Discovery, design, and synthesis of anti-metastatic lead phenylmethylene hydantoins inspired by marine natural products

Article abstract of DOI:10.1016/j.bmc.2008.12.053

The Red Sea sponge Hemimycale arabica afforded the known (Z)-5-(4-hydroxybenzylidene)-hydantoin (1), (R)-5-(4-hydroxybenzyl)hydantoin (2), and (Z)-5-((6-bromo-1H-indol-3-yl)methylene)-hydantoin (3). The natural phenylmethylene hydantoin (PMH) 1 and the sy

Full text of DOI:10.1016/j.bmc.2008.12.053

Bioorganic & Medicinal Chemistry 17 (2009) 1731–1738
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery, design, and synthesis of anti-metastatic lead phenylmethylene
hydantoins inspired by marine natural products
Mudit Mudit a, Mohammad Khanfar a, Anbalagan Muralidharan a, Shibu Thomas a, Girish V. Shah a,
Rob W. M. van Soest b, Khalid A. El Sayed a,
*
a Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of Louisiana at Monroe, 1800 Bienville Drive, Monroe, LA 71201, USA
b Zoological Museum, University of Amsterdam, PO Box 94766, 1090 GT Amsterdam, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
The Red Sea sponge Hemimycale arabica afforded the known (Z)-5-(4-hydroxybenzylidene)-hydantoin
(1), (R)-5-(4-hydroxybenzyl)hydantoin (2), and (Z)-5-((6-bromo-1H-indol-3-yl)methylene)-hydantoin
(3). The natural phenylmethylene hydantoin (PMH) 1 and the synthetic (Z)-5-(4-(ethylthio)benzyli-
dene)-hydantoin (4) showed potent in vitro anti-growth and anti-invasive properties against PC-3M
prostate cancer cells in MTT and spheroid disaggregation assays. PMHs 1 and 4 also showed significant
anti-invasive activities in orthotopic xenograft and transgenic mice models. To study the effect of elec-
tronic and lipophilic parameters on the activity, a wide array of several substituted aldehydes possessing
Received 25 October 2008
Revised 12 December 2008
Accepted 14 December 2008
Available online 29 December 2008
Keywords:
Red Sea sponge Hemimycale arabica
Phenylmethylene hydantoins
Prostate cancer
electron-withdrawing (+
r
), lipophilic (+
p
), electron-donating (ꢀ
r
), and less lipophilic substituents (ꢀ
p)
were used to synthesize several PMHs. Few des-phenylmethylenehydantoins and 2-thiohydanoins were
also synthesized and the anti-invasive activities of all compounds were evaluated. Comparative molecu-
lar field analysis (CoMFA) was then used to study the 3D QSAR. Predictive 3D QSAR model with conven-
tional r2 and cross validated coefficient (q2) values up to 0.910 and 0.651 were established. In conclusion,
PMH is a novel antimetastatic lead class with potential to control metastatic prostate cancer.
Ó 2008 Elsevier Ltd. All rights reserved.
Anti-metastatic
Spheroid disaggregation
Comparative molecular field analysis
3D QSAR
1. Introduction
species was never investigated. Liquid chromatography of this ex-
tract afforded the known (Z)-5-(4-hydroxybenzylidene)-hydantoin
Prostrate cancer is the most prevalent cancer form in men and
constitutes the second leading cause of cancer deaths in men in
United States.1 Later stages of prostate cancer progress to life-
threatening, androgen-independent and fatal metastatic forms.2
Patient progression at metastatic stage of disease is very poor,
and currently few options are available for the treatment at this
stage.1,2 Therefore, morbidity and mortality can be remarkably re-
duced by attenuation of metastasis.
The sponge genus Hemimycale (family Mycalidae) is well known
for its bioactive secondary metabolites especially biogenetically
complex guanidine alkaloids.3,4 Ptilomycalin A1 has a unique poly-
cyclic guanidine skeleton with a spermidine group linked to a 16-
hydroxyhexadecanoic acid moiety.3–5 This novel guanidine alka-
loid possesses remarkable antifungal, antiviral, and antitumor
activities.3–5
(1),6 (R)-5-(4-hydroxybenzyl)hydantoin (2),7 and (Z)-5-((6-bromo-
1H-indol-3-yl)methylene)-hydantoin (3).8 PMH 1 was reported as
a natural product from the Red Sea sponge Laxosubrites sp. without
any biological activity.9
The natural PMH 1 and the synthetic 4 showed potent in vitro
anti-growth and anti-invasive properties against PC-3M prostate
cancer cells in MTT and spheroid disaggregation assays.10 PMH 1
and 4 also showed anti-invasive activity in orthotopic xenograft
of PC-3M cells in nude mice model.10 They decreased orthotopic
tumor growth and inhibited the formation of tumor micrometasta-
ses in distant organs without apparent cytotoxic effects at the test
doses.10 PMH 4 also showed potent activity in LPB-Tag mice trans-
genic model.10 Prostate tumors of LPB-Tag mice display significant
similarities with human tumors including tumor heterogeneity,
neuroendocrine features and relatively slower tumor growth.10
PMH 4 remarkably reduced growth of primary tumors and their
metastasis in reproductive organs, decreased morbidity and in-
creased survival of the LPB-Tag mice. Therefore, PMH 1 and 4 were
suggested as potent attenuators of the disassembly of junctional
complexes which are key events in the progression of a localized
Ethanolic extract of abundant shallow water Red Sea sponge
H. arabica was targeted because it inhibited the proliferation and
invasion of the highly metastatic human prostate cancer PC-3M
cell line. Interestingly, the chemistry of this particular Hemimycale
prostate tumor to its metastatic form.10 PMH
novel leads appropriate for future development as treatment for
1 and 4 are
* Corresponding author. Tel.: +1 318 342 1725; fax: +1 318 342 1737.
E-mail address: elsayed@ulm.edu (K.A.El. Sayed).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.053

1732
M. Mudit et al. / Bioorg. Med. Chem. 17 (2009) 1731–1738
metastatic prostate cancer and a chemopreventive agent.10 This
study represents the first structure–activity relationship study of
phenylmethylene hydantoins as a novel anti-metastatic class.
The cyclic imide hydantoins are well investigated for their anti-
convulsant activity.11,12 Various pharmacological activities were
reported for hydantoins including fungicidal, herbicidal, anti-
inflammatory, anti-HIV, analgesic, cannabinoid receptor-1 (CB-1),
5HT, purine P-2X receptor antagonism, platelet aggregation inhibi-
tion, anti-arrhythmic and antihypertensive, anti-diabetic, neuro-
protective, HDL/cholesterol modulating, antiviral, and growth
hormone secretagogue.12 Phenytoin and PMHs were previously re-
ported to possess anticonvulsant properties.11,12 It was also estab-
lished that for the anticonvulsant activity hydantoin nucleus and
phenyl ring in PMH served the basic structural requirement.12 Sev-
eral PMHs were synthesized earlier for investigating the influence
of various substitutions in the phenyl ring of PMH. Maximal elec-
troshock seizure (MES) assay was used for the evaluation of anti-
convulsant activity for these compounds, and response score of
2.5 was considered equivalent to its effective dose.11
liferation, invasion, and junctional complexes destabilization of PC-
3M cells.10–12
Eight new compounds (4-11) were synthesized along with
thirty-one other known PMHs (12-42) using the base-catalyzed
condensation reaction of hydantoin with different substituted
benzaldehydes in the presence of ethanolamine (Scheme 1).11,12
O
O
O
R2
9
NH
NH
O
R1
NH
O
3
HN
HN
HN
HN
1
R3
R4
HO
5
7
O
11
2
3
Br
R5
3`
O
S
S
R2
N
1`
R1
a
b
c
N
HO
O
O
Occasionally, hydantoin analogs were reported to show antitu-
mor activities. Hydantoins were shown to modulate Pg–P and inhi-
bit MMPs.12 Substituted diisopropylbenzylidene hydantoins and 2-
thiohydantoins were patented in 1987 for their tyrosine kinase
inhibitory and antiallergic activities.13 These compounds were po-
O
e
d
The HREIMS data of 4 showed a molecular ion peak at m/z
248.0622, suggesting the molecular formula C12H12N2O2S. 1H and
13C NMR data (Tables 1 and 2) analysis indicated that 4 is (Z)-5-
(4-(ethylthio)benzylidene)imidazolidine-2,4-dione. The olefinic
proton singlet at d 6.36 which correlated with the methine carbon
at d 108.2 was assigned H-6. Proton H-6 showed 3J-HMBC correla-
tions with the amide carbonyl C-4 (d 166.5) and the aromatic
methine carbons C-8 and C-12 (d 130.4), confirming the phenylm-
ethylene hydantoin entity of 4. H-6 also showed 2J-HMBC correla-
tions with the quaternary carbons C-5 and C-7 (d 137.8 and 128.3,
respectively). Protons H-8 and H-12 showed COSY correlations
with protons H-9 and H-11, respectively (Table 1). Protons H-8
and H-12 also showed 3J-HMBC correlations with the quaternary
carbon C-10 (d 130.5). The methyl triplet H3-20 (d 1.23) showed
COSY coupling with the methylene quartet H2-10 (d 3.02). H2-10
also showed 3J-HMBC correlation with the quaternary aromatic
carbon C-10 (d 130.5). The later carbon was assigned based on its
3J-HMBC correlation with protons H-8 and H-12.
The HREIMS data of 5 showed a molecular ion peak at m/z
238.0559 and suggested a molecular formula of C11H8F2N2O2. 1H
and 13C NMR data of 5 (Tables 1 and 2) confirmed the identity of
2,3-difluoro-4-methylbenzylidene moiety. The olefinic proton sin-
glet H-6 showed 3J-HMBC correlations with the quaternary aro-
matic C-8 (d 148.1) and the methine aromatic C-12 (d 124.4).
Proton H-12 showed 3J-HMBC correlations with the quaternary
aromatic carbons C-8 and C-10 (d 127.3). The methyl singlet H3-
10 (d 2.29) showed a 2J-HMBC correlation with the quaternary aro-
matic C-10 (d 127.3) and 3J-HMBC correlations with the quaternary
aromatic carbon C-9 (d 149.0) and the methine aromatic carbon C-
11 (d 126.7). It is worth noting that the 8,9-difluoro substitution
remarkably affected the splitting of aromatic protons and carbons.
In addition to expected o-coupling (J = 7.6 Hz), each of H-11 and H-
12 also showed 4J-coupling (J = 4.4 Hz) with C-9 and C-8 fluoro
moieties, respectively. Each of carbons C-7 and C-10 splitted as
doublet (J = 9.9 and 13.8 Hz, respectively) due to 2J-coupling with
C-8 and C-9 fluoro groups, respectively. Similarly, each of carbons
tent at 100 l
M dose in guinea pigs.13 2-[4-[(5-Oxo-2-thioxo-4-imi-
dazolidinylidene)-methyl]phenoxy] propionic acid and related
analogs were patented for their ability to inhibit substrate binding
by extracellular signal-regulated kinases by binding docking do-
mains.14 This compound blocked the interaction of extracellular
signal-regulated kinases (ERK kinases) with substrate proteins,
and hence considered as a possible treatment for neoplasms asso-
ciated with abnormal ERK kinase activity.14 2-[4-[(5-Oxo-2-thioxo-
4-imidazolidinylidene)-methyl]phenoxy] propionic acid and its
analogs bind with the ERK1 or ERK2 kinase, inhibit phosphoryla-
tion, and suppresses cell proliferation in vitro.14 Peptide hydantoin
analogs were patented for their ability to inhibit thrombocyte
aggregation, metastasis, and osteoclast binding to bone surfaces.15
Though not typically hydantoin, but structurally related to 1, the
imidazol-2-amine, 5-chloro-N-(4-methylphenyl)-4-(phenylmeth-
ylene) was patented for its in vitro anti-melanoma activity.15 Some
1-phenethyl and 5-(E)-benzylidene hydantoins inhibited EGFR
autophosphorylation and polyGAT phosphorylation and were
found to inhibit the growth and proliferation of human A-431
cells.16 Therefore, these hydantoins were considered good scaffolds
for future design of tyrosine kinase inhibitors.16
Limited sustainable supply of most bioactive natural products is
the main obstacle that may hinder further development as drug
leads.17–19 Feasible regioselective and cost effective synthesis of
the pharmacologically active marine natural product 1 not only
secured adequate supply for in vitro and animal studies but it also
allowed the synthesis of several related analogs for the structure-
activity relationship study.11 Several PMHs were previously
synthesized to establish the anticonvulsant structure-activity rela-
tionship of this class.11,12,20–22 This study reports the synthesis of
forty PMHs and the novel report of their anti-invasive and junc-
tional complexes stabilization activities. Only PMHs with ED-
MES(2.5) > 200 mg/kg in MES assay were selected to avoid the
possibility of potential central nervous system effects.11 3D-QSAR
Model was established and validated.
O
O
H
NH
O
O
NH
O
HN
NaHCO3/Aq. EtOH
Ethanolamine
2. Results and discussion
HN
+
R
R
Reflux
Three known hydantoins (1–3) were isolated from the Red Sea
sponge H. arabica. Among these, (Z)-5-(4-hydroxybenzylid-
ene)hydantoin (1) was found to be the most active one against pro-
H
Scheme 1. General synthetic scheme of phenylmethylene hydantoins.11

M. Mudit et al. / Bioorg. Med. Chem. 17 (2009) 1731–1738
1733
Table 1
1H NMR data of compounds 4–7a
Position
dH
4
5
6
7
6
6.36, s
6.31, s
6.19, s
6.50, s
7
—
—
—
—
8
9
7.56, d (8.4)
7.28, d (8.4)
—
—
—
7.83, br s
—
7.72, m
10
11
12
10
20
30
40
50
60
—
—
7.43, m
7.41, m
7.34, m
—
7.42, 2H, d (8.8)
7.33, 2H, dd (8.8, 8.0)
7.48, m
7.33, 2H, dd (8.8, 8.0)
7.42, 2H, d (8.8)
7.60, d (7.7)
7.41, m
7.60, d (7.7)
—
7.74, 2H, d (7.4)
7.51, m
7.41, m
7.28, d (8.4)
7.56, d (8.4)
3.02, q (7.4)
1.23, t (7.4)
—
—
—
—
7.12, dd (7.6, 4.4)
7.45, dd (7.6, 4.4)
2.29, 3H, s
—
—
—
—
—
7.51, m
7.74, 2H, d (7.4)
a
In DMSO, 400 MHz. Coupling constants (J) are in Hertz.
The 1H and 13C NMR data of 8 and 9 (Tables 3 and 4) as well as
their HREIMS suggested close similarity to 1 with an additional 2-
thienyl moiety. The location of this new thienyl group whether at
C-8 in 8 or at C-10 in 9 was based on HMBC data. The quaternary
carbon C-20 (d 141.5) of the thiophene ring in 8 was assigned based
on its 3J-HMBC correlation with the aromatic proton H-9. Proton H-
5’ (d 7.62) showed 3J-HMBC correlations with C-20 and C-30, in addi-
Table 2
13C NMR data of compounds 4–7a
Position
dC
4
5
6
7
2
4
5
6
7
8
9
10
11
12
10
20
30
40
50
60
156.6, qC
166.5, qC
137.8, qC
108.2, CH
128.3, qC
130.4, CH
127.8, CH
130.5, qC
127.8, CH
130.4, CH
26.0, CH2
14.6, CH3
—
156.8, qC
166.3, qC
131.4, qC
97.9, CH
121.3, qC
148.1, qC
149.0, qC
127.3, qC
126.7, CH
124.4, CH
14.6, CH3
—
156.2, qC
165.9, qC
129.3, qC
107.4, CH
140.5, qC
142.5, qC
129.5, CH
128.3, CH
128.1, CH
130.6, CH
131.1, qC
129.0, CH
130.1, CH
129.1, CH
130.1, CH
129.0, CH
156.4, qC
166.2, qC
128.8, qC
108.8, CH
140.3, qC
128.0, CH
141.4, qC
129.0, CH
128.2, CH
129.0, CH
134.2, qC
127.6, CH
129.4, CH
128.2, CH
129.4, CH
127.6, CH
2
tion to a J HMBC correlation with C-4’. Proton H-30 (d 7.51)
showed a 3J-HMBC correlation with C-8 (d 134.5) which confirmed
the position of thiophene ring.
The 1H and 13C NMR data of 10 (Tables 3 and 4) along with its
HREIMS suggested similarity to 1 with an additional 2-(furan-2-
yl) functionality. The carbon C-20 of furan ring showed 3J-HMBC
correlations with Proton H-9 of benzene ring, and also with pro-
tons H-40 and H-50. Connection of the furan ring to the benzene ring
—
—
—
—
3
was also confirmed by the J-HMBC correlation of H-30 with the
—
—
—
quaternary aromatic C-8 (d 130.0).
The IR spectrum of 11 showed a mmax band at 2235 cmꢀ1, sug-
gesting the presence of cyano functionality.25 The 1H and 13C
NMR data of 11 (Tables 3 and 4) along with its HREIMS suggested
similarity to 1 with an additional 9-cyano functionality. The qua-
ternary carbon at d 119.1 was assigned as the cyano carbon C-10
based on its 3J-HMBC correlation with proton H-10 (d 7.74).
The loss of cell–cell adhesion is associated with tumor metasta-
sis in several epithelial cancers including prostate, breast and renal
carcinomas.26,27 Thus compounds which strengthen cell–cell adhe-
sion can potentially serve as possible therapies for advanced pros-
tate cancer.28 PC-3M prostrate cancer cells used in this study were
a
In DMSO, 100 MHz. Carbon multiplicities were determined by APT experiments.
qC = quaternary, CH = methine, CH2 = methylene, CH3 = methyl carbons.
C-8 and C-9 splitted as doublet of doublets (J = 247.0, 13.0 and
242.0, 11.9 Hz, respectively) due to 1J and 2J coupling with C-8
and C-9 fluoro groups.23,24
The 1H and 13C NMR data of 6 and 7 (Tables 1 and 2) as well as
their HREIMS suggested close similarity to 1 with an additional
phenyl moiety. The location of this phenyl group whether at C-8
in 6 or at C-9 in 7 was based on HMBC and COSY data.
Table 3
1H NMR data of compounds 8–11a
Position
dH
8
9
10
11
6
7
6.45, s
—
6.43, s
—
6.55, s
—
6.39, s
—
8
—
7.68, 2H, m
—
8.12, s
9
7.68, m
7.40, m
7.18, m
7.42, m
—
7.66, 2H, m
—
7.66, 2H, m
7.68, 2H, m
—
—
7.59, dd (5.1, 1.1)
7.16, dd (5.1, 3.7)
7.62, dd (3.7, 1.1)
—
7.69, dd (1.8, 7.5)
7.43, ddd (7.7, 7.6, 1.5)
7.39, ddd (7.7, 7.6, 1.5)
7.62, dd (7.5, 1.8)
—
—
10
11
12
10
20
30
40
50
60
7.74, br d (8.0)
7.58, dd (7.7, 7.7)
7.89, br d (8.0)
—
—
—
—
—
—
—
—
7.51, m
7.17, m
7.67, m
—
6.67, dd (3.3, 0.8)
6.65, dd (3.3, 1.8)
7.85, dd (1.8, 0.8)
—
a
In DMSO, 400 MHz. Coupling constants (J) are in Hertz.

1734
M. Mudit et al. / Bioorg. Med. Chem. 17 (2009) 1731–1738
Table 4
cancer. To establish the structure–activity relationship of this class,
a wide array of several substituted aldehydes possessing electron-
withdrawing (+ ), lipophilic (+ ), and less
), electron-donating (ꢀ
lipophilic substituents (ꢀ ) were used to synthesize diverse PMHs
13C NMR data of compounds 8–11a
r
p
r
Position
dC
p
8
9
10
11
4-42, which were then tested for their anti-metastatic activity.
Spheroid disaggregation assay was selected to asses this activ-
ity.29,32–34 Since tumor cells are generally released in clumps, at-
tach to the preferred extracellular matrix (ECM), and then
radially migrate in all directions, spheroid disaggregation assay
was considered a better in vitro model of tumor metastasis com-
pared to the linear invasion assays.29,32–34 To avoid repeated addi-
tion of exogenous CT during long incubation period of 48 h, PC-3M-
CT+ cells were used because they secrete large amounts of CT and
have CTR.29 Most known PMHs including 1 and 12-42 showed no
significant cytotoxic effects in the CNS or other organs when tested
2
4
5
6
7
8
9
10
11
12
10
20
30
40
50
156.2, qC
165.9, qC
130.0, qC
107.1, CH
131.4, qC
134.5, qC
128.0, CH
129.2, CH
128.5, CHb
128.7, CH
—
156.3, qC
166.1, qC
128.4, qC
108.2, CH
132.6, qC
130.7, CH
126.0, CH
134.0, qC
126.0, CH
130.7, CH
—
156.1, qC
165.9, qC
79.7, qC
159.2, qC
170.6, qC
130.6, qC
105.8, CH
134.9, qC
132.5, CH
112.5, qC
131.9, CH
130.4, CH
134.6, CH
119.1, qC
—
107.2, CH
130.4, qC
130.0, qC
127.7, CH
128.5, CH
129.1, CH
130.1, CH
—
152.0, qC
112.6, CH
111.2, CH
130.1, CH
141.5, qC
130.7, CH
128.6, CHb
130.0, CH
143.2, qC
126.8, CH
129.2, CH
124.9, CH
—
—
—
in animals at doses >100
observable toxic effects on prostate cancer cells at doses up to
200 M in spheroid disaggregation assays (no observed dead cells)
or up to 50 M in MTT assays. We tested the anti-invasive effects
of PMHs at 200 M in spheroid disaggregation assays to observe
l
M.11 Moreover, PMH 1 did not display
l
a
In DMSO, 100 MHz. Carbon multiplicities were determined by APT experiments.
l
qC = quaternary, CH = methine carbons.
b
Interchangeable.
l
(1) anti-invasive effects of these compounds at that dose relative
to the PMH 1; and (2) to ensure that the compound does not have
significantly greater cytotoxic effects. If a PMH was not active at
this particular dose it was deemed inactive. Both 1 and 4 inhibited
PC-3M-CT+ spheroid disaggregation and cell migration in a dose-
derived from liver metastasis of PC-3 xenografts.29,30 Studies have
shown that calcitonin (CT) significantly increases tumor growth
and metastasis of PC cell lines.29,30 These effects may be mediated
by the disruption of cell-cell adhesion.29,30 A dose of 50 nM CT was
optimal for stimulation of PC-3M cell proliferation and invasion.10
dependent manner with EC50 of 150 and 30 lM, respectively
(Fig. 1). As depicted in Figure 1, the spheroid is represented by a
core sphere, from which the cells migrate radially to expand the
area occupied by cells (as depicted by arrow). Addition of PMH 1
or related PMHs does not affect the core spheroid, but remarkably
reduce the surface area increase caused by spheroid disaggregation
with time, and migration of cells in all directions (represented by
arrows). The results showed that spheroid disaggregation as well
as migration of cells declined in the presence of PMH derivatives
in a dose-dependent manner, and the core spheroid was un-
A 50 lM dose of PMH 1 remarkably reduced CT-stimulated prolif-
eration increase and displayed minimal cytotoxic effect on PC-3M
cells as suggested by no decrease in cell viability in MTT assay.10
The same dose of PMH 1 significantly increased transepithelial
resistance of (TER) diluent and CT-treated PC-3M cultures, suggest-
ing that 1 promotes tight junctions (TJs) formation and completely
reverses the actions of CT on TER.10 PMH 1 also decreased baseline
and abolished CT-induced increase in paracellular permeability of
polarized PC-3M cell layers.10 This effect further supported the fact
that 1 promotes TJs formation.10
Intraperitoneally-administered to orthotopic PC-3M-CT+ xeno-
grafts in nu/nu nude mice, PMHs 1 and 4 significantly reduced
and localized the tumor mass in the abdominal cavity and pre-
vented the formation of distant metastases at a dose of 5 or
changed even after 48 h when incubated with 100 lM PMH or
greater. This was not associated with cytotoxicity since PMH 1
did not affect cell viability in MTT assay. The anti-metastatic activ-
ities of synthetic PMHs 4-42 using spheroid disaggregation assay
are shown in Tables 5 and 6. Comparative molecular field analysis
(CoMFA) was used to study the 3D QSAR.35,36 Predictive 3D QSAR
model with conventional r2 and cross validated coefficient (q2) val-
ues up to 0.910 and 0.651, respectively, were established. The
results of the CoMFA studies are summarized in Table 7. CoMFA-
derived QSAR shows a good q2 and therefore indicates a consider-
able predictive and correlative capacity of the model. Analysis
yielded q2 of 0.651 (3 pc) and a conventional r2 of 0.91. A high
bootstrapped (100 runs) r2 of 0.933 (0.036) adds a high confidence
limit to this analysis.
1 lg/g body weight/day, respectively, compared to diluent-treated
mice in a 60-days in vivo study.10,31,32 Both 1 and 4 remarkably de-
creased the metastasis of PC-3M-CT+ cells in most organs, and re-
duced size of tumor cell colonies in organs where metastases were
present.10,31,32 Three equally divided intraperitoneal doses of 2
lg
of 4/g body weight/week remarkably reduced growth of primary
tumors and their metastasis in reproductive organs, decreased
morbidity and increased survival of LPB-Tag transgenic mice.10
Figure 2 displays a plot of predicted versus actual activities of
PMHs training set used in building a QSAR model, and the error
bars depict the standard error. In this analysis both steric and elec-
trostatic fields contribute to the QSAR equation by 70.6% and
29.4%, respectively. This suggested that variation in biological
2.1. Molecular modeling and CoMFA analysis
Based on the aforementioned results, PMHs are considered po-
tential leads for the control and prevention of metastatic prostate
Figure 1. Spheroid disaggregation of PC-3M-CT+ prostate cells after 24 h of incubation with different doses of 4. VC = Vehicle control. Magnification = 100x.

M. Mudit et al. / Bioorg. Med. Chem. 17 (2009) 1731–1738
1735
Table 5
Training Set
Actual (200 l
M dose) and predicted activity of training seta
250
Tested compound
Actual activityb
(lm)
Predicted activityc
(lm)
1
4
5
7
8
9
79
42
18
167
70
67
38
185
62
106
96
68
112
96
115
27
114
109
109
94
70
81
124
122
74
200
150
100
50
15.5
175
76
50
39
195
58
125
90
89
130
91
117
29
94
112
115
89
87
65
145
106
90
11
12
13
14
15
17
18
20
22
23
24
26
31
32
33
34
35
36
37
38
39
41
42
0
0
50
100
150
200
250
Actual activity (um)
Figure 2. Predicted versus actual activity of selected PMHs training set; standard
errors are shown as error bars.
Test Set
200
150
100
50
91
82
86
135
138
92
69
92
161
134
0
a
Migration value for control group 180 lm.
Measured from spheroid disaggregation assay.
Calculated using the PLS equation of CoMFA.
0
50
100
150
200
b
c
Actual Activity (um)
Figure 3. Predicted versus actual activity of selected PMHs test set; standard errors
are shown.
Table 6
Actual (200 l
M dose) and predicted activity of test seta
The leave-one-out cross-validation method might lead to high
q2 values which do not necessarily reflect a general predictability
of the models. Therefore, cross-validation was performed using
two groups (leave-half-out). In this method, 50% of the PMHs were
randomly selected and a model was generated, which was then
used to predict the activity of the remaining 50% of compounds.
The random formation of the cross-validation groups may have
an effect on the results and therefore cross-validation was per-
formed 100 times for all the analyses. The mean q2 value was
0.613 which is slightly lower compared to the values obtained with
the leave-one-out method. In no case were q2 values negative. The
obtained results suggest that there is a good internal consistency in
the underlying data set. Figure 4 shows the superposition of the
PMHs training and test sets.
Tested compound
Actual activityb
(lm)
Predicted activityc
(lm)
6
56
62
10
28
29
30
40
186
116
26
87
55
160
122
43
99
79
a
Migration value for control group 185 lm.
Measured from spheroid disaggregation assay.
Calculated from the PLS equation of CoMFA.
b
c
Table 7
To visualize the CoMFA steric and electrostatic fields from PLS
analysis, contour maps of the product of the standard deviation
associated with the CoMFA column and coefficient (SD ꢁ coeff) at
each lattice point were generated (Fig. 5). The contour maps are
plotted as percentage contribution to the QSAR equation and are
associated with the differences in biological activity. The most ac-
tive PMH 4 is displayed in the contour maps of QSAR model (Fig. 5).
The regions of high and low steric tolerance are shown in green
and yellow polyhedra, respectively (Fig. 5A). Areas of high steric
bulk tolerance (80% contribution) were observed near the p-posi-
tion of the benzylidene group in 4. The enhanced activity of PMHs
4, 9, and 29 may also be attributed to the presence of bulky groups
in p-position, which is surrounded by green contours in steric field
plot. The sterically unfavored yellow regions were observed near
the o-position of benzylidine group at the opposite direction of
N-1 of hydantoin moiety.
Summary of CoMFA results
Statistical parameter
Value
q2
0.651
0.910
0.115
3
84.092
0.000
0.706
0.294
0.686
r2 Conventional
Std. error
No. of comp.
F value
P value
Steric contrib.
Electrostatic contrib.
r2pred
r2bs (SD)
0.933 (0.036)
activity of inhibitors is dominated by differences in steric (van der
Waals) interactions with the hydantoins active site. This analysis
predicted the activities of the molecules of test set with r2Pred of
0.686. Figure 3 shows the plot of predicted versus observed activ-
ities of the test set.35,36
CoMFA electrostatic fields are shown as blue (more positive
charge) and red (more negative charge) polyhedra in Figure 5B.

1736
M. Mudit et al. / Bioorg. Med. Chem. 17 (2009) 1731–1738
phenyl ring of 4. Therefore, compounds possessing electronegative
groups near these regions (high electron density) may show more
activity. The distal part of the benzylidene group have an electro-
negative group with prominent red contour, which is consistent
with the observation that the three most active PMHs with migra-
tion values less than 30 lm (22, 29 and 4) posses oxygen, nitrogen
and sulfur atoms, respectively in the p-position. These compounds
have H-accepting properties that could contribute to hydrogen
bonding interaction near this position. Blue contours reside around
the upper right-hand and upper left-hand corners of 4, which brace
the hydrogen atoms of the aromatic and ethylene moieties, indicat-
ing that electropositive groups near this area are predicted to in-
crease the activity. This could also be a possible indication that H-
bond donor groups in this position could be favored. The red con-
tours near the p-position of the benzylidene group coincide with
the sterically favorable green contours. Therefore, substituents at
these positions should be electron-donating but with high steric
bulk for better activity. Blue contour at the o-position of benzyli-
dene group was observed. Therefore, a low electron density in this
area will have a positive effect on the anti-metastatic activity. Due
to the low substitution variability in the training set around the
hydantoin ring, no contours were observed in this area.
Figure 4. Superposition of PMHs training and test sets using field fit alignment.
In conclusion, PMH is a novel anti-metastatic lead class that can
be easily, regioselectively, and cost effectively synthesized. These
advantages bode well for the emergence of future PMHs as poten-
tial therapeutics for the control and prevention of metatstatic pros-
tate cancer.
3. Experimental
3.1. General experimental procedures
IR spectra were recorded on a Varian 800 FT-IR spectrophotom-
eter. Optical rotation measurements were carried out on a Rudolph
Research Analytical Autopol III polarimeter. TLC analyses were car-
ried out on precoated silica gel 60 F254 500
MeOH/CHCl3 (1:9) as a developing solvent. For CC, Si gel 60 (parti-
cle size 63–200
m) was used. The 1H and 13C NMR spectra were
lm TLC plates, using
l
recorded in DMSO-d6, using TMS as an internal standard, on a JEOL
Eclipse NMR spectrometer operating at 400 MHz for 1H and
100 MHz for 13C. The HREIMS experiments were conducted at
the University of Michigan on a Micromass LCT spectrometer.
Molecular modeling and CoMFA studies were performed using
the SYBYL 8.0 suit of programs (Tripos Discovery Informatics, St.
Louis, Missouri) installed on a DELL desktop workstation equipped
with a 1.8 GHz IntelÒ XeonÒ processor running the Red Hat Enter-
prise Linux (version 4) operating system. The chemical structures
of hydantoin derivatives were sketched in SYBYL 8.0 and assigned
Gasteiger partial charges and energy minimized using Energy Force
Field to a final root-mean-square gradient of 0.01 kcal/mol.
3.2. Animal material
The sponge Hemimycale arabica was collected at a depth of
ꢀ10 m in June 2003 using SCUBA from Desha, 12.4 miles south
of Hurghada at the Egyptian Red Sea coast. The sponge is dark blue
from the exterior with greenish yellow linings at its interior. En-
crusted form of this sponge has crumbly texture with smooth sur-
face, and it usually builds thin layer of crust and covers an area
approximately equal to 15 cm. Its thickness varies between several
mm to 2.5 cm and hence its growth forms are sometimes more
cushion-like. Areolated pore fields were present on the surface of
sponge which forms minor depressions, and oscules are also pres-
ent which are raised in to the small tubes having 1–5 mm in diam-
eter and are irregularly distributed over the entire surface. A
*
Figure 5. Graphical representation of CoMFA STDEV COEFF analysis based field fit
alignment. (A) Steric contour plots: Sterically favored (80% contribution level) are
represented by green polyhedra predicted to increase activity. Sterically disfavored
(10% contribution level) are represented by yellow polyhedra predicted to decrease
activity. (B) Electrostatic contour plots: Negative charge-favored areas (80%
contribution level) are represented by red polyhedra. Negative charge-disfavored
areas (20% contribution level) are represented by blue polyhedra.
Though the electrostatic field contributions are less, a small change
in electrostatic interaction will have a considerable effect on the
activity. Red contour was observed near m- and p-positions of the

M. Mudit et al. / Bioorg. Med. Chem. 17 (2009) 1731–1738
1737
voucher was deposited in the collections of the Zoological Museum
of the University of Amsterdam under the registration number
ZMAPOR19764.
using PC-3M spheroid disaggregation assay. The activity was ex-
pressed as migration distance in m (Tables 5 and 6). The
l
(ꢀlogEC50) which is the concentration required to decrease migra-
tion distance by 50% was used in CoMFA.
To evaluate the predictability of the generated 3D QSAR model,
PMHs were divided into training set and test set. Each set pos-
sessed PMH analogs with a similar range of anti-metastatic activ-
ity. Thus, the test set is the true representative of the training
set. This was achieved by arbitrarily setting aside 6 compounds
as a test set with a regularly distributed anti-metastatic activity.
The mean (standard deviation) of the activity of the training and
test set was 96.32 (40.04) and 87.67 (57.18), respectively. Tables
5 and 6 show the actual and predicted activity for both training
and test sets, respectively.35,36 The predicted activities of training
and test set values were obtained by multiplying the values of each
descriptor for a particular row generated by CoMFA by its corre-
sponding coefficient from the PLS equation in the model.35
3.3. Extraction and isolation
About 1 kg frozen sponge was blended with EtOH and filtered.
The blended sponge material was extracted with EtOH at room tem-
perature (4 ꢁ 2 L). Ethanolic extracts were pooled and then evapo-
rated under vacuum. The dried extract (ꢂ32.0 g) was then
subjected to Si gel 60 (1 kg, <63 lm) medium pressure liquid chro-
matography (MPLC) using CHCl3–MeOH gradient elution to afford
several fractions containing phenylmethylene hydantoins. Pooled
fractions were further chromatographed on Si gel 60 using CHCl3–
MeOH gradient elution. Final purification was achieved on C-18 Si
gel (Bakerbond, Octadecyl, 40 lm) with H2O–MeOH gradient elu-
tion or Sephadex LH-20 using CHCl3–MeOH gradient elution.
3.6.1. Alignment rule
3.4. Preparation of semisynthetic phenylmethylene hydantoins
The alignment rule, that is, molecular conformation and orien-
tation, is one of the most sensitive input areas for CoMFA stud-
ies.35,36 The SYBYL QSAR rigid body field fit command was used
for this alignment. Field fit uses a Simplex algorithm in SYBYL that
minimizes the differences in steric and electrostatic fields averaged
over all the lattice grid points to find the best fit. One of the most
active PMHs 4 was used as a reference compound, on which other
molecules were aligned. This fit is dependent on the similarity and
initial orientations of the molecules. Values of the steric and elec-
trostatic fields were truncated at 30 kcal/mol. The aligned PMHs
are shown in Figure 4.
Hydantoin (1.0 gm) was dissolved in 10 mL H2O while heating
at 70 °C on oil bath with continuous stirring (Scheme 1).11 After
complete dissolution, the pH was adjusted to 7.0 using saturated
NaHCO3 solution. The temperature was then raised to 90 °C after
the addition of 0.9 mL ethanolamine.11 An equimolar quantity of
the corresponding aldehyde solution in 2–5 mL EtOH was added
drop wise with continuous stirring.11 The reaction was kept under
reflux for approximately 5–8 h. The reaction was monitored by TLC
every hour after a yellow or white precipitate is formed. After com-
plete depletion of the starting material, the mixture was cooled
and the precipitate was filtered and washed with EtOH/H2O (1:5)
before recrystallization from EtOH.11 Reaction yield range from
60-90%, based on the nature of the used aldehyde.
3.6.2. Partial least square (PLS) analysis
The partial least squares algorithm was used in conjugation with
the cross-validation (leave-one-out) option to obtain the optimum
number of components which were used to generate the final CoM-
FA model without cross-validation.35,36 The result from a cross-val-
idated analysis was expressed as q2, which is defined as
3.5. Spheroid disaggregation assay
P
P
2
q2 = 1 ꢀ PRESS/ (Y ꢀ Ymean
)
where PRESS = (Y ꢀ YPred)2. The q2
Spheroids were prepared from single cell suspension of PC-3M
prostate cancer cell line as described before.29,30,33,34 In brief,
5 ꢁ 104/mL cells in RPMI 1640 serum-free medium were placed
on 96-well low-attachment tissue culture plates. The plates were
rocked on a gyrorotatory shaker in a CO2 incubator at 37 °C for 2
can take up values in the range from 1, suggesting a perfect model,
to less than 0, where the errors of prediction are greater than the er-
ror from assigning each compound mean activity of the model.
Cross validated PLS analysis using the leave-one-out procedure em-
ployed 6 components, in which each compound is systematically
excluded from the set and its activity was predicted by a model de-
rived from the rest of the compounds. Thus cross validation by the
leave-one-out method evaluates the model not by how it best fits
the data but by how it best predicts the data.35,36 The optimum
number of components was defined as that which yielded the high-
est q2 value, which normally had the smallest RMS error of predic-
tion (SEP). Equal weights were assigned to the steric and
electrostatic descriptors using the CoMFA_STD scaling option. The
final PLS analysis was then performed using the optimum number
of components with no cross-validation. The final PLS analysis
was then performed using the optimum number of components
with no cross-validation. To obtain the statistical confidence limits
on the analysis, PLS analysis using 100 bootstrap (bs) groups with
the optimum number of components was performed.35,36
days, at the end of which the spheroids measuring 150–300 lm
in diameter (ꢂ4 ꢁ 104 cells/spheroid) were formed. A single spher-
oid was then placed in the center of each well of ECM-coated 24-
well microplate in 200 l
L of serum-free medium.29,30,33,34 Previous
studies determined that 1 h is an appropriate time for spheroids to
begin adhering to an ECM. Thus t = 0 was set as 1 h from initial
plating, so that if the plate was not disturbed, the spheroids would
not move from their location at the time of plating. Spheroids were
photographed digitally at t = 0, cultured at 37 °C for 48 h, and then
re-photographed. The spheroids were then fixed, stained with Diff-
QuikTM (Dade Behring, Newark, DE) and examined under light
microscopy. The diameter of the area covered with cells migrated
from the spheroids was measured in a microscope calibrated with
a stage and ocular micrometer. The radial distance of migration
was calculated after subtraction of the mean initial spheroidal
diameter at t = 0. PMHs 1 and 4 were tested at multiple doses rang-
3.6.3. Predictive r2 values
ing from 0 to 500 lM and each dose was tested in triplicate. Values
shown represent the average percent increase in surface area of
The predictive ability was determined from a set of 6 com-
pounds that were not included in the training set (Table 6). These
compounds were aligned, and their activities were predicted by
PLS analysis. The predictive r2 (r2pred) value was based on PMHs
of the test set only and is defined as where SD is the sum of the
squared deviations between the anti-metastatic activities of the
test set and mean activity of the training set molecules and PRESS
spheroids.29,30,33,34
3.6. Design of 3D QSAR model
A data set of 35 PMH derivatives was used for this study. The
anti-metastatic activity for these compounds was determined

1738
M. Mudit et al. / Bioorg. Med. Chem. 17 (2009) 1731–1738
is sum of the squared deviation between predicted and actual
activity values for every molecule in the test set.35,36
Acknowledgments
This investigation was made possible through the support of
NIH Grants P20RR16456 from the BRIN Program of the National
Center for Research Resources, and R01CA96534 (G.V.S.).
3.7. (Z)-5-(4-(Ethylthio)benzylidene)imidazolidine-2,4-dione (4)
Yellow amorphous solid, IR mmax (CHCl3) 3382, 1721, 1645,
1592 cmꢀ1 1H and 13C NMR see Tables 1 and 2; HREIMS m/z
;
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dione (8)
Yellow amorphous solid, IR mmax (CHCl3) 3441, 3227, 1771,
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Yellow amorphous solid, IR mmax (CHCl3) 3383, 3317, 2235,
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;
IMS m/z 213.0530 [M]+ (calcd for C11H7N3O2, 213.0538).