Old drug scaffold, new activity: Thalidomide-correlated compounds exert different effects on breast cancer cell growth and progression

Article abstract of DOI:10.1002/cmdc.201600629

Thalidomide was first used for relief of morning sickness in pregnant women and then withdrawn from the market because of its dramatic effects on normal fetal development. Over the last decades, it has been used successfully for the treatment of several pathologies, including cancer. Many analogues with improved activity have been synthesized and tested. Herein we report some effects on the growth and progression of MCF-7 and MDA-MB-231 breast cancer cells by a small series of thalidomide-correlated compounds, which are very effective at inducing cancer cell death by triggering TNFα-mediated apoptosis. The most active compounds are able to drastically reduce the migration of breast cancer cells by regulation of the two major proteins involved in epithelial–mesenchymal transition (EMT): vimentin and E-cadherin. Moreover, these compounds diminish the intracellular biosynthesis of vascular endothelial growth factor (VEGF), which is primarily involved in the promotion of angiogenesis, sustaining tumor progression. The multiple features of these compounds that act on various key points of the tumorigenesis process make them good candidates for preclinical studies.

Full text of DOI:10.1002/cmdc.201600629

Accepted Article
Title: Old Drug Scaffold, New Activity: Thalidomide Correlated
Compounds Exerting Different Effects on Breast Cancer Cell
Growth and Progression
Authors: Alessia Carocci, alessia catalano, Domenico Iacopetta, and
Maria Stefania Sinicropi
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Old Drug Scaffold, New Activity: Thalidomide Correlated Compounds
Exerting Different Effects on Breast Cancer Cell Growth and Progression
Dr. Domenico Iacopetta,a,§ Dr. Alessia Carocci,b,§ Prof. Maria Stefania Sinicropi,a,* Dr. Alessia
Catalano,b,* Prof. Giovanni Lentini,b Dr. Jessica Ceramella,a Dr. Rosita Curcio,a Prof. Maria
Cristina Caroleo.a
aDepartment of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036,
Arcavacata di Rende, Italy
bDepartment of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", 70126, Bari, Italy.
§These authors contributed equally to the paper.
*Corresponding authors:
Prof. Sinicropi Maria Stefania,
Department of Pharmacy, Health and Nutritional Sciences
University of Calabria
87036, Arcavacata di Rende, Italy
Phone: +39 0984 493200
E-mail: s.sinicropi@unical.it
Prof. Catalano Alessia
Department of Pharmacy-Drug Sciences
University of Bari "Aldo Moro"
70126, Bari, Italy
Phone: +39 080 5442745
E-mail: alessia.catalano@uniba.it
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Abstract
Thalidomide was firstly used as morning sickness relief in pregnant women and then withdrawn
from market because of its dramatic effects on fetal normal development. Over the last decades, it
has been successfully used for the care of several pathologies, including cancer. Many analogues
with improved activity were synthesized and tested. Herein, we report some effects on MCF-7 and
MDA-MB-231 breast cancer cells growth and progression of a small series of thalidomide
correlated compounds, very effective in inducing cancer cells death by triggering TNFα-mediated
apoptosis. Moreover, the most active compounds are able, as well, to reduce drastically the
migration of breast cancer cells, through the regulation of the two major proteins involved in
epithelial-mesenchymal transition (EMT), vimentin and E-cadherin. They diminish the
intracellular biosynthesis of vascular endothelial growth factor (VEGF), primarily involved in the
promotion of angiogenesis that sustain tumor progression. The multiple features possessed by
these compounds, acting on different key-points of tumorigenesis process, make them good
candidates for preclinical studies.
Keywords: Thalidomide; Antitumor activity; TNFα; Apoptosis; Angiogenesis.
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Introduction
Thalidomide is a glutamic acid derivative synthesized in Germany in 1954 and marketed in 46
countries in the late 1950’s and early 1960’s as morning sickness relief in pregnant women.[1]
However, a massive increase of newborns with severe malformations (deafness, blindness, limb
growth defects) was registered after thalidomide use, so that it was subsequently withdrawn in
1962.[2] Over the last few decades the interest in this old drug has been renewed, because of its
efficacy in several important diseases as, for instance, the erythema nodosum leprosum,[3] multiple
myeloma,[4] breast cancer,[5] refractory Crohn’s disease[6] and HIV-related diseases.[7] It became
clearer that thalidomide exerted multifaceted properties, leading many research groups to
synthesize several derivatives and to study their effects, mostly in cancer research. Cancer growth
and progression need fundamental changes, e.g. in energy metabolism pathways, in nutrient
uptake and in several factors, leading to a phenotypic heterogeneity within subpopulations of
cancer cells.[8,9] These changes allow a higher ability to grow even in unsuited micro-
environmental conditions (nutrient depletion, hypoxia).[10] In 1994, D'Amato et al.[11] reported that
thalidomide was able to inhibit angiogenesis, induced in a rabbit cornea model, shedding light on
the anti-angiogenic role responsible for teratogenicity. Remarkably, angiogenesis is essential for
cancer growth, progression and metastasis and, amongst the pro-angiogenic factors, vascular
endothelial growth factor (VEGF) plays a key role because of its up-regulation in tumors.[12]
Furthermore, many literature data showed that thalidomide inhibits tumor necrosis factor alpha
(TNFα) production, which plays a dual role in cell proliferation, stimulating tumor cell growth or
inducing cancer cell death.[13] Starting from thalidomide, as a lead compound, several analogues
have been developed, sharing and augmenting the antiproliferative, anti-angiogenic, and TNFα
reducing properties of the parent compound.[14–18] Another interesting thalidomide effect is the
ability to inhibit the epithelial-mesenchymal transition (EMT), a dynamic process enabling
polarized epithelial cells to assume a mesenchymal phenotype with enhanced migratory and
invasive capabilities.[19] EMT is associated with early stages of carcinogenesis, cancer invasion
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and recurrence and is characterized by the reduction of epithelial cell junction proteins, including
E-cadherin, α-catenin, claudins, occludin, combined with an increased expression of mesenchymal
markers, such as N-cadherin, vimentin and fibronectin. In this context, it is of great interest to
design and synthesize thalidomide analogues able to exert polyhedric effects on cancer cells
proliferation.[20,21] In this paper, we report the anti-tumor activity of some thalidomide analogues
and correlated compounds (Figure 1), which are able to trigger cell death by TNFα-mediated
apoptosis in two breast cancer cell lines, MCF-7 and MDA-MB-231, without affecting the
proliferation of non-tumoral MCF-10a cells. Moreover, some of these compounds are able to
reduce cancer cell migration, regulating the expression of two important proteins involved in
epithelial-mesenchymal transition (EMT), namely E-cadherin and vimentin. We proved that these
compounds possess a better activity and may represent a more effective drugs interfering with
different breast tumor targets, thus offering a valid alternative in cancer treatment.
Chemistry.
[24]
Compounds 1,7[22] 2,[23] 3,6
(Figure 1) were prepared as described elsewhere. Chiral
compounds (3,4,6,8) were prepared in their racemic forms. Compound 4 was prepared as reported
in Scheme 1. Compound 10 was readily prepared by the selectively Boc-protection of piperazine-
2-carboxylic acid (9) at the 4-position, followed by Cbz-protection at the 1-position.[25] Then, 10
was reacted with 2,6-dimethylaniline in the presence of IIDQ (2-isobutoxy-1-isobutoxycarbonyl-
1,2-diihydroquinoline) to afford the corresponding carboxamide 11.[26] Selective Cbz-deprotection
of 11 with triethylsilane and palladium chloride[27] gave 12, which was converted into the N-
benzyl derivative 13 by reaction with benzyl bromide.[28] Boc-deprotection of 13 and conversion
of the resulted amine into the corresponding hydrochloride salt was performed with gaseous HCl
as previously described.[29] Compound 5 was synthesized as reported in Scheme 2 by reacting
methyl imidazole-4-carboxylate (14) with 2,6-dimethylaniline according to a procedure reported
in the literature.[30] Compound 8 was synthesized as reported in Scheme 3. The amino alcohol 15
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was protected with phthalic anhydride[31] to give the phthalimido alcohol 16 which was converted
into the corresponding bromo derivative 8 by treatment with PBr3.
Results and discussion
Antiproliferative activity
Thalidomide analogues were evaluated for their antiproliferative activities against two human
breast cancer cell lines, namely estrogen receptor positive (ER+) MCF- and triple negative (ER-,
PR-, and HER-2/Neu not amplified) MDA-MB-231 cells.[32] Cells were subjected to 72 hours of
continuous exposure to the tested compounds and, after that, their viability was measured by 3-
(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium (MTT) test.[33,34] Thalidomide was used as
reference molecule in these assays. The IC50 values, i.e. the concentrations of the compounds
producing the 50% of growth inhibition, are reported in Table 1. As shown, thalidomide exerted
only a very poor antitumor activity in both the breast cell lines used, showing slight effects only at
doses higher than 500 µM, whereas a very impressive activity was recorded with the synthesized
compounds. Particularly, compounds 3 and 8 were the most active, with IC50 values of 47 ± 1 µM
and 40.3 ± 0.8 µM towards MCF-7 cells and 56.5 ± 1.3 µM and 37.2 ± 1.0 µM towards MDA-
MB-213, respectively. The other compounds also showed interesting antiproliferative effects, even
though to a lesser extent (see Table 1). Once established that our compounds possessed an
improved antitumor activity with respect to thalidomide, we tested all the considered compounds
on non-malignant breast epithelial cells, MCF-10a, in order to establish whether a cytotoxic effect
could be exerted. We found that none the considered compounds produced toxic effects on the
viability of normal breast epithelial cells up to 500 µM. The improved ability of our compounds to
affect the proliferation of MCF-7 cells and, most importantly, of the highly aggressive and
metastatic MDA-MB-231, without affecting non-tumoral breast epithelial cells MCF-10a,
prompted us to investigate more deeply the properties of the most active compounds.
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Cancer breast cells death by TNFα-mediated apoptosis
In previous reports, thalidomide was found to suppress TNFα mRNA expression.[35] In this regard,
literature data have clarified the complex interplay existing between TNFα and apoptosis,
highlighting that TNFα can promote apoptotic cell death through the activation of several caspases
and the mitochondrial damage, a pattern known as extrinsic apoptotic pathway. Hence, we
investigated the ability of compounds 3 and 8 to modulate TNFα cell levels by
immunofluorescence analysis. MCF-7 cells were exposed for 24 h to the two different compounds,
at the concentrations equal to their own IC50 values. Then cells were processed for
immunofluorescence procedure using an antibody raised against TNFα vehicle-treated cells were
used as a control. Immunofluorescence analysis of MCF-7 cells treated with the compounds 3 and
8 revealed a three- and two-fold increase of TNFα expression, respectively (Figure 2A and B).
Furthermore, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) staining of the
same samples highlighted nuclei containing condensed DNA, frequently associated with
apoptosis. It is known that the activation of the extrinsic apoptotic pathway implies an increased
mitochondrial permeability, followed by the cytosolic release of cytochrome C, a small protein
associated with the inner membrane of the intact mitochondria, that may act as apoptogenic
factor.[36] Therefore, we further investigated the subcellular localization of cytochrome C in MCF-
7 cells treated with compounds 3 or 8, using an antibody raised against cytochrome C (Figure 3A).
It was remarkable that cytochrome C was released in the cytosol of the treated cells, as it can be
evidenced by the increased and diffused fluorescence (Figure 3A and B), whereas in the vehicle-
treated cells is still localized in the mitochondrial network. Moreover, TUNEL assay conducted on
MCF-7 cells treated for 24 h with the compounds 3 or 8 revealed a green fluorescence in the cell
nuclei, which indicates DNA fragmentation correlated with the apoptotic process (Figure 4).
Taken together, our data highlight that compounds 3 and 8 are able to promote cancer breast cells
death by increasing TNFα synthesis that, in turn, promotes cytochrome C release from
mitochondria and, therefore, apoptosis.
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Effects of compounds 1-8 on breast cancer cells migration
We tested the ability of compounds 1–8 to affect cell migration of MCF-7 and MDA-MB-231
cells by using a simple, low-cost and well-developed in vitro method, i.e. the wound-healing
assay. Cells were plated and cultured to form a monolayer, then scratched to form a wound (see
Experimental section) and treated with compounds 1–8 at the concentrations corresponding to
their calculated IC50 (see Table 1) for 48 and 72 hours, respectively for MDA-MB-231 and MCF-7
cells. These different endpoint times were experimentally determined, considering that MDA-MB-
231 cells possess a higher growth rate than the MCF-7 cells and tend to give greater metastases in
vivo.[37] In MCF-7 cells compounds 2, 3, 7 and 8 produced the higher effect, preventing the total
wound closure, with percentages of about 52, 77, 72 and 69% (Figure 5A). In MDA-MB-231
cells, the above mentioned compounds 2, 3, 7 and 8 produced a significant lack of a total wound
closure, respectively of about 11, 25, 27 and 25% but, differently from MCF-7 cells, compound 6
exhibited an impressive effect in prevent wound closure, of about 32% (Figure 5B). The other
compounds had no significant effects in such experiments. In both breast cancer cell lines,
compound 2 was the most effective in preventing the wound closure (Figure 3A and B) with
respect to the other active compounds, but it possess a lesser efficacy in reducing cell viability, as
demonstrated in our previous experiments (see Table 1). Overall, our data highlighted that
compounds 3 and 8 showed a good activity in diminishing cancer cell migration (see Figure 5A
and B, Figure 6), together with the lowest IC50 values (Table 1). Thus, we chose these two
compounds for further investigations.
Compounds 3 and 8 hamper EMT decreasing breast cancer cells invasiveness
We firstly tried to better understand the role of compounds 3 and 8 in breast cancer cells invasion.
Indeed, during EMT, malignant cells reorganize their cytoskeleton, expressing mesenchymal
markers as vimentin, and lose inter-cellular adhesion molecules such as E-cadherin, which is
considered as a diagnostic biomarker in breast cancer.[38] In this regard, we tested the ability of
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compounds 3 and 8 to modulate vimentin and E-cadherin levels in MCF-7 cells. Cells were
exposed for 24 h to the compounds, used at the concentrations equal to their IC50 values,
processed and subjected to immunofluorescence analysis using antibodies against vimentin and E-
cadherin. Vehicle-treated cells were used as a control. Immunofluorescence microscopy analysis
showed that both compounds 3 and 8 strongly decreased vimentin levels (Figure 7A and B), at the
same time, they significantly increased E-cadherin levels (Figure 8A and B), mostly at
cytoplasmic membrane, where cell-cell interactions occur. These data indicate that compounds 3
and 8 may play a role in inhibiting EMT process, through the regulation of vimentin and E-
cadherin expression, which are mainly involved in cancer invasion and metastases dissemination.
Compounds 3 and 8 are able to inhibit VEGF expression in cancer breast cells
Thalidomide is known to be a potent inhibitor of angiogenesis, an essential process in cancer cell
proliferation, extracellular matrix invasion and metastasis. In 2006, Komorowski et al.[39] reported
that this drug could inhibit VEGF secretion in human endothelial cell line, decreasing
neovascularization and VEGF can act as an autocrine or paracrine factor in the invasiveness of
several cell types via receptor binding. On this basis, we tested by immunofluorescence studies
whether compounds 3 and 8 could interfere with VEGF expression in MCF-7 cells. With this aim,
MCF-7 cells were treated for 24 hours with compounds 3 or 8 (used at concentrations equal to
their IC50 values), and then subjected to immunofluorescence staining using an antibody raised
against VEGF (Figure 9A). In such experiments, vehicle-treated cells were used as a control
(CRTL). Interestingly, the fluorescent cytoplasmic signal correlated with the presence of VEGF
was remarkably decreased in MCF-7 cells treated with both compounds 3 and 8, with respect to
the vehicle-treated cells (Figure 9A and B), indicating an interference with VEGF biosynthesis.
This feature has been correlated, as well, with apoptosis induction in ER-positive and triple
negative breast cancer cells, as already reported.[40] Additionally, in MCF-7 cells treated with
compound 8, the decrease of VEGF-associated fluorescence is accompanied by an evident change
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in subcellular localization (see Figure 9A, white arrows), which is no more evidently localized in
the perinuclear area, as in the vehicle-treated cells, but diffused as well in the nuclei. This
difference could indicate a decreased biosynthesis at the endoplasmic reticulum network and, at
the same time, an interaction with nuclear receptors. VEGF nuclear accumulation may play
several and still poorly understood roles, however Li et al.[41] suggested a role in coagulation and
fibrinolysis pathways, affecting vascular endothelial cellular physiology independently of its
growth stimulation. The latter observation contrast with the results obtained by scratch and MTT
assays, but further studies are needed in order to shed light on this feature. Summing up, our
observations highlight the anti-angiogenic potential of these compounds, which represent one of
the most desired feature of drugs used for the treatment of breast cancer.
Conclusions
The library of thalidomide correlated compounds reported in this paper consists of three newly
synthesized compounds (4, 5 and 8) and other already reported five thalidomide analogues (1, 2, 3,
6, and 7). The studied compounds have shown some interesting properties on breast cancer cells,
particularly the estrogen-positive (ER+) MCF-7 and the triple negative MDA-MB-231 cells.
Compared with their lead molecule, thalidomide, some of these compounds have shown a higher
antitumor activity and, particularly, compounds 3 and 8 possess IC50 values almost ten-fold lower
than thalidomide. Besides, no significant cytotoxic effects have been noticed regarding MCF-10A
normal breast cells viability. The antitumor effects have been ascribed to an intracellular increase
of TNFα biosynthesis, which we believe is responsible for a protection mechanism (paracrine
effect) against the abnormal proliferation of surrounding breast cancer cells. Indeed, this cytokine
may trigger the apoptotic process through the mitochondrial pathway, given that a massive release
of cytochrome c from the mitochondrial compartment to the cytosol has been detected by
immunofluorescence assays. Cancer cell death by apoptosis has been definitely proved by TUNEL
assay. Besides these effects on the breast cancer cells viability, compounds 3 and 8, chosen to
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further investigate other properties, are also able to decrease cancer cells migration and invasion,
as demonstrated by wound healing assays. These features reside in the regulation of two major
proteins involved in the EMT, i.e. vimentin and E-cadherin, which allow tumours cells to detach
from the original site and disseminate through the blood vessels. The inhibition of vimentin
biosynthesis and the increase of E-cadherin expression, induced by our compounds, represent a
very interesting features, making cancer cell less prone to invasion and metastasis. Finally,
compounds 3 and 8 may play a role in regulating VEGF expression in breast cancer cells, given
that we have noticed a fall of VEGF intracellular levels and, as well, a different subcellular
localization in cancer cells treated with compound 8. In this regard, other studies are needed in
order to better understand the precise mechanism responsible for the shown effects. However, we
believe that these compounds possess a high potential, because of their multiple properties toward
different aspects of breast cancer cell biology. Indeed, besides to their selective killing effect on
cancer cells, the interference with the migration and invasion processes, which are the main cause
of death for breast cancer patients, represent a peculiar and promising property. Lastly, the role
played by our compounds in decreasing VEGF levels, mainly involved in the angiogenesis
pathway responsible for promoting cancer survival, complete the sight of the multiple anticancer
properties, exerted by our compounds. We are confident that our research will be seminal and
encourage other studies on the design, development and biological evaluation of thalidomide
correlated compounds.
Experimental section
Chemistry. All chemicals were purchased from Sigma-Aldrich or Lancaster in the highest quality
commercially available. Solvents were reagent grade unless otherwise indicated. Yields refer to
purified products and were not optimized. The structures of the compounds were confirmed by
routine spectrometric analyses. Only spectra for compounds not previously described are given.
Melting points were determined on a Gallenkamp melting point apparatus in open glass capillary
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tubes and are uncorrected. 1H NMR and 13C NMR spectra were recorded on a Varian VX Mercury
1
13
spectrometer operating at 300 and 75 MHz for H and C, respectively, or an Agilent 500 MHz
1
operating at 500 and 125 MHz for H and 13C, respectively, using CDCl3 and DMSO-d6 as
solvents. Chemical shifts are reported in parts per million (ppm) relative to solvent resonance:
CDCl3, δ 7.26 (1H NMR) and δ 77.0 (13C NMR); DMSO-d6, δ 2.48 (1H NMR) and δ 39.9 (13C
NMR). J values are given in Hz. The following abbreviations are used: s-singlet, d-doublet, t-
triplet. Gas chromatography (GC)/mass spectroscopy (MS) was performed on a Hewlett-Packard
6890–5973 MSD at low resolution. Elemental analyses were performed on a Eurovector Euro EA
3000 analyzer and the data for C, H, N were within ± 0.4 of theoretical values (Table S1).
Chromatographic separations were performed on silica gel columns by flash chromatography
(Kieselgel 60, 0.040–0.063 mm, Merck, Darmstadt, Germany) as previously described.[42]
N-(2,6-Dimethylphenyl)piperazine-1-[(benzyloxy)carbonyl]-4-(tert-
butoxycarbonyl)piperazine-2-carboxamide (11). IIDQ (0.27 mL, 0.92 mmol), 2,6-
dimethylaniline (0.99 mL, 0.85 mmol) and Et3N (0.16 mL, 1.15 mmol) were successively added to
a stirring solution of compound 10 (0.28 g, 0.77 mmol) in CHCl3 (26 mL). The reaction mixture
was heated under reflux for 6 h. The solvent was removed under reduced pressure and the residue,
taken up with EtOAc, was washed three times with 2 N HCl, twice with 2 N NaOH, and then
dried over anhydrous Na2SO4. Flash chromatography (eluent EtOAc/petroleum ether 4:6) of the
residue gave 90 mg (25% yield) of 11 as a slightly yellowish oil: GC/MS (70 eV) m/z (%) 367 (M+
–100, <1), 91 (100).
tert-Butyl 3-[(2,6-dimethylphenyl)carbamoyl]piperazine-1-carboxylate (12). A suspension of
compound 11 (80 mg, 0.17 mmol), triethylsilane (0.11 mL, 0.68 mmol), triethylamine (17 μL,
0.12 mmol), and PdCl2 (9.03 mg, 0.051 mmol) in dichloromethane (1 mL) was heated at reflux for
3 h. The reaction mixture was quenched with saturated aqueous ammonium chloride solution and
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extracted with ether several times. The combined organic phases were washed with water and then
brine, dried, and concentrated in vacuo to give 45 mg (79%) of a slightly yellowish oil: GC/MS
(70 eV) m/z (%) 333 (M+, 3), 129 (100).
tert-Butyl 4-benzyl-3-(2,6-dimethylphenylcarbamoyl)piperazine-1-carboxylate (13). To a
stirring solution of compound 12 (0.15 g, 0.45 mmol) in dioxane (8 mL), a solution of K2CO3
(0.18 g, 1.30 mmol) in H2O (8 mL) was added. The reaction mixture was heated to 70 °C, and
then, benzyl bromide (63 μL, 0.53 mmol) was added dropwise. The heating was continued for 45
min. Then, the dioxane was removed under reduced pressure and the aqueous residue was taken
up with EtOAc and extracted with 2 N HCl. The aqueous phase was made alkaline with 2 N
NaOH and extracted twice with EtOAc. The combined organic layers were dried over anhydrous
Na2SO4 and concentrated under vacuum to give 90 mg (47%) of a yellow oil: GC/MS (70 eV) m/z
(%) 323 (M+ –100, 2), 175 (100).
1-Benzyl-N-(2,6-dimethylphenyl)piperazine-2-carboxamide
Hydrochloride
(4.HCl).
Compound 4 as hydrochloride salt was obtained by saturating a solution of compound 13 in
anhydrous Et2O with gaseous HCl and stirring it at room temperature for 15 min. Removal of the
solvent under reduced pressure gave a white solid (67%) which was recrystallized from
EtOH/Et2O to afford white crystals: mp 235–237 °C (abs EtOH/Et2O); anal. calcd for C12H13N3O:
C 65.70, H 7.90, N 11.49, found: C 65.35, H 7.68, N 11.22.
N-(2,6-Dimethylphenyl)-1H-imidazole-4-carboxamide (5). To a stirring suspension of 60%
NaH (0.27 g, 6.67 mmol) in dry dioxane (10 mL) in N2 atmosphere, 2,6-dimethylaniline (0.72 g,
5.95 mmol) was added and the mixture brought to reflux. Then, a solution of methyl imidazole-4-
carboxylate (14, 0.30 g, 2.30 mmol) in dry DMF (13 mL) was added dropwise in an ice bath. After
5 h the mixture was poured into ice and extracted with EtOAc. The organic layer was then
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evaporated in vacuo. Purification by column chromatography gave 0.26 g (53%) of 5 as a slightly
yellowish solid: mp 212–214 °C; 1H NMR (500 MHz, DMSO-d6): δ 2.18 (s, 6H, CH3), 3.77 (br s,
1H, NH), 7.05–7.15 (m, 3H, Ar), 8.57 (s, 1H, Ar), 9.18 (s, 1H, Ar), 10.5 (s, exch D2O, 1H, NH);
13C NMR (125 MHz, DMSO-d6): δ 18.6 (2C), 121.6 (1C), 127.6 (1C), 128.2 (1C), 128.3 (2C),
134.2 (2C), 135.9 (1C), 136.5 (1C), 156.1 (1C); GC/MS (70 eV) m/z (%) 215 (M+, 27), 121 (100);
anal. calcd for C12H13N3O: C 66.96, H 6.09, N 19.52, found: C 66.62, H 5.89, N 19.20.
2-(1-Hydroxy-3-methylbutan-2-yl)-1H-isoindole-1,3(2H)-dione (16).
A mixture of 2-amino-3-methylbutan-1-ol 15 (5 mmol), phthalic anhydride (5 mmol) and
triethylamine (0.5 mmol) in 10 mL of toluene is heated under reflux in a flask fitted with a Dean–
Stark tube for 3 h. During this period, the temperature of the oil bath is maintained at about 130°C
and water separates. All volatile matter are then evaporated under vacuum and the solid residue is
taken up with EtOAc and washed with 2N HCl, NaHCO3, and H2O. The organic phase is dried
(Na2SO4) and concentrated under vacuum to give 0.56 g of 16 as a yellow oil (48%): GC/MS (70
eV) m/z (%) 233 (M+, 2), 202 (100).
2-(1-Bromo-3-methylbutan-2-yl)-1H-isoindole-1,3(2H)-dione (8).
PBr3 (1.7 mmol) was carefully added to 16 (1.5 mmol) at 0 °C. The reaction mixture was stirred
for 2 h at 0 °C and for 6 h at room temperature, then poured onto ice and extracted with AcOEt.
The organic layer was washed with brine, dried over Na2SO4, and the solvent was evaporated in
vacuo to give a pale-yellow oily residue. Purification by column chromatography (eluent
EtOAc/hexane 2:8) gave 0.30 g (66%) of 8 as a white solid: 1H NMR (500 MHz, CDCl3): δ 0.91
(d, J = 6.4 Hz, 3H, CH3), 1.08 (d, J = 6.4 Hz, 3H, CH3), 2.36–2.46 (m, 1H, CHCH3), 3.75–3.83
(m, 1H, CHN), 4.10–4.22 (m, 2H, CH2), 7.68–7.78 (m, 2H, Ar), 7.82–7.90 (m, 2H, Ar); 13C
NMR (125 MHz, CDCl3): δ 20.3 (1C), 20.4 (1C), 30.4 (1C), 31.5 (1C), 59.6 (1C), 123.4 (2C),
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131.5 (2C), 134.1 (2C), 168.4 (2C); GC/MS (70 eV) m/z (%) 296 (M+, 1), 252 (100); anal. calcd
for C13H14N2OBr: C 52.72, H 4.76, N 4.73, found: C 52.73, H 4.99, N 4.43.
Biology
Cell cultures. The three cell lines used in this work have been purchased from American Type
Culture Collection (ATCC, Manassas, VA, USA). MCF-7 human breast cancer cells, estrogen
receptor (ER) positives, were maintained in Dulbecco’s Modified Eagle’s Medium/Nutrient
Mixture F-12 Ham (DMEM/F12), supplemented with 10% Foetal Bovine Serum (FBS) and 100
U/ml penicillin/streptomycin, as previously described.[43] MCF-10a human mammary epithelial
cells, were cultured in DMEM/F12 medium, supplemented with 5% Horse Serum (HS, (Eurobio,
Les Ullis, Cedex, France), 100 U/ml penicillin/streptomycin, 0.5 mg/ml hydrocortisone, 20 ng/ml
hEGF (human epidermal growth factor), 10 μg/ml insulin and 0.1 mg/ml cholera enterotoxin
(Sigma–Aldrich, Milano, Italy). MDA-MB-231 human breast cancer cells, known as triple
negative cells (i. e. not overexpressing human epidermal growth factor receptor 2, or HER2,
estrogen and progesterone receptors), were cultured in DMEM/F12, supplemented with 5% FBS,
1% L-glutamine and 100 U/ml penicillin/streptomycin. Cells were maintained at 37 ºC in a
humidified atmosphere of 95% air and 5% CO2 and periodically screened for contamination.[44]
Thalidomide or its analogues were dissolved in dimethylsulfoxide (DMSO) (Sigma, St. Louis,
Missouri, USA) and opportunely diluted in DMEM/F12 medium in order to obtain the working
concentrations.
Cell viability. MDA-MB-231 and MCF-7 cells were grown in complete medium and, before being
treated, they were starved in serum free medium for 24 h, to allow cell cycle synchronization.
Then cells were grown in phenol red-free medium supplemented with 1% DCC (destran coated
charcoal treated) FBS. Cells were treated with increasing concentrations (1, 5, 10, 25, 50, 100 and
500 μM) of each compound for 72 h. Untreated cells were supplemented with the DMSO (final
14
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10.1002/cmdc.201600629
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concentration 0.1%) and used as a control.[45] Cell viability was assessed using the 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reagent (MTT), according to the
manufacturer’s protocol (Sigma-Aldrich, Milan, Italy), as previously described.[46] For each
sample mean absorbance, measured at 570 nm, was expressed as a percentage of the control and
plotted versus drug concentration to determine for each cell line the IC50 values (i.e. drugs
concentrations able to reduce cell viability of 50% with respect to the control), using GraphPad
Prism 5 Software (GraphPad Inc., San Diego, CA). Mean values of three independent experiments
carried out in triplicate and standard deviations (SD) are shown.
Wound-healing assay. MCF-7 and MDA-MB-231 cells were plated on 6-well plates and cultured
in full medium to produce confluent monolayers. They were wounded in a line using a standard
200-µl pipette sterile tip, then washed with PBS to remove cell debris before incubation with
different concentrations of each compound at its IC50, as indicated in table 1. Images at time zero
(t = 0 h) were acquired to record the initial area of the wound, and the recovery of the wounded
monolayer due to cells migration toward the scratched area was estimated at 48 and 72 h (t = Δ h).
Images were captured using an inverted microscope equipped with digital camera (Leica DM
6000). The migration of cells toward the wounds was expressed as percentage of wound closure:
% of wound closure = [(At = 0 h – At = Δ h)/At = 0 h] × 100%,
Where, At =0h is the area of wound measured immediately after scratching, and At=Δh is the area
of wound measured 48 or 72 h after scratching. Vehicle-treated cells were used as a control. The
collected images were analysed using Leica Application Suite X (LAS X) software. Each
experiment was performed three times, and each treatment was conducted in three replicates.
Representative images have been shown.
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ChemMedChem
Tunel assay. Apoptosis was detected by the TUNEL assay, according to the guidelines of the
manufacturer (CF™488A TUNEL Assay Apoptosis Detection Kit, Biotium, Hayward, CA, USA),
with some modifications.[47] In brief, cells were grown on glass coverslips, after treatment, they
were washed 3 times with PBS, then methanol-fixed at –20 °C for 15 min. Fixed cells were
washed 3 times with 0.01% (V/V) Triton X-100 in PBS and incubated with 100 µL of TUNEL
equilibration buffer for 5 min, then it was removed and 50 µL of TUNEL reaction mixture
containing 1 µL of terminal deoxynucleotidyl transferase (TdT) were added to each sample and
incubated in a dark and humidified chamber for 2 h at 37 ºC. Samples were washed 3 times with
ice cold phosphate-buffered saline (PBS) containing 0.1% Triton X-100 and 5 mg/mL bovine
serum albumin (BSA). DAPI (0.2 µg/mL) counterstain was performed for 10 min at 37 ºC in dark
and humidified conditions. Cells were then washed 3 times with cold PBS, adding one drop of
mounting solution, then they were observed and imaged under a fluorescence microscope (Leica
DM 6000) (20X magnification) with excitation/emission wavelength maxima of 490 nm/515 nm
(CFTM488A) or 350 nm/460 nm (DAPI). Images are representative of three independent
experiments.
Immunofluorescence. For immunocytochemistry, cells were grown on coverslips in full media,
then serum-starved for 24 h for the indicated time with examined compounds. Then they were
PBS-washed and fixed with cold methanol (15 min/–20 °C) and washed 3 times (10 min/room
temperature) with cold PBS containing 0.01 % TritonX-100. After incubation (30 min/room
temperature) with blocking solution (PBS, 2% BSA), they were incubated with primary antibodies
diluted in blocking solution (4°C/overnight). The primary antibody raised against E-Cadherin
(4065) and the antibody raised against vimentin (3932) were purchased from Cell Signaling (Cell
Signaling technology, MI, Italy) and both used at 1:100 dilution, anti-TNFα (52B83) and anti-
VEGF (A-20), acquired from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), were used at
1:50 and 1:100 dilutions, respectively, furthermore the purified mouse anti-cytochrome C
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10.1002/cmdc.201600629
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(556433) was purchased from BD Biosciences and used at 1:100 dilution. Coverslips were then
washed 3 times with PBS, then fixed cells were incubated with the secondary antibodies Alexa
Fluor® 568 conjugate goat-anti-mouse or Alexa Fluor® 488 conjugate goat-anti-rabbit (both used
at 1:500 dilution and acquired from Thermo Fisher Scientific, MA USA). Nuclei were stained
using DAPI (Sigma) for 10 min at a concentration of 0.2 μg/mL then washed 3 times with PBS.
Fluorescence was detected using a fluorescence microscope (Leica DM 6000). LASX software
was used to acquire and process all images.[48]
Statistical analysis
Data were analysed for statistical significance (p<0.001) using One-way ANOVA followed by
Dunnett's test performed by Graph Pad Prism 5. Standard deviations (SD) are shown.
Acknowledgment. This work was supported by MIUR to M.S. Sinicropi. We would like to thank
Dr. Noemi Muià for her technical support.
Abbreviations used
ANOVA,
Analysis
of
Variance;
DAPI,
2-(4-amidinophenyl)-6-indolecarbamidine
dihydrochloride; DMEM/F12, Dulbecco’s Modified Eagle Medium F12; EMT, epithelial-
mesenchymal transition; FBS, Fetal Bovine Serum; IIDQ, 2-isobutoxy-1-isobutoxycarbonyl-1,2-
diihydroquinoline; MCF-7, Michigan Cancer Foundation-7; MCF-10A, Michigan Cancer
Foundation-10A; MDA-MB-231, M.D. Anderson Metastatic Breast-231; MTT, Methylthiazol
Tetrazolium Assay; TNFα, tumor necrosis factor alpha; TUNEL, Terminal deoxynucleotidyl
transferase dUTP nick end labeling; VEGF, vascular endothelial growth factor.
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Figure 1. Structures of thalidomide and correlated compounds
N N
O
O
O
O
HN
N
N
N
HN
N
O
O
O
O
CN
Thalidomide
1
2
Bn
N
O
Cl
O
N
H
N
Cl
O
N
H
N
N
H
N
O
O
H.
HCl
3
4
5
N N
O
Br
O
O
N
N
N
O
N
N
O
O
O
6
7
8
20
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10.1002/cmdc.201600629
ChemMedChem
Figure 2. A) Immunofluorescence analysis of TNFα levels in MCF-7 cells. Cells were treated for
24 h with compounds 3 and 8 or vehicle (CTRL), then processed as described in Experimental
section. Images were acquired at 20X magnification. Panel A: DAPI; Panel B: Alexa Fluor 568;
Panel C: Overlay. Images are representative of three separate experiments. B) Fluorescence
quantification is shown. *** p<0.001.
A
B
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10.1002/cmdc.201600629
ChemMedChem
Figure 3. A) Immunofluorescence analysis of cytochrome c. MCF-7 cells were treated for 24 h
with compounds 3 or 8, or with vehicle (CTRL). Images were acquired at 63X magnification.
Panel A: DAPI; Panel B: Alexa Fluor 568; Panel C: Overlay. Images are representative of three
separate experiments. B) Fluorescence quantification is shown. *** p<0.001.
A
B
22
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10.1002/cmdc.201600629
ChemMedChem
Figure 4. Apoptosis detection by TUNEL assay on MCF-7 breast cancer cells. Cells were treated
for 24 h with compounds 3 and 8, then cold methanol fixed and subjected to TUNEL procedure.
Then, cells have been washed, dyed with DAPI and observed and imaged under an inverted
fluorescence microscope (20X magnification). Panels A: CFTM488A; Panels B: DAPI; Panels C:
Overlay. Representative fields are shown.
23
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10.1002/cmdc.201600629
ChemMedChem
Figure 5. Wound-healing assay conducted on MCF-7 (A) and MDA-MB-231 (B) cells treated
with compounds 1-8. Wound closure was monitored recording the area of the cell-free wound at
time 0h and 72h for MCF-7 and at time 0h and 48h for MDA-MB-231, by the use of an inverted
microscope. The wound healing effect was estimated as the percentage of wound closure
calculated as reported in material and method section. Vehicle-treated (CTRL) cells were used as a
control. *** p<0.001.
A
B
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10.1002/cmdc.201600629
ChemMedChem
Figure 6. Wound-healing assay conducted on MCF-7 (A) and MDA-MB-231 (B) cells plated on
six-well plates and treated with compounds 3 or 8 and vehicle (CTRL). Wound closure was
monitored at times 0h and 72h for MCF-7 and at times 0h and 48h for MDA-MB-231, by the use
of an inverted microscope (5X magnification). The dotted red lines define the areas lacking cells.
A
B
25
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10.1002/cmdc.201600629
ChemMedChem
Figure 7. Immunofluorescence analysis of vimentin. A) MCF-7 cells were treated for 24h with
compounds 3, 8 or vehicle (CTRL). Images were acquired at 20X magnification. Panel A: DAPI;
Panel B: Alexa Fluor® 488; Panel C: Overlay. Images are representative of three separate
experiments. B) Fluorescence quantification, *** p<0.001.
A
B
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ChemMedChem
Figure 8. Immunofluorescence analysis of E-cadherin. A) MCF-7 cells were treated for 24h with
compounds 3, 8 or vehicle (CTRL). Images were acquired at 20X magnification. Panel A: DAPI;
Panel B: Alexa Fluor® 488; Panel C: Overlay. Images are representative of three separate
experiments. B) Fluorescence quantification, *** p<0.001.
A
B
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10.1002/cmdc.201600629
ChemMedChem
Figure 9. Immunofluorescence analysis of VEGF. A) MCF-7 cells were treated for 24 h with
compounds 3, 8 or vehicle (CTRL). White arrows indicate the different subcellular localization of
VEGF in MCF-7 cells treated with compound 8, with respect to the vehicle treated cells (CTRL).
Images were acquired at 20X magnification. Panel A: DAPI; Panel B: Alexa Fluor® 488; Panel C:
Overlay. Images are representative of three separate experiments. B) Fluorescence quantification,
*** p<0.001.
A
A
B
C
CTRL
3
8
B
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ChemMedChem
Scheme 1. Synthesis of compound 4
CBZ
H
N
CBZ O
N
O
H
N
COOH
N
COOH
iii
N
H
N
H
i
ii
.
2HCl
N
H
N
N
N
t-BOC
t-BOC
t-BOC
9
10
11
12
iv
Bn
N
O
Bn
N
O
v
N
H
N
H
N
N
.
t-BOC
H HCl
4
13
Reagents and conditions: (i) Boc-ON, benzylchloroformate, dioxane/water, rt; (ii) 2,6-dimethylaniline, IIDQ, Et3N,
CHCl3, reflux; (iii) Et3SiH, PdCl2, Et3N, CH2Cl2, reflux; (iv) BnBr, K2CO3, dioxane/water, 70 °C; (v) g. HCl, anhyd
Et2O, rt.
29
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