N-carbamidoyl-4-((3-ethyl-2,4,4-trimethylcyclohexyl)methyl)benzamide enhances staurosporine cytotoxic effects likely inhibiting the protective action of Magmas toward cell apoptosis

Article abstract of DOI:10.1021/jm5000535

We recently demonstrated that Magmas overexpression protects GH-secreting rat pitutitary adenoma cell lines from apoptosis by inhibiting cytochrome c release from mitochondria after treatment with staurosporine, strongly suggesting a role of Magmas in preventing apoptosis. The aim of this study was to produce a drug that, by inhibiting Tim16, may sensitize chemoresistant tumor cell to proapoptotic stimuli. We synthesized six compounds and challenged their sensitizing effects toward the proapoptotic effects of staurosporine in the TT cell line, derived from a human medullary thyroid carcinoma. We found that compound 5, devoid of the planarity in the aliphatic part of the lead 1, is not cytotoxic but enhances the proapoptotic effects of staurosporine by reducing MMP activation. Compound 5 may be useful for cancer treatment in association with chemotherapeutic drugs, possibly allowing a reduction of the chemotherapeutic agent effective dose.

Full text of DOI:10.1021/jm5000535

Article
pubs.acs.org/jmc
N‑Carbamidoyl-4-((3-ethyl-2,4,4-
trimethylcyclohexyl)methyl)benzamide Enhances Staurosporine
Cytotoxic Effects Likely Inhibiting the Protective Action of Magmas
toward Cell Apoptosis
Maria Chiara Zatelli,†,‡,∥ Teresa Gagliano,†,∥ Michela Pela,§ Sara Bianco,§ Valerio Bertolasi,§
̀
Federico Tagliati,† Remo Guerrini,‡,§ Ettore degli Uberti,†,‡ Severo Salvadori,‡,§ and Claudio Trapella*,‡,§
†Section of Endocrinology, Department of Medical Sciences, University of Ferrara, 44121 Ferrara, Italy
‡Laboratorio in Rete del Tecnopolo Tecnologie delle Terapie Avanzate (LTTA), University of Ferrara, 44121 Ferrara, Italy
§Department of Chemical and Pharmaceutical Sciences, University of Ferrara, Via Fossato di Mortara 17, 44121 Ferrara, Italy
S
* Supporting Information
ABSTRACT: We recently demonstrated that Magmas overexpression protects GH-secreting rat pitutitary adenoma cell lines
from apoptosis by inhibiting cytochrome c release from mitochondria after treatment with staurosporine, strongly suggesting a
role of Magmas in preventing apoptosis. The aim of this study was to produce a drug that, by inhibiting Tim16, may sensitize
chemoresistant tumor cell to proapoptotic stimuli. We synthesized six compounds and challenged their sensitizing effects toward
the proapoptotic effects of staurosporine in the TT cell line, derived from a human medullary thyroid carcinoma. We found that
compound 5, devoid of the planarity in the aliphatic part of the lead 1, is not cytotoxic but enhances the proapoptotic effects of
staurosporine by reducing MMP activation. Compound 5 may be useful for cancer treatment in association with
chemotherapeutic drugs, possibly allowing a reduction of the chemotherapeutic agent effective dose.
G1 phase of the cell cycle,3 supporting the hypothesis that
Magmas may play a role in tumor development by protecting
INTRODUCTION
■
Magmas (mitochondria-associated protein involved in gran-
neoplastic cells from apoptosis and by promoting cell
ulocyte-macrophage colony-stimulating factor signal trans-
proliferation. The protective role of Magmas toward apoptotic
duction) gene encodes for the mitochondrial protein Tim 16,
a member of the translocase complex TIM23, located in the
stimuli was also confirmed in GH-secreting rat pituitary
adenoma cell lines, where Magmas overexpression protects
mitochondrial inner membrane. Tim 16 drives proteins from
from apoptosis by inhibiting staurosporine-induced cytochrome
the intermembrane space into the mitochondrial matrix by
c release from mitochondria.4 These data point to a role for
functionally interacting with Tim 14, a protein participating in
the TIM23 complex.1
Magmas in tumor resistance to proapoptotic stimuli (such as
chemotherapic drugs), indicating that Magmas inhibition may
Magmas was found to be overexpressed in prostate cancer,
suggesting a role for this gene in tumor development.2 In
represent a successful strategy to overcome chemoresistance. A
consolidated in vitro model of chemoresistance is represented
keeping with these findings, we previously demonstrated that
Magmas is overexpressed in human pituitary adenomas as well
by the TT cell line, derived from a human medullary thyroid
carcinoma (MTC), that displays resistance to chemotherapeu-
tic drugs and to apoptotic stimuli.5−7 TT cells display an
as in ACTH-secreting mouse pituitary adenoma cell lines
compared to their respective human and murine normal
counterparts. We found that Magmas silencing sensitizes
ACTH-secreting mouse pituitary adenoma cell lines to
proapoptotic stimuli and induces an accumulation in the G0/
Received: January 10, 2014
Published: April 24, 2014
© 2014 American Chemical Society
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activating RET mutation8 that leads to constitutive p38MAPK
signaling that, in turn, confers resistance to apoptosis.9 MTC is
an aggressive tumor arising from thyroid parafollicular C cells
that, to date, is orphan of an effective medical therapy, since
classic chemotherapy or radiation therapy is seldom effective in
controlling disease progression.10
We therefore aimed at synthesizing a chemical inhibitor of
Tim 16, starting from a previously synthesized compound
(referred to as compound 1)11 (Scheme 1), evaluating its
capability of sensitizing a chemoresistant cell line, the TT cells,
to proapoptotic stimuli.
reaction with urea in the presence of trifluoromethylboronic
acid as a catalyst.12
To better understand the role of planarity in the aliphatic
portion of the molecule, we decided to reduce both endocyclic
and exocyclic double bonds by catalytic hydrogenation as
depicted in Scheme 3, obtaining compound 5. The same
reduction was done for the corresponding urea 4 to obtain
compound 6.
a
Scheme 3
Scheme 1
RESULTS
■
Synthesis. The synthesis of compound 1, which was
demonstrated to reduce yeast proliferation in vitro, was
achieved as previously reported.11 On the basis of these data,
we decided to perform a SAR study of the molecule,
investigating in particular the polar function (guanidine) and
the planarity of its aliphatic portion. As depicted in Scheme 2,
a
Conditions: (a) EtOH/EtOAc 1:1, C/Pd, H2, 1 atm, 12 h, 97% for 5,
82% for 6.
a
Scheme 2
Magmas Expression in TT Cell Line. The TT cell line was
characterized for Magmas expression. As shown in Figure 1A,
Tim 16, the protein encoded by the Magmas gene, is expressed
in TT cells, with the expected molecular weight (13,8 kDa), to
a greater extent compared to normal thyroid tissue (NT).
As shown in Figure 1B, Tim 16 protein levels were not
influenced in TT cells by treatment with each compound alone
or in combination with staurosporine.
Effects of Compounds on Cell Viability and Apopto-
sis. We evaluated the ability of the synthesized compounds
(compounds 1, 2, 3, 4, 5, and 6) to influence TT cell viability.
As shown in Figure 2A, compound 1 significantly (P < 0.01)
reduced cell viability at 10 μM concentration. On the contrary,
compounds 2, 3, 4, and 5 did not influence this parameter. In
order to verify whether the reduction in cell viability was due to
the induction of apoptosis, caspase 3/7 activation was
evaluated. As shown in Figure 2B, 10 μM compound 1
significantly (P < 0.01) induced caspase activation, while
compounds 2, 3, 4, and 5 did not significantly modify this
parameter in TT cells. These results indicate that compound 1
at higher concentration reduces cell viability and promotes
apoptosis in TT cells. On the contrary, all the other compounds
did not affect these parameters in basal conditions.
a
Conditions: (a) DMF, CDI, NH4OH, 24 h, Y = 20%; (b) toluene,
urea, 3,4,5 trifluoromethylphenylboronic acid, reflux, 24 h, Y = 45%.
we started from acid derivative 2 to prepare the corresponding
primary amide 3 and urea 4. The amide preparation was
achieved by carbonyldiimidazole activation of the correspond-
ing carboxylate and subsequent reaction with concentrated
ammonia solution to yield amide 3 after crystallization from
diethyl ether. The urea derivative 4 was obtained from acid 2 by
Effects of Compounds in Promoting Apoptotic
Stimuli Action. To investigate the possible role of Magmas
inhibitors in enhancing apoptosis activation under apoptotic
stimuli, TT cells were treated with compounds 1, 2, 3, 4, and 5
at 5 or 10 μM with or without 50 nM staurosporine.
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Figure 1. (A) Total proteins were isolated from TT cells and from a pool of normal thyroid tissues (NT), and Western blot analysis for Tim 16
protein expression was performed. β-Actin is shown as loading control. (B) TT cells were treated with compounds 1−5 (C1, C2, C3, C4, C5) at 10
μM alone or in combination with 50 nM staurosporine (S) for 24 h and then harvested. Total proteins were isolated, and Western blot analysis for
Tim 16 protein expression was performed. β-Actin is shown as loading control.
< 0.01) enhanced the inhibitory effects of staurosporine on cell
viability. To verify whether the reduction in cell viability was
associated with apoptotic mechanisms, apoptosis activation was
evaluated by measuring caspase 3/7 activity in the same
conditions. As shown in Figure 3A (right panel), staurosporine
significantly (P < 0.01) induced caspase 3/7 activation (200%
vs control cells). In line with the results on cell viability,
compound 1 at 10 μM, but not at 5 μM, significantly induced
caspase 3/7 activation (by ∼300%; P < 0.01 vs control). In
addition, 5 and 10 μM compound 1 significantly (P < 0.01)
enhanced the stimulatory effects of staurosporine on caspase
activation by 40% and 400%, respectively.
As shown in Figure 3B (left panel), 5 and 10 μM compound
2 did not significantly modify cell viability nor significantly
enhanced staurosporine antiproliferative effects. Accordingly, 5
and 10 μM compound 2 did not significantly modify caspase 3/
7 activity nor significantly enhanced staurosporine-induced
apoptosis activation (Figure 3B, right panel). Similar results
were obtained when TT cells were challenged with compound
3 (Figure 3C) and compound 4 (Figure 3D), concerning both
cell viability (left panels) and caspase activation (right panels).
In keeping with previous results, compound 5 alone did not
affect cell viability but was capable of enhancing the inhibitory
effects of staurosporine on cell viability at both concentrations
tested (by 12% and 20%, respectively; P < 0.05) (Figure 3E, left
panel). Similarly, compound 5 alone did not affect caspase 3/7
activation but was capable of potently enhancing the
stimulatory effects of staurosporine on this parameter
Figure 2. Effects of compounds 1, 2, 3, 4, and 5 on cell viability and
apoptosis. TT cells were treated with increasing doses (1−10 μM) of
compound 1 (black line) or compound 2, 3, 4, or 5 (gray line),
renewing the treatment every 24 h. After 72 h, cell viability was
measured by ATPlite assay (A) and apoptosis activation was measured
by caspase 3/7 assay (B). ∗∗ indicates P < 0.01 vs control cells.
(+140% and +330% with 5 and 10 μM, respectively; P <
0.01) (Figure 3E, right panel).
These data indicate that both compound 1 and compound 5
are capable of sensitizing TT cells to the proapoptotic effects of
staurosporine. However, while compound 1 displays a cytotoxic
action, the latter is not exerted by compound 5, which, per se,
does not significantly alter basal TT cell viability and caspase 3/
7 activation.
As shown in Figure 4A, staurosporine significantly reduced
MCF12A cell viability (by 53%; P < 0.01). In keeping with
previous results, compound 5 at 5 and 10 μM did not
significantly affect cell viability and did not enhance the
As shown in Figure 3A (left panel), staurosporine
significantly reduced TT cell viability (by 63%; P < 0.01). In
keeping with previous results, compound 1 at 10 μM, but not at
5 μM, significantly reduced cell viability (by ∼70%; P < 0.01 vs
control). In addition, 5 and 10 μM compound 1 significantly (P
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Figure 3. Effects of compounds 1, 2, 3, 4, and 5 at 5 and 10 μM in combination with staurosporine 50 nM on cell viability and apoptosis. Left panels:
TT cells were treated without or with 5 or 10 μM indicated compounds (white bars) alone or in combination with 50 nM staurosporine (black bars).
Treatments were renewed every 24 h, and cell viability was evaluated after 72 h. Right panels: TT cells were treated with 5 or 10 μM indicated
compounds (white bars) alone or in combination with 50 nM staurosporine (black bar). Treatments were renewed every 24 h, and caspase 3/7
activity was evaluated after 72 h. ∗∗ indicates P < 0.01 vs control cells. # indicates P < 0.05 and ## indicates P < 0.01 vs staurosporine-treated cells.
antiproliferative effects of staurosporine. In addition, Tim16
protein levels are much lower in MCF12A cells compared to
TT cells (Figure 4B).
Effects of Compounds 1 and 5 on Mitochondrial
Membrane Potential. We then evaluated the ability of
Magmas inhibitors to influence mitochondrial membrane
potential (MMP). Since compounds 2, 3, and 4 did not
display any sensitizing effect toward the proapoptotic action of
staurosporine, MMP was tested in TT cells only under
treatment with compound 1 and compound 5. TT cells were
treated with 50 nM staurosporine alone or in combination with
either 10 μM compound 1 or 10 μM compound 5 and then
submitted to MMP evaluation by using Jc-1 assay. As shown in
Figure 5, staurosporine alone dramatically reduced red
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show that, as expected, staurosporine reduces MMP, a
phenomenon observed also under treatment with compound
5 but not with compound 1, indicating that indeed compound
5 is acting at mitochondrial level. Compound 6 was also tested
in MMP assay with no significant results (see Supporting
Information page S17)
DISCUSSION AND CONCLUSION
■
The evidence that Magmas down-regulation sensitizes pituitary
cells to proapoptotic stimuli prompted the search for chemical
compounds that may be effective in reducing Magmas function.
The availability of such compounds may indeed represent a
powerful therapeutic tool to enhance tumor sensitivity to drugs
inducing apoptosis, thereby contrasting the proliferative tumor
potential. Our study provides the first evidence that a
compound likely inhibiting Magmas protein, compound 5, is
capable of sensitizing chemoresistant cells to proapoptotic
stimuli without affecting cell viability per se. The latter evidence
is of crucial importance for our study, since our aim was to
identify a molecule devoid of toxic effects that could overcome
resistance to proapoptotic stimuli. However, we cannot exclude
that compound 5 may reduce cell viability and/or increase
apoptosis in the cell line model at later time points. However,
the lack of planarity of compound 5 does not make this
compound a good candidate for DNA intercalation, which is,
on the contrary, very likely for compound 1. Indeed, we show
that compound 1 has direct cytotoxic effects, in keeping with
previous evidence showing that compound 1 inhibits cell
proliferation in yeast.11
Figure 4. (A) Effects of compound 5 at 5 and 10 μM in combination
with staurosporine 50 nM on MCF12A cell viability. (B) Total
proteins were isolated from MCF12A and from TT cells, and Western
blot analysis for Tim 16 protein expression was performed. β-Actin is
shown as loading control.
fluorescence consistent with a reduction in MMP. Compound 1
alone slightly affected MMP, as shown by the persistence of red
fluorescence, consistent with a conserved MMP. On the
contrary, TT cells treated with compound 5 showed a prevalent
green fluorescence, indicating a drastic decrease in MMP. The
latter was observed also after combined treatment with
staurosporine and compound 1 or compound 5. These data
Figure 5. Mitochondrial membrane potential (MMP) evaluation. TT cells were treated with 50 nM staurosporine and with 5 or 10 μM compound 1
or compound 5 alone or in combination. The red fluorescence corresponds to Jc-1 aggregates, indicating MMP activation. Green fluorescence
indicates the presence of Jc1 monomer, corresponding to a fall in MMP.
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We here expand the SAR study of putative Magmas
inhibitors by evaluating the importance of the guanidine
residue for these compounds as chemosensitizers. Our study
demonstrates that the guanidine moiety is crucial for the latter
activity, since compounds 2, 3, and 4, which are lacking this
functionality, do not affect basal cell viability and apoptosis in
TT cells and do not enhance the proapoptotic effects of a
renown apoptotic stimulus, such as staurosporine. Therefore,
these data indicate that the guanidine moiety is fundamental for
the chemosensitizing effects of Magmas inhibitors. We then
evaluated the importance of planarity in the aliphatic portion of
the molecule by studying the effects of compound 5. Our
results show that compound 5, where planarity is lost, is indeed
devoid of any cytotoxicity but is capable of enhancing the
proapoptotic effects of staurosporine, indicating a better safety
profile while the same chemical modification in the urea
compound 6 did not elicit any activity. These data are in line
with previous studies demonstrating that Magmas inhibition
confers sensitivity to proapoptotic stimuli,3,4 suggesting that
these compounds may be useful in association with classical
chemotherapeutic drugs for cancer treatment. This strategy
would allow a reduction of the chemotherapeutic agent
effective dose, with a consequent decrease in side effect
development.
EXPERIMENTAL SECTION
■
All the reagents and solvents were of the highest quality available or
were freshly distilled (Sigma-Aldrich, Milan, Italy). Melting points
(uncorrected) were measured with a Buchi-Tottoli apparatus. 1H, 13C,
and bidimensional NMR spectra were recorded on a Varian 400 MHz
instrument unless otherwise noted. Chemical shifts are given in ppm
(δ) relative to the solvent, and coupling constants are in hertz. The
abbreviation used are the following: s = singlet, d = doublet, t = triplet,
q = quartet, dd = double doublet, m = multiplet, td = triple doublet, dt
= double triplet, ddd = double double doublet. MS analyses were
performed on a ESI Micromass ZMD 2000. HRMS results were
recorded using a ESI-Q-TOF mass spectrometer (Agilent Technolo-
gies). Infrared spectra were recorded on a PerkinElmer FT-IR
Spectrum 100 spectrometer. Flash chromatography was carried out
on a silica gel (Merck, 230−400 mesh). HPLC analyses were
performed using Beckman system Gold 168 using a C18 Jupiter
column (150 mm × 4.6 mm, 5 μm) and water/acetonitrile/TFA
eluent. Preparative HPLC results were recorded in a Waters Delta
Prep 4000, using a C18 Jupiter column (250 mm × 30 mm, 15 μm)
and water/acetonitrile/TFA eluent. The purity of all compounds was
determined by HPLC (Beckman system Gold 168) and was greater
than 95%.
Synthesis. N-[4-(3-Ethyl-2,4,4-trimethylcyclohex-2-
enylidenemethyl)benzoyl]guanidine (1). The synthesis was as
reported in ref 11a (1): C20H27N3O mp = 125−130 °C. HRMS
[MH]+ = 326.22 193. HRMS [MH]+ calcd = 326.222 69. H NMR
1
(400 MHz, acetone-d6) δ 8.04 (br, 3H, NH), 7.98 (d, 2H, J = 8 Hz,
CHar), 7.25 (d, 2H, J = 8 Hz, CHar), 6.50 (s, 1H, CHCq), 3.40 (br,
NH), 2.63−2.60 (m, 2H, CH2-CH2-Cq), 2.25 (q, 2H, J = 7.6 Hz,
CH2-CH3), 1.91 (s, 3H, CH3-C), 1.49−1.47 (m, 2H, CH2-CH2-
C), 1.08 (s, 6H, 2CH3), 1.04 (t, 3H, J = 7.6 Hz, CH2-CH3). 13C
NMR (100 MHz, acetone-d6) δ 173.13 (NH-CO-Ar), 160.62 (NH2-
CNH), 147.94 (CH3CH2C), 141.41(2CqAr), 141.14 (CH2-Cq
CH), 130.05 (2CHAr), 129.32 (2CHAr), 128.10 (CH3-C), 122.30
(CqAr-CHCq), 39.68 (CH2-CH2-C), 36.46 (CH3-Cq-CH3),
28.02 (2CH3-Cq), 24.98 (CH2-CH2-C), 23.39 (CH2-CH3), 15.48
(CH3-C), 15.17 (CH2-CH3).
The evidence that Tim 16 is overexpressed in neoplastic
tissues2,3 suggests that this protein may represent a cancer
biomarker, even if further studies are needed to support this
hypothesis. However, the evidence that Tim 16 overexpression
confers resistance to proapoptotic stimuli4 supports a role for
this protein in cancer survival and progression.
Magmas protein lays in the mitochondrial inner membrane,
where it takes part in the TIM23 complex, which in turn is
fundamental for mitochondrial function preservation.13 It has
been previously demonstrated that mitochondrial perturbation
is critical for apoptosis in TT cells7 that express Tim 16, the
protein encoded by Magmas. In addition, we have previously
shown that Magmas overexpression enhances MMP and
prevents the inhibitory effects of staurosporine on this
parameter, indicating that Magmas is indeed involved in
MMP control.4 Therefore, the evidence that treatment with
compound 5 reduces MMP in TT cells provides further
support for a mitochondrial involvement in the sensitizing
effects of Magmas inhibitors. In addition, our results indicate
that planarity is not important for the mitochondrial activity of
the synthesized compounds, since compound 1 does not have a
significant impact on MMP, in which perturbation, on the
contrary, may be important for the sensitizing effects of
Magmas inhibitors toward apoptotic stimuli. Our results
indicate that compound 5 interacts with Magmas at the
mitochondrial level, even if they do not rule out other
possibilities. However, the hypothesis that compound 5 acts by
influencing Tim 16 is supported by the evidence that
compound 5 does not enhance the antiproliferative effects of
staurosporine in the MCF12A cells, where Tim 16 is expressed
at a very low level. Previous evidence showed that Magmas may
also have a cytoplasmic localization,4 even if a cytoplasmic
function for this protein is not known yet. Therefore, further
studies are necessary to demonstrate the mitochondrial
localization of compound 5 and its possible disruption of the
TIM23 complex.
4-(3-Ethyl-2,4,4-trimethylcyclohex-2-enylidenemethyl)benzoic
Acid (2). The synthesis was as reported in refs 11b and 11c (2):
C19H24O2, mp = 150−155 °C. ESI [MH]+ = 285.21. H NMR (400
1
MHz, MeOD) δ 7.84 (d, 2H, J = 7.6 Hz, CHar), 7.20 (d, 2H, J = 7.6
Hz, CHar), 6.41 (s, 1H, CHCq), 2.54−2.52 (m, 2H, CH2-CH2-
Cq), 2.18 (q, 2H, J = 7.6 Hz, CH2-CH3), 1.85 (s, 3H, CH3-C),
1.45−1.42 (m, 2H, CH2-CH2-Cq), 1.03 (s, 6H, CH3-C-CH3), 1.02
(t, 3H, J = 7.6 Hz, CH2-CH3). 13C NMR (100 MHz, MeOD) δ 148.59
(CH3CH2C), 142.91 (CO-CqAr), 142.13 (CH2-CqCH), 134.92
(CqAr-CHCq), 130.12 (2CHAr), 129.75 (2CHAr), 128.41 (Cq-
CH3), 122.17 (CqAr-CHCq), 40.10 (CH2-CH2-C), 36.80 (CH3-
Cq-CH3), 28.14 (2CH3), 25.31 (CH2-CH2-C), 23.73 (CH2-CH3),
15.48 (CH3-C or CH3-CH2), 15.21 (CH3-CH2 or CH3-C). IR:
cm−1 2958.76 C−H stretching, 1669.14 CO stretching, 1589.06
CC stretching, 961−610 C−H bending aromatic ring. X-ray crystal
data: C H O ; triclinic; space group P1; Z = 2; a = 7.1637(2) Å, b =
̅
19 24
2
7.6937(2) Å, c = 14.8938(4) Å, α = 82.749(1)°; β = 81.158(1)°, γ =
88.023(1)°; V = 804.54(4) Å3; Mo Ka radiation θmax = 27.5°; 3635
unique reflections measured; 2892 observed reflections [I > 2σ(I)];
final R index = 0.0518 (observed reflections), Rw = 0.1518 (all
reflections).
4-(3-Ethyl-2,4,4-trimethylcyclohex-2-enylidenemethyl)benzamide
(3). To a stirred solution of (2) (60 mg, 0.21 mmol) in DMF (5 mL),
carbonyldiimidazole (CDI) (37.4 mg, 0.23 mmol) and concentrated
NH4OH (1.2 equiv) were added. After 24 h at room temperature, the
reaction was monitored by mass spectrometry. The solvent was
evaporated in vacuo, and the residue was crystallized from ether and
centrifuged to yield the product as a yellow solid (3) (yield 20%). 3:
C19H25NO; mp over 220 °C. ESI [MH]+ = 284.25. HRMS [MH]+ =
284.293 53. HRMS [MH]+ calcd = 284.2009. H NMR (400 MHz,
1
DMSO) δ 7.81 (m, 4H, 2CHar and NH2), 7.18 (d, 2H, J = 8.2 Hz,
2CHar), 6.41 (s, 1H, CHPh), 2.54−2.51 (m, 2H, CH2-CH2-Cq),
2.15 (q, 2H, J = 7.5 Hz, CH2-CH3), 1.83 (s, 3H, CH3-C), 1.43−1.40
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(m, 2H, CH2-CH2-Cq), 1.03 (s, 6H, CH3-C-CH3), 1.00 (t, 3H, J =
7.6 Hz, CH2-CH3). 13C NMR (100 MHz, DMSO) δ 169.85 (NH2-
Western Blot Analysis. Human normal thyroid tissue was
obtained from patients undergoing surgery for thyroid cancer and
collected as previously described.17,18 The tissues were disrupted by
using TissueRaptor (Qiagen, Milano, Italy) according to the
manufacturer’s instruction. For immunoblotting, human normal
thyroid tissue and cell lines were dissolved in RIPA buffer (Pierce,
Rockford, IL) and treated as previously described.19 Briefly samples
were kept in ice for 30 min and then centrifuged for 10 min. The
supernatant, containing the proteins, was then transferred to a new
tube, and protein concentration was measured by BCA protein assay
reagent kit (Pierce, Rockford, IL, USA), as previously described.4 For
protein evaluation, lysates were fractionated on 10% SDS−PAGE, as
previously described,20 and transferred by electrophoresis to nitro-
cellulose transfer membrane (PROTRAN, Dassel, Germany).
Membranes were incubated with 1:1000 polyclonal rabbit anti-Tim
16 (PRIMM, Milano, Italy). Anti-rabbit horseradish peroxidase-
conjugated IgG antibody (Dako Italia, Milano, Italy) was then used
at 1:5000, and binding was revealed using enhanced chemilumines-
cence (Pierce). The blots were then stripped and used for further
blotting with 1:1000 polyclonal rabbit anti-human β-actin (Cell
Signaling, Beverly, MA, USA).
CO-Ar), 146.81 (CH3CH2C), 139.27 (CO-CqAr), 138.76 (CqAr
-
CH), 137.38 (CHCq-CH2), 129.28 (CHAr), 128.76 (CHAr), 128.00
(CHAr), 126.69 (Cq-CH3), 125.04 (CHAr), 121.40 (CqAr-CHCq),
38.33 (CH2-CH2-C), 35.43 (CH3-Cq-CH3), 27.47 (2CH3), 23.78
(CH2-CH2-C), 22.31 (CH2-CH3), 14.92 (CH3-C or CH3-CH2),
14.73 (CH3-CH2 or CH3-C). IR: 2966.39 C−H stretching benzene,
1583.04, 1543.12 N−H bending.
[4-(3-Ethyl-2,4,4-trimethylcyclohex-2-enylidenemethyl)benzoyl]-
urea (4). To a solution of 2 (80 mg, 0.282 mmol) in azeotropic
toluene (5 mL), urea (3.61 mg, 0.282 mmol) and the catalyst 3,4,5-
trifluoromethylphenylboronic acid (3.61 mg, 0.310 mmol) were added.
The mixture was stirred at reflux for 24 h. After this time, the reaction
was monitored by TLC (AcOEt/EtPt 0.2:9.8) and mass spectrometry.
At the end of the reaction toluene was removed in vacuo. The residue
was solubilized with AcOEt and washed with NaHCO3 (5%). The
organic phase was dried and evaporated to dryness. The crude product
was purified by preparative HPLC. 4: C20H26N2O2, ESI [MH]+
=
327.14. HRMS [MH]+ = 327.205 20. HRMS [MH]+ calcd = 327.2073.
1H NMR (400 MHz, CDCl3) δ 8.05 (d, 2H, J = 8.4 Hz, CHar), 7.36
Viable Cell Number Assessment. Variations in viable cell
number were assessed by the ATPlite kit (PerkinElmer Life Sciences,
MA, USA), seeding 2 × 104 cells/well in 96-well white plates, as
previously described,21 and treated with the indicated compounds for
72 h. Control cells were treated with vehicle alone (0.1% DMSO).
After incubation, the revealing solution was added and the luminescent
output (relative luminescence units, RLU) was recorded using the
Envision multilabel reader (PerkinElmer, Monza, Italy). Results are
expressed as mean value SE percent RLU vs vehicle-treated control
cells from three independent experiments in six replicates.
Caspase Activity Assay. Caspase activity was measured by using
Caspase-Glo 3/7 assay (Promega, Milano, Italy), as previously
described.22 Briefly, 2 × 104 cells/well were seeded in 96-well white-
walled plates and treated with the indicated compounds for 72 h. Then
caspase-Glo 3/7 reagent was added at room temperature directly to
the cell culture plates, which were shaken at 12.5g for 30 s, incubated
for 1 h, and then measured for luminescent output (RLU) by the
Envision multilabel reader (PerkinElmer). Results are expressed as
mean value SE percent RLU vs vehicle-treated control cells from
three independent experiments in six replicates.
Mitochondrial Membrane Potential Evaluation. TT cells were
treated without or with 50 nM staurosporine and 5 or 10 μM
compound 1 or compound 5, alone or in combination. MMP was then
evaluated by employing the JC-1 mitochondrial membrane potential
assay kit, following manufacturer instruction (Cayman Europe, Tallin,
Estonia). Briefly, MMP was determined by incubating the cells with
JC-1 dye at 37 °C for 30 min. The fluorescence intensity was observed
by fluorescent microscopy (Nikon Eclipse T1000, Tokio, Japan), as
previously described.23
Statistical Analysis. Concerning the results of cell viability and
caspase 3/7 activation experiments, a preliminary analysis was carried
out to determine whether the data sets conformed to a normal
distribution, and a computation of homogeneity of variance was
performed using Bartlett’s test. The results were compared within each
group and between groups using ANOVA. If the F values were
significant (P < 0.05), Student’s paired or unpaired t test was used to
evaluate individual differences between mean values. P values of <0.05
were considered significant. For all the other experiments, Student’s
paired or unpaired t test was used to evaluate individual differences
between mean values and P values of <0.05 were considered
significant.
(d, 2H, J = 8.2 Hz, CHar), 6.47 (s, 1H, Ph-CHCq), 2.75−2.51 (m,
2H, CH2-CH2-C), 2.23 (q, 2H, J = 7.5 Hz, CH2-CH3), 1.92 (s, 3H,
CH3-C), 1.56−1.44 (m, 2H, CH2-CH2-C), 1.15−1.02 (m, 9H,
3CH3). 13C NMR (100 MHz, CDCl3) δ 171.44 (NH-CO-Ph), 149.51
(NH2-CO-NH), 149.24 (CH3CH2C), 144.95 (CO-CqAr), 142.72
(CH2-CqCH), 130.02 (2CHAr), 129.38 (2CHAr), 127.14 (CH3-
C), 126.32 (CqAr-CH), 120.62 (CqAr-CHCq), 38.92 (CH2-
CH2-C), 36.05 (CH3-Cq-CH3), 27.78 (2CH3-Cq), 24.49 (CH2-
CH2-C), 23.10 (CH2-CH3), 15.37 (CH3-C), 14.87 (CH2-CH3).
N-[4-(3-Ethyl-2,4,4-trimethylcyclohexylmethyl)benzoyl]guanidine
(5). To a solution of 1 (1.5 g, 4.8 mmol) in EtOH/AcOEt (1:1) (20
mL) was added 10% Pd/C, and the mixture was stirred under a
hydrogen atmosphere (1 atm) until the theoretical amount of
hydrogen gas was consumed. The reaction was monitored by mass
spectrometry. At the end of the process the mixture was filtered, and
the filtrate was concentrated. The residue was recrystallized from
diethyl ether to afford 1.5 g (97% yield) of 5: C20H31N3O; mp = 118−
121 °C. ESI [MH]+ = 330.25. HRMS [MH]+ = 330.253 08. HRMS
1
[MH]+ calcd = 330.2545. H NMR (400 MHz, MeOD) δ 7.94−7.90
(m, 2H, CHar), 7.30−7.23 (m, 2H, CHar), 3.43 (s, 1H, NH guanidine),
3.01 (dd, 1H, J = 13.2 Hz, J = 5.0 Hz, -CH-Bn), 2.62 (ddd, 2H, J =
21.8 Hz, J =13.4 Hz, J = 7.6 Hz, -CH2-Ph), 1.26−0.8 (m, 20H,
aliphatic). 13C NMR (100 MHz, MeOD) δ 171.55 (NH-CO-Ph),
168.64 (NH2-CNH), 146.85 (CqAr), 131.42 (CqAr), 129.39 (CHAr),
129.28 (CHAr), 128.84 (CHAr), 127.95 (CHAr), 60.11, 52.72, 38.44,
31.45, 27.16, 27.00, 23.93, 19.43, 18.80, 13.91.
[4-(3-Ethyl-2,4,4-trimethylcyclohexylmethyl)benzoyl]urea (6). To
a solution of 4 (0.1 g, 0.302 mmol) in EtOH/AcOEt (1:1) (5 mL) was
added 10% Pd/C, and the mixture was stirred under a hydrogen
atmosphere (1 atm) until the theoretical amount of hydrogen gas was
consumed. The reaction was monitored by mass spectrometry. At the
end of the process the mixture was filtered, and the filtrate was
concentrated. The residue was recrystallized from diethyl ether to
afford compound 6 in 82% yield: C20H30N2O2; mp = 160−165 °C.
1
HRMS [MH]+ = 331.237 60. HRMS [MH]+ calcd = 331.2386. H
NMR (400 MHz, DMSO) δ 7.84 (d, 2H, J = 8.3 Hz, CHar), 7.30 (d,
2H, J = 8.2 Hz, CHar), 3.50 (m, 2H, Ph-CH2-Cy), 1.74 (m, 1H, CH
aliphatic), 1.55−0.71 (m, 20H, aliphatic). 13C NMR (100 MHz,
DMSO) δ 167.21 (NH-CO-Ar), 146.63 (NH2-CO-NH), 129.29
(CqAr), 129.12 (CHAr), 129.06 (CHAr), 128.90 (CHAr), 128.16 (CqAr),
128.12 (CHAr), 69.95, 51.95 (CH3-Cq-CH3), 51.00, 43.94, 42.05,
32.38, 27.73, 22.40, 18.62, 11.99, 8.30.
Cell Line Culture. TT cells were purchased from ATCC
(American Type Culture Collection, Manassas, VA, USA; ATCC
CRL-1803) and grown in F12 medium (Euroclone, Milano, Italy),
supplemented with 10% fetal bovine serum, at 37 °C in a humidified
atmosphere with 5% CO2, as previously described.14,15
ASSOCIATED CONTENT
■
S
* Supporting Information
NMR and HRMS characterization of compounds 1−6 and
molecular formula strings in csv format. This material is
available free of charge via the Internet at http://pubs.acs.org.
Complete crystallographic data have been deposited with the
The nontumorigenic breast epithelial MCF-12A cell line was
purchased from the ATCC and grown as previously described.16
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Journal of Medicinal Chemistry
Article
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Cambridge Crystallographic Data Centre as supplementary
publications number CCDC 970130. Copies of that data can be
obtained, free of charge, via www.ccdc.cam.ac.uk./conts/
retrieving.html or on application to CCDC, 12 Union Road,
Cambridge CB2 1EZ, U.K. [fax: +44(0)-1223-336033, e-mail
deposit@ccdc.cam.ac.uk].
(9) Mazumdar, M.; Adhikary, A.; Chakraborty, S.; Mukherjee, S.;
Manna, A.; Saha, S.; Mohanty, S.; Dutta, A.; Bhattacharjee, P.; Ray, P.;
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AUTHOR INFORMATION
Corresponding Author
■
*Phone: +39 0532 455924. Fax: +39 0532 455953. E-mail:
claudio.trapella@unife.it.
Author Contributions
∥A.C.Z. and T.G. contributed equally to this work.
(11) (a) Jubinsky, P. T.; Short, M. K.; Ghanem, M.; Das, B. C.
Design, synthesis, and biological activity of novel Magmas inhibitors.
Bioorg. Med. Chem. Lett. 2011, 21, 3479−3482. (b) Das, B. C.;
Kabalka, G. W. Design and synthesis of (E)-4-((3-ethyl-2,4,4-
trimethylcyclohex-2-enylidene)methyl)benzoic acid. Tetrahedron Lett.
2008, 49, 4695−4696. (c) Das, B. C.; Mahalingam, S. M.; Mohapatra,
P. Design and synthesis of guanidine-containing novel retinoids.
Tetrahedron Lett. 2009, 50, 5860−5863.
(12) Maki, T.; Ishihara, K.; Yamamoto, H. Arylboronic acid-catalyzed
direct condensation of carboxylic acids with ureas. Synlett 2004, 8,
1355−1358.
(13) Meier, S.; Neupert, W.; Herrmann, J. M. Conserved N-terminal
negative charges in the Tim17 subunit of the TIM23 translocase play a
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from the Italian Ministry of
University and Research (Grants FIRB RBAP11884M,
RBAP1153LS, and RBFR109SBM for C.T.), Fondazione
Cassa di Risparmio di Ferrara, and Associazione Italiana per
la Ricerca sul Cancro (AIRC) in collaboration with Laboratorio
in Rete del Tecnopolo ‘‘Tecnologie delle Terapie Avanzate’’
(LTTA) of the University of Ferrara.
ABBREVIATIONS USED
■
(14) Zatelli, M. C.; Tagliati, F.; Taylor, J. E.; Piccin, D.; Culler, M. D.;
degli Uberti, E. C. Somatostatin, but not somatostatin receptor
subtypes 2 and 5 selective agonists, inhibits calcitonin secretion and
gene expression in the human medullary thyroid carcinoma cell line,
TT. Horm. Metab. Res. 2002, 34, 229−233.
Magmas, mitochondria-associated protein involved in gran-
ulocyte-macrophage colony-stimulating factor signal trans-
duction; ACTH, adrenocorticotropic hormone; MTC, medul-
lary thyroid carcinoma; GH, growth hormone; NT, normal
thyroid tissue; MMP, mitochondrial membrane potential
(15) Zatelli, M. C.; Piccin, D.; Tagliati, F.; Bottoni, A.; Luchin, A.;
degli Uberti, E. C. SRC homology-2-containing protein tyrosine
phosphatase-1 restrains cell proliferation in human medullary thyroid
carcinoma. Endocrinology 2005, 146, 2692−2698.
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