One of the attractive properties of artemisinins is their extremely fast-killing capability, quickly relieving malaria symptoms. Nevertheless, the unique benefits of these medicines are now compromised by the prolonged parasite clearance times and the increasing frequency of treatment failures, attributed to the increased tolerance of Plasmodium falciparum to artemisinin. This emerging artemisinin resistance threatens to undermine the effectiveness of antimalarial combination therapies. Herein, we describe the medicinal chemistry efforts focused on a cGMP-dependent protein kinase (PKG) inhibitor scaffold, leading to the identification of novel chemical entities with very potent, similar to artemisinins, fast-killing potency against asexual blood stages that cause disease, and activity against gametocyte activation that is required for transmission. Furthermore, we confirm that selective PKG inhibitors have a slow speed of kill, while chemoproteomic analysis suggests for the first time serine/arginine protein kinase 2 (SRPK2) targeting as a novel strategy for developing antimalarial compounds with extremely fast-killing properties.
One of the attractive properties of artemisinins is their extremely fast-killing capability, quickly relieving malaria symptoms. Nevertheless, the unique benefits of these medicines are now compromised by the prolonged parasite clearance times and the increasing frequency of treatment failures, attributed to the increased tolerance of Plasmodium falciparum to artemisinin. This emerging artemisinin resistance threatens to undermine the effectiveness of antimalarial combination therapies. Herein, we describe the medicinal chemistry efforts focused on a cGMP-dependent protein kinase (PKG) inhibitor scaffold, leading to the identification of novel chemical entities with very potent, similar to artemisinins, fast-killing potency against asexual blood stages that cause disease, and activity against gametocyte activation that is required for transmission. Furthermore, we confirm that selective PKG inhibitors have a slow speed of kill, while chemoproteomic analysis suggests for the first time serine/arginine protein kinase 2 (SRPK2) targeting as a novel strategy for developing antimalarial compounds with extremely fast-killing properties.
Malaria, an infectious
disease caused by parasites of the genus Plasmodium (Plasmodium falciparum and Plasmodium vivax are responsible
for most of the clinical cases), is a major healthcare challenge,
especially in developing countries. According to the 2018 World Health
Organization (WHO) global malaria report, in 2017, there were an estimated
219 million cases of malaria, an increase of about 8 million cases
over 2015, with deaths reaching 435 000, a number similar to
the previous year. It is clear that the steep decline in mortality
and disease burden observed between 2000 and 2015 has now been replaced
by a plateau. Even more disturbing is the fact that of these people,
more than two-thirds were children under 5 years of age and expectant
mothers.[1] Malaria control programs are
currently focused on two pillars, namely, disease prevention by vector
control and disease treatment with artemisinin-combination therapies
(ACTs).[2,3] Artemisinins (1, Chart ) are extremely fast-killing
agents, quickly relieving malaria symptoms. Nevertheless, the unique
properties of these medicines are compromised by prolonged parasite
clearance times and the increasing frequency of treatment failures,
attributed to the increased tolerance of P. falciparum to artemisinin.[4−7] These emerging problems have started to raise concerns about the
effectiveness of this widely administered class of antimalarial drugs.[8,9] As a consequence, the development of new bioactive molecules endowed
with novel mechanisms of action has garnered the attention of both
academia and industry. However, the biggest challenge in developing
medicines to replace artemisinins is the identification of new chemical
entities displaying parasite killing kinetics as fast as artemisinins.
Such an achievement is a very tough and demanding task, given that
no compound, to the best of our knowledge, with fast-killing properties
similar to or better than those of artemisinin derivatives (1 and artesunate, Chart ) has been reported in the literature thus far.
Chart 1
Structures of Artemisinin-Based Drugs and Compounds with Potent Inhibitory
Activity against PKG (2–6)
The cyclic GMP-activated serine–threonine protein
kinase,
PKG, has been shown to play an essential role in all of the key stages
of the complex parasite life cycle, including blood stage replication
in the human host as well as gametogenesis and ookinete motility in
the mosquito vector.[10−12] In addition, it has been shown to be key for sporozoite
motility, liver cell invasion, and late liver stage development.[13−15] In the blood stages, PKG regulates the release of proteins from
apical organelles and the mobilization of calcium required for merozoite
egress and invasion.[10] Using phosphoproteomics,
PKG has also been shown to act as a crucial signaling hub in a number
of the malaria parasite’s core processes required for egress
and invasion.[16] Thus, it can be inferred
that targeting PKG is a tractable and multifaceted strategy for malaria
intervention, and developing PKG inhibitors should be considered as
a component of a promising alternative approach to combat malaria.The in vivo proof of principle of using PKG inhibitors against
malaria has been established recently, where an imidazopyridine PKG
inhibitor (2, Chart ) was able to clear infection in the GSK P. falciparum humanized mouse model and block transmission.[17,18] The development of these compounds was based on structure–activity
relationship (SAR) studies using the imidazopyridine compound 3 (Chart )
as a lead. Compound 3 was originally developed by Merck
for the treatment of coccidiosis caused by Eimeria infection,[19] with compound 4 serving as the starting point.[20] Thiazoles
(e.g., compounds 5 and 6, Chart ) constitute another class of
PKG inhibitors,[21] identified in the context
of scaffold-hopping approaches conducted on the pyrrole analogue 3 (Chart ).[20]Despite the very promising antimalarial
potential of PKG inhibitors,
parasite reduction ratio (PRR) studies using the most potent and selective
imidazopyridine and thiazole derivatives 2 (in a previous
study)[17] and 5 (in the context
of this study, Chart ), respectively, clearly showed that both analogues suffer from slow
parasite killing kinetics. Mindful of the aforementioned, the aim
of this study was to refine those structural determinants to provide
the thiazole pharmacophore with fast-killing activity through the
application of molecular diversity-oriented SAR and (bio)isosterism
approaches. Toward this end, state-of-the-art medicinal chemistry
strategies accompanied by cell-based assays and chemoproteomic approaches
were applied. The present research delineates the optimization and
the mode of action of a novel series of thiazole derivatives endowed
with fast-killing properties which are similar to or slightly better
than artesunate (Chart ), the best fast-killing drug available so far. It aspires at the
same time to create novel chemotype leads for further development,
with the ultimate goal of identifying novel fast-killing agents with
“druglike” properties against malaria.
Results and Discussion
Chemistry
The reference compounds 2–5 were synthesized
according to previously published procedures.[17−21] The new thiazole derivatives designed were synthesized
following
modified procedures described in the literature (scheme –). Special emphasis was
placed on the development of routes that are amenable to parallel
synthesis for the exploration of extended SAR studies. First, a library
of 52 compounds (6–57) with a molecular diversity
at the 2-position of the pyrimidine ring and 2-position of the core-thiazole
ring were made (Schemes and ). Starting
from 4-methyl-2-(methylthio/chloro)pyrimidines a1 and a2, deprotonation followed by reaction with 4-fluoro-N-methoxy-N-methylbenzamide b gave the respective ketones c1 and c2,
which upon α-chlorination with sulfuryl chloride in chloroform
afforded the chlorides d1 and d2, respectively.[22,23] Condensation of d1 and d2 with either
numerous different substituted thioamides or thiourea in refluxing
ethanol produced the corresponding 2-substituted thiazoles of structure e, g, and j (Schemes and ).[22,23] The thioether group of e1 was subsequently oxidized to furnish derivatives of formula f (Scheme ).[22,23] An SNAr reaction between f and
different (hetero)aromatic or aliphatic amines afforded the majority
of the final compounds. Where the SNAr reaction was found to be inefficient,
a Buchwald-type C-N palladium-catalyzed cross-coupling was performed
using the respective chlorides e2 (Scheme ).[24] A few derivatives
(9–18) were synthesized by slightly modifying
the aforementioned synthetic procedure (Scheme ).
Scheme 1
Synthetic Procedure Followed for the
Preparation of the Thiazole
Derivatives 6–8 and 19–57
Reagents and conditions: (i)
LDA, THF, −78 °C to rt, (ii) SO2Cl2, CHCl3, 0 °C to rt, (iii) EtOH, reflux, (iv) Oxone,
MeOH:H2O, rt, (v) NaH, THF, rt or iPrOH,
catalytic HCl/dioxane, 120 °C or DMSO, 100 °C (depending
on amine’s basicity), (vi) Pd2(dba)3,
XantPhos, t-BuOK, toluene, reflux, (vii) LiOH·H2O, THF:EtOH:H2O, reflux.
Scheme 3
Synthetic Route Used for the Preparation of the Thiazole
Derivatives 58–60
Reagents
and conditions: (i)
chloroacetaldehyde, acetone, reflux, (ii) NBS, DMF, rt, (iii) LDA,
−78 °C to rt, (iv) Pd(OAc)2, XPhos or cataCXiumA,
CsCO3, THF:H2O or toluene:H2O, reflux,
(v) NIS, catalytic CF3COOH, CH3CN, rt or I2, n-BuLi, THF, −78 °C to rt,
(vi) Pd(PPh3)4, CuI, DMF, 70 °C, (vii)
Pd2(dba)3, XantPhos, t-BuOK,
toluene, reflux, (viii) 4 M HCl/dioxane, dioxane, rt.
Scheme 2
Synthetic Procedure Followed for the Preparation of the Thiazole
Derivatives 9–18
Reagents
and conditions: (i)
EtOH, reflux, (ii) CuCl2, t-BuONO, CH3CN, rt, (iii) morpholine or pyrrolidine, THF, rt, (iv) m-CPBA, CH2Cl2, rt, (v) 1-Boc-4-(4-aminophenyl)piperazine,
TFA, iPrOH, reflux, (vi) TFA or 4 N HCl/dioxane, iPrOH, reflux, (vii) RCOCl or RSO2Cl, Et3N, CH2Cl2, or HATU, DIPEA, DMF.
Synthetic Procedure Followed for the
Preparation of the Thiazole
Derivatives 6–8 and 19–57
Reagents and conditions: (i)
LDA, THF, −78 °C to rt, (ii) SO2Cl2, CHCl3, 0 °C to rt, (iii) EtOH, reflux, (iv) Oxone,
MeOH:H2O, rt, (v) NaH, THF, rt or iPrOH,
catalytic HCl/dioxane, 120 °C or DMSO, 100 °C (depending
on amine’s basicity), (vi) Pd2(dba)3,
XantPhos, t-BuOK, toluene, reflux, (vii) LiOH·H2O, THF:EtOH:H2O, reflux.
Synthetic Procedure Followed for the Preparation of the Thiazole
Derivatives 9–18
Reagents
and conditions: (i)
EtOH, reflux, (ii) CuCl2, t-BuONO, CH3CN, rt, (iii) morpholine or pyrrolidine, THF, rt, (iv) m-CPBA, CH2Cl2, rt, (v) 1-Boc-4-(4-aminophenyl)piperazine,
TFA, iPrOH, reflux, (vi) TFA or 4 N HCl/dioxane, iPrOH, reflux, (vii) RCOCl or RSO2Cl, Et3N, CH2Cl2, or HATU, DIPEA, DMF.Regarding the synthesis of analogues 58–60 (Scheme ), the 4-bromo-2-(4-piperidinyl)thiazole analogue l was initially built from N-Boc-piperidinethioamide.
Thiazole ring formation followed by bromination at 5-position using
NBS gave k,[25] which subsequently
afforded l under halogen-dance conditions.[26] A Suzuki cross-coupling reaction between l and the substituted trifluoroborates produced analogues
of formula m, which upon iodination and Stille cross-coupling
reactions furnished n and o, respectively.
A Buchwald cross-coupling[24] between o and 4-amino-2-methylpyridine or 4-aminopyrimidine followed
by Boc-deprotection under acidic conditions finally led to the final
products 58–60. The isostere of the thiazole derivative 28A, oxazole analogue 28B, was made according
to the synthetic procedure depicted in Scheme S1.
Synthetic Route Used for the Preparation of the Thiazole
Derivatives 58–60
Reagents
and conditions: (i)
chloroacetaldehyde, acetone, reflux, (ii) NBS, DMF, rt, (iii) LDA,
−78 °C to rt, (iv) Pd(OAc)2, XPhos or cataCXiumA,
CsCO3, THF:H2O or toluene:H2O, reflux,
(v) NIS, catalytic CF3COOH, CH3CN, rt or I2, n-BuLi, THF, −78 °C to rt,
(vi) Pd(PPh3)4, CuI, DMF, 70 °C, (vii)
Pd2(dba)3, XantPhos, t-BuOK,
toluene, reflux, (viii) 4 M HCl/dioxane, dioxane, rt.
SAR Studies and Identification of Compounds with Fast-Killing
Properties
Our efforts toward developing novel antimalarial
compounds with potent fast-killing properties were first focused on
performing SAR exploration using compound 6(21) as the starting point (Chart ). The in vitro inhibition of recombinant
PKG activity as well as the antiparasitic activity of each compound
synthesized were evaluated using a kinase inhibition assay and a cell-based P. falciparum asexual blood stage growth inhibition
assay, respectively. In parallel, several compounds were subjected
to a preliminary characterization of the main physicochemical parameters
potentially affecting the “druglike” properties of a
bioactive molecule (lipophilicity, aqueous solubility, cytotoxicity,
cardiotoxicity, plasma protein binding, permeability, and metabolic
clearance). Our ultimate goal was to determine those structural and
molecular features conferring fast-acting potency as well as favorable
biopharmaceutical properties at the same time, and to come up with
a promising lead compound that would be amenable to further optimization/development.The first structural part of compound 6 investigated
was the substitution at position 2 of the thiazole ring (Table ). It seems that the
substitution at this position does not significantly affect the inhibition
of PKG activity since both the unsubstituted amino derivatives 11–13 and other structural motifs (7, 9, 14, 16–18) are well tolerated,
with the exception of the amide derivative 15. On the
other hand, 2-substitution plays a significant role in the antiplasmodial
activity exhibited in cells (Pf EC50)
since the unsubstituted analogues 11 and 12 are less potent compared to 7 and 6, respectively.
The pyrrolidine (10) and acetyl (14) derivatives
displayed similar cell potency, while other acetyl (15, 16) and sulfonamide (17, 18) derivatives either did not improve or abolished potency. It was
also concluded from this SAR study that the basic nitrogen of piperidine
of reference compounds 6 and 7 is crucial
for cell potency as decrease of its basicity significantly reduced
potency (8). Of note, the most potent analogues of this
library (7, 10, and 14) showed
a very promising metabolic stability (clearance in mouse, rat, and
human liver microsomes), permeability (artificial membrane permeability,
AMP), and cardiotoxicity (hERG inhibition) profile,
whereas their cytotoxicity liability was subject to further optimization.
In general, in the AMP permeability assay, compounds with values >0.05
× 10–5 cm/s are considered high permeable,
between 0.01 and 0.05 × 10–5 moderate permeable,
whereas <0.01 × 10–5 cm/s low permeable.[27]
Table 1
SAR Studies Conducted
at Position
2 of the Thiazole Ring (R) and Piperidine Ring (X)
With the objective of
improving both antiparasitic activity and
cytotoxicity, our interest then focused on investigating the impact
of the amino substitution of the pyrimidine ring of 6 and 7 on their biopharmaceutical profile. Toward this
end, the bulky and lipophilic 4-piperazinyl-phenyl ring was replaced
with heteroaromatic substituents endowed with molecular diversity
(Table ). From the
experimental data shown in Table , it can be deduced that although all compounds tested
are very potent PKG inhibitors in vitro, the activity in cells is
strictly dependent on their electrostatic and shape/steric complementarities.
The most active compounds that emerged were the pyrimidine and 2-methylpyridine
derivatives 28A and 31, respectively. Notably,
in the case of 28A, the number and the position of nitrogen
atoms in the heteroaromatic ring are of paramount importance since
the respective pyridine (25), pyrazine (26), and pyridazine (27) analogues exhibited much lower
cell activity (Table ). In addition, small substituents at the pyrimidine ring (29, 30) did not favor an increase in potency,
while isosterism (different five-membered aromatic rings, 19–23) was not applicable in this series, with the result that 19–23 offered at least 10-fold decreased potency. Moreover, in a scaffold-hopping
attempt, the thiazole core ring was replaced by its respective isostere
oxazole (28B, Table ). Interestingly, this modification in spite of the
better solubility achieved (226 μM), resulted in decreased PKG
inhibitory activity (IC50 = 89.1 nM), abolishing at the
same time the antiparasitic activity (EC50 > 5 μM).
Regarding 31, small substituents around the 2-methylpyridine
ring (32, 34) were not detrimental, without
however offering any significant improvement in potency. Finally,
other (poly)substituted pyridine derivatives (35–40) did not give the desired result. After further evaluating the most
potent analogues of this library (28A and 31), it was found that the incorporation of pyrimidine and 2-methylpyridine,
besides increasing potency (compared to reference compound 6), also delivered molecules with very good solubility, permeability,
and metabolic stability, although at the expense of potent inhibition
against the cardiac ion channel hERG (IC50 values for 28A and 31 are 0.6 and 1.0
μM, respectively, Table ). Moreover, the cytotoxicity/cell activity safety windows
of both analogues were significantly improved (36 and 25 for compounds 28A and 31, respectively, compared to 6.8 and
5.4 for the reference compounds 6 and 7,
respectively).
Table 2
SAR Studies Conducted at Position
2 of the Pyrimidine Ring of the Thiazole Scaffold (Aromatic Substitution,
Ar)
Another desirable pharmacological feature of this
series concerns
their potential to act as transmission-blocking agents (Tables and 2). The capability of compounds to prevent mature male and female
gametocytes from activation and progressing to extracellular gametes
(both of which are considered indicators of gametocyte functionality)
was estimated using specialized bioassays.[28] The activation of male gametocytes to differentiate into mature
microgametes was evaluated by measuring levels of exflagellation (extrusion
of rapidly waving flagellated microgametes from the infected erythrocyte),
whereas the activation of female gametocytes was based on the specific
expression of the Pfs25 protein at the surface of the female gamete
following activation. The potent transmission-blocking effect exerted
by the majority of the compounds can likely be ascribed to the powerful
specific PKG inhibitory activity exhibited by the analogues tested.
It has been previously demonstrated that PKG is essential for gametocyte
activation and their transformation to gametes.[12]31, being one of the most promising compounds
of this series, endowed with a balanced physicochemical and cell activity
profile, also potently inhibited the activation of both male and female
gametocytes with EC50 values of 0.30 and 0.40 μM,
respectively.Notwithstanding the liability of inhibiting hERG, 28A and 31 were subsequently
tested in terms
of their fast-acting properties in a parasite reduction ratio (PRR)
study, using artesunate (fast rate of killing), pyrimethamine (moderate
rate of killing), and atovaquone (slow rate of killing) for comparison.
Surprisingly, both compounds exhibited an extremely fast-killing effect,
displaying 87 and 93% clearance of parasites in the first 24 h, respectively.
As depicted in Figure , this effect is similar to or slightly better than artesunate (Figure ), the best fast-killing
drug available so far.
Figure 1
PRR study with compounds 28A and 31.
Artesunate (fast rate of killing), pyrimethamine (moderate rate of
killing), and atovaquone (slow rate of killing) have also been included.
PRR study with compounds 28A and 31.
Artesunate (fast rate of killing), pyrimethamine (moderate rate of
killing), and atovaquone (slow rate of killing) have also been included.Encouraged by the unprecedented fast-killing potency
of 31, which is accompanied by a favorable aqueous solubility,
permeability,
and metabolic stability profile (Table ), this compound was initially tested for other (besides hERG) secondary pharmacology-related liabilities (Table ). It was found that
no agonistic or antagonistic activity was exerted against several
receptors and ion channels tested, while 31, except for
inhibiting acetylcholinesterase (IC50 = 0.40 μM),
did not exhibit any inhibitory activity against human monoamine oxidase
A (MAOA), phosphodiesterase, and organic anion transport polypeptide
OATP1B1 (Table ).
Table 3
In Vitro Evaluation of Compound 31 Effect
against Other Enzymes, Ion Channels, and Receptors
protein
activity
(μM)
human monoamine oxidase
A (MAOA) inhibitor
IC50 > 100
β2 adrenoreceptor
human agonist
EC50 > 100
β2 adrenoreceptor
human antagonist
IC50 > 100
human PXR (NR1I2) agonist
EC50 > 50
human KCNQ1/KCNE1 (Kv7.1/MinK) blocker
IC50 > 25
human aryl hydrocarbon receptor
(AhR) agonist
EC50 > 50
organic anion transport
polypeptide OATP1B1 inhibitor
IC50 > 30
acetylcholinesterase
(AChE)
inhibitor
IC50 = 0.40
phosphodiesterase 3A (PDE3A)
inhibitor
IC50 > 100
human CaV1.2 (L-type) calcium channel blocker
IC50 > 30
Our approach
then focused on the identification of a more developable
fast-killing agent, with similar or better antiparasitic activity
in cells, and even further reduced cytotoxicity and/or potency against hERG. Our strategy to this end was based on the reduction
of the numbers of aromatic rings of 31, on the grounds
that the more aromatic rings a compound has, the more chance there
is for this agent to exert toxicity. For synthetic reasons, our efforts
were first focused on replacing the 2-methylpyridine heteroaromatic
ring of 31 with a large number of nonaromatic/aliphatic
groups (Table ). Compound 5 (Chart )
was included for comparison. According to the data depicted in Table , these structural
modifications resulted in a great improvement of cytotoxicity (41–57), which was however counterbalanced by the significant
drop (10- to 50-fold) in the antiplasmodial potency observed. Although
the permeability of all derivatives was maintained at acceptable levels
(Table , AMP values),
none of the newly synthesized compounds 41–57 displayed
better activity than 5, 28A, and 31.
Table 4
SAR Studies Conducted at Position
2 of the Pyrimidine Ring of the Thiazole Scaffold (Nonaromatic Substitution,
R)
Additionally, PRR studies conducted with 5 clearly
showed that it is a slow-acting compound (Figure ), rendering the heteroaromatic substitution
at this specific position of the thiazole-pyrimidine scaffold a key
player toward the identification of fast-killing antimalarial compounds.
Figure 2
PRR study
with compounds 5 and 28A. Artesunate
(fast rate of killing), pyrimethamine (moderate rate of killing),
and atovaquone (slow rate of killing) have also been included.
PRR study
with compounds 5 and 28A. Artesunate
(fast rate of killing), pyrimethamine (moderate rate of killing),
and atovaquone (slow rate of killing) have also been included.After having experimentally confirmed that position
2 of the pyrimidine
ring is not amenable to aliphatic/nonaromatic substitution, furnishing
slow-acting analogues, a preliminary exploration of position 4 of
the thiazole ring was performed. By use of similar fundamental design
principles, the lipophilic para-fluorophenyl group
of 28A and 31 was replaced by the smaller
cyclopropyl group endowed with an sp2 character (Table ). Somewhat surprisingly, despite
the outstanding improvement in cytotoxicity noted by 58 (EC50 = 43.7 μM), the antiplasmodial potency was
decreased by 23-fold (EC50 = 3.45 μM) compared to 28A (EC50 = 0.15 μM). Intriguingly, the same
group preserved (compound 59, IC50 = 0.27
μM) the antiparasitic activity of 31 (IC50 = 0.16 μM), attenuating at the same time by 5-fold the hERG inhibitory activity and slightly (2.5-fold) the cytotoxicity
(Table ). Compound 59 also showed very good solubility, permeability, and metabolic
clearance properties (Table ). Finally, the potency was maintained (EC50 =
0.19 μM) and the cytotoxicity (EC50 = 20 μM,
cytotoxicity/cell activity safety window = 105) was yet further improved
by substituting the 4-position of the thiazole ring with the piperidinylmethyl
group (compound 60, Table ).
Table 5
Preliminary SAR Studies Conducted
at Position 4 of the Thiazole Ring (R)
Furthermore, PRR studies
using compounds 59 and 60 proved that both
analogues are also endowed with potent
fast-killing properties (with 59 displaying a better
fast-killing profile than 60), exhibiting an effect similar
to that of artesunate (Figure ).
Figure 3
PRR study with compounds 59 and 60. Artesunate
(fast rate of killing), pyrimethamine (moderate rate of killing),
and atovaquone (slow rate of killing) have also been included.
PRR study with compounds 59 and 60. Artesunate
(fast rate of killing), pyrimethamine (moderate rate of killing),
and atovaquone (slow rate of killing) have also been included.
Additional Target Identification by Chemoproteomics
With the purpose of providing new insights into the mechanism of
action of this series and specifically to investigate whether these
potent fast-killing kinetics are attributed to a PKG-selective or
an off-target (other than PKG) or a synergistic effect, assays utilizing
the P. falciparum PKG gatekeeper mutant
line (T618Q, Table )[17] followed by chemoproteomic approaches
were deployed. Initially, representative thiazole derivatives (26, 28A, 31, 32) were
evaluated in the aforementioned transgenic cell line to determine
whether PfPKG is their primary target in the asexual
blood stages. In this transgenic cell line, the threonine residue
at position 618 of the PfPKG catalytic site, which
functions as a gatekeeper residue forming a small lipophilic pocket
which is essential for the binding of small molecule inhibitors in
the active site of the enzyme, has been replaced by the larger glutamine
residue. As a consequence, entry to this small lipophilic pocket has
been disrupted in the mutant line, preventing inhibitor binding to
the catalytic site of PKG.[17,29] Compound 5, which has been demonstrated by previous studies to exhibit high
levels of potency against PKG in vitro and a remarkable selectivity
over other human kinases,[21] was also included
for comparison.
Table 6
Inhibitory Activity of Representative
Compounds against Plasmodium falciparum WT and Transgenic PKG T618Q Cell Lines at 48 and 72 h
The results
presented in Table show that the potent inhibitory activity of 5 against
parasite growth (EC50 = 110 nM) in the
WT cell line at 72 h is significantly reduced (40-fold) in the T618Q
mutant line (at 72 h). These experimental data strongly confirm that
the potent antiparasitic activity in cells for this derivative is
attributed to its selective inhibitory activity against PKG. Notably,
the fact that 5 also exhibits a significantly decreased
activity in the WT cell line at 48 h (EC50 = 1.02 μM)
compared to 72 h (10-fold, Table ) substantiates the PRR studies performed with 5 (Figure ), classifying this analogue as a slow-acting agent. In particular,
the loss of activity of 5 in the P. falciparum PKG gatekeeper mutant line (T618Q) at both 48 and 72 h (Table ) further demonstrates
that selective PKG inhibition is accompanied by the exhibition of
slow parasite killing kinetics. In contrast, all of the new thiazole
derivatives tested exerted a similar effect in both WT and mutant
cell lines both at 48 and 72 h (Table ), a signature of off-target (other than PKG) activity
conferring the potent fast-killing properties of this series.The above-mentioned results motivated us to investigate in more
detail the molecular mechanism through which this powerful fast kill
rate is exerted. Therefore, a chemoproteomic approach (Kinobeads profiling)
was applied to identify the target(s) inhibited by one of the most
potent derivatives of the series developed (31, Table ). Compound 50 (Table ), exhibiting an in vitro inhibitory potency against PKG (IC50 = 12.6 nM) comparable to 31, but a much weaker
antiplasmodial activity in cells (EC50 = 4.03 μM),
was also included to have a reliable monitoring framework in terms
of the proteins whose inhibition is accompanied by potent antiparasitic/antimalarial
activity and a fast-killing effect. In that way, any potential misinterpretation
of the results could be avoided. Kinobeads represent a selection of
immobilized promiscuous ATP-competitive kinase inhibitors,[30,31] to affinity capture potential kinase target proteins from a P. falciparum protein extract. Using this approach,
the activity of the compounds against 54 endogenous P. falciparum kinases was analyzed (Figure a). To explore additional potential
kinase (Figure b)
and nonkinase targets (Table S1), the compounds
were attached to a bead matrix via their amine moiety and pull down
assays were performed. All of the experiments were performed either
without or with excess of 31 or 50 to identify
target proteins for which capture is competitively inhibited. Both
compounds were added in concentrations between 0.08 and 20 μM
aiming at establishing a competition-binding curve and determining
a half-maximal inhibitory concentration (IC50). The IC50 values obtained in this kind of experiment are representative
of target affinity but are also affected by the target protein affinity
for the bead-immobilized ligands. The latter effect can be deduced
by determining the apparent dissociation constants (Kdapp), which are largely nondependent on the
bead ligand, thus representing the depletion of the target proteins
by the beads.[32] The proteins captured by
the beads were finally quantified by using isotope tagging of tryptic
peptides and analyzed by LC-MS/MS.[32]
Figure 4
Chemoproteomics
profiling of compounds 31 and 50. (a) Both
compounds were profiled on Kinobeads, which represent
a combination of immobilized promiscuous ATP-competitive kinase inhibitors,
in a P. falciparum protein extract.
A total of 54 P. falciparum kinases
were analyzed. The concentration of the “free” compounds
used for competition, compound 50 and compound 31, was between 0.08 and 20 μM over six samples. (b)
Compounds 50 and 31 were profiled with a
bead matrix generated by immobilizing either compound 50 or compound 31 to beads, and competed with the respective
“free” analogue over six concentrations between 0.08
and 20 μM. The Heatmaps show the protein kinases affected by
any of the two compounds in two independent experiments, respectively.
The values shown are apparent pKd values
(blue: decreasing apparent pKd values;
white: no competition; gray: protein not identified).
Chemoproteomics
profiling of compounds 31 and 50. (a) Both
compounds were profiled on Kinobeads, which represent
a combination of immobilized promiscuous ATP-competitive kinase inhibitors,
in a P. falciparum protein extract.
A total of 54 P. falciparum kinases
were analyzed. The concentration of the “free” compounds
used for competition, compound 50 and compound 31, was between 0.08 and 20 μM over six samples. (b)
Compounds 50 and 31 were profiled with a
bead matrix generated by immobilizing either compound 50 or compound 31 to beads, and competed with the respective
“free” analogue over six concentrations between 0.08
and 20 μM. The Heatmaps show the protein kinases affected by
any of the two compounds in two independent experiments, respectively.
The values shown are apparent pKd values
(blue: decreasing apparent pKd values;
white: no competition; gray: protein not identified).Based on our pharmacological results, both 31 (Table ) and 50 (Table ) are potent
PKG inhibitors in vitro, but only 31 exhibits a strong
effect in the P. falciparum growth
inhibition assay at 48 h. Consequently, proteins preferentially binding
to 31 and not to 50 are more likely to be
potential targets that could lead to fast killing of the parasites.
Capture experiments using Kinobeads showed that besides PKG, both 31 and 50 also inhibit calcium-dependent protein
kinase 4 (CDPK4) with Kdapp values of 50 and 60 nM, respectively (Figure ). Both compounds were also shown to bind
to CDPK1 (Figure ),
with 31 exhibiting a much more potent inhibitory activity
than 50 (Kdapp = 0.05 μM and Kdapp = 2.1 μM for 31 and 50, respectively).
The importance of CDPK1 in the viability of P. falciparum during the erythrocytic and sporozoite stages, as well as its regulating
role in parasite motility during egress and invasion in response to
calcium transitions have been particularly underlined elsewhere.[33] Three additional proteins which showed competition
by both 31 and 50 were not considered as
efficacy targets: (a) zinc finger (CCCH type) protein (Table S1), as the apparent Kdapp values were high (3.9 μM for 31 versus 12.6 μM for 50), (b) glycogen
synthase kinase 3 (GSK3, Figure ) (Kdapp values
of 0.09 versus 5.3 μM for 31 and 50, respectively), and (c) casein kinase 1 (CK1, Figure b) (Kdapp values of 0.15 versus 1.5 μM for 31 and 50, respectively), as a second PfPKG inhibitor with activity against P. falciparum was tested and did not show any activity against GSK3 and CK1 (data
not shown). As a follow-up to the above experiments, comparison of 31 and 50 revealed a difference in binding to
only one protein, the serine/arginine protein kinase 2 (SRPK2 or CLK2, Figure a).
The Kdapp values of 31 and 50 for SRPK2 are 0.19 μM and >20 μM,
respectively. Notably, when tested on the immobilized compounds (Figure b),
SRPK2 was found to not bind to 50 attached to beads,
which suggests no affinity for the compound. In contrast, SRPK2 was
found to bind to 31 attached to beads, with 31 strongly competing the protein with an apparent Kd value of 0.05 μM.Altogether, the results
of our target identification experiments
suggest SRPK2 as a protein kinase target in which the potent fast-killing
properties of the most active derivatives developed herein could be
attributed to. Although we cannot rule out a synergistic SRPK2/CDPK1,4/PKG
effect, such a scenario seems to be less likely due to the inhibitory
effect also displayed by 50 against CDPK1, CDPK4, and
PKG. Therefore, our study highlights for the first time SRPK2 targeting
as a tractable approach for the development of potential fast-killing
antimalarial drugs. Recent global kinomic and phospho-proteomic analyses
of the humanmalaria parasite P. falciparum have emphasized the necessity of SRPK2 in parasite proliferation
during the erythrocytic asexual cycle.[34,35] A transcriptomic
study by Hoeijmakers et al.[36] indicates
the expression profile of SRPK2 (27–45 h post invasion) is
more extensive than that of PKG (32–45 h post invasion) in
blood stages, which supports the idea that inhibition of SRPK2 might
mediate the fast kill phenotype of compound 31 since
it is likely active against a greater proportion of the 48 h blood
stage cycle. We have previously assumed that the slow kill phenotype
of specific PKG inhibitors is due to the narrow window of expression
just prior to egress and invasion. Previous work has implicated Plasmodium
SRPKs (CLKs) such as SRPK2 (CLK2) in the regulation of alternative
splicing of mRNA.[37] CLK1 and CLK2 (both
located in the nucleus)[34] are orthologues
of a yeast SR protein kinase Sky1p that phosphorylates SR proteins
which bind to RNA and play a key role in RNA splicing. SR kinases
phosphorylate SR proteins in a serine/arginine-rich domain, thereby
influencing their activity and localization. CLK1–CLK4 have
been shown to phosphorylate SR protein orthologues[34,38] and are expressed in blood stages and gametocytes.[38] It is therefore possible that breakdown of the regulation
of mRNA splicing by inhibition of SRPK2 contribute significantly to
the fast kill phenotype of compound 31. There is a precedent
for targeting SRPK/CLK to treat disorders such as Duchenne muscular
dystrophy[39] and Alzheimer’s disease[40] either by disrupting alternative splicing or
by correcting the aberrant splicing observed in some diseases.[37] During the review process of the current article,
an interesting research paper was published, highlighting CLK3 as
a multistage cross-species malarial target, the inhibition of which
could offer both a prophylactic and transmission-blocking effect.[41]Collectively, starting from “hits”
(4–6, Chart ) acting through a well-validated target
(PKG) playing a crucial
role in all of the key stages of the complex parasite life cycle but
offering a low parasite killing rate, we were able to develop novel
powerful fast kill entities comparable to artemisinins by refocusing
on a kinase polypharmacology strategy. Whole transcriptome and kinome
screens suggest that Plasmodiuminfection dramatically
alters signaling networks within both the circulation and hepatocytes.[42−46] Recent evidence also suggests that signaling alterations in infected
cells may affect the response of cells to extrinsic stimuli and provide
new targets for therapeutic intervention which are unique to infected
cells.[47] Given that kinases are critical
enzymes in cell signaling, protein regulation, cellular transport,
secretory processes, and many other cellular pathways in malaria transmission,
infection, and spread, the development of bioactive molecules targeting
multiple kinases has the potential to offer a superior effect compared
to a single agent. In addition, targeting simultaneously more than
one parasite component may limit the development of resistance to
a single therapeutic. The exploitation of the polypharmacology of
kinase inhibitors has already become a major focus for the development
of more efficient anticancer therapeutics and is currently a relatively
untapped resource for the repurposing of drugs for use against malaria
and other infectious diseases.[48] In addition,
it has long been recognized that Plasmodium protein kinases are attractive
targets for antimalarial chemotherapy.[49,50] The Plasmodium
kinome is made up of between 65[51] and 99
protein kinases,[52] comprising a single
PKG and a family of four SRPK-like kinases (CLKs).[53]
Conclusions and Future Plans
In
this article, the development of powerful fast-acting agents
with killing rates similar to or better than artemisinins is described,
and the structural and molecular characteristics providing such unique
properties are highlighted. Compounds 31, 59, and 60 could be considered promising lead compounds
for further optimization in the search for identifying novel antimalarial
agents with new mechanisms of action and a strong fast-killing profile
which are missing from the therapeutic arsenal against malaria. In
parallel, the present study confirms that selective PKG inhibition
is accompanied by low parasite killing rates, while it brings to light
for the first time the tractability of targeting SRPK2/PKG in developing
powerful fast-kill chemotypes with curative and transmission-blocking
properties against malaria. Our efforts are currently being focused
on further refinement of the structural features of 31, 59, and 60, aiming at extracting a new
generation of fast-kill antimalarial chemical entities with an optimal
developability profile. Elucidation of the involvement of SRPK2 inhibition
in mediating the fast-kill phenotype of these compounds is also underway
and will utilize recombinant expression and immunoprecipitation of
the native kinase from parasite extracts, as described elsewhere.[38]
Experimental Section
General
Chemistry Information
All starting materials
were purchased from commercial sources and used as received or synthesized
via literature procedures. Solvents were dried using a commercial
solvent purification system and stored under nitrogen. All final compounds
were characterized by 1H NMR spectroscopy and LCMS. 1H NMR spectra were recorded on a Bruker Avance 400 MHz spectrometer
at 293 K. Purity was determined by HPLC (Acquity UPLC BEH C18 1.7
μ 2.1 mm × 50 mm) at 35 °C. All compounds tested present
a purity >95%, except for a couple of derivatives that presented
a
purity of >90%. Method: acetate NH4 25 mM + 10% ACN
at
pH 6.6/ACN, 0–0.2 min 100:0; 0.2–1.0 min 10:90; 1.0–1.8
min 10:90; 1.8–2.0 min 100:0. Flow: 0.8 mL/min. The UV detection
wavelength was 254 and 210 nm. Positive-ion mass spectra (high-resolution
mass spectroscopy) was acquired using a QSTAR Elite (AB Sciex Instruments)
mass spectrometer, equipped with a turbospray source, over a mass
range of 250–700, with a scan time of 1 s. The elemental composition
was calculated using Analyst QS 2.0 software.Compounds 5–8 were synthesized using previously described procedures.[21]
Synthesis of Final Compounds 9 and 10
To a microwave vial containing a magnetic
stirring bar
were added compounds of formula i (0.38 mmol), 1-Boc-4-(4-aminophenyl)piperazine
(116 mg, 0.42 mmol), iPrOH (4 mL), and trifluoroacetic
acid (TFA, 44 μL, 0.57 mmol). The vial was capped and stirred
in a Biotage microwave reactor at 105 °C for 2 h. Additional
TFA (44 μL, 0.57 mmol) was added, and the reaction mixture further
stirred in the microwave reactor at 105 °C for 2 h to afford
the Boc-protected analogues of 9 and 10.
To the resulting crude mixtures were then added 4 N HCl in dioxane
(6 mL). The reaction mixtures were stirred at room temperature for
3 h and concentrated under reduced pressure. The crude materials were
purified by semipreparative HPLC to furnish the desired final products.
To a round-bottom flask containing a magnetic stirring bar were
added compound j (1.0 g, 3.26 mmol), 1-Boc-4-(4-aminophenyl)piperazine
(1.09 g, 3.91 mmol), iPrOH (20 mL), and trifluoroacetic
acid (TFA, 0.30 mL, 3.91 mmol). The reaction mixture was stirred at
100 °C for 2 h. Additional TFA (0.30 mL, 3.91 mmol) was added
and the reaction mixture was further stirred at 100 °C for 2
h, concentrated under reduced pressure, diluted with EtOAc, and washed
with saturated NaHCO3 solution. The organic phase was dried
over Na2SO4, filtered, and concentrated under
reduced pressure. The crude material was purified on silica gel cartridge
(0–100% EtOAc in cyclohexane) to give the Boc-protected derivative
of 12 (1.17 g, 66%) as a yellow solid. 1H
NMR (400 MHz, DMSO-d6) δ 9.26 (s,
1H), 8.05 (d, J = 5.36 Hz, 1H), 7.61–7.52
(m, 6H), 7.27 (t, J = 8.90 Hz, 2H), 6.87 (d, J = 9.13 Hz, 2H), 6.20 (d, J = 5.36 Hz,
1H), 3.46 (t, J = 4.90 Hz, 4H), 3.00 (t, J = 5.09 Hz, 4H), 1.42 (s, 9H). MS: m/e 548 (MH+). To a stirred solution of the Boc-protected
carbamate (100 mg, 0.18 mmol) in anhydrous 1,4-dioxane (8 mL) was
added 4 N HCl in dioxane (8 mL). The reaction mixture was stirred
at room temperature for 4 h, concentrated and passed through an SCX-2
cartridge. The impurities were first eluted using MeOH, and the desired
product was subsequently eluted using 7 N NH3 in MeOH solution.
The resulting solution was concentrated under reduced pressure, and
the crude material was purified by semipreparative HPLC. Bright yellow
solid. Yield = 40%. 1H NMR (400 MHz, DMSO-d6) δ 9.22 (s, 1H), 8.05 (d, J =
5.34 Hz, 1H), 7.60–7.53 (m, 6H), 7.28 (t, J = 8.90 Hz, 2H), 6.84 (d, J = 9.05 Hz, 2H), 6.20
(d, J = 5.34 Hz, 1H), 5.76 (s, 1H), 3.01–2.91
(m, 4H), 2.89–2.77 (m, 4H). MS: m/e 448 (MH+). Purity was determined as >95%
by
HPLC (292 nm). R: 0.96 min (Acquity UPLC
BEH C18 1.7 μm, 3 mm × 50 mm, acetate NH4 25
mM + 10% ACN at pH 6.6/ACN).
To a microwave vial containing a magnetic stirring bar
were added
compound j (70 mg, 0.23 mmol), 4-morpholinoaniline (41
mg, 0.23 mmol), iPrOH (4 mL), and 4 N HCl in dioxane
(57 μL, 0.23 mmol). The vial was capped and stirred in a Biotage
microwave reactor at 170 °C for 30 min. The reaction mixture
was concentrated under reduced pressure and purified by semipreparative
HPLC. Pale brown solid. Yield = 4%. 1H NMR (400 MHz, DMSO-d6) δ 9.25 (s, 1H), 8.06 (d, J = 5.36 Hz, 1H), 7.61–7.53 (m, 6H), 7.28 (t, J = 8.92 Hz, 2H), 6.87 (d, J = 9.11 Hz, 2H), 6.21
(d, J = 5.35 Hz, 1H), 3.75 (t, J = 4.74 Hz, 4H), 3.04 (t, J = 4.78 Hz, 4H). MS: m/e 449 (MH+). Purity was determined
as >95% by HPLC (296 nm). R: 1.13
min
(Acquity UPLC BEH C18 1.7 μm, 3 mm × 50 mm, acetate NH4 25 mM + 10% ACN at pH 6.6/ACN).
To a stirred solution
of tert-butyl 4-[4-({4-[2-amino-4-(4-fluorophenyl)-1,3-thiazol-5-yl]pyrimidin-2-yl}amino)phenyl]piperazine-1-carboxylate
(100 mg, 0.18 mmol) in anhydrous CH2Cl2 (10
mL) were added acetyl chloride (9 μL, 0.13 mmol) and dry triethylamine
(18 μL, 0.13 mmol). The reaction mixture was stirred at room
temperature for 4 h under N2 atmosphere. Additional acetyl
chloride (3.9 μL, 0.055 mmol) and triethylamine (7.6 μL,
0.055 mmol) were added and the mixture was further stirred at room
temperature overnight and concentrated under reduced pressure to give
the crude BOC-protected 14 (MS: m/e 590 (MH+)), which was dissolved in anhydrous
1,4-dioxane (8 mL) and 4 N HCl in dioxane solution (8 mL). The mixture
was stirred at room temperature for 4 h, concentrated under reduced
pressure, and the residue was purified by semipreparative HPLC. Orange
solid. Yield = 22%. 1H NMR (400 MHz, DMSO-d6) δ 12.44 (s, 1H), 9.50 (s, 1H), 8.67 (br s, 2H),
8.21 (d, J = 5.26 Hz, 1H), 7.64–7.58 (m, 4H),
7.31 (t, J = 8.90 Hz, 2H), 6.92 (d, J = 9.10 Hz, 2H), 6.40 (d, J = 5.26 Hz, 1H), 3.26
(br s, 8H), 2.20 (s, 3H). MS: m/e 490 (MH+). Purity was determined as >95% by HPLC (258
nm). R: 0.95 min (Acquity UPLC BEH C18
1.7 μm, 3 mm × 50 mm, 0.1% formic acid in water/0.1% formic
acid in ACN).
To a stirred solution
of tert-butyl 4-[4-({4-[2-amino-4-(4-fluorophenyl)-1,3-thiazol-5-yl]pyrimidin-2-yl}amino)phenyl]piperazine-1-carboxylate
(80 mg, 0.15 mmol) in anhydrous CH2Cl2 (10 mL)
were added cyclopropanecarbonyl chloride (13.2 μL, 0.15 mmol)
and dry triethylamine (20.3 μL, 0.15 mmol). The reaction mixture
was stirred at room temperature for 18 h under N2 atmosphere.
The reaction mixture was concentrated under reduced pressure to give
the Boc-protected carbamate of 15 (MS: m/e 616 (MH+) as a crude residue, which
was dissolved in anhydrous 1,4-dioxane (8 mL) and 4 N HCl in dioxane
solution (8 mL). The resulting mixture was stirred at room temperature
for 4 h, concentrated under reduced pressure, and the crude material
was purified by semipreparative HPLC. Bright yellow solid. Yield =
11%. 1H NMR (400 MHz, DMSO-d6) δ 8.67 (br s, 2H), 8.29 (d, J = 5.52 Hz,
1H), 7.77 (br s, 2H), 7.55 (dd, J = 5.60, 8.77 Hz,
2H), 7.29 (t, J = 8.89 Hz, 2H), 7.07 (d, J = 8.98 Hz, 2H), 6.99 (d, J = 9.02 Hz,
2H), 6.64 (d, J = 5.52 Hz, 1H), 3.36–3.34
(m, 4H), 3.27–3.21 (m, 4H), 1.97–1.91 (m, 1H), 0.94–0.90
(m, 2H), 0.88–0.82 (m, 2H). MS: m/e 516 (MH+). Purity was determined as >95%
by
HPLC (254 nm). R: 0.93 min (Acquity UPLC
BEH C18 1.7 μm, 3 mm × 50 mm, 0.1% formic acid in water/0.1%
formic acid in ACN).
To a stirred solution
of 1-methylpiperidine-4-carboxylic acid (13 mg, 0.09 mmol) in anhydrous
DMF (10 mL) under N2 atmosphere were added HATU (52 mg,
0.14 mmol), DIPEA (48 μL, 0.27 mmol), and tert-butyl 4-[4-({4-[2-amino-4-(4-fluorophenyl)-1,3-thiazol-5-yl]pyrimidin-2-yl}amino)phenyl]piperazine-1-carboxylate
(50 mg, 0.09 mmol). The resulting mixture was stirred at room temperature
for 2 h and then heated at 60 °C for 72 h. In addition, 1-methylpiperidine-4-carboxylic
acid (13 mg, 0.09 mmol), HATU (35 mg, 0.09 mmol), and DIPEA (32 μL,
0.18 mmol) were added and the mixture was further stirred at 60 °C
for 48 h. LCMS showed product to starting material ratio as 1:1. Hence,
NaH (60% dispersion in mineral oil, 7.3 mg, 0.18 mmol) was added with
the mixture being stirred at room temperature for further 4 h. The
reaction mixture was diluted with water and extracted with EtOAc.
The combined organic extracts were dried over Na2SO4, filtered, and concentrated under reduced pressure to give
the Boc-protected 16 as a pale brown solid (MS: m/e 673 (MH+)). This pale brown
solid was dissolved in 4 N HCl in dioxane (10 mL) and stirred at room
temperature for 4 h, concentrated under reduced pressure, and purified
by semipreparative HPLC. Orange solid. Yield = 17%. 1H
NMR (400 MHz, DMSO-d6) δ 12.63 (s,
1H), 9.48 (s, 1H), 9.43 (br, 1H), 8.73 (br s, 2H), 8.23 (d, J = 5.26 Hz, 1H), 7.62–7.57 (m, 4H), 7.31 (t, J = 8.89 Hz, 2H), 6.92 (d, J = 9.07 Hz,
2H), 6.42 (d, J = 5.24 Hz, 1H), 3.26 (s, 8H), 3.04–2.95
(m, 2H), 2.83–2.76 (m, 3H), 2.54 (s, 3H), 2.14–2.08
(m, 2H), 1.91–1.78 (m, 2H). MS: m/e 573 (MH+). Purity was determined as >95%
by
HPLC (260 nm). R: 0.86 min (Acquity UPLC
BEH C18 1.7 μm, 3 mm × 50 mm, 0.1% formic acid in water/0.1%
formic acid in ACN).
To a stirred solution
of tert-butyl 4-[4-({4-[2-amino-4-(4-fluorophenyl)-1,3-thiazol-5-yl]pyrimidin-2-yl}amino)phenyl]piperazine-1-carboxylate
(150 mg, 0.27 mmol) and Et3N (46 μL, 0.33 mmol) in
anhydrous CH2Cl2 (25 mL) at 0 °C under
N2 atmosphere was added 1-propanesulfonyl chloride (32
μL, 0.28 mmol). The reaction mixture was stirred at 0 °C
for 30 min and then allowed to warm to room temperature and stirred
at room temperature for 5 h. In addition, Et3N (46 μL,
0.33 mmol) was added and the mixture was stirred under reflux for
a further 2 h and concentrated under reduced pressure to give the
Boc-protected carbamate of 17 (160 mg) as a yellow oil
(MS: m/e 654 (MH+)).
The oil was dissolved in anhydrous 1,4-dioxane (3 mL) and 4 N HCl
in dioxane (7 mL), stirred at room temperature for 4 h, and concentrated
under reduced pressure. The crude residue was purified by semipreparative
HPLC. Orange solid. Yield = 47%. 1H NMR (400 MHz, DMSO-d6) δ 13.08 (br s, 1H), 9.52 (s, 1H), 8.80
(br s, 2H), 8.16 (d, J = 5.32 Hz, 1H), 7.66 (dd, J = 5.40, 8.82 Hz, 2H), 7.59 (d, J = 9.12
Hz, 2H), 7.41 (t, J = 8.86 Hz, 2H), 6.92 (d, J = 9.12 Hz, 2H), 6.10 (d, J = 5.30 Hz,
1H), 3.25 (s, 8H), 3.06 (t, J = 7.59 Hz, 2H), 1.73
(sextet, J = 7.50 Hz, 2H), 0.99 (t, J = 7.48 Hz, 3H). MS: m/e 554 (MH+). Purity was determined as >95% by HPLC (274 nm). R: 0.85 min (Acquity UPLC BEH C18 1.7 μm,
3 mm × 50 mm, 0.1% formic acid in water/0.1% formic acid in ACN).
To a stirred solution
of tert-butyl 4-[4-({4-[2-amino-4-(4-fluorophenyl)-1,3-thiazol-5-yl]pyrimidin-2-yl}amino)phenyl]piperazine-1-carboxylate
(80 mg, 0.15 mmol) in anhydrous CH2Cl2 (10 mL)
were added 4-chlorosulfonyl-piperidine-1-carboxylic acid tert-butyl ester (41 mg, 0.15 mmol) and dry triethylamine (20.3 μL,
0.15 mmol). The reaction mixture was stirred at room temperature for
18 h under N2 atmosphere. In addition, 4-chlorosulfonyl-piperidine-1-carboxylic
acid tert-butyl ester (41 mg, 0.15 mmol) and dry
Et3N (20.3 μL, 0.15 mmol) were added. The mixture
was stirred under reflux overnight, concentrated under reduced pressure,
and purified on silica gel cartridge (0–100% EtOAc in cyclohexane,
then 0–20% MeOH in EtOAc) to give the di-Boc-protected 18 (MS: m/e 795 (MH+) as a pale brown solid. This solid was subsequently dissolved
in anhydrous 1,4-dioxane (8 mL) and 4 N HCl in dioxane solution (8
mL), stirred at room temperature for 4 h, and concentrated under reduced
pressure. The crude residue was purified by semipreparative HPLC.
Burgundy solid. Yield = 42%. 1H NMR (400 MHz, DMSO-d6) δ 13.26 (br s, 1H), 9.52 (s, 1H), 8.76
(br s, 3H), 8.46–8.31 (m, 1H), 8.18 (d, J =
5.28 Hz, 1H), 7.65 (dd, J = 5.40, 8.66 Hz, 2H), 7.59
(d, J = 9.02 Hz, 2H), 7.43 (t, J = 8.82 Hz, 2H), 6.92 (d, J = 9.06 Hz, 2H), 6.11
(d, J = 5.26 Hz, 1H), 3.41–3.38 (m, 2H), 3.33–3.29
(m, 1H), 3.26 (br s, 8H), 2.99–2.89 (m, 2H), 2.20–2.15
(m, 2H), 1.90–1.79 (m, 2H). MS: m/e 595 (MH+). Purity was determined as >90%
by
HPLC (274 nm). R: 0.83 min (Acquity UPLC
BEH C18 1.7 μm, 3 mm × 50 mm, 0.1% formic acid in water/0.1%
formic acid in ACN).
General Procedure for the Removal of Ethyl
Carbamate Group:
Synthesis of the Final Compounds 19–40
In a microwave vial, the respective N-ethyl
carbamate-protected derivatives 61−82 (0.067 mmol), LiOH·H2O (0.042 mg, 1.01 mmol), THF
(1 mL), ethanol (1 mL), and water (1 mL) are added and the mixture
is stirred at 105 °C overnight. EtOAc (15 mL) and an aqueous
solution of NaOH (1 N, 5 mL) are added, the two phases are separated,
the aqueous phase is washed with EtOAc (5 mL), and the combined organic
phases are washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to afford
the desired compounds, which are purified by semipreparative HPLC
to afford pure materials.
General Procedure for the Removal of Ethyl Carbamate Group:
Synthesis of the Final Compounds 41–57
In a microwave vial, the respective N-ethyl
carbamate-protected derivatives 83−99 (0.067 mmol), LiOH·H2O (0.042 mg, 1.01 mmol), THF
(1 mL), ethanol (1 mL), and water (1 mL) are added and the mixture
is stirred at 105 °C overnight. EtOAc (15 mL) and an aqueous
solution of NaOH (1 N, 5 mL) are added, the two phases are separated,
the aqueous phase is washed with EtOAc (5 mL), and the combined organic
phases are washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to afford
the desired compounds, which are purified by preparative HPLC to afford
pure materials.
In a microwave vial with a stirring bar, tert-butyl 4-(5-(2-chloropyrimidin-4-yl)-4-cyclopropylthiazol-2-yl)piperidine-1-carboxylate
or tert-butyl 4-(5-(2-chloropyrimidin-4-yl)-4-(piperidin-1-ylmethyl)thiazol-2-yl)piperidine-1-carboxylate
(o) (0.048 mmol), 4-aminopyrimidine or 2-methyl-4-aminopyridine
(0.059 mmol), Pd2(dba)3 (0.003 mmol), Xantphos
(0.005 mmol), and potassium tert-butoxide (0.095
mmol) are put and air is removed under reduced pressure. Then, anhydrous
and degassed toluene (1.5 mL) is added and the reaction mixture is
refluxed for 2 h under nitrogen. The reaction is monitored by TLC.
Toluene is distilled off under reduced pressure, and the residue is
purified on silica gel cartridge eluted with EtOAc:iPrOH 95:5, affording the desired compounds as yellow solids (72–88%
yield), which were used directly in the next step. The Boc-protected
thiazole derivatives of the previous step (0.042 mmol) dissolved in
dry dioxane (0.6 mL) are treated with 4 N HCl/dioxane (0.84 mmol),
and the mixture is stirred at room temperature for 1.5 h, after which
LCMS showed the complete consumption of the starting material. Dioxane
is distilled off, the residue is washed with diethyl ether, and is
dried in vacuum to give the desired hydrochloride salt as a yellow
solid.
Plasmodium falciparum 3D7A and NF54 strains (from
the Malaria Research and Reference Reagent Resource Center MR4) were
grown in complete medium [RPMI 1640 (Sigma), 25 mM HEPES and NaHCO3)] supplemented with 2 g/L d-sucrose, 0.3 g/L l-glutamine, and 0.150 mM hypoxanthine and with 5 g/L AlbuMAX
II. Parasitized red blood cells (RBC) with 3D7A P.
falciparum strain, (0.5% parasitemia, 2% hematocrit)
in RPMI-1640, 5% AlbuMAX, and 5 μM hypoxanthine were exposed
to threefold serial dilutions of the compounds (nine serial dilutions
5 μM as maximal concentration). After incubating the plates
for 24 h at 37 °C and 5% CO2/5%O2/90% N2, [3H]-hypoxanthine (0.2 μCi to each well,
from a stock solution of 3H-hypoxanthine of 0.025 μCi/μL
in RPMI-1640) was added and the incubation of the plates was continued
for another 24 h period. Thereafter, parasites were harvested on a
glass fiber filter, filters were dried, and the incorporation of [3H]-hypoxanthine was determined using melt-on scintillator
sheets. A microbeta counter was used for measuring radioactivity,
while data normalization was performed by incorporation of the positive
control (compound-free parasitized red blood cells). IC50 values were determined using Grafit 7 program.
In Vitro PKG
Assay
The expression and purification
of recombinant PKG as well as the PKG inhibitory activity assay were
performed as previously described (see also the Supporting Information).[54]
Male/Female
Gametocyte Functional Viability Assay
(A)
Gametocyte production. Gametocyte cultures were produced as previously
described (see also the Supporting Information).[28] (B) Dual gamete formation assay.
Gametocyte activation was triggered by reduced temperature and the
addition of ookinete medium containing xanthurenic acid supplemented
with the antibody anti-Pfs25-Cy3 at a final concentration
of 1/2000 (from 1 mg/mL stock). Plates were analyzed to detect exflagellation
centers. “Activated” cultures were then incubated (protected
from light) at 26 °C for 24 h (in a thermo regulated incubator)
to increase the fluorescent signal emitted by female gametes. The
plates were then analyzed to record female activated gametes.[28] (C) Measured parameters. The activation of male
gametes is based on light change detections provoked by flagella movements
causing movement of surrounding cells. A 10-frame video was taken
and subsequently analyzed to determine the changes in cell position
based on pixels change. The activation of female gametes was based
on the detection of fluorescent Cy3-Anti Pfs25 antibody (as primary
parameter), followed by selection of events according to their size,
roundness, and the intensity of the fluorescence (see also the Supporting Information).[55]
In Vitro Parasite Reduction Ratio
In vitro PRR testing
was conducted as previously described.[56] The limiting dilution technique was used to quantify the number
of parasites remaining viable after drug treatment. P. falciparum strain 3D7A (Malaria Research and Reference
Reagent Resource Center, MR4, BEI Resources; Cat. No. MRA-102) was
treated with a 10 × EC50 (antiparasitic activity in
cells) drug concentration. Parasites were treated for 120 h. Parasite
samples were collected from the treated culture every 24 h (24, 48,
72, 96, and 120 h time points). The number of viable parasites was
determined by counting the number of wells with growth after 21 and
28 days, using [3H]-hypoxanthine incorporation (see also
the Supporting Information).
Chemoproteomics-Target
Identification Experiments
Kinobeads
were prepared as described.[31,32] Sepharose beads were
derivatized with 31 or 50 at a concentration
of 1 mM as described.[30] The chemoproteomic
affinity capturing experiments were performed as previously described.[32] The experimental setup was such that 10 samples
are measured in parallel (TMT 10-plex)[57] to generate values for the affinity of the beads to the bound proteins
and to generate IC50 values in a single experiment. Apparent
dissociation constants were determined by taking into account the
protein depletion by the beads.[32] Proteins
were digested according to a modified single-pot solid-phase sample
preparation (SP3) protocol.[58,59] Peptides were labeled
with isobaric mass tags (TMT10, Thermo Fisher Scientific, Waltham,
MA) using the 10-plex TMT reagents, enabling relative quantification
of 10 conditions in a single experiment.[57,60] LC-MS/MS measurements on Q Exactive Orbitrap or Orbitrap Fusion
Lumos mass spectrometers (Thermo Fisher Scientific) was performed
as described elsewhere.[61] Analytical procedures
and raw data tables for the chemoproteomics experiments can be found
in the Supporting Information files 1 and 2, respectively.
Authors: Laura M Sanz; Benigno Crespo; Cristina De-Cózar; Xavier C Ding; Jose L Llergo; Jeremy N Burrows; Jose F García-Bustos; Francisco-Javier Gamo Journal: PLoS One Date: 2012-02-23 Impact factor: 3.240
Authors: Sónia S Albuquerque; Céline Carret; Ana Rita Grosso; Alice S Tarun; Xinxia Peng; Stefan H I Kappe; Miguel Prudêncio; Maria M Mota Journal: BMC Genomics Date: 2009-06-17 Impact factor: 3.969
Authors: Miguel Prudêncio; Cristina D Rodrigues; Michael Hannus; Cécilie Martin; Eliana Real; Lígia A Gonçalves; Céline Carret; Robert Dorkin; Ingo Röhl; Kerstin Jahn-Hoffmann; Adrian J F Luty; Robert Sauerwein; Christophe J Echeverri; Maria M Mota Journal: PLoS Pathog Date: 2008-11-07 Impact factor: 6.823
Authors: Shams Ul Mahmood; Huimin Cheng; Sreedhar R Tummalapalli; Ramappa Chakrasali; Rammohan R Yadav Bheemanaboina; Tamara Kreiss; Agnieska Chojnowski; Tyler Eck; John J Siekierka; David P Rotella Journal: RSC Med Chem Date: 2019-12-16
Authors: Rammohan R Yadav Bheemanaboina; Mariana Laureano de Souza; Mariana Lozano Gonzalez; Shams Ul Mahmood; Tyler Eck; Tamara Kreiss; Samantha O Aylor; Alison Roth; Patricia Lee; Brandon S Pybus; Dennis J Colussi; Wayne E Childers; John Gordon; John J Siekierka; Purnima Bhanot; David P Rotella Journal: ACS Med Chem Lett Date: 2021-11-15 Impact factor: 4.632
Authors: Aurélia C Balestra; Konstantinos Koussis; Natacha Klages; Steven A Howell; Helen R Flynn; Marcus Bantscheff; Carla Pasquarello; Abigail J Perrin; Lorenzo Brusini; Patrizia Arboit; Olalla Sanz; Laura Peces-Barba Castaño; Chrislaine Withers-Martinez; Alexandre Hainard; Sonja Ghidelli-Disse; Ambrosius P Snijders; David A Baker; Michael J Blackman; Mathieu Brochet Journal: Sci Adv Date: 2021-03-24 Impact factor: 14.136
Authors: Andrew J Jezewski; Ann M Guggisberg; Dana M Hodge; Naomi Ghebremichael; Gavin Nicholas John; Lisa K McLellan; Audrey Ragan Odom John Journal: PLoS Pathog Date: 2022-09-14 Impact factor: 7.464
Chart 1
Scheme 1
Scheme 3
Scheme 2
Figure 1
Figure 2
Figure 3
Figure 4