Antileishmanial activity evaluation of bis-lawsone analogs and DNA topoisomerase-I inhibition studies

Article abstract of DOI:10.3109/14756366.2013.765413

For the development of potent novel antileishmanial agents, 3,3′-(arylmethylene)bis(2-hydroxynaphthalene-1,4 dione) derivatives were synthesized from lawsone and evaluated for cytotoxicity on Leishmania donovani promastigotes as well as on leishmanial DNA

Full text of DOI:10.3109/14756366.2013.765413

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, 2014; 29(2): 185–189
2014 Informa UK Ltd. DOI: 10.3109/14756366.2013.765413
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ORIGINAL ARTICLE
Antileishmanial activity evaluation of bis-lawsone analogs and DNA
topoisomerase-I inhibition studies
Garima Sharma1, Sayan Chowdhury2, Suman Sinha1,3, Hemanta K. Majumder2, and S. Vasanth Kumar1
2
1Department of Chemistry, School of Science and Humanities, Karunya University, Coimbatore, India, Molecular Parasitology Laboratory,
3
Department of Infectious Diseases & Immunology Division, Indian Institute of Chemical Biology, Jadavpur, Kolkata, India, and School of
Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia
Abstract
Keywords
For the development of potent novel antileishmanial agents, 3,30-(arylmethylene)bis(2-
hydroxynaphthalene-1,4 dione) derivatives were synthesized from lawsone and evaluated for
cytotoxicity on Leishmania donovani promastigotes as well as on leishmanial DNA topoisomerase-
I. Enzyme inhibition studies were conducted with simultaneous and preincubation conditions.
Total inhibition is compared to camptothecin (CPT), which was taken as positive control on both
the systems of enzyme inhibition. The range of activity varied from 37.5 to 70 mM in simultaneous
assay and 13–16 mM in preincubation assay. Furthermore, when evaluated against L. donovani
promastigotes, the synthesized compounds exhibited the activity ranging from 2 to 14 mM. The
results revealed that all the compounds exhibit promising antileishmanial activity.
Antileishmanial activity, camptothecin,
DNA topoisomerase-I, lawsone,
naphthoquinone
History
Received 10 September 2012
Revised 26 December 2012
Accepted 7 January 2013
Published online 27 March 2013
Introduction
two characteristics: (i) the ability to cleave and reseal the
phosphodiester backbone of DNA in two successive trans-
esterification reactions and (ii) once a topoisomerase is cleaved
the DNA intermediate is formed, the enzyme allows the severed
DNA end to come apart, opening a gate for the passage of another
Leishmaniasis, a protozoan parasitic disease, is currently a global
health problem occurring with visceral, cutaneous and mucocuta-
neous manifestations. Clinical implications of this disease range
from the disfiguring skin lesions in the case of cutaneous
single or double stranded DNA segment9,10
.
leishmaniasis to the often fatal visceral leishmaniasis (VL)1,2
.
There are two facts which were accumulated together from
literature to form the genesis of the present work. NSC 117027, a
NCI repository compound has been shown to inhibit calf thymus
DNA topoisomerase-I by Yves Pommier and co-workers11.
Few natural compounds containing naphthoquinone moiety like
diospyrin and isodiospyrin12–14 and non-naphthoquinone moiety
like camptothecin (CPT)15,16 and etoposide have been proved to
be potent antileishmanial agents by binding to DNA topoisom-
erases (Figure 1). Unlike the prototypic human topoisomerase-I
poison CPT; isodiospyrin does not induce human topoisomerase
I–DNA covalent complexes. Isodiospyrin antagonizes CPT-
induced, human topoisomerase I-mediated DNA cleavage.
Diospyrin, on the other hand exerts its inhibitory effect by binding
with the enzyme and stabilizing the topoisomerase I–DNA
‘‘cleavable complex’’. Inspired from the above-mentioned facts,
we attempted to develop naphthoquinone-based compounds with
cytotoxic potential to L. donovani cells which can potentially
target DNA topoisomerase-I. The compounds mentioned in the
present work are synthesized from a natural compound lawsone
or 2-hydroxy-1,4-naphthoquinone, which is a phytochemical
constituent found in Lawsonia inermis. Since a long time, this
plant is known to the people residing in Indian subcontinent due
to its usage in making of skin and hair dye. All the compounds
evaluated in the current work showed significant results in cell-
based assay. As far as leishmanial DNA topoisomerase-I inhib-
ition activity is concerned, both preincubation and simultaneous
Visceral leishmaniasis (VL), also known as kala-azar, caused by
Leishmania donovani, is often associated with a marked suppres-
sion of the cell-mediated immune response of the host, leading to
severe morbidity, even mortality, if left untreated3. According to the
World Health Organization [http://www.who.int/leishmaniasis/en/
], leishmaniasis threatens about 350 million men, women and
children in 88 countries around the world. As many as 12 million
people are believed to be currently infected with about 1 to 2 million
estimated new cases occurring every year4. It has to be noted that in
spite of development of antimonial drugs, amphotericin B, pent-
amidine isethionate, sodium stibogluconate and miltefosine, there
has been observed a rapid growth of resistance in leishmaniasis5,6
.
Among the various biological catalysts which are established
as cellular therapeutic targets, DNA topoisomerases are of
particular importance. DNA topoisomerases are ubiquitous
enzymes that play a pivotal role in modulating the dynamic
nature of DNA secondary or higher order structures and thus
support essential functions inside cells. These functions relate
mainly to nucleic acid metabolism, namely replication, transcrip-
tion, recombination and repair7,8. All known topoisomerases share
Address for correspondence: S. Vasanth Kumar, Department of
Chemistry, School of Science and Humanities, Karunya University,
Coimbatore-641114, India. Tel: +91 422 2614480: Fax: 91 422 2615615.
E-mail: kumar2359@yahoo.com

186 G. Sharma et al.
J Enzyme Inhib Med Chem, 2014; 29(2): 185–189
Figure 1. Chemical structures of diospyrin, isodiospyrin, camptothecin, etoposide and NSC 117027.
assays were conducted to understand the mechanism of action. Liquid culture media
The results demonstrated in the current work again proves that
Liquid culture media was prepared using M199 powder (Sigma
Aldrich, St. Louis, MO) 1.1% and HEPES buffer (pH 7.5) 20 mM.
Buffered media was filter sterilized by passing through 0.22 mm
pore-sized filter (Nalgene, Rochester, NY) and then heat inacti-
vated fetal calf serum 10% (GIBCO-Invitrogen, Carlsbad, CA),
Penicillin 100 U/ml and Streptomycin 100 mg/ml were added. This
media was used for liquid culture of L. donovani promastigotes.
naphthoquinone scaffolds are potent against L. donovani cells and
these compounds can be novel leads towards the development of
better inhibitors of leishmaniasis.
Materials and methods
Chemistry
All chemicals used in this work were purchased from Sigma
Aldrich Chemical Company (Bangalore, India). Solvents were
used as obtained from the supplier (Merck Chemical Company,
Bangalore, India) or redistilled as necessary. Thin layer chroma-
tography was performed on readymade sheets from Merck
Chemical Company. Melting points were measured on capillary
melting point apparatus and were uncorrected. Mass spectra were
recorded on a Agilent ESI-Q-TOF 6520 (New Delhi, India), with
Drug solutions
All compounds, CPT, were dissolved in 100% DMSO at 20 mM
concentration. All drug solutions were kept at room temperature.
The final concentration of DMSO was kept at 4% and 0.5% for all
in vitro and cellular experiments, respectively.
Parasite culture and maintenance
1
a resolution of 1 000 000 ppm. H NMR spectra and 13C NMR
The L. donovani strain AG83 promastigotes were grown at 22 ꢀC
in Rays modified media and in M199 liquid media supplemented
with 10% fetal calf serum as described previously19.
were recorded in DMSO with 400 MHz on a BRUKER Advance
liquid state NMR (Bangalore, India) using Trimethylsilane as
internal standard and the chemical shifts (ꢀ) are reported in ppm.
IR spectra were recorded on a SHIMADZU FT-IR 8400-S
spectrophotometer (Mumbai, India).
Agarose gel electrophoresis
Agarose gel electrophoresis was carried out for the separation of
DNA. Gel was prepared from 1.25 g of agarose, 125 ml of distilled
water and 2.4 ml of TAE. Then agarose was boiled in hot water
bath until the solution became clear and subsequently it was
cooled to about 50–55ꢀ C. The melted agarose solution was
poured into the casting tray and was cooled until it became solid
and the gel was loaded in the electrophoresis chamber with
1 ꢁ TAE buffer at a constant voltage of 5–6 V/cm for 2–8 h.
Different concentrations of compounds were added with enzyme
and DNA control. DNA was stained by soaking in 0.5 mg/ml of
ethidium bromide solution, visualized in U.V. transilluminator
and photographed.
Synthesis of compounds
The compounds were synthesized in aqueous medium according
to the procedure by Ayoob Bazgir and co-workers17. Detailed
experimental procedures and characterization data of synthesized
compounds 3a–e (Figure 2) can be found in the supplementary
information. Lawsone dimer has been procured as such from
Sigma Aldrich Chemical Company.
Biological screening
Agarose (A6013), ficoll-400, polyvinyl-pyrollidone (PVP-360),
M199, vitamins, amino acids CPT, 3-(4, 5-dimethylthiazol-2-yl)-
2, 5-diphenylterazolium bromide (MTT) and other chemicals
were purchased from Sigma Aldrich (St. Louis, MO). pBlue script
(SK) DNA was purchased from Novagen (Madison, WI, USA).
Topoisomerase LDTOP1LS was obtained according to an already
published procedure18.
Plasmid relaxation assay
The type 1 DNA topoisomerase was assayed by decreased
mobility of the relaxed isomers of supercoiled pBlue script (SK)
DNA in agarose gel. Relaxation assay was carried out as

DOI: 10.3109/14756366.2013.765413
Study of bis-lawsone analogs against leishmanial cells and DNA topoisomerase-I 187
Figure 2. Synthetic scheme of compounds with lawsone (1), different aromatic aldehydes (2), in refluxing condition/H2O/LiCl/12 h and their chemical
structures from 3a–e with lawsone dimer.
described previously with LdTOPILS serially diluted in the inhibitor, LdTOP1LS relaxes supercoiled plasmid DNA com-
relaxation buffer (25 mM Tris-HCl pH 7.5, 5% glycerol, 0.5 mM pletely after 30 min of incubation21. Screening of all the above-
DTT, 10 mM MgCl2, 2.5 mM EDTA and 150 mg/ml BSA) mentioned compounds on relaxation activity of LdTOP1LS were
supercoiled pBluescript (SKþ) DNA and 50 mM KCl. The carried out using 2% (v/v) of DMSO. All six compounds were
amount of supercoiled monomer DNA band fluorescence after initially screened at 200 mM concentrations. It was observed that
ethidium bromide (EtBr) (0.5 mg/ml) staining was quantitated by four compounds, 3a, 3b, 3c and 3d were showing inhibition
using GEL Doc 2000 from Biorad (Hercules, CA, USA).
against DNA topoisomerase-I of L. donovani at a concentration
of 200 mM. Compound 3e and lawsone dimer failed to show
any inhibition. It implies that only compounds 3a–d could be
potentially effective against DNA topoisomerase-I of L. donovani
and were further investigated.
Antileishmanial activity in vitro
The effect of drug on the viability of L. donovani AG83 cells was
determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylterazo-
lium bromide (MTT) assay20. The cells at the exponential phase
were collected and transferred into 24-well plate (approximately
4 ꢁ 105 to 106 cells/well). The cells were then incubated for 48 h
in the presence of various concentrations of compounds 3a–e and
lawsone dimer individually. After incubation, the cells were
centrifuged, and the supernatant was aspirated. The cell pellet was
washed twice with PBS (1X) and was finally suspended in 100 ml
of PBS (1ꢁ) in 96-well plates. Ten micro liters of MTT solution
(10 mg/ml) were added to each sample of 96-well plates and
samples were incubated for 4 h. After incubation, 100 ml of stop
solution (stock: 4963 ml of isopropanol and 17 ml of 12 N HCl)
was added and kept for 20 to 25 min at room temperature. The
optical density was taken at A492 on an ELISA reader (Multiskan
EX; Thermo Fisher Scientific, Waltham, MA).
Simultaneous assay of four compounds with CPT as
standard
In simultaneous relaxation assay, CPT concentrations were 50 and
100 mM, whereas concentration of compounds were 50, 100 and
200 mM. CPT showed 100% inhibition at a concentration of
100 mM. Compound 3a showed total inhibition at a concentration
of 100 mM and the others, i.e. compound 3b, 3c and 3d, were
showing total inhibition at a concentration of 200 mM (Figure 3A,
lane 7, 11, 14, 17). The IC50 for the compounds 3a–d were
calculated using the non-linear equation for sigmoidal response in
Graph Pad Prism ver. 5.00 (La Jolla, CA) and are listed in (Table 1).
Preincubation assay of four compounds with CPT as
standard
Results
To investigate whether these compounds interact with the enzyme
(without DNA substrate), LdTOP1LS was preincubated with
compounds 3a–d at different concentrations for 5 min at 37 ꢀC
before the addition of substrate DNA (Figure 3B). Inhibitory
effects of these compounds in preincubation condition were
compared with the inhibition by these compounds incubated
simultaneously with the enzyme (LdTOP1LS) and supercoiled
DNA in the relaxation reaction (Figure 3A). In the preincubation
assay, CPT concentrations were 50 and 100 mM, whereas various
Initial screening of all compounds
The effect of these chemically synthesized derivatives (3a–e) and
lawsone dimer on the unusual bi-subunit type IB topoisomerase of
L. donovani (LdTOP1LS) were examined by plasmid relaxation
assays as described in the section ‘‘Materials and methods’’. The
relaxation assays were carried out under standard assay conditions
where the plasmid DNA and the enzyme were present at a molar
ratio of 3:1. Under this reaction condition, in the absence of any

188 G. Sharma et al.
J Enzyme Inhib Med Chem, 2014; 29(2): 185–189
Table 2. EC50 values of the compounds of L. donovani promastigotes.
Compounds
EC50 (mM)
3a
3b
3c
3d
2 ꢂ 0.74
6 ꢂ 2.1
2 ꢂ 0.13
9 ꢂ 1.6
3e
Lawsone dimer
CPT
2.5 ꢂ 0.43
14 ꢂ 3
5.8 ꢂ 0.82
EC50 ꢂ S.D. for each of the tested compounds.
CPT is used as a positive control.
Figure 3. Inhibition of catalytic activity of LdTOP1LS by derivatives
bis-naphthoquinone 3,30-(arylmethylene)bis(2-hydroxynaphthalene-1,4
dione) analogs. (A) Relaxation of super coiled pBS (SKþ) DNA with
reconstituted LdTOP1LS at a molar ratio of 3:1. Lane 1, 90 fmol of pBS
(SKþ) DNA; lane 2, same as lane 1, but simultaneously incubated with
30 fmol of LdTOP1LS for 30 min at 37 ꢀC; lane 3, same as lane 2, but
in the presence of 2% (v/v) DMSO; lanes 4–5 same as lane 2, but in the
presence of 50 and 100 mM of CPT, respectively; lanes 6–17 same as lane
2, but in the presence of 50, 100, 200 mM of compounds 3a, 3b, 3c
and 3d, respectively. Positions of super coiled monomer (SM) and relaxed
and nicked monomer (RL/NM) are indicated. (B) Preincubation of
LdTOP1LS with respective inhibitors followed by the addition of DNA.
Lane 1, 90 fmol of pBS (SKþ) DNA; lane 2, same as lane 1, but the
enzyme was pre-incubated with 2% (v/v) of DMSO; lanes 4–5, same as
lane 2, but the enzyme was preincubated with 50 and 100 mM of CPT, Figure 4. Quantitative representation for percentage of promastigotes
respectively; lanes 6–17, same as lane 2, but the enzyme was preincubated survival of L. donovani in vitro in the presence of compounds 3a–e and
with 25, 50, 100 mM of compounds 3a–e, respectively. Reactions were lawsone dimer.
stopped by the addition of SDS to a final concentration of 0.5% and
electrophoresed in 1% agarose gel.
Graph Pad Prism using sigmoidal dose–response (variable slope
model) equation. Cytotoxic assay of all the compounds tested
revealed that the EC50 (the half maximal effective concentra-
tion) of compounds 3a, 3b, 3c, 3d, 3e and lawsone dimer obtained
were 2, 6, 2, 9, 2.5 and 14 mM (Table 2 and Figure 4), respectively.
It has to be noted that the compound 3e and lawsone dimer though
did not inhibit DNA topoisomerase-I enzyme yet were active
against L. donovani promastigotes. Both compound 3e and
lawsone dimer exhibited a considerable amount of cytotoxicity
for L. donovani promastigotes.
Table 1. Inhibitory concentration of the compounds on LdTOPILS.
IC50 (mM)
Compounds
Simultaneous
Preincubation
3a
3b
3c
3d
51 ꢂ 9.23
37.5 ꢂ 5.91
74 ꢂ 13.4
16 ꢂ 3.42
17 ꢂ 4.2
14 ꢂ 2.2
70 ꢂ 9.04
13 ꢂ 2.31
3e
Lawsone dimer
CPT
No inhibition
No inhibition
4.2 ꢂ 2.04
No inhibition
No inhibition
2.8 ꢂ 1.1
Discussion
The results shown here demonstrate the antileishmanial activity of
3,30-(arylmethylene) bis(2-hydroxynaphthalene-1,4 dione) deriva-
tives for the first time. Among the two methods used by us for
the DNA topoisomerase-I enzyme assay, in simultaneous assay
the compounds 3a–d showed IC50 in the range of 37.5–70 mM,
whereas compound 3b, the N-dimethyl amino derivative,
exhibited highest activity among all. Additionally, preincubation
assay was also conducted for the dual purpose of knowing
whether the compounds were able to inhibit the enzyme as such
and to investigate whether the compounds work with the same
mechanism as diospyrin, which is also a binapthoquinone and it
inhibits topoisomerase-I and stabilizes the topoisomerase I–DNA
‘‘cleavable complex’’12. In our case, we notice that all the
IC50 ꢂ S.D. for each of the tested compounds.
Compound 3e and lawsone dimer showed no inhibition.
CPT is used as a positive control in both methods.
concentrations of compounds were 25, 50 and 100 mM. CPT
showed 100% inhibition at 100 mM concentration. The com-
pounds showed total inhibition at 3a ¼ 100; 3b ¼ 100; 3c ¼ 100
and 3d ¼ 50 mM concentration, respectively (Figure 3B, lane 8,
11, 14, 16).
Cytotoxic effect against L. donovani promastigotes in vitro
The EC50 was calculated according to the already published compounds except the lawsone dimer and the trimethoxy
procedure21. The results shown are the means of three independ- derivative (compound 3e) were active on both the simultaneous
ent experiments with S.E. The fitted lines were generated with and preincubation assays. The preliminary results reported here

DOI: 10.3109/14756366.2013.765413
Study of bis-lawsone analogs against leishmanial cells and DNA topoisomerase-I 189
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molecules for further studies.
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Acknowledgements
GS and SVK thank the management and administration of Karunya
University for their encouragement and support.
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Declaration of interest
The authors report no declarations of interest.
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