New alkylated benzoquinone from Iris nepalensis

Article abstract of DOI:10.1080/14786419.2012.763124

Chemical investigation of chloroform extract of rhizomes of Iris nepalensis yielded new alkylated 1,4-benzoquinone derivative (1). The structure of Compound 1 was established by analysis of spectroscopic data. Compound 1 was evaluated for cytotoxic activi

Full text of DOI:10.1080/14786419.2012.763124

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New alkylated benzoquinone from Iris
nepalensis
Mudasir A. Tantryab, Khalid Ghazanfarb & Ummer R. Zargarc
a National Center for Natural Products Research, Research
Institute of Pharmaceutical Sciences, School of Pharmacy, The
University of Mississippi, MS38677, USA
b Drug Standardisation Research Unit, Regional Research Institute
of Unani Medicine, NaseemBagh, Srinagar, 190006, KashmirIndia
c Centre of Research for Development, University of Kashmir,
Srinagar, 190006, KashmirIndia
Published online: 31 Jan 2013.
To cite this article: Mudasir A. Tantry, Khalid Ghazanfar & Ummer R. Zargar (2013) New alkylated
benzoquinone from Iris nepalensis, Natural Product Research: Formerly Natural Product Letters,
27:20, 1832-1836, DOI: 10.1080/14786419.2012.763124
To link to this article: http://dx.doi.org/10.1080/14786419.2012.763124
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Natural Product Research, 2013
Vol. 27, No. 20, 1832–1836, http://dx.doi.org/10.1080/14786419.2012.763124
New alkylated benzoquinone from Iris nepalensis
Mudasir A. Tantrya,b*, Khalid Ghazanfarb and Ummer R. Zargarc
aNational Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of
Pharmacy, The University of Mississippi, MS 38677, USA; bDrug Standardisation Research Unit, Regional
Research Institute of Unani Medicine, NaseemBagh, Srinagar 190006, Kashmir, India; Centre of
c
Research for Development, University of Kashmir, Srinagar 190006, Kashmir, India
(Received 9 May 2012; final version received 21 November 2012)
Chemical investigation of chloroform extract of rhizomes of Iris nepalensis yielded
new alkylated 1,4-benzoquinone derivative (1). The structure of Compound 1 was
established by analysis of spectroscopic data. Compound 1 was evaluated for cytotoxic
activities against human cancer cell lines A549, HL-60, HCT116 and ZR-75.
Compound 1 showed least cytotoxicity against HL-60, HCT116 and ZR-75-30.
Keywords: Iris nepalensis; alkylated 1,4-benzoquinone; cytotoxic activity
1. Introduction
The genus Iris belongs to the family Iridaceae, comprising over 300 species, 16 of which are found
in western Himalaya. Iris nepalensis (Himalayan iris) is a fully hardy perennial herbaceous bulb
which blooms with white, blue and purple flowers in late spring and early summer, and it takes ,1
year to flower. It grows well in semi-shade and direct sun, and prefers medium levels of water. The
flowers are butterfly shaped. It looks best in spring and summer. (The Wealth of India 1959; Ali &
Mathew 1993). Iridaceae family has been used to treat cold, flu, malaria, toothache and bruising
(Lin et al. 2002). Previous phytochemical investigations on Iris plants have resulted in the isolation
of a variety of secondary metabolites, which mainly include flavonoids, isoflavonoids and their
glycosides, benzoquinones, triterpenoids, stilbeneglycoside and phenolics (Seki et al. 1994; Atta-
ur-Rahman et al. 2003; Fang et al. 2007). The isoflavonoids were found to be characteristic of the
Iris rhizomes (Williams et al. 1997), and the C-glycosylflavones, xanthone glycosides as well as
flavonoid aglycones were the main classes of phenolic constituents in the leaves (Blinova &
Kalyupanova 1974; Blinova et al. 1977; Fujita & Inoue 1982; Pryakhina et al. 1984; Minami et al.
1996). Several coumaronochromones (ayamenin A–E) were obtained as stress metabolites from
the Iris species (Hanawa et al. 1991a). Iris species have an immense medicinal importance and are
used in the treatment of cancer, inflammation, bacterial and viral infections (Hanawa et al. 1991b).
The compounds isolated from these species were reported to have piscicidal, antineoplastic,
antioxidant, anti-tumor, antiplasmodial and antituberculosis properties (Hideyuki et al. 1995;
Miyake et al. 1997; Bonfils et al. 2001). I. nepalensis grows somewhat as an ornamental plant in the
Kashmir Valley, alongside roads and in-between trees of apple gardens. As a part of a programme
to investigate bioactive constituents from plants, we investigated the chemical profile of
chloroform extract of rhizomes of I. nepalensis, which led to the isolation of Compound 1 as
*Corresponding author. Email: mtantry@olemiss.edu
The work of Mudasir Tantry was authored as part of his official duties as an Employee of the United States Government and is therefore a
work of the United States Government. In accordance with 17 USC. 105, no copyright protection is available for such works under US Law.
Khalid Ghazanfar and Ummer R. Zargar hereby waive their right to assert copyright, but not their right to be named as co-authors in the article.

Natural Product Research
1833
alkylated 1,4-benzoquinone and is being reported as new. The structural characterisation of 1 was
achieved by NMR techniques, including chemical derivatisation.
2. Results and discussion
The chloroform extract of rhizomes of I. nepalensis was subjected to repeated chromatographic
separation which led to the isolation of Compound 1.
Compound 1 was obtained as red viscous oil with definite molecular formula C53H94O4,
displayed in HR-ESI-MS at m/z 795.7074 [M þ H]þ. The UV and IR absorptions of 1 were
indicative of the presence of 1,4-benzoquinone moiety (Lin et al. 2011). The 1H NMR spectrum
displayed the signals for one methoxyl (dH 3.81) and two alkenyl side chains [dH 1.99 (4H, m),
2.21 (2H, t), 2.23 (2H, m), 5.30 (2H, m), 5.32 (2H, m), 0.81 (3H, t), 0.86 (3H, t)]. The 13C NMR
spectrum of 1 suggested the presence of one methoxyl group (dC 56.2), substituted
p-benzoquinone ring [dC 185.2 (C-1), 144.2 (C-2), 152.4 (C-3), 184.3 (C-4), 145.1 (C-5), 158.2
(C-6)], and a group of side chain methylenes at dC 28.3–30.5, signals for olefinic signals at dC
128.4, 128.5 and 128.6. The resonance signals of methylene groups adjacent to double at dC 27.9
each (for C-21, C-24, C-21 and C-24) in the 13C NMR confirmed the Z-geometry of double
bonds in the side chains of the molecule. The problem of solving length of the two side chains
and the position of double bonds was achieved by chemical derivatisation. The acetylated
product of Compound 1 was subjected to ozonolysis to obtain aldehyde adduct which displayed
molecular ion peak in HR-ESI-MS at m/z 673.4896 [M þ H]þ (Figure 1). The chemical shift
assignments of 1 were confirmed by HMBC experiments (Figure 2) and finally Compound 1 was
named nepaloquinone A (Figure 3).
Compound 1 was evaluated for their cytotoxicity against four human cancer cell lines using
the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) method (Zheng et al.
2011) (Table 1). Relative cytotoxicity of Compound 1 against cell lines A549, HCT116, HL-60
and ZR-75-30 is depicted in Table 1.
O
O
O
H3CO
H3CO
H3CO
OHC
CHO
15
Acetylation
Ozonolysis
5
5
15
15
OH
OAc
OAc
5
15
5
15
15
O
O
O
1, m/z 795.7074 [M + H]+
2, m/z 837.7336 [M + H]+
3, 673.4896 [M + H]+
Figure 1. Reaction illustrating aldehyde adduct formation from 1 by ozonolysis.
O
H3CO
OH
O
1
Figure 2. Significant HMBC ({) and H–1H DQF-COSY (———) correlations of Compound 1.
O
1
23
7
H3CO
29
6
2
22
22'
5
3
29'
OH
4
23'
7'
O
Figure 3. Structure of Compound 1.

1834
M.A. Tantry et al.
Table 1. In vitro cytotoxicity of Compound 1 against four human cancer cell lines [IC50 (mg/mL),
mean ^ SD, n ¼ 3].
Cell lines
Compound
A549
HCT116
HL-60
ZR-75
1
.83
10 ^ 0.0115
10 ^ 1.1002
0.012 ^ 0.0020
34 ^ 1.1205
0.008 ^ 0.0010
31 ^ 1.1001
0.013 ^ 0.0011
Doxorubicina
a Positive control.
3. Experimental
3.1 General
NMR spectra were recorded on a Varian 600 NMR spectrometer using TMS as internal standard.
Chemical shifts were reported in d units and coupling constants (J) in Hz. ESI-MS and HR-ESI-
MS were obtained on Agilent Series 1100 SL mass spectrometer. IR spectra were recorded using
KBr pellet on a Bruker Tensor 27 FT-IR spectrometer. Column chromatography was carried out
by using silica gel (40 mm mesh, JT Baker), ODS silica gel and reversed-phase RP-C18, C-8
silica (Polarbond; JT Baker). Medium pressure liquid chromatography was carried out on
Biotage, Inc., Horizon MPLC System. TLC analysis was carried out on silica gel 60 F254 plates
(Merch, USA) and spots on TLC plates were observed under UV light (254/365 nm). Reagents
p-anisaldehyde–H2SO4 (Sigma-Aldrich, USA), FeCl3 solution, 10% H2SO4 in ethanol and
water were sprayed, followed by heating, for the detection of spots. All HPLC experiments were
carried out on a Shimadzu SIL-10A with auto injector, SCL-10A system with Activation
GoldPak 100 5 mm ODS 250 £ 10 mm or Waters C18 300 £ 8 mm 5 mm semi-preparative
columns. All solvents used for extractions and chromatographic separations were of analytical
grade with the exception of HPLC grade solvents used for HPLC separations.
3.2 Plant material
The rhizomes of I. nepalensis were collected from Zainapore, Shopian, apple gardens (Kashmir
Valley) and were identified by the taxonomist at Centre for Biodiversity and Taxonomy
Biodiversity (CBT), University of Kashmir, Srinagar, India. A voucher specimen (No.: IN-R-09-
0017) of the plant has been deposited at the same centre.
3.3 Extraction and isolation
The rhizomes of I. nepalensis (1.2 kg) were refluxed with chloroform (4 L) at its boiling point for
10 h. The extract was concentrated in vacuum to give a residue (82 g), which was
chromatographed on a silica gel column with a gradient elution system of hexanes–chloroform
(9:1 ! 1:9) to obtain 10 fractions. Fraction 8 was chromatographed on Sephadex LH-20
(CHCl3–MeOH, 1:1) isocratically to give four fractions. Fraction 8-2a was subjected to reverse-
phase HPLC (MeOH–H2O, 75:25) to obtain Compound 1 (6.8 mg).
3.3.1 Compound 1
Reddish viscous oil; UV (CHCl3): lmax (log 1): 272 (4.42) nm; IR (KBr) nmax: 3397, 2925, 2832,
1681, 1638, 1598, 1448, 1359, 1227, 1143, 1051 cm21; HR-ESI-MS: m/z 795.7074 [M þ H]þ
(Calcd. for C53H94O4H); 1H NMR (600 MHz, CDCl3, dH, J/Hz): 3.81 (s, 6-OCH3), 2.19 (t, H-7),
1.15–1.29 (o, H-8–H-20), 1.98 (m, H-21), 5.30 (m, H-22), 5.30 (m, H-23), 1.99 (m, H-24),
1.15–1.29 (m, H-25–H-27), 1.15–1.29 (m, H-28), 0.81 (t, H-29, J ¼ 6.4 Hz), 2.23 (m, H-70),

Natural Product Research
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1.15–1.29 (m, H-80 –H-200), 1.98 (m, H-210), 5.32 (m, H-220), 5.32 (m, H-230), 1.99 (m, H-240),
1.15–1.29 (m, H-250 –H-270), 1.15–1.29 (m, H-280), 0.86 (t, H-290, J ¼ 6.2 Hz); 13C NMR
(150 MHz, CDCl3, dC):185.2 (C-1), 144.2 (C-2), 152.4(C-3), 184.3 (C-4), 145.1 (C-5), 158.2 (C-
6), 56.2 (ZOCH3) 26.3 (C-7), 28.3–30.5 (C-8–C-20), 27.9 (C-21), 128.5 (C-22), 128.4 (C-23),
27.9 (C-24), 28.3–30.5 (C-25–C-27), 23.5 (C-28), 14.1 (C-29), 27.2 (C-70), 28.3–30.5(C-80 –
200), 27.9 (C-210), 128.6 (C-220), 128.6 (C-230), 27.9 (C-240), 28.3–30.5 (C-250 –C-270), 23.4
(C-280), 14.1 (C-290).
3.4 Cytotoxicity assay
Cytotoxicity assays against human cancer cell lines in vitro were carried out by a modified MTT
method. Compound 1 was evaluated for its cytotoxicity against human lung carcinoma (A549),
colon carcinoma (HCT116), blood cancer (HL-60) and breast carcinoma (ZR-75) cell lines.
Doxorubicin was used as a positive control and IC50 values of the tested compounds were
calculated from averages of three experiments.
3.5 Aldehyde adduct formation
Compound 1 (3.5 mg) was added to a mixture of C5H5N (0.5 mL) and Ac2O (1.0 mL) and
magnetically stirred for 8 h. The reaction mixture was then dried under helium to get an
acetylated product 2 (m/z 837.7336). Ozone gas was bubbled into the CHCl3 solution of 2 at
2608C until the solution acquired a peculiar colour. Excess of ozone was removed by bubbling
the solution with Ar. The reaction mixture was stirred using magnetic stirrer at 2608C for 4 h
and quenched with dimethyl sulphide to obtain an aldehyde adduct 3 (m/z 673.4896).
Acknowledgement
MT thanks the Center for Scientific and Industrial Research (CSIR), New Delhi, India, for financial
assistance for his Postdoctoral Research.
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