Optimization of chloronitrobenzamides (CNBs) as therapeutic leads for human African trypanosomiasis (HAT)

Article abstract of DOI:10.1021/jm301687p

We previously reported the discovery of the activity of chloronitrobenzamides (CNBs) against bloodstream forms of Trypanosoma brucei. Herein we disclose extensive structure-activity relationship and structure-property relationship studies aimed at identification of tractable early leads for clinical development. These studies revealed a promising lead compound, 17b, that exhibited nanomolar potency against T. brucei (EC ₅₀ = 27 nM for T. b. brucei, 7 nM for T. b. rhodesiense, and 2 nM for T. b. gambiense) with excellent selectivity for parasite cells relative to mammalian cell lines (EC₅₀ > 25 μM). In addition compound 17b displayed suitable physiochemical characteristics and microsomal stability (t_1/2 > 4 h for human and mouse) to justify pursuing in vivo studies.

Full text of DOI:10.1021/jm301687p

Article
pubs.acs.org/jmc
Optimization of Chloronitrobenzamides (CNBs) as Therapeutic Leads
for Human African Trypanosomiasis (HAT)
Jong Yeon Hwang,† David Smithson,† Fangyi Zhu,† Gloria Holbrook,† Michele C. Connelly,†
Marcel Kaiser,‡,§ Reto Brun,‡,§ and R. Kiplin Guy*,†
†Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis,
Tennessee 38105-3678, United States
‡Department of Medical Parasitology, Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland
§University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland
S
* Supporting Information
ABSTRACT: We previously reported the discovery of the
activity of chloronitrobenzamides (CNBs) against bloodstream
forms of Trypanosoma brucei. Herein we disclose extensive
structure−activity relationship and structure−property rela-
tionship studies aimed at identification of tractable early leads
for clinical development. These studies revealed a promising
lead compound, 17b, that exhibited nanomolar potency
against T. brucei (EC50 = 27 nM for T. b. brucei, 7 nM for
T. b. rhodesiense, and 2 nM for T. b. gambiense) with excellent
selectivity for parasite cells relative to mammalian cell lines (EC50 > 25 μM). In addition compound 17b displayed suitable
physiochemical characteristics and microsomal stability (t1/2 > 4 h for human and mouse) to justify pursuing in vivo studies.
brucei brucei.14 The preliminary structure−activity relationship
INTRODUCTION
■
(SAR) studies reported in the previous paper identified the
The eukaryotic protozoan parasite Trypanosoma brucei causes
electrophilic chloronitrobenzamide group as essential for
human African trypanosomiasis (HAT), a major health concern
potency and demonstrated that the para-chloro substituent on
in sub-Saharan Africa with an estimated annual morbidity
the aniline improved ligand efficiency relative to the original lead
exceeding 50000 cases and 60 million at risk of infection.1 HAT is
and had improved potency compared to the methyl or methoxy
caused by two subspecies of trypanosomes (Trypanosoma brucei
substituent (Figure 2). Because of our prior work with a related
gambiense and Trypanosoma brucei rhodesiense) that are vectored
scaffold active against the thyroid hormone receptor, we are
between human and cattle hosts by the bite of infected tsetse
aware that this series is weakly electrophilic and in the context of
flies. The two subspecies vary in the severity of disease, with T.
an organized binding site with a properly positioned nucleophile
can form covalent complexes.15−17 We presume the compounds
brucei gambiense causing primarily a chronic disease and T. brucei
rhodesiense causing primarily an acute disease.2 There are two
are acting as irreversible inhibitors of a critical enzyme in the
parasite.
The first-generation CNBs showed moderate potency against
stages of HAT: First (early) stage that is characterized by a
dispersed hemolymphatic infection, and second (late) stage that
is characterized by infection of the central nervous system.
T. brucei brucei (EC50 = 1.5 μM). This led us to test our existing
Second stage HAT is fatal if left untreated. Currently available
collection of chloronitrobenzamides (CNBs) bearing various
drugs for first stage disease are suramin and pentamidine and for
aryl (2) and heteroaryl anilines (3). The preliminary SAR
developed from that study prompted us to carry out more
extensive studies using novel compounds designed to map the
second stage disease melarsoprol and eflornithine (Figure 1).1
All of these therapies were established more than 20 years ago,
and the current drugs have serious side effects, poor clinical
effect, and are hampered by emerging drug resistance.3,4
Recently, a new drug, nifurtimox, was developed as a
combination therapy with eflornithine for second stage T. b.
gambiense.5 There remains an urgent need for new drugs that
possess good safety, exhibit efficacy against both disease forms,
and are easy to administer.6,7 Recently, several groups, including
ours, have reported the use of phenotypic screening to identify
new inhibitors of T. brucei spp. growth.8−13
elements that are responsible for antitrypanosomal activity. Here
we report the result from these studies and the finding that the
series shows promise for further development.
RESULTS AND DISCUSSION
■
Testing Existing CNBs to Define Preliminary SAR. A set
of 231 CNBs from our in-house collection was screened, as
We previously reported that chloronitrobenzamides (CNBs,
Figure 2, 1) block the proliferation of cultured Trypanosoma
Received: November 15, 2012
Published: March 13, 2013
© 2013 American Chemical Society
2850
dx.doi.org/10.1021/jm301687p | J. Med. Chem. 2013, 56, 2850−2860

Journal of Medicinal Chemistry
Article
Figure 1. Currently available drugs for the treatment of African trypanosomiasis.
Figure 2. Initial active CNB (1) and in-house collection (2 and 3).
previously described, to test their potency in blocking
proliferation of bloodstream stage T. brucei brucei after 48 h of
treatment.14,18 The identity and purity of all stocks were
confirmed by UPLC/MS as correct and >90%, respectively,
prior to use. The CNBs were clustered into five representative
subgroups based on the identity of the aryl aniline.
Representative compounds from each group are summarized
in Figure 3. Compounds not presented were either weaker than
those shown or inactive.
Overall, most compound groups contained some relatively
potent compounds and there were no strong structure−activity
relationships; activity varied by less than 15-fold through the
entire set. Thus the data was most useful in focusing subsequent
synthetic work and no driving trends in structure−activity
relationships were uncovered. In group 1, three phenyl CNBs
(4a−4c), with 3-CF3, 3-CF3-6-Cl, and 3-Cl-4-F substituents,
respectively, showed submicromolar activity (EC50 = 0.95, 0.52,
and 0.95 μM, respectively). Group 2 contained benzoxazolyl-
CNBs bearing substituted phenyl moieties. The most potent
compound in group 2 was 5a, with a 4-fluorophenyl substituent
(EC50 = 0.64 μM). Group 3 contained substituted phenyl-
thiazolyl-CNBs. Three compounds in group 3 (6a−6c) showed
good inhibitory potency (EC50 = 0.37, 0.4, and 0.54 μM,
respectively). Group 4 contained benzothiazolyl-CNBs. Two
compounds with methoxy substitution (7a and 7b) showed
submicromolar activity (0.5 and 0.98 μM, respectively). The
closely related imidazolyl-CNB (7e) was inactive. Group 5
contained varied heteroaryl substituted phenyl rings and had
highly variable activity, which we termed phenylheteroarylchlor-
onitrobenzamides (PCNBs). Compound 8a, bearing a benzox-
azolyl group at the 3-position on the phenyl ring, showed good
antitrypanosomal potency (EC50 = 0.29 μM). However, the SAR
in this subset was strong and trends were not clear from available
compounds with meta-substituted benzoxazoles (8a) being
significantly more potent than para-substituted 8c (6.1 μM)
but with closely related analogues being essentially inactive (8b).
The pyridinoxazolyl-PCNB (8d) showed relative weak potency
(3.1 μM), and replacement of the benzoxazole with
benzimidazole (8e) was tolerated. Thus, overall the studies
suggested there could be benefit in potency from extending the
aryl group to include a second or third aryl ring, but that such
substitutions would need to be carefully explored, both for
optimal potency and for negative impact on physiochemical
properties.
Refining SAR of CNBs. On the basis of our data, we
synthesized a limited number CNB analogues to further explore
SAR and structure−property relationships (SPR). All CNBs
studied in this paper were synthesized using amide-coupling
reactions with acids or acid chlorides and amines. Generally,
compounds were purified to greater than 95% purity, as judged
by UPLC/UV/ELSD, and the identity confirmed by MS and
NMR.19 The antitrypanosomal activity of all compounds was
evaluated against Trypanosoma brucei brucei using our standard
proliferation inhibition assay. The growth inhibitory potency of
all CNBs was also tested against four human cell lines using our
standard assay: Raji, a Burkitt’s lymphoma derived line; BJ, a
foreskin fibroblast derived line; HEK 293, an embryonic kidney
fibroblast derived line; and HepG2, a hepatocellular carcinoma
derived line. Kinetic aqueous solubility and permeability were
also measured using standard high throughput methods. The
data are summarized in Table 1.
2851
dx.doi.org/10.1021/jm301687p | J. Med. Chem. 2013, 56, 2850−2860

Journal of Medicinal Chemistry
Article
Figure 3. Representative subtypes of tested pre-existing CNB collection. The compounds existed in the corporate collection and were tested without
resynthesis from existing DMSO stocks. The identity and purity of all stocks were confirmed as correct and >90%, respectively, prior to use. In all
subsequent studies, all compounds were resynthesized prior to testing; for compounds tested from both sources, there is some variance in potency,
presumably due to compound precipitation during storage for the DMSO stocks. We report data from pre-existing stocks in this figure and data from
resynthesized materials below.
In the first study, small electron withdrawing groups on the
“inner” ring provided the best potency and this trend was further
explored. For example, two additional electron-withdrawing
substituted phenyl CNBs in group 1, 4-Cl-3-CF3 (9a) and di-CF3
(9b) phenyl CNBs, were roughly equivalent to the initial lead
compound (1). Several similar molecules in group 3 with
electron-withdrawing substitutents on the phenyl ring attached
to the thiazole (10a−10c) all displayed weak potency compared
to their original hits (6a−6c). In the group 4 series, all electron-
withdrawing substituted compounds (11a−11c) gave weak
inhibitory potency. These data suggested that potency was not
strongly driven by the electron withdrawing character of
substitutents.
In group 5, we synthesized benzothiazole substituted PCNBs
with the heterocycle attached to an “interior” phenyl ring.
Among benzothiazolyl-PCNBs (12a−12d), the meta compound
2852
dx.doi.org/10.1021/jm301687p | J. Med. Chem. 2013, 56, 2850−2860

Journal of Medicinal Chemistry
Article
Table 1. Activities and Physical Properties of Synthesized CNBs
a
b
c
Values are the mean of six replicates with errors reported as the 95% confidence interval. Values are the mean of three replicates. Solubility and
d
e
permeability (PAMPA) were measured in PBS buffer (at pH 7.4). The values within parentheses were measured at pH 3.0. DL, detection limit. nd,
not determined. These values were previously reported.14 Remeasured simultaneously with other compounds in this table.
f
g
2853
dx.doi.org/10.1021/jm301687p | J. Med. Chem. 2013, 56, 2850−2860

Journal of Medicinal Chemistry
Article
Table 2. Anti-trypanosomal Activity against T. b. rhodesiense and T. b. gambiense Strains
trypanosomal activity (EC50, μM)
a
b
b
T. b. brucei
T. b. rhodesiense
T. b. gambiense
compd
221
STIB900 wt
STIB930
DAL 898R
KO48
c
melarsoprol
nd
0.0072
0.0006
3.8
0.0067
0.0007
0.42
0.012
0.0009
4.4
0.014
0.051
2.6
pentamidine
0.012
d
5a
6.0 2.1
d
8a
0.48 0.07
1.1 0.2
2.5 1.2
0.37
1.8
0.21
0.62
1.6
0.46
1.3
9a
0.82
10b
12b
1.5
0.56
1.6
0.63
0.17
0.19 0.03
0.16
0.11
0.20
a
b
Values are the mean of six replicates with errors reported as the 95% confidence interval. Values are mean of two replicates; the individual values
c
d
differed less than 50%. nd, not determined. Compound was resynthesized and re-evaluated in this study.
Table 3. Activities and Physical Properties of Substituted Benzoxazolyl- and Benzothiazolyl-PCNBs
EC50 (μM)
a
b
b
b
b
c
c
no.
X
R
T. b. brucei
HEK
HEPG2
Raji
BJ
solubility (μM)
PAMPA (× 10−6cm/s)
8a
O
O
O
O
O
O
S
H
0.48 0.07
0.02 0.01
0.12 0.02
0.22 0.04
0.30 0.06
0.17 0.02
0.19 0.03
0.006 0.001
0.68 0.14
0.12 0.036
3.6 1.6
>25
>14
>25
>25
>25
>25
>25
>14
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>14
>25
>25
>25
>25
>25
>14
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>14
>25
>25
>25
>25
>25
>14
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>14
>25
>25
>25
>25
>25
>14
>25
>25
>25
>25
>25
>25
>25
>25
>25
0.47
0.31
0.09
0.09
45
<DL
274
105
214
366
3
13a
13b
13c
13d
13e
12b
14a
14b
14c
14d
14e
14f
14g
14h
14i
2-Me
2-MeO
3-Me-4-OH
2-F
d
<DL
4-F
0.27
0.58
<DL
<DL
<DL
0.26
0.43
0.023
<DL
0.49
0.10
0.41
H
S
2-Me
6-Me
2-MeO
4-MeO
3-Me-4-OH
4-OH
2-F
41
S
<DL
<DL
<DL
48
S
S
S
>10
S
>33
<DL
191
17
S
0.16 0.02
0.25 0.15
0.24 0.05
0.32 0.06
S
4-F
S
2-Cl
<DL
14j
S
4-Cl
3
a
b
c
Values are the mean of six replicates with errors reported as the 95% confidence interval. Values are the mean of three replicates. Solubility and
d
permeability (PAMPA) were measured in PBS buffer (at pH 7.4). DL, detection limit.
(12b) stood out with significantly improved antitrypanosomal
potency, more than 5-fold more potent than the para compound
(12a). This follows the same trend that was observed with
benzoxazolyl-PCNBs (8a and 8c). The ortho-oriented product
(12c) was inactive. The N-methyl substitution (12d) reduced
activity. Several sterically similar analogues had poor potency,
including the benzimidazole (12e) and the biphenyl compounds
(12f and 12g). Because this subseries showed both a significant
improvement in potency relative to the original lead and a strong
SAR, it was selected for further optimization that will be
discussed below.
selectivity index for T. brucei. Meanwhile, solubility and
permeability of CNBs were evaluated in the PBS buffer (pH
7.4) to provide early structure−property relationships. CNBs
generally possessed poor solubility (<1.5 μM) and permeability.
The most potent compound from the preliminary studies, 12b,
was also tested in acidic conditions (pH 3), but no improvement
was seen compared to neutral conditions (0.51 μM for solubility
and 51 × 10−6 cm/s for PAMPA). No correlation was observed
between cellular activity and either solubility or permeability.
Because we have previously seen high species differences with
antitrypanosomals, we evaluated selected CNBs against T. b.
gambiense and rhodesiense, the subspecies causing human disease
(Table 2). All compounds showed antitryanosomal potency in
these species similar to that seen with T. b. brucei. This suggests
In general, most of CNBs had little or no mammalian
cytotoxicity. The most potent compound (12b) was not toxic in
the testing concentration range, indicating that it had a high
2854
dx.doi.org/10.1021/jm301687p | J. Med. Chem. 2013, 56, 2850−2860

Journal of Medicinal Chemistry
Article
a
Scheme 1
a
Reagents and conditions: (a) amines, diisopropylethylamine, dichloromethane, rt, overnight; (b) R1R2NH, diisopropylethylamine, tetrahydrofuran,
rt, overnight; (c) LiOH, H2O, THF, rt, 3 h; (d) amine, PyBOP, diisopropylethylamine, tetrahydrofuran, rt, overnight; (e) R4X, K2CO3,
dimethylformamide, rt, overnight.
that there is effectively no species variation in sensitivity to the
CNBs. Benzothiazolyl-PCNB (12b) showed significant potency
against T. b. brucei (0.19 μM EC50) and all other strains (0.16 μM
for STIB900 wt, 0.11 μM for STIB930, 0.20 μM for DAL898R,
and 0.17 μM for KO48). Benzoxazolyl-PCNB (8a) also
displayed submicromolar activity in all species. Others had
moderate antitrypanosomal activity. In general, STIB930 was
slightly more sensitive against PCNBs than other species.
Focus on the PCNBs. These studies suggested that expanded
examination of the SAR within the benzoxazolyl and
benzothiazolyl PCNB series was warranted, especially studying
effects of substituents on the phenyl ring (B-ring) (Table 3).
Substitution of the PCNB phenyl ring dramatically affected
antitrypanosomal potency in both benzoxazolyl (13) and
benzothiazolyl-PCNBs (14) in a parallel manner. Introducing a
2-methyl substituent (13a and 14a) significantly increased
potency (20 nM and 6 nM EC50 values, respectively), whereas a
6-methyl substituent (14b) decreased potency. 4-Hydroxy and
methoxy substituents (14d−14f) led decreased activity. The
fluoro (13d−13e and 14g−14h) or chloro (14i−14j)
substituted compounds were almost equipotent with unsub-
stituted compounds. Although most of compounds in this series
showed very good inhibitory potency, this series had very poor
solubility. The most potent compounds (13a and 14a) in this
series showed particularly poor solubility and permeability. No
compound exhibited significant growth inhibitory potency
against any mammalian cell line.
retaining potency. 4-Fluoro-2-chloro-4-nitrobenzoyl chloride
(15) was reacted with benzothiazolyl anilines in the presence
of DIEA to give the corresponding compounds 16 (Scheme 1).
Compounds 16 were treated with amines to afford the
corresponding products 17a−17k. Hydroxyl and alkoxyl
compounds 17n−17p were synthesized using an amide coupling
reaction with 4-hydroxy-3-chloro-5-nitrobenzoic acid 18,
followed by alkylation with alkyl halides. Ester compounds 17j,
17k, and 17p were hydrolyzed using LiOH to afford the
corresponding acids 17l, 17m, and 17q, respectively. All
compounds were purified and tested as described above and
relevant data are summarized in Table 4.
Interestingly, piperazines (17a−17c) and methylpiperazines
(17d−17f) showed good antitrypanosomal potency (0.027−
0.19 μM EC50 range), whereas others (17g−17m and 17o−17q)
gave slightly weaker potency (0.22−0.80 μM EC50 range).
Hydroxy compound 17n was inactive in this series (>33 μM
EC50). Among the examined subseries, the piperazine 17b with
2-methyl substituent on the B-ring exhibited the best potency
(0.027 μM EC50). No compounds were toxic in the mammalian
cell lines, thus maintaining high selectivity index. However, most
of the compounds in this series had poor aqueous solubility in
both neutral (pH 7.4) and acidic conditions (pH 3.0). The
exception was compound 17d, whose solubility was drastically
increased (46 μM) in acidic media (pH 3.0). The permeability of
the PCNBs in this series was also poor. There was no correlation
between potency and solubility or permeability. Thus, none of
these potentially solubilizing groups provided a strong enhance-
ment of solubility while retaining activity. However, further
attention was focused on the piperizine subseries, which had the
potential for formation of strong acid salts during formulation.
Optimization of the A-Ring of PCNBs. Next, we explored
substituent effects on the A-ring of PCNBs. In particular, we
attempted to utilize polar groups, such as amines and the
carboxylic acids, to improve aqueous solubility of PCNBs while
2855
dx.doi.org/10.1021/jm301687p | J. Med. Chem. 2013, 56, 2850−2860

Journal of Medicinal Chemistry
Article
Table 4. Activities and Physical Properties of Benzothiazolyl-PCNBs Bearing Solubilizing Groups
a
b
c
Values are the mean of six replicates with errors reported as the 95% confidence interval. Values are the mean of three replicates. Solubility and
d
permeability (PAMPA) were measured in PBS buffer (at pH 7.4). The values within parentheses were measured at pH 3.0. DL, detection
limitation. nd, not determined. Compounds were sorted by EC50 value against T. b. brucei.
e
f
2856
dx.doi.org/10.1021/jm301687p | J. Med. Chem. 2013, 56, 2850−2860

Journal of Medicinal Chemistry
Article
Table 5. Summary of Pharmacological Properties of the Selected PCNBs
a
b
Values are the mean of six replicates with errors reported as the 95% confidence interval. Values are the mean of two replicates; the individual
c
d
values differed less than 50%. nd, not determined. Compounds were sorted by EC50 value against T. b. brucei.
To ensure that species selectivity was maintained, several
potent PCNB analogues from this series were tested against T. b.
rhodesiense (STIB900 wt) and T. b. gambiense (STIB930) strains
(Table 5). The 2-methyl substituted compounds (14a, 17b, and
17e) that were most potent against T. b. brucei were also strongly
potent against both T. b. rhodesiense (0.013, 0.007, 0.011 μM,
respectively) and T. b. gambiense (0.036, 0.002, and 0.001 μM,
respectively). Other compounds also exhibited moderate activity
against all T. brucei species. Thus the pan activity against all T.
brucei subspecies, a desirable property in a lead, was maintained.
Meanwhile, several active compounds were tested in human
and mouse liver microsome models to aid in prediction of
metabolic stability. Generally, the PCNBs have reasonable
stability in both human and mouse microsomal models. The
exception was compound 12b, with t1/2 = 0.18 h in mouse
microsomes. Of the current PCNBs, compound 17b had the best
balance of properties including very good antitrypanosomal
potency (27 nM against T. b. brucei, 7 nM against T. b. rhodesiense,
and 2 nM against T. b. gambiense) and good to excellent stability
2857
dx.doi.org/10.1021/jm301687p | J. Med. Chem. 2013, 56, 2850−2860

Journal of Medicinal Chemistry
Article
Hydrolysis of Ester 17l, 17m, and 17q. A mixture of ester (10 mg, 1
equiv) and LiOH (1.3 equiv) in H2O/tetrahydrofuran solution (0.5 mL,
1/9 v/v) was stirred at room temperature for 3 h. The mixture was
acidified to pH 3−4 by addition of 1 M aq HCl solution and extracted
with ethyl acetate. The organic layer was concentrated in vacuo to give
the corresponding acid.
Synthesis of Compound 17n. A mixture of compound 21 (100 mg,
0.46 mmol), 3-(benzo[d]thiazole-2yl)aniline (125 mg, 0.552 mmol),
PyBOP (287 mg, 0.552 mmol), and diisopropylethylamine (0.12 mL,
0.69 mmol) in tetrahydrofuran (0.5 mL) was stirred at room
temperature overnight. The reaction mixture was diluted with ethyl
acetate and washed with water. The organic layer was dried over MgSO4,
concentrated in vacuo, and purified by flash column chromatography
(SP1, Biotage, Silica gel) to give compound 17n (89 mg, 45% yield).
Synthesis of Compound 17o−17p. A solution of compound 17o
(30 mg, 1 equiv) and K2CO3 (1.5 equiv) in dimethylformamide (0.3
mL) was treated with the indicated alkyl halide (1.15 equiv) and stirred
at room temperature overnight. The reaction mixture was poured into
water and extracted with ethyl acetate. The organic layer was dried,
concentrated, and purified by flash column chromatography (SP1,
Biotage, silica gel) to give the product.
Trypanosoma brucei brucei Proliferation Inhibition Assay.
Culture adapted T. brucei brucei were grown at 37 °C, 5% CO2, in HMI-9
medium (HyClone) supplemented with penicillin/streptomycin (50
μg/mL, Invitrogen, Carlsbad, CA), 10% heat inactivated FBS (Omega
Scientific), and 10% Serum Plus (JHR Biosciences) to a density of 1 ×
106 cells/mL and then diluted to 1 × 104 cells/mL. Then 30 μL of the
diluted culture was added to each well of a white polycarbonate flat
bottom sterile 384-well tissue culture treated plate (Greiner) and 60 nL
inhibitor in DMSO (preprepared dilution series) was added by pin-
transfer using slotted hydrophobic coated pins (V&P Scientific, San
Diego, CA). Plates were incubated for 48 h at 37 °C, 5% CO2, then
equilibrated at room temperature for 30 min before addition of 30 μL of
Cell Titer Glo (Promega) to each well. Plates were shaken on an orbital
shaker for 2 min at 500 rpm. Luminescence was read after 8 min on an
Envision plate reader (Perkin-Elmer). All T. brucei work was performed
in a BSL2 tissue culture room under an IBC approved protocol.
Trypanosoma brucei gambiense or rhodesience Proliferation
Inhibition Assay. T. b. rhodesiense (STIB 900) parasites were cultured
at 37 °C under a humidified 5% CO2 atmosphere in Minimum Essential
Medium (MEM) with Earle’s salts, supplemented according to the
protocol of Baltz22 with the following modifications: 0.2 mM 2-
mercaptoethanol, 1 mM Na-pyruvate, 0.5 mM hypoxanthine, and 15%
heat-inactivated horse serum as supplement. T. b. gambiense strains
(STIB930, DAL 898R, and K048) were grown in HMI-9 medium
supplemented with 15% heat-inactivated fetal bovine serum (FBS) and
5% human serum.
The compounds were tested in a serial drug dilution assay in order to
determine the IC50 values by using the Alamar Blue assay. Serial drug
dilutions were prepared in 96-well microtiter plates containing
appropriate culture medium as described above for each parasite strain,
and wells were inoculated with either 2000 bloodstream forms for T. b.
rhodesiense assay or 10000 trypanosomes for T. b. gambiense assay.
Cultures were incubated for 70 h at 37 °C under a humidified 5% CO2
atmosphere. After this time, 10 μL of resazurin (12.5 mg resazurin
(Sigma) dissolved in 100 mL of phosphate buffered saline) was added to
each well. The plates were incubated for an additional 2−4 h for T. b.
rhodesiense and an additional 4−6 h for T. b. gambiense isolates. The
plates were read in a Spectramax Gemini XS microplate fluorescence
scanner (Molecular Devices) using an excitation wavelength of 536 nm
and an emission wavelength of 588 nm. The IC50 values were calculated
by linear regression from the sigmoidal dose inhibition curves using
SoftmaxPro software.
in both microsomal models (>4 h for both human and mouse),
although its solubility is not optimal.
CONCLUSION
■
This paper describes completion of lead validation of the CNBs,
including SAR studies aimed at improving the antitrypanosomal
potency and SPR studies aimed at improving development
potential. Initially, an existing set of CNB analogues was tested
against bloodstream Trypanosoma brucei brucei. Additionally, the
species selectivity was established for select active compounds
against T. b. gambiense and T. b. rhodesiense. Among the active
compounds, the benzoxazolo-substituted PCNBs demonstrated
good growth inhibitory potency against all Trypanosoma brucei
species and good selectivity for parasite over host cells. Then we
explored substituent effects on both the A- and B-rings of the
benzoxazole or benzothiazole subseries of PCNBs. These SAR
studies showed that benzothiazole analogues generally gave
better activity than benzoxazole analogues and that the 2-methyl
substituted B-ring of PCNBs showed the best potency against T.
b. brucei strain. In addition, piperazine substitution on the A-ring
was tolerated and significantly improved solubility over the
parental compounds although not to the level desired. Among
the compounds produced in these studies, 17b exhibited best
balance of properties: maximum potency against all T. brucei
species, acceptable metabolic stability in both human and rat
microsome models, modest solubility, and reasonable perme-
ability. These properties suggest it is reasonable to pursue in vivo
experiments. While all PCNBs generated in this study showed
high selectivity against a set of mammalian cell lines, solubility
and permeability ranged from poor to modest. The compounds
clearly fall into biopharmaceutics classification system (BCS)
classes II and IV. Formulations addressing this liability will be
critical to further development.20,21
EXPERIMENTAL SECTION
■
Chemistry. All materials were obtained from commercial suppliers
and used without further purification. All solvents used were dried using
an aluminum oxide column. Thin-layer chromatography was performed
on precoated silica gel 60 F254 plates. Purification of intermediates was
carried out by normal phase column chromatography (SP1 [Biotage],
silica gel 230−400 mesh). NMR spectra were recorded on a Bruker 400
MHz, and NMR peaks were assigned by MestReNova (5.2.2). Identity
of all final compounds was confirmed by proton NMR and by mass
spectrometry. The purity of all final compounds was assessed using LC/
MS/UV/ELSD using a Waters Acquity UPLC with the purity being
assigned as the average determined by UV/ELSD (see Supporting
Information for details).19 With three exceptions, the purity of all
compounds was >95% prior to biological testing. In those three cases
(Supporting Information), the low purity assessment was driven by a
highly UV active impurity that had very low ELSD signal (<2%). As
these compounds were not critical for conclusions, this was deemed
acceptable.
General Synthesis of CNBs. To a solution of amine (0.091 mmol)
and diisopropylethylamine (0.109 mmol) in dichloromethane (1 mL)
was added 2-chloro-5-nitrobenzoyl chloride (0.091 mmol) in DCM (0.5
mL). The reaction was stirred at rt for 3 h. After concentration, the
reaction mixture was purified by flash column chromatography (SP1,
Biotage, Silica gel) or recrystallization. NMR and MS of all final
compounds are available in the Supporting Information.
Amination of Compound 16 to Compound 17. A mixture of
compound 16 (30 mg, 1 equiv), amines (1.1 equiv), and
diisopropylethylamine (1.5 equiv) in tetrahydrofuran (0.5 mL) was
stirred at room temperature overnight. The reaction mixture was
concentrated in vacuo and purified by flash column chromatography
(SP1, Biotage, Silica gel) to give the corresponding compound 17.
Cytotoxicity Assay. Cells were grown to 80% confluence, collected,
and plated in 25 μL of media per well in 384-well plates (Costar 3712).
Compounds were diluted and transferred to cells by a pin tool (V&P
Scientific) and the plates incubated for 72 h at 37 °C in 5% CO2.
CellTiter-Glo (Promega) detection reagent was added following the
manufacturer’s instructions, and luminescence was measured using an
EnVision (PerkinElmer) plate reader.
2858
dx.doi.org/10.1021/jm301687p | J. Med. Chem. 2013, 56, 2850−2860

Journal of Medicinal Chemistry
Article
Liver Microsomal Stability. First, 0.633 mL of mouse liver
microsome (20 mg/mL, female CD9 mice, Fisher Scientific, no.
NC9567486) or human liver microsome (200 pooled mix gender,
Fisher Scientific no. 50−722−552) was mixed with 0.051 mL of 0.5 M
EDTA solution and 19.316 mL of potassium phosphate buffer (0.1M,
pH 7.4, 37 °C) to make 20 mL of liver microsome solution. One part of
10 mM DMSO compound stock was mixed with four parts of
acetonitrile to make 2 mM diluted compound stock in DMSO and
acetonitrile. Then 29.1 μL of diluted compound stock was added to 2.3
mL of liver microsomal solution and vortexed to make microsomal
solution with compound. Then 180 μL of the microsomal solutions with
different compounds were dispensed into respective rows of a 96-well
storage plate (pION Inc., MA, no. 110323). For 0 h time point, 450 μL
of precooled (4 °C) internal standard (10 μM warfarin in methanol) was
added to the first three columns before the reaction starts. Then 1.25 mL
of microsome assay solution A (Fisher Scientific, no. NC9255727) was
combined with 0.25 mL of solution B (Fisher Scientific, no.
NC9016235) in 3.5 mL of potassium phosphate buffer (0.1 M, pH
7.4). Finally, 45 μL of this A + B solution was added to each well of the
96-well storage plate (reaction plate). Liquid in the first three columns
was moved to another storage plate (quenched plate). The reaction
plate was then sealed and incubated at 37 °C, shaken at a speed of 60
rpm, and 0.5, 1, 2, and 4 h time points were taken. At each time point,
450 μL of precooled internal standard was added to three rows in the
reaction plate and the liquid was then transferred to the quenched plate.
The quenched plate was then centrifuged (model 5810R, Eppendorf,
Westbury, NY) at 4000 rpm for 20 min. Then 200 μL of supernatant was
transferred to a 96-well plate and analyzed by UPLC-MS (Waters Inc.,
Milford, MA). The compounds and internal standard were detected by
SIR. The log peak area ratio (compound peak area/internal standard
peak area) was plotted against time, and the slope was determined to
calculate the elimination rate constant [k = (−2.303) × slope]. The half-
life (hour) was calculated as t(1/2) = 0.693/k.
Data Analysis. Curves were fit to Titration-response data using
GraphPad Prism 4.03 (GraphPad Software). IC50 values were obtained
by fitting data to the following equation: (sigmoidal dose response
̂
(variable slope)): y = bottom + (top −bottom)/(1 + 10((LogIC50 − x)
× Hill slope)), where x is the logarithm of concentration and y is the
response.
ASSOCIATED CONTENT
* Supporting Information
■
S
Supporting data including characterization data for final
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: (901) 595-5714. Fax: (901) 595-5715. E-mail: kip.
guy@stjude.org.
Author Contributions
Conceived and designed the experiments: J.Y.H. and R.K.G.
Performed the experiments: J.Y.H., D.S., F.Z., G.H., M.C.
Analyzed the data: J.Y.H. and R.K.G. Wrote the paper: J.Y.H. and
R.K.G.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the American Lebanese Syrian
Associated Charities (ALSAC) and St. Jude Children’s Research
Hospital (SJCRH). We thank Monica Cal (Swiss TPH) for
technical assistance with the T. b. gambiense assays.
Solubility. The solubility assay was carried out on Biomek FX lab
automation workstation (Beckman Coulter, Inc.). Ten μL of compound
stock was added to 190 μL of 1-propanol to make a reference stock plate.
Then 5 μL from this reference stock plate was mixed with 70 μL of 1-
propanol and 75 μL of phosphate buffered saline (PBS, pH 7.4) to make
the reference plate and the UV spectrum (250−500 nm) of the reference
plate was measured using a SPECTRAmax PLUS plate reader
(Molecular Devices). Then 6 μL of 10 mM test compound stock was
added to 600 μL of PBS in a 96-well storage plate and mixed. The
storage plate was sealed and incubated at room temperature for 18 h.
The suspension was then filtered through a 96-well filter plate (pION
Inc.). Then 75 μL of filtrate was mixed with 75 μL of 1-propanol to make
the sample plate for UV spectroscopic analysis. A single experiment was
performed in triplicate for each compound. Solubility was calculated
using uSOL Evolution software based on the AUC (area under the
curve) of the UV spectrum of the sample plate and the reference plate.
Permeability Assay. The parallel artificial membrane permeability
assay (PAMPA) was carried out on a Biomek FX lab automation
workstation (Beckman Coulter, Inc.). First, 3 μL of test compound stock
(10 mM in DMSO) was mixed with 600 μL of SSB (system solution
buffer, pH 7.4 or 4, pION Inc.) to dilute the test compound. Then 150
μL of diluted test compound in SSB was transferred to a UV plate
(pION Inc.), and the UV spectrum was measured on a SPECTRAmax
PLUS plate reader (Molecular Devices) to establish a reference plate.
The membrane on a preloaded PAMPA sandwich (pION Inc.) was
painted with 4 μL of GIT lipid (pION Inc.). The acceptor chamber was
then filled with 200 μL of ASB (acceptor solution buffer, pION Inc.),
and the donor chamber was filled with 180 μL of test compound diluted
in SSB. The PAMPA sandwich (donor and acceptor chamber) was
assembled, placed on the Gut-box (pION Inc.), and stirred for 30 min.
The aqueous boundary layer was set to 40 μM for stirring, and the UV
spectrum (250−500 nm) of the donor and the acceptor chambers were
read. A single experiment was performed in triplicate for each
compound. The permeability coefficient was calculated using PAMPA
Evolution 96 Command software (pION Inc.) based on the AUC of the
reference, donor, and acceptor plates.
ABBREVIATIONS USED
■
CNB, chloronitrobenzamide; HAT, human African trypanoso-
miasis; PCNB, phenylheteroarylchloronitrobenzamide
REFERENCES
■
(1) Jacobs, R. T.; Nare, B.; Phillips, M. A. State of the art in African
trypanosome drug discovery. Curr. Top. Med. Chem. 2011, 11, 1255−
1274.
(2) Welburn, S. C.; Maudlin, I. Priorities for the elimination of sleeping
sickness. Adv. Parasitol. 2012, 79, 299−337.
(3) Delespaux, V.; de Koning, H. P. Drugs and drug resistance in
African trypanosomiasis. Drug Resist. Update 2007, 10, 30−50.
(4) Rodgers, J. Human African trypanosomiasis, chemotherapy and
CNS disease. J. Neuroimmunol. 2009, 211, 16−22.
(5) Priotto, G.; Kasparian, S.; Mutombo, W.; Ngouama, D.;
Ghorashian, S.; Arnold, U.; Ghabri, S.; Baudin, E.; Buard, V.; Kazadi-
Kyanza, S.; Ilunga, M.; Mutangala, W.; Pohlig, G.; Schmid, C.;
Karunakara, U.; Torreele, E.; Kande, V. Nifurtimox−eflornithine
combination therapy for second-stage African Trypanosoma brucei
gambiense trypanosomiasis: a multicentre, randomised, phase III,
noninferiority trial. Lancet 2009, 374, 56−64.
(6) Fevre, E. M.; Wissmann, B. V.; Welburn, S. C.; Lutumba, P. The
burden of human African trypanosomiasis. PLoS Negl. Trop. Dis. 2008, 2,
e333.
(7) Simarro, P. P.; Jannin, J.; Cattand, P. Eliminating human African
trypanosomiasis: where do we stand and what comes next? PLoS Med.
2008, 5, e55.
(8) Kuettel, S.; Zambon, A.; Kaiser, M.; Brun, R.; Scapozza, L.;
Perozzo, R. Synthesis and evaluation of antiparasitic activities of new 4-
[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine derivatives. J. Med.
Chem. 2007, 50, 5833−5839.
(9) Bakunov, S. A.; Bakunova, S. M.; Wenzler, T.; Barszcz, T.;
Werbovetz, K. A.; Brun, R.; Tidwell, R. R. Synthesis and antiprotozoal
2859
dx.doi.org/10.1021/jm301687p | J. Med. Chem. 2013, 56, 2850−2860

Journal of Medicinal Chemistry
Article
activity of cationic 2-phenylbenzofurans. J. Med. Chem. 2008, 51, 6927−
6944.
(10) Bolognesi, M. L.; Lizzi, F.; Perozzo, R.; Brun, R.; Cavalli, A.
Synthesis of a small library of 2-phenoxy-1,4-naphthoquinone and 2-
phenoxy-1,4-anthraquinone derivatives bearing anti-trypanosomal and
anti-leishmanial activity. Bioorg. Med. Chem. Lett. 2008, 18, 2272−2276.
(11) Ismail, M. A.; Arafa, R. K.; Wenzler, T.; Brun, R.; Tanious, F. A.;
Wilson, W. D.; Boykin, D. W. Synthesis and antiprotozoal activity of
novel bis-benzamidino imidazo[1,2-a]pyridines and 5,6,7,8-tetrahydro-
imidazo[1,2-a]pyridines. Bioorg. Med. Chem. 2008, 16, 683−691.
(12) Papanastasiou, I.; Tsotinis, A.; Kolocouris, N.; Prathalingam, S. R.;
Kelly, J. M. Design, synthesis, and trypanocidal activity of new
aminoadamantane derivatives. J. Med. Chem. 2008, 51, 1496−1500.
(13) Weis, R.; Seebacher, W. New bicyclic amines: synthesis and SARs
of their action against the causative organisms of malaria and sleeping
sickness. Curr. Med. Chem. 2009, 16, 1426−1441.
(14) Hwang, J. Y.; Smithson, D.; Connelly, M.; Maier, J.; Zhu, F.; Guy,
K. R. Discovery of halo-nitrobenzamides with potential application
against human African trypanosomiasis. Bioorg. Med. Chem. Lett. 2010,
20, 149−152.
(15) Hwang, J. Y.; Attia, R. R.; Carrillo, A. K.; Connelly, M. C.; Guy, R.
K. Synthesis and evaluation of methylsulfonylnitrobenzamides
(MSNBAs) as inhibitors of the thyroid hormone receptor−coactivator
interaction. Bioorg. Med. Chem. Lett. 2013, 23, 1891−1895.
(16) Hwang, J. Y.; Attia, R. R.; Zhu, F.; Yang, L.; Lemoff, A.; Jeffries, C.;
Connelly, M. C.; Guy, R. K. Synthesis and evaluation of sulfonylni-
trophenylthiazoles (SNPTs) as thyroid hormone receptor−coactivator
interaction inhibitors. J. Med. Chem. 2012, 55, 2301−2310.
(17) Hwang, J. Y.; Huang, W.; Arnold, L. A.; Huang, R.; Attia, R. R.;
Connelly, M.; Wichterman, J.; Zhu, F.; Augustinaite, I.; Austin, C. P.;
Inglese, J.; Johnson, R. L.; Guy, R. K. Methylsulfonylnitrobenzoates, a
new class of irreversible inhibitors of the interaction of the thyroid
hormone receptor and its obligate coactivators that functionally
antagonizes thyroid hormone. J. Biol. Chem. 2011, 286, 11895−11908.
(18) Mackey, Z. B.; Baca, A. M.; Mallari, J. P.; Apsel, B.; Shelat, A.;
Hansell, E. J.; Chiang, P. K.; Wolff, B.; Guy, K. R.; Williams, J.;
McKerrow, J. H. Discovery of trypanocidal compounds by whole cell
HTS of Trypanosoma brucei. Chem. Biol. Drug Des. 2006, 67, 355−363.
(19) Lemoff, A.; Yan, B. Dual detection approach to a more accurate
measure of relative purity in high-throughput characterization of
compound collections. J. Comb. Chem. 2008, 10, 746−751.
(20) Lindenberg, M.; Kopp, S.; Dressman, J. B. Classification of orally
administered drugs on the World Health Organization Model List of
Essential Medicines according to the biopharmaceutics classification
system. Eur. J. Pharm. Biopharm. 2004, 58, 265−278.
(21) Wu, C. Y.; Benet, L. Z. Predicting drug disposition via application
of BCS: transport/absorption/elimination interplay and development
of a biopharmaceutics drug disposition classification system. Pharm. Res.
2005, 22, 11−23.
(22) Baltz, T.; Baltz, D.; Giroud, C.; Crockett, J. Cultivation in a
semidefined medium of animal infective forms of Trypanosoma brucei, T.
equiperdum, T. evansi, T. rhodesiense and T. gambiense. EMBO J. 1985, 4,
1273−1277.
2860
dx.doi.org/10.1021/jm301687p | J. Med. Chem. 2013, 56, 2850−2860