Marine natural products-inspired phenylmethylene hydantoins with potent in vitro and in vivo antitumor activities via suppression of Brk and FAK signaling

Article abstract of DOI:10.1039/c4ob00553h

Breast and prostate cancers are among the most common cancers worldwide with devastating statistics for the metastatic, chemotherapy- and radiotherapy-resistant phenotypes. Novel therapies interfering with new and/or multiple pathways involved in the pathology of cancer are urgently needed. Preliminary results showed that the marine natural product Z-4- hydroxyphenylmethylene hydantoin (PMH, 1) and its 4-ethylthio-analog (SEth, 2) promoted tight junction formation and showed anti-invasive and anti-migratory activities in vitro against metastatic prostate cancer cells and inhibited tumor growth and micrometastases in distant organs in orthotopic and transgenic mice models. This study focuses on the design and synthesis of second-generation PMHs with enhanced antitumor activities. A series of substituted benzaldehydes was selected based on earlier SAR studies and reacted with hydantoin to yield 11 new compounds 3-13. Compounds were evaluated for their antiproliferative, antimigratory and anti-invasive properties in vitro against the human mammary and prostate cancer cell lines MDA-MB-231 and PC-3, respectively. A Western blot analysis of the most active analog 7 showed its ability to suppress the expression of the total levels of c-Met and FAK, with subsequent reduction of their phosphorylated (activated) levels in MDA-MB-231 cells. In addition, 7 also inhibited Brk, paxillin and Rac1 phosphorylation. 7 was formulated using hydroxypropyl β-cyclodextrin (HPCD) to improve its solubility and was further evaluated in a nude mice xenograft model using MDA-MB-231/GFP cells. PMH 7 reduced breast tumor growth and suppressed Ki-67, CD31, p-Brk and p-FAK expression in tumor samples. Thus, 7 is a potential lead for the control of invasive breast malignancies.

Full text of DOI:10.1039/c4ob00553h

Organic &
Biomolecular Chemistry
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PAPER
Marine natural products-inspired phenylmethylene
hydantoins with potent in vitro and in vivo
antitumor activities via suppression of Brk and
FAK signaling†
Cite this: Org. Biomol. Chem., 2014,
12, 5295
Asmaa A. Sallam,a Mohamed M. Mohyeldin,a Ahmed I. Foudah,a Mohamed R. Akl,a
Sami Nazzal,a Sharon A. Meyer,b Yong-Yu Liua and Khalid A. El Sayed*a
Breast and prostate cancers are among the most common cancers worldwide with devastating statistics
for the metastatic, chemotherapy- and radiotherapy-resistant phenotypes. Novel therapies interfering
with new and/or multiple pathways involved in the pathology of cancer are urgently needed. Preliminary
results showed that the marine natural product Z-4-hydroxyphenylmethylene hydantoin (PMH, 1) and its
4-ethylthio-analog (SEth, 2) promoted tight junction formation and showed anti-invasive and anti-
migratory activities in vitro against metastatic prostate cancer cells and inhibited tumor growth and micro-
metastases in distant organs in orthotopic and transgenic mice models. This study focuses on the design
and synthesis of second-generation PMHs with enhanced antitumor activities. A series of substituted
benzaldehydes was selected based on earlier SAR studies and reacted with hydantoin to yield 11 new
compounds 3–13. Compounds were evaluated for their antiproliferative, antimigratory and anti-invasive
properties in vitro against the human mammary and prostate cancer cell lines MDA-MB-231 and PC-3,
respectively. A Western blot analysis of the most active analog 7 showed its ability to suppress the
expression of the total levels of c-Met and FAK, with subsequent reduction of their phosphorylated
(activated) levels in MDA-MB-231 cells. In addition, 7 also inhibited Brk, paxillin and Rac1 phosphorylation.
7 was formulated using hydroxypropyl β-cyclodextrin (HPCD) to improve its solubility and was further
evaluated in a nude mice xenograft model using MDA-MB-231/GFP cells. PMH 7 reduced breast tumor
growth and suppressed Ki-67, CD31, p-Brk and p-FAK expression in tumor samples. Thus, 7 is a potential
lead for the control of invasive breast malignancies.
Received 13th March 2014,
Accepted 22nd May 2014
DOI: 10.1039/c4ob00553h
www.rsc.org/obc
metastatic status remain largely unknown, lines of evidence
have shown that aberrant receptor tyrosine kinase (RTK)
1. Introduction
Breast cancer is the second most common cancer among signaling plays a crucial role.3 Understanding the signaling
American women after skin cancers.1 About 1 in 8 (12%) pathways involved in establishing a metastatic phenotype in
women in the US will develop invasive breast cancer during cancer is fundamental for understanding the pathology and
their lifetime.1 Similarly, prostate cancer is the most frequently treatment of the disease.4 Aberrant tyrosine kinase signaling,
diagnosed malignancy in adult men from Western countries.2 whether by stimulation of growth factor receptors or intra-
About 1 in 6 men will be diagnosed with prostate cancer cellular tyrosine kinase expression, has been shown to contri-
during their lifetime.1 Although the precise molecular mecha- bute to various steps of tumor development and progression,
nisms underlying the transformation of these cancers from including metastasis.4
the primary, treatment-responsive to the resistant and highly
c-Met is a receptor tyrosine kinase (RTK) that acts as the
receptor for its only known ligand, hepatocyte growth factor
(HGF) or scatter factor (SF).3,5 c-Met is expressed mainly in
cells of mesenchymal origin, although some epithelial cancer
cells appear to express both HGF and c-MET.3 The HGF–c-MET
axis contributes a critical physiological function in embryo-
genesis, angiogenesis, and wound healing.5 However, improper
HGF–c-MET interaction may confer proliferative, survival, and
invasive/metastatic abilities of cancer cells.3,5 The HGF–c-MET
aDepartment of Basic Pharmaceutical Sciences, School of Pharmacy, University of
Louisiana at Monroe, Monroe, Louisiana 71201, USA. E-mail: elsayed@ulm.edu;
Fax: +318-342-1737; Tel: +318-342-1725
bDepartment of Toxicology, School of Pharmacy, University of Louisiana at Monroe,
Monroe, Louisiana 71201, USA
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ob00553h
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signaling cascade has been repeatedly shown to be dysregu- non-receptor tyrosine kinases, including c-Met, Brk and FAK,
lated in a variety of tumors such as lung, kidney, head & neck, and their activation/overexpression in a variety of carcinomas,
breast, prostate and colorectal cancers.5–7 Increased HGF–c- such as breast and prostate cancers, is of particular interest.
MET signaling in these tumors correlates with poor patient Interfering with these kinases is an attractive therapeutic strat-
outcomes.5,7 In addition, phosphorylated c-MET has also been egy towards the management of those cancers.
shown to be an important predictor of tumor aggressiveness,
metastatic potential, and poor survival.5
Natural products have proven to be the most reliable
sources of new therapeutic entities.19,20 In the years
Breast tumor kinase (Brk), also known as PTK6, is an intra- 01/1981–12/2010, over 50% of the 1355 New Chemical Entities
cellular tyrosine kinase related to Src family kinases that is (NCEs) were natural products, natural product derivatives/
typically expressed in differentiated epithelial cells of the skin analogs or synthetic compounds based on natural product
and the gastrointestinal tract.4,8–10 In the normal breast epi- pharmacophores, making them the most consistently success-
thelium, Brk is low or undetectable, but the protein is over- ful sources of drug leads, both historically and currently.21,22
expressed in up to 86% of breast tumors, with the highest levels We previously reported the potent antiproliferative, antimigra-
in advanced tumors, suggesting that Brk expression is related tory and anti-invasive properties of (Z)-5-(4-hydroxybenzyli-
to carcinogenesis.9,10 Its expression levels increase in associ- dene)-hydantoin (PMH, 1, initially isolated from the marine
ation with the carcinoma content of breast tumors, tumor sponge Hemimycale arabica) and a number of its semisynthetic
grade, and invasiveness.8–10 Melanoma, lymphoma, ovarian, and synthetic analogs against prostate cancer cells, PC-3 and
prostate, and colon cancers can also exhibit overexpressed PC-3M.23–26
and/or mis-localized Brk.10 Although the expression is not sig-
nificantly altered in prostate cancer, Brk translocates from the in vitro assays followed by in vivo testing in two mice
nucleus to the cytoplasm during tumor progression.4,11 models.23–26 The marine natural product 1 and its synthetic
Focal adhesion kinase (FAK), non-receptor tyrosine analog (Z)-5-(4-(ethylthio)benzylidene)-imidazolidine-2,4-dione
The unique activities of PMHs were validated using several
a
kinase, is an important intermediary of growth factor signal- (SEth, 2) significantly increased transepithelial resistance
ing, cell survival, proliferation, adhesion, migration, and inva- (TER) of calcitonin (CT)-treated PC-3M cells, reversed CT
sion.12,13 FAK has been shown to regulate cell migration and action on TER, and abolished CT-induced increase in para-
invasion through distinct pathways by promoting the dynamic cellular permeability of polarized PC-3M cell layers.23,24 Com-
regulation of focal adhesion and peripheral actin polymeriz- pounds 1 and 2 promoted tight junctions (TJs) formation and
ation, as well as the matrix metalloproteinases (MMPs)- showed anti-invasive and anti-migratory activities against
mediated extracellular matrix (ECM) degradation.14 Tyrosine metastatic prostate cancer cells in various in vitro assays. 1 and
phosphorylation of FAK also triggers downstream signaling 2 showed prominent anti-metastatic activity in orthotopic
events, including phosphorylation of paxillin, which is xenografts of PC-3M cells in a nude mice model, inhibiting
required for the cytoskeleton reorganization to facilitate cell tumor growth and formation of tumor micrometastases in
metastasis.14 Elevated FAK expression has been observed in a distant organs.23,24 They also showed potent anti-metastatic
number of human cancer cell lines and is well correlated activity in a LPB-Tag transgenic mice model, reducing the
with tumor development and/or the maintenance of tumor growth of primary tumors and their metastasis in reproductive
phenotype.12,15
organs, decreasing morbidity and increasing the mice survival
Paxillin is a multidomain adaptor protein primarily func- average.24 1 and 2 reduced the total CD44 and CD44 v7–10
tioning as a molecular scaffold that provides multiple docking expression in PC-3M cells, which could partly justify their anti-
sites at the plasma membrane for an array of signaling, metastatic activity.27,28 Activity levels (IC50) of 1 and 2 were
adaptor, and structural proteins.4 Through these interactions, 139.2 and 51.4 μM, respectively. Subsequent optimizations
paxillin is involved in a variety of physiological functions, afforded PMHs which inhibited the migration of PC-3 cells
including matrix organization, cell motility, tissue remodeling, with an IC50 range of 4.2–21.8 μM.26,29 Multivariate analysis on
metastasis, gene expression, cell survival, and proliferation.16 PMHs was characterized by 14 physicochemical descriptors
Paxillin undergoes tyrosine phosphorylation in response to representing their lipophilicity, size, and electronic pro-
various physiological stimuli and integrin-mediated cell perties.29 Inspection of the variable importance projection
adhesion events.4 Although a large number of stimuli induce (VIP) plot and descriptor’s correlation coefficients revealed the
tyrosine phosphorylation of paxillin, only a few tyrosine importance of size and lipophilic parameters in the following
kinases have been reported to phosphorylate paxillin, includ- order: MA (molecular area) < MV (molecular volume) < BC
ing the focal adhesion kinase (FAK), Src family kinases, the (bond count) < c log P. The MV, BC, and c log P were directly
proto-oncogene c-Abl, Brk and Csk.4,16 Phosphorylation related to the activity, while MA was inversely related to the
regulates adaptor molecule binding that ultimately co- activity. The c log P and MV were the most influential descrip-
ordinates multiple complex cell signaling pathways, including tors.29 CoMFA analysis of 35 synthetic PMHs revealed the fol-
survival, proliferation, differentiation, migration, adhesion lowing: (1) areas of high steric bulk tolerance near the
and invasion.4,16–18
p-position of the benzylidene group in 2 were observed
To improve cancer therapy, new potential therapeutic and therefore the activity can be significantly enhanced by
targets are required. The potential role of receptor and bulky groups in this position. (2) Bulky groups are sterically
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Scheme 2 General
hydantoins.23,25,26,30
synthetic
scheme
of
phenylmethylene
analogs of 1 were generated (3–13, Scheme 2). Generally,
selected benzaldehydes had an electronegative group at the
para-position possessing varying degree of bulkiness, with or
without other substituents at the ortho- and/or meta-positions.
A para-positioned electronegative group was previously shown
to be an essential mediator of the activity.23,26,29
The HRESIMS data of 3 showed a molecular ion peak at m/z
251.0462 [M
−
H]−, suggesting the molecular formula
C11H9FN2O4. 1H and 13C NMR (Tables S1 and S4, ESI†) indi-
cated that 3 is (Z)-5-(9-methoxy-10-hydroxy-11-fluorobenzyli-
dene)imidazolidine-2,4-dione. The olefinic proton singlet H-6
at δH 6.25 showed 3J-HMBC correlations with the amide carbonyl
C-4 (δC 166.5) and the aromatic methine carbons C-8 and
C-12 (δC 109.9 and 109.7, respectively), confirming the phenyl-
methylene hydantoin entity of 3. Geometrical isomerism (E/Z
isomers) would be possible around the exocyclic Δ5,6 system
due to its restricted rotation. However, the used reaction con-
ditions regioselectively afforded the Z-geometry as confirmed
by spectral data and previous literature.23,30,31 Generally, the
use of N-unsubstituted hydantoins as a starting parent for
the synthesis of PMHs usually affords their Z-isomer due to
the greater steric repulsion in the E-analog.30 Additionally, the
chemical shift of the most diagnostic olefinic proton H-6 was
downfield shifted in the 1H NMR spectra (δH ∼ 6.40 in most
compounds), which further confirmed the Z-orientation of the
Δ5,6 system. The expected chemical shift of H-6 in the E-PMHs
would be more upfield shifted to ∼6.20–6.30 ppm. The down-
field shift of H-6 in the Z-PMHs is attributed to the anisotropic
effect of the spatially nearby C-4 carbonyl group, which creates
a deshielding cone.31 The 1H and 13C NMR data of compounds
4–13 (Tables S1–S5, ESI†) and their HRESIMS data were used
to confirm their identity in a similar fashion.
Scheme 1 Structures of the known and new PMHs 1–13.
unfavorable near the o-position and therefore it should not
possess any bulky groups. (3) Electronegative (high electron
density) groups, including alkyl substituted O, N, or S groups,
near the m- and p-positions may show better activity. (4) Low
electron density groups at the o-position can improve the
activity.29 A pharmacophore model for PMHs using the DIS-
tance COmparison technique (SYBYL’s DISCOtech) was also
reported.26,29 Pharmacophoric elements forming this model
included hydrogen-bond donor (HBD) atoms, hydrogen-bond
acceptor (HBA) atoms, and hydrophobic centers. The final
model with the highest score showed 3 HBA ligands, 2 HBD
ligands, and 2 hydrophobic centers.26,29
This study reports the synthesis of 11 new PMH analogs
(3–13, Scheme 1) designed based on SAR features described
above and their evaluation in vitro and in vivo. To expand the
therapeutic scope of PMHs, the new compounds were
additionally tested against the highly metastatic mammary
MDA-MB-231 cancer cells. All compounds were tested by using
the MTT proliferation assay, the wound-healing assay (WHA)
and the Cultrex® BME cell invasion assay. A Western blot ana-
lysis of the most active 7 was conducted to evaluate possible
molecular targets. A xenograft model was then selected using
human breast cancer MDA-MB-231/GFP cells to assess the
in vivo antitumor potential of 7. To enhance its druglikeness
and in vivo solubility, 7 was formulated using 5-hydroxypropyl-
β-cyclodextrin (HPCD).
2.2. Biological evaluation and structure–activity relationship
(SAR)
2.2.1. Cytotoxic activity against the non-tumorigenic
MCF10A epithelial cell line. In order to determine the selec-
tive activity and cytotoxicity of the new compounds to malig-
nant cells, the MTT assay using the non-tumorigenic human
breast cell line MCF10A was conducted. All compounds were
nontoxic up to concentrations higher than their IC50 values in
subsequent in vitro assays, suggesting their good selectivity
towards the malignant cells (Fig. 1, Table 1).
2. Results and discussion
2.1. Chemistry
Using a regioselective and cost-effective condensation reaction
2.2.2. Antiproliferative activity. In the proliferation assay,
of hydantoin and substituted benzaldehydes,23,25,26 11 new PMHs 6, 7, 10 and 11 showed the most promising activity in
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or a p-fluoro substituted phenyl (7) ring attached at the para-
position of the benzylidene moiety. This additional ring may
be involved in π–π stacking with the target receptor, thus
anchoring the molecule in a position that allows for better
ligand–receptor interaction and hence better activity. The loss
of para-fluoro substitution, compound 8, caused a more drastic
(almost five-fold) reduction in activity in MDA-MB-231 cells
than in PC-3 cells. On the other hand, bulkiness at the para-
position, compounds 4, 5 and 9, had a more detrimental
effect on the activity in PC-3 cells (Table 2). Compound 5 was
active (IC50 4.6 µM) against the breast cancer MDA-MB-31 cells
but only marginally active against PC-3 prostate cancer cells
(IC50 24.9 µM). The known 2 proved to be more active (IC50 8.8
µM) against PC-3 cells than MDA-MB-231 cells (IC50 18.8 µM),
whereas 1 was only marginally active (IC50 ∼36.0 µM) in both
cell lines.
Fig. 1 Selective cytotoxicity of the most active PMHs SEM. PMHs 1, 7,
10, and 11 showed no cytotoxicity to the non-tumorigenic human
mammary epithelial cells MCF10A at concentrations up to 60 µM. Error
bars indicate the SEM for n = 3 per compound.
2.2.3. Antimigratory activity. In the wound-healing assay,
compounds were more potent as PC-3 cell migration inhibi-
tors, with most compounds showing IC50 values <15 µM
(Table 2). Compounds 7, 10 and 11 showed the highest antimi-
gratory activity with IC50 values of 1.3, 2.4, and 3.6 µM, respect-
ively (Table 2). The potency against MDA-MB-231 cells in the
same assay was lower, based on their higher IC50 values
(Table 2). Nevertheless, compound 7 was the most active with
an IC50 of 15.9 µM. This difference in potency can be attribu-
ted to various reasons such as a lower binding affinity to
migration targets, binding to targets of lower importance to
this particular cell line’s migration or even different molecular
targets dictating the migratory potential of these two cell lines.
2.2.4. Anti-invasive activity. The dose selected for testing
in the Cultrex® BME cell invasion assay was based on the
overall performance of compounds in the previous assays. 10 µM
and 20 µM were selected as the optimal test doses for PC-3
and MDA-MB-231 cells, respectively. None of the compounds
showed significant activity at the selected concentrations
against PC-3 cells. Previous activity level of 1 and 2 in invasion
assay models was >50 μM doses.23,24 In MDA-MB-231 cells, the
most active compounds were 7 and 10 which allowed only 15.6
and 25.3% invasion, respectively.
Table 1 % Survival of normal mammary epithelial cells MCF10A treated
with 40 µM each of PMHs 1–13
Compound
% Cell survival (40 µM)
1
2
3
4
5
6
7
8
92.9 6.5
88.6 8.2
90.5 5.3
87.6 8.9
92.0 7.1
89.3 9.2
91.5 6.4
78.4 3.7
92.1 11.1
79.3 5.6
78.6 5.4
86.1 7.9
85.0 6.4
9
10
11
12
13
Table 2 Antiproliferative and antimigratory activities of PMHs 1–13
against PC-3 and MDA-MB-231 cell lines
Antiproliferative activity
(IC50, µM)
Antimigratory activity
(IC50, µM)
Compound
PC-3
MDA-MB-231
PC-3
MDA-MB-231
2.2.5. Western blot analysis. In the Western blot analysis,
compound 7 was evaluated against multiple targets following
72 hour treatment of MDA-MB-231 cells (Fig. 2). Results
showed that 7 caused a marked reduction of total c-Met and FAK
protein expression and a subsequent reduction of their phos-
phorylated (active form) levels in a dose-dependent manner. The
effect on total protein level may be related to the ability of the
compound to either reduce the protein expression (negatively) or
enhance proteasomal degradation (positively). In addition, treat-
ment with 7 showed a dose–response decrease in Brk, paxillin
and Rac1 phosphorylation with no or little effect on their total
levels. Inhibition of phosphorylation may be a consequence of
upstream effects or direct interactions. These results are of sig-
1
2
3
4
5
6
7
8
35.7
8.8
24.8
66.3
24.9
7.3
35.8
18.8
11.7
10.4
4.6
3.8
3.8
17.6
10.9
9.6
7.2
18.4
13.4
—
—
46.5
43.4
40.4
20.1
32.6
21.5
15.9
41.4
>50
>50
>50
16.1
>50
27.5
20.8
14.9
12.3
1.3
10.2
7.2
2.4
3.6
19.4
5.3
6.8
11.7
40.9
6.5
7.0
17.2
38.0
9
10
11
12
13
both prostate and breast cancer cell lines with IC50 values <10 µM nificance since these proteins are strongly implicated in the
(Table 2). In MDA-MB-231 cells, 6 and 7 were the most active pathophysiology of a variety of carcinomas, including breast
compounds, each with an IC50 value of 3.8 µM. Both com- and prostate cancers. Blocking one or more of these proteins is
pounds possess either a p-fluoro substituted oxybenzyl ring (6) beneficial for the management of metastatic cancer forms.
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Fig. 3 (a) In vivo activity of 7 formulated in HPCD. It slows the pro-
gression of tumor in an orthotopic nude mouse model as compared to
the vehicle (DMSO) control. A paired-samples t-test was conducted to
compare tumor volume in the control group and the HPCD7-treated
group. A p-value of 0.0091 indicates a significant difference between
the two groups. (b) No significant change in body weight was observed
among treated animals, indicating the safety of the formulation. Error
bars indicate standard error of the mean (SEM) for n = 5.
Dosing (10 mg kg−1 i.p., 3 times per week) started 5 days post-
inoculation and continued for 4 weeks.
Growth of breast tumor was compared between non-treated
animals (DMSO control group) and animals receiving com-
pound 7. Tumor progression was followed by direct measure-
ment of tumor volume starting at day 14 post-inoculation.
Fig. 3a indicates that treatment with 7 slowed the progression
of tumor and by the end of the 5-week study the average tumor
volume in the treatment group was about 50% of that in the
DMSO control group. Moreover, treatment had no effect on
mice weight or their gross phenotype, indicating that 7 did not
exert toxic effects in treated mice (Fig. 3b).
Fig. 2 Western blot analysis of c-Met, phospho-c-Met, FAK, phospho-
FAK, Brk, phospho-Brk, paxillin, phospho-paxillin, Rac1 and phospho-
Rac1 after exposure of MDA-MD-231 cells to 0, 10, and 20 μM treat-
ments of 7 for 72 h. β-Tubulin was used as a loading control. Scanning
densitometric analysis was performed on all blots done in triplicate and
the integrated optical density of each band was normalized to the
corresponding β-tubulin, as shown in the bar graphs to the right of their
respective Western blot images. Vertical bars in the graph indicate
the normalized integrated optical density of bands visualized in each
lane SEM.
Immunohistochemical analysis showed that 7, compared to
the DMSO-control group, was capable of suppressing Ki-67 and
CD-31 expression, indicating its ability to suppress both mitosis
and new vessel formation, respectively (Fig. 4a and b). Ki-67 is a
2.2.6. In vivo antitumor activity of 7. The new PMH analog nuclear protein expressed only in proliferating cells, with peak
7 was considered the most promising hit and was further eval- concentrations in the G2 and M phases of the cell cycle.34,35
uated in vivo to assess its antitumor potential. An orthotopic CD-31 is a validated endothelial cell marker shown to be a sen-
nude mouse model was selected and the human breast cancer sitive and specific indicator of endothelial differentiation.33,35
cell line MDA-MB-231/GFP was used. Because of solubility In addition, 7 caused attenuation of p-Brk and p-FAK levels in
issues, 7 was formulated using hydroxypropyl β-cyclodextrin tumor samples (Fig. 4c and d), further supporting the Western
(HPCD).32,33 The formula (HPCD7) helped us to improve 7’s blot analysis results discussed earlier. These results strongly
solubility based on the lack of precipitation in the animal per- suggest the potential of the PMH class, represented by 7, to be
itoneum cavity upon intraperitoneal (i.p.) administration. used in future to control invasive breast cancer.
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(Walnut, CA). The antibody for p-Brk was purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-rabbit
and goat anti-mouse secondary antibodies were purchased
from PerkinElmer Biosciences (Boston, MA). Hepatocyte
growth factor (HGF) was purchased from PeproTech Inc.
(Rocky Hill, NJ).
4.2. General experimental procedures
TLC analysis was carried on precoated Si gel 60 F254 500 μm
TLC plates (EMD Chemicals), and CHCl3–MeOH (9 : 1) was
1
used as the developing system. H and 13C NMR spectra were
recorded in DMSO-d6 on a JEOL Eclipse-ECS NMR spectro-
meter operating at 400 MHz for 1H NMR and 100 MHz for
13C NMR. High-resolution ESIMS experiments were conducted
using a JEOL JMS-T100 LP AccuTOF LC-Plus equipped with an
ESI source (JEOL Co. Ltd, Tokyo, Japan). ESI-MS detection was
set using the negative ion mode; needle voltage was set at
−2000 V; and the ring lens and orifice 1 and 2 voltages were
set at −10, −35, and −7 V, respectively. Nitrogen was used as
the nebulizing and desolvation gas, and pressure was
maintained constant at 0.608 MPa. Desolvation chamber and
orifice 1 temperatures were set to 250 °C and 120 °C, res-
pectively. Results were obtained using Mass Center software,
MS-56010MP (JEOL).
4.3. Chemical synthesis
4.3.1. Preparation of synthetic PMHs.23,25,31 A two-neck
round bottom flask was used to dissolve hydantoin (1.0 g) in
10 mL H2O by heating at 70 °C in an oil bath with continuous
stirring. A saturated solution of NaHCO3 was used to maintain
the pH at 7.0. Ethanolamine (0.9 mL) was then added and the
temperature was raised to 90 °C. An equimolar quantity of sub-
stituted benzaldehyde dissolved in 10 mL EtOH was then
added dropwise. The mixture was kept under reflux for 10 h.
The reaction was monitored by TLC every hour and the end-
point of the reaction was visualized by the formation of a pre-
cipitate. The mixture was then cooled to 4 °C, and the
precipitate was filtered, washed with EtOH–H2O (1 : 5), and
then recrystallized from EtOH. Yields of the product ranged
from 60 to 90%, based on the nature of the individual benz-
aldehyde used.
4.3.2. (Z)-5-(9-Methoxy-10-hydroxy-11-fluorobenzylidene)-
imidazolidine-2,4-dione (3). Yellow amorphous solid, 1H and
13C NMR see Tables S1 and S4 in ESI;† HRESIMS m/z 251.0462
[M − H]− (calcd for C11H8FN2O4, 251.0468).
4.3.3. (Z)-5-(10-(Dimethylamino)-11-cyano-12-fluorobenzyl-
idene)imidazolidine-2,4-dione (4). White amorphous solid,
1H and 13C NMR see Tables S1 and S4 in ESI;† HRESIMS m/z
273.0783 [M − H]− (calcd for C13H10FN4O2 273.0788).
Fig. 4 Immunostaining of sections obtained from vehicle-treated
(control) and 7-treated (10 mg kg−1 per day, 3 times per week) mice
against Ki-67 (mitosis marker), CD31 (endothelial marker), p-Brk and
p-FAK antibodies.
3. Conclusions
Phenylmethylene hydantoin is an interesting prostate and
breast cancer proliferation, migration and invasion inhibitory
entity inspired by marine natural products. The structural sim-
plicity and synthetic feasibility of PMHs render them appropri-
ate for future preclinical optimizations. This is the first report
of activity of this class in breast cancer. The most active analog
7 showed promising in vitro and in vivo activities without
notable toxicities. Its ability to interfere with multiple signal-
ing pathways known to play a role in cancer metastasis quali-
fies it as an interesting lead for future investigations.
4. Experimental
4.1. Chemicals, reagents, and antibodies
4.3.4. (Z)-5-(10-(tert-Butylthio)benzylidene)imidazolidine-
2,4-dione (5). Yellow amorphous solid, 1H and 13C NMR see
All materials were purchased from Sigma-Aldrich (St. Louis, Tables S1 and S4 in ESI;† HRESIMS m/z 275.0850 [M − H]−
MO), unless otherwise stated. All antibodies were purchased (calcd for C14H15N2O2S 275.0854).
from Cell Signaling Technology (Beverly, MA), unless otherwise
4.3.5. (Z)-5-(9-Nitro-10-(5′-fluorobenzyloxy)benzylidene)-
stated. The antibody for Brk was obtained from Abnova imidazolidine-2,4-dione (6). Yellow amorphous solid, 1H and
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13C NMR see Tables S1 and S4 in ESI;† HRESIMS m/z 356.0689 Briefly, cells in exponential growth were plated on a 96-well
[M − H]− (calcd for C17H11FN3O5 356.0683). plate at a density of 1 × 104 cells per well (6 wells per group),
4.3.6. (Z)-5-((4′-Fluorobiphenyl-10-yl)methylene)imidazol- and allowed to attach overnight at 37 °C under 5% CO2 in a
1
idine-2,4-dione (7). Yellow amorphous solid, H and 13C NMR humidified incubator. Complete growth medium was then
see Tables S1 and S4 in ESI;† HRESIMS m/z 281.0724 [M − H]− replaced with 100 µL of RPMI medium (GIBCO-Invitrogen, NY)
(calcd for C16H10FN2O2 281.0726).
supplemented with 5% FBS, containing various doses of the
4.3.7. (Z)-5-(10-(3′-Fluoro-7′-chlorobenzyloxy)benzylidene)- specific test compound, and incubation resumed at 37 °C
imidazolidine-2,4-dione (8). White amorphous solid, 1H and under 5% CO2 for 72 h. Cells in all groups were fed fresh
13C NMR see Tables S2 and S4 in ESI;† HRESIMS m/z 345.0448 treatment medium every other day. Viable cell count was deter-
[M − H]− (calcd for C17H11ClFN2O3 345.0442).
mined using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetra-
4.3.8. (Z)-5-(10-(1′,1′,2′,2′-Tetrafluoroethoxy)benzylidene)- zolium bromide (MTT) colorimetric assay.38 The absorbance of
imidazolidine-2,4-dione (9). White amorphous solid, 1H and each sample was measured at λ 570 nm on a microplate reader
13C NMR see Tables S2 and S5 in ESI;† HRESIMS m/z 303.0397 (BioTek, VT, USA). The number of cells per well was calculated
[M − H]− (calcd for C12H9F4N2O3 303.0393).
against a standard curve prepared at the start of each exper-
4.3.9. (Z)-5-(10-(Trifluoromethylthio)benzylidene)imidazol- iment by plating various numbers of cells (in the range
idine-2,4-dione (10). Yellow amorphous solid, 1H and 13C NMR 1000–60 000 cells per well), as counted by a hemocytometer.
see Tables S2 and S5 in ESI;† HRESIMS m/z 287.0107 [M − H]− The IC50 value for each compound was calculated by nonlinear
(calcd for C11H6F3N2O2S 287.0102).
regression (curve fit) of log (concentration) versus the % survi-
4.3.10. (5Z,5′E)-5,5′-(Perfluoro-7,10-phenylene)bis(methan- val, implemented in GraphPad Prism version 5.0 (GraphPad
1-yl-1-ylidene)diimidazolidine-2,4-dione (11). Yellow amorphous Software, La Jolla, CA , USA). The % cell survival was calculated
solid, 1H and 13C NMR see Tables S2 and S5 in ESI;† HRESIMS as follows: % cell survival = (cell no.treatment/cell no.DMSO) ×
m/z 369.0249 [M − H]− (calcd for C14H5F4N4O4 369.0247).
4.3.11. (Z)-5-(10-Hydroxy-11,12-difluorobenzylidene)imidazol-
100%.
4.4.2. Wound-healing assay (WHA). The WHA is a simple
idine-2,4-dione (12). Yellow amorphous solid, 1H and 13C NMR method for evaluating directional cell migration in vitro.39 The
see Tables S3 and S5 in ESI;† HRESIMS m/z 239.0263 [M − H]− assay was conducted as described previously.38 Briefly, cells
(calcd for C10H5F2N2O3 239.0268).
were plated on sterile 24-well plates and allowed to form a con-
4.3.12. (Z)-5-(9,10-Dimethoxy-12-fluorobenzylidene)imidazol- fluent monolayer per well (>90% confluence) overnight.
idine-2,4-dione (13). Yellow amorphous solid, 1H and 13C NMR Wounds were then inflicted in each cell monolayer using a
see Tables S3 and S5 in ESI;† HRESIMS m/z 265.0621 [M − H]− sterile 200 µL pipette tip. The media was removed and cells
(calcd for C12H10FN2O4 265.0625).
were washed twice with PBS and once with fresh RPMI
medium. Test compounds at the desired concentrations were
prepared in fresh medium (0.0% or 0.5% FBS) and were added
4.4. In vitro activities
All cell lines, prostate cancer PC-3, breast cancer MDA-MB-231 to wells in triplicate. The incubation was carried out for 24 h,
and normal breast MCF10A, were purchased from ATCC after which the medium was removed and cells were washed,
(Manassas, VA). PC-3 and MDA-MB-231 cells were maintained fixed and stained using Diff-Quick™ staining (Dade Behring
in RPMI 1640 medium (GIBCO-Invitrogen, NY) supplemented Diagnostics, Aguada, Puerto Rico). Cells which migrated across
with 10% fetal bovine serum (FBS) and glutamine (2 mmol L−1), the inflicted wound were counted under the microscope in at
and containing penicillin G (100 U mL−1) and streptomycin least five randomly selected fields (magnification: 400X).
(100 µg mL−1). MCF10A cells were maintained in Dulbecco’s
4.4.3. Cultrex® BME cell invasion assay. This assay was
modified Eagle’s medium (DMEM)/F12 containing 5% horse conducted according to the protocol provided with the kit.40
serum, 1% penicillin–streptomycin, 0.5 µg mL−1 hydro- Briefly, about 50 μL of basement membrane extract (BME) coat
cortisone, 100 ng mL−1 cholera toxin, 10 µg mL−1 insulin, and (1X for MDA-MB-231 and 0.5X for PC-3 cells) was added per
20 ng mL−1 epidermal growth factor (rhEGF). Cells were incu- well. After overnight incubation at 37 °C in a 5% CO2, 50 000
bated at 37 °C in a humidified incubator under 5% CO2.
per 50 μL of cells in fresh RPMI medium was added per well to
A stock solution of each compound was prepared in DMSO the top chamber. Test compounds were prepared at 6× the
at a concentration of 25 mM for all assays. Appropriate media desired concentrations (60 and 120 μM) and 10 μL of each
(serum-free, 0.5% FBS or 5% FBS) were used to prepare com- compound was added in triplicate to achieve the final test con-
pounds at their final concentrations for each assay. The centrations (10 and 20 μM). 150 μL of RPMI medium, contain-
vehicle (DMSO) control was prepared by adding the maximum ing 10% FBS and penicillin–streptomycin as well as
volume of DMSO used in preparing test compounds to the fibronectin (1 μL mL−1) and N-formyl-met-leu-phe (10 nM) as
appropriate media type such that the final DMSO concen- chemoattractants, was then added to the lower chamber.
tration never exceeded 0.2%.
Plates were re-incubated at 37 °C in 5% CO2 for 24 h after
4.4.1. MTT (proliferation assay). The antiproliferative which the top and bottom chambers were aspirated and
activity of test compounds was evaluated on the human pros- washed with wash buffer supplemented with the kit. 100 μL of
tate cancer cell line PC-3 and the breast cancer cell line cell dissociation/calcein-AM solution was added to the bottom
MDA-MB-231 using the procedure described previously.36,37 chamber and incubated at 37 °C in 5% CO2 for 1 h. The cells
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internalize calcein-AM, and the intracellular esterases cleave At 48 h post-inoculation, the mice were randomly divided into
the acetomethylester (AM) moiety to generate free calcein. two groups: (i) the vehicle-treated control group (n = 5) and
Fluorescence of the samples was determined at λexcitation (ii) the HPCD7-treated group (n = 5). Treatment (3 times per
485 nm and λemission 528 nm using an ELISA plate reader week) started 5 days post-inoculation with intraperitoneal (i.p.)
(BioTek, VT, USA). The numbers of cells that invaded through administered vehicle control (DMSO–saline) or 10 mg kg−1
the BME coat were calculated using a standard curve and % HPCD7. The HPCD7 formula was prepared as follows: 4 g of
invasion of different treatments was compared relative to the hydroxypropyl β-cyclodextrin was dissolved in 20 mL distilled
DMSO control.
water (1 : 5 ratio). Compound 7 was then added to this solu-
4.4.4. Western blot analysis. A Western blot analysis was tion, the vial sealed and autoclaved for 15–30 minutes to
performed according to the method previously described.41 achieve a final concentration of 0.5 mg mL−1. Mice were moni-
Briefly, MDA-MD-231 cells were initially plated at a density of tored daily for general wellbeing, and tumor volume and body
1 × 106 cells per 100 mm culture plate, and allowed to attach weight were measured prior to each dose (3 times per week).
overnight in RPMI-1640 medium containing 10% FBS. Cells Tumor volume (V) was calculated using the formula V = (L × W2)/2,
were then washed with PBS and incubated with vehicle control where L is the length in mm and W is the width in mm of
or treatment in serum-free media for 3 days in culture. At the tumors as measured using a caliper. All mice were sacrificed
end of the treatment period, cells were lysed in RIPA buffer on day 33 post-inoculation, and the tumors were excised and
(Qiagen Sciences Inc., Valencia, CA). Protein concentration was weighed. Some tumor tissues were stored at −80 °C until
determined by the BCA assay (Bio-Rad Laboratories, Hercules, total protein extraction for Western blot analysis and others
CA). Equivalent amounts of protein were electrophoresed on were stored in 70% ethanol at RT for immunohistochemistry
SDS-polyacrylamide gels. The gels were then electroblotted studies.
onto PVDF membranes. These PVDF membranes were then
4.4.6. Immunohistochemistry. The tumor specimens were
blocked with 2% BSA in 10 mM Tris-HCl containing 50 mM processed with the use of alcohols and xylene and then infil-
NaCl and 0.1% Tween 20, pH 7.4 (TBST), and then incubated trated in paraffin wax using the Excelsior™ ES Tissue Pro-
with specific primary antibodies against Brk (Abnova, CA) and cessor. Paraffin sections were dewaxed in xylene, rinsed in
p-Brk (Santa Cruz, CA) and incubated overnight at 4 °C. At the grade alcohol, and rehydrated in water and then were placed
end of the incubation period, membranes were washed in citric buffer (PH 6.0) and treated in a microwave oven with
5 times with TBST and then incubated with the respective horse- high power for 3 min and 10% goat serum for 30 min. Sub-
radish peroxide-conjugated anti-rabbit or anti-mouse second- sequently, antibodies with appropriate dilution were applied
ary antibodies (PerkinElmer Biosciences, MA) in 2% BSA in on the sections as follows: CD31 (Pierce Product #PA5-32321;
TBST for 1 h at room temperature followed by rinsing with 1 : 50 dilution, 1 h at rt), Ki-67 (Cell Signalling Product #9027;
TBST 5 times. Blots were then visualized by chemilumines- 1 : 150 dilution, 1 h at rt), p-Brk (Bioss Product #bs-12890R;
cence according to the manufacturer’s instructions (Pierce, 1 : 100, 1 h at rt), and p-FAK (Cell Signalling Product #8556;
Rockford, IL, USA). Images of protein bands from all treatment 1 : 100, 1 h at rt). Following that, secondary antibodies
groups within a given experiment were acquired using a Kodak (Ventana Multimer Anti-Rb-HRP Product #760-4311; 24 min at
Gel Logic 1500 Imaging System (Carestream Health Inc., New rt) were applied. Signals were developed with Vector ImmPACT
Haven, CT, USA). The visualization of β-tubulin (Cell Signaling DAB Product #SK-4105 for 8 min at rt. The sections were
Technology, MA) was used to ensure equal sample loading in finally counterstained using a hematoxylin solution for 1 min
each lane. All experiments were repeated at least three times at rt.
and a representative Western blot image from each experiment
is shown in Fig. 2.
4.4.5. Xenograft studies.42 All animal experiments were
approved by the Institutional Animal Care and Use Committee,
University of Louisiana at Monroe, and were handled in strict
accordance to good animal practice as defined by the NIH
guidelines. Athymic nude mice (Foxn1nu/Foxn1+, 4–5 weeks,
female) were purchased from Harlan (Indianapolis, IN). Mice
had free access to drinking water and pelleted rodent chow
(no. 7012, Harlan/Teklad, Madison, WI) and were acclimated
to animal house facility conditions at a temperature of
18–25 °C, with a relative humidity of 55 to 65% and a 12 h
light/dark cycle, for at least one week prior to the experiments.
MDA-MB-231/GFP human breast cancer cells were cultured
and resuspended in serum-free DMEM medium. After anesthe-
sia, cell suspensions (1 × 106 cells per 20 μL) were inoculated
into the second mammary gland fat pad just beneath the
nipple of each animal to generate orthotopic breast tumors.
Acknowledgements
The Louisiana Campuses Research Initiative (LaCRI) is
acknowledged for financial support (ELD041). The Louisiana
Board of Regents is also acknowledged for the support of the
new HRMS facility (LEQSF(2013-14)-ENH-TR-26).
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