Novel bisnaphthalimidopropyl (BNIPs) derivatives as anticancer compounds targeting DNA in human breast cancer cells

Article abstract of DOI:10.1039/c6ob01850e

Bisnaphthalimidopropyl (BNIP) derivatives are a family of compounds that exert anti-cancer activities in vitro and, according to previous studies, variations in the linker sequence have increased their DNA binding and cytotoxic activities. By modifying th

Full text of DOI:10.1039/c6ob01850e

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DOI: 10.1039/C6OB01850E
Organic & Biomolecular Chemistry
ARTICLE
Novel Bisnaphthalimidopropyl (BNIPs) derivatives as anticancer
compounds targeting DNA in human breast cancer cells
Maria Kopsidaa , Gemma A. Barrona, b, Giovanna Bermanob, Paul Kong Thoo Lina and Marie Gouaa
Received 00th January 20xx,
Accepted 00th January 20xx
Bisnaphthalimidopropyl (BNIP) derivatives are a family of compounds that exert anti-cancer activities in vitro and, according
to previous studies, variations in the linker sequence have increased their DNA binding and cytotoxic activities. By modifying
the linker sequence of Bisnaphthalimidopropyl diaminodicyclohexylmethane (BNIPDaCHM), a previously synthesised BNIP
derivative with anti-cancer properties, three novel BNIP derivatives were designed. Bisnaphthalimidopropyl-
DOI: 10.1039/x0xx00000x
www.rsc.org/
piperidylpropane (BNIPPiProp),
a
structural isomer of BNIPDaCHM, bisnaphthalimidopropyl ethylenedipiperidine
shorter linker chain, and (trans(trans))-
dihydrobromide (BNIPPiEth), an isoform of BNIPDaCHM with
a
bisnaphthalimidopropyl diaminodicyclohexylmethane (trans,trans-BNIPDaCHM), a stereoisomer of BNIPDaCHM, were
successfully synthesised (72.3-29.5 % yield) and characterised by nuclear magnetic resonance spectroscopy (NMR) and mass
spectrometry (MS). Competitive displacement of ethidium bromide (EtBr) and UV binding studies were used to study the
interactions of BNIP derivatives with Calf Thymus DNA. The cytotoxicity of these derivatives was assessed against human
breast cancer MDA-MB-231 and SKBR-3 cells by MTT assay. Propidium iodide (PI) flow cytometry was conducted in order to
evaluate the cellular DNA content in both breast cancer cell lines before and after treatment with BNIPs. The results showed
that all novel BNIPs exhibit strong DNA binding properties in vitro, and strong cytotoxicity, with IC50 values in the range of
0.2-3.3 µM after 24 hour drug treatment. Two of the novel BNIP derivatives, BNIPPiEth and trans,trans-BNIPDaCHM,
exhibited greater cytotoxicity against the two breast cancer cell lines studied, compared to BNIPDaCHM. By synthesising
enantiopures and reducing the length of the linker sequence, the cytotoxicity of the BNIP derivatives was significantly
improved compared to BNIPDaCHM, while maintaining DNA binding and bis-intercalating properties. In addition, cell cycle
studies indicated that trans,trans-BNIPDaCHM, the most cytotoxic BNIP derivative, induced sub-G1 cell cycle arrest,
indicative of apoptotic cell death. Based on these findings, further investigation is under way to assess the potential efficacy
of trans,trans-BNIPDaCHM and BNIPPiEth in treating human breast cancer.
Keywords: Bisnaphthalimides, DNA binding, cytotoxicity, cell cycle.
breast cancers respond better to anti-oestrogen (endocrine)
therapies, such as tamoxifen and exemestane, by inhibiting the
Introduction
Breast cancer is the most commonly occurring cancer in women, with
incidence rates approaching 1.38 million cases per year worldwide1.
Breast cancer, depending on whether it develops in response to the
hormone oestrogen, is divided in two categories: oestrogen
dependent (ER+) and oestrogen independent (ER-) cancer2. ER+
effect of oestrogen and decreasing the uncontrolled proliferation of
breast cancer cells2. On the other hand, ER- breast cancers are more
invasive and less responsive to current standard-of-care treatment
regimes, such as fluorouracil, epirubicin and doxorubicin, which do
not selectively target breast cancer cells hence leading to severe side
effects3. Over the last few decades, there have been numerous
attempts to develop novel breast cancer-specific therapies that will
act on specific molecular targets, increasing selectivity and
potentially reducing treatment resistance and side effects4. DNA was
one of the first biochemical targets identified in anti-cancer
a School of Pharmacy and Life Sciences, Robert Gordon University, Garthdee Road,
Aberdeen, AB10 7GJ, Scotland, UK
bCentre for Obesity Research and Education (CORE), Faculty of Health and Social
Care, Robert Gordon University, Garthdee Road, Aberdeen, AB10 7GJ, Scotland,
UK
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therapeutics5, 6, 7, which lead to a new generation of agents with compared to BNIPPiProp and was synthesised in order to assess the
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improved efficacy and solubility. In the 1990’s naphthalimido effect of a shorter linker chain on cytotoDxiOciIt:y10a.1n0d39D/CN6AOBb0in18d5in0Eg
compounds, such as mitonafide, were found for the first time to be properties. In parallel, it is still unknown whether the cytotoxicity of
highly active against cervical cancer and leukaemia cells, however, a BNIPDaCHM is associated with the existence of the three isomers in
Phase I clinical trial revealed that mitonafide treatment at doses its
structure,
therefore
(trans(trans))-4,4’-Methylenebis
above 118 mg/m2 ×5 days lead to central nervous system toxicity8. (cyclohexylamine), the only commercially available stereoisomer
Subsequently, bisnaphthalimido compounds were synthesised with precursor required to synthesise trans,trans-BNIPDaCHM the latter
improved therapeutic properties. They had also overcome the dose- being , the third BNIP derivative used in this study. The synthesis of
limiting toxicity issues9, however, their aqueous insolubility limited trans,trans-BNIPDaCHM lead to the investigation into the
their potential use as anti-cancer agents10. Kong Thoo Lin and importance of this stereoisomer compared to the mixture of three
Pavlov10
designed
and
synthesised
a
number
of stereoisomers present in BNIPDaCHM in relation to DNA binding
bisnaphthalimidopropyl derivatives, by incorporating natural affinities, cytotoxicity in MDA-MB-231 and SKBR-3 breast cancer cell
polyamines and, diamino or triamino alkyl chains into the lines and the possible mode of cell death via cell cycle analysis were
bisnaphthalimide structure. This led to improved aqueous solubility, studied for the three novel compounds. By using two cell lines which
as well as anti-cancer activity11, 12. Additional alterations to the linker are unresponsive to currently available anti-cancer regimes, it is
were performed to confirm that BNIP moiety was crucial for in vitro possible to extend our knowledge on BNIP derivative cytotoxicity
anti-cancer activity. One such alteration was the introduction of a against different breast cancer cells types and to gain more
bicyclohexylmethane group (in the linker chain). This tended to information about their mode of action.
reduce the flexibility of the linker but it enhanced its DNA binding
properties, resulting in the synthesis of BNIPDaCHM (Figure 1).
BNIPDaCHM contained a pseudo-asymmetrical centre with a mixture
of three isomers (cis,cis; trans,trans and cis,trans) (indicated by
asterisks, Figure 1)13, 14. This linker chain modification resulted in a
more cytotoxic BNIP derivative against triple negative breast cancer
cells MDA-MB-231 (IC50 value 6.8 µM), compared to chemotherapy
drug doxorubicin (IC50 value 14.4 µM), after 24 hours treatment13.
With regards to selectivity, non-tumourigenic breast epithelial MCF-
10A cells were found to be less responsive to BNIPDaCHM (IC50 value
6.06 µM) than doxorubicin 13
.
In the present work, we describe for the first time, the synthesis and
characterisation of three novel BNIP derivatives that were designed
by considering several modifications to the structure of BNIPDaCHM
(Figure 1). The first BNIP derivative, bisnaphthalimidopropyl-
piperidylpropane dihydrobromide (BNIPPiProp) is a structural isomer
of BNIPDaCHM that consists of only one species (enantiopure). The
aim of synthesising BNIPPiProp was to investigate whether
cytotoxicity and DNA binding properties differ among structural
isomers and how the position of the ring structure in the linker chain
Figure 1. Chemical structure of BNIP derivatives: BNIPDaCHM with its three
stereo isomers, bisnaphthalimidopropyl-piperidylpropane (BNIPPiProp),
bisnaphthalimidopropyl- ethylenedipiperidine (BNIPPiEth) and (trans(trans))-
4,4’Methylenebis(cyclohexylamine) (trans,trans-BNIPDaCHM).
affects
cytotoxicity.
The
second
BNIP
derivative,
bisnaphthalimidopropyl- ethylenedipiperidine (BNIPPiEth), consists
of one carbon less between the two piperidine ring structures,
2 | J. Name., 2012, 00, 1-3
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performed
by
reacting
trans,trans-N4V,ieNw4-AdrtiimcleeOsintliynle-
DOI: 10.1039/C6OB01850E
dicyclohexylmethane with toluenesulfonyloxypropylnaphthalimide
in DMF (37.0% yield). For the final step, trans,trans-
bisnaphthalimido-dimesityl-dicyclohexylmethane was dissolved in
DCM, followed by treatment with hydrobromic acid/glacial acetic
acid (HBr/g.CH3CO2H). All new compounds were fully characterised
by NMR and high resolution mass spectrometry (see experimental
section). The melting point of trans,trans-BNIPDaCHM was found to
be 120 - 125 oC indicating good purity. In contrast, BNIPDaCHM
o
exhibited a higher melting point range (105 - 130 C) compared to
trans,trans-BNIPDaCHM. BNIPPiProp and BNIPPiEth had melting
o
o
points in the range of 160 - 170 C and 120 - 125 C, respectively,
indicating their high purity.
According to matched molecular pair (MMP) analysis, which has
been broadly used in the last few years to investigate the effects of
hydrogen bond donors/acceptors and rotatable bonds on the
melting point of drug-like compounds15, 16, 17, has shown that an
increase in rotatable bonds leads to a decrease of the melting
point18. Therefore, the existence of the three isomers on
Scheme 1. Synthetic pathway of bisnaphthalimidopropyl-piperidylpropane
(BNIPPiProp)
and
bisnaphthalimidopropyl-
ethylenedipiperidine BNIPDaCHM increases its flexibility and leads to a low melting point,
compared to the three novel compounds.
dihydrobromide (BNIPPiEth).
Results and discussion
Chemical synthesis
The synthesis of bisnaphthalimidopropyl-dipiperidyl-propane and
ethane free bases was carried out by reacting 1,3-bis-(4-piperidyl)
propane
or
1,3-bis-(4-piperidyl)ethane
and
toluenesulfonyloxypropylnaphthalimide in tetrahydrofuran (THF)
under reflux with subsequent addition of cesium carbonate (Scheme
1). The corresponding BNIPPiProp and BNIPPiEth dihydro chloride
and bromide salts were prepared by their treatment with
HBr/g.CH3CO2H and conc. HCl (72.3% and 29.5% yield), respectively.
The synthetic strategy of trans,trans-BNIPDaCHM (Scheme 2) was
based on methods previously developed in our group for the
synthesis of BNIPDaCHM13. Here the single isomer trans, trans-4,4’-
methylenebiscyclohexylamine was used as the starting material. The
synthesis of trans,trans-N4,N4-dimesityl-dicyclohexylmethane was
carried
out
by
reacting
trans,trans-4,4’- Scheme
2.
Synthetic
pathway
of
(trans(trans))-4,4’-
Methylenebis(cyclohexylamine) (trans,trans-BNIPDaCHM).
methylenebis(cyclohexylamine), with 2-mesitylenesulfonyl chloride
(Mts-Cl) in anhydrous pyridine (21.1% yield). N-Alkylation was
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On the other hand, BNIPPiProp, with the longest linker sequence, has BNIPPiEth, which has the shortest linker with two piperidine rings
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DOI: 10.1039/C6OB01850E
the highest melting point (160 - 170 oC), due to the increase of attached to an ethyl group, had the lowest C50 value. The shorter
hydrogen bond donors/acceptors that stabilize the crystal lattice18.
length of the linker chain, as well as the incorporation of a nitrogen
atom within the cyclohexane ring, compared to BNIPDaCHM,
resulted in increased DNA binding. On the other hand, the
incorporation of the nitrogen atom within the cyclohexane ring was
not found to improve the binding properties of BNIPPiProp (3.9 ± 0.3
µM), compared to BNIPDaCHM and BNIPPiEth, confirming that the
length of the bridging alkyl linkers is crucial and affects significantly
BNIP binding to DNA duplexes21.
DNA binding studies
Competitive displacement of ethidium bromide from Calf Thymus
DNA
Competitive displacement of ethidium bromide (EtBr) with BNIPs
from Calf Thymus DNA was carried out to investigate their DNA
interactions. EtBr is a well known DNA structural probe and
intercalating dye that exerts fluorescence once it binds to DNA19. A
compound with higher DNA binding affinity than EtBr either
displaces EtBr or breaks the DNA secondary structure, resulting in
fluorescence quenching and a decrease in fluorescence intensity20.
All three novel BNIP derivatives competitively displaced EtBr from
Calf Thymus DNA duplexes. For each derivative, a range of
concentrations (0-7 µM) were tested and the corresponding C50
values calculated. All BNIP derivatives displaced EtBr with C50 values
ranging from 1.1 to 5.6 µM confirming their high DNA binding affinity
(Table 1). BNIPDaCHM was included in the following studies in order
to evaluate and compare its binding properties to the novel BNIP
derivatives.
The trans,trans-BNIPDaCHM, a stereoisomer of BNIPDaCHM, gave a
C50 value of 5.6 ± 0.2 µM. BNIPDaCHM, which consists of three
isomers, gave a C50 value of 2.3 ± 0.1 µM, which indicated that in
absence of cis,cis or/and cis,trans, trans,trans-BNIPDaCHM was not
able to achieve as high DNA-binding interactions as BNIPDaCHM.
Based on the above results, the planar structure of BNIPDaCHM and
its mixture of three isomers was found to improve its interacting
properties within the DNA base pairs, compared to trans,trans-
BNIPDaCHM. This was confirmed since trans,trans-BNIPDaCHM
resulted in a lower C50 value compared to BNIPDaCHM, revealing that
each of the three or more than one (trans,trans/cis,trans or
trans,trans/cis,cis) isomers co-existing in BNIPDaCHM, are involved
in the intramolecular complexes/interactions with DNA. By isolating
one of its isomers (trans,trans-BNIPDaCHM), the DNA binding affinity
was decreased (p<0.01). The planar structure of BNIPDaCHM allows
it to fit between the base pairs and in parallel, its rotational freedom
within the plane of the aromatic rings, may allow the exposure of
more than one intercalating sidechain to DNA22.
The order of their binding affinity to Calf Thymus DNA from highest
to lowest was BNIPPiEth (1.1 ± 0.2 µM), BNIPDaCHM (2.3 ± 0.1 µM),
BNIPPiProp (3.9 ± 0.3 µM) and trans,trans-BNIPDaCHM (5.6 ± 0.2
µM).
BNIP derivatives
Mean ± SD
C50 value (µM)
UV binding studies
Calf Thymus DNA alone
BNIPDaCHM
-
The binding of the novel BNIP derivatives with Calf Thymus DNA was
also studied by UV spectroscopy. A continuous decrease in UV
absorption was observed at 260 nm, within the range of drug
concentrations (0 - 7µM) investigated. The apparent binding
constants for the compounds under study, were calculated from the
intercept and the slope by plotting A0/ (A-A0) against BNIP derivative
concentrations23, where A0 and A correspond to the absorbance
values in the absence and presence of each BNIP compound (Figure
2a), respectively. Binding constant values K for the BNIP derivatives
range between 3.25 x 104 - 12.23 x 104 (Figure 2b), and indicate that
all BNIP derivatives interact with the DNA helix. The highest binding
2.3±0.1
BNIPPiProp
3.9±0.3 *
1.1±0.2 *
5.6±0.2 **
BNIPPiEth
trans,trans-BNIPDaCHM
Table 1. Competitive displacement of EtBr from Calf Thymus DNA. Effect of
different BNIP concentrations (0-7 µM) on % fluorescence intensity compared
to Calf Thymus DNA alone. C50 value: concentration of each BNIP derivative
required to cause a 50% decrease on fluorescence intensity of DNA-EtBr
complex. Data are the mean ± SD of three independent experiments (n=3).
*P<0.05, **P<0.01, compared to BNIPDaCHM.
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Furthermore, they have been reported to induce strand cleavage,
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a.
DOI: 10.1039/C6OB01850E
allowing the electron transfer and formation of hydrogen bonding
between the nitrogen atoms and the nucleobases (excluding
guanine) of the minor groove25, suggesting that the high K binding
constant of trans,trans-BNIPDaCHM is obtained not only via
intercalation, but also via binding on the major and minor groove of
DNA.
Biological studies
Cytotoxicity
BNIP derivatives
BNIPDaCHM
BNIPPiProp
K constant
12.2 x 104
3.3 x 104
Cytotoxicity evaluation of BNIPDaCHM, BNIPPiProp, BNIPPiEth and
trans,trans-BNIPDaCHM was performed by using MTT assay26 against
MDA-MB-231 and SKBR-3 cells (Table 2). After 24 hours treatment,
all novel BNIP derivatives, exhibited strong cytotoxicity with IC50
values ranging from 1.4 μM to 3.3 μM in MDA-MB-231 cells (Table
2), compared to previously synthesised BNIPs or DNA intercalating
drugs (doxorubicin) that have been tested against the same cell line,
with IC50 values ranging from 4.9 μM to 12.7 μM13. In particular,
trans,trans-BNIPDaCHM exhibited the lowest IC50 value of 1.4 μM,
BNIPPiEth an IC50 of 1.8 μM and BNIPPiProp an IC50 of 3.3 μM. A
similar pattern of cytotoxicity was found for SKBR-3 cells, although
the IC50 values were between 0.2 - 0.7 μM (Table 2).
b.
BNIPPiEth
10.9 x 104
11.4 x 104
trans,trans-BNIPDaCHM
Figure 2.a) Competitive displacement of EtBr from Calf Thymus DNA. Plot of
A0/ (A-A0) versus 1/CBNIP of the interaction between BNIP derivatives and Calf
Thymus DNA. b) K constant values of BNIPP derivatives after UV binding
studies.
constant was observed with BNIPDaCHM(12.23 x 104), followed by
trans,trans-BNIPDaCHM (11.38 x 104) and BNIPPiEth (10.85 x 104).
The lowest K binding constant was observed for BNIPPiProp (3.25 x
104). This outcome is in agreement with the competitive
displacement of EtBr studies (Table 1), highlighting the importance
of linker chain length in achieving strong DNA binding interactions.
In addition, the UV absorption studies revealed that trans,trans-
BNIPDaCHM, the less effective derivative in displacing EtBr from
DNA, obtained a high binding constant (11.38 x 104), compared to
BNIPDaCHM (12.23 x 104: highest K binding constant). This suggests
that trans,trans-BNIPDaCHM exhibits lower intercalation capacity
than the other two isomers present in the BNIPDaCHM. However,
the trans,trans isomer has similar DNA affinity to the mixture of
isomers present in BNIPDaCHM as demonstrated by their binding
µM)
IC values (
(Mean ± SEM)
SKBR-3
50
BNIP derivatives
MDA-MB-231
BNIPDaCHM
2.3± 0.1
0.4±0.1
BNIPPiProp
BNIPPiEth
3.3± 0.1 **
1.8±0.1 *
0.7±0.1 *
0.3±0.1 *
trans,trans-BNIPDaCHM
1.4± 0.1 **
0.2±0.0 *
Table 2. Cytotoxicity of BNIP derivatives against MDA-MB-231 and SKBR-3
cells. MDA-MB-231 and SKBR-3 cells were treated with different BNIP
constant (Figure 2b). Previous molecular modelling studies have concentrations (0-10 μM) for 24 hours at 37 °C. IC50 values correspond to the
concentration required to reduce cell growth by 50% compared to control
revealed that for the most stable conformation of bis-1,8-
cells. Data presented as mean ± SEM of 3 independent experiments (n=3).
naphthalimide in presence of DNA, the naphthalimide rings obtain
*P<0.05, **P<0.01, compared to BNIPDaCHM.
an antiparallel orientation and are detected in the major groove24.
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trans,trans-BNIPDaCHM (1.4 μM) was more active (p<0.01) than BNIPDaCHM (1µM) (31.3% and 29.4% decrease, both p<0.05) (Figure
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BNIPDaCHM (2.3 μM) against MDA-MB-231 cells, showing that the 3a). The above results indicate that both BNIPDaCHM
DOI: 10.1039/C6OB018a5n0dE
existence of a single isomer in the linker sequence results in a more trans,trans-BNIPDaCHM induced sub-G1 cell cycle arrest to a greater
cytotoxic compound, compared to a compound which contains a extent than camptothecin. Camptothecin is a well known positive
mixture of isomers. BNIPPiEth was more cytotoxic (p<0.05) than control for sub-G1 arrest30, 31 and for inducing apoptosis32, 33
BNIPDaCHM, suggesting that the shorter length of the linker chain,
.
Therefore, this suggests that BNIPDaCHM and trans,trans-
BNIPDaCHM could use similar mechanisms of action compared to
camptothecin and may trigger apoptotic cell death in MDA-MB-231
and SKBR-3 human breast cancer cells.
as well as the incorporation of the nitrogen atom within the
cyclohexane ring, not only improved the binding properties of
BNIPPiEth, but enhanced significantly its in vitro cytotoxicity, too.
Regarding BNIPPiProp, the derivative with the longest linker
sequence and the highest IC50 value, it was found that the length of
the linker chain length plays an important role in the functionality
and effectiveness of a BNIP derivative. In addition, all the derivatives
followed a similar pattern of cytotoxicity in both cell lines however,
they appeared more cytotoxic against SKBR-3 cells. This may be due
to the different mutational and tumorigenic statuses.
a.
MDA-MB-231 cells are triple negative breast cancer cells (TNBC)
(oestrogen receptor negative (ER-), progesterone receptor negative
(PR-), human epidermal growth factor receptor 2 negative HER2-)
with four gene mutations being reported (BRAF, CDKN2A, KRAS and
TP53) and a fast-growing basal B tumour classification (49.5%
proliferation rate)27, which makes them less responsive to anti-
cancer treatments, such as docetaxel or carboplatin, compared to
cells that are hormone receptor positive27. On the other hand, SKBR-
3 cells are double negative breast cancer cells (DNBC) (ER-, PR-,
HER2+) without gene mutations and with a luminal tumour
classification (35.2% proliferation rate)27, resulting in a better
response to anti-cancer treatments compared to TNBCs, which is in
agreement with our findings.
b.
Cell cycle analysis
Cell cycle distribution of MDA-MB-231 and SKBR-3 cells after cell
synchronisation was studied using flow cytometry following PI
staining28 with the most active compound trans,trans-BNIPDaCHM
(Table 2), together with BNIPDaCHM (mixture of isomers) that has
been reported to induce cell cycle instability29.
Figure 3. Cell cycle distribution of MDA-MB-231 and SKBR-3 cells after BNIP
treatment. a) Quantification of MDA-MB-231 and b) SKBR-3 cell cycle profiles
by flow cytometry following PI staining after 24 hours treatment with
BNIPDaCHM, trans,trans-BNIPDaCHM and camptothecin. DMSO/dH2O (50%
v/v) was used as the solvent control. The percentage of the cell population in
sub-G1, G1, S and G2/M were calculated from histograms of linear FL-2 plots
in the ungated regions (10,000 events). Data are mean ± SEM of three
independent experiments (n=3), conducted in duplicates. *P<0.05, compared
to solvent control.
An increase in the proportion of MDA-MB-231 cells in sub-G1 phase
(122.3%, 139.3% and 142.2% increase, respectively) was exhibited
relative to the control, whilst in G1 phase, the cell population was
decreased after treatment with BNIPDaCHM (1µM) and trans,trans-
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Similar experiments were carried out with synchronised SKBR-3 cells aluminium plates (EMD/Merck) in chloroform/methanol (95:5).
View Article Online
DOI: 10.1039/C6OB01850E
(Figure 3b), where the cell population was significantly increased in NMR was recorded on a Bruker 400 Ultrashield spectrometer
1
sub-G1 phase only after treatment with BNIPDaCHM (136.4%) and operating at 400.1 MHz for H and 100.6 MHz for 13C. Accurate
camptothecin (210.2%), but not for trans,trans-BNIPDaCHM, mass spectra were obtained by were obtained on Thermo
demonstrating that the effect of the two BNIP derivatives was Scientific LTQ Orbitrap XL or Waters Xevo G2-S analytical
different in each cell line and that the existence of one isomer has a instruments (EPSRC National Mass Spectrometry Service Centre
different effect on cell cycle distribution. Therefore, it is suggested at Swansea University, Swansea).
that the mechanisms of cell death among trans,trans-BNIPDaCHM
and BNIPDaCHM may differ, even though they belong to the same Synthesis of bisnaphthalimidopropyl-dipiperidyl-propane base
family of compounds.
1,3-bis-(4-piperidyl)propane (1.19 x10-3mol, 0.25g) and
toluenesulfonyloxypropylnaphtalamide (2.39 x10-3mol, 0.98g)
were dissolved in tetrahydrofuran (THF) (6 mL). Using a reflux
Conclusions
o
condenser, the reaction was stirred at 50 C for 15 minutes and
In this study, we have shown that three BNIP derivatives,
BNIPPiProp, BNIPPiEth and trans,trans-BNIPDaCHM could have
potential as anti-cancer agents. DNA binding affinity was
confirmed as each BNIP derivative had the ability to competitively
displace EtBr from DNA and quenched UV absorption in presence
after the addition of caesium carbonate (3.069 x10-3 mol, 1 g), the
reaction was left to stir overnight at 50oC. The reaction was
monitored with TLC and once complete, the solution was poured
into icy water (100 mL). A precipitate was formed and after
vacuum filtration, the product was dried in a vacuum oven at 45oC
overnight. The crude product (base of BNIPPiProp) was
recrystallized from ethanol and the pure product was
characterised by 1H-NMR (64.8% yield).
of DNA in
a dose dependent manner. Linker sequence
modifications showed stronger cytotoxic effects against MDA-
MB-231 and SKBR-3 cells, compared to the parental compound
BNIPDaCHM. In addition, cell cycle analysis showed that
trans,trans-BNIPDaCHM induced sub-G1 arrest in MDA-MB-231
cells, but not in SKBR-3 cells, suggesting that its mode of action
could be cell line dependent, in contrast to BNIPDaCHM that
shows a similar trend in cell cycle arrest in both cell lines. This
study has confirmed that BNIPPiProp, BNIPPiEth and trans,trans-
BNIPDaCHM have the ability to interact with DNA, intercalate and
stabilise the double helix, and exhibit better cytotoxic activities,
compared to previously synthesised BNIP derivatives. Further
research is ongoing into the mode of DNA damage, cell death or
pathway deregulations in human breast cancer cell lines.
1H-NMR (CDCl3): δH 8.53 – 8.51 (2H, CH aromatic protons), 8.14 –
8.12 (2H, CH aromatic protons), 7.70 – 7.66 (2H, CH aromatic
protons), 4.17 – 4.14 (2, CH2 protons), 2.84 – 2.82 (2H, CH2
protons), 2.41 – 2.37 (2H, CH2 protons), 1.91-1.83 (2H, CH2
protons), 1.79-1.74 (2H, CH2 protons), 1.51-1.48 (2H, CH2
protons), 1.71-1.09 (H, CH proton) ppm (parts per million).
Synthesis of BNIPPiProp salt
The base of BNIPPiProp (1.459 x10-3 mol, 1 g) was dissolved in
DCM (20mL) and HBr/CH3CO2H (2 mL) was added slowly. The
reaction was stirred for 2 hours at room temperature and
reaction completion was monitored by TLC yielding a precipitate.
The precipitate was filtered by vacuum filtration and washed with
DCM (30 mL) and ether (10 mL). The BNIPPiProp salt was dried
under negative pressure in a vacuum oven set at 45oC for 2 hours
(72.3% yield).
Experimental
Chemical synthesis
BNIPPiProp, BNIPPiEth and trans,trans-BNIPDaCHM were
synthesised according to the methods (with some modifications)
previously described by Kong Thoo Lin and Pavlov10.The chemical
structure, purity and stability of the derivatives were confirmed
by TLC, NMR, MS and melting point determination. All reagents
were purchased from Fisher Scientific or Sigma-Aldrich, unless
otherwise stated. TLC was performed on silica gel 60 F254
1H-NMR (CDCl3): δH 8.53 – 8.51 (2H, CH aromatic protons), 8.14 –
8.12 (2H, CH aromatic protons), 7.70 – 7.66 (2H, CH aromatic
protons), , 4.69 (2H, CH2 protons), 4.17 – 4.14 (2H, CH2 protons),
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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Journal Name
2.84 – 2.82 (2H, CH2 protons), 2.41 – 2.37 (2H, CH2 protons), 1.91- 1H-NMR (CDCl3): δH 8.52 – 8.50 (2H, CH aromatic prVoiteownAsrt)ic,le8.O1n3lin–e
DOI: 10.1039/C6OB01850E
1.83 (2H, CH2 protons), 1.79-1.74 (2H, CH2 protons), 1.51-1.48 8.11 (2H, CH aromatic protons), 7,69 – 7.65 (2H, CH aromatic
(2H, CH2 protons), 1.71-1.09 (H, CH proton) ppm.
protons), 4.18 – 4.14 (2H, CH2 protons), 2.83 – 2.80 (2H, CH2
13C-NMR (CDCl3): δH 164.24 (C=O), 131.87 (CH aromatic), 131.60 protons), 2.40 – 2.36 (2H, CH2 protons), 1.90-1.83 (2H, CH2
(C aromatic), 131.24 (CH aromatic), 127.75 (C aromatic), 126.94 protons), 1.79-1.71 (2H, CH2 protons), 1.48 (2H, CH2 protons), 1.2
(CH aromatic), 122.80 (C aromatic), 56.58 (CH2), 54.03 (CH2), (H, CH protons) and 1.00-0.80 (2H, CH2 protons) ppm.
39.10 (CH2 aromatic), 36.77-35.69 (CH), 32.34 (CH2), 25.33 (CH2)
and 23.76 (CH2) ppm.
Synthesis of BNIPPiEth
Mass spectrum (HRMS), m/z = 685.3739 (M + H)+ C43H48N4O4 BNIPPiEth (0.1 g) was dissolved in DCM (20 mL). Then, conc. HCl
requires 685.3748 (M + H)+.
(1.5 mL) was added dropwise and the solution stirred at room
temperature for 60 minutes, which resulted in the formation of a
pale, blue precipitate. The latter was filtered and washed with
Synthesis of 4,4-Ethylenedipiperidine
4,4-Ethylenedipiperidine dihydrochloride (7.428x10-4 mol, 0.2 g) ether (50 mL) and afterwards with ethanol (50 mL). The product
was dissolved in distilled water (2 mL), Sodium hydroxide (2 M, 1 was left under negative pressure in a vacuum oven at 45 oC for 60
mL) was added, which resulted in the formation of a precipitate. minutes (29.5% yield).
The pH was 14. The resulting was transferred into a separating
funnel (100 mL) followed by extraction with DCM (300 mL). The 1H-NMR (CDCl3): δH 8.42 – 8.38 (2H, CH aromatic protons), 7,82 –
organic layer was collected, dried with sodium sulfate and 7.78 (2H, CH aromatic protons), 4.05 – 4.01 (2H, CH2 protons), 2.7
filtered. The solvent was evaporated by a rotary film evaporator. (2H, CH2 protons), 2.44 – 2.43 (2H, CH2 protons), 2.05 (2H, CH2
The final white solid product was left to dry under negative protons), 1.70-1.67 (2H, CH2 protons), 1.35-1.32 (H, CH protons)
pressure in a vacuum oven at 45oC for 30 minutes (90.6% yield). and 1.090 (2H, CH2 protons) ppm.
1
The synthesis of the free base was confirmed by H-NMR.
13C-NMR (CDCl3): δH 164.10 (C=O), 134.81 (CH aromatic), 131.76
(C aromatic), 131.18 (CH aromatic), 127.92 (C aromatic), 127.68
1H-NMR (CDCl3): δ 2.98 – 2.95 (2H, CH2 protons), 2.52 – 2.45 (2H, (CH aromatic), 122.59 (C aromatic), 55.40 (CH2), 52.27 (CH2),
CH2 protons), 1.8 (NH), 1.60 – 1.57 (2H, CH2 protons), 1.25 – 1.21 37.79 (CH), 29.43 (CH2) 23.04 (CH2) and 22.54 (CH2) ppm.
(H, CH protons) and 1.19 – 1.16 (2H, CH2 protons) ppm.
Mass spectrum (HRMS), m/z = 671.5372 (M + H)+ C42H46N4O4
requires 671.3592 (M + H)+.
Synthesis of BNIPPiEth base
4,4 Ethylenedipiperidine (5.044 x10-4 mol, 0.1 g) was reacted with Synthesis of N-(3-hydroxypropyl)naphthalimide
toluenesulfonyloxypropyl-naphthalamide (1.001 x10-3 mol, 0.41 1,8-naphthalic anhydride (0.050 mol, 10 g) was dissolved in
g, 2.01 excess). Caesium carbonate (3.069 x10-3 mol, 1 g) was dimethylformamide (DMF) (140 mL). Once the 1,8-naphthalic
added in the reaction. All the reagents were dissolved in THF (6 anhydride was totally dissolved, 3-amino-1-propanol (0.050 mol,
mL) and the solution refluxed overnight at 60oC. The reaction was 3.75 g) and 1,8-diazabicylo[5.4.0]undec-7-ene (DBU) (13 mL)
monitored using TLC. Once the reaction was complete, the were added. The reaction was left to stir for 5 hours at 85°C.
solution was poured into icy water (100 mL), which resulting in Reaction completion was monitored with TLC and once
the formation of a precipitate. The precipitate was filtered using completed; the solution was poured into icy water (200 mL) while
a Buchner funnel and the product was left to dry under negative stirring with a glass rod to form a precipitate. The precipitate was
pressure in a vacuum oven at 45oC for 60 minutes (72.3% yield). filtered and washed with water. The pure product was
The crude product was recrystallised from ethanol and the pure characterised using 1H-NMR. Yield was calculated as: 53.9%.
product was characterised by 1H-NMR.
8 | J. Name., 2012, 00, 1-3
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1H-NMR (CDCl3): δH 8.66 – 8.63 (2H, aromatic H, doublet, J=7.2 excess) were dissolved in DMF (8 mL). AfterwarVdisew, AerxticcleesOsnlionef
DOI: 10.1039/C6OB01850E
Hz), 8.29 – 8.25 (2H, aromatic H, doublet, J=8.4 Hz), 7.83 – 7.78 caesium carbonate (1.13 g, 3.5x10-3 mol, 2.5 g excess) was added
(2H, aromatic H, doublet, J=8.0 Hz), 4.41-4.38 (3H, triplet, J=5.6 slowly. The reaction was left to stir for 48 hours at 60°C. After TLC
Hz), 3.64-3.59 (2H, CH2, multiplet) 3.23 (OH, singlet) and 2.06 – confirmed the reaction was complete, the solution was poured
2.00 (2H, CH2 multiplet) ppm.
into icy water (150 mL) to form a precipitate. After vacuum
filtration and several washes with water, the product was left to
dry under negative filtration in a vacuum oven at 45°C for 3 hours
Synthesis of toluenesulfonyloxypropylnaphthalimide
N-(3-hydropropyl)naphthalimide (0.0196 mol, 5.0 g) was (92.2 % yield). The product was recrystallised from ethanol and
dissolved in anhydrous pyridine (70 mL), whilst stirring on ice. characterised by NMR.
Once the N-(3-hydroxypropyl)naphthalimide was completely
dissolved, p-toluenesulfonyl chloride (Ts-Cl) (0.0394 mol, 7.51 g, 1H-NMR (CDCl3): δH 8.50 – 8.47 (2H, aromatic H), 8.18 – 8.15 (2H,
2.01 excess) was slowly added to the reaction. The reaction was aromatic H), 7.72 – 7.67 (2H, aromatic H), , 6.87 (2H, CH-Mts H),
left at 4°C overnight. The reaction was monitored using TLC and 6.55 – 6.53 (2H, CH-Mts H), 3.94 – 3.92 (2H, CH2), 3.12 – 3.10 (2H,
once it was complete, the solution was poured into icy water (200 CH2), 2.87-2.80 (2H, CH2), 2.35-2.32 (3H, CH3), 2.05-2.04 (3H, CH3),
mL) to form a precipitate which was filtered and washed 1.61-1.58 (H, CH), 1.61 (2H,CH2) 1.49-1.37 (2H, CH) and 1.04 (2H,
thoroughly with water. The crude product was recrystallised from CH2) ppm.
ethanol (68.2% yield).
13C-NMR (CDCl3): δH 163.87-162.76 (C=O), 134.36 (C aromatic),
131.40-130.74 (C aromatic), 127.75 (C aromatic), 35.75 (CH2),
1H-NMR (CDCl3): δH 8.59 – 8.57 (2H, aromatic H, doublet, 8.2 Hz), 30.41 (CH2), 21.97 (CH3) and 20.32 (CH3) ppm.
8.25 – 8.23 (2H, aromatic H, triplet, 7.2 Hz), 7.81 – 7.76 (2H,
multiplet), 7.30 (2H, aromatic H, doublet, 1.2 Hz), 4.27-4.20 (2H, Synthesis of bisnaphthalimidopropyl-diamino-
CH2, multiplet), 2.44 (3H, CH3 , singlet) and 2.19 – 2.12 (2H, CH2, dicyclohexylmethane dihydro-bromide salt
multiplet) ppm.
Bisnaphthalimido-dimesityl-dicyclohexylmethane
(3.813x10-4
mol, 0.4 g) was dissolved in dichloromethane (DCM) (8 mL).
Afterwards, hydrobromic acid/glacial acetic acid (HBr/CH3CO2H)
Synthesis of N4,N4-dimesityl-dicyclohexylmethane
4,4’-methylenebis(cyclohexylamine) (4.75x10-3 mol, 1.0 g) was (1 mL) was added drop wise. The reaction was stirred overnight
dissolved in anhydrous pyridine (10 mL) and left to stir for 15 at room temperature. TLC was used to confirm that the reaction
minutes. 2-mesitylenesulfonyl chloride (Mts-Cl) (9.56x10-3 mol; was complete. The precipitate formed, was filtered by vacuum
2.1 g) was added. The reaction was left to stir overnight at room filtration and washed with DCM (15 mL) and ether (5 mL). The
temperature and TLC confirmed completion of reaction. The final product was dried under negative pressure in a vacuum oven
solution was poured into icy water (150 mL) with the formation set at 45°C for 3 hours (37.5% yield).
of a precipitate. The latter was filtered by vacuum filtration, left
to dry in a vacuum oven set at 45°C for 24 hours (43.3% yield).
1H-NMR (CDCl3): δH 8.32 – 8.25 (2H, aromatic Hs), 7.73 – 7.65 (2H,
aromatic Hs), 3.70 – 3.64 (2H, CH2 protons), 3.13 (2H, CH2
1H-NMR (CDCl3): δH 7.30 (NH), 6.97 (4H, CH aromatic protons), protons), 2.90-2.30 (3H, CH3 protons), 2.09-2.02 (3H, CH3
2.99 (4H, CH protons), 2.67-2.66 (6H, CH3-Mts protons) and 1.73- protons), 1.83-1.80 (H, CH proton), 1.43-1.33 (2H, CH2 protons)
1.49 (CH2 and cyclohexane protons) ppm.
ppm.
13C-NMR(CDCl3): δ 163.87-162.76 (C=O), 134.36 (C aromatic),
131.40-130.74 (C aromatic), 127.75 (C aromatic), 35.75 (CH2) and
30.41 (CH2) ppm.
Synthesis of protected bisnaphthalimido-dimesityl-
dicyclohexylmethane
N4,N4-dimesityl-dicyclohexylmethane (6.968x10-4 mol, 0.4 g) and Mass spectrum (HRMS), m/z = 685.3764 (M + H)+ C43H48N4O4
toluenesulfonyloxypropylnaphthalimide (1.400x10-3 mol, 2.01 requires 685.3748 (M + H)+.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
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ARTICLE
Synthesis of trans,trans-N4,N4-dimesityl-dicyclohexylmethane
Journal Name
2.34 (H, CH protons), 2.16-2.15 (2H,CH2 protons) V1ie.w84Art(ic2leHO,nClinHe
DOI: 10.1039/C6OB01850E
trans,trans-4,4’-methylenebis(cyclohexylamine) (2.38x10-4 mol, protons) and 1.73-1.71 (2H, CH2 protons) ppm.
0.05 g) was added in anhydrous pyridine (1.5 mL) and left to stir 13C-NMR (CDCl3): δH 141.93(C=O), 138.76 (C aromatic), 135.17 (C
for 15 minutes with warming. After dissolution, 2- aromatic), 131.95 (C aromatic), 43.72 (CH2), 33.93-31.99 (CH2),
mesitylenesulfonyl chloride (Mts) (4.76x10-4 mol; 0.10 g) was 23.02 (CH3) and 20.97 (CH3) ppm.
added. The reaction was left to stir overnight at room
temperature and TLC confirmed reaction completion. The Synthesis of trans,trans-bisnaphthalimidopropyl-diamino-
solution was poured into icy water (10 mL) and stirred with a glass dicyclohexylmethane dihydro-bromide salt
rod until the formation of a precipitate. The suspension was trans,trans-bisnaphthalimido-dimesityl-dicyclohexylmethane
centrifuged and was washed 3 times with distilled water and the (2.38x10-5 mol, 25mg) was dissolved in dichloromethane (DCM)
precipitate was left to dry under negative pressure in a vacuum (1.0 mL). Afterwards, hydrobromic acid/glacial acetic acid
oven set at 45°C for overnight (21% yield). The product was (HBr/CH3CO2H) (0.2mL) was added slowly. The reaction was
afterwards characterised by 1H-NMR.
stirred overnight at room temperature. TLC was used to confirm
that the reaction was complete. The suspension formed was
1H-NMR (CDCl3): δH 7.31 (NH), 6.98 (4H, CH aromatic protons), transferred to Eppendorf tubes and centrifuged, was with DCM
3.87 (4H, CH protons), 2.67 (6H, CH3-Mts protons), 2.33 (3H, CH3- (1.0 mL) and ether (1.0 mL). The final product was dried under
Mts protons), 1.84-1.82 (CH2-cyclohexane ring), 1.65-1.62 (CH2- negative pressure in a vacuum oven set at 45°C for 3 hours to give
cyclohexane ring), 1.16-1.10 (CH-cyclohexane ring) and 0.99-0.95 the product as a white solid (8.3 mg, 41% yield).
(CH2 cyclohexane protons) ppm.
1H-NMR (CDCl3): δ 8.53 – 8.49 (2H, CH aromatic protons), 4.16 –
Synthesis of protected trans,trans-bisnaphthalimidopropyl
4.12 (2H, CH aromatic protons), 3.42 (2H, CH aromatic protons)
3.04 (2H, CH2 protons), 2.94 (2H, CH2 protons), 2.51-2.49 (3H, CH3
dimesityl-dicyclohexylmethane
trans,trans-N4,N4-dimesityl-dicyclohexylmethane
(6.44x10-5 protons), 2.09-1.99 (3H, CH3 protons), 1.75-1.72 (H, CH proton),
mol, 0.037 g) and toluenesulfonyloxypropylnaphthalimide 1.31-1.28 (2H, CH2 protons) ppm.
(1.29x10-4 mol, 0.053 g, 2.01 excess) were dissolved in DMF (1 13C-NMR(CDCl3): δ 164.26 (C=O), 131.82-131.23 (C aromatic),
mL). Afterwards, excess of caesium carbonate (n = 3.07x10-4 mol, 127.99-127.75 (C aromatic), 122.66 (C aromatic), 42.45 (CH2) and
0.1 g, 5.0 excess) was added slowly. The reaction was left to stir 40.61-37.61 (CH2) ppm.
for 48 hours at 60°C. After TLC confirmed the reaction was Mass spectrum (HRMS), m/z = 685.3732 (M + H)+ C43H48N4O4
complete, the solution was poured into icy water (10 mL) to form requires 685.3748 (M + H)+.
a precipitate. The suspension was centrifuged and was washed
twice with distilled water. The product was left to dry under Ethidium Bromide (EtBr) Fluorescence Displacement studies
negative pressure? in a vacuum oven at 45°C for 24 hours. The BNIPDaCHM, BNIPPiProp, BNIPPiEth and trans,trans-BNIPDaCHM
crude product (59 mg, 88% yield) was purified using column working solutions (100 µM) were prepared from stock solutions
chromatography and the final product (25 mg, 37% yield) was (10 mM in 50% DMSO/H20) and were further diluted to a final
characterised by 1H-NMR.
concentration of 50 µM in 0.01 M saline sodium citrate (SSC)
buffer. Calf Thymus DNA (0.5 g) was dissolved in 0.01 M SSC buffer
1H-NMR (CDCl3): δH 8.65 – 8.59 (2H, CH aromatic protons), 8.30 – (100 mL). EtBr solution (200 µM) was prepared by dissolving 3.94
8.25 (2H, CH aromatic protons), 7.84 – 7.78 (2H, CH aromatic mg of EtBr in distilled water (50 mL) and was further diluted in
protons), 6.65 – (4H, CH-Mts protons), 4.41 – 4.38 (2H, CH2 0.01 SSC buffer to give the final concentration of 20 µM. Test
protons), 4.06 – 4.04 (2H, CH2 protons), 3.71-3.68 (2H, CH2 solutions were prepared by adding varying volumes of SSC buffer,
protons), 2.68 (3H, CH3 protons), 2.46-2.44 (3H, CH3 protons), Calf Thymus DNA solution, EtBr solution and BNIP derivative
solution. The final solutions were thoroughly mixed and analysed
10 | J. Name., 2012, 00, 1-3
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at 510 nm (excitation) and 520 nm (emission) using a Shimadzu consisted of six replicates per plate. Curves were used to
View Article Online
RF-5301 spectrophotometer. The C50 values were determined as represent the percentage growth inhibitioDnOoI:f1M0.1D0A39-M/CB6O-2B30118a5n0dE
the concentration (µM) required to decrease the fluorescence of SKBR-3 cells treated with BNIP derivatives, compared to
DNA bound EtBr by 50%.
DMSO/H2O control that represented 100% cell viability. IC50
values were defined as the drug concentration that reduces
absorbance compared to control values by 50%.
UV binding studies
BNIPDaCHM, BNIPPiProp, BNIPPiEth and BNIPDaCHM working
solutions (100 µM) were prepared from their stock solutions (10 Cell cycle analysis
mM) as before, and were further diluted to 20 µM final MDA-MB-231 and SKBR-3 cells (1 x 106 cells per T75 flask) cells
concentration in 0.01 M SSC buffer. Calf Thymus DNA and 0.01 were washed with PBS and serum free medium added in the
SSC buffer were prepared as described before (EtBr Fluorescence flasks in order to achieve cell synchrony. The cells were incubated
Displacement studies). Test solutions were prepared by adding in serum free medium for 24 hours. BNIP derivatives (IC25
Calf Thymus DNA solution (1 mL) in a quartz cuvette and BNIP concentrations) were added and incubated for 24 hours, at 37°C.
derivative solution (100 µL) was added. The final solutions were After 24 hours treatment, the medium was removed and
thoroughly mixed and analysed at 260 nm using an Agilent 8453 collected. The cells were washed twice with PBS. Both washes
UV-visible spectrophotometer. The values of apparent binding were collected and the cells trypsinised, mixed with the collected
constants (K) were calculated from the intercept and slope by washes and centrifuged at 2,500 rpm for 5 minutes at 4°C. The
plotting A0/ (A-A0) against the BNIP derivative concentrations, supernatant of each sample was discarded, the pellet was
where A0 and A correspond to the absorbance values in absence resuspended in PBS (1 mL) and centrifuged at 2,500 rpm for 5
and presence of a compound.
minutes at 4°C, the supernatant was discarded and the pellet
resuspended in PBS (100 µL). Then, 70% (v/v) ice-cold ethanol
(900 µL) was added and samples incubated for 2 hours at -20°C.
Cell culture maintenance
MDA-MB-231 (ECACC, Public Health England, UK, 92020424) and The cells were centrifuged at 5,000 rpm for 5 minutes at 4°C, the
SKBR-3 (ATCC, HTB-30) cells were maintained in Roswell Park supernatant was discarded and the pellet resuspended in PBS (1
Memorial Institute 1640 medium (RPMI-1640) (containing mL), followed by re-centrifugation at 5,000 rpm for 5 minutes at
GlutaMAX-1 with 25 mM HEPES), supplemented with 10% (v/v) 4 °C. The supernatant was discarded once more and the pellet
Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin (10,000 resuspended in PBS (500 µL) and DNA extraction buffer (0.2 M
µg/mL). Cells were grown at 37oC (5% CO2).
Na2HPO4, 4 mM citric acid, pH 7.8, 500 µL) and incubated for 5
minutes at room temperature. The extracts were centrifuged at
5,000 rpm for 5 minutes at 4 °C, the supernatant was removed
5- and the pellets were resuspended in DNA staining solution (0.2
Cytotoxicity
Colourimetric
3-(4,
5-Dimethylthiazol-2-yl)-2,
diphenyltetrazodium bromide (MTT) assay was performed to mg/mL Ribonuclease A (DNAse-free) and 20 µg/mL propidium
access the growth/inhibitory effects of each BNIP compound. iodide (PI) in PBS). The samples were incubated for 30 minutes at
MDA-MB-231 and SKBR-3 cells (7.5 x 103 cells/100 μL) were 4°C in the dark and PI nuclei were examined by flow cytometry
treated with different concentrations (0-10 μM) of BNIP (EXPO32 ADC XL 4 color, Beckman Coulter, UK). For cell cycle
derivatives. After 24 hour treatment, sterile-filtered MTT solution analysis, EXPO32 ADC analysis software (Beckman Coulter, UK)
(1 mg/mL) was added to each well. After 4 hour incubation at 37 was used to record and analyse 10,000 single events. The
oC, the MTT solution was removed and formazan crystals percentage of cells with DNA content in sub-G1, G1, S and G2/M
solubilised in DMSO. The plates were shaken for 20 minutes at phases was calculated from histograms of linear FL-2 plots (575
room temperature and absorbance measured at 560 nm nm) in the gated region.
(Synergy/HT, BIOTEK, UK). For each compound, three
independent experiments were carried out and each treatment
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
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Journal Name
Statistics
10. P. Kong Thoo Lin and V. Pavlov, Bioorg Med. CheVmie.wLAerttitc.l,e2O0n0lin0e,
DOI: 10.1039/C6OB01850E
Three independent experiments were conducted and each
experiment was comprised of at least two internal replicates.
Data are presented as mean ± SD or mean ± SEM. Statistical
analysis was performed by using an unpaired Student’s t-test.
Statistically significant data were detailed when P<0.05).
10, 1609-1612.
11. V. Pavlov, P. Kong Thoo Lin and V. Rodilla, Chem-Biol Inter,
2001, 137, 15-24.
12. J. Oliveira, L. Ralton, J. Tavares, A. Codeiro-Da-Silva, C.S.
Bestwick, A. MacPherson and P. Kong Thoo Lin, Bioorg. Med.
Chem., 2007, 15, 541-545.
Acknowledgements
13. G.A. Barron, G. Bermano, A. Gordon and P. Kong Thoo Lin, Eur.
J. Med. Chem., 2010, 45, 1430-1437.
The authors acknowledge the Robert Gordon University for financial
support, and the EPSRC National Mass Spectrometry Service Centre
at Swansea University, Swansea for mass spectral analysis.
14. R. Lima, G.A. Barron, J. Grabowska, G. Bermano, S. Kaur, N. Roy,
M. Vasconcelos and P. Kong Thoo Lin, Anti-Cancer Agents Med.
Chem., 2013, 3, 414-421.
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