Structurally simple inhibitors of lanosterol 14α-demethylase are efficacious in a rodent model of acute Chagas disease

Article abstract of DOI:10.1021/jm900030h

We report structure-activity studies of a large number of dialkyl imidazoles as inhibitors of Trypanosoma cruzi lanosterol-14α-demethylase (L14DM). The compounds have a simple structure compared to posaconazole, another L14DM inhibitor that is an anti-Chagas drug candidate. Several compounds display potency for killing T. cruzi amastigotes in vitro with values of EC ₅₀ in the 0.4-10 nM range. Two compounds were selected for efficacy studies in a mouse model of acute Chagas disease. At oral doses of 20-50 mg/kg given after establishment of parasite infection, the compounds reduced parasitemia in the blood to undetectable levels, and analysis of remaining parasites by PCR revealed a lack of parasites in the majority of animals. These dialkyl imidazoles are substantially less expensive to produce than posaconazole and are appropriate for further development toward an anti-Chagas disease clinical candidate.

Full text of DOI:10.1021/jm900030h

J. Med. Chem. 2009, 52, 3703–3715
3703
Structurally Simple Inhibitors of Lanosterol 14r-Demethylase Are Efficacious In a Rodent
Model of Acute Chagas Disease
Praveen Kumar Suryadevara,†,‡ Srinivas Olepu,†,‡ Jeffrey W. Lockman,†, Junko Ohkanda, Mandana Karimi,|
Christophe L. M. J. Verlinde,§ James M. Kraus,‡ Jan Schoepe,§,| Wesley C. Van Voorhis,| Andrew D. Hamilton,*,
Frederick S. Buckner,*,| and Michael H. Gelb*,‡,§
Departments of Chemistry, Biochemistry, and Medicine, UniVersity of Washington, Seattle, Washington 98195, Department of Chemistry,
Yale UniVersity, New HaVen, Connecticut 06520
ReceiVed January 12, 2009
We report structure-activity studies of a large number of dialkyl imidazoles as inhibitors of Trypanosoma
cruzi lanosterol-14R-demethylase (L14DM). The compounds have a simple structure compared to
posaconazole, another L14DM inhibitor that is an anti-Chagas drug candidate. Several compounds display
potency for killing T. cruzi amastigotes in vitro with values of EC50 in the 0.4-10 nM range. Two compounds
were selected for efficacy studies in a mouse model of acute Chagas disease. At oral doses of 20-50 mg/kg
given after establishment of parasite infection, the compounds reduced parasitemia in the blood to undetectable
levels, and analysis of remaining parasites by PCR revealed a lack of parasites in the majority of animals.
These dialkyl imidazoles are substantially less expensive to produce than posaconazole and are appropriate
for further development toward an anti-Chagas disease clinical candidate.
Introduction
cholesterol salvaged from its host, enzymes of the parasite sterol
biosynthetic pathway offer potential targets for the development
of drugs. Urbina and others have shown that a number of
inhibitors of fungal L14DM, including the recently developed
antifungal drug posaconazole, are potent inhibitors of T. cruzi
L14DM and are able to cure mice suffering from acute and
chronic Chagas disease.5 The use of posaconazole to treat
Chagas disease is being considered, however its manufacturing
costs may limit widespread use especially in the long term
treatment of the chronic disease.6
Chagas disease causes the third largest parasitic disease
burden in the world and the largest in the Western hemisphere,
currently affecting 16-18 million people throughout Central
and South America.1 The disease is caused by the parasite
Trypanosoma cruzi (T. cruzia), which is able to invade a wide
variety of host cells. The vaccine prospects for preventing
Chagas disease are not promising because the pathogen has
developed complex immune evasion techniques to allow for
persistent infection.2 Drug therapy options for Chagas disease
are limited. The principal drugs are benznidazole and nifurtimox,
which have modest efficacy during the acute phase of the disease
and are not effective for treatment of the chronic, life-threatening
stage. Both of these nitroheterocyclic drugs are poorly tolerated
by adults. In short, more effective and better-tolerated anti-
Chagas disease drugs are greatly needed.3
Sterol biosynthesis is a complex enzymatic pathway, which
produces membrane lipids for many eukaryotic organisms.
Mammals produce cholesterol as their primary sterol, fungi
produce ergosterol, and T. cruzi produces a mixture of ergosterol-
like sterols that contain various alkyl substituents at C24.4 Sterol
synthesis in mammals, yeast, and T. cruzi go through the
common intermediate lanosterol, which is formed in several
steps from acetyl-CoA. The first of the postlanosterol processing
steps is the removal of the methyl group at C14 by lanosterol
14R-demethylase (cytochrome P450 subfamily 51) (L14DM).
Because the parasite apparently cannot survive solely on
In our studies of inhibitors of protein farnesyltransferase
as antiparasite agents such as 1 (Figure 1), we found that
compounds such as 2, which lack the methionyl group, do
not inhibit parasite protein farnesyltransferase and yet display
potent activity in blocking the growth of T. cruzi amastigote
stage (intracellular) parasites.7 Further studies showed that
compounds in this class led to the accumulation of lanosterol
in amastigotes and to the formation of unusual sterols that
are predicted to result from the blockade of the lanosterol
14R-demethylation step.7 Compounds in this class were also
found to bind to recombinant T. cruzi L14DM, causing a
spectral shift that is consistent with coordination of the
unsubstituted imidazole nitrogen to the heme iron of this
cytochrome P450.7 Because the methyl ester of 2 is rapidly
hydrolyzed by enzymes in serum, we went on to prepare
derivatives including 3 (Figure 1) that displayed desirable
pharmacokinetic properties in mice.7 Administration of a
single dose of 3 to mice at 30 mg/kg by oral gavage led to
a maximal drug concentration in the plasma of 16 µM after
1 h, and 3 was lost with a serum half-life of ∼4 h.
Administration of 3 at 50 mg/kg, twice per day for 14 days,
to T. cruzi-infected mice reduced parasitemia in blood to
∼1% of the level seen in untreated mice.7 These encouraging
results along with the low cost of goods anticipated for this
new class of L14DM inhibitors, which lack stereogenic
centers, prompted us to carry out extensive structure-activity
studies of 3 in an effort to maximize potency against T. cruzi
amastigotes while maintaining respectable pharmacokinetic
properties. The synthesis of analogues of 3 was guided using
* To whom correspondence should be addressed. For medicinal chem-
istry: (M.H.G.) phone, 206-543-7142; fax, 206-685-8665; E-mail, gelb@
chem.washington.edu; or (A.D.H.) phone, 203-432-5570; fax, 203-432-3221;
E-mail, Andrew.Hamilton@yale.edu. For F.S.B.: phone, 206-598-9148; fax,
206-685-8681; E-mail, fbuckner@u.washington.edu.
†
These authors contributed equally to this study.
Department of Chemistry, University of Washington.
Department of Biochemistry, University of Washington.
Department of Medicine, University of Washington.
‡
§
|
Department of Chemistry, Yale University.
a Abbreviations: EC50, concentration of compound that causes 50%
reduction in parasite growth in vitro; L14DM, lanosterol 14R-demethylase;
T. cruzi, Trypanosoma cruzi.
10.1021/jm900030h CCC: $40.75
2009 American Chemical Society
Published on Web 05/22/2009

3704 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
SuryadeVara et al.
Figure 1. Dialkyl imidazole-based L14DM inhibitors.
Scheme 1. Synthesis of the Dialkylimidazoles Containing the Methoxycarbonyl Groupa
a Reagents and conditions: (a) TrtCl, Et3N, DMF, rt, 90-94%; (b) RC6H4CH2Br, CH3CN, 60 °C, 24 h, 70-75%; (c) PhB(OH)2, Pd(OAc)2, K2CO3,
acetone/H2O, 85-89%; (d) KMnO4, pyr/H2O, 100%; (e) SOCl2, MeOH, reflux, 80%; (f) SnCl2 ·2H2O, EtOAc, reflux, 6 h, 86%; (g) AcOH, 5, MeOH, 4 Å
mol sieves, NaCNBH3, overnight, rt, 60-70%.
a model of the structure of T. cruzi L14DM templated on
the X-ray structure of L14DM from Mycobacterium tuber-
culosis.8 The present article discloses the synthesis and
structure-activity relationship studies of a series of dialkyl
substituted imidazole derivatives as potent L14DM inhibitors
for Chagas chemotherapy.
a ketone functional group are shown in Scheme 3. After several
unsuccessful attempts, we found that the Fries rearrangement
worked well to install the ketone group.12,13 The phenolic
hydroxyl group was conveniently activated as a triflate ester to
generate 18a-c, followed by the usual Suzuki cross coupling
to produce 19a-c.
Non acyl-containing functional groups were introduced into
the inhibitor scaffold in place of the methoxycarbonyl group
using a Sandmeyer reaction14-16 (22b,c, Scheme 4a) by heating
the aryl halide with the secondary amine (22d,e) or reductive
amination with formaldehyde (22f). Incorporation of small
heterocycles into the dialkyl imidazole scaffold is shown in
Scheme 4b using the appropriate aryl bromide in the presence
of KOAc and catalytic Pd(PPh3)4 in DMAC.17-19
Results and Discussion
Chemistry. Synthetic procedures are illustrated in Schemes
1-7. Scheme 1 shows the synthesis of dialkyl imidazoles
containing a methoxycarbonyl group and also shows the
synthetic steps used to make most of the compounds reported
in this study, the first of which is alkylation of the N-tritylated
imidazole carboxaldehyde 4 to give the N-alkylated imidazole
5.9,10 The second piece, the substituted aniline 9, contains a
phenyl-phenyl bond, which is made using the Suzuki cross
coupling, followed by standard functional group transforma-
tion.11 The two inhibitor pieces are joined by reductive
amination to give 10a-l.10
Preparation of dialkyl imidazoles containing the benzothiazole
appendage is shown in Scheme 5.The key reaction is treatment
of the aryl bromide with benzothiazole in the presence of
Pd(PPh3)4 and KOAc in DMAC (Heck reaction). The latter
proceeded in poor yield, so we developed a second method
starting with benzoic acid derivative 7, which was converted
to the acid chloride and then condensed with 2-amino-thiophenol
using pyridine in xylene.20 The addition of PTSA to the reaction
mixture considerably improved the overall yield up to 70%.
Scheme 2 shows the preparation of dialkyl imidazoles
containing additional acyl groups (esters and amides). It is based
on standard functional group transformations and the general
methods outlined in Scheme 1. Dialkyl imidazoles containing

Inhibitors of Lanosterol 14R-Demethylase in Chagas Disease
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3705
Scheme 2. Preparation of Dialkylimidazoles Containing Esters and Amidesa
a Reagents and conditions: (a) SOCl2, ROH or ROH, EDCI, HOBt, CH2Cl2, rt, 24 h, 90% or SOCl2, NH2R, Et3N, CH2Cl2, 0 °C, rt, 2-4 h, 85%; (b)
SnCl2 ·2H2O, EtOAc, reflux, 86-90%; (c) AcOH, 5, MeOH, 4 Å mol sieves, NaBH3CN, overnight, rt, 60%.
Scheme 3. Synthesis of Dialkylimidazoles Containing Ketonesa
a Reagents and conditions: (a) (i) (RCO)2O, pyridine, 1.5 h, rt; (ii) AlCl3, 120 °C, overnight, 40%. (b) Tf2O, pyridine, rt, overnight, 96%. (c) PhB(OH)2,
Pd(PPh3)4, K2CO3, DME/EtOH/H2O, 54%. (d) (i) NaOH, i-PrOH/H2O, reflux, overnight, 81%; (ii) AcOH, 5, MeOH, 4 Å mol sieves, NaBH3CN, overnight,
rt, 20-40%.
Scheme 6a shows the synthesis of additional dialkyl imida-
zoles containing different substituents on each of the phenyl
groups using methods derived mainly from Scheme 1. Scheme
6b shows the route to a dialkyl imidazole containing a 3,4-
diphenyl unit, which was installed via Suzuki cross coupling.
Finally, Scheme 7 shows the route for preparing dialkyl
imidazoles with an ortho-amino group on the benzyl unit
attached to the imidazole nitrogen. Several compounds in this
series were prepared as we found that the addition of this amino
group improved antiparasite potency. The route is derived from
Scheme 1. We also developed a large scale synthesis of 44a
and 44d. In this case, all reaction products were purified by
recrystallization except for 41 and 42.
Molecular Modeling and Structure-Activity Relationship
Studies. We made use of the homology model of the T. cruzi
L14DM in complex with tipifarnib based on the M. tuberculosis
enzyme structure earlier described.21 Design and docking studies
were carried out with the FLO/QXP program suite, version
0602.21 In each case, amino acid residues within 11 Å of
tipifarnib were included in the binding site model for Metropolis
Monte Carlo searches and energy minimization procedures.
Details of the procedures were earlier described.21
L14DM. The scaffold of all compounds in this paper consists
of a dialkyl imidazole, where one substituent is benzyl or
biphenyl and the other is biphenylamine. The imidazole nitrogen
binds to the heme iron, and the two substituents occupy mainly
hydrophobic clefts. The anilino fragment of the scaffold is
surrounded by several hydrophobic residues, Tyr 77, Phe 84,
and Ala 265, but the amino group does not interact directly
with the enzyme, thus acting as a spacer.
To study the effect of substitutions on biological activity, we
explored the possibility with various functional groups by varying
the size, polarity, and position on the phenyl ring (Table 1).
Compounds described in this report were generated from previous
compounds shown to have potent activity against T. cruzi cells.
The benzyl substituent on the imidazole fits in a hydrophobic cleft
created by Phe 264, Leu 330, and Val 435 and allows for small
substituents in the ortho and meta positions only on one side of
the aromatic ring and for larger substituents in the para position.
Small meta substituents (10c,d,e) also contact Val 435. Various
hydrophobic para substituents are tolerated (10c,d,e,h) consistent
with the lipophilic character of the cleft’s extension defined by
Tyr 77, Met 80, Ile 183, and Met 434. Not surprisingly, the
p-phenyl substituent (10h) is the most potent, with an EC50 of 0.5
nM because of its size. The t-butyl substitution (10k) leads to poor
activity, as it is too bulky for the narrow cleft. Hydrophilic
substituents in the para position (10b,i) are incompatible with the
To understand the structure-activity relationship of the
various modifications of the dialkyl imidazoles, we docked the
various compounds into the homology model of the T. cruzi

3706 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
SuryadeVara et al.
Scheme 4. (a) Synthesis of Dialkylimidazoles Containing Non-Acyl Functional Groups; (b) Synthesis of Dialkylimidazoles Containing
Heterocyclesa
a Reagents and conditions: (a) (i) Br2, AcOH, rt, 1 h, 76%; (ii) PhB(OH)2, Pd(OAc)2, K2CO3, acetone/H2O, reflux, 86%. (b) NaNO2, H2SO4, AcOH; (i)
CuBr2, HCl, 71%; (ii) CuCl2, HCl, 75%. (c) Zn(CN)2, Pd(PPh3)4, DMF, 100 °C, overnight, 60-65%. (d) Piperidine, 100 °C, 2 h, 90%. (e) Pyrrolidine, 80
°C, 2-3 h, 80-90%. (f) HCHO, H2SO4, NaBH4, THF, 10-30 °C, 0.5 h, 65-70%. (g) SnCl2 ·2H2O, EtOAc, reflux, 6 h, 76-80%. (h) AcOH, 5, MeOH, 4
Å mol sieves, NaBH3CN, overnight, rt, 60-65%. (i) Heterocycle, Pd(PPh3)4, KOAc, DMAC, 160 °C, overnight, 32-35%.
Scheme 5. Synthesis of Dialkylimidazoles Containing a Benzothiazole Groupa
a Reagents and conditions: (a) PhR1B(OH)2, Ba(OH)2 ·8H2O, Pd(PPh3)4, DME-H2O (5:1), reflux, 90%. (b) NaNO2, H2SO4, AcOH, CuBr2, HCl, 71%. (c)
Benzothiazole, Pd(PPh3)4, KOAc, DMAC, 160 °C, overnight, 32-35%. (d) SnCl2 ·2H2O, EtOAc, reflux, 6 h, 76-80%. (e) AcOH, 5, MeOH, 4 Å mol sieves,
NaBH3CN, overnight, rt, 60-65%.

Inhibitors of Lanosterol 14R-Demethylase in Chagas Disease
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3707
a
Scheme 6. (a) Synthesis of Dialkylimidazoles with Various Substitutions on the Two Phenyl Groups; (b) Synthesis of a
Dialkylimidazole Bearing a 3,4-Diphenyl Unitb
a Reagents and conditions: (a) R1B(OH)2, Pd(OAc)2, K2CO3, acetone/H2O, 89%; (b) SnCl2 ·2H2O, EtOAc, reflux, 2 h 90%; (c) AcOH, 5, MeOH, 4 Å mol
sieves, NaBH3CN, rt, overnight, 80%. b Reagents and conditions: (a) Tf2O, pyridine, rt, overnight, 96%; (b) PhB(OH)2, Pd(PPh3)4, K2CO3, DME/EtOH/H2O,
82%; (c) SnCl2 ·2H2O, EtOAc, reflux, 2 h, 90%; (d) AcOH, 5, MeOH, 4 Å mol sieves, NaBH3CN, rt, overnight, 40%.
Scheme 7. Synthesis of Dialkylimidazoles with an ortho-Amino Groupa
a Reagents and conditions: (a) PhB(OH)2, Ba(OH)2 ·8H2O, Pd(PPh3)4, DME/H2O (5:1), reflux, 90%; (b) NBS, CCl4, overnight, reflux, 60%; (c) 4, CH3CN,
60 °C, overnight, 55%; (d) AcOH, 23b, 23c, 26a, 26d, 14a, 4 Å mol sieves, MeOH, NaCNBH3, overnight, 60%; (e) SnCl2 ·2H2O, EtOAc, reflux, 2 h,
80-82%.
lipophilic character of the cleft. Only one ortho substituent appears
to be tolerated (10i), projecting an amino group toward His 268
and making a hydrogen bond with the imidazole. Hydrogen bond
acceptors (10b) or hydrophobic groups (10c,d,e) in the ortho
position would desolvate the imidazole and are expected to have
very poor activity.
We achieved considerable potency in the ester series of
analogues (Table 1), but these compounds lack metabolic

3708 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
SuryadeVara et al.
Table 1. Activities of Compounds Showing General Structure I against T. cruzi Amastigotes and Murine Fibroblast Cells (Scheme 1)a
para
meta
ortho
compd
R
T. cruzi EC50 (nM) fibroblasts EC50 (nM) T. cruzi EC50 (nM) fibroblasts EC50 (nM) T. cruzi EC50 (nM) fibroblasts EC50 (nM)
10a
10b
10c
10d
10e
10f
10g
10h
10i
H
80
100
5
5
20
25
25
0.5, 0.9, 1.0
250
ND
ND
21, 25
>1000
>10000
50000
25000
>10000
ND
>10000
>1000
>10000
ND
NO2
CH3
Cl
Br
OMe
CN
Ph
NH2
F
C(CH3)3
2,4-F2
30
20
20
20
ND
100
ND
130
150
475
>10000
>10000
>10000
>10000
ND
>10000
ND
>10000
>1000
>1000
100-1000
1000
100-1000
100
ND
200
ND
5
ND
ND
10000
10000
10000
10000
ND
25000
ND
25000
ND
10j
10k
10l
ND
>1000
ND
a Multiple values for the same compound indicate independent determinations.
Table 2. Activities of Compounds Showing General Structure II against
T. cruzi Amastigotes and Murine Fibroblast Cells (Scheme 2,3,4)
stability in rodents because of hydrolases that are prevalent both
extra- and intracellularly. Schematic changes have been made
on the basic skeleton by docking into the homology model. On
the basis of the previous data (Table 1), we decided to maintain
the biphenyl group on the imidazole and modify the ester
functionality. Our first approach involved the synthesis of
various esters and amides in anticipation of reducing the risk
of plasma hydrolysis. We further explored the series and
undertook a systematic survey of structure-activity relationships.
Various substituents coming off the para position of the
anilino fragment in the scaffold (Table 2) can make limited
contacts with Tyr 77 and Phe 84 but mainly point into the
solvent surrounding the enzyme. This explains why almost all
substitutions are tolerated in this position. While the esters
(15a,b,c) have good activity in vitro, they are liable to hydrolysis
in vivo, hence we began efforts to find nonhydrolyzable
replacements such as halogens, ketones, amides, and various
ring systems. In particular, we investigated ester replacements
of our previous compounds and assessed their viability by
molecular modeling. We observed that there was sufficient room
in the hydrophobic binding pocket, and we introduced a variety
of heterocycles at the para position to the biphenyl aniline
system. We determined that substituting the ester with other
functional groups, such as benzothiazole, chloro, or methoxy,
which can fit into the hydrophobic binding pocket, was needed
for potency as well as stability. Pharmacokinetic stability was
examined on selected analogues from Table 2. Compounds 27a
and 27d showed good pharmacokinetic profile in mice,7 but
these compounds are relatively ineffective against cultured T.
cruzi (Table 2). In the hope of maintaining these improved
pharmacokinetic properties, we focused on further modifications
to increase potency. In particular, the benzothiazole from the
structure-activity study in Table 2 was retained and the
biphenyl was replaced with substituted benzyl groups on
the imidazole. Branched p-substituents on the benzyl, isopropyl
(32a), or methylsulfonyl (32e) do not fit in the narrow
hydrophobic cleft (Table 3). As to the m-phenyl substituent on
the anilino fragment of the scaffold, various residues lining the
pocket are flexible and can easily adopt alternative rotamers as
compd
15a COOEt
15b COOiPr
15c COOCy
16a C(dO)NHCH3 2.4, 3.0, 3.8, 5.1
16b C(dO)N(CH3)2 9.3, 14
R
EC50 T. cruzi (nM) EC50 Fibroblasts (nM)
10
>10000
>10000
>10000
>1000
>1000
>1000
>10000
>10000
>10000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>10000
>10000
>1000
>10000
>10000
>10000
50
10
16c
C(dO)N(CH2)5 24, 39
20a C(dO)CH3
20b C(dO)Et
40
40
40
6, 7
1.3, 1.8
23, 35
22, 41
20, 26
7, 9
9, 9, 22, 28, 28
100
40
20c
C(dO)Pr
24b Cl
24c
CN
24d 1-piperidine
24e
24f
1-pyrrolidine
N(CH3)2
24g OCH3
27a 5-thiazole
27b 2-pyrrole
27c
2-benzofuran
27d 2-benzothiazole 19, 7, 7, 15, 14, 22, 21
27e
27f
2-benzoxazole
3-benzisoxazole 10
50
100
27g 3-anthranil
they are not packed by the other residues Arg 96 and Met 97.
It is not surprising that the methyl group of 32g and 32h can
make extra contacts and afford a slightly better activity. These
structural differences do not significantly affect the binding
behavior, as shown in Table 3.

Inhibitors of Lanosterol 14R-Demethylase in Chagas Disease
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3709
Table 3. Activities of Compounds Showing General Structure III
against T. cruzi Amastigotes and Murine Fibroblast Cells (Scheme 5)
Table 5. Activities of Compounds Showing General Structure V against
T. cruzi Amastigotes and Murine Fibroblast Cells (Scheme 7)
EC50 T.
cruzi (nM)
EC50 fibroblasts
(nM)
compd
R
R1
32a
32b
32c
32d
32e
32f
p-isopropyl
p-ethyl
p-chloro
o,p-difluoro
p-methyl sulfonyl
p-(2-toluyl)
p-Ph
H
H
H
H
H
H
540, 860
110, 190
250
420, 630
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
compd
R
EC50 T. cruzi (nM) EC50 fibroblasts (nM)
44a
44b
44c
44d
44e
44f
Cl
CN
OCH3
1.2, 1.5, 2.1
1.5, 2.6
>1000
>1000
>1000
>1000
>1000
>1000
0.8, 2.1
120, 180
31, 42
2-benzothiazole 0.4, 0.6, 0.7, 1.1, 1.4
5-thiazole
C(dO)NHCH3
32g
32h
2-methyl
2, 3-dimethyl 64, 101
10, 13
12, 26
p-Ph
Table 4. Activities of Compounds Showing General Structure IV
against T. cruzi Amastigotes (EC50) and Murine Fibroblast Cells (nM)
(Scheme 6)
makes hydrophobic interactions with Tyr 77 and Phe 84. The
predicted binding mode is shown in Figure 2.
Binding of 44a and 44d to Recombinant T. cruzi
L14DM in Vitro. We tested the binding of key compounds
44a and 44d to recombinant T. cruzi L14DM by monitoring
the difference in the visible spectrum that occurs when the
imidazole nitrogen coordinates to the heme iron (Soret band
shift) (Figure 3). Both compounds were found to bind tightly
to L14DM, with a maximal difference spectrum obtained when
the amount of inhibitor approached the total amount of enzyme
in solution. This indicates that the equilibrium dissociation
constant for the L14DM-inhibitor complex is ,2.2 µM, the
concentration of enzyme used in the assay. It was not possible
to obtain accurate values of the dissociation constants because
the use of lower enzyme concentrations gives rise to spectral
signals that are not significantly above the noise. The spectral
shift observed (type II difference spectrum7) is consistent with
direct coordination of the imidazole nitrogen of the inhibitors
to the heme-iron. Thus, it is expected that these compounds
compete with lanosterol for binding to L14DM, but this was
not established with kinetic studies.
EC50 T.
cruzi (nM)
EC50 fibroblasts
(nM)
compd
R
R1
35a
35b
35c
35d
35e
35f
35g
35h
35i
35j
35k
35l
35m Ph
35n
39
NO2 3-Ph
CH3 3-Ph
100
100
80
10, 29, 23, 20
360, 400
>1000
230, 310
550
10000
10000
10000
10000
>750
>1000
>1000
>1000
>1000
>1000
>1000
>10000
>10000
>10000
>10000
Cl
Ph
H
3-Ph
3-Ph
3-Ph
H
H
H
3-CH(CH3)2
3-(o-toluyl)
3-(m-toluyl)
H
3-(o,m-dimethyl phenyl) 260, 300
H
H
Ph
3-(3′-pyridyl)
3-(4′-pyridyl)
4-COOCH3
2-Ph
600, 760
520, 510
80
80
200
Pharmacokinetics and Activity of Compounds in the
Murine Model of Chagas Disease. Two compounds with
potent activity against T. cruzi in vitro, 44a and 44d, were
subjected to single dose (50 mg/kg by oral gavage) pharmaco-
Ph
Ph
4-Ph
3-Ph; 4-Ph
80
Using the structure-activity from Table 1, we decided to
explore further combinations of substitutions of the two-phenyl
rings of the scaffold in the absence of the methyl ester
substituent. Para substitutions on the benzyl recapitulate our
findings from Table 1, with 35d being the most active. None
of the substitutions on the anilino fragment were as active as
the m-phenyl. Finally we combined the most active substituents
of Tables 1-4. All compounds exhibit EC50s against T. cruzi
in the 1-25 nM range (Table 5). We focused our attention
toward the ortho amino substituted compound 10i, which is the
analogue in this series showing the best potency against cultured
T. cruzi. The combination of these observations resulted in a
series of compounds possessing inhibition in the 1-2 nM range
against T. cruzi (Table 5), and these structural differences
significantly affect the potency. Among these the most active
is 44d, which projects the amino-substituted biphenyl in the
hydrophobic cleft that has a uniquely placed histidine for
forming a hydrogen bond. It also possesses a benzothiazole that
Figure 2. Homology model of T. cruzi L14DM in complex with
44d.

3710 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
SuryadeVara et al.
Figure 4. Efficacy of compounds in mice infected with T. cruzi. Mice
in groups of six were given compounds or vehicle by oral gavage twice
per day from days 7-27 postinfection. Parasitemia was quantified on
wet mounts of fresh blood. All vehicle treated mice were dead by day
16. Dramatic suppression of parasitemia was observed in all compound
treated groups, with 44d at 50 mg/kg showing the most suppression
along with posaconazole. The mice tolerated all of the treatments
without apparent side effects.
Table 7. Mice Parasitemia by PCR
PCR parasite detection
after 100 days post
infection (no. of mice)
dose
(mg/kg)
compd
44d
44d
44a
50
20
50
20
2 of 6
5 of 6
6 of 6
0 of 6
Figure 3. Difference spectra for the binding of 44a (top panel) and
44d (bottom panel) to recombinant T. cruzi L14DM. Compounds were
added to 2.2 µM enzyme in increments of 0.2 µM. Maximum difference
spectra were obtained with equimolar enzyme and compound. The
absorbance around 360 nm in the spectra with 44d is attributed to the
compound alone. Binding of compound to enzyme caused an increase
in absorbance at ∼440 nm and a decrease at ∼390 nm.
posaconazole
postinfection. Parasitemia was monitored by microscopic ex-
amination of blood through 97 days postinfection. All mice in
the vehicle group developed overwhelming parasitemia and were
dead by day 16 post-infection. All drug-treated groups mani-
fested a low level of parasitemia in the post-treatment period
that gradually declined to levels that were microscopically
undetectable by the end of the experiment (Figure 4). The
differences in parasitemia between the four compound-treated
groups were not statistically significant at any of the time points.
At 100 days, the mice were sacrificed, and 200 µL of whole
blood was subjected to PCR for detection of T. cruzi. Parasitemia
was suppressed below PCR detectability in all the posaconazole
treated mice and in 4 of 6 mice treated with 44d at 50 mg/kg.
Although parasitemia was dramatically suppressed by 44d at
20 mg/kg and by 44a at 50 mg/kg, most or all of these mice
had detectable parasites by PCR (Table 7). It is uncertain
whether or not the PCR negative mice are absolutely cured of
the T. cruzi infection due to the intermittent nature of parasitemia
in chronically infected animals. Future studies will also employ
the approach of giving immunosuppression to the mice at the
termination of the experiment to more definitively determine if
the mice are parasitologically cured.24
Table 6. Single Oral Dose Pharmacokinetics of T. cruzi L14DM
Inhibitors
44a
44d
compd
mouse 1, 2, 3 ave ( SD mouse 1, 2, 3 ave ( SD
Cmax (µg/mL) 2.8, 2.4, 1.9
2.4 ( 0.5 5.8, 8.6, 7.2
0.8 ( 0.3 1.0, 2.0, 2.0
7.2 ( 1.4
1.7 ( 0.6
Tmax (h)
0.5, 1.0, 1.0
6.7, 6.7, 5.6
AUC0-5h
(µg·h/mL)
6.3 ( 0.6 24.7, 32.4, 29.0 28.7 ( 3.9
kinetic studies on Balb/c mice in groups of three. Table 6 gives
the data summary, and plasma concentration-versus-time plots
for each mouse are provided as Supporting Information. For
44a, the peak average blood level of 2.4 µg/mL (Cmax) was
obtained in 0.8 h (Tmax) (average from 3 mice). Values for 44d
are Cmax ) 7.2 µg/mL and Tmax ) 1.7 h. We collected plasma
drug concentration data out to 5 h and obtained AUC0-5h of
6.3 and 28.7 µg·h/mL for 44a and 44d, respectively. Accurate
terminal elimination half-lifes were not obtained, but the data
show that the half-life is in excess of 3 h for 44a (Supporting
Information). For 44d, significant drug loss was not observed
out to 5 h (Supporting Information), showing that this compound
is more stable than 44a in mice. These values are not very
different from the published terminal phase half-life of posa-
conazole of 7-9 h in mice and rats.22 A long elimination half-
life, as exhibited by these compounds, is believed to be
important for successful elimination of the slowly dividing T.
cruzi during chronic infection.23
The mice appeared to tolerate the drug treatments without
apparent side effects. All mice in the four drug-treated groups
survived to the termination of the experiment at day 100.
Weights were monitored on a weekly basis, and all treated
animals gradually gained weight from the completion of drug
treatment until the end of the experiment (data shown in
Supporting Information).
Conclusions
The efficacies of the 44a and 44d in the murine model of
Chagas disease were compared to vehicle and posaconazole
(Figure 4). The compounds or vehicle were administered to a
group of 6 mice at the indicated doses twice per day for 21
consecutive days to mice by oral gavage from days 7-27
From this study have come several new inhibitors of T. cruzi
L14DM that are among the most potent inhibitors of in vitro T.
cruzi amastigote growth known to date (subnanomolar to low
nanomolar potency). Two of the most promising compounds,

Inhibitors of Lanosterol 14R-Demethylase in Chagas Disease
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3711
mL of water. The mixture was heated to 90 °C, and KMnO4 (14.2
g, 90 mmol) was added in portions. The mixture was refluxed for
5 h. The black solid was filtered off, and the filtrate was acidified
with 6 N HCl. The mixture was cooled in an ice bath and the white
precipitate was collected (7 3.1 g, 85%); mp 175-176 °C. 1H NMR
CDCl3) δ 8.25-8.33 (m, 2H, aryl), 8.08 (d, J ) 8.9 Hz, 1H, aryl),
7.41-7.51 (m, 3H, aryl), 7.31-7.39 (m, 2H, aryl). MS m/z 243.2
(M + H)+.
5-Nitro-biphenyl-2-carboxylic Acid Methyl Ester (8). A solution
of compound 7 (2.84 g, 11.7 mmol) in methanol (35 mL) was
treated with SOCl2 (4.16 g, 35 mmol) dropwise at 0 °C. The mixture
was heated to reflux for 6 h and then cooled to rt. Upon transfer,
spontaneous crystallization occurred. The residual solvent was then
filtered off, and the resulting solid was left to dry overnight to give
8 (2.28 g, 76%). 1H NMR CDCl3) δ 8.25 (m, 2H), 7.92 (d, J ) 8.4
Hz, 1H), 7.43 (m, 3H), 7.32 (dd, 2H), 3.67 (s, 3H, COOCH3). MS
m/z 258.1 (M + H)+.
5-Amino-biphenyl-2-carboxylic Acid Methyl Ester (9). A mixture
of 5-nitro-biphenyl-2-carboxylic acid methyl ester 8 (2.0 g, 7.8
mmol) and SnCl2.2H2O (8.8 g, 39 mmol) in EtOAc (75 mL) was
stirred at reflux under nitrogen for 2.5 h. Upon cooling, saturated
NaHCO3 (150 mL) was added. The organic layer was removed,
and the aqueous layer washed with EtOAc (2 × 100 mL). The
combined organic layers were dried (Na2SO4) and concentrated to
dryness to give 9 as a white solid (1.53 g, 86%). 1H NMR CDCl3)
δ 7.80 (d, J ) 8.4 Hz, 1H), 7.27-7.37 (m, 5H, aryl), 6.63 (dd, J
) 8.4, 2.1 Hz, 1H), 6.56 (d, J ) 8.4 Hz), 3.59 (s, 3H, COOCH3).
MS m/z 228.3 (M + H)+.
44a and 44d, were chosen for studies in mice and display
excellent pharmacokinetic properties including oral activity and
reasonable stability in mouse plasma. These two compounds
are efficacious in a mouse model of acute Chagas disease with
efficacy comparable to that of posaconazole. This new class of
L14DM inhibitors should be much cheaper to produce than
posaconazole. Further studies are in progress to explore the
animal toxicology profile of these compounds, as well as their
efficacy in a chronic model of Chagas disease in mice, and
efficacy studies in larger animals.
Experimental Section
Synthesis of Compounds. Unless otherwise indicated, all
anhydrous solvents were commercially obtained and stored under
nitrogen. Reactions were performed under an atmosphere of dry
nitrogen in oven-dried glassware and were monitored for complete-
ness by thin layer chromatography (TLC) using silica gel 60 F-254
1
(0.25 mm) plates with detection with UV light. H NMR spectra
were recorded on dilute solutions in CDCl3, CD3OD, or DMSO-d6
at 300 MHz. Chemical shifts are reported in parts per million (δ)
downfield from tetramethylsilane (TMS). Coupling constants (J)
are reported in Hz. Electron spray ionization mass spectra were
acquired on an Bruker Esquire LC00066.
Flash chromatography was carried out with silica gel (40-63
µm). Preparative reverse phase HPLC was performed on an
automated Varian Prep Star system using a gradient of 20% MeOH
to 100% MeOH (with 0.1% trifluoroacetic acid) at 12 mL/min over
30 min using a YMC S5 ODS column (20 mm × 100 mm, Waters
Inc.). All compounds tested on parasites were purified to a single
peak by HPLC as described above. HPLC purified compounds were
submitted to 1H NMR analysis, and compounds were submitted to
testing on parasites if the 1H NMR spectrum was free of detectable
noncompound peaks (estimated purity at least 95%).
1-Triphenylmethyl-4-imidazole Carboxaldehyde (4). To a 1
L three-necked round-bottomed flask with an addition funnel was
added 3H-imidazole-4-carbaldehyde (12 g, 0.125 mol), trityl
chloride (38.3 g, 0.137 mol), and acetonitrile (400 mL). The mixture
was stirred at rt to give a slurry. Triethylamine (30 mL, 0.215 mol)
was added dropwise over 20 min. After the addition was complete,
the reaction mixture was stirred at rt for 20 h. Hexane (40 mL)
and water (400 mL) were added. The slurry was stirred for 30 min
and filtered. The cake was washed with water (3 × 100 mL) and
dried in a vacuum oven at 50 °C for 20 h to give 4 as a white solid
(39.8 g, 94%). 1H NMR (CDCl3) δ 9.81 (s, 1H), 7.54 (s, 1H), 7.46
(s, 1H), 7.29 (m, 10H), 7.04 (m, 5H). MS m/z 339.4 (M + H)+.
General Procedure for the Synthesis of 3-Alkyl-3H-imidazole-
4-carbaldehyde (5). A solution of compound 4 (1.5 mmol) in
acetonitrile (10 mL) was treated with alkyl bromide (1.5 mmol) at
rt and heated to 60 °C and stirred overnight under nitrogen. The
reaction mixture was cooled and concentrated under vacuum, and
the resulting paste was triturated with acetone (20 mL) and stir-
red for 2-3 h. The resulting solid was isolated by filtration and
extracted with CH2Cl2 (2 × 25 mL) and washed with saturated
NaHCO3. The organic layers were combined, dried over Na2SO4,
and concentrated to give 5 as a yellow solid.6
General Procedure for Reductive Amination.10 To a solution
of 9a-l (0.32 mmol) and 5 (0.32 mmol) in MeOH (6 mL) was
added 0.4 g of 4 Å molecular sieves. The solution was stirred at rt
under nitrogen for 1 h, after which acetic acid (4.4 mmol) was
added. After 5 min, NaCNBH3 (0.64 mmol) was added in portions.
The mixture was stirred at rt under nitrogen overnight. The reaction
mixture was mixed with CH2Cl2 and saturated NaHCO3. The
aqueous layer was extracted with CH2Cl2, after which the combined
organic layers were washed with brine. The organic layer was dried
with Na2SO4, and the solvent was removed by evaporation. The
crude product was purified by preparative HPLC to give 10 a-l.
See Supporting Information for p-10b, p-10c, p-10d, p-10e,
p-10f, p-10 h, p-10i, p-10k, m-10b, m-10c, m-10d, m-10e, m-10g,l,
m-10i, m-10j, o-10b, o-10c, o-10d, o-10e, o-10g, and o-10i.
Compounds 11a-b were prepared by following the procedure
described for the synthesis of 8 (Supporting Information).
Representative Amidation. A solution of compound 7 (1.75 g,
7.2 mmol) in SOCl2 (3 mL) was heated to reflux for 2 h and then
cooled to rt and concentrated under vacuo to give a pale-white solid.
The crude acid chloride (1.87 g, 6.7 mmol) was dissolved in CH2Cl2
(25 mL) and was added to a stirred solution of the amine (6.7 mmol)
and triethylamine (15.4 mmol) in CH2Cl2 at 0 °C, and the mixture
was stirred for 4 h at rt, diluted with H2O and extracted with CH2Cl2
(2 × 30 mL) and concentrated under vacuo to provide 12a-c.
Compounds were submitted to nitro reduction conditions as
described for the synthesis of 9.
Compounds 13a-c were prepared by following the procedure
described for the synthesis of 9 (Supporting Information).
Compounds 15a-c were prepared from 13 and 5 by following
the procedure described for the synthesis of 10 (Supporting
Information).
Compounds 16a-c were prepared via reductive amination of
14a-c with 5 following the procedure described previously
(Supporting Information).
Representative Acylation and Fries Rearrangement. A mixture
of 3-aminophenol (1.09 g, 10 mmol) and butyryl anhydride (4.1
mL, 25 mmol) in pyridine (10 mL) was stirred at rt for 1.5 h. The
reaction mixture was diluted with EtOAc (60 mL), and the organic
layer washed with sat. NaHCO3 (2 × 60 mL), 10% HCl (2 × 60
mL), dried (Na2SO4), and concentrated, and to the resulting oil was
added 1.5 mL of 1,2-dichlorobenzene and AlCl3 (2.67 g, 20 mmol)
and the solution was stirred at 120 °C overnight under nitrogen.
The resulting tar was added to EtOAc (60 mL) and washed with
2-Methyl-5-nitro-biphenyl (6). To a solution of 4-nitro-2-
bromotoluene (6.48 g, 30 mmol) and phenyl boronic acid (3.84 g,
31.5 mmol) in 70 mL of acetone was added 85 mL of water,
potassium carbonate hydrate (12.4 g, 75 mmol, 2.5 equiv), and
Pd(OAc)2 (0.33 g, 5% equiv). The mixture was refluxed for 10 h
and then cooled. The deep-black solution was extracted with ether
and 3 N HCl. The ether fraction was passed through a layer of
celite. After evaporating solvent, the residue was dried and then
recrystallized from methanol to give flake crystals7 (4.60 g, 88%);
mp 77-78 °C. 1H NMR (CDCl3) δ 8.09-8.11 (m, 2H, aryl),
7.40-7.49 (m, 4H, aryl), 7.30-7.33 (m, 2H, aryl), 2.37 (s, 3H).
MS m/z 213.3 (M + H)+.
5-Nitro-biphenyl-2-carboxylic Acid (7). 2-Methyl-5-nitro-biphe-
nyl (3.2 g, 15 mmol) was dissolved in 15 mL of pyridine and 30

3712 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
SuryadeVara et al.
water (3 × 60 mL) and brine (60 mL). The organic layers combined
and dried over Na2SO4 and concentrated. The resulting tan solid
was recrystallized from EtOAc to yield 17a-c as a brown solid
(1.04 g, 40%) (Supporting Information).
and then washed with brine, dried (Na2SO4), filtered, and
concentrated. The residue was purified by flash column chro-
matography using 30% EtOAc/hexane to give 97 mg of cyano
compound 22c (60%).
Representative Triflation. To solution of compound 17a-c (249
mg, 1.0 mmol) in pyridine (3 mL) was added Tf2O (0.20 mL, 1.2
mmol) dropwise at 0 °C and the mixture was stirred at rt overnight.
The solution was diluted with EtOAc (10 mL) and washed with
water and brine. The organic layers were combined, dried (Na2SO4),
and concentrated under vacuo to get 18a-c as brown oil (365 mg,
96%) (Supporting Information).
Representative Suzuki Cross Coupling. To a mixture of 18 (193
mg), phenylboronic acid (101 mg, 0.83 mmol, 1.4 equiv) and
Pd(PPh3)4 (69 mg, 10 mol %) in dimethoxyethane (5 mL) was added
ethanol (0.5 mL) and K2CO3 (0.59 mL of 2 M in H2O, 2 equiv),
and the solution was stirred at reflux overnight under nitrogen. The
solvent was removed under vacuum, and the resulting solid was
taken up into CH2Cl2 (30 mL) and washed with brine (30 mL).
The solvent was removed by evaporation, and the resulting black
solid was purified by column chromatography using a gradient of
1:1 Hex:EtOAc yielded a white solid (81 mg, 54%).
Preparation of Compounds 22d-e. Compounds 22d-e were
prepared from 22a (1.0 g, 3.5 mmol) and 4.3 mmol of the
corresponding amine. Starting materials were mixed together and
heated at 100 °C for 2-3 h followed by usual workup to give the
required nitro intermediate compounds.
Dimethyl-(5-nitro-biphenyl-2-yl)-amine (22f). A solution of 21
(0.5 g, 2.33 mmol) and sodium borohydride (0.43 g, 11.6 mmol)
in THF (25 mL) was added dropwise to an efficiently stirred
solution of 3 M sulfuric acid (11.5 mL, 35 mmol) and 35% aq
formaldehyde (9.3 mmol) in THF (5 mL) at 10-15 °C. After
the first half of the addition, the mixture is acidified with 3 M
H2SO4 (11.5 mL, 35 mmol) and then stirred for 1 h. To the
resultant mixture, water (20 mL) was added followed by the
addition of aq KOH to raise the pH to about 9-10. The organic
phase was separated and the aqueous phase was extracted with
ether (2 × 100 mL). The combined organic layers were washed
with saturated NaCl and dried over sodium sulfate. The ether
layer was evaporated to dryness, and the crude material was
purified by flash chromatography eluting at 10% EtOAc to afford
22f, 0.45 g (80%).
See Supporting Information for 19a-c.
Representative N-Acyl Deprotection and Reductive Amina-
tion. Biphenyl 19 (180 mg, 0.58 mmol) was dissolved in 5 mL
i-PrOH, and NaOH (232 mg, 5.8 mmol in 0.5 mL H2O) was added.
The solution was heated to reflux overnight, after which the solvent
was removed under vacuum. The resulting solid was taken up into
EtOAc (60 mL) and washed with sat. NaHCO3 (60 mL), water (60
mL), and brine (2 × 60 mL). The organic layer was dried over
sodium sulfate to yield an orange oil, (112 mg, 81%). The resulting
aniline was coupled to 5 as described for compound 10.
Synthesis of 23a-g. Compounds were prepared as described for
compound 9.
Synthesis of 24b-f. Compounds were prepared as described for
compound 10. See Supporting Information for 24b-g.
Representative Heterocyclic Heck Reaction: 3-(5-Nitro-biphe-
nyl-2-yl)-benzo[d]isoxazole (25f). A mixture of 2-phenyl-4-nitro-
bromobenzene (22a, 556 mg, 2.0 mmol), Pd(PPh3)4 (116 mg,
0.1 mmol), and potassium acetate (294 mg, 3.0 mmol, 1.5 equiv)
were flushed with nitrogen for 5 min after which N,N-
dimethylacetamide (5 mL) was added. The solution was flushed
with nitrogen for an additional 5 min and 1,2-benzisoxazole (0.24
mL, 286 mg, 2.4 mmol, 1.2 equiv) was added. The solution was
stirred at 160 °C overnight. The solution was added to ether
(150 mL) and washed with water (3 × 100 mL). The organic
layer was dried with sodium sulfate and concentrated to yield a
black oily solid, which was purified by column chromatography
4:1 (hexane:EtOAc) to obtain the crude product (206 mg), which
was used without further purification.
See Supporting Information for 20a-c.
2-Phenyl-4-nitrobromobenzene (22a). Sodium nitrite (2.66 g,
38.5 mmol, 1.1 equiv) was added in portions to 21 mL of
concentrated sulfuric acid at rt. The suspension was cooled to
10 °C and acetic acid (22 mL) was added dropwise. The mixture
was stirred for 20 min at 10 °C, and 2-phenyl-4-nitroaniline (7.56
g, 35 mmol) was added in portions over 30 min. The solution
was stirred for 2 h. at 10 °C, and water (15 mL) was added to
clear the suspension. The solution was stirred for 1 h at rt, and
cupric bromide (13.07 g, 56 mmol, 1.6 equiv) in 27 mL of 2M
HCl was added slowly. The resulting black sludge was stirred
for 20 min at rt and 1 h at 60 °C. The solution was added to
ether (200 mL) and washed with water (3 × 100 mL) and brine
(150 mL). The organic layer was dried with sodium sulfate and
concentrated to give an orange solid, which was purified by
recrystallization from methanol to yield a red solid (6.94 g, 71%).
1H NMR (CDCl3) δ 8.20 (d, J ) 2H, 1H, ortho to NO2), 8.06
(dd, J ) 2 and 9 Hz, 1H, ortho to NO2), 7.86 (d, J ) 9 Hz, 1H,
ortho to Br), 7.41-7.49 (m, 5H, Ar).
See Supporting Information for 25a-c and 25e-g.
2-(5-Nitro-biphenyl-2-yl)-benzothiazole (25d). A large scale
synthesis of this intermediate was carried out as follows. A
mixture of 7 (2 g, 8.23 mmol) and SOCl2 (20 mL) was refluxed
for 3 h. SOCl2 was removed under reduced pressure to give the
acid chloride. The resulting 5-nitro-biphenyl-2-carbonyl chloride
(2 g, 7.6 mmol) was added to a mixture of 2-aminothiophenol
(0.82 mL, 7.6 mmol) and pyridine (0.61 mL, 7.6 mmol) in
p-xylene (60 mL). The contents were stirred at room temperature
for 1 h, then p-TsOH · H2O (7.2 g, 38.0 mmol) was added and
the reaction mixture was stirred at reflux. After 12 h, the reaction
was cooled, extracted with CH2Cl2 (2 × 100 mL). The combined
organic layers were washed with saturated NaHCO3 (2 × 50
mL) and brine (100 mL) and dried over Na2SO4. Evaporation
of organics resulted in a green solid, which was recrystallized
from EtOAc to obtain 2-(5-nitro-biphenyl-2-yl)-benzothiazole
25d as a white solid (1.7 g, 70%). 1H NMR (CDCl3) δ 8.30-8.35
(m, 2H), 8.28 (d, 1H, J ) 2 Hz), 8.07 (d, 1H, J ) 7 Hz), 7.72
(d, 1H, J ) 9 Hz), 7.33-7.49 (m, 7 H).
Synthesis of 2-Chloro-5-nitro-biphenyl (22b). Synthesis of 22b
performed as described for 22a. The procedure for the large-
scale synthesis of 22b is as follows. A mixture of phenylboronic
acid (1.51 g, 10.05 mmol), tetrakis(triphenylphosphine)pal-
ladium(0) (0.35 g, 0.30 mmol, 3 mol %), and K2CO3 (2.76 g,
20.0 mmol) in 10 mL (4:1) dry toluene/EtOH was stirred at room
temperature under argon for 15 min. To this mixture was added
3-bromo-4-chloronitrobenzene 16 (2.37 g, 10.0 mmol) and the
mixture refluxed for 24 h. The contents were cooled and filtered.
The filtrate was diluted with toluene and washed with 10% citric
acid (2 × 20 mL), 1 M NaHCO3 (2 × 20 mL), and brine (2 ×
20 mL). The combined organic layers were evaporated to give
a brown oil, which was recrystallized from hot hexane to give
22b as a pale-yellow solid (1.56 g, 88%).
5-Nitro-biphenyl-2-carbonitrile (22c). To a solution of 22a (0.2
g, 0.72 mmol) in DMF (5 mL) was placed under high vacuum
for 15 min. The solution was purged with Ar for 15 min. While
purging was continued, ZnCN2 (101 mg, 0.86 mmol) and
Pd(PPh3)4 (83 mg, 0.072 mmol) were added. The reaction
mixture was heated at 100 °C under Ar for 18 h and cooled to
rt and added to H2O. The mixture was extracted with EtOAc
Synthesis of Compounds 26a-g. Compounds were prepared
from 25a-g as described for the synthesis of 9.
Synthesis of Compounds 27a-g. Reductive amination with 26
and 5 was performed as for compound 10. See Supporting
Information for 27a-g.
Synthesis of Compounds 32a-h. Preparation of compounds
32a-h were performed as described for 10. See Supporting
Information for 32a-h.

Inhibitors of Lanosterol 14R-Demethylase in Chagas Disease
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3713
Preparation of Compounds 35a-n. Compounds were prepared
from 34a-n and 5 under reductive amination conditions as
performed previously. See Supporting Information for 35a-k.
3,4-Diphenylnitrobenzene (37). The synthesis was carried out
according to the procedure for 19. H NMR (acetone-d6): δ 7.58
(dd, 1 H, J ) 3 and 9 Hz), 7.51 (d, 1 H, J ) 3 Hz), 7.00 (d, 1 H,
J ) 9 Hz), 6.54-6.58 (m, 5 H), 6.48-6.51 (m, 4 H).
3,4-Diphenylaniline (38). The synthesis was carried out using
37 according to the procedure described for 9. 1H NMR (acetone-
d6) δ 7.02-7.20 (m, 11 H), 7.71-7.75 (m, 2 H).
(3-Biphenyl-4-ylmethyl-3H-imidazol-4-ylmethyl)-[1,1′;2′,1′′]ter-
phenyl-4′-yl-amine (39). The reaction was carried out using 38 and
5 according to the procedure described for 10. Yellow solid, 40%;
mp 195-199 °C. 1H NMR (acetone-d6) δ 7.68 (s, 1 H), 7.59-7.62
(m, 4 H), 7.44 (t, 2 H, J ) 7 Hz), 7.35 (tt, 2 H, J ) 2 and 8 Hz),
7.26 (d, 2 H, J ) 8 Hz), 7.121-7.17 (m, 5 H), 7.06-7.12 (m, 4
H), 7.01-7.04 (m, 2 H), 6.98 (s, 1 H), 6.72 (dd, 1 H, J ) 2 and 8
Hz), 6.64 (m, 1 H), 5.40 (s, 2 H), 4.31 (s, 2 H). 13C NMR (CDCl3)
δ 146.65, 141.79, 141.47, 141.20, 138.87, 135.03, 131.58, 129.86,
129.71, 129.25, 128.85, 128.72, 127.81, 127.73, 127.59, 127.24,
127.03, 126.45, 125.74, 115.19, 112.34, 48.68, 38.29. HRMS [FAB
M + H]+ (C35H30N3) calcd, 492.2439; found, 492.2440.
and 4 Å molecular sieves. The resulting mixture was stirred at
room temperature under argon for 0.5 h. To this, NaCNBH3 (0.4
g, 6.15 mmol) was added and then stirring continued overnight.
The resulting solid was separated and dissolved in EtOAc (100
mL). The undissolved material was filtered off, and the organic
layer was evaporated to give 43a as a yellow solid (0.715 g,
70%). 1H NMR 300 MHz (CD3OD) δ 9.05 (s, 1H), 8.41 (d, J )
2.1 Hz, 1H), 7.80 (dd, J ) 1.8, 8.1 Hz, 1H), 7.70 (s, 1H),
7.45-7.65 (m, 5H), 7.25-7.35 (m, 5H), 7.15 (d, J ) 8.4 Hz,
1H), 7.05 (d, J ) 8.4 Hz, 1H), 6.52 (dd, J ) 2.4, 8.4 Hz, 1H),
6.35 (d, J ) 2.4 Hz, 1H), 6.0 (s, 2H), 4.42 (s, 2H)). MS m/z
495.5 (M + H+).
1
See Supporting Information for 43b, 43c, 43e, and 43f.
(6-Benzothiazol-2-yl-biphenyl-3-yl)-[3-(3-nitro-biphenyl-4-ylm-
ethyl)-3H-imidazol-4-ylmethyl]-amine (43d). A large scale syn-
thesis based on previous work25 was carried out as follows. To
a solution of 26d (1.1 g, 3.67 mmol) and 42 (1.12 g, 3.67 mmol)
in CH2Cl2 (40 mL) was added TiCl4 (1 M solution in dichlo-
romethane) (1.83 mL, 1.83 mmol) dropwise at rt, and the mixture
was stirred for 30 min. To this was added NaCNBH3 (276 mg,
4.4 mmol) in MeOH (6 mL), the resulting mixture was stirred
overnight, and the solvents was removed by evaporation. The
resulting crude material was dissolved in CH2Cl2 (150 mL) and
sat. NaHCO3 (50 mL) was added. The organic layer was
separated and dried (Na2SO4) to obtain a yellow solid, which
4-Methyl-3-nitro-biphenyl (40). A large scale synthesis of 40
was carried out as follows. A mixture of 4-bromo-2-nitrotoluene
(1.0 g, 4.6 mmol), phenyl boronic acid (0.6 g, 5.09 mmol), and
Ba(OH)2 · 8H2O (3.2 g, 9.2 mmol) in 15 mL of DME:H2O (5:1)
was stirred under Ar for 15 min. To this, Pd (PPh3)4 (0.53 g,
0.46 mmol) was added and the resulting solution was refluxed
for overnight. The reaction was cooled and diluted with EtOAc
(30 mL) and washed with NaHCO3 and brine. The resulting
organic layer was filtered through celite, dried over (Na2SO4)
and evaporated to give a brown solid, which was recrystallized
1
was recrystallized from EtOH to afford 43d (1.52 g, 70%). H
NMR 300 MHz (CD3OD) δ 9.1 (s, 1H), 8.42 (d, J ) 2.1 Hz
1H), 7.87 (d, J ) 8.1 Hz, 1H), 7.71-7.81 (m, 4H), 7.57-7.62
(m, 2H), 7.24-7.49 (m, 8H), 7.13-7.18 (m, 2H), 6.95 (d, J )
8.4 Hz, 1H), 6.65 (dd, J ) 2.4, 8.4 Hz, 1H), 6.40 (d, J ) 2.4
Hz, 1H), 6.0 (s, 2H), 4.6 (s, 2H). MS m/z 594.4 (M + H+).
Preparation of Compounds 44a-e. Compounds were synthe-
sized by following the procedure described for 9.
1
from hexane to obtain a white solid (0.89 g, 90%). H NMR
(CDCl3) δ 8.23 (d, J ) 1.8 Hz, 1H), 7.75 (dd, J ) 7.8, 1.8 Hz,
1H), 7.63 (m, 1H), 7.60 (d, J ) 1.2 Hz, 1H), 7.53-7.45 (m,
2H), 7.44-7.39 (m, 2H) 2.65 (s, 3H). MS m/z 214.2 (M + H+).
4-Bromomethyl-3-nitro-biphenyl (41). A large scale synthesis
of 41 was carried out as follows. A solution of 2-methyl-3-
nitrobipyhenyl (40, 0.382 g, 1.79 mmol), NBS (0.333 g, 1.87
mmol), and a few crystals of benzoyl peroxide in CCl4 (15 mL)
was refluxed for 36 h. The mixture was cooled and treated with
benzene (50 mL). The resulting solution was filtered and
evaporated to dryness in vacuo. The crude material was
chromatographed (hexane/EtOAc, 20:1) to give 41 (0.31 g, 60%).
1H NMR (CDCl3) δ 8.30 (d, J ) 2.0 Hz, 1H), 7.86 (dd, J ) 8.0,
2.0 Hz, 1H), 7.69-7.64 (m, 4H), 7.53 (d, J ) 1.8 Hz,1H), 7.48
(m, 1H), 4.91 (s, 2H). MS m/z 293.1 (M + H+).
(6-Chloro-biphenyl-3-yl)-[3-(3-amino-biphenyl-4-ylmethyl)-3H-
imidazol-4-ylmethyl]-amine (44a). A large scale synthesis was
carried out as follows. To a solution of 43a (0.5 g, 1.01 mmol)
in EtOAc (75 mL) was added SnCl2 · 2H2O (1.13 g, 5.05 mmol),
and the mixture was stirred at reflux for 3 h. Upon cooling,
saturated NaHCO3 (100 mL) was added until the pH was neutral,
and the liquid was filtered through a pad of celite. The organic
layer was separated, and the aqueous layer was extracted with
EtOAc (2 × 100 mL). The combined organic layers were washed
with brine and dried over Na2SO4. Evaporation of the solvent
resulted in crude material 44a, which was recrystallized from
isopropanol (0.374 g, 80%). Off-white solid; mp 188-190 °C.
1H NMR (CD3OD) δ 8.70 (s, 1H), 7.51-7.58 (m, 3H),
7.32-7.47 (m, 8H), 7.23 (d, J ) 8.1 Hz 1H), 7.14 (d, J ) 1.8
Hz 1H), 7.10 (d, J ) 8.1 Hz 1H), 6.98 (dd, J ) 1.8, 7.8 Hz,
1H), 6.67 (dd, J ) 2.4, 8.7 Hz, 1H), 6.59 (d, J ) 2.4 Hz, 1H),
5.45 (s, 2H), 4.50 (s, 2H). MS m/z 465.5 (M + H+).
3-(3-Nitro-biphenyl-4-ylmethyl)-3H-imidazole-4-carbaldehyde (42).
A large scale synthesis of 42 was carried out as follows. 1-Trityl-
4-imidazole carboxaldehyde 4 (0.5 g, 1.5 mmol) and bromo
compound 41 (0.43 g, 1.5 mmol) were stirred in acetonitrile
(10 mL) at 60 °C under nitrogen overnight. The solvent was
removed by evaporation, and the resulting paste was triturated
with acetone (20 mL). The resulting solid was isolated by
filtration and extracted with CH2Cl2 and saturated NaHCO3. The
organic layer was dried over Na2SO4 and evaporated to dryness.
The residue was purified by column chromatography (EtOAc/
See Supporting Information for 44b-f.
Preparation of Posaconazole. Noxafil (Schering-Plough), was
purchased from a pharmacy, and the active compound was
purified from the suspension by organic extraction and flash
chromatography. The liquid formulation (12.5 mL) was added
to a 1 L separating funnel and diluted with water (300 mL) and
then extracted with EtOAc (2 × 500 mL) and the organic layer
was separated. The aqueous layer was extracted with CH2Cl2 (3
× 300 mL). The combined organic layers were evaporated to
dryness, and the solid obtained was purified by flash column
chromatography eluting with 3% methanol in CH2Cl2 to yield
425 mg (85%).
1
MeOH, 9.5: 0.5) to give 42 as a solid 0.2 g. H NMR (CDCl3)
δ 9.75 (s, 1H, -CHO), 8.36 (d, J ) 1.8 Hz, 1H), 7.95 (s, 1H),
7.89 (s, 1H), 7.51 (dd, J ) 8.1, 1.8 Hz, 1H), 7.59-7.55 (m,
2H)), 7.50-7.41 (m, 3H), 6.84 (d, J ) 8.1 Hz,1H), 5.95 (s, 2H).
MS m/z 308.3 (M + H+).
Preparation of compounds 43a-f. The compounds were pre-
pared by treating 23b,c, 26a,d, and 14a with 42 under reductive
amination conditions as performed previously for 10.
(6-Chloro-biphenyl-3-yl)-[3-(3-nitro-biphenyl-4-ylmethyl)-3H-
imidazol-4-ylmethyl]-amine (43a). A large scale synthesis was
carried out as follows. To a mixture of 6-chloro-biphenyl-3-
ylamine 26a (0.5 g, 2.46 mmol) and 3-(3-Nitro-biphenyl-4-
ylmethyl)-3H-imidazole-4-carbaldehyde 42 (0.72, 2.46 mmol)
in methanol (25 mL) was added acetic acid (0.35 mL, 4.92 mmol)
T. cruzi and Murine Fibroblast Growth Inhibition Assays.
Compounds were screened against the ꢀ-galactosidase expressing
the Tulahuen strain of T. cruzi in 96-well tissue culture plates
as described previously.25,26 The Tulahuen strain originated in
Chile and is grouped with the more common TCII phylogenetic
lineage of T. Cruzi. In this assay, T. cruzi proliferates as
intracellular amastigotes within murine 3T3 fibroblasts.
Compounds were screened in triplicate to determined values
of EC50. Standard errors within assays were consistently less

3714 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
SuryadeVara et al.
(2) Buckner, F. S. Van Voorhis. W. C. Immune response to Trypanosoma
cruzi: Control of Infection and Pathogenesis of Chagas Disease. Ed;
Cunningham, M. W., Fujinami, R. S., Eds.; Lippincott Williams &
Wilkins: Philadelphia, 2000; Vol. 569, p 591.
(3) Urbina, J. A.; Docampo, R. Specific chemotherapy of Chagas disease:
controversies and advances. Trends Parasitol. 2003, 19, 495–501.
(4) Goad, L. J.; Berens, R. L.; Marr, J. J.; Beach, D. H.; Holz, G. G. The
activity of ketoconazole and other azoles against Trypanosoma cruzi:
biochemistry and chemotherapeutic action in vitro. Mol. Biochem.
Parasitol. 1989, 32, 179–190.
(5) Ferraz, L. M.; Gazzinelli, T. R.; Alves, O. R.; Urbina, A. J.; Romanha,
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than that of benznidazole. Antimicrob. Agents Chemother. 2007, 51,
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than 15%. Compounds were separately screened against murine
3T3 fibroblast cells to determine the EC50 values against these
host cells. Growth of the 3T3 fibroblasts was quantified by
the resazurin assay as previously described.25 Although some
compounds were yellow-colored as solids, there was no effect
of the diluted compounds on the colorimetric readouts.
Pharmacokinetic Studies in Mice. Compounds were suspended
at 10 mg/mL in 20% (w/v) Trappsol hydroxypropyl ꢀ-cyclo-
dextrin (pharmaceutical grade) (CTD, Inc.) and administered to
BALB/c mice (7-8 week females weighing approximately 20 g)
by oral gavage in a volume of 100 µL. Thus, the mice received
a dose of 50 mg/kg. At timed intervals, 40 µL of tail blood was
collected in heparinized capillary tubes. Plasma was separated
and frozen for later analysis according to the method previously
reported.27 Individual traces of plasma drug concentration versus
time for each mouse are provided as Supporting Information.
Efficacy Studies in Mice. BALB/c mice (7-8 week females)
were infected with 1 × 104 T. cruzi trypomastigotes (Tulahuen
strain) by subcutaneous injection. By 7 days postinfection, every
mouse had microscopically observable parasites on slides of
peripheral blood. On day 7 postinfection, mice (in groups of 6)
began receiving treatments by oral gavage twice per day for 21
days. Compounds were administered in a volume of 100 µL per
dose using the vehicle, 20% (w/v) Trappsol hydroxypropyl
ꢀ-cyclodextrin (pharmaceutical grade) (CTD, Inc.). Parasitemia
was monitored by placing 5 µL of tail blood under a coverslip
and counting 50 high-powered fields. Mice that were premorbid
from progressive infection were euthanized. All surviving mice
were sacrificed on day 100 postinfection, and ∼200 µL of blood
from cardiac puncture was taken for PCR analysis of parasitemia.
L14DM Binding Studies. Expression of T. cruzi L14DM in
E. coli and purification will be reported elsewhere. Binding
reactions contained 2.2 µM T. cruzi L14DM (concentration
determined from the Soret peak, absorbance at 420 nm minus
absorbance at 490 nm using an extinction coefficient of 111
mM-1 cm-1) in 1 mL of 50 mM sodium phosphate, pH 7.5, 300
mM NaCl, 10% glycerol. Binding of inhibitors was monitored
by difference spectroscopy (340-600 nm) in which 1 mL of
the above protein solution was placed in the sample and reference
cuvettes. After setting the difference spectrum to zero, 1 µL
aliquots of inhibitor stock solution (200 µM in DMSO) was
added to the sample compartment, and 1 µL of DMSO only was
added to the reference cuvette. The inhibitor concentration was
varied from 200 to 2000 nM.
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infections. AdV. Exp. Med. Biol. 2008, 625, 61–80.
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PCR Detection of T. cruzi Parasitemia. Extraction of T. cruzi
DNA and PCR amplification of DNA was performed according
to previously published methods.28-30 Briefly, 200 µL of the
whole was mixed with 200 µL of 6 M guanidine HCl-0.2 M
EDTA and stored at 4 °C. Samples were boiled for 15 min and
extracted with an equal volume of phenol:chloroform (1:1) and
then extracted with an equal volume of chloroform:isoamyl
alcohol (24:1), followed by ethanol precipitation and resuspen-
sion in 20 µL of H2O. Precipitated DNA (2 µL of a 1:20 dilution)
was subjected to PCR in a volume of 50 µL for 39 cycles using
the S35 and S36 primers that amplify a 330-bp minicircle
sequence.30 Then 6 µL of the PCR products were separated on
2% agarose gel and visualized with ethidium bromide.
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Acknowledgment. This work was supported by grants from
the National Institutes of Health (AI070218 and CA67771).
(22) Rachel, C.; Sudhakar, P.; Mark, L.; Josephine, L.; Vijay, B. Pharma-
cokinetics, safety, and tolerability of oral posaconazole administered
in single and multiple doses in healthy adults. Antimicrob. Agents
Chemother. 2003, 47, 2788–2795.
Supporting Information Available: Pharmacokinetic data for
compounds 44a and 44d and mice weight data. This material is
available free of charge via the Internet at http://pubs.acs.org.
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vitro and in vivo activities of ravuconazole on Trypanosoma cruzi,
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