Discovery and Optimization of 5-Amino-1,2,3-triazole-4-carboxamide Series against Trypanosoma cruzi

Article abstract of DOI:10.1021/acs.jmedchem.7b00463

Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, is the most common cause of cardiac-related deaths in endemic regions of Latin America. There is an urgent need for new safer treatments because current standard therapeutic options, benznidazole and nifurtimox, have significant side effects and are only effective in the acute phase of the infection with limited efficacy in the chronic phase. Phenotypic high content screening against the intracellular parasite in infected VERO cells was used to identify a novel hit series of 5-amino-1,2,3-triazole-4-carboxamides (ATC). Optimization of the ATC series gave improvements in potency, aqueous solubility, and metabolic stability, which combined to give significant improvements in oral exposure. Mitigation of a potential Ames and hERG liability ultimately led to two promising compounds, one of which demonstrated significant suppression of parasite burden in a mouse model of Chagas' disease.

Full text of DOI:10.1021/acs.jmedchem.7b00463

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Article
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Discovery and Optimization of 5‑Amino-1,2,3-triazole-4-carboxamide
Series against Trypanosoma cruzi
Stephen Brand,† Eun Jung Ko,† Elisabet Viayna,† Stephen Thompson,† Daniel Spinks,† Michael Thomas,†
Lars Sandberg,† Amanda F. Francisco,‡ Shiromani Jayawardhana,‡ Victoria C. Smith,† Chimed Jansen,†
Manu De Rycker,† John Thomas,† Lorna MacLean,† Maria Osuna-Cabello,† Jennifer Riley,†
Paul Scullion,† Laste Stojanovski,† Frederick R. C. Simeons,† Ola Epemolu,† Yoko Shishikura,†
Sabrinia D. Crouch,§ Tania S. Bakshi,∥ Christopher J. Nixon,∥ Iain H. Reid,⊥ Alan P. Hill,⊥
Tim Z. Underwood,⊥ Sean J. Hindley,⊥ Sharon A. Robinson,# John M. Kelly,‡ Jose M. Fiandor,§
Paul G. Wyatt,† Maria Marco,§ Timothy J. Miles,*,§ Kevin D. Read,*,† and Ian H. Gilbert*,†
†Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee,
Sir James Black Centre, Dundee DD1 5EH, U.K.
‡Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, U.K.
§Diseases of the Developing World, GlaxoSmithKline, Calle Severo Ochoa 2, 28760 Tres Cantos, Madrid, Spain
∥GlaxoSmithKline, 1250 South Collegeville Road, PO Box 5089, Collegeville, Pennsylvania 19426-0989, United States
⊥Medicines Research Centre, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K.
#David Jack Centre for R&D, GlaxoSmithKline, Park Road, Ware, Hertfordshire SG12 0DP, United Kingdom
S
* Supporting Information
ABSTRACT: Chagas’ disease, caused by the protozoan parasite Trypanosoma cruzi, is the most common cause of cardiac-related
deaths in endemic regions of Latin America. There is an urgent need for new safer treatments because current standard therapeutic
options, benznidazole and nifurtimox, have significant side effects and are only effective in the acute phase of the infection with
limited efficacy in the chronic phase. Phenotypic high content screening against the intracellular parasite in infected VERO cells
was used to identify a novel hit series of 5-amino-1,2,3-triazole-4-carboxamides (ATC). Optimization of the ATC series gave
improvements in potency, aqueous solubility, and metabolic stability, which combined to give significant improvements in oral
exposure. Mitigation of a potential Ames and hERG liability ultimately led to two promising compounds, one of which
demonstrated significant suppression of parasite burden in a mouse model of Chagas’ disease.
food or drink,7 organ donation,8 breast feeding,9 and the
congenital route are widely documented. Increasingly, due to
migration of Latin American immigrants who unknowingly
carry the parasite in their blood, patient numbers are growing
INTRODUCTION
Chagas’ disease,1 caused by the protozoan parasite Trypanoso-
■
ma cruzi is a leading cause of cardiac related deaths in endemic
regions of Central and South America.2,3 It is estimated that
in nonendemic countries including Australia, Canada, Japan,
5−8 million people are currently infected with the parasite,
many of them unknowingly, and that the disease causes over
Spain, and the United States.10
10,000 deaths per year.4 The parasite is primarily transmitted
The disease has two distinct phases, both of which can be
via the faeces of the blood-sucking “kissing” bug5 (triatomine
hard to detect. There is a short acute phase, during which blood
parasite levels are relatively high, which although typically
bug of the Reduviid order), which proliferates in poor housing
conditions. The parasite enters the body through the bite site
wound or through mucosal membranes, although cases of
transmission through blood transfusions,6 eating contaminated
Received: March 30, 2017
© XXXX American Chemical Society
A
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associated with mild symptoms such as fever, body aches,
malaise, and vomiting, causes death in approximately 5% of
diagnosed children. This is followed by the chronic indeterminate
or “silent” stage11 which may span decades,12 during which infec-
ted people may spontaneously clear the infection or reduce it to
undetectable levels and not present clinical symptoms. However, if
left untreated, up to 30% of chronically infected people will go on
to suffer irreversible damage to the heart, esophagus, and/or
colon,13 ultimately leading to severe disability or death.14
The nitro compounds benznidazole (BNZ, 1) and nifurtimox
(2), which are not FDA approved, are the most commonly used
drugs to treat T. cruzi infection.15 However, they cause signifi-
cant side effects16 and are only proven to be effective in the
acute phase of the infection with limited efficacy in the
chronic phase of the infection.17−19 Furthermore, in patients
with Chagas’ cardiomyopathy although trypanocidal therapy
with benznidazole (1) does significantly reduce serum parasite
detection, after five years there is no significant effect on cardiac
clinical deterioration.20 There is therefore an urgent need for
affordable drugs which have a superior safety and efficacy
profile, which do not require hospitalization, and are suitable
for tropical climates.
Twelve hit series were originally identified which met our
own progression criteria as well as being in-line with Drugs
for Neglected Diseases initiative (DNDi) requirements.32,33
Compound 3 was the most potent representative of a series of
compounds containing the 5-amino-1,2,3-triazole-4-carboxamide
(ATC) core and had submicromolar activity (pEC50 > 6), which
was confirmed upon retesting resynthesized material. Literature
searching indicated the ATC core was highly novel, with very
little precedent in medicinal chemistry programmes.
This series was prioritized for optimization based on a
comparison of the activity, selectivity, and ligand efficiency
profiles of all 12 trypanocidal hit series identified from this
screen. Although compound 3 only had moderate potency
against the parasite, it had high ligand efficiency (LE = 0.35),
good lipophilic ligand efficiency34,35 (LLE = (pEC50
−
cLogD) = 5.4), and cell selectivity (>100-fold against VERO
and HepG2 cells). The compound had poor metabolic stability
when incubated with mouse liver microsomes and poor kinetic
aqueous solubility. The aim was therefore to identify a more
potent drug-like compound with an acceptable physicochemical
and DMPK profile suitable for evaluation in acute and chronic
animal models of Chagas’ disease.
STRUCTURE−ACTIVITY RELATIONSHIPS AND HIT
HIT DISCOVERY
■
■
TO LEAD STUDIES
To identify new chemical start points, a phenotypic screen was
performed on a small diversity collection,21 a nonbiased diversity
library containing 15659 lead-like compounds, using a high-
content assay which measures inhibition of proliferation of
intracellular transgenic Trypanosoma cruzi parasites (Silvio
X10/7 clone A1 strain) in VERO cells.22 Compounds were
initially tested at a single concentration (15 μM) over 3 days.
The potency of suitable hits (n = 495), defined as those which
exhibited >80% inhibition of parasite growth and <30% inhi-
bition of growth of VERO cells, was then determined by
10-point dose response (potency range 0.0025−50 μM).
Compounds were manually clustered into series according to
their core scaffolds, and those series containing exemplars
with desirable properties, i.e., potency (pEC50 > 6), cellular
ligand efficiency23,24 (LE > 0.25), cell selectivity (>100-fold
over VERO and HepG2 cells), and calculated lipophilicity
(cLogD < 3), were progressed. All series which were not
cidal, or which had been the subject of previously reported
studies, were subsequently excluded.25−31 The screening cascade
is shown in Figure 1.22
We first explored which of the pharmacophoric elements of the
ATC scaffold were associated with activity by performing some
simple modifications and deletions, and these indicated the core
was not susceptible to optimization through a scaffold hopping
approach. For example, a compound where the amino group
related to 3 was replaced with H, Me, Cl, or NMe2 were inactive
(see Supporting Information, Table S1). This could suggest that
the intramolecular hydrogen bond between the NH2 and amide
carbonyl oxygen might be required for maintaining the bioactive
conformation or that the NH2 group makes a specific interaction
with the molecular target. Modifications of the central triazole
to either an imidazole or pyrazole also led to loss of activity
(see Supporting Information, Table S2)
Optimization of this series therefore focused on adjustment
of peripheral substituents around this core. Rapid exploration
of structure−activity relationships (SAR) was made possible
because analogues could be synthesized expediently either via
cyclization of an azide with an α-cyano amide36 (7, X = NR2R3)
or by Lewis-acid catalyzed (AlMe3) amination37 of the ethyl
ester (8), prepared by cyclization of an azide (6) with ethyl-2-
cyanoacetate (see Scheme 1). The azide intermediates were
conveniently accessed either by reaction of alkyl or benzyl
halides with sodium azide or from the corresponding primary
amine by diazo transfer with 1H-imidazole-1-sulfonyl azide
hydrochloride.38
Compound 3 would likely have poor oral exposure due to its
poor metabolic stability (high intrinsic clearance when incubated
with mouse liver microsomes, Cli = 6.4 mL min−1 g−1) and was
therefore not progressed to in vivo pharmacokinetics. Metabolite
ID following incubation with mouse liver microsomes (at 0.5 and
5.0 μM for 30 min) indicated rapid formation of a principal
metabolite arising from oxidation on the benzyl subunit, assumed
to be the corresponding sulfoxide 16 which is significantly less
active, followed by further oxidation on the benzamide ring.
Work therefore focused on modifications at the para-position of
the benzyl ring to improve the metabolic stability and probe for
additional potency (see Table 1). This revealed that activity was
very significantly dependent upon the nature of the substituent.
Figure 1. Screening cascade and biological and physicochemical profile
of screening hit 3.
B
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Scheme 1. Generalized Synthetic Routes to 5-Amino-1,2,3-triazole-4-carboxamides
Table 1. Modifications to the N-Benzyl Substituent
a
pEC50 values are shown as mean values from two or more EC50 determinations. Standard deviation is typically within 2-fold from the EC50.
b
c
Cli when incubated with liver microsomes; mL min−1 g−1. Kinetic aqueous solubility at pH 7.4 measured by nephelometry.
For instance, removal of the thiomethyl reduced activity
hydrophobic binding pocket. Indeed, cyclopropyl (12), −OCF3
(13), and pyrazole (14) groups all offered good activity which
reiterated this trend, with the most potent compound from
this series being the para-tert-butyl-α-methylbenzyl-substituted
more than 10-fold (i.e., 10) while replacing the SMe with the
larger isopropyl group improved activity by 10-fold (i.e., 11,
pEC50 = 7.4), suggesting para substituents might be occupying a
C
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Figure 2. Metabolism of compound 11.
compound (15). In general, substituents investigated at the para-
position of the benzyl ring improved metabolic stability com-
pared to the thiomethyl substituent.
[1.63ChiLogD + 0.396], i.e., PFI = [1.63ChiLogD + 0.396] +
number of aromatic rings).
Because substituents at the benzyl para-position of 3
appeared to access a “sweet spot” at the target, efforts were
focused first on modifying functionality in this position, seeking
to strike a balance between the degree of lipophilicity required
for good potency while minimizing LogD and/or number of
aromatic rings (nAr), to help improve solubility. Even though
solubility and metabolic stability were generally improved, a
range of more polar modifications were found to significantly
reduce activity, including: polar replacements of the iPr group
of 11 (i.e., 16 and 17), replacement of the benzyl ring with
para-functionalized saturated ring systems (i.e., 21, 22 and 23),
or methylhydroxylation of the benzylic position of 11 (i.e., 24).
Introduction of heteroatoms into the aromatic ring only led
to a reduction in activity (i.e., 18 and 19). Compound 20
(morpholine substituent), however, appeared to represent a
promising lead because it had acceptable potency, selectivity,
and moderate clearance, significantly reduced PFI relative to 11
(7.0 vs 8.9) and the pendant morpholine offered the additional
advantage of using salt preparation to maximize solubility.44
Investigation of alternative substitution patterns around the
benzyl group showed that although a similar level of potency
was maintained when the morpholine substituent was moved
to the meta-position, a decrease in metabolic stability was
observed compared to 20 (i.e., S31 see Supporting Information,
Table S4). Moving the morpholine substituent to the ortho-
position led to a significant loss in potency (i.e., S32 see
Supporting Information, Table S4). Attempts to improve the
profile of 20 with respect to potency, metabolic stability, and
hERG inhibition, either by using alternative amines to the
morpholine or applying different linkages between the morpho-
line and the triazole core, did not yield more suitable
compounds and again demonstrated the apparent trend for
substituents with higher polarity in this position to worsen
potency (see Supporting Information, Table S3 and S4).
It was important to ensure that activity was not being driven
predominantly by nonspecific hydrophobic interactions because
this can frequently lead to compounds with poor physicochem-
ical properties. The aim was to maintain LLE > 5 and cLogD < 3.
In fact, calculated values of LogD and LLE (Stardrop version
6.0, www.optibrium.com) appeared to be well within acceptable
limits even for the most active compounds; all compounds
in Table 1 have cLogD < 2.2, and those with pEC50 > 6 have
LLE > 5. However, it was hypothesized that cLogD might
be underestimating the true lipophilicity, due to the effects of
intramolecular hydrogen bonding (in this case between the
NH2 and the amide carbonyl oxygen) and the effects of the
pKa of the molecules.39 Indeed comparison of LogD measured
chromatographically with calculated LogD (i.e., ChiLogD40,41
vs cLogD) of early compounds in this series suggested an
underprediction (i.e., for 11 ChiLogD = 3.4, and cLogD = 1.7;
for 13 ChiLogD = 3.6, and cLogD = 2.1; see Supporting
Information, Figure S3 for a plot of ChiLogD vs cLogD).
The higher than predicted lipophilicity was of concern as
developability of compounds is negatively correlated with high
lipophilicity. For example, the poor oral exposure of 11 and 13
(see below) is probably due to low solubility, which is likely to
be driven in part by high lipophilicity.
Compounds 11 and 13 exhibited very poor exposure in
mouse when dosed orally at 10 mg free base/kg (AUC0−8h
=
3200 ng·min mL−1 and 2300 ng·min mL−1; Cmax = 17 and
13 ng/mL, respectively, see Supporting Information, Figures S5
and S6). In addition, 11, when administered intraperitoneally
(10 mg/kg), had very low blood exposure (<100 ng/mL) albeit
with a long half-life, likely consistent with the slow release of
a poorly soluble compound (aqueous solubility of 11 = 14 μM,
determined by both kinetic nephelometry and chemilumines-
cent nitrogen detection methods) from the peritoneal cavity.
For both compounds, the poor oral exposure was likely driven
by poor absorption due to the low solubility and moderate/
high first pass metabolism (Cli in mouse liver microsomes
3.2 and 2.9 mL min−1g−1, respectively).
LEAD OPTIMIZATION
■
Compound 20 exhibited a promising profile: cellular activity
and selectivity were in line with our compound progression
criteria and DMPK properties were reasonable; this included
good thermodynamic solubility in physiologically relevant
media, simulated intestinal fluid in the fasted state (FaSSIF =
264 μg/mL for the HCl salt). The compound demonstrated
moderate metabolic stability, with no significant off-target
pharmacology (only one alert at pIC50 > 5 in the GSK
enhanced cross-screen panel (eXP) of enzymes, receptors, ion
channels, and other liability targets)45,46 and low inhibition
of human CYP450s (IC50 for CYP1A2, 2C19, 2C9, 2D6, 3A4
isoforms >10 μM). There were, however, two significant
potential safety issues: (1) moderate inhibition of the hERG47
potassium channel (IC50 = 24 μM, determined by Q-patch48),
a potential cause of cardiac arrhythmia, and (2) the potential
Further analysis showed there was evidence of hydrolysis
of the amide in vivo of compound 11 to give the acid 25
(and presumably the corresponding aniline, 26; Figure 2),
which was detected in blood at approximately 25% of the level
of parent (see Supporting Information, Figure S7).
In addition to high lipophilicity and low solubility,
another potential concern over developability is the number
of aromatic rings.42 The property forecast index,43 PFI =
ChromLogDpH7.4 + number of aromatic rings, was therefore
used as a parameter to guide optimization of aqueous solubility.
(The ChromLogDpH7.4 is related to the chromatographically
determined LogD through the equation ChromLogDpH7.4
=
D
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Table 2. Optimization of the Oxadiazole Bioisostere
a
pEC50 values are shown as mean values from two or more EC50 determinations. Standard deviation is typically within 2-fold from the EC50.
b
c
d
Cli when incubated with liver microsomes; mL min−1 g−1. Kinetic aqueous solubility at pH 7.4 measured by nephelometry. Kinetic aqueous
solubility at pH 7.4 measured by nephelometry of the HCl salt.
for release in vivo of 3-methoxy aniline, which is known to be
a potent mutagen and carcinogen.49
of the amide oxygen possessed promising activity, with pEC50 >
6.0 (see Supporting Information, Table S5 and Scheme S4 for
details of synthesis and activity of these). Investigation of the
effect of substitution on the phenyl ring of oxadiazole S44
(see Supporting Information, Table S5) showed that ortho
substitution gave a 5-fold improvement in potency compared
to no substituent, whereas no improvement in potency was
observed for meta substituted analogue (i.e., S45 and S46 see
Supporting Information, Table S5).
Oxadiazole 27 was ultimately identified as the most
promising bioisostere of the amide bond on the basis of its
activity and PFI. Although this modification largely retained the
activity of 20 and effectively discharged the risk associated with
in vivo release of a mutagenic aniline, compound 27 had much
lower metabolic stability when incubated with mouse liver
microsomes, prompting examination of additional modifica-
tions to the oxadiazole substituent which might address this
(see Table 2). A variety of different analogues were prepared
in which a mimic or replacement of the 3-methoxyphenyl
group of compound 11 were investigated. This included
aromatic rings with a methoxy substituent, heteroaromatics
(five-membered and six-membered) and aliphatics (Table 2
and Supporting Information, Table S6). Compound 27
and analogues 34−41 were synthesized by base-promoted
cyclization of a substituted oxadiazolyl acetonitrile 33 with
4-(4-(azidomethyl)benzyl)morpholine 30 (Scheme 2). Azide
30 could be prepared in three steps from commercially available
Although there was some evidence for hydrolysis of 11, there
was no evidence for release of 3-methoxyaniline from 20 after
oral dosing in mouse or after incubation in mouse plasma at
37 °C for 5 h. However, it was thought prudent to identify an
improved lead which negated the mutagenesis risk associated
with undetected release of the aniline in vivo. It was also
recognized that the aniline makes an important contribution
to binding because truncation of the aniline of 11 to the simple
primary amide (CONH2, S39) abolished activity (see Supporting
Information, Table S5). Two complementary strategies were
therefore adopted primarily focused upon mitigation of the
Ames risk; identifying bioisosteres of the amide bond which
could not be hydrolyzed to give an aniline and identification of
Ames negative anilines which retained activity. Improvement
of the hERG safety margin was also sought by attempting
to modulate the lipophilicity of the benzamide portion of the
molecule.
Evaluation of Amide Bioisosteres. Compound 11 was
initially used as a synthetically convenient template to explore
amide replacements. Classical topological-based isosteric
replacements of the amide functionality,50 such as sulphone
(S40), ketone (S41), and hydroxyethyl (S42) (see Supporting
Information, Table S5), were not effective surrogates of the
parent amide in this system. However, replacement with biaryl
and fused bicyclic heterocycles which retained a HBA in place
E
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a
Scheme 2. Synthesis of Oxadiazole 27 and Analogues in Table 2
a
Reagents: (i) NaN3, DMF; (ii) MnO2, DCM; (iii) morpholine, NaB(OAc)3H, CHCl3; (iv) N-hydroxyamidine, 1,4-dioxane; (v) NaOMe, MeOH,
then add azide 30.
a
Scheme 3. Synthesis of Amides in Table 3
a
Reagents: (i) ethylcyanoacetate, NaOEt, EtOH; (ii) amine “RH” (see Table 3), AlMe3, toluene.
(4-(bromomethyl)phenyl)methanol 28 (see Scheme 2), and
acetonitrile intermediates 33 were prepared in one step by
condensation of 3-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxopropa-
nenitrile 31 with the appropriate amidoxime 32.
priority was conducting an Ames test on the constituent amine
unless they were already reported as Ames negative.
Only directly linked aromatic or heteroaromatic amides had
the desired level of activity (i.e., pEC50 > 6). Benzylic compounds
were inactive (i.e., compare N-benzyl analogue 44 with N-phenyl
compound 43, Table 3). The most active nonaromatic system
was the adamantyl amide 45 (pEC50 = 5.5), while all others were
substantially inactive, including the closest fully saturated
analogue of 20 (i.e., 46). N-Methylation of the amide, which is
known to improve the hydrolytic resistance of aniline amides
such as phenacetin,51 in this case resulted in significantly reduced
activity (i.e., compare 47 with 20).
The meta-methoxy of 20 could be replaced with a variety
of alternative groups including −OCF3 (S70), CF3 (S71),
acetylene (S72) (see Supporting Information, Table S7), and
chloro (48) while retaining activity and solubility, but of
these only the former had marginally better metabolic stability
than the benchmark compound 20. 3-Methyl (49) and 3-fluoro
analogues (50) were of similar potency to 20, and highly
selective, but they did not offer an advantage with respect to
metabolic stability. Conversely, anilides with substantially more
polar groups in the meta-position (for example CH2NMe2
(S73), SO2Me (S74), and amide (S75) (see Supporting
Information, Table S7) were at least 100-fold less active.
Although there is evidence to suggest heteroaromatic amines
have a reduced risk of being mutagenic52 and that increases in
polarity can also reduce hERG inhibition,53 it was unfortunate
that pyridine or pyrimidine analogues of 20 had lower activity
(i.e., 51, 52, and 53) and were not considered as suitable
leads.
The addition of a methoxy group intended to mimic that of
20 (i.e., 34), improved activity and reduced binding to hERG
(>30 μM), but metabolic stability was not improved. Metabolic
stability could be significantly improved through reductions in
PFI, i.e., by replacing the oxadiazole phenyl with various cyclic
ethers or hydroxylated cycloalkyl groups (i.e., 35 and 36,
see Supporting Information, Table S6 for further examples),
although these compounds were unfortunately inactive. Com-
pounds where the methoxyphenyl ring was replaced with a
2-methoxy-substituted heterocycle generally had better meta-
bolic stability (i.e., 37, 38, 39, 40), although only compound 37
offered improved potency combined with good metabolic
stability. Moreover, because hERG inhibition was also low
(IC50 > 30 μM), this compound represented the best lead from
this series.
Identification of an Alternative Amide Derived from
an Ames Negative Amine. Alternative amines to 3-methoxy
aniline were also examined as a complementary strategy for
addressing the pro-mutagenic potential of 20. There are several
factors involved in activation of an aromatic amine to the
actual DNA-reactive nitrenium species, structure−mutagenicity
relationships are poorly understood, and there is a paucity of
literature information on the Ames status of amines. Therefore,
because amides could be conveniently prepared by direct
substitution of ethyl ester 42 with an amine under Lewis
acid conditions (AlMe3), a prospective approach was adopted
using parallel synthesis to identifying suitable alternatives
(see Scheme 3). A diverse library of amides was prepared and
evaluated, excluding amines known to be Ames positive. For final
compounds showing good potency and FaSSIF solubility, a high
Applying an inductive (−I) effect by addition of fluorine
atoms at various positions of the meta-methoxy anilide of 20
would be considered a logical approach to reducing Ames
risk because electron density of the parent arylamine is one
F
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Table 3. Optimization of the Amide Substituent
G
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Table 3. continued
a
b
pEC50 values are shown as mean values from two or more EC50 determinations. Standard deviation is typically within 2-fold from the EC50. Cli
c
d
when incubated with liver microsomes; mL min−1 g−1. Kinetic aqueous solubility at pH 7.4 measured by nephelometry. Kinetic aqueous solubility
at pH 7.4 measured by nephelometry of the HCl salt.
Table 4. Comparison of the Activity and Pharmacokinetic Profile of Optimized Amides 48, 58, 59, and Oxadiazole 37 with Early
Leads 11 and 20
T. cruzi
VERO
pEC50
mouse
a
mouse PPB
(% bound)
hERG IC50
Cmax
FaSSIF
CYP-3A4
pEC50
compd
11
pEC50
Cli
(μM)
AUC (0−8 h) (ng·min/mL) (ng/mL)
PFI
8.9
7.0
7.2
7.0
6.8
7.2
(μg/mL)
7.4
7.0
7.8
7.1
7.4
7.1
<4.3
<4.3
4.5
3.2
3.6
3.7
4.7
3.4
3.3
nd
80
88
93
69
66
nd
24
3200
17
nd
nd
b
20
260000
250000
370000
300000
190000
1300
710
264
<4.4
<4.4
<4.4
<4.4
<4.4
48
17
38
b
58
<4.3
<4.3
<4.3
23
1600
1300
1300
577
b
59
113
100
b
37
>30
18
a
b
Cli when incubated with liver microsomes; mL min−1 g−1. Solubility in simulated fasted intestinal fluid (FaSSIF) was determined on the
monohydrochloride salts.
recognized factor influencing bioactivation by aromatic oxidation.54
Surprisingly, potency was very sensitive to the position of
fluorination; while a fluorine atom either meta- or para- to the
methoxy actually improved activity by 7-fold (55 and 54),
fluorine in the ortho- position reduced activity by more than
10-fold (56 and 57). A similar trend was observed in difluoro
analogues (see Supporting Information, Table S7), which were
all significantly less potent than 20. Given that additional
functionalities meta- to the methoxy could enhance potency, it
was found that addition of a 5-methoxy (i.e., 58) improved
activity without detriment to metabolic stability and selectivity.
This 3,5-dimethoxy arrangement was also significantly active in a
pyrimidine system (59). The constituent amines of amides 48,
58, and 59 (i.e., 3-chloroaniline, 3,5-dimethoxy aniline, and
2,6-dimethoxypyrimidin-4-amine) as well as parent compounds
(58 and 59 only) were shown to be Ames-negative in the
presence or absence of rat liver S9 fraction. On the basis of
having high potency (pEC50 > 7), selectivity against VERO cells
(>100-fold), hERG (>30 μM), and having resolved the Ames
liability, the amides 48, 58, and 59 and the oxadiazole 37 were
selected for further profiling.
Figures S8−S12). These were selected as the most promising
examples of the various series, in terms of potency, selectivity,
metabolic stability, and solubility. All compounds had oral
exposure that was significantly improved compared to that
of early lead compound 11, presumably as a consequence of
improved solubility.
The ability of compounds from this series to kill parasites
rather than merely inhibiting their replication was confirmed
using a static-cidal assay on the intracellular amastigote form of
the parasite.22 The “rate of kill” of T. cruzi intracellular parasites
was also measured and compared to nifurtimox (2) (which
has a very rapid onset (0−24 h) of parasite killing) and
posaconazole (9) (which has a slower lag (24−48 h) before
killing occurs (Figure 3). The rate of kill of ATC series
compounds 11, 20, 48, 58, 59 (amides), and 37 (oxadiazole)
was slower than nifurtimox. With nifurtimox, parasite growth
stopped immediately and decreased rapidly after 24 h. However,
with compound 58, parasite growth continued for 24 h before
starting to decrease. Compound 58 gave a comparable rate of
kill to posaconazole, a potent inhibitor of the important sterol
biosynthesis enzyme of T. cruzi, lanosterol-14α-demethylase55
(TcCYP-51).
CELL BIOLOGY AND PHARMACOLOGY OF THE
ATC SERIES
Our high throughput screening efforts have revealed a
relatively high proportion of hit series whose apparent target
is TcCYP-51. This is a mode of action which needed to be
deprioritized, given the recent disappointing phase 2 clinical
trial results with TcCYP51 inhibitors posaconazole and E1224
■
Pharmacokinetic parameters for 20, 37, 48, 58, and 59 were
determined in female NMRI mouse following a single
10 mg/kg oral dose (see Table 4 and Supporting Information,
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T. cruzi is a genetically diverse parasite which has been
assigned six discrete typing units (DTU). It is important to
consider strain diversity64 to ensure compounds from any given
series are not just active against the Silvio X10/7 clone A1
(DTU Tc1) strain used in routine screening. Thus, it was
confirmed that representative compounds 20, 58, 48, and 59 all
showed activity of pEC50 > 6 against a panel of key T. cruzi
discrete typing units including YUAM, M6241 Clone 6, Era
Clone 2, and Tula Clone 2 (DTU Tc II, III, IV, and VI,
respectively). However, the slow-growing PAH179 Clone 5
(Tc V) strain was significantly less sensitive to compounds
from this series (data not shown).
A representative set of compounds from this series was also
tested against Leishmania donovani, Trypanosoma brucei, and
Plasmodium falciparum parasites in standard assays and were
found to be essentially inactive (pEC50 < 5.0).
Figure 3. Rate of kill of compound 58 compared to nifurtimox (2)
(fast kill) and posaconazole (9) (slow-kill).
Efficacy in Acute and Chronic Mouse Models of
Chagas’ Disease. There are several mouse models of Chagas’
disease currently being used, although it is not clear which of
these will be better at predicting clinical success, given the lack
of clinically effective compounds to back-translate. Conse-
quently, a BALB/c mouse model of acute and chronic Chagas’
disease was chosen that could at least reproduce posaconazole
(9) and benznidazole (1) clinical outcome. This model uses
T. cruzi (clone CL Brener), which express a red-shifted luciferase
reporter that emits tissue-penetrating orange-red light when
luciferin is injected.65 Compound 58 was selected for further
evaluation in this model (acute and chronic) as an exemplar
of the series to establish proof-of-concept. This compound had
the best total exposure and significantly better FaSSIF solubility
(the phosphate prodrug of ravuconazole)18,19 and the significant
number of series against this target currently being devel-
oped.56−61 It is unlikely, however, that the mode of action of the
ATC series is via TcCYP-51 because, in spite of containing a
common CYP inhibitor motif (the 1,2,3-triazole ring),
trypanocidal activity does not correlate with TcCYP-51 enzyme
inhibition (see Figure 4) measured using a TcCYP51 inhibition
assay.62 Nevertheless, there remains the remote possibility
that this series is exerting activity through mechanism-based
inhibition63 of TcCYP-51, i.e., via bioactivation to a suicide
substrate, and time-dependency has not been investigated to
rule this out.
Figure 4. Relationship between parasite activity (T. cruzi pEC50) and inhibition of the TcCYP-51 enzyme for compounds in the ATC series (blue)
compared to a training set of known antifungal azole CYP-51 inhibitors (red) (see Supporting Information, Table S8 for structures and IC50 data).
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Figure 5. Parasite load, as determined by bioluminescent imaging, in the acute mouse model.65 (a) Benznidazole (1) dosed at 100 mg/kg u.i.d. for
21 days. (b) Posaconazole (9) at 20 mg/kg u.i.d. for 20 days. (c) 58 dosed at 50 mg/kg b.i.d. for 21 days. Vehicle control is shown as red line.
and relatively similar potency compared to alternative com-
pounds profiled in detail, such as compound 59 (Table 4).
When 58 was evaluated (50 mg/kg b.i.d. orally for 20 days),
against both the acute (treatment initiated 14 days post-
infection) and chronic stages (97 days post-infection) of the
infection, sterile cure, as seen for benznidazole (1), was not
achieved. In both the acute and chronic models with 58, the
parasite burden was considerably reduced but not eliminated
(Figures 5−7). Following the “chronic” experiment, ex vivo
imaging indicated that most of the parasites were found in the
colon (Figure 7).
dosing. A summary of structure−activity findings are shown in
Figure 8.
We have shown that significant issues with this series, i.e.,
poor oral exposure, hERG activity, and potential mutagenicity
could be addressed to obtain compounds with an improved safety
profile suitable for evaluation in a murine model of Chagas’
disease. Compounds 58 and 59 were the most promising in
terms of potency, PFI, oral exposure (AUC), FaSSIF solubility,
and hERG.
This compound series appears to have a different molecular
mode of action to posaconazole. However, it showed a similar
rate of kill against the parasites as posaconazole (Figure 3).
In addition, as for posaconazole, it showed significantly reduced
activity against a slow growing strain of T. cruzi. There is
currently no evidence that rate of kill correlates with in vivo
activity, in particular as dosing in the in vivo efficacy studies
is much longer than the 4 days used in the rate of kill
experiment. However, by comparing such data across different
chemical series, it may be possible to determine which in vitro
experiments are predictive of in vivo efficacy. Hence, we
thought it is important to share this data with others working in
the field.
This compound series demonstrated significant reduction of
parasite burden in mouse models of acute and chronic Chagas’
disease but was unable to deliver sterile cures under the
conditions used. Current focus is on trying to understand the
reasons for this: whether it is driven by pharmacokinetic (tissue
distribution) or mechanistic considerations. It is possible that
the slow rate of kill of the compound against the parasite and
the reduced activity against slow growing strains of T. cruzi may
be predictive of a noncurative in vivo outcome. This needs
further investigation.
Figure 6. Parasite load, as determined by bioluminescent imaging, in
the chronic mouse model65 following treatment with 58 at 50 mg/kg
b.i.d. for 21 days (gray shaded area) compared to vehicle control.
Parasite rebound was observed at day 147 following immune
suppression with cyclophosphamide (pink shaded area) (Experimental
Section).
While this compound series itself is not progressible toward
the clinic, it is providing valuable learnings in our ultimate goal
of developing new medicines for Chagas’ disease.
Bioanalysis of spot blood sampling from the infected animals
in each study demonstrated that blood-free levels of 58 were
maintained at least 2 × EC90 for the duration of dosing (21
days b.i.d.), once a correction for plasma protein binding had
been applied.
EXPERIMENTAL SECTION
■
General Experimental Information. Chemicals and solvents
were purchased from the Aldrich Chemical Co., Fluka, ABCR,
VWR, Acros, Fisher Chemicals, and Alfa Aesar and were used as
received unless otherwise stated. Air- and moisture-sensitive reactions
were carried out under an inert atmosphere of argon in oven-dried
glassware. Analytical thin-layer chromatography (TLC) was performed
on precoated TLC plates (layer 0.20 mm silica gel 60 with fluorescent
indicator UV254, from Merck). Developed plates were air-dried
and analyzed under a UV lamp (UV254/365 nm). Flash column
chromatography was performed using prepacked silica gel cartridges
CONCLUSIONS
■
In this paper, the discovery and optimization of a novel com-
pound series is described which demonstrates potent activity
against the T. cruzi parasite and parasite knockdown in murine
models of acute and chronic Chagas’ disease following oral
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Figure 7. Time-course bioluminescence ventral and dorsal images of mice (n = 6) treated with compound 58 (50 mg/kg b.i.d; 21 days) in the
chronic model (left). Ex-vivo imaging of organs from mouse 5 at the end of the experiment (day 159; right) indicates that the residual parasites were
localized primarily in the colon.
Prototypical Procedure for the Cyclization Reaction between an
α-Cyanoacetic Ester or Amide with an Azide Formed in Situ:
Preparation of 5-Amino-N-(3-methoxyphenyl)-1-[(4-pyrazol-1-
ylphenyl)methyl]triazole-4-carboxamide (14). Sodium azide (253 mg,
3.89 mmol) was added to a stirred solution of 1-[4-(chloromethyl)-
phenyl]pyrazole (250 mg, 1.30 mmol) in DMF (8.0 mL) at rt, then
the slurry was warmed to 80 °C for 1 h. The reaction was then
concentrated in vacuo, the residue partitioned between water (10 mL)
and EtOAc (10 mL), and the organic phase separated, dried (MgSO4),
and concentrated. The residual oil was then immediately dissolved in
EtOH (8.0 mL), 2-cyano-N-(3-methoxyphenyl)acetamide (250 mg,
1.30 mmol) added, followed by finely ground sodium hydroxide
(52 mg, 1.30 mmol), and the reaction heated to 80 °C for 1 h in the
Figure 8. Summary of the structure−activity relationships for the ATC
microwave. The cooled reaction was then concentrated in vacuo, the
residue partitioned between 1 M HCl (25 mL) and EtOAc (25 mL),
the organic phase separated, dried (MgSO4), and concentrated in vacuo
to give a residue, which was triturated from EtOAc, collected by
filtration, and dried under vacuum to give the title compound as a white
powder (285 mg, 0.73 mmol, 56%). 1H NMR (500 MHz, DMSO-d6):
δ 10.03, (s, 1H), 8.48 (s, 1H), 7.85 (d, J = 8.3 Hz, 2H), 7.75 (d, J =
1.0 Hz, 1H), 7.56 (t, J = 1.9 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.37 (d,
J = 8.3 Hz, 2H), 7.20 (t, J = 8.6 Hz, 1H), 6.68 (s, 2H), 6.63 (dd, J =
2.1 Hz, 8.2 Hz, 1H), 6.55 (t, J = 2.0 Hz, 1H), 5.51 (s, 2H), 3.73 (s, 3H).
HRMS (m/z): [M + H]+ calcd for C20H20N7O2, 390.1673; found,
390.1666.
4-[[4-(Azidomethyl)phenyl]methyl]morpholine (30). Sodium azide
(14.5 g, 223.5 mmol) was added portionwise into a solution of
[4-(chloromethyl)phenyl]methanol (25.0 g, 159.6 mmol) in DMF
(800 mL) at rt, and the reaction was then warmed to 60 °C behind a
blast shield for 3 d. The reaction mixture was then allowed to cool to rt
then concentrated in vacuo at minimum pressure behind a blast shield
with water bath temperature of <60 °C. The residual oil was then
diluted with brine (300 mL) and Et2O (600 mL), and the organic
phase separated, washed with brine (3 × 100 mL), dried (MgSO4),
and concentrated in vacuo to give 27.9 g of crude azide as a colorless
series.
(230−400 mesh, 40−63 μm, from SiliCycle) using a Teledyne ISCO
Combiflash Companion or Combiflash Retrieve. 1H NMR, 13C NMR,
19F NMR, and 2D NMR spectra were recorded on a Bruker Avance
DPX 500 spectrometer (1H at 500.1 MHz, 13C at 125.8 MHz, 19F at
470.5 MHz). Chemical shifts (δ) are expressed in ppm recorded using
the residual solvent as the internal reference in all cases. Signal splitting
patterns are described as singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m), broad (b), or a combination thereof. Coupling
constants (J) are quoted to the nearest 0.1 Hz. LC-MS analyses were
performed with either an Agilent HPLC 1100 series connected to a
Bruker Daltonics MicrOTOF or an Agilent Technologies 1200 series
HPLC connected to an Agilent Technologies 6130 quadrupole
LC/MS, where both instruments were connected to an Agilent
diode array detector. All assay compounds had a measured purity of
≥95% as determined using this analytical LC-MS system (TIC and
UV). High resolution electrospray measurements were performed
on a Bruker Daltonics MicrOTOF mass spectrometer. Microwave-
assisted chemistry was performed using a Biotage Initiator microwave
synthesizer. Purity (>97%) and molecular mass were confirmed by
HPLC and high resolution mass spectrometry.
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oil. This was then taken up into DCM (750 mL) at rt, and MnO2
(50.0 g, 574.7 mmol) added portionwise. The slurry was stirred at
40 °C for 48 h before filtration through a pad of Celite. The filtrate
was concentrated in vacuo to give 23.9 g of a colorless oil, which was a
single component on TLC. The oil was then immediately taken up
into chloroform (1000 mL) and treated with morpholine (25.8 g,
296.1 mmol), 20 g of 4 Å molecular sieves were added, and the
solution was stirred at 40 °C for 2 h prior to removal of the sieves by
filtration. The solution was then cooled to 0 °C and treated portion-
wise with sodium triacetoxyborohydride (50.0 g, 235.9 mmol) with
careful monitoring of the temperature, such that it did not rise above
5 °C. After 3 h, the reaction was allowed to warm to room temperature
prior to warming to 45 °C for 48 h. The reaction was then quenched
by careful addition of 2N NaOH (aq) (250 mL) with stirring, the
organic phase separated, and the aqueous phase extracted with
chloroform (3 × 100 mL). The combined extracts were dried
(MgSO4), filtered, and concentrated in vacuo to give a clear oil which
contained desired product contaminated with approximately 10%
N-acetyl morpholine as impurity. Chromatography (SiO2, 330 g pre-
packed column eluting with EtOAc) allowed straightforward separa-
tion to give the title compound as a colorless oil (28.6 g, 120.7 mmol,
Et2O (100 mL) and treated dropwise with 2 M HCl in Et2O (7.0 mL)
and the slurry stirred at rt for 18 h prior to collection by filtration
under a nitrogen atmosphere and washing with Et2O to give the title
compound as an off-white powder (3.45 g, 6.70 mmol, 77%). 1H NMR
(500 MHz, DMSO-d6): δ 9.77 (s, 1H), 7.65 (d, J = 8.2 Hz, 2H), 7.34
(d, J = 8.2 Hz, 2H), 7.14 (d, J = 7.3 Hz, 2H), 6.89 (s, br, 2H), 6.22 (t,
J = 7.3 Hz, 1H), 5.54 (s, 2H), 4.31 (s, 2H), 3.96−3.74 (m, 4H),
3.73 (s, 6H), 3.23−3.16 (m, 2H), 3.11−3.01 (m, 2H). HRMS (m/z):
[M + H]+ calcd for C23H28N6O4, 453.2245; found, 453.2263.
Prototypical Procedure for Formation of an Azide from an
Amine with 1H-Imidazole-1-sulfonyl Azide Hydrochloride: Prepara-
tion of 2-Azido-2-(4-isopropylphenyl)ethanol (60), Used in the
Synthesis of 24. 1H-Imidazole-1-sulfonyl azide hydrochloride (1.54 g,
8.93 mmol) was added portionwise to a well-stirred slurry of 2-amino-
2-(4-isopropylphenyl)ethanol (1.0 g, 5.58 mmol) and potassium
carbonate (1.54 g, 11.16 mmol) in MeOH (10.0 mL) at rt and the
reaction left for 18 h. The mixture was then concentrated in vacuo,
partitioned between DCM (25 mL) and water (25 mL), the organic
phase separated, dried (MgSO4), and concentrated to give a yellow oil
which was purified by flash column chromatography (SiO2, 1:10 to 1:1
EtOAc:hexanes) to give the title compound as a clear oil (435 mg,
1
1
2.12 mmol, 38%). H NMR (500 MHz, CDCl3): δ 7.48 (s, 4H), 4.67
76% overall yield for 3 steps), which was stored at −18 °C. H NMR
(t, J = 6.5 Hz, 1H), 3.77 (dd, J = 5.9 Hz, 5.9 Hz, 2H), 2.98−2.91 (m,
1H), 1.97 (dd, J = 5.8 Hz, 5.8 Hz, 1H), 1.28 (d, J = 6.9 Hz, 6H).
2-Cyano-N-(3-methoxyphenyl)acetamide (61). A slurry of 3-(3,5-
dimethylpyrazol-1-yl)-3-oxo-propanenitrile (5.0 g, 30.6 mmol) sus-
pended in toluene (50 mL) was treated with 3-methoxyaniline (3.77 g,
30.6 mmol), and the mixture heated to 80 °C for 4 h. The resulting
solution was allowed to cool to rt, and the solid formed was removed
by filtration, washed with Et2O, and dried in vacuo to give the title
(500 MHz, DMSO-d6): δ 7.38 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 8.4 Hz,
2H), 4.35 (s, 2H), 3.75−3.72 (t, J = 4.6 Hz, 4H), 3.53 (s, 2H), 2.47
(t, J = 4.6 Hz, 4H).
Caution: Some azides are known to be explosive. Thermal hazard
screening was carried out on azide 30. Differential scanning
calorimetry indicated that the temperature at which heat flow exceeds
0.01 W/g = 180 °C. Simple impact test was negative, hot plate tests
were inconclusive, and oxygen balance test deemed the compound
to be low risk. Further tests should be considered if the proposed
processing or drying temperature is above 80 °C.
1
compound as a white powder (4.97 g, 25.9 mmol, 84%). H NMR
(500 MHz, DMSO-d6): δ 10.30 (s, 1H), 7.26−7.22 (m, 2H), 7.08
(dd, J = 1.4 Hz, 7.7 Hz, 1H), 6.69 (dd, J = 2.2 Hz, 7.9 Hz, 1H), 3.90 (s,
2H), 3.74 (s, 3H).
Ethyl 5-Amino-1-(4-(morpholinomethyl)benzyl)-1H-1,2,3-tria-
zole-4-carboxylate (42). A stirred solution of sodium ethoxide
(6.45 g, 94.71 mmol) in EtOH (100 mL) at 0 °C was treated
dropwise with ethyl 2-cyanoacetate (9.74 g, 86.10 mmol). After
10 min, a solution of 4-[[4-(azidomethyl)phenyl]methyl]morpholine
(10.0 g, 43.05 mmol) in EtOH (20 mL) was added dropwise, then
the reaction was allowed to warm to rt before being heated to reflux
for 3 h. After cooling to rt, the reaction was diluted by addition of
water (200 mL) and EtOAc (200 mL), the organic phase was
separated, the aqueous layer was extracted with EtOAc (3 × 50 mL),
and the combined extracts were dried (MgSO4) and concentrated
in vacuo to give a residual solid which was triturated with Et2O,
collected by filtration, and dried in vacuo to give the title compound
T. cruzi in Vitro Intracellular Assay. Compounds were dispensed
into black 384-well assay plates (Corning) by acoustic dispensing
(LabCyte ECHO). For potency determinations, 10-point one in three
dilution curves were generated, with a top concentration of 50 μM.
VERO cells were infected overnight with T. cruzi trypomastigotes
(X10/7) in T225 tissue culture flasks at a multiplicity of infection of 5.
Next, any remaining free trypomastigotes were washed away with
serum-free MEM media and the infected VERO cells were harvested
by trypsinization. The infected VERO cells were then plated into
384-well plates containing the compounds to be tested at 4000 cells
per well in MEM media with 1% FCS. After 72 h incubation at
37 °C in the presence of 5% CO2, the plates were fixed with
4% formaldehyde for 20 min at room temperature and stained with
5 μg/mL Hoechst 33342. The plates were imaged on a PerkinElmer
Operetta high-content imaging system using a 20× objective. Images
were analyzed using the Columbus system (PerkinElmer). The
image analysis algorithm first identified the VERO nuclei, followed
by demarcation of the cytoplasm and identification of intracellular
amastigotes. This algorithm reported mean number of parasites per
VERO cell and total number of VERO cells.
TcCYP-51 Assay. An 11-point dose−response curve of test com-
pound was created using an Echo acoustic dispenser and 200 nL added
to a low volume 384-well assay plate. Two dose−response curves for
posaconazole were added to this plate as a positive control. To the
assay plate, 10 μL of an incubation mixture (containing recombinantly
expressed T. cruzi CYP51 (final assay conc 37 pmol/mL) and Vivid
BOMCC (purchased from Invitrogen, final assay conc 100 μM) was
added. Then 10 μL of regenerating cofactor mixture (final conc
2.7 mM glucose-6-phosphate, 0.2 mM NADP, 0.6 units glucose-6-
phosphate dehydrogenase and 2% NaHCO3) is added, and the
reaction is stored at ambient temperature for 1 h. After this time, 2 μL
of a stop solution (10 uM posaconazole) was added to terminate any
subsequent activity, and the plate was read on a fluorescence plate
reader (exc 405 nm, em 450 nm). The % decrease in production of
fluorescent product CHC from BOMCC is measured in the presence
of each concentration of test compound to provide an IC50 plot.
1
(7.20 g, 20.64 mmol, 48%) as a white powder. H NMR (500 MHz,
DMSO-d6): δ 7.29 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 8.0 Hz, 2H), 6.57
(s, 2H), 5.42 (s, 2H), 4.26 (q, J = 7.1 Hz, 2H), 3.55 (t, J = 4.6 Hz,
4H), 3.43 (s, 2H), 2.32 (t, J = 4.2 Hz, 4H), 1.29 (t, J = 7.1 Hz, 3H).
m/z (ES+, 70 V) 346.1 [MH+].
Prototypical Procedure for the Lewis-Acid Catalyzed Direct
Amidation of an Ester with an Amine: Preparation of 5-Amino-N-
(3,5-dimethoxyphenyl)-1-[[4-(morpholinomethyl)phenyl]methyl]-
triazole-4-carboxamide Hydrochloride (58). To a well stirred
suspension of ethyl 5-amino-1-[[4-(morpholinomethyl)phenyl]-
methyl]triazole-4-carboxylate (3.0 g, 8.60 mmol) and 3,5-dimethox-
yaniline (2.66 g, 17.37 mmol) in toluene (250 mL) at rt was added
trimethylaluminum (2 M in toluene, 10.0 mL, 20.0 mmol). Fuming on
addition of the trimethylaluminum was noted. The reaction mixture
was then heated to 80 °C for 18 h with periodic monitoring by LCMS
and tlc (the reaction gradually became a solution). The reaction was
then cooled to rt, quenched carefully by slow addition of 2N NaOH
(aq) (150 mL) and the ensuing biphasic mixture extracted with EtOAc
(3 × 50 mL), allowing 10−20 min for each separation due to turbidity.
The organic phase was washed with brine (100 mL), then saturated
aqueous Rochelle’s salt (100 mL), dried (MgSO4), filtered, and
concentrated in vacuo. The resulting oil was taken up into 100 mL of
1:1 EtOAc/Et2O and stirred at rt for 3 days, during which time a
precipitate was formed. Filtration and copious washing with cold
diethyl ether gave 3.26 g of the free base. This was then taken up into
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Kinetic Aqueous Solubility Assessment. The aqueous solubility
of test compounds was measured using laser nephelometry.
Compounds were subject to serial dilution from 10 to 0.5 mM in
DMSO. An aliquot was then mixed with Milli-Q water to obtain an
aqueous dilution plate with a final concentration range of 250−12 μM,
with a final DMSO concentration of 2.5%. Triplicate aliquots were
transferred to a flat-bottomed polystyrene plate, which was immediately
read on the NEPHELOstar (BMG Lab Technologies). The amount of
laser scatter caused by insoluble particulates (relative nephelometry
units, RNU) was plotted against compound concentration using a
segmental regression fit, with the point of inflection being quoted as the
compounds aqueous solubility (μM).
Solubility of Solid Compounds in Fasted Simulated
Intestinal Fluid. This experiment determines the solubility of solid
compounds in fasted simulated intestinal fluid (FaSSIF) at pH 6.5
after 4 h equilibration at room temperature. Then 1 mL of FaSSIF
buffer (3 mM sodium taurocholate, 0.75 mM lecithin in sodium
phosphate buffer at pH 6.5) was added to manually weighed 1 mg of
solid compound in a 2 mL HPLC autosampler vial. The resulting
suspension is shaken at 900 rpm for 4 h at room temperature and then
transferred to a Multiscreen HTS, 96-well solubility filter plate. The
residual solid was removed by filtration. The supernatant solution was
quantified by HPLC-UV using single-point calibration of a known
concentration of the compound in DMSO. The dynamic range of the
assay was 1−1000 ug/mL.
Measurement of ChiLogD. A fast gradient, reverse-phase HPLC
method was used to determine the chromatographic hydrophobicity
index (ChiLogD7.4) as devised by Camurri and Zaramella40 and Valko
et al.41 By plotting the retention time of a set of reference compounds
against known Chi values, the Chi value of test compounds can
be calculated according to their retention time. Test compounds
were prepared as 0.5 mM solutions in 50:50 acetonitrile:water and
analyzed by reversed-phase HPLC-UV (wavelength 254 nm) using a
Phenomenex Luna C18 100 Å 150 mm × 4.6 mm 5 μm column with
a gradient of aqueous phase (50 mM ammonium acetate (pH 7.4))
and mobile phase (acetonitrile).
Intrinsic Clearance (Cli) Experiments. Test compound (0.5 μM)
was incubated with female CD1 mouse liver microsomes (Xenotech
LLC; 0.5 mg/mL 50 mM potassium phosphate buffer, pH 7.4) and the
reaction started with addition of excess NADPH (8 mg/mL 50 mM
potassium phosphate buffer, pH 7.4). Immediately, at time zero,
then at 3, 6, 9, 15, and 30 min, an aliquot (50 μL) of the incubation
mixture was removed and mixed with acetonitrile (100 μL) to stop
the reaction. Internal standard was added to all samples, the samples
centrifuged to sediment precipitated protein, and the plates then
sealed prior to UPLCMSMS analysis using a Quattro Premier XE
(Waters Corporation, USA). XLfit (IDBS, UK) was used to calculate
the exponential decay, and consequently the rate constant (k) from the
ratio of peak area of test compound to internal standard at each time
point. The rate of intrinsic clearance (Cli) of each test compound was
then calculated using the equation Cli (mL/min/g liver) = k × V ×
microsomal protein yield, where V (mL/mg protein) is the incubation
volume/mg protein added and microsomal protein yield is taken as
52.5 mg protein/g liver. Verapamil (0.5 μM) was used as a positive
control to confirm acceptable assay performance.
plasma or buffer were transferred to micronic tubes (Micronic BV, The
Netherlands) and the composition in each tube balanced with control
fluid (50 μL), such that the volume of buffer to plasma was the same.
Sample extraction was performed by the addition of 200 μL of
acetonitrile containing an appropriate internal standard. Samples were
allowed to mix for 1 min and then centrifuged at 3000 rpm in 96-well
blocks for 15 min (Allegra X12-R, Beckman Coulter, USA), after
which 150 μL of supernatant was removed to 50 μL of water. All sam-
ples were analyzed by UPLC-MS/MS on a Quattro Premier XE mass
spectrometer (Waters Corporation, USA). The unbound fraction was
determined as the ratio of the peak area in buffer to that in plasma.
All Animal Studies Were Ethically Reviewed and Carried out in
Accordance with Animals (Scientific Procedures) Act 1986 and the
GSK/Dundee University/LSHTM Policy on the Care, Welfare, and
Treatment of Animals
Mouse Pharmacokinetics. Test compound (11) was dosed as a
bolus solution intraperitoneally at 10 mg free base/kg (dose volume,
10 mL/kg; dose vehicle, 5% DMSO/40% PEG400/55% saline) to
female NMRI mice (n = 3) or dosed orally by gavage as a solution at
10 mg free base/kg (11 [suspension], 13, 20, 37, 48, 58, and 59) to
female NMRI mice (n = 3/dose level) or as suspension at 50 mg
of free base/kg (58 and 59) to female balb/c mice (dose volume,
10 mL/kg; dose vehicle, 5% DMSO/40% PEG400/55% distilled
water, 37 and 59; 10% DMSO/40% PEG400/50% distilled water, 20,
48, and 58; or 0.5% w/v hydroxypropylmethylcellulose with 0.4% v/v
Tween 80 and 0.5% v/v benzyl alcohol, 11 (10 mg free base/kg),
58 and 59 (50 mg free base/kg). Blood samples were taken from each
mouse at 5, 15, and 30 min, 1, 2, 4, 6, 8, and 24 (58 and 59 only)
hours postdose and mixed with two volumes of distilled water. After
suitable sample preparation, the concentration of test compound in
blood was determined by UPLC/MS/MS using a Quattro Premier XE
(Waters, USA). Pharmacokinetic parameters were derived from the
mean blood concentration time curve using PK solutions software v
2.0 (Summit Research Services, USA).
In Vivo Testing and Bioluminescence Imaging. Female
BALB/c (Charles River; UK), aged 8−12 weeks, were infected with
bioluminescent T. cruzi (CL Brener) expressing the firefly luciferase
PpyRE9h gene.65 Typically, 104 bloodstream trypomastigotes (BTs) in
0.2 mL of PBS were first used to infect SCID mice via intraperitoneal
(ip) inoculation. Parasitaemic blood from these mice was obtained
2−3 weeks later and adjusted to 5 × 103 BTs mL−1 with PBS. The
infection of BALB/c mice was by ip inoculation with 1 × 103 BTs.
Treatment was initiated 14 (acute stage) or 97 (chronic stage) days
postinfection. Drugs were administered by oral gavage (∼200 μL), and
vehicle only (0.5% w/v hydroxypropylmethylcellulose with 0.4% v/v
Tween 80 and 0.5% v/v benzyl alcohol) was administered to control
mice. Following the first daily dose on day 1 and day 21, a blood sample
was taken from all drug treated mice predose, then 30 min, 1, 2, 4, and
8 h postdose, mixed with two volumes of distilled water and analyzed for
test compound as for mouse pharmacokinetics above. To detect residual
infection, mice were immunosuppressed with cyclophosphamide
(200 mg/kg) by ip injection every 4 days for a maximum of three doses.
For in vivo imaging, mice were injected ip with 150 mg/kg
D-luciferin in Dulbecco’s Ca2+/Mg2+ free PBS, then anaesthetized
using 2.5% (v/v) isofluorane in oxygen. Images were obtained using
an IVIS Lumina II system (Caliper Life Science) 10−20 min after
D-luciferin administration. The detection threshold was established
from uninfected mice. Organs/tissue samples were assessed by ex vivo
imaging as described previously.65
Plasma Protein Binding Experiments. In brief, a 96-well
equilibrium dialysis apparatus was used to determine the free fraction in
mouse plasma for each compound (HT Dialysis LLC, Gales Ferry, CT).
Membranes (12−14 kDaA cutoff) were conditioned in deionized water
for 60 min, followed by conditioning in 80:20 deionized water:ethanol
for 20 min and then rinsed in isotonic buffer before use. Female CD1
mouse plasma was removed from the freezer and allowed to thaw
on the day of experiment. Thawed plasma was then centrifuged
(Allegra X12-R, Beckman Coulter, USA), spiked with test compound
(final concentration 10 μg/mL), and 150 μL aliquots (n = 6 replicate
determinations) loaded into the 96-well equilibrium dialysis plate.
Dialysis vs isotonic buffer (150 μL) was carried out for 5 h in a
temperature controlled incubator at ca. 37 °C (Barworld scientific Ltd.,
UK) using an orbital microplate shaker at 100 rpm (Barworld scientific
Ltd., UK). At the end of the incubation period, 50 μL aliquots of
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jmedchem.7b00463.
This contains synthetic details for all compounds in the
main body of the paper, supplementary data tables, more
information on ADMET and pharmacology (PDF)
Molecular formula strings (CSV)
M
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(13) Koberle, F. Chagas’ disease and Chagas’ syndromes: the
pathology of American trypanosomiasis. Adv. Parasitol. 1968, 6, 63−
116.
(14) Prata, A. Clinical and epidemiological aspects of Chagas’ disease.
Lancet Infect. Dis. 2001, 1, 92−100.
AUTHOR INFORMATION
Corresponding Authors
*For I.H.G.: phone, +44 1382 386 240; E-mail, i.h.gilbert@
dundee.ac.uk.
*For T.J.M.: phone, +34 650 349 771; E-mail, tim.j.miles@gsk.
com.
*For K.D.R.: phone, +44 1382 388 688; E-mail, k.read@
dundee.ac.uk.
■
(15) Bern, C.; Montgomery, S. P.; Herwaldt, B. L.; Rassi, A., Jr.;
Marin-Neto, J. A.; Dantas, R. O.; Maguire, J. H.; Acquatella, H.;
Morillo, C.; Kirchhoff, L. V.; Gilman, R. H.; Reyes, P. A.; Salvatella, R.;
Moore, A. C. Evaluation and treatment of Chagas disease in the
United States: a systematic review. JAMA, J. Am. Med. Assoc. 2007,
298, 2171−2181.
(16) Castro, J. A.; de Mecca, M. M.; Bartel, L. C. Toxic side effects of
drugs used to treat Chagas’ disease (American trypanosomiasis). Hum.
Exp. Toxicol. 2006, 25, 471−479.
(17) Le Loup, G.; Pialoux, G.; Lescure, F. X. Update in treatment of
Chagas’ disease. Curr. Opin. Infect. Dis. 2011, 24, 428−434.
(18) Molina, I.; Gomez i Prat, J.; Salvador, F.; Trevino, B.; Sulleiro,
E.; Serre, N.; Pou, D.; Roure, S.; Cabezos, J.; Valerio, L.; Blanco-Grau,
A.; Sanchez-Montalva, A.; Vidal, X.; Pahissa, A. Randomized trial of
posaconazole and benznidazole for chronic Chagas’ disease. N. Engl. J.
Med. 2014, 370, 1899−1908.
(19) Torrico, F., E1224results of proof of concept clinical trial in
patients with chronic indeterminate Chagas disease. Proceedings of the
62nd Annual Meeting of the American Society of Tropical Medicine and
Hygiene, Washington, DC, November 13−17, 2013, 2013.
(20) Morillo, C. A.; Marin-Neto, J. A.; Avezum, A.; Sosa-Estani, S.;
Rassi, A.; Rosas, F.; Villena, E.; Quiroz, R.; Bonilla, R.; Britto, C.; Guhl,
F.; Velazquez, E.; Bonilla, L.; Meeks, B.; Rao-Melacini, P.; Pogue, J.;
Mattos, A.; Lazdins, J.; Rassi, A.; Connolly, S. J.; Yusuf, S.
Investigators, B. Randomized trial of benznidazole for chronic Chagas’
cardiomyopathy. N. Engl. J. Med. 2015, 373, 1295−1306.
(21) Brenk, R.; Schipani, A.; James, D.; Krasowski, A.; Gilbert, I. H.;
Frearson, J.; Wyatt, P. G. Lessons learnt from assembling screening
libraries for drug discovery for neglected diseases. ChemMedChem
2008, 3, 435−444.
(22) De Rycker, M.; Thomas, J.; Riley, J.; Brough, S. J.; Miles, T. J.;
Gray, D. W. Identification of trypanocidal activity for known clinical
compounds using a new Trypanosoma cruzi hit-discovery screening
cascade. PLoS Neglected Trop. Dis. 2016, 10, e0004584.
(23) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand efficiency: a
useful metric for lead selection. Drug Discovery Today 2004, 9, 430−
431.
ORCID
Manu De Rycker: 0000-0002-3171-3519
Ian H. Gilbert: 0000-0002-5238-1314
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Funding for this work was provided by the Wellcome Trust
(nos. 092340 and 100476). We thank Gina MacKay for
performing HRMS analyses and for assistance with performing
other NMR and MS analyses, Alastair Pate and Francesco
Gastaldello for data management, Lorna Campbell and Irene
Hallyburton for performing HepG2 assays and plasmodium
parasite assays, and Dr. Stephen Patterson for critical evaluation
of the manuscript.
ABBREVIATIONS USED
■
ATC, 5-amino-1,2,3-triazole-4-carboxamide; BNZ, benznida-
zole; PFI, property forecast index; eXP, GSK enhanced cross-
screen panel
REFERENCES
■
(1) Rassi, A., Jr.; Rassi, A.; Marin-Neto, J. A. Chagas disease. Lancet
2010, 375, 1388−1402.
(2) Bocchi, E. A. Heart failure in South America. Curr. Cardiol. Rev.
2013, 9, 147−156.
(3) Martins-Melo, F. R.; Alencar, C. H.; Ramos, A. N., Jr.;
Heukelbach, J. Epidemiology of mortality related to Chagas’ disease
in Brazil, 1999−2007. PLoS Neglected Trop. Dis. 2012, 6, e1508.
(4) Bonney, K. M. Chagas disease in the 21st century: a public health
success or an emerging threat? Parasite 2014, 21, 11.
(5) Giddings, O. K.; Eickhoff, C. S.; Smith, T. J.; Bryant, L. A.; Hoft,
D. F. Anatomical route of invasion and protective mucosal immunity
in Trypanosoma cruzi conjunctival infection. Infect. Immun. 2006, 74,
5549−5560.
(6) Nickerson, P.; Orr, P.; Schroeder, M. L.; Sekla, L.; Johnston, J. B.
Transfusion-associated Trypanosoma cruzi infection in a non-endemic
area. Ann. Intern. Med. 1989, 111, 851−853.
(7) Nobrega, A. A.; Garcia, M. H.; Tatto, E.; Obara, M. T.; Costa, E.;
Sobel, J.; Araujo, W. N. Oral transmission of Chagas’ disease by
consumption of acai palm fruit, Brazil. Emerging Infect. Dis. 2009, 15,
653−655.
(8) Nowicki, M. J.; Chinchilla, C.; Corado, L.; Matsuoka, L.; Selby,
R.; Steurer, F.; Mone, T.; Mendez, R.; Aswad, S. Prevalence of
antibodies to Trypanosoma cruzi among solid organ donors in southern
california: a population at risk. Transplantation 2006, 81, 477−479.
(9) Norman, F. F.; Lopez-Velez, R. Chagas disease and breast-
feeding. Emerging Infect. Dis. 2013, 19, 1561−1566.
(24) Hopkins, A. L.; Keseru, G. M.; Leeson, P. D.; Rees, D. C.;
Reynolds, C. H. The role of ligand efficiency metrics in drug discovery.
Nat. Rev. Drug Discovery 2014, 13, 105−121.
(25) Kaiser, M.; Maser, P.; Tadoori, L. P.; Ioset, J. R.; Brun, R.
Antiprotozoal activity profiling of approved drugs: a starting point
toward drug repositioning. PLoS One 2015, 10, e0135556.
(26) Avelar, L. A. A.; Camilo, C. D.; de Albuquerque, S.; Fernandes,
W. B.; Goncalez, C.; Kenny, P. W.; Leitao, A.; McKerrow, J. H.;
Montanari, C. A.; Orozco, E. V. M; Ribeiro, J. F. R.; Rocha, J. R.;
Rosini, F.; Saidel, M. E. Molecular design, dynthesis and trypanocidal
activity of dipeptidyl nitriles as cruzain inhibitors. PLoS Neglected Trop.
Dis. 2015, 9, e0003916.
(27) Menezes, J. C. L.; Vaz, L. B. A.; de Abreu Vieira, P. M.; da Silva
Fonseca, K. D.; Carneiro, C. M.; Taylor, J. G. Synthesis and anti-
Trypanosoma cruzi activity of diaryldiazepines. Molecules 2015, 20, 43−
51.
(28) Germain, A. R.; Carmody, L. C.; Dockendorff, C.; Galan-
Rodriguez, C.; Rodriguez, A.; Johnston, S.; Bittker, J. A.; MacPherson,
L.; Dandapani, S.; Palmer, M.; Schreiber, S. L.; Munoz, B.
Identification of small-molecule inhibitors of Trypansoma cruzi
replication. Bioorg. Med. Chem. Lett. 2011, 21, 7197−7200.
(29) Keenan, M.; Chaplin, J. H.; Alexander, P. W.; Abbott, M. J.;
Best, W. M.; Khong, A.; Botero, A.; Perez, C.; Cornwall, S.;
Thompson, R. A.; White, K. L.; Shackleford, D. M.; Koltun, M.;
Chiu, F. C. K.; Morizzi, J.; Ryan, E.; Campbell, M.; von Geldern, T.
W.; Scandale, I.; Chatelain, E.; Charman, S. A. Two analogues of
(10) Coura, J. R.; Vinas, P. A. Chagas disease: a new worldwide
challenge. Nature 2010, 465, S6−7.
(11) Dias, J. C. The indeterminate form of human chronic Chagas’
disease a clinical epidemiological review. Rev. Soc. Bras. Med. Trop.
1989, 22, 147−156.
(12) Bustamante, J. M.; Rivarola, H. W.; Fernandez, A. R.; Enders, J.
E.; Fretes, R.; Palma, J. A.; Paglini-Oliva, P. A. Indeterminate Chagas’
disease: Trypanosoma cruzi strain and re-infection are factors involved
in the progression of cardiopathy. Clin. Sci. 2003, 104, 415−420.
N
DOI: 10.1021/acs.jmedchem.7b00463
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
fenarimol show curative activity in an experimental model of Chagas’
disease. J. Med. Chem. 2013, 56, 10158−10170.
Bernasconi, P., Eds.; Humana Press: Totowa, NJ, 2009; Vol. 565,
209−223.
(30) Neitz, R. J.; Chen, S.; Supek, F.; Yeh, V.; Kellar, D.; Gut, J.;
Bryant, C.; Gallardo-Godoy, A.; Molteni, V.; Roach, S. L.; Chatterjee,
A. K.; Robertson, S.; Renslo, A. R.; Arkin, M.; Glynne, R.; McKerrow,
J.; Siqueira-Neto, J. L. Lead identification to clinical candidate
selection: drugs for Chagas’ disease. J. Biomol. Screening 2015, 20,
101−111.
(49) Haworth, S.; Lawlor, T.; Mortelmans, K.; Speck, W.; Zeiger, E.
Salmonella mutagenicity test-results for 250 chemicals. Environ.
Mutagen. 1983, 5, 3−49.
(50) Brown, N. Bioisosteres and scaffold hopping in medicinal
chemistry. Mol. Inf. 2014, 33, 458−462.
(51) Junge, W.; Krisch, K.; Conney, A. The carboxylesterases/
amidases of mammalian liver and their possible significance. Crit. Rev.
Toxicol. 1975, 3, 371−434.
(31) Nwaka, S.; Besson, D.; Ramirez, B.; Maes, L.; Matheeussen, A.;
Bickle, Q.; Mansour, N. R.; Yousif, F.; Townson, S.; Gokool, S.; Cho-
Ngwa, F.; Samje, M.; Misra-Bhattacharya, S.; Murthy, P. K.; Fakorede,
F.; Paris, J. M.; Yeates, C.; Ridley, R.; Van Voorhis, W. C.; Geary, T.
Integrated dataset of screening hits against multiple neglected disease
pathogens. PLoS Neglected Trop. Dis. 2011, 5, e1412.
(32) Katsuno, K.; Burrows, J. N.; Duncan, K.; van Huijsduijnen, R.
H.; Kaneko, T.; Kita, K.; Mowbray, C. E.; Schmatz, D.; Warner, P.;
Slingsby, B. T. Hit and lead criteria in drug discovery for infectious
diseases of the developing world. Nat. Rev. Drug Discovery 2015, 14,
751−758.
(33) Chatelain, E. Chagas disease drug discovery: toward a new era. J.
Biomol. Screening 2015, 20, 22−35.
(34) Edwards, M. P.; Price, D. A. Role of physicochemical properties
and ligand lipophilicity efficiency in addressing drug safety risks. Annu.
Rep. Med. Chem. 2010, 45, 380−391.
(35) Freeman-Cook, K. D.; Hoffman, R. L.; Johnson, T. W.
Lipophilic efficiency: the most important efficiency metric in medicinal
chemistry. Future Med. Chem. 2013, 5, 113−115.
(36) Kiselyov, A. S.; Semenova, M.; Semenov, V. V. (1,2,3-Triazol-4-
yl)benzenamines: synthesis and activity against VEGF receptors 1 and
2. Bioorg. Med. Chem. Lett. 2009, 19, 1344−1348.
(52) Borosky, G. L. Quantum-chemical studies on mutagenicity of
aromatic and heteroaromatic amines. Front. Biosci., Scholar Ed. 2013,
S5, 600−610.
(53) Jamieson, C.; Moir, E. M.; Rankovic, Z.; Wishart, G. Medicinal
chemistry of hERG optimizations: highlights and hang-ups. J. Med.
Chem. 2006, 49, 5029−5046.
(54) Shamovsky, I.; Ripa, L.; Borjesson, L.; Mee, C.; Norden, B.;
Hansen, P.; Hasselgren, C.; O’Donovan, M.; Sjo, P. Explanation for
main features of structure-genotoxicity relationships of aromatic
amines by theoretical studies of their activation pathways in
CYP1A2. J. Am. Chem. Soc. 2011, 133, 16168−16185.
(55) Lepesheva, G. I.; Villalta, F.; Waterman, M. R. Targeting
Trypanosoma cruzi sterol 14alpha-demethylase (CYP51). Adv.
Parasitol. 2011, 75, 65−87.
(56) Gunatilleke, S. S.; Calvet, C. M.; Johnston, J. B.; Chen, C. K.;
Erenburg, G.; Gut, J.; Engel, J. C.; Ang, K. K.; Mulvaney, J.; Chen, S.;
Arkin, M. R.; McKerrow, J. H.; Podust, L. M. Diverse inhibitor
chemotypes targeting Trypanosoma cruzi CYP51. PLoS Neglected Trop.
Dis. 2012, 6, e1736.
(57) Keenan, M.; Alexander, P. W.; Chaplin, J. H.; Abbott, M. J.;
Diao, H.; Wang, Z.; Best, W. M.; Perez, C. J.; Cornwall, S. M.; Keatley,
S. K.; Thompson, R. C.; Charman, S. A.; White, K. L.; Ryan, E.; Chen,
G.; Ioset, J. R.; von Geldern, T. W.; Chatelain, E. Selection and
optimization of hits from a high-throughput phenotypic screen against
Trypanosoma cruzi. Future Med. Chem. 2013, 5, 1733−1752.
(58) Friggeri, L.; Scipione, L.; Costi, R.; Kaiser, M.; Moraca, F.;
Zamperini, C.; Botta, B.; Di Santo, R.; De Vita, D.; Brun, R.;
Tortorella, S. New promising compounds with in vitro nanomolar
activity against Trypanosoma cruzi. ACS Med. Chem. Lett. 2013, 4,
538−541.
(59) Andriani, G.; Amata, E.; Beatty, J.; Clements, Z.; Coffey, B. J.;
Courtemanche, G.; Devine, W.; Erath, J.; Juda, C. E.; Wawrzak, Z.;
Wood, J. T.; Lepesheva, G. I.; Rodriguez, A.; Pollastri, M. P.
Antitrypanosomal lead discovery: identification of a ligand-efficient
inhibitor of Trypanosoma cruzi CYP51 and parasite growth. J. Med.
Chem. 2013, 56, 2556−2567.
(60) Alonso-Padilla, J.; Rodriguez, A. High throughput screening for
anti-Trypanosoma cruzi drug discovery. PLoS Neglected Trop. Dis. 2014,
8, e3259.
(37) Levin, J. I.; Turos, E.; Weinreb, S. M. An alternative procedure
for the aluminium-mediated conversion of esters to amides. Synth.
Commun. 1982, 12, 989−993.
(38) Goddard-Borger, E. D.; Stick, R. V. An efficient, inexpensive,
and shelf-stable diazotransfer reagent: imidazole-1-sulfonyl azide
hydrochloride. Org. Lett. 2007, 9, 3797−3800.
(39) Mannhold, R.; Dross, K. Calculation procedures for molecular
lipophilicity: a comparative study. Quant. Struct.-Act. Relat. 1996, 15,
403−409.
(40) Camurri, G.; Zaramella, A. High-throughput liquid chromatog-
raphy/mass spectrometry method for the determination of the
chromatographic hydrophobicity index. Anal. Chem. 2001, 73,
3716−3722.
(41) Valko, K.; Nunhuck, S.; Bevan, C.; Abraham, M. H.; Reynolds,
D. P. Fast gradient HPLC method to determine compounds binding
to human serum albumin. Relationships with octanol/water and
immobilized artificial membrane lipophilicity. J. Pharm. Sci. 2003, 92,
2236−2248.
(42) Leeson, P. D.; Young, R. J. Molecular property design: does
everyone get it? ACS Med. Chem. Lett. 2015, 6, 722−725.
(43) Young, R. J.; Green, D. V. S.; Luscombe, C. N.; Hill, A. P.
Getting physical in drug discovery II: the impact of chromatographic
hydrophobicity measurements and aromaticity. Drug Discovery Today
2011, 16, 822−830.
(44) Bastin, R. J.; Bowker, M. J.; Slater, B. J. Salt selection and
optimization procedures for pharmaceutical new chemical entities. Org.
Process Res. Dev. 2000, 4, 427−435.
(45) Bowes, J.; Brown, A. J.; Hamon, J.; Jarolimek, W.; Sridhar, A.;
Waldron, G.; Whitebread, S. Reducing safety-related drug attrition: the
use of in vitro pharmacological profiling. Nat. Rev. Drug Discovery
2012, 11, 909−922.
(46) Kargl, J.; Brown, A. J.; Andersen, L.; Dorn, G.; Schicho, R.;
Waldhoer, M.; Heinemann, A. A selective antagonist reveals a potential
role of G protein−coupled receptor 55 in platelet and endothelial cell
function. J. Pharmacol. Exp. Ther. 2013, 346, 54−66.
(47) Priest, B. T.; Bell, I. M.; Garcia, M. L. Role of hERG potassium
channel assays in drug development. Channels 2008, 2, 87−93.
(48) Jones, K. A.; Garbati, N.; Zhang, H.; Large, C. H. In High
Throughput Screening: Methods and Protocols, 2n ed.; Janzen, W. P.,
(61) Pena, I.; Pilar Manzano, M.; Cantizani, J.; Kessler, A.; Alonso-
Padilla, J.; Bardera, A. I.; Alvarez, E.; Colmenarejo, G.; Cotillo, I.;
Roquero, I.; de Dios-Anton, F.; Barroso, V.; Rodriguez, A.; Gray, D.
W.; Navarro, M.; Kumar, V.; Sherstnev, A.; Drewry, D. H.; Brown, J.
R.; Fiandor, J. M.; Julio Martin, J. New compound sets identified from
high throughput phenotypic screening against three kinetoplastid
parasites: an open resource. Sci. Rep. 2015, 5, 8771.
(62) Riley, J.; Brand, S.; Voice, M.; Caballero, I.; Calvo, D.; Read, K.
D. Development of a fluorescence-based Trypanosoma cruzi CYP51
inhibition assay for effective compound triaging in drug discovery
programmes for Chagas’ disease. PLoS Neglected Trop. Dis. 2015, 9,
e0004014.
(63) Kalgutkar, A. S.; Obach, R. S.; Maurer, T. S. Mechanism-based
inactivation of cytochrome P450 enzymes: chemical mechanisms,
structure-activity relationships and relationship to clinical drug-drug
interactions and idiosyncratic adverse drug reactions. Curr. Drug
Metab. 2007, 8, 407−447.
(64) Zingales, B.; Miles, M. A.; Moraes, C. B.; Luquetti, A.; Guhl, F.;
Schijman, A. G.; Ribeiro, I. Drug discovery for Chagas’ disease should
O
DOI: 10.1021/acs.jmedchem.7b00463
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
consider Trypanosoma cruzi strain diversity. Mem. Inst. Oswaldo Cruz
2014, 109, 828−833.
(65) Francisco, A. F.; Lewis, M. D.; Jayawardhana, S.; Taylor, M. C.;
Chatelain, E.; Kelly, J. M. Limited ability of posaconazole to cure both
acute and chronic Trypanosoma cruzi infections revealed by highly
sensitive in vivo imaging. Antimicrob. Agents Chemother. 2015, 59,
4653−4661.
P
DOI: 10.1021/acs.jmedchem.7b00463
J. Med. Chem. XXXX, XXX, XXX−XXX