Triazole-based, optically-pure metallosupramolecules; highly potent and selective anticancer compounds

Article abstract of DOI:10.1039/d0cc02429e

Functionalised triazole aldehydes are used in the highly selective self-assembly of water-compatible, optically pure, low symmetry Fe(ii)- and Zn(ii)-based metallohelices. Sub-micromolar antiproliferative activity is observed against various cancerous cel

Full text of DOI:10.1039/d0cc02429e

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Triazole-based, optically-pure metallosupramolecules; highly
potent and selective anticancer compounds†
Hualong Song,a Nicola Rogers,*,a Viktor Brabec,*,c Guy Clarkson,a James Coverdale,a Hana
Received 00th January 20xx,
Accepted 00th January 20xx
Kostrhunova,c Roger M. Phillips,b Miles Postings, a Samantha L. Shepherdb and Peter Scott*,a
DOI: 10.1039/x0xx00000x
2
1a-e
Functionalised triazole aldehydes are used in the highly selective
self-assembly of water-compatible, optically pure, low symmetry
Fe(II)- and Zn(II)-based metallohelices. Sub-micromolar
antiproliferative activity is observed against various cancerous cell
lines, accompanied by excellent selectivity versus non-cancerous
cells and potential for synergistic combinatorial therapy with
cisplatin.
R
N
N
O
N
N
+ 3
3
O
N
H2N
2 M2+
4+
O
N
N
N
N
Pyridines and other heterocyclic donor ligands are ubiquitous in
multinuclear metal coordination structures1-7 despite the often
cumbersome syntheses of the ligands and the lack of functional
diversity. The 1,2,3-triazoles, available in various forms via
copper(I)-catalysed alkyne-azide cycloaddition/click (CuAAC)
chemistry,8 have recently been employed as ligands in a range
of potential applications.9-15 However, since these heterocycles
are both weaker σ-donors and π-acceptors than e.g. pyridines,12
the metal complexes are inherently less stable to hydrolysis or
competition with coordinating counter-ions such as chloride. In
this context, few triazole-derived metallo-supramolecules have
been reported. Petitjean and co-workers synthesized a family
N
N
R
H
H
N
N
M
N
N
N
M
N
N
N
O
R
O
N
N
N
N
R
(Rc,Fe)-HHT-[Fe2L3a3]Cl4 (R = H)
RC,DFe,HHT-[Fe2L3b3]Cl4 (R = CN)
RC,DFe,HH -[Fe2L3c3]Cl4 (R = OCH3)
RC,DFe,HH -[Fe2L3d3]Cl4 (R = F)
RC,DFe,HH -[Fe2L3e3]Cl4 (R = COOH)
(
,
Sc

Fe)-HHT-[Fe2L3a3]Cl4 (R = H)
-[Fe2L3b3]Cl4 (R = CN)
-[Fe2L3c3]Cl4 (R = OCH3)
-[Fe2L3d3]Cl4 (R = F)
-[Fe2L3e3]Cl4 (R = COOH)
(Rc,Zn)-HHT-[Zn2L3a3][ClO4]4 (R = H)
RC,DFe,HHT-[Zn2L3b3][ClO4]4 (R = CN)
RC,DFe,HHT-[Zn2L3c3][ClO4]4 (R = OCH3)
-[Zn2L3d3][ClO4]4 (R = F)
of M2L3 (M = Fe2+and Ni2+) helicates based on pyridyl-1,2,3-
triazoles.16,
Crowley and co-workers’ Fe(II) systems18
17
RC,DFe,HHT
necessarily have weakly-coordinating counter-ions, so there is Scheme 1 Synthesis of triazole-imine/bipyridine derived triplex metallohelices.
Inter-strand H-bonds indicated by red dashed lines.
little if any solubility in water, and while matters were
addressed successfully by using inert metals,19,20 no significant
oxidation; MnO2 was a more effective in our hands than
biological activity was found. As with most helicates, all the
pyridinium chloro-chromate,24 2-iodoxy-benzoic acid,25 or Jones
above systems are racemic.
reagents.26
We noticed that the triazole aldehydes such as 1 (Scheme 1)
had been little exploited in ligand synthesis,21-23 and to our
knowledge not at all in the metallo-supramolecular arena. The
compounds 1a-e were thus synthesised from propargyl alcohol
Given the challenges noted above in the coordination
chemistry of triazoles we were surprised to find that mixing
aldehydes
1
with enantiomers of 2-([2,2'-bipyridin]-5-
ylmethoxy)-1-phenylethan-1-amine6 (2) in appropriate
proportions with divalent metal cations directly gave the new
optically pure assemblies shown in Scheme 1; reactions were
essentially quantitative according to 1H NMR experiments.
Water-soluble enantiomers [Fe2L3a-e3]Cl4 were synthesised
using Fe(II) chloride, while Zn(II) perchlorate gave the
isostructural analogues [Zn2L3a-d3][ClO4]4.
and the respective benzyl azides using CuAAC, followed by
a. Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
b. School of Applied Sciences, University of Huddersfield, Huddersfield, HD1 3DH.
c. The Czech Academy of Sciences, Institute of Biophysics, Kralovopolska 135, CZ-
61265 Brno, Czech Republic.
†
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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While some of our metallohelices have high antimicrobial
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27
potency,7,
here no significant activDitOyI:a10g.a10in3s9t/DS0.CCA0u24re2u9Es
(ATCC29213) and E. coli (ATCC25922) was observed (minimum
inhibitory concentration ≥ 128 g mL-1 for enantiomers of
[Fe2L3a3]Cl4). Pleasingly, the same compounds were also inactive
in red blood cell haemolysis at concentrations up to 256 g mL-
1 (Table S3, S4, ESI).
The panel of ten Fe(II) compounds (Scheme 1) were initially
screened for cancer antiproliferative potency against the
human colorectal cancer cells with wild-type p53 (HCT116
p53+/+), and for comparison against non-cancerous human
epithelial retinal pigment cells (ARPE-19). We observed very
high activity against HCT116 p53+/+ with IC50 < 500 nM (cf. IC50
of cisplatin = 3.5±1.5 M) for most complexes, with the sole
exception of the tricarboxylic acid enantiomer Δ-[Fe2L3e3]Cl4.
The Ʌ enantiomers were marginally more active than Δ, and the
most potent compound was Ʌ-[Fe2L3a3]Cl4 with IC50 191 ± 10 nM.
In the above screens we noted that complexes [Fe2L3a3]Cl4
were surprisingly stable in water and biological media. In pH 1.5
buffer the compound has t1/2 of ca 4.3 h (Fig. S20, ESI)
comparable with that for Hannon’s “cylinder” of ca 1.4 h at pH
~1.6 At neutral pH, time-dependent photoabsorbance
measurements of Ʌ-[Fe2L3a3]Cl4 solutions (100 μM) reveal a loss
of only 18% intensity at 480 nm (MLCT absorption) over one
week (Fig. S19, ESI). We further tested the IC50 of -
[Fe2L3a3]Cl4 complexes that had been pre-incubated in cell
Fig. 1 1H (500 MHz, CD3CN, 298 K) NMR spectrum of of (Rc,ΔZn)-HHT-[Zn2L3a3][ClO4]4
The presence of three unique ligand environments in the 1H
spectra (e.g. Fig. 1; Fig. S1-S11, ESI) indicates the formation in
each case of a single antiparallel (head-to-head-to-tail, HHT) i.e.
“triplex”6 configuration of the new triazole-containing
metallohelices, with no detectable HHH species or other
diastereomers. Given that components 2 are optically pure, we
know that the new self-assembled compounds are single
enantiomers. This selectivity is thus even greater than that
observed (ca 99% dr) in the related architectures of this kind
based solely on pyridines.6 Signals for the imine protons Ha and
triazole Hc were observed at low fields (9.4-7.8 ppm). Two bpy
protons Hb, assigned by 2D NMR experiments, also appear in
this region as a result of inter-strand H-bonding as shown in
Scheme 1. Two sets of phenyl ring protons Hd and He are also at
unusual chemical shifts (6.8–5.8 ppm) as a result of strong
through-space shielding from the bpy unit of an adjacent ligand.
Methanolic solutions of [Fe2L3a3]Cl4 enantiomers were
o
medium for 24 h (37 C) and observed identical responses to
fresh solutions (Table S2, ESI). The potencies of the ligand
precursors 1a and 2, as well as FeCl2 against HCT116 p53++ were
measured; we found that 1a has minimal activity (IC50 > 70 μM)
and FeCl2 induced no measurable antiproliferative response in
concentrations ≤ 100 M (Table S1 and Fig. S21, ESI), whilst the
two precursor amine enantiomers 2 are both less potent than
their respective metallohelix (by a factor of ca 2 per mole of
ligand).
analysed using circular dichroism absorption spectroscopy,
which show equal and opposite differential extinction
coefficients, typical of enantiomeric pairs. (Fig.S18 ESI).
Significant enantiomeric differences in potency were
observed in the ARPE-19 cells; the Ʌ enantiomers (average IC50
= 1.5 µM) were more potent than the Δ enantiomers (average
IC50 = 6.8 µM) (Fig. 2b). The ligand precursor 1a has modest
antiproliferative activity (IC50 > 89 µM) towards ARPE-19 cells
while the amine enantiomers 2 have IC50 < 10 µM, and both are
more potent than their respective -[Fe2L3a3]Cl4 metallohelix,
per mole of ligand.
Selectivity index (SI), defined as the mean IC50 of ARPE-19
divided by that of HCT116 p53++, are shown in Fig. 2(c).
Excluding for the moment the [Fe2L3e]Cl4 compounds, we see
that the Δ enantiomers exhibit substantially better selectivity (SI
17-34) than Λ enantiomers (SI 5-10). Notably the compound Δ-
[Fe2L3a3]Cl4 has five times the selectivity of its amine precursor
(R)-2. In contrast, enantiomers [Fe2L3e]Cl4 are poorly selective;
the three carboxylic acid groups present in these compounds
will be substantially deprotonated at cell pH. These
observations suggest that the cationic charge is important for
the overall selectivity
Fig. 2 IC50 concentrations of metallohelices [Fe2L3a-e3]Cl4 and ligand precursor (S/R)-
1 against (a) HCT116 p53++ cancer cell line; (b) ARPE-19 (noncancerous cell line). (c)
Selectivity indices i.e. [mean IC50 (ARPE-19) / mean IC50 (HCT116 p35+/+).
Due to the high potency and high selectivity Δ-[Fe2L3a3]Cl4
was selected for screening against a variety of cancer cell lines
of different tissue origins. As seen in Table 1, Δ-[Fe2L3a3]Cl4
2 | J. Name., 2012, 00, 1-3
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Table 2 Combination of Δ-[Fe2L3a3]Cl4, and cisplatin (molar ratio 1:1) were tested with the
showed excellent activity throughout the panel of employed
cell lines with nanomolar IC50 values.
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MTT assay to assess the antiproliferative effect of this combination (IC50, µM) on
DOI: 10.1039/D0CC02429E
cancerous cells. The cells were treated for 72 hours. The results are expressed as the
mean along with the range of the combination indices (CIs, in parentheses) from at least
three independent experiments. CIs were calculated using the CompuSyn software
(Combo SynInc, City, State, USA).
Table 1 Δ-[Fe2L3a3]Cl4, and cisplatin were tested with the MTT assay to assess the
compounds’ antiproliferative effect (IC50, µM) on cancerous and non-cancerous cells. The
cells were treated for 72 hours. The results are expressed as mean ± SD from at least
three independent experiments. a The cells were treated for 96h
Cancerous cell line
Mean CI (range)
Δ-[Fe2L3a3]Cl4+cisplatin (1:1)
0.47 (0.39-0.55)
Cell line
HCT116 p53++
(colon)
MDA-MB-231
(breast)
IC50 (µM)
Δ-[Fe2L3a3]Cl4
HCT116 p53++ (colon)
MDA-MB-231 (breast)
MCF-7 (breast)
cisPt
9 ± 1
0.61 (0.27-0.74)
0.69 (0.46-0.96)
0.5 ± 0.2
0.24 ± 0.05
0.15 ± 0.01
0.8 ± 0.2
HeLa (cervical)
0.75 (0.43-1.08)
29 ± 8
15 ± 1
MCF-7
(breast)
HeLa
The most sensitive cell line was MCF-7 (breast cancer), with
IC50 as low as 151 nM. Δ-[Fe2L3a3]Cl4 is 20 times more active than
cisplatin against HCT116 p53+/+ (colon cancer) and HeLa
(cervical cancer), and 100 times more active than cisplatin
against both MCF-7 and MDA-MD-231 breast cancer cell lines.
The selectivity indices of the six cancerous cell lines in Table
1 with respect to the non-cancerous cell lines ARPE-19 and
MRC-5 pd30 are plotted in Fig. 3. The metallohelix Δ-[Fe2L3a3]Cl4
far outperforms cisplatin across the board, ranging from 5 (HeLa
vs MRC-5 pd30) to 42 (MCF-7 vs ARPE-19) for Δ-[Fe2L3a3]Cl4,
compared with SI < 1 for the majority of the cell lines screened
with cisplatin.
18 ± 4
(cervical)
A2780
0.33 ± 0.01
0.36 ± 0.02
3.7 ± 0.9
3.2 ± 0.5
21 ± 2
(ovarian)
A2780cisR
(ovarian)
MRC-5 pd30
(lung)
aARPE-19
(retinal)
10 ± 3
6.3 ± 0.8
6.4 ± 0.9
Cisplatin is an important agent in the treatment of
numerous malignancies, but its clinical use is limited by side
effects and intrinsic or acquired resistance. Therefore, there is
need for alternatives to cisplatin or for the development of new
treatment procedures, such as combined therapy with other
compounds28, 29 The new metallohelix Δ-[Fe2L3a3]Cl4 exhibited
almost identical activity towards cisplatin-sensitive ovarian
cancer cell line A2780 and its cisplatin-resistant partner
A2780cisR (Table 1), whereas, the resistance factor for cisplatin
is 6.6. Thus, we investigated the effect of combined treatment
of cisplatin and Δ-[Fe2L3a3]Cl4 in four different cell lines (Table
2), at equimolar concentrations (1:1). The data analysis to
determine the combination index (CI) of the used combination
treatment was carried out using the CompuSyn software
(Combo SynInc, City, State, USA). CI < 0.9 is considered as
synergism, CI > 1.1 is considered as antagonism, and CI within
the range (0.9 – 1.1) indicates additive effect. In all probed cell
lines, the mean combination index ranged from 0.47 to 0.75
indicating synergism. In HCT116 p53++ and MDA-MB-231 all
values showed significant synergism while in MCF-7 and HeLa,
the individual CI values fell within the range from synergism to
additive effect.
In this context we make two further mechanistic
observations. First, Real-time Cell Analysis,30 which has
previously been employed to characterize the overall effect of
small compounds on cell growth and morphology, shows a very
different profile for [Fe2L3a3]Cl4 to that of cisplatin in A2780 cells
(Fig. S22, ESI). Secondly, preliminary linear dichroism studies
indicate that the enantiomers [Fe2L3a3]Cl4 are rare examples of
DNA groove-binding metallohelices (logKapp ca 6) (Fig. S23), but
that the orientations of the two enantiomers are different (Fig.
Fig. 3 Selectivity Indices of -[Fe2L3a3]Cl4 for six cancerous cell lines vs two non-cancerous
cell lines – i.e. [mean IC50 (non-cancer cell line)] / [mean IC50 (cancer cell line), compared
with cisplatin.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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11.
12.
13.
14.
15.
16.
R. A. Vasdev, D. Preston and J. D. Crowley, DVaieltwoAnrtTicrleaOnsn.li,ne
S24). This could be the origin of the large enantiomer effects in
selectivity described above.
2017, 46, 2402-2414.
DOI: 10.1039/D0CC02429E
B. Schulze and U. S. Schubert, Chem. Soc. Rev., 2014, 43,
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P. A. Scattergood, A. Sinopoli and P. I. Elliott, Coord.
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Overall we conclude that the new metallohelices reported
here have a number of appealing features. The systems self-
assemble with exceptionally high stereoselectivity to single
enantiomers, whilst the water-soluble Fe compounds are
surprisingly stable to hydrolysis, thus enabling reliable testing in
the biological media, and they are also available with a range of
functional groups. The chemical diversity available via the
triazole aldehydes 1 is far more readily accessible than the
equivalent pyridines or other similar heterocycles, and we hope
that this will allow rapid development of new functionalised
17.
18.
metallo-supramolecular
architectures.
The
current
metallohelices are exceptionally potent against cancer cell lines,
with IC50 values comfortably sub-micromolar, and there is
pronounced selectivity versus the non-cancer cells tested. The
combination therapy tests demonstrate that Δ-[Fe2L3a3]Cl4 and
cisplatin act synergistically; this could decrease the drug dosage
whilst maintaining/increasing efficacy, attenuating toxicity and
slowing down the development of drug resistance.
19.
20.
21.
22.
23.
24.
25.
26.
Acknowledgement
PS thanks the China Scholarships Council and University of
Warwick for a PhD Scholarship, and the Institute of Advanced
Study, Warwick for an Early Career Fellowship (HS). The
research of VB and HK was supported by the Czech Science
Foundation (Grant 18-09502S).
G. L. Goud, S. Ramesh, D. Ashok and V. P. Reddy, Russ. J.
Gen., 2016, 86, 1419-1423.
P. S. Rao, C. Kurumurthy, B. Veeraswamy, G. S. Kumar, Y.
Poornachandra, C. G. Kumar, S. B. Vasamsetti, S.
Kotamraju and B. Narsaiah, Eur. J. Med. Chem., 2014, 80,
184-191.
Conflicts of interest
There are no conflicts to declare.
27.
D. H. Simpson, A. Hapeshi, N. J. Rogers, V. Brabec, G. J.
Clarkson, D. J. Fox, O. Hrabina, G. L. Kay, A. K. King, J.
Malina, A. D. Millard, J. Moat, D. I. Roper, H. Song, N. R.
Waterfield and P. Scott, Chemical Science, 2019, 10, 9708-
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