is apparent at the R1 and R2 positions.
Hydrogen is preferred at the R3 position and -H and morpholine
are preferred at the R4 position. Compounds 5k and 5m show a
reasonable balance of anti-malarial activity and cytotoxicity,
approaching the desired 50-fold window.

.
ACCEPTED MANUSCRIPT
X
X
F
H
N
N
N
R3
(a)
(b)
+
R3
R3
X
NO2
O2N
2a-c
R3=CH3, X=NCH3, R3=H, X=NCH3, R3=H, X=O
H2N
3a-c
a
b
c
Scheme 1. General scheme for the synthesis of anilines 5a-c. Reagents and conditions for 3a: (a) K2CO3, DMSO, rt, 4-6 h, (b) Pd/C, H2, MeOH, rt, overnight.
Cl
N
R2
R3
R2
R2
R3
HN
N
R1
HN
N
(a)
(b)
N
R1
R4
R1
+
+
N
N
Cl
NH2
NH2
NH
Cl
4a-i
5a-r
Scheme 2. Synthetic approach used for the preparation of compounds 1a, 5a-r. Reagents and conditions for; 5a: (a) DIPEA, n-BuOH, 110 oC overnight, (b)
DIPEA, DMF, rt, 4 h; 5i, (a) DIPEA, n-BuOH, 110 oC overnight, (b) TFA, isopropanol, overnight.
Table 3
Activities and yields of compounds a1, 5a-r.
P.f. K1 IC50
(ꢀM)
Vero Cytotox.
ID
R1
R2
R3
R4
%yielda
clogPd
MWTd
(%/50 ꢀM)
CN
CN
o-CONH2
Me
Me
Me-piperazine
Me-piperazine
25
23
4.7
3.1
443
479
3.93
94
91
1a
5a
m-SO2NH2
5.56
3-Me & 4-
Methylpiperazine
CN
SO2NH2
H
15
3.1
NAe
33
5b
479
478
535
315
333
484
418
403
390
454
440
427
389
374
361
290
433
320
378
284
CN
CN
CN
CONH2
OMe
H
m-SO2CH3
m-SO2NHtBu
m-Me
Me
Me
Me
Me
Me
Me
H
Me-piperazine
Me-piperazine
H
26
39
50b
48c
91
67
56
11
70
62
75
80
82
79
1.5
20
-
4.4
5.4
5.1
3.4
3.1
4.8
4.3
4.2
3.3
2.8
2.7
5.2
4.7
4.6
5.2
4.0
3.9
4.1
2.8
4.72
4.56
NAe
92
31
40
96
88
92
91
68
66
80
35
94
94
74
94
25
45
-
5c
5d
5e
5f
m-Me
H
NAe
m-SO2NH2
o-CONH2
o-CONH2
o-CONH2
m-SO2NH2
m-SO2NH2
m-SO2NH2
m-Me
Me-piperazine
Me-piperazine
Me-piperazine
morpholine
Me-piperazine
Me-piperazine
morpholine
Me-piperazine
Me-piperazine
morpholine
H
5.26
1.06
1.50
4.39
3.86
7.25
8.97
0.647
0.431
NAe
5g
5h
5i
H
H
H
5j
H
Me
H
5k
5l
H
H
H
5m
5n
5o
5p
5q
5r
H
Me
H
H
m-Me
H
m-Me
H
H
m-Me
Me
H
NAe
H
4-phenylmorpholine
morpholine
NAe
Chloroquine
0.57
0.07
0.00254
Mefloquine
-
Dihydroartemisinine
-
-
b
a%yield obtained from di-substitution reaction at 2- and 4- position using condition c%yield obtained from oxidation reaction of compound 7.dCalculated
logarithm of the octanol/water distribution coefficient using JChem Version 14.9.100.707. eCompound not active.

Table 4
4. Experimental Details
ACCEPTED MANUSCRIPT
Activity of compounds selected for kinase selectivity and further cytotoxicity
assessment.
4.1. Chemistry
Reagents and solvents obtained from commercial suppliers
were used without further purification. All reactions were
monitored by TLC aluminum/silica gel plate with UV light
visualization. Column chromatography was performed using
silica gel 60 (40-63 µm). 1H NMR spectra were recorded on both
Varian instrument at 400 MHz and on a Bruker instrument at 500
MHz. Chemical shifts were reported in ppm (δ) using the residual
solvent line as the internal standard. Mass spectra (MS) were
performed on Agilent 1100 HPLC instrument coupled to a
LC|MSD Trap mass spectrometer, in ESI(+) mode or APCI
mode. Compounds showed a purity ≥ 95% as determined from
the corresponding UV absorbance HPLC chromatogram.
IC50, μM
JAK3
>100
4.1
EC50, μM
Vero
ID
JAK2
>100
0.126
0.129
0.05
EGFR
>150
>150
>100
>150
11.0
A549
> 100
4.2
5d
5k
5m
5n
5o
45.4
>100
37.0
2.0
7.1
9.4
1.8
3.5
0.14
2.1
1.0
7.1
2.3.3. Protein kinase activity
Compounds 1a, 5a-r were evaluated for their in vitro
inhibition of human kinases JAK2, JAK3 and EGFR (Table 4).
Inhibition was determined by using an Antibody BeaconTM Tyrosine
Kinase Assay Kit (A-35725; Invitrogen, USA). Compounds 5k,m-o
display sub µM inhibition at hJAK2 kinase, µM inhibition at hJAK3
and all compounds display >10 µM inhibition at hEGFR.
4.2. Preparation of anilines 3a-c
4.2.1. 3-methyl-4-(4-methylpiperazin-1-yl)aniline (3a)
A mixture of 1-fluoro-2-methyl-4-nitro-benzene (3.22 mmol),
1-methylpiperazine (3.63 mmol) and potassium carbonate (3.83
mmol) in dimethyl sulfoxide (4 ml) were stirred and heated to
100 °C for 4 hours. The resultant reaction solution was cooled
down to room temperature, added water (50 ml) to the reaction
mixture, filtered off the solid residue, washed with water (50 ml),
and air-dried to give the desired product of 1-methyl-4-(2-
methyl-4-nitrophenyl-piperazine (2a) as a brown solid, 77%
Compounds 5m and 5o, the most potent anti-malarials, but also
the most cytotoxic of compounds are more active at the human
kinases assessed. 5o is a confirmed inhibitor of all three protein
kinases while compound 5n inhibits JAK2 at 0.05 µM. In contrast,
5d shows minimal inhibition of the kinases and also displays good
cytotoxicity at both the Vero and A549 cell lines.
1
yield. H NMR (500 MHz, Chloroform-d) δ 8.04 (d, J=8.3 Hz,
2H), 7.01 (d, J=8.4 Hz, 1H), 3.07 (t, J=4.8 Hz, 4H), 2.67-2.57
(m, 4H), 2.39 (s, 3H), 2.37 (s, 3H). [M+H]+, ESI m/z 236.29.
MW: 235.28 g/mol.
Inhibiting multiple Pf kinase targets without harming human
cells will be a challenge, particularly since many kinase
compounds come from human anti-cancer projects.20 Yet, there is
considerable safety and tolerability information in relation to
kinase inhibition profiles that could be leveraged to develop
molecules with a more targeted pharmacology. Furthermore,
tackling host-parasite target interactions is also a feature of
Afterwards, the obtained compound, 2a was hydrogenated.
Pd/C (2.78 mmol) was added to a stirred solution of 1-methyl-4-
(2-methyl-4-nitrophenyl-piperazine (5.53 mmol) in methyl
alcohol (15 ml) in N2 atmosphere. Following this it was
hydrogenated overnight by maintaining the reaction flask in an
atmosphere of H2 gas (balloon). The resultant was filtered
thorough celite plate and concentrated under vacuum. The crude
product was purified with silica-gel column chromatography
(5%MeOH/DCM, 1%NH3) to yield the desired product, 3a as a
established DHFR anti-malarials. Methotrexate,
a hDHFR
inhibitor used in cancer treatment, also has potential as a
PfDHFR anti-malarial if used at lower doses.51
1
brown solid, 87% yield. H NMR (500 MHz, DMSO-d6) δ 6.74
3. Conclusions
(d, J=8.4 Hz, 1H), 6.38 (d, J=2.7 Hz, 1H), 6.34 (dd, J=8.4, 2.7
Hz, 1H), 4.61 (s, 2H), 2.68 (t, J=4.8 Hz, 4H), 2.41 (s, 4H), 2.20
(s, 3H), 2.10 (2, 3H). [M+H]+, ESI m/z 206.23. MW: 205.30
g/mol.
We identified a series of diamino-pyrimidines anti-malarial
from mining the screening data reported by GSK.26 We
performed the design, synthesis and testing of new analogs to
reveal additional SAR with respect to the K1 strain, cytotoxicity
at the Vero and A549 cell lines as well as inhibition at
representative human kinases.
4.2.2. 4-(4-methylpiperazin-1-yl)aniline (3b)
A solution of 4-fluoronitrobenzene (7.1 mmol) and potassium
carbonate (8.52 mol) in dimethyl sulfoxide (2 ml) was stirred at
room temperature for 0.5 hour. 1-methylpiperazine (7.1 mmol)
was added dropwise in the mixture solution. The resulting
reaction mixture was stirred at room temperature for 6 hours.
The mixture was then poured into cold water. A yellow
precipitate formed and was collected to give 1-methyl-4-(4-
nitrophenyl)piperazine (2b), 93% yield. 1H NMR (400 MHz,
CDCl3) δ 8.16 – 8.09 (m, 2H), 6.86 – 6.80 (m, 2H), 3.53 – 3.46
(m, 4H), 2.70 – 2.60 (m, 2H), 2.42 (s, 3H). [M+H]+, APCI m/z
222.1. MW: 221.26 g/mol.
Sixteen new compounds have been synthesized and evaluated
against the K1 resistant Pf strain. The compounds displayed
activity comparable to chloroquine. We identified 5n and 5o as
having sub µM anti-malarial activity. These compounds were
were also identified as being potent human kinase inhibitors as
well as being cytotoxic. Compounds 5d and 5k were identified as
µM inhibitors with improved cytotoxicity and human kinase
selectivity.
Results suggest series has potential but further work needed to
explore the strain dependence and maximize the anti-
malarial/potency window. Additional work is required to
establish the molecular target(s) of this compound class.
Following this it was hydrogenated using the same procedure
as compound 2a to yield the desired product, 3b as a brown solid,
100% yield. 1H NMR (400 MHz, DMSO) δ 6.69 – 6.64 (m, 2H),
6.50 – 6.44 (m, 2H), 4.53 (s, 1H), 2.91 – 2.85 (m, 4H), 2.45 –

4c was obtained as a side product from the reaction of 4b,
2.37 (m, 4H). methyl proton overlaps with AtheCsColEvePntTpEeaDk. MANUSCRIPT
[M+H]+, APCI m/z 192.2. MW: 191.27 g/mol.
whereby substitution at the 2-position occurred. Purified by
silica-gel column chromatography (10%EtOAc/hexane), a beige
solid, yield 35%, 1H NMR (500 MHz, DMSO-d6) δ 10.56 (s, 1H),
8.79 (s, 1H), 7.99 (t, J = 1.8 Hz, 1H), 7.80 (ddd, J = 8.1, 2.2, 1.1
Hz, 1H), 7.67 (dt, J = 7.9, 1.4 Hz, 1H), 7.60 (t, J = 7.9 Hz, 1H),
7.40 (s, 2H). MS-ESI: m/z 310.06 [M+H]+. MW: 309.73 g/mol.
4.2.3. 4-(morpholin-4-yl)aniline (3c)
A mixture of 1-bromo-4-nitrobenzene (5.0 mmol), morpholine
(5.0 mol) and potassium carbonate (6.0 mmol) in dimethyl
sulfoxide (2 ml) were stirred and heated to 100 °C for 7 hours.
The resultant reaction solution was allowed to cool to room
temperature. The mixture solution was extracted with EtOAc.
The organic layer was collected, washed with brine, dried over
Na2SO4, filtered, and dried under reduced pressure. The crude
product was purified with silica-gel column chromatography
(30% EtOAc/hexane) to give the desired product of 4-(4-
nitrophenyl)morpholine, 2c as a yellow solid, 47% yield. 1H
NMR (400 MHz, CDCl3) δ 8.18 – 8.09 (m, 2H), 6.86 – 6.79 (m,
2H), 3.88 – 3.82 (m, 4H), 3.39 – 3.31 (m, 4H).
4.3.4. 2-chloro-4-(3-methylsulfonylanilino)pyrimidine-5-
carbonitrile (4d)
3-(methylsulfonyl)aniline hydrochloride is used as an
aminobenzene reagent, followed the general procedure and
purified by silica-gel column chromatography (5%MeOH/DCM)
1
to yield 4d as a yellow solid, yield 13%, H NMR (500 MHz,
DMSO-d6) δ 11.14 (s, 1H), 8.99 (s, 1H), 8.31 (s, 1H), 7.97 (dt, J
= 6.4, 2.4 Hz, 1H), 7.69 – 7.62 (m, 2H), 3.21 (d, J = 1.4 Hz, 3H).
MS-ESI: m/z 309.0 [M+H]+. MW: 308.74 g/mol.
Following this compound 2c was hydrogenated using the
same procedure as compound 2a to yield the desired product, 3c
as a purple solid, 97% yield. 1H NMR (400 MHz, DMSO) δ 6.70
– 6.65 (m, 2H), 6.51 – 6.47 (m, 2H), 4.57 (s, 2H), 3.71 – 3.65 (m,
4H), 2.91 – 2.84 (m, 4H). [M+H]+, APCI m/z 179.1. MW: 178.23
g/mol.
4.3.5. N-tert-butyl-3-[(2-chloro-5-cyano-pyrimidin-4-
yl]benzenesulfonamide (4e)
N-t-butyl-3-aminobenesulfonamide is used as an
aminobenzene reagent, followed the general procedure and
purified
by
silica-gel
column
chromatography
(0-
1
5%MeOH/DCM) to yield 4e as a beige solid, yield 30%, H
NMR (500 MHz, DMSO-d6) δ 11.06 (s, 1H), 8.94 (s, 1H), 8.21
(s, 1H), 7.85 – 7.81 (m, 1H), 7.56 (dd, J = 2.6, 1.4 Hz, 1H), 7.55
(d, J = 4.6 Hz, 2H), 1.12 (s, 9H). MS-ESI: m/z 366.08 [M+H]+.
MW: 365.84 g/mol.
4.3. General procedure for the preparation of intermediate
compounds 4a-e
DIPEA (0.86 mmol) was added to the solution of 2,4-
dichloropyrimidine-5-carnonitrile (0.57 mmol) in DMF (0.5 ml)
and stirred at room temperature for 10 minutes. A solution of
aminobenzene reagent corresponding to desired product (0.57
mmol) in DMF (0.5 ml) was added dropwise to the reaction
mixture and stirred for 1 hour. The resultant was evaporated
under vacuum and extracted with EtOAc. The organic layer was
collected, washed with brine, dried over MgSO4, filtered, and
dried under reduced pressure. 4c was obtained from the reaction
of 4b, whereby substitution at the 2- position occurred.
4.4. General procedure for the preparation of intermediate
compounds 4f
4.4.1. 3-[(2-chloro-5-methoxypyrimidin-4-yl)amino]benzene-1-
sulfonamide (4f)
3-aminobenzenesulfonamide (3.07 mmol) and DIPEA (8.38
mmol) was added to the mixture of 2,4-dichloro-5-
methoxypyrimidine (2.79 mmol) in 1,4-dioxane (15 ml). The
reaction mixture was stirred at 100°C for 3 days. The solvent was
evaporated under reduce pressure and extracted with EtOAc. The
organic layer was collected, washed with brine, dried over
MgSO4, filtered, and dried under reduced pressure. The crude
product was purified with column chromatography (5%
MeOH/DCM) to give the desired product, 4f as a light yellow
solid with a 22% yield, 1H NMR (500MHz, DMSO-d6) δ 9.58 (s,
1H), 8.29-8.24 (m, 1H), 8.00 (s, 1H), 7.99-7.95 (m, 1H), 7.56-
7.52 (m, 2H), 7.34 (s, 2H), 3.95 (s, 3H). MS-ESI: m/z 315.28
[M+H]+. MW: 314.75 g/mol.
4.3.1. 2-[(2-chloro-5-cyano-pyrimidin-4-yl)amino]benzamide
(4a)
2-aminobenzamide is used as an aminobenzene reagent,
followed the general procedure and purified by silica-gel column
chromatography (25%EtOAc/hexane) to yield 4a as a yellow
solid, yield 41%, 1H NMR (500 MHz, DMSO-d6) δ 12.23 (s, 1H),
8.95 (s, 1H), 8.40 (d, J = 8.4 Hz, 1H), 8.31 (s, 1H), 7.84 (dd, J =
7.9, 1.5 Hz, 1H), 7.80 (s, 1H), 7.58 (t, J = 7.6 Hz, 1H), 7.21 (t, J
= 7.6 Hz, 1H), MS-ESI: m/z 273.67 [M+H]+. MW: 273.68 g/mol.
4.3.2. 3-[(2-chloro-5-cyano-pyrimidin-4-
yl)amino]benzenesulfonamide (4b)
4.5. General procedure for the preparation of compounds 4g-4i
4.5.1. 2-[(2-chloropyrimidin-4-yl)amino]benzamide (4g)
2-aminobenzensulfonamide is used as an aminobenzene
reagent, followed the general procedure and purified by silica-gel
column chromatography (10%EtOAc/hexane) to yield 4b as a
A mixture of 2,4-dichloropyrimidine and 2-aminobenzamide
in 0.1M HCl was stirred at 100°C for 3 hrs. The precipitated
obtained was filtered and the product was washed with water and
recrystallized in methanol. The crystal product was collected and
air-dried to give the desired intermediate compound, 4g as a light
1
beige solid, yield 32%, H NMR (500 MHz, DMSO-d6) δ 11.07
(s, 1H), 8.95 (s, 1H), 8.20 (s, 1H), 7.89 – 7.83 (m, 1H), 7.61 –
7.55 (m, 2H), 7.39 (s, 2H). MS-ESI: m/z 309.89 [M+H]+. MW:
309.73 g/mol.
1
green solid, with a 39% yield. H NMR (400 MHz, DMSO-d6) δ
11.24 (s, 1H), 8.18 (dd, J = 11.3, 7.1 Hz, 3H), 7.76 (dd, J = 7.9,
1.5 Hz, 1H), 7.70 (s, 1H), 7.56 – 7.50 (m, 1H), 7.17 (td, J = 7.7,
1.1 Hz, 1H), 6.87 (d, J = 5.9 Hz, 1H). MS-APCI: m/z, 249.1
[M+H]+. MW: 248.67 g/mol.
4.3.3. 3-[(4-chloro-5-cyano-pyrimidin-4-
yl)amino]benzenesulfonamide (4c)

4.5.2. 3-[(2-chloropyrimidin-4-yl)amino]benzenesulfonamide
(4h)
4.6.3. 3-[[5-cyano-4-[3-methyl-4-(4-methylpiperazin-1-
yl)aniline]pyrimidin-2-yl]amino]benzenesulfonamide (5b)
ACCEPTED MANUSCRIPT
A
solution
of
2,4-dichloropyrimidine,
3-
Compound 3a as the amino-intermediate and 4c as the chloro-
intermediate, following the general procedure. Purified by silica-
gel column chromatography (5%MeOH/DCM, 1%NH3), a beige
aminobenzenesulfonamide and DIPEA in n-BuOH was stirred at
110 °C for 13 hrs. The reaction mixture was cooled down to
room temperature, concentrated under reduce pressure, and
extracted with EtOAc. The organic layer was collected, washed
with brine, dried over Na2SO4, filtered, and dried under reduced
pressure. The crude product was purified with column
chromatography (10% MeOH/DCM) to give the desired
intermediate compound, 4h as a beige solid, with a 48% yield. 1H
NMR (400 MHz, DMSO-d6) δ 10.31 (s, 1H), 8.23 (d, J = 5.9 Hz,
1H), 8.03 (d, J = 1.8 Hz, 1H), 7.90 (d, J = 7.9 Hz, 1H), 7.54 (tt, J
= 4.6, 2.9 Hz, 2H), 6.80 (s, 1H). MS-APCI: m/z 285.1 [M+H]+.
MW: 284.72 g/mol.
1
solid, yield 15%, H NMR (500 MHz, DMSO-d6) δ 9.67 (s, 1H),
8.48 (s, 1H), 8.35 (d, J = 2.5 Hz, 1H), 7.71 (dt, J = 6.6, 2.3 Hz,
1H), 7.51 (dd, J = 5.0, 2.8 Hz, 2H), 7.30 (d, J = 7.0 Hz, 2H), 6.74
(d, J = 8.4 Hz, 1H), 6.41 (d, J = 2.4 Hz, 1H), 6.34 (dd, J = 8.5,
2.6 Hz, 1H), 4.66 (s, 2H), 2.72 (d, J = 5.4 Hz, 4H), 2.16 (s, 3H).
MS-ESI: m/z 479.12 [M+H]+. MW: 478.58 g/mol.
4.6.4. 2-[3-methyl-4-(4-methylpriperazin-1-yl)aniline]-4-(3-
methylsulfonylanilino)pyrimidine-5-carbonitrile (5c)
Compound 3a as the amino-intermediate and 4d as the chloro-
intermediate, following the general procedure. Purified by silica-
gel column chromatography (0-10%MeOH/DCM, 1%NH3), a
yellow solid, yield 26%, 1H NMR (500 MHz, DMSO-d6) δ 10.12
(s, 1H), 9.42 (s, 1H), 8.52 (s, 1H), 8.10 (dd, J = 8.0, 2.1 Hz, 1H),
7.98 (s, 1H), 7.50 (dt, J = 7.8, 1.3 Hz, 1H), 7.37 (s, 1H), 7.30 (d,
J = 11.7 Hz, 2H), 7.02 (d, J = 8.4 Hz, 1H), 3.11 (s, 3H), 2.85 (t, J
= 4.6 Hz, 4H), 2.24 (s, 4H), 2.22 (s, 3H), 1.98 (s, 1H). MS-ESI:
m/z 478.25 [M+H]+. MW: 477.59 g/mol.
4.5.3. 2-chloro-N-(3-methylphenyl)pyrimidin-4-amine (4i)
4i was prepared using the same procedure as 4h, with a 25
hours of reaction time. A desired intermediate product 4i was
1
obtained as beige solid, with a 65% yield, H NMR (400 MHz,
DMSO-d6) δ 9.94 (s, 1H), 8.13 (dd, J = 5.8, 3.2 Hz, 1H), 7.40 (d,
J = 7.8 Hz, 1H), 7.34 (s, 1H), 7.25 (t, J = 7.8 Hz, 1H), 6.92 (d, J
= 7.3 Hz, 1H), 6.75 – 6.72 (m, 1H), 2.30 (s, 3H), MS-APCI: m/z
220.1 [M+H]+. MW: 219.67 g/mol.
4.6.5. N-tert-butyl-3-(5-cyano-2-(3-methyl-4-(4-methylpiperazin-
1-yl)phenylamino)pyrimidin-4-ylamino)benzene-sulfonamide
(5d)
4.6. General procedure for the synthesis of compound 1a, 5a-d
A chloro-intermediate compound (4a-e) (0.22 mmol) and
DIPEA (0.33 mmol) were dissolved in DMF (0.5 ml). A solution
of amino-intermediate compound (3a-c) (0.2 mmol) in DMF (0.5
ml) was then added dropwise into the solution. The reaction
mixture was stirred at room temperature for 4 hours. The
resultant solution was concentrated under vacuum, and extracted
with EtOAc. The organic layer was collected, washed with brine,
dried over MgSO4, filtered, and dried under reduced pressure.
Compound 3a as the amino-intermediate and 4e as the chloro-
intermediate, following the general procedure. Purified by silica-
gel column chromatography (5%MeOH/DCM, 1%NH3), a beige
solid, yield 39%, 1H NMR (500 MHz, DMSO-d6) δ 10.06 (s, 1H),
9.38 (s, 1H), 8.49 (s, 1H), 8.00 (d, J = 8.2 Hz, 1H), 7.90 (s, 1H),
7.44 – 7.40 (m, 2H), 7.29 (d, J = 16.0 Hz, 3H), 7.02 (d, J = 8.5
Hz, 1H), 2.85 (t, J = 4.7 Hz, 4H), 2.25 (s, 3H), 2.22 (s, 3H), 1.09
(s, 9H). 13C NMR (101 MHz, DMSO) δ 164.71, 162.44, 160.56,
159.89, 158.68, 147.38, 144.62, 139.53, 133.06, 132.33, 122.64,
119.88, 119.15, 118.21, 117.79, 116.86, 116.39, 53.00, 50.92,
46.34, 35.66, 29.88, 21.80, 17.43, 13.93. MS-ESI: m/z 535.24
[M+H]+. MW: 534.68 g/mol.
4.6.1. 2-[[5-cyano-2-[3-methyl-4-(4-methylpiperazin-1-
yl)aniline]pyrimidin-4-yl]amino]benzamide (1a)
Compound 3a as the amino-intermediate and 4a as the chloro-
intermediate, following the general procedure. Purified by silica-
gel column chromatography (5%MeOH/DCM, 1%NH3) to give
4.7. General procedure for the synthesis of compound 5e-g
4.7.1. 2,4-bis(3-methylanilino)pyrimidine-5-carbonitrile (5e)
1
the desired product, 1a with a 25% yield as a beige solid. H
NMR (500 MHz, DMSO-d6) δ 11.67 (s, 1H), 9.51 (s, 1H), 8.49
(s, 1H), 8.39 (d, J = 8.5 Hz, 1H), 8.20 (s, 1H), 7.74 (dd, J = 7.9,
1.5 Hz, 1H), 7.69 (s, 1H), 7.33 (d, J = 2.5 Hz, 1H), 7.29 – 7.24
(m, 1H), 7.20 (t, J = 8.0 Hz, 1H), 7.03 (dd, J = 16.6, 8.2 Hz, 2H),
2.87 (t, J = 4.7 Hz, 4H), 2.25 (s, 3H), 2.24 (s, 3H). MS-ESI: m/z
443.13 [M+H]+. MW: 442.53 g/mol.
3-methylaniline (0.29 mmol) and DIPEA were added to a
solution of 2,4-dichloropyrimidine-5-carbonitrile (0.29 mmol) in
DMF (5 ml). The reaction mixture was stirred at room
temperature for 4.5 hrs. The reaction mixture was then extracted
with EtOAc. The organic layer was collected, washed with brine,
dried over MgSO4, filtered, and dried under reduced pressure to
yield the desired product, 5e which disubstituted reaction at 2-
1
4.6.2. 3-[[5-cyano-2-[3-methyl-4-(4-methylpiperazin-1-
and 4-position occurred, with a 50% yield as a white solid. H
yl)aniline]pyrimidin-4-yl]amino]benzenesulfonamide (5a)
NMR (500 MHz, DMSO-d6) δ 9.80 (s, 1H), 9.40 (s, 1H), 8.48 (s,
1H), 7.40 (s, 1H), 7.36 (q, J = 18.1, 10.4 Hz, 4H), 7.30 (t, J = 8.1
Hz, 1H), 7.24 (t, J = 8.0 Hz, 1H), 7.09 (d, J = 7.6 Hz, 1H), 7.03
(d, J = 4.9 Hz, 3H), 6.99 (d, J = 7.7 Hz, 1H), 6.78 (d, J = 7.4 Hz,
1H), 2.28 (s, 3H), 2.14 (s, 3H). MS-ESI: m/z 316.31 [M+H]+.
MW: 315.38 g/mol.
Compound 3a as the amino-intermediate and 4b as the chloro-
intermediate, following the general procedure. Purified by silica-
gel column chromatography (5%MeOH/DCM, 1%NH3), a beige
solid, yield 23%, 1H NMR (500 MHz, DMSO-d6) δ 10.07 (s, 1H),
9.38 (s, 1H), 8.50 (d, J = 1.3 Hz, 1H), 7.99 (d, J = 8.1 Hz, 1H),
7.90 (s, 1H), 7.45 – 7.40 (m, 1H), 7.31 (s, 3H), 7.27 (s, 2H), 7.02
(d, J = 8.2 Hz, 1H), 2.85 (t, J = 4.7 Hz, 4H), 2.25 (s, 3H), 2.23 (s,
3H). MS-ESI: m/z 479.20 [M+H]+. MW: 478.58 g/mol.
4.7.2. 2,4-bis(3-methylanilino)pyrimidine-5-carboxamide (5f)
5e (0.09 mmol), hydrogen peroxide solution (0.86 mmol) and
1M NaOH (0.17 mmol) in methyl alcohol (1 ml) were stirred to
40°C for 24 hrs. The reaction mixture was concentrated under

11.1, 4.1 Hz, 1H), 6.87 (d, J = 9.1 Hz, 2H), 6.17 (d, J = 5.7 Hz,
vacuum and extracted with EtOAc. The oArgaCniCc ElaPyeTr EwDas MANUSCRIPT
collected, washed with brine, dried over MgSO4, filtered, and
dried under reduced pressure. The crude product was purified by
column chromatography (0-10%MeOH/DCM, 1%NH3) to give
5f, with a 48% yield as a beige solid. 1H NMR (500 MHz,
DMSO-d6) δ 11.52 (s, 1H), 9.55 (s, 1H), 8.69 (s, 1H), 7.99 (s,
1H), 7.51 (s, 2H), 7.47 (dd, J = 8.4, 2.1 Hz, 1H), 7.40 (s, 1H),
7.21 (t, J = 7.8 Hz, 1H), 7.13 (t, J = 7.8 Hz, 1H), 6.90 (dd, J =
7.4, 1.7 Hz, 1H), 6.83 – 6.79 (m, 1H), 2.27 (s, 3H), 2.22 (s, 3H).
MS-ESI: m/z 334.13 [M+H]+. 333.40 g/mol.
1H), 3.09 – 3.03 (m, 4H), 2.48 – 2.44 (m, 4H), 2.22 (s, 3H). MS-
APCI: m/z, 404.3 [M+H]+. MW: 403.49 g/mol.
4.8.3. 2-[(2-{[4-(morpholin-4-yl)phenyl]amino}pyrimidin-4-
yl)amino]benzamide (5j)
Compound 3c as the amino-intermediate and 4g as the chloro-
intermediate, following the general procedure. Purified by silica-
gel column chromatography (5%MeOH/DCM, 1%NH3), a white
solid, yield 11%, 1H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H),
9.02 (s, 1H), 8.57 (d, J = 8.4 Hz, 1H), 8.21 (s, 1H), 8.03 (d, J =
5.7 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.66 (s, 1H), 7.54 (d, J =
9.0 Hz, 2H), 7.45 (t, J = 7.1 Hz, 1H), 7.04 (t, J = 7.6 Hz, 1H),
6.88 (d, J = 9.0 Hz, 2H), 6.18 (d, J = 5.7 Hz, 1H), 3.78 – 3.72 (m,
4H), 3.07 – 3.01 (m, 4H), MS-APCI: m/z, 391.3 [M+H]+. MW:
390.45 g/mol.
4.7.3. 3-[2-methoxy-5-[3-methyl-4-(4-methylpiperazin-1-yl)1-
aniline]aniline]benzenesul-fonamide (5g)
Compound 3a was dissolved in tetrahydrofuran (0.4 ml) and
treated with a solution of 1M HCl (0.33 mmol) in ethyl ether.
The mixture was stirred for 15 minutes and then concentrated
under vacuum. The residue was then treated with 4f (0.16 mmol)
and 2-propanol (0.8 ml). The suspension was stirred at 100°C for
5 days. The reaction mixture was added to a NaHCO3 solution
(10 ml) and extracted with EtOAc. The organic layer was
collected, washed with brine, dried over MgSO4, filtered, and
dried under reduced pressure to give the desired product, 5g with
a 91% yield as a beige solid. 1H NMR (500 MHz, Methanol-d4) δ
8.32 (t, J = 2.0 Hz, 1H), 8.04 (ddd, J = 8.2, 2.2, 1.0 Hz, 1H), 7.71
(s, 1H), 7.58 (dt, J = 7.9, 1.3 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H),
7.33-7.35 (m, 2H), 7.01 (d, J = 8.2 Hz, 1H), 3.92 (s, 3H), 2.96 (t,
J = 4.9 Hz, 4H), 2.76 (s, 4H), 2.46 (s, 3H), 2.26 (s, 3H). MS-ESI:
m/z 484.23 [M+H]+. MW: 483.59 g/mol.
4.8.4. 3-[(2-{[3-methyl-4-(4-methylpiperazin-1-
yl)phenyl]amino}pyrimidin-4-yl)amino]benzene-1-sulfonamide
(5k)
Compound 3a as the amino-intermediate and 4h as the chloro-
intermediate, following the general procedure. Purified by silica-
gel column chromatography (5%MeOH/DCM, 1%NH3), a brown
1
solid, yield 70%, H NMR (400 MHz, DMSO-d6) δ 9.63 (s, 1H),
8.94 (s, 1H), 8.25 (d, J = 8.3 Hz, 1H), 8.03 (d, J = 5.7 Hz, 1H), 7.89
(s, 1H), 7.45 (ddd, J = 7.7, 7.2, 1.5 Hz, 4H), 7.37 (s, 2H), 6.95 (d, J =
8.7 Hz, 1H), 6.21 (d, J = 5.7 Hz, 1H), 2.80 (t, J = 4.5 Hz, 4H), 2.24
(s, 3H), 2.20 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 160.27,
159.58, 156.38, 145.46, 144.50, 140.64, 135.80, 131.82, 129.41,
122.46, 122.02, 118.84, 118.58, 117.85, 116.19, 98.65, 55.25, 51.56,
45.81, 40.19, 40.15, 39.98, 39.94, 39.73, 39.52, 39.31, 39.10, 38.89,
17.65. MS-APCI: m/z 454.2 [M+H]+. MW: 453.57 g/mol.
4.8. General procedure for the synthesis of compounds 5h-5r
A
chloro-intermediate (4g-i) (0.32 mmol) and amino-
intermediate (3a-c) (0.32 mmol) were combined in a round
bottom flask with 5 ml of isopropanol and catalytic amounts of
TFA. The reaction mixture was stirred at 80°C overnight. The
reaction was then cooled down to room temperature, neutralized
with 1 M NaOH, and evaporated under vacuum to give a crude
product. The crude product was purified by silica-gel column
chromatography to yield a desired product.
4.8.5. 3-[(2-{[4-(4-methylprperazin-1-
yl)phenyl]amino)pyrimidin-4-yl)amino]benzene-1-sulfonamide
(5l)
Compound 3b as the amino-intermediate and 4h as the chloro-
intermediate, following the general procedure. Purified by silica-
gel column chromatography (10%MeOH/DCM, 1%NH3), a beige
1
solid, yield 62%, H NMR (400 MHz, DMSO-d6) δ 9.60 (s, 1H),
8.89 (s, 1H), 8.19 (d, J = 8.3 Hz, 1H), 8.01 (d, J = 5.7 Hz, 1H),
7.93 (s, 1H), 7.52 (d, J = 9.0 Hz, 2H), 7.44 (ddd, J = 11.1, 7.9,
4.7 Hz, 2H), 7.37 (s, 1H), 6.88 (d, J = 9.1 Hz, 1H), 6.18 (d, J =
5.7 Hz, 1H), 3.07 (t, J=3.2Hz 3H), 2.26 (s, 3H). MS-APCI: m/z
440.2 [M+H]+. MW: 439.54 g/mol.
4.8.1. 2-[(2-{[3-methyl-4-(4-methylpiperazin-1-
yl)phenyl]amino}pyrimidin-4-yl)amino]benzamide (5h)
Compound 3a as the amino-intermediate and 4g as the chloro-
intermediate, following the general procedure. Purified by silica-
gel column chromatography (5-10%MeOH/DCM, 1%NH3), a
1
beige solid, yield 67%, H NMR (400 MHz, DMSO-d6) δ 11.10
(s, 1H), 9.05 (s, 1H), 8.57 (d, J = 8.3 Hz, 1H), 8.21 (s, 1H), 8.04
(d, J = 5.7 Hz, 1H), 7.77 – 7.73 (m, 1H), 7.66 (s, 1H), 7.52 (d, J =
2.3 Hz, 1H), 7.46 – 7.37 (m, 2H), 7.04 (t, J = 7.0 Hz, 1H), 6.93
(s, 1H), 6.20 (d, J = 5.7 Hz, 1H), 2.80 (t, J = 4.6 Hz, 4H), 2.23 (s,
3H), 2.20 (s, 3H). Another set of 4 protons is overlapped under
water peak. MS-APCI: m/z, 418.3 [M+H]+. MW: 417.52 g/mol.
4.8.6. 3-[(2-{[4-(morpholin-4-yl)phenyl]amino}pyrimidin-4-
yl)amino]benzene-1-sulfonamide (5m)
Compound 3c as the amino-intermediate and 4h as the chloro-
intermediate, following the general procedure. Purified by silica-
gel column chromatography (5-10%MeOH/DCM, 1%NH3), a
1
purple solid, yield 75%, H NMR (400 MHz, DMSO-d6) δ 9.62 (s,
1H), 8.92 (s, 1H), 8.18 (d, J = 8.5 Hz, 1H), 8.01 (d, J = 5.7 Hz, 1H),
7.93 (s, 1H), 7.53 (d, J = 9.0 Hz, 2H), 7.47 (t, J = 7.8 Hz, 1H), 7.42
(dt, J = 7.8, 1.4 Hz, 1H), 7.37 (s, 2H), 6.88 (d, J = 9.1 Hz, 2H), 6.19
(d, J = 5.7 Hz, 1H), 3.77 – 3.68 (m, 4H), 3.06 – 2.99 (m, 4H). 13C
NMR (101 MHz, DMSO-d6) δ 160.29, 159.69, 156.38, 146.10,
144.48, 140.64, 133.05, 129.37, 122.57, 120.76, 118.59, 116.18,
115.62, 98.46, 66.20, 49.35, 40.19, 40.15, 39.98, 39.94, 39.73, 39.52,
39.31, 39.10, 38.89. MS-APCI: m/z 427.2 [M+H]+. MW: 426.50
g/mol.
4.8.2. 2-[(2-{[4-(4-methylpiperazin-1-
yl)phenyl]amino}pyrimidin-4-yl)amino]benzamide (5i)
Compound 3b as the amino-intermediate and 4g as the chloro-
intermediate, following the general procedure. Purified by silica-
gel column chromatography (5%MeOH/DCM, 1%NH3), a brown
solid, yield 56%, 1H NMR (400 MHz, DMSO-d6) δ 11.07 (s, 1H),
8.99 (s, 1H), 8.57 (d, J = 8.5 Hz, 1H), 8.20 (s, 1H), 8.01 (t, J =
5.2 Hz, 1H), 7.76 (dd, J = 7.9, 1.5 Hz, 1H), 7.65 (s, 1H), 7.52 (d,
J = 9.0 Hz, 2H), 7.44 (dd, J = 11.4, 4.3 Hz, 1H), 7.03 (dd, J =

(m, 4H), 3.06 – 2.99 (m, 4H), MS-APCI: m/z, 434.3 [M+H]+. MW:
4.8.7. 2-N-[4-(3,4-dimethylpiperazin-1-yl)phenAyl]C-4-CNE-(3P- TED MANUSCRIPT
432.53 g/mol.
methylphenyl)pyrimidine-2,4-diamine (5n)
Compound 3a as the amino-intermediate and 4i as the chloro-
intermediate, following the general procedure. Purified by silica-
gel column chromatography (5%MeOH/DCM, 1%NH3), a brown
4.9. Biological Assays
1
solid, yield 80%, H NMR (400 MHz, DMSO-d6) δ 9.20 (s, 1H),
4.9.1. Anti-Malarial Activity
8.90 (s, 1H), 7.96 (d, J = 5.7 Hz, 1H), 7.48 (dd, J = 15.8, 8.0 Hz,
4H), 7.16 (t, J = 7.8 Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 6.80 (d, J =
7.5 Hz, 1H), 6.15 (d, J = 5.7 Hz, 1H), 2.78 (d, J = 4.3 Hz, 4H), 2.27
(s, 3H), 2.23 (s, 3H), 2.18 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ
160.44, 159.71, 155.99, 145.29, 140.07, 137.77, 136.01, 131.78,
128.48, 122.65, 122.01, 120.26, 118.72, 117.79, 116.96, 98.28,
55.28, 51.63, 45.87, 40.20, 40.15, 39.99, 39.94, 39.73, 39.52, 39.31,
39.10, 38.89, 21.23, 17.58. MS-APCI: m/z 389.4 [M+H]+. MW:
388.52 g/mol.
Multidrug resistant Plasmodium falciparum (K1 strain) were
cultivated as previously described.43,52 Briefly, P. falciparum
were cultivated in RPMI-1640 medium supplement with 25 mM
HEPES, 25 mM NaHCO3, 10% heat-activated human serum and
3% erythrocytes. The culture is incubated at 37oC in a humidified
incubator with 3% CO2 and daily passaged to fresh medium
containing erythrocyte to maintain parasite growth. The in vitro
antimalarial activity of the test peptides were tested against
multidrug resistant Plasmodium falciparum (K1strain) using a
semi-automated microdilution technique.42 200 ꢀl of early ring
state parasite mixture (1% parasitemia, 1.5% Hct) were added
into each well of a 96 well plate. Then, 25 ꢀl tested peptide were
added in duplicate. Plates were incubated in CO2 incubator for 24
hours, then 25 ꢀl of medium containing 0.5 ꢀCi [3H]
hypoxanthine (Perkin Elmer, USA) were added and incubated for
18-20 hours. The levels of incorporated radioactive labeled
hypoxanthine, indicating parasite growth, were determined by
using the TopCount microplate scintillation counter (Packard,
USA). Dihydroartemisinine chloroquine and mefloquine were
used as positive control and 0.5% DMSO was used as negative
controls. The antimalarial activity was expressed as the inhibitory
concentration (IC50), representing the concentration of drug that
reduced 50% of parasite growth. Data fitting was performed
using SOFTMax Pro (Molecular Devices, USA).
4.8.8. 4-N-(3-methylphenyl)-2-N-[4-(4-methylpiperazin-1-
yl)phenyl]pyrimidine-2,4-diamine (5o)
Compound 3b as the amino-intermediate and 4i as the chloro-
intermediate, following the general procedure. Purified by silica-
gel column chromatography (5%MeOH/DCM, 1%NH3), a yellow
1
solid, yield 82%, H NMR (400 MHz, DMSO-d6) δ 9.18 (s, 1H),
8.85 (s, 1H), 7.94 (d, J = 5.7 Hz, 1H), 7.59 – 7.47 (m, 3H), 7.43 (d, J
= 6.6 Hz, 1H), 7.16 (t, J = 7.7 Hz, 1H), 6.85 (d, J = 8.9 Hz, 2H), 6.79
(d, J = 7.1 Hz, 1H), 6.13 (d, J = 5.7 Hz, 1H), 3.04 (t, J = 2.8 Hz 4H),
2.46 (t, J = 4.4 Hz, 4H), 2.27 (s, 3H), 2.22 (s, 3H). 13C NMR (101
MHz, DMSO-d6) δ 160.45, 159.85, 155.99, 145.97, 140.11, 137.77,
132.96, 128.43, 122.57, 120.79, 120.19, 116.89, 115.84, 98.03,
54.73, 48.98, 45.79, 40.19, 40.15, 39.98, 39.94, 39.77, 39.73, 39.52,
39.31, 39.10, 38.89, 21.31. MS-APCI: m/z 375.3 [M+H]+. MW:
374.49 g/mol.
4.9.2. Cytotoxicity
4.8.9. 4-N-(3-methylphenyl)-2-N-[4-(morpholin-4-
The percent cytotoxicity against the Vero cell line (ATCC
CCL-81) at 10 µM employed the method described by Hunt and
co-workers.44 The GFP-expressing Vero cell line was generated
by stably transfecting the Vero cells with pEGFP-N1 plasmid
Clontech) and maintained in minimal essential medium
supplemented with 10% heat inactivated fetal bovine serum, 2
yl)phenyl]pyrimidine-2,4-diamine (5p)
Compound 3c as the amino-intermediate and 4i as the chloro-
intermediate, following the general procedure. Purified by silica-
gel column chromatography (5%MeOH/DCM, 1%NH3), a brown
1
solid, yield 78%, H NMR (400 MHz, DMSO-d6) δ 9.18 (s, 1H),
8.88 (s, 1H), 7.95 (d, J = 5.7 Hz, 1H), 7.58 – 7.51 (m, 3H), 7.44 (d, J
= 8.1 Hz, 1H), 7.16 (dd, J = 10.1, 5.5 Hz, 1H), 6.88 – 6.83 (m, 2H),
6.80 (d, J = 7.5 Hz, 1H), 6.14 (d, J = 5.7 Hz, 1H), 3.78 – 3.69 (m,
4H), 3.05 – 2.97 (m, 4H). MS-APCI: m/z 362.3 [M+H]+. MW:
361.45 g/mol.
mM
L-glutamine, 1 mM sodium pyruvate, 1.5 g/L sodium
bicarbonate, and 0.8 mg/mL geneticin at 37°C in an incubator
with 5% CO2. Vero cell suspension (45 ꢀL, 3.3×104 cells/mL)
was added into each well of 384-well plates containing 5 ꢀL of
test compounds. Plates were incubated in a CO incubator at 37
2
°C for 4 days. Fluorescence signals were measured using
SpectraMaxM5 microplate reader (Molecular Devices, U.S.A.).
Fluorescence signals were measured using SpectraMax M5
microplate reader (Molecular devices, U.S.A.) in the bottom-
reading mode at the excitation and emission wavelengths of 530
and 590 nm. Ellipticine was used as the positive control and
0.5% DMSO as the negative control.
4.8.10. N,N’-bis(3-methylphenyl)pyrimidine-2,4-diamine (5q)
5q was a by-product obtained from the double substitution
reaction from the aniline used to produce 4i. Purified by silica-gel
column chromatography (10% MeOH/DCM), a beige solid, yield
1
2%, H NMR (400 MHz, DMSO-d6) δ 9.25 (s, 1H), 9.05 (s, 1H),
7.99 (d, J = 5.7 Hz, 1H), 7.53 (d, J = 7.1 Hz, 3H), 7.46 (s, 1H), 7.18
(t, J = 7.8 Hz, 1H), 7.14 – 7.07 (m, 1H), 6.82 (d, J = 7.5 Hz, 1H),
6.73 (d, J = 7.4 Hz, 1H), 6.20 (d, J = 5.7 Hz, 1H), 2.28 (s, 3H), 2.24
(s, 3H), MS-APCI: m/z 291.2 [M+H]+. MW: 290.37 g/mol.
Dose response curves for a subset of compounds were
determined in Vero (ATCC CCL-81) and A549 (ATCC CCL-
185) cells. The MTT based method described by Mosman el al
was employed.45 Cells were cultured in DMEM medium with
10% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml
streptomycin at 37oC in a humidified atmosphere with 5% CO2.
The cells were cultured in complete DMEM for 16-18 h, then
cultured with compound for 72h. Each well was charged with
DMEM containing 3-[4,5-dimehyl-2-thiazolyl]-2,5- diphenyl-
2H-tetrazolium bromide (MTT) (5 mg/ml in normal sodium), and
then cells were incubated for 3 h. The medium was carefully
decanted and 50 ꢀl of dimethyl sulfoxide was added. The plate
was shaken mechanically for 5 min, and then absorbance
readings at a wavelength of 570 nm were performed on a Sunrise
4.8.11. 2-N,4-N-bis[4-(morpholin-4-yl)phenyl]pyrimidine-2,4-
diamine (5r)
5r was
a
by-product from the elimination of 2-
aminobenzamide on the 2-position of compound 5j. Purified by
silica-gel column chromatography (5%MeOH/DCM, 1%NH3), a
1
yellow solid, yield 20%, H NMR (400 MHz, DMSO-d6) δ 10.13 (s,
1H), 9.02 (s, 1H), 8.51 (d, J = 8.9 Hz, 1H), 8.06 (d, J = 5.6 Hz, 1H),
7.94 (dd, J = 8.0, 1.6 Hz, 1H), 7.60 – 7.49 (m, 3H), 7.15 – 7.08 (m,
1H), 6.86 (d, J = 9.1 Hz, 2H), 6.26 (d, J = 5.7 Hz, 1H), 3.76 – 3.71

Tarning, J.; Taylor, W. J.; Yeung, S.; Woodrow, C. J.; Flegg, J. A.;
microplate reader (Tecan). Geftinib was used as the positive
control and 0.5% DMSO as the negative control.
ACCEPTED MANUSCRIPT
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manner except for the following differences in reagents: For JAK2
kinase assays, the reaction contained 10ng JAK2 enzyme, 0.1 mg/ml
ploly (Glu:Tyr) substrate and 0.1 mg/ml ATP. For JAK3 kinase
assays, the reaction contained 3ng JAK3 enzyme, 0.1 mg/ml poly
(Glu:Tyr) substrate and 0.1 mg/ml ATP.
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1X kinase assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1
mM EGTA, 0.01% Brig and 2mM DTT), 0.0267 mg/ml poly
(Glu:Tyr), 6.7 nM anti-phosphotyrosine antibody, 0.33 nM Oregon
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at 30oC in a fluorescence microplate reader using excitation at 485
nm and emission at 535 nm using an Infinite F200 fluorescence
microplate reader (Tecan). The IC50 data was obtained by fitting
using Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA).
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The authors are grateful for financial support provided by the
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acknowledges support from the Kasetsart University Research
and Development Institute (KURDI) and the Thailand Research
Fund (TRF, RSA570080). OP would like to acknowledge a
scholarship provided by the Development and Promotion of
Science and Technology Talents Project Grant (DPST). MPG
and OP would like to thank Prof. S. Ruchiriwat and Dr P.
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Glenn Noronha; Chi Ching Mak; Jianguo Cao;
List of Captions
Joel Renick; Andrew McPherson; Binqi Zeng; Ved P. Pathak; Daniel
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HTS data.26 Pf 3D7 (IC50, µM) and HepG2 cytotoxicity (% at 10 µM) are
reported for: TCMDC-134115 (1a), TCMDC-134116 (1b), TCMDC-141384
(1c) and TCMDC-141383 (1d).
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Johnson, N. W.; Bryan, D. L. Anthranilamide
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ACCEPTED MANUSCRIPT
Highlights
Eighteen 2,4 diamino-pyrimidines have prepared and tested at the Pf. K1 strain.
Cytotoxicities in Vero and A549 cells, as well as selected kinase activities, were assessed.
5n and 5m are sub-µM inhibitors, with activity comparable to Chloroquine
5d and 5k, are µM inhibitors of Pf, but with improved cytotoxicity