Discovery of halo-nitrobenzamides with potential application against human African trypanosomiasis

Article abstract of DOI:10.1016/j.bmcl.2009.11.022

A series of halo-nitrobenzamide were synthesized and evaluated for their ability to block proliferation of Trypanosoma brucei brucei. A number of these compounds had significant activity against the parasite, particularly 2-chloro-N-(4-chlorophenyl)-5-nitrobenzamide 17 which exhibited low micromolar inhibitory potency against T. brucei and selectivity towards both malaria and mammalian cells.

Full text of DOI:10.1016/j.bmcl.2009.11.022

Bioorganic & Medicinal Chemistry Letters 20 (2010) 149–152
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of halo-nitrobenzamides with potential application against human
African trypanosomiasis
*
Jong Yeon Hwang, David Smithson, Michele Connelly, Julie Maier, Fangyi Zhu, Kiplin R. Guy
St Jude Children’s Hospital, Department of Chemical Biology and Therapeutics, 262 Danny Thomas Place, Memphis, TN 38105-3678, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of halo-nitrobenzamide were synthesized and evaluated for their ability to block proliferation of
Trypanosoma brucei brucei. A number of these compounds had significant activity against the parasite,
particularly 2-chloro-N-(4-chlorophenyl)-5-nitrobenzamide 17 which exhibited low micromolar inhibi-
tory potency against T. brucei and selectivity towards both malaria and mammalian cells.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 19 September 2009
Revised 3 November 2009
Accepted 5 November 2009
Available online 14 November 2009
Keywords:
Trypanosomiasis
HAT
Inhibitor
The protozoan parasite Trypanosoma brucei causes Human Afri-
can trypanosomiasis (HAT), a major health concern in sub-Saharan
Africa with an estimated 50,000 cases and 60 million at risk of
infection.1 Several drugs such as Melarsoprol and Eflornithine
(DFMO) have been developed to treat HAT (Fig. 1).2,3 However
these drugs used have serious side effects, poor clinical effect,
and emerging drug resistance (Fig. 1).4–7 Therefore, there is urgent
need for new drugs against HAT with low toxicity.8,9
As part of our ongoing search for novel chemical entities with
antitrypanosomal or antimalarial effects, all compounds synthe-
sized in our laboratory or purchased by our laboratory are routinely
assayed to detect such activity. Compound 3 emerged as a hit com-
pound inhibiting T. brucei proliferation in this campaign. It has two
distinct features: a potentially electrophilic center and a hydropho-
bic side chain. The halo-nitro substituted benzene structure is well
known as an electrophile. Thus, compound 3 might be forming a
covalent bond with nucleophiles such as cysteine and lysine in the
binding pocket of its target.10 Compound 3 inhibited T. brucei prolif-
by amide or ester coupling reactions induced with polymer bound
EDC (Scheme 1). All compounds were evaluated for their ability to
inhibit proliferation of cultured T. brucei brucei and Plasmodium fal-
ciparum (3D7 strain). Additionally, the growth inhibitory activity
against HepG2 cells was determined to set a cellular therapeutic
index. The solubility and passive permeability of the small mole-
cules were also investigated to support the cell based assay results
and to guide the earliest stages of discovery towards tractable
compounds (Fig. 3).
First we explored putative nucleophilic ring group of hit com-
pound 3 to determine if the 2-fluoro-5-nitrobenzamide moiety
F
OH
NH2
N
S
F
NH2
As
S
N
H2N
O
H2N
N
N
OH
H
1
2
eration with fairly good potency (EC50 = 1.5
tivity against malaria (3D7 strain, EC50 = 7.3
l
M) but had poor selec-
M) and mammalian
l
Figure 1. Melarsoprol 1 and eflornithine 2.
cells (HepG2, EC50 = 2.5 lM). In addition compound 3 had poor sol-
ubility (<0.1
l
M) and permeability (<1 Â 10À6 cm/s). Therefore we
began optimizing this chemical structure to improve both pharma-
cological properties and selectivity. Here we report this new class
of T. brucei inhibitors (Fig. 2).
NO2
All of compounds investigated in this report were synthesized
from commercially available anilines or phenols and benzoic acid
Nucleophilic group
H
Hydrophobic chain
N
O
F
* Corresponding author. Tel.: +1 901 595 5714; fax: +1 901 595 5715.
E-mail address: kip.guy@stjude.org (K.R. Guy).
Figure 2. Chemical structure of hit compound 3.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.022

150
J. Y. Hwang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 149–152
O
X
X
NH2
EDC-polymer
H
HO
N
THF, rt, 12h
NO2
NO2
R
O
R
O
X
X
O
HY
EDC-polymer
THF, rt, 12h
OH
Y
NO2
R
NO2
R
X=F an d Cl
Y=NH and O
Scheme 1. General synthetic route for halo-nitrobenzene esters and amides.
F
NO2
NO2
F
H
N
H
N
H
N
O
NO2
O
F
O
C6H13
C6H13
C6H13
3
4
5
NO2
NO2
H
N
H
N
H
N
NO2
O
Cl
O
Cl
C6H13
O
Cl
C6H13
C6H13
7
8
6
NO2
O2N
H
N
O2N
H
N
Cl
Cl
H
N
O
Cl
O
C6H13
O
C6H13
C6H13
9
10
11
NO2
Cl
O
Cl
NO2
O
O
O
NO2
N
N
H
H
C6H13
Cl
C6H13
C6H13
14
13
Figure 3. Halo-nitrobenezene compounds investigated.
12
was essential for inhibition T. brucei proliferation (Table 1). 4-Flu-
oro-3-nitrobenzamide compound 4 showed weak T. brucei inhibi-
against HepG2 cells (EC50 >50
l
M). However, compound 6 also
showed poor solubility (<0.1 lM) and modest permeability
tory activity (EC50 = 24
(EC50 = 10.3 M) in HepG2 cells compared to original hit com-
pound 3. 3-Fluoro-5-nitrobenzamide compound 5 also showed
weak T. brucei inhibitory activity (EC50 = 22.5 M) but no toxicity
in HepG2 cells (EC50 >50 M). All of fluoro-nitrobenzamide com-
pounds 3–5 lacked growth inhibitory activity against malaria.
l
M) and relatively low cytotoxicity
(7 13 Â 10À6 cm/s). Other chloro-nitrobenzamide compounds 8-
l
11 showed weak T. brucei inhibitory activity (10–30 lM). Reverse
halo-nitrophenyl amides 12 and 13 and ester 14 were also investi-
gated. 4-Chloro-5-nitrophenyl amide 12 showed moderate activity
l
l
(EC50 = 6.0
activity and weak activity (EC50 >50 and 24.0
All of this series showed good selectivity against malaria (EC50
l
M) but compounds 13 and 14 showed no inhibitory
l
M), respectively.
However, all of these compounds had poor solubility (<0.1 lM)
and permeability (<1 Â 10À6 m/s). Next we investigated whether
fluorine is replaceable with chlorine. 2-Chloro-5-nitro benzamide
compound 6 showed not only inhibitory activity similar to initial
>15
lM) and HepG2 cells (EC50 >50 lM) and poor solubility and
permeability (<0.1
l
M and <1 Â 10À6 cm/s, respectively). All com-
pounds in this series showed relatively high A log P, regardless of
hit 3 against T. brucei (EC50 = 1.0
lM), but also high selectivity
structure.11

J. Y. Hwang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 149–152
151
Table 1
Activities and pharmacological properties of halo-nitrobenzamides
Compound
Yield (%)
IC50 versus T. brucei
IC50 versus P. falcip
IC50 versus HepG2
Solubility (
l
M)
Permeability 10À6 cm/s
A log P
3
4
5
6
7
1.5 0.4
24 1.2
22.5 2.5
1.0 0.1
>50
19.0 3.6
29.8 1.4
20.8 4.8
11.9 0.7
>50
7.3 1.0
>15
>15
>15
>15
>15
>15
>15
>15
2.5 0.5
10.3 7.7
>50
>50
>50
>50
>50
>50
>50
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<1
<1
<1
7
<1
5.94
5.48
5.48
5.48
5.94
5.94
5.94
5.94
5.94
5.94
5.94
5.94
86
68
75
40
66
23
63
64
55
64
99
13
8
9
82 142
3
5
10
11
12
13
14
<1
<1
<1
<1
<1
>15
>15
>15
>50
>50
>50
6.0 1.3
24.0 1.9
NO2
Cl
NO2
NO2
H
H
N
H
N
N
O
O
16
Cl
O
15
Cl
Cl
O
17
O
NO2
NO2
NO2
N
H
N
H
N
N
O
19
Cl
O
18
Cl
O
Cl
20
Figure 4. 2-Chloro-5-nitrobenezamide compounds.
Table 2
Activities and pharmacological properties of 2-chloro-5-nitrobenzamides
Compound
Yield (%)
IC50 versus T. brucei
IC50 versus P. falcip.
IC50 versus HepG2
Solubility (
lM)
Permeability Pe, 10À6 cm/s
A log P
15
16
17
18
19
20
94
65
51
49
51
56
7.7 0.6
19.2 1.0
1.5 1.0
>50
27.0 3.1
24.5 13.7
>15
>15
>15
>15
>15
>15
>50
>50
>50
>50
>50
>50
5.7 0.1
9.9 0.5
2.9 0.1
3.9 0.2
7.5 0.4
5.9 0.2
363 56
248 38
536 87
186.5 50
418 27
386 21
3.66
3.16
3.84
3.18
3.79
3.43
Next we explored variation of the hydrophobic side chain to
determine if it was possible to improve the poor physicochemical
properties of the original series. The results are summarized in Fig-
ure 4 and Table 2. 4-Methylphenylamide 15 and 4-methoxyphe-
nylamide 16 exhibited moderate activity against T. brucei
17 showed the most balance of potency, selectivity, and physico-
chemical properties.
In summary we have reported the synthesis and evaluation of a
series of halo-nitrobenzamides made to explore the SAR of a hit se-
lected from screening for inhibitors of T. brucei proliferation. The
optimized inhibitor exhibited potent activity against T. brucei pro-
liferation and selectivity against malaria and mammalian cells.
Although the mechanism of action of these series remains unclear
the halo-nitrobenzamides warrant further examination as leads for
the therapy of human African trypanosomiasis.
proliferation (EC50 = 7.7
lM
and 19.2
l
M, respectively), and
showed improvement of solubility (5.7
l
M and 9.9 M, respec-
l
tively) and permeability (363 Â 10À6 cm/s and 248 Â 10À6 cm/s,
respectively). 4-chloroaniline 17 showed not only good inhibitory
activity against T. brucei (EC50 = 1.5 lM) but also reasonable solu-
bility (2.9
l
M) and permeability (536 Â 10À6 cm/s) without change
Acknowledgements
of selectivity against HepG2 (EC50 >50 lM). Benzylamine 18 was
inactive against T. brucei and cyclohexyemthylamine 19 and 4-
methoxyphenylpiperazine 20 showed moderate activity
(EC50 = 27.0 lM and 24.5 lM, respectively). This entire series also
showed good selectivity against malaria and mammalian cells.
The replacement of the hexyl chain resulted in reduced A log P
(3.16–3.84) and improved solubility (30–90-fold). In contrast to
the earlier series, all compounds showed reasonable permeability.
In addition, the electron withdrawing chloro group improved anti-
trypanosomal activity. Conclusively chloro-substituted compound
This work was supported by the American Lebanese Syrian
Associated Charities (ALSAC) and St. Jude Children’s Research
Hospital.
Supplementary data
Experimental procedures and characterization data for all final
compounds are available. Supplementary data associated with this

152
J. Y. Hwang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 149–152
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