Synthesis, anticancer evaluation and docking study of 3- benzyloxyhydantoin derivatives

Article abstract of DOI:10.2174/1573406411666150708111631

A series of 3-benzyloxyhydantoin derivatives were designed and synthesized by introducing hydroxyurea pharmacophore into hydantoin rigid scaffold. The cytotoxic activities of the target compounds were evaluated in vitro against three cancer cell lines. Co

Full text of DOI:10.2174/1573406411666150708111631

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Medicinal Chemistry, 2016, 12, 37-47
37
Synthesis, Anticancer Evaluation and Docking Study of 3-
Benzyloxyhydantoin Derivatives
Jun Liu1, Kai Zhang1, Xi Mai*,1, Jinjin Wei1, Yijing Liao*,1, Ying Zhong1,2, Yang Liu1, Lihua Feng1
and Chao Liu1
2
1School of Pharmacy, Nanchang University, Nanchang, China; Center for
Drug Evaluation, Jiang Xi Province, Nanchang, China
Abstract: A series of 3-benzyloxyhydantoin derivatives were designed and synthe-
sized by introducing hydroxyurea pharmacophore into hydantoin rigid scaffold. The
cytotoxic activities of the target compounds were evaluated in vitro against three can-
cer cell lines. Compounds 5b, 5c, 5e, 5g, 6c and 6g displayed high activity on all of the
three cancer cell lines and the most promising compounds were 5g, 6g with IC50 values
of 0.04 and 0.01μM. Binding of derivatives for the ribonucleotide reductase (RR) was
investigated by use of molecular docking studies. Our findings show that
modification at the C5 position of hydantoin with isopropyl or isobutyl was favorable
to increasing binding affinity to the active site of the RR receptor and antiproliferative
activity.
Keywords: Molecular modeling, ribonucleotide reductase, anticancer, synthesis, MTT Assay, benzyloxyhydantoin derivatives.
1. INTRODUCTION
Hydantoins and their derivatives have been investigated
for more than 140 years [12] and the hydantoin is a common
5-membered ring containing a reactive cyclic urea core
which is present in various biologically active compounds
[13]. Hydantoin derivatives possess a variety of pharmacol-
ogical and biological activities, including antitumor [14-17],
antimicrobial [18, 19], anti-inflamatory [20], antiplatelet
[21], antiarrythmia [22, 23], antidiabetes [24, 25] and anti-
HIV [26]. In particular, they have also been found to act as
antagonists of leukocyte cell adhesion and allosteric inhibi-
tors of the protein–protein interaction [27] and show antipro-
liferative effects toward human carcinoma cells [28]. So the
derivatives of hydantoin have attracted much interest in drug
discovery [29].
As we all know, because of its low cure rate and high
mortality, tumor has become one of the most terrible dis-
eases and is also one of the leading causes of death around
the world [1-3]. Leukemia is a malignant disorder of blood
progenitor cells affecting a significant segment of the popu-
lation, especially children. In fact, leukemia is the most
frequent childhood cancer, with 26% of all cases, and
20% mortality [4]. For the therapy of leukemia various drugs
such as hydroxyurea (HU), cyclophosphamide, vincristine,
doxorubicin,
cisplatin,
cytarabine,
mercaptopurine,
methotrexate, imatimb, ect are available in the market. Long
term use of these drugs causes several side effects such as
arrest of bone marrow, nephrotoxicity, neurotoxicity and
gastrointestinal reaction [5]. In addition, the treatments for
leukemia may become ineffective and complex as patients
develop resistance to front-line therapies [6]. Therefore, it is
essential to search for new chemotherapeutic or adjuvant
agents for the treatment of leukemia. The past two decades
have seen a dramatic change in cancer treatment paradigms.
For example, clofarabine, a ribonucleotide reductase(RR)
inhibitor, is one of such major advances [7]. Eukaryotic ri-
bonucleotide reductase catalyzes nucleoside diphosphate
conversion to deoxynucleoside diphosphate. Crucial for rap-
idly dividing cells, RR is an attractive therapeutic target of
cancer, and over the past years, many chemotherapeutics
inhibiting different subunits of RR have been developed and
tested clinically for their anticancer activities [8-11].
We have recently reported preliminary results on the an-
tiproliferative action by 3-benzyloxyhydantoins [30]. The
results unraveled that 3-benzyloxyhydantoin analogs pos-
sessed potent antitumor activity against the human leukemia
cell line K562 and murine leukemia cell line L1210. Inspired
by these results, we have designed a set of new series of
more constrained 3-benzyloxyhydantoin derivatives contain-
ing different substituents on the benzene ring of benzyloxy
and different side chains at C5 position of hydantoin (hydro-
gen, methyl, isobutyl, isopropyl) and synthesized in a con-
venient route. Computational docking studies were per-
formed to help explaining the possible interactions that
might take place between the proposed tested derivatives and
the RR enzyme in comparing to hydroxyurea (HU) as a lead
compound. HU, which quenches tyrosine radicals in the ac-
tive site of RR and inhibits DNA synthesis in proliferating
cells [31], has been widely used in the treatment of many
neoplastic diseases [32], such as chronic, resistant, myelo-
*Address correspondence to these authors at the School of Pharmacy,
Nanchang University, 461 Nanchang 330006, China; Tel: 086-
13970866198; Fax: 086- 791-86239556; E-mails: maixi123@aliyun.com,
jingzi5027@126.com
1875-6638/16 $58.00+.00
© 2016 Bentham Science Publishers

38 Medicinal Chemistry, 2016, Vol. 12, No. 1
Liu et al.
.
HCl
NH2
O
Cl
O
R
+
O
O2N
1 a-g
a:R=H, b:R=4-CH3, c: R=4-Br, d:R=4-F
(i)
.
e: R=4-Cl, f: R=2-Cl, g: R=3-Cl
H
O
O
O
N
(ii)
R
(iii)
R
O
O
R
O
N
O
N
HN
O2N
HN
2 a-g
O
O
6 a-g
3 a-g
(v)
(iv)
O
O
R
R
O
O
N
N
HN
HN
O
O
5 a-g
4 a-g
Scheme 1. Reagents and conditions: (i) pyridine, dry dichloromethane, reflux; (ii) alanine methyl ester hydrochloride, dry triethylamine, dry
dichloromethane, rt; (iii) glycine methyl ester hydrochloride, dry triethylamine, dry dichloromethane, rt; (iv) leucine methyl ester hydrochlo-
ride, dry triethylamine, dry dichloromethane, rt; (v) valine methyl ester hydrochloride, dry triethylamine, dry dichloromethane, rt.
cytic leukemia [33-35], cervical carcinoma [36], oesophageal
carcinoma [37] ect. The benzyloxyhydantoin derivatives
designed in this study are also potential HU analogues which
contain benzyloxy substituted hydroxyurea pharmacophore
(3- benzyloxy- 1, 3- diamino- 2- ketone moiety in hydantoin
ring). Hence, they may be arranged to yield potent and hope-
fully selective anticancer drugs by introducing hydroxyurea
pharmacophore into hydantoin rigid scaffold. The target
compounds were evaluated for their anticancer activity in
vitro against human leukemia cell line K562, murine leuke-
mia cell line L1210 and human laryngreal carcinoma cell
line HEP-2 by the 3-(4, 5-dimethylthiahiazol-2-y1)-2, 5-
diphenytetrazolium bromide (MTT) assay. Meanwhile, the
technology of molecular docking was used to target com-
pounds, which revealed many useful informations.
Fig. (1). An ORTEP-3 projection, showing the atomic labelling and
the 30% probability ellipsoids of the compound 4e.
2. RESULTS AND DISCUSSION
2.1. Chemistry
All the target compounds were fully characterized on the
basis of spectroscopic techniques (1H NMR, 13C NMR, IR
and MS). In addition, the structure of 4e was also confirmed
by X-ray diffraction analysis. The good quality crystal of 4e
was grown by the slow evaporation technique using acetic
ester and petroleun ether (4:1) as solvent. (Fig. 1) showed the
ORTEP diagram with 30% probability, main crystallo-
graphic parameters were presented in supplementary files. In
the title crystal structure, the two carbonyl bond lengths were
1.204(3) (C1=O2), 1.218(2) (C2=O3) respectively, which
was in the mean value range of 1.19 - 1.23 Å found for car-
bonyl bonds [42,43], but the latter was longer than former.
The reason of this phenomenon was probably that the elec-
tron donation from the ring N atoms to the carbonyl groups
The target compounds were synthesized following the
procedures as shown in Scheme 1. The key starting com-
pounds O-benzyl-hydroxylamine analogues 1a-1g were pre-
pared by the procedures reported by us [30, 38]. The O-
benzyl- hydroxylamine analogues were treated with 4-
nitrophenyl chloroformate in dry dichloromethane (DCM) to
give 2a-2g [30, 38-41]. Finally, the synthesis of the hydan-
toin derivatives 3a-3g, 4a-4g, 5a-5g and 6a-6g were accom-
plished by reaction of amino acids methyl ester hydrochlo-
ride with 2a-2g in dry dichloromethane in the presence of
anhydrous triethylamine (Scheme 1).

Synthesis, Anticancer Evaluation and Docking Study
Medicinal Chemistry, 2016, Vol. 12, No. 1 39
Table 1. In vitro cytotoxicity results of target compounds
N2, C1, C2, C3). The benzene plane forms a dihedral angle
of 47.14° with the hydantoin plane. The N-H…O and C-
H…O hydrogen bonds link the molecules into a continous
parallel structure.
against L1210, K562 and HEP-2 cancer cell lines.
IC50(μM)
Compound No.
2.2. Antitumor Evaluation
L1210 Cells
K562 Cells
HEP-2 Cells
The in vitro antitumor activity of the synthesized com-
pounds 3a-3g, 4a-4g, 5a-5g, 6a-6g against three different
cancer cell lines, including human leukemia cell line K562,
murine leukemia cell line L1210 and human laryngreal car-
cinoma cell line HEP-2, were assayed by the improved MTT
method [45] and the results are listed in Table 1.
3a
3b
22.19
>200
21.63
10.96
>200
>200
3c
3d
3e
3f
>200
25.24
15.12
21.82
46.94
25.08
>200
21.10
25.29
34.24
9.50
12.95
22.39
>200
19.14
3.80
20.91
>200
>200
>200
>200
0.048
>200
>200
>200
116.8
>200
>200
78.86
69.63
0.15
From the obtained results we concluded that all target
compounds showed moderate to excellent cytostatic potency
against the different cancer cells compared to the reference
compound HU. In general, most of them displayed good
inhibitory effects on L1210 and K562, especially 3a, 3d, 3f,
3g, 4c, 4e, 4f, 5b, 5c, 5d, 5e, 5g, 6b, 6c, 6f and 6g which
demonstrated high activity on both L1210 and K562 cell
lines. Compounds 3c, 4a, 5c, 5e, 5f, 5g, 6a, 6c, 6g showed
significant activity on HEP-2 cell with IC50 values of 20.91,
0.048, 0.15, 22.84, 0.10, 0.04, 0.70, 17.48, 0.01μmol/L re-
spectively. Interestingly, the substitute R of most potent
compounds (5c, 5e, 5f, 5g, 6g) against HEP-2 cell were bro-
mine and chlorine. Hence, it was anticipated that substitution
of halogens substituents, such as chlorine, bromine on the
benzene ring may be essential for increasing antitumor activ-
ity. In general, the compounds with hydrophobic isopropyl
and isobutyl groups at the C-5 position of hydantoin demon-
strated better activity, especially the compounds 5b, 5c, 5e,
5g, 6c and 6g showed highly active to inhibit proliferation of
all the three cancer cell lines.
3g
4a
4b
4c
4d
4e
4f
>200
4.24
12.49
>200
8.49
5.67
4g
5a
5b
5c
5d
5e
5f
>200
>200
4.26
9.87
>200
10.58
9.70
10.99
31.01
3.56
7.83
nd
2.3. Docking Study
10.02
15.99
15.67
>200
3.96
22.84
0.10
Similar to our previous docking study, the subsite B in
Saccharomyces cerevisiae R1 (ScR1) domain of RR (PDB
id- 2ZLF) [38, 46] was selected as the active site. Molecular
modeling simulation study demonstrated that there were
good complementarities between the target derivatives and
the hydrophobic pocket of the enzymatic cavity (Fig. 2).
Binding energies of all tested hydantoin compounds were in
range from -5.44 to -6.85 kcal/mol and were less than HU
(Table 2).
>200
7.87
5g
6a
6b
6c
6d
6e
6f
0.04
10.84
6.10
0.70
>200
17.48
>200
>200
nd
3.46
7.39
7.23
>200
>200
13.60
17.82
7.27
The LEU716, ARG717, GLN692, LYS723, SER726,
MET727, LYS693, ILE696 and TYR730 of RR protein were
found to be directly interacted with the target compounds.
Most of the compounds, such as 3a in (Fig. 2), were oriented
in the active site of the protein in a way that placed the aro-
matic ring into the hydrophobic pocket comprising the resi-
dues MET727, LYS693, ILE696 and TYR730. However, the
compounds 5a, 5c, 6a and 6g were oriented in a way that
placed the hydantoin ring into the above hydrophobic pocket
and all of them showed good binding energy which were -
6.16, -6.52, -6.56, -6.56 respectively. This may be attributed
to lipophilic substituents: isopropyl and isobutyl groups at
C5 position of hydantoin which comfirms the preference of
the lipophilic substituents on the hydantoin ring. These de-
rivatives showed high activity on HEP-2 cell lines with IC50
values of 78.86, 0.15, 0.70, 0.01μmol/L respectively, espe-
cially 6g exhibited the best activity on HEP-2 cell lines.
5.59
5.30
6g
7.62
0.01
HU
21.67
192.0
a) nd=not determined
would be expected to lengthen the C=O bonds and shorten
the ring C-N bonds, C2=O3 being affected from both sides
while C1=O2 being affected from only one side [44]. This
was also reflected in the ranking order of bond lengths of
hydantoin ring which was C1-C3 [1.492(3)]> C3-N2
[1.446(3)] > C2-N1 [1.399(3)] > C1-N1 [1.368(3)] > C2-N2
[1.330(3)]. There are two significant planar parts in the struc-
ture: the benzene and the hydantoin plane (O1, O2, O3, N1,

40 Medicinal Chemistry, 2016, Vol. 12, No. 1
Liu et al.
Fig. (2). The docking modes of compounds 3a, 6g at ScR1. The compounds are color coded as carbon beige (3a) and nattier blue (6g), nitro-
gen blue, oxygen red and hydrogen white. (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of the article.)
Fig. (3). Plausible binding modes for compound 3a in the binding site of ScR1. The compound is color coded as carbon dim gray, nitrogen
blue, oxygen red and hydrogen green. The hydrogen bonds are drawn in black dash line. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of the article).
According to Table 2, similar H-bonds have been ob-
served for compounds 3a-3g and 4a-4g except for 4c. The
hydrogen bonding interactions of representative compound
3a were showed in (Fig. 3), two hydrogen bonds were identi-
fied. One is a H-bond formed between the C1- carbonyl
oxygen of hydantoin and amide proton from ARG717 in RR.
The other bond was formed between the C3- carbonyl oxy-
gen of hydantoin and amide proton from GLN692 in en-
zyme. However, 5a-5g and 6a-6g showed more diversified
hydrogen bonding interaction with more kinds of amino acid
including LYS693, LYS723, SER691, LEU716 besides
GLN-692 and ARG-717. The binding modes of representa-
tive compound 5g in (Fig. 4) revealed key hydrogen binding
interactions: the H-bond of the oxygen of C3- carbonyl in
hydantoin with amide hydrogen of LYS 693 and the H-bond
of the hydrogen in 4-imino group of hydantoin with the the
oxygen in SER691.
Better affinities to the active site of RR were gained
by the compounds 5a-5g, 6a-6g which have better antitumor

Synthesis, Anticancer Evaluation and Docking Study
Medicinal Chemistry, 2016, Vol. 12, No. 1 41
Table 2. In Silico docking results.
Compound No.
Binding Energy(Kcal/mol)
H-bond
HU
3a
3b
3c
3d
3e
3f
-3.09
-6.35
-5.77
-6.68
-5.82
-6.35
-5.95
-6.31
-5.44
-5.99
-6.31
-6.53
-6.04
-5.81
-5.94
-6.16
-6.61
-6.52
-6.12
-6.32
-6.64
-6.56
-6.19
-6.85
-6.08
-5.95
-6.46
-6.37
-6.56
GLN692(1.942Å), GLN692(2.216Å), ARG717(1.821Å)
GLN692(1.651Å), ARG717(1.830Å)
GLN692(1.675Å), ARG717(1.836Å)
GLN692(1.701Å), ARG717(1.822Å)
GLN692(1.663Å), ARG717(1.849Å)
GLN692(1.660Å), ARG717(1.975Å)
GLN692(1.656Å), ARG717(1.904Å)
GLN692(1.657Å), ARG717(1.832Å)
GLN692(1.763Å), ARG717(1.809Å)
GLN692(1.786Å), ARG717(2.020Å)
GLN692(1.730Å), LEU716(1.781Å)
GLN692(2.284Å), ARG717(1.911Å)
GLN692(1.676Å), ARG717(1.835Å)
GLN692(2.108Å), ARG717(1.955Å)
GLN692(1.689Å), ARG717(1.853Å)
LYS693(2.406Å), SER691(2.302Å)
GLN692(1.759Å)
3g
4a
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
5e
5f
LYS723(2.211Å), ARG717(2.558Å)
GLN692(1.650Å)
GLN692(1.807Å), LEU716(2.064Å)
LYS693(2.363Å), SER691(2.305Å)
LYS693(2.300Å), SER691(2.247Å)
GLN692(2.271Å)
5g
6a
6b
6c
6d
6e
6f
GLN692(1.655Å), ARG717(1.871Å)
GLN692(2.465Å)
GLN692(1.775Å), ARG717(1.767Å)
GLN692(1.686Å), ARG717(1.950Å)
GLN692(1.830Å)
6g
LYS723 (2.035Å)
Fig. (4). Plausible binding modes for compound 5g in the binding site of ScR1. The compound is color coded as carbon dim gray, nitrogen
blue, oxygen red and hydrogen green. The hydrogen bonds are drawn in black dash line.ꢀ(For interpretation of the references to color in this
figure legend, the reader is referred to the web version of the article).

42 Medicinal Chemistry, 2016, Vol. 12, No. 1
Liu et al.
activity. They showed binding energy less than -6.00 except
for compound 6d and compound 6b exhibited the highest
affinity and attained the score of ꢀ6.85 kcal/mol. This is
probably the reason that compound 6b displayed the best
inhibitory effects on K562 with IC50 values of 3.96, yet,
compound 6d exhibited inactive on K562 and HEP-2. The
active compounds on all of the three cancer cell lines (5b, 5c,
5e, 5g, 6c and 6g) fitted well in the active site of RR and
attained the score of -6.61 to -6.08 kcal/mol. This suggested
that the introduction of isopropyl and isobutyl groups was
favorable not only to increasing antiproliferative activity, but
also to increasing binding affinity to the active site of the RR
receptor.
1. All complexes pictures were rendered employing the
UCSF Chimera software [49].
4.2. Chemistry
Melting points were determined using a capillary method
and were uncorrected. Infrared spectra were recorded on a
1
Shimadzu FTIR 8400 spectrometer (KBr pellets). H- NMR
and 13C-NMR spectra were recorded on either a Bruker AV
400 MHz or a Bruker AV 600 MHz spectrometer. Chemical
shifts (ꢁ values) and coupling constants (J values) were re-
ported in parts per million (ppm) and Hertz (Hz), respec-
tively. Chemical shifts were relative to TMS except for sol-
vents that were used, and the signals were quoted as s (sin-
glet), d (doublet), t (triplet), q (quartet) and m (multiplet).
The mass spectra (EI or ESI) were recorded on either a Ag-
ilent 1100 MSD/TOF or on a Waters 2695 LC-ZQ4000.
Crystal data were collected by a Bruker APEX- area-
detector diffractometer. Column chromatography was carried
out on silica gel (200-300 mesh). Unless otherwise stated,
materials were obtained from commercial suppliers and puri-
fied according to the methods of chemical reagents. Alanine
methyl ester hydrochloride, glycine methyl ester hydrochlo-
ride, leucine methyl ester hydrochloride and valine methyl
ester hydrochloride were prepared following the procedures
reported by Zeng [50].
3. CONCLUSION
In summary, a series of 3-benzyloxyhydantoin deriva-
tives was designed and synthesized by a convenient method.
The anticancer activity of the target compounds was esti-
mated by studying their inhibitory effects on human leuke-
mia cell line K562, murine leukemia cell line L1210 and
human laryngreal carcinoma cell line HEP-2. Binding of 3-
benzyloxyhydantoin derivatives for the RR was investigated
by use of molecular docking studies. Some of the prepared
compounds showed potent antitumor activities against dif-
ferent cancer cell lines. Structure activity relationship (SAR)
analysis and molecular moleding studies revealed that
slightly larger lipophilic substituents such as isopropyl and
isobutyl at C5 position of hydantoin were favorable to in-
creasing binding affinity to the active site of the RR receptor
and antiproliferative activity. In particular, the promising
compounds 5b, 5c, 5e, 5g, 6c and 6g, which were the com-
pounds of 5-isopropyl and 5-isobutyl benzyloxyhydantoin
series, displayed high activity on all of the three cancer cell
lines. These ndings have encouraged us to continue the
development and testing of novel hydantoin derivatives and
to conduct further studies to investigate SAR and their
mechanisms of action.
4.3. General Procedure for the Synthesis of 1-(4-
Nitrophenyl)- N- benzyloxy Carbamate Analogues 2a-2g
The synthesis of 1-(4-nitrophenyl)- N- benzyloxy carba-
mate analogues 2a-2g has been described by us in previous
papers and the spectroscopic data for compounds 2a-2e and
2g are in agreement with those in the papers [30, 39]. The
spectroscopic data of 2f were as follows.
4.3.1. 4-Nitrophenyl-N-[O-(2-chlorobenzylhydroxy)] carba-
mate (2f)
O-(2-Chlorobenzyl)hydroxylamine hydrochloride (19.3g,
0.1mol) was reacted according to general procedure to give
2f as a white solid (23.1g, 72%): mp 182°C-183°C; IR
(KBr): ꢀ = 3267, 3074, 2870, 1732, 1543, 1481, 752 cm-1;
MS (ESI): m/z [M+Na]+ 345.08.
4. EXPERIMENTAL
4.1. Docking Studies
Docking studies were performed using a free Autodock
4.0 [47]. The X-ray structures of ScR1-P6 complex (Protein
Data Bank ID: 2ZLF) was retrieved from the Protein Data
Bank [38]. The three-dimensional structures of the proposed
compounds were constructed and energy minimizations were
performed with the Chem-Draw/Chem3D [48]. A grid of 40,
40, and 40 points in x-, y-, and z-direction, respectively, with
grid spacing of 0.375Å was built centered on the center of
mass of the catalytic site of considered receptor. The popula-
tion size was set to 150 and the individuals were initialized
randomly. Docking was performed with the Lamarckian Ge-
netic Algorithm using a medium of 2,500,000 energy evalua-
tions per docking. The maximum number of top individual
the automatically survived set to 1, maximum number of
generations to 27,000, mutation rate to 0.02 and crossover
rate to 0.8. The number of GA runs was set to 100. Calcu-
lated AutoDock binding free energies were used to score the
ligands. The top scoring (most negative, thus favorable to
binding) of the ligands were selected and arranged in Table
4.4. General Procedure for the Synthesis of Hydantoin
Analogues 3a-3g
Alanine methyl ester hydrochloride (9 mmol) was sus-
pended in dry DCM (20 ml), dry triethylamine (36 mmol)
was added slowly. 4-Nitrophenyl -N -(O -benzylhydroxy)
carbamate (2a-2g) (9mmol) was dissolved in dry DCM (40
ml) and then added dropwise in the solution. The mixture
was stirred at room temperature for 12 h. Upon completion,
the reaction mixture was successively washed with 1 M Na-
HCO3 (3ꢂ30ml), 1M HCl (30ml) and H2O (2ꢂ30 ml). The
DCM layer was dried by anhydrous MgSO4, filtered and
concentrated under vacuum to give yellow oil substance,
which was purified by crystallization from anhydrous
ether/petroleun ether (4:1) to afford the expected com-
pounds.

Synthesis, Anticancer Evaluation and Docking Study
Medicinal Chemistry, 2016, Vol. 12, No. 1 43
4.4.1. 3-Benzyloxy-5-methylhydantoin (3a)
4.4.7. 3-(3-Chlorobenzyloxy)-5-methylhydantoin (3g)
Yield: 36%; m.p.: 104-107°C; IR (KBr): ꢀ = 3314, 1771,
1724, 1416, 760, 706 cm-1; 1H-NMR (400 MHz, CDCl3): ꢀ =
7.48 (d, J = 3.0 Hz, 3H), 7.37 (d, J = 2.5 Hz, 2H), 6.61 (s,
1H), 5.13 (s, 2H), 3.96 (d, J = 6.9 Hz, 1H), 1.34 ppm (d, J =
6.9 Hz, 3H); 13C-NMR (100 MHz, CDCl3): ꢀ = 169.38,
154.23, 133.62, 130.40, 129.81, 128.88, 77.43, 51.39, 17.73
ppm; HRMS-ESI: m/z calcd. for C11H13N2O3 [M+H]+:
221.0926, found: 221.0918.
Yield: 27%; m.p.: 108~110°C; IR (KBr): ꢀ = 3294, 1774,
1724, 1574, 1402, 1477, 1369, 891, 802 cm-1; 1H-NMR (400
MHz, CDCl3): ꢀ = 7.51 (s, 1H), 7.41-7.28 (m, 3H), 6.05 (s,
1H), 5.13 (s, 2H), 4.06 (s, 1H), 1.43 ppm (s, 3H); 13C-NMR
(100 MHz, CDCl3): ꢀ = 168.83, 153.54, 135.26, 134.37,
129.87, 129.56, 127.93, 77.03, 51.09, 17.45 ppm. HRMS-
ESI: m/z calcd. for C11H12ClN2O3 [M+H]+: 255.0536, found:
255.0526.
4.4.2. 3-(4-Methylbenzyloxy)-5-methylhydantoin (3b)
4.5. General Procedure for the Synthesis of Hydantoin
Analogues 4a-4g
Yield: 17%; m.p.: 90-92°C; IR (KBr) cm-1: ꢀ = 3329,
1740, 1709, 1423, 810, 750 cm-1; 1H-NMR (400 MHz,
CDCl3): ꢀ = 7.38 (s, 2H), 7.19 (s, 2H), 6.32 (s, 1H), 5.10 (s,
2H), 4.00 (s, 1H), 2.36 (s, 3H), 1.38 ppm (s, 3H); 13C-NMR
(100 MHz, CDCl3): ꢀ = 168.92, 153.73, 139.46, 130.28,
130.17, 129.21, 77.04, 51.02, 21.33, 17.45 ppm; HRMS-ESI:
m/z calcd. for C12H15N2O3 [M+H]+: 235.1083, found:
235.1078.
Glycine methyl ester hydrochloride (9 mmol) and 4-
nitrophenyl - N - (O -benzylhydroxy) carbamate (2a-2g)
(9mmol) were reacted according to the procedure of 3a-3g to
give 4a-4g as a white solid.
4.5.1. 3-Benzyloxyhydantoin (4a)
Yield: 20%; m.p.: 107~109°C; IR (KBr): ꢀ = 3248, 1728,
1450, 1201, 741, 698 cm-1; 1H-NMR (400 MHz, CDCl3): ꢀ =
7.49 (s, 3H), 7.38 (s, 2H), 6.57 (s, 1H), 5.12 (s, 2H), 3.86
ppm (s, 1H); 13C-NMR (100 MHz, CDCl3): ꢀ = 166.04,
154.99, 133.67, 130.23, 129.82, 128.96, 77.44, 44.79 ppm;
HRMS-ESI: m/z calcd. for C10H11N2O3 [M+H]+: 207.0770,
found: 207.0763.
4.4.3. 3-(4-Bromobenzyloxy)-5-methylhydantoin (3c)
Yield: 43%; m.p.: 134-136°C; IR (KBr): ꢀ = 3346, 1732,
1705, 1593, 1489, 1420, 812, 720 cm-1; 1H-NMR (400 MHz,
CDCl3): ꢀ = 7.51 (d, J = 8.2 Hz, 2H), 7.37 (d, J=8.1 Hz, 2H),
6.11 (s, 1H), 5.09 (s, 2H), 4.02(d, J = 6.9 Hz, 1H), 1.39 ppm
(d, J=7.0 Hz, 3H); 13C-NMR (100 MHz, CDCl3): ꢀ = 168.78,
153.39, 132.35, 131.75, 131.53, 123.72, 77.02, 51.05, 17.48
ppm; HRMS-ESI: m/z calcd. for C11H12BrN2O3 [M+H]+:
299.0031, found: 299.0028.
4.5.2. 3-(4-Methylbenzyloxy)hydantoin (4b)
Yield: 28%; m.p.: 118~121°C; IR (KBr): ꢀ = 3263, 1709,
1447, 1201, 813, 730 cm-1; 1H-NMR (400 MHz, CDCl3): ꢀ =
7.45 (s, 2H), 7.26 (s, 2H), 5.15 (s, 2H), 4.52 (s, 1H), 3.94 (s,
2H), 2.43 ppm (s, 3H); 13C-NMR (100 MHz, CDCl3): ꢀ =
169.56, 157.47, 142.28, 133.24, 132.91, 132.05, 80.58,
43.96, 23.92 ppm; HRMS-ESI: m/z calcd. for C11H13N2O3
[M+H]+: 221.0926, found: 221.0917.
4.4.4. 3-(4-Fluorobenzyloxy)-5-methylhydantoin (3d)
Yield: 18%; m.p.: 98-100°C; IR (KBr) : ꢀ = 3306, 1736,
1713, 1620, 1585, 1497, 920, 768 cm-1; 1H-NMR (400 MHz,
CDCl3): ꢀ = 7.50 (t, J = 7.3 Hz, 1H), 7.35-7.48 (m, 1H), 7.16
(t, J = 14.9 Hz, 1H), 7.07 (t, J = 18.1 Hz, 1H), 6.53 (s, 1H),
5.20 (s, 2H), 4.01-3.99 (m, 1H), 1.38 ppm (d, J=7.0 Hz, 3H);
13C-NMR (100 MHz, CDCl3): ꢀ = 168.92, 153.65, 132.46,
131.65, 131.57, 124.30, 120.85, 115.41, 77.04, 51.06, 17.33
ppm; HRMS-ESI: m/z calcd. for C11H12FN2O3 [M+H]+:
239.0832, found: 239.0823.
4.5.3. 3-(4-Bromobenzyloxy)hydantoin (4c)
Yield: 18%; m.p.: 160~162°C; IR (KBr): ꢀ = 3256, 1732,
1
1701, 1450, 1201, 802, 730 cm-1; H-NMR (600 MHz, CD-
Cl3): ꢀ = 7.52 (d, J=8.4 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H),
5.38 (s, 1H), 5.10 (s, 2H), 3.94 ppm (s, 2H); 13C-NMR (100
MHz, CDCl3): ꢀ = 163.97, 158.48, 132.83, 130.75, 130.49,
129.57, 122.57, 121.96, 76.84, 40.64 ppm; HRMS-ESI: m/z
calcd. for C10H10BrN2O3 [M+H]+: 284.9875, found:
284.9869.
4.4.5. 3-(4-Chlorobenzyloxy)-5-methylhydantoin (3e)
Yield: 14%; m.p.: 136~137°C; IR (KBr): ꢀ = 3344, 1709,
1597, 1489, 802, 690 cm-1; 1H-NMR (600 MHz, CDCl3): ꢀ =
7.44 (dd, J1=6.6 Hz, J2=1.8 Hz, 2H), 7.36 (dd, J1=6.6 Hz,
J2=1.8 Hz, 2H), 5.50 (s, 1H), 5.12 (s, 2H), 4.04-4.01 (m,
1H), 1.41 ppm (d, J=7.2 Hz, 3H); 13C-NMR (150 MHz,
CDCl3): ꢀ = 168.68, 153.17, 135.48, 131.78, 131.33, 128.77,
77.02, 51.03, 17.55 ppm; HRMS-ESI: m/z calcd. for
C11H13ClN2O3 [M+H]+: 255.0536, found: 255.0533.
4.5.4. 3-(4-Fluorobenzyloxy)hydantoin (4d)
Yield: 12%; m.p.: 74~76°C; IR (KBr): ꢀ = 3306, 1747,
1720, 1651, 1551, 1512, 1201, 833, 700 cm-1; 1H-NMR (600
MHz, CDCl3): ꢀ = 7.48 (dd, J1=8.5 Hz, J2=5.4 Hz, 1H), 7.39
(dd, J1= 8.4 Hz, J2=5.4 Hz, 1H), 7.10-7.06 (m, 2H), 6.15 (s,
1H), 5.11 (s, 1H), 4.81 (s, 1H), 4.02 (d, J=4.8 Hz, 1H), 3.92
ppm (s, 1H); 13C-NMR (150 MHz, CDCl3): ꢀ = 164.22,
159.54, 154.05, 131.91, 130.97, 129.26, 115.66, 115.53,
77.03, 41.30 ppm; HRMS-ESI: m/z calcd. for C10H10FN2O3
[M+H]+: 225.0675, found: 225.0687.
4.4.6. 3-(2-Chlorobenzyloxy)-5-methylhydantoin (3f)
Yield: 22%; m.p.: 148~149°C; IR (KBr): ꢀ = 3329, 1740,
1709, 1402, 760 cm-1; 1H-NMR (600 MHz, CDCl3): ꢀ = 7.59
(dd, J1=7.0 Hz, J2=2.2 Hz, 1H), 7.40 (dd, J1=7.6 Hz, J2=1.7
Hz, 1H), 7.34-7.30 (m, 2H), 5.75 (s, 1H), 5.29 (dd, J1=13.2
Hz, J2=11.4 Hz, 2H), 4.03 (q, J1= 13.8 Hz, J2=7.2 Hz, 1H),
1.42 ppm (d, J= 6.6 Hz, 3H); 13C-NMR (150 MHz, CDCl3):
ꢀ = 168.71, 153.35, 134.81, 131.99, 131.60, 130.69, 129.59,
126.99, 77.03, 51.05, 17.42 ppm; HRMS-ESI: m/z calcd. for
C11H12ClN2O3 [M+H]+: 255.0536, found: 255.0531.
4.5.5. 3-(4-Chlorobenzyloxy)hydantoin (4e)
Yield: 17%; m.p.: 158~159°C; IR (KBr): ꢀ = 3260, 1732,
1
1701, 1450, 1201, 806, 730 cm-1; H-NMR (600 MHz, CD-
Cl3): ꢀ = 7.45 (d, J=4.8 Hz, 2H), 7.37 (dd, J1=6.6 Hz, J2=1.8

44 Medicinal Chemistry, 2016, Vol. 12, No. 1
Liu et al.
Hz, 2H), 5.43 (s, 1H), 5.12 (s, 2H), 3.94 ppm (d, J=1.2 Hz,
2H); 13C-NMR (150 MHz, CDCl3): ꢀ = 165.15, 153.74,
135.48, 131.80, 131.20, 128.83, 77.23, 44.35 ppm; HRMS-
ESI: m/z calcd. for C10H10ClN2O3 [M+H]+: 241.0380, found:
241.0372.
77.24, 53.76, 40.68, 24.80, 23.00, 21.47 ppm; HRMS-ESI:
m/z calcd. for C15H21N2O3 [M+H]+: 277.1552, found:
277.1535.
4.6.3. 3-(4-Bromobenzyloxy)-5-isobutylhydantoin (5c)
Yield: 30%; m.p.: 108~111°C; IR (KBr): ꢀ = 3190, 2955,
1778, 1736, 1470, 1202, 810, 710 cm-1; 1H-NMR (400 MHz,
CDCl3): ꢀ = 7.52 (s, 2H), 7.39 (s, 2H), 5.73 (s, 1H), 5.10 (s,
2H), 3.98 (s, 1H), 1.73 (s, 2H), 1.45 (s, 1H), 0.96 ppm (s,
3H), 0.95 ppm (s, 3H); 13C-NMR (100 MHz, CDCl3): ꢀ =
168.51, 153.34, 132.41, 131.74, 131.55, 123.71, 77.34,
53.83, 40.72, 24.88, 22.97, 21.65 ppm; HRMS-ESI: m/z
calcd. for C14H18BrN2O3 [M+H]+: 341.0501, found:
341.0483.
4.5.6. 3-(2-Chlorobenzyloxy)hydantoin (4f)
Yield: 20%; m.p.: 118~119°C; IR (KBr): ꢀ = 3337, 1732,
1
1709, 1439, 1201, 890, 760 cm-1; H-NMR (600 MHz, CD-
Cl3): ꢀ = 7.60 (t, J=7.2 Hz, 1H), 7.41 (dd, J1=7.2 Hz, J2=1.8
Hz, 1H), 7.34-7.31 (m, 2H), 5.94 (s, 1H), 5.29 (s, 2H), 3.93
ppm (d, J=6.0 Hz, 2H). 13C-NMR (150 MHz, CDCl3): ꢀ =
165.26, 154.09, 134.66, 131.83, 131.57, 130.68, 129.62,
127.08, 77.03, 44.42 ppm; HRMS-ESI: m/z calcd. for
C10H10ClN2O3 [M+H]+: 241.0380, found: 241.0373.
4.6.4. 3-(4-Fluorobenzyloxy)-5-isobutylhydantoin (5d)
4.5.7. 3-(3-Chlorobenzyloxy)hydantoin (4g)
Yield: 12%; m.p.: 109~110°C; IR (KBr): ꢀ = 3240, 2963,
1778, 1732, 1512, 1423, 1210, 829, 710 cm-1; 1H-NMR (600
MHz, CDCl3): ꢀ = 7.50-7.47 (m, 2H), 7.08-7.05 (m, 2H),
6.01 (s, 1H), 5.11 (s, 2H), 3.99-3.96 (m, 1H), 1.74-1.71 (m,
2H), 1.45(dd, J1=9.6Hz, J2=8.4Hz, 1H), 0.97(d, J=6.6 Hz,
3H), 0.94 ppm (d, J=6.6 Hz, 3H); 13C-NMR (150 MHz, CD-
Cl3): ꢀ = 168.59, 164.24, 162.59, 153.64, 132.05, 129.28,
115.61, 115.47, 77.03, 53.82, 40.68, 24.86, 22.98, 21.60
ppm; HRMS-ESI: m/z calcd. for C14H18FN2O3 [M+H]+:
281.1301, found: 281.1307.
Glycine methyl ester hydrochloride (1.13g, 9 mmol) and
4- nitrophenyl-N-[O-(3-chlorobenzylhydroxy)] carbamate
(2.90g, 9mmol) (2g) were reacted to give crude product as
yellow oil substance. The crude product was purified by sili-
ca gel chromatography using ethyl acetate/ n-hexane (2:5) as
eluent, solvent was eliminated from the elution under redu-
ced pressure and then recrystallized in ether/petroleun ether
(4:1) to give 4g as a white solid. Yield: 24%; m.p.: 68~70°C;
IR (KBr): ꢀ = 3306, 1747, 1720, 1651, 1547, 1439, 1210,
1
795, 710 cm-1; H-NMR (600 MHz, CDCl3): ꢀ = 7.51 (s,
4.6.5. 3-(4-Chlorobenzyloxy)-5-isobutylhydantoin (5e)
1H), 7.48 (s, 1H), 7.39-7.35 (m, 2H), 6.23 (s, 1H), 5.10 (s,
2H), 3.93 ppm (d, J=6.0 Hz, 2H); 13C-NMR (150 MHz, CD-
Cl3): ꢀ = 165.37, 154.14, 137.11, 134.59, 130.05, 129.52,
129.01, 127.78, 77.25, 41.31 ppm; HRMS-ESI: m/z calcd.
for C10H10ClN2O3 [M+H]+: 241.0380, found: 241.0371.
Yield: 19%; m.p.: 104~106°C; IR (KBr): ꢀ = 3221, 2959,
1774, 1724, 1663, 1431, 1201, 814, 719 cm-1; 1H-NMR (600
MHz, CDCl3): ꢀ = 7.44 (dd, J1= 6.6 Hz, J2=1.8 Hz, 2H), 7.35
(dd, J1=6.6 Hz, J2=2.4 Hz, 2H), 5.90 (s, 1H), 5.11 (s, 2H),
3.98-3.96 (m, 1H), 1.75-1.70 (m, 2H), 1.46-1.43(m, 1H),
1.01(d, J=6.6 Hz, 3H) 0.95 ppm (dd, J1=6.6 Hz, J2=6.0 Hz,
3H); 13C-NMR (150 MHz, CDCl3): ꢀ = 168.65, 153.82,
135.45, 131.86, 131.30, 128.75, 77.25, 53.83, 40.66, 24.81,
23.00, 21.60 ppm; HRMS-ESI: m/z calcd. for C14H18ClN2O3
[M+H]+: 297.1006, found: 297.0996.
4.6. General Procedure for the Synthesis of Hydantoin
Analogues 5a-5g
Leucine methyl ester hydrochloride (9 mmol) and 4-
nitrophenyl - N - (O -benzylhydroxy) carbamate (2a-2g)
(9mmol) were reacted as described for 3a-3g. Purification by
recrystallization from AcOEt and petroleum ether to give 5a-
5g as a white solid.
4.6.6. 3-(2-Chlorobenzyloxy)-5-isobutylhydantoin (5f)
Yield: 21%; m.p.: 85~86°C; IR (KBr): ꢀ = 3190ꢀ2959,
1774, 1728, 1431, 1201, 748 cm-1; 1H-NMR (600 MHz,
CDCl3): ꢀ = 7.60-7.58 (m, 2H), 7.42-7.39 (m, 2H), 5.98 (d,
J=6.0 Hz, 1H), 5.27(t, J=16.2 Hz, 2H), 3.99-3.96 (m, 1H),
1.76-1.71 (m, 2H), 1.48 (dd, J1=8.4 Hz, J2=8.4 Hz, 1H), 0.97
(d, J=6.6 Hz, 3H), 0.94 ppm (d, J=6.6 Hz, 3H); 13C-NMR
(150 MHz, CDCl3): ꢀ = 168.48, 153.52, 134.81, 132.10,
130.86, 129.67, 127.08, 126.97, 77.23, 53.81, 40.63, 24.87,
22.99, 21.59 ppm; HRMS-ESI: m/z calcd. for C14H18ClN2O3
[M+H]+: 297.1006, found: 297.0992.
4.6.1. 3-Benzyloxy-5-isobutylhydantoin (5a)
Yield: 17%; m.p.: 106~108°C; IR (KBr): ꢀ = 3233, 2954,
1782, 1736, 1420, 1202, 745, 698 cm-1; 1H-NMR (600 MHz,
CDCl3): ꢀ = 7.50(dd, J1=1.2 Hz, J2=3.6 Hz, 2H), 7.38(t,
J=1.8 Hz, 3H), 5.75(s, 1H), 5.15(s, 2H), 3.97-3.94 (m, 1H),
1.75-1.68 (m, 2H), 1.45-1.40 (m, 1H), 0.96 (d, J=6.6 Hz,
3H), 0.93 ppm (d, J=6.6 Hz, 3H); 13C-NMR (100 MHz, CD-
Cl3): ꢀ = 168.47, 153.33, 133.33, 130.09, 129.44, 128.52,
77.23, 53.79, 40.72, 29.72, 24.92, 22.97 ppm. HRMS-ESI:
m/z calcd. for C14H19N2O3 [M+H]+: 263.1396, found:
263.1380.
4.6.7. 3-(3-Chlorobenzyloxy)-5-isobutylhydantoin (5g)
Yield: 28%; m.p.: 98~100°C; IR (KBr): ꢀ = 3240ꢀ2955,
4.6.2. 3-(4-Methylbenzyloxy)-5-isobutylhydantoin (5b)
1
1728, 1427, 1201, 787, 709 cm-1; H-NMR (600 MHz, CD-
Yield: 13%; m.p.: 103~104°C; IR (KBr): ꢀ = 3233, 2959,
1782, 1732, 1466, 1202, 810, 740 cm-1; 1H-NMR (600 MHz,
CDCl3): ꢀ = 7.37 (t, J= 8.4 Hz, 2H), 7.18 (d, J=7.8 Hz, 2H),
6.16 (s, 1H), 5.10 (s, 2H), 3.96-3.93 (m, 1H), 2.36 (s, 3H),
1.74-1.66 (m, 2H), 1.44-1.39 (m, 1H), 0.96 (d, J=6.6 Hz,
3H), 0.93 ppm (d, J=6.6 Hz, 3H); 13C-NMR (150 MHz, CD-
Cl3): ꢀ = 168.66, 153.89, 139.41, 130.29, 130.17, 129.19,
Cl3): ꢀ = 7.48 (d, J=6.6 Hz, 1H), 7.41 (d, J=7.4 Hz, 1H),
7.37-7.31 (m, 2H), 5.67 (s, 1H), 5.11 (d, J=6.0 Hz, 2H), 3.98
(dd, J1=9.3 Hz, J2=3.1 Hz, 1H), 1.76-1.71 (m, 2H), 1.46 (t, J
=9.0 Hz, 1H), 0.97 (d, J=6.0 Hz, 3H), 0.95 ppm (d, J=6.0
Hz, 3H); 13C-NMR (100 MHz, CDCl3): ꢀ = 168.53, 153.56,
135.34, 134.36, 129.86, 129.30, 129.03, 127.95, 77.33,
53.87, 40.71, 24.79, 22.90, 21.79 ppm; HRMS-ESI: m/z

Synthesis, Anticancer Evaluation and Docking Study
Medicinal Chemistry, 2016, Vol. 12, No. 1 45
calcd. for C14H18ClN2O3 [M+H]+: 297.1006, found:
297.0999.
4.7.5. 3-(4-Chlorobenzyloxy)-5-isopropylhydantoin (6e)
Yield: 14%; m.p.: 100-101°C; IR (KBr): ꢀ = 3252, 2966,
1786, 1728, 1493, 1408, 1201, 853, 805 cm-1; 1H-NMR (600
MHz, CDCl3): ꢀ = 7.40 (d, J=1.7 Hz, 1H), 7.36 (dd, J1=7.3
Hz, J2=1.4 Hz, 1H), 7.34-7.32 (m, 1H), 7.30 (d, J=7.2 Hz,
1H), 5.80 (d, J=7.2 Hz, 1H), 5.10 (dd, J1=14.4 Hz, J2=10.2
Hz, 2H), 3.88 (dd, J1=3.6 Hz, J2=1.8 Hz, 1H), 2.21-2.18 (m,
1H), 1.00 (d, J=6.6 Hz, 3H), 0.86 ppm (d, J=6.6 Hz, 3H);
13C-NMR (150 MHz, CDCl3): ꢀ = 167.77, 154.60, 135.38,
132.43, 131.34, 130.55, 128.86, 123.64, 77.06, 60.39, 30.08,
18.30, 15.88 ppm; HRMS-ESI: m/z calcd. for C13H16ClN2O3
[M+H]+: 283.0849, found: 283.0840.
4.7. General Procedure for the Synthesis of Hydantoin
Analogues 6a-6g
Valine methyl ester hydrochloride (9 mmol) and 4- nitro-
phenyl-N-[O-(3-chlorobenzylhydroxy)] carbamate (9mmol)
(2a-2g) were reacted as described for 3a-3g to give crude
product as yellow oil substance. The crude product was firs-
tly purified by recrystallization from ether and petroleun
ether, and then recrystallized from acetone and water to af-
ford the expected compounds.
4.7.6. 3-(2-Chlorobenzyloxy)-5-isopropylhydantoin (6f)
4.7.1. 3-Benzyloxy-5-isopropylhydantoin (6a)
Yield: 24%; m.p.: 111~113°C; IR (KBr): ꢀ = 3233, 2959,
1774, 1724, 1477, 1427, 1201, 764, 680 cm-1; 1H-NMR (600
MHz, CDCl3): ꢀ = 7.39 (d, J=1.2 Hz, 1H), 7.36 (d, J=1.2 Hz,
1H), 7.34-7.33 (m, 1H), 7.33-7.30 (m, 1H), 6.61 (s, 1H),
5.10 (dd, J1=13.2 Hz, J2=10.2 Hz, 2H), 3.88 (dd, J1=4.2 Hz,
J2=1.2 Hz, 1H), 2.21-2.18 (m, 1H), 1.02 (d, J=7.2 Hz, 3H),
0.86 ppm (d, J=7.2 Hz, 3H); 13C-NMR (150 MHz, CDCl3): ꢀ
= 167.65, 154.45, 135.37, 134.35, 129.86, 129.81, 129.49,
127.84, 77.04, 60.42, 30.12, 18.32, 15.92 ppm; HRMS-ESI:
m/z calcd. for C13H16ClN2O3 [M+H]+: 283.0849, found:
283.0835.
Yield: 11%; m.p.: 91~92°C; IR (KBr): ꢀ = 3248, 2970,
1771, 1720, 1462, 1416, 1201, 748, 698 cm-1; 1H-NMR (600
MHz, CDCl3): ꢀ = 7.50 (t, J1=9.6 Hz, 2H), 7.37 (t, J1=6.6
Hz, 3H), 5.37 (s, 1H), 5.15 (t, J =15.6 Hz, 2H), 3.84 (dd,
J1=1.2 Hz, J2=1.2 Hz, 1H), 2.18-2.15 (m, 1H), 0.98 (d, J=6.6
Hz, 3H), 0.94 ppm (d, J=6.6 Hz, 3H); 13C-NMR (100 MHz,
CDCl3): ꢀ = 167.64, 154.30, 135.23, 133.44, 129.93, 129.37,
128.80, 128.52, 77.03, 57.43, 52.13, 29.90, 18.59 ppm;
HRMS-ESI: m/z calcd. for C13H17N2O3 [M+H]+: 249.1239,
found: 249.1228.
4.7.7. 3-(3-Chlorobenzyloxy)-5-isopropylhydantoin (6g)
4.7.2. 3-(4-Methylbenzyloxy)-5-isopropylhydantoin (6b)
Yield: 25%; m.p.: 73~74°C; IR (KBr): ꢀ = 3256, 2970,
1771, 1720, 1410, 1201, 791, 687 cm-1; 1H-NMR (600 MHz,
CDCl3): ꢀ = 7.43(dd, J1=4.2 Hz, J2=1.8 Hz, 2H), 7.35 (dd,
J1=1.8 Hz, J2=4.2 Hz, 2H), 6.13 (s, 1H), 5.10 (dd, J1=15.0
Hz, J2=10.8 Hz, 2H), 3.86 (dd, J1=4.2 Hz, J2=1.2 Hz, 1H),
2.20-2.17 (m, 1H), 1.00 (d, J=7.2 Hz, 3H), 0.85 ppm (d,
J=6.6 Hz, 3H); 13C-NMR (150 MHz, CDCl3): ꢀ = 167.58,
154.27, 135.37, 134.36, 130.30, 129.49, 128.65, 127.85,
77.03, 60.40, 30.10, 18.32, 15.91 ppm. HRMS-ESI: m/z
calcd. for C13H16ClN2O3 [M+H]+: 283.0849, found:
283.0856.
Yield: 15%; m.p.: 74-76 °C; IR (KBr): ꢀ = 3233, 2962,
1771, 1724, 1462, 1416, 1201, 802, 695 cm-1; 1H-NMR (600
MHz, CDCl3): ꢀ = 7.38 (d, J=7.8 Hz, 2H), 7.18 (d, J=8.4 Hz,
2H), 5.26 (s, 1H), 5.10 (dd, J1= 10.2 Hz, J2=9.6 Hz, 2H),
3.84 (d, J=3.6 Hz, 1H), 2.35 (s, 3H), 2.17 (t, J=3.6 Hz, 1H),
0.98 (d, J=6.6 Hz, 3H), 0.84 ppm (d, J=7.2 Hz, 3H); 13C-
NMR (150 MHz, CDCl3): ꢀ = 167.55, 154.08, 139.37,
130.36, 130.06, 129.20, 77.02, 60.29, 30.05, 21.33, 18.36
ppm; HRMS-ESI: m/z calcd. for C14H19N2O3 [M+H]+:
263.1396, found: 263.1389.
4.7.3. 3-(4-Bromobenzyloxy)-5-isopropylhydantoin (6c)
4.8. Cytotoxicity Assay in Vitro
Yield: 12%; m.p.: 70-73°C; IR(KBr): ꢀ = 3287, 2966,
1790, 1724, 1462, 1408, 1157, 849, 798 cm-1; 1H-NMR (600
MHz, CDCl3): ꢀ = 7.51 (t, J=9.0 Hz, 2H), 7.38 (d, J=8.4 Hz,
2H), 6.13 (d, J=6.6 Hz, 1H), 5.08 (dd, J1=9.6 Hz, J2=4.2 Hz,
2H), 3.87 (dd, J1=4.2 Hz, J2=1.2 Hz, 1H), 2.21-2.16 (m, 1H),
1.00 (d, J=6.6 Hz, 3H), 0.85 ppm (d, J=6.6 Hz, 3H); 13C-
NMR (150 MHz, CDCl3): ꢀ = 167.78, 154.63, 132.45,
131.72, 131.47, 123.65, 77.08, 60.41, 30.10, 18.31, 15.91
ppm; HRMS-ESI: m/z calcd. for C13H15BrN2O3 [M]+:
326.0266, found: 326.0261.
The human leukemia cell line K562, murine leukemia
cell line L1210 and human laryngreal carcinoma cell line
HEP-2 were purchased from Nanjing Keygen Biotech. Co.,
Ltd., China. The three cell lines were cultured in RPMI-1640
medium supplemented with 10% heat-inactivated fetal bo-
vine serum (FBS), 80 IU /ml penicillin G and 100 IU /ml
streptomycin sulfate in a 5% CO2-95% humidity incubator at
37°C. Each compound was dissolved with DMSO to obtain a
serial concentration of 1ꢁ10-3, 1ꢁ10-4, 1ꢁ10-5, 1ꢁ10-6 and
1ꢁ10-7M. The improved MTT assay was chosen to sdeter-
mine the IC50 of the target compounds 3a-3g, 4a-4g, 5a-5g
and 6a-6g. Tumor cells (1ꢁ106 cells /ml) and the test sam-
ples were inoculated in 96-well culture plates with the quan-
tity of 90μl /well and 10μl /well respectively. After cultured
for 48 h, 20μl of MTT (5mg /mL) was added to each well
and then the mixture was incubated for 4h. After that, the
cultured cells were mixed with 100μl of triple solution (10%
SDS, 5% isobutanol, 0.01 M HCl ) and incubated for 10 h at
37°C. Finally the absorbance of each well was measured at
570 nm. Each experiment was performed at least 3 times.
4.7.4. 3-(4-Fluorobenzyloxy)-5-isopropylhydantoin (6d)
Yield: 15%; m.p.: 97-100°C; IR (KBr): ꢀ = 3132, 1728,
1427, 1218, 941, 763 cm-1; 1H-NMR (400 MHz, CDCl3): ꢀ =
7.52 (s, 1H), 7.37 (s, 1H), 7.17 (s, 1H), 7.08 (s, 1H), 5.72 (s,
1H), 5.22 (s, 2H), 3.86 (s, 1H), 2.18 (s, 1H), 1.00 (s, 3H),
0.87 ppm (s, 3H); 13C-NMR (100 MHz, CDCl3): ꢀ = 167.63,
154.26, 132.29, 131.45, 130.27, 127.58, 124.23, 115.52,
77.04, 60.37, 30.12, 18.27, 16.02 ppm; HRMS-ESI: m/z
calcd. for C13H16FN2O3 [M+H]+: 267.1145, found: 267.1139.

46 Medicinal Chemistry, 2016, Vol. 12, No. 1
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CONFLICT OF INTEREST
The authors confirms that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
The financial support from the National Nature Science
Fundation of China (Grant No. 81360469), the program of
Science and Technology Project of Jiangxi Province
(20122BBG70094-2) and the Nature Science Fundation of
Jiangxi Province (20122BAB205038) are gratefully ac-
knowledged. The authors also thank Center of Analysis and
Testing of Nanchang University for assistance with the MS
and NMR testing of compounds.
[15]
[16]
[17]
SUPPLEMENTARY MATERIAL
[18]
[19]
[20]
Main crystallographic parameters of 4e. 1H-NMR and
13C-NMR for target compouds 3a-3g, 4a-4g, 5a-5g and 6a-
6g.
Supplementary material is available on the publishers
Web site along with the published article.
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Received: January 28, 2015
Revised: July 03, 2015
Accepted: July 03, 2015