A new generation of ferrociphenols leads to a great diversity of reactive metabolites, and exhibits remarkable antiproliferative properties

Article abstract of DOI:10.1039/c7sc04213b

Organometallic compounds bearing the redox motif [ferrocenyl-ene-phenol] have very promising antiproliferative properties which have been further improved by incorporating pertinent substituents able to engender new mechanisms. Here we show that novel ferrociphenols bearing a hydroxypropyl chain exhibit strong antiproliferative effects, in most cases much better than those of cisplatin, tamoxifen, or of previously described ferrociphenols devoid of this terminal OH. This is illustrated, in the case of one of these compounds, by its IC₅₀ values of 110 nM for MDA-MB-231 triple negative breast cancer cells and of 300 nM for cisplatin-resistant A2780cisR human ovarian cancer cells, and by its GI₅₀ values lower than 100 nM towards a series of melanoma and renal cancer cell lines of the NCI-60 panel. Interestingly, oxidative metabolism of these hydroxypropyl-ferrociphenols yields two kinds of quinone methides (QMs) that readily react with various nucleophiles, such as glutathione, to give 1,6- and 1,8-adducts. Protonation of these quinone methides generates numerous reactive metabolites leading eventually to many rearrangement and cleavage products. This unprecedented and fully characterized metabolic profile involving a wide range of electrophilic metabolites that should react with cell macromolecules may be linked to the remarkable profile of antiproliferative activities of this new series. Indeed, the great diversity of unexpected reactive metabolites found upon oxidation will allow them to adapt to various situations present in the cancer cell. These data initiate a novel strategy for the rational design of anticancer molecules, thus opening the way to new organometallic potent anticancer drug candidates for the treatment of chemoresistant cancers.

Full text of DOI:10.1039/c7sc04213b

View Article Online
View Journal
Chemical
Science
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: Y. Wang, P. M.
Dansette, P. Pigeon, S. Top, M. J. McGlinchey, D. Mansuy and G. Jaouen, Chem. Sci., 2017, DOI:
10.1039/C7SC04213B.
This is an Accepted Manuscript, which has been through the
Volume
7 Number 1 January 2016 Pages 1–812
Royal Society of Chemistry peer review process and has been
accepted for publication.
Chemical
Science
Accepted Manuscripts are published online shortly after
www.rsc.org/chemicalscience
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 2041-6539
EDGE ARTICLE
Francesco Ricci et al.
Electronic control of DNA-based nanoswitches and nanodevices
rsc.li/chemical-science

Page 1 of 21
Chemical Science
View Article Online
DOI: 10.1039/C7SC04213B
A new generation of ferrociphenols leads to a great
diversity of reactive metabolites, and exhibits
remarkable antiproliferative properties
Yong Wang,1,2 Patrick M. Dansette,3 Pascal Pigeon,1,2 Siden Top, 2* Michael J. McGlinchey,4 Daniel
Mansuy,3* and Gérard Jaouen1,2*
1PSL, Chimie ParisTech, 11 rue Pierre et Marie Curie, Fꢀ75005 Paris, France
2Sorbonne Universités, UPMC Univ Paris 6, UMR 8232 CNRS, IPCM, place Jussieu, Fꢀ75005 Paris,
France
3Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS,
Université Paris Descartes, PRES Paris Cité Sorbonne, 45 rue des Saints Pères, 75270 Paris Cedex 06,
France
4UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4,
Ireland
Eꢀmail: gerard.jaouen@upmc.fr, siden.top@upmc.fr, Daniel.Mansuy@parisdescartes.fr
1

Chemical Science
Page 2 of 21
View Article Online
DOI: 10.1039/C7SC04213B
Abstract
Organometallic compounds bearing the redox motif [ferrocenylꢀeneꢀphenol] have very promising
antiproliferative properties which have been further improved by incorporating pertinent substituents
able to engender new mechanisms. Here we show that novel ferrociphenols bearing a hydroxypropyl
chain exhibit strong antiproliferative effects, in most cases much better than those of cisplatin,
tamoxifen, or of previously described ferrociphenols devoid of this terminal OH. This is illustrated, in
the case of one of these compounds, by its IC50 values of 110 nM for MDAꢀMBꢀ231 triple negative
breast cancer cells and of 300 nM for cisplatinꢀresistant A2780cisR human ovarian cancer cells, and by
its GI50 values lower than 100 nM towards a series of melanoma and renal cancer cell lines of the NCIꢀ
60 panel. Interestingly, oxidative metabolism of these hydroxypropylꢀferrociphenols yields two kinds of
quinone methides (QMs) that readily react with various nucleophiles, such as glutathione, to give 1,6ꢀ
and 1,8ꢀadducts. Protonation of these quinone methides generates numerous reactive metabolites
leading eventually to many rearrangement and cleavage products. This unprecedented and fully
characterized metabolic profile involving a wide range of electrophilic metabolites that should react
with cell macromolecules may be linked to the remarkable profile of antiproliferative activities of this
new series. Indeed, the great diversity of unexpected reactive metabolites found upon oxidation will
allow them to adapt to various situations present in the cancer cell. These data initiate a novel strategy
for the rational design of anticancer molecules, thus opening the way to new organometallic potent
anticancer drug candidates for the treatment of chemoresistant cancers.
Keywords. antitumor agents, glutathione, ferrocifens, bioorganometallics, quinone methides
2

Page 3 of 21
Chemical Science
View Article Online
DOI: 10.1039/C7SC04213B
Introduction
Despite recent advances in the treatment of certain cancers (for example, controlled targetting of the
tumour, nanocapsule formulations, new biopharmaceutical agents with specific activity) the number of
fatalities linked to cancers whose outcomes remain poor (e.g. melanoma, pancreatic and ovarian cancer,
tripleꢀnegative breast cancer, glioma etc.), and which do not respond well to proapoptotic stimuli,
continues to rise.1ꢀ4 This has encouraged exploration of the potential role of inorganic compounds in this
area, following the success of the coordination complexes of Pt, often in oxidation state +II, for which
DNA is a primary but not the only target, and which are now in regular use.5ꢀ8 Owing to a number of
known issues with these complexes (relatively narrow therapeutic window, serious resistance and
toxicity problems), recent research has turned towards organometallic complexes, principally those of
gold and the Fe, Ru, Os triad, a novel approach that is now undergoing exponential growth.9ꢀ19 These
compounds often target the mitochondrial system or inhibit kinases or redox proteins overexpressed in
cancer cells. Thus, they provide a new channel that offers a complementary solution to that of the
platinum derivatives, bringing different targets and mechanisms of action into play.
Bioactive ferrocenes have provided a number of examples over the last few years to illustrate this
approach, using a variety of synthetic strategies.20ꢀ31 The ferrociphenols stand out from the crowd of
current candidates owing to the advanced state of biological studies in the area, and also because of the
rich variety of their mechanisms of action.32ꢀ36 This richness is due to their ability to generate the redox
motif {ferrocenylꢀeneꢀphenol} in the cancer cell. The motif’s activity can be modulated by various
substitutions, thus permitting a diversity of possible targets, an effect that is known to delay or prevent
phenomena of drug resistance to a given biomolecule.
In particular we have been able to show that hydroxytamoxifen 1, known for its antagonist effect on
MCFꢀ7 hormoneꢀdependent (ERα+) breast cancer cells and for its lack of activity on hormoneꢀ
independent (ERαꢀ) MDAꢀMBꢀ231 breast cancer cells, becomes strongly antiproliferative on both
3

Chemical Science
Page 4 of 21
View Article Online
DOI: 10.1039/C7SC04213B
ERα+ and ERαꢀ cells when modified to become the ferrociphenols 2a (Figure 1).37 The IC50 of 2a on
MDAꢀMB231 (ERαꢀ) cells is 0.5 µM, and a cytostatic effect occurs via senescence.38, 39 This
mechanism represents an alternative strategy to apoptosis in halting cell proliferation, and bypasses the
problem of the resistance of cancers to proapoptotic stimuli. In addition, in those cancer cells for which
apoptosis is possible, the apoptotic pathway is still operative. Moreover, compound 2a interacts with a
redox target overexpressed in certain cancers such as thioredoxine reductase.40
The basic chain in 2a can be removed to give the ferrociphenol, 2b, whose cytostatic effect on MDAꢀ
MBꢀ231 cells is comparable to that of 2a. Subsequently, we were able to show that quinone methides 2-
QM, arising from the initial oxidation of ferrocene FeII to the corresponding ferricinium FeIII species,
are active metabolites of compounds 2 (Figure 1).41ꢀ43 These compounds are active on cancer cells but
not on healthy cells (astrocytes, melanocytes) where the redox effect is not observed.44, 45 The
effectiveness of these two compounds, 2a and 2b, delivered by lipid nanocapsules (LNCs), has been
studied on experimental subcutaneous models of tripleꢀnegative breast cancer and malignant glioma,
respectively. In both cases, a significant slowing of tumor progression was observed, confirming the
antiproliferative activity of these compounds.46ꢀ48
Figure 1. Hydroxytamoxifen 1, ferrociphenols 2 and their derived quinone methides 2-QM,
hydroxypropylꢀferrociphenols 3.
In our search for a new series of molecules surpassing the antiproliferative effect of the above
products we have recently obtained promising results by introducing functional groups at the end of the
alkyl chain.49 In particular, the hydroxypropyl derivative 3b (Figure 1) exhibited exceptional
4

Page 5 of 21
Chemical Science
View Article Online
DOI: 10.1039/C7SC04213B
antiproliferative activity against inter alia liver hepatocellular carcinoma cells (HepG2) and triple
negative breast cancer cells (MDAꢀMBꢀ231) with IC50 values of 0.07 and 0.11 µM, respectively.49
Chemical oxidation of 3a/3b yielded an unprecedented tetrahydrofuranꢀsubstituted quinone methide
(QM), 4a/4b, via an internal cyclization of the hydroxylꢀalkyl chain (Figure 2). The ferrocenyl group in
3 plays a key role not only as an intramolecular reversible redox “antenna” but also as a stabilized
carbenium ion “modulator”.49 Moreover, the presence of the oxygen heterocycle in 4 enhances its
stability and leads to a unique chemical evolution profile, leading to products 5, 6, 7, and 8 (Figure 2)
that were characterized spectroscopically (NMR, HRMS) or by Xꢀray crystallography.49
To further explore the promising antiproliferative properties of compounds 3a and 3b and their
unique evolution profile upon oxidation, we have studied the fate of these compounds upon chemical
oxidation or after metabolism by liver microsomes, in the presence of nucleophiles such as thiols that
are present in proteins and nucleic acids. The antiproliferative properties of compounds 3 and their
metabolites towards breast cancer cells were evaluated. Moreover, we have compared the activities of
3b, 2b and cisplatin towards ovarian cancer cell lines sensitive or resistant to cisplatin. The
antiproliferative effects of 3b were also evaluated by the National Cancer Institute on the NCIꢀ60
human tumor cell line panel.50 The obtained data showed a quite remarkable profile of antiproliferative
activities of 3b, and the formation of a surprisingly great diversity of reactive metabolites upon
metabolic oxidation of 3b.
Results
Chemical oxidation of the hydroxypropyl-ferrociphenols, 3a and 3b. To compare the behaviour of
the acyclic QMs, such as 2b-QM, and the tetrahydrofuranꢀsubstituted QMs 4a and 4b, we have
explored the effects of several oxidants (including Ag2O, MnO2, H2O2) on their precursors 3a and 3b.
As shown in Figure 2, generation of the QMs 4, followed by addition of 1N HCl aqueous solution in
acetone, not only led to the previously reported molecules 5-8, but also to two new products, 9, the 1,6ꢀ
5

Chemical Science
Page 6 of 21
View Article Online
DOI: 10.1039/C7SC04213B
adduct of water to 4, and 10, the product of ferrocenyl migration in a pinacolꢀtype rearrangement
(Figure S1). In contrast, protonation of 4b with HCl in dry diethyl ether yielded only two major
products, 5b (35 %) and 6b (47 %). Compounds 7 and 8 were major products only when the protonation
was carried out in acetone, a wellꢀknown solvent for triplet state photosensitized processes.51
HO
OH
HO
O
1N HCl in
acetone
Fe
Fe
OH
12b (~1%)
OH
OH
OH
11b (trace to 10 %)
O
OH
O
O
Ag2O
OH
3b
HO
OH
acetone
Fe
Fe
O
Fe
O
OH
1N HCl in
acetone
OH
9b (18 %)
5b (10 %)
6b (28 %)
Fe
OH
OH
O
OH
O
O
OH
4b (> 90%)
Fe
Fe
7b (25 %)
8b (23 %)
10b (18%)
OH
OH
Figure 2. Quinone methides 4b and 11b from the oxidation of hydroxypropylꢀferrociphenols 3b and
subsequent protonation (similar results were obtained in the case of 3a). Yields are based on starting
compound 3b, but note that cleavage of the CꢀC bond of 9b yields both 7 and 8.
The novel products 10, arising from the protonation of 4, can come from a 1,2ꢀmigration of the
ferrocenyl moiety, thus generating a cation stabilized by the adjacent oxygen in the fiveꢀmembered ring
(Figure S1). These rearrangement products, in which the 2,3ꢀdihydrofuran ring and the ferrocenyl
moiety are attached to the same carbon center, were unequivocally characterized spectroscopically, and
by Xꢀray crystallography in the case of 10b whose structure appears in Figure 3.
6

Page 7 of 21
Chemical Science
View Article Online
DOI: 10.1039/C7SC04213B
O2
C19
O3
C16
C1
C25
C6
C2
O1
C3
Fe
C5
C4
C11
Figure 3. Molecular structure of 10b (thermal ellipsoids are shown at 50%).
In addition to the products described above, the hydroxyꢀalkenyl quinone methides 11 (Figure 2),
1
analogous to the vinylic 2-QM, were also formed, and identified by HPLC and H NMR, even though
their existence was somewhat transient. Thus, in the case of 11b for instance, the appearance of a
multiplet and a triplet at 1.60 and 6.43 ppm, respectively, indicated the formation of a double bond
within the starting hydroxypropyl group (Figure S3). The yield of 11b (varying from trace to 10%,
relative to 4b, as monitored by 1H NMR) was dependent on the concentration of its precursor 3b (from
2 to 100 mM), and the quantity of oxidant (one to five equivalents of Ag2O). Indene product 12b
resulting from an acidꢀcatalyzed cyclisation of the hydroxyꢀalkenyl quinone methide 11b, as previously
found in the case of vinylic quinone methides, 2-QM,52 (Figure S2) was also formed in low amounts
upon oxidation of 3b. The identities of the new QM, 11b, and its corresponding indene, 12b, were also
confirmed by their characteristic UVꢀVIS spectra (λmax ~560 nm and ~320 nm for vinyl QMs and indene
derivatives, respectively.53).
In this case, we found for the first time that chemical oxidation of the hydroxypropylꢀferrociphenol, 3,
yields two kinds of quinone methides. Since the tetrahydrofuranꢀQMs 4, and the hydroxyalkenylꢀQMs
11, arise from a common precursor, a ferrocenyl cation 13 (Figure 4) initially generated by a twoꢀ
electron oxidation of 3, the observed 4/11 ratio is the result of a competition between intramolecular
7

Chemical Science
Page 8 of 21
View Article Online
cyclization with subsequent deprotonation to form 4, versus deprotonation to generDaOteI: 1t0h.1e039c/oC7nSjCu0g42a1t3eBd
carbonꢀcarbon double bond in 11. This ratio and the proportions of the observed products 5-12 were
greatly dependent on the nature of the used oxidant and acid, and on the amounts of oxidant relative to 3.
Reactions of the tetrahydrofuran-substituted QMs 4a and 4b toward nucleophiles. The reactions of
this new type of electrophilic intermediate with various nucleophiles, including thiols such as
mercaptoethanol (ME), NꢀacetylꢀLꢀcysteine methyl ester (NACM) and glutathione (GSH), were studied.
Compounds 3a or 3b were first treated with silver oxide to generate 4a or 4b at room temperature and,
after removal of excess Ag2O, the nucleophiles were added. In each case, progress of the reaction was
monitored by TLC or HPLC.
Methanol reacted via 1,6ꢀaddition to yield the pinacolꢀtype methyl ethers, 14, in good yield (Figure 4).
By contrast, ME in acetone gave a poor yield of the 1,6ꢀadducts, 15. However, both the rate of reaction
and the yield of 15 were greatly enhanced by addition of NaOH to form the thiolate. Analogous
reactions with NACM and glutathione in the presence of NaOH led to the corresponding 1,6ꢀadducts, 16
and 17, respectively, in very good yields (Figure 4). The adducts 14-16 were characterized by 1H NMR
spectroscopy and mass spectrometry. Complete purification of 17 was not possible because of the high
polarity of the GS function, and a crude sample was obtained.
When treated under more physiological conditions (50 mM phosphate buffer, pH 7.4, 37 °C), 4b led
after 30 min to the aforementioned products 5b-10b in low to moderate yields. Under these conditions,
the halfꢀlife of 0.15 mM 4b was approximately 25 min. However, incubation of 4b under these
conditions in the presence of ME, NACM or GSH gave the corresponding 1,6ꢀadducts, 15b, 16b or 17b,
respectively, in yields greater than 80%. In the presence of these thiols, the halfꢀlife of 4b, as monitored
by UVꢀVIS spectroscopy, was markedly reduced to 1.5, 3.2 and 2.3 min, respectively. It is noteworthy
that all these 1,6ꢀadducts were progressively transformed into 4ꢀhydroxyꢀ1ꢀferrocenylbutanꢀ1ꢀone 7 and
8

Page 9 of 21
Chemical Science
View Article Online
diarylmethanone 8 (Figure S4ꢀ8). However, they were stable for at least a week in theDaObI:s10e.n10c3e9/Co7fSCa0i4r2a13nBd
light.
Figure 4. 1,6ꢀ and 1,8ꢀadducts to quinone methides 4 and 11.
Oxidation of compounds 3a and 3b by horseradish peroxidase (HRP). Incubation of 3a and 3b with
HRP and H2O2 for 30 min (0.25 mM 3, 0.1% HRP and 4 equivalents of H2O2) led to products that have
been observed upon chemical oxidation of 3a and 3b and that were found to derive from quinone
methides 4 and 11. The proportions of these products varied as a function of the used conditions
(3/HRP/H2O2 ratios, reaction time) (Tables S2ꢀ3). However, compounds 7 and 8 were always the major
oxidation products, and the products deriving from the hydroxyalkenylꢀQM 11 were only formed in
trace amounts. When low amounts of HRP and H2O2 were used (0.1 mM 3, 0.02% HRP and one
equivalent of H2O2) their total yield increased to about 10%.
Oxidative metabolism of compounds 3a and 3b by liver microsomes. Incubation of 3a or 3b with rat
liver microsomes in the presence of NADPH, the reducing agent necessary for the activity of
microsomal monooxygenases, led to the formation of all the products that were observed upon oxidation
of 3a or 3b by chemical oxidants or HRP and H2O2 (Tables S2ꢀ3). They were characterized by
comparison of their HPLC retention times, and also their MS and MSꢀMS characteristics, with those of
authentic samples obtained by chemical synthesis. None of these metabolites were observed under
identical incubations without NADPH. Incubations of 3a and 3b with human liver microsomes under
9

Chemical Science
Page 10 of 21
View Article Online
DOI: 10.1039/C7SC04213B
identical conditions led to results very similar to those obtained with rat liver microsomes. Ketones 7
and 8 were always the major products and represented more than 40% of all metabolites, whereas
dihydropyran 5 was formed in lower amounts and compounds 6, 9 and 10 were formed in trace amounts.
These data showed that microsomal oxidation of 3 mainly led to products deriving from QM 4.
However, minor formation of metabolites 12 and 18, that derived from the hydroxyalkenyl quinone
methide 11, showed that both QM 4 and 11 were formed as initial oxidation products. It is noteworthy
that a decrease of the microsomal proteins concentration in the incubate led to an increase of the ratio of
18 relative to all other metabolites (from less than 5 % to about 10 % when decreasing the protein
concentration by a factor 1.7). This result is to be compared to analogous data obtained in oxidation of 3
with chemical oxidants or HRP and H2O2, and indicates that the intermediate formation of QM 4 is the
major pathway occurring in oxidation of 3, but that the relative importance of the formation of QM 11 is
greater under milder oxidizing conditions (use of lower oxidant or oxidation catalyst amounts).
Incubation of ferrociphenols 3a and 3b with rat liver microsomes in the presence of NADPH and
various thiols, led to QMꢀthiol adducts, as well as the metabolites we have already observed under
identical conditions in the absence of thiols (Tables S2ꢀ3). All the 1,6ꢀ and 1,8ꢀthiol adducts (15-17 and
19-21, respectively, Figure 4) arising from their corresponding QM precursors were detected in trace to
moderate amounts, and were characterized by comparison of their HPLC retention times and MS
spectra with those of authentic samples. The 1,6ꢀadducts are less polar and exhibit longer retention
times than their 1,8ꢀadduct counterparts; moreover, their mass spectral fragmentation patterns were also
significantly different (Table S4). In microsomal incubations of 3 in the presence of ME or NACM,
ketones 7 and 8 were still the major products and represented more than 40% of all metabolites, whereas
the proportions of 1,6ꢀthiol adducts relative to all metabolites remained below 10% and only low
amounts of 1,8ꢀthiol adducts were observed under these conditions. Interestingly, incubation of 3 with
liver microsomes, in the presence of NADPH and GSH, gave 17 as a major GSH adduct (40% of all
metabolites) with a minor amount of the 1,8ꢀGSH adduct 21. Upon increasing the protein concentration,
10

Page 11 of 21
Chemical Science
View Article Online
DOI: 10.1039/C7SC04213B
the relative proportion of 7 and 8 increased and 17 was still formed in an about 5%~10% yield relative
to all metabolites whereas 21 was only formed in trace amounts (Tables S2ꢀ3). Accordingly, incubation
of authentic 1,6ꢀthiol adducts 15-17 with liver microsomes and NADPH mainly led to 7 and 8. The
stability of these 1,6ꢀthiol adducts towards microsomal oxidation is as follows: 17 > 16 > 15, suggesting
that the size and polarity of the thiol substituents adjacent to the tetrahydrofuran ring play an important
role in their microsomal oxidation into 7 and 8.
Antiproliferative effects of 3a and 3b and their oxidation metabolites against TNBC MDA-MB-
231 cells. The antiproliferative activities of compounds 3a and 3b, and of the products derived from
their oxidation in the presence or absence of thiols, against hormoneꢀrefractory breast cancer MDAꢀMBꢀ
231 cells, are shown in Table 1. Compounds 3a and 3b were the most active, with IC50 values of 1.1 and
0.11 ꢁM, respectively. However, the transient intermediates appearing during their oxidative
metabolism, the quinone methides 4, their water 1,6ꢀadducts 9, and their thiol 1,6ꢀadducts, 15 and 16,
were also active with IC50 values between 2 and 10 ꢁM; these data are in sharp contrast with the very
low activity of the final major metabolites 7 and 8 that exhibited IC50 values above 100 ꢁM. These IC50
values of intermediates 4, 9, 15 and 16 are quite remarkable, especially if one takes into account their
instability in the assay medium, and possible problems of cell penetration. This suggests that they
should have potent antiproliferative effects when they are formed inside the cell, close to important cell
targets, upon oxidative metabolism of 3.
Table 1. IC50 values for selected ferrocenyl compounds towards MDAꢀMBꢀ231 cells.
Compound
2b
IC50 (ꢁM) a
0.64 ± 0.06
1.16 ± 0.02
1.89 ± 0.08
8.17 ± 1.56
6.12 ± 0.97
7.51 ± 0.52
4.36 ± 0.26
Compound
IC50 (ꢁM) a
2.03 ± 0.79
4.14 ± 1.33
≈ 150
5b b
6b b
3a b
4a (QM) b
7 b
8b b
≈ 295
6a
9.92 ± 0.78
9.60 ± 1.29
9a (1,6-OH)
15a (1,6-ME)
16a (1,6-NACM)
10b
9b (1,6-OH)
14b (1,6-OMe)
3.07 ± 0.01
11

Chemical Science
Page 12 of 21
View Article Online
DOI: 10.1039/C7SC04213B
3b b
0.11 ± 0.02
4.39 ± 1.47
2.36 ± 0.14
15b (1,6-ME)
16b (1,6-NACM)
4b (QM) b
2.43 ± 0.4
(a) Measured after 5 days of culture (mean of two independent experiments ± SD); (b) Values taken
from ref. 49.
Cytotoxicity studies of compounds 2b and 3b against cisplatin-sensitive and cisplatin-resistant
human ovarian cancer cell lines. The cytotoxic activities of the vinyl derivative, 2b, and the
hydroxypropyl complex, 3b, were further evaluated against two human ovarian cancer cell lines,
cisplatinꢀsensitive A2780 and cisplatinꢀresistant A2780cisR cells (Tables 2). Their activities toward a
healthy human lung fibroblast cell line, MRCꢀ5, were also measured. The A2780cisR variant is known
to encompass all of the known major mechanisms of resistance to cisplatin:54 decreased drug transport,
enhanced DNA repair/damage tolerance, elevated cellular thiol (GSH) level, and blockage of cellꢀdeath
pathways. As shown in Table 2, 2b and 3b showed a potent antiproliferative activity not only against
wildtype A2780, but also against resistant A2780cisR cells, with a resistance factor smaller than one,
contrary to cisplatin (CDDP) that exhibits a resistance factor of about ten.55 It is noteworthy that 3b
showed a 7ꢀfold greater cytotoxicity than 2b against A2780 and A2780cisR cells, and a 38ꢀfold greater
cytotoxicity than cisplatin against A2780cisR cells. These observations suggest that compound 3b may
be much less affected by high cellular GSH levels or other physiological adaptations occurring in
resistant cancer cells. Moreover, 3b is approximately sixteen times less toxic towards normal human
cells MRCꢀ5 than towards A2780 cancer cells, whereas 2b and cisplatin have lower selectivity factors
of approximately 2 and 8.5, respectively.
Table 2. IC50 values for compounds 2b, 3b and cisplatin (CDDP) towards human ovarian cancer cells
and normal human fibroblast cells.
IC50 (ꢁM) a
IC50 (ꢁM) a
MRCꢀ5
Compound
RF b
SF c
A2780
A2780cisR
2.3 ± 0.7
3.5 ± 0.94
0.66
0.57
9.6
7.28 ± 0.39
8.54 ± 0.32
16.2 ± 0.6
2.1
16.1
8.5
2b
3b
0.53 ± 0.01 0.30 ± 0.03
1.2 ± 0.20 11.5 ± 0.3
CDDP d
12

Page 13 of 21
Chemical Science
View Article Online
DOI: 10.1039/C7SC04213B
(a) Measured after 72 hrs of culture (mean of three independent experiments ± SD); (b) RF, Resistance
Factor: IC50 toward A2780cisR / IC50 toward A2780 ratio; (c) SF, Selectivity Factor: IC50 toward MRCꢀ
5 / IC50 for A2780 ratio; (d) Values taken from ref. 55.
Antiproliferative effects of 2b and 3b on the NCI cell line panel. The antiproliferative effects of 3b
were further evaluated by the National Cancer Institute on the NCIꢀ60 human tumor cell line panel,50
which consists of approximately 60 cell lines within nine tumor type subpanels. They were compared to
those found for compound 2b,56 tamoxifen (TAM) and cisplatin (CDDP) (Figure 5 and Table S5ꢀ6). The
cells were treated for 48 h at five concentrations ranging from 0.01 to 100 ꢁM. Three endpoints were
calculated: GI50 (50% growth inhibition concentration), TGI (100% growth inhibition concentration),
and LC50 (50% lethal concentration). Compounds 2b and 3b exhibited a broad spectrum of activity over
a 3ꢀlog range of GI50, which means that these compounds are not indiscriminately cytotoxic against
cellular growth. Compounds 2b and 3b (mean GI50 around 0.5 ꢁM and 0.4 ꢁM respectively) are
approximately tenꢀfold and thirtyꢀfold more potent (in terms of GI50 values) than tamoxifen and
cisplatin, respectively (Table S6). Compound 3b shows a high potency towards a wide range of cancer
cell lines, with a particularly high activity against renal, melanoma and CNS cancer cell lines. Thus, its
GI50 values for M14, SKꢀMELꢀ5, UACCꢀ62 (3/8 melanoma subꢀlines) and 786ꢀ0, A498, ACHN, CAKIꢀ
1 (4/8 renal cancer subꢀlines) were found to be lower than 100 nM. Renal cancers are often
chemoresistant because of the high expression of multiꢀdrug resistant (MDR1) protein, and elevated
level of Pꢀglycoprotein in renal cancer cells.57, 58 Melanoma is a highly aggressive neoplasm and has a
poor response to conventional chemotherapy,59 meaning that intrinsic drug resistance is also a serious
problem in melanoma treatment. The high potency of compound 3b towards the phenotypes of renal
cancer and melanoma is promising for the use of such organometallic complexes for the treatment of
such resistant cancers.
13

Chemical Science
Page 14 of 21
View Article Online
DOI: 10.1039/C7SC04213B
GI50
Class
Cell line
MCF7
3b
2b
TAM CDDP
Class
Cell line
3b
2b
TAM CDDP
SF-268
SF-295
< 0.1
0.1-0.5
0.5-2
2-10
MDA-MB-
Breast
HS 578T
BT-549
SF-539
CNS
SNB-19
T-47D
SNB-75
U251
> 10
A549/ATCC
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
786-0
COLO 205
HCC-2998
HCT-116
HCT-15
KM12
Lung
Colon
SW-620
IGROV1
OVCAR-3
OVCAR-4
A498
ACHN
Ovarian
OVCAR-5
OVCAR-8
SK-OV-3
CAKI-1
Renal
RXF 393
SN12C
NCI/ADR-
TK-10
LOX IMVI
M14
UO-31
CCRF-CEM
HL-60(TB)
MOLT-4
RPMI-8226
SR
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
MDA-MB-435
Melanoma
Leukemia
Prostate
PC-3
DU-145
Figure 5. Heat map for GI50 values of 3b, 2b, TAM and CDDP. The deep red color indicates the highest
activity, whereas the deep blue color represents the lowest activity.
Discussion
The novel ferrociphenols 3 bearing a terminal OH group in their alkyl moiety exhibit strong
antiproliferative properties, very frequently much better than those of ferrociphenols 2, cisplatin or
tamoxifen. This is illustrated, in the case of 3b, by its IC50 values of 110 nM for MDAꢀMBꢀ231 breast
cancer cells (Table 1) and of 530 nM for A2780 human ovarian cancer cells (Table 2), and by its GI50
values lower than 100 nM towards a series of renal cancer and melanoma cells (Figure 5). Interestingly,
compound 3b is quite active towards cisplatinꢀresistant A2780cisR ovarian cancer cells (Table 2).
Ferrociphenols 3 also exhibit a remarkable profile of metabolic oxidation, when compared to
ferrociphenols 2. This is due to the formation, after their twoꢀelectron oxidation, of a new kind of QMs
14

Page 15 of 21
Chemical Science
View Article Online
4, involving an oxygenꢀcontaining heterocycle, in addition to the vinyl QMs 11 (FiguDrOeI:41)0,.10a3n9a/Cl7oSgCo04u2s13Bto
those formed upon oxidation of ferrociphenols 242 (Figure 1 and S2). To the best of our knowledge, this
is the first case involving such two chemotypes of QM pathways from a single substrate, whether in
organic or organometallic chemistry. We found that this occurred not only upon oxidation by chemical
oxidants such as Ag2O, but also by enzymatic systems such as HRP in the presence of H2O2, or
cytochrome P450ꢀdependent monooxygenases that are present in liver microsomes. Evolution of QMs 4
in the incubation medium leads to a surprisingly great diversity of reactive intermediates and final
metabolites. After protonation, they lead to rearrangement products such as 5, 6, and 10, and pinacolꢀ
type products 9 (Figure 2). In incubations performed in the presence of an added nucleophile, such as
thiols ME, NACM, or GSH, this nucleophile can compete with H2O for reaction with protonated 4 to
give 1,6ꢀthiol adducts 15-17 (Figure 4). The cleavage of the central CꢀC bond of intermediates 9, 14, 15,
16, and 17 further leads to the final stable products 7 and 8. The mechanism of this CꢀC bond cleavage
remains to be determined. However, one might tentatively propose two possible mechanisms. Firstly,
photocleavage of the CꢀC bond leading to a radical pair has been already proposed (Figure S1).51
Another viable mechanism could be a oneꢀelectron oxidation of the phenol function, followed by betaꢀ
cleavage of the intermediate radical leading to a radical alpha to the ferrocene ring and a new quinone,
the hydrolysis of which would lead to 8. Further oxidation of the latter radical would lead to 7 (Figure 6).
The particularly high yields of 7 and 8 observed upon oxidation of 3 by liver microsomes in the
presence of NADPH would be in favour of the second mechanism, because the radicals formed by oneꢀ
electron oxidation of the phenol function of compounds 9 and 15-17 by highꢀvalent Fe=O cytochrome
P450 species would be generated inside the enzyme active site, and be efficiently oxidized by the FeIVꢀ
OH intermediate to give 7 and 8.
15

Chemical Science
Page 16 of 21
View Article Online
DOI: 10.1039/C7SC04213B
Figure 6. Possible mechanism for the oxidative cleavage of ferrocenyl pinacolꢀtype compounds by liver
microsomes in the presence of NADPH.
Comparison of the oxidative metabolism of the hydroxypropylꢀferrociphenols 3 with that of
ferrociphenols 2 reveals that a much greater number of reactive, electrophilic intermediates are formed
in the former case. This includes the tetrahydrofuranꢀQMs, 4, and all the cationic intermediates involved
in the formation of 5, 6 and 9. Upon protonation, QMs 4 were found to react with a variety of oxygenꢀ
and sulfurꢀcontaining nucleophiles, such as water, methanol, mercaptoethanol, NꢀacetylꢀLꢀcysteine
methyl ester and glutathione, to give the corresponding 1,6ꢀadducts 9, 14, 15, 16, and 17. When formed
inside the cell, these QMs should covalently react with protein and nucleic acid nucleophiles. The
radical intermediates thought to be involved in the formation of 4ꢀhydroxyꢀ1ꢀferrocenylbutanꢀ1ꢀone, 7,
and the diaryl ketones, 8, from 9 and 17 could also react with cell macromolecules. Moreover,
compounds 5, 6, 9, and 15-17 themselves were also found to have significant antiproliferative properties
against breast cancer cells (Table 1).
It is thus likely that the remarkable antiproliferative properties of ferrociphenols 3 are linked to the
great diversity of reactive intermediates and metabolites formed during their oxidative metabolism. In
that regard, the interesting activity of 3b towards human ovarian A2780cisR cisplatin resistant cancer
cells (Table 2), whose resistance mechanism would refer to an elevated cellular thiol (GSH) level, could
be related to a high level of GSH adduct, 17b, that should exhibit antiproliferative activity. In a more
general manner, the great diversity of reactive metabolites formed upon oxidative metabolism of 3
should allow them to be more aggressive and cytotoxic for cancer cells that are known to have a higher
16

Page 17 of 21
Chemical Science
View Article Online
DOI: 10.1039/C7SC04213B
level of oxidative stress than normal cells. This great diversity of reactive metabolites that could bind to
various cell targets should allow them to adapt themselves to various situations existing in cancer cells
(Figure 7). It is wellꢀknown that some chemotherapy drugs exert their anticancer effects by forming a
diversity of reactive metabolites; typically, 5ꢀfluorouracil is widely used in the treatment of a range of
solid tumors, and its mechanism involves the intracellular conversion to several active metabolites.60, 61
The effective cure of acute promyelocytic leukemia by the inorganic drug arsenic trioxide (As2O3) is
also believed to involve its in vivo conversion to yield a diversity of active metabolites.62
cytotoxicity
Figure 7. The diversity of reactive intermediates and metabolites involved in the oxidation of
ferrociphenols 3.
Further optimization concerning in vivo toxicity, mode of administration, pharmacokinetics and
pharmacodynamics are currently in progress to maximize the full potential of the hydroxypropylꢀ
ferrociphenols for clinical application. The hydroxypropylꢀferrociphenols 3 not only yield a great
diversity of reactive species under metabolic oxidation, but should also be capable of finding the
preferential metabolic pathway to accommodate the redox microenvironment of cells. This appears to be
the first report of small molecules that are able to achieve a kind of "selfꢀregulation" in terms of the
virtual redox microenviroment. This unprecedented metabolic profile may initiate a new strategy for the
17

Chemical Science
Page 18 of 21
View Article Online
rational design of anticancer molecules based on prodrugs, thus opening the wDaOyI: 1t0o.103n9e/Cw7SCp04o2t1e3nB t
organometallic drug candidates for the treatment of chemoresistant cancers.
Acknowledgements. Y.W. thanks the PGG foundation, PSL University and Feroscan for financial
support, and we thank Geoffrey Gontard (IPCM, CNRS UMR 8232) for the Xꢀray structure
determinations, Jérôme Bignon (Plateforme CIBI, ICSN, Gif) for cytotoxicity tests, the National Cancer
Institute Developmental Therapeutics Program for in vitro testing, Barbara McGlinchey for linguistic
assistance and Fatima MechtaꢀGrigoriou (Insitut Curie, Inserm U830) for helpful disscussions.
Supplementary Information. Experimental procedures for syntheses and biological evaluation,
supplementary figures 1ꢀ8 and tables 1ꢀ6, Xꢀray crystallographic data, cif file. This material is available
free of charge via the Internet at http://www.rsc.org.
References
1.
2.
3.
N. S. Gandhi, R. K. Tekade and M. B. Chougule, J. Control. Release, 2014, 194, 238-256.
E. Hutchinson, Molecular Oncology, 2014, 8, 1-8.
F. X. Gu, R. Karnik, A. Z. Wang, F. Alexis, E. Levy-Nissenbaum, S. Hong, R. S. Langer and O. C. Farokhzad,
Nano Today, 2007, 2, 14-21.
4.
M. O. Palumbo, P. Kavan, W. H. Miller, Jr., L. Panasci, S. Assouline, N. Johnson, V. Cohen, F. Patenaude, M.
Pollak, R. T. Jagoe and G. Batist, Front. Pharmacol., 2013, 4, 57.
5.
6.
B. Rosenberg and L. Vancamp, Cancer Res., 1970, 30, 1799-1802.
B. Lippert, Cisplatin : Chemistry and Biochemistry of a Leeding Anticancer Drug, John Wiley and Sons,
New York, 1999.
7.
8.
S. Dhar and S. J. Lippard, Proc. Natl. Acad. Sci. USA, 2009, 106, 22199-22204.
P. Zhang and P. J. Sadler, J. Organomet. Chem., 2017, 839, 5-14.
9.
N. P. E. Barry and P. J. Sadler, Chem. Commun., 2013, 49, 5106-5131.
A. A. Nazarov, C. G. Hartinger and P. J. Dyson, J. Organomet. Chem., 2014, 751, 251-260.
G. Gasser, I. Ott and N. Metzler-Nolte, J. Med. Chem., 2011, 54, 3-25.
C. G. Hartinger, N. Metzler-Nolte and P. J. Dyson, Organometallics, 2012, 31, 5677-5685.
V. Brabec, S. E. Howson, R. A. Kaner, R. M. Lord, J. Malina, R. M. Phillips, Q. M. A. Abdallah, P. C.
McGowan, A. Rodger and P. Scott, Chem. Sci., 2013, 4, 4407-4416.
10.
11.
12.
13.
14.
15.
16.
M. Dörr and E. Meggers, Current Opinion Chem. Biol., 2014, 19, 76-81.
L. Oehninger, R. Rubbiani and I. Ott, Dalton Trans., 2013, 42, 3269-3284.
M. A. Cinellu, I. Ott and A. Casini, in Bioorganometallic Chemistry, eds. G. Jaouen and M. Salmain, Wiley-
VCH, 2015, ch. 4, pp. 117-140.
17.
18.
19.
20.
21.
K. D. Mjos and C. Orvig, Chem.Rev., 2014, 114, 4540-4563.
J. J. Soldevila-Barreda, I. Romero-Canelón, A. Habtemariam and P. J. Sadler, Nat. Commun., 2015, 6, 6582.
G. Jaouen and N. Metzler-Nolte, Medicinal Organometallic Chemistry, Vol. 32, Springerlink ed., 2010.
S. S. Braga and A. M. S. Silva, Organometallics, 2013, 32, 5626-5639.
B. Balaji, B. Balakrishman, S. Perumalla, A. A. Karande and A. R. Chakravarty, Eur. J. Inorg. Chem., 2015, 8,
1398-1407.
18

Page 19 of 21
Chemical Science
View Article Online
DOI: 10.1039/C7SC04213B
22.
23.
24.
O. Payen, S. Top, A. Vessieres, E. Brule, M. A. Plamont, M. J. McGlinchey, H. Muller-Bunz and G. Jaouen, J.
Med. Chem., 2008, 51, 1791-1799.
K. Kowalski, L. Szczupak, L. Oehninger, I. Ott, P. Hikisz, A. Koceva-Chyla and B. Therrien, J. Organomet.
Chem., 2014, 772, 49-59.
J. Amin, I. S. Chuckowree, M. Wang, G. J. Tizzard, S. J. Coles and J. Spencer, Organometallics, 2013, 32,
5818-5825.
25.
26.
P. James, J. Neudoerfl, M. Eissmann, P. Jesse, A. Prokop and H.-G. Schmalz, Org, Lett,, 2006, 8, 2763-2766.
H. V. Nguyen, A. Sallustrau, J. Balzarini, M. R. Bedford, J. C. Eden, N. Georgousi, N. J. Hodges, J. Kedge, Y.
Mehellou, C. Tselepis and J. H. R. Tucker, J. Med. Chem., 2014, 57, 5817-5822.
A. Mooney, R. Tiedt, T. Maghoub, N. O'Donovan, J. Crown, B. White and P. T. M. Kenny, J. Med. Chem.,
2012, 55, 5455-5466.
27.
28.
29.
S. B. Deepthi, R. Trivedi, L. Giribabu, P. Sujitha and C. G. Kumar, Dalton Trans., 2013, 42, 1180-1190.
S. Daum, V. F. Chekhun, I. N. Todor, N. Y. Lukianova, Y. V. Shvets, L. Sellner, K. Putzker, J. Lewis, T. Zenz, I. A.
M. de Graaf, G. M. M. Groothuis, A. Casini, O. Zozulia, F. Hampel and A. Mokhir, J. Med. Chem., 2015, 58,
2015-2024.
30.
31.
32.
P. F. Salas, C. Herrmann, J. F. Cawthray, C. Nimphius, A. Kenkel, J. Chen, C. de Kock, P. J. Smith, B. O.
Patrick, M. J. Adam and C. Orvig, J. Med. Chem., 2013, 56, 1596-1613.
R. Pulukkody, R. B. Chupik, S. K. Montalvo, S. Khan, N. Bhuvanesh, S.-M. Lim and M. Y. Darensbourg,
Chem. Commun., 2017, 53, 1180-1183.
P. Messina, E. Labbe, O. Buriez, E. A. Hillard, A. Vessieres, D. Hamels, S. Top, G. Jaouen, Y. M. Frapart, D.
Mansuy and C. Amatore, Chem. Eur. J., 2012, 18, 6581-6587.
33.
34.
35.
36.
G. Jaouen, A. Vessieres and S. Top, Chem.Soc. Rev., 2015, 44, 8802-8817.
S. Top, J. Tang, A. Vessieres, D. Carrez, C. Provot and G. Jaouen, Chem. Commun., 1996, 955-956.
G. Jaouen, S. Top, A. Vessieres, G. Leclercq and M. J. McGlinchey, Curr. Med. Chem., 2004, 11, 2505-2517.
S. Top, A. Vessieres, G. Leclercq, J. Quivy, J. Tang, J. Vaissermann, M. Huche and G. Jaouen, Chem. Eur. J.,
2003, 9, 5223-5236.
37.
G. Jaouen and S. Top, in Advances in Organometallic Chemistry and Catalysis: The Silver/Gold Jubilee
International Conference on Organometallic Chemistry Celebratory Book, ed. A. J. L. Pombeiro, Wiley,
2014, ch. 42, pp. 563-580.
38.
39.
40.
A. Vessieres, C. Corbet, J. M. Heldt, N. Lories, N. Jouy, I. Laios, G. Leclercq, G. Jaouen and R. A. Toillon, J.
Inorg. Biochem., 2010, 104, 503-511.
C. Bruyere, V. Mathieu, A. Vessières, P. Pigeon, S. Top, G. Jaouen and R. Kiss, J. Inorg. Biochem., 2014,
141, 144-151.
A. Citta, A. Folda, A. Bindoli, P. Pigeon, S. Top, A. Vessieres, M. Salmain, G. Jaouen and M. P. Rigobello, J.
Med. Chem., 2014, 57, 8849-8859.
41.
42.
E. Hillard, A. Vessières, L. Thouin, G. Jaouen and C. Amatore, Angew. Chem. Int. Ed., 2006, 45, 285-290.
Y. Wang, M.-A. Richard, S. Top, P. M. Dansette, P. Pigeon, A. Vessières, D. Mansuy and G. Jaouen, Angew.
Chem. Int. Ed., 2016, 55, 10431-10434.
43.
D. Hamels, P. M. Dansette, E. A. Hillard, S. Top, A. Vessieres, P. Herson, G. Jaouen and D. Mansuy, Angew.
Chem. Int. Ed., 2009, 48, 9124-9126.
44.
45.
Q. Michard, G. Jaouen, A. Vessieres and B. A. Bernard, J. Inorg. Biochem., 2008, 102, 1980-1985.
E. Allard, C. Passirani, E. Garcion, P. Pigeon, A. Vessières, G. Jaouen and J.-P. Benoit, J. Control. Release,
2008, 130, 146-153.
46.
47.
48.
49.
50.
E. Allard, D. Jarnet, A. Vessieres, S. Vinchon-Petit, G. Jaouen, J. P. Benoit and C. Passirani, Pharm. Res.,
2011, 27, 56-64.
A. L. Laine, E. Adriaenssens, A. Vessieres, G. Jaouen, C. Corbet, E. Desruelles, P. Pigeon, R. A. Toillon and C.
Passirani, Biomaterials, 2013, 34, 6949-6956.
A. L. Laine, A. Clavreul, A. Rousseau, C. Tétaud, A. Vessieres, E. Garcion, G. Jaouen, R. A. Toillon and C.
Passirani, Nanomedicine: NBM, 2014, 10, 1667-1677.
Y. Wang, P. Pigeon, S. Top, M. J. McGlinchey and G. Jaouen, Angew. Chem. Int. Ed., 2015, 54, 10230-
10233.
R. H. Shoemaker, Nat. Rev. Cancer, 2006, 6, 813-823.
19

Chemical Science
Page 20 of 21
View Article Online
DOI: 10.1039/C7SC04213B
51.
52.
I. G. Gut, P. D. Wood and R. W. Redmond, J. Am. Chem. Soc., 1996, 118, 2366-2373.
M.-A. Richard, D. Hamels, P. Pigeon, S. Top, P. M. Dansette, H. Z. S. Lee, A. Vessières, D. Mansuy and G.
Jaouen, ChemMedChem, 2015, 10, 981-990.
53.
V. Scalcon, A. Citta, A. Folda, A. Bindoli, M. Salmain, I. Ciofini, S. Blanchard, J. D. Cazares-Marinero, Y.
Wang, P. Pigeon, G. Jaouen, A. Vessieres and M. P. Rigobello, J. Inorg. Biochem., 2016, 165, 146-151.
D. Wang and S. J. Lippard, Nat. Rev. Drug Discov., 2005, 4, 307-320.
I. Romero-Canelon, L. Salassa and P. J. Sadler, J. Med. Chem., 2013, 56, 1291-1300.
M. Görmen, P. Pigeon, S. Top, E. A. Hillard, M. Huché, C. G. Hartinger, F. de Montigny, M.-A. Plamont, A.
Vessières and G. Jaouen, ChemMedChem, 2010, 5, 2039-2050.
54.
55.
56.
57.
58.
59.
60.
61.
H. T. Cohen and F. J. McGovern, N. Engl. J. Med., 2005, 353, 2477-2490.
W. T. Bellamy, Annu. Rev. Pharmacol. Toxicol., 1996, 36, 161-183.
D. Grossman and D. C. Altieri, Cancer Metastasis Rev., 2001, 20, 3-11.
D. B. Longley, D. P. Harkin and P. G. Johnston, Nat. Rev. Cancer, 2003, 3, 330-338.
P. Álvarez, J. A. Marchal, H. Boulaiz, E. Carrillo, C. Vélez, F. Rodríguez-Serrano, C. Melguizo, J. Prados, R.
Madeddu and A. Aranega, Expert Opin. Ther. Pat., 2012, 22, 107-123.
62.
G.-Q. Chen, L. Zhou, M. Styblo, F. Walton, Y. Jing, R. Weinberg, Z. Chen and S. Waxman, Cancer Res.,
2003, 63, 1853-1859.
20

Page 21 of 21
Chemical Science
View Article Online
DOI: 10.1039/C7SC04213B
TOC
Two chemotypes of quinone methide pathways from a single substrate are reported, which may be linked to its
remarkable antiproliferative activity.
21