A comparative study of antitumor activities and DNA cleavage on a class of dehydroabietylamine derivatives

Article abstract of DOI:10.1691/ph.2013.3575

A series of novel dehydroabietylamine derivatives containing tricyclic diterpene structures were synthesized. The antitumor activities of these compounds against L02, Hey-1B and HepG2 cellswere investigated. Significant activitywas discovered for fourteen analogs. Meanwhile these compounds exhibit DNA cleavage activities on plasmid DNA (Escherichia coli), which depend on the Schiff base structure and the substituent of the aromatic moiety. Our findings present further information on the relationship between the chemical structure, biological function and DNA cleavage characteristics.

Full text of DOI:10.1691/ph.2013.3575

ORIGINAL ARTICLES
College of Chemical Engineering1, Nanjing Forestry University, Nanjing; Bozhou Vocational and Technolege College2,
Bozhou, China; Department of Chemistry3, Cleveland State University, Cleveland, USA; School of Life Sciences4,
Northeast Normal University, Changchun, China
A comparative study of antitumor activities and DNA cleavage on a class of
dehydroabietylamine derivatives
Chao-Xiang Liu1,2, Zhong-Xiang Lin1, Xing Yu1, Zhou Lu1, Ai-Min Zhou3, Yong-Li Bao4
Received February 18, 2013, accepted April 12, 2013
Zhong-Xiang Lin, College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, P. R. China
Linzhongxiang36@gmail.com
Pharmazie 68: 861–865 (2013)
doi: 10.1691/ph.2013.3575
A series of novel dehydroabietylamine derivatives containing tricyclic diterpene structures were synthe-
sized. The antitumor activities of these compounds against L02, Hey-1B and HepG2 cells were investigated.
Significant activity was discovered for fourteen analogs. Meanwhile these compounds exhibit DNA cleavage
activities on plasmid DNA (Escherichia coli), which depend on the Schiff base structure and the substituent
of the aromatic moiety. Our findings present further information on the relationship between the chemical
structure, biological function and DNA cleavage characteristics.
compounds were characterized by IR, MS, and 1H NMR spectra.
In FT-IR spectral analysis of the compounds 2, 3 and 4a show
broad peak in the range 3350 − 3422 cm−1 for NH2, intensity
peaks for NO2 stretching in the range 1500 − 1521 cm−1. The
1H NMR spectra of the compounds 2, 3 and 4a showed the
protons of the aromatic ring appear 7. 08 ppm (s, 1H), 7. 64
(s, 1H) for 2 and 7. 55 ppm (s, 1H) for 3. Those Schiff bases
for compounds showed sharp peaks for nitrile grouping stretch-
ing in the range 1580 − 1650 cm−1 and the protons of methine
1. Introduction
Many bioactive tricyclic diterpenes, such as dehydroabiety-
lamine, have become an important area of natural product
chemistry. Dehydroabietylamine derivatives have been reported
to show constant anticancer activity, Schiff bases and their metal
complexes are under investigation (Son et al. 2005; Gigante
et al. 2003). Anticancer drugs cause cell death through different
mechanisms and their cytotoxicity is related to their interactions
with DNA (Gu and Wang 2010; Cui et al. 2010). Although dehy-
droabietylamine derivatives synthesis and associated antitumor
activity have been persistently investigated (Savluchinske-Feio
et al. 2006; Fonseca et al. 2004; Tashima et al. 2006; Wada
1
proton in Schiff bases is in the range δ8.00 − 8.50 ppm in H
NMR for compounds 5, 6, 7 and 9. The mass spectra of all
compounds exhibited molecular ion peaks at their respective
molecular weight which confirmed their structures.
ˇ
et al. 1985; Sepuılveda et al. 2005; Faneyte et al. 2004; Cortez
et al. 2001; Harper and Elledge 2007), any relationship between
DNA cleavage activity and structure of these tricyclic diterpenes
derivatives have not been reported so far.
2.2. Antitumor activity
We report the synthesis, DNA cleavage and antitumor activi-
ties for newer Schiff bases derived from dehydroabietylamine
(White et al. 1997; Dervan and Edelson 2003; Egli and Pal-
lan 2010). The Schiff bases are capable of generating reactive
species under alkaline pH conditions to induce DNA damage
and inhibitory action against L02 (normal human hepatocyte
cell line), HepG2 (human liver carcinoma cell line) and Hey-
1B (human ovarian carcinoma cell line) in vitro (Heinecke and
Melander 2010). These observations attracted our considerable
attention due to their importance in the design of probes for
DNA structures (Tian et al. 2007; Rossi et al. 2005), because
DNA cleavage by natural or designed molecules has long been
recognized as a potential way to affect cell proliferation.
The antitumor activities of the compounds were determined by
the activity of each compound against L02, Hey-1B and HepG2
cells. Initially, the compounds were tested for cytotoxic activity
by the MTT assay method. The compounds were tested at differ-
ent concentrations to find out the fifty percent growth inhibitory
doses (IC50) against these cells. Partial compounds showed
antiproliferative effects in the range of 10 to 50 ␮M. Some of the
most interesting compounds showed excellent activity at lower
concentrations (Table).
Among these compounds, 5a, 5e − f, 7a, 7d − c, 7m, 7r − s,
8d − e and 9a − b showed potential activities against L02, Hey-
1B and HepG2 cells. Apparently, the antitumor activity of these
compounds may be related to their functional group (C N) and
substituent groups in the aromatic aldehyde. Compounds with
a hydroxyl substituent in the ortho position of the phenyl group
have shown greater antitumor activity than other compounds
without a hydroxyl substituent. The presence of nitro groups
on the 12 and 14 position on the aromatic ring is not impor-
tant for determining the biological activity of the compounds
5a, 7a and 9a. Among these compounds, 7r − s and 8d − e
2. Investigations, results and discussion
2.1. Synthesis of the compounds
The synthesis of dehydroabietylamine derivatives was per-
formed as outlined in the Scheme. All the synthesized
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ORIGINAL ARTICLES
Scheme: Reagents and conditions: (a) H2SO4/HNO3, 10 h, 0–5 ◦C, 87%; (b) (CF3CO2)O, HNO3, 3 h, 0–5 ◦C, 76%; (c) Aldehyde/EtOH, refulx, 5 h, 85%; (d) NaHB4, r.t. (97%);
(e) HNO3-HAc, 4 h, 0–5 ◦C, 69% or N2H4H2O-EtOH, refulx, 6 h H2SO4-NaNO2, 71%; (f) N2H4H2O-EtOH, reflux, 6 h, 78%.
showed good cytotoxicity against HepG2 and Hey-1B cancer
cells, but substituent groups of Br or OCH3 in the ortho or para
position of pyridine ring of compounds 8a − c reduced anti-
tumor activity. This may be because the introduction of these
groups decreases the toxicity and chemotherapeutic index (Vli-
etinck et al. 1995; Montogomery 1959). The results have also
demonstrated that the introduction of functional group (N—C)
in compound 7r and 8a led to less active compounds. The com-
pounds 7l, 7p − q, 8a and 8c with an electron-withdrawing
group on the position of phenyl ring had little or no activity
against HepG2 and Hey-1B cancer cells. Meanwhile, compound
5e displayed antiproliferative effect against Hey-1B cell at con-
centrations of less than 10 ␮M. The cytotoxic effect for Hey-1B
and PC-3 was evaluated after treated with different concentra-
tions of compound 5e for 24 h. As indicated in Figs. 1 and
2, the typical morphology of apoptosis such as cells shrink-
age and membrane blebbing was observed under an inverted
phase-contrast microscopy, compared to untreated control cells.
Obviously, compound 5e induced Hey-1B cell apoptosis in a
dose-dependent manner.
2.3. DNA cleavage activity
Aftertreatmentwithcompounds7l, 7o, 7q, theplasmidDNAhas
been cleaved completely; compound 8a with the introduction of
a functional group (N—C) showed no effect on DNA cleavage.
After treatment with compounds 4a − b, 5a − f, 6a − j, 7e − k,
7m − n, 8b, 9f, the Form I (supercoiled DNA) can be partially
862
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Fig. 1: Compound 5e induces apoptosis in Hey-1B after treatment for 24 hours.
Fig. 2: Compound 5e induces apoptosis in PC-3 cells after treatment for 24 hours.
converted to Form II; other compounds can convert Form I into
Form II and Form III simultaneously (Fig. 3). We also exam-
ined the DNA-cleaving activity of sectional compounds in the
presence of metal ions. Apart from compound 9b, the com-
pounds 5e, 7a − d, 9a with hydroxyl groups on the phenyl ring
showed no DNA cleaving activity in the presence of Fe(II) or
Fe(III) in comparison with control groups. Fe ions inhibited
hydroxyl radical mediated or other radical damage to pBR322
plasmid DNA (Meneghini 1997). Compounds 7l, 7p − q with an
electron-withdrawing group at the meta position of phenyl ring
displayed very good DNA-cleaving activity in the presence of
Fe irons, which may imply some form of nucleophilic reaction
mechanism for the initiation of cleavage as shown in Figs. 4 and
5 (Taj et al. 2011).
Table: IC50 of compounds
Compd
L02 IC (␮g/ml)
HepG2 IC (␮g/ml)
Hey-1B IC (␮g/ml)
50
50
50
5a
5d
5e
5f
7a
7c
7d
7m
7r
7s
5.43
−
8.14
−
20.33
2326
19.17
21.37
6.37
−
5.39
7.36
5.61
5.34
5.57
5.68
5.86
5.56
9.99
6.58
6.05
5.58
5.02
9.22
7.67
6.64
7.94
6.11
6.72
7.28
6.95
7.96
5.71
8.44
−
7.40
−
−
8d
8e
9a
9b
19.77
−
2.4. Conclusion
−
−
A series of new dehydroabietylamine derivatives were synthe-
sized to further elucidate the relationships between structure
and activity. The substituent group in the Schiff base struc-
Pharmazie 68 (2013)
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3. Experimental
3.1. Chemistry
Dehydroabietylamine was converted into 12,14-dinitro-dehydroabietyl-
amine 2 by using a HNO3–H2SO4 mixture for 10 h at 0 ◦C and 12-nitro-
dehydroabietylamine 3 by (CF3CO)2O–HNO3 mixture for 3 h at 0 ◦C. The
ethanol solution of compound 1–3 with different substituted aromatic alde-
hydesaffordedthecorrespondingsubstitutedtheSchiffbase5a–f(Zhaoetal.
2007; Sui et al. 2011), 7a–s and 9a–g according to the literature, respectively.
Compound 8a–e is a reduzate from the corresponding Schiff base through
NaBH4. Dehydroabietylamine was reacted with nitric acid mixture for 4 h in
thepresenceofaceticanhydridetoprovidecompound4a, whichwasreduced
using hydrazine hydrate for 6 h under refluxing to give 12-amino-acetyl
dehydroabietylamine. The diazotization of 12-amino-acetyl dehydroabiety-
lamine with H2SO4–NaNO2 and hydrolysis reaction gave the compound
4b. Condensation reaction of 12-amino-acetyl dehydroabietylamine with
different substituted aromatic aldehydes provided compounds 6a-6j.
3.2. Antitumor activity
HepG2, L02 and Hey-1B cells were seeded in 96-well plates, respectively.
Cells were diluted in medium to a density of 5 × 104 viable cells per ml.
Using a DMSO control group, cells were treated with test compounds
(10 ␮g/ml) the following days, respectively. All cells were incubated and
maintained 44 h in a humidified atmosphere at 37 oC and 5% CO2. MTT
was used to assess the viability of cells following treatment. Aliquots of
20 ml of stock MTT solution (5 ␮g/ml) were added to each well and incu-
bated with the cells for 4 h. Following incubation the medium was removed
and 100 ml of DMSO added to solubilize the formazan crystals. The OD
value was measured at 570 nm by a microplate reader (Model 550, Bio-
Rad, USA). The relative cell viability was calculated as absorbance of
compound-treated/absorbance of control.
3.3. DNA cleavage activity
Fig. 3: DNA cleavage activity of compounds FormII (Oc DNA), FormIII (Lin DNA).
The compounds have been studied using pBR322 supercoiled plasmid
DNA (Form I) as a substrate in a medium of 50 ␮M Tris-HCl/NaCl buffer
(pH = 7.4) under physiological conditions. Reactions were carried out for
3 h at 37 ◦C in 20 ␮l (total volume) of 50 ␮M Tris-HCl/NaCl buffer (2.5%
DMF), containing 5 ␮l pBR322 supercoiled DNA (0.25 ␮g/␮l), 50 ␮M of
each dehydroabietylamine derivative or 10 ␮M Fe irons. The DNA cleavage
fragmentwasanalyzedbyagarosegelelectrophoresis. Afterelectrophoresis,
the gels were illuminated with UV light and photographed.
Acknowledgements: The generous financial support of the Natural Science
Foundation of China (31170536) was gratefully acknowledged.
Fig. 4: DNA cleavage activity of compounds in the presence of Fe(II).
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