Synthesis and macrofilaricidal activity of substituted 2-hydroxy/5-hydroxy/ 2-methyl-1,4-naphthoquinones

Article abstract of DOI:10.1002/ddr.21065

Preclinical Research Lymphatic filariasis is a disfiguring disease caused by parasitic worms that destroy the human lymphatic system leading to substantial morbidity. The current drug of choice for the treatment of filariasis is diethylcarbamazine and ivermectin with albendazole which are only effective against the microfilaria, leaving the adult worm unaffected, requiring the development of "adulticidal drugs." Thirty amino substituted 2-hydroxy/5-hydroxy/2-methyl-1,4-naphthoquinones were synthesized via the reaction of 2-hydroxy/5-hydroxy/2-methyl-1,4-naphthoquinones with different primary and secondary amines. Compounds 1-30 were evaluated for in vitro antifilarial activity against the adult bovine filarial worm Setaria digitata as assessed by worm motility and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) reduction assays. The mutagenecity, tumerogenecity, irritantancy, reproductive toxicity, drug score, druglike, and cLogP properties were calculated using OSIRIS property predictor. Ten compounds showed macrofilaricidal activity with ED₅₀ values ranging between 0.086 and 7.6 μM. Taking into account the biological effects and the promising drug-like profiles of these compounds, these represent valid leads for the development of antifilarial agents against adult filarial worm.

Full text of DOI:10.1002/ddr.21065

DRUG DEVELOPMENT RESEARCH 74 : 216–226 (2013)
Research Article
Synthesis and Macrofilaricidal Activity of Substituted
2-Hydroxy/5-Hydroxy/2-Methyl-1,4-Naphthoquinones
Twinkle Karunan, Nisha Mathew,* Lakshmy Srinivasan, and Kalyanasundaram Muthuswamy
Vector Control Research Centre (Indian Council of Medical Research), Indira Nagar,
Pondicherry-605006, India
Strategy, Management and Health Policy
Enabling
Preclinical Development Clinical Development
Technology,
Genomics,
Proteomics
Preclinical
Research
Toxicology, Formulation
Drug Delivery,
Pharmacokinetics
Phases I-III
Regulatory, Quality,
Manufacturing
Postmarketing
Phase IV
ABSTRACT
Lymphatic filariasis is a disfiguring disease caused by parasitic worms that destroy the
human lymphatic system leading to substantial morbidity. The current drug of choice for the treatment
of filariasis is diethylcarbamazine and ivermectin with albendazole which are only effective against the
microfilaria, leaving the adult worm unaffected, requiring the development of “adulticidal drugs.” Thirty
amino substituted 2-hydroxy/5-hydroxy/2-methyl-1,4-naphthoquinones were synthesized via the reac-
tion of 2-hydroxy/5-hydroxy/2-methyl-1,4-naphthoquinones with different primary and secondary
amines. Compounds 1–30 were evaluated for in vitro antifilarial activity against the adult bovine
filarial worm Setaria digitata as assessed by worm motility and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) reduction assays. The mutagenecity, tumerogenecity, irritantancy, repro-
ductive toxicity, drug score, druglike, and cLogP properties were calculated using OSIRIS property
predictor. Ten compounds showed macrofilaricidal activity with ED50 values ranging between 0.086 and
7.6 mM. Taking into account the biological effects and the promising drug-like profiles of these com-
pounds, these represent valid leads for the development of antifilarial agents against adult filarial worm.
Drug Dev Res 74 : 216–226, 2013.
© 2013 Wiley Periodicals, Inc.
Key words: macrofilaricide; filariasis; naphthoquinone; Setaria digitata; ADME
INTRODUCTION
therapy of different diseases [Watt and Breyer-
Brandwijik, 1962; Duke, 1985; Gafner et al., 1996].
Systematic studies of naphthoquinone derivatives have
led to the discovery of compounds with anticancer
[O’Brien, 1991; Lamson and Plaza, 2003; Taper et al.,
2004], antitumor [Lin et al., 1989], antifungal [Gershon
and Shanks, 1975; Gafner et al., 1996; Tandon et al.,
2004; Chung and Mi, 2005], antibacterial [Machado
Lymphatic filariasis (LF) is a disfiguring disease
caused by parasitic worms that damage the human
lymphatic system leading to morbidity (http://www.
filariasis.org—Global Alliance to Eliminate Lymphatic
Filariasis). The parasitic worms include Wuchereria
bancrofti, Brugia malayi, and Brugia timori that are
transmitted by mosquito vectors. The drugs currently
used for the treatment of LF are diethyl carbamazine
and ivermectin with albendazole. These treatments are
not effective in fully killing the longer-lived adult worm
The authors declare no conflicts of interest.
*Correspondence to: Nisha Mathew, Department of
and therefore are intended at reducing transmission Chemistry, Vector Control Research Centre (Indian Council of
Medical Research), Indira Nagar, Pondicherry-605006, India.
E-mail: nishamathew@yahoo.com
and pathology. This requires the necessity for the devel-
opment of an “adulticidal” drug.
Received 22 December 2012; Accepted 13 January 2013
Derivatives of 1,4-naphthoquinones are exten-
sively distributed in nature with many plants containing
Published online in Wiley Online Library (wileyonlinelibrary.
these substances being used in folk medicine for the com). DOI: 10.1002/ddr.21065
© 2013 Wiley Periodicals, Inc.

MACROFILARICIDAL ACTIVITY OF NAPHTHOQUINONES
217
et al., 2003; Medina et al., 2004], antiviral [Rastogi and were taken in glass capillary tubes on Melting point
Dhawan, 1990], molluscicidal [Celso et al., 2008], and apparatus (Techno Instruments Pvt. Ltd, Bangalore,
antithrombotic [Jin et al., 2004] activities. The inhibi- India) and were corrected using KSPII (KRUSS,
1
tory activity of certain naphthoquinones on HIV-1 pro- GmbH, Hamburg, Germany). HNMR spectra were
tease has also been decribed [Brinworth and Fairlie, recorded on a Bruker 400 MHz NMR spectrometer
1995; Mazumder et al., 1996]. The antiparasitic effects (Billerica, MA), chemical shifts are reported in parts per
of naphthoquinones against Trypanosoma cruzi million (ppm) relative to tetramethylsilane (TMS), and
[Salmon-Chemin et al., 2001], Toxoplasma gondii, spin multiplicities are given as s (singlet), d (doublet), dd
Leishmania sp. [Touraire et al., 1996], and Plasmodium (doubledoublet), t(triplet), q(quartet), orm(multiplet).
sp. [Bullock et al., 1970; Lin et al., 1991] have been The FT-IR spectrum was taken in accordance with the
studied, while the hydroxyl-1,4-napthoquinone, atova- KBr disc technique on Shimadzu FT-IR model 8300
quone [Williams and Clark, 1998] has been approved by (Kyoto, Japan). Chromatographic separation was per-
the Food and Drug Administration for the treatment of formed on glass columns with silica gel 60 (230–400
pneumonia due to Pneumocystis carinii.
mesh, Merck). Compound purity was determined using
The development of new antifilarial drugs based on HPLC (Thermo Finnigan, San Jose, CA) composed of
new molecular targets present in adult worms is a chal- Spectra System P4000, solvent delivery system Spectra
lenging task [Nisha et al., 2007]; however, it is important System AS3000, autosampler, and photodiode array
as there is no adulticidal drug available for killing the detector SN4000. Output signals were supervised via a
adult filarial worms. Polyamines have macrofilaricidal Chromquest 4.0 chromatography workstation (Thermo
activity and represent a nucleus which may be used for Finnigan, San Jose, CA). A 3 mm, Supelcosil ABZplus
synthesis of effective compounds [Kinnamon et al., analytical column (Sigma-Aldrich, St. Louis, MO)
1999]. N-alkyl amines have been studied as the pharma- (150 ¥ 4.6 mm) and a mobile phase combination of
cophore for antifilarial drug development [Srivastava acetonitrile-water (70 : 30) at a flow rate of 1 mL/min at
et al., 2000]. Aminoquinones are used as medicines 40°C and detection at 280 nm were utilized for the
[Elslager et al., 1970; Kallmayer and Tappe, 1987) and analysis. Retention times were recorded for all the com-
herbicides [William and Anja, 2001].
pounds. The purity of target compounds was Ն95%.
The product formed from the reaction of amine Thin layer chromatography (TLC) was executed with
with various quinones has significant scope for investi- F254 (Merck, GmbH, Darmstadt, Germany) coated alu-
gation. Earlier we have reported the antifilarial poten- minium sheets. Synthesis was done in a combinatorial
tial of plumbagin and substituted 1,4-naphthoquinones library synthesizer, Miniblock XT (Mettler-Toledo
[Nisha et al., 2002, 2010; Lakshmy et al., 2009] that Bohdan, New Jersey). Absorbance measurements were
suggested that this chemical class is worthy of additional prepared using Spectra-Max Plus (Molecular Devices,
investigation. The present work provides a prelimin- Sunnyvale, CA, USA) with SoftmaxPro software. All
ary account of results obtained from the reaction incubations were completed in a New Brunswick Scien-
of 2-hydroxy/5-hydroxy/2-methyl-1,4-naphthoquinone tific CO2 incubator (Edison, NJ, USA).
with primary and secondary amines and their screening
against bovine filarial worm Setaria digitata (Nema-
toda: Filariodea) for antifilarial activity. Absorption, dis-
tribution, metabolism, excretion, and toxicity (ADME/
tox) are key properties that need to be considered early
on any drug development project and this has been
done using the FAFDrugs online program developed
by Maria et al. [2006]. The prediction of properties such
as mutagenecity, tumerogenecity, irritant, reproductive
toxicity, drug score, druglike, and cLogP was also done
using the online program OSIRIS Property Explorer
developed by Sander [2001].
Synthesis of Substituted Naphthoquinones
To a stirred solution of substituted amine
(6.96 mM) in ethanol (4 mL), substituted naptho-
quinone (2.32 mM) was added slowly in 2 mL of dich-
loromethane (DCM). Stirring was continued for 5–6 h
at room temperature [Nisha et al., 2010]. The color of
the reaction mixture was changed from yellow to deep
black. The reaction was monitored by TLC. Chromato-
graphic purification of the crude product was carried
out by using column chromatography. The analytical
and spectroscopic data for the synthesized compounds
described here are shown in Table 1.
MATERIALS AND METHODS
General Procedures
In Vitro Screening for Antifilarial Activity Against
S. digitata
Chemicals and solvents purchased from Sigma-
Aldrich (St. Louis, MO) or Merck (Mumbai, India) were
Adults of the cattle filarial parasite of S. digitata
used without additional purification. Melting points were used to screen macrofilaricidal activity. Adult
Drug Dev. Res.

218
KARUNAN ET AL.
Drug Dev. Res.

MACROFILARICIDAL ACTIVITY OF NAPHTHOQUINONES
219
Drug Dev. Res.

220
KARUNAN ET AL.
Drug Dev. Res.

MACROFILARICIDAL ACTIVITY OF NAPHTHOQUINONES
221
female S. digitata worms collected from the peritoneal incubation period, the worms were further incubated
cavity of freshly slaughtered cattle were washed with for 30 min individually in phosphate-buffered saline
normal saline (0.85%) to free them from extraneous (pH 7.4, 0.5 mL) containing MTT (0.25 mg/mL). A
material and transferred to Dulbecco’s modified Eagle control was set up with untreated adult females but
medium (DMEM) containing 0.01% streptopenicillin exposed to DMSO as described previously. At the end
and supplemented with 10% heat-inactivated fetal calf of the MTT incubation, worms were transferred to a
serum and were used within an hour as reported earlier microliter plate containing 400 mL of spectroscopic-
[Nisha et al., 2002, 2010; Lakshmy et al., 2009].
grade DMSO and equilibrated at room temperature
for 1 h, with occasional gentle shaking to extract the
color developed. The absorbance of the resulting for-
mazan solution was then determined at 492 nm in a
microplate spectrophotometer relative to DMSO blank.
Compounds showing greater than 50% inhibition in
formazan formation with respect to control at
0.1 mg/mL were considered effective and were further
screened at lower concentrations to generate ED50
values (the dose that gives a 50% response, determined
by a sigmoid plot obtained by plotting the logarithm of
the dose on the x-axis and the percentage response on
the y-axis).
Worm Motility Assay
Stock solutions of compounds 1–30 were pre-
pared in dimethylsulfoxide (DMSO)/ethyl alcohol
depending upon the solubility of the compound at
30 mg/mL. For the assays, the compounds were further
diluted to the appropriate concentration using com-
plete assay medium. The DMSO/alcohol concentration
in the medium was kept below 1%. Preliminary screen-
ing was done at a concentration of 0.1 mg/mL. A simul-
taneous control with an equal volume of the vehicle in
the DMEM was included. Two adult female S. digitata
worms were introduced into each Petri dish with three
replicates for both test and control. Worms were incu-
bated at 37°C for 24 and 48 h in an incubator. After the
incubation period, the number of immobilized worms
Calculated ADME Properties of Substituted
1,4-Naphthoquinones
FAFDrugs is an online service that allows users
was counted. Immediately after counting, the worms to process compounds via simple ADME/tox filtering
were washed twice with fresh medium and transferred rules, for example, molecular weight, polar surface
to another set of Petri dishes containing fresh medium, area, logP, or number of rotatable bonds. Compounds
without the test solution, to assess whether any of the 1–30 were transformed to SMILES coordinates to
immobile worms regained motility. If the worms did not generate these calculated ADME properties. The
revive, the condition was considered irreversible and parameters were either in compliance with Lipinski’s
the concentration lethal. Each experiment was repeated rule of five or set as default. The prediction of prop-
twice.
erties such as mutagenecity, tumerogenecity, irritant,
reproductive toxicity, drug score, druglike, and cLogP
was done using the online program OSIRIS Property
Explorer available in the Organic Chemistry Portal
[Sander, 2001]. cLogP data were acquired using the
OSIRIS property explorer that uses the Chou and
Jurs (1979] algorithm, based on computed atom
contributions.
MTT-Formazan Colorimetric Assay for Viability
of Worms
Compounds 1–30 were further screened for
viability of adult S. digitata through an (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bro-
mide) (MTT) reduction assay [Comley et al., 1989].
Yellow MTT is reduced to purple formazan by mito-
chondrial enzymes present in living cells. This reduc-
tion takes place only when mitochondrial reductase
RESULTS
Thirty amino substituted 1,4-naphthoquinones
enzymes are active, and therefore conversion is used were synthesized by the reaction between alcoholic
as a measure of viable (living) cells. During the assay, solutions of substituted amines and substituted 1,4-
the formazan formed is extracted with DMSO and is naphthoquinones in DCM followed by chromato-
quantified. As the values of absorption correlate with graphic purification. The yield was found to vary from
formazan formation, worm viability was estimated as 13.5 to 57.6%.
percentage inhibition in formazan formation relative to
The results of preliminary screening of com-
control worms. Adult female worms were used for pounds 1–30 at 0.1 mg/mL for antifilarial activity in
this assay. After the exposure of the worms to the vitro against adult bovine filarial worm S. digitata by
compounds (0.1 mg/mL) in DMEM at 24 and 48 h worm motility assay by visual observation are shown in
Drug Dev. Res.

222
KARUNAN ET AL.
TABLE 2. In Vitro Macrofilaricidal Activity of Compounds 1–30 Against S. digitata Adult by Worm Motility and MTT Reduction Assays
% MTT reduction at 0.1 mg/mL Ϯ SE (n = 12)
Worm motility at 0.1 mg/mL
24 h 48 h
+++ +++
ED50 value mM
List of
compounds
24 h
48 h
24 h
48 h
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
75.52 Ϯ 0.94
86.41 Ϯ 0.24
82.15 Ϯ 0.15
83.72 Ϯ 1.01
67.24 Ϯ 0.28
77.99 Ϯ 0.77
20.49 Ϯ 2.25
18.26 Ϯ 2.50
27.36 Ϯ 2.64
46.26 Ϯ 0.13
23.99 Ϯ 1.90
31.42 Ϯ 3.05
24.36 Ϯ 1.16
69.31 Ϯ 1.85
36.00 Ϯ 2.04
31.41 Ϯ 1.96
30.58 Ϯ 2.53
61.93 Ϯ 1.10
62.33 Ϯ 0.54
25.40 Ϯ 0.60
72.47 Ϯ 0.57
73.73 Ϯ 0.73
85.65 Ϯ 0.55
86.49 Ϯ 0.61
80.42 Ϯ 0.97
90.78 Ϯ 0.33
24.45 Ϯ 3.01
66.38 Ϯ 1.11
88.68 Ϯ 0.65
34.29 Ϯ 1.19
87.01 Ϯ 0.23
90.28 Ϯ 0.27
91.85 Ϯ 0.11
96.04 Ϯ 0.27
91.50 Ϯ 0.48
97.03 Ϯ 0.82
40.45 Ϯ 0.48
39.28 Ϯ 0.93
48.64 Ϯ 0.18
64.90 Ϯ 0.93
44.50 Ϯ 0.86
56.03 Ϯ 1.68
48.28 Ϯ 0.99
88.19 Ϯ 0.38
48.57 Ϯ 0.49
34.12 Ϯ 1.68
37.87 Ϯ 2.80
74.36 Ϯ 0.24
72.53 Ϯ 0.34
38.61 Ϯ 0.35
85.0 Ϯ 0.45
85.27 Ϯ 0.24
90.29 Ϯ 0.21
93.87 Ϯ 0.44
86.01 Ϯ 1.84
92.2 Ϯ 0.37
44.72 Ϯ 0.29
71.30 Ϯ 0.29
96.93 Ϯ 0.91
46.54 Ϯ 0.57
30.61
3.85
18.21
28.4
136.3
116.13
–
5.78
0.086
2.43
0.37
2.42
1.84
–
+++
+++
+++
+++
+++
+
+++
+++
+++
+++
+++
+
+
+
–
–
–
–
–
–
–
–
–
–
–
–
+
+
+
+
++
+
+
+
++
+
+++
+
+
+++
+
+
49.88
–
12.53
–
–
–
–
–
+
+
++
++
+
+++
+++
+
93.26
277.29
–
52.44
29.37
–
+++
+++
+++
+++
+++
+++
+
+++
+++
+
+++
+++
+++
+++
+++
+++
+
+++
+++
+
131.95
214.0
36.5
61.6
101.0
8.04
–
13.5
26.9
7.3
7.6
44.2
4.85
–
16.50
12.34
–
14.70
2.64
–
+ Active worms; ++ Sluggish worms; +++ Immotile or dead worms.
Table 2. Worm motility assay results showed that the Calculated ADME properties of substituted 1,4-
worms treated with compounds 1, 2, 3, 4, 5, 6, 14, Naphthoquinones by FAF-Drugs online program and
18, 19, 21–26, 28, and 29 were completely paralyzed OSIRIS property explorer are given in Table 3.
or dead after 48 h incubation, whereas with com-
pounds 7–13, 15–17, 27, and 30, the worms showed
DISCUSSION
active or sluggish movement even after 48 h incuba-
tion. The results of in vitro macrofilaricidal screening
at 0.1 mg/mL by MTT reduction assay are also shown
There is an obvious requirement for the develop-
in Table 2. Seventeen compounds showed greater than ment of efficient, complementary chemotherapeutic
50% inhibition in formazan formation. No macrofilari- move that results in a long-term decline of the
cidal activity was observed for compounds 7–9, 11, 13, pathology-inducing worm stages, for example, adult
15–17, 20, 27, and 30 at 0.1 mg/mL (<50% reduction worms in lymphatic filariasis or to a macrofilaricidal
in MTT assay) while 10 and 12 showed moderate effect. An exhaustive review of literature [Nisha and
activity. The results of the motility assay were con- Kalyanasundaram, 2007] illustrated that considerable
firmed by the MTT assay results. The effective com- efforts have been focused on developing an effective
pounds were further screened at lower concentrations and safe drug that could kill or permanently sterilize
to get ED50 values and the results are given in Table 2. adult filarial worms. Although many of the studied com-
Ten compounds viz., 2, 4, 6, 5, 3, 29, 26, 1, 23, and pounds showed promising activity, none reached the
24, showed promising macrofilaricidal activity with final stage as an adulticidal drug either due to toxici-
ED50 values of 0.086, 0.37, 1.84, 2.42, 2.43, 2.64, 4.85, ty or poor absorption and other practical reasons.
5.78, 7.3, and 7.6 mM, respectively, at 48 h incubation. Many plant-derived compounds are also being studied
Drug Dev. Res.

MACROFILARICIDAL ACTIVITY OF NAPHTHOQUINONES
TABLE 3. OSIRIS Calculations for Compounds 1–30
223
Comp
MW
M
T
I
RT
cLogP
Drs
Ars
FB
RB
DL
DS
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
231
231
245
273
327
272
215
229
229
243
243
272
300
356
271
325
270
269
255
227
246
245
274
302
358
272
255
241
213
231
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
R
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
1.71
1.65
2.11
2.91
3.13
1.07
2.23
2.69
2.63
3.15
3.09
1.94
2.81
4.67
3.9
4.12
2.05
3.38
3.06
2.42
1.73
2.46
1.25
2.12
3.98
1.36
2.99
2.67
2.03
1.34
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
2
2
2
2
1
1
1
1
1
4
4
4
4
4
5
3
3
3
3
3
4
4
4
3
3
4
3
3
3
4
4
5
5
5
5
3
3
3
4
3
2
3
4
4
1
2
3
2
4
3
5
7
11
4
4
1
2
2
2
4
4
5
7
11
1
2
13
13
13
13
19
19
13
13
13
13
13
13
13
13
13
19
19
19
18
16
13
13
13
13
13
19
19
18
16
13
-0.41
-0.05
0.29
-0.64
0.95
0.62
0.66
0.67
0.32
0.58
0.94
0.57
0.58
0.62
0.44
0.61
0.88
0.84
0.45
0.28
0.51
0.92
0.36
0.40
0.54
0.61
0.75
0.93
0.89
0.69
0.94
-0.38
0.42
0.61
0.67
G
G
G
G
G
G
G
G
G
G
R
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
5.1
-0.95
-0.54
-0.19
-2.36
0.07
2.54
3.86
-0.09
-0.85
0.80
4.89
-6.36
-3.65
-0.96
-0.68
1.01
5.91
7.15
3.18
8.31
-5.84
-3.10
-0.39
-0.17
2
2
4
MW, Molecular weight; M, mutagenic; T, tumorigenic; I, irritant; RT, reproductive toxicity; R, red; G, green; Drs, hydrogen donors; Ars, hydrogen
acceptors; FB, flexible bonds; RB, rigid bonds; ClogP, fragment based prediction of logP (octanol / water); DL, drug likeness; DS, drug score donors.
for macrofilaricidal activity [Chatterjee et al., 1992; [O’Brien, 1991]. 1,4-naphthoquinone pharmacophore
Singh et al., 1994; Nisha et al., 2008]. Our recent is known to impart cytotoxity in a number of drugs,
work on alkylamino-1,4-naphthoquinones resulted in for example, streptonigrin [McBride et al., 1966],
few macrofilaricidal lead molecules [Nisha et al., actinomycins [Reich et al., 1962], mitomycins [Keyes
2010].
et al., 1991], and 2-hydroxynaphthoquinone derivatives
The 1,4-naphthoquinone scaffold has received [Hatzigrigoriou et al., 1993]. In addition to imparting
attention as a pharmacophore for the design of antitu- antifungal and cytotoxic activity, 1,4-naphthoquinones
mor and antimalarial agents [Baggish and Hill, 2002; exhibit significant antiparasitic activity [Williams and
Tandon et al., 2004]. The mechanism of action of naph- Clark, 1998; Lanfranchi et al., 2012]. Introduction of
thoquinones has not been completely elucidated. nitrogen in two different positions of the naphtho-
Exceptional biological activity is imparted on the 1,4- quinone core, at C-5 and at C-8 of menadione through
naphthoquinone pharmacophore due to the presence of a two-step, straightforward synthesis based on the regi-
two carbonyl groups that have the ability to accept elec- oselective hetero-Diels–Alder reaction, improved the
trons to produce the corresponding radical anion or solubility of polysubstituted 1,4-naphthoquinone deriva-
di-anion species, as well as their acid-base properties tives. The antimalarial and the antischistosomal acti-
[Tandon et al., 2004]. Atovaquone, a hydroxyl-1,4- vities of these polysubstituted aza-1,4-naphthoquinone
napthoquinone is an antiparasitic drug that selectively derivatives were evaluated and led to the selection of
targets the mitochondrial respiratory chain of the distinct compounds for antimalarial versus antischisto-
malaria parasite [Fry and Pudney, 1992; Baggish and somal action [Lanfranchi et al., 2012]. The structure–
Hill, 2002]. The 1,4-naphthoquinone structure is activity relationships (SARs) revealed that the presence
common in many natural products linked with antifun- of nitrogen-containing substituents at the 2-position was
gal, antibacterial, antiviral, and antitumour activities associated with an increase in activity—specifically, the
Drug Dev. Res.

224
KARUNAN ET AL.
presence of an arizidinyl substituent at the 2-position of at C3 position of the quinone ring showed macrofilari-
the naphthoquinone ring was associated with a sub- cidal activity with ED50 values 14.7 and 2.64 mM,
stantial enhancement in antimalarial activity, while respectively.
compounds with non-arizidinyl nitrogen-containing
substituents at the 2-position demonstrated even greater of the compounds using FAFDrugs ADME/tox filtering
activity [Lin et al., 1991]. and OSIRIS Property Explorer showed that all the
The results generated for ADME/tox properties
Our current research on substituted 1,4- compounds are in agreement with Lipinski’s rule of five
napthoquinones has resulted in 10 antifilarial molecules and passed through the filter. In general, an orally
of which two (2 and 4) showed excellent macrofilari- active drug has: not more than five hydrogen bond
cidal activity in vitro against adult bovine filarial worm donors (OH and NH groups), not more than 10 hydro-
S. digitata. The ED50 values for the most effective gen bond, acceptors (notably N and O), a molecular
macrofilaricidal compounds 2 and 4 were 0.086 and weight under 500, a LogP under 5. These features are
0.37 mM, respectively. SAR studies were carried out to referred to as Lipinski’s rule of five and can be used as
find out the influence of chemical structure modifica- a rule of thumb to indicate whether a molecule is likely
tion on macrofilaricidal activity. Comparison of the to be orally bioavailable (bioactive). Molecular property
macrofilaricidal activity of compounds 1–30 showed the evaluation of the analogs was done using the Osiris
following trend in killing the adult S.digitata in vitro: Property Explorer (Table 3). The chemical structure of
2 > 4 > 6 > 5 > 3 > 29 > 26 > 1 > 23 > 24. SAR studies the lead compound was entered and various drug-
showed that when position 2 of the quinone ring relevant properties like druglike, drug score, etc., were
was occupied by a hydroxyl group, the macrofilaricidal calculated. Prediction results were assessed and color
activity was more compared with a methyl group in coded. Properties with high risks of undesired effects
the same position along with isopropylamino and 4- like mutagenicity or a poor intestinal absorption are
(methylpentan-2-yl)amino substitution at position 3. shown in red (R). Whereas green (G) indicates drug-
Our earlier studies [Nisha et al., 2010] showed that like conform behavior. The toxicity predicted by
1,4-napthoquinone with propylamino, isopropylamino, OSIRIS property explorer clearly shows that the amino
isobutylamino, 1,3-dimethylbutylamino, and piperazi- substituted compounds have less toxicity in terms of
nylamino substitutions in the quinone ring exhibited mutagenicity, tumorigenicity, reproductive toxicity.
macrofilaricidal activity with ED50 values of 36, Red indicates unfavorable toxicity. Compounds 4 and
3.6, 3.1, 0.91, and 1.2 mM, respectively. In the pre- 15 with 4-methylpentan-2-ylamino substitution may be
sent study, an enhancement in macrofilaricidal acti- irritants as indicated by the red color. A positive value
vity was observed with a hydroxyl substitution at for druglikeness indicates that the molecule contains
position 2 of the quinone ring, along with similar predominantly fragments that are present in commer-
substituted amino groups at position 3 except for pip- cial drugs. The “druglikeness” was improved in most of
erazinylamino substitution (ED50 values were 5.78, the substituted compounds except in the case of cyclo-
0.086, 2.43, 0.37, and 1.84 mM, respectively, for hexylamino substitution. Toxicity assessment using
3-propylamino, 3-isopropylamino, 3-isobutylamino, Osiris revealed that except for 4 and 15, all other com-
3-(1,3-dimethylbutylamino), and 3-piperazinylamino pounds had a good calculated ADME profile which
substituted 2-hydroxy-1,4-napthoquinone). However, a minimizes the toxicity risk of napthoquinone analogs in
methyl substitution at position 2 instead of hydroxyl humans.
group has reduced the macrofilaricidal activity as
In conclusion, our studies have shown that amino
seen in compounds 7–21. Only compounds with substituted 1,4-napthoquinones exhibit antifilarial
3-(dibutylamino)propylamino (14), cyclohexylamino activity against adult filarial worms and the activity is
(18), cyclopentylamino (19), and 2-methoxyethylamino enhanced by the presence of hydroxyl group at position
(21) groups at C3 position showed macrofilaricidal 2 of the quinone ring and the activity is diminished by
activity with ED50 ranging between 12.53 and the presence of a methyl group. The compounds 2, 3, 5,
52.44 mM. When the hydroxyl group was in the 5th and 6 may be exploited as leads for the development of
carbon of the 1,4-napthoquinone, compounds with sub- effective macrofilaricidal agents.
stitutions of butylamino (22), 3-(dimethylamino) propy-
lamino (23), 3-(diethylamino)propylamino (24),
3-(dibutylamino)propylamino (25), and piperaziny-
ACKNOWLEDGMENT
lamino (26) groups at C3 exhibited macrofilaricidal
activity ED50 ranging between 4.85 and 26.9 mM.
The authors are grateful to Dr. P. Jambulingam,
As observed in the case of compounds 28 and 29, Director, Vector Control Research Centre, Pondi-
cyclopentylamino and cyclopropylamino substitutions cherry, for his encouragement during the study.
Drug Dev. Res.

MACROFILARICIDAL ACTIVITY OF NAPHTHOQUINONES
225
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FUNDING
Dr. Nisha Mathew expresses sincere gratitude to
the Department of Science and Technology (DST),
Government of India for the funding [Grant SR/SO/
HS-83/2005] to conduct this study.
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