Substituted 2-phenylimidazopyridines: A new class of drug leads for human African trypanosomiasis

Article abstract of DOI:10.1021/jm401178t

A phenotypic screen of a compound library for antiparasitic activity on Trypanosoma brucei, the causative agent of human African trypanosomiasis, led to the identification of substituted 2-(3-aminophenyl)oxazolopyridines as a starting point for hit-to-lead medicinal chemistry. A total of 110 analogues were prepared, which led to the identification of 64, a substituted 2-(3-aminophenyl)imidazopyridine. This compound showed antiparasitic activity in vitro with an EC₅₀ of 2 nM and displayed reasonable druglike properties when tested in a number of in vitro assays. The compound was orally bioavailable and displayed good plasma and brain exposure in mice. Compound 64 cured mice infected with Trypanosoma brucei when dosed orally down to 2.5 mg/kg. Given its potent antiparasitic properties and its ease of synthesis, compound 64 represents a new lead for the development of drugs to treat human African trypanosomiasis.

Full text of DOI:10.1021/jm401178t

Article
pubs.acs.org/jmc
Substituted 2‑Phenylimidazopyridines: A New Class of Drug Leads
for Human African Trypanosomiasis
Hari Babu Tatipaka,† J. Robert Gillespie,‡ Arnab K. Chatterjee,∥,∞ Neil R. Norcross,†
Matthew A. Hulverson,‡ Ranae M. Ranade,‡ Pendem Nagendar,† Sharon A. Creason,‡ Joshua McQueen,†
Nicole A. Duster,‡ Advait Nagle,∥ Frantisek Supek,∥ Valentina Molteni,∥ Tanja Wenzler,⊥,# Reto Brun,⊥,#
Richard Glynne,∥ Frederick S. Buckner,*,‡ and Michael H. Gelb*,†,§
†
‡
§
Departments of Chemistry, Medicine, and Biochemistry, University of Washington, Seattle, Washington 98195, United States
∥Genomics Institute of the Novartis Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, United States
⊥Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
#University of Basel, 4003 Basel, Switzerland
S
* Supporting Information
ABSTRACT: A phenotypic screen of a compound library for
antiparasitic activity on Trypanosoma brucei, the causative agent
of human African trypanosomiasis, led to the identification of
substituted 2-(3-aminophenyl)oxazolopyridines as a starting
point for hit-to-lead medicinal chemistry. A total of 110
analogues were prepared, which led to the identification of 64,
a substituted 2-(3-aminophenyl)imidazopyridine. This com-
pound showed antiparasitic activity in vitro with an EC50 of 2
nM and displayed reasonable druglike properties when tested
in a number of in vitro assays. The compound was orally bioavailable and displayed good plasma and brain exposure in mice.
Compound 64 cured mice infected with Trypanosoma brucei when dosed orally down to 2.5 mg/kg. Given its potent antiparasitic
properties and its ease of synthesis, compound 64 represents a new lead for the development of drugs to treat human African
trypanosomiasis.
toxicity. An additional concern is the increasing incidence of
melarsoprol resistant strains.2
INTRODUCTION
■
Two protozoan agents cause human African trypanosomiasis
(HAT): Trypanosoma brucei gambiense and Trypanosoma brucei
rhodesiense. Natural transmission occurs in 36 countries of sub-
Saharan Africa by the bite of infected tsetse flies (WHO Fact
Sheet No. 259). After the initial cutaneous inoculation, early
stage (hemolymphatic) infection occurs. In the form occurring in
West and Central Africa, (Gambian HAT), the first stage can last
for months to years before progressing to second stage infection
in the central nervous system. In the form occurring in East and
Southern Africa (Rhodesian HAT), the first stage lasts only a few
weeks to months before causing late-stage disease. Once the
parasites enter the central nervous system (second stage), the
patients suffer neurological effects that culminate in coma and
death if untreated.
Better HAT drugs are desperately needed especially for second
stage HAT.1 The recent introduction of nifurtimox−eflornithine
combination therapy for Gambian HAT allows for the use of
fewer doses of eflornithine, but these still need to be given by iv
injections, and the treatment remains very expensive. Melarso-
prol, a trivalent arsenical, is the only drug effective for second
stage Rhodesian HAT. Unfortunately, toxic encephalopathy
occurs in 5−18% of patients with associated mortality of 3−12%
from treatment itself. Because second stage HAT has 100%
mortality, melarsoprol remains as first line treatment despite this
Better drugs are also desirable for treating first stage disease.
Current treatment options pentamidine and suramin must be
given by injection, although most patients with early stage HAT
are able to take oral medicines. Both drugs have considerable
toxicity and are unsafe in pregnancy.3−5
Two promising drug candidates have recently entered early
clinical development for treating HAT: a nitroheterocyle
fexinidazole and a benzoxaborole SCYX-7158.6,7 These
candidates follow on the heels of the diamidine drug,
pafuramidine (DB-289), where efficacy was established in
phase III studies of HAT; however, nephrotoxicity observed in
an expanded phase I safety trial led to abandonment.8 As
evidenced by pafuramidine, the attrition rate of clinical drug
candidates is high (nearly 90% upon entering phase I),9
underscoring the importance of maintaining an adequate
pipeline of drug candidates.
Received: October 13, 2013
© XXXX American Chemical Society
A
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Table 1. 2-(3-Aminophenyl)oxazolopyridines Containing a N-Linked 2-Furanoyl Group
a
b
compd
R1
R2
EC50 (μM)
CC50 (μM)
cde
,
,
1
H
Cl
0.22
>50
>50
>50
>50
>50
>50
>50
c
c
c
c
c
c
2
H
F
0.12
3
H
Me
CN
OMe
0.43
4
H
0.4
5
H
>10
6
H
4-fluorophenyl
>50
7
H
3,4-difluorophenyl
>50
8
H
4-pyridyl
>20
c
c
9
Br
CN
F
F
F
F
F
F
F
0.23
>50
>50
10
11
12
13
14
15
0.38
c
phenyl
0.04 (0.08)
0.05 (0.1)
0.05
7
d
e
4-fluorophenyl
3-chlorophenyl
4-MeO-phenyl
4-phenylphenyl
>50, 32
c
>50
0.17
c
0.42
>50
f
pentamidine
1.7
g
d
e
quinacrine
3.9, 9.8
g
c
podophyllotoxin
0.014
a
Concentration of compound required to inhibit growth by 50% (EC50) of T. brucei strain BF427 or T. brucei rhodesiense (numbers in parentheses).
b
c
d
Concentration of compound required to inhibit growth by 50% (CC50) of mammalian cell lines. Rat myoblasts (L6). Human lymphoblasts
e
f
g
(CRL-8155). Human hepatocytes (HepG2). Control compound for T. brucei assays: average EC50 was 1.74 0.15 SEM (n = 57 assays). Control
compounds for cytotoxicity assays, average CC50 and SEM: for CRL-8155 cells, 3.92 0.91 μM (n = 8 assays); for HepG2 cells, 9.78 μM 1.27 μM
(n = 10 assays); for L6 myoblasts, 0.014.3 0.00065 μM (n = 15 assays).
compounds having a EC50 on T. brucei of less than 100 nM and a
further 446 compounds having a EC50 of less than 500 nM.
Properties of Lead Compound 1. Lead compound 1 was
selected from the available hits based on ease of synthesis,
including a lack of stereogenic carbons, as well as druglike
features including low MW and Lipinski rule-of-five compliance.
Compound 1 blocks the growth of T. brucei in vitro with an EC50
of 220 nM (Table 1). All compounds in this study were tested for
anti-parasite activity on the cattle virulent T. brucei brucei strain
BF427 in vitro, and selected examples were also tested on the
human virulent T. brucei rhodesiense strain STIB900. In all cases,
EC50 values for both parasite strains were within a factor of 2−3
of each other. Compound 1 was not cytotoxic to mammalian
cells, as the EC50 was >50 μM for rat myoblasts (L6 cells), human
lymphoblasts (CRL-8155 cells), and human hepatocytes
(HepG2 cells). The compound was reasonably soluble in
aqueous buffer (20 μM at pH 7.4 and 22 μM at pH 2.0)
(Supporting Information Table 1).
Supporting Information Table 1 summarizes in vitro ADME
studies and pharmacokinetics (PK) studies in mice. When
injected ip into mice, analysis of brain and plasma compound
levels 15 and 60 min postinjection showed a brain-to-plasma
concentration ratio of 0.54 and 0.38, respectively, indicating
exposure into the central nervous system. We also measured
permeability of 1 across a tight monolayer of Madin−Darby
canine kidney cells that have been transfected with the human
MDR1 (P-glycoprotein) drug efflux pump gene (MDCK-MDR1
cells), and permeability was measured in the presence and
absence of the MDR1 inhibitor GF-120918 to gauge whether 1 is
a substrate for the efflux pump.10−12 Values of Papp in the absence
RESULTS AND DISCUSSION
■
A high throughput screen was designed to identify new small
molecules with antiparasitic activity toward T. brucei within a
library of 700 000 compounds. This library was assembled with a
particular focus on druglike properties and structural diversity.
The library had been previously profiled in more than 60 other
high throughput screens, which included both biochemical and
cell-based assays against human and pathogen targets and has
been an excellent source of attractive starting points for
medicinal chemistry in these other programs. Further, the screen
history of this library also allowed rapid identification and
elimination of compounds with a “frequent hitter” profile.
Compounds from this library were tested for inhibitory activity
on T. brucei at 3.6 μM. The screen yielded 3889 primary hits
(0.6% hit rate) that inhibited growth by >50%. Data from more
than 95% of the assay plates had Z′ > 0.6, using DMSO as the
negative control and 1 μM pentamidine as the positive control.
Primary hits from the screen were further characterized using a
dose−response assay format to determine the EC50. In parallel,
cytotoxicities of these compounds were determined against a
proliferating human hepatoma cell line (Huh7). The final set of
confirmed hits consisted of compounds that had EC50 < 3.6 μM
against T. brucei, as well as a limited or nonobservable Huh7
cytotoxicity (CC50 > 10 μM or SI > 10; SI = CC50/EC50). The
final set of confirmed T. brucei hits consisted of 1035 inhibitors.
Of this set, over 95% of the identified hits did not carry a
“frequent hitter” liability (identified as a hit in >5 screens out of a
total of 60−65 screens). The 1035 confirmed and selective hits
could be grouped into about 115 distinct scaffolds, with 144
B
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and presence of GF-120918 were 417 and 362 nm/s,
respectively. The corresponding value for propanolol, a readily
permeable, non-MDR1 substrate comparator compound, was
444−469 nm/s in the presence and absence of GF-120918. The
additional comparator compound amprenavir is highly perme-
able by passive diffusion and is also a potent MDR1 substrate and
shows Papp values of 252 and 27 nm/s in this assay. The high
permeability of 1 and its poor tendency to be pumped by MDR1
in this in vitro assay are consistent with central nervous system
exposure in mice.
Scheme 2. Synthesis of Substituted 2-(3-
Aminophenyl)oxazolopyridines Containing 6-Substituents
a
A liability of 1 is its poor metabolic stability in liver
microsomes. The half-life for loss of 1 in mouse and human
liver microsomes was 0.8 and 6.4 min, respectively. A PK study
was carried out by dosing 1 by oral gavage in mice at 50 mg/kg.
The Cmax value in plasma was low, 0.06 μM (Tmax was 30 min).
The area under the plasma concentration−time curve (AUC)
was 187 min·μM. The half-life for elimination from the plasma
was 53 min, and the plasma clearance was 98 mL min−1 kg−1. The
low exposure of 1 in mice is consistent with the high rate of
metabolism seen in liver microsomes. In vitro metabolite
identification studies in liver microsomes showed a major
metabolite due to amide hydrolysis of the 4-chloroanilide group
(not shown). Given this result, we tested the hydrolysis product
(the aniline derived from 1) and found no inhibition of T. brucei
growth in vitro when tested up to 20 μM. Thus, the anilide and
not its hydrolysis product was causing the antiparasitic activity. It
could be possible that 1 kills parasites by acylating its biological
target (given that the 4-chloroaniline is a relatively good leaving
group); however, studies with analogues where the anilide is
replaced with a urea (described below) suggest that this
mechanism is unlikely. In summary, 1 was selected for hit-to-
lead optimization because of good whole-cell activity on T. brucei,
no cytotoxicity on mammalian cell lines, reasonable exposure
into the central nervous system, and acceptable solubility.
Moving forward, the goal was to design and synthesize chemical
analogues to further optimize the antitrypanosomal potency
while improving metabolic stability.
a
Reagents and conditions: (a) N,N′-disuccinimidyl carbonate, THF,
rt; (b) DMF, Br2, rt; (c) NaOH, H2O, reflux 5 h; (d) as in Scheme 1;
(e) K2CO3, R3-boronic acid, tetrakis(triphenylphosphine)Pd(0)
(catalytic), toluene, ethanol, reflux.
Structure−Activity Studies of Analogues of 1. Table 1
shows a number of analogues of 1 and their antiparasitic activity
on T. brucei in vitro. Replacement of the aryl chlorine (R2) with
fluorine (2) leads to a 2-fold improvement in potency. In
contrast, replacement with methyl (3) or cyano (4) reduces
potency about 2-fold, and replacement by methoxy (5) abrogates
activity. Replacement of chloro with various aryl groups (6−8)
abrogates anti-parasite activity.
When the 6-position (R1) of the oxazolopyridine ring in
compound 2 is substituted with Br or CN, there is a 2- to 3-fold
decrease in potency (9, 10) with respect to 2, but in contrast a
phenyl group improves potency, with or without halogen
substitution at the 3 or 4 position of the phenyl ring (11, 12,
13). When the phenyl is substituted with a 4-methoxy, there is no
potency gain (14) and larger substitutions lead to a decrease in
potency (15).
Table 2 shows the effect of substituting or replacing the 2-
furanoyl group with other moieties. While methylation of the 2-
furan ring at the 3- and 5-position is tolerated (16, 17), larger
substitutions such as benzofuran (18) are not tolerated. The 3-
furoyl group is equally tolerated (19), but other amides including
aliphatics, aromatics, and heteroaromatics lead to a mild to
profund decline in activity (20−29) including closely related
tetrahydrofuran (22) and oxazole (24). Also inactives are
sulfonamides (30, 31), while thioamide retains some activity
(32). Interestingly, while methylation at the amide nitrogen is
not tolerated (33), acylation is (34, 35). Complete removal of
the amide (36) and either monobenzylation or dibenzylation
lead to loss and decrease of activity, respectively (37, 38). In an
effort to identify additional R1 moieties with increased metabolic
stability, the replacement of the furanoyl amide with ureas was
explored. Terminally monosubstituted and disubstituted ureas
(39−44) were investigated, and we discovered that some of the
disubstutued ureas are tolerated with the pyrrolidine urea (43)
showing slightly better potency than the similar 2-furanoyl
derivatives.
Synthesis of 1 and Its Analogues. Scheme 1 shows the
synthetic route to make 1 and its analogues (substituted 2-(3-
Scheme 1. Synthesis of Substituted 2-(3-
a
Aminophenyl)oxazolopyridines
a
Reagents and conditions: (a) 2-amino-3-hydroxypyridine, polyphos-
phoric acid, 160 °C, 5 h; (b) SnCl2·2H2O, EtOAc, reflux; (c) R2COCl,
pyridine, cat. DMAP, rt or R2CO2H, EDCI, HOBt, CH2Cl2, rt.
aminophenyl)oxazolopyridines). The substituted 3-nitrobenzoic
acid is condensed with 2-amino-3-hydroxypyridine to generate
the oxazole ring. When R1 is chloro, it can be replaced with
phenyl or substituted phenyl by Suzuki cross coupling (not
shown). Reduction of the nitro group is followed by acylation.
Scheme 2 shows the preparation of compounds containing a
phenyl substituent at the 6-position of the oxazolopyridine ring.
2-Amino-3-hydroxy-5-bromopyridine is made in three steps and
then converted into the oxazolopyridine as in Scheme 1. Bromine
at the 6-position is replaced by phenyl or substituted phenyl by
Suzuki cross coupling.
Additional structure−activity relationships came from a study
of the analogues shown in Scheme 3 where changes at the core
were explored. The results show that the oxazolopyridine bicycle
cannot be easily replaced with a monocycle (45−49). Moreover,
the N at the 4-position of the oxazolopyridines is essential for
activity, as it cannot be replaced with C or moved to the 5-
position (50−57).
An alternative to the oxazolopyridine core came by replacing
the oxygen with a nitrogen in the bicycle, leading to the
imidazopyridine core (Table 3). These analogues were made as
C
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a
Table 2. 2-(3-Aminophenyl)oxazolopyridines Containing Variations of the N-Linked Group
b
c
compd
R1
R2
R3
EC50 (μM)
CC50 (μM)
d
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
5-methylfuran-2-carbonyl
3-methylfuran-2-carbonyl
benzofuran-2-carbonyl
3-furanoyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
0.20
0.1
>50
d
F
>50
d
F
>10
0.15
>20
(>50)
>10
7.1
>50
d
F
>50
d
acetyl
F
>50
trifluoroacetyl
Cl
F
tetrahydrofuran-2-carbonyl-
benzoyl
d
Cl
F
>50
d
oxazole-5-carbonyl-
2-thiophenoyl
1.9
>50
d
Cl
F
1.5
>50
d
3-pyridinecarbonyl-
1-methyl-1H-imidazole-5-carbonyl
pyrazine-2-carbonyl-
N-methylpyrrole-2-carbonyl-
methylsulfonyl
7.0
>50
d
F
>20
0.9
>50
d
F
>50
d
F
1.1
>50
d
Cl
Cl
F
6.1
>50
d
phenylsulfonyl
>20
0.41
>10
0.5
>50
d
2-furancarbothioyl-
2-furanoyl
>50
d
methyl
F
>50
2-furanoyl
2-acetyl
F
def
, ,
2-furanoy
2-furanoyl
F
0.12
>20
>20
1.1
>50
H
H
Cl
F
benzyl
H
d
d
d
d
d
benzyl
benzyl
H
F
>50
>50
>50
>50
>50
>50
methylcarbamoyl
isopropylcarbamoyl-
phenylcarbamoyl-
dimethylcarbamoyl-
1-pyrrolidinoyl-
F
3.8
H
Cl
Cl
Cl
Cl
Cl
1.0
H
12.0
0.4
H
ef
,
H
0.09
1.9
1-piperidinoyl-
H
a
b
Controls are shown at bottom of Table 1. Concentration of compound required to inhibit growth by 50% (EC50) of T. brucei strain BF427 or T.
c
d
brucei rhodesiense (numbers in parentheses). Concentration of compound required to inhibit growth by 50% (CC50) of mammalian cell lines. Rat
myoblasts (L6). Human lymphoblasts (CRL-8155). Human hepatocytes (HepG2).
e
f
Scheme 3
in Scheme 1 starting from the appropriately substituted 2,3-
diaminopyridines (not shown). Compound 58 showed com-
parable potency to 2, and chloro and fluoro substitution at C6
(R2) of the core was also beneficial (59, 60). As expected from
the previous SAR, chloro substitution at C5 (61) led to an
inactive compound and it was shown that chloro substitution was
also tolerated at C7 (62). In general the SAR for the
imidazopyridines was found to be similar to that for the
oxazolopyridines (only selected compounds are shown). The
pyrrolidine urea was a good replacement for the furanoyl amide,
as was the case for the oxazolopyridines (63). An expected
improvement in potency was further achieved by adding a phenyl
group at the 6 position of the core which led to compound 64
with an EC50 against T. brucei of 2 nM. A wide variety of
substituents can be added to this 6-phenyl group, and the phenyl
can also be replaced with several heterocyclic groups with
maintenance of anti-parasite potency (65−86).
Compound 64 was studied in detail. It produced a very steep
dose response curve for antiparasitic activity on T. brucei in vitro
with an EC90 of 2 nM, and complete growth inhibition was
D
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Journal of Medicinal Chemistry
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a
Table 3. 2-(3-Aminophenyl)imidazopyridines Containing a N-Linked 2-Furanoyl Group
b
c
compd
R1
R2
EC50 (μM)
CC50 (μM)
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
2-furanyl
H
0.2
df
,
e
2-furanyl
Cl
0.07
0.2
>50, 1.7
2-furanyl
F
2-furanyl
5-Cl
7-Cl
Cl
10
2-furanyl
0.12
0.05
0.002
0.01
0.005
0.002
0.005
0.003
0.002
0.03
0.02
0.01
0.004
0.002
0.004
0.005
0.01
0.004
0.01
0.005
0.02
0.005
0.03
0.06
0.04
d
ef
,
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
N-pyrrolidinyl
15, >50
e
f
phenyl
24, 30
e
f
3-methoxyphenyl
2-methoxyphenyl
3-Cl-phenyl
3.6, 35
e
f
13, >50
e
f
2.8, >50
2-chlorophenyl
3-acetylphenyl
e
f
3-Me-phenyl
5, 10
e
f
f
f
3-trifluoromethoxyphenyl
3-methyl-4-fluorophenyl
3-NH2-phenyl
11, >50
e
>50
e
f
5, 40
e
3-furanyl
25, >50
e
f
3-thiophenyl
6, >50
e
2-thiophenyl
9
e
3-pyridyl
14, >50
e
f
5-(2-chloropyridyl)
4-(2-chloropyridyl)
5-(3-methylpyridyl)
5-(2-methoxypyridyl)
5-(3-pyrrolidino)
5-(3-chloropyrimidinyl)
5-pyrimidinyl
3.8, >50
ef
,
>50
>50
ef
,
ef
,
>50
e
f
8, >50
e
f
f
38, >50
e
12, >50
e
f
5-(2-methoxypyrimidinyl)
5-(2-chloropyrimidinyl)
3.9, 9.8
d
14.3
a
b
Controls are shown at bottom of Table 1. Concentration of compound required to inhibit growth by 50% (EC50) of T. brucei strain BF427 or T.
c
d
brucei rhodesiense (numbers in parentheses). Concentration of compound required to inhibit growth by 50% (CC50) of mammalian cell lines. Rat
myoblasts (L6). Human lymphoblasts (CRL-8155). Human hepatocytes (HepG2).
e
f
observed. Cytotoxicity toward mammalian cells was minimal
(24−30 μM, Table 3). Along with other analogues, it was
characterized in in vitro ADME assays (Supporting Information
Table 1) and showed aqueous solubility at pH 7.4 of 8.1 μM
which increased to 32 μM at pH 2.0, presumably because of
imidazole ring protonation. Compound 64 was metabolized
upon incubation with mouse and human liver microsomes in
vitro with half-lives of 10 and 31 min, respectively. This
represented a significant improvement in metabolic stability over
the initial lead 1, presumably because the latter is more sensitive
to hydrolysis. Compound 64 was a modest inhibitor of hepatic
cytochrome P450 3A4-isoform in vitro with an IC50 of 1.5 μM.
Oral gavage dosing of 64 in mice at 50 mg/kg gave a maximal
concentration in the plasma (Cmax of 4.3 μM) with a time to Cmax
(Tmax) of 30 min. The plasma concentration versus time curve is
provided as Figure 1 of Supporting Information. The area under
the plasma concentration versus time curve (AUC) after oral
dosing was 1125 min·μM. The half-life for elimination from the
plasma was 204 min, and the plasma clearance (Clp) was 14.8 mL
min−1 kg−1. The in vivo clearance was substantially lower than
what was expected from the in vitro metabolism in liver
microsmes likely because of the high plasma protein binding
(95% in mouse plasma). Compound 64 showed excellent
permeability across MDCK-MDR1 cells and was not a significant
substrate for the MDR1 efflux pump (Papp = 401 and 503
(+MDR1 inhibitor) nm/s). Consistent with this is the significant
distribution of 64 into mouse brain, with a brain-to-plasma
concentration ratio 60 min after ip dosing of 0.5 and 1.1 (two
independent mouse experiments). PK studies of 64 in rats are
summarized in Supporting Information Figure 2 and Table 2.
Briefly, rats were dosed by iv injection and by oral gavage and the
bioavailability of 64 was 35% upon delivery of a 21.3 mg/kg oral
dose. The apparent volume of distribution during steady state
(VSS) was 2.4 L/kg. PK data in mice for additional compounds
are provided in Supporting Information Table 2.
Analysis of Efficacy of 64 in Parasite-Infected Mice.
Given the good physiochemical and PK properties of 64, we
decided to test it for efficacy in a mouse model of acute HAT
infection. Mice were injected with T. brucei rhodesiense on day 0,
and 64 was given by oral gavage 48 h after parasite injection. Mice
were dosed for a total of 5 days using the dosing regimens shown
in Table 4. Parasitemia in the blood and survival were followed
out to 60 days. Cures in all mice were obtained with once and
twice dosing at 20 and 50 mg/kg (Table 4). With 5 mg/kg
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a
overnight at 37 °C, the tube was centrifuged at 1000g for 20 min at 37
°C. An amount of 7 mL of supernatant was transferred to a new tube,
and buffer was removed in a Speed-Vac concentrator. Then 1 mL of
distilled water was added to the residue, and the sample was
concentrated to dryness as above. To the residue was added 0.3 mL
of water and 0.3 mL of methanol. The amount of compound was
determined by HPLC using a C18 reverse-phase column. HPLC of a
standard amount of test compound was used to convert the UV peak
area to moles of compound. In some cases, solubility was carried out in
0.01 M HCl.
Distribution of Compounds between Mouse Plasma and
Brain. Mice (in groups of 3) were injected with test compounds (5 mg/
kg ip) and sacrificed at the indicated times for collection of plasma and
brain. Compound was dissolved in 0.4 mL of dosing solution (7%
Tween 80, 3% ethanol, 5% DMSO, 0.9% saline) for ip injections. The
brains were weighed and immediately frozen, then later homogenized in
acetonitrile using a Dounce homogenizer. Prior to animal studies,
recovery of test compound was carried out by adding a known amount
to a mouse brain in the test extraction solvent and performing the
homogenization. Compound recovery was determined by liquid
chromatography/tandem mass spectrometry analysis relative to a
standard compound amount. Blood was taken from the same mice in
heparinized capillary tubes (Fisherbrand) for determination of
compound concentration in plasma. The concentration of compound
in the brain was obtained by dividing the moles of compound in the
brain by the brain volume (obtained from the brain weight assuming 1 g
is 1 mL) and correcting for the brain vasculature volume of 2.5% by
weight.
In Vitro Parasite Growth Arrest Assay. Compounds were tested
for antiparasitic activity on T. brucei brucei (strain BF427) as described.13
Cells were tested in triplicate in the presence of serial dilutions of
compound, and growth is quantified with AlamarBlue.14 Pentamidine
isothionate (Aventis, Dagenham, U.K.) was included in each assay as a
positive control (EC50 = 1.2 0.3 nM).
Mammalian Cell Cytotoxicity Assay. Selected compounds were
tested for cytotoxicity at University of Washington against CRL-8155
cells (human lymphoblasts) and HepG2 cells (human hepatocellular
carcinoma) as previously described (PMID, 22720744). Briefly, cells
were exposed to serial dilutions of compounds for 48 h and cytotoxicity
was assayed using the AlamarBlue (Life Technologies). Each dilution
was assayed in quadruplicate with standard error of the mean values
averaging <15%. Concentrations causing 50% growth inhibition (CC50)
were calculated by nonlinear regression using GraphPad Prism software
(San Diego, CA). L6 rat myoblasts were tested by similar methods at the
Swiss TPH as previously described (PMID, 12512078).
In Vitro Liver Microsome Studies. Liver microsome metabolism
studies were carried out as described.15,16 Compounds were incubated
with liver microsomes (mouse or human), and samples were extracted at
time points for liquid chromatography/tandem mass spectrometry
analysis. Reference drugs dextromethorphan and testosterone were used
to standardize each assay.
Inhibition of Liver Cytochrome P450s. Inhibition of human
cytochrome P450 (3YP3A4 isoform) by test compounds was measured
with a commercial kit (BD Biosciences) according to the manufacturer’s
instructions.
Permeability across Monolayers of MDCKII-MDR1 Cells. This
assay uses Madin−Darby canine kidney cells that were transfected with
the human MDR1 (P-gp) gene.10−12 Permeability across these
monolayers was measured in triplicate as described.17 The assay was
performed with and without the addition of GF-120918, an inhibitor of
the MDR1 efflux pump to determine if the compound is a pump
substrate. Propranolol was used as a permeable, non-MDR1 substrate
control, and amprenavir was used as a permeable, MDR1 substrate
control.
Table 4. Efficacy of 64 in T. brucei Infected Mice
b
expt
dose (mg/kg)
doses per day
cured
day of euthanasia
1
1
1
1
1
2
2
2
2
2
2
50
20
5
2
2
2
1
2
1
2
1
2
2
2
5/5
5/5
5/5
5/5
0/5
5/5
5/5
2/5
2/5
0/5
0/5
60
60
60
60
4
50
Veh
20
5
60
60
5
13, 18, 27, 60, 60
16, 16, 20, 60, 60
4, 4, 9, 11, 13
4
2.5
1
Veh
a
Mice were infected on day 0 and treated for 5 consecutive days
beginning 48 h after infection when patent parasitemia was detectable.
Mice that cleared parasites and remained parasite free through day 60
b
after infection were declared cured.
dosing, all mice were cured with twice daily dosing, and 2 of 5
mice were cured with once daily dosing. Twice daily dosing at 2.5
mg/kg cured 2 of 5 mice, and twice daily dosing with 1 mg/kg
cured 0 of 5 mice. These results show that 64 is highly efficacious
in a mouse model of acute trypanosomiasis.
Mice dosed with 64 orally at 50 mg/kg twice per day for 14
days had no difference in weight change compared to vehicle
treated mice and no observed clinical toxicity, indicating good
tolerability of the compound above fully efficacious doses.
CONCLUDING REMARKS
■
A high throughput phenotypic screen successfully identified the
oxazolopyridines as antiparasitic agents against T. brucei.
Medicinal chemistry optimization to improve potency and PK
properties led to the discovery of the imidazopyridines as a novel
class of potent antiparasitic T. brucei compounds. The substituted
2-phenylimidazopyridine 64 is a highly potent antiparasitic agent
against T. brucei and cures parasite-infected mice when given
orally when dosed down to 2.5 mg/kg. Thus, 64 represents a
significant lead compound for the development of a new drug
class to treat HAT. Studies are in progress to further optimize the
druglike properties of 64 and to explore its potential in the
treatment of second stage (central nervous system involvement)
HAT. Additional toxicology studies are also in progress as part of
a preclinical drug development program.
EXPERIMENTAL SECTION
■
The synthesis of all compounds is given in Supporting Information. All
target compounds were purified by preparative HPLC and are judged to
be >95% pure.
Phenotypic Screen for T. brucei Growth Arrest Compounds.
To screen compound libraries for antiparasitic activity on T. brucei, we
employed the bloodstream form of the well-characterized T. brucei
isolate, Lister 427. During the screen, the parasite was grown in 1536-
well plates in 5.5 μL of HMI-9 medium in the presence of library
compounds. All wells including negative controls contained a final of
0.4% DMSO. After incubation of the plates at 37 °C for 48 h, the parasite
density was determined using the CellTiter-Glo reagent (Promega), a
firefly luciferase assay system that measures the amount of cellular ATP
present in plate wells.
PK Studies in Mice. Test compound was administered to mice by
oral gavage followed by blood sampling at intervals of 30, 60, 120, 180,
240, and 360 min. Compound was dosed orally at 50 mg/kg in 0.2 mL of
dosing solution (7% Tween 80, 3% ethanol, 5% DMSO, 0.9% saline).
Experiments were performed with groups of three mice per
compound as published.17,18 Plasma was separated and extracted with
Solubility Measurement. A glass tube containing 8 mL of buffer
(20 mM ammonium acetate, pH 7.4) was placed in a 37 °C water bath.
The test compound was added from a DMSO stock solution to give the
desired concentration of compound with 1% DMSO final concen-
tration. The tube was incubated at 37 °C for 5−10 min. Sufficient
compound was used to give a visibly cloudy solution. After incubation
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acetonitrile for measurements of compound concentrations by liquid
chromatography/tandem mass spectrometry.
Notes
The authors declare no competing financial interest.
PK Studies in Rats. Test compound was administered to Sprague−
Dawley jugular canulated rats (Charles River) by either oral gavage or iv
injection followed by blood sampling from the jugular vein at 5, 15, 30,
60, 90, 120, 240, 360, 480, 720, and 1440 min. The oral dose was
administered to each rat at 20 mg/kg at time = 0 in a 1 mL volume of
dosing solution (7% Tween 80, 3% EtOH, 5% DMSO, 09.% saline). The
iv injections were administered at 5 mg/kg from time = 0 to time = 3 min
in a 1 mL volume of dosing solution, and blood was sampled at the same
time points via the jugular vein. Experiments were performed with
groups of two rats each for the oral and iv dosing. Plasma was separated
and extracted with acetonitrile for measurements of compound
concentrations by liquid chromatography/tandem mass spectrometry.
PK calculations were performed using Phoenix WinNonlin software
(Pharsight).
ACKNOWLEDGMENTS
■
This work was supported by grants from the National Institutes
of Health (Grants AI054384, AI070218, and AI106850) and The
Consortium for Parasitic Drug Development (CPDD).
ABBREVIATIONS USED
■
AUC, area under the plasma concentration−time curve; Clp,
plasma clearance; HAT, human African trypanosomiasis;
MDCK, Madin−Darby canine kidney cells; MDCK-MDR1,
MDCK cells transfected with human P-glycoprotein; PK,
pharmacokinetics
Plasma Protein Binding. Dialysis membrane sheet (MW cutoff 3.5
kDa, HTDialysis catalog no. 1135) was soaked for 1 h in distilled water
and then in 20% ethanol for 30 min. The membrane was clamped
between two Teflon plates containing a row of opposing wells. Test
compound in DMSO was added to 0.12 mL of serum to give 9 μM. A
small aliquot was taken as a 100% recovery standard, and the solution
was placed on one side of the membrane. The well on the other side of
the membrane was charged with an equal volume of dialysis buffer (14.2
g/L Na2HPO4, 8,77 g/L NaCl, pH 7.4), and the block was placed on an
orbital shaker for 18 h at 37 °C. An aliquot was taken from each side of
the membrane. One-fourth volume of acetonitrile was added to each
aliquot, and the samples were centrifuged to precipitate protein. Test
compound in the supernatants was quantified by liquid chromatog-
raphy/tandem mass spectrometry to determine the concentration on
each of the membrane and the total recovery of test compound from the
device. A control dialysis was carried out with dialysis buffer on both
sides of the membrane to ensure that equilibration of test compound
across the membrane was achieved. The fraction of compound bound to
protein was calculated as bound/(unbound + bound), and the value was
taken only if compound recovery was >50% (recovery was 84% for 64).
Anti-Parasite Efficacy Studies in Mice. Studies were carried out
using the standard operating procedure used by WHO screening
centers.19 The studies were done in compliance with University of
Washington IACUC approved protocol. Female Swiss-Webster mice
age 6−8 weeks in groups of 5 were infected with 2 × 104 T. brucei
rhodesiense STIB 900 strain on day 0, then administered compound (in
three dose levels) or vehicle for 5 days. Treatments were administered
orally at a dose and schedule anticipated to maintain plasma
concentrations well above the EC50 (see Table 4). The first dose was
48 h after parasite injection, and dosing was 12 or 24 h apart. Dosing was
carried out by oral gavage as for PK studies (see above). Parasitemia was
monitored for 60 days by microscopic analysis of blood collected from
tail bleeds. Cures were defined by sustained clearance of microscopic
parasitemia through the end of the 60-day observation period. Mice
were euthanized when high levels of parasitemia were evident on
peripheral blood slides.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis of compounds and pharmacokinetics data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*F.S.B.: phone, 206-616-9214; e-mail, fbuckner@u.washington.
■
edu.
*M.H.G.: phone, 206-543-7142; e-mail, gelb@chem.
washington.edu.
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Structure-based design of submicromolar, biologically active inhibitors
of trypanosomatid glyceraldehyde-3-phosphate dehydrogenase. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 4273−4278.
Present Address
∞A.K.C.: California Institute for Biomedical Research, 11119 N.
Torrey Pines Road, No. 100, La Jolla, CA 92037.
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NOTE ADDED IN PROOF
■
Just prior to the submission of our manuscript, Baell and co-
workers reported on the initial structure−activity relationships
centered around compound 1. This is the result of an
independent phenotypic screen to discover anti-T. brucei
scaffolds.20
H
dx.doi.org/10.1021/jm401178t | J. Med. Chem. XXXX, XXX, XXX−XXX