n-alkylamino analogs of Vitamin K3: Electrochemical, DFT and anticancer activity of their oxidized and one electron reduced form

Article abstract of DOI:10.1016/j.molstruc.2018.11.024

Synthesis, characterization and anticancer activity of reduced form of n-alkylamino analogs of Vitamin K3 (1Na to 8Na) are undertaken. Molecular structure and molecular association of oxidized form of 2-ethylamino-3-methyl-1,4-naphthoquinone (2) and 2-hexylamino-3-methyl-1,4-naphthoquinone (6) were studied by single crystal X-ray analysis. 2 crystallize in monoclinic C2/c and 6 in P2₁ space group. The reduced form of eight homologated analogs of 2-(n-alkylamino)-3-methyl-1,4-naphthoquinone (1Na to 8Na) were synthesized using sodium metal as reductant at 0 °C in methanol. The formations of naphthosemiquinone radical in 1 to 6 have been confirmed from their EPR spectra. Polycrystalline powder X-band EPR spectra of 1 to 6 shows signals ～2.0020 ± 0.0026 at 133 K. Anticancer activity of 2-(n-alkylamino)-3-methyl-1,4-naphthoquinone (1Na to 8Na) and one electron reduced forms have been evaluated against breast cancer (HeLa) cell line, 1 and 1Na showed promising anticancer activity.

Full text of DOI:10.1016/j.molstruc.2018.11.024

Accepted Manuscript
n-alkylamino analogs of vitamin K3: Electrochemical, DFT and anticancer activity of
their oxidized and one electron reduced form
Dattatray Chadar, Priyabrata Banerjee, Sourav Kr. Saha, Sujit Bhand, Rajendra Patil,
Sunita Salunke-Gawali
PII:
S0022-2860(18)31328-0
DOI:
https://doi.org/10.1016/j.molstruc.2018.11.024
MOLSTR 25853
Reference:
To appear in: Journal of Molecular Structure
Received Date: 8 May 2018
Revised Date: 7 July 2018
Accepted Date: 7 November 2018
Please cite this article as: D. Chadar, P. Banerjee, S.K. Saha, S. Bhand, R. Patil, S. Salunke-Gawali,
n-alkylamino analogs of vitamin K3: Electrochemical, DFT and anticancer activity of their oxidized
and one electron reduced form, Journal of Molecular Structure (2018), doi: https://doi.org/10.1016/
j.molstruc.2018.11.024.
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ACCEPTED MANUSCRIPT
1 to 8 series
1Na to 8Na Series
100
80
60
40
20
0
100
80
60
40
20
0
0
0
25
50
100
50
100
200
Compound (µg/mL)
Compound (µg/mL)
Compound (µg/mL)
Oxidized from of vitamin K3 analogs
Reduced from of vitamin K3 analogs

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n-alkylamino analogs of vitamin K3: Electrochemical, DFT and anticancer
activity of their oxidized and one electron reduced form
Dattatray Chadara, Priyabrata Banerjeeb,c, Sourav Kr. Sahab,c, Sujit Bhanda, Rajendra Patild,
Sunita Salunke-Gawalia*
aDepartment of Chemistry, Savitribai Phule Pune University, Pune 411007, Maharashtra India
bSurface Engineering & Tribology Group, CSIR-Central Mechanical Engineering Research
Institute, Mahatma Gandhi Avenue, Durgapur 713209, West Bengal, India
cAcademy of Scientific and Innovative Research, CSIR-CMERI Campus, Durgapur 713209, West
Bengal, India
dDepartment of Biotechnology, Savitribai Phule Pune University, Pune 411007, Maharashtra
India
Abstract
Synthesis, characterization and anticancer activity of reduced form of n-alkylamino analogs of
Vitamin K3 (1Na to 8Na) are undertaken. Molecular structure and molecular association of
oxidized form of 2-ethylamino-3-methyl-1,4-naphthoquinone (2) and 2-hexylamino-3-methyl-
1,4-naphthoquinone (6) were studied by single crystal X-ray analysis. 2 crystallize in
monoclinic C2/c and 6 in P21 space group. The reduced form of eight homologated analogs of 2-
(n-alkylamino)-3-methyl-1,4-naphthoquinone (1Na to 8Na) were synthesized using sodium
metal as reductant at 0 °C in methanol. The formations of naphthosemiquinone radical in 1 to 6
have been confirmed by EPR spectra. Polycrystalline powder X-band EPR spectra of 1 to 6
shows signals ~ 2.0020 ± 0.0026 at 133 K. Anticancer activity of 2-(n-alkylamino)-3-methyl-1,4-
naphthoquinone (1Na to 8Na) and one electron reduced forms have been evaluated against
breast cancer (HeLa) cell line, 1 and 1Na showed promising anticancer activity.
Key words: aminonaphthoquinone, vitamin K3, semiquinone, catechol, anticancer activity
1

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1.
Introduction
Vitamin K is an essential cofactor in humans for the synthesis of several proteins that
play a role in regulation of normal coagulation. In addition growing evidence has shown that
Vitamin K is also a coenzyme of glutamic acid γ-carboxylase, which converts the glutamic acid
residue into γ-carboxyglutamic acid (in Vitamin K cycle) in the molecule osteocalcin which is
implicated in bone mineralization and calcium ion homeostasis [1-3]. Vitamin K deficiency
affects calcium homeostasis, which leads to vascular calcification and bone disorders. Currently,
Vitamin K is widely used to treat osteoporosis in several Asian countries [4]. Vitamin K3(2-
methyl-1,4-naphthoquinone) is a synthetic version of Vitamin K that can be converted into the
active form of Vitamin K2 inside the animal body [5], it’s giving the same biological properties
as Vitamin K3. As a synthetic compound, Vitamin K3 is more stable and inexpensive than
natural Vitamin K. Vitamin K3 is approved as a drug by FDA (Food and Drug Administration)
[6]. It affects the redox activity of thiols and thiol containing compounds and calcium
homeostasis. Most important pharmacological property of vitamin K3 is anticancer activity, it
induces oxidative stress in cancer cells by production of hydroxyl radicals and DNA strand
breaks [7,8].
Quinones are redox active and they form one electron reduction product as semiquinone
and two electron reduction leads to formation of hydroquinone. Both these reactions leads to
consumption of superoxide radicals which is essential for cancer cell homeostasis [9,10].
Aminonaphthoquinones are integral part of several natural antibiotics [11-12]. Several analogs of
aminonaphthoquinones are cytotoxic to cancer cell lines [13-16]. n-alkylamino-1,4-naphthoquinone
posses potential antibacterial, antifungal and anticancer agents [17-19]. Redox potential plays key role in
cytotoxicity of quinone. So as to compare the antiproliferative activity of the redox forms of the n-
alkylamino derivatives of vitamin K3, in this investigation reduced form of eight n-alkylamino derivatives
(1 Na to 8 Na) been synthesized chemically using sodium metal as a reductant. Anti-proliferative activity
of a total 16 compounds viz., oxidized 1 to 8 and one electron reduced 1Na to 8Na have been studied
against HeLa cell line. The redox potentials of oxidized form derivatives were evaluated by cyclic
voltammetry studies. Molecular structure and molecular interactions were evaluated by single crystal X-
ray diffraction studies. It was observed that oxidized form of the compounds are effective than the one
electron reduced form.
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where,
O
O
8
5
1: R=H ; 2: R=CH3;
3:R=C2H5, 4: R=C3H7
5: R=C4H9; 6: R=C5H11;
7: R=C6H13; 8: R=C7H15
9
1
NH
7
6
CH2R
2
CH3
3
10
4
Scheme 1 Molecular formula of 1 to 8.
2. Experimental section
2.1 Materials and Methods
The materials used for synthesis, vitamin-K3 (2-methyl-1,4-naphthoquinone), ethyl
amine solution (70%), propylamine (99%), butylamine (99%), pentylamine (99%), hexylamine
(99%), heptylamine (99%) and octylamine (99%) were purchased from Sigma-Aldrich and used
as received. Methyl amine solution (40%) was obtained from Loba chemicals; sodium metal,
toluene and methanol were obtained from Merck Chemicals. Solvents were distilled by standard
methods [20] and dried wherever necessary.
The X-band EPR spectra were recorded in solid state and liquid state at liquid nitrogen
temperature (133 K) with VARIAN, E112 EPR SPECTROMETER with microwave frequency
9.5 GHz and modulation of frequencies from 100 to 15 Hz at SAIF-IIT Powai, Mumbai, India.
The concentration of Na+ was determined by flame photometry on SYNTRONIC flame
photometer 128. A. R grade sodium chloride used as standard.
2.2 Synthesis of reduced form of 1 to 8
2-(n-alkylamino)-3-methyl-1, 4-naphthoquinone about 0.200 g was taken in one neck
round bottom flask. About 10 ml dry methanol was added to completely dissolve the powder.
The solution was stirred under nitrogen atmosphere in Dewar flask at 0°C. Calculated amount of
sodium metal (~0.045g to 0.030g) was added to it. The reaction was kept for 3 hrs with constant
stirring at 0°C on ice bath; further continues stirring to 24 hrs. Dark red colored precipitate was
formed, which was transferred to beaker for evaporation of solvent. The powder obtained further
washed with diethyl ether and dried in vacuum.
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2.3 Single crystal X-ray diffraction studies
X-Ray Crystallographic Data Collection and Refinement of the Structures for 2 and 6
Single X-ray crystal structure data of compounds 2 and 6 were collected on a Bruker D8
Venture PHOTON 100 CMOS diffractometer equipped with a INCOATEC micro-focus source
1ꢀs with graphite monochromatic MoKα radiation ( λ = 0.71073 Å) operating at 50 kV and
30mA. The program SAINT [21] was used for integration of diffraction profiles and absorption
correction was made with SADABS [22] program. Both the structures were solved by SIR 92
[23] and refined by full matrix least square method using SHELXL-2014 [24]) and WinGX
system, Ver 2013.3[25]. All the non-hydrogen atoms were located from the difference Fourier
map and refined anisotropically. All the hydrogen atoms were fixed by HFIX and placed in ideal
positions and included in the refinement process using riding model with isotropic thermal
parameters. All the crystallographic and structure refinement data of 2 and 6 are summarized in
Table 1. Selected bond lengths and angles are given in Table S16 to Table S21 (in ESI†). The
crystallographic information file is deposited with the CCDC numbers 1446868 for 2 and
1446870 for 6.
2.4 Cyclic Voltammetry
Electrochemical measurements were performed with CHI 6054E electrochemical
analyzer equipped with a commercial platinum disc electrode (CHI Instruments, USA, 2 mm
diameter), AgNO3 wire and platinum wire loop which serve as working, reference and counter
electrodes, respectively. After fixing the electrodes to the cell 0.513 g of tetra butyl ammonium
perchlorate (100 mM in 15 ml solution) was transferred to cell through high purity argon gas.
The blank or controlled voltammogram were acquired in tetra butyl ammonium perchlorate–
DMSO mixture prior to measurements. Sample dispersed in small amount of solvent injected in
cell for further measurement (analytic concentration 5 mg/15 ml). At the end of each set of
experiments the potentials were calibrated with respect to the normal hydrogen electrode (NHE)
using the ferrocene as internal standard.
2.5 DFT Studies
The exploration of the molecular properties of the studied inhibitor molecules have been
executed by the quantum chemical calculations. Herein, ORCA programme package, module
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version 2.7.0 has been employed for all quantum chemical calculations [26]. The hybrid B3LYP
functional level of DFT has been used for the geometry optimization of the studied molecules
[27-34] All-electron Gaussian basis sets were developed by the Ahlrichs group [35]. In this
calculation, triple-ζ quality basis sets, TZV (P), along with one set of polarization functions on
the N, O atoms have been employed [35]. Whereas, slightly smaller polarized split-valence SV
(P) basis sets were used for atoms like carbon and hydrogen. This basis set is of double-ζ quality
in the valance region and had a polarizing set of d functions on the non hydrogen atoms. In all
the calculations, self consistent field (SCF) were converged firmly with 10-8 Eh in energy, 10-7 Eh
in density change and 10-7 in maximum element of Direct Inversion of the Iterative Subspace
(DIIS) error vector. For the sake of accuracy the effects of solvent have been included in the
present study. Here, COSMO model was applied in order to incorporate the effect of solvent
(Acetonitrile) in this calculation. Canonical orbital's and density plots were obtained using
Molekel.
2.6 Anticancer activity
The anticancer study of the synthesized compounds (1Na to 8Na) as well as their oxidized
analogs (1 to 8) against HeLa cell line were evaluated by cytotoxicity method using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Hela cells (100 mL of a 103
cell/mL) were seeded in each well of 96-well culture plate, in RPMI 1640 supplemented with
10% FCS culture medium and incubated at 37 °C in a 5% CO2 incubator for 24 h. After
incubation, test compounds with serial concentration (0-200 µg/mL) were added to each well.
The cells were further incubated for 24 h, followed by addition MTT solution (5 mg/mL) to each
well. The absorbance of each well was read at 550 nm by a micro plate spectrophotometer.
3. Results and discussion
The synthesis of 2-(n-alkylamino)-3-methyl-1,4-naphthoquinone (1 to 8) have been
performed by using Vitamin K3 and corresponding primary amines at room temperature (26°C);
where n-alkyl is methyl; 1, ethyl; 2, propyl; 3, butyl; 4, pentyl; 5, hexyl; 6, heptyl; 7, octyl; 8.
The molecular structural study of 1 and 3 compounds has been confirmed by single crystal X-ray
diffraction analysis [17]. In this investigation one electron reduction of n-alkylamino derivatives
of Vitamin K3 (1Na to 8Na) have been synthesized. The mechanism for one electron reduction
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with sodium metal of n-alkylamino derivatives of Vitamin K3 is as shown in Scheme 2. The n-
alkylamino derivatives of Vitamin K3 are oxidized 1,4-dicarbonyl quinone (I).
Naphthosemiquinone radical is generated by one electron reduction from sodium metal. The
concentration of Na+ was determined by flame photometry (Table S22 in ESI†). The mole ratio
of Na+ was observed as 1:1 in 1Na to 6Na. Na+ was not detected in 7 and 8; probably no
reduction occurred in these compounds.
3.1 Single crystal X-ray diffraction studies
ORTEP diagrams for 2 and 6 were shown in Fig.1. The crystal data and hydrogen
bonding geometries are presented in Table 1 and Table 2 respectively. 2 crystallize in monoclinic
space group C2/c while 6 in P21. The carbonyl bond distances are similar to those observed for
n-alkylamino-1,4-naphthoquinones [36]. One of the carbonyl namely C(4)-O(2) is 0.02Å longer
than the other quinonoid carbonyl [37], because of intramolecular hydrogen bonding C-HꢁꢁꢁO
hydrogen bonding. 2 and 6 is in vicinity to four neighboring molecules via N-HꢁꢁꢁO, C-HꢁꢁꢁO and
C-Hꢁꢁꢁπ interaction (Fig.S1 in ESI †and Fig.S2 in ESI †).
6

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Table 1 Crystal data and structure refinement for 2 and 6.
Parameters
CCDC
2
6
1446868
1446870
Formula
Formula weight
Crystal system
Space group
a /Å
C26H26N2O4
430.49
Monoclinic
C2/c
32.938(5)
4.522(5)
14.844(5)
90.00
C17H21NO2
271.35
Monoclinic
P21
7.568(5)
4.720(5)
20.796(5)
90.00
b /Å
c /Å
α (°)
β (°)
94.650(5)
90
2204(3)
4
1.300
0.088
90.236(5)
90
742.9(9)
2
1.212
0.079
γ(°)
V(Å3)
Z
ρ(g cm-3)
ꢀ(mm-1)
F(000)
T(K)
912
298
292
292
λ(Mo Kα)(Å)
Θmin(°)
Θmax(°)
Total data
Unique data
Rint
0.71073
2.5
28.3
26710
2726
0.074
0.71073
2.7
28.4
31552
3661
0.147
Data[I>2σ(I)]
1416
2867
aR1
0.0666
0.1921
1.05
0.0625
0.1684
1.10
wR2
S
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Table 2 Hydrogen bond geometries for 2 and 6 molecules.
Com
2
D-HꢁꢁꢁA
D-H(Ǻ) HꢁꢁꢁA(Ǻ) DꢁꢁꢁA(Ǻ)
D-HꢁꢁꢁA(°)
136.3
128.8
138.2
113.5
109.9
120.0
133.7
131.4
141.5
113.5
109.9
N(11)-H(11)ꢁꢁꢁO(2) (i)
C(13)-H(13C)ꢁꢁꢁO(2) (i)
C(14)-H(14C)ꢁꢁꢁC(4) (ii)
N(11)-H(11)ꢁꢁꢁO(1) (intra)
0.859
0.959
0.960
0.859
2.198
2.649
2.823
2.115
2.242
2.558
2.628
2.654
2.811
2.119
2.249
2.881(3)
3.335(5)
3.595(5)
2.580(3)
2.723(4)
3.079(4)
3.339(4)
3.375(4)
3.612(5)
2.568(4)
2.732(4)
C(14)-(H14A)ꢁꢁꢁO(2) (intra) 0.960
N(11)-H(11)ꢁꢁꢁO(2)(iii)
C(5)-H(5)ꢁꢁꢁO(1)(iii)
0.860
0.930
0.970
0.960
0.860
6
C(13)-H(13B)ꢁꢁꢁO(2) (iii)
C(18)-(H18B)ꢁꢁꢁC(4) (iv)
N(11)-H(11)ꢁꢁꢁO(2) (intra)
C(18)-(H18A)ꢁꢁꢁO(2) (intra) 0.960
i)
x,-y,-1/2+z (ii) x,-1+y,z(iii) -1+x,y,z(iv) x,-1+y,z
i)
ii)
Fig.1. ORTEP plots of i) 2 and ii) 6. The ellipsoids were drawn with 50% of probability.
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Fig.2. Polymeric chain of 2 down b-axis (left). Butterfly like arrangement of 2 molecules down
c-axis (right), hydrogen were not shown for clarity.
Fig.3. Molecular packing of 6 down b-axis.
A beautiful butterfly like arrangement was observed to 2. The oxygen’s were the eyes of
the butterfly while there were two tentacles the C(3)-methyl and n-ethyl side chain (Fig.2
(right)). Polymeric chain can be seen down b-axis to 2 molecules formed via N(11)-H(11)ꢁꢁꢁO(2),
which was supported by C-HꢁꢁꢁO interaction of the –ethyl side chain (C(13)-H(13C)ꢁꢁꢁO(2))
(Fig.2 (left)). The neighboring polymeric chains were hold together by C-Hꢁꢁꢁπ interactions
(Fig.S3 in ESI†). Except unsubstituted derivative viz. 2-ethylamino-1,4-naphthoquinone (LH-2)
9

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[18, 38], all other analogs structures for example -Cl(L-2) [19, 39], –Br(L-2Br) [40] and –CH3(2)
substituted derivatives showed one dimensional polymeric chain formed by N-HꢁꢁꢁO
intermolecular hydrogen bonding interaction. LH-2 formed a dimer by N-HꢁꢁꢁO and C-HꢁꢁꢁO
interactions [26]. The torsion angle C(2)-N(11)-C(12)-C(13) differs in other derivatives, it was
observed as 90.4(4)° in 2, -176.1(1)° in LH-2, -78.4(9)° in L-2 and -82.4(6)° in L-2Br.
Moreover the bond angle C(2)-N(11)-C(1ff2) observed in 2 is 132.4(2)°, which is similar to L-
2(131.6(5)°) and L-2Br(132.1(4)°), while it was reduced to 122° in LH-2, that could differed its
molecular interactions.
The molecules of 6 formed a polymeric chain via N(11)-H(11)ꢁꢁꢁO(2) and C(5)-
H(5)ꢁꢁꢁO(1) and are also stabilized by C(13)-H(13B)ꢁꢁꢁO(2) of the side chain alkyl group(Fig.3).
The orientation of n-alkyl groups present on the same side of polymeric chain and the
neighboring chains related to each other by two fold symmetry.
3.2 EPR studies
X-band polycrystalline powder EPR spectra of 1 to 6 showed sharp signals near ~g = 2
(Fig.S4 to Fig.S15 in ESI†, Table 3) and a weak signal near g ~ 4.2 (Fig.4 for compound 4),
which confirms the presence of radical in these compounds.
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Table 3 Gyromagnetic ratios (g) of 1 to 6.
Comp.
code
giso (133K)
Half field Check
Solid state (133K)
Solid state
Liquid state
2.0026
2.0020
2.0026
2.0029
2.0023
2.0029
2.0029
2.0029
2.0023
2.0026
2.0020
2.0026
4.25
4.24
4.21
4.22
4.25
-
1
2
3
4
5
6
Half Field Check (2000/2000)
Fig.4. Polycrystalline powder, X-band EPR spectra of 4 at 133 K.
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3.3 Electrochemical study
Table 4 Cyclic Voltammetry data of 1 to 8 at 0.1 V/s in DMSO solvent.
Comp Conc. Potential (E) Vs NHE (V)
1/2 (I) EPc(II) EPa(II)
Code
(mM)
EPc(I)
-0.789
-0.821
-0.784
-0.682
-0.792
-0.691
-0.693
-0.709
EPa(I)
E
E1/2 (II)
∆Ep(I)
∆Ep(II)
-1.067 0.182
1.65
1.55
1.45
1.37
1.29
1.23
1.16
1.11
-0.628 0.160
-0.629 0.192
-0.657 0.127
-0.611 0.071
-0.649 0.143
-0.617 0.073
-0.630 0.063
-0.708
-0.724
-0.720
-0.646
-0.720
-0.654
-0.664
-0.672
-1.249
-1.344
-1.324
-1.324
-1.331
-1.339
-1.329
-
-1.158
-1.241
-1.241
-1.257
-1.252
-1.266
-1.261
-
1
2
3
4
5
6
7
8
-1.137 0.208
-1.158 0.166
-1.190 0.136
-1.173 0.158
-1.194 0.151
-1.195 0.134
-0.635
0.074
-
-
O
O
H
O
H
N
N
H
R -0.65 to -0.72 V
1e-
N
R -1.15 to -1.26 V
1e-
R
O
I
O
II
O
III
Catechol
Quinone form
Semiquinone
Scheme 3.
Various redox forms of 2-(n-alkylamino)-3-methyl-1,4-naphthoquinone, I-Quinone form, II-
Semiquinone, III-Catechol.
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1.5x10-5
1.0x10-5
4
3
2
1
IIc
Ic
5.0x10-6
0.0
IIa
-5.0x10-6
Ia
-1.5 -1.0 -0.5 0.0 0.5 1.0
E(V) Vs NHE
1.5x10-5
1.0x10-5
8
7
6
5
IIc
Ic
5.0x10-6
0.0
IIa
-5.0x10-6
Ia
-1.5 -1.0 -0.5 0.0 0.5 1.0
E(V) Vs NHE
Fig.5. a) Cyclic voltammogram of 1 to 4 at 0.1 V/s and b) Cyclic voltammogram of 5 to 8 at 0.1
V/s in DMSO solvent with tetrabutyl ammonium perchlorate as an electrolyte.
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Fig.5 showed overlay cyclic voltammogram of 1 to 8, that are recorded at 0.1 Vs−1 scan
rate in DMSO containing 0.1M tetra butyl ammonium perchlorate (TBAP) as supporting
electrolyte and the electrochemical data is presented in Table 4. Ferrocene was used as internal
standard and the reported potential were corrected to normal hydrogen electrode (NHE). The
exhaustive figures and data of all compounds is presented in Fig S16 to Fig.S31 in and Table S1
to S15 in ESI†. A reversible couple (peak Ic and Ia in Fig.5) observed ∼E1/2 = −0.69 ± 0.4 V was
assigned to one electron reduction of naphthoquinone to naphthosemiquinone in 1 to 8. ꢂEp of 1
to 8 was observed to be ∼61–192 mV and the ratios for ipa/ipc have been plotted. The values for
ipc and ipa were found to be closer to unity for peak Ic and Ia for all compounds. This reveals
that the one electron reduction is a favorable process in 1 to 8 (Scheme 3). An irreversible
cathodic peak, IIc (∼−1.2 V) was observed varying reduction potentials in all compounds
studied, it was assigned to one electron reduction of naphthosemiquinone to catechol. Weak
anodic peak is observed in positive potential region in 4 to 6 that was assigned oxidation of
reduced species formed during redox cycling. There was no trend followed i.e. either increase or
decrease in the redox potential in the homologues series, 1 to 8 and higher. Cyclic Voltammetry
studies revealed that all the aminonaphthoquinone derivatives can be reduced by one electron to
form naphthosemiquinone radical species in solution.
3.4 DFT Studies
Electron donor-accepter (D-A) ability is mainly depends on the frontier molecular
orbitals (FMO’s) of the molecules. The two main FMO’s are highest occupied molecular orbital
(HOMO) and lowest unoccupied molecular orbital (LUMO). On the basis of FMO theory,
HOMO and LUMO of the reacting species are mainly associated with the transition of electrons.
HOMO is the orbital from where molecules donate its electron i.e; oxidation whereas, LUMO is
the orbital where back donating electrons are accommodates i.e.; reduction. Thus, those
molecules which have lower EHOMO and ELUMO values easily oxidized and reduced from their
neutral state.
The obtained optimized geometric structures, electron density distribution in the HOMO
and LUMO orbitals for 1, 2, 3 and 6 in neutral state are depicted in Fig.6 and their FMO’s
energies are tabulated in Table 5. It is seen from the DFT obtained geometry optimized structure
of the neutral form of 2 and 6 molecules that naphthalene-1,4-dione ring is planar in nature,
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whereas aliphatic ring connected to it have non-planar orientation. These obtained findings have
to correlate with the crystal structure of 2 and 6 molecules. It is clearly seen from ORTEP plots
of 2 and 6 (vide Fig. 1) that naphthalene-1,4-dione ring is planar in nature and aliphatic chain
connected to it have non-planar orientation. Thus, DFT optimized geometric structures in
agreement with the structure obtained from single crystal XRD. It is seen that theoretically
obtained bond distances (Table S23 and Table S24 in ESI†) match with the bond distances
obtained from the crystal structure (Table S16 and Table S19 in ESI†). Thus, DFT optimized
geometric structures corroborate well with XRD analysis.
Now, if we analyzed electron density distribution it is seen that HOMO electron density
is mainly distributed on the quinone ring as well as nitrogen atom connecting both quinone ring
and the aliphatic chain of varying length for 1, 2, 3, 6. Whereas, LUMO electron density is
mainly distributed on the naphthalene-1,4-dione ring and N atom connected to it. Thus, it is
concluded that by these sections of the molecules electron donation and acceptance are mainly
occurred. From Table 5, it is also seen that all the molecules have almost similar ELUMO values.
Thus it is quite impossible to judge which one reduced easily and which are not.
From the redox study it is seen that one and two electron reduction of 1, 2, 3, 6 molecules
has been very prominent. Therefore it is quite essential to analyze electron density distribution in
the HOMO and LUMO of one and two electron reduced species of the molecules. In this regard,
we have also carried out DFT calculation for one reduced and two electron reduced species of 1,
2, 3, 6 molecules. The optimized geometric structure along with HOMO and LUMO plots of the
one and two reduced form of 1, 2, 3, 6 species are shown in Fig.7 and Fig.8. It is seen that,
electron density distribution in HOMO and LUMO of one electron reduced species are quite
different than that of the neutral species. HOMO electron density is mainly distributed over the
quinone ring and LUMO electron density distributed over naphthalene-1,4-dione ring. It is
further confirmed from spin density plot of 1, 2, 3, 6 molecules that one unpaired electron is
mainly localized on quinone ring of the molecules. In minute observation, it is also seen from
spin density plot that two oxygen atom in quinone ring have appreciable localized electron
density whereas in neutral form there is no such electron clouds available. Thus, C-O bond
distances in one electron reduced form should be elongated than that of the neutral form of the
molecule. The C-O bond distance of 3 ligand in neutral form is 1.2283 Å and 1.2434 Å
respectively, whereas in one electron reduced form bond distances changes to 1.2781 Å and
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1.2733 Å respectively. Thus, it can be concluded that one electron reduced species is formed and
mainly localized on the quinone ring of the molecule. It is also seen from Table 5 that after one
electron reduction EHOMO and ELUMO values are also changes from their neutral state and among
the four theoretical studied molecules 3 has the lowest ELUMO value.
Two electron reduced form of 1, 2, 3, 6 molecule generates (1−··, 2−··, 3−·· and 6−··)
species. The optimized geometric structure along with HOMO and LUMO plots of the two
electron reduced form of 1, 2, 3, 6 species are shown in Fig.8. From the figure it is seen that
electron density distribution in the HOMO and
LUMO are mainly distributed over the whole molecular skeleton of naphthalene-1,4-
dione ring. It is further more confirmed form the spin density plot that 2nd unpaired electron is
distributed over the naphthalene-1,4-dione ring of the molecule. Therefore, form the DFT
analysis of neutral, one electron reduced and two electron reduced form of the molecules it is
concluded that oxidation-reduction mainly occurred from the aromatic section of the molecules.
n-alkyl chain connected to the aromatic ring does not participated in oxidation and reduction
process.
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1
2
HOMO
LUMO
HOMO
LUMO
3
6
HOMO
LUMO
HOMO
LUMO
Fig.6 Optimized geometries, HOMO, LUMO of neutral form of 1, 2, 3 and 6.
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HOMO
LUMO
Spin density
1
2
3
6
Fig.7. Optimized geometries, HOMO, LUMO and spin density of one electron reduced form of
1, 2, 3 and 6.
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Spin density
HOMO
2
LUMO
2
3
6
Fig.8. Optimized geometries, HOMO, LUMO and spin density of two electron reduced form of
1, 2, 3 and 6.
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Table 5 Calculated quantum chemical parameters of the studied inhibitors in neutral form one
reduction form and two reduction form.
Inhibitors EHOMO
(eV)
ELUMO
(eV)
One electron
reduction
ELUMO
(eV)
Two electron
reduction
ELUMO
(eV)
EHOMO (eV)
EHOMO (eV)
-5.9100
-3.0176
-3.0185
-3.0257
-3.0459
-3.7933
-0.5519
-0.5336
-0.5539
-0.5308
-1.1928
0.9882
1.0373
0.9951
1.0435
1
2
3
6
-5.9197
-5.8932
-5.9598
-3.7623
-3.7994
-3.7569
-1.1669
-1.1900
-1.1640
3.5 Anticancer activity
The anticancer potentials of 1 to 8 and 1Na to 8Na series of compounds have been
evaluated by rapid colorimetric assay. The anticancer study is performed by using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
a
yellow water-soluble
tetrazolium salt. MTT is reduced in the mitochondria of metabolically active cells by succinate
dehydrogenase to yield a water insoluble purple formazan crystal, which can be
spectrophotometrically measured upon solubilization. When the HeLa cells were incubated with
different concentrations of 1 to 8 and 1Na to 8Na series of compounds and incubated for 24 hrs,
it was found that the percentage of live cells decreased. In presence of 1 to 8 series of
compounds, the viability of HeLa cells were unaltered till 100 µg/mL for all except 1 (Fig.9). In
presence of 1, the viability of cells decreased linearly with increase in concentrations and it
decreased to 50 % at 200 µg/mL (Fig.9). Thus, it seems that amongst all 1 to 8 series
compounds, 1 show relatively better anticancer activity.
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1 to 8 series
100
80
60
40
20
0
0
25
50
100
LM-1 LM-2 LM-3 LM-4 LM-5 LM-6 LM-7 LM-8
1
2
3
4
5
6
7
8
Compound (µg/mL
)
Fig.9. MTT assay plots of 1 to 8 against HeLa cell line.
1Na to 8Na Series
100
80
60
40
20
0
0
50
100
200
1Na 2Na
3Na 4Na
5Na 6Na 7Na 8Na
Compound (µg/mL)
Fig.10. MTT assay plots of 1Na to 8Na against HeLa cell line.
Similarly, in presence of 1 to 8 series of compounds, all compounds showed an anticancer
activity and 1 showed better anticancer activity over others member of the series (Fig.9 and
Fig.10). It seems that 1 and its sodium derivative 1Na are showing a better anticancer activity
over other members and its respective sodium derivatives. Anticancer activity in quinones is
proposed to occur via their radical formation ability. However this not the prime reason to the
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series of compounds studied in present investigation. The anticancer activity would also depend
on how these molecules interact with biomolecules. Small molecular frame work and π-π
stacking ability are the probable reasons for better anticancer activity of 1 and 1Na.
4. Conclusions
Molecular structures and molecular interactions of ethyl (2) and hexyl (6) derivatives 2-(n-
alkylamino)-3-methyl-1,4-naphthoquinone (1 to 8) have been described. One electron reduced
forms 1Na to 8Na has been synthesized chemically using sodium metal as reducing agent.
Radical signatures of the reduced compounds are confirmed by X-band EPR spectroscopy. The
redox potentials of oxidized form of homologated series compound 1 to 8 are determined from
cyclic voltammetry studies in DMSO. There is no trend was observed (increase or decrease) in
redox potential with increase in n-alkyl chain of the homologated series 1 to 8. The neutral, one
electron reduced (naphthosemiquinone) and two electron reduced form (catechol) of 1 to 8 were
optimized by DFT studies and it is concluded that oxidation-reduction mainly occurred from the
quinonoid ring of the compounds, n-alkyl chain connected to the aromatic ring does not
influence the oxidation and reduction process. Anticancer activity of oxidized (1 to 8) and one
electron reduced form (1Na to 8Na) compounds have been evaluated on breast cancer (HeLa)
cell line, all compounds show anticancer activity. 1 and 1Na showed promising anticancer
activity among all.
Acknowledgements
SSG grateful to RGYI scheme of Department of Biotechnology, New Delhi, India for Ref. No.
(BT/PR6565/GBD/27/456/2012). Thanks to Dr. Bharat Ugale and Dr. C. M. Nagaraja ,
Department of Chemistry, Indian Institute of Technology, Ropar, Rupanagar-140001, Punjab,
India for SXRD data.
†Electronic Supplementary Information (ESI)
Single crystal X-ray structure figures Fig.S1 to Fig.S3. Figures of EPR spectra from Fig.S4
through Fig.S15. Figures of Cyclic Voltammogram from Fig.S16 through Fig.S31.
Electrochemical data from Table S1 through Table S15. Crystallography tables Table S16
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through Table S21. Flame photometry data Table S22. Bond distances from DFT Table S23 and
Table S24. Crystallographic data have been deposited with the Cambridge Crystallographic Data
Centre and may be obtained on request quoting the deposition number CCDC numbers 1446868
for 2, 1446869 for 5 and 1446870 for 6, from the CCDC, 12 Union Road, Cambridge CB21EZ,
UK (Fax: +44 1223 336 033; E-mail address: deposit@ccdc. cam.ac.uk).
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•
•
•
•
•
Reduced form of aminonaphthoquinones of vitamin K3 synthesized chemically
Radical signatures are confirmed by EPR studies
Redox behavior is studied by cyclic voltammetry
Compounds were optimized to three different redox forms by DFT
Anticancer activity evaluated for oxidized and reduced form of the compounds