2,4-Diaminothienopyrimidines as orally active antimalarial agents

Article abstract of DOI:10.1021/jm401760c

A novel series of 2,4-diaminothienopyrimidines with potential as antimalarials was identified from whole-cell high-throughput screening of a SoftFocus ion channel library. Synthesis and structure-activity relationship studies identified compounds with potent antiplasmodial activity and low in vitro cytotoxicity. Several of these analogues exhibited in vivo activity in the Plasmodium berghei mouse model when administered orally. However, inhibition of the hERG potassium channel was identified as a liability for this series.

Full text of DOI:10.1021/jm401760c

Article
pubs.acs.org/jmc
2,4-Diaminothienopyrimidines as Orally Active Antimalarial Agents
Diego Gonzalez Cabrera,† Claire Le Manach,† Frederic Douelle,† Yassir Younis,† Tzu-Shean Feng,†
́
Tanya Paquet,† Aloysius T. Nchinda,† Leslie J. Street,† Dale Taylor,‡ Carmen de Kock,‡ Lubbe Wiesner,‡
Sandra Duffy,§ Karen L. White,∥ K. Mohammed Zabiulla,⊥ Yuvaraj Sambandan,⊥ Sridevi Bashyam,⊥
David Waterson,▽ Michael J. Witty,▽ Susan A. Charman,∥ Vicky M. Avery,§ Sergio Wittlin,▲,○
and Kelly Chibale*,†,■
†Department of Chemistry and ■Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch
7701, South Africa
‡Division of Clinical Pharmacology, Department of Medicine, University of Cape Town, Observatory 7925, South Africa
§Discovery Biology, Eskitis Institute, Griffith University (Nathan Campus), Don Young Road, Brisbane, Queensland 4111, Australia
∥Centre for Drug Candidate Optimisation, Monash University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052,
Australia
⊥Syngene International Ltd., Biocon Park, Plot No. 2 & 3, Bommasandra IV Phase, Jigani Link Road, Bangalore 560099, India
▽Medicines for Malaria Venture, International Center Cointrin, Route de Pre-
́
Bois 20, Post Office Box 1826, 1215 Geneva,
Switzerland
▲Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland
○University of Basel, 4002 Basel, Switzerland
S
* Supporting Information
ABSTRACT: A novel series of 2,4-diaminothienopyrimidines
with potential as antimalarials was identified from whole-cell
high-throughput screening of a SoftFocus ion channel library.
Synthesis and structure−activity relationship studies identified
compounds with potent antiplasmodial activity and low in
vitro cytotoxicity. Several of these analogues exhibited in vivo
activity in the Plasmodium berghei mouse model when
administered orally. However, inhibition of the hERG
potassium channel was identified as a liability for this series.
analogues. Of these, 118 displayed greater than 80% inhibition
INTRODUCTION
■
of parasite growth at the 1.82 μM screening concentration. The
overall hit rate from the SFI06 library was high at 21.3%. In
comparison, screening libraries for the recently described
thiazole5 and aminopyridine series6 exhibited significantly
lower hit rates of 1.3% and 1.8%, respectively. As with other
SoftFocus libraries, the rationale behind choosing the SoftFocus
ion channel library for our high-throughput screening (HTS)
campaign was that molecules contained in this target-focused
library were designed with a particular protein target or protein
family in mind. This is on the premise that, relative to
conventional libraries, fewer compounds needed to be screened
in a SoftFocus ion channel library in order to obtain hits. We
have previously provided several advantages offered by
SoftFocus libraries.7
The rapid and widespread emergence of resistance to most
marketed and commonly used antimalarial drugs has led to
treatment failures and, as a consequence, to a rising concern
over malaria control and/or eradication.1 This is especially
worrying in sub-Saharan Africa and southeast Asia where this
infectious disease, typically caused by the Plasmodium
falciparum organism, continues to inflict a tremendous burden.
With ∼40% of the world’s population being at risk of
contracting this life-threatening parasitic disease,2 there is an
urgent need for novel and structurally diverse classes of
compounds to develop effective and affordable drugs against
malaria.3
One of the libraries identified from screening 36 608
compounds from the BioFocus DPI (BFDPI) SoftFocus library
against 3D7 (sensitive) and Dd2 (multidrug resistant) strains of
P. falciparum, in an image-based whole-cell assay,4 was
designated SFI06 (SoftFocus ion channel) and contained 554
Received: November 14, 2013
Published: January 21, 2014
© 2014 American Chemical Society
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The active compounds from the SFI06 series were identified
as thienopyrimidines 1 (Figure 1). Analysis of the structure−
replacement of the aminomethyl moiety at position 2 with
other alkylamino substituents, such as an isopropylamino
group, led to a significant loss of potency (4 vs 8), although
basic alkylamino side chains were tolerated at this position and
also at the 4-position. Finally, replacement of a benzyl group
with an aniline moiety at position 4 resulted in a significant
decrease in antiplasmodial activity (7 vs 9).
On the basis of good in vitro antiplasmodial potency and
favorable absorption, distribution, metabolism, and excretion
(ADME) properties, and in order to assess the antimalarial
potential of this antiplasmodial chemotype, three analogues 4,
5, and 8 were selected for in vivo oral efficacy evaluation in the
Plasmodium berghei mouse model.8 Interestingly, 4 and 5
exhibited significant antimalarial activity at 4 × 50 mg/kg, with
87% and 95% reduction in parasitemia albeit with a modest
survival time of 15 days. At this initial stage, these data were
encouraging when it was considered that these analogues were
chosen as representatives for hit validation with no additional
optimization and/or chemical modification (Table 2).
Figure 1. Thienylpyrimidine (1) and quinazoline (2) structures.
activity relationship (SAR) from the HTS data as well as from
synthesized representative thienopyrimidines 1 and the related
quinazolines 2 (Figure 1) led to the conclusion that the 6-aryl-
thienopyrimidine fragment was required for potent in vitro
activity and that monosubstituted amines were the preferred
substituents at the 2- and 4-positions. Despite this limitation, a
range of aliphatic, aromatic, and heteroaromatic amine side
chains were tolerated. However, more polar H-bonding groups
(for example, amides) generally decreased activity.
Accordingly, a number of active analogues were selected for
resynthesis and confirmatory testing against multidrug resistant
(K1) and sensitive (NF54) strains of P. falciparum and counter-
screened for cytotoxicity in the L6 mammalian cell line assay
(Supporting Information). Several SAR trends were observed
from the compounds tested (Table 1). First, the phenyl moiety
at position 6 was essential for potent activity (3 vs 4). Second,
Although related quinazoline9 and pyrimidine10 compounds
with antiplasmodial activity were known, this was the first time
that excellent in vivo antimalarial efficacy and mean survival
time (MSD) of 2,4-diaminothienopyrimidines at low doses had
been observed. It is noteworthy that 2,4-diaminothieno[2,3-
d]pyrimidines have previously been reported as antifolates and
antimalarials.11 These differ structurally from the current SFI06
series compounds as they lack alkyl substituents on the 2- and
4-amino positions and contain a carbocyclic or heterocyclic ring
fused at the 5,6-position. In addition, 4 displayed fast killing
kinetics in the recently developed speed of action and stage-
specificity assays.12 This contrasts with the slower killing
kinetics of dihydrofolate reductase inhibitors such as pyrimeth-
amine. Fast-acting derivatives not only allow maximization of
therapeutic efficacy but also potentially minimize the
emergence of drug resistance, which is an ideal characteristic
for new antimalarial drugs. On the basis of these initial
promising results, it was concluded that 2,4-diaminothienopyr-
imidine compounds presented themselves as an attractive series
for a hit-to-lead (H2L) and lead optimization (LO) program.
Herein we describe a comprehensive assessment of
antiplasmodial activity, SAR analyses, antimalarial efficacy, and
pharmacokinetic (PK) properties in parallel with human ether-
Table 1. In Vitro Activity of Representative Compounds
against Sensitive and Multidrug-Resistant Strains of
a
Plasmodium falciparum
̀
a-go-go-related gene (hERG) inhibition studies to examine the
potential cardiotoxicity risk posed by this novel antimalarial
chemotype.
We began exploring the SAR based on 4, by modifying the
heterocyclic core to determine the optimum structural
framework needed for potent antiplasmodial activity (Table
3). Variation in the position of the sulfur atom in the scaffold to
give reverse thienopyrimidines (10 and 11) or replacement of
the thiophene ring with the corresponding furan ring (12) led
to a significant loss of activity, indicating the importance of the
thieno[3,2-d]pyrimidine core for potency.
After investigating the consequence of varying the hetero-
cyclic scaffold and using the initial hit compound 4 as starting
point, we decided to explore the SAR around the 6-aryl
substituent.
RESULTS AND DISCUSSION
■
Chemistry. Scheme 1 summarizes the seven-step synthesis
of 2,4-diaminothienopyrimidine compounds from commercially
available reagents. Reaction of starting material 13 with
trichloroacetyl isocyanate in tetrahydrofuran (THF) and
a
Mean from n values of ≥2 independent experiments with multidrug-
b
resistant (K1) and sensitive (NF54) strains of P. falciparum. Data
from Gonzalez Cabrera et al.5
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Table 2. In Vivo Antimalarial Efficacy for Single and Multiple Doses of Selected Compounds in Plasmodium berghei-Infected
a
Mice
a
Compounds were dissolved or suspended in a nonsolubilizing, standard suspension vehicle (hydroxypropyl methylcellulose, HPMC), except for 4,
5, and 8, which were dosed in 70/30 Tween 80/ethanol, diluted before administration 10× with water. bMSD = mean survival time (in days). cData
from Gonzalez Cabrera et al.5 dMice were euthanized on day 4 in order to prevent death otherwise occurring at day 6.
subsequent bromination afforded substrate 14 in good yield
(91%). Treatment of 14 with ammonia in CH3OH furnished
intermediate 15, which cyclized under basic conditions.
Subsequent chlorination with POCl3 produced key dichloro
intermediate 16 in excellent yield (88%). Derivatives 4−9 and
17−43 were then accessed via two consecutive N-substitution
reactions in the presence of the relevant amine, followed by a
Suzuki cross coupling13 reaction with commercially available
boronic acids.
Biology. In Vitro Antiplasmodial Activity. The activity of
the newly synthesized analogues was determined against
multidrug-resistant (K1) and sensitive (NF54) strains, with
chloroquine and artesunate as the reference drugs in all the
experiments. Nineteen of these compounds showed potent
antiplasmodial activity in the low nanomolar range (IC50 NF54
< 100 nM), with five analogues having IC50 values below 20 nM
(Table 4).
exhibited a 3-fold increase in potency compared to 4 and better
activity than chloroquine against both NF54 and K1 strains. 25,
which contained a fluoro substituent, showed moderate
potency.
Varying the position of the substituents from para to meta on
the phenyl ring seemed to have relatively little effect on
antiplasmodial activity (17, 21, and 23 vs 18, 20, and 22,
respectively). Similarly, introducing an ortho substituent, as in
33, had little adverse effect on potency.
The potencies of 30, 31, and 32 indicated that aliphatic and
heterocyclic rings were tolerated. It is noteworthy that the
presence of the morpholino group in 30 gave rise to one of the
most potent compounds of the series (IC50 NF54 = 7 nM).
Disubstituted derivatives 33 and 34 retained good potency,
while 35 significantly lost activity, presumably due to the
presence of the polar EWG 4-methanesulfonyl substituent.
Attempts to replace the phenyl ring with heterocyclic or
electron-deficient bicyclic groups led to a drop in potency (36,
39, and 38). However, antiplasmodial activity was regained by
the introduction of a less polar benzoxadiazole group as shown
Electron-withdrawing groups (EWG), such as -CF3 (17)
were well-tolerated and resulted in potent compounds. In
contrast, groups such as methylsulfone (29) were less active
compared to 4. One of the most potent analogues, 21,
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In Vitro ADME Profiling. Along with the extensive SAR
studies toward optimizing the potency of this series, some of
the most active analogues were evaluated for their phys-
icochemical properties and in vitro metabolic stability in human
liver microsomes. In general, most of the newly synthesized
compounds had good solubility under acidic (pH 2) or neutral
(pH 6.5) conditions, with the exception of 30, which showed
poor solubility at both pH values, and 19, 26, 31, and 40, which
were poorly soluble at pH 6.5. The partition coefficients were
generally moderate to high, with log D7.4 values ranging from
2.1 to 4.3 (Table 4).
Table 3. In Vitro Activity against Sensitive and Multidrug-
Resistant Strains of Plasmodium falciparum and Solubility
a
Most of the derivatives displayed low to moderate rates of
degradation in human liver microsomes. On the basis of the in
vitro intrinsic clearance values, low to moderate in vivo hepatic
clearance would be expected (Table 5). Unfortunately 30, one
of the most potent of the series, showed a high rate of
degradation in the microsomal incubations.
In addition to studies of their stability in human liver
microsomes, key compounds were investigated in mouse and
rat liver microsomes. Generally, metabolic stability of the
derivatives was comparable across the three species (data not
shown).
In Vivo Efficacy Studies. Compounds that displayed potent
in vitro antiplasmodial activity (IC50 < 100 nM) and good
metabolic stability were evaluated for in vivo activity in the P.
berghei mouse model.8 Parasitemia reduction and MSD for
multidose regimens are reported in Table 2. Following oral
administration of 4 × 50 mg/kg, analogues 17 and 23 reduced
parasitemia by more than 99% and prolonged MSD to 14 and
23 days, respectively. However, none of the treated animals
were completely cured. Dosing the compounds at 4 × 3 mg/kg
and 4 × 10 mg/kg was less effective, with <40% percent
reduction in parasitemia compared to untreated infected mice.
Despite their potent in vitro activity against P. falciparum,
analogues such as 20 were inferior in terms of parasitemia
reduction as well as MSD.
a
Mean from n values of ≥2 independent experiments with multidrug-
b
resistant (K1) and sensitive (NF54) strains of P. falciparum. Data
from Gonzalez Cabrera et al.5 cEstimates from nephelometry.
dEstimates from HPLC-DAD-MS. Dash indicates that the value was
not measured.
for 37, which exhibited a 2-fold improvement in activity
compared to 4.
Introduction of more polar carboxamide groups to the
phenyl ring resulted in considerable loss of activity, albeit for
the acetyl-containing analogue 40 the decrease was relatively
moderate.
a
Scheme 1. Synthetic Approach to 2,4-Diaminothienopyrimidine Analogues
a
Reagents and conditions: (i) trichloroacetyl isocyanate, THF, 0 °C to rt, 2 h, 91%; (ii) bromine, acetic acid, 0−80 °C, 14 h, 65%; (iii) ammonia,
CH3OH, 0 °C to rt, 30 min, 76%; (iv) t-BuOK, DMF, rt, 14 h, 99%; (v) POCl3, N,N-dimethylaniline, 130 °C, 14 h, 88%; (vi) R1 = appropriate
amine, Na2CO3, ethanol, rt, 14 h, 93−99%; (vii) R = appropriate amine, in a sealed tube, THF, 100 °C, 14 h, 78−83%; (viii) R2 = appropriate
boronic acid, Pd(PPh3)2Cl2, K2CO3, DMF, 90 °C, 14 h, 24−82%.
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Table 4. In Vitro Activity against Sensitive and Multidrug-Resistant Strains of Plasmodium falciparum and Physicochemical
a
Properties
a
Mean from n values of ≥2 independent experiments with multidrug-resistant (K1) and sensitive (NF54) strains of P. falciparum. bValue measured
5
by the chromatographic glogD technique. Data from Gonzalez Cabrera et al. dEstimates from nephelometry. Estimates from HPLC-DAD-MS.
c
e
Dash indicates that the value was not measured.
In Vivo Pharmacokinetic Studies. In an attempt to
rationalize the in vivo efficacy data, PK studies on 4 were
conducted in male Sprague-Dawley rats following oral (po) and
intravenous (iv) administration. In addition, mouse exposure
studies of 17 and 25 were performed at a single dose of 50 mg/
kg. No sign of toxicity was observed when 4 was administered
intravenously at a dose of 5 mg/kg or orally at a dose of 20 mg/
kg. The in vivo plasma clearance in rats after iv dosing was high
(154 mL·min−1·kg−1), much higher than the expected hepatic
blood flow in rats (CLplasma 67.6 mL·min−1·kg−1). Ex vivo
evaluation of the whole blood to plasma partitioning ratio (B/
P) indicated that 4 distributes extensively into erythrocytes (B/
P = 3.8). By use of this value to convert the plasma clearance to
blood clearance, a value for CLblood was estimated to be 40 mL·
min−1·kg−1, which is reasonably consistent with the microsome
predicted clearance (predicted CLblood = 27.9 mL·min−1·kg−1)
based on studies in rat liver microsomes. The high B/P is also
one of the reasons for the extremely high plasma volume of
distribution (139 L/kg); however, the blood volume of
distribution (37 L/kg) was also very high. This trend for
high volume of distribution is commonly seen with dibasic
compounds and was presumably responsible for the long in
vivo half-life of 21 h (Table 6). Furthermore, 4 had a moderate
oral bioavailability (34%) likely limited by first-pass metabolism
and consistent with the in vivo blood clearance being 41% of
the hepatic blood flow in rats.
Mouse Exposure Studies. Plasma concentrations in
Plasmodium berghei-infected mice were measured after a single
oral dose of 50 mg/kg and followed for 24 h. Compound 17
showed significant in vivo exposure, with a higher plasma
concentration and prolonged exposure profile compared to 25
(Table 7). Thus, the in vivo efficacy displayed by 17 can be
rationalized on the basis of better drug exposure and higher in
vitro potency [IC50 = 32 nM (K1) and 29 nM (NF54)]
compared to 25 [IC50 = 92 nM (K1) and 42 nM (NF54)].
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Table 5. In Vitro Metabolic Stability in Human Liver Microsomes
a
compd
degrad half-life (min)
in vitro CLint [μL·min−1·(mg of protein)−1
]
microsome-predicted EH (human)
4
344.9
124
31.1
179
238
230
>250
181
115
15
5
14
0.22
0.47
0.78
0.35
0.29
0.30
17
18
19
21
22
23
24
29
30
31
32
33
34
36
37
38
39
40
41
55.8
10
7
8
b
<7
<0.28
10
0.35
0.46
0.87
0.58
0.53
0.43
15
119
36
48
99.7
130
>100
>100
>250
25.3
52.2
21
18.4
13
b
<17
<17
<7
<0.48
b
<0.48
b
<0.28
68.7
33.3
81
0.81
0.67
0.76
0.49
100
17
a
b
Predicted hepatic extraction ratio based on in vitro intrinsic clearance (CLint). No measurable degradation of the parent compound was observed;
hence, the clearance parameters could not be determined. EH was considered to be <0.28 or <0.48.
Table 6. Pharmacokinetic Parameters for 4 in Male Sprague-
Dawley Rats following Intravenous and Oral Administration
Table 7. Plasma Concentrations of 17 and 25 in Plasmodium
berghei-Infected Mice following Administration of a Single
a
50 mg/kg Oral Dose
a
a
parameter
iv 4
oral 4
measured dose (mg/kg)
apparent t1/2 (h)
4
19
44
c
21
plasma CL (mL·min−1·kg−1
)
154
139
41
37
c
plasma Vss (L/kg)
c
b
blood CL (mL·min−1·kg−1
)
b
blood Vss (L/kg)
Cmax (μM)
0.008
105
125
34
Tmax (min)
c
AUC0−∞ (μM·min)
bioavailability (%)
77
c
a
b
Values are the mean from two animals. Calculated by dividing
plasma CL or plasma Vss by the blood to plasma partitioning ratio
c
(3.8). Dash indicates that the value was not measured or was not
relevant.
a
Values are the mean from two animals. Sample time postdose is
shown in hours.
The pharmacokinetic/pharmacodynamic (PK/PD) relation-
ship for this series is difficult to interpret. As with many
lipophilic dibasic compounds, the SFI06 series is characterized
by very high volumes of distribution, which leads to very low
blood levels but long apparent half-lives. In addition,
compounds such as 4 are readily metabolized by demethylation
on both side chains. However, the desmethyl compounds also
retain intrinsic activity and would likely contribute to the
overall efficacy of the compounds.
In Vitro hERG Activity. Six analogues of the series were
tested for their activity against the hERG K+ channel via in vitro
IonWorks patch clamp electrophysiology. As observed in Table
8, 4 turned out to be a moderate inhibitor of the hERG channel
(IC50 = 8.5 μM). Unfortunately any changes made in the
phenyl ring did not improve the hERG profile to desired levels,
and all showed potential for hERG K+ channel blockade at
physiologically relevant concentrations (IC50 values in 1−10
μM range).
CONCLUSION
■
From a high-throughput screen of 36 608 compounds, a novel
series of 2,4-diamino-6-arylthienopyrimidines was identified.
Nineteen of the newly synthesized compounds showed activity
in the low nanomolar range (IC50 < 100 nM) against
multidrug-resistant (K1) and sensitive (NF54) strains of P.
falciparum, with five analogues having an IC50 below 20 nM.
Compounds 4, 17, and 23 also suppressed the parasitemia and
increased MSD in vivo in the P. berghei mouse model when
administered orally at 4 × 50 mg/kg. However, no curative
effect was observed at the tested dose. Pharmacokinetic studies
on 4 revealed a high volume of distribution, with moderate
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1H), 3.84 (s, 3H); LC-MS (APCI) m/z = 346.6 [(M + H)+] (anal.
calcd for C9H7Cl3N2O4S+, m/z = 345.59); LC-MS 99.9%.
Table 8. hERG Inhibition Data
To a solution of methyl 3-[3-(2,2,2-trichloroacetyl)ureido]-
thiophene-2-carboxylate (40 g, 0.12 mol) in acetic acid (400 mL) at
0 °C was added dropwise bromine (74.0 g, 0.46 mol), and then the
mixture was stirred at 80 °C overnight. The reaction mixture was
concentrated under reduced pressure, and the remaining residue was
filtered and washed with diethyl ether (2 × 50 mL) to yield 14 (32 g,
65%) as a white solid. 1H NMR (400.2 MHz, DMSO-d6) δ = 11.67 (br
s, 1H), 9.63 (br s, 1H), 8.16 (s, 1H), 3.78 (s, 3H); LC-MS APCI m/z
= 422.8 [(M + H)+] (anal. calcd for C9H6BrCl3N2O4S+, m/z =
421.83); LC-MS 92.9%.
To an ice-cooled suspension of 14 (32 g, 0.08 mol) in CH3OH
(300 mL) was added ammonia, until the suspension became clear. The
solution was then stirred at rt for 30 min, during which time a white
solid precipitated. The white solid was filtered and washed with
CH3OH (2 × 50 mL) to give 15 (16 g, 76%). 1H NMR (400.2 MHz,
DMSO-d6) δ = 9.21 (s, 1H), 8.04 (s, 1H), 6.90 (s, 2H), 3.81 (s, 3H);
LC-MS APCI m/z = 278.8 [(M + H)+] (anal. calcd for
C7H7BrN2O3S+, m/z = 277.94); LC-MS 93.1%. To an ice-cooled
solution of 15 (16 g, 0.06 mol) in N,N-dimethylformamide (DMF)
(300 mL) was added potassium t-butoxide, and the mixture was stirred
at rt overnight. The reaction mixture was concentrated under reduced
pressure, and deionized water (500 mL) was added to the remaining
residue. The solution was then acidified with 50% concentrated
H2SO4, during which time a white solid precipitated. The solid was
filtered and washed with deionized H2O (2 × 25 mL) and CH3OH (2
× 25 mL) to furnish 6-bromothieno[3,2-d]pyrimidine-2,4-diol (14 g,
blood clearance after iv administration and moderate
bioavalability. Additionally, all of the more active compounds
exhibited high affinity for the hERG K+ channel. These
liabilities will be addressed along with further investigation of
the SAR in a lead optimization campaign.
1
99%). H NMR (400.2 MHz, DMSO-d6) δ = 11.60 (br s, 1H), 11.32
(br s, 1H), 7.03 (s, 1H); LC-MS APCI m/z = 245.0 [M+] (anal. calcd
for C6H3BrN2O2S+, m/z = 245.91); LC-MS 99.0%.
To a solution of 6-bromothieno[3,2-d]pyrimidine-2,4-diol (14 g,
0.06 mol) in POCl3 (300 mL) was added N,N-dimethylaniline (4.1 g,
0.03 mol), and the resulting solution was stirred at 130 °C overnight.
The reaction mixture was concentrated under reduced pressure, and
CH3OH (10 mL) was added to the remaining residue. The white solid
was filtered and washed with CH3OH (2 × 5 mL) to give 16 (14 g,
EXPERIMENTAL SECTION
■
General Comments on Experimental Data. Chloroform
(CHCl3) and tetrahydrofuran (THF) solvents were analytical-grade,
without stabilizer; ethyl acetate, hexane, and dichloromethane were
distilled. Unless stated otherwise, all other solvents and reagents were
purchased from commercial sources and used without further
purification. Column chromatography was carried out on silica gel
60 (Fluka), particle size 0.063−0.2 mm (70−230 mesh ASTM), as the
stationary phase. Analytical thin-layer chromatograpjy (TLC) was
performed on silica on TLC aluminum foils, 20 cm × 20 cm, with
fluorescent indicator (200 μm thick, Fluka) visualized under UV light.
1
88%). H NMR (400.2 MHz, DMSO-d6) δ = 8.06 (s, 1H); LC-MS
APCI m/z = 282.8 [(M + H)+] (anal. calcd for C6HBrCl2N2S+, m/z =
281.84); LC-MS >99.9%.
General Procedure 1 for Synthesis of 6-Bromo-N4-[3-
(dimethylamino)propyl]-N2-methylthieno[3,2-d]pyrimidine-
2,4-diamine 3. To a solution of 16 (5 g, 0.02 mol) in ethanol (150
mL) were added Na2CO3 (3.7 g, 0.04 mol) and 3-(dimethylamino)-
propylamine (2 g, 0.02 mol). The solution was then stirred at rt for 4
h, after which time it was concentrated under reduced pressure. The
remaining residue was dissolved in ethyl acetate, washed with brine (3
× 15 mL), dried over Na2SO4, and concentrated in vacuo. The residue
was subjected to column chromatography on silica gel, with CH2Cl2/
CH3OH in a 9.8:0.2 (v/v) ratio as eluent, to furnish N1-(6-bromo-2-
chlorothieno[3,2-d]pyrimidin-4-yl)-N3,N3-dimethylpropane-1,3-dia-
1
Routine H and 13C NMR spectra were recorded on either a Varian
Mercury-300 (1H 300.1 MHz, 13C 75.5 MHz) or Bruker-400 (1H
400.2 MHz, 13C 100.6 MHz) instrument. Spectra were recorded at
ambient temperature, unless otherwise stated. Chemical shifts (δ) are
reported in parts per million from low to high field and referenced to
residual solvent. Standard abbreviations indicating multiplicity are used
as follows: br s = broad singlet, d = doublet, m = multiplet, q = quartet,
quint = quintet, s = singlet, t = triplet. In many cases deuterated
1
mine (4.4 g, 72%). H NMR (400.2 MHz, DMSO-d6) δ = 8.53 (br s,
1
dimethyl sulfoxide (DMSO-d6) was used as a solvent and H was
1H), 7.57 (s, 1H), 2.91 (t, J = 6.9 Hz, 2H), 2.53 (t, J = 6.9 Hz, 2H),
2.34 (s, 6H), 1.79 (t, J = 6.9 Hz, 2H).
referenced to 2.500 ppm for the quintuplet downfield methyl signal.
13C was referenced to the methyl carbon septuplet at 39.52 ppm.
Atmospheric pressure chemical ionization (APCI) mass spectrometry
was carried out by the services at the Centre for Drug Candidate
Optimisation and Syngene. LC purity traces were performed by one of
the methods shown in Supporting Information.
N1-(6-Bromo-2-chlorothieno[3,2-d]pyrimidin-4-yl)-N3,N3-dime-
thylpropane-1,3-diamine (4.4 g, 0.013 mol) was added into a sealed
tube containing methylamine in THF, 2.0 M (150 mL), and the
mixture was stirred at 95 °C overnight. The reaction mixture was
concentrated under reduced pressure, and the remaining residue was
subjected to column chromatography on silica gel, with CH2Cl2/
CH3OH in a 9.8:0.2 (v/v) ratio as eluent, to give 3 (3.6 g, 83%) as a
white solid. 1H NMR (400.2 MHz, DMSO-d6): δ = 7.12 (s, 1H), 3.61
(t, J = 6.9 Hz, 2H), 3.24 (t, J = 6.9 Hz, 2H), 3.05 (d, J = 4.8 Hz, 3H),
2.93 (s, 6H), 2.04 (t, J = 6.9 Hz, 2H); 13C NMR (100.6 MHz, DMSO-
d6) δ = 161.2, 159.3, 156.0, 125.3, 120.7, 107.2, 55.5, 42.4, 36.8, 26.8,
25.2; Anal. RP-HPLC tR = 9.58 min (method 1A, purity 99.0%); LC-
MS APCI m/z = 344.2 [M+] (anal. calcd for C12H18BrN5S+, m/z =
344.28).
Purity was determined by HPLC or LC-MS, and all compounds
were confirmed to have >95% purity.
Synthesis of 6-Bromo-2,4-dichlorothieno[3,2-d]pyrimidine.
To a solution of 13 (20 g, 0.13 mol) in THF (200 mL) at 0 °C was
added dropwise trichloroacetyl isocyanate (23.9 g, 0.13 mol), and then
the mixture was stirred at rt for 2 h. The reaction mixture was
concentrated under reduced pressure. The remaining residue was
filtered and washed with diethyl ether to furnish methyl 3-[3-(2,2,2-
trichloroacetyl)ureido)]thiophene-2-carboxylate (40 g, 91%) as a
white solid. 1H NMR (400.2 MHz, DMSO-d6) δ = 12.04 (br s,
1H), 11.56 (br s, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.0 Hz,
General Procedure
2
for Synthesis of N4 -[3-
(Dimethylamino)propyl]-N2-methyl-6-phenylthieno[3,2-d]-
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pyrimidine-2,4-diamine 4. To a solution of 3 (0.2 g, 0.58 mmol) in
1,4-dioxane (2 mL) was added phenylboronic acid (0.1 g, 0.70 mmol).
The mixture was thoroughly degassed with nitrogen for 15 min, after
which time Pd(PPh3)2Cl2 (20 mg, 0.03 mmol) was added, followed by
(1 M) aqueous K2CO3 (0.61 mL, 0.61 mmol). The reaction mixture
was stirred at 90 °C overnight and extracted with ethyl acetate (4 × 15
mL). The combined organic layers were washed with brine (3 × 10
mL), dried over Na2SO4, and concentrated in vacuo. The remaining
residue was subjected to column chromatography on silica gel, with
dichloromethane/methanol in a 8.5:1.5 (v/v) ratio as eluent, to furnish
4 as a white solid (0.13 g, 63%). 1H NMR (400.2 MHz, DMSO-d6) δ
= 8.28 (s, 1H), 7.75 (d, J = 6.8 Hz, 2H), 7.53−7.36 (m, 2H), 6.32 (br
s, 1H), 3.45−3.42 (m, 2H), 2.81 (s, 3H), 2.66−2.61 (m, 2H), 2.39 (s,
6H) 1.89−1.79 (m, 2H); 13C NMR (100.6 MHz, DMSO-d6) δ =
165.1, 162.2, 157.2, 147.7, 133.9, 129.7, 129.5, 126.3, 120.1, 105.5,
56.5, 44.3, 38.6, 28.6, 26.2; Anal. RP-HPLC tR = 8.59 min (method 1B,
purity 98.2%); LC-MS APCI m/z 341.2 [M+] (anal. calcd for
C18H23N5S+, m/z = 341.17).
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H.-P.; Gobert, J. G.; Rogers, W. O.; Coppee, J.-Y.; Fandeur, T.;
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in vitro susceptibility of Plasmodium falciparum isolates to artesunate,
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H.; Se, Y.; Schaecher, K.; Smith, B. L.; Socheat, D.; Fukuda, M. M.;
Consortium, A. R. C. S. Evidence of artemisinin-resistant malaria in
Western Cambodia. New Engl. J. Med. 2008, 359, 2619−2620.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Additional text and one figure with details of characterization of
selected compounds and procedures used for in vitro and in
vivo antimalarial studies as well as in vitro metabolism and
mouse exposure studies. This material is available free of charge
via the Internet at http://pubs.acs.org.
́
(5) Gonzalez Cabrera, D.; Douelle, F.; Feng, T.-S.; Nchinda, A. T.;
Younis, Y.; White, K. L.; Wu, Q.; Ryan, E.; Burrows, J. N.; Waterson,
D.; Witty, M. J.; Wittlin, S.; Charman, S. A.; Chibale, K. Novel orally
active antimalarial thiazoles. J. Med. Chem. 2011, 54, 7713−7719.
AUTHOR INFORMATION
Corresponding Author
*Phone +27-21-6502553; fax +27-21-6505195; e-mail Kelly.
Chibale@uct.ac.za.
́
(6) Younis, Y.; Douelle, F.; Feng, T.-S.; Gonzalez Cabrera, D.; Le
■
Manach, C.; Nchinda, A. T.; Duffy, S.; White, K. L.; Shackleford, D.
M.; Morizzi, J.; Mannila, J.; Katneni, K.; Bhamidipati, R.; Zabiulla, K.
M.; Joseph, J. T.; Bashyam, S.; Waterson, D.; Witty, M. J.; Hardick, D.;
Wittlin, S.; Avery, V.; Charman, S. A.; Chibale, K. 3,5-Diaryl-2-
aminopyridines as a novel class of orally active antimalarials
demonstrating single dose cure in mice and clinical candidate
potential. J. Med. Chem. 2012, 55, 3479−3487.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(7) Paquet, T.; Gordon, R.; Waterson, D.; Witty, M. J.; Chibale, K.
Antimalarial aminothiazoles and aminopyridines from phenotypic
whole cell screening of a SoftFocus library. Future Med. Chem. 2012, 4
(18), 2265−2277.
(8) Charman, S. A.; Arbe-Barnes, S.; Bathurst, I. C.; Brun, R.;
Campbell, M.; Charman, W. N.; Chiu, F. C. K.; Chollet, J.; Craft, J. C.;
Creek, D. J.; Dong, Y.; Matile, H.; Maurer, M.; Morizzi, J.; Nguyen, T.;
Papastogiannidis, P.; Scheurer, C.; Shackleford, D. M.; Sriraghavan, K.;
Stingelin, L.; Tang, Y.; Urwyler, H.; Wang, X.; White, K. L.; Wittlin, S.;
Zhou, L.; Vennerstrom, J. L. Synthetic ozonide drug candidate OZ439
offers new hope for a single-dose cure of uncomplicated malaria. Proc.
Natl. Acad. Sci. U.S.A. 2011, 108, 4400−4405.
(9) (a) Madapa, S.; Tusi, Z.; Mishra, A.; Srivastava, K.; Pandey, S. K.;
Tripathi, R.; Puri, S. K.; Batra, S. Search for new pharmacophores for
antimalarial activity. Part II: Synthesis and antimalarial activity of new
6-ureido-4-anilinoquinazolines. Bioorg. Med. Chem. 2009, 17, 222−234.
(b) Mishra, A.; Srivastava, K.; Tripathi, R.; Puri, S. K.; Batra, S. Search
for new pharmacophores for antimalarial activity. Part III: Synthesis
and bioevaluation of new 6-thioureido-4-anilinoquinazolines. Eur. J.
Med. Chem. 2009, 44, 4404−4412. (c) Verhaeghe, P.; Azas, N.;
Gasquet, M.; Hutter, S.; Ducros, C.; Laget, M.; Rault, S.; Rathelot, P.;
Vanelle, P. Synthesis and antiplasmodial activity of new 4-aryl-2-
trichloromethyl quinazolines. Bioorg. Med. Chem. Lett. 2008, 18, 396−
■
We thank Medicines for Malaria Venture (MMV) for financial
support for this research (Project MMV09/0002). We thank
Dr. Jeremy N. Burrows (MMV) and Dr. David Hardick
(BioFocus) for helpful discussions. The University of Cape
Town, South African Medical Research, and South African
Research Chairs initiative of the Department of Science and
Technology, administered through the South African National
Research Foundation, are gratefully acknowledged for support
(K.C.). We thank Christian Scheurer, Petros Papastogiannidis,
and Jolanda Kamber for assistance in performing the
antimalarial assays.
ABBREVIATIONS USED
HTS, high throughput screen; SAR, structure−activity relation-
ship; MSD, mean survival time; H2L, hit to lead; LO, lead
■
̀
optimization; PK, pharmacokinetics; hERG, human ether-a-go-
go-related gene; rt, room temperature; THF, tetrahydrofuran;
DMF, N,N-dimethylformamide; EWG, electron-withdrawing
group; EDG, electron-donating group; p.o, per os (oral
administration); iv, intravenous administration; AUC, area
under the curve; TLC, thin-layer chromatography; HPLC,
high-pressure liquid chromatography; TMS, tetramethylsilane;
MMV, Medicines for Malaria Venture
̀
401. (d) Kabri, Y.; Azas, N.; Dumetre, A.; Hutter, S.; Laget, M.;
Verhaeghe, P.; Gellis, A.; Vanelle, P. Original quinazoline derivatives
displaying antiplasmodial properties. Eur. J. Med. Chem. 2010, 45,
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616−622. (e) Verhaeghe, P.; Dumetre, A.; Castera-Ducros, C.; Hutter,
S.; Laget, M.; Fersing, C.; Prieri, M.; Yzombard, J.; Sifredi, F.; Rault, S.;
Rathelot, P.; Vanelle, P.; Azas, N. 4-Thiophenoxy-2-trichloromethy-
quinazolines display in vitro selective antiplasmodial activity against the
human malaria parasite Plasmodium falciparum. Bioorg. Med. Chem.
Lett. 2011, 21, 6003−6006. (f) Malmquist, N. A.; Moss, T. A.;
Mecheri, S.; Scherf, A.; Fuchter, M. J. Small-molecule histone
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