Molecular structures and biological evaluation of 2-chloro-3-(n-alkylamino) -1,4-napthoquinone derivatives as potent antifungal agents

Article abstract of DOI:10.1016/j.molstruc.2013.11.029

Derivatives of 2-chloro-3-(n-alkylamino)-1,4-naphthoquinone {n-alkyl: methyl; L-1, ethyl; L-2, propyl; L-3 and butyl; L-4} have been synthesized and characterized by elemental analysis, FT-IR, ¹H NMR, UV-visible spectroscopy, LC-MS and single crystal X-ray diffraction studies. Antifungal activity of L-1 to L-4 has been evaluated against Candida tropicalis, Candida albicans and Cladosporium herbarum. The intramolecular hydrogen bonding affects the N-H vibrational frequency in L-2 (3273 cm^-1). The single crystal X-ray structure reveal that L-1 and L-3 crystallizes in triclinic P-1, whereas L-2 crystallizes in orthorhombic Pca2₁ space group. An extensive intra and intermolecular hydrogen bonding interactions were observed in L-1 to L-3 which leads to molecular association. Intramolecular N-Ha?O hydrogen bonding were observed in L-1 to L-3. Moreover π-π stacking interactions were observed between the quinonoid rings of L-1 and L-3, however no such interactions were observed in L-2. An electrochemical study showed molecular association of L-1 to L-4 in DMSO solution. Compounds L-1 to L-4 were found to be potent antifungal agents against all the three strains, especially against C. tropicalis. Amongst these promising antifungal candidates, L-1 showed better activity compared to the clinically administered antifungal drug Amphotericin B and Nitrofurantoin with MIC = 1.25 μg ml^-1 and MIC = 0.025 μg ml^-1 respectively against C. albicans. Structure and activity relationship (SAR) study suggest a Log P value of ～2.0 and the cyclic voltammetry studies reveals additional chemical processes for L-1, which exhibits maximum activity against all fungal strains.

Full text of DOI:10.1016/j.molstruc.2013.11.029

Journal of Molecular Structure 1059 (2014) 68–74
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Molecular structures and biological evaluation of 2-chloro-3-
(n-alkylamino)-1,4-napthoquinone derivatives as potent
antifungal agents
Omkar Pawar a, Ashwini Patekar b, Ayesha Khan b, Laxmi Kathawate a, Santosh Haram a, Ganesh Markad a,
Vedavati Puranik c, , Sunita Salunke-Gawali a,
⇑
⇑
a Department of Chemistry, University of Pune, Pune 411007, India
b Institute of Bioinformatics & Biotechnology, University of Pune, Pune 411007, India
c Center for Material Characterization, National Chemical Laboratory, Pune 411007, India
h i g h l i g h t s
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2-chloro-3-(n-alkylamino)-1,4-naphthoquinones are synthesized and characterized.
Single crystal X-ray structures reveal molecular association by hydrogen bondings.
Molecular associations of aminonaphthoquinones are revealed by cyclic voltammetry.
Antifungal activity is studied against C. tropicalis, C. albicans and C. herbarum.
Methyl derivative shows promising antifungal activity.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2013
Received in revised form 8 November 2013
Accepted 9 November 2013
Available online 17 November 2013
Derivatives of 2-chloro-3-(n-alkylamino)-1,4-naphthoquinone {n-alkyl: methyl; L-1, ethyl; L-2, propyl; L-
3 and butyl; L-4} have been synthesized and characterized by elemental analysis, FT-IR, 1H NMR, UV–vis-
ible spectroscopy, LC-MS and single crystal X-ray diffraction studies. Antifungal activity of L-1 to L-4 has
been evaluated against Candida tropicalis, Candida albicans and Cladosporium herbarum. The intramolecu-
lar hydrogen bonding affects the N–H vibrational frequency in L-2 (3273 cmÀ1). The single crystal X-ray
structure reveal that L-1 and L-3 crystallizes in triclinic P-1, whereas L-2 crystallizes in orthorhombic
Pca21 space group. An extensive intra and intermolecular hydrogen bonding interactions were observed
in L-1 to L-3 which leads to molecular association. Intramolecular N–HÁ Á ÁO hydrogen bonding were
Keywords:
p–p Stacking
Aminonaphthoquinone
Antifungal activity
LogP
observed in L-1 to L-3. Moreover p–p stacking interactions were observed between the quinonoid rings
of L-1 and L-3, however no such interactions were observed in L-2. An electrochemical study showed
molecular association of L-1 to L-4 in DMSO solution. Compounds L-1 to L-4 were found to be potent anti-
fungal agents against all the three strains, especially against C. tropicalis. Amongst these promising anti-
fungal candidates, L-1 showed better activity compared to the clinically administered antifungal drug
Naphthosemiquinone
Amphotericin
B and Nitrofurantoin with MIC = 1.25 l l
g mlÀ1 and MIC = 0.025 g mlÀ1 respectively
against C. albicans. Structure and activity relationship (SAR) study suggest a LogP value of ꢁ2.0 and
the cyclic voltammetry studies reveals additional chemical processes for L-1, which exhibits maximum
activity against all fungal strains.
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
undergoing chemotherapy and organ transplant recipients taking
immunosuppressive drugs [1,2]. Azole derivatives including fluco-
The increase in the incidence of fungal infections may be attrib-
uted primarily to increasing number of critically ill and immune
compromised patients, including those with AIDS, cancer patients
nazole and itraconazole are widely used in clinical settings, how-
ever there are major weaknesses in their spectra, potency, safety
and pharmacokinetic properties. The widespread and increasing
resistance of bacterial and fungal pathogens to commonly used
antibiotics and chemotherapeutics has provided the necessary
impetus to find alternative drugs and/or therapies to which micro-
organisms will not develop resistance [3]. Cladosporium herbarum
⇑
Corresponding authors. Tel.: +91 2025601397; fax: +91 2025693981
(S. Salunke-Gawali).
E-mail addresses: veda.puranik@gmail.com (V. Puranik), sunitas@chem.unipu-
ne.ac.in (S. Salunke-Gawali).
0022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2013.11.029

O. Pawar et al. / Journal of Molecular Structure 1059 (2014) 68–74
69
is an important allergenic fungal species that has been reported to
cause allergic diseases in nearly all climatic zones, with 5–30% of
the allergic population demonstrating IgE sensitization against
moulds. Sensitization to Cladosporium may often be associated
with severe or life-threatening asthma and less frequently with
chronic urticaria and atopic eczema [4,5]. Upper respiratory symp-
toms occur with exposure to Cladosporium, but asthma symptoms
are more prevalent [6,7]. The term candidiasis often is used to de-
scribe an infection caused by the yeast like fungus Candida albicans
[8]. Species of Candida other than C. albicans, however, also have
the potential to cause infection, particularly in patients who are
immunologically or physiologically compromised. Candida tropi-
calis has emerged as a potentially dangerous opportunistic fungus
isolated from patients in critical care unit [9].
L-1 to L-4 have been investigated by electrochemical studies. The
antifungal activities have been evaluated against C. tropicalis, C.
albicans and C. herbarum. The derivatives from L-1 to L-4 have been
found to be active against all the three fungal strains. The structure
activity correlation suggested that the lipophilicity of all the syn-
thesized compounds were appropriate for drug uptake, of which
the compound L-1 was found to possess the highest activity. The
electrochemical studies also suggests of a reversible electron trans-
fer associated with some other chemical processes that leads to
highest cytotoxicity towards the fungal strains.
2. Experimental
2.1. General materials and methods
The clinical importance of 1,4-naphthoquinones has stimulated
enormous interest in this class of compounds [10,11]. In most of
the cases, the biological activity of naphthoquinones has been re-
lated to their ability to accept one or two electron to form the cor-
responding radical anion or radical dianion [12–14]. The electron
accepting ability of given naphthoquinones can be modified by di-
rectly adding a substituent to the naphthoquinone system. The
synthesis of alkyl amino derivatives of 1,4-naphthoquinones is of
prime importance, since these compounds exhibit strong antibac-
terial [15], antimalarial and antitumor activity [16]. In addition
to this, the amino substituted 1,4-naphthoquinone moiety is a
key component of molecular framework of many natural products
and has been used as a synthetic key intermediate for the synthesis
of several compounds with important biological applications [17].
The ‘amino’ and ‘thioether’ substituted derivatives of 1,4-naphtho-
quinones have extremely rich biological activities because of their
redox potentials [17]. Several studies have also demonstrated that
the naphthoquinone derivatives can also exhibit marked activity
against fungi [15–17]. ‘Arylthio’ substituted derivatives are known
to have activity against Candida species [16]. The widespread bio-
logical activities of substituted 1,4-naphthoquinones are related to
their ability to form radicals. The stability of these radicals will de-
pends upon the functional groups attached to different positions of
the 1,4-naphthoquinone moiety. Single crystal X-ray diffraction
studies of 1,4-naphthoquinone derivatives also shows that the
molecules forms three dimensional polymeric network through
The materials used viz. 2,3-dichloro-1,4-naphthoquinone,
methyl amine solution (40%), ethyl amine solution (70%), propyl
amine and butyl amine were purchased from Sigma–Aldrich. The
reactant 2,3-dichloro-1,4-naphthoquinone was recrystallized from
methanol and used for further reactions. The solvents used such as
dichloromethane, toluene, methanol were of analytical grade and
were purchased from Merck Chemicals, India. The solvents used
were distilled in nitrogen atmosphere by standard methods [26]
and dried wherever necessary. FT-IR spectra of the compounds
have been recorded between 4000 and 400 cmÀ1 as KBr pellets
on SHIMADZU FT 8400 spectrometer and 1H NMR of ligands are re-
corded in DMSO-d6 as well as in CDCl3, on Varian Mercury
300 MHz with TMS (tetramethylsilane) used as a reference. UV–
visible spectra of all compounds were recorded on SHIMADZU
UV 1650 in DMSO and in solid state. Elemental analysis has been
performed on Thermo Finnigan EA 1112 Flash series Elemental
Analyzer. Mass of the derivatives L-1 to L-4 were determined using
LC-MS 2010-eV (Make SHIMADZU). Melting points of the deriva-
tives L-1 to L-4 were determined using melting point apparatus
(Make-METTLER) and were corrected using DSC (Differential Scan-
ning Calorimetry) technique (Make-METTLER, STAR SW 8.10).
2.2. Synthesis
C–HÁ Á ÁO and O–HÁ Á ÁO hydrogen bonding and
p–p stacking interac-
All four derivatives (L-1 to L-4) of 2-chloro-3-(n-alkylamino)-
1,4-naphthoquinones have been synthesized from 2,3-dichloro-
1,4-naphthoquinone and using the respective primary amines
[27]. 1 g of 2,3-dichloro-1,4-naphthoquinone (4.4 mM) was dis-
solved in 25 ml of dichloromethane and stirred for 15 min, fol-
lowed by drop wise addition of alkylamine solution (5.2 mM).
The reaction mixture was stirred for 24 h at room temperature
(26 °C). The progress of the reaction and the purity of the ligands
were monitored by thin layer chromatography. Red color solids
were obtained on evaporation of the solvent. The crude product
was purified by column chromatography using toluene: methanol
(9:1) as the mobile phase. The crystals for X-ray analysis were ob-
tained by recrystallization in appropriate solvents.
tions of the quinonoid rings [18–25]. Molecular associations
formed by intra and intermolecular hydrogen bonding may lead
to stabilization of radical species and the redox cycling determines
the biological activity of the substituted 1,4-naphthoquinone
derivatives.
O
NHR
L-1: R=CH3
L-2: R=C2H5
Cl
Spectroscopic data for L-1 and L-4 are represented in the Sup-
plementary material (in ESI ).
L-3: R=C3H7
O
L-4: R=C4H9
2.2.1. Characterization of 2-chloro-3-ethylamino-1,4-
naphthoquinone; L-2
.
Red crystals, 0.817 g (78.8%). m.p.:129–130 °C. Anal. Data Calcd.
for C12H10NO2Cl: C, 61.16; H, 4.28; N, 5.94. Found C, 60.87; H, 4.19;
N, 5.73; FT-IR(KBr) vmax/cmÀ1 1676, 3273, 721. 1H NMR (300 MHz,
CDCl3) d: 1.34 (3H, t, J = 6.8 Hz), 3.91 (2H, m, J = 6.8 Hz), 6.03 (br s,
1H, D2O ex.), 7.62 (1H, t, J = 6.8 Hz), 7.73 (1H, t, J = 7.4 Hz), 8.03 (1H,
d, J = 7.5 Hz), 8.16 (1H, d, J = 7.5 Hz); UV–visible, kmax: 470 nm; LC-
MS (EI): m/z 235.95 (M+ + H).
Aminonaphthoquinone derivatives, 2-chloro-3-(n-alkylamino)-
1,4-naphthoquinone (n-alkyl: methyl; L-1, ethyl; L-2, propyl; L-3
and butyl; L-4) are synthesized and characterized by various ana-
lytical tools (Figs. S1–S3, in ESI ) in the present investigation.
The molecular associations of L-1 to L-3 were revealed by single
crystal X-ray diffraction studies. The radical formation abilities of

70
O. Pawar et al. / Journal of Molecular Structure 1059 (2014) 68–74
2.2.2. Characterization of 2-chloro-3-propylamino-1,4-
naphthoquinone; L-3
Dc = 1.392 g/cc,
l
(Mo K
a
) = 0.309 mmÀ1, 5794 reflections mea-
(I)], R value 0.0451, wR2 = 0.1111. Larg-
sured, 2098 unique [I > 2
r
Red crystals, 0.856 g (78%), m.p.: 115 °C; Anal. Data Calcd. for
est diff. peak and hole 0.231 and À0.262 e ÅÀ3
.
C13H12NO2Cl: C,62.53; H,4.84; N,5.61. Found C, 61.82; H, 4.64; N,
5.40; FT-IR(KBr) vmax/cmÀ1 1634, 1682, 3310, 719; 1H NMR
(300 MHz, CDCl3) d: 1.01 (t, 3H J = 7.4 Hz), 1.70 (m, 2H,
J = 11.9 Hz), 3.81 (q, 2H, J = 6.0 Hz), 6.10 (br.S, 1H, D2O ex.), 7.61
(t, 1H, J = 7.2 Hz), 7.72 (t, 1H, J = 6.8 Hz), 8.02 (d, 1H, J = 6.0 Hz),
8.14 (d,1H, J = 7.2 Hz); UV–visible, kmax: 471 nm; LC-MS (EI): m/z
249.69 (M+ + H).
2.4. Cyclic voltammetric studies
The electrochemical measurements were performed with the
help of Metrohm Potentiostat/Galvanostat (Model Autolab PGSTAT
100). A commercial Pt disc electrode (CHI Instruments, USA, 2-mm
diameter), Ag wire, and Pt wire loop were used as working, qau-
sireference and counter electrodes respectively. The voltammetric
measurements were carried out in an indigenously developed vac-
uum electrochemical cell having a special provision to transfer the
analyte with minimum exposure to the laboratory atmosphere
[29]. After fixing the electrodes to the cell 0.239 g of tetra butyl
ammonium perchlorate (typically 100 mM in 5 mL solution) was
transferred and vacuum dried in situ at 80 °C for an hour. The cell
was cooled down to room temperature and brought to atmo-
spheric pressure by relieving the vacuum through high purity ar-
gon gas. 5 mL of predried solvent was injected into the cell
through silicon septum under argon atmosphere. The blank or con-
trolled voltammograms were acquired in tetra butyl ammonium
perchlorate-DMSO mixture prior to the measurements. Sample
dispersed in small amount of solvent injected in the cell for further
measurement (analyte concentration 1 mg/mL). At the end of each
set of experiments the potentials were calibrated with respect to
the normal hydrogen electrode (NHE) using ferrocene as an inter-
nal standard.
2.3. X-ray crystal structure analysis of L-1 to L-3
Data for all three compounds (L-1 to L-3) were collected at
T = 293 K, on SMART APEX CCD Single Crystal X-ray diffractometer
using Mo-Ka radiation (k = 0.7107 Å) to a maximum h range of
25.00°. Crystal to detector distance 6.05 cm, 512 Â 512 pixels/
frame, Oscillation/frame À0.3°, maximum detector swing an-
gle = À30.0°, beam centre
= (260.2, 252.5), in plane spot
width = 1.24, SAINT integration and SADABS correction applied.
The structures were solved by direct methods using SHELXTL. All
the data were corrected for Lorentzian, polarization and absorption
effects. SHELX-97 (ShelxTL) was used for structure solution and full
matrix least squares refinement on F2 [28]. Hydrogen atoms were
included in the refinement as per the riding model. The refine-
ments were carried out using SHELXL-97.
2.3.1. Single crystal analysis of 2-chloro-3-methylamino-1,4-
naphthoquinone; L-1
Single crystals of the compound were grown by slow evapora-
tion of the solution in dichloromethane. Dark red colored plate of
approximate size 0.41 Â 0.28 Â 0.08 mm3, was used for data col-
lection. Quadrant data acquisition. Total scans = 4, total
2.5. Antifungal activity
2.5.1. Agar disc diffusion assay
Fungal strains C. tropicalis NCIM 3548, C. albicans NCIM 3557
and C. herbarum NCIM 1112 procured from National Chemical Lab-
oratory, Pune, India. The susceptibility of synthesized compounds
was tested by agar disc diffusion assay. Commercially available
frames = 2424, exposure/frame = 5.0 s/frame,
25.00°, completeness to of 25.0° is 97.5%.
M = 221.63. Crystals belong to triclinic, space group P-1,
a = 7.1438(8), b = 7.4626(8), c = 9.1504(10) Å, = 84.839(2)°,
h
range = 2.29–
h
C11H8ClNO2,
a
discs (9 mm diameter) preloaded with fluconazole (25 lg/disc)
b = 76.708(2)°,
cc, (Mo K
c
= 86.574(2)°, V = 472.44(9) Å3, Z = 2, Dc = 1.558 g/
were used as positive control. The concentration of drugs to be
were determined by first performing preliminary experiments to
determine the optimal concentration that produced inhibition
zones which can be conveniently measured on the 100 Â 15 mm
plate (Fisher Scientific Co, KY, USA). Stock solution of each drug
was prepared using dimethyl sulfoxide: 1 mg/mL. Blank paper
discs (6 mm diameter; Sparks, MD) were loaded with 5, 10, 15,
l
a
) = 0.378 mmÀ1, 3312 reflections measured, 1625 un-
ique [I > 2
r
(I)], R value 0.0620, wR2 = 0.1535. Largest diff. peak
and hole 0.549 and À0.723 e Å–3
.
2.3.2. Single crystal analysis of 2-chloro-3-ethylamino-1,4-
naphthoquinone; L-2
Single crystals of the compound were grown by slow evapora-
tion of the solution in methanol. Red colored plate of approximate
size 0.55 Â 0.11 Â 0.05 mm3 was used for data collection. Hemi-
sphere data acquisition. Total scans = 3, total frames = 1271, expo-
sure/frame = 15.0 s/frame, h range = 2.49–23.49°, completeness to
h of 23.49° is 100%. C12H10ClNO2, M = 235.66. Crystals belong to
20 lL of the prepared stock solutions to obtain the desired drug
concentration of L-1 to L-4 per disc. All the fungi were sub-cultured
on potato dextrose agar (PDA).
Discs containing the test agents were applied to the surface of
inoculated plates. Plates were inverted and incubated at 30 °C for
48 h to allow for fungal growth. Inhibition zone diameters (IZD)
were measured in cm. All isolates were run in triplicate and the
standard deviations determined.
Orthorhombic,
c = 16.3347(18) Å, V = 1105.4(2) Å3, Z = 4, Dc = 1.416 g/cc,
) = 0.328 mmÀ1
4384 reflections measured, 1618 unique
Pca21,
a = 14.8140(17),
b = 4.5680(5),
l
(Mo
Ka
,
[I > 2
r
(I)], R value 0.0647, wR2 = 0.1489. Largest diff. peak and hole
2.5.2. Radical scavenging activity (NBT-SOD activity)
The compounds L-1 to L-4 was assayed for the SOD activity
using the alkaline DMSO- NBT method developed by Beauchamp
and Fridovich [30].
0.300 and À0.203 e ÅÀ3
.
2.3.3. Single crystal analysis of 2-chloro-3-propylamino-1,4-
naphthoquinone; L-3
Single crystals of the compound were grown by slow evapora-
tion of the solution in methanol. Red colored plate of approximate
size 0.28 Â 0.20 Â 0.03 mm3, was used for data collection. Quad-
2.5.3. Minimum inhibitory concentration assay (MIC)
Using the multipipettor, dispense 100
into all the wells of a microtitre plate. To this medium 100
appropriate 2Â antibiotic solutions were added into the wells in
column 1. Using the multipipette preset at 100 L, the antibiotics
was mixed into the wells of column 1 by sucking up and down
6–8 times. 100 L of mixture was withdrawn from column 1 and
was added to column 2. This makes the column 2 a twofold dilu-
lL of medium is taken
lL of
rant data acquisition. Total scans = 4, total frames = 2424,
h
range = 1.99–25.00°, completeness to h of 25.0° is 99.8%. C13H12-
ClNO2, M = 249.70. Crystals belong to triclinic, space group P-1,
l
a = 7.3022(5), b = 7.9970(6), c = 11.0164(8) Å,
a
= 68.8010(10)°,
Z = 2,
l
b = 84.3960(10)°, = 84.6730(10)°,
c
V = 595.76(7) Å3,

O. Pawar et al. / Journal of Molecular Structure 1059 (2014) 68–74
71
tion of column 1 and the procedure is repeated to column 3 and so
on. The plates were incubated at 30 °C and were scanned with an
ELISA reader.
is similar to that observed for the amino substituted derivatives
of 1,4-naphthoquinone [16–21]. The C(3)–N–C(11) bond angle ob-
served in L-1, L-2 and L-3 are 131.0(3)°, 131.5(5)° and 132.0(1)°
respectively (Table S2, ESI ) and these bond angles are greater than
the classical value for sp3 hybridized nitrogen atom. L-1 and L-3
3. Results and discussion
shows strong
cent chains of the quinonoid moiety, whereas such
p
–
p
interactions through C(2) and C(4) of the adja-
interac-
p–p
Kuo et al studied the antiplatelet activity of 2-substituted-3-
chloro-1,4-naphthoquinones, wherein the compounds were syn-
thesized using benzene as a solvent [31]. The reaction of 2,3-di-
tions were not observed in L-2. L-1 to L-3 shows intramolecular
hydrogen bonding between N–HÁ Á ÁO(2) (Table 1).
An ORTEP view of L-1 molecule is shown in Fig. 1. This molecule
crystallizes in a triclinic P-1 space group. Molecules of L-1 form
ladder like polymeric chains, via intermolecular hydrogen bonding
of N–HÁ Á ÁO interactions. The adjacent chains are held together by
chloro-1,4-naphthoquinone
with
n-alkyl
amines
in
dichloromethane at room temperature results in the formation of
2-chloro-3-(n-alkylamino)-1,4-naphthoquinone. The derivatives
L-1 to L-4, can also be obtained with yields more than 90% by car-
rying out the reactions at room temperature without using any or-
ganic solvent and by mixing equimolar concentrations of 2,3-
dichloro-1,4-naphthoquinone and the respective n-alkylamines.
FT-IR spectra (Fig. S1 in ESI ) shows mN–H frequency at
3333 cmÀ1 in at L-1, 3273 cmÀ1 in L-2, at 3308 cmÀ1 in L-3 and
at 3311 cmÀ1 in L-4. This frequency reflects on the extent of inter
and intra molecular hydrogen bonding in the derivatives L-1 to
L-3. The derivative L-2 differs structurally from rest of the ligands.
The mC=O frequency increased by ꢁ4 cmÀ1 in both L-1 and L-3
(1680 cmÀ1) by ꢁ2 cmÀ1 in L-2 (1678 cmÀ1) and decreased by
ꢁ2 cmÀ1 in L-4, as compared to 2,3-dichloro-1,4-naphthoquinone
(1676 cmÀ1). The mC–N and mC=C vibrations were observed at
ꢁ1599 3 cmÀ1 and 1570 cmÀ1 respectively in L-1 to L-4. The par-
anaphthoquinone vibrations (ꢁ1265 5 cmÀ1) show shifts to-
wards lower frequencies as compared to 2,3-dichloro-1,4-
naphthoquinone (1274 cmÀ1). This decrease is due to hydrogen
bonding effect of C(4)-O(2) with neighbouring molecules. mC–Cl
vibration is observed at 721 cmÀ1 for L-1 to L-4.
p–p interactions forming dimeric chains. The dimeric chains inturn
are held together (Fig. 2a) by C–HÁ Á ÁO interactions (Table 1). The
orientations of the chains are opposite to one another and all the
chlorine atoms lie on same side in the chain.
Molecules of L-2 are oriented in such a way that they form a
butterfly-like structure when viewed down the ‘a’ axis, where chlo-
rines represents eyes of the butterfly (Fig. 3a). The L-2 molecules
form linear chains via N–HÁ Á ÁO interactions (Fig. 3b). All the chlo-
rine atoms lie on same side of chain similar to L-1.
Molecules of L-3 also form a ladder like polymeric chain via
intermolecular N–HÁ Á ÁO and C–HÁ Á ÁO hydrogen bonding. However,
the polymeric chains with opposite orientations are
p–p stacked
and shows intermolecular N–HÁ Á ÁO interactions. The difference be-
tween the ladder like chains in L-1 and L-3 is that, the dimeric
chains in L-1 are further linked through C–HÁ Á ÁO interactions,
whereas in L-3, the dimeric chains in L-3 shows close contacts with
CHÁ Á ÁC (Fig. 2b).
Overlay of the naphthoquinone framework from L-1 to L-3 is
shown in Fig. 4. Overlapping of L-1 to L-3 molecules showed that
the naphthoquinone framework overlaps, while differing slightly
in the orientation of the alkyl side chains.
The UV–visible spectra of L-1 to L-4 (Fig. S3 in ESI ) showed the
naphthoquinone bands in the UV region at around 291–297 nm
330–337 nm are assigned to the
p ?
pà transition of the benzenoid
Single crystal X-ray structures reveals intra and intermolecular
ring and the quinonoid ring respectively. In addition, a third lower
energy transition appeared in the visible region ꢁ472 nm, which is
typical for amino substituted quinones [32]. The lower energy
absorption ꢁ470 nm is assigned to the charge transfer (CT) transi-
tion and the weak n ? pà transition.
hydrogen bonding interactions in L-1 to L-3 and
p–p stacking
interaction in L-1 and L-3. This unique feature of L-1 to L-3 will en-
hance their biological activity by interacting with DNA via hydro-
gen bonding and p–p stacking interactions [34,35].
3.2. Cyclic voltammetry studies
3.1. X-ray diffraction studies
Cyclic voltammogram of L-1 to L-4 are shown Fig. 5. Scan rate
variation plots are presented in Figs. S4, S7, S9, S12, S15 (in ESI ).
Cyclic voltammetric data is represented in Tables S3-S7 (in ESI ).
Peak, Ic corresponds to reduction of naphthoquinone to naph-
The crystal structure data of L-1 to L-3 reveals that the quino-
noid and the aromatic bond distances are similar to those observed
in several 1,4-naphthoquinone derivatives [18–25]. The carbonyl
C(1)-O(1) and C(4)-O(2) bond distances are 1.232 Å and 1.217 Å
respectively in L-1 and L-3, 1.216 Å and 1.225 Å respectively in L-
2 (Table S1 in ESI ). These distances are similar and typically rep-
resent the 1,4-naphthoquinone carbonyl [16–25,33]. The C(3)–N
bond distance is 1.331 Å in L-1, 1.345 Å in L-2, 1.393 Å in L-3 and
thosemiquinone [36–38] (Table 2).
DEp values corresponding to
peak I and peak II in L-2 to L-4 is more than 60 mV, which could
be attributed to the molecular association in solution through
hydrogen bonding interaction and it has also been proved by single
crystal X-ray structures of L-1 to L-3, wherein the ligands possess
inter and intra molecular hydrogen bonding. No shift was observed
in the solid state UV–visible spectra of L-1 to L-4 as compared to
the bands observed in DMSO [Fig. S3 in ESI ], which implies that
the hydrogen bonding is not disturbed in solution of all the ligands.
Unlike hydrogen bonded hydroxy naphthoquinones the reduction
of L-1 to L-4 from naphthoquinone to catechol involves two or
more steps and reduced intermediate products may be resulted
during reduction [39–41]. Peak III is a result of oxidation of such
intermediate products. Scan rate variation in specific potential
range [Fig. S5 in ESI ] showed that peak III in L-1 is indeed related
to peak I i.e. naphthoquinone to naphthosemiquinone reduction.
Peak III is observed only in L-1 and L-4. The ratios for ipa/ipc have
been plotted [Fig. S18 in ESI ] and the values for ipc and ipa are
found to be closer to unity for peak I and II of compounds L-2 to
L-4. However, the peak current deviates from one, which could
Table 1
Hydrogen bond geometries for L-1 to L-3 in (Å).
D–HÁ Á ÁA
D–H (Å)
D–A (Å)
D–HÁ Á ÁA (Å)
\D-H-A (°)
L-1
N–H(0)Á Á ÁO(2)i
0.86
0.86
0.93
0.86
3.187(6)
3.030(6)
3.319(6)
2.582(6)
2.567(3)
2.551(4)
2.664(3)
2.134(3)
116.2(3)
129.8(2)
128.1(2)
112.1(2)
N–H(0)Á Á ÁO(1)i
C(8)–H(8)Á Á ÁO(1)ii
N–H(0)Á Á Á(O2)intra
L-2
L-3
N–H(0)Á Á ÁO(1)iii
0.86
0.86
2.850(5)
2.156(5)
2.197(3)
2.595(7)
132.5(3)
111.3(3)
N–H(0)Á Á ÁO(2)intra
C(8)–H(8)Á Á ÁO(1)iv
0.93
0.86
0.86
0.86
3.370(8)
3.200(5)
3.112(8)
2.581(5)
2.684(5)
2.559(4)
2.546(6)
2.115(5)
131.2(4)
131.2(4)
124.3(4)
113.4(4)
N–H(0)Á Á ÁO(1)v
N–H(0)Á Á ÁO(2)iv
N–H(0)Á Á ÁO(2)intra
Symmetry operators (i) 1Àx, 1Ày, 2Àz; (ii) À1Àx, 2Ày, 2Àz; (iii) À1/2, 1Ày, 1/2 + z;
(iv) À1 + x, y, z; (v) 1Àx, Ày, 1Àz.

72
O. Pawar et al. / Journal of Molecular Structure 1059 (2014) 68–74
Fig. 1. ORTEP plot of (a) L-1, (b) L-2 and (c) L-3. The ellipsoids were drawn with 50% probability.
Fig. 2. Hydrogen bonding and p–p stacking interaction in (a) L-1 and (b) L-3.
Fig. 3. (a) Packing of L-2 molecules in unit cell in down ‘a’ axis in which molecules form butterfly like structure, and (b) linear chains of L-2 formed by intermolecular N–HÁÁÁO
hydrogen bonding.
be due to some additional chemical processes [42]. The linear
½
regression of ip vs
m
plots (Figs. S5, S8a, S10, S13, S16 in ESI ) sug-
gested that the charge transfer is diffusion controlled.
All the compounds were found to possess pro-oxidant activity,
which can be attributed to the redox cycling of the quinone moie-
ties. It is observed that the compounds interacted with the super-
oxide radicals and instead of quenching the radical generation was
increased as compared to the control. Thus, ROS (reactive oxygen
species) generation considerably increased in the reaction. This
can also be attributed to the redox cycling properties of L-1 to L-
4, which was also supported by the cyclic voltammetric studies.
Fig. 4. Overlays of L-1, L-2 and L-3.
3.3. Antifungal activity
nificant zone of clearance (Fig. S19 in ESI ). However, the data ob-
tained by this method was not consistent, and hence, the
microdilution method was followed.
Table 3 depicts the diameter of clearance zone for compounds
L-1 and L-3, whereas compounds L-2 and L-4 did not show any sig-

O. Pawar et al. / Journal of Molecular Structure 1059 (2014) 68–74
73
Ia
L-2
L-1
IIa
III
10 µA
10 µA
a)
Ic
Ic
IIc
IIc
1
0
-1
-2
1
0
-1
-2
E(V) Vs NHE
E(V) Vs NHE
L-4
Ia
Ia
L-3
III
IIa
IIa
10 µA
10 µA
Ic
IIc
Ic
IIc
1
0
-1
-2
1
0
-1
-2
E(V) Vs NHE
E (V) Vs NHE
Fig. 5. Cyclic voltammogram of L-1 to L-4.
Table 2
Half peak potentials and logP values of L-1 to L-4.
picts the MIC values obtained for the compounds L-1 to L-4 [Fig. S7
in ESI ]. Compounds L-1 to L-4 exhibited fungicidal activity to-
wards all the tested fungi, although the agar diffusion assay exhib-
ited no significant data for compounds L-2 and L-4. Tran et al have
shown that substituted 1,4-naphthoquinone possesses potent anti-
fungal activity against C. albicans, when the C-3 position is replaced
by a chlorine atom [43]. The MIC values for the compounds are de-
picted in Table 4. The MIC values for the compounds L-1 to L-4,
E1/2
E1/2
LogP
I
II
L-1
L-2
L-3
L-4
À0.776595
À0.569075
À0.587080
À0.516853
À1.270978
À1.181245
À1.203768
À1.102065
2.059
2.435
2.938
3.497
ranged from 0.025 lg/ml to 1.25 lg/ml for all the strains. Each
experiment was carried out twice and performed in duplicate.
Vulvovaginal candidiasis (VVC) is a mucosal infection caused by
Candida species. C. albicans, a dimorphic commensal organism of
the genital and gastrointestinal tract, is the causative agent of
3.3.1. Minimum inhibitory concentration assay
The CLSI approved microdilution method (M38-A2) [7] was
used to determine the minimum inhibitory concentration (MIC)
of the test compounds against the three fungal isolates. Table 4 de-
Table 3
Agar disc diffusion assay of L-1 and L-4 (1 mg/mL stock).
No
Fungus
Compound
Diameter of clearance zone (cm)
5
l
L
10
lL
15
l
L
20 lL
1.
2.
3.
Cladosporium herbarum
Candida tropicalis
Candida albicans
L-1
L-3
1.6
1.1
1.7
1.1
1.8
1.1
2.3
1.1
L-1
L-3
0.4
0.6
0.6
0.6
0.75
0.6
0.9
0.6
L-1
L-2
L-3
L-4
1.7
1.1
0.9
0.7
1.8
1.2
1.0
0.8
2.0
1.3
1.1
0.8
2.2
1.3
1.1
0.8
Table 4
MIC values for L-1 to L-4 (lg/mL).
L-1 0.0025
L-2 0.0025
L-3 0.0025
L-4 0.0025
Amphotericin B 0.0025
Nitrofurantoin 0.0025
Cladosporium herbarum
Candida tropicalis
Candida albicans
0.078
0.488
0.025
1.25
0.024
0.85
1.25
0.024
2.5
1.25
0.039
1.25
1.25
1.25
1.25
1.25
1.25
1.25

74
O. Pawar et al. / Journal of Molecular Structure 1059 (2014) 68–74
VVC in about 85–90% of patients with positive vaginal fungal cul-
tures [44]. The remainders of the cases are caused by non Candida
species, the most common of which are Candida glabrata and C.
tropicalis [45]. Since the compounds L-1 to L-4 possess excellent
fungicidal activity in vitro and in vivo outcome requires further
investigation.
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