Carbamoyl Triazoles, Known Serine Protease Inhibitors, Are a Potent New Class of Antimalarials

Article abstract of DOI:10.1021/acs.jmedchem.5b00434

(Graph Presented) Screening of the GSK corporate collection, some 1.9 million compounds, against Plasmodium falciparum (Pf), revealed almost 14000 active hits that are now known as the Tres Cantos Antimalarial Set (TCAMS). Followup work by Calderon et al.

Full text of DOI:10.1021/acs.jmedchem.5b00434

Article
pubs.acs.org/jmc
Carbamoyl Triazoles, Known Serine Protease Inhibitors, Are a Potent
New Class of Antimalarials
†
†
†
†
́
Matthew McConville, Jorge Fernandez, Inigo Angulo-Barturen, Noemi Bahamontes-Rosa,
́
̃
Lluis Ballell-Pages,† Pablo Castaneda,† Cristina de Cozar,† Benigno Crespo,† Laura Guijarro,†
́
̃
†
†
†
†
́
María Belen Jimenez-Díaz, Maria S. Martínez-Martínez, Jaime de Mercado, Angel Santos-Villarejo,
́
́
Laura M. Sanz,† Micol Frigerio,‡ Gina Washbourn,‡ Stephen A. Ward,§ Gemma L. Nixon,‡
Giancarlo A. Biagini,§ Neil G. Berry,‡ Michael J. Blackman,∥ Felix Calderon,*,†,⊥ and Paul M. O’Neill*,‡
́
́
†Tres Cantos Medicines Development Campus, DDW, GlaxoSmithKline, Severo Ochoa 2, 28760 Tres Cantos, Spain
‡Department of Chemistry, University of Liverpool, Liverpool L69 7ZD, United Kingdom
§Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom
∥Division of Parasitology, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom
S
* Supporting Information
ABSTRACT: Screening of the GSK corporate collection, some 1.9 million compounds, against Plasmodium falciparum (Pf),
revealed almost 14000 active hits that are now known as the Tres Cantos Antimalarial Set (TCAMS). Followup work by
Calderon et al. clustered and computationally filtered the TCAMS through a variety of criteria and reported 47 series containing
a total of 522 compounds. From this enhanced set, we identified the carbamoyl triazole TCMDC-134379 (1), a known serine
protease inhibitor, as an excellent starting point for SAR profiling. Lead optimization of 1 led to several molecules with improved
antimalarial potency, metabolic stabilities in mouse and human liver microsomes, along with acceptable cytotoxicity profiles.
Analogue 44 displayed potent in vitro activity (IC50 = 10 nM) and oral activity in a SCID mouse model of Pf infection with an
ED50 of 100 and ED90 of between 100 and 150 mg kg−1, respectively. The results presented encourage further investigations to
identify the target of these highly active compounds.
been discovered in Southeast Asia.9−12 The issue of resistance
INTRODUCTION
■
coupled with the demand of a newly accepted set of
Malaria, the disease caused by parasites of the Plasmodium
genus, results in approximately 0.7 million deaths per annum
antimalarial target product profiles13 has prompted the search
for antimalarials with novel mechanisms of action that can be
and is responsible for some 660 million clinical cases
worldwide.1 Malaria affects some of the most vulnerable people
in the world, particularly children and pregnant women in
resource-poor regions such as sub-Saharan Africa and South
deployed for treatment of all malaria species for prophylaxis
and transmission blocking.
Screening of the GSK corporate collection, some 1.9 million
compounds, against Plasmodium falciparum (Pf), revealed
East Asia. The current frontline treatments for falciparum
almost 14000 active hits that are now known as the Tres
malaria, the most deadly species, are artemisinin based
Cantos Antimalarial Set (TCAMS).14 Further work by
Calderon et al. filtered the TCAMS through a variety of
criteria (activity, physicochemical properties, similarity indices,
molecular weight, functional groups, scaffold novelty, etc.) and
reported 47 series containing a total of 522 compounds.15 From
these compounds, we chose the carbamoyl triazole TCMDC-
combination therapies.2 These consist of the coadministration
of a highly efficacious but rapidly cleared artemisinin derivative3
(dihydroartemisinin, artemether, or artesunate) with another,
longer acting secondary agent, typically lumefantrine or
piperaquine.2 A current area of global concern in malaria is
the emergence of resistance to antimalarial drugs, including the
artemisinins, which renders them ineffective.4−8 Recent reports
have confirmed that parasites with reduced susceptibility to
artemisinins, reflected in a slow parasite clearance rate, have
Received: March 18, 2015
© XXXX American Chemical Society
A
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134379 (1, Figure 1) as a starting point for further
development. Carbamoyl triazoles have been extensively
Table 1. In Vitro and In Vivo Data for Compound 1
measurement
value
18.2/1.6
a
Clint mL−1 min−1 g−1 (m/h)
in vitro t1/2 min (m/h)
3.8/>30
58
b
HSA binding (%)
CYP3A 4VG/4VR IC50 (μM)
>50/31.6
4.82 (5.8)
>100
c
ChromlogD (PFI)
Tox50 HepG2 (μM)
d
P. berghei, 2 doses, 50 mg kg−1
ED50 > 50 mg kg−1
a
b
c
(mouse/human). Human serum albumin. PFI = ChromlogD + no.
d
of aromatic rings. p.o.
(chemoluminescent nitrogen detection kinetic solubility
(CLND) ≥ 500 μM), passive permeability (PAMPA) >300
nm/s). In vitro pharmacological profiling which involves the
screening of 1 against a broad range of human targets
(receptors, ion channels, enzymes, and transporters) in order
to identify specific molecular interactions that may cause
adverse drug interaction in humans revealed no major concerns.
Cytotoxicity against HepG2 cells was shown to have IC50 > 100
μM. A QPatch assay was negative for hERG inhibition. High
clearance in mouse liver microsomes (Clint = 18.2 mL min−1
g−1) demonstrated that metabolic stability was likely to provide
an optimization challenge in this series. However, it should be
noted that the values in human liver microsomes were
significantly lower (Clint = 1.6 mL min−1 g−1). From a
medicinal chemistry point of view, the compound was also
attractive as it offers several sites for modification with high
synthetic tractability.
Figure 1. Biologically active carbamoyl triazoles.
reported as herbicides,16 fungicides,17 and insecticides,18
particularly in the patent literature. Triazamate (Aphistar, 2)
is a commercial insecticide used in the control of aphids on
various crops and kills insects by inhibition of acetylcholine
esterase (AChE). Carbamoyl triazoles such as 3 and the related
thiocarbamoyl triazole 4 were shown to be effective at reducing
sephadex-induced eosinophilia in rats when administered both
po and ip.19 4 was shown to have low toxicity (LD50 > 2000 mg
kg−1) in mice. Jacobsen et al. reported carbamoyl triazoles were
potent inhibitors of hormone sensitive lipase (HSL), an
esterase responsible for regulation of fatty acid metabolism.20
They found that, although selective over hepatic, lipoprotein,
pancreatic, and butyrylcholine esterases, the compounds were
still inhibitors of acetylcholine esterase (AChE). Given 5’s
similariy to 2, this is hardly surprising. However, by exchanging
an N-Me (5a) for N-Ph (5b), they were able to improve the
selectivity for HSL over AChE by more than 2 orders of
magnitude, demonstrating that designing in selectivity is
possible for these compounds. A pseudoreversible mechanism
of inhibition involving acylation of the active site serine is
proposed. Cravatt et al. showed that carbamoyl triazoles are
remarkably selective for the serine protease class of enzyme.21
Furthermore, they were able to demonstrate excellent
selectivity for a single serine protease in the mouse brain
proteome after synthesizing a series of carbamoyl 1,2,3-triazole
analogues. AA74−1 was able to completely and selectively
inhibit the serine hydrolase APEH in vivo at ip doses of 0.4 mg
kg−1. These studies show the potential of the carbamoyl
triazoles to be developed as selective and potent pharmaceutical
compounds.
There was, however, some cause for concern with respect to
plasma/blood stability and consequent in vivo efficacy given the
instability of the hit compound in both plasma and blood
(Figure 2). Addition of protease/esterase inhibitors showed
Figure 2. Mouse blood stability assays for 1 with and without various
esterase/protease inhibitors.
that PMSF, a known, nonselective serine protease inhibitor, was
able to significantly increase the mouse blood stability of the hit
compound. This evidence indicates that proteases/esterases are
the likely culprits for the instability.
RESULTS AND DISCUSSION
■
1 was attractive for several reasons. It is highly potent in vitro
(IC50 = 49 nM) against Pf, and it represents a chemical scaffold
that is unique to known antimalarial compounds (Table 1). 1 is
a potent inhibitor of the serine protease dipeptidyl peptidase IV
(DPP-IV) (IC50 = 9 nM).22 Serine proteases are a relatively
unexplored class of enzyme for antimalarial compounds, so
there is potential to exploit novel enzyme targets in this
class.23−28 The likely mechanism of action is irreversible
inhibition by carbamoylation of nucleophilic serine residues in
the active site. Carbamoyl triazoles are reported to increase
glucose tolerance in mice, through DPP-IV inhibition, with oral
doses as low as 1 mg kg−1.22 1 has physicochemical properties
favorable for oral drugs (ChromlogD = 4.9, solubility
A preliminary evaluation of the in vivo efficacy (p.o.
administration) of 1 against a P. berghei murine model of
malaria infection showed a lack of oral efficacy (ED50 > 50 mg
kg−1). Given the efficacy of related compounds with respect to
glucose tolerance and the high potency of 1 in vitro against Pf,
this result was surprising. The reason for this discrepancy could
be explained by the different sites of action for glucose
tolerance and antiplasmodial effects. DPP-IV is responsible for
the deactivation of glucagon-like peptide-1 (GLP-1).29 GLP-1
stimulates insulin and inhibits glucagon secretion. It follows
that inhibition of DPP-IV increases the level of active GLP-1
B
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and, through the action of GLP-1 on insulin and glucagon,
lowers blood glucose (increased glucose tolerance). The
interaction of DPP-IV/GLP-1 takes place predominantly on
the brush border epithelium of the small intestine.30 As a
consequence, orally administered DPP-IV inhibitors do not
have to enter the systemic circulation to exert their effect on
glucose tolerance. On the other hand, to effectively affect
malaria parasites, compounds must pass through the brush
border epithelium, into the systemic circulation and finally into
erythrocytes where the Plasmodium targets are located. Given
that 1 is a likely irreversible inhibitor of DPP-IV (and other
proteases) and shows low stability in blood, it can be seen that
the difference in oral efficacy between plasmodial infection and
glucose tolerance is explicable through the differing sites of
action. The challenge was, therefore, to moderate the reactivity
of the carbamoyl group in such a way that the antiplasmodial
activity is retained, plasma/blood stability is increased, and
clearance is reduced. This could conceivably be achieved by
altering the steric and electronic properties of the carbamoyl
group.
Further in vitro assays allowed us to gain more information
with regard to the antiplasmodial effects of 1. The parasite
reduction ratio (PRR) assay is used to measure the killing
kinetics of compounds against Plasmodium falciparum.31 Several
parameters can be determined from the assay and the killing
profile compared to known antimalarial compounds previously
reported. The assay is run at 10 × IC50 of the compound to be
studied. In vitro PRR represents the decrease in viable parasites
by a drug treatment over one life cycle (48 h for Pf), and it is a
direct measurement of the killing rate of an antimalarial drug.
Figure 3 shows that, for 1, log10 PRR = 3.1, PCT99.9% = 48 h,
Figure 4. Key SAR features of carbamoyl triazoles.
and, therefore, reactivity of the carbamoyl group. This change is
also attractive from a tractability point of view as the sulfones
are accessible from the sulfides via simple oxidation. (iii) The R
group bonded to linker Y. Although remote from the carbamoyl
group and unlikely to influence the reactivity directly, the R
group should have profound effects on activity by way of
mediating the interaction with protease active sites. The S1/S1′
sites of proteases play a vital role in substrate recognition/
binding, and therefore groups remote to the reactive center are
key to inhibitor activity.
As mentioned previously, 1 was attractive from a medicinal
chemistry point of view due to the high tractability offered by
its structure. Scheme 1 shows the synthetic approach used to
Scheme 1. Overview of Chemistry for Synthesis of 1 and
Analogues
prepare the analogues tested. Nucleophilic substitution of aryl
bromides 6 by mercaptotriazole 7 with potassium carbonate in
DMF yielded the sulfides 8 in good to excellent yields. Reaction
of 8 with various carbamoyl chlorides yielded the carbamoyl
sulfide analogues of type 9, which were converted to sulfones
10 in good yield by oxidation with mCPBA. Alternatively, the
sulfides 8 were oxidized to the triazole sulfones 11 with
mCPBA in EtOAc. Reaction of 11 with triphosgene in DCM
generates a carbamoyl chloride that was reacted in situ with
secondary amines to yield analogues of sulfone 10.
Figure 3. PRR assay of 1 (black) compared to that of artemesinin
(blue), atovaquone (red), pyrimethamine (green), and chloroquine
(purple).
Table 2 shows the results obtained for a series of analogues
exploring the steric properties around the carbamoyl group.
However, the N-Me Pr substitution pattern was found to be
and there is no lag phase. 1 has a profile between that of
previously reported profiles for pyrimethamine and chlor-
oquine31 and is classified as a moderate killer.
i
optimal (1, vide supra). Substitutions with low steric demand
such as those in 13−15 exhibited nanomolar levels of in vitro
activity. Sterically demanding N-substituents such as those in
16−18 were inactive in vitro, most likely due to a decreased
reactivity toward the target enzymes. The pyrrolidine analogue
19 showed a similar activity to 1, while piperidine 21 was less
active. The N-benzyl compound 22 has a moderate level of
activity, 23 and 24, containing a N-phenyl groups had poor
activity.
To determine the SAR, a range of analogues were
synthesized and tested in vitro against Pf. Three main areas
were identified for SAR exploration (Figure 4): (i) The
secondary amide substituents of the carbamoyl group, R′ and
R″. Various combinations of aryl, alkyl, and benzyl were
considered in order to control the steric and electronic
properties of the carbonyl group thought to be key to the
serine protease inhibiting activity. (ii) The linker group (Y)
separating the triazole ring from the varying R groups. Varying
between the electron donating sulfide and electron withdrawing
sulfone allows dramatic changes in the electronic properties
The sulphonyl group is strongly electron withdrawing and as
such imparts enhanced leaving group ability onto the triazole.
The oxidation level of the sulfur atom, therefore, presents an
C
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compound’s polarizability and electronegativity and is con-
sistent with the proposed antimalarial mechanism of action, i.e.,
carbonyl reactivity with protein nucleophiles.
Table 2. In Vitro Pf (3D7) IC50s for a Range of Carbamoyl
Triazoles
a
The S-substituent was also varied from the original exo-
norbornane (Table 3). Cyclohexyl substitution provided 28, a
Table 3. In Vitro Pf (3D7) IC50s for Carbamoyl Triazoles
a
Containing Different S-Substituents
a
Atovaquone was used as positive control with IC50 = 0.001 μM
a
Atovaquone was used as positive control with IC50 = 0.001 μM
opportunity to temper the reactivity of the carbamoyl group. A
range of sulfide analogues were synthesized and their in vitro
activities compared with those of the corresponding sulfones.
Although differences in activity were observed, it was not
possible to establish any SAR conclusions regarding the
preferred oxidation state of the sulfur atom from these results.
Quantum mechanical calculations were performed in order
to investigate if differences in antimalarial activity were
attributable to the oxidative state of the sulfur atom [(II) and
(VI)] (Table 2). Given the proposed mechanism of action of
this chemotype, we hypothesized that electronic structure
calculations might be able to capture aspects of a compound’s
reactivity and relate those to measured IC50s. Several
descriptors derived from the calculations were investigated
including LUMO, electronegativity, and softness (see Support-
ing Information (SI) for a complete list). A good linear
correlation (r2 = 0.79) was found between the “softness” of the
molecules and the measured in vitro IC50 values (Figure 5).
The model passed tests for validity (see SI) and had a good 5-
compound endowed with slightly improved potency. The
bulky, lipophillic 2-adamantyl group provided the highly potent
29. Aromatic groups attached directly to sulfur 30−33 resulted
in lower activity except the highly potent 31. The S-benzyl
compounds 34 and 35 were considerably less active than the
corresponding norbornyl analogues 19 and 20.
The C-5 of the triazole also offers an opportunity to increase
steric demand around the carbamoyl group; however, the 5-Me
compound 36 proved to be inactive (Figure 6).
fold cross validation r2 value of 0.65
0.07 indicative of a
Figure 6. In vitro Pf (3D7) IC50 for 36.
robust model. The softness of a molecule is a measure of a
To assist in analyzing and interpreting the SAR associated
with the compounds synthesized, QSAR models were
developed for the quantitatively measured IC50 values. Initially,
models were built using the quantum mechanical approach as
described above, however, none of the models explored using a
genetic algorithm multiple linear regression approach were
satisfactory (data not shown). An alternative nonlinear
approach was employed in which models were constructed
using random forests32 within a multiple cross-validation
scheme33 to select the optimal parameter (mtry) to use with
the molecular descriptors34 (for further computational details,
see the SI). We adopted this approach of using multiple
training and test sets as every sample may be needed for the
Figure 5. Plot of experimental log(1/IC50) vs predicted log(1/IC50)
using calculated molecular softness.
D
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model,32 and previous studies have indicated the inadequacies
of using single test set to assess predictive power.35 Indeed,
often the performance estimates superior to a single test set
because they evaluate many alternate versions of the data.33
The multiple cross validation approach showed that an mtry
value of 109 was optimal with (r2 = 0.94 0.20). Applying this
model gave an excellent overall fit with the antimalarial activity
(Figure 7A, r2 = 0.92). The top 10 most influential descriptors
A screen of several, structurally diverse analogues was then
undertaken to establish the metabolic stability in both mouse
and human liver microsomes (Table 5) The di-isopropyl-
Table 5. In Vitro Clearance and Half Lives in Mouse and
Human Liver Microsomes for Carbamoyl Triazoles
mouse
human
in vitro Clint (mL in vitro t1/2 in vitro Clint (mL in vitro t1/2
compd
min−1 g−1
)
(min)
min−1 g−1
)
(min)
18
19
20
22
22.0
>30
>30
4.0
<5
<5
3.1
1.0
1.5
1.5
22.3
>30
>30
>30
<5
19.4
containing 18 showed an in vitro intrinsic clearance in mouse
liver microsomes of 22.0 mL−1 min−1 g−1, very similar to that of
1. The pyrrolidine analogues 19 and 20 both exhibit higher
clearance (>30 mL min−1 g−1) than 1. These values all
correspond to in vitro t1/2 values of <5 min. This data suggests
that high clearance in mouse liver microsomes is a common
feature across the series of compounds containing exclusively
aliphatic N-substituents. However, all of these compounds
showed much lower clearance in human liver microsomes. 22
showed an improved clearance in the mouse microsomes of 4.0
mL min−1 g−1 which corresponds to an in vitro t1/2 of almost 20
min.
With the improved metabolic stability and reduced clearance
exhibited by 22, it was decided to investigate the SAR around
the benzyl group with a view to obtaining higher potency. A
series of benzyl analogues were synthesized, and the results are
shown in Table 6. Addition of a chlorine atom in the 4- (37) or
2- (38) position led to a slight reduction in activity compared
to 22, 3-Cl analogue 39 maintained the same activity.
Trifluoromethyl substitution (40−42) led to a reduction in
activity with 40 and 39 being significantly less active than 22. 3-
OMe analogue 43 showed an improvement in potency over 22
and the 4-OMe 44 was extremely potent in the low nanomolar
range (10 nM). The 2,4-dimethoxy substituted 45 was also
highly potent (30 nM). Fluoro substituted compounds 46−48
showed similar activity to 22 with the position of F-substitution
having little effect on the activity. Introduction of a heterocycle
such as pyridine in 49 (86 nM) was well tolerated and slightly
more potent than 22. While 4-SO2Me had negligible impact on
potency (50), 4-Ac and 4-tBu analogues were both highly active
displaying IC50 values of 50 and 16 nm, respectively. The 3-CN
benzyl substituted 53 and the bicyclic tetrahydroisoquinoline
54 were inactive. It was previously observed that the
diisopropyl analogue 17 was significantly more stable in
blood compared to 1 (Figure S2), presumably a result of the
highly sterically demanding substitution around the carbamoyl
group. Because of the potency exerted by the 4-OMe benzyl
group and the stabilizing ability of bulky aliphatic groups, 55
and 56 were synthesized containing cyclopropyl and isopropyl
groups, respectively. However, the in vitro activities of these
compounds were too low to warrant further investigation.
The two most potent compounds, 44 and 52, were then
evaluated for microsomal stability. 44 (Clint = 3.0 mL min−1
g−1; t1/2 = 22.6 min) showed a similar clearance value to that of
22 (vide supra) in mouse liver microsomes, and 52 had a lower
metabolic stability under the same conditions (Clint = 7.8 mL
min−1 g−1; t1/2 = 9.0 min). The ChromLogD (pH 7.4) for 44
and 52 were 5.72 and 7.69, respectively. 44 was significantly
Figure 7. (A) Plot of experimental log(1/IC50) vs predicted log(1/
IC50) of the entire quantitative data set. (B) Relative importance of the
top 10 most influential descriptors in the model.
are shown in Figure 7B (definitions of descriptors are provided
in the SI). Most of these descriptors are challenging to interpret
directly, however the utility of the robust QSAR developed
would be of value in future virtual screening studies to inform
and prioritize synthesis and testing.
Further insight into the behavior of the carbamoyl triazoles
comes from observations regarding the 48 vs 72 h IC50 values,
and the results are shown in Table 4. However, none of the
compounds tested showed any significant difference in IC50
between the two time points.
Table 4. In vitro Pf IC50 at 48 and 72 h for Carbamoyl
a
Triazoles
Pf IC50 (μM) (SD)
compd
48 h
72 h
1
0.016 ( 0.001)
>5 (NA)
0.028 ( 0.004)
> 5 (NA)
17
18
19
22
23
1.428 ( 0.006)
0.037 ( 0.002)
0.247 ( 0.086)
2.686 ( 0.879)
0.537 ( 0.185)
0.037 ( 0.002)
0.379 ( 0.196)
2.713 ( 0.237)
a
Atovaquone was used as positive control with 48/72 h IC50s: 0.001
μM (48 h)/0.0003 μM (72 h)
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a
Table 6. In Vitro IC50s for N-Benzyl Substituted Carbamoyl Triazoles vs the 3D7 Strain of Plasmodium falciparum
a
Atovaquone was used as positive control with 48 h IC50: 0.001 μM.
more soluble (CLND) than 52, where maximum concen-
trations were measured at 125 and 28 μM, respectively.
Cytotoxicity assays against HepG2 cells revealed IC50 > 100
μM for 44 and = 75.2 μM for 52.
Given the slightly higher potency, reduced in vitro clearance,
adequate toxicological profile, and superior physicochemical
properties of 44, an in vivo oral efficacy study was initiated with
this compound. The experiment was carried out in the GSK Pf
malarial serine proteases examined to date, PfSUB1 (Pf
subtilisin like serine protease-1). This enzyme is released into
the parasitophorous vacuole where it activates a family of
protease-like proteins called SERA, leading to rupture of the
vacuole and host cell membrane.37−39 Inhibition of PfSUB1 is a
promising approach to block propagation of a malarial infection
by preventing the egress of merozoites from the red blood
cell.39 1, 12, 18, 34, and 44 were screened against this target,
and none of the azole compounds showed inhibitory activity
against this serine protease. The positive control NF2114
compound showed good inhibition, with an IC50 of ∼3 μM
(SI). This result indicates that the novel molecules prepared
here, and the known serine protease inhibitor carbamoyl
triazole 1 are likely targeting novel uncharacterized plasmodial
serine protease enzymes.
humanized mouse model of infection in a standard four-day
36
́
́
assay as described by Jimenez-Diaz. In this assay, 44 was
efficacious against P. falciparum in vivo showing an ED90 ≈ 128
mg kg−1 (Figure 8A) and clear phenotypic effects on parasites
remaining in blood (Figure 8B) were observed. Interestingly,
44 was not detected in peripheral blood of infected humanized
mice. This apparent disconnect between the in vivo efficacy and
levels in blood of 44 could be related to the instability of the
compound in blood.
CONCLUSIONS
■
To further characterize the profiles of 1 and 44, we
conducted a phenotypic assay to assess the effects of the two
compounds on the parasite life cycle (Figure 9). In the
presence of 1 and 44, the parasites could be found as primarily
young trophozoites at both 24 and 48 h after drug treatment.
Parasites are arrested in young trophozoite stage comparable to
atovaquone-treated parasites included as control.
The carbamoyl triazole series (1) belonging to the TCAMS is a
promising new compound class for antimalarial agents that
potentially work by inhibition of novel serine protease targets
other than PfSUB1. Analogue synthesis led to several
compounds with low nanomolar in vitro activity. The N-benzyl
series contains several highly potent compounds with low
nanomolar activity such as 44, 45, 51, and 52. These new
molecules display a greatly improved metabolic stability over
The final part of our study was focused on establishing if this
chemotype had the potential to target the best characterized
F
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b00434.
Molecular formula strings (XLSX)
General experimental procedures and characterization
data for all synthesized compounds (PDF)
AUTHOR INFORMATION
Corresponding Authors
*For F.C.: phone, +44 793108881; e-mail, felix.r.calderon-
romo@gsk.com.
■
*For P.M.O.: phone, + 44 151 794 3553; e-mail, pmoneill@liv.
ac.uk.
Present Address
⊥For F.C.: Immuno-inflammation Therapy Area Unit, GSK,
Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK.
Notes
The human biological samples were sourced ethically and their
research use was in accord with the terms of the informed
consents. All animal studies were ethically reviewed and carried
out in accordance with European Directive 2010/63/EU and
the GSK Policy on the Care, Welfare and Treatment of
Animals.
Figure 8. Efficacy of 44 against the human malaria parasite in the GSK
Pf humanized mouse model. (A) Parasitemia in individual mice treated
with different doses of 44 during the efficacy assay. Each open symbol
represents an individual mouse treated with 44. Solid symbols are the
average of n = 4 mice treated with vehicle (solid circle) and n = 2
treated po with chloroquine at 10 mg·kg−1 (solid square). (B)
Microscopic and flow cytometry analysis of P. falciparum present in
peripheral blood of mice treated with vehicle or 44 at 217 mg·kg−1.
Samples were taken 96 h after starting the treatment. Flow cytometry
dot plots from samples of peripheral blood show P. falciparum-infected
human erythrocytes (polygonal region). Animals treated with vehicle
show healthy nonsynchronized asexual stages, whereas the parasites
present in 44-treated animals are altered rings, abnormal trophozoites
with hemozoin granules and pyknotic cells.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the Tres Cantos OpenLab
Foundation for funding (project TC042), registered as a
limited company no. 7301222, registered as a charity in
́
England and Wales no. 1142577. We also thank Dr. Barbara
Villa Marcos for assistance with proofreading the manuscript
and Dr. Tony Mete for helpful discussions around protease
inhibitor drug design.
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