Synthesis, characterization, in vitro anticancer activity, and docking of Schiff bases of 4-amino-1,2-naphthoquinone

Article abstract of DOI:10.1007/s00044-012-0150-7

A series of Schiff bases of 4-amino-1,2-naphthoquinone were synthesized, purified, characterized, and evaluated for cytotoxicity against a panel of human cancer cell lines (Hep-G₂, MG-63, and MCF-7). The cells were dosed with these Schiff bases at varying concentrations, and cell viability was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Significant anticancer activities were observed in vitro for some members of the series and compounds 4-(3,4,5-trimethoxybenzylideneamino) naphthalene-1,2-dione (S10) as well as 4-(4-hydroxy-3-methoxybenzylideneamino) naphthalene-1,2-dione (S13) are active cytotoxic agents against different cancer cell lines with IC₅₀ values in the range of 5.91-9.98 μM. The structures of synthesized compounds were established by spectroscopic (FT-IR, ¹H NMR, ¹³C NMR) and elemental analysis. To study the molecular basis of interaction and affinity of binding of the target molecules, all the compounds were docked into the ATPase domain of Topoisomerase-II (TP-II) by using Schroedinger molecular modeling software package. Docking experiments showed a good correlation between their predicted glide scores and the observed IC₅₀ values of synthesized compounds. Structure-activity relationships indicated that presence of electron donating groups on phenyl ring of Schiff bases enhances the activity but Schiff base with electron withdrawing substituents on phenyl ring shows diminished activity.

Full text of DOI:10.1007/s00044-012-0150-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:1604–1617
DOI 10.1007/s00044-012-0150-7
ORIGINAL RESEARCH
Synthesis, characterization, in vitro anticancer activity, and
docking of Schiff bases of 4-amino-1,2-naphthoquinone
•
S. Shukla R. S. Srivastava S. K. Shrivastava
•
•
•
A. Sodhi Pankaj Kumar
Received: 6 February 2012 / Accepted: 8 June 2012 / Published online: 30 June 2012
Ó Springer Science+Business Media, LLC 2012
Abstract A series of Schiff bases of 4-amino-1,2-naph-
thoquinone were synthesized, purified, characterized, and
evaluated for cytotoxicity against a panel of human cancer
cell lines (Hep-G2, MG-63, and MCF-7). The cells were
dosed with these Schiff bases at varying concentrations, and
cell viability was measured by a 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay. Significant
anticancer activities were observed in vitro for some
members of the series and compounds 4-(3,4,5-trimeth-
oxybenzylideneamino)naphthalene-1,2-dione (S10) as well
as 4-(4-hydroxy-3-methoxybenzylideneamino)naphtha-
lene-1,2-dione (S13) are active cytotoxic agents against
different cancer cell lines with IC50 values in the range of
5.91–9.98 lM. The structures of synthesized compounds
donating groups on phenyl ring of Schiff bases enhances the
activity but Schiff base with electron withdrawing substitu-
ents on phenyl ring shows diminished activity.
Keywords Hep-G2 ꢀ MG-63 ꢀ MCF-7 ꢀ Cytotoxicity ꢀ
Schiff bases ꢀ Docking
Introduction
According to World Cancer Report from the International
Agency for Research on Cancer, cases of cancer doubled
globally between 1975 and 2000, will double again by
2020, and will nearly triple by 2030 (Mulcahy, 2008).
Therefore various research initiatives are going on world-
wide for the treatment of malignancy with the objective to
discover some novel potent and effective antineoplastic
agents, particularly those interacting with novel biological
targets. Mass screening programs of natural products by the
National Cancer Institute have identified the quinone as an
important pharmacophoric moiety for cytotoxic activity
(Driscoll et al., 1974; Liu et al., 2004).
1
were established by spectroscopic (FT-IR, H NMR, 13C
NMR) and elemental analysis. To study the molecular basis
of interaction and affinity of binding of the target molecules,
all the compounds were docked into the ATPase domain of
¨
Topoisomerase-II (TP-II) by using Schrodinger molecular
modeling software package. Docking experiments showed a
good correlation between their predicted glide scores and the
observed IC50 values of synthesized compounds. Structure–
activity relationships indicated that presence of electron
Literature survey described a variety of naturally occur-
ring and synthetic 1,2-naphthoquinones receiving a great
deal of attention for their anticancer activity. Among them
some naturally occurring 1,2-naphthoquinones include
rhinacanthone (Thirumurugan et al., 2000; Siripong et al.,
2006, 2009; Kongkathip et al., 2003), beta lapachone (Dubin
et al., 2001; Lai et al., 1998; Pardee et al., 2002), mansonones
(Wang et al., 2004), etc. (Fig. 1a–c) as well as synthetic 1,
2-naphthoquinones include 4-hydroxy-3-methyl-1,2-naph-
thoquinone thiosemicarbazone (HM-NQTS, Saha et al.,
2002), 1,2-naphthoquinone-2-thiosemicarbazone (NQTS,
Chen et al., 2004), 4-alkyl/aryl-1,2-naphthoquinones thio-
semicarbazones and their copper complex (Afrasiabi et al.,
S. Shukla ꢀ R. S. Srivastava (&) ꢀ S. K. Shrivastava
Department of Pharmaceutics, I.T., Banaras Hindu University,
Varanasi 221005, India
e-mail: rssriitbhu@yahoo.com; shubpharma@rediffmail.com
A. Sodhi
School of Biotechnology, Faculty of Science,
Banaras Hindu University, Varanasi 221005, India
P. Kumar
School of Pharmaceutical Science, Siksha ‘O’ Anusandhan
University, Bhubaneshwar, Orrisa, India
123

Med Chem Res (2013) 22:1604–1617
1605
Fig. 1 Structures of some
potent natural and synthetic
anticancer 1,2-naphthoquinone
analogues
2004) (Fig. 1d–f). Our research group had also explored and
reported a series of 1,2-naphthoquinone derivatives having
antiproliferative activity (Shukla et al., 2012). Several 1,
2-napthoquinone derivatives can induce DNA-TOPO II-
mediated cleavages of DNA, and this effect is crucial for the
cytotoxicity of these compounds (Frydman et al., 1997).
Schiff bases also gained importance because of the
physiological and pharmacological activities associated
with them. Compounds containing azomethine group
(–C=N–) in the structure are known as Schiff bases, which
are usually synthesized by the condensation of primary
amines and active carbonyl groups. Various schiff bases
are found to have anticancer activity (Hu et al., 2008;
Sharma et al., 1998; Pathak et al. 2000; Xu and Xu, 2000).
Therefore, these above-mentioned results prompted us
to continue our investigation towards synthesis of Schiff
bases after introducing amino group in 1,2-naphthoquinone
ring system in order to achieve new lead compounds for
future development as anticancer agents and docking
studies were conducted to get an insight about their binding
preferences at the active site of the receptor (ATPase
domain of Topoisomerase-II). In this context, new series
of 4-(arylideneamino)naphthalene-1,2-dione (S01–S16)
and 4-(cyclohexylidene/cyclopentylidine amino)naphtha-
lene-1,2-dione (S17, S18) were synthesized and their
anticancer activity was evaluated.
amino)naphthalene-1,2-dione (S17, S18) were prepared
by following the synthetic pathway depicted in Fig. 2.
According to previously reported method, the starting
material 1,2-naphthoquinone was conveniently prepared by
oxidation of 1-amino-2-naphthol hydrochloride, which was
obtained by reduction of water soluble dye b naphthol
orange (Furniss et al., 1995; Vogel’s Text Book Of Prac-
tical Organic Chemistry). The synthesized 1,2-naphtho-
quinone aminated with sodium azide in acetic acid and
gave 4-amino-1,2-naphthoquinone (Fieser and Hartwell,
1935). Then Schiff bases (S01–S16) were prepared by
condensing substituted aryl aldehydes with 4-amino-1,
2-naphthoquinone in suitable solvent (methanol/ethanol) in
the presence of few drops of glacial acetic acid as catalyst.
Compounds (S17, S18) were synthesized by the above
method using cyclohexanone/cyclopentanone as starting
material. The chemical structures of the synthesized com-
pounds were established by spectroscopic (FT-IR, 1H
NMR, 13C NMR) and elemental analysis.
Experimental
All reactions were monitored by thin layer chromatography
(TLC) using silica gel G (Spectrochem Pvt. Ltd., Mumbai).
The plates were developed by exposing to iodine chamber.
Melting points were determined by BARNSTEAD/
Electrothermal Stuart-SMP10, open capillary melting point
apparatus and were uncorrected. IR spectra were recorded
on KBr disks using SHIMADZU Infrared-spectro-
photometer, FTIR-8400S. The 1H NMR and 13C NMR
spectra were measured by JEOL, AL300, FT-NMR spec-
trophotometer with Operating frequency, 300 MHz at
temperature 25 °C using DMSO-d6/CDCl3 as solvent
containing TMS as internal standard and reported chemical
Materials and methods
Chemistry
The target compounds 4-(arylideneamino)naphthalene-1,
2-dione (S01–S16) and 4-(cyclohexylidene/cyclopentylidine
123

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Med Chem Res (2013) 22:1604–1617
Fig. 2 Scheme for synthesis of 4-amino-1,2-naphthoquinone and its
Schiff bases, reagents, and conditions (i) Na2S2O4, stirring at
40–50 °C, SnCl2 in conc. HCl with 100 ml water added, and cooled
to 0 °C. (ii) FeCl3 in conc. HCl with crushed ice and added with
stirring. (iii) Sodium azide in water and glacial acetic acid, heated to
40 °C. (iv, a) Aromatic aldehydes, glacial acetic acid, ethanol/
methanol, reflux 6–10 h (iv, b) Cyclic ketones, conc. H2SO4, ethanol/
methanol, reflux 8–10 h
shift in d values (ppm). C, H, N analysis has been per-
formed with Exeter Analytical Inc., USA, CE-440 ele-
mental analyzer.
after 2 h the reaction product was crystallized from water
and it formed beautiful deep red needles.
M.P. 172–173 °C, yield 80 %, TLC (benzene:chloro-
form, 2:8) Rf: 0.73. IR (KBr, tmax cm-1): 3367.82 (N–H
str.), 1683.91, 1576.54 (C=O str. of carbonyl group),
3115.14 (Aromatic –C–H str.); 1H NMR (DMSO-d6,
300 MHz) d (ppm): 11.46 (s, 2H, NH2), 6.77 (s, 1H, eth-
ylene proton), 7.48–8.02 (m, 4H, Ar–H); 13C NMR
(DMSO-d6) d (ppm): 101 (C3, ethylene proton), 127.44–
143.34 (Aromatic C, C5–C10), 174.64, 182.21 (C1–C2,
C=O); Anal. Calc. for C10H7NO2: C, 69.36; H, 4.07; N,
8.09. Found: C, 69.41; H, 4.02; N, 8.12 (cf. Fig. 2).
Procedure for preparation of 4-amino-1,
2-naphthoquinone (ANQ)
Solution of 0.01 mol of 1,2-naphthoquinone in 15 ml of
glacial acetic acid at 40 °C was treated with a solution of
0.017 mol of sodium azide in 5 ml of water. Gas was
evolved; the solution became reddish brown in color, and
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Med Chem Res (2013) 22:1604–1617
1607
0
(Aromatic-C, C5–C10 C1 –C6 ), 156.25 (C=N–), 178.58,
0
General procedure for synthesis of schiff bases
[4-(arylideneamino)naphthalene-1,2-dione (S01–S16)]
180.04 (C1–C2, C=O) (cf. Fig. 2).
A mixture of equimolar quantities of substituted aryl
aldehydes (0.01 mol) and 4-amino-1,2-naphthoquinone
(0.01 mol) in ethanol/methanol (20 ml) was refluxed on a
water bath for 6–10 h in the presence of few drops of
glacial acetic acid as catalyst. The progress of reaction was
monitored by TLC at appropriate time interval. After
completion of reaction, the solution was cooled, separated
solid was filtered and washed with ice-cold water and
dried. Finally, the product thus obtained was recrystallized
from ethyl acetate and ethanol in different proportions
depending on the nature of the compound.
4-(4-Hydroxybenzylideneamino)naphthalene-1,2-dione
(S04)
TLC (methanol:chloroform, 1:9) Rf: 0.73. IR (KBr, tmax
cm-1): 3138.79, 3056.69 (Aromatic C–H str.), 1621.83
(C=N, azomethine), 1703.65, 1683.33 (C=O str. of car-
1
bonyl group), 3451.27 (O–H str.); H NMR (DMSO-d6,
300 MHz) d (ppm): 6.71 (s, 1H, C=CH– group), 7.12–7.91
(m, 8H, Ar–H), 8.21 (s, 1H, –CH=N– group), 9.61 (s, 1H,
–OH); 13C NMR (DMSO-d6) d (ppm): 123.99–140.16
0
0
(Aromatic-C, C5–C10 C1 –C6 ), 151.20 (C=N–), 179.14,
Physicochemical data and results of elemental analysis
of the synthesized Schiff bases are listed in Table 1. The
spectral data of all the compounds are given below.
182.81 (C1–C2, C=O) (cf. Fig. 2).
4-(4-Methoxybenzylideneamino)naphthalene-1,2-dione
(S05)
4-(Benzylideneamino)naphthalene-1,2-dione (S01)
TLC (methanol:chloroform, 1:9) Rf: 0.57. IR (KBr, tmax
cm-1): 3085.88 (Aromatic C–H str.), 2935.43 (C–H str.,
O–CH3 group), 1641.28 (C=N, azomethine), 1705.54,
TLC (methanol:chloroform, 2:8) Rf: 0.62. IR (KBr, tmax
cm-1): 1638.84 (C=N, azomethine), 3159.83 (Aromatic C–H
str.), 1701.39, 1688.49 (C=O str. of carbonyl group), 1538.54
1
1678.87 (C=O str. of carbonyl group); H NMR (DMSO-
d6, 300 MHz) d (ppm): 3.81 (s, 3H, OCH3), 6.57 (s, 1H,
C=CH– group), 7.19–7.95 (m, 8H, Ar–H), 8.07 (s, 1H,
HC=N group); 13C NMR (DMSO-d6) d (ppm): 55.67
1
(Aromatic C=C str.); H NMR (DMSO-d6, 300 MHz) d
(ppm): 6.52 (s, 1H, C=CH– group), 7.38–7.95 (m, 9H, Ar–H),
8.34 (s, 1H, –CH=N– group); 13C NMR (DMSO-d6) d (ppm):
0
(OCH3), 121.19–150.3 (Aromatic-C, C5–C10 C1 –C6 ),
0
0
124.52–138.72 (Aromatic-C, C5–C10 C1 –C6 ), 139.78 (C=N)
0
154.90 (C=N–), 177.87, 179.51 (C1–C2, C=O) (cf. Fig. 2).
180.43, 181.23 (C1–C2, C=O) (cf. Fig. 2).
4-(2-Chlorobenzylideneamino)naphthalene-1,2-dione
(S06)
4-(4-Nitrobenzylideneamino)naphthalene-1,2-dione (S02)
TLC methanol:chloroform, 2:8) Rf : 0.67. IR (KBr, tmax
cm-1): 1631.83 (C=N, azomethine), 3129.50 (Aromatic
C–H str.), 1359.50 (N=O str.) 1707.69, 1679.30 (C=O str.
of carbonyl group); 1H NMR (DMSO-d6, 300 MHz) d
(ppm): 6.44 (s, 1H, C=CH– group), 7.33–8.07 (m, 8H,
Ar–H), 8.53 (s, 1H, –CH=N– group); 13C NMR (DMSO-
TLC methanol:chloroform, 1:9) Rf: 0.61. IR (KBr, tmax
cm-1): 3117.98, 3053.67 (Aromatic C–H str.), 1617.22
(C=N, azomethine), 1709.19, 1682.82 (C=O str. of carbonyl
group), 1053.17 (C–Cl str.); 1H NMR (DMSO-d6, 300 MHz)
d (ppm): 6.62 (s, 1H, C=CH– group), 7.40–7.90 (m, 8H,
Ar–H), 8.71 (s, 1H, HC=N– group); 13C NMR (DMSO-d6) d
0
0
d6) d (ppm): 123.99–140.16 (Aromatic-C, C5–C10 C1 –C6 ),
158.10 (C=N–), 179.14, 182.81 (C1–C2, C=O) (cf. Fig. 2).
0
(ppm): 121.85–135.96 (Aromatic-C, C5–C10 C1 –C6 ),
0
154.85 (C=N–),181.04, 180.58 (C1–C2, C=O) (cf. Fig. 2).
4-(3-Bromobenzylideneamino)naphthalene-1,2-dione (S07)
4-(2,4-Dichlorobenzylideneamino)naphthalene-1,2-dione
(S03)
TLC (methanol:chloroform, 1:9) Rf: 0.63. IR (KBr, tmax
cm-1): 3108.54, 3023.18 (Aromatic C–H str.), 1636.47
(C=N, azomethine), 1702.91, 1679.52 (C=O str. of qui-
none), 771.55 (C–Br str.); 1H NMR (DMSO-d6, 300 MHz)
d (ppm): 6.64 (s, 1H, C=CH– group), 7.22–8.37 (m, 8H,
Ar–H), 8.81 (s, 1H, HC=N– group); 13C NMR (DMSO-d6)
TLC (methanol:chloroform, 1:9) Rf: 0.71. IR (KBr, tmax
cm-1): 3115.93, 3033.61 (Aromatic C–H str.), 1634.63
(C=N, azomethine), 1705.25, 1681.66 (C=O str. of car-
bonyl group), 1107.19, 1046.55 (C–Cl str.); 1H NMR
(DMSO-d6, 300 MHz) d (ppm): 6.62 (s, 1H, C=CH–
group), 7.26–8.08 (m, 7H, Ar–H), 8.59 (s, 1H, –CH=N–
group); 13C NMR (DMSO-d6) d (ppm):122.14–135.06
0
0
d (ppm): 123.12–142.62 (Aromatic-C, C5–C10 C1 –C6 ),
157.52 (C=N–), 180.27, 181.78 (C1–C2, C=O) (cf. Fig. 2).
123

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Med Chem Res (2013) 22:1604–1617
Table 1 Analytical and physicochemical data of the synthesized compounds
O
O
O
N
O
O
O
N
C
N
R'
R
S18
S17
S01-S16
Compounds
R
H
R1
Molecular formula M.W.a M.P. (°C)b Yield (%) % Analysis of C H and N found (calc)c
C, H, N
S01
C17H11NO2
C17H10N2O4
C17H9Cl2NO2
261
306
329
154–156
142–143
133–134
51
65
71
78.11 (78.15), 4.21 (4.24), 5.33 (5.36)
66.69 (66.67), 3.27 (3.29), 9.12 (9.15)
61.82 (61.84), 2.77 (2.75), 4.26 (4.24)
S02
S03
H
H
NO2
Cl
Cl
S04
S05
S06
H
H
H
C17H11NO3
C18H13NO3
C17H10ClNO2
277
291
295
141–143
171–173
158–159
67
44
57
73.66 (73.64), 3.99 (4.00), 5.07 (5.05)
74.24 (74.22), 4.48 (4.50), 4.79 (4.81)
69.07 (69.05), 3.45 (3.41), 4.71 (4.74)
OH
OCH3
Cl
S07
S08
S09
H
H
H
C17H10BrNO2
C18H13NO2
C19H16N2O2
339
275
304
183–185
147–149
127–129
78
63
74
60.05 (60.02), 2.93 (2.96), 4.09 (4.12)
78.50 (78.53), 4.73 (4.76), 5.11 (5.09)
74.95 (74.98), 5.33 (5.30), 9.17 (9.20)
Br
CH3
N
OCH3
OCH3
OCH3
S10
H
C20H17NO5
351
139–141
72
68.39 (68.37), 4.91 (4.88), 4.03 (3.99)
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Med Chem Res (2013) 22:1604–1617
1609
Table 1 continued
Compounds
R
R1
Molecular formula M.W.a M.P. (°C)b Yield (%) % Analysis of C H and N found (calc)c
C, H, N
OCH3
S11
H
C19H15NO4
C17H10N2O4
C18H13NO4
321
306
307
167–169
179–181
147–148
69
41
59
71.05 (71.02), 4.68 (4.71), 4.33 (4.36)
66.64 (66.67), 3.31 (3.29), 9.12 (9.15)
70.31 (70.35), 4.24 (4.26), 4.58 (4.56)
OCH3
S12
S13
H
H
O2N
OCH3
OH
S14
S15
H
H
C17H11NO3
277
300
123–125
189–190
77
62
73.67 (73.64), 3.98 (4.00), 5.08 (5.05)
76.01 (75.99), 4.06 (4.03), 9.35 (9.33)
HO
C19H12N2O2
N
H
S16
H
C19H13NO2
287
204–205
78
79.39 (79.43), 4.59 (4.56), 4.91 (4.88)
S17
S18
a
–
–
–
–
C16H15NO2
15H13NO2
253
239
152–153
168–170
64
55
75.85 (75.87), 5.94 (5.97) 5.57 (5.53)
75.28 (75.30), 5.51 (5.48), 5.88 (5.85)
C
Molecular weight (M.W.) of the compound
b
c
Melting point (M.P.) of the compounds at their decomposition
Elemental analysis for C, H, and N were within 0.04 % of the theoretical value
4-(4-Methylbenzylideneamino)naphthalene-1,2-dione
(S08)
azomethine), 1721.40, 1681.49 (C=O str. of carbonyl
1
group); H NMR (DMSO-d6, 300 MHz) d (ppm): 3.29,
3.31 (d, 6H, CH3), 7.11–7.83 (m, 8H, Ar–H), 8.27 (s, 1H, –
HC=N– group); 13C NMR (DMSO-d6) d (ppm): 43.2,
0
44.19 (2CH3–), 111.57–136.96 (Aromatic-C, C5–C10 C1 –
TLC (ethylacetate:hexane, 7:3) Rf: 0.54. IR (KBr, tmax
cm-1): 2889.61 (C–H str. of CH3 group), 3048.84 (Aro-
matic C–H str.), 1633.90 (C=N, azomethine), 1701.73,
0
C6 ), 148.05 (C=N–), 179.66, 180.14 (C1–C2, C=O) (cf.
1
1669.80 (C=O str. of carbonyl group); H NMR (DMSO-
Fig. 2).
d6, 300 MHz) d (ppm): 2.88 (s, 3H, –CH3), 6.61(s, 1H,
C=CH– group), 7.08–7.89 (m, 8H, Ar–H), 8.18 (s, 1H,
HC=N– group); 13C NMR (DMSO-d6) d (ppm): 21.10
4-(3,4,5-Trimethoxybenzylideneamino)naphthalene-1,
2-dione (S10)
0
(–CH3), 101.03–134.25 (Aromatic-C, C5–C10 C1 –C6 ),
0
152.10 (C=N–), 179.58, 182.21 (C1–C2, C=O) (cf. Fig. 2).
TLC (methanol:chloroform, 1:9) Rf: 0.49. IR (KBr, tmax
cm-1): 3184.91, 3124.54, 3089.52 (Aromatic C–H str.),
2924.18, 2854.58, 2804.17 (C–H str., O–CH3 groups),
1632.91 (C=N, azomethine), 1701.27, 1687.47 (C=O str. of
carbonyl group); 1H NMR (DMSO-d6, 300 MHz) d (ppm):
3.86 (t, 9H, OCH3), 7.11–7.96 (m, 6H, Ar–H), 8.23 (s, 1H,
HC=N–group); 13C NMR (DMSO-d6) d (ppm): 57.14,
58.66, 58.94 (OCH3), 116.36–139.11 (Aromatic-C, C5–C10
4-(4-(Dimethylamino)benzylidene amino)naphthalene-1,
2-dione (S09)
TLC (ethylacetate:hexane, 7:3) Rf: 0.51. IR (KBr, tmax
cm-1): 1338.64 (–C–N stretch), 2881.06 (C–H str. of CH3
group), 3066.35 (Aromatic C–H str.), 1620.26 (C=N,
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Med Chem Res (2013) 22:1604–1617
0
C1 –C6 ), 149.87 (C=N–), 177.82, 182.14 (C1–C2, C=O)
0
0
C1 –C6 ), 153.97 (C=N–), 176.58, 182.15 (C1–C2, C=O)
0
(cf. Fig. 2).
(cf. Fig. 2).
4-((1H-indol-3-yl)methyleneamino)naphthalene-1,2-dione
(S15)
4-(3,4-Dimethoxybenzylideneamino)naphthalene-1,2-dione
(S11)
TLC (methanol:chloroform, 1:9) Rf: 0.68. IR (KBr, tmax
cm-1) 3109.35 (Aromatic C–H str.), 1620.26 (C=N str.,
azomethine), 1703.40, 1682.25 (C=O str. of carbonyl
group), 3290.67 (–N–H str.); 1H NMR (DMSO-d6,
300 MHz) d (ppm): 11.51 (s, 1H, Indole NH), 7.07–7.82
(m, 9H, Ar–H), 8.48 (s, 1H, –HC=N– group);13C NMR
(DMSO-d6) d (ppm): 111.58–136.70 (Aromatic-C), 147.11
(C=N–), 179.56, 180.47 (C1–C2, C=O) (cf. Fig. 2).
TLC (methanol:chloroform, 1:9) Rf: 0.52. IR (KBr, tmax
cm-1): 3071.49 (Aromatic C–H str.), 2873.59, 2804.76
(C–H str., O–CH3 groups), 1638.25 (C=N, azomethine),
1707.48, 1668.13 (C=O str. of carbonyl group);1H NMR
(DMSO-d6, 300 MHz) d (ppm): 3.88 (d, 6H, OCH3) 6.91–
7.81 (m, 7H, Ar–H), 8.28 (s, 1H, –HC=N– group); 13C
NMR (DMSO-d6) d (ppm): 55.57, 55.81 (OCH3), 113.31–
0
147.11 (Aromatic-C, C5–C10 C1 –C6 ), 154.11 (C=N–),179.87,
0
181.51(C1–C2, C=O) (cf. Fig. 2).
4-((3-Phenylallylidene)amino)naphthalene-1,2-dione (S16)
4-(2-Nitrobenzylideneamino)naphthalene-1,2-dione (S12)
TLC (methanol:chloroform, 1:9) Rf: 0.51. IR (KBr, tmax
cm-1): 3164.19, 3051.49 (Aromatic C–H str.), 2962.76,
2834.59 (C–H str. of allyl group), 1640.12 (C=N str.,
azomethine), 1711.24, 1683.52 (C=O str. of carbonyl
TLC (methanol:chloroform, 1:9) Rf: 0.65. IR (KBr, tmax
cm-1): 3142.11, 3123.59 (Aromatic C–H str.), 1346.24
(–N=O str.), 1635.69 (C=N, azomethine), 1718.39, 1682.34
(C=O str. of carbonyl group); 1H NMR (DMSO-d6,
300 MHz) d (ppm): 6.92 (s, 1H, C=CH– group), 7.60–8.61
(m, 8H, Ar–H), 8.82 (s, 1H, –HC=N– group); 13C NMR
(DMSO-d6) d (ppm): 123.10–140.06 (Aromatic-C, C5–C10
1
group); H NMR (DMSO-d6, 300 MHz) d (ppm): 7.50–
8.29 (m, 9H, Ar–H), 8.61 (s, 1H, –HC=N– group), 6.63–
6.82 (dd, 2H, –CH=CH–); 13C NMR (DMSO-d6): d (ppm):
123.06–137.46 (Aromatic-C), 152.12 (N=CH– group)
174.60, 182.14 (C1–C2, C=O) (cf. Fig. 2).
0
C1 –C6 ), 155.07 (C=N–),178.34, 182.31 (C1–C2, C=O) (cf.
0
Fig. 2).
General procedure for synthesis of schiff bases from
cyclic ketones [4-(cyclohexylidene/cyclopentylidine
amino)naphthalene-1,2-dione (S17–S18)]
4-(4-Hydroxy-3-methoxybenzylideneamino)naphthalene-
1,2-dione (S13)
For the preparation of schiff bases with cyclic ketones,
ethanolic solution of 4-amino-1,2-naphthoquinone (0.01 mol)
was taken in 100-ml round bottom flask, to this equimolar
(0.01 mol) ethanolic solution of respective cyclic ketones
were added and few drops of conc. H2SO4 were added as
catalyst. The solution was allowed to reflux on a water bath
for 8–10 h. The solution was poured on to the crushed ice,
neutralized with K2CO3 and kept aside. The precipitate
thus separated was collected by filtration.
TLC (methanol:chloroform, 1:9) Rf: 0.69. IR (KBr, tmax
cm-1): 3059.20, 3032.24 (Aromatic C–H str.), 1638.73
(C=N, azomethine), 1713.25, 1668.13 (C=O str. of carbonyl
group), 3484.12 (O–H str.), 2871.90 (C–H str., O–CH3
group); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 10.24 (br,
s, 1H, –O–H group), 3.80 (s, 3H, OCH3), 6.96–7.72 (m, 7H,
Ar–H), 8.27 (s, 1H, –HC=N– group); 13C NMR (DMSO-d6)
d (ppm): 56.62 (OCH3), 127.29–139.72 (Aromatic-C,
0
C5–C10 C1 –C6 ), 147.73 (C=N–), 178.29, 180.17(C1–C2, C=O)
0
(cf. Fig. 2).
4-(Cyclohexylideneamino)naphthalene-1,2-dione (S17)
4-(2-Hydroxybenzylideneamino)naphthalene-1,2-dione
(S14)
TLC (ethylacetate:hexane, 7:3) Rf: 0.62. IR (KBr, tmax
cm-1): 3121.23–3038.31 (Aromatic C–H str.), 2932.28–
2848.81 (C–H str. of cylohexyl group), 1619.83 (C=N str.,
azomethine), 1713.27, 1679.61 (C=O str. of carbonyl
TLC (methanol:chloroform, 1:9) Rf: 0.71. IR (KBr, tmax
cm-1): 3128.23, 3041.81 (Aromatic C–H str.), 1635.84
(C=N, azomethine), 1707.48, 1681.13 (C=O str. of car-
1
group); H NMR (DMSO-d6, 300 MHz) d (ppm): 1.80–
2.37 (m, 10H, cyclohexylidene–H), 6.50 (s, 1H, C=CH–
proton, C4), 7.40–8.09 (m, 4H, Ar–H); 13C NMR (DMSO-
d6) d (ppm): 23.57–34.51 (Cyclohexyl-C), 125.94–136.16
(Aromatic-C), 150.65 (C=N group), 181.04, 180.58 (C1–C2,
C=O) (cf. Fig. 2).
1
bonyl group), 3481.23 (O–H str.); H NMR (DMSO-d6,
300 MHz) d (ppm): 10.18 (s, 1H, –O–H group), 7.20–7.89
(m, 8H, Ar–H), 8.43 (s, 1H, –HC=N– group); 13C NMR
(DMSO-d6) d (ppm): 119.45–136.16 (Aromatic-C, C5–C10
123

Med Chem Res (2013) 22:1604–1617
1611
4-(Cyclopentylideneamino)naphthalene-1,2-dione (S18)
preferences at the active site of the receptor. The compu-
¨
tation was carried out using Schrodinger 2011 molecular
TLC (ethylacetate:hexane, 7:3) Rf: 0.57.IR (KBr, tmax cm-1):
3087.14, 3058.54 (Aromatic C–H str.), 2924.17, 2843.88 (C–
H str. of cylopentyl group), 1637.73 (C=N str., azomethine),
modeling software package. Docking was performed by
¨
using the Glide 5.7 integrated with Maestro 9.2 (Schro-
dinger, LLC, 2011) interface on the Linux operating
system.
1
1702.29, 1677.47 (C=O str. of carbonyl group); H NMR
(DMSO-d6, 300 MHz) d (ppm), 1.46–1.75 (m, 8H, cyclop-
entylidene–H), 6.61 (s, 1H, C=CH– proton, C4), 7.40–8.61
(m, 4H, Ar–H); 13C NMR (DMSO-d6) d (ppm): 23.66–31.50
(Cyclopentyl-C), 123.93–134.20 (Aromatic-C), 143.37
(C=N group), 177.60, 179.14 (C1–C2, C=O) (cf. Fig. 2).
Crystal structure selection
The starting coordinates of the human Topoisomerase-II
ATPase (TP-II)–AMP-PNP complex [PDB:1ZXM] was
taken from the Protein Data Bank (www.rcsb.org) and
further modified for docking calculations.
Anticancer activity
Maintenance of cancer cell lines
Preparation of ligands
The cell lines MCF-7 (human breast cancer), Hep-G2 (liver
sarcoma), MG-63 (Osteosarcoma), were obtained from
National Centre for Cell Sciences Repository, Pune, India.
The cells were maintained in Dulbecco’s minimum
essential medium (DMEM) with 10 % fetal calf serum and
0.1 % antibiotic solution (complete medium-CM) at 37 °C
in humidified air containing 5 % CO2 in the CO2 incubator,
Thermo Scientific Heraeus Cytoperm.
A compound library of synthesized Schiff bases were built
¨
on Maestro 9.2 build panel and minimized in Schrodinger
using the parental structure of doxorubicin as a template by
¨
Lig Prep 2.5 version v25111 (Schrodinger, LLC., USA)
which generates a single low energy 3D structure with
correct chiralities for each successfully processed input
structure based upon the OPLS 2005 molecular mechanics
force field.
Preparation of samples for cell line testing
Docking
The compounds S01–S18 were dissolved in DMSO and then
diluted with complete medium-CM to obtain different con-
centrations of the compounds (1, 2.5, 5, 10, 15, 20, 25 lM).
The protein (TP-II complex) was optimized with the
‘‘protein preparation wizard’’ workflow by subjecting a
cycle of constrained minimization steps allowing a maxi-
˚
mum root mean square deviation (RMSD) of 0.30 A from
¨
the original structure. For Glide (Schrodinger) calculations,
Testing of compounds on various cell lines
¨
TP-II complex was imported to Maestro (Schrodinger), the
The cells were seeded in a 96-well plate at the density of
20,000 cells per well for MCF-7, 25,000 cells per well for
Hep-G2 and 15,000 cells per well for MG-63 and allowed
to attach for 24 h. A range of concentrations of the com-
pounds diluted in CM were added to the cells and incu-
bated at 37 °C in a CO2 incubator for 48 h. Triplicate wells
were prepared for each individual dose. After the incuba-
tion, the cells were visualized using an Olympus micro-
scope. All experiments were carried out in laminar flow
hoods, Laminar Flow Ultraclean Air Unit, Microfilt, India.
After completion of treatment, MTT solution was added to
the monolayers and incubated for 4 h at 37 °C. The MTT
reaction was terminated by the addition of 0.04 N HCl in
isopropanol. The MTT formazan formed was measured on
ELISA plate reader at 540 nm.
co-crystallized ligands were identified and removed from
the structure. The ligands were docked with the binding
site using the ‘standard precision’ Glide algorithm in
Schrodinger.
Pharmacokinetic predictions
The molecules were also subjected to Qikprop analysis to
predict the in silico pharmacokinetic properties. The QikProp
3.4 (V34111) program predicts both physically significant
descriptors and pharmaceutically relevant properties.
Result and discussion
Chemistry
In silico studies
IR spectrum of synthesized compounds showed C=O stretching
bands in the range of 1,710–1,580 cm-1 that confirm the pres-
ence of carbonyl groups in quinone ring system. In 13C NMR
Molecular docking studies were carried out on the syn-
thesized compounds to get insight about their binding
123

1612
Med Chem Res (2013) 22:1604–1617
presence of peaks at 174.64 –182.41 also confirm the presence
of two carbonyl groups in 1,2-naphthoquinone ring system.
The FT-IR spectra of Schiff bases of 4-amino-1,2-naph-
thoquinone showed absorption bands at 3,030–3,170 cm-1
for aromatic C–H and at 1,590–1,650 cm-1 for azomethine
group (–CH=N–). Formation of Schiff bases also confirmed
Table 2 Docking score and IC50 of compounds S1–S18 in MCF-7,
Hep-G2, and MG 63 cell lines
Compounds
Docking score
IC50 (lM)
MCF-7
Hep-G2
MG-63
NQ
-4.34
-5.23
-6.07
-6.16
-5.33
-6.82
-5.80
-5.92
-5.27
-5.90
-6.71
-7.84
-6.66
-6.21
-7.96
-5.68
-6.11
-5.84
-5.35
-5.45
-8.06
16.92
15.89
[25
by the disappearance of N–H stretching (3,130–3,400 cm-1
)
ANQ
S01
S02
S03
S04
S05
S06
S07
S08
S09
S10
S11
S12
S13
S14
S15
S16
S17
S18
Dox
14.8
15.68
24.6
1
13.75*
14.74*
14.66*
11.12**
11.16**
14.28*
14.63*
12.61*
10.59**
7.65***
10.86**
14.4*
11.47*
15.61ns
15.73ns
8.92**
9.03**
12.10*
12.19*
10.43*
7.72**
5.91**
9.15**
11.82*
6.37**
11.04*
10.14*
10.63*
7.37**
7.77**
1.69
16.17ns
24.97ns
24.31ns
13.64*
12.71*
17.12ns
17.34ns
15.05ns
12.91*
9.69**
13.31*
17.01ns
9.98**
16.05ns
13.85*
14.84ns
11.52*
11.88*
3.17
of 4-amino-1,2-naphthoquinone. H NMR also showed a
characteristic singlet peak in the range of 8.07–8.82 d ppm
which also confirms the presence of azomethine proton
(–CH=N–). The appearance of multiplets at d 6.9–8.6 con-
firms presence of aromatic protons. Moreover, 13C NMR
spectra showed peak in the range of 143.37–160.00 ppm
which was due to azomethine (–CH=N–) carbon. The mul-
tiplets at d 3.8–3.9 ppm indicated the presence of –OCH3
groups of the benzene ring which was also confirmed by
presence of peaks in the range of d 55.57–58.94 ppm in 13
C
NMR spectra of schiff bases having methoxy groups.
Resonating position of the azomethine proton is strongly
affected by the electro-negativity of the substituent’s group
on adjoining benzene ring. It is noteworthy that the
downfield chemical shift (d 8.58–8.82 ppm) corresponds to
the azomethine proton was observed in compounds having
electron withdrawing substituents (Cl, Br, NO2) due to
de-shielding of proton while upfield chemical shift (d 8.07–
8.43 ppm) was observed in compounds with electron
donating substituents (OCH3, OH) due to shielding of
proton.
8.24**
13.25*
11.33**
12.9*
9.3**
9.64**
2.67
The bold values revealed the focus on compounds having more sig-
nificant activity against all the cell lines
IC50 (lM): dose of the compound which caused 50 % reduction of
cell survival. Values were calculated from dose–response curves for
triplicate dose of each compound
Anticancer activity
Level of significance checked by using one way ANOVA followed by
Dunnett’s t test * P \ 0.05, ** P \ 0.01, *** P \ 0.001, ns: non-
significant
The cytotoxicity of compounds S01–S18 was evaluated
against human breast adenocarcinoma (MCF-7), liver sar-
coma (Hep-G2), and Osteosarcoma (MG-63) cell lines.
Doxorubicin was taken as reference compound. The IC50
values derived from in vitro screening studies revealed that
most of the compounds possess significant cytotoxicity
against MCF-7 and Hep-G2 cancer cell lines (Table 2).
However, a lesser activity was observed against MG-63
cancer cell line. Morphological changes in cells were
observed on treatment with compounds and microscopic
images after treatment with compound S10 are shown in
Fig. 3. The results are represented graphically in Figs. 4, 5,
6, and 7. Compounds S09, S10, S11, S13, S17, and S18
showed significant cytotoxicity against all the cancer cells
with IC50 values in the range of 5.91–13.31 lM. Com-
pounds S01, S06, S07, S08, S14, and S16 were found to be
significant for MCF-7 and Hep-G2 but non-significant for
MG-63. Compounds S02 and S03 were found to be least
active in the series.
conversion of amino group to azomethine group via schiff
base formation enhances the activity. Schiff bases having
electron donating substituents on phenyl ring and schiff
bases of cyclic ketones were found to be more active than
Schiff bases with electron withdrawing substituents.
Statistical analysis
Statistical analysis was performed using Graph Pad prism
software, version 5 (Graph Pad software) by using non-
linear regression analysis, IC50 values were calculated from
each set of triplicate wells.
Docking study
Structure–activity relationship (SAR) studies from the
results of the antiproliferative activity revealed that
Molecular docking studies were conducted to explore
the interaction between target (ATPase domain of
123

Med Chem Res (2013) 22:1604–1617
1613
Fig. 3 Morphological changes
in MCF-7 cells incubated for
48 h (a) negative control (b)
with 10 lM concentration of
compound S10. Morphological
changes in Hep-G2 cells
incubated for 48 h (c) negative
control (d) with 10 lM
concentration of compound S10.
Morphological changes in
MG-63 cells incubated for 48 h
(e) negative control (f) with
10 lM concentration of
compound S10. The cells were
observed under phase contrast
microscope 9100
Fig. 4 Effect of concentration (1–25 lM) of compounds S09, S10,
S11, S13, and doxorubicin on viability of human breast cancer
(MCF-7) cell line
Fig. 5 Effect of concentration (1–25 lM) of compounds S09, S10,
S11, S13, and doxorubicin on viability of Osteosarcoma (MG-63) cell
line
Topoisomerase-II) and synthesized compounds. Glide cal-
culations were carried out with Impact version v57111 and
the G (Glide)-scores are shown in Table 2.
As per glide prediction, compounds S04, S09, S10, S11,
S13, were nicely accommodated in the ATPase domain of
Topoisomerase-II and showed good interaction with TP-II.
123

1614
Med Chem Res (2013) 22:1604–1617
These results were matching with wet lab findings. It is
evident that the compounds S10 and S13, which were
predicted in silico to be the most active, has been observed
the most active. As seen in Fig. 8, the carbonyl groups of
1,2-naphthoquinone ring system in S10 as the hydrophilic
head is involved in hydrogen bonds formation with Asn
150 and Ser 149 and nitrogen of azomethine linkage of
schiff base showed hydrogen bonds interaction with Ser
149. Compound S13 was having strong H bonds interac-
tions with Asn 150, Ser 148, Ser 149, and Gly 164 (Fig. 9).
As seen in Fig. 10 the standard drug doxorubicin showed
hydrophilic binding interaction with Arg 98, Tyr 186, and
Ser 148 and having the glide score -8.06, which is com-
parable with glide scores of compounds S10 (-7.84) and
S13 (-7.96).
Fig. 6 Effect of concentration (1–25 lM) of compounds S09, S10,
S11, S13, and doxorubicin on viability of liver sarcoma (Hep-G2) cell
line
Some compounds like S17 and S18 did not exhibit good
glide scores but found to have significant activity. It is
possible that these compounds may act through a different
mechanism, and further studies are required to elucidate
their mechanism of action.
Pharmacokinetic predictions of the best fit molecules
Unfavorable ADME and toxicity properties have been
identified as a major cause of failure for candidate mole-
cules in drug development. Therefore with the objective of
increasing the success rate of compounds reaching devel-
opment, the in silico ADME prediction studies were per-
formed on QikProp module of the software for ligands
having comparable G-scores with doxorubicin (Sengupta
et al., 2007). The best-fit ligands were neutralized before
being used by QikProp and significant ADME properties
were predicted for the best-fit molecules (Ligand Nos. S04,
S09, S10, S11, and S13), consisting of principal descriptors
and physiochemical properties with analysis of the log P
Fig. 7 Comparative IC50 values of compound S09, S10, S11, S13,
S17, S18, and doxorubicin against MCF-7, Hep-G2, and MG-63 cell
lines
Fig. 8 Binding pose for
compound S10 within the
ATPase domain of
Topoisomerase-II, active site
amino acid residues, i.e.,
Asparagine (ASN) 150 and
Serine (SER) 148 bind with
oxygen of 1,2-naphthoquinone
ring system, Serine (SER) 149
bind with nitrogen of
azomethine linkage of Schiff
base. Hydrogen bondings are
shown as dashed yellow lines
(Color figure online)
123

Med Chem Res (2013) 22:1604–1617
1615
Fig. 9 Binding pose for
compound S13 within the
ATPase domain of
Topoisomerase-II, i.e., ASN 150
and SER 148 bind with oxygen
of carbonyl group of 1,2-
naphthoquinone ring system,
SER 149 bind with nitrogen of
azomethine linkage and GLY
164 bind with hydroxyl group of
Schiff base
Fig. 10 Binding pose for
standard drug doxorubicin
which shows binding interaction
with Arginine (ARG) 98,
Tyrosine (TYR) 186, and Serine
(SER) 148 residue at the active
site of ATPase domain of
Topoisomerase-II
(octanol/water), % human oral absorption, CNS activity,
and permeability through Madin-Darby Canine Kidney
(MDCK) cells in nm/s, etc. (MDCK cells are considered to
be a good mimic for the blood–brain barrier) (cf. Tables 3, 4).
All the compounds were found to be non-toxic for CNS,
value of permeability through MDCK cells is \500 in all
cases so the compounds will not cross blood–brain barrier
which is desirable. The predicted drug likeliness of the
synthesized compounds follow the Lipinski ‘‘Rule of
Five’’, all four parameter values for a compound, i.e.,
Log P \ 5, H-bond donors\5, H-bond acceptors\10 and
molecular weight \500 suggested that the compounds
might have good absorption or permeability properties
(Moorthy et al., 2009).
Conclusion
Schiff bases of 4-amino-1,2-naphthoquinone were synthe-
sized and characterized successfully. All the synthesized
compounds gave satisfactory analytical and spectroscopic
data, which were in full accordance with their depicted
structures. The results of in vitro antiproliferative screening
of synthesized compounds revealed that the compounds
S04, S05, S09, S10, S11, S13, S15, S17, and S18 showed
significant cytotoxicity against all the cancer cell lines.
Most of the compounds having significant cytotoxicity are
also found to have nice glide scores which reveal the
support of wet lab findings with docking studies. On the
basis of in silico ADME studies, we concluded that these
123

1616
Med Chem Res (2013) 22:1604–1617
Table 3 Pharmacokinetic prediction of the best fit compounds by QikProp 3.4
Comp code
Percentage human
oral absorption
QPlogPo/w
QPPMDCK
CNS
#Stars
DonorHB
AccptHB
Rule of five
violation
S04
S09
S10
S11
S13
80.22
85.34
86.62
82.87
84.34
1.8
2.1
2.2
1.9
2.0
107.80
168.43
179.43
159.27
153.98
-2
-2
-2
-2
-2
0
0
0
0
0
1
0
0
0
1
5.75
6
0
0
0
0
0
7.25
6.5
6.5
Table 4 QikProp properties and descriptors
S. No.
1
Descriptor
Description
Recommended range
% Human oral
absorption
It predicts human oral absorption on 0–100 % scale. The prediction is based on a
quantitative multiple linear regression model. This property usually correlates
well with human oral-absorption
[80 % is high \25 % is
poor
2
3
QPlog Po/w
QPPMDCK
Predicted octanol/water coefficient
-2.0–6.5
Predicted apparent MDCK cell permeability in nm/s. MDCK cells are
considered to be a good mimic for the blood–brain barrier. QikProp predictions
are for non-active transport
\25 poor, [500 great
4
5
CNS
Predictive Central Nervous Activity on a -2 (inactive) to ?2 (active) scale
-2 to ?2
#Stars
Number of property or descriptor values that fall outside the 95 % range of
similar values for known drugs. A large number of stars suggest that a
molecule is less drug-like than molecules with few stars. The following are
some of properties and descriptors are included in the determination of #stars:
Molecular weight, dipole, QPlogPw, QPlogPo/w, QlogS, solvent accessible
surface area (SASA), etc.
0–5
6
7
8
DonorHB
AccptHB
Estimated number of hydrogen bonds that would be donated by the solute to
water molecules in an aqueous solution. Values are averages taken over a
number of configurations, so they can be non-integer
0.0–6.0
Estimated number of hydrogen bonds that would be accepted by the solute from
water molecules in an aqueous solution. Values are averages taken over a
number of configurations, so they can be non-integer
2–20
Rule of five
Number of violations of Lipinski’s rule of five. The rules are: mol_MW \ 500,
Q Plog Po/w \ 5, donorHB B 5, accptHB B 10. Compounds that satisfy these
rules are considered drug-like
Maximum is 4
Chen J, Huang YW, Liu G, Afrasiabi Z, Sinn E, Padhye S, Ma Y
(2004) The cytotoxicity and mechanisms of 1,2-naphthoquinone
thiosemicarbazone and its metal derivatives against MCF-7
human breast cancer cells. Toxicol Appl Pharmacol 197:40–48.
doi:10.1016/j.taap.2004.02.004
compounds can be used for second generation of devel-
opment and needed for further modifications to obtain
more effective anticancer compounds.
Acknowledgments The authors are grateful to The Head, Depart-
ment of Chemistry, Faculty of Science, Banaras Hindu University
Driscoll JS, Hazard GF, Wood HB, Goldin A (1974) Structure
antitumor activity relationships among quinone derivatives.
Cancer Chemother Rep 2 4(2):1–362
(BHU) Varanasi, India for providing the facilities of 1H NMR and 13
C
NMR spectroscopy. One of the author, Shubhanjali Shukla likes to
thank Jawaharlal Nehru Memorial Fund, New Delhi for providing
financial assistance to continue the research.
Dubin M, Fernandez Villamil SH, Stoppani AO (2001) Cytotoxicity
of beta-lapachone, an naphthoquinone with possible therapeutic
use. Medicina (Buenos Aires) 61(3):343–350
Fieser LF, Hartwell JL (1935) The reaction of hydrazoic acid with the
naphthoquinones. J Am Chem Soc 57:1482–1484. doi:10.1021/
ja01311a030
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