New 5-aryl-1H-imidazoles display in vitro antitumor activity against apoptosis-resistant cancer models, including melanomas, through mitochondrial targeting

Article abstract of DOI:10.1021/jm400287v

We designed and synthesized 48 aryl-1H-imidazole derivatives and investigated their in vitro growth inhibitory activity in cancer cell lines known to present various levels of resistance to proapoptotic stimuli. The IC₅₀ in vitro growth inhibitory concentration of these compounds ranged from >100 μM to single digit μM. Among the most active compounds, 2i displayed similar in vitro growth inhibition in cancer cells independent of the cells' levels of resistance to proapoptotic stimuli and was found to be cytostatic in melanoma cell lines. Compound 2i was then tested by the National Cancer Institute Human Tumor Cell Line Anti-Cancer Drug Screen, and the NCI COMPARE algorithm did not reveal any correlation between its growth inhibition profiles with the NCI database compound profiles. The use of transcriptomically characterized melanoma models then enabled us to highlight mitochondrial targeting by 2i. This hypothesis was further confirmed by reactive oxygen production measurement and oxygen consumption analysis.

Full text of DOI:10.1021/jm400287v

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Article
New 5-aryl-1H-imidazoles display in vitro antitumor
activity against apoptosis-resistant cancer models,
including melanomas, through mitochondrial targeting
Véronique Mathieu, Emilie Van Den Berge, Justine Ceusters, Tomasz Konopka, Antonin Cops, Céline
Bruyère, Christine Pirker, Walter Berger, Tran trieu-Van, Didier Serteyn, Robert Kiss, and Raphael Robiette
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400287v • Publication Date (Web): 11 Jul 2013
Downloaded from http://pubs.acs.org on July 14, 2013
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New 5ꢀarylꢀ1Hꢀimidazoles display in vitro antitumor activity against
apoptosisꢀresistant cancer models, including melanomas, through
mitochondrial targeting
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Véronique Mathieu,*,† Emilie Van Den Berge,‡ Justine Ceusters, § Tomasz Konopka,# Antonin Cops†,
Céline Bruyère†, Christine Pirker, ¶ Walter Berger,& Tran Trieu-Van,‡ Didier Serteyn, § Robert Kiss†
and Raphaël Robiette*,‡
†Laboratoire de Toxicologie, Faculté de Pharmacie, Université Libre de Bruxelles (ULB), Boulevard du
Triomphe, CP205/1, Bꢀ1050 Brussels, Belgium.
‡Institute of Condensed Matter and Nanosciences, Université catholique de Louvain, Place Louis
Pasteur 1 box L4.01.02, Bꢀ1348 LouvainꢀlaꢀNeuve, Belgium.
§ Center for Oxygen, Research and Development, Institute of Chemistry B6a, University of Liège, Sart
Tilman, 4000 Liège, Belgium.
# BioSystems, BioModeling and BioProcesses Group, Université Libre de Bruxelles, CP165/61, Bꢀ1050
Brussels, Belgium.
¶ Department of Thoracic Surgery, Medical University Vienna, Vienna, Austria.
& Department of Medicine I, Institute of Cancer Research, Medical University Vienna, Vienna, Austria.
RECEIVED DATE
AUTHOR INFORMATION
* Phone: +32ꢀ2ꢀ6505179. Eꢀmail: vemathie@ulb.ac.be (V.M.; biology). Phone: +32ꢀ10ꢀ479176. Fax:
+32ꢀ10ꢀ474168. Eꢀmail: raphael.robiette@uclouvain.be (R.R.; chemistry).
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ABSTRACT
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We designed and synthesized 48 arylꢀ1Hꢀimidazole derivatives and investigated their in vitro growth
inhibitory activity in cancer cell lines known to present various levels of resistance to proꢀapoptotic
stimuli. The IC50 in vitro growth inhibitory concentration of these compounds ranged from >100ꢁM to
single digit ꢁM. Among the most active compounds, 2i displayed similar in vitro growth inhibition in
cancer cells independently of the cells’ levels of resistance to proꢀapoptotic stimuli, and was found to be
cytostatic in melanoma cell lines. Compound 2i was then tested by the National Cancer Institute Human
Tumor Cell Line AntiꢀCancer Drug Screen, and the NCI COMPARE algorithm did not reveal any
correlation between its growth inhibition profiles with the NCI database compound profiles. The use of
transcriptomically characterized melanoma models then enabled us to highlight mitochondrial targeting
by 2i. This hypothesis was further confirmed by reactive oxygen production measurement and oxygen
consumption analysis.
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INTRODUCTION
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Since the beginning of anticancer chemotherapy in the 1950s, the survival rate associated with several
cancer types has been markedly improved, e.g. testicular cancers or several types of leukemia and
lymphoma that can even be cured.1, 2 In contrast, despite the enrichment of the chemical libraries used in
oncology for over half a century with natural as well as synthetic compounds, other cancer types remain
associated with a dismal prognosis due to their acquired (e.g. multidrug resistance phenotype)3, 4 or
innate (intrinsic apoptosis resistance) 5, 6 resistance to chemotherapeutic insults. Most of the compounds
used to treat cancer patients are cytotoxic and proꢀapoptotic agents.7 Cancers associated with dismal
prognoses, include, but are not limited to, glioma,8 advanced melanoma,9, 10 nonꢀsmall cell lung
carcinoma,11 head and neck carcinoma,12 esophageal carcinoma13 and pancreatic carcinoma.14
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Arylꢀ1Hꢀimidazoles are key chemical structures that display a wide range of biological and
pharmacological activities.15ꢀ17 Biologically active arylꢀ1Hꢀimidazoles include compounds with potent
in vitro antifungal activity, inhibitors of βꢀglucosidase, molecules with antiꢀinflammatory properties,
substances exhibiting activin receptorꢀlike kinase 5 (ALK5) inhibitory activity, antagonists of NPY5
receptors, potent Na+ channel blockers and molecules with antitubulin and antiproliferative activities
against cancer cells.15ꢀ17 In a previous report, we demonstrated that 5ꢀarylꢀ1Hꢀimidazoles embedded in a
macrocycle via a connection at the C2 and orthoꢀaryl positions (1) display interesting binding activities
toward biological targets that are important actors in the central nervous system such as the A3
adenosine (h) receptor, dopamine D1 (h) receptor and chlorine channel (GABAꢀgated).18
Here, we report on (i) the identification and optimization of 5ꢀarylꢀ1Hꢀimidazoles as potential anticancer
agents, (ii) the characterization of the in vitro cytostatic versus cytotoxic antiꢀcancer activity of these
compounds, and (iii) the partial deciphering of their mechanism of action in melanoma cells. The
revealed mechanism led us to highlight the targeting by this type of compound of mitochondrial
metabolism, which is known to be reprogrammed in cancer cells.19 The original mechanism of action we
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identified for the most active compounds under study opens chemistry to several new antiꢀcancer
avenues.
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CHEMISTRY
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The synthesis of all studied 2ꢀalkoxyꢀ5ꢀarylꢀ1Hꢀimidazole derivatives starts from intermediate 5, which
was prepared using a highly regioselective methodology previously developed in our lab (Scheme 1).20
The strategy for forming the macrocycle is based on three key steps: a nucleophilic aromatic
substitution, a Suzuki coupling and a ring closure metathesis reaction.18 The nucleophilic aromatic
substitution places the first arm on the scaffold (R1), while the Suzuki coupling allows the second alkene
chain (R2) to be introduced. Some boronic acids used in this latter step were synthesized from
corresponding bromo derivatives (see the SI). Finally, the two last steps are the ring closure metathesis,
using the secondꢀgeneration Grubbs catalyst, and the hydrogenation of the resulting double bond. This
strategy allowed us to obtain a series of macrocycles (1a-j), as well as intermediates, during the
synthesis (2a-aa, 4a) (Table 1). We also synthesized imidazoles missing the alkene chain on C(2) of the
imidazole (6) or the aryl group (14) as well as an imidazole bearing the aryl group in 4 position (15) to
investigate the influence of these structural motifs on the in vitro growth inhibition activity of various
cancer cell lines.
It is important to mention that a detailed NMR and HPLC study revealed that some of our macrocycles
(1) are chiral.21 Indeed, due to their cyclophaneꢀtype structure, macrocycles 1a-f are planar chiral
molecules. In contrast, their superior analogues 1h-j isomerize rapidly at room temperature. Given the
importance of chirality for biological activities, we separated the two enantiomers of 1a to separately
test their in vitro growth inhibition activity in cancer cells.
We decided to also synthesize the corresponding 2ꢀamidoimidazoles to investigate the influence of the
electronic properties of the imidazole ring on in vitro cancer cell growth inhibition. We first planned to
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prepare these derivatives by applying the same methodology as the synthesis of the 2ꢀalkoxyꢀimidazole
derivatives (1). However, our attempts to achieve the nucleophilic aromatic substitution of the sulfone
group by an amine were unsuccessful. Thus, we turned our efforts to the implementation of the
methodology developed by the E. Van der Eycken group.22, 23 This reaction consists of the coupling of
an αꢀbromoꢀketone (8) with a 2ꢀaminopyrimidine under microwave heating, followed by the addition of
hydrazine. The application of this strategy to our compounds led to 2ꢀaminoꢀimidazole 9 at moderate
yields (Scheme 2). The placing of the second alkene chain was then performed by acid coupling to form
an amide (10). Finally, the ring closure metathesis reaction followed by direct hydrogenation of the
resulting double bond led to macrocycle 11.
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BIOLOGY
Determination of the in vitro growth inhibitory activity of the 5-aryl-1H-imidazoles. We used an
MTT colorimetric assay to evaluate the in vitro growth inhibitory effects of the 48 compounds
synthesized and listed in Table 1. These in vitro growth inhibitory effects were determined in each
cancer cell line by calculating the concentration that decreased the growth of this cancer cell line by
50% after 72 h of culture in the presence of the drug of interest (the IC50 in vitro growth inhibitory
index, SI Table 1). This assay was performed on various cancer cell lines that we analyzed based on
their display of sensitivity or resistance to proꢀapoptotic stimuli. Four cancer cell lines displaying
sensitivity to proꢀapoptotic stimuli were thus analyzed: the mouse B16F10 melanoma,24 the human
prostate PCꢀ3, 25 the breast MCFꢀ7,25 and the colon LoVo26 carcinoma cell lines. In the same manner,
four human cancer cell lines that displayed various levels of resistance to proꢀapoptotic stimuli were
also analyzed: the human U37327, 28 and T98G glioma27 the SKMELꢀ28 melanoma24 and the A549 nonꢀ
small cell lung carcinoma (NSCLC) 29 cell lines (SI Table 1).
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Among the 48 compounds tested, the IC50 value range included nonꢀactive compounds (defined by
mean IC50 values > 100 ꢁM) to compounds with single digit ꢁM IC50 values. No correlation between the
IC50 values and calculated log P values could be found (see the SI).
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Interestingly, the most active compounds, i.e., 2h, 2i, 2q, 2r and 2z, displayed similar in vitro growth
inhibitory activity in the various cancer cell lines analyzed regardless of the cell line level of resistance
to proꢀapoptotic stimuli (Table 1). Thus, it is unlikely that the in vitro growth inhibitory effects of this
group of compounds related to proꢀapoptotic effects, a hypothesis that we experimentally investigated as
detailed below.
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The mean IC50 concentration on cancer cells lines of 2i (6ꢁM) chosen among the 5 most potent
compounds was further compared to its IC50 concentrations determined on immortal and primary nonꢀ
cancerous cell lines. While immortal cells were as sensitive to 2i as cancer cells (data not shown),
NHDF and NHLF normal fibroblasts revealed to be 6 and 4 times less sensitive to 2i respectively
(NHDF: IC50 =36ꢁM; NHLF: IC50=22ꢁM). This result indicates that 2i could display some
bioselectivity towards highly proliferative and metabolically active cells.
2i displays cytostatic and not cytotoxic anti-cancer effects in melanoma cells. We made use of
computerꢀassisted phaseꢀcontrast microscopy (quantitative videomicroscopy) to study the effects of 2i
on two melanoma cell lines, i.e. the apoptosisꢀsensitive B16F10 mouse model and the apoptosisꢀ
resistant SKMELꢀ28 human model 24 at their respective IC50 in vitro growth inhibitory concentrations as
determined by means of a colorimetric MTT assay. While the global growth of each of these two
melanoma cell populations (SKMELꢀ28, Fig. 1A; B16F10, data not shown) was decreased by
approximately 50% between 48 h and 72 h of culture (Fig. 1B), no cell death occurred according to the
morphological analyses provided by quantitative videomicroscopy (Fig. 1A). In contrast, morphological
changes emphasizing increases in cell size were observed, and 2i displayed cytostatic rather than
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cytotoxic effects (Fig. 1A). These results were further confirmed by flow cytometry determination of
cell cycle kinetics (Fig. 1C) and activation of apoptosis (Fig. 1D). Indeed, the data in Fig. 1C reveal that
2i did not modify the cell cycle kinetic profiles or activate apoptosis (Fig. 1D) in human SKMELꢀ28
melanoma cells. Similar results were obtained with the B16F10 melanoma cells (data not shown). The
fact that no significant modifications were observed in B16F10 and SKMELꢀ28 melanoma cell cycle
kinetics suggests that the 2iꢀinduced cytostatic effects are unrelated to a specific cell cycle phase.
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Evaluation of the in vitro growth inhibitory concentration of 2i in the NCI 60 cancer cell line
panel. Compound 2i was analyzed by the National Cancer Institute in a panel of 60 cancer cell lines at
10 ꢁM (Fig. 2, revealed with the permission of the NCI). The data obtained revealed that the residual
growth ranged between 13 to 106% depending on the cell line analyzed (Fig. 2). The NCI 50% growth
inhibitory concentration determination using the 60 models equals 4 ꢁM, a value which fits perfectly
with our data above (6 ꢁM, Table 1).
We then compared the 2i in vitro growth inhibitory response profiles of the NCI panel of 60 cancer cell
lines to the full NCI database compound profile set using the COMPARE algorithm developed by the
NCI 30, 31 as we have successfully done with other types of compounds.32 We wanted to thus determine
the “Compare Correlation Coefficients” (the CCC index) with respect to the > 763,000 compounds
already present in the NCI database. The best CCC index we obtained was below 0.7, suggesting that
the mechanisms of action of 2i as a potential anticancer agent should be distinct from those mechanisms
of action, at least in terms of in vitro cancer cell growth inhibition, of the > 763,000 compounds already
present in the NCI database.
Transcriptomic comparison of melanoma primary cultures that display different sensitivity levels
to 2i highlights the mitochondria as a potential target for this compound. Because our group is
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interested in identifying novel types of compounds to overcome, at least partly, the intrinsic resistance
of melanoma cells to cytotoxic compounds in general and to proꢀapoptotic compounds in particular, we
had developed a collection of human melanoma primary cultures characterized at their genomic and
transcriptomic levels that we decided to use as further explained in the discussion to try to decipher, at
least partly the mechanism(s) by which 2i exerts its growth inhibition. The IC50 growth inhibitory
concentrations of 2i on the 11 previously characterized human melanoma primocultures33 appeared to be
as heterogeneous (Fig. 3) as the in vitro profiling results conducted using the NCI panel of 60 cell lines
(Fig. 2). Thus, this panel of 11 melanoma primocultures did not modify the conclusions that could be
drawn from the NCI 60 cell line panel in terms of 2iꢀinduced in vitro growth inhibition of cancer cells.
The in vitro growth inhibition profiling of 2i on this panel of 11 primocultures revealed that three cell
lines appeared rather resistant (VMꢀ1, VMꢀ23 and VMꢀ30 cell lines further labeled as group 1; mean
IC50 ± SEM: >86 ± 11 ꢁM), while 5 cell lines were more sensitive (VMꢀ7, VMꢀ8, VMꢀ21, VMꢀ24 and
VMꢀ47; further labeled group 2; mean IC50 ± SEM = 6 ± 2 ꢁM) when comparing the mean IC50 value
calculated for these 11 models, i.e., approximately 35 ꢁM (the dotted line in Fig. 3). Using the Agilent
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mRNA arrayꢀrelated data already available for these 11 primary melanoma cultures, we proceeded
with bioinformatic comparison between groups 1 and 2 at the gene mRNA expression level to identify
potential genes that could explain, at least partly, the difference in sensitivity observed for 2i in terms of
in vitro growth inhibition of these 11 melanoma primocultures.
As the up/downꢀregulated gene lists were quite extensive, we performed enrichment analysis using
EASE software.34 It appeared that mitochondrial oxidoꢀreductive metabolism could drive 2iꢀinduced in
vitro growth inhibition of cancer cells. We only took into account EASE scores with p values < 0.005
(Table 2). Indeed, both the cellular component categories and the biological processes that differentiate
the high (group 2) from the low (group 1) responsive melanoma cell lines to 2i relate to the
mitochondria and, more particularly, to its inner membrane with a focus on the energetic pathways and
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oxidoꢀreductase activity (Table 2). The gene list symbols, names and functions are detailed in SI Table 2
as provided by the EASE annotation system.
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Interestingly, group 1 cell lines, which are less sensitive to compound 2i, generally overexpress these
genes at the mRNA level. Therefore, it appears that mitochondrial activity could drive the 2i response,
and we experimentally checked this hypothesis as detailed below.
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Evaluation of the effects of 2i on mitochondria in SKMEL-28 melanoma cells. The mitochondria in
untreated and 2iꢀtreated cells were visualized by fluorescent microscopy using MitoTracker®. Fig. 4A
illustrates the typical morphological distribution and fluorescence intensity of SKMELꢀ28 cells left
untreated or treated for 24 h with 5 ꢁM 2i. No major modification could be observed while an increase
in cell size was again observed (see Fig. 1). Further analyses revealed that 2i treatment induced a timeꢀ
dependent increase in the oxygen reactive species content of the cells as shown in Fig. 4B. To validate
that 2i targets the mitochondrial metabolism, we made use of oxygraphic measurement. Interestingly,
the direct addition of 10 ꢁM 2i compound in the oxygraphic chamber containing SKMElꢀ28 cells has no
impact on the cellular oxygen consumption (data not shown), while pretreatment of SKMELꢀ28 cells for
72 h with 10 ꢁM 2i induced a marked increase in the subsequent oxygen consumption. These results
were confirmed in the U373 apoptosisꢀresistant glioma model (data not shown). Thus, it appears that 2i
affects mitochondrial metabolism in an indirect manner, the mechanism of which remains to be
deciphered.
DISCUSSION AND CONCLUSIONS
The panel of 48 compounds synthesized and tested in the current study allows some conclusions to be
drawn concerning structureꢀactivity relationships (Fig 5). First, the nonꢀcyclized compounds (2 and 10)
were systematically more active than their respective macrocyclic structure (1, 7 and 11). The aryl group
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is found to be crucial for the biological activity since compound 14 which misses this motif shows no or
a very low growth inhibition activity. The displacement of the aryl group in position 4 of the imidazole
(see compound 15) is also detrimental to the biological activity. Concerning the substitution pattern, the
obtained IC50 for compounds 1b-f,h and 2b-f,h,r-w indicate that substitution of the aryl group (R3, R4
and/or R5 ≠ H) leads to a decrease of inhibition activity (except for R3 = iꢀPr, 2r, which has no
significant effect on the activity). The nature of the R2 chain does not significantly influence the
biological activity, as compounds with a methyl group in that position (R2 = CH3) presented similar
activities to those possessing a longer chain (compare for instance 2i and 2q) (Fig. 5).
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In contrast, the nature of substituent in position 2 of the imidazole (R1) appears to have marked impact
on the growth inhibition activity in cancer cell line models. Indeed, variations in the chain length (see
2a,g,i-j) induce large difference in IC50, with an optimal chain length of six carbon atoms (R1 =
(CH2)4CH=CH2) (Fig. 5). The inclusion of fluorine (2n) or oxygen (2y) atoms in this alkene chain (R1)
is detrimental to its activity. However, the terminal unsaturation is important for activity (compare, for
instance, 2k,l,o,x with 2a,g,i-j,m,p,q,z,aa), with the best results being obtained with a terminal double
bond or a phenyl group (2a-j,p-w,z,aa) (Fig. 5).
The synthesis and biological evaluation of amide derivatives 10 and 11 indicated that 2ꢀalkoxy and 2ꢀ
amido imidazole derivatives possess very similar inhibition activities (compare 10 with 2i).
Biological evaluations of one of the most active compounds, 2i, indicated a potential First In Class
molecule, at least when comparing its in vitro growth inhibitory profiles in 60 cancer cell lines with the
>763,000 NCI molecule database. We obtained a COMPARE correlation coefficient < 0.7. To attempt
to partly decipher the mechanism of action through which 2i induces in vitro growth inhibitory activity
in cancer cells, we made use of genetically wellꢀdefined models as previously done by other groups.35ꢀ37
In the present study, we compared the transcriptomic characterization of highly versus less sensitive
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melanoma models to compound 2i, and this approach enabled us to highlight the mitochondria as a
potential target candidate for 2i. Indeed, the gene set enrichment analysis revealed 43 genes coding for
proteins related to mitochondrial location (27/43 proteins, 10/27 mitochondrial membraneꢀrelated
proteins) and/ or energetic and redox balance (31/43 proteins), two biological processes in which
mitochondria play key roles (Table 2; SI table 2). Such mitochondria targeting is of clinical interest.
Indeed, the targeting of the electron transfer chain (ETC) in the mitochondria by elesclomol produced
therapeutic benefits in a phase II clinical trial in melanoma patients. 38 The experimental data that led to
these clinical trials were obtained by using homozygote mutated yeast strain comparisons between
sensitive and resistant models.37
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The reprogramming of mitochondrial metabolism in cancer cells has been studied since 1923 when Otto
Warburg described the aerobic glycolysis of these cells,39 but no single compound on the market to date
is known to specifically target this organelle function. This reprogramming has been recognized as one
of the hallmarks of cancer19 candidates for antiꢀcancer targeting to avoid, as best at least, nonꢀspecific
adverse effects due to effects on normal cells.40 In cancer cells and in melanoma in particular, the
tricarboxylic acid Krebs (TCA) cycle is decoupled from glycolysis,41 which produces approximately
50% of the needed ATP, while the remaining 50% is provided through the TCA cycle fed with other
carbon sources than glucose, such as glutamine in particular.41, 42 This decoupling is favorable for
biosynthesis, energetic and redox balances. The equilibrium results of the balance between production
and scavenging of ROS. Production is mainly due to the 1 to 4% of oxygen in the electron transfer chain
.-
that is incompletely reduced in O2 and further in H2O2, while scavenging is performed by the four antiꢀ
oxidant defenses, glutathione oxidases and glutaredoxins that use reduced glutathione as an electron
donor and the thioredoxins and peroxiredoxins that use NADPH as an electron donor.40, 43 The
equilibrium level of cancer cells is known to be higher compared with normal cells, making cancer cells
more sensitive to oxidative stress.40 Here, we demonstrated that 2i treatment for 72 h induced an
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increased respiration rate and, more precisely, O2 consumption rates along with marked ROS production
(Figure 4). These results fit with our findings that the expression levels of genes of the ETC, e.g.
COX5B, COX6C, COX17, FDXR, NDUFA5, and NDUFA6 and more generally of the oxidoꢀreductase
balance, i.e., ACADS, AMID, BLVRB, COX5B, COX6C, DECR1, FADS2, FDXR, FLJ10661, FMO4,
HSD17B12, IDH3A, MDH1, NDUFA5, NDUFA6, QDPR, RRM2B, SEPW1, TXN, UQCRB, and
VAT1, are associated with differences in 2iꢀmediated in vitro growth inhibitory activities (Table 2; SI
Table 2). Such a high metabolic rate and mitochondrial dysfunction can lead to increased ROS
production and inefficient ATP generation. Because cancer cells display increased energy needs, the
perturbation of mitochondrial respiration and/or of glycolysis is an attractive avenue when developing
new drugs. The sensitization of cancer cells to death has already been shown with metformin, which
inhibits oxidative phosphorylation, and with 2ꢀdeoxyglucose or lonidamine, which inhibit hexokinase.44
Moreover, the oxidation status of cysteine residues of proteins regulates the duration and intensity of
transduction signals to the nucleus. Interestingly, the signaling pathways modulated by glutathione and
thioredoxins are not the same, and the outcome of this regulation will depend on the local context.45
Increased levels of ROS can thus lead to apoptosis, cell cycle deregulation, senescence or tumorigenesis.
Indeed, the cell cycle has been shown to be synchronized with the respiration cycle through the
oxidative status tuning of the PI3K/Akt/FoxO3a pathway.45 In the present study, we demonstrated that
disruption of mitochondrial functions and increased intracellular ROS content is associated with
cytostatic effects in cancer cells (Figure 1). Therefore, our compound differs from elesclomol, which
induces apoptosis as a consequence of massive ROS production through ETC perturbation that depends
directly on its copperꢀbinding properties.37 As 2i treatment does not directly alter O2 consumption, we
hypothesize the existence of indirect and different mechanisms of action than those observed with
elesclomol and that remain to be deciphered.
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In conclusion, we have synthesized fortyꢀeight 5ꢀarylꢀ1Hꢀimidazoles, among which several compounds
displayed single digit ꢁM 50% growth inhibitory concentration against various cancer models, including
apoptosisꢀresistant ones such as melanomas. Interestingly, the mechanism by which these compounds
(at least 2i) exert their antiꢀcancer cytostatic activities does not correlate with any compound from the
NCI database. In contrast, we discovered that these compounds could induce perturbations of
mitochondrial energetic metabolism, leading to increased O2 consumption and reactive oxygen species
production. Because this metabolism is known to be reprogrammed in cancer cells, these compounds
could represent a new way to combat apoptosisꢀresistant cancer types.
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EXPERIMENTAL SECTION
CHEMISTRY
Flash chromatography was performed on silica gel (230ꢀ400 mesh). TLC was performed on aluminumꢀ
backed silica plates that were developed using standard visualizing agents: UV fluorescence (254 and
1
366 nm), KMnO4. NMR spectra were recorded at 300 or 500 MHz for H NMR and at 75 MHz for 13C
NMR. Chemical shifts (δ) are given in part per million downfield from internal TMS. Reactions under
microwave heating were conducted in a sealed tube using MicroSYNTH equipment (Milestone Srl). The
temperature of the reaction mixture was monitored via an infrared sensor. Infrared spectra were
recorded using a Shimadzu FTIRꢀ8400S spectrometer. Mass spectra were recorded using a Finnigan
MAT LCQ mass spectrometer. HRMS were recorded by the MAPS laboratory at the University College
of London. The synthesis and characterization of compounds 1a-e,g,i-j, 2a-e,g,i-j, 6a-b and 7a have
been previously reported.18, 20, 21 The general procedures and key analytical data for 1i are reported
below with full data for all derivatives in the Supporting Information. The purity of intermediates was
measured by 1H and 13C NMR. The purity of the final compounds was determined by analytical reverseꢀ
HPLC (Xbridge C18). The measured purity was >95%.
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General procedure for alkylation of bromophenols. DBU (9.70 mmol, 1.5 equiv) was added
dropwise to a solution of corresponding bromophenol (6.47 mmol, 1 equiv) in DMF (10 ml). The
mesylate (8.415 mmol, 1.3 equiv) was added dropwise to this solution and then the tꢀbutylammonium
iodine (0.198 mmol, 0.03 equiv). The mixture was heated at 70°C for 7 hours and then stirred at room
temperature overnight. The solvent was evaporated under reduced pressure. Ether (100 mL), water (15
ml) and a solution of HCl 1 N (5 mL) were added. The phases were separated, and the organic phase
was washed with water (2×20 mL). The combined aqueous phases were extracted with ether (2×30 mL).
The organic phases were collected, dried (MgSO4), and concentrated in vacuo.
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Characterization of 2-bromo-1-(hex-5-en-1-yloxy)benzene (12a)
1
Colorless oil; Yield: 1.47 g (91%); H NMR (300 MHz, CDCl3):
δ 7.53 (dd, 1H, J =1.6, 7.9 Hz), 7.24
(ddd, 1H, J = 1.6, 7.4, 8.3 Hz), 6.88 (dd, 1H, J = 1.3, 8.3 Hz), 6.82 (dt, 1H, J = 1.4, 7.6 Hz), 5.89ꢀ5.78
(m, 1H), 5.01ꢀ4.99 (m, 2H), 4.03 (t, 2H, J = 6.4 Hz), 2.20ꢀ2.10 (m, 2H), 1.90ꢀ1.80 (m, 2H), 1.70ꢀ1.60
(m, 1H); 13C NMR (75 MHz, CDCl3):
δ 155.6, 138.7, 133.5, 128.5, 121.8, 114.9, 113.3, 112.4, 69.1,
33.5, 28.7, 25.4; IR: 3074, 2941, 2858, 1535, 1500, 1491, 1369, 1244, 1142, 912 cmꢀ1; CIꢀMS m/z: 254;
HRMS calcd for C12H15O79Br: 254.0301, found: 254.0314.
General procedure for the synthesis of boronic acids. The corresponding alkylated bromophenol
(4.68 mmol, 1 equiv) in THF (20 ml) was cooled down to ꢀ78°C. nꢀBuLi (2.5 M in hexane, 6.24 mmol,
1.3 equiv) was added dropwise. The mixture was stirred at ꢀ78°C for 1 hour and then the triisopropyl
borate (9.35 mmol, 2 equiv) was added dropwise. The mixture was stirred overnight. Ethyl acetate (10
ml) and an aqueous solution of HCl 1 N (7 ml, until pH = 1) were added. The phases were separated,
and the combined aqueous phases were extracted with ethyl acetate (3× 20 mL). The organic phases
were collected, dried (MgSO4), and concentrated in vacuo. The crude product was purified by flash
chromatography over silica with a mixture of cyclohexane/ethyl acetate as an eluent.
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Characterization of 2-(hex-5-en-1-yloxy)phenylboronic acid (3a)
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Colorless oil; Yield: 945 mg (84 %); H NMR (300 MHz, CDCl3):
δ 7.87 (dd, 1H, J = 7.3, 1.7 Hz),
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7.45ꢀ7.40 (m, 1H), 7.03 (t, 1H, J = 7.3 Hz), 6.90 (d, 1H, J = 8.3 Hz), 6.04 (s, 2H), 5.89ꢀ5.75 (m, 1H),
5.08ꢀ4.98 (m, 2H), 4.09 (t, 2H, J = 6.6 Hz), 2.19ꢀ211 (m, 2H), 1.90ꢀ1.80 (m, 2H), 1.65ꢀ1.55 (m, 2H); 13C
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NMR (300 MHz, CDCl3): δ 164.1, 138.2, 137.0, 132.9, 121.3, 115.3, 110.9, 68.3, 33.4, 28.7, 25.4; IR:
3409, 3074, 2926, 2854, 1641, 1599, 1576, 1487, 1448, 1340, 1225, 910 cmꢀ1; CIꢀMS m/z: 220, 219;
HRMS calcd for C12H17BO3: 220.1265, found: 220.1265.
General procedure for SNAr: alcohol (10.40 mmol, 5 equiv) was added to a solution of sodium
hydride (60 wt% suspension in mineral oil, 9.58 mmol, 4.6 equiv.) in dry THF (7 mL), under argon. The
solution was stirred until no more gas evolution was observed and then added to a solution of 1ꢀmethylꢀ
5ꢀiodoꢀ2ꢀphenylsulfonylimidazole (5) (2.08 mmol, 1 equiv.) in dry THF (35 mL). The reaction mixture
was transferred into a sealed tube and heated with microwaving for 8 h at 80 °C. Ethyl acetate (50 mL)
and saturated sodium chloride solution (30 mL) were successively added. The organic phase was further
washed twice with a saturated sodium chloride (30 mL). The combined aqueous phases were extracted
with ethyl acetate (20 mL). The combined organic phases were dried over MgSO4, and concentrated
under reduced pressure. The crude product was purified by flash chromatography over silica using a
mixture of hexane/ethyl acetate as eluent.
Characterization of 5-iodo-1-methyl-2-(hex-4-en-1-yloxy)-1H-imidazole (4a)
Yellow oil; Yield : 402 mg were obtained from 0.6 g of 5 (76 %); 1H NMR (300 MHz, CDCl3):
δ 6.73
(s, 1H), 5.87ꢀ5.74 (m, 1H); 5.05ꢀ4.95 (m, 2H); 4.32 (t, 2H, J = 6.5 Hz), 3.33 (s, 3H), 2.15ꢀ2.05 (m, 2H),
1.83ꢀ1.74 (m, 2H), 1.57ꢀ1.48 (m, 2H); 13C NMR (75 MHz, CDCl3):
δ
154.0, 138.6, 130.4, 115.1, 69.8,
63.7, 33.5, 31.4, 28.7, 25.3; IR: 3074, 2937, 2858, 1541, 1496, 1475, 1253, 1142, 910 cmꢀ1; ESIꢀMS
m/z: 307; HRMS calcd for C10H16IN2O: 307.0307, found: 307.0298.
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General procedure for Suzuki coupling: Tetrakis (triphenylphosphino) palladium (0.012 mmol, 0.03
equiv.) and 4 (0.342 mmol, 1 equiv.) in dimethoxyethane (4 mL) were introduced in a microwave tube.
Boronic acid (3) (0.376 mmol, 1.1 equiv.) in dimethoxyethane (4 mL), water (4 mL), and an aqueous
solution of sodium carbonate (20%, 3 equiv.) were successively added. The resulting mixture was then
degassed for 30 min by means of a flow of argon and placed in a microwave oven at 105 °C and 200 W
for 1 h. Ethyl acetate (100 mL) and water (50 mL) were added. The phases were separated, and the
aqueous phases were extracted with ethyl acetate (50 mL). The organic phases were combined, washed
with a saturated aqueous solution of NaCl (50 mL), dried over MgSO4, and concentrated under reduced
pressure. The crude product was purified by flash chromatography using CH2Cl2/2ꢀpropanol 97/3 as an
eluent.
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Characterization of 2-(hex-3-en-yloxy)-5-[2-(hex-5-en-1-yloxy)phenyl]-1-methyl-1H-imidazole (2i)
Yellow oil; Yield: 166 mg were obtained from 235 mg of 12’ (76 %); 1H NMR (300 MHz, CDCl3):
δ
7.32 (td, 1H, J = 8.1, 1.8 Hz), 7.25 (dd, 1H, J = 8.1, 1.8 Hz), 6.97 (dt, 1H, J = 7.4, 0.9 Hz), 6.94 (d,
1H, J = 8.1 Hz), 6.61 (s, 1H), 5.90ꢀ5.71 (m, 2H); 5.07ꢀ4.94 (m, 4H); 4.38 (t, 2H, J = 6.7 Hz), 3.97 (t,
2H, J = 6.6 Hz), 3.25 (s, 3H), 2.18ꢀ2.02 (m, 4H), 1.88ꢀ1.70 (m, 4H), 1.62ꢀ1.42 (m, 4H); 13C NMR (75
MHz, CDCl3):
δ 157.0, 153.7, 138.8, 138.7, 132.2, 129.8, 126.7, 121.8, 120.9, 120.2, 115.1, 112.4, 69.5,
68.5, 33.7, 33.6, 29.7, 28.92, 28.87, 25.52, 25.51; IR: 3074, 2935, 2858, 1639, 1580, 1497, 1244, 1142,
995, 800 cmꢀ1; ESIꢀMS m/z: 355; HRMS calcd for C22H31N2O2: 355.2386, found: 355.2374.
General procedure for RCM reaction. Imidazole 2 (0.266 mmol, 1 equiv) and 1,2ꢀdichloromethane
(70 ml) were introduced in a flask under argon. The mixture was heated at 95°C (reflux). A Grubbs
catalyst (second generation) (0.013 mmol, 0.05 equiv) was added. After one day, a second fraction of
Grubbs catalyst (0.007 mmol, 0.025 equiv) was added, and the solution was stirred at reflux for 24
hours. The mixture was cooled down until room temperature and potassium isocyanate (20 mg) was
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added. After one hour of stirring, the solvent was evaporated. The crude product was filtrated by flash
chromatography over silica with a mixture of cyclohexane/ethyl acetate as an eluent and directly
engaged to the next step.
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General procedure for hydrogenation. Imidazole 7 (0.44 mmol, 1 equiv), ethanol (10 ml) and ethyl
acetate (10 ml) were added in a flask. The catalyst Pd/C 10% (0.0222 mmol, 0.05 equiv.) was added and
the mixture was placed under H2(g) atmosphere. After 2 h 30, the mixture was filtrated and concentrated
under vacuum. The crude product was purified by flash chromatography over silica with a mixture of
cyclohexane/ethyl acetate as an eluent.
Characterization of 23-methyl-8,19-dioxa-21,23-diazatricyclo[18.2.1.02,7]tricosa-1(22),2,4,6,20-
pentaene (1i)
Colorless oil; Yield : 106 mg were obtained from 145 mg of 7i (49 % over the two steps; purity = 90%);
1H NMR (300 MHz, CDCl3):
δ 7.34 (td, 1H, J = 7.7, 1.8 Hz), 7.27 (dd, 1H, J = 7.4, 1.8 Hz), 6.98 (dt,
1H, J = 7.4, 1.0 Hz), 6.92 (d, 1H, J = 8.2 Hz), 6.56 (s, 1H), 4.81 (dt, 1H, J = 10.5, 2.9 Hz); 4.20 (dt, 1H,
J = 7.1, 2.9 Hz), 4.00ꢀ3.96 (m, 1H); 3.88 (dt, 1H, J = 7.7, 3.0 Hz,), 3.23 (s, 3H), 1.98ꢀ1.91 (m, 1H),
1.80ꢀ1.60 (m, 4H), 1.58ꢀ1.20 (m, 11H); 13C NMR (75 MHz, CDCl3):
δ 157.6, 153.3, 132.4, 130.2,
126.8, 121.2, 121.0, 120.4, 112.4, 69.7, 69.0, 29.7., 29.5, 28.6, 28.2; 28.1, 27.6, 27,4, 26,4, 25.9; IR:
2927, 2854, 1579, 1537, 1492, 1448, 1377, 1244, 1142, 1053, 991, 800 cmꢀ1; ESIꢀMS m/z: 329; HRMS
calcd for C20H29N2O2: 329.2229, found: 329.2231.
General procedure for the formation of fumaric salts. The imidazole compound (1 equiv.) was
dissolved in ethanol (0.1 M). A solution of fumaric acid (1 equiv.) in ethanol (0.05 M) was added. The
mixture was stirred overnight and then concentrated under reduced pressure. The correct 1:1 ratio was
verified by 1H NMR.
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BIOLOGY
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Cell lines and compounds. Seven human and one mouse cancer cell lines were obtained from either the
European Collection of Cell Cultures (ECACC; Salisbury, UK) or the American Type Culture
Collection (ATCC; Manassas, VA). The two glioma cell lines were the U373 (ECACC code 89081403)
and T98G (ATCC code CRLꢀ1690) astroglioma cell lines. The carcinoma cell lines were the A549 non
small cell carcinoma cell line (ATCC code CCL185), the MCFꢀ7 mammary breast carcinoma cell line
(ATCC code HTBꢀ22), the Lovo colon cancer cell line (ATCC code CCLꢀ229) and the PCꢀ3 prostate
cancer cell line (ATCC code CRLꢀ1435). The melanoma cell lines were the mouse B16F10 cells (ATCC
code CRLꢀ6475) and the human SKMELꢀ28 cells (ATCC code HTBꢀ72). The two immortal nonꢀ
cancerous cell lines were the HBL100 human breast epithelial cells (330178) and the HaCat human
keratinocyte cells (300493) that were purchased from Cell Lines Service (Eppelheim, Germany). The two
human normal primary cell lines were from fibroblast origin (NHDF and NHLF: normal human dermal
fibroblasts and normal human lung fibroblasts respectively; codes CCꢀ2509 and CCꢀ2512) and were
purchase from Lonza (Braine L’Alleud, Belgium).
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The human melanoma primary cultures were obtained as previously described with full genomic and
transcriptomic characterization.33
All biological assays were performed on fumaric salts (see above).
Determination of the IC50 growth inhibitory concentration. We made use of a colorimetric MTT
assay to determine the concentration that reduced the whole cell population by 50% after 72 h of
exposure to the compound as described previously.32 This test is based on the ability of living cells to
reduce 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyl tetrazolium bromide (MTT) salt in purple formazan
crystals via mitochondrial succinate dehydrogenase enzymatic activity. Each experimental condition
was run in six replicates.
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Quantitative videomicroscopy. The effects of tested compounds on cell morphology, cell proliferation
and cell death were assessed with the global view system developed in our lab and described
previously.32, 46 Briefly, each cell culture field was photographed every 4 minutes over a 72ꢀh period to
be compressed in a short 1ꢀminute movie. Global growth (GG) is the ratio between the number of cell
present on the picture at one time point and the number of cell present at time 0. Each condition was
tested in three replicates. The global growth ratio (GGR) is calculated by dividing the treated global
growth value by the global growth value of the control condition. In that case, the control GGR is 1.
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Flow cytometry
Apoptosis and cell cycle profile analyses. The 2iꢀinduced effects on cell cycle kinetics and apoptosis
were evaluated by means of flow cytometry with the double propidium iodide and TUNEL staining by
using the APOꢀDirect™ kit according to the manufacturer instructions (BD Biosciences, Erembodegem,
Belgium) as described previously.47
Intracellular reactive oxygen species measurements. DCFHꢀDA (2’ꢀ7’ꢀdichlorodihydrofluorescein
diacetate, SigmaꢀAldrich, Bornem, Belgium) is a cellꢀpermeant indicator for reactive oxygen species
(ROS). This chemically reduced and acetylated form of DCF, once inside the cell, is deacetylated by
intracellular esterases that yield the dye retained intracellularly in a charged form. The oxidation of this
later probe by ROS can be detected by monitoring the increase in fluorescence by flow cytometry.
Briefly, after cell exposure to the drug, the cells are washed twice and stained for 1 h at 37°C with 20
ꢁM DCFHꢀDA in RPMI without phenol red medium.48 After two washing steps, cells are detached by
trypsin and fluorescence was measured with a Cell Lab Quanta cytometer (Beckman Coulter, Analis,
Suarlée, Belgium). The experiments were conducted once in quadruplicate.
Mitochondria fluorescent staining. We made use of MitoTracker® Green FM (Invitrogen, Merelbeke,
Belgium) to highlight mitochondria. Briefly, SKMELꢀ28 cells were seeded on glass coverslips 48 h
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before treatment with 2i at 5 ꢁM for 3 to 48 h. After two washing steps, the mitochondria were stained
with 100nM of MitoTracker® Green FM in RPMI without phenol red for 30 min. After two rinses, the
cells were visualized under a Zeiss Axio fluorescent microscope (Zeiss, Oberkochen, Germany). Three
coverslips per experimental condition were analyzed.
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Oxygraphy. The cellular ROUTINE respiration, reflecting the aerobic metabolic activity under routine
culture conditions (with the physiological substrates in culture medium) of SKMElꢀ28 and U373 cancer
cells were recorded by highꢀresolution respirometry with a Clark electrode, at 37 °C (OROBOROS
INSTRUMENTS, Innsbruck, Austria). The oxygraph was calibrated as recommended with the assay
Dulbecco Modified Eagle’s Medium containing glucose (1 g/L) and pyruvate (110 mg/L) without
phenol red (Gibco, Invitrogen, Belgium). Five million cells, preꢀincubated during the last 72 h with 2i
(10ꢀ5 M) or not (control cells), diluted in assay medium, were transferred into each respiratory chamber
of the oxygraph. The measurement was performed under continuous stirring and started just after the
closing of the chamber. The slopes of O2 consumption, representing the routine respiration of the cells,
were calculated with the Oroboros oxygraph software (DatLab 4.0, OROBOROS DatLab software,
Innsbruck, Austria). Data are presented in relative values (%) in reference to the control group, for
which the routine respiratory rate was considered 100%.
Bioinformatic analyses. We normalized the measured expression values via a multiplicative
transformation chosen so that approximately the same number of probes appeared relatively
overexpressed in each of the arrays. We evaluated the multiplicative factors using the whole array as
well as several subsets of probes and used this information to estimate uncertainties in expression due to
normalization.
After normalization, we scored probes according to their ability to separate lowꢀ and high response cell
lines. We adopted an orderꢀbased scoring system closely linked to the wellꢀknown MannꢀWhitney
statistic. In contrast to the usual approach where a cellꢀline would be represented by a single number, we
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used the normalized expression values together with their lower/upper uncertainty levels in the
calculation. The addition of uncertainty information increases the resolution of the score and helps
distinguish between marginally and strongly different expression levels, even in small group sizes. For
groups with three and five cell lines, the scoring system allows for 135 distinct values. We reported
them in a signed format with values ± 1 indicating perfect separation and higher absolute values
indicating progressively worse separation.
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We compiled a short list of genes by picking probes with absolute score values ≤ 5. Finally, we analyzed
enrichment of the short gene lists in different Gene Ontology categories using the EASE (Expression
Analysis Systematic Explorer) software package (version 2.0) as previously described.32 Briefly, EASE
ranks functional gene clusters by means of the statistical overꢀrepresentation of individual genes in
specific categories relative to all the genes in the same category on the microarray.
Acknowledgment. This work was supported by the Fonds de la Recherche Scientifique– FNRS (F.R.S.ꢀ
FNRS). R.R. and R.K. are, respectively, Associate Researcher and Director of research of the F.R.S.ꢀ
FNRS. This article is dedicated to Professor Jacqueline MarchandꢀBrynaert on
the occasion of her 65th birthday.
Supporting Information Available. Full analytical and spectral data for all synthesized compounds as
well as tables of COMPARE analysis and bioinformatic comparisons’ data between group 1 and 2. This
material is available free of charge via the Internet at http://pubs.acs.org.
Corresponding authors’ informations
*V Mathieu (biology): Phone: +32ꢀ2ꢀ6505179. Eꢀmail: vemathie@ulb.ac.be ; R Robiette (chemistry):
Phone: +32ꢀ10ꢀ479176. Fax: +32ꢀ10ꢀ474168. Eꢀmail: raphael.robiette@uclouvain.be.
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Abbreviations
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DBBA = 5,5ꢀdibromobarbituric acid
MW = microwave heating
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LEGENDS TO THE FIGURES
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Figure 1: 2i exerts cytostatic rather than cytotoxic effects on melanoma cells. A: quantitative
videomicroscopy illustrations of 2iꢀinduced effects on human SKMELꢀ28 melanoma cells after 72 h of
treatment (5 ꢁM). B: Videomicroscopic analysis was conducted twice in triplicate and quadruplicate,
respectively. The global growth ratio was calculated, and data are presented as the mean ± SEM. C: Cell
cycle analysis was performed on SKMELꢀ28 cells treated with 5 ꢁM 2i for 24, 48 and 72 h,
respectively. D: Apoptosis measurement by TUNEL staining of SKMELꢀ28 cells treated for 24, 48 and
72 h with 5 ꢁM 2i. The manufacturer provided negative and positive controls (Ct – and Ct +,
respectively), and we also used internal controls with PCꢀ3 human prostate cancers cells left untreated or
treated for 72 h with 1 ꢁM narciclasine. The results are presented as the mean ± SEM (quadruplicate
except for the kit controls, which are presented as the result in singulate).
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Figure 2: Characterization of the effects induced by 10 µM of 2i in the NCI 60-cell-line panel.
Figure 3: IC50 values of 2i compound on 11 human primary melanoma cultures. The dotted line
corresponds to the mean IC50 value obtained with these 11 primary cultures after 2iꢀtreatment for 72 h as
revealed by the colorimetric MTT assay. Experiment was conducted once in sextuplicate. * indicates
that the IC50 is over 100 ꢁM, i.e., the maximal concentration tested.
Figure 4: 2i-induced effects on mitochondria. A: Mitochondrial staining with a fluorescent
MitoTracker: fluorescence distribution and intensity was not altered by 2i treatment over a 24 h period.
B: ROS production over time was analyzed by flow cytometry in SKMELꢀ28 cells treated with 5 ꢁM 2i.
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