Synthesis, analysis and biological evaluation of novel indolquinonecryptolepine analogues as potential anti-tumour agents

Article abstract of DOI:10.1039/c5ob02408k

A small library of cryptolepine analogues were synthesised incorporating halogens and/or nitrogen containing side chains to optimise their interaction with the sugar-phosphate backbone of DNA to give improved binding, interfering with topoisomerase II hen

Full text of DOI:10.1039/c5ob02408k

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Organic and Biomolecular Chemistry
ARTICLE
Synthesis, analysis and biological evaluation of novel
indolquinonecryptolepine analogues as potential anti-tumour
agents.
Received 00th January 20xx,
Accepted 00th January 20xx
A. Le Gresley, V. Gudivaka, S. Carrington, A. Sinclair and J. E. Brown
A small library of cryptolepine analogues were synthesised incorporating halogens and/or nitrogen containing side chains
to optimise their interaction with the sugar-phosphate backbone of DNA to give improved binding, interfering with
topoisomerase II hence enhancing cytotoxicity. Cell viability, DNA binding and Topoisomerase II inhibition is discussed for
these compounds. Fluorescence microscopy was used to investigate the uptake of the synthesised cryptolepines into the
nucleus. We report the synthesis and anti-cancer biological evaluation of nine novel cryptolepine analogues, which have
greater cytotoxicity than the parent compound and are important lead compounds in the development of novel potent
and selective indoloquinone anti-neoplastic agents.
DOI: 10.1039/x0xx00000x
www.rsc.org/
Introduction
Cl
CH3
N+
Cryptolepine (1) has been known for some years to possess activity
as an anticancer agent not only by intercalating into DNA but also
by inhibiting the topoisomerase II. This has led to a number of
derivatives being synthesised incorporating halogens at various ring
positions.1,2,3,4,5 Comparison of anti-cancer activity of cryptolepines
with chloro, bromo and iodo atoms at the 11 position showed
considerable variation, with 11-bromo showing an improved
cytotoxicity profile and the 11-chlorinated compound showing a
reduction in the activity compared to the parent compound in the
tested cell lines. Of those synthesized, 7,11dichlorocryptolepine (2)
showed the highest cytotoxic activity against MAC15a (colon
adenocarcinoma) cells with an IC50 value of 14.4 ± 2.8 μM (after 1
hour exposure).7,8
CH3
N+
N
H
N
H
Cl
1
2
Within this manuscript, we report the synthesis of novel
cryptolepineaminoalkyl- and amidoalkyl- derivatives along with
halogenated cryptolepines, including 11-iodocryptolepine. These
compounds were screened for anti-cancer activity in vitro against
cell lines MCF-7, A549 and DLD-1 using doxorubicin as positive
control, data was also obtained pertaining to rates of cellular
uptake and cellular localisation studies. The previously unreported
biophysical and in vitro testing of halogenated cryptolepines and
novel derivatives are discussed in an effort to clarify structure
activity relationship and address the inconsistencies vis mode of
action within the literature.
Whilst the literature is generally in agreement that cryptolepines
are an interesting target for anti-cancer research, the mode of
action is still not that well understood.9 For example, it is observed
that the cytotoxicity of cryptolepine is linked to its ability to inhibit
the topoisomerase II enzyme so it would be expected that bromo
and iodo compounds would show greater topoisomerase II
inhibition owing to their increased lipophilicity. Topoisomerase II
assay results of these 11-halogenated compounds showed they are
responsible for greater inhibition compared to the parent molecule
1, but surprisingly the 11-chloro derivative showed the highest
topoisomerase II inhibition despite its lower in vitrocyototoxicity.
10,11,12It was hyptothesised that this could be due to the changes in
drug-DNA interactions or drug uptake based on the structure and
size of the molecule.13
Materials and methods
Synthetic chemistry
The synthetic techniques used in the synthesis of the chosen targets
are reasonably well known and details of key coupling and
quaternisation reactions are given in more detail under general
procedures. Full spectroscopic data and detailed methodology is
provided in the supplementary information. Scheme 1 indicates the
varied routes taken to achieve the synthesis of a series of amido,
amino and halo-derivatised cryptolepines.
School of Life, Chemical and Pharmaceutical Sciences, SEC Faculty, Kingston
University, Kingston-upon-Thames, KT1 2EE, UK
Electronic Supplementary Information (ESI) available: Synthetic details and
spectroscopic data. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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Journal Name
R'
Cl
NH
H
N
3a
R = Me R' = CH(CH3)2
H
N
R = Me R' = n-Butyl
R = Me R' = n-Pentyl
R = Me R' = CH2CH2NHCH2CH2O
3b
3c
3d
Proc. D
RNH
2
3a-e
+
+
+
N
R
–
R = Et R' = n-Butyl
3e
N
R
–
Cl
Cl
Br
O
Br
N
H
N
H
N
H
N
Proc. B
a
4a
+
N
N
H
–
Cl
CH3
Br
I
H
N
H
N
Proc. B
4b
N
+
N
–
Cl
CH3
O
Cl
Cl
N
H
N
H
N
H
N
Proc. B / Proc A
b
4c-d
N
H
+
N
–
Cl
R''
R'' = Me
R'' = Et
4c
4d
CO H
2
O
NR'''
O
NR'''
H
N
H
N
H
N
Proc. C
Proc. A
R'''NH2
5a-c
+
N
N
+
N
–
Cl
CH3
R''' = CH(CH3)2
R''' = n-Butyl
R''' = CH2CH2NHCH2CH2OH
5a
5b
5c
Scheme 1. Synthetic route for the cryptolepine derivatives. (a) POBr3 PBr3, 16hrs, reflux (b) POCl3, PCl5, 4 hrs reflux. General procedures are
given as Proc. A-D and are discussed in more depth in the methods section: (A) methyl/ethyl triflate, 34hrs, RT (B) MeI, Sulfolane 16hrs RT (C)
COMU, Et3N, DCM 16hrs RT (D) EtOAC , amine, 16 hrs, reflux.
2 | J. Name., 2012, 00, 1-3
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Instrumentation
ARTICLE
General procedure for amide coupling reaction C (5a-5c)
NMR spectra were recorded on Bruker Avance III 400 two-channel To finely ground 10H-indolo[3,2-b]quinoline-11-carboxylic acid,
FT-NMR spectrometer (AV400). It is equipped with a 5 mm BBFO+ dichloromethane and DMF were added and the mixture was stirred
probe (broadband multinuclear, auto-tune, with Z-gradients). for 15 min. NEt3was added drop wise under continuous stirring
Spectra were recorded using TopSpin 3.0 pl4, © 2010 Brüker before adding COMU to the solution, which was then stirred for 10
BioSpin, control and processing software and Icon NMR 4.5.1 Build min. The desired amine was added drop wise and the reaction
51 for TopSpin 3.0 automation software.Mass spectra of mixture was stirred for 16 hours at room temperature.
compounds were obtained using a Waters LCT-TOF- MS instrument
using ESI selective mass detector and Agilent 5973
The reaction mixture was added to ethyl acetate (100 ml) and
extracted with aqueous 1MHCl solution (2x30 ml) and aqueous 1M
GC-MS. High resolution mass spectra of compounds were obtained sodium bicarbonate solution (2x30 ml). Then the organic layers
from Medac labs, UK (BS EN ISO9001:2008) using Waters LCT were collected and dried using magnesium sulphate and
Premier (ES-ToF)/Acquity i-Class mass spectrometer. Infrared evaporated in vacuo to give the crude quindoline amide, which was
spectra were recorded on Thermo Scientific™ Nicolet™ iS™5 FT-IR purified by column chromatography over silica gel using ethyl
Spectrometer. Melting points were measured using Gallenkamp acetate:hexane (1:1) and dried in vacuo to give desired
digital Melting-point apparatus 5A 6797. Up to 350 °C (Division: 0.1 alkylamidoquindoline
°C. 220 V)Unless otherwise stated, all the chemicals used in the
synthesis were purchased from Sigma Aldrich, UK.
General procedure for 11-chloro substitution D (3a-3e)
General Procedures
To
a solution of 11-Chloro-5-methyl(or ethyl)-10H-indolo[3,2-
b]quinolin-5-ium chloride (4c) in ethyl acetate, the desired amine
Specific details of intermediate synthesis and spectroscopic data was added and the contents of the flask were heated to reflux for
are found in supporting information. The below general procedures 16 hours. The reaction mixture was cooled down to room
outline the final synthetic steps for the cryptolepine derivatives as temperature and the precipitate was collected by filtration and
indicated in table 1.
washed with diethyl ether and dried in vacuo to give yellow
coloured crystals.
General procedure for quaternisation A (4d, 5a-5c)
Biological methods
Under dry conditions, 10H-indolo[3,2-b]quinoline (or its 11-Halo
derivative) and methyl/ethyl triflate were stirred in anhydrous General
toluene for 24 hours at room temperature. The reaction mixture
was poured in diethyl ether and the resulting yellow precipitate was
RPMI culture medium, DMEM culture medium, Foetal bovine se-
collected by filtration. Aqueous ammonia solution (3.3% v/v) was
added to the precipitate and the mixture was stirred to convert the
product to its free base form. Then the mixture was extracted using
chloroform (4 x 75ml). The collected organic layers were combined
and evaporated in vacuo to give the crude product which was then
purified by column chromatography on silica gel by using DCM:
methanol: NH4OH (5:1:0.1) as the eluent. Finally the free base was
converted to its hydrochloride salt by neutralising with 0.1 M
methanolic hydrochloric acid (5ml) and then solution was
evaporated in vacuo to give the desired N5-methyl or N5-ethyl
cryptolepine derivative, as a yellow coloured solid.
rum(FBS), penicillin-streptomycin solution ((10,000 U/mL penicillin,
10 mg/mL streptomycin), L-glutamine (200 mM), Trypsin, Trypan
blue solution, MTT, Doxorubicin, Dimethyl sulfoxide (DMSO)
(biological grade) were obtained from Sigma Aldrich, UK. 96 well
culture plates, T75ml flasks, and pipettes were purchased from
Fischer Scientific, UK. Plate reader used to scan the 96 well plates
was Lab-tech LT-4000. The Incubator used to grow the cells was
from Thermo Scientific, UK. The human cell lines, MCF-7, A549 and
DLD-1 cells were kindly provided by the Institute of Cancer
Therapeutics, Bradford University, Bradford, UK.
Cell line culturing
General procedure for quaternisation B (1&4a-4c)
The three cell lines were cultured in RPMI medium supplemented
with 10% heat inactivated foetal bovine serum, 10,000 U/mL
penicillin, 10 mg/mL streptomycin and 200mM L-glutamine. Cells
were grown in 95% humidified air mixed with 5% carbon dioxide at
a fixed temperature of 37 °C. Exhausted growth medium was
replaced with fresh growth medium every two days. When the cells
reached 90% confluence, they were sub-cultured. Firstly, in a
laminar flow chamber, the media was removed and the cell were
washed with 15 ml of PBS and then 2.5 ml of trypsin-EDTA solution
was added then incubated for 5-7 min to allow the cell splitting to
takes place. Then cells were observed under a light microscope
(20x) to check if they were detached. If needed they were tapped
gently to detach from each other. Fresh growth medium (10 ml)
Methyl iodide was added drop wise to a solution of sulfolane and
11-Bromo-10H-indolo[3,2-b]quinoline and stirred under nitrogen
for 16 hours at 50 ºC. The reaction mixture was cooled to room
temperature then ether (25 ml) and methanol (5 ml) were added to
induce precipitation. Diethyl ether (25 ml) was added and then the
mixture was filtered and the solid was dried in vacuo. The solid was
mixed with aqueous sodium carbonate solution (10% w/v, 30 ml)
followed by extraction with chloroform (4x30 ml). The organic
layers were combined and dried using magnesium sulfate and
filtered. The solvent was removed in vacuo to concentrate the
solution which was then treated with a solution of methanolic
hydrochloric acid (0.1% v/v) drop wise to give hydrochloride salt of
cryptolepine as a bright yellow coloured solid.
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Journal Name
was added and the suspension was aspirated using a pasture Cryptolepine and its analogues were initially dissolved in DMSO and
pipette. For further culturing 2ml of that suspension was taken and then diluted to reach the desired concentration. Serial dilutions
added to 20 ml of fresh growth medium in a T75 flask and were carried out to produce a concentration range of 100µM to
incubated.
0.01μM. The maximum DMSO concentration did not exceed 0.1%
v/v. Buffer (2 μL complete reaction buffer = buffer A + buffer B in
1:1 ratio), kDNA (0.25 μg/mL, 1μL), bovine serum albumin (30
μg/mL, 1 μL), distilled water (to make up to 20 μL), drug to be
tested in desired concentration (1 μL) were pipetted out to a
Eppendorf tube and incubated for 30 minutes at 37°C
Growth curves
Growth curves were plotted on three cell lines MCF7, A549 and
DLD-1 to observe the growth characteristics of the cells. The MTT
assay was used to determine the growth of the cells over the course
of time. Cells were seeded in a series at varying densities (500,
1,000, 2000, 4000, 8000, 16000, 32000 and 64000) at a final volume
of 200µL and incubated for 96 hours. Two-hundred microliters of
the growth medium without cells served as the blank for the assay.
Following incubation, the medium was removed and 25µL of the
MTT reagent (concentration) was added to each well and incubated
for 3 hours. The purple formazan crystals were solubilised with
The reaction was then stopped by adding 4μL of stopping buffer
and 2 μL of protein kinase. The mixture was then incubated for
digestion for 30 minutes. The reaction products were then loaded
on to the ethidium bromide-agarose gel and electrophoresis was
carried out at 65V for 2.5 hours in TAE buffer. After the separation,
the gel was visualised under UV illumination.
100μL of DMSO and gentle shaking. Plates were then left in the Fluorescence microscopy
dark for an hour and were then scanned at 570nm using a plate
reader. A graph was plotted with the average absorbance on the Y-
axis and cell count on the X-axis. This was replicated thrice on the
same plate in three rows.
Using BD-Falcon 8 chambered culture slides, A549 cells were grown
in RPMI medium without any indicator. A cell seeding density of
4000 cells per well was determined as optimum and was used for
fluorescence microscopy. After 17 hours of incubation, the chamber
slides were taken out and the medium was carefully removed and
loaded with the desired concentration of the drug solution in RPMI
medium and further incubated for 1 hour. Cells were gently washed
with PBS (37ºC), stained with DAPI nuclear stain (5 mg/ml) and
shielded using a fluoro-shield solution to protect from photo
bleaching. A cover slip was placed on the slide ensuring no air
bubbles formed and sealed. Drug localisation in the cells was
observed under the microscope at x40 magnification. Firstly, images
were captured under normal light at x40 magnification and then
using fluorescent filters, the blue cell nuclei were captured. By
changing the filter to green again the whole cells were observed to
see the drug localisation. Finally both the nuclei and whole cell
images were overlayed using the Leica microscopy imaging
software.
MTT/ATP Assay
DMSO (biological grade) was used to dissolve cryptolepine and its
analogues to reach a drug concentration of 10 mM (stock solution
A). Serial dilutions of stock solution A were prepared (100µM,
10µM, 1µM, 0.1µM, 0.01µM and 0.001µM) at a final volume of
10mL. All of the drug solutions made were stored at -20ºC and
thawed when required for the MTT assay experiment. The final
concentration of DMSO in the working stocks did not exceed 0.1%
v/v. Twenty thousand cells were seeded per well and treatment
applied after 24 hours. After X hours of treatment, the medium
was removed (including control cell-free wells) and the MTT assay
carried out as described in the previous section.
After MTT treatment purple coloured crystals were observed under
the microscope then medium from each well was carefully removed
and 100μL of DMSO were added to dissolve the precipitate and the
In vitro drug-uptake studies
plate was left in the dark for an hour. The obtained raw data was Cryptolepine and its derivatives were dissolved in methanol and the
then analysed using MS-Excel (2010) software and the IC50 values solutions were scanned using a UV spectrophotometer and the λ-
were calculated using the GraphPad Prism software (version 6.0).
max was recorded. Using these wavelength readings, samples were
excited using a fluorescent spectrophotometer to record their
emission wavelengths.
Topoisomerase II inhibition studies
Cells were grown in a 96 well plate for 24 hours in phenol red-free
RPMI medium at a seeding density of 20,000 cells per well. Seven
rows of 3 columns (7x3=21 wells) were loaded with cell suspension
for incubation. Twenty four hours post incubation, media from the
first row of the cells was removed and loaded with 100μL of drug
solution (solvent = media) with a concentration of 10μM and the
plate was placed back in to the incubator. After 30 min. the plate
was removed from the incubator and the media from the second
row was removed and loaded with 100μL of drug solution (10μM).
The same procedure was repeated for the remainder of the rows
every 30 minutes, leaving the last row with no treatment
(blank).After the treatments were complete, the drug solutions
were gently removed and 100μL of DMSO was added to the wells
A topoisomerase II assay kit was purchased from Topogen, Florida,
USA (Topogen, 2005) - kDNA, decatenated kDNA marker, Linear
kDNA marker, gel loading buffer, protein kinase Buffer A: Mixture of
Tris-HCl (pH-8) (0.5M), NaCl (1.50 M), MgCl2 (100 mM),
Dithiothreitol (5mM), bovine serum albumin (BSA) (300µg /mL).
Buffer B: 20mM ATP in water: Drugs: Cryptolepine and its analogues
Eppendorf tubes, Micro pipettes and pipette tips,
A grade
glassware. 1% Agarose gels were prepared (0.75g agarose in 75mL
Tris Buffer) and ethidium bromide (0.5µg/mL) added to the
solution. The solution was allowed to solidify at room temperature
for an hour prior to loading. Running Buffer (TAE buffer): 50x
concentration: Tris base (242 g), glacial acetic acid (57.1 mL) and of
0.5 M EDTA (100 mL). Diluted to 1x
4 | J. Name., 2012, 00, 1-3
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and shaken gently for 30 minutes. The positive control wells were All the concentrations were recorded using the Carry Eclipse
filled with 100μL of 10μM drug solution.
software and the integral graphs were plotted for each ligand
concentration measured. The decrease in percentage of
fluorescence intensity was measured for each ligand concentration
and hence the binding efficiency of the ligand was interpreted.
Cells were lysed using Triton X-100 (a non-ionic surfactant which
increases the cell permeation) and the drug was extracted using
methanol. The plate was then scanned using a fluorescence plate
reader measuring the intensity of the fluorescence from each well Results – Synthesis
(25 scans for each well). The 8th was filled with 10μM drug solution,
which served as a positive control for the experiment. Using a
In addition to probing the halocryptolepine activity, alkylamino side
positive control (100% drug solution), data was analysed and
compared to generate the report of percentage drug uploaded into
the cells over the period of three hours.
chains were attached to increase lipophilicity and improve overall
drug uptake. For the same reason alkylamino side chains with
different lengths were selected to see if there is any correlation
between side chain length and the drug uptake in the cell lines in
vitro.
Drug-DNA binding assay
Duplex DNA (DS26) and quadruplex DNA (AG23) oligonucleotides 11-Chlorocryptolepine served as the key intermediate to make the
were purchased from Eurogenetic, Belgium. Fluorometric titrations derivatives with amino linkages at 11-position via nucleophilic
were performed on Varian-Cary Eclipse fluorescence spectrometer substitution (See general procedure D). In the derivatives with
(scans at 24,000 nm/min, radiation source is Xenon flash lamp with amino linkages, it is unlikely that the secondary amine present on
room light immunity) with carry eclipse spectrophotometric the side chain can be protonated at physiological pH but it is
software. Sodium cacodylate was purchased from Sigma Aldrich, possible that the positive charge arising from the pyridinium
UK. Made using 0.2M sodium cacodylate and 0.2M HC1Thiazole nitrogen (Figure 1) can resonate to transfer the positive charge to
orange was purchased from Sigma Aldrich, UK. Cacodylate buffer the side chain. This structure can potentially better interact with
was made by dissolving sodium cacodylate crystals (42.8 gm) in the negatively charged phosphate backbone of DNA which should
distilled water (1L) and the pH was adjusted to 7.3 using aqueous ultimately lead to improved binding interactions.
HCl solution (0.2 M).The G-quadruplexes were made by heating the
22AG nucleotide in the cacodylate buffer (10mM, pH 7.3), 100mM
KCl at 90°C for five minutes and then the solution was cooled in ice
amido linkages, the positive charge will not be able to move to the
to form G-quadruplexes by intermolecular folding. This duplex
nucleotide sequence was made by heating DS26 in the cacodylate
how having an amide bond between side chains and the aromatic
buffer (10mM, pH 7.3), 100mM KCl at 90°C for five minutes and
then slowly cooling over a period of 6 hours. Once these DNA
solutions were made, the concentrations were measured using the
UV-VIS spectrophotometer at 260nm using their extinction
coefficient values.
Unlike derivatives with amino linkages, in the derivatives with
aromatic ring (Figure 1). So these compounds were made to see
Compound
Yield (%)
N/A
61a
1
3a
3b
3c
3d
3e
4a
4b
4c
4d
5a
5b
5c
68a
Cacodylate buffer containing 100mM of KCl was used for all the
dilutions to get desired concentrations. In a 3mL capacity cuvette,
1mL of thiaozole orange solution and 1mL of DS26 solution were
pipetted and then the volume was adjusted to 3mL using the blank
buffer. The fluorescence was measured using the fluorimeter. This
served as a control for the titration. For the ligand binding
experiments, first 1mL of thiozole orange and 1 mL of DS26 solution
were put into a cuvette and then the total concentration of ligand
was made to 0.375μM by adding the desired ligand and the volume
was made up using the cacodylate buffer. The solution was allowed
to equilibrate for 3 min and then the fluorescence intensity was
measured.
67a
23a
35*a
11
65
56
74*
83a
91a
12a
The same was repeated for each fluorometric titration by increasing
the concentration of ligand to 0.75 μM, 1.5 μM, 3.0 μM, 4.5 μM, 7.5
μM in the cell and keeping the total volume of the cell constant at
3mL.The same procedure was carried out for the G-quadruplex
sequence with the only difference being the concentration of
thiazole orange used which was compared to DS26. The reason for
this is that quadruplex sequence has a smaller number of binding
sites compared to the duplex sequence of DS26.During the titration,
depending on the activity of the ligand, a gradual fall in the
fluorescence was seen as the concentration of the ligand increased.
Table 1. Yields for synthesised halo-, alkylamino- and alkylamido
analogues. * denotes ethyl quaternary ammonium salt rather than
methyl, which appears for the rest. Details of the synthetic
methodology and supporting spectroscopic data are available in
supporting information. a denotes the synthesis compounds first
reported in this paper.
system of cryptolepine will affect the cytotoxicity and the in vitro
drug uptake in cancer cells.
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Cell Viability
After exposing cells to 1 μM or higher It was observed under the
microscope that cells started changing morphology. As the
concentration increased, more cells were lysed and more cell debris
was observed. Compounds 3b and 3c showed a higher level of
activity compared to 3a suggesting that having an alkylamino side
chain is structurally significant. In particular, compound 3b showed
10 times greater activity than the parent compound 1 with an IC50
value of 0.63±0.51 μM. Interestingly 3c derivative showed a greater
activity than 3b for A549 and DLD-1 cells with IC50 values of
0.83±0.28 μM and 2.4±0.96 respectively.
Figure 1.Possible resonance structure for 11-aminocryptolepine
derivatives.
Results - In vitro and biophysical testing
Three different cancer cell lines: MCF-7 (breast cancer) A549 (lung
cancer) DLD-1 (colon cancer) were used to test the anticancer
activity of the synthesised cryptolepine analogues. In addition to
ease of culture and laboratory maintenance, choosing these cell
lines make it possible to compare the results with previous
14a-c
literature.
All three cell lines incubated with cryptolepine 1 for
96 hours, showed anti-proliferative activity. The greatest activity
was against A459 cells with an IC50 value of 0.47±0.12 μM compared
to MCF-7 and DLD-1 cells on which it gave IC50 values of 5.9±0.69
μM and 1.25±0.77 μM respectively (Table 2), which are in
agreement with similar experiments carried out by Gokcek et al on
A-549 and DLD-1 cells and reported IC50 values of 0.55 ± 0.05 μM
and 1.44 ± 0.002 μM.15From the dose response curve it is observed
that no significant activity was seen until a 0.10 μM concentration
of cryptolepine was applied. After that there was a gradual
downward trend until 1.00 μM. Once the concentration reached 10
μM, the percentage cell survival was essentially zero.
Figure 2. Dose-response curve for 1 against MCF 7 (-o-), A549 ( )
and DLD-1 (∆) cancer cell lines
Unsurprisingly, these results indicate the dependence of the activity
in the specific cell line being tested, however the results of testing
against cancer cell lines suggest that alkylamino amino side chains
Compound A549 Cells
IC50 (µM)
MCF-7 Cells DLD-1 Cells Topoisomerase DS26
22AG
Binding
(Kd)
IC50 (µM)
IC50 (µM)
II inhibition
Binding
(Kd)
MIC (µM)
1
0.47 ±0.12
20.38±2.01 17.92±2.31 37.05±2.36
5.90±0.69
1.25±0.77
0.5
-
0.25
-
-
1.62±0.62
2.51±0.33
0.79±0.37
1.26±0.26
2.51±0.58
1.55±0.55
-
0.10±0.06
0.40±0.20
-
2.45±0.36
39.81±5.17
5.01±1.85
6.31±3.15
3.98±1.53
26.92±6.96
-
0.32±0.10
0.63±0.02
-
3a
3b
3c
3d
3e
4a
4b
4c
4d
5a
5b
5c
1.05±0.15
0.63±0.51
4.253±1.88 2.4±0.96
12.23±1.20
12.58±2.12 43.59±4.72 26.73±3.92
2.85±0.87
0.83±0.28
10.41±0.75 6.77±2.03
5.16±1.25
0.28±0.1
2.15±0.41
0.1±0.02
3.35±1.62
0.34±0.05
-
42.83±3.74 62.87±2.71 81.58±2.61 0.25
15.24±2.20 21.00±3.62 31.84±3.86
42.64±3.17 31.72±2.56 75.01±2.92 >100
21.41±1.76 7.26±1.24
12.38±2.16 9.15±1.56
-
-
-
4.14±0.89
15.21±3.23
-
-
2.00±0.74
-
3.55±1.04
-
Table 2.In vitro and biophysical test results for the synthesised derivatives 3a-3e, 4a-4d and 5a-5c for comparison with cryptolepine1.
MIC for topoisomerase II inhibition refers to the minimum inhibititory concentration as per Bonjean et al10
6 | J. Name., 2012, 00, 1-3
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may be useful in forming electrostatic interactions with the The topoisomerase II activities obtained from both compounds 3b
negatively charged DNA backbone.16,17 Addition of a hydrophilic and 4c are significantly higher than that of the parent compound.
motif as in the case of 3d failed to improve upon the anticancer
activity, however it was shown to still be superior to the more
lipophilic 3b derivative.
It is possible that the lack of correlation between Topoisomerase II
inhibition and cytotoxicity may be due to differences in the
pharmacokinetics profile of the compounds in so drug uptake was
The derivative 3d is actually similar to mitoxantrone and showed investigated.
greatest activity on the breast cancer cells with an IC50 value of
6.77±2.03 μM. This selectivity was also observed when Von Hoff
Fluorescence microscopy
and his team tested mitoxantrone on 20 different tumour cell lines,
where greatest activity against breast cancer (MCF-7) cells
compared to DLD-1 cells was observed.18a,19The anti-proliferative
Fluorescence microscopy was used to investigate whether the
derivatives were reaching the nucleus by crossing the cellular
membrane and nuclear envelope. For this experiment A549 cells
activity shown by 4b was most potent, with 4a showing the second
highest activity among all the halogenated compounds tested. The
were selected by observing the morphology under a microscope.
11 halogenated analogues followed a downward trend of activity
Though the cultured cells are in monolayers, MCF-7 and DLD-1 cells
from 11-iodo to the 11-chloro derivative indicating the importance
tend to form clusters in the culture plate whereas A549 cells are
of a lipophilic group in the 11 position. The importance of halogen
evenly spread. Out of the compounds scanned, 4a-4c and 3b
interactions has been reported by Sarwar et al where the potential
emitted high enough fluorescence intensity to facilitate
for encapsulation augments the role of halogen interactions, which
are negligible in comparison with the bulk solvent interactions.18b
fluorescence microscopy. Derivative 3b was selected as one of the
most active and 4c was included with view to identifying the reason
for its low cytotoxicity despite high topoisomerase II activity.
This Van der Waals interaction between iodine and amino
Fluorescence microscopic pictures were taken for both DAPI and
hydrogens is important (3.43 ± 0.33 A) for optimal binding and
the cryptolepine derivatives by exciting them at their respective
selectivity of drug towards that particular DNA sequence.18a
A
excitation wavelengths and then overlaying them to identify co-
localisation.22, 23
gradual decrease in the DNA-binding has been similarly observed
from iodo to fluoro analogues of Calicheamicine. This could mean
that the presence of Iodine atom at 11- position may have been
involved in similar molecular interactions which were contributing
for the DNA binding efficiency of 4b.
Co-localisation of DAPI and 3b in the nucleus of A549 cells indicated
that although 3b was spread all over the cell, high concentrations
are localised at nucleus, which was clearly seen in the pictures as
more bright fluorescence was arising from the nucleus of the cell
which is co-localised with DAPI.
Derivatives 5a-5c showed comparatively less activity than the other
sets. Amongst the amido-derivatives, 5c showed the highest activity
whereas the 5a derivative showed lowest activity. Regardless of
whether it is an amino derivative and an amido derivative, the
isopropyl group reduced the activity significantly, possibly due to
the steric hindrance imparted by the isopropyl functional group.
Amongst all the molecules synthesised, derivatives with 11-
alkylamino linkages and 11-halogenated compounds showed
comparatively greater activity than compounds having 11-amido
side chains.
Topoisomerase II inhibition studies
Cryptolepine has been tested for topoisomerase II activity in the
past using both of the methods outlines above and it has been
proposed that cryptolepine acts as a topoisomerase II poison.20, 21 A
random derivative was selected from each set for Topoisomerase II
testing. When cryptolepine1 was tested for its topoisomerase II
activity it gave a minimum inhibitory concentration value (MIC) of
0.5 μM. The same result was reported by Bonjean and his team on
supercoiled plasmid DNA obtained from Escherichia coli.10
Cryptolepine3b showed the highest topoisomerase II inhibition of
the compounds tested and also the highest overall cytotoxicity. This
continues to support the link between the two. The analogue 5a
gave much lower topoisomerase II activity than the parent
compound; however the 4c derivative failed to confirm this
correlation between Topoisomerase II inhibition and cytotoxicity.
Figure 3.Showing co-localisation of DAPI and drug in the nucleus of
A549 cells. Magnification: 40x, green: 3b (A), blue: DAPI nuclear dye
(B), Overlay of both 3b and DAPI (C)
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to decrease reaching to a 20% at 3 hours this could be due the
oozing of cellular contents along with drug from ruptured cell
membranes of dying cells. This high rapid percentage drug uptake in
to the cells may be the reason for high activity of 3b.
Figure 4. Showing co-localisation DAPI and drug in the nucleus of
A549 cells. Magnification: 40x, green: 11-chloro cryptolepine, blue:
DAPI nuclear dye, Overlay of both drug and DAPI
The same was observed with 4c but the fluorescence intensity
seemed to be lower than 3b (Figure 4). This is supported by similar
experiments conducted by Carl et al to see the cellular localisation
of fluorescent zinc complexes using DAPI as a counterstain.24
These drug localisation experiments only gave information about
whether the drug is present in the nucleus or not. By measuring the
amount of drug taken up by the cells after treatment for particular
time will give quantitative information about the rate of drug
uptake in the cells.
Figure 5. Showing the rate of drug uptake of a range of cryptolepine
derivatives in A549 cells
In the case of 4c, the percentage drug uptake only reached 25% in
the first 30 minutes and then increased to 40% after 2 hours then it
started to decline and reached about 30% after three hours. This
lower uptake rate might be the reason behind the lower
cytotoxicity of 4c. Another important issue in the drug uptake
profile of this compound was that the intracellular concentration of
4c did not fall as quickly as 3b. This may be due to lower cytotoxicity
of the 4c.
In vitro drug uptake studies
To carry out the drug uptake studies A549 cells were selected
because, after drug treatment, they make evenly spread
monolayers. MCF-7 and DLD-1 cells tend to form clusters in the
culture plates this was observed under microscope while
performing cytotoxicity assays. Based on the observed lack of cell
expansion the images suggest apoptosis as the pathway and this is
supported by the literature.24
11-Isopropylaminocryptolepine 3a gave a poor uptake profile with a
percentage uptake of 8% in the first thirty minutes which is 6 times
lower compared to 3b and approximately 4 times lower than the
parent compound 1. The maximum drug uptake for 3a of 18% was
seen at 2.5 hours which is 3 times lower than 3b.
Drug uptake was determined using the method of Kunwar et al.26
Fluorescence intensity was measured and compared using
a
positive control of the same drug solution and result was expressed
as percentage drug uptake. Cryptolepine derivatives from each set
were chosen at random. The amount of fluorescence exhibited by
each drug can be different, so the same drug was taken as positive
control (without any cells being present-100%) percentage drug
uptake was calculated. This percentage drug uptake was directly
proportional to the amount of drug present in the cells.
Drug-DNA Binding
Since cryptolepines have the potential to act as an anticancer agent
by intercalating into DNA, it was felt prudent to investigate binding
efficiencies to see whether the novel derivatives showed improved
binding with respect to 1. The aim was also to better understand
the mode of action to address the counterintuitive relationship
between for example, the high topoisomerase II activity, yet low
cytotoxicity for 4c.
Looking at the in vitro drug uptake results, 3b was taken up into the
cells more quickly than any other compounds tested reaching 40%
within first 30 min after drug treatment, then it continued to rise up
to 60% in 1.5 hours. After 1.5 hours percentage drug uptake started
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Fluorescence displacement assay was selected as method of choice Conclusions
for the current project for its robustness and the fact that we could
use different types of DNA sequences (duplex, quadruplex) to test a
The purpose of this study was to attach halogens (Cl, Br and I) and
amino/amido- alkyl side chains at the 11-position of cryptolepine
and then investigate these molecules for anti-cancer activity using
established in vitro cytotoxicity assay and biophysical studies
group of random cryptolepine derivatives covering the different
classes synthesised.
Using this fluorimetric titration assay, cryptolepine derivatives were including DNA binding assays, topoisomerase II assays, fluorescence
tested for their selectivity and efficiency of DNA binding. This assay microscopy and quantitative drug uptake studies.
was done using two types of DNA sequences; one was a duplex-
forming DNA (DS26) and the other was quadruplex-forming DNA
Of the novel derivatives synthesised the 11-aminoalkyl and 11-halo
(22AG).
cryptolepines (3a-3d&4a-4c resp.) showed greater activity than
those with 11-amido side chains (5a-5c).Overall, the 11-iodo 4b and
22AG is an oligonucleotide which is similar to the human telomeric the 11-butylamino 3b derivative showed the highest activity (Table
sequence. It is a repeat of the [5’-AG3(T2AG3)3-3’] which forms G 2). The reason for the enhanced cytotoxicity of 3b could be the
quadruplex DNA. DS26 is a self-complementary DNA sequence 5’- lipophilic alkyl side chain which may facilitate faster drug-uptake by
CA2TCG2ATCGA2T2CGATC2GAT2G-3’] which form a duplex DNA.24 the cells.
The gradual decrease in the fluorescence arising from displacement
of thiazole orange from quadruplex and duplex DNA is used to
Topoisomerase II results reinforced the link between the
profile the binding
concentration.
upon gradual increase of the ligand
cytotoxicity and topoisomerase II inhibition. Surprisingly, despite its
lower cytotoxicity, the 11-chloro derivative 4c showed the highest
topoisomerase II activity with an MIC value of 0.25 μM. When
Reference to table 2 shows that the 11-halocryptolepines 4b&4c analysing its drug uptake profile, the percentage drug uptake was
had the best DNA binding overall with slightly more selectivity lower than both the 11-butylamino derivative3b and parent
towards duplex DNA over G-quadruplex DNA. The 11-iodo cryptolepine1, reaching an intracellular concentration of only 25%
compound 4b showed the highest DNA binding constant in both in the first 30 minutes. In contrast to this despite its lower
duplex and quadruplex sequences. This reinforces the cytotoxicity topoisomerase II activity, 11-iodocryptolepine 4b (MIC = 0.5 μM)
data as high cytotoxicity was shown by the 11-iodo compound 4b . gave
a
higher cytotoxicity value compared to the 11-
Interestingly the 11-chloro derivative 4c showed better DNA chlorocryptolepine.
binding efficiency than parent molecule despite having a poorer
cytotoxicity profile.
Fluorescence microscopic images showed that, although 3b
was dispersed throughout the cell, high concentrations were
The 11-alkylamino derivatives 3a-3e showed better DNA binding localised in the nucleus. Despite this also being observed in 11-
than 1, especially the molecules 3b and 3c with the 11-butylamino chlorocryptolepine 4c, the amount of drug localised in the
and pentylamino side chains. The same pattern of DNA binding for nucleus is dramatically lower than for 3b. This indicates not
this class of cryptolepine derivatives was observed for the only that a smaller amount of the 4c is being taken up by the
cytotoxicity data. The 11-isopropylamino derivative 3a showed cells but also that it is not reaching the nucleus effectively. We
binding efficiency to G-quadruplex which is 16 times lower than its have also shown that 5-ethyl cryptolepines (3e&4d) have
duplex DNA affinity. When compared to 1, 3b and 3c derivatives substantially lower anti-cancer activity that 5-methyl
showed higher binding efficiency to duplex DNA, implying that the derivatives and make unlikely lead compounds.
compounds with 11-alkylamino side chains prefer duplex DNA over
quadruplex DNA.
We report the synthesis, characterisation and in vitro testing of two
new classes of cryptolepine derivatives and have considered their
Even though derivative 3d with the aminoalcohol side chain did not interaction with topoisomerase II and as DNA intercalators. In
show particularly strong binding to duplex DNA, its quadruplex addition, we highlight that whilst topoisomerase II inhibition is a
binding efficiency is considerably higher than derivatives 3a-3c. potentially important mode of action for cryptolepine anti-cancer
More importantly there is not much selectivity of 3d for duplex or activity, the direct intercalation of duplex DNA as well as cell
quadruplex DNA.
permeability significantly influence the anti-cancer profile of any
such derivative. This is important in deciding what potential
anticancer motifs derivatives of this class of molecule will require to
enhance activity and selectivity. Our group is currently working on
the combination of halogenations and substitution of the
crypolepine with amine bearing moieties
Derivative 5b was selected from the 11-alkylamido class as it
showed higher cytotoxicity compared to the other 11-alkyl amido
derivatives. It showed less binding efficiency towards both duplex
and quadruplex DNAs when compared to 1 but it showed better
quadruplex binding than the 11-alkylamino derivatives implying
that it prefers G-quadruplex structures than duplexes. Overall In conclusion, this work has shown that novel amino and
cryptolepines appear to bind preferentially to duplex DNA over G- halogenated cryptolepine analogues have greater in vitro
quadruplex DNA, this may be because of its selectivity towards GC cytotoxicity than the parent compound and are important lead
rich sequences present in the duplex DNA.
compounds in the development of novel potent and selective
indoloquinone anti-neoplastic agents. Further synthesis of
derivatives which incorporate the amino and halogen groups
may yet result in enhanced activity and potential selectivity for
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G-quadruplex DNA, leading to indirect telomerase inhibition,
which are currently being investigated.
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Peron for excellent support in NMR interpretation. A special
thanks go to Dr Eleanor Polycarpou for proof reading and
useful discussions on the biological aspects.
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