Ferrocenyl 2,5-Piperazinediones as Tubulin-Binding Organometallic ABCB1 and ABCG2 Inhibitors Active against MDR Cells

Article abstract of DOI:10.1021/acsmedchemlett.6b00046

The tubulin-microtubule system is a common target of many anticancer drugs. However, the use of chemotherapeutics frequently leads to the development of a clinically relevant phenomenon of multidrug resistance (MDR). One of the basic mechanisms involved in MDR involves elevated expression and/or activity of several ATP-binding cassette superfamily members (ABC transporters) which are normally responsible for the efflux of xenobiotics or secondary metabolites outside the cell. Here we present the synthesis and biological characteristics of ferrocenyl analogues of plinabulin, i.e. one of the so-called "spindle poisons". We found that replacement of the phenyl group of plinabulin by the ferrocenyl moiety turns this compound into a potent inhibitor of ABCB₁ and ABCG₂, thus making it possible to overcome the multidrug resistance phenomenon. We also demonstrated that the alkyl group attached to the imidazole moiety of ferrocenyl analogues of plinabulin strongly affects their potency to inhibit tubulin polymerization.

Full text of DOI:10.1021/acsmedchemlett.6b00046

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Letter
Ferrocenyl 2,5-piperazinediones as tubulin-binding organometallic
ABCB1 and ABCG2 inhibitors active against MDR cells
Anna Wieczorek, Andrzej B#au#, Janusz Zakrzewski, Blazej Michal Rychlik, and Damian Plazuk
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00046 • Publication Date (Web): 22 Feb 2016
Downloaded from http://pubs.acs.org on February 23, 2016
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Ferrocenyl 2,5-piperazinediones as tubulin-binding organometallic
ABCB1 and ABCG2 inhibitors active against MDR cells
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Anna Wieczorek,a,b Andrzej Błauż,b Janusz Zakrzewski,a Błażej Rychlik,b* Damian Plażuka*
aDepartment of Organic Chemistry, Faculty of Chemistry, University of Łódź, Tamka 12, 91-403 Łódź, Poland. Fax:
(+48)42 6786583 ; Tel: (+48)42 6355760
bCytometry Lab, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University
of Łódź, Pomorska 141/143, 90-236 Łódź, Poland
KEYWORDS: ferrocene, cancer, ABCB1 inhibitor, ABCG2 inhibitor, multidrug resistance, bioorganometallic chemistry
ABSTRACT: The tubulin-microtubule system is a common target of many anticancer drugs. However, the use of
chemotherapeutics frequently leads to the development of a clinically relevant phenomenon of multidrug resistance
(MDR). One of the basic mechanisms involved in MDR involves elevated expression and/or activity of several ATP-
binding cassette superfamily members (ABC transporters) which are normally responsible for the efflux of xenobiotics or
secondary metabolites outside the cell. Here we present the synthesis and biological characteristics of ferrocenyl
analogues of plinabulin, i.e. one of the so-called ‘spindle poisons’. We found that replacement of the phenyl group of
plinabulin by the ferrocenyl moiety turns this compound into a potent inhibitor of ABCB1 and ABCG2, thus making it
possible to overcome the multidrug resistance phenomenon. We also demonstrated that the alkyl group attached to the
imidazole moiety of ferrocenyl analogues of plinabulin strongly affects their potency to inhibit tubulin polymerization.
Microtubules are cytoskeleton elements composed of
α- and β-tubulin heterodimers which are involved in a
number of cellular process e.g. cell motility, intracellular
transport and mitosis. Compounds interacting with the
dynamic tubulin-microtubule system are used as
chemotherapeutic drugs against many types of
neoplasms, including ovarian, breast, head & neck and
pancreatic cancers.1 Widely used are taxanes, e.g.
paclitaxel2 and its semisynthetic analogue docetaxel,3
which can induce polymerization of tubulins and
stabilize microtubules. Another example are the Vinca
alkaloids vincristine and vinblastine, which promote
depolymerization of the microtubule back to α- and β-
tubulin.4 Some other naturally occurring inhibitors of
tubulin polymerization, e.g. colchicine,5 are not used in
therapy due to their high toxicity.
cells..10 Plinabulin is currently being studied in a phase II
clinical trial on non-small cell lung cancer.11
One of the biggest problems in cancer chemotherapy
is the development of resistance to anticancer drugs.
During the first course of chemotherapy, drugs kill only
drug-sensitive cancer cells, while some cancer cells
survive and the tumor begins to grow again.
Additionally, the mechanism by which these survivor
cells have escaped the therapy is enhanced by negative
selection, so all tumor cells become drug-resistant and
the treatment fails. Although there are multiple
mechanisms underlying this phenomenon, one of the
most important of these seems to involve the
transmembrane transport proteins of the ATP-binding
cassette superfamily (ABC transporters).12 In normal
cells, these proteins are responsible for the efflux of
xenobiotics or secondary metabolites outside the
cytoplasm. Their overexpression in cancer cells leads to
resistance to many anticancer drugs, e.g. cisplatin,
colchicine, vinblastine, paclitaxel, doxorubicin and other
small molecules, such as protein kinase inhibitors.13
Thus, it is important to develop new compounds which
will be able to not only kill cancer cells at low
concentrations but also to overcome the multidrug-
resistant phenotype (MDR) of cancer cells.
Because the use of taxanes or other anticancer drugs
leads to many side effects,6 there is an ongoing search
for natural or synthetic compounds which could
preserve their anticancer activity while being less
threatening to normal cells and tissues. One of the most
promising potent anticancer drug-candidates is
plinabulin,7 a synthetic analogue of naturally occurring
9
2,5-piperazinedione – phenylahistin (Figure 1).8,
It
binds to the colchicine binding site of tubulin and thus
induces a collapse of the tumor vasculature, reducing
the blood supply to the cancer cells and, consequently,
leading to the death of tumor cells. These effects are
accompanied by its direct cytotoxic action on cancer
Tremendous progress in the bioorganometallic
chemistry of anticancer, antimalarial and antibacterial
compounds has been observed in recent years. The
presence of an metal atom which can exist in various
oxidation states allows to synthesize a large number of
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new types of molecules with properties that are different
groups attached to imidazole moieties 2a-d. We also
synthesized
from those of typical organic compounds.14 Depending
on the metal-ligand bond nature, it is possible to
develop bioactive organometallic compounds which
contain covalent metal-carbon bonds and/or labile
ligands.15-20 Pioneering studies in this field were carried
1
2
3
4
5
6
7
8
a ferrocene-containing conjugate which is structurally
similar to plinabulin but is devoid of imidazole moiety 4,
as well as an imidazole conjugate without ferrocenyl
moiety 5d (Figure 1). Synthesis of the desired
compounds was performed by modified standard
procedure (Scheme 1). First, glycine anhydride was
refluxed with an excess of acetic anhydride to give 1,4-
diacetylpiperazine-2,5-dione 3. Due to the instability of
the crude product (catalyzed by traces of acetic acid), it
was purified by silica gel chromatography. In the next
17
out by Jaouen and co-workers for ferrocene, which is
one of the metallocenes. Ferrocene alone exhibits only
slight cytotoxic activity, whereas conjugated with
biologically active compounds it provides them with
added value, e.g. which leads to some promising
anticancer agents.17, 21 One of the possible explanations
for its activity is based on reversible cellular oxidation of
the ferrocenyl moiety to a cytotoxic ferrocenium ion
accompanied by the formation of reactive oxygen
species (ROS), e.g. hydroxyl radicals. ROS are capable of
damaging DNA and other important biological
molecules, thus leading to cellular injury and death.22-24
It has been showed that organometallic compounds are,
in some cases, able to overcome drug resistance (this
was observed, for example, in cancer25 and parasite
[Plasmodium] cells26). Vock et al. showed that the
ruthenium half-sandwich complexes of a known MDR
modulator are able to inhibit the activity of the ABCB1
protein more efficiently than the purely organic form of
the MDR modulator.27
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step,
a
condensation
reaction
of
3
with
ferrocenecarboaldehyde in a mixture of tert-butanol and
DMF, using potassium tert-butanolate as a base, gave
the desired 4 as dark red crystals in 79% yield.
Unfortunately, further condensation with commercially
available 1H-imidazole-4-carboaldehyde under various
reaction conditions failed, and only the
Scheme 1. Synthesis of the ferrocenyl analogues of
plinabulin
products of decomposition of the starting 4 or a trace
amount of product 2a were obtained (e.g. DMF as a
solvent, caesium carbonate as a base, room temperature
or 50 °C, reaction time 2-24h – only a trace of 4 were
Figure 1. Structure of phenylahistin, plinabulin (top),
and its ferrocenyl analogues (2a-d) and half-plinabulin
analogues (4, 5d) synthesized herein.
detected (by TLC); when the condensation reaction was
performed at 90 °C, instead of RT, for 2.5 h, the desired
product was obtained in 3% yield). We assumed that
either no reaction occurred or the reaction yield was low
due to the presence of an acidic amide proton in 4.
Thus, we decided to re-acetylate the nitrogen atom of
the amide. Similarly to the synthesis of 3, we refluxed 4
with an excess of acetic anhydride but only unreacted 4
was recovered. The deprotonation reaction of 4 with
potassium bis(trimethylsilyl)amide followed by reaction
with acetyl chloride also resulted in recovery of an
unreacted substrate. Finally, we tried to re-acetylate 4 in
reaction with an excess of acetic acid and DIC as the
coupling agent in the presence of DMAP. Depending on
the reaction time, we were able to isolate either a
desired
Herein we report the synthesis of ferrocenyl analogues
of plinabulin and the systematic study of their cytotoxic
activity against a panel of human cancer lines, including
their
ABC-transporter
overexpressing
multidrug
resistance variants. We also studied the ability of novel
compounds to interfere with tubulin polymerization
and stability as well as their influence on the cell cycle.
Finally, we demonstrated here that some of these
compounds are potent inhibitors of MDR proteins.
To test the influence of plinabulin phenyl group
replacement by the ferrocenyl moiety on biological
activities towards cancer cells, we synthesized
ferrocene-plinabulin analogues with various alkyl
2
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Table 1. Cytotoxic properties of plinabulin, its ferrocenyl analogues 2a-2d and half-analogues 4 and 5a expressed as IC50. Mean values and
95%-confidence intervals (lower line, parentheses) are given in µM. Data of at least 3 independent experiments.
1
Cell line
NPI-2358
2a
2b
2c
2d
4
5d
2
3
4
5
0.034
(0.021-0.056)
34.11
31.43
(21.59-45.76)
67.74
9.65
(7.52-12.38)
31.42
16.65
(11.42-24.26)
19.83
SW620
> 100
> 100
> 100
SW620C
SW620D
SW620E
SW620M
SW620V
MCF7
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
6
7
8
9
(12.96-89.77)
(47.79-96.02)
72.04
(25.24-39.10)
24.23
(16.11-24.40)
21.77
> 100
(51.25-101.30)
64.05
(19.14-30.68)
24.57
(16.38-28.94)
18.64
52.45
(23.90-115.10)
0.132
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(42.79-95.88)
43.53
(20.34-29.69)
23.54
(11.67-29.78)
32.69
(0.024-0.744)
(32.52-58.27)
(13.04-42.48)
27.95
(20.48-52.17)
13.68
> 100
> 100
> 100
> 100
(9.99-78.24)
43.50
(5.26-35.60)
42.61
23.79
(7.33-77.29)
(17.83-106.10)
92.95
(10.41-174.40)
HepG2
> 100
> 100
(46.33-186.50)
re-acetylated 6 in 40% yield (the reaction stopped after 1
h) or its rearrangement product (the reaction stopped
after 24 h).28 Unfortunately, compound 6 was not stable
enough to perform the next condensation reaction.
Therefore we decided to reverse the order of the
condensation steps. First, in the reaction of 3 with 5-
alkyl-1H-imidazole-4-carboaldehydes in DMF in the
presenceof caesium carbonate at 35 °C for 4 h, 5a-d were
prepared in moderate yield (38-41%). The next
condensation stepof 5a-d with ferrocenecarboaldehyde
in the presence of caesium carbonate carried out at 90
°C for 2 h gave the required ferrocenyl analogues of
plinabulin 2a-d in a moderate to good yield (40-61%;
and 15.6–25% overall yield after two condensation steps).
The structures of the synthesized compounds were
confirmed by 1D and 2D NMR spectra, mass
spectrometry and by elemental analysis (See Supporting
Information for more detailed information).
range) to the SW620 and SW620M lines, while its
toxicity is rather moderate (IC50 values at tens of
micromolar levels in SW602C, SW620E and MCF7 cells)
or even negligible in other cell lines. This was somehow
unexpected, as NPI-2358 was shown to be highly active
against several cell lines of different background,
including also multidrug-resistant cells.29
The ferrocenyl analogues of plinabulin 2c and 2d –
both with a bulky alkyl group at the imidazole moiety –
were found to be equally effective against all cell lines
that were tested, although their toxicity (IC50 in the ten
micromolar range) differed from line to line, being the
highest for SW620 and the lowest for HepG2 cells. What
is interesting to note is that none of the MDR lines was
less sensitive to any of these compounds. Compound 2b
was moderately toxic to almost all the cell lines except
for SW620V, MCF7 and HepG2, while compound 2a and
both half-analogues were non-toxic in the range studied.
It seemed feasible that both the ferrocene and imidazole
moieties (the latter was substituted by a bulky alkyl
group) are required for 2,5-piperazinedione to be
biologically active, thus we discontinued further
analyses of 4 and 5d at this point.
The cytotoxic properties of the synthesized
compounds were determined in MCF7 (human
mammary
hepatocellular cancer) and SW620 (human colon
adenocarcinoma) cell lines (Table 1). We also employed
a panel of drug-resistant cell lines obtained by a gradual
selection of SW620 sensitive cells with common
chemotherapeutics. Depending on the selecting agent,
they were designed as SW620C – selected with cisplatin,
SW620D – doxorubicin, SW620E – etoposide, SW620M
adenocarcinoma),
HepG2
(human
To confirm the cytotoxicity results and to have some
insight into the mode of action of the synthesized
compounds, we performed a cell cycle analysis in cells
exposed to NPI-2358 and its organometallic analogues
2a-2d (Table S1). We observed a high pre-G0/G1
(apoptotic) peak and significantly elevated G2/M peak
values for plinabulin, thus indicating that cells exposed
to these compounds are unable to proceed with division
(due to their inability to form a mitotic spindle with
impaired microtubules) and die. Exposure to
compounds 2c and 2d resulted in a similar pattern of
DNA
–
methotrexate and SW620V
–
vincristine. We
previously characterized these cells with respect to
multidrug-resistant protein phenotype (at mRNA,
protein and activity level, submitted) and found that
ABCG2 is the transporter overexpressed in the SW620C
line, ABCC1 is the main MDR protein of SW620M, while
SW620D, -E, and –V cells highly overexpress ABCB1. We
found that plinabulin is highly toxic (at a nanomolar
3
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1
2
3
Table 2. Direct effect of 2d on multidrug resistance protein transport activity. Mean values and standard deviations are presented.
Averaged data of 3 independent experiments. Effects of NPI-2358 and 2d on the SW620 and MDCKII cell lines do not differ from the
control, thus they are not shown here for the sake of clarity.
4
Calcein accumulation assay (ABCB1 activity) [AU/min]
5
6
7
8
SW620V (ABCB1-
overexpressing)
SW620V+10 µM NPI-
2358
SW620
43,851 ± 7,175
MDCKII
SW620+ 10 µM VER
43,811 ± 7,499
SW620V+10 µM VER
SW620V+10 µM 2d
806 ± 484
MDCKII-MDR1
2,207 ± 198
18,975 ± 2,013
805 ± 467
21,497 ± 2,160
MDCKII-MDR1+10
µM VER
MDCKII-MDR1+10
µM NPI-2358
MDCKII-MDR1+10
µM 2d
MDCKII+ 10 µM VER
62,837 ± 1,973
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62,902 ± 1,959
32,691 ± 634
11,498 ± 351
24,328 ± 1,776
PheA accumulation assay (ABCG2 activity) [AU/min]
SW620C (ABCG2-
overexpressing)
SW620C+30 µM
KO143
SW620C+10 µM NPI-
2358
SW620
5,645 ± 856
MDCKII
SW620+30 µM KO143
SW620C+10 µM 2d
6,167 ± 939
2,063 ± 273
MDCKII-BCRP
6,942 ± 273
7,586 ± 1,151
1,611 ± 161
6,080 ± 2,504
MDCKII+ 30 µM
KO143
MDCKII-BCRP+30
µM KO143
MDCKII-BCRP+10
µM NPI-2358
MDCKII-BCRP+10
µM 2d
10,647 ± 326
11,675 ± 429
12,682 ± 450
7,845 ± 257
12,216 ± 373
BCECF extrusion assay (ABCC1 activity) [AU×min]
SW620M (ABCC1-
overexpressing)
SW620M+100 µM
PRB
SW620M+10 µM NPI-
2358
SW620
SW620+100 µM PRB
SW620M+10 µM 2d
19,557 ± 98
19,517 ± 98
17,113 ± 121
19,187 ± 214
17,353 ± 123
17,210 ± 114
distribution in the analyzed cells. Compounds 2a and 2b
exhibited a slight, albeit significant, increase of the
G2/M peak value, although it was unaccompanied by
modulatory activities (except for some effect in
MDCKII-MDR1 cells), which supports previous reports.29
25000
the presence of the apoptotic peak
.
20000
To check whether chemical modifications alter the
tubulin-binding properties of ferrocenyl plinabulin
analogues, we performed a tubulin polymerization
inhibition study (Figure 2). In the range studied
(between 1 and 100 µM), both compounds with the
bulky group at the imidazole moiety (2c and 2d) tended
to decrease the tubulin polymerization rate, while
compounds 2a and 2b were rather moderate modulators
of this process. The overall potency of plinabulin
ferrocenyl analogues was at least an order of magnitude
lower than that of vincristine and NPI-2358 itself. When
analyzing the toxicity profile of 2d (Table 1), we found
that it was significantly active in ABCB1-overexpressing
SW620D, -E and –V lines, especially when taking into
account the moderate or even lack of effect of plinabulin
on these lines. Thus, we decided to check the potential
of 2d to modulate MDR protein activity in real-time
transport experiments. We compared the results
obtained in SW620 cell line panel with commercially
available hABCB1- and hABCG2-transfected MDCKII
cells. The results are summarized in Table 2. Compound
2d clearly exhibited the properties of a low-specificity
inhibitor of MDR proteins. It inhibited ABCB1 transport
activity (measured by calcein accumulation assay30) to
the same extent as verapamil, which is an established
inhibitor of this protein.31 Its activity against ABCG2 was
also notable, as accumulation of pheophorbide A
increased in 2d-treated SW620C or MDCKII-BCRP cells
to the same level as in the presence of Ko143 - a specific
inhibitor of ABCG2.32 No effect on the ABCC1 transport
properties was observed as the BCECF efflux was
undisturbed by the presence of 2d. It is worth
mentioning that NPI-2358 showed none of these
2d
15000
10000
5000
0
2c
2b
2a
NPI-2358
VINC
DMSO
1
10
100
[ꢀM]
Figure 2. Influence of plinabulin and its ferrocenyl
analogues 2a-2d on tubulin polymerization rate. Data of
a representative experiment. VINC – vincristine used as
a positive control. DMSO is shown for reference (its
concentration was constant in all samples).
We were also interested whether the inhibitory effect
of 2d could also be observed in a toxicity assay.
Therefore, we exposed MDR cells to increasing
concentrations of doxorubicin or mitoxantrone at 100
nM of 2d (Table S2). Again, we used 10 µM verapamil
and 20 µM KO143 as MDR protein-specific controls.
Modulators exerted no effect on control cell lines while
2d consistently sensitized all MDR cell lines tested to
doxorubicin (completely restoring the sensitivity level of
parental cells in case of SW620C and MDCKII-BCRP).
Effect of 2d on mitoxantrone resistance could be
observed for both SW620C and MDCKII-BCRP
(although here to a lesser extent) further supporting the
hypothesis of the ability of this compound to modulate
ABCG2 activity.
We found that a simple replacement of the phenyl
group of plinabulin by a ferrocenyl moiety leads to new
compounds of unexpected biological properties. Our
results clearly showed that both the ferrocenyl and
imidazole moieties are required to maintain the activity
of the synthesized compounds. The bulkiness of the
imidazole-bound alkyl moiety strongly influences the
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cytotoxic profile of these compounds, as the isopropyl
Abbreviations
BCECF, 2',7'-bis
carboxyfluorescein; Ko143, (3S,6S,12aS)-1,2,3,4,6,7,12,12a-
octahydro-9-methoxy-6-(2-methylpropyl)-1,4-
1
2
3
and tert-butyl derivatives showed the highest
cytotoxic/antiproliferative activities. The effect of the
imidazole-bound alkyl type is important also in the
(2-carboxyethyl)-5-(and-6)-
4
5
6
7
8
9
aspect
of
tubulin
polymerization
inhibition.
dioxopyrazino[1',2':1,6]pyrido[3,4-b]indole-3-propanoic
acid 1,1-dimethylethyl ester; MDR, multidrug resistance;
NPI-2358, plinabulin; PheA, pheophorbide A; PRB,
probenecid; ROS, reactive oxygen species; VER,
verapamil; VINC, vincristine
Additionally, replacement of the phenyl group of
plinabulin by the ferrocenyl moiety not only
significantly enhances its toxicity against multidrug-
resistant cell lines but also, quite unexpectedly, turns
the ferrocenyl analogues of plinabulin into potent
inhibitors of ABCB1 and ABCG2. Co-administration of
vincristine and the ferrocenyl analogue of plinabulin
should exert a synergic effect not only in terms of
microtubule-related cytotoxicity but also by overcoming
the multidrug-resistance barrier formed by ABCB1 or
ABCG2 proteins, thus opening up novel therapeutic
possibilities. We are currently investigating such a
hypothesis in in vitro systems.
References
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
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59
60
1.
targeted chemotherapy, Future Med. Chem., 2013, 5, 339.
2. Schiff, P. B.; Fant, J.; Horwitz, S. B., Promotion of
microtubule assembly in vitro by taxol, Nature, 1979, 277, 665.
3. GuBritte-Voegelein, F.; Guenard, D.; Lavelle, F.; Le
Seligmann, J.; Twelves, C., Tubulin: An example of
Goff, M.-T.; Mangatal, L.; Potier, P.; J. Med. Chem. 1991, 34,
992.
4.
Passarella, D.; Giardini, A.; Peretto, B.; Fontana, G.;
ASSOCIATED CONTENT
Sacchetti, A.; Silvani, A.; Ronchi, C.; Cappelletti, G.; Cartelli, D.;
Borlak, J.; Danieli, B., Inhibitors of tubulin polymerization:
Synthesis and biological evaluation of hybrids of vindoline,
anhydrovinblastine and vinorelbine with thiocolchicine,
podophyllotoxin and baccatin III, Bioorg. Med. Chem., 2008, 16,
6269.
Supporting Information. Experimental procedure
for synthesis, cell culture, cytotoxicity testing, cell cycle
analysis, flow cytometry assays of MDR protein activity,
interference with tubulin polymerization and copy of 1H
and 13C NMR spectra, Table S1 and S2. The Supporting
Information is available free of charge on the ACS
Publications website at DOI:.
5.
Jordan, M. A., Mechanism of action of antitumor
drugs that interact with microtubules and tubulin, Curr. Med.
Chem. Anti-cancer agents, 2002, 2, 1.
6.
Ojima, I., Guided Molecular Missiles for Tumor-
AUTHOR INFORMATION
Targeting Chemotherapy—Case Studies Using the Second-
Generation Taxoids as Warheads, Acc. Chem. Res., 2007, 41,
108.
*Corresponding Authors:
*(D.P.) E-mail: damplaz@uni.lodz.pl
*(B.R.) E-mail: brychlik@biol.uni.lodz.pl
Author Contributions
7.
Mita, M. M.; Spear, M. A.; Yee, L. K.; Mita, A. C.;
Heath, E. I.; Papadopoulos, K. P.; Federico, K. C.; Reich, S. D.;
Romero, O.; Malburg, L.; Pilat, M.; Lloyd, G. K.; Neuteboom, S.
T. C.; Cropp, G.; Ashton, E.; LoRusso, P. M., Phase 1 first-in-
human trial of the vascular disrupting agent plinabulin (NPI-
2358) in patients with solid tumors or lymphomas, Clin. Cancer
Res., 2010, 16, 5892.
Anna Wieczorek was responsible for the synthesis of
the investigated compounds and tubulin polymerization
assay and participated in cytotoxicity, cell cycle and
transport assays. Andrzej Błauż was responsible for the
supervision of cytotoxicity, cell cycle and transport
assays, he performed the resistance reversal assay.
Contributions of both Authors were equal and
indispensable for this work; Błażej Rychlik was
responsible for assay choice and design, analysis of the
biological data and overall supervision of the biological
portion of this project; Janusz Zakrzewski and Damian
Plażuk designed the project. All authors wrote their
respective parts of the manuscript, which was finally
compiled by Anna Wieczorek, Błażej Rychlik and
Damian Plażuk. All authors have given approval to the
final version of the manuscript.
8.
Kanoh, K.; Kohno, S.; Katada, J.; Takahashi, J.; Uno, I.,
(-)-phenylahistin arrests cells in mitosis by inhibiting tubulin
polymerization, J. Antibiotics, 1999, 52, 134.
9.
Kanoh, K.; Kohno, S.; Asari, T.; Harada, T.; Katada, J.;
Muramatsu, M.; Kawashima, H.; Sekiya, H.; Uno, I., (-)-
phenylahistin: A new mammalian cell cycle inhibitor produced
by Aspergillus ustus, Bioorg. Med. Chem. Lett., 1997, 7, 2847.
10.
Singh, A. V.; Bandi, M.; Raje, N.; Richardson, P.;
Palladino, M. A.; Chauhan, D.; Anderson, K. C., A novel
vascular disrupting agent plinabulin triggers JNK-mediated
apoptosis and inhibits angiogenesis in multiple myeloma cells,
Blood, 2011, 117, 5692.
11.
Mayer, A. M. S.; Glaser, K. B.; Cuevas, C.; Jacobs, R. S.;
Kem, W.; Little, R. D.; McIntosh, J. M.; Newman, D. J.; Potts, B.
C.; Shuster, D. E., The odyssey of marine pharmaceuticals: a
current pipeline perspective, Trends Pharmacol. Sci., 2010, 31,
255.
Funding Sources
This work was supported by the National Science
Centre
Poland
(contract
No.
UMO-
2011/01/B/ST5/03933). Analysis of the multidrug-
resistance protein activity of synthesized compounds
was financed by the Polish POIG grant 01.01.02-10-
005/08 TESTOPLEK, supported by the EU through the
European Regional Development Fund.
12.
Kunjachan, S.; Rychlik, B.; Storm, G.; Kiessling, F.;
Lammers, T., Multidrug resistance: Physiological principles
and nanomedical solutions, Adv. Drug Deliver. Rev., 2013, 65,
1852.
13.
Persidis, A., Cancer multidrug resistance, Nat
Biotech, 1999, 17, 94.
Notes
The authors declare no competing financial interest.
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14.
chemistry
Hartinger, C. G.; Dyson, P. J., Bioorganometallic
From teaching paradigms to medicinal
multidrug-resistant human KB carcinoma membrane vesicles
and inhibit drug binding to P-glycoprotein, J. Biol. Chem., 1987,
1
2
3
4
5
6
7
-
applications, Chem. Soc. Rev., 2009, 38, 391.
15. Babak, M. V.; Plażuk, D.; Meier, S. M.; Arabshahi, H.
J.; Reynisson, J.; Rychlik, B.; Błauż, A.; Szulc, K.; Hanif, M.;
Strobl, S.; Roller, A.; Keppler, B. K.; Hartinger, C. G., Half-
Sandwich Ruthenium(II) Biotin Conjugates as Biological
Vectors to Cancer Cells, Chem. –Euro. J., 2015, 21, 5110.
262, 2166.
32.
Allen, J. D.; van Loevezijn, A.; Lakhai, J. M.; van der
Valk, M.; van Tellingen, O.; Reid, G.; Schellens, J. H.; Koomen,
G. J.; Schinkel, A. H., Potent and specific inhibition of the
breast cancer resistance protein multidrug transporter in vitro
and in mouse intestine by a novel analogue of fumitremorgin
C, Mol. Cancer. Ther., 2002, 1, 417.
16.
Hartinger, C. G.; Metzler-Nolte, N.; Dyson, P. J.,
8
9
Challenges and Opportunities in the Development of
Organometallic Anticancer Drugs, Organometallics, 2012, 31,
5677.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table of Contents (TOC)
17.
Top, S.;Tang, J.; Vessieres, A.; Carrez, D.; Provot, C.;
Jaouen, G., Ferrocenyl hydroxytamoxifen: a prototype for a new
range of oestradiol receptor site-directed cytotoxics, Chem.
Commun., 1996, 955.
18.
derivatives in cancer research, New J. Chem., 2011, 35, 1973.
19. Liu, Z.; Habtemariam, A.; Pizarro, A. M.; Fletcher, S.
A.; Kisova, A.; Vrana, O.; Salassa, L.; Bruijnincx, P. C. A.;
Clarkson, G. J.; Brabec, V.; Sadler, P. J., Organometallic half-
sandwich iridium anticancer complexes, J. Med. Chem., 2011,
54, 3011.
20.
anticancer compounds, J. Med. Chem., 2011, 54, 3.
21. Plazuk, D.; Vessieres, A.; Hillard, E. A.; Buriez, O.;
Ornelas, C., Application of ferrocene and its
Gasser, G.; Ott, I.; Metzler-Nolte, N., Organometallic
Labbe, E.; Pigeon, P.; Plamont, M. A.; Amatore, C.; Zakrzewski,
J.; Jaouen, G., A [3]ferrocenophane polyphenol showing a
remarkable antiproliferative activity on breast and prostate
cancer cell lines, J. Med. Chem., 2009, 52, 4964.
22.
Hillard, E.; Vessières, A.; Le Bideau, F.; Plazuk, D.;
Spera, D.; Huché, M.; Jaouen, G., A series of unconjugated
ferrocenyl phenols: Prospects as anticancer agents,
ChemMedChem, 2006, 1, 551.
23.
Antitumoral ferrocenes, Organometallics, 2013, 32, 5626.
24. Vessières, A., Metal carbonyl tracers and the
ferrocifen family: Two facets of bioorganometallic chemistry, J.
Organomet. Chem., 2013, 734, 3.
25.
Braga, S. S.; Silva, A. M. S., A new age for iron:
Kater, B.; Hunold, A.; Schmalz, H.-G.; Kater, L.;
Bonitzki, B.; Jesse, P.; Prokop, A., Iron containing anti-tumoral
agents: unexpected apoptosis-inducing activity of a ferrocene
amino acid derivative, J. Cancer Res. Clin. Oncol., 2011, 137, 639.
26.
Dubar, F.; Khalife, J.; Brocard, J.; Dive, D.; Biot, C.,
Ferroquine, an Ingenious Antimalarial Drug –Thoughts on the
Mechanism of Action, Molecules, 2008, 13, 2900.
27.
Lagopoulos, L.; Juillerat-Jeanneret, L.; Sava, G.; Scopelliti, R.;
Dyson, P. J., Development of ruthenium antitumor drugs that
overcome multidrug resistance mechanisms, J. Med. Chem.,
2007, 50, 2166.
Vock, C. A.; Ang, W. H.; Scolaro, C.; Phillips, A. D.;
28.
Wieczorek, A.; Plazuk, D.; Zakrzewski, J.; Makal, A.;
Woźniak, K., Synthesis and unusual ring transformation of 1-
acyl-3-(ferrocenylmethylidene)-piperazine-2,5-diones,
Organomet. Chem., 2013, 745-746, 373.
29.
A.; Kiso, Y.; Hayashi, Y.; Neuteboom, S. T. C., NPI-2358 is a
tubulin-depolymerizing agent: In-vitro evidence for activity as
a tumor vascular-disrupting agent, Anti-Cancer Drugs, 2006,
17, 25.
J.
Nicholson, B.; Lloyd, G. K.; Miller, B. R.; Palladino, M.
30.
Holló, Z.; Homolya, L.; Davis, C.W.; Sarkadi, B.;
Calcein accumulation as a fluorometric functional assay of the
multidrug transporter., Biochim Biophys Acta, 1994, 1191, 384.
31.
Cornwell, M. M.; Pastan, I.; Gottesman, M. M.,
Certain calcium channel blockers bind specifically to
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Figure 1. Structure of phenylahistin, plinabulin (top), and its ferrocenyl analogues (2a-d) and half-plinabulin
analogues (4, 5d) synthesized herein
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Scheme 1. Synthesis of the ferrocenyl analogues of plinabulin
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Figure 2. Influence of plinabulin and its ferrocenyl analogues 2a-2d on tubulin polymerization rate. Data of a
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