Novel naphthoquinone derivatives: Synthesis and activity against human African trypanosomiasis

Article abstract of DOI:10.1016/j.bmcl.2012.12.075

A series of naphthoquinone derivatives has been synthesized and tested for its biological activity against human African trypanosomiasis. The use of reverse micellar medium not only enhanced the conversion rate, but also showed selectivity towards mono-co

Full text of DOI:10.1016/j.bmcl.2012.12.075

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1420–1423
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel naphthoquinone derivatives: Synthesis and activity against
human African trypanosomiasis
⇑
Bhupesh S. Samant , Chikomborero Chakaingesu
Natural Product and Medicinal Chemistry (NPMC) Research Group, Division of Pharmaceutical Chemistry, Faculty of Pharmacy, Rhodes University, Grahamstown 6140, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of naphthoquinone derivatives has been synthesized and tested for its biological activity against
human African trypanosomiasis. The use of reverse micellar medium not only enhanced the conversion
rate, but also showed selectivity towards mono-coupled product in aryl chloride–aniline coupling reac-
tions. Two derivatives of naphthoquinone (9b and 9c) exhibited potent activity against Trypanosoma bru-
cei in vitro with low cytotoxicity.
Received 30 August 2012
Revised 17 December 2012
Accepted 21 December 2012
Available online 4 January 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Human African trypanosomiasis
Sleeping sickness
Naphthoquinone derivatives
Micellar media
The Human African trypanosomiasis (HAT), normally known as
sleeping sickness, is a perilous and neglected parasitic disease. It is
prevalent in at least 36 sub Saharan Africa countries. Most (96%)
cases of HAT are caused by the Trypanosoma brucei gambiense
protozoa, which is transmitted from infected animals to humans
by bites of blood sucking tsetse flies (Glossina genus).1–3 The med-
ications currently used for treatment are unsafe and inefficient; for
example, treatment with melarsoprol (a trivalent arsenical deriva-
tive) often results in fatal encephalopathy, agranulocytosis, and
myocardial damage.4,5 There is a dearth of effective treatment
regimens to fight against HAT. The negligence towards this disease
engenders a demand for new research projects to produce drugs
which are effective against the disease as well as safe for human
use.
example of a naphthoquinone that is in use as an antimalarial
agent. It is believed that it interferes with the mitochondrial respi-
ratory chain of the Plasmodium sp.12 Because of their positive
antimalarial activities; we tested the naphtaquinones against
HAT and performed a structural activity relationship study. In a
continuation of our ongoing research focused on novel chemical
entities with antimalarial or antitrypanosomal activities we aim
on synthesizing various bioactive compounds; examples include
derivatives of hydroxypyrid-2-ones,13 febrifugine,14 and fexinidaz-
ole,15
.
Our previous structure–activity relationship study13
revealed the following requirements for bioactivity against T. brucei:
an electron withdrawing moiety, an aromatic ring (good source of
electrons), an electronegative halogen and its position in the mol-
ecule. We decided to design a molecule which included a halogen
at the 2nd position and an electron donating and/or withdrawing
substitution on the aromatic rings (X, R1 and R2, respectively in
Scheme 1). In the proposed molecule, substitution of R1 and R2
on various positions aid in our understanding of the effect of sub-
stitution on activity against T. brucei. Hence, we decided to synthe-
size a series of substituted naphthoquinone derivatives (Scheme 1).
Mital et al. synthesized various 2-substituted 1,4-naphthoqui-
none derivatives by using 2-bromonaphthalene as the starting
material in a Heck coupling reaction followed by oxidation.10 Sim-
ilarly, some scientists used palladium catalyzed Suzuki cross cou-
pling reaction of aryl chlorides and Boronic acids.16 Tandon et al.
have done exceptional work in chemoselective coupling reactions
by using water as a solvent.17–19 However, coupling reaction in
water (Tandon process) has limitation of substrate specificity.18
The catalytic amination of aryl halides, called as Buchwald–
Hartwig reaction, has been proved to be an useful method for
preparing a variety of arylamines.20–25
During research conducted on the chemical genetics of Plasmo-
dium falciparum, potential anti-malaria agents, such as naphtho-
quinones, were discovered through use of screening techniques
such as high through-put screening (Scheme 1).6
A process development study of transition metal catalyzed
coupling reactions is one of the most important areas for organic
scientists because of the applications of these reactions in pharma-
ceuticals;7 especially in synthesizing bioactive compounds to fight
against various diseases such as parasitic diseases. One such class
of drugs is naphthoquinones which has shown anti-malarial,
anti-cancer, anti-diabetic, anti-fungal, anti-bacterial and anti-
inflammatory activities since 1969,8–10 until recently, where
epoxy-1,4-naphthoquinones have also been used as inhibitors of
human leukemia (THP1) cell proliferation.11 Atovaquone is an
⇑
Corresponding author. Tel.: +27 46 6037426; fax: +27 46 6361205.
E-mail address: B.Samant@ru.ac.za (B.S. Samant).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.075

B. S. Samant, C. Chakaingesu / Bioorg. Med. Chem. Lett. 23 (2013) 1420–1423
1421
O
O
O
The application of the process to derivatives of 2,3-dichloro-
naphthalene-1,4-dione with different substituted anilines is shown
in Table 2, in which electron withdrawing and donating groups on
aniline and electron withdrawing group (NO2) on 2,3-dichloro-
naphthalene-1,4-dione gave good yields. The substitution pattern
(2nd, 3rd and 4th position) on aniline does not appear to affect
the yield.
N
N
N
NH
O
Scaffold
Core
The synthesized compounds were evaluated on the basis of
their ability to inhibit cell proliferation of T. brucei rhodesiense in
culture.34 The growth inhibitory activity against L-6 rat skeletal
muscle myoblast cells was determined to establish a cellular ther-
apeutic index.35 The relationship between toxicity of naphthoqui-
none derivatives and substituted functional groups on the
structure is still unknown and perplexing. Some naphthoquinone
derivatives have shown to possess haemolytic activity and cause
nephrotoxicity.36,37 Hence, before proposing naphthoquinone
derivatives as biologically active compounds, along with activity
R2
X
O
O
O
NH2
R1
R1
X
X
Retrosynthesis
R2
NH
O
Proposed Molecule
R2
determination, in vitro toxicological studies becomes
prerequisite.
a
Scheme 1. Retrosynthetic approach for naphthoquinone derivatives. R1 and
R2 = electron withdrawing or donating functional groups; X = halogen.
Compounds with mono-substitution (b and c) showed higher T.
brucei rhodesiense inhibitory activity compared to di-substituted
isomers (a). The presence of an electron-withdrawing chlorine
group and phenyl amine ring with high electron density at two
adjoining carbon atoms in the structure seems to be the main rea-
son for this activity. This also showed the importance of selectivity
towards mono-substituted product in coupling reaction. Com-
pounds 1a and 1b showed effective T. brucei inhibitory activity
and low cytotoxicity (Table 2, entry 1). However, compounds 2–4
showed lower inhibition activity compared to 1 but had low cyto-
toxicity (Table 2, entries 3–8). The decrease in the T. brucei inhibi-
tory activity was attributed to the presence of an electron-donating
group (Me) on the phenyl amine ring.
The design of novel, eco-friendly and cost effective synthetic
processes for use in industry is an important goal. One of the best
approaches involves the use of micellar micro-reactors, which
possess a controlled balance of amphiphilicity and the ability to
affect selectivity towards particular products.26–32 In this study,
we present the catalytic coupling reaction of anilines and aryl ha-
lides (Buchwald–Hartwig reaction) for synthesizing substituted
naphthoquinones using reverse micellar micro-reactors.
We coupled 2,3-dichloronaphthalene-1,4-dione (RCl) and
aniline (R0NH2) using NaOH, L (10%), and [cinnamylPdCl]2 (5%), in
surfactant (sodium lauryl sulphate, SLS)–toluene as the reaction
media and we not only observed a high % conversion but also
enhancement in the selectivity towards 1b (Table 1, entry 3).33
The selectivity towards 1b was very surprising because substitu-
tion of the activating group NHR on 1b was expected to enhance
the rate of formation of di-coupled 1a product.
The above reaction suggest that in the presence of reverse
micellar media (60 mM SLS surfactant) gave good yield of coupling
reaction between aniline and 2,3-dichloronaphthalene-1,4-dione
with reduced quantities of Pd catalyst and L (5 mol % Pd and
10 mol % L). It was also observed that the presence of surfactant
micelles enhanced the % conversion and the selectivity towards
the mono-coupled 1b product of the reaction. Gas chromatography
technique was employed to determine the exact % conversion to
product 1, and results were further confirmed by using 1H and
13C NMR spectroscopy.
On comparing the T. brucei inhibitory activity of compounds 2–
4 and 5–7 there is no correlation between the activity and the po-
sition of electron-donating group on phenyl amine ring (Table 2,
entries 3–8 and 9–17).
To study the effect of electron withdrawing groups (NO2 and
CF3) on the T. brucei inhibitory activity, we tested compounds 5–
11 (Table 2, entries 9–27). We found that the presence of electro-
phile NO2 on naphthalene-1,4-dione ring (with electron donating
Me group on phenyl amine ring) in compounds 5–7 increased T.
brucei inhibitory activity with a slight increase in cytotoxicity com-
pared to compounds 2–4. Surprisingly, compounds 8a and 8b with
a CF3 (electron withdrawing group) at the 2nd position on phenyl
amine ring (without the presence of Me group on phenyl amine
ring) showed T. brucei inhibitory activity with average cytotoxicity
(Table 2, entries 18 and 19). Also the presence of NO2 on naphtha-
lene-1,4-dione ring and CF3 on phenyl amine ring in compounds
Table 1
Synthesis of 2-chloro-3-(phenylamino)naphthalene-1,4-dione
O
O
O
O
H
Cl
N
Cl
Ph
ArNH2
Cl
NH
Ph
NH
Ph
O
O
1b
1a
Entry
Reaction conditions
% Total conversiona (% selectivity)
Isolated yield (%)
1
2
3
Pd (10 mol %), L (20 mol %), THF (5 mL), 24 h, 70 °C
Pd (10 mol %), L (20 mol %), Toluene (5 mL), 24 h, 110 °C
Pd (5 mol %), L (10 mol %), Toluene (5 mL), SLS (60 mM), 5 h, 20 °C
35, 1a (90), 1b (10)
2
62, 1a (7), 1b (93)
1a (27), 1b (5)
—
1a (3), 1b (55)
a
Determined by gas chromatograph. Each experiment was done three times.

1422
B. S. Samant, C. Chakaingesu / Bioorg. Med. Chem. Lett. 23 (2013) 1420–1423
Table 2
Scope of the reaction in micellar micro-reactors and pharmacological properties of synthesized naphthoquinones
O
O
O
O
O
H
N
H
N
NH2
Cl
Cl
Cl
R2
R2
Cl
NH
NH
R2
R1
O
R1
R1
O
R1
O
R2
R2
a
b
c
Entry
Substrate
% Total conversiona
(% selectivity)
Isolated yield (%)
IC50 versus T. bruceib
(l
M)
IC50 versus L-6 rat skeletal
myoblast cellsb
(lM)
1
2
3
4
5
6
7
8
1a, R1 = H, R2 = H
1b, R1 = H, R2 = H
62 (7)
3
55
5
56
3
55
4
58
3
25
25
4
23
26
3
25
26
2
57
4
0.67 0.1
0.17 0.1
378 15
417 20
>450
>450
>450
>450
>450
>450
186 10
195 10
210 10
172 10
186 10
169 10
195 10
214 10
223 10
292 15
307 15
62 (93)
65 (10)
65 (90)
65 (8)
65 (92)
66 (9)
66 (91)
58 (8)
58 (45)
58 (47)
60 (9)
60 (44)
60 (47)
60 (7)
60 (46)
60 (47)
62 (5)
2a, R1 = H, R2 = 2-Me
2b, R1 = H, R2 = 2-Me
3a, R1 = H, R2 = 4-Me
3b, R1 = H, R2 = 4-Me
4a, R1 = H, R2 = 3-Me
4b, R1 = H, R2 = 3-Me
5a, R1 = NO2, R2 = 2-Me
5b, R1 = NO2, R2 = 2-Me
5c, R1 = NO2, R2 = 2-Me
6a, R1 = NO2, R2 = 4-Me
6b, R1 = NO2, R2 = 4-Me
6c, R1 = NO2, R2 = 4-Me
7a, R1 = NO2, R2 = 3-Me
7b, R1 = NO2, R2 = 3-Me
7c, R1 = NO2, R2 = 3-Me
8a, R1 = H, R2 = 2-CF3
8b, R1 = H, R2 = 2-CF3
9a, R1 = NO2, R2 = 2-CF3
9b, R1 = NO2, R2 = 2-CF3
9c, R1 = NO2, R2 = 2-CF3
10a, R1 = H, R2 = 4-CF3
10b, R1 = H, R2 = 4-CF3
20
18
24
20
21
18
7
3
3
3
3
3
3
1
1
1
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
4
3
12
8
6
2
1
1
15
10
11
2
2
2
0.45 0.1
0.15 0.1
0.09 0.01
0.07 0.01
0.05 0.01
62 (95)
60 (9)
88
75
95
5
5
5
60 (45)
60 (46)
61 (11)
61 (89)
58 (8)
24
24
4
52
3
2
0.2
276 10
292 10
0.8 0.1
11a, R1 = NO2, R2 = 4-CF3
11b, R1 = NO2, R2 = 4-CF3
11c, R1 = NO2, R2 = 4-CF3
4
2
0.5
0.2
97
5
58 (44)
58 (48)
23
25
121 10
115 10
0.8 0.2
a
Determined by gas chromatograph.
IC50 values are means standard deviations; each experiment was done three times.
b
100
90
80
70
60
50
40
9a–c showed highest T. brucei inhibitory activity in the series with
low cytotoxicity (Table 2, entries 20–22). The reason for this
enhancement in T. brucei inhibitory activity may be the balance
of strong electron withdrawing groups in the structure that is,
CF3 on phenyl amine ring, NO2 on naphthalene ring and Cl on
1,4-dione ring.
To evaluate the importance of the position of CF3 group on phe-
nyl amine ring with respect to the inhibition of T. brucei cell prolif-
eration, we compared the activities of compounds 9 and 11 (or 8
and 10) (Table 2, entries 20–27). The decrease in the T. brucei inhib-
itory activities of compounds with CF3 at the 4th position in phenyl
amine ring (compounds 10 and 11) supported the postulation that
the positioning of a strong electron withdrawing groups in the
structure of naphthoquinones an important factor for T. brucei
inhibitory activity.
The exact mechanism of action for this series of naphthoqui-
nones against T. brucei is still unknown. However, to get glimpse
about the relationship of the structure and the activity we evalu-
ated the % conversion of compounds 2,3-dichloronaphthalene-
1,4-dione, 1a, 9b and 9c in the presence of cysteine in ringer solu-
tion38 (Fig. 1). The higher rate of consumption of compounds 9b
and 9c than 2,3-dichloronaphthalene-1,4-dione and 1a indicates
the comparison of the structures with respect to activity. The pres-
ences of a phenyl amine ring (a good source of electron density)
with strong electrophile CF3 group in the structure of 9c, may cause
protonation at C4 carbonyl group to form reactive intermediate
which may react with a nucleophile (i.e., cysteine and lysine); sim-
ilar protonation was observed previously in the case of lawsone
0
2
4
6
8
10
12
Time (h)
Figure 1. The consumption study of compounds 2,3-dichloronaphthalene-1,4-
dione ( ), 1a ( ), 9b ( ) and 9c ( ) (1 mmol) in the presence of cysteine (5 mmol)
in Ringer solution at 37 °C. % Conversion was determined by gas chromatograph.
acetate.39 The presence of electrophilic groups like Cl, NO2 and
CF3 (at the 2nd position in phenylamine ring) in the structure
may cause an increase in electrophilicity of the ring. These groups
may also act as good leaving groups in the binding step of the cys-
teine thereby, conferring activity of 9c against T. brucei.
It seems that these compounds are affecting against polyamine
biosynthesis40 in the parasite. If activities of compounds 9 and 11

B. S. Samant, C. Chakaingesu / Bioorg. Med. Chem. Lett. 23 (2013) 1420–1423
1423
30. Samant, B. S.; Bhagwat, S. S. J. Chin. Catal. 2011, 32, 231.
31. Samant, B. S.; Kabalka, G. W. Chem. Commun. 2011, 47, 7236.
32. Samant, B. S.; Kabalka, G. W. Chem. Commun. 2012, 48, 8658.
compared, then it would appear that the presence of CF3 at 4th po-
sition on phenylamine ring may not act as good leaving group as
compared to the CF3 at 2nd position due to less steric hindrance.
Hence, the position at which these electrophilic groups reside in
the structure effect on the T. brucei inhibitory activity.
In conclusion, coupling of substituted RCl and anilines has been
achieved using reverse micellar media. It resulted in selectivity to-
wards mono-coupled substituted naphthoquinones. Compounds
9b and 9c showed excellent T. brucei inhibitory activity with low
cytotoxicity. It seems that the electron withdrawing group such
as NO2, CF3 and Cl and electron density rich groups (phenylamine
ring and aromatic ring) at particular positions on the naphthoqui-
none as the backbone structure are important to T. brucei inhibi-
tory activity.
33. L (93.30 mg, 0.20 mmol, 20 mol %), and (cinnamylPdCl)2 (52 mg, 0.10 mmol,
10 mol %) were added in 2.5 mL solution of SLS (100 mM) in toluene. This
mixture was stirred under reflux for 5 min, under nitrogen atmosphere in
50 cm3 glass round bottom flask with cylindrical shape, 1 cm length magnetic
stirrer. The following mixture was then added; 2,3-dichloro-1,4-
naphthoquinone (227 mg, 1 mmol, 1 equiv), aniline (0.1 mL, 2 mmol, 2 equiv)
and powdered sodium hydroxide (44 mg, 1.1 mmol) in 2.5 mL solution of SLS
(100 mM) in toluene. Reaction mixture was stirred for 5 h. A solution of CTAB
(100 mM) in toluene (5 mL) was added to the reaction mixture which was
agitated for an additional 10 min. The mixture was filtered through a plug of
Celite, the filtrate concentrated under reduced pressure, and then purified by
flash chromatography on silica gel using hexane:ethyl acetate 9:1 to give 2,3-
bis(phenylamino)naphthalene-1,4-dione (1a); yield: 10.2 mg (3% yield), 2-
chloro-3-(phenylamino)naphthalene-1,4-dione (1b) yield: 161.3 mg (57%
yield). The experiments were performed in replicates of three. The variation
in the results from the reported average values was within 0.75%.
34. Parasites T. brucei rhodesiense were grown in mice to subsaturation density
(3 ꢀ 108 cells/mL of blood). One milliliter of infected blood was collected with
0.2 vol of citrate glucose anticoagulant and 0.1 M sodium citrate/0.04 M
glucose at pH 7.7 and was diluted in 9 mL of HMI-9 medium. Samples were
centrifuged at 200 rpm for 5 min at room temperature. The supernatant with
trypanosomes was transferred to a culture flask and incubated at 37 °C for 2 h
to settle remaining blood cells. Trypanosomes in the supernatant were then
cultured in an atmosphere of 5% CO2 at 37 °C, in HMI-9 medium supplemented
Acknowledgments
Authors like to thank Rhodes University Joint Research Commit-
tee (JRC) (Rhodes University JRC grant number 35047) and Sandisa
Imbew fund grant for providing financial support for this work.
Authors thank, Marvel Pharmaceutical Co. Ltd, India, for helping
in the biological work. Authors also like to thank Professor G. W.
Kabalka (University of Tennessee, USA) for helpful discussions.
with 50 lM streptomycin/penicillin, 10% heat inactivated FBS and 10% Serum
Plus to a density of 1 ꢀ 106 cells/mL. Cultures were fed daily by adding fresh
medium (2.5 mL) after removing an equal volume and routinely diluted 1:10 to
1:20 daily to maintain densities in the range of 104 cells/mL. Cell proliferation
tests were performed in 96-well tissue culture white polycarbonate flat bottom
References and notes
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Fluorescence development was measured and expressed as percentage of the
control. Data were transferred into the graphic program Softmax Pro
(Molecular Devices) to calculate IC50 values.
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plates with the protein binding dye sulforhodamine
B (SRB). Series of
compounds were serially diluted and added to empty wells of the 96-well
plate. The wells were immediately seeded with the cell line L-6 (rat skeletal
muscle myoblasts) at 2 ꢀ 103 cells/100
lL, 50 lL per well in Dulbecco’s
modified eagle medium (DMEM) supplemented with 10% heat inactivated
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added after stirring. Plates were incubated at 37 °C then equilibrated at room
temperature for 5 min and performed gas chromatography analysis. We also
tried to do this study in the presence of HMI-9 medium; however, due to the
presence of various amino acids in that medium, extraction and analysis of %
conversion for compounds became very complicated.
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