SUPPLEMENTARY MATERIAL
Two new phenolic compounds from Ficus rumphii and their antiproliferative activity
Mehtab Parveena*a, Ali Mohammed Mallaa, Mahboob Alama, Faheem Ahmada, P. S. Pereira
Silvab and Manuela Ramos Silvab
a
Division of Natural Products, Department of Chemistry, Aligarh Muslim University, Aligarh,
202002, India.
b
CEMDRX Physics Department, University of Coimbra, 3004–516 Coimbra, Portugal.
Abstract
Two new compounds (2) and (4), along with two known compounds (1) and (3) were isolated for
the first time from 95% ethanolic extract of the leaves of Ficus rumphii. Their structures were
elucidated on the basis of chemical and physical evidences (Elemental analysis, UV, IR, 1H
NMR,
13
C NMR and Mass spectra) and comparison with literature compounds. Structural
authentication of compound (4) was further validated by single crystal x-ray analysis and DFT
calculations. The compounds (1-4) were screened for in vitro cytotoxicity against cancer and
non-cancer cells and also tested for genotoxicity (comet assay). Compound (2) and (4) displayed
significant activity against HL-60 with IC50 values of 3.3 µM and 2.3 µM respectively. The
results revealed that compound (4) has better prospectus to act as cancer chemotherapeutic
candidate which warrants further in vivo anticancer investigations.
Keywords: Ficus rumphii; Phenolic compounds; X- ray study; Anticancer activity
* Corresponding author E-mail address: mehtab.organic2009@gmail.com (Mehtab Parveen)
1
Experimental
1. Stigmast-5-en-3-yl acetate (1):
Petroleum ether-benzene (1:1) fraction yielded compound (1). Crystallization from hexane-ether
gave white crystals (160 mg), m.p. 117-118 oC [lit. mp 118–119 oC] (McCarthy et al., 2005).
Anal. Calc. for C31H52O2; C, 81.52; H, 11.48; found: C, 81.54; H, 11.47. IR (KBr) ν cm-1: 1730
(acetate carbonyl), 1460 (C═C), 1250 (C–O). 1H NMR (400 MHz, DMSO, δ, ppm): 0.67-2.32
(m, 47H), 2.03 (s, 3H, CH3CO), 4.57-4.63 (m, 1H, 3α-H), 5.37-5.38 (d, 1H, H-6, j= 4.5 Hz).
13
C
NMR (100 MHz, DMSO, δ, ppm): 11.98 (CH3), 15.43 (CH3), 17.57 (CH3), 18.25 (CH3), 18.77
(CH3), 19.31 (CH3), 19.83 (CH3), 20.53, 21.46, 23.03, 24.28, 26.0, 27.76, 28.24, 29.10 (CH2- C),
30.25, 30.54, 31.43, 31.88, 32.41 (CH), 33.91, 36.15 (CH2- C), 36.57 (quaternary C), 39.70
(CH2), 42.29 (quaternary C), 45.80, 49.99, 56.05, 56.66, 73.98 (CH), 122.65 (═CH), 139.63 (C5), 170.57 (acetate carbonyl). ESI-MS (m /z): 456.407 [M+H] +• (C31H52O2).
2. 3-acetyl-2H-chromen-2-one (3):
Benzene-ethylacetate (6:4) fraction furnished compound (3). Crystallization from chloformmethanol gave yellowish white solid (85 mg), m.p 121oC [lit. 121-123] (Wang, Guan, & He,
2011). Anal. Calc. for C11H8O3; C, 70.21; H, 4.29; found: C, 70.23; H, 4.28. IR (KBr) ν cm-1:
1737 (lactone carbonyl), 1681 (CH3CO), 1610 (α, β- C═C
γ-pyrone),
1554, 1451 (C═C). 1H NMR
(400 MHz, DMSO, δ, ppm): 2.73 (s, 3H, CH3), 7.27-7.39 (m, 2H, aromatic), 7.64-7.68 (m, 2H,
aromatic), 8.52 (s, 1H, H-4).
13
C NMR (100 MHz, DMSO, δ, ppm): 30.63, 116.70, 118.23,
124.46, 125.01, 130.25, 134.44, 147.55, 155.31, 159.27, 195.55. ESI-MS (m /z): 188.053
[M+H]+•.
3. Crystal structure determination
A crystal of (4) with a block habit and having approximate dimensions of 0.56 mm  0.44 mm 
0.20 mm was glued on a glass fibre and mounted on a Bruker Apex II diffractometer. The
diffraction data was collected at room temperature 293(2) K using graphite monochromated Mo
Kα (λ= 0.71073 Å). Data reduction was performed with
APEX II (Bruker APEX2 & SAINT, 2003). Lorentz and polarization corrections were
applied. Absorption correction was applied using SADABS (Sheldrick, 2003). The
crystallographic structure was solved by direct methods (SHELXS-97) (Bruker APEX2 &
2
SAINT, 2003). Refinements were carried out with SHELXL-97 package (Sheldrick, 2008). All
refinements were made by full-matrix least-squares on F2, with anisotropic displacement
parameters for all non-hydrogen atoms. All the hydrogen atoms were located in a difference
Fourier synthesis, placed at calculated positions and then, included in the structure factor
calculation in a riding model using SHELXL-97 defaults. The final least-squares cycle was based
on 1376 observed reflections [I>2σ(I)], 154 variable parameters, converged with R = 0.0340 and
wR = 0.0976. Additional information to the structure determination is given in (Table S1).
Hydrogen bonding geometric data is shown in (Table S4) and selected structural parameters
have been shown (Table S5). Although the molecule is achiral, the structure crystallized in a
chiral space group but, because of the weak anomalous scattering at the Mo wavelength, the
absolute structure could not be determined from the X-ray data.
4. DFT calculations
The geometry optimization was performed using the PC GAMESS/Firefly QC package
(Granovsky, GAMESS/Firefly, 2009), which is partially based on the GAMESS (US) source
code (Schmidt et al., 1993), starting from the experimental X-ray geometry. The calculation was
performed within density functional theory (DFT) using B3LYP (Becke three–parameter Lee–
Yang–Parr) for exchange and correlation, which combines the hybrid exchange functional of
Becke (Becke, 1988; Becke, 1993) with the correlation functional of Lee, Yang and Parr (Lee,
Yang, & Parr, 1988). The calculation was performed with an extended 6–31G (d,p) basis set.
Tight conditions for convergence of both the self-consistent field cycles and the maximum
density and energy gradient variations were imposed (10–5 atomic units). At the end of this
geometry optimization we conducted a Hessian calculation to guarantee that the final structure
corresponds to a true minimum, using the same level of theory as in the geometry optimization.
5. Cytotoxicity assay (MTT)
Cells and cell culture conditions
The newly isolated compounds (1-4) were evaluated for anticancer activity towards a panel of
four cancer cell lines: A549 (Human lung carcinoma cells), HepG2 (Human hepatic carcinoma
cells), HL-60 (Human leukemia cells), MDA-MB-361 (Ductal breast carcinoma cells) and two
non-cancerous cell lines: HEK- 293 and MCF-10A. Human cancer cell lines were procured from
3
National Cancer Institute, Frederick, U.S.A. The cells lines HL-60, A549 and MDA-MB-361
were grown in RPMI (Roswell Park Memorial Institute medium) medium whereas HepG2 cell
lines were maintained in EMEM (Eagle's minimal essential medium) medium.
6. MTT Assay
In vitro evaluation of anticancer activity of the isolated compounds (1-4) was carried out using 3(4, 5-dimethylthiozol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The complete
growth mediums were supplemented with 10% (v/v) heat- inactivated fetal bovine serum,
penicillin (100 units/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), pyruvic acid, 37%
NaHCO3 and 2-mercaptoethanol (50 µM) at 37 oC in an atmosphere of 95% humidified air
containing 5% CO2 .Cells were cultured in culture medium in a 75 cm2 tissue culture flask (BD
Falcon). Cultivation and experimental incubation were performed in a cell incubator at 37°C,
90% air humidity and 5% CO2. Cells grown in logarithmic phase were treated with tested
botanical dissolved in DMSO while the untreated control cultures received only the vehicle
(DMSO, < 0.5%v/v).The growth inhibitory effect of PS-20 compound towards various cells
lines was measured by means of MTT (3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay .The cleavage and conversion of the soluble yellowish MTT to the insoluble
purple formazan by active mitochondrial dehydrogenase of living cells has been used to develop
an assay system for measurement of cell proliferation. Exponentially growing cells were
harvested and plated in 96 wells cultures were treated with increasing concentrations (0-15 µM)
of compounds (1-4) for 48 and 24 hours. 20 µl of MTT solution (2.5 mg/ml) was added to each
well and incubated at 37 ºC for 3 hours before the termination of assay. The plates were
centrifuged and the supernatant was discarded while the MTT- formazan crystals were dissolved
in 100µL of DMSO. The OD at 570 nm with reference wavelength of 620 nm was recorded with
a microplate reader (Bio-Rad Instruments).
7. Comet assay (single cell gel electrophoresis)
To assess the genotoxic effect of the isolated compounds (1-4), comet assay was performed in
HL-60 cells. HL-60 (1×106) cells were treated with three different concentrations 10, 25 and
50µg/mL of isolated compounds (1-4) for 24 h. The cells were then washed and 200 µL of cell
suspension in low melting agarose (LMA) was layered on to the labelled slides precoated with
4
agarose (1.5%). The slides were placed on ice for 10 min and submerged in lysis buffer (2.5%
NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO and 1% Troton X-100) at 4oC at pH 10 for
more than 1 h. The slides were then equilibrated in alkaline buffer (30 mM NaOH, 1mM EDTA)
at 4oC at pH 13, electrophoresed at 0.86 V/cm at 4oC, neutralized, washed and dried. At the time
of capturing the images, the slides were stained with ethidium bromide (EtBr, 150 µL 1X) and
cover slips were placed over them. For visualization of DNA-damage EtBr-stained slides were
observed under 209 objectives of a fluorescent microscope (Olympus BX-51, Japan). The images
of 50–100 randomly selected cells were captured per slide using a CCD camera.
5
Table captions:
Table S1.
Crystallographic data and structure refinement of compound (4)
Table S2.
Anticancer activity of isolated compounds (1-4)
Table S3.
GI50 values shown by isolated compound (1-4) and doxorubicin against noncancerous cell lines
Table S4.
H-bond geometry (Å,º) in compopund (4)
Table S5.
Comparison of selected geometrical parameters for compound (4) as determined
by X-ray diffraction and from DFT calculation (Å,°)
6
Table S1. Crystallographic data and structure refinement of compound (4)
Empirical formula
Formula weight
C14 H19 NO2
Temperature (K)
233.30
293(2)
Wavelength (Å)
0.71073
Crystal system
Orthorhombic
Space group
P212121
a (Å)
7.5046(2)
b (Å)
11.6821(3)
c (Å)
14.0434(4)
α (deg.)
β (deg.)
γ (deg.)
Volume (Å3)
Z
Calculated density (g/cm3)
Absorption coefficient (mm–1)
F(000)
90
90
90
1231.18(6)
4
1.259
0.084
Crystal size (mm)
504
0.56  0.44  0.20
ө range for data collection (deg.)
3.08 –25.76
Index ranges
–9<h<9, –14<k<14, –17<l<17
Reflections collected/unique
23422 /1376 [R(int) = 0.0313]
Completeness to ө= 25.00º
Refinement method
99.8 %
Full–matrix least–squares on F2
Data/restraints/parameters
1376/0/154
Goodness–of–fit on F2
Final R indices [I> 2σ(I)]
1.054
R1 =0.0340 wR2 = 0.0976
R indices (all data)
R1 = 0.0390 wR2 = 0.1018
Largest diff. peak and hole (e Å – 3)
0.146 and –0.174
7
Table S2. Anticancer activity of isolated compounds (1-4)
IC50 (µM)a
Compounds
Cancer cell lines
A549
HePG2
HL-60
MDA-MB-361
1
9.3
˃10
LA
9.8
2
3.9
2.5
3.3
2.4
3
7.2
4.2
5.8
7.5
4
3.7
2.4
2.3
1.9
Doxorubicin
3.61
2.17
3.13
2.03
(Standard)
aIC
50
= Concentration of the compound that inhibits 50% of cell growth.
IC50 values are mean of three experiments in replicate; LA = Less active
Table S3. GI50 values shown by isolated compound (1-4) and doxorubicin against non-cancerous
cell lines
Compounds
GI50 (µM)a
Non-cancerous cell lines
HEK-293
MCF10A
1
72.3
77.5
2
95.7
˃100
3
˃100
98.5
4
85.6
78.9
67.3
81.5
Doxorubicin
aGI
50 = Molar concentration causing 50% growth inhibition of non-cancerous cells.
Values are the mean of triplicates of at least two independent experiments
8
Table S4. H-bond geometry (Å,º) in compopund (4)
D-H
H...A
D...A
D-H...A
O1—H1...O2i
0.82
1.92
2.7391(17)
179.3
C14—H14B...O2 (intra)
0.97
2.33
2.746(3)
105.2
(symmetry code i: -x, y+1/2, -z+1/2)
Table S5. Comparison of selected geometrical parameters for (4) as determined by X-Ray
diffraction and from DFT calculation (Å,°)
Experimental
DFT
O1-C1
1.363(2)
1.370
O2-C9
1.233(2)
1.229
N1-C9
1.337(2)
1.376
N1-C10
1.470(3)
1.461
N1-C14
1.469(2)
1.465
C6-C7
1.514(2)
1.515
C7-C8
1.517(2)
1.528
C8-C9
1.519(2)
1.533
C9-N1-C14
120.94(16)
119.29
O2-C9-N1
121.57(15)
121.62
C1-C6-C7
118.27(15)
118.28
C6-C7-C8-C9
177.32(17)
179.87
C7-C8-C9-N1
174.89(19)
179.20
C9-N1-C10-C11
125.2(2)
126.18
C9-N1-C14-C13
-124.4(2)
-125.92
9
Figure captions:
Figure S1 (a) X-ray structure of compound 4.
Figure S1 (b) Packing diagram for 4 viewed along a axis. Hydrogen bonds are shown as dashed
lines (Mercury, version 3.1) (Macrae et al., 2006).
Figure S2 Detection of DNA damage in HL-60 cells. Treated cells (24 h) were coated over
agarose gel, lysed, electrophoresed in alkaline buffer and stained with propidium iodide. Control
cells were treated with DMSO alone. The DNA fragmentation resulting in a comet-like
appearance in cells treated with doxorubicin and compounds (1-4).
Figure S3 Graph comparing the effect of isolated natural compounds on the tail length in comet
assay. There was a concentration-dependent increase in the apoptotic DNA fragmentation and
hence an increase in tail length in all the compounds except compound 1. The extent of damage
caused by doxorubicin (0.1 µM) was more when compared to the isolated compounds (1-4).
Compound 4 caused maximum DNA damage in the comet assay.
Figure S4 Comparison of the molecular conformation of 4, as established from the X-ray study
(red) with the optimized geometry (blue). Software used for visualization: VMD, version 1.9.1
(Humphrey, Dalke, & Schulten (1996).
10
Figure S1 (a)
Figure S1 (b)
11
Doxorubicin (0.1μM)
Control (Untreated)
Comp. 1 (50µg/mL)
Comp. 2 (50µg/mL)
Comp. 3 (50µg/mL)
Comp. 4 (50µg/mL)
Figure S2
12
Control ═ DMSO treated cells
Dox ═ Doxorubicin (Standard)
4 ═ Blue (10μg/mL), Red (25µg/mL),
Green (50μg/mL)
2 ═ Blue (10μg/mL), Red (25µg/mL),
Green (50μg/mL)
3 ═ Blue (10μg/mL), Red (25µg/mL),
Green (50μg/mL)
1 ═ Blue (10μg/mL), Red (25µg/mL),
Green (50μg/mL)
Figure S3
Figure S4
13
1
H-NMR spectrum of compound 2 (in DMSO-d6)
14
1
H-NMR spectrum of compound 4 (in DMSO-d6)
15
13
C-NMR spectrum of compound 2 (in DMSO-d6)
16
13
C-NMR spectrum of compound 4 (in DMSO-d6)
17
References
Becke, A.D. (1988).
Density-fnnctional exchange-energy approximation with correct asymptotic
behaviour. Physical Review A, 38, 3098-3100.
Becke, A.D. (1993) Density-functional thermochemistry. III. The role of exact exchange. Journal
Chemical Physics, 98, 5648-5652.
Bruker APEX2 and SAINT (2003). Bruker AXS Inc., Madison, Wisconsin, USA,
Granovsky A.A., GAMESS/Firefly, P.C. (2009). Version 7.1.G.wwwhttp://classic.chem.msu.su/
gran/gamess/index.html.
Lee, C., Yang, W., Parr, R.G. (1988). Development of the Colic-Salvetti correlation-energy formula into a
functional of the electron density. Physical Review B, 37, 785-789.
McCarthy, F.O., Chopra, J., Ford, A., Hogan, S.A., Kerry, J.P., O’Brien, N.M., Ryan, E., Maguirea, A.R.
(2005). Synthesis, isolation and characterization of β-sitosterol and β-sitosterol oxide derivatives.
Organic & Biomolecular Chemistry, 3, 3059-3065.
Schmidt, M.W., Baldrige, K.K., Boatz, J.A., Elbert, S.T., Gordon, M.S., Jensen, J.J., Koseki, S.,
Matsunaga, N., Nguyen, K.A., Sue, S., Windus T.L., Dupuis, M., Montgomery, J.A. (1993).
General atomic and molecular electronic structure system. Journal Computational Chemistry,
14, 1347-1363.
Sheldrick G.M. (2008). A short history of SHELX. Acta Crystallographica Section A, 64, 112-122.
Sheldrick G.M., (2003). SADABS, University of Göttingen, Germany.
Wang, C.H., Guan, Z., He, Y.H. (2011). Biocatalytic domino reaction: synthesis of 2H-1-benzopyran-2one derivatives using alkaline protease from Bacillus licheniformis. Green Chemistry, 13, 20482054.
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