Molecular structures and biological activities of (N)-n-alkylammonium 2-chloro-3-oxido-1,4-naphthoquinone salts

Article abstract of DOI:10.1016/j.molstruc.2017.05.083

Single crystal X-ray structures and vibrational spectra of (N)-n-alkylammonium 2-chloro-3-oxido-1,4-naphthoquinone salts (alkyl = methyl to octyl, CS-1 to CS-8) possessing X-H?Y (X = N, C and Y[dbnd]O, Cl) hydrogen bonding and diverse noncovalent interactions have been characterized. Except for the CS-2 and CS-7 rest of the compounds facilitate π?π and Cl?π interactions. The compound CS-3 showed the presence of Cl?O interactions. Electronic structure and spectral characteristics of obtained are in consonance with the density functional theory. These complexes showed remarkable antiproliferative and antifungal activities.

Full text of DOI:10.1016/j.molstruc.2017.05.083

Journal of Molecular Structure 1145 (2017) 309e320
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Molecular structures and biological activities of (N)-n-
alkylammonium 2-chloro-3-oxido-1,4-naphthoquinone salts
Dinkar Choudhari a, Dipali N. Lande a, Aditi Bagade a, Shridhar P. Gejji a,
,
*
Debamitra Chakravarty b, Kisan M. Kodam a, Sunita Salunke-Gawali a
a Department of Chemistry, Savitribai Phule Pune University, Pune 411007, India
b Central Instrumentation Facility, Department of Chemistry, Savitribai Phule Pune University, Pune 411007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Single crystal X-ray structures and vibrational spectra of (N)-n-alkylammonium 2-chloro-3-oxido-1,4-
naphthoquinone salts (alkyl ¼ methyl to octyl, CS-1 to CS-8) possessing X-H/Y (X ¼ N, C and Y]O,
Cl) hydrogen bonding and diverse noncovalent interactions have been characterized. Except for the CS-2
Received 10 April 2017
Received in revised form
16 May 2017
Accepted 17 May 2017
Available online 20 May 2017
and CS-7 rest of the compounds facilitate p/p and Cl/p interactions. The compound CS-3 showed the
presence of Cl/O interactions. Electronic structure and spectral characteristics of obtained are in
consonance with the density functional theory. These complexes showed remarkable antiproliferative
and antifungal activities.
Keywords:
Lawsone
© 2017 Elsevier B.V. All rights reserved.
Hydrogen bonding
n-alkylamine
Antiproliferative activity
Hydroxynaphthoquinone
1. Introduction
leaves and widely used in cosmetic industry and explored in
pharmacological applications [8]. Phthiocol (2-hydroxy-3-methyl-
Biologically active quinones representing naturally occurring
secondary metabolites and are found in a variety of plants, fungi,
bacterial and insect families attracted significant attention in the
literature. Amongst these hydroxynaphthoquinones find applica-
tions in medicines, pigmentations, cosmetics, agrochemicals and
other functional chemicals for quite some time [1]. Naph-
thoquinones exhibit wide range biological activities viz., antibac-
terial, fungicidal, antiinflammatory, antiviral, antiparasitic,
antitrypanosome, antiplasmodial agent, antiproliferative, anti-
cancer and antimalarial [2e7]. The toxicological and pharmaco-
logical effects of hydroxy naphthoquinone displaying strong
dependence on the chemical structure in particular, the number
and positions of hydroxy groups, significantly influence the
chemical, physical, redox or biological properties. Hydroxynaph-
thoquinones for example, lawsone, phthiocol, plumbagin, lapha-
chol, juglone are naturally occurring and possess wide range of
pharmacological applications (Fig. 1). Lawsone (2-hydroxy-1,4-
1,4-naphthoquinone) is synthesized by tubercular bacteria in hu-
man intestine [9]. Lapachol (2-hydroxy-3-(2-methyl-1-propenyl)-
1,4-naphthoquinone) extracted from lapacho tree and shows
cytotoxic activity against several human cancers, among other
biological properties [10]. Plumbagin (5-hydroxy-2-methyl-1,4-
naphthoquinone) is also known as Chinese medicine and it is iso-
lated from plants of the plumbago family, that shows a wide range
of pharmacological activities, including anticancer, antimicrobial,
etc [11]. Juglone (5-hydroxy-1,4-naphthoquinone) is
a dye
commonly used in food and cosmetic industry, with herbicidal
properties [12]. 2-chloro-3-hydroxy-1,4-naphthoquinone is a syn-
thetic derivative of lawsone, its successful synthesis [13] dates back
to 1924. Hydroxynaphthoquinones are sparingly soluble in water;
their solubility can be increased by salt formation. Deprotonation of
hydroxyl group occurs in basic medium and the anionic forms
generated trigger keto-enol tautomerism in solutions which ren-
ders biological activity [14,15] to these systems. In the present
endeavor X-ray single crystal structures of (N)-n-alkylammonium
2-chloro-3-oxido-1,4-naphthoquinone salts were elucidated
following their successful syntheses by n-alkyl amines (CS-1 to CS-
8 displayed in Fig. 2). The structures were further derived using the
density functional theoretic calculations. The earlier investigations
naphthoquinone) is
a natural colorant obtained from heena
* Corresponding author.
E-mail address: sunitas@chem.unipune.ac.in (S. Salunke-Gawali).
http://dx.doi.org/10.1016/j.molstruc.2017.05.083
0022-2860/© 2017 Elsevier B.V. All rights reserved.

310
D. Choudhari et al. / Journal of Molecular Structure 1145 (2017) 309e320
carried out with the Thermo Finnigan EA 1112 Flash series.
Elemental Analyzer and on Elementar Vario EL III. The HR-MS
spectra were recorded on Bruker impact HD with the ESI source.
Gas chromatograph mass spectrums (GC-MS) were recorded on
Shimadzu, GC-MS-QP5050.
2.2. Synthesis of 2-chloro-3-hydroxy-1,4-
naphthoquinone:chlorolawsone
Modified procedure [21] has been used for synthesis of chlor-
olawsone. Recrystallized 2,3-dichloro-1,4-naphthoquinone (0.5 g,
2.2 mmol) have been added in 10 ml of water. To this suspension,
10 ml aqueous solution of KOH (0.247 g, 4.4 mmol) was added with
constant magnetic stirring. This reaction mixture was heated for
3 hrs at 70 ꢁC. Red color solution was obtained. Unreacted dichlone
was extracted with dichloromethane from aqueous reaction
mixture and was acidified by adding a few drops of concentrated
hydrochloric acid till pH of the reaction mixture becomes pH ¼ 2.
Yellow color residue obtained was filtered and washed with diethyl
ether and dried over vacuum and subsequently purified by column
chromatography and eluted with the ethyl acetate/pet-ether (1:9).
Fig. 1. Naturally occurring hydroxynaphthoquinones.
2.3. Synthesis of salts of 2-chloro-3-oxido-1,4-naphthoquninone
(N)-n-alkylammonium salts: CS-1 to CS-8
Recrystallization of 2-chloro-3-hydroxy-1,4-naphthoquinone
(0.5 g, 2.2 mmol) was carried out by dissolving with 15 ml of
dichloromethane (Scheme 1). The mixture was stirred for about
15 min. To this solution, corresponding amines (0.1 ml methyl (CS-
1)), (0.29 ml ethyl (CS-2)), (0.485 ml propyl (CS-3)), (0.475 butyl
(CS-4)), (0.40 ml pentyl (CS-5)), (0.412 hexyl (CS-6)), (0.428 heptyl
(CS-7)), (0.476 octyl amine (CS-8)) solutions were added by drop
wise. The reaction mixture was stirred for 24 h at the room tem-
perature (26 ꢁC) with constant magnetic stirring till completion of
the reaction that is monitored on TLC. Red colored precipitate thus
obtained was filtered and washed with dichloromethane followed
by diethyl ether and the residue was dried in vacuum. Subsequently
X-ray quality crystals for all these compounds were obtained after
recrystallization of solid products in the methanol.
Fig. 2. Molecular structures of CS-1 to CS-8.
on n-alkylamino derivatives of 1,4-naphthoquinones have
demonstrated that these derivatives possess distinct molecular
interactions and biological properties [16e19]. The present in-
vestigations focus on how varying alkyl chain of (N)-n-alky-
lammonium cations in chlorolawsonote anion brings forth the
distinct molecular interactions which influence their antifungal
and anticancer activities. Antifungal activity of CS-1 to CS-6 was
evaluated against Saccharomyces cerevisiae and Candida albicans.
Antiproliferative activity was evaluated against breast cancer cell
line (MDA-MB-231) and lung cancer cell line (L-132, HeLa
derivative).
2.3.1. Analytical data of 2-chloro-3-hydroxy-1,4-naphthoquinone:
cholorolawsone
Yellow solid, Yield: 0.4 g (87.14%). FT-IR (KBr, cmꢀ1): 3269, 1668,
1639, 1585, 1454, 1363, 1330, 1298, 1273, 1219, 1128, 1008, 856, 727,
682, 599, 538. 1H NMR (400 MHz, DMSO-d6,
d(ppm)): 8.04 (d,
2. Experimental section
J ¼ 7.5 Hz, 1H), 8.02 (d, J ¼ 7.84 Hz, 1H), 7.86 (td, J ¼ 7.3, 1.3 Hz, 1H),
7.8 (m, 1H).13C NMR (100 MHz DMSO-d6 (ppm)): 180, 178, 158, 135,
133, 132, 130, 126, 126, 126. UVeVis: (methanol, lmax, nm): 287, 321,
473. GC-MS (m/z): calcd. for C11H10ClNO3: 208.60; Found: 208.
2.1. Materials and methods
2,3-dichloro-1,4-naphthoquinone, ethyl amine solution (70%),
propylamine (99%), butylamine (99%), pentylamine (99%), hexyl-
amine (99%), heptylamine (99%) and octylamine (99%) were pur-
chased from Sigma-Aldrich, Methyl amine solution (40%) from Loba
chemicals, KOH pellets obtained from Merck Chemicals. Analytical
grade dichloromethane, methanol, pet ether, ethyl acetate solvents
were purchased from Merck Chemicals. The solvents were distilled
by standard methods [20] and dried wherever necessary. FT-IR
spectra were recorded between 4000 and 400 cmꢀ1 as KBr pel-
lets on the Shimadzu FT 8400 Spectrophotometer. Melting points of
compounds were determined using (Make- METLER). 1H
(400 MHz) and 13C (100 MHz) NMR of compounds are recorded in
DMSO-d6, on Varian 400 MHz NMR instrument using the TMS
(tetramethylsilane) as the reference. UVeVisible spectra of all
compounds in methanol were recorded from 200 nm to 800 nm on
Shimadzu UV 1800 Spectrophotometer. Elemental analysis was
2.3.2. Analytical data of methyl ammonium-3-chlorolawsonate:
CS-1
Red solid, Yield: 0.55 g. (79.82%). Anal. data calcd. for
C11H10ClNO3: C, 55.13; H, 4.21; N, 5.84. Found: C, 55.39; H, 4.57; N,
5.90. FT-IR (KBr, cmꢀ1): 3022, 2991, 2980, 1676, 1621, 1516, 1377,
1269, 1155, 1001, 837, 736, 555. 1H NMR (400 MHz, DMSO-d6,
d
(ppm)): 7.91 (d, J ¼ 7.6 Hz, 1H), 7.80 (d, J ¼ 7.5 Hz, 1H), 7.73 (s, 3H),
7.69 (m, 1H), 7.56 (t, J ¼ 7.5 Hz, 1H), 2.42 (s, 3H).13C NMR (100 MHz,
DMSO-d6, d (ppm)): 25, 113,125, 125, 130, 131, 134, 135,167, 174,184.
UVeVis: (methanol, lmax, nm): 468.
2.3.3. Analytical data of ethyl ammonium-3-chlorolawsonate: CS-2
Red solid, Yield: 0.51
12H12ClNO3: C, 56.81; H, 4.77; N, 5.52 Found: C, 56.63; H, 4.52; N,
5.52. FT-IR (KBr, cmꢀ1): 3045, 3010, 2982, 1681, 1579, 1527, 1500,
g (83.46). Anal. data calcd. for
C

D. Choudhari et al. / Journal of Molecular Structure 1145 (2017) 309e320
311
Scheme 1. Synthesis of (N)-n-alkylammonium 2-chloro-3-oxido-1,4-naphthoquinone.
1317, 1269, 1153, 829, 729, 553. 1H NMR (400 MHz, DMSO-d6,
7.69 (t, J ¼ 7.5 1H), 7.56 (t, J ¼ 7.5, Hz, 1H), 1.54 (t, 2H), 1.27 (m, 7H),
0.84 (t, 3H). 13C NMR (100 MHz DMSO-d6,
(ppm)): 184, 173, 167,
135, 134, 131, 131, 125, 125, 113, 39, 31, 27, 25, 22, 14. UVeVis:
(methanol, lmax nm): 468.
d
(ppm)): 7.91 (d, J ¼ 7.6 Hz, 1H), 7.79 (d, J ¼ 7.6 Hz, 1H), 7.76 (s, 3H),
d
7.68 (td, J ¼ 7.5, 1.3 Hz, 1H), 7.55 (td, J ¼ 7.5, 1.2 Hz, 1H), 2.86 (q,
J ¼ 7.3 Hz, 2H), 1.16 (t, J ¼ 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6,
d
(ppm)): 184, 174, 167, 135, 134, 131, 131, 125, 125, 113, 34, 13.
UVeVis: (methanol, lmax, nm): 470. HRMS (m/z): calcd. for
C
2.3.8. Analytical data of heptyl ammonium-3-chlorolawsonate: CS-
7
12H12ClNO3 [MþH]þ: 253.68, found: 254.0575.
Red solid, Yield: 0.41
17H20ClNO3: C, 63.06; H, 6.85; N, 4.33. Found: C, 63.48; H, 6.73; N,
4.39%. FTIR (KBr, cmꢀ1): 2956, 2981, 2906, 1676, 1585, 1514, 1504,
1269, 1222, 1145, 837, 555. 1H NMR (DMSO-d6, 400 MHz,
(ppm)):
g (87.58%). Anal. data calcd. for
2.3.4. Analytical data of propyl ammonium-3-chlorolawsonate: CS-
3
C
Red solid. Yield: 0.556
g (86.33%). Anal. data calcd. for
d
C
13H14ClNO3: C, 58.32; H, 5.27; N, 5.23. Found: C, 58.51; H, 5.24; N,
7.90 (d, J ¼ 7.2 Hz, 1H), 7.79 (d, J ¼ 7.5 Hz, 1H), 7.73 (s, 3H), 7.76 (t,
J ¼ 7.5 Hz, 2H), 2.83e2.74 (m, 2H), 1.52 (m, J ¼ 7.6 Hz), 1.22 (m,
J ¼ 7.6 9H), 0.86 (t, J ¼ 6.7 Hz, 3H). 13C NMR (100 MHz, DMSO-d6,
5.35%. FT-IR (KBr, cmꢀ1): 3043, 2987, 2962, 1681, 1589, 1521, 1315,
1269, 1155, 999, 835, 727, 637, 555. 1H NMR (400 MHz, DMSO-d6,
d
(ppm)): 7.90 (d, J ¼ 7.6 Hz, 1H), 7.76 (d, J ¼ 7.6 Hz, 1H), 7.68 (t,
d
(ppm)): 184, 174, 167, 135,134, 131, 130, 126, 125, 113, 39, 31, 28, 27,
J ¼ 7.3 Hz, 1H), 7.55 (t, J ¼ 7.2 Hz, 1H), 7.76 (S, 3H), 2.78 (t, J ¼ 7.5 Hz,
26, 22, 14: UVeVis: (methanol, lmax, nm): 466.
2H), 1.57 (m, 2H), 0.91 (t, J ¼ 7.4 Hz, 3H). 13C NMR (100 MHz, DMSO-
d6, d (ppm)): 184, 176, 167, 135, 134, 131, 130, 126, 125, 113, 41, 20, 11.
2.3.9. Analytical data of octyl ammonium-3-chlorolawsonate: CS-8
UVeVis; (methanol, lmax, nm): 468. HRMS (m/z): calcd. for
C
Red solid, Yield: 0.45
g (93.75%). Anal. data calcd. for
13H14ClNO3 [MþH]þ: 267.71, Found: 268.93.
C18H24ClNO3: C, 63.99; H, 7.16; N, 4.15. Found: C, 64.35; H, 7.45; N,
4.36. FT-IR (KBr, cmꢀ1): 3138, 3064, 3049, 1678, 1610, 1585, 1516,
1373, 1267, 1219, 1153, 997, 829, 734, 559. 1H NMR (400 MHz,
2.3.5. Analytical data of butyl ammonium-3-chlorolawsonate: CS-4
Red solid, Yield: 0.570 (84.07%). Anal. data calcd. for
C14H16ClNO3: C, 59.68; H, 5.72, N, 4.97. Found: C, 60.13; H, 6.00, N:
5.01%. FT-IR (KBr, cmꢀ1): 3136, 3064, 3014, 1678, 1621, 1585, 1523,
1516, 1269, 1220, 1153, 921, 827, 736, 559. 1H NMR (400 MHz,
g
DMSO-d6,
d
(ppm)): 7.91 (d, J ¼ 7.3 Hz, 1H), 7.90 (s, 3 H), 7.80 (d,
J ¼ 7.4 Hz, 1H), 7.69 (t, J ¼ 7.4 Hz, 1H), 7.56 (t, J ¼ 7.4 Hz,1H), 2.81 (m,
2H), 1.54 (m, 2H), 1.25 (m, 10H), 0.81 (d, J ¼ 3H). 13C NMR (100 MHz,
DMSO-d6, d (ppm)): 184,174,167,135,134,131,130,126,125,113, 38,
DMSO-d6,
d
(ppm)): 7.90 (d, J ¼ 7.79 Hz, 1H), 7.81 (s, 3H), 7.79 (d,
31, 28, 27, 26, 22, 14. UVeVis: (methanol, lmax, nm): 468.
J ¼ 7.8 Hz,1H), 7.68 (t, J ¼ 7.6 Hz,1H), 7.55 (d, J ¼ 7.5 Hz,1H), 2.81 (m,
2H), 1.55 (m, 2H), 1.33 (m, 2H), 0.88 (t, J ¼ 7.3, Hz, 3H). 13C NMR
2.4. X-ray crystallographic data collection and refinement of the
structures
(100 MHz, DMSO-d6, d (ppm)): 184, 173, 167, 135, 134, 131, 130, 125,
126, 113, 40, 29, 19, 13: UVeVis: (methanol, lmax, nm): 468. HRMS
(m/z): calcd. for C14H16ClNO3 [MþH] þ: 281.08, found: 282.08.
X-ray quality red crystals of CS-1 to CS-8 were obtained after
evaporation of methanol solvent of the respective salt. Data for all
the compounds has been collected on D8 Venture PHOTON 100
CMOS diffractometer using graphite monochromatized Mo-Ka ra-
2.3.6. Analytical data of pentyl ammonium-3-chlorolawsonate: CS-
5
Red solid, Yield: 0.4
g (86.37%). Anal. data calcd. for
diation (
l
¼ 0.7107 Å) with exposure/frame ¼ 10 s. The X-ray
C
15H18ClNO3: C, 60.91; H, 6.13; N, 4.74, Found: C, 61.02; H, 6.58; N,
generator was operated at 50 kV and 30 mA. An initial set of cell
constants and an orientation matrix were calculated from total of
24 frames. The optimized strategy used for data collection con-
4.76. FT-IR (KBr, cmꢀ1): 3144, 3036, 2955, 1680, 1516, 1471, 1375,
1267,1220,1039, 999, 829, 732, 673, 557. 1H NMR (400 MHz, DMSO-
sisted of different sets of 4 and
u u.
scans with 0.5ꢁ steps in 4/
d6,
d
(ppm)): 7.90 (d, J ¼ 7.6 Hz, 1H), 7.79 (d, J ¼ 7.5 Hz, 1H), 7.72 (s,
3H), 7.67 (m, 1H), 7.54 (m, 1H), 2.51 (m, 2H), 1.53 (m, J ¼ 7.9 Hz, 2H),
Crystal to detector distance was 5.00 cm with 512 ꢂ 512 pixels/
frame, Oscillation/frame ꢀ0.5ꢁ, maximum detector swing
angle ¼ ꢀ30.0ꢁ, beam centre ¼ (260.2, 252.5), in plane spot
width ¼ 1.24. Data integration was carried out by Bruker SAINT
Program and empirical absorption correction for intensity data
were carried out by Bruker SADABS. The programs are integrated in
APEX II package [22]. The data were corrected for Lorentz and po-
larization effect. The structure was solved by Direct Method using
SHELX-97 [23]. The final refinement of the structure was carried out
by full-matrix least-squares techniques with anisotropic thermal
data for non-hydrogen atoms on F2. The non-hydrogen atoms were
1.29 (m, J ¼ 7.9, 4H), 0.87 (t, J ¼ 6.7 Hz, 3H). 13C NMR (100 MHz,
DMSO-d6, d (ppm)): 184, 174, 167, 135,134, 131, 131, 125, 125, 113, 39,
28, 27, 22, 14. UVeVis: (methanol, lmax, nm): 468.
2.3.7. Analytical data of hexyl ammonium-3-chlorolawsonate: CS-6
Red solid, Yield: 0.521
g (87.71%). Anal. data calcd. for
C
16H20ClNO3: C, 62.03; H, 6.51; N, 4.52. Found: C, 62.28; H, 6.34; N,
4.64. FT-IR (KBr, cmꢀ1): 3136, 3059, 3049, 1678, 1608, 1587, 1525,
1317, 1269, 1155, 989, 829, 736, 559. 1H NMR (DMSO-d6, 400 MHz,
d
(ppm)): 7.92 (d, J ¼ 7.6 Hz, 1H), 7.91 (s, 3H), 7.81 (d, J ¼ 7.6 Hz, 1H),

312
D. Choudhari et al. / Journal of Molecular Structure 1145 (2017) 309e320
refined anisotropically, whereas the hydrogen atoms were refined
at the calculated positions as riding atoms with isotropic
displacement parameters. Molecular diagrams were generated
using ORTEP-3 [24] and Mercury programs [25]. Structural calcu-
lations were performed using the SHELXTL [23] and PLATON [26].
were maintained in Leibovit's 15 medium and L-132 cells were
maintained in DMEM medium at 37 ꢁC with 5% CO2. The varying
concentrations of (5, 25, 50, 100, and 200 mg/ml) compounds were
incubated with 2 ꢂ 105 cells/ml of MDA-MB-231 cell line for 48 h
and L-132 cell line for 24 h in CO2 incubator (Nuaire, USA). After
incubation, culture media was removed from the 96 well culture
plates and cells were washed with phosphate buffered saline till the
2.5. Density functional theory
color of compounds was removed, 40
and incubated for 3 h in CO2 incubator. After incubation, the for-
mazan crystals formed in wells were solubilized in 100 l of DMSO
mg of MTT was added per well
Geometry optimizations of naphthoquinone derivatives were
carried out within the framework of Density Functional Theory
(DFT) with the use of Gaussian 09 suite of programs [27]. The hybrid
M06-2X exchange correlation functional in conjunction with 6-
311 þ G(d,p) basis set was employed. Net atomic charges are
derived from natural bond order analysis. Stationary point struc-
tures thus derived were confirmed to be the local minima on the
potential energy surfaces through the vibrational frequency cal-
culations. All the normal vibrational frequencies of these stationary
point structures turned out to be real. The normal vibrations were
assigned by visualizing the atomic displacements around their
equilibrium (mean) positions with the help of GAUSSVIEW-5 pro-
gram [28]. Frontier orbitals were computed within the same
framework of theory.
m
and kept for 5e10 min for complete dissolution of formazan and
then absorbance was measured at 580 nm on multimode plate
reader (Multimode plate reader, Enspire, Perkin Elmer) [30].
3. Results and discussion
3.1. FT-IR, 1H and 1C NMR UVeVisible studies
FT-IR spectra of CS-1 to CS-8 revealed a broad band between
3250 and 2500 cmꢀ1 (Fig. S1 in ESIy) that arises from the over-
lapped bands of the eNH as well as eCH stretching's. The carbonyl
frequency was increased by18 cmꢀ1 while p-naphthoquinone fre-
quency was decreased by ~20 cmꢀ1 when compared with free
chlorolawsone (Table 1). 1H and 13C chemical shifts are assigned
from the 1H and 13C and DEPT experiments (Fig. S2-S10, Tables S2
and S3 in ESIy). The proton chemical shifts of the ammonium
2.6. Antimicrobial activity
The antifungal activity was studied against Saccharomyces cer-
evisiae NCIM 3559 and Candida albicans ATCC 10231. Compounds
were dissolved in DMSO with a stock concentration of 20 mg/ml
which were further serially diluted in sterile distilled water. Reac-
cation were observed in ~
d
¼ 7.73e7.91 ppm region (Table S2 in
ESIy). As may also be observed the 13C chemical shifts (in ppm)
follows the order: C(2) (113) < C(3) (167) < C(4) (174) < C(1)
(184 ppm) as displayed in Table S3 (in ESIy) of the supporting in-
formation. Thus C(3), C(4) reveal significant deshielded signals
compared to the free chlorolawsone. The UVevisible spectra for CS-
1 to CS-8 along with the chlorolawsone were recorded with
4 ꢂ 10ꢀ4 M concentration in methanol (Fig. S11). The peak centered
at ~468 nm in UVevisible spectra of all compounds was assigned
using time dependent DFT computations.
tion volume was maintained at 100
well consisted of varying concentration of test compound, 67.5
of resazurin (BDH) along with fungal cultures were added. Volume
was made up to 100
l with 3.3ꢂ autoclaved Sabouraud's medium
ml in sterile 96 well plates. Each
mg
m
(Hi-Media, India). A column of wells with all solutions except fungal
cultures was taken a negative control. All the solutions except the
test compound were also taken as a negative control. DMSO control
consisted of different concentrations of DMSO utilized without
addition of the test compounds to examine toxicity by DMSO.
Commercially available antibiotic, amphotericin B was used as
positive control. The 96 well plates were incubated at 37 ꢁC for 48 h.
All the experiments were carried out in triplicate. Color change
from blue to pink was assessed visually. The concentration that
accompanies the color change was taken as the MIC value [29].
3.2. Single crystal X-ray diffraction studies
ORTEP plots of CS-1 to CS-8 are portrayed in Fig. 3. The crystal
structure data is summarized in Table 2. Moreover, the hydrogen
bonding parameters are given in Table S34 (in ESIy). As may readily
be noticed CS-1 crystallizes in triclinic space group P-1 while CS-4
and CS-8 in P1. CS-2, CS-3, CS-5 and CS-7 crystallize in monoclinic
space group P21/c and CS-6 reveals the P21 space group. The bond
distances C(1)eO(1), C(3)eO(2), and C(4)eO(3) respectively, are
observed to be ~1.24 Å, ~1.26 Å and ~1.211 Å [14,31e35]. As revealed
further from the X-ray data the carbonyl bond distances are closer
to those observed in the anionic form of hydroxynaphthoquinone
[35]. A delocalization of electron density on the quinonoid moiety
hence, can be inferred.
2.7. Antiproliferative activity
The antiproliferative activities were examined using the MTT [3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide]
assay on a MDA-MB-231 (breast cancer) and L-132 (lung cancer,
HeLa derivative) cell lines. The MTT assay was used to assess the
anti-proliferative activity of these compounds. MDA-MB-231 cells
Table 1
Prominent stretches in salts CS-1 to CS-8 from DFT studies, the experimental values are given in parenthesis.
Compounds
nOH
nNH
nNHþ nCH
n4(C1]O1)
n4(C4]O2)
n1(C]C)
Chlorolawsone
CS-1
CS-2
CS-3
CS-4
CS-5
CS-6
CS-7
CS-8
3456(3272)
e
1696(1659)
1678(1678)
1678(1678)
1675(1678)
1676(1678)
1677(1678)
1685(1678)
1681(1678)
1677(1678)
1676
1670
1671
1666
1672
1669
1679
1672
1672
(1582)
e
e
e
e
e
e
e
3364
3362
3353
3362
3347
3366
3351
3351
2422(3015)
2369(2979)
2384(2961)
2369(2955)
2410(2944)
2371(2955)
2460(2920)
2351(2913)
1584(1582)
1583(1582)
1588(1588)
1587(1580)
1582(1588)
1590(1594)
1579(1582)
1587(1594)

D. Choudhari et al. / Journal of Molecular Structure 1145 (2017) 309e320
313
Fig. 3. ORTEP plots of CS-1 to CS-8. The ellipsoids were drawn with 50% probability.

Table 2
Crystallography parameter of CS-1 to CS-8.
Identification code
CS-1
CS-2
CS-3
CS-4
CCDC
1,504,336
1,504,330
C12H12ClNO3
253.68
200(2) K
1.54178 Å
Monoclinic
P 21/c
1,504,334
C13H14ClNO3
267.70
100(2) K
1.54178 Å
1,504,329
C14H16ClNO3
281.73
296(2) K
0.71073 Å
Triclinic
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
Unit cell
C11H10ClNO3
239.65
296(2) K
0.71073 Å
Triclinic
P -1
Monoclinic
P 21/c
P 1
a ¼ 7.382(8) Å,
b ¼ 8.611(8) Å,
c ¼ 9.270(8) Å
a ¼ 13.2023(5) Å a ¼ 90ꢁ.
b ¼ 10.6791(5) Å b ¼ 102.524(2)ꢁ.
c ¼ 8.1788(4) Å
a ¼ 7.2690(4)Å,
b ¼ 15.4708(8)Å
c ¼ 11.5339(6)Å
a ¼ 5.0099(5)Å,
b ¼ 8.2750(8)Å,
c ¼ 9.1878(10)Å
Dimensions
a
b
g
¼ 98.46(5)ꢁ,
¼ 109.34(4)ꢁ,
¼ 106.58(5)ꢁ
b
¼ 102.524(2)ꢁ
b
¼ 97.628(2)ꢁ,
a
g
¼ 112.829(3)ꢁ,
b
¼ 96.095(3)ꢁ,
ꢁ
¼ 90.889(3)
.
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta range for data collection
Index ranges
Reflections collected
Independent reflections
Completeness to
513.5(8) Å3
1125.68(9) Å3
1285.59(12) Å3
348.41(6) Å3
1
2
4
4
1.550 Mg/m3
1.497 Mg/m3
1.383 Mg/m3
1.343 Mg/m3
0.277 mmꢀ1
148
0.361 mmꢀ1
2.989 mmꢀ1
2.646 mmꢀ1
248
528
560
0.139 ꢂ 0.109 ꢂ 0.093 mm3
0.231 ꢂ 0.137 ꢂ 0.071 mm3
0.42 ꢂ 0.19 ꢂ 0.06 mm3
0.28 ꢂ 013 ꢂ 0.07 mm3
3.113e24.986ꢁ.
5.379e68.288ꢁ.
4.810e68.307ꢁ.
2.676e28.420ꢁ.
ꢀ8 ꢃ h<¼8, 10 ꢃ k<¼10, ꢀ11 ꢃ l<¼11
10950
ꢀ15 ꢃ h<¼15, ꢀ12 ꢃ k<¼12, ꢀ9 ꢃ l<¼9
9708
ꢀ8 ꢃ h<¼8, ꢀ18 ꢃ k<¼18, ꢀ13 ꢃ l<¼13
13393
ꢀ6 ꢃ h<¼6, ꢀ11 ꢃ k<¼10, ꢀ12 ꢃ l<¼12
7388
1782 [R(int) ¼ 0.0746]
2012 [R(int) ¼ 0.0757]
2338 [R(int) ¼ 0.0421]
3287 [R(int) ¼ 0.0318]
q
¼ 25.242ꢁ
96.0%
97.5%
99.0%
99.9%
Refinement method
Full-matrix least-squares on F2
1782/0/147
Full-matrix least-squares on F2
2012/0/156
Full-matrix least-squares on F2
2338/0/165
Full-matrix least-squares on F2
3287/3/174
Data/restraints/parameters
Goodness-of-fit on F2
1.067
1.087
1.007
1.059
Final R indices [I > 2
R indices (all data)
Extinction coefficient
s
(I)]
R1 ¼ 0.0558, wR2 ¼ 0.1630
R1 ¼ 0.0885, wR2 ¼ 0.2030
n/a
R1 ¼ 0.0775, wR2 ¼ 0.2085
R1 ¼ 0.0981, wR2 ¼ 0.2245
n/a
R1 ¼ 0.0324, wR2 ¼ 0.0833
R1 ¼ 0.0358, wR2 ¼ 0.0864
n/a
R1 ¼ 0.0438, wR2 ¼ 0.0822
R1 ¼ 0.0722, wR2 ¼ 0.0929
n/a
Largest diff. peak and hole
0.432 and ꢀ0.614 e.Åꢀ3
0.547 and ꢀ0.568 e.Åꢀ3
0.245 and ꢀ0.337 e.Åꢀ3
0.188 and ꢀ0.224 e.Åꢀ3
Identification code
CS-5
CS-6
CS-7
CS-8
1,504,335
C15H18ClNO3
295.75
1,504,332
C16H20ClNO3
309.78
1,504,331
C17H22ClNO3
323.80
1,504,333
C18H20ClNO3
333.80
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
200(2) K
1.54178 Å
Monoclinic
P 21/c
296(2) K
1.54178 Å
Monoclinic
P 21
296(2) K
1.54178 Å
Monoclinic
P 21/c
296(2) K
1.54178 Å
Triclinic
P 1
Unit cell dimensions
a ¼ 4.8891(3) Å
b ¼ 15.3434(9) Å
c ¼ 19.7154(12) Å
a ¼ 8.0710(4) Å
b ¼ 4.8513(3) Å
c ¼ 20.3990(10) Å
a ¼ 10.101(2) Å
b ¼ 15.606(3) Å,
c ¼ 11.531(2) Å
a ¼ 5.0846(2) Å,
a
¼ 92.780(2)ꢁ
b ¼ 8.2238(4) Å,
b
¼ 94.069(3)ꢁ
b
¼ 100.119(2)ꢁ
b
¼ 112.871(9)ꢁ
b
¼ 90.898(2)ꢁ
c ¼ 10.5386(5) Å,
g
¼ 90.756(2)ꢁ
Volume
1475.23(15) Å3
786.30(7) Å3
1674.8(6) Å3
440.05(3) Å3

D. Choudhari et al. / Journal of Molecular Structure 1145 (2017) 309e320
315
Table 3
Molecular interactions in CS-1 to CS-8 (interaction √ ¼ present, ✕ ¼ absent).
Compound C/A A/A NeH/Cl CeH/O CeH/Cl Cl / p/p / p
CS-1
CS-2
CS-3
CS-4
CS-5
CS-6
CS-7
CS-8
4
5
3
3
4
4
3
4
3
e
2
2
2
2
e
2
✓
✕
✓
✓
✕
✓
✓
✓
✓
✓
✓
✓
✓
✓
✕
✕
✓
✓
✕
✕
✕
✓
✓
✕
p
✕
p
Cl /
p
/
/
p
p
, Cl/O
p
,
p
/
p
/ p
Cl /
✕
p
Cl /
p
,
p
/
p
C ¼ n- alkyl ammonium.
A ¼ 3-chloro-2-oxido-1,4-naphthoquinone.
Chlorolawsone anion and n-alkyl ammonium cation hydrogen
bonding and
p
-
p
stacking interactions (Fig. S16 in ESIy) are dis-
played in Table 3. All compounds reveals strong NeH/O in-
teractions in addition to a variety of NeH/Cl, CeH/O, CeH/Cl
interactions. Except for CS-2 and CS-7 the
p/p and Cl/p in-
teractions (Table 3) are also noticed. Besides the CS-3 crystal
structure shows the presence of Cl/O interactions.
Stair case like polymeric chain of CS-1 molecules is formed via
NeH/O and CeH/O interaction (Fig. 4a). As shown in Fig. 4a the
crystal structure of CS-1 shows the cation is bound to two anions
and vice a versa via hydrogen bonding interactions. Further a
dimeric chain observed in the crystal structure further facilitated
by NeH/O interactions in addition to C(8)/C(8)(1-x,1-y,1-z,
3.346 Å) stacking. Polymeric chain of CS-2 extends via NeH/O
and CeH/O interactions, which further links to nearby polymeric
chain via NeH/O and CeH/Cl (1 þ x, y, ꢀ1þz) interactions
displayed in Fig. 4b down the c-axis.
Molecular packing of CS-3 is shown in Fig. 4c down the a-axis.
With the reverse orientation of the molecules the benzenoid and
quinonoid rings exhibit p/p stacking with their separation being
3.534 Å. Polymeric chain of cation and anion of CS-4 are formed
via NeH/O, CeH/O and CeH/Cl interaction as shown in Fig. 4d
down the a-axis. Besides Cl/p interactions are observed in CS-4
anions which are evidenced from the C(4)/Cl(1) (3.370(3)
Å, ꢀ1þx, y, z) and C(5)/C(1) close contacts (3.355(5) Å, ꢀ1þx, y,
z). Polymeric network of CS-5 is formed via NeH/O and CeH/O
interactions, as displayed in Fig. 4e. In addition to these in-
teractions Cl/p interactions between the centroid of quinonoid
ring to chlorine (3.519 Å, ꢀ1þx, y, z) are observed. Polymeric chain
in CS-6 molecules extends by NeH/O, NeH/Cl, CeH/O and
CeH/Cl interactions where the neighboring polymeric chains are
linked by C(6)eH(6)/O(3) (1-x, þ1/2 þ y, 1-z) as shown in Fig. 4f.
Unlike CS-5, CS-6 also showed Cl/p interaction of chlorine to
centroid of the quinonoid ring (3.438 Å, x, ꢀ1þy, z). CS-7 is void of
p
/p
stacking or Cl/ interactions. Tetrameric unit of CS-7
p
comprises of two cations and two anions which is facilitated
through NeH/O interaction arising from O(2) and O(3) oxygens
those are further linked by NeH/O interactions of O(1) (Fig. 4g).
Molecular packing of CS-8 shows polymeric chain comprising of
alternate cation and anion formed via the NeH/O interaction
along the b-axis wherein the anionic chain reveals Cl/
p in-
teractions with the quinonoid ring of the adjacent anion (3.453 Å,
1 þ x, y, z). The C(5) and C(1) close contacts also can be inferred as
displayed in Fig. 4h.
3.3. DFT investigations
Optimized structures of chlorolawsone and CS-1 to CS-8 are
shown in Fig. 5. Atomic labeling scheme as well as net atomic
charges are also given along with. An isomer with the hydroxyl
placed at C(3) position is turned out to be of the lowest energy.

316
D. Choudhari et al. / Journal of Molecular Structure 1145 (2017) 309e320
Fig. 4. Molecular packing of a) CS-1 down c axis, b) CS-2 down c axis, c) CS-3 down c-axis, d) CS-4 down b-axis, e) CS-5 molecule, f) Polymeric chain s of CS-6 down b-axis, g) CS-7
down a-axis, h) CS-8.

D. Choudhari et al. / Journal of Molecular Structure 1145 (2017) 309e320
317
Fig. 6. MESP mapped density of chlorolawsone.
alkyl chain length has no significant effect on characteristic
stretching vibrational frequency. Fig. 8 illustrates characterize the
charge distribution in the methyl complex. Frontier orbital anal-
ysis clearly shows that HOMO and LUMO resides over the naph-
thoquinonoid moiety. Interestingly the HOMO and LUMO for the
rest of molecules are strikingly similar as shown in Fig. S17 (in
ESIy) of the supporting information. MESP isosurfaces in these
systems are also displayed.
3.4. Antifungal activity
The compounds CS-1 to CS-6 showed inhibition against
S. cerevisiae than C. albicans. Standard antifungal amphotericin
B showed MIC of 1.25 mg/ml for both the strains (Table 4).
Antifungal activities of CS-3 and CS-4 against S. cerevisiae were
better than amphotericin B with MIC values being the least for
the CS-3. The MIC value decreased from 2.5 mg/ml for CS-1 to
0.009 mg/ml for CS-3 and 0.625 mg/ml for CS-4, further it
increased to 2.5 mg/ml for the CS-5. In C. albicans the MIC
value was 10 mg/ml for CS-1, which is same i.e. 5 mg/ml for
the CS-2 to CS-6 compounds.
A variety of controls were used in this study, negative control
which did not contain any test compound exhibited pink color. Blue
colored indicator dye resazurin was reduced to resofurin which
subsequently was reduced to hydroresofurin by oxidoreductases
from live cells giving pink color. The control without microbial
culture showed blue color since the resazurin was not reduced.
DMSO control at all concentrations showed pink color that renders
non-toxicity to microbes used.
Fig. 5. Isomers of chlorolawsone (isomer ‘a’ is more stable).
The charge distribution is characterized by the molecular elec-
trostatic potential (MESP) mapped topography as illustrated in
Fig. 6. It is thus evident that effective electron rich regions (red)
are located around the domain of lone pair residing on the oxygen
atom centers whereas the large electron deficient regions are
characterized near the vicinity of hydroxyl functionalities; which
is consistent with net atomic charges derived from the NBO
analysis. The molecular structures comprising of varying alkyl
chain and amide group was obtained which is reported in Fig. 7.
The interactions between nitrogen lone pair and the hydroxyl
proton may further be inferred. A proton transfer is evident with
concomitant increase of O(2)eH(2) bond distance (up to
~0.0656 Å) in all these systems. Interestingly the proton transfer
which results in the disappearance of ~3456 cmꢀ1 vibration in 2-
chloro-3-hydroxy-1,4-naphthoquinone, the parent compound. A
new intense band in the range of 2351 cmꢀ1- 2422 cmꢀ1 can
readily be noticed. Subsequently C(3)eO(2) carbonyl stretching
was observed near ~1678 cmꢀ1. Noteworthy enough an increasing
3.5. Antiproliferative activity
The first four members (carbon 1 to 4) of homologated (N)-n-
alkylamino1,4-naphthoquinone derivatives showed enhanced bio-
logical activity as compared the rest of the members [16e19].
Cohesive interactions are observed in n-alkylamines in five or more
carbon atoms; the biological activities are thus hindered by such
molecular interactions. Hence we studied antiproliferative activity
of first four members of (N)-n-alkylammonium 2-chloro-3-oxidio-
1,4-naphthoquinone salts, CS-1 to CS-4. Compounds CS-1 to CS-4
showed antiproliferative activity against MDA-MB-231 breast
cancer cell line. CS-1 showed the minimum IC50 of 37.6 3.1
with increasing carbon chain-length the IC50 value increases for CS-
2 (51.0 13.5 g/ml) and CS-3 (58.2 10.4 g/ml). A decrease of
47.6 2.5 g/ml in the CS-4 was also noticed. On the other hand,
mg/ml,
m
m
m
antiproliferative activity against lung cancer cell line L-132 (HeLa
derivative) showed lower activity than the standard methotrexate.

318
D. Choudhari et al. / Journal of Molecular Structure 1145 (2017) 309e320
Fig. 7. Optimized geometries of CS-1 to CS-8.
The compounds, CS-2 showed maximum IC50 of 150 5.2
CS-1 showed 124 0.54 g/mL, CS-3 exhibited 105 12.8
and CS-4 showed least IC50 value of 95 1.24 g/mL (Table 5). The
m
g/mL,
4. Conclusions
m
mg/mL
m
A
family of (N)-n-alkylammonium 2-chloro-3-oxido-1,4-
DMSO control showed negligible antiproliferative activity. The
antiproliferative studies further demonstrate that these com-
pounds are more effective on breast cancer MDA-MB-281 cell line
than lung cancer L-132 cell line.
naphthoquinone salts have been synthesized and characterized
using the single crystal X-ray diffraction in conjunction with the
density functional theory. Diverse molecular interactions in CS-1 to
CS-8 are analyzed. The antifungal activities points to CS-1 to CS-6

D. Choudhari et al. / Journal of Molecular Structure 1145 (2017) 309e320
319
Fig. 8. Frontier orbitals in CS-1 to CS-8.

320
D. Choudhari et al. / Journal of Molecular Structure 1145 (2017) 309e320
Table 4
Appendix A. Supplementary data
Antifungal activity of CS-1 to CS-6 (MIC values are in mg/ml).
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.molstruc.2017.05.083.
Samples
Minimum inhibitory concentration (MIC) mg/ml
Saccharomyces cerevisiae
Candida albicans ATCC
NCIM 3559
10231
CS-1
CS-2
2.5
2.5
10
5
References
CS-3
CS-4
CS-5
CS-6
Amphotericin B
(Standard)
0.009
0.625
2.5
1.25
1.25
5
5
5
5
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Anti-proliferative activity (IC50 values) of CS-1 to CS-4 on breast cancer (MDA-MB-
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Breast cancer cell line
(MDA-MB-231)
Lung cancer cell line
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CS-2
CS-3
CS-4
37.6 3.1
51.0 13.5
58.2 10.4
47.6 2.5
18.4 3.2
124 0.5
150 5.2
105 12.8
95 1.2
Methotrexate (standard)
6.2 1.2
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possibly more than one mechanistic pathways led to cytotoxic ac-
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hitherto compounds restricts their interactions with DNA. Alter-
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redox cycling of naphthoquinones, their optimum size and non
covalent interacting modes with DNA are key factors, which
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SSG grateful to RGYI scheme of Department of Biotechnology,
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thankful to UGC for meritorius fellowship. SPG acknowledges
support from the Research Project (37(2)/14/11/2015-BRNS) from
the Board of Research in Nuclear Sciences (BRNS), India. DNL is
thankful to Savitribai Phule Pune University for the award of
research fellowship through the University of Potential excellence
scheme from the University Grants Commission, New Delhi, India.
SPG and DNL thank the Center for Development of Advanced
Computing (CDAC), Pune for providing National Param Super-
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