Design, structure-activity relationship and in vivo efficacy of piperazine analogues of fenarimol as inhibitors of Trypanosoma cruzi

Article abstract of DOI:10.1016/j.bmc.2013.01.050

A scaffold hopping exercise undertaken to expand the structural diversity of the fenarimol series of anti-Trypanosoma cruzi (T. cruzi) compounds led to preparation of simple 1-[phenyl(pyridin-3-yl)methyl]piperazinyl analogues of fenarimol which were investigated for their ability to inhibit T. cruzi in vitro in a whole organism assay. A range of compounds bearing amide, sulfonamide, carbamate/carbonate and aryl moieties exhibited low nM activities and two analogues were further studied for in vivo efficacy in a mouse model of T. cruzi infection. One compound, the citrate salt of 37, was efficacious in a mouse model of acute T. cruzi infection after once daily oral dosing at 20, 50 and 100 mg/kg for 5 days.

Full text of DOI:10.1016/j.bmc.2013.01.050

Bioorganic & Medicinal Chemistry 21 (2013) 1756–1763
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, structure–activity relationship and in vivo efficacy
of piperazine analogues of fenarimol as inhibitors
of Trypanosoma cruzi
a
a
a
b,
Martine Keenan a, , Paul W. Alexander , Hugo Diao , Wayne M. Best , Andrea Khong , Maria Kerfoot b,
R. C. Andrew Thompson b, Karen L. White c, David M. Shackleford c, Eileen Ryan c, Alison D. Gregg c,
Susan A. Charman c, Thomas W. von Geldern d, Ivan Scandale d, Eric Chatelain d
⇑
a Epichem Pty Ltd, Murdoch University Campus, South Street, Murdoch, Western Australia 6150, Australia
b Department of Parasitology and Veterinary Sciences, Murdoch University, South Street, Murdoch, Western Australia 6150, Australia
c Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Victoria 3052, Australia
d Drugs for Neglected Diseases initiative (DNDi), 15 Chemin Louis Dunant, 1202 Geneva, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A scaffold hopping exercise undertaken to expand the structural diversity of the fenarimol series of anti-
Trypanosoma cruzi (T. cruzi) compounds led to preparation of simple 1-[phenyl(pyridin-3-yl)methyl]pip-
erazinyl analogues of fenarimol which were investigated for their ability to inhibit T. cruzi in vitro in a
whole organism assay. A range of compounds bearing amide, sulfonamide, carbamate/carbonate and aryl
moieties exhibited low nM activities and two analogues were further studied for in vivo efficacy in a
mouse model of T. cruzi infection. One compound, the citrate salt of 37, was efficacious in a mouse model
of acute T. cruzi infection after once daily oral dosing at 20, 50 and 100 mg/kg for 5 days.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 15 November 2012
Revised 16 January 2013
Accepted 23 January 2013
Available online 31 January 2013
Keywords:
Trypanosoma cruzi
Chagas disease
Fenarimol
Inhibition
1. Introduction
time the amastigote form of T. cruzi has caused considerable dam-
age to the heart and/or gastrointestinal tract via infiltration of soft
Chagas disease, caused by the protozoan parasite Trypanosoma
cruzi, is a major neglected parasitic disease endemic in South and
Central America with an increasing number of cases reported in
countries such as USA, Spain and Japan.1,2 Research into new ther-
apies to treat Chagas disease must overcome a number of difficul-
ties arising from the complex nature of the parasite’s life cycle3 and
its extensive impact on the body.4 Following infection with T. cruzi
parasites, blood-form trypomastigotes enter cells and rapidly pro-
liferate in amastigote form, causing acute illness with mild symp-
toms lasting for a few weeks. Treatment is effective at this point in
60–85% of cases,5 but acute infection can also cause mortality in a
small number of patients.6 Parasites remain in the body as the dis-
ease progresses to an asymptomatic phase during which time pa-
tients may not be aware they are carriers of the parasite and
may never develop the chronic form of the disease. Chronic Chagas
disease can take as long as 15–20 years to present, during which
tissues and stimulation of immune responses.7 A recent epidemio-
logical study of mortality related to Chagas disease in Brazil be-
tween 1999 and 2007 reported 97% of diagnosed Chagas related
deaths were due to the chronic form of the disease.6 T. cruzi is
genetically highly diverse and is divided into six discrete typing
units (DTUs)8 which have the potential to vary in susceptibility
to drug treatment. The current standard of care, benznidazole, is
given at a high dose for 30–60 days, has undesirable side effects
leading to poor patient compliance and has a limited and contro-
versial impact against chronic Chagas disease.5 The protracted nat-
ure of the illness, extent of parasite distribution into the body,
monitoring of disease progression and determination of cure add
complexity to clinical evaluation of existing and promising new
treatments.1,5
CYP51 is a well-validated drug target for the inhibition of T. cru-
zi and a major drug target in the cytochrome P450 family.9 The en-
zyme is an important part of sterol biosynthesis in eukaryotes,
with inhibition causing a breakdown in membrane structural
integrity, fluidity and permeability leading to deleterious down-
stream effects.10 Pioneering work by Urbina et al. brought anti-
fungal CYP51 inhibitors to the forefront of T. cruzi drug discovery
⇑
Corresponding author. Tel.: +61 8 9360 7216; fax: +61 8 9360 7699.
E-mail address: martine.keenan@epichem.com.au (M. Keenan).
Current address: School of Medicine and Pharmacology, The University of
Western Australia, Nedlands 6009, Western Australia.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.050

M. Keenan et al. / Bioorg. Med. Chem. 21 (2013) 1756–1763
1757
R1
N
X
N
establishing the cross-reactivity of leading azole antifungal sterol
biosynthesis inhibitors against T. cruzi in vitro and in vivo.11–14
More active second generation antifungals posaconazole and a rav-
uconazole pro-drug are the first compounds to enter clinical trials
for the treatment of Chagas disease in more than 40 years. Other
compounds targeting T. cruzi CYP51 include the tipifarnib ser-
ies,15–18 dialkyl imidazoles,19 LP10,20 and indomethacin amides,21
with current hit generation efforts potentially fueling further re-
search.22 The recently reported crystal structure of T. cruzi CYP51
with bound posaconazole and fluconazole is an important step for-
ward in the ongoing story of this target opening up the opportunity
for structure based drug design to address potential issues of drug
resistance.23,24
X
O
R2
R2
N
N
N
N
R2
R2
6 X = OH
7 X = Cl
8
N
N
3
4 X = BOC
5 X = H
Scheme 1.
amide analogues 28–42; isocyanates gave piperazinyl–
carbamates 44,45, 47, 48, 51, 53–56 and chloroformates gave pip-
erazinyl–carbonates 43, 46, 49, 50 and 52. Chloro intermediate 7
was also reacted with pre-functionalised piperazine analogues to
give the desired target analogues of 3 directly and to prepare
R1 = aryl analogues 57–66. As representative examples of the
synthetic methodology, Schemes 2 and 3 show the preparation of
[4-chlorophenyl(pyridin-3-yl)methyl]piperazine analogues 24, 29,
46–47 and 62, respectively. Compounds were prepared in racemic
form and synthetic transformations are unoptimised.
We recently identified fenarimol (1), a fungicide used in agricul-
ture since the mid-1970s, as having moderate in vitro activity
against T. cruzi (IC50 350 nM) following a targeted hit generation
campaign25 utilizing
a phenotypic whole organism in vitro
screen.26 Fenarimol inhibits fungal CYP51 and is classified within
the pyrimidine sub-class of fungal CYP51 inhibitors.27 The ability
of 1 to kill T. cruzi could occur by a similar mechanism and the out-
come of mechanistic studies will be reported in due course. Struc-
ture–activity relationship (SAR) studies focused on improving
potency and establishing in vivo efficacy arrived at optimised tri-
aryl analogue 2, which was orally active in a stringent in vivo
mouse model of acute T. cruzi infection. In a general effort to ex-
pand the chemical diversity of the fenarimol series and assess
the subsequent impact of a scaffold hop on activity, selectivity over
CYP3A4, solubility, oral exposure and ultimately in vivo efficacy,
one aromatic ring of the triaryl pharmacophore of 1 and 2 was re-
placed with piperazine to give a new 1-(diarylmethyl)piperazinyl
scaffold (3) which could be further derivatised on the piperazine
N-4 atom (Fig. 1).
2.2. Biological evaluation
The new [phenyl(pyridin-3-yl)methyl]piperazinyl derivatives
13–66 were tested for their ability to inhibit T. cruzi in vitro in a
whole parasite assay. Specifically, compounds were incubated with
T. cruzi Tulahuen strain TcVI (a strain transfected with the b-galac-
tosidase gene).26 Parasites in the extra cellular trypomastigote
form were added to the plate wells seeded with L6 cells (rat skel-
etal myoblasts) and incubated for 48 h to allow infection to estab-
lish before addition of test compounds and a further 4-day
incubation period, exposing compounds to both the extracellular
trypomastigote and intracellular amastigote forms of the parasite.
An IC50 value was recorded corresponding to the concentration of
compound required to inhibit 50% of parasite growth. Compound
cytotoxicity was evaluated in L6-cells as a counter-screen. The
compounds in this series were determined to be non-cytotoxic
with selectivity ratios for T. cruzi/cytoxicity generally >100.
Compounds with good activity or those of structural interest
were further evaluated for their propensity to undergo in vitro oxi-
dative metabolism by hepatic microsomes and to inhibit CYP3A4, a
key drug metabolising enzyme inhibited by most azole-derived
CYP51 antifungals. Solubility in phosphate buffer at pH 6.5 was
also determined.
2. Materials and methods
2.1. Chemistry
The retro synthesis of scaffold 3 is illustrated in Scheme 1. Benz-
ophenones (8) were selected as convenient precursors due to their
ease of synthesis from classical Friedel–Craft reactions, palladium
catalysed acylations or addition of aryl organometallic reagents
to Weinreb amide derivatives of carboxylic acids.25 Straightfor-
ward reduction with sodium borohydride gave diarylmethanols
(6) which were also prepared directly in gram quantities by addi-
tion of aryl Grignard or aryllithium species to aromatic aldehydes
at low temperature. As both aromatic groups could be introduced
as either the organometallic or aldehyde entity, the choice of react-
ing partners was determined by availability of reagents and reac-
Suitable compounds with low to moderate predicted hepatic
extraction ratios (Eh) were further evaluated in vivo by measure-
ment of the oral exposure in non-infected Swiss outbred mice
and subsequently, for in vivo efficacy in a mouse model of acute
T. cruzi infection.25
tivity of pendent substituents. Conversion of
6
to the
corresponding chloro derivative 7 on reaction with thionyl chloride
was followed by substitution with t-butyl piperazine-1-carboxyl-
ate to give BOC-protected scaffold-4. Simple acid mediated depro-
tection gave 5 in overall good yields.
3. Results and discussion
Reaction of 5 with acid chlorides gave piperazinyl-amide ana-
logues 13–27; with sulfonyl chlorides gave piperazinyl-sulfon-
The exploration of the SAR was greatly facilitated by the tracta-
ble synthesis of the target compounds allowing each region of the
pharmacophore to be investigated independently (Fig. 2).
R1
CN
3.1. Structure–activity relationship (SAR)
N
Exploration of the heterocycle and aromatic ring R2 was guided
by the results of previous SAR studies on the triaryl series, which
had established 3-pyridyl and a handful of 4-substituted phenyl
motifs as optimal for activity and in vitro oxidative metabolic sta-
bility (and thus oral exposure).25 5-Pyrimidinyl analogues of sev-
eral compounds were made in this study in order to confirm that
Cl
N
N
OH
OH
R2
N
N
1
2
CF3
N
3
Cl
Figure 1. Fenarimol (1), 2 and proposed scaffold (3).

1758
M. Keenan et al. / Bioorg. Med. Chem. 21 (2013) 1756–1763
R1
N
O
O
X
N
24
29
*
O
S
*
O
X
N
N
1,2
Cl
3, 4
Cl
5a-d
O
O
Ph
46
47
*
*
N
N
Cl
Cl
N
N
NH
Ph
O
9
X=OH
10 X=Cl
24,29,46,47
11 X=BOC
12 X=H
Scheme 2. (1) NaBH4, MeOH, RT, 48 h, (97%); (2) 9, SOCl2, Tol, reflux, 18 h, (94%); (3) 10, BOC-piperazine, NEt3, KI, AcCN, 80 °C, 48 h, (80%). (4) 11, TFA, DCM, MeOH, rt, 18 h,
(quant.) (5) (a) 12, CH3COCl, DCM, Et3N, 0 °C–RT, 1 h, (17%), (b) 12, PhSO2Cl, DCM, Et3N, 0 °C–rt, 2 h (83%), (c) 12, PhOCOCl, DCM, Et3N, 0 °C–rt, 12 h (56%), (d) 12, PhN@C@O,
DCM, Et3N, 0 °C–rt, 45 min, (70%).
Table 1
Ph
R1 = alkyl and aryl amide (COR3)
N
c
d
Cmpd
HETa
R2b
R3
Tc IC50
(lM)
L6 IC50 (lM)
Cl
13
14
15
16
17
18
19
20
21
22
23
24
25
26
3-Pyr
3-Pyr
3-Pyr
3-Pyr
3-Pyr
3-Pyr
3-Pyr
3-Pyr
3-Pyr
5-Pyrim
5-Pyrim
3-Pyr
3-Pyr
3-Pyr
3-Pyr
B
D
B
B
C
F
B
A
B
B
B
A
E
E
E
tBu
tBu
cPr
0.019
0.024
0.026
0.028
0.030
0.035
0.036
0.068
0.070
0.081
0.171
0.240
0.254
0.281
0.513
0.0007
1.15
60
73
>100
62
59
>100
93
61
N
Cl
N
cHex
tBu
iPr
10
62
Cl
N
Hete
Ph
Scheme 3. 10, 1-phenylpiperazine, DMF, K2CO3, 150 °C, microwave, 30 min (24%)
Ph
tBu
cPr
69
>100
>100
>100
>100
>100
>100
>100
>100
O
O
S
O
1
O
R
5
R
N
Me
tBu
cPr
R1 =
R2 =
Ar
R
3
4
*
*
R
*
*
X
X = O, NH
27
Hete
Table 1 Table 2
Table 3 Table 4
fPosa
gBnz
N
X
X
*
*
*
*
2
a
R
HET: 3-pyr (3-pyridine), 5-pyrim (5-pyrimidine).
A (4-chlorophenyl), B (4-chloro-2-fluorophenyl), C (4-trifluoromethylphenyl), D
(2-fluoro-4-trifluoromethylphenyl), E (4-benzonitrile), F (4-ipropoxyphenyl).
N
b
OiPr
CF
Cl
CN
3
3
c
A
B
X=H
X=F
C
D
X=H
X=F
E
F
Tc = T. cruzi, values are the mean of at least two experiments.
L6 cells (rat myoblasts from skeletal muscle) for assessment of cytotoxicity.
19, 27 Het = 2-(4-methyl-1,3-thiazole).
Posa (posaconazole).
d
e
f
Figure 2. Scope of SAR investigation.
g
Bnz (benznidazole).
SAR trends remained true in the new piperazine series. The bulk of
the SAR exploration was thus focused on establishing the best R1
group.
Table 2
R1 = alkyl and aryl sulfonamide (SO2R4)
In general, across all variations of R1, the in vitro activity of the
new piperazinyl compounds was very encouraging with activities
in the low nM range. Tables 1–4 show selected analogues from
the amide series (Table 1), sulfonamide series (Table 2), carba-
mate/carbonate series (Table 3) and aryl series (Table 4). The com-
pounds are ordered by in vitro activity starting with the most
active analogue and a twofold difference in IC50 value was consid-
ered to reflect an actual difference in potency. Activity data for ref-
erence compounds posaconazole and benznidazole has been
included in Table 1. Posaconazole is very active in vitro, in contrast
c
Cmpd
HETa
R2b
R4
Tc IC50
(
lM)
L6 IC50d
(lM)
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
3-Pyr
3-Pyr
3-Pyr
3-Pyr
5-Pyrim
3-Pyr
5-Pyrim
3-Pyr
3-Pyr
3-Pyr
3-Pyr
3-Pyr
3-Pyr
5-Pyrim
5-Pyrim
B
A
A
A
B
A
D
D
A
B
C
A
E
Ph
Ph
0.008
0.009
0.010
0.012
0.018
0.024
0.025
0.028
0.028
0.035
0.077
0.110
0.365
0.769
0.880
42
43
56
80
cHex
Ph(4-iPr)
Ph(4-iPr)
Ph(4-Cl)
Me
Me
iPr
Me
Me
Me
Me
Me
Me
>100
23
>100
77
>100
>100
>100
>100
>100
>100
>100
to the lM activity level of benznidazole.
Considering each different R1 in turn, for R1 equal to amide (Ta-
ble 1) bulky alkyl amido analogues 13–18 were more potent than
benzamides 20 and 21. Thiazole 19 showed good activity. Pyrimid-
inyl analogues were less potent than the corresponding pyridyl
compounds (e.g., 15 cf 23) and there was a slight preference for
2-F containing R2 analogues (B and D) and a definite mismatch
with R2 = 4-benzonitrile (E).
C
A
a
HET: 3-pyr (3-pyridine), 5-pyrim (5-pyrimidine).
b
A (4-chlorophenyl), B (4-chloro-2-fluorophenyl), C (4-trifluoromethylphenyl), D
(2-fluoro-4-trifluoromethylphenyl), E (4-benzonitrile), F (4-ipropoxyphenyl).
c
Tc = T. cruzi, values are the mean of at least two experiments.
L6 cells (rat myoblasts from skeletal muscle) for assessment of cytotoxicity.
d
In contrast, the sulfonamide series (Table 2) showed a prefer-
ence for aryl sulfonamides over alkyl analogues, and simple ben-
zenesulfonamides 28 and 29 were very active. Additional Cl or
iPr substituents did not enhance activity. Methyl sulfonamides
34, 35 and 37–42 provided an interesting sub-set of analogues
with surprisingly active and equipotent pyridyl and pyrimidinyl
lead compounds, and a clear preference for 2-F containing R2 ana-
logues B and D (34 cf. 41 and 35 cf. 38). Similar to the sulfonamide

M. Keenan et al. / Bioorg. Med. Chem. 21 (2013) 1756–1763
1759
Table 3
R
Table 5
1 = alkyl and aryl carbonates/carbamates (COXR5 X=O/NH)
Representative in vitro metabolism, solubility and CYP selectivity data
c
d
a
a
Cmpd
HETa
R2b
R5
Tc IC50
(l
M)
L6 IC50
(
lM)
Cmpd
EH
<0.2
0.59
Sol.b
CYPc
Cmpd
EH
Sol.b
CYPc
43
44
45
46
47
48
49
50
51
52
53
54
55
56
3-Pyr
5-Pyr
5-Pyrim
3-Pyr
3-Pyr
5-Pyrim
3-Pyr
3-Pyr
5-Pyrim
5-Pyrim
5-Pyrim
3-Pyr
5-Pyrim
3-Pyr
B
B
B
A
A
B
C
B
A
B
B
A
C
E
OPh
NHPh
NHPh(4-iPr)
OPh
NHPh
NHPh(4-Cl)
OtBu
OtBu
NHPh(4-Cl)
OtBu
NHtBu
NMe2
0.005
0.006
0.007
0.008
0.009
0.010
0.017
0.020
0.022
0.024
0.054
0.120
>1
59
45
62
56
67
34
56
43
59
64
>100
>100
>100
>100
24
15
22
19
14
17
13
16
39
37
>100
50–100
25–50
50–100
12.5–25
12.5–25
12.5–25
6.3–12.5
12.5–25
12.5–25
7.4
1.8
7
2.8
3.9
2.3
1
3.9
3.6
1.3
28
35
36
29
34
48
53
50
52
66
0.71
0.74
0.76
0.79
0.84
0.56
0.69
0.75
0.76
0.9
<1.6
0.6
1
25–50
12.5–25
<1.6
25–50
3.1–6.3
50–100
3.1–6.3
12.5–25
3.1–6.3
0.63
0.65
0.66
0.68
0.73
0.89
0.53
0.65
0.81
1.9
5.6
0.3
6.9
1.9
2.9
25
a
Microsome predicted hepatic extraction ratio (EH) calculated from in vitro data
in human liver microsomes.
NHcPr
NHcPr
b
kinetic solubility at pH 6.5 in phosphate buffer (lg/mL) determined by
>1
nephelometry.
a
c
HET: 3-pyr (3-pyridine), 5-pyrim (5-pyrimidine).
IC50 (lM) for the inhibition of testosterone-b-hydroxylation by CYP3A4.
b
A (4-chlorophenyl), B (4-chloro-2-fluorophenyl), C (4-trifluoromethylphenyl), D
(2-fluoro-4-trifluoromethylphenyl), E (4-benzonitrile), F (4-ipropoxyphenyl).
c
Tc = T. cruzi, values are the mean of at least two experiments.
L6 cells (rat myoblasts from skeletal muscle) for assessment of cytotoxicity.
d
potential drug-drug interactions for combination therapy).28 Re-
sults for selected compounds are shown in Table 5.
Table 4
As is often the case, the most active compounds do not neces-
sarily possess the best ‘drug-like’ properties. From the amide ser-
ies, methyl analogue 24 (T. cruzi IC50 240 nM) was very stable to
oxidation by microsomes, had excellent solubility and only moder-
ate activity at CYP3A4. Higher alkyl and more active analogues 15,
19, 14 and 17 (T. cruzi IC50 <40 nM) had acceptable profiles. The
very active benzenesulfonyl analogues 28 and 29 (T. cruzi
IC50<10 nM) had disappointing profiles with Eh >0.7 suggesting
they would have poor exposure profiles in vivo. Carbamates/car-
bonates 48, 53, 50 and 52 had mixed profiles with no clear stand-
out compounds, whilst compounds in the aryl series were highly
metabolised and poorly soluble (data not shown). Pyrazinyl ana-
logue 66 had minimal cross-reactivity with CYP3A4, but was 10-
fold less active than leading pyridyl compounds and thus of no real
interest. Not enough comparative data was collected to determine
the influence of R2 on in vitro ADME and physicochemical proper-
ties, but previous studies with the triaryl series showed that the
rate of oxidative metabolism could be reduced by the inclusion
of 4-trifluoromethylphenyl.25
R1 = aryl (Ar)
HETa
R2b
Ar
Tc IC50
(lM)
L6 IC50 (lM)
c
d
Cmpd
57
58
59
60
61
62
63
64
65
66
3-Pyr
3-Pyr
3-Pyr
3-Pyr
3-Pyr
3-Pyr
5-Pyr
5-Pyrim
5-Pyrim
2-Pyra
D
B
A
B
F
A
E
B
B
C
Hete
Ph
0.017
0.018
0.020
0.026
0.028
0.034
0.050
0.065
0.078
0.240
51
58
61
59
77
67
>100
60
63
62
Ph(2-OMe)
2-pyrimidine
Ph(2-OMe)
Ph
2-thiazole
Ph(2-OMe)
Ph
Ph(2-OMe)
a
HET: 3-pyr (3-pyridine), 5-pyrim (5-pyrimidine), 2-pyra (2-pyrazine).
A (4-chlorophenyl), B (4-chloro-2-fluorophenyl), C (4-trifluoromethylphenyl), D
b
(2-fluoro-4-trifluoromethylphenyl), E (4-benzonitrile), F (4-ipropoxyphenyl).
c
Tc = T. cruzi, values are the mean of at least two experiments.
L6 cells (rat myoblasts from skeletal muscle) for assessment of cytotoxicity.
57 Het = 5-(3-methyl-1,2,4-oxadiazole).
d
e
set, aromatic carbamates and carbonates (Table 3) were potent and
more active than even bulky, lipophilic t-butyl analogues and pyr-
idyl and pyrimidinyl analogues were equipotent. No activity
advantage of carbamate-NH vs carbonate-O or for 2-F containing
R2 analogues was evident within the most potent aryl derivatives
43–48.
3.3. Evaluation of oral exposure
The systemic exposures of 24 and 37 (citrate salt) were deter-
mined in non-infected male Swiss outbred mice after single oral
doses of 20, 50 and 100 mg/kg administered by gavage. Both com-
pounds exhibited rapid absorption after oral administration, with
maximum concentrations attained approximately 15 min post-
dose. No adverse reactions or compound-related side effects were
observed following oral administration of either compound at the
dose levels examined. The apparent half-lives of 37 (T1/2 = 2.1–
2.9 h) and 24 (T1/2 = 1.7–2.7 h) were similar, however the AUC for
37 over the 7.5 h post-dose sampling period was higher than that
The R1 equal to aryl (Table 4) series shared an activity level sim-
ilar to the amide series. The set was very limited in scope, but quite
electronically diverse analogues such as 57, 58 and 60 were essen-
tially equiactive suggestive of a flat SAR and thus non-optimal
binding interactions. This can be understood by considering that
the most active sulphonamide/carbonate/carbamate series have
an aromatic ring extended into chemical space whereas the di-
rectly linked N-aryl analogues are shorter and linear and probably
do not reach into the same binding pocket.
for 24 at each comparable dose (37: AUC0–7.5 h
20 mg/kg, 60.2 at 50 mg/kg and 43.7 at 100 mg/kg. 24: AUC0–7.5 h
M h) 2.3 at 20 mg/kg, 6.2 at 50 mg/kg and 9.4 at 100 mg/kg)
(lM h) 7.2 at
(l
(see Supplementary data for oral exposure profiles). Full-scan LC-
MS data were collected for selected plasma samples to identify po-
tential metabolites. Putative products of oxygenation and amide
hydrolysis were detected for 24, and products of oxygenation
and N-dealkylation were observed for 37. Potential metabolites
having molecular weights consistent with the products of glucu-
ronidation, sulphation or oxygenation followed by conjugation or
ring opening were not detected.
3.2. Assessment of in vitro ADME and CYP3A4 inhibition profiles
Alongside the assessment of activity, compounds were screened
in vitro for oxidative stability in human liver microsomes (to assess
the likelihood of rapid hepatic clearance limiting systemic expo-
sure after oral administration), solubility at pH 6.5 in phosphate
buffer and selectivity for T. cruzi over CYP3A4 (a potentially limit-
ing characteristic of the azole series and important for minimising

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M. Keenan et al. / Bioorg. Med. Chem. 21 (2013) 1756–1763
3.4. Evaluation of in vivo efficacy
A
Compounds 24 and 37 were evaluated for in vivo efficacy in a
mouse model of acute T. cruzi infection following doses of 20, 50
and 100 mg/kg. Female Swiss mice (n = 5 per group) were infected
with 25,000 blood form trypomastigotes of T. cruzi (Tulahuen
strain/Tc VI). Compound dosing commenced on day 8 post infec-
tion (pi) and each mouse received the indicated dosage of com-
pound in a single, once daily administration by oral gavage for 5
consecutive days. Blood parasitemia was evaluated on days 8, 9,
12 and 14 pi and expressed as ‘number of trypomastigotes/mL of
blood’. Compound efficacy was indicated by a decrease in parasite-
mia levels in comparison to vehicle-treated mice on day 14 pi.
B
Fig. 4. Survival following compound treatment. Animals were administered (A) 24
or (B) 37 once daily for 5 days at different concentrations. Percent survival relative
to treatment with posaconazole (100 mg/kg) or vehicle only is shown.
Posaconazole (NoxafilÒ) treated mice (50 and 100 mg/kg) were
used as a positive control alongside the vehicle only treated group.
Compared to vehicle-treated mice, 24 was unable to signifi-
cantly reduce blood parasitemia at all doses tested (Fig. 3A) but
was able to improve survival. All 24-treated mice survived to the
end of the treatment period (day 14) whereas 60% of vehicle-trea-
ted mice did not survive past day 12 (Fig. 4A). In contrast, 37-trea-
ted mice had significantly improved reduction in parasitemia when
dosed at 100, 50 and 20 mg/kg (96%, 95% and 73%) compared to
vehicle-treated on day 14, respectively) (Fig. 3B). All 37-treated
mice had improved survival outcomes relative to vehicle (4:5 mice
at 20 mg/kg; 5:5 at 50 mg/kg and 4:5 at 100 mg/kg survived to the
end of the experiment) (Fig. 4B). This was comparable to the posi-
tive control, posaconazole, which reduced blood parasitemia to be-
low detectable limits by day 14 pi in both 50 mg/kg and 100 mg/kg
treated groups (Fig. 3C), with 3:5 mice (50 mg/kg) and 4:5 mice
(100 mg/kg) surviving to the end of the experiment (Fig. 4).
4. Conclusions
Simple 1-[phenyl(pyridin-3-yl)methyl]piperazinyl analogues of
fenarimol were investigated for their ability to inhibit T. cruzi in vi-
tro in a whole parasite assay and further studied for in vivo efficacy
in a mouse model of acute T. cruzi infection. A range of analogues
bearing amide, sulfonamide, carbamate/carbonate and aryl substi-
tuents appended to [phenyl(pyridin-3-yl)methyl]piperazine scaf-
folds 3 exhibited low nM activities in vitro. Two compounds, 24
and 37, gave good plasma exposures in mice following oral admin-
Figure 3. Efficacy of compounds on blood parasitemia in vivo. Animals were
administered (A) 24, (B) 37 or (C) posaconazole once daily for 5 days at different
concentrations. Parasitemia per ml blood was evaluated relative to vehicle-treated
animals. indicates animal death due to parasite burden/morbidity. Error bars
represent SEM.

M. Keenan et al. / Bioorg. Med. Chem. 21 (2013) 1756–1763
1761
istration and were tested for efficacy in vivo. Compound 37 was
able to significantly reduce parasitemia in a mouse model of acute
T. cruzi infection after once daily oral dosing at 20, 50 and 100 mg/
kg for 5 days, consistent with a higher potency and overall superior
plasma concentration profile compared to 24 which was not effica-
cious in vivo. The tractable synthesis of the target compounds, oral
exposure profiles and demonstrated in vivo efficacy of 37 makes
the 1-[phenyl(pyridin-3-yl)methyl]piperazinyl series an attractive
target for further optimisation.
1H), 8.46 (dd, J = 4.8, 1.4 Hz, 1H), 7.60–7.76 (m, 1H), 7.21–7.36
(m, 5H), 5.85 (d, J = 2.3 Hz, 1H), 3.27 (br s, 1H).
5.2.2. Preparation of chloro diarylmethyl intermediates (7)
from diarylmethanol intermediates (6)
5.2.2.1. 3-[Chloro(4-chlorophenyl)methyl]pyridine (10).
A
mixture of 9 (2.5 g, 11 mmol), thionyl chloride (5.2 g, 44 mmol)
and toluene or dichloromethane (10 mL) was stirred at room tem-
perature for 2 h with a drying tube or heated under reflux over-
night. On cooling, the mixture was concentrated and azeotroped
with toluene (2 Â 10 mL) to afford 10 as a white solid (2.5 g,
94%). 1H NMR (300 MHz, CHLOROFORM-d) d 8.63 (d, J = 2.0 Hz,
1H), 8.57 (dd, J = 4.7, 1.3 Hz, 1H), 7.69–7.76 (m, 1H), 7.24–7.39
(m, 5H), 6.11 (s, 1H).
5. Experimental
5.1. General
Reagents were purchased from commercial suppliers and used
without further purification. Commercially available anhydrous
solvents were used and stored under nitrogen, unless indicated
otherwise. Reactions involving moisture sensitive reagents were
conducted under an atmosphere of dry nitrogen in glassware dried
with a heat-gun. Thin layer chromatography (TLC) using silica gel
Merck 60 F254 plates and detection with UV light was used to
monitor reactions. 1H NMR spectra were recorded in CDCl3, CD3OD
or DMSO-d6 solutions on a Varian 200, Brucker 300 or Varian
400 MHz machine. Chemical shifts are reported in parts per million
(d) downfield of tetramethylsilane (TMS). GC–MS were acquired on
an Agilent 5973 network machine. LC-MS were acquired on an Ap-
plied Biosystems/MDS Sciex API-2000 system. Flash chromatogra-
5.2.3. Preparation of diarylmethylpiperazine intermediates (4
and 5) from chloro diarylmethyl intermediates (7)
5.2.3.1.
(12).
1-[(4-Chlorophenyl)(pyridin-3-yl)methyl]piperazine
To a dry 100 mL round bottomed flask fitted with a
condenser and a stirrer bar was added 10 (5 g, 21 mmol). This
was dissolved in anhydrous acetonitrile (40 mL) and t-butyl
piperazine-1-carboxylate (5.9 g, 31.5 mmol) was added followed
by triethylamine (anhydrous, 5.9 mL, 42 mmol) and a catalytic
amount of potassium iodide (dried by heat-gun under vacuum).
The reaction was heated at 80 °C for 48 h. The reaction mixture
was cooled, concentrated and the residue partitioned between
dichloromethane (100 mL) and
a saturated aqueous sodium
phy was carried out with silica gel (0.04–0.06
lm, 230–400 mesh),
carbonate solution (100 mL). The aqueous phase was re-extracted
with dichloromethane (2 Â 50 mL), the organic phases combined,
dried (sodium sulfate), filtered and concentrated to give a crude or-
ange oil. Purification by Flashmaster II chromatography (eluent
ethylacetate in dichloromethane 0–30%) gave BOC-intermediate
reverse phase silica gel (C18 35-70 m) or on a Flashmaster II sys-
l
tem using cartridges prepared in-house. Microwave irradiations
were conducted using a Biotage initiator. All compounds tested
in the in vitro and in vivo biological screens were purified to
>95% purity as determined by analysis of HPLC on an Agilent
1100 series machine, fitted with a C8 reverse phase Agilent zorbax
11 as
a
semi-pure solid (16.1 g, 80%). 1H NMR (400 MHz,
CHLOROFORM-d) d 8.62 (br s, 1H), 8.47 (d, J = 3.9 Hz, 1H), 7.68
(d, J = 7.8 Hz, 1H), 7.17–7.38 (m, 5H), 4.28 (s, 1H), 3.36–3.48 (m,
4H), 2.25–2.39 (m, 4H), 1.43 (s, 9H). 11 (3.4 g, 8.8 mmol) was dis-
solved in dichloromethane (40 mL) and methanol (10 drops) was
added. The mixture was cooled to 0 °C and trifluoroacetic acid
(6.5 mL, 87.7 mmol) was added drop wise. The mixture was stirred
for 1 h at 0 °C, allowed to come to room temperature overnight,
concentrated and azeotroped with dichloromethane to give 12
(3.7 g) as the crude trifluoroacetic acid salt which was used with-
out further purification. 1H NMR (400 MHz, DMSO-d6) d 8.87 (br
s, 1H), 8.64–8.77 (m, 2H), 8.32 (d, J = 7.8 Hz, 1H), 7.81 (dd, J = 7.6,
5.7 Hz, 1H), 7.39–7.48 (m, 4H), 4.86 (s, 1H), 3.13 (br. s., 4H), 4
protons obscured by DMSO peak.
eclipse DB-LB 4.6 x 150 mm 5 lm column (flow rate 1.2 mL/min;
method 1: 20% acetonitrile in water, increasing to 60% acetonitrile
over 10 min; method 2: 50% acetonitrile in water increasing to 90%
acetonitrile over 10 min; method 3: 70% acetonitrile in water in-
creasing to 95% acetonitrile over 10 min); 1H NMR spectra (ac-
counting for non-compound peaks and residual solvent) and in
some cases LC-MS or GC–MS. Fenarimol [60168-88-9] (1) was pur-
chased from commercial sources and posaconazole was a gift from
the CDCO.
5.2. Synthesis of compounds
The preparation of [4-chlorophenyl(pyridin-3-yl)methyl]piper-
azine analogues 24, 29, 46, 47 and 62 is provided as a representa-
tive example of the synthetic methodology. The synthesis of
compound 37 is also described. Methods and data for other re-
ported compounds is available in the Supplementary data.
5.2.4. Preparation of diarylmethylpiperazine analogue libraries
(3) from diarylmethylpiperazine intermediates (5) method A
5.2.4.1. Amides from acid chlorides. 1-{4-[(4-Chlorophenyl)
(pyridin-3-yl)methyl]piperazin-1-yl}ethan-1-one
(24).
Compound 12 (3.7 g) was dissolved in anhydrous di-
chloromethane (30 mL) and the solution cooled to 0 °C. Triethyla-
mine (5 mL, 36 mmol) was added drop wise with stirring. A
further 5 mL of triethylamine was added, followed by drop wise
addition of acetyl chloride (1.0 mL, 14.4 mmol) and the mixture al-
lowed to stir for 45 min, poured onto aqueous sodium carbonate
solution (20 mL) and extracted with chloroform (3 Â 40 mL). The
organic phases were combined, dried (sodium sulfate), filtered
and concentrated to give a dark orange oil which was purified by
Flashmaster II chromatography (eluent chloroform in methanol)
to give 24 (485 mg, 17%) as a hygroscopic colourless solid. 1H
NMR (300 MHz, CHLOROFORM-d) d 8.64 (br s, 1H), 8.49 (d,
J = 2.4 Hz, 1H), 7.71 (d, J = 7.9 Hz, 1H), 7.19–7.40 (m, 5H), 4.30 (s,
5.2.1. Preparation of diarylmethanol intermediates (6) from
benzophenones25
5.2.1.1. (4-Chlorophenyl)(pyridin-3-yl)methanol (9).
To a
solution of 3-(4-chlorobenzoyl)pyridine (3 g, 14 mmol) in metha-
nol (20 mL) was slowly added sodium borohydride (0.72 g,
20 mmol). The mixture was stirred at room temperature under ni-
trogen for 48 h, then concentrated, diluted with water (40 mL) and
extracted with chloroform (40 mL). The organic layer was dried, fil-
tered and the filtrate concentrated to afford 9 as a white solid (3 g,
97%). 1H NMR (300 MHz, CHLOROFORM-d) d 8.54 (d, J = 2.0 Hz,

1762
M. Keenan et al. / Bioorg. Med. Chem. 21 (2013) 1756–1763
1H), 3.62 (apparent triplet, J = 4.9 Hz, 2H), 3.47 (apparent triplet,
J = 4.9 Hz, 2H), 2.28–2.48 (m, 4H), 2.06 (s, 3H); HPLC 96.1% at
230 nm; GCMS m/z = 329.
clear oil. 1H NMR (300 MHz, CHLOROFORM-d) d 8.61–8.75 (m, 1H),
8.42–8.56 (m, 1H), 7.68–7.85 (m, 1H), 7.20–7.42 (m, 7H), 6.76–7.00
(m, 3H), 4.34 (s, 1H), 3.17–3.28 (m, 4H), 2.47–2.67 (m, 4H); HPLC
>99% at 230 nm; GCMS m/z = 363.
5.2.4.2. Sulfonamides from sulfonyl chlorides. 1-(Benzenesulfo-
nyl)-4-[(4-chlorophenyl)(pyridin-3-yl)methyl]piperazine
5.2.5. Preparation of 1-[(4-chloro-2-fluorophenyl)(pyridin-3-
yl)methyl]-4-methanesulfonylpiperazine (37). (4-Chloro-2-
fluorophenyl)(pyridin-3-yl)methanol (67)
(29).
Compound 12 (250 mg, 0.24 mmol) was dissolved in di-
chloromethane (2.5 mL) under an atmosphere of nitrogen and ben-
zenesulfonyl chloride (0.46 mL, 2 equiv) was added and the
mixture cooled to 0 °C. Triethylamine (0.6 mL) was added and
the mixture stirred for 2 h then allowed to warm to room tempera-
ture, poured onto a saturated aqueous sodium carbonate solution
(20 mL) and extracted with dichloromethane (3 Â 15 mL). The or-
ganic phases were combined, dried (magnesium sulfate), filtered,
concentrated and purified by Flashmaster II chromatography (elu-
ent ethylacetate in dichloromethane) to give 29 as an off-white
powder (86 mg, 83%). 1H NMR (300 MHz, CHLOROFORM-d) d
8.57 (br s, 1H), 8.45 (br s, 1H), 7.74–7.78 (m, 2H), 7.54–7.68 (m,
4H), 7.24–7.26 (m, 4H) 7.16–7.22 (m, 1H), 4.28 (s, 1H) 3.02–3.05
(m, 4H), 2.44–2.48 (m, 4H); HPLC >99% at 230 nm; LCMS
[M+H]+ = 428.2.
To a solution of (4-chloro-2-fluorochlorophenyl)(pyridin-3-yl)-
methanone (15.6 g, 66.2 mmol) in methanol (400 mL) cooled to
0 °C was slowly added sodium borohydride (4.5 g, 119 mmol) por-
tion wise. The mixture was stirred at room temperature under ni-
trogen for 24 h, then diluted with water (400 mL) and extracted
into dichloromethane (4 Â 300 mL). The organic layer was dried,
filtered and concentrated. Purification by silica plug (eluent di-
chloromethane then ethyl acetate) gave 67 as a white powder
(11.3 g, 72%). 1H NMR (300 MHz, CHLOROFORM-d) d 8.60 (d,
J = 2.1 Hz, 1H), 8.48 (dd, J = 4.8, 1.8 Hz, 1H), 7.71–7.67 (m, 1H),
7.50 (t, J = 8.1 Hz, 1H), 7.29–7.25 (m, 1H), 7.20–7.17 (m, 1H), 7.07
(dd, J = 9.9, 1.8 Hz, 1H), 6.13 (d, J = 3.4 Hz, 1H), 3.05 (d, J = 3.4 Hz,
1H); HPLC >99% @230 nm.
5.2.4.3. Carbonates from chloroformates. Phenyl 4-[(4-chloro-
phenyl)(pyridin-3-yl)methyl]piperazine-1-carboxylate
5.2.5.1.
(68).
3-[Chloro(4-chloro-2-fluorophenyl)methyl]pyridine
A solution of 67 (11.3 g, 47.5 mmol) in dichloromethane
(46).
A solution of 12 (0.3 g) in dichloromethane (3 mL) was
(140 mL) was cooled to 0 °C and thionyl chloride (5.86 mL,
80.8 mmol) added. The reaction mixture was allowed to warm to
room temperature over 2 h. The mixture was concentrated, re-ex-
tracted into dichloromethane and washed with an aqueous solu-
tion of sodium carbonate. The aqueous layer was treated with
salt and re-extracted with dichloromethane (3 Â 100 mL). The or-
ganic layers were combined, dried (magnesium sulfate), filtered
and concentrated to give 68 as a crude light brown oil (11.2 g,
93%). 1H NMR (300 MHz, CHLOROFORM-d) d 8.65 (s, 1H), 8.56 (d,
J = 3.6, 1H), 7.78–7.74 (m, 1H), 7.49 (t, J = 8.4 Hz, 1H), 7.32 (dd,
J = 8.1, 4.8 Hz, 1H), 7.22–7.18 (m, 1H), 7.12 (dd, J = 8.1, 1.8 Hz,
1H), 6.37 (s, 1H).
cooled to 0 °C and triethylamine (1.5 mL) was added, followed by
phenyl chloroformate (0.07 mL, 0.58 mmol). The mixture was al-
lowed to warm to room temperature overnight, then 10 mL of a sa-
turated solution of ammonium chloride and 10 mL of
dichloromethane were added. The organic phase was separated
and the aqueous phase re-extracted with dichloromethane
(2 Â 10 mL). The organic phases were combined, dried (magne-
sium sulfate) filtered and concentrated. The crude material was
purified by Flashmaster II chromatography (eluent ethyl acetate
in hexanes) to give 46 (66 mg, 56%) as an off-white solid. 1H
NMR (400 MHz, CHLOROFORM-d) d 8.66 (br s., 1H), 8.50 (br s.,
1H), 7.71 (d, J = 7.8 Hz, 1H), 7.23–7.40 (m, 7H), 7.20 (d, J = 7.0 Hz,
1H), 7.09 (d, J = 8.2 Hz, 2H), 4.34 (s, 1H), 3.50–3.79 (m, 4H), 2.34–
2.55 (m, 4H); HPLC 96.9% at 230 nm; LCMS [M+H]+ = 408.1.
5.2.5.2.
1-[(4-Chloro-2-fluorophenyl)(pyridin-3-yl)methyl]pi-
Compound 68 (11.2 g, 43.7 mmol) was dis-
perazine (69).
solved in anhydrous acetonitrile (100 mL) and t-butyl piperazine-
1-carboxylate (24.4 g, 0.13 mmol) was added followed by triethy-
lamine (anhydrous, 12.2 mL, 87.4 mmol) and a catalytic amount
of potassium iodide (dried by heat gun under vacuum). The reac-
tion was heated at 80 °C for 48 h (an additional 12 mL triethyla-
mine and 5 g of t-butyl piperazine-1-carboxylate were added
after 24 h). The reaction mixture was cooled, concentrated and
the residue partitioned between dichloromethane (100 mL) and a
saturated solution of sodium carbonate (100 mL). The aqueous
phase was re-extracted with dichloromethane (3x100 mL), the or-
ganic phases combined, dried (sodium sulfate), filtered and con-
centrated to give a crude off-white solid (16.3 g, 92%). The crude
material (4 g, 9.85 mmol) was dissolved in dichloromethane
(40 mL) and methanol (1 mL) was added. The mixture was cooled
to 0 °C and trifluoroacetic acid (10 mL, 135 mmol) was added drop
wise. The mixture was stirred for 4 days, concentrated and azeo-
troped with dichloromethane to give 69 (8.5 g) as the crude tri-
fluoroacetic acid salt as an orange oil which was used without
further purification. 1H NMR (200 MHz, CHLOROFORM-d) (Free
base) d 8.64 (s, 1H), 8.42–8.51 (m, 1H), 7.69 (br d, J = 7.7 Hz, 1H),
7.52 (t, J = 8.1 Hz, 1H), 7.17–7.29 (m, 1H), 7.09–7.17 (m, 1H), 7.03
(dd, J = 1.8, 9.9 Hz, 1H), 4.68 (s, 1H), 2.84–3.03 (m, 4H), 2.32–2.52
(m, 4H).
5.2.4.4. Carbamates from isocyanates. 4-[(4-Chlorophenyl)(pyr-
idin-3-yl)methyl]-N-phenylpiperazine-1-carboxamide
(47).
Compound 12 (0.3 g, 0.29 mmol) was dissolved in di-
chloromethane (3.0 mL) and the solution was cooled to 0 °C.
Triethylamine (2.0 mL) was added, followed by phenyl isocyanate
(52 mg, 0.44 mmol) and the mixture stirred for 30 min, poured into
a saturated solution of sodium chloride (20 mL) and extracted with
dichloromethane (2 Â 10 mL). The organic phases were combined,
dried (magnesium sulfate) filtered and concentrated. The crude
material was purified by Flashmaster II chromatography (eluent
ethyl acetate in hexanes) to give 47 (83 mg, 70%) as a white solid.
1H NMR (400 MHz, DMSO-d6) d 8.63 (s, 1H), 8.40–8.52 (m, 2H),
7.81 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 8.6 Hz, 2H), 7.39–7.44 (m, 3H),
7.36 (dd, J = 7.6, 4.9 Hz, 1H), 7.21 (t, J = 7.8 Hz, 2H), 6.92 (t,
J = 7.2 Hz, 1H), 4.54 (s, 1H), 3.42–3.52 (m, 4H), 2.23–2.38 (m,
4H); HPLC >99% at 230 nm; LCMS [M+H]+ = 407.3.
5.2.4.5.
Aryl
derivatives.
1-[(4-Chlorophenyl)(pyridin-3-
mixture of 10
yl)methyl]-4-phenylpiperazine (62).
A
(0.20 g, 0.7 mmol), N,N-dimethylformamide (2 mL), potassium car-
bonate (0.5 g, 2.1 mmol) and 1-phenylpiperazine (0.57 g,
3.5 mmol) was heated at 150 °C for 30 min in a microwave. The
mixture was diluted with water (40 mL) and extracted into ethyl
acetate (40 mL). The organic layer was separated, washed with
water (40 mL), dried, and concentrated. The crude residue was pur-
ified by Flashmaster II chromatography to give 62 (60 mg, 24%) as a
5.2.5.3.
methanesulfonylpiperazine (37).
mmol) was dissolved in a solution of dichloromethane (40 mL)
1-[(4-Chloro-2-fluorophenyl)(pyridin-3-yl)methyl]-4-
Compound 69 (5 g, 5.8

M. Keenan et al. / Bioorg. Med. Chem. 21 (2013) 1756–1763
1763
containing triethylamine (5 mL) at 0 °C. Methanesulfonyl chloride
(0.9 mL, 11.6 mmol) was added drop wise and the mixture stirred
for 2 h then allowed to warm to room temperature, poured onto a
saturated solution of ammonium chloride (20 mL) and extracted
with dichloromethane (3 Â 30 mL). The organic phases were
combined, dried (magnesium sulfate), filtered, concentrated and
purified by preparative HPLC (eluent 30% acetonitile in water) to
give 37 as an off-white solid (690 mg, 31%). 1H NMR (300 MHz,
CHLOROFORM-d) d 8.64–8.68 (m, 1H), 8.48–8.54 (m, 1H), 7.63–
7.72 (m, 1H), 7.47 (t, J = 8.0 Hz, 1H),7.21–7.25 (m, 1H), 7.11–7.18
(m, 1H), 7.02–7.10 (m, 1H), 4.76 (s, 1H), 3.27 (apparent triplet,
J = 4.8 Hz, 4H), 2.81 (s, 3H), 2.44–2.62 (m, 4H); HPLC 97.4% at
230 nm; LCMS [M+H]+ = 384.1.
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.2013.01.050.
References and notes
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General methods for the in vitro T. cruzi assay for determina-
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Acknowledgments
DNDi is grateful to its donors, public and private, who have
provided funding to DNDi since its inception in 2003. With the
support of these donors, DNDi is well on its way to achieving
the objectives of building a robust pipeline and delivering 6–8
new treatments by 2014. A full list of DNDi’s donors can be found
at http://www.dndi.org/index.php/donors.htmL?ids=8. For the
work described in this paper, DNDi received financial support
from the following donors: Department for International Develop-
ment (DFID)/UNITED KINGDOM, GiZ on behalf of the Government
of the Federal Republic of Germany/GERMANY, Ministry of For-
eign and European Affairs (MAEE)/FRANCE, Spanish Agency for In-
ternational Development Cooperation (AECID)/SPAIN, Swiss
Agency for Development and Cooperation (SDC)/SWITZERLAND,
Medecins Sans Frontieres (Doctors without Borders)/INTERNA-
TIONAL and a Swiss foundation. The donors had no role in study
design, data collection and analysis, decision to publish, or pre-
paration of the manuscript.
The authors thank Harriet Newson and Sarah Keatley for help in
preparing the manuscript and Jason Chaplin and Michael Abbott
for additional chemistry support.
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Kerfoot, M.; Khong, A.; Nguyen, T.; McManus, J. D.; Morizzi, J.; Ryan, E.;
Scandale, I.; Thompson, R. A.; Wang, S. Z.; White, K. L. J. Med. Chem. 2012, 55,
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paration of intermediates to final compounds, spectroscopic/analy-
tical data and oral exposure profiles for 24 and 37 is available)
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