New derivatives of hydantoin as potential antiproliferative agents: Biological and structural characterization in combination with quantum chemical calculations

Article abstract of DOI:10.1007/s00706-013-1149-6

Two new series of hydantoin derivatives, 3-(4-substituted benzyl)-5,5-diphenyl- and 3-(4-substituted benzyl)-5-ethyl-5-phenylhydantoins, were synthesized and their antiproliferative activity was tested against human colon cancer HCT-116 and breast cancer

Full text of DOI:10.1007/s00706-013-1149-6

Monatsh Chem
DOI 10.1007/s00706-013-1149-6
ORIGINAL PAPER
New derivatives of hydantoin as potential antiproliferative agents:
biological and structural characterization in combination
with quantum chemical calculations
•
•
•
•
ˇ
•
´
Sleem Hmuda Nemanja Trisovic Jelena Rogan Dejan Poleti
ˇ
•
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Zeljko Vitnik Vesna Vitnik Natasa Valentic Biljana Bozic
•
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´
ˇ ´
ˇ´
Gordana Uscumlic
´
Received: 24 September 2013 / Accepted: 26 December 2013
Ó Springer-Verlag Wien 2014
Abstract Two new series of hydantoin derivatives, 3-(4-
substituted benzyl)-5,5-diphenyl- and 3-(4-substituted
benzyl)-5-ethyl-5-phenylhydantoins, were synthesized and
their antiproliferative activity was tested against human
colon cancer HCT-116 and breast cancer MDA-MB-231
cell lines. The presence of different substituents on both
hydantoin and benzyl moieties changed the antiprolifera-
tive activity of the investigated hydantoins, whereby most
of the compounds showed superior antiproliferative activ-
ity against MDA-MB-231 than against the HCT-116 cell
line. The structure of three compounds was studied by
single-crystal X-ray diffraction. The general structural
characteristic is the presence of N–HÁÁÁO hydrogen bonds
in crystal packings. The molecular geometry and bonding
features of the investigated hydantoins in the ground states
were calculated using the density functional method. The
relationship between structure and antiproliferative activity
was discussed. The data presented in this investigation
afford guidelines for the preparation of new hydantoin
derivatives with greater antiproliferative activity.
Keywords Hydantoin Á Antiproliferative activity Á
X-ray structure determination Á Quantum chemical
calculations Á Natural bond orbital analysis
Introduction
Derivatives of hydantoin (imidazolidine-2,4-dione) repre-
sent pharmacologically important compounds that are
employed, among other uses, as anticonvulsants, antiar-
rhythmics, antimicrobial agents, and skeletal muscle
relaxants [1]. Phenytoin (5,5-diphenylhydantoin) is an
example of a well-known drug that was introduced in the
treatment of epilepsy in the late 1930s, but its versatile
biological effects still attract considerable attention within
the scientific community.
Somehydantoin derivatives havealreadybeenidentifiedas
antitumor agents. Spiromustine, a spirohydantoin mustard,
rapidly penetrates the blood–brain barrier and directs the drug
delivery to brain tumors [2]. On the other side, nilutamide [3-
(4-nitro-3-trifluoromethylphenyl)-5,5-dimethylhydantoin] is
a potent antiandrogen used primarily in the treatment of
prostate cancer in advanced stages [3]. Lipophilic derivatives
of hydantoin bearing cycloalkyl, phenyl, or benzhydril sub-
stituents show inhibitory activity against several cancer cell
lines [4]. The antiproliferative effects of diazaspiro bicyclo
hydantoins have been determined on the human leukemia cell
lines K562 and CEM [5]. It has been demonstrated that the
cytotoxic activity of the compounds with a substituent in
position 3 increases in the order: alkene[ ester [ ether.
Fluorinated spirohydantoins have been reported to induce
cytotoxicity in chronic myelogenous leukemia by inhibiting
the cell growth via interfering with DNA replication [6]. 1-(2-
Arylethyl)-5-arylidenehydantoins inhibit the proliferation of
the lung cancer cell line A549 via a dual mechanism: blocking
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-013-1149-6) contains supplementary
material, which is available to authorized users.
ˇ
´
´
S. Hmuda Á N. Trisovic Á J. Rogan Á D. Poleti Á N. Valentic Á
ˇ´
´
G. Uscumlic (&)
Faculty of Technology and Metallurgy, University of Belgrade,
Belgrade, Serbia
e-mail: goca@tmf.bg.ac.rs
ˇ
Z. Vitnik Á V. Vitnik
Department of Chemistry, IChTM-Institute of Chemistry,
Technology and Metallurgy, University of Belgrade,
Belgrade, Serbia
ˇ ´
B. Bozic
Faculty of Biology, University of Belgrade, Belgrade, Serbia
123

S. Hmuda et al.
EGFR tyrosine kinase activity and inducing genomic DNA
damage [7]. 5-Arylidenehydantoins show potent in vitro and
in vivo antigrowth and anti-invasive properties against the
prostate cancer cell line PC-3M [8]. A b-carboline hydantoin
has been found to inhibit phosphodiesterase 5 and the prolif-
eration of the colon cancer cell line HC-29 [9].
analysis. In addition, the X-ray crystal structure determi-
nation for compounds 2a, 2e, and 2g is presented.
Results and discussion
3-(2-Oxo-2-arylethyl)-5,5-diphenylhydantoins
have
Antiproliferative activity
recently been reported to possess selective activity against
the renal cancer cell lines A498 and UO-31, whereby the
derivative bearing the piperidine moiety shows additionally
strong activity against melanoma and breast cancer cell
lines MDA-MB-435 and MCF7, respectively [10].
Molecular docking of the most active compound into the
active sites of EGFR and V600E-B-RAF kinase has also
been studied. It has been demonstrated that the hydantoin
ring binds in the enzyme-active site through the formation
of hydrogen bonds, while the phenyl groups in position 5
are responsible for the arene p–p interactions. We have
previously shown that in a series of 3-alkyl-5,5-diph-
enylhydantoins, the derivative with a benzyl group has a
significant antiproliferative effect on the colon cancer cell
line HCT-116 [11]. This compound also exhibits antibac-
terial activity against the Gram-positive bacterium
Enterococcus faecalis and the Gram-negative bacteria
Escherichia coli ATCC 25922 and Escherichia coli (clini-
cally isolated). Using 3-benzyl-5,5-diphenylhydantoin as a
leading compound, the present research has been focused
on the effects of structural modifications on both hydantoin
and benzyl moieties on the antiproliferative activity.
Cytotoxic evaluation has been the first step in the biolog-
ical characterization of the investigated compounds. The
examination of cytotoxic effects (non-specific cell killing)
on the viability of unstimulated and lipopolysaccharide
(LPS)-stimulated rat peritoneal macrophages has been
conducted by MTT assay. All investigated hydantoin
derivatives are non-toxic to normal rat peritoneal macro-
phages (data not shown).
The antiproliferative activity of compounds 1a–1h and
2a–2g has been tested against cancer cell lines HCT-116
and MDA-MB-231. With the exception of compounds 1d
(0.1 and 0.01 lmol dm-3) and 1h (1 and 0.1 lmol dm-3),
all compounds exhibit significant antiproliferative effects
against human breast cancer cells MDA-MB-231 in the
investigated concentration range (Table S1 in Supplemen-
tary Material). As can be seen, the highest antiproliferative
activity has been observed by compounds 1b–1d and 1h
against MDA-MB-231. These compounds show a better
antiproliferative activity with respect to the unsubstituted
derivative 1a (Fig. 2). Furthermore, 5,5-diphenylhydanto-
ins (series 1) exhibit a higher antiproliferative activity
when compared to 5-ethyl-5-phenylhydantoins (series 2).
This may be attributed, at first sight, to the introduction of
the additional phenyl group in position 5, which contributes
to the formation of a strong lipophilic shield around the
hydantoin moiety. Concerning the compounds with an
alkyl group as the benzyl substituent X, compound 1b with
the methyl group shows less than 50 % inhibition of MDA-
MB-231 cell proliferation in the highest concentration
(100 lmol dm-3) and has a better effect when compared to
the compound 1h with the tert-butyl group. Compounds
having a halogen substituent as X (1c: X = Cl; 1d:
X = Br) have a superior antiproliferative capacity than the
other compounds in series 1.
In this study, two series of 3-(4-substituted benzyl)-5,5-
diphenyl- and 3-(4-substituted benzyl)-5-ethyl-5-phenyl-
hydantoins (Fig. 1) have been synthesized and their anti-
proliferative activity has been evaluated against cell lines
MDA-MB-231 (human breast adenocarcinoma) and HCT-
116 (human colon cancer), as well as non-cancerous, nor-
mal rat peritoneal macrophage controls. This has further
motivated a detailed theoretical analysis of substituent
effects on the geometry, electronic structure, and frontier
molecular orbitals (MOs) of the investigated hydantoin
derivatives. The stability of compound 2a arising from
hyperconjugative interactions and charge delocalization
has been studied by using natural bond orbital (NBO)
Compounds 1a–1h and 2a–2f exhibit significant anti-
proliferative activity against the human colon cancer cells
HCT-116 only in the highest investigated concentration
(100 lmol dm-3), and this inhibition is lower compared to
the inhibition of cells in MDA-MB-231 (Fig. 2 and Table
S2 in Supplementary Material). The strongest antiprolif-
erative activity has been observed by compounds 1b and
1c.
It can be concluded that all investigated hydantoin
derivatives show satisfying inhibitory activity against the
investigated cancer cell lines without non-specific
Fig. 1 Chemical structures of the investigated hydantoin derivatives
123

New derivatives of hydantoin
Fig. 2 The effect of the
investigated hydantoins at the
concentration of
100 lmol dm-3 on the
inhibition of proliferation HCT-
116 and MDA-MB-231 cell
lines (*p \ 0.05 vs non-treated
cells)
cytotoxic effect. Because the investigated compounds are
non-toxic to normal rat peritoneal cells, the pronounced
antiproliferative activity against MDA-MB-231 indicates
their significant anticancer potential for breast cancer cells.
Also, the results of this study indicate a better antiprolif-
erative potential of 5,5-diphenylhydantoins bearing an
alkyl (methyl or tert-butyl) or halogen substituent in the
benzyl moiety. In particular, compounds 1b, 1c, and 1d
may represent good starting points for the development of
new, specific antiproliferative hydantoins. It has been
reported that compounds 1c and 1g show significant inhi-
bition of the renal cancer cell lines (A498 and UO-31,
31–59 % at the concentration of 10 lmol dm-3) [10],
while the replacement of the benzyl moiety with a
phenacyl group results in derivatives with somewhat
decreased antitumor activity against different cell lines,
including the human colon cancer HCT-15 cell lines. A
great inhibition of the breast cancer MCF7 and melanoma
cancer MDA-MB-435 cell lines is achieved by the intro-
duction of a basic piperidine unit as the substituent X. It is
assumed that this derivative of hydantoin is located well in
the biological target.
The general structural feature of 2a, 2e, and 2g is that of
crystal packing governed by only one hydrogen N–HÁÁÁO
bond in each case (Table 2) and numerous weak C–HÁÁÁN/
O contacts between neighboring molecules that stabilize
the crystal lattices. In 2a, hydrogen bonds between a pair of
enantiomers make R22(8) centrosymmetric dimers (Fig. 4a).
The dimers further built double-layers parallel to the ab-
plane that are linked by C–HÁÁÁN/O and van der Waals
interactions. The existence of centrosymmetric dimers in
the crystal packing of 2a may well influence differences in
its pharmacokinetic properties, when compared with 2e and
2g.
Four crystallographically different molecules of 2e, A,
B, C, and D, make two pairs of zigzag chains parallel to the
c-axes in the crystal packing (Fig. 5). In one pair, A and B
(an enantiomeric pair), and in another, C and D molecules
are connected through N–HÁÁÁO hydrogen bonds (Table 2).
A pair of zigzag chains formed by one stereoisomer par-
allel to the b-axes (Fig. 6) exists in 2g.
Quantum chemical calculations
The geometry of the investigated compounds was opti-
mized according to density functional theory (DFT) using
B3LYP/6-311G(d,p) level, and selected geometric param-
eters are listed in Tables 1 and 3. The bonds in the
hydantoin ring change very slightly upon changing the
substituents R and X. The N1–C5 and C4–C5 single bonds
in the molecules from series 1 are longer than those in the
Crystal structures of 2a, 2e, and 2g
The molecular structures of the compounds 2a, 2e, and 2g
are shown in Fig. 3. The bond distances and angles are as
expected for hydantoin derivatives and are very similar
mutually (Table 1). Also, the bond distances are in good
accordance with the values calculated by DFT (Table 1).
Four chemically identical, but crystallographically differ-
ent molecules, A, B, C, and D, exist in the asymmetric unit
of 2e (Fig. 3). It is interesting to note that the orientation of
the A, B, and D molecules are nearly the same, while the
molecule C is in the opposite direction.
˚
molecules from series 2 (about 0.010 and 0.005 A,
respectively). The N1–C2 and N3–C4 bond lengths of
˚
around 1.37 A exhibit double-bond character and indicate
strong stabilizing conjugation of the N1 and N3 atoms with
the carbonyl groups. In the connecting chain, the N2–C14
bond becomes slightly longer with increasing electron-
123

S. Hmuda et al.
Fig. 3 ORTEP-like
representations of asymmetric
unit of the compounds: 2a (a),
2e (b), and 2g (c) with atomic
numbering schemes.
Displacement ellipsoids are
drawn at the 50 % probability
level and the H atoms are shown
as small spheres of arbitrary
radii
donating ability of the benzyl substituent X, while the
opposite is observed for the C14–C15 bond. With excep-
tion of 2g, the values of torsion angle C4–C5–C6–C7 in the
molecules from series 1 are about 10° smaller than those in
the molecules from series 2. This should be ascribed to the
steric hindrance when two phenyl groups neighbor each
other.
structures of the investigated compounds can easily be
affected by packing effects, which modify the geometry
[12]. Thus, the conformation observed in the single crystal
may differ from that at the biological target site.
The molecular orbital calculation of the investigated
hydantoin derivatives has also been performed according to
DFT. The energy of HOMO and LUMO are respectively a
rough measure of the electron-donating and electron-with-
drawing ability of a molecule [13]. Changing the
substituents R and X modifies the energy gap between the
HOMO and LUMO localized on different parts of the
molecular skeleton (Table 4; Figs. 7, 8). According to the
calculations, the electron density of the HOMO for the
compounds 1a, 1c, 2a, and 2c spreads over the whole
The selected structural parameters of the experimental
crystal structures for compounds 2a, 2e, and 2g are also
compared with computed geometries in Table 1. The main
differences between the experimental structures and
structures computed by the DFT method result from the
different spatial orientation of the phenyl and benzyl
groups relative to the hydantoin moiety. The solid-state
123

New derivatives of hydantoin
molecule, while the electron density of the LUMO is shifted
toward the central hydantoin moiety and the phenyl
group(s) in position 5. When substituent X is an electron-
donating type, the HOMO is mostly localized on the benzyl
moiety and the LUMO is to a large extent delocalized over
the hydantoin and phenyl rings. When X is a strong elec-
tron-withdrawing substituent, the molecular orbital diagram
is considerably different; the HOMO is mostly localized on
the phenyl group(s) in position 5, while the LUMO is on the
benzyl acceptor moiety (compounds 1f and 2f).
As expected, the electron-donating substituents X lead to
high-lying HOMO, the highest energy being obtained for
X = OCH3 in both series of compounds, while electron-
withdrawing substituents lead to low-lying LUMOs, the
lowest energy being when X = NO2. The replacement of
one of the phenyl groups in position 5 with an ethyl group
leads to the stabilization of the LUMO for compounds 2a–
2d and 2g with respect to the corresponding derivatives
from series 1. The energy of HOMO remains almost
unchanged. On the other hand, the HOMO level for com-
pounds 2e and 2f is stabilized when compared with the
HOMO for 1e and 1f, while the energy of the LUMO is
almost unaffected. The net result is that the HOMO–LUMO
energy gap is somewhat smaller (by *0.10 eV) for the
compounds in series 1 than for the compounds in series 2.
NBO analysis
A useful feature of the NBO method is that it gives
information about interactions in both filled and virtual
orbital spaces that can improve the analysis of intra- and
intermolecular interactions. Delocalization of electron
density between occupied Lewis-type (bonding or lone
pair) NBO orbitals and formally unoccupied (anti-bonding
Fig. 4 a Centrosymmetric dimers in 2a; b crystal packing of
compound 2a showing layers parallel to the ab-plane. Intermolecular
H bonds are represented by dashed lines
Fig. 5 Crystal packing of
compound 2e viewed along
b-axes. Intermolecular H bonds
are represented by dashed lines
123

S. Hmuda et al.
Fig. 6 Zigzag chains parallel to
b-axes in the crystal packing of
compound 2g. Intermolecular H
bonds are represented by dashed
lines
˚
Table 1 Selected structural parameters (bond distances/A and torsion angles/°) obtained by theoretical calculations and single-crystal X-ray study for the
investigated hydantoins 2a–2g
2a (X-ray)
2a
2b
2c
2d
2e* (X-ray)
2e
2f
2g (X-ray)
2g
N1–C2
1.334(2)
1.461(2)
1.229(2)
1.395(2)
1.372(2)
1.207(2)
1.541(2)
1.462(2)
1.509(2)
94.0(2)
1.368
1.459
1.209
1.412
1.374
1.210
1.552
1.465
1.516
1.368
1.366
1.459
1.209
1.413
1.374
1.210
1.552
1.464
1.515
1.367
1.334
1.447
1.221
1.394
1.362
1.217
1.538
1.466
1.502
97.4
1.365
1.460
1.209
1.414
1.375
1.210
1.551
1.462
1.516
1.365
1.460
1.209
1.414
1.375
1.210
1.551
1.462
1.517
1.340(2)
1.462(2)
1.221(2)
1.397(2)
1.371(2)
1.208(2)
1.530(2)
1.466(2)
1.508(2)
100.8(2)
63.5(2)**
-42.0(2)**
152.3(1)
1.368
1.458
1.209
1.412
1.373
1.211
1.552
1.467
1.513
N1–C5
1.459
1.209
1.412
1.373
1.210
1.552
1.466
1.515
1.460
1.209
1.413
1.374
1.210
1.552
1.464
1.515
C2–O2
C2–N3
N3–C4
C4–O4
C4–C5
N3–C14
C14–C15
C4–N3–C14–C15
N3–C14–C15–C16
N1–C5–C6–C7
C4–C5–C6–C7
86.4
85.7
86.6
85.9
86.8
87.1
86.5
135.2(2)**
-2.2(2)
108.7(1)
87.9
-9.3
120.5
87.7
-9.6
120.9
85.9
-9.4
120.6
86.1
-8.5
119.7
62.8
85.6
-9.2
120.4
85.8
-9.5
120.6
90.2
-9.8
121.1
-13.3
125.4
* Bond distances are calculated as mean values of distances in individual A, B, C, and D molecules. Torsion angles are calculated as mean values of angles
using the corresponding absolute values in A, B, C, and D molecules. The negative sign of the mean N1–C5–C6–C7 torsion angle is assigned according to
the matching enantiomer
** The sign of the torsion angle is selected according to the matching enantiomer
or Rydberg) non-Lewis NBO orbitals corresponds to a
stabilizing donor–acceptor interaction. The larger the E(2)
(energy of hyperconjugative interactions) value, the more
intensive is the interaction between electron donors and
electron acceptors.
molecule. The intramolecular interactions are formed by the
orbital overlap between bonding C–C, C–N, C–H, and C=O
antibonding orbital, which results in intramolecular charge
transfer (ICT) causing stabilization of the molecule. These
interactions are observed due to an increase in the electron
density (ED) in C–C, C–N, C–H, and C=O antibonding
orbitals that weakens the respective bonds. The strong
intramolecular hyperconjugative interaction of the r and p
NBO analysis has been performed on the molecule 2a at
the DFT level in order to elucidate the intramolecular
interactions and delocalization of electron density within the
123

New derivatives of hydantoin
Table 2 Hydrogen bond geometry in 2a, 2e, and 2g
˚
d(D–H)/A
˚
˚
D–HÁÁÁA
d(HÁÁÁA)/A
d(DÁÁÁA)/A
\(DHA)/°
2a
N1–H1ÁÁÁO2a
2e
0.93
1.92
2.837(2)
170
N1A–H1ÁÁÁO2B
N1B–H18ÁÁÁO2Ab
N1C–H35ÁÁÁO2Dc
N1D–H52ÁÁÁO2C
2g
0.86
0.86
0.86
0.86
2.17
1.98
2.05
2.09
2.847(6)
2.837(5)
2.764(6)
2.931(6)
136
171
140
167
N1–H1ÁÁÁO2d
Symmetry codes: -x, -y ? 1, -z; x, -y ? 1/2, z ? 1/2; x, -y ? 3/2, z - 1/2; -x ? 2, y - 1/2, -z ? 3/2
0.86
2.07
2.900(2)
165
a
b
c
d
˚
Table 3 Selected structural parameters (bond distances/A and torsion angles/°) obtained by theoretical calculations for the investigated hyd-
antoins 1a–1h
1a
1b
1c
1d
1e
1f
1g
1h
N1–C2
1.372
1.469
1.209
1.410
1.374
1.208
1.557
1.466
1.516
1.372
1.469
1.209
1.409
1.373
1.208
1.558
1.467
1.515
1.371
1.469
1.209
1.410
1.374
1.208
1.558
1.465
1.515
1.371
1.369
1.470
1.209
1.411
1.375
1.208
1.557
1.462
1.516
1.369
1.470
1.209
1.411
1.375
1.208
1.557
1.462
1.517
1.373
1.469
1.209
1.409
1.373
1.208
1.559
1.468
1.514
1.372
1.468
1.209
1.410
1.374
1.208
1.558
1.467
1.515
N1–C5
1.469
1.209
1.411
1.374
1.208
1.558
1.464
1.516
C2–O2
C2–N3
N3–C4
C4–O4
C4–C5
N3–C14
C14–C15
C4–N3–C14–C15
N3–C14–C15–C16
N1–C5–C6–C7
C4–C5–C6–C7
84.8
85.5
86.6
85.8
86.7
87.2
85.6
86.4
83.7
-2.9
107.3
83.8
-2.7
107.6
83.8
-2.2
107.9
85.0
-1.3
108.8
82.7
-3.1
107.0
83.0
-2.9
107.2
86.5
-0.9
109.2
86.2
-1.0
109.1
electrons of C–C to the anti C–C bond of the hydantoin ring
leads to stabilization of some part of the ring, as evident from
Table S3 (Supplementary material). For example: the
intramolecular hyperconjugative interaction of r(C4–N3)
distributes to r*(N3–C2), C2–O2, and N1–C5, leading to
stabilization of 2.85–4.48 kJ mol-1. The same kind of
interaction is calculated in the r-type bonding of C4–C5 with
r-type antibonding of C5–C6, N3–C14, and N1–H orbitals,
as shown in Table S3.
NBO analysis clearly manifests the evidences of the
intramolecular charge transfer in molecule 2a from p(C–C)
and p*(C–C) in the phenyl ring in position 5 and the phenyl
ring of the benzyl moiety, as shown in Table S3, that clearly
shows a large stabilization energy of about 84 kJ mol-1. The
maximum energy delocalization takes place in the p–p*
transition. The orbital overlap in phenyl rings between p(C–
C) and p*(C–C) results in an intramolecular charge transfer
causing stabilization of the whole system.
The interaction energy, related to the resonance in the
molecule, is electrons withdrawing to the ring through r*
of N3–C2, C2–N1 and C4–N3,and the C4–C5 bond from
the lone pairs LP(2)O2 and LP(2)O4, respectively, which
leads to moderate stabilization energy of 92–109 kJ mol-1
(Table S3). The most important interaction energies of lone
pairs LP(1)N1 ? p*(C2–O2), LP(1)N3 ? p*(C2–O2),
and LP(1)N3 ? p*(C4–O4), are 241.83, 206.91, and
248.48 kJ mol-1, respectively.
Rule of five and drug-like properties
To identify which of the investigated compounds should be
examined in detail, their drug-like properties have been
considered based on the ‘rule of five’, as well as the number
of rotable bonds and polar surface area (Table 5). This
empirical rule states that if a compound satisfies any two of
the following rules, it is likely to exhibit poor in vivo
123

S. Hmuda et al.
permeability: (1) molecular weight [500, (2) number of
hydrogen bond donors [5, (3) number of hydrogen bond
acceptors[10, and (4) calculated value of the logarithm of
the octanol–water partition coefficient, log P [ 5 [14]. In
addition, reduced molecular flexibility (the number of
halogen substituents yields the highest values. As expected,
the replacement of one of the phenyl groups in position 5
with an ethyl group results in a decreased lipophilicity of
compounds in series 2 with respect to the corresponding
derivatives in series 1. Generally, lipophilicity (expressed
as log P) is directly related to the change in the Gibbs
energy of solvation of a molecule between octanol and
water; its dipolarity/polarizability and, especially, hydrogen
bond basicity favor partitioning into water and thus
decrease lipophilicity, whereas molecular size favors octa-
nol [16, 17]. In a previous paper [18], we have shown that a
linear dependency exists between log P values and hydro-
gen bond basicity for the derivatives bearing moderate
electron-donating and electron-withdrawing substituents X
at the benzyl moiety (1a–1d, 1g, 2a–2d), whereas a strong
electron-withdrawing substituent (1f and 2f) significantly
modifies the solvation characteristics of the molecule. It
might be stated that more lipophilic compounds show
higher inhibition, namely an increase in the antiproliferative
activity is observed for compounds 1b, 1c, and 1d with log
P in a range from 4.69 to 5.25. Furthermore, the investi-
gated compounds contain a moderate number of the
rotatable bonds, and thus somewhat larger conformational
changes upon binding to a receptor are possible. A aarger
polar surface area indicates that reduced absorption can be
expected. Evidently, the investigated hydantoin derivatives
meet these empirical criteria and potentially qualify for the
pharmacodynamic phase of the drug development.
2
˚
rotatable bonds \8) and low polar surface area (\140 A )
are important predictors of good bioavailability [12, 15].
The investigated hydantoin derivatives are highly lipo-
philic compounds with lipophilicity between 3.36 and 6.12.
A trend is observed: the strong electron-donating and
electron-withdrawing groups (i.e., NO2, CN, OCH3)
decrease lipophilicity, while the introduction of an alkyl or
Table 4 B3LYP/6-311G(d,p) HOMO and LUMO energies of the
investigated hydantoins
Compound
E(HOMO)/eV
E(LUMO)/eV
E(HOMO)
- E(LUMO)/eV
1a
1b
1c
1d
1e
1f
-6.82
-6.63
-6.86
-6.79
-7.02
-7.04
-6.14
-6.64
-6.87
-6.62
-6.87
-6.77
-7.14
-7.17
-6.11
-0.87
-0.84
-0.96
-0.95
-1.73
-2.62
-0.81
-0.83
-0.77
-0.74
-0.87
-0.87
-1.71
-2.60
-0.72
5.95
5.79
5.90
5.84
5.29
4.42
5.34
5.82
6.11
5.88
5.99
5.90
5.43
4.57
5.38
1g
1h
2a
2b
2c
2d
2e
2f
Conclusion
In this study, two new series of hydantoin derivatives were
prepared and characterized. The in vitro antiproliferative
2g
Fig. 7 The molecular orbitals
and HOMO–LUMO energy
gaps for 1a, 1b, 1c, and 1f
123

New derivatives of hydantoin
Fig. 8 The molecular orbitals
and HOMO–LUMO energy
gaps for 2a, 2b, 2c, and 2f
Table 5 Evaluation of drug candidates
2
˚
Polar surface area/A
Compound
Molecular weight
Clog P
Hydrogen bonds
Rotatable bonds
Violations
Donorsa
Acceptorsb
1a
342.39
356.42
376.84
421.29
367.40
387.39
372.42
398.50
294.34
308.37
328.79
373.24
319.36
339.35
324.37
\500
4.41
4.69
4.97
5.24
4.55
4.45
4.13
6.12
3.64
3.92
4.20
4.47
3.78
3.68
3.36
\5
1
1
4
4
4
4
69.26
68.17
68.61
68.97
124.9
161.9
85.08
68.48
68.07
68.20
68.20
68.33
126.2
159.9
84.92
\140
0
0
0
1
0
1
0
1
0
0
0
0
0
1
0
B1
1b
1c
1
4
4
1d
1
4
4
1e
1
5
4
1f
1
7
5
1g
1
5
5
1h
1
4
5
2a
1
4
4
2b
1
4
4
2c
1
4
4
2d
1
4
4
2e
1
5
4
2f
1
7
5
2g
1
5
5
Ideal compound
\5
\10
\8
a
A donor indicates any O–H or N–H group
b
An acceptor indicates any O or N including those in donor groups
activity of the investigated hydantoin derivatives was
evaluated against two human cancer cell lines: HCT-116
(colon carcinoma) and MDA-MB-231 (breast cancer).
Most of the investigated compounds show superior anti-
proliferative activity against MDA-MB-231 than against
HCT-116 cell line, whereby those in series 1 possess higher
potencies than the compounds in series 2.
The crystal structure of compounds 2a, 2e, and 2g was
resolved by X-ray diffraction. Crystal packings are mainly
governed by hydrogen N–HÁÁÁO bonds between neighboring
molecules. In 2a, hydrogen bonds make R22(8) centrosym-
metric dimers along the c-axis. In other two compounds, N–
HÁÁÁO bonds between neighboring molecules built zigzag
chains connected further by van der Waals forces only.
123

S. Hmuda et al.
Table 6 Synthesis of the investigated hydantoin derivatives
Compound
Yield/%
M.p./°C
Found
Reported
1a
1b
1c
1d
1e
1f
49
55
48
52
48
42
56
50
62
56
68
72
58
68
74
145–146
110–112
140–142
130–133
98–100
145–146 [21]
110–112 [18]
140–142 [18]
130–133 [18]
–
174–177
122–125
142–144
138–140
164–167
165–168
170–173
141–143
174–176
147–149
174–177 [18]
–
1g
1h
2a
2b
2c
2d
2e
2f
–
138–140 [22]
164–167 [18]
165–168 [18]
170–173 [18]
–
DFT calculations were used to obtain the molecular
geometries and bonding features of the investigated com-
pounds. The main differences between the experimental
structures and computed geometries result from the dif-
ferent orientation of the phenyl and benzyl groups relative
to the hydantoin moiety. This can be attributed to the
packing effects that influence the conformation of real
molecules. A detailed analysis of the effect of substituents
on both hydantoin and benzyl moieties on the frontier
molecular orbitals was also performed.
174–176 [18]
–
2g
derivatives is presented. Elemental analysis was realized
using an Elemental Vario EL III microanalyzer, and the
results were found to be in good agreement ( 0.3 %) with
the calculated values. FT-IR spectra were recorded on a
Bomem MB 100 spectrometer in the form of KBr pellets.
1H and 13C NMR spectra were recorded on a Varian
Gemini 200 spectrometer at 200 MHz for the 1H NMR and
50 MHz for the 13C NMR spectra. The spectra were
recorded at room temperature in CDCl3 and DMSO-d6. The
chemical shifts are expressed in ppm, referring to TMS
By assessing their different physicochemical properties,
guidelines for the design of further derivatives were
achieved. Regarding the biological profile, compounds 1b,
1c, and 1d represent starting points for the development of
new, specific antiproliferative hydantoins. Further studies
should also examine their antiproliferative potential against
additional cell lines.
1
(d(H) = 0 ppm) in the H NMR and the residual solvent
signal
(CDCl3:
d(C) = 77 ppm;
DMSO-d6:
d(C) = 39.5 ppm) in the 13C NMR spectra. The coupling
constants J(H,H) are expressed in Hz.
Materials and methods
Synthesis
4-[(2,5-Dioxo-4,4-diphenyl-1-imidazolidinyl)methyl]-
benzonitrile (1e, C23H17N3O2)
Commercially available materials were used without fur-
ther purification. The investigated hydantoin derivatives
were synthesized following the procedure presented in the
synthesis scheme (Scheme 1). Phenytoin was commer-
cially available, while 5-ethyl-5-phenylhydantoin was
synthesized according to the method of Bucherer and Lieb
[19]. The investigated compounds were obtained in the
reaction between the starting hydantoins and 4-substituted
benzyl chlorides in the presence of K2CO3, as described
previously [20].
White crystalline solid; yield: 48 %; m.p.: 98–100 °C; IR
(KBr): m = 3,248 (NH), 1,748 (C=O), 1,706 (C=O) cm-1
;
1H NMR (200 MHz, DMSO-d6): d = 9.88 (s, 1H, NH),
7.78 (d, 2H, J = 8 Hz, –C6H4–), 7.40 (d, 2H, J = 8 Hz, –
C6H4–), 7.40–7.30 (m, 10H, 2 9 C6H5), 4.75 (s, 2H, N–
CH2–) ppm; 13C NMR (50 MHz, DMSO-d6): d = 173.3
(C4), 155.3 (C2), 142.3, 139.7, 132.9, 129.0, 128.6, 128.5,
126.9, 118.9, 110.8, 69.7 (C5), 41.5 ppm.
3-(4-Methoxybenzyl)-5,5-diphenyl-2,4-imidazolidinedione
(1g, C23H20N2O3)
The chemical structures and the purities of the synthe-
sized hydantoin derivatives were confirmed by melting
White crystalline solid; yield: 56 %; m.p.: 122–125 °C; IR
1
points, elemental analysis, FT-IR, H and 13C NMR spec-
(KBr): m = 3,339 (NH), 1,765 (C=O), 1,706 (C=O) cm-1
;
tra, and agree well with the reported data (Table 6). Herein,
1H NMR (200 MHz, DMSO-d6): d = 9.73 (s, 1H, NH),
7.43–7.30 (m, 10H, 2 9 C6H5), 7.18 (d, 2H, J = 8 Hz,
the characterization of 1e, 1g, 1h, 2e, and 2g as novel
123

New derivatives of hydantoin
˚
–C6H4–), 6.87 (d, 2H, J = 8 Hz, –C6H4–), 4.56 (s, 2H, N–
CH2–), 3.70 (s, 3H, –OCH3) ppm; 13C NMR (50 MHz,
DMSO-d6): d = 173.3 (C4), 158.9, 155.5 (C2), 139.9,
129.2, 128.9, 128.8, 128.5, 126.8, 114.2, 69.4 (C5), 55.3,
41.2 ppm.
(k = 0.71073 A) was used, while Cu Ka radiation
˚
(k = 1.5418 A) was applied for single-crystals 2e and 2g. A
multi-scan correction for absorption was applied in all
cases. The structures were solved by direct methods (SIR92
[23] for 2a and 2g and SIR2004 [24] for 2e) and refined on
F2 by full-matrix least-squares using the programs SHEL-
XL97 [25] and WinGX [26]. For all three compounds, non-
hydrogen atoms were refined anisotropically. Hydrogen
atoms were placed at geometrically calculated positions and
refined using a riding model with fixed C–H and N–H dis-
tances. A test of intensity statistics indicated that the
selected crystal of 2e was a pseudo-merohedral twin with a
twin law (1 0 0/0 -1 0/0 0 -1). Therefore, a new reflection
file (HKLF5) with detwinned data was generated using the
program PLATON [27]. Selected crystallographic data and
refinement results are listed in Table 7.
3-(4-tert-Butylbenzyl)-5,5-diphenyl-2,4-imidazolidinedione
(1h, C26H26N2O2)
White crystalline solid; yield: 50 %; m.p.: 142–144 °C; IR
(KBr): m = 3,260 (NH), 1,771 (C=O), 1,713 (C=O) cm-1
;
1H NMR (200 MHz, DMSO-d6): d = 9.72 (s, 1H, NH),
7.44–7.35 (m, 10H, 2 9 C6H5), 7.33 (d, 2H, J = 8.6 Hz, –
C6H4–), 7.16 (d, 2H, J = 8.6 Hz, –C6H4–), 4.61 (s, 2H, N–
CH2–), 1.23 (s, 9H, C(CH3)3) ppm; 13C NMR (50 MHz,
DMSO-d6): d = 173.3 (C4), 155.5 (C2), 150.2, 139.9,
133.8, 128.8, 128.4, 127.2, 126.8, 69.4 (C5), 41.4, 34.4,
31.3 ppm.
Crystallographic data for the structures in this paper
were deposited in the Cambridge Crystallographic Data
Centre as supplementary publication numbers CCDC
959932-959934. These data can be obtained free of charge
from http://www.ccdc.cam.ac.uk/data_request/cif, by
emailing data_request@ccdc.cam.ac.uk, or by contacting
the Cambridge Crystallographic Data Centre, 12, Union
Road, Cambridge CB2 1EZ, UK; Fax: ?44-1223-336033.
4-[(4-Ethyl-2,5-dioxo-4-phenyl-1-imidazolidinyl)methyl]-
benzonitrile (2e, C19H17N3O2)
White crystalline solid; yield: 58 %; m.p.: 141–143 °C; IR
(KBr): m = 3,259 (NH), 1,759 (C=O), 1,709 (C=O) cm-1
;
1H NMR (200 MHz, DMSO-d6): d = 9.21 (s, 1H, NH),
7.81 (d, 2H, J = 7.8 Hz, –C6H4–), 7.54 (d, 2H,
J = 7.8 Hz, –C6H4–), 7.46–7.31 (m, 5H, C6H5), 4.67 (s,
2H, N–CH2–), 2.24–1.90 (m, 2H, –CH2–), 0.78 (t, 3H,
J = 7.3 Hz, –CH3) ppm; 13C NMR (50 MHz, DMSO-d6):
d = 174.8 (C4), 156.0 (C2), 142.4, 138.7, 132.8, 128.9,
128.5, 128.3, 125.7, 118.9, 110.7, 67.4 (C5), 41.2, 31.6,
8.2 ppm.
Biological characterization
Compounds and solutions
MTT was dissolved (5 mg cm-3) in phosphate buffered
saline (pH 7.2) and filtered (0.22 lm) before use. The
RPMI 1,640 cell culture medium, fetal bovine serum
(FBS), and MTT were purchased from Sigma Chemical
Company, USA.
5-Ethyl-3-(4-methoxybenzyl)-5-phenyl-2,4-
imidazolidinedione (2g, C19H20N2O3)
White crystalline solid; yield: 74 %; m.p.: 147–149 °C; IR
(KBr): m = 3,269 (NH), 1,765 (C=O), 1,706 (C=O) cm-1
;
1H NMR (200 MHz, DMSO-d6): d = 9.07 (s, 1H, NH),
7.54–7.31 (m, 5H, C6H5), 7.18 (d, 2H, J = 8 Hz, –C6H4–),
6.86 (d, 2H, J = 8 Hz, –C6H4–), 4.48 (s, 2H, N–CH2–),
3.70 (s, 3H, –OCH3), 2.16–1.89 (m, 2H, –CH2–), 0.73 (t,
3H, J = 7.3 Hz, –CH3) ppm; 13C NMR (50 MHz, DMSO-
d6): d = 174.7 (C4), 158.9, 156.2 (C2), 139.0, 129.3,
129.0, 128.8, 128.2, 125.7, 114.1, 67.1 (C5), 55.2, 41.0,
31.7, 8.1 ppm.
Cell lines
Human colon cancer HCT-116 and human breast cancer
MDA-MB-231 cell lines were maintained in a monolayer
culture using a nutrient medium RPMI 1,640 with 10 %
FBS and antibiotics.
Treatment of peritoneal macrophages for evaluation
of cytotoxic effect
Stock solutions of compounds were prepared in DMSO and
dissolved in the corresponding medium to the required
working concentrations. The compounds were evaluated for
their cytotoxic effects on rat peritoneal macrophages by the
MTT assay during 24 h. Rat peritoneal macrophages (10,000
cells per well) were seeded into wells of a 96-well, flat-
bottomed microtiter plate in 0.1 cm3 of medium with or
without LPS. After 24 h of incubation, 0.1 cm3 of the
investigated compound was added to cells in the final con-
centration (0.01, 0.1, 1, 10, 50, and 100 lmol dm-3), except
in the control wells where only medium was added to the
Crystal structure determination
Single crystals of 2a, 2e, and 2g were obtained by slow
evaporation (30 days at -5 °C) from a diluted ethanol (2a)
or ethyl acetate solution (2e and 2g). Suitable colorless,
single crystals of 2a, 2e, and 2 g were chosen for the
structure determination. X-ray single-crystal diffraction
data were collected on an Oxford Gemini S diffractometer
equipped with a CCD detector at 293(2) K. In the case of
single-crystal 2a, monochromatized Mo Ka radiation
123

S. Hmuda et al.
Table 7 Crystallographic data and structure refinement for compounds 2a, 2e, and 2g
Compound
2a
2e
2g
Molecular formula
M/g mol-1
C18H18N2O2
294.34
C19H17N3O2
319.36
C19H20N2O3
324.37
Crystal system
Space group
Triclinic
P1
Monoclinic
P21/c
Monoclinic
P21/c
˚
a/A
8.3857(2)
8.8113(5)
11.2005(8)
88.679(15)
77.885(12)
74.171(11)
0.45 9 0.12 9 0.09
777.96(7)
2
43.1188(8)
10.1104(2)
15.3109(3)
90
8.7665(4)
8.1607(3)
23.2077(9)
90
˚
b/A
˚
c/A
a/°
b/°
c/°
90.002(5)
90
92.179(4)
90
Crystal size/mm3
0.37 9 0.10 9 0.09
6,674.8(2)
16
0.17 9 0.08 9 0.07
3
˚
V/A
1,659.10(12)
4
Z
qc/g cm-3
l/mm-1
F/000
1.257
1.271
1.299
0.083
0.682
0.718
312
2,687
688
h range/°
Range of h, k, l
1.86–25.68
5.02–70.07
3.81–70.06
-10 B h B 9, -7 B k B 10,
-13 B l B 12
-51 B h B 52, -4 B k B 12,
-18 B l B 18
-9 B h B 10, -9 B k B 4,
-27 B l B 28
Reflections collected/unique
Data/restraints/parameters
7,405/2,912 (Rint = 0.0258)
38,960/12,623 (Rint = 0.0294)
12,623/0/866
5,690/3,141 (Rint = 0.0183)
3,141/0/221
2,912/0/203
Observed I [ 2r(I) reflections 2,174
8,413
2,545
R indices [I [ 2r(I)]
R indices (all data)
GOF on F2
R1 = 0.0420, wR2 = 0.1092a
R1 = 0.0987, wR2 = 0.2808b
R1 = 0.1265, wR2 = 0.3167
1.035
R1 = 0.0404, wR2 = 0.1029c
R1 = 0.0514, wR2 = 0.1102
1.038
R1 = 0.0577, wR2 = 0.1178
0.997
-3
˚
Dqmax, Dqmin/eA
0.174, -0.141
0.670, -0.283
0.162, -0.186
a
w = 1/[r2(Fo2) ? (0.0685P)2], where P = (Fo2 ? 2Fc2)/3
b
c
w = 1/[r2(Fo2) ? (0.1759P)2 ? 4.7267P], where P = (Fo2 ? 2Fc2)/3
w = 1/[r2(Fo2) ? (0.0489P)2 ? 0.2351P], where P = (Fo2 ? 2Fc2)/3
cells. The effects of the investigated compounds on the
survival of peritoneal macrophages was determined 24 h
later by the MTT test [28], modified by Ohno and Abe [29].
Briefly, 0.02 cm3 of MTT dye (5 mg cm-3) was added to
each well. After incubation for further 3 h, 0.1 cm3 of 10 %
SDS (sodium dodecyl sulfate) was added to extract the
insoluble product formazan resulting from conversion of the
MTT dye by viable cells. The number of viable cells in each
well is proportional to the intensityofthe absorbance of light,
which was then read in an ELISA plate reader at 570 nm.
the investigated compound was added to cells in the final
concentration (0.01, 0.1, 1, 10, 50, and 100 lmol dm-3),
except in the control wells, where only medium was added
to the cells. The effect of the investigated compounds on
cancer cell survival was determined 24 h later by the MTT
test described in the previous section. The antiproliferative
effect of the compounds was expressed as a percentage of
inhibition of untreated cell proliferation. It was calculated
as 100 % minus the ratio between the absorbance of each
dose of the compounds and the absorbance of the untreated
control cells 9 100.
Treatment of cell lines for antiproliferative in vitro
screening
Computational details
The target cells HCT-116 (10,000 cells per well) and
MDA-MB-231 (100,000 cells per well) were seeded in
triplicate into wells of a 96-well, flat-bottomed microtiter
plate in 0.1 cm3 medium. Twenty-four hours later, after the
cell adaptation and adherence for both cell lines, 0.1 cm3 of
The geometry of the investigated hydantoins was fully
optimized with the DFT B3LYP/6-311G(d,p) method
without any constraint on the geometry using Gaussian 09
program package [30]. The validity of the optimized
123

New derivatives of hydantoin
ˇ
´
ˇ ´
ˇ ´
´
´
´
11. Trisovic N, Bozic B, Obradovic A, Stefanovic O, Markovic S,
geometry was confirmed by frequency calculations, which
gave real values for all the obtained frequencies. The
frontier molecular orbital energies and HOMO–LUMO
energy gaps were also calculated with the same methods.
Natural bond orbital (NBO) analysis [31] was performed
using the NBO 3.1 program, as implemented in the
Gaussian 09 package [30] at the B3LYP/6-311G(d,p) level
in order to understand various second-order interactions
between the filled orbitals of one subsystem and vacant
orbitals of another subsystem, which is a measure of the
intramolecular delocalization of electron density (ED). The
second-order perturbation theory analysis of the Fock
matrix in the NBO basis of molecule 2a was carried out to
evaluate the donor–acceptor interactions.
ˇ
´
ˇ´
´
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Acknowledgments This work has been performed within the
framework of the research projects Nos. ON 172013, III 45007, and
ON 172035, supported by the Ministry of Education, Science and
Technological Development of the Republic of Serbia.
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