Hydantoin derivatives of L- and D-amino acids: Synthesis and evaluation of their antiviral and antitumoral activity

Article abstract of DOI:10.3390/11110837

3,5-Disubstituted hydantoin (1,3-imidazolidinedione) derivatives 5a-h were prepared by base induced cyclization of the corresponding N-(1- benzotriazolecarbonyl)-L-and D-amino acid amides 4a-h. Compounds 5a-h were evaluated for their cytostatic and antivi

Full text of DOI:10.3390/11110837

Molecules 2006, 11, 837-848
molecules
ISSN 1420-3049
http://www.mdpi.org
Full Paper
Hydantoin Derivatives of L- and D-amino acids: Synthesis and
Evaluation of Their Antiviral and Antitumoral Activity
Zrinka Rajic 1, Branka Zorc 1,*, Silvana Raic-Malic 2, Katja Ester 3, Marijeta Kralj 3, Krešimir
Pavelic 3, Jan Balzarini 4, Erik De Clercq 4 and Mladen Mintas 2,*
1 Department of Medicinal Chemistry, Faculty of Pharmacy and Biochemistry, A. Kovačića 1, HR-
10000 Zagreb, Croatia; E-mail: zrajic@pharma.hr
2 Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, Marulićev trg
20, HR-10000 Zagreb, Croatia; E-mail: silvana.raic@fkit.hr
3 Division of Molecular Medicine, Ruđer Bošković Institute, Bijenička cesta 54, HR-10000 Zagreb;
E-mails: kester@irb.hr, mhorvat@irb.hr, pavelic@irb.hr
4 Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-
3000 Leuven, Belgium; E-mail: jan.balzarini@rega.kuleuven.ac.be, erik.declercq@rega.kuleuven.be
* Authors to whom correspondence should be addressed; Branka Zorc: bzbz@pharma.hr; Phone:
(+385) 1 4856202, Fax: (+385) 1 4856201; Mladen Mintas: mladen.mintas@fkit.hr; Phone: (+385) 1
4597214, Fax: (+385) 1 4597224
Received: 6 October 2006; in revised form: 25 October 2006 / Accepted: 25 October 2006 /
Published: 1 November 2006
Abstract: 3,5-Disubstituted hydantoin (1,3-imidazolidinedione) derivatives 5a-h were
prepared by base induced cyclization of the corresponding N-(1-benzotriazolecarbonyl)-L-
and D-amino acid amides 4a-h. Compounds 5a-h were evaluated for their cytostatic and
antiviral activities. Among all the compounds evaluated, 3-benzhydryl-5-isopropyl
hydantoin (5a) showed a weak but selective inhibitory effect against vaccinia virus (EC50 =
16 µg/mL; selectivity index: 25). 3-Cyclohexyl-5-phenyl hydantoin (5g) showed inhibitory
activity against cervical carcinoma (HeLa, IC50 = 5.4 µM) and breast carcinoma (MCF-7,
IC50 = 2 µM), but also cytotoxic effects towards human normal fibroblasts (WI 38). On the
contrary, the 3-benzhydryl-5-phenyl substituted hydantoin derivative 5h showed moderate
inhibitory activity towards HeLa, MCF-7, pancreatic carcinoma (MiaPaCa-2), lung
carcinoma (H 460) and colon carcinoma (SW 620) (IC50 = 20−23 µM), but no effect on WI
38.

Molecules 2006, 11
Keywords: Hydantoin, L- and D-amino acids, antiviral activity, antitumoral activity.
838
Introduction
Hydantoin (1,3-imidazolidinedione) derivatives display diverse and interesting pharmacological
properties. Several such derivatives (phenytoin, mephenythoin, norantoin, methetoin, ethotoin,
fosphenytoin) are well-known anticonvulsive drugs [1,2]. Other 5-substituted hydantoins like 5,5-
dithienylhydantoin, 5,5-dipyridylhydantoin, spirothiohydantoin, thiohydantoin and dithiohydantoins
also possess anticonvulsive activity [3]. Hydantoin derivatives can also be found as antiarrhythmics
(azimilide), antimicrobial agents (nitrofurantoin), skeletal muscle relaxants (dantrolene) and
nonsteroidal antiandrogens (nilutamide), while allantoin is used as a keratolytic, astringent, wound
remedy, antacid and antipsoriatic drug [2]. Hydantoins also exhibit antidepressant, antiviral and
antithrombotic activities, as well as inhibitory activity against some enzymes (human aldose reductase
and human leucocyte elastase) [4]. Finally, some herbicides (spirohydantoin, thioxohydantocidin),
fungicides (clodantoin) and insecticides also have the hydantoin skeleton in their structure [5,6].
During our previous research a series of hydantoins were synthesized and evaluated for their antiviral
and cytostatic activities [7,8]. The best pharmacological activities were achieved with lipophilic
hydantoin derivatives bearing cycloalkyl, phenyl or benzhydryl substituents. This led us to prepare and
pharmacologically evaluate a series of the novel structural analogues of these lead compounds in order
to assess their antiviral and antitumoral potential. The results are reported in this paper.
Results and Discussion
Chemistry
A series of 3,5-disubstituted hydantoins 5a-h were prepared by intramolecular cyclization of N-[(1-
benzotriazolecarbonyl (Btc)]-amino acid amides 4a-h, following our previously described procedures
[7−9] (Scheme 1). All these hydantoins are new compounds, except 5d and 5g, whose syntheses have
been previously described [9−11], although their cytostatic and antiviral activities have not been
reported until now. Synthesis of the N-Btc-amino acid amides 4a-h and their precursors N-Btc-amino
acid derivatives 2a-d and the corresponding chloride derivatives 3a-d were also described in our
previous reports [12]. Syntheses of the compounds 4a-h and 5a-h were carried out under mild reaction
conditions and the products were obtained in high yields. In the preparation of the amides 4a-h, an
equimolar ratio of the N-Btc-amino acid chloride derivatives 3a-d and the corresponding amine was
crucial in order to avoid formation of ureido amides. Our previous research results had shown that 3-
cyclohexanemethyl-5-phenylhydantoin possessed marked antiviral activity (i.e. against cytomegalo-
virus) [7]. Based on that lead compound we have now prepared some novel hydantoins containing one
or two lipophilic substituents at positions 3 and 5. Three amino acids of the L-series (L-valine, L-
leucine and L-phenylalanine) and one D-amino acid (D-phenylglycine) and highly lipophilic amines
(cyclopentyl-, cyclohexyl-, cyclohexanemethyl- or benzhydrylamines) were used as the starting
reagents. As the chiral centre was not involved in chemical transformations of the L- and D-amino acid

Molecules 2006, 11
839
precursors (Scheme 1) these configurations should be retained in the target hydantoin molecules [7].
The validity of this assumption has been proven by X-ray crystallographic analysis of the
stereostructure of the hydantoin derivative of L-valine [8].
Scheme 1. Synthesis of hydantoin derivatives of L- and D-amino acids, 5a-f and 5g,h, respectively
H
R
H
R
R
H
OH
OH
Cl
(i)
(ii)
BtcCl
+
BtcHN
H2N
BtcHN
O
O
O
1
2
3
a
b
c
d
1-3
i-Pr i-Bu Bn Ph
R
(iii)
O
H
O
N
R
H
R
N
N
(iv)
NHR'
BtcCl
=
HN
NR'
BtcHN
O
O
5
f
4
a
b
c
d
e
g
h
4, 5
i-Pr i-Bu
-CHPh2 -CHPh2
Bn Bn
Bn
Bn
-CHPh2
Ph Ph
-CHPh2
R
R'
Reagents and conditions: (i) anhydrous dioxane, rt, 24 h; (ii) SOCl2, rt, 24 h or reflux, 1 h; (iii)
amine, Et3N, anhydrous toluene, rt, 24 h; (iv) Na2CO3, acetone, rt, 2 h.
1
The structures of the newly synthesized compounds were deduced from the analysis of their H-
13
and C-NMR and IR spectra, and confirmed by elemental analysis. The IR spectra of the hydantoin
derivatives (Table 1) showed characteristic bands at 3421−3227 (NH), 1775−1760 and 1718−1698
13
(CO) cm-1. The chemical shifts in 1H- and C-NMR spectra (Tables 2 and 3) are in concordance with
the proposed structures of the novel compounds and also consistent with previously described
analogous hydantoin derivatives [7].
Table 1. Analytical data of the novel N-Btc-L-valine benzhydrylamide (4a) and 3,5-
disubstituted hydantoin derivatives 5a-h.
H
O
R
H
NHBtc
H
N
HN
NR'
CHPh2
O
O
4a
5a-h
Yield
(%)
mp
Molecular
formula (Mr)
C25H25N5O2
(427.25)
Compd.
4a
R
R'
IR (KBr) νmax (cm-1)
(°C)
3287, 2966, 1713, 1650,
1556, 1524
−
−
48
86
155−160
C19H20N2O2
(308.37)
3249, 3063, 3030, 2963,
2930, 1772, 1711, 1417
5a
oil

Molecules 2006, 11
840
Table 1. Cont.
Yield
mp
Molecular
formula (Mr)
C20H22N2O2
(322.40)
Compd.
5b
R
R'
IR (KBr) νmax (cm-1)
(%)
(°C)
3421, 3265, 3110, 2956,
1773, 1711, 1422
88
139−141
C15H18N2O2
(258.32)
3258, 3091, 2959, 2868,
1760, 1718, 1431
5c
5d
5e
73
85
80
121−124
119−120
150−153
C16H20N2O2
(272.34)
3270, 3093, 2926, 2856,
1768, 1707, 1436
C17H22N2O2
(286.37)
314, 3030, 2924, 2852,
1768, 1698, 1454, 1428
3269, 3029, 2924, 1774,
1711, 1419
C23H22N2O2
(356.42)
5f
5g
5h
29
37
24
76 (decomp.)
180−184
C15H18N2O2
(258.32)
3227, 3098, 3037, 2927,
2856, 1765, 1705, 1429
3230, 3093, 2825, 1774,
1712, 1454, 1423
C22H18N2O2
(342.39)
172−177
1
Table 2. H-NMR spectral data of the novel N-Btc-L-valine benzhydrylamide (4a) and
3,5-disubstituted hydantoin derivatives 5a-h.
H
O
R
5
H
NHR
3
5
NHR'
1''
HN
1
NR'
6
2
3
7
O
O
4a
5a-h
Compd.
4a
R
R'
1H-NMR (DMSO-d6) δ ppm
4''
5''
3'
2'' 3''
9.19 (d, 1H, 3, J = 8.4 Hz), 8.68 (d, 1H, 1'', J = 8.5 Hz), 8.25−7.14 (m, 14H,
arom.), 6.20 (d, 1H, 2'', J = 8.5 Hz), 4.49 (d, 1H, 2, J = 7.9 Hz), 2.34−2.23
(m, 1H, 5), 0.96−0.70 (m, 6H, 6, 7)
6''
6''
6''
O
2'
4
9''
8''
10''
7''
14''
13''
N
5'
11''
N
N
12''
4''
5''
2'' 3''
5a
5b
5c
5d
5e
8.40 (s, 1H, 1), 7.38−7.23 (m, 10H, arom.), 6.35 (s, 1H, 2''), 4.07−4,06 (d,
1H, 5, J = 3.3 Hz), 2,08−1,97 (m, 1H, 6, J = 3.4 Hz), 0.93−0.91 (d, 3H, 7,
J = 6.9 Hz), 0.72−0.70 (d, 3H, 8, J = 6.8 Hz)
7
8
9''
6
8''
10''
7''
14''
13''
11''
12''
4''
5''
2'' 3''
8.51 (s, 1H, 1), 7.38−7.22 (m, 10H, arom.), 6.34 (s, 1H, 2''), 4.20−4.16 (q,
1H, 5, J = 4.1 Hz), 1.79−1.74 (m, 1H, 7, J = 4.7 Hz), 1.56−1.36 (m, 2H, 6,
J = 4.7 Hz), 0.88−0.85 (2d, 6H, 8, 9, J = 4.1 Hz)
8
9
9''
7
6
8''
10''
7''
14''
13''
11''
12''
9
8
3''
8.18 (s, 1H, 1), 7.28−7.13 (m, 5H, arom.), 4.30−4.27 (t, 1H, 5, J = 4.4 Hz),
4.09−4.02 (m, 1H, 2'', J = 7.7 Hz), 2.94−2.93 (d, 2H, 6, J = 4.2 Hz),
1.67−1.42 (m, 8H, 3''−6'')
4''
5''
7
6
2''
2''
10
11
9
12
8
6''
3'' 4''
8.15 (s, 1H, 1), 7.28−7.13 (m, 5H, arom.), 4.30−4.27 (t, 1H, 5, J = 4.4 Hz),
3.52−3.44 (m, 1H, 2'', J = 3.5 Hz), 2.94−2.92 (t, 2H, 6, J = 3.5 Hz),
1.85−0.94 (m, 10H, 3''−7'')
7
7
6
6
5''
10
10
11
9
7'' 6''
4''
12
8
5''
8.21 (s, 1H, 1), 7.27−7.14 (m, 5H, arom.), 4.39−4.36 (t, 1H, 5, J = 4.2 Hz),
3.05−2.87 (m, 4H, 6, 2''), 1.61−0.50 (m, 11H, 3''−8'')
6''
2'' 3''
8''
7''
11
12

Molecules 2006, 11
841
Table 2. Cont.
Compd.
R
R'
1H-NMR (DMSO-d6) δ ppm
4''
5''
2'' 3''
9
8
8.44 (s, 1H, 1), 7.30−6.73 (m, 15H, arom.), 6.13 (s, 1H, 2''), 4.53−4.50 (t, 1H,
5, J = 4.3 Hz), 3.07−2.94 (m, 2H, 6)
6''
7
6
9''
8''
10''
7''
10
5f
14''
13''
11
8
12
7
11''
12''
3'' 4''
5g
8.64 (s, 1H, 1), 7.44−7.29 (m, 5H, arom.), 5.13 (s, 1H, 5), 3.81−3.70 (m, 1H,
2'', J = 3.8 Hz), 2.12−1,02 (m, 10H, 3''−7'')
6
2''
5''
9
10 11
7'' 6''
4''
5''
2'' 3''
8
7
6''
6
9''
8''
7''
9
10''
5h
14''
13''
8.91 (s, 1H, 1), 7.43−7.20 (m, 15H, arom.), 6.39 (s, 1H, 2''), 5.34 (s, 1H, 5)
10 11
11''
12''
13
Table 3. C-NMR spectral data of the novel N-Btc-L-valine benzhydrylamide (4a) and
3,5-disubstituted hydantoin derivatives 5a-h.
H
O
R
5
4
H
NHR
5
NHR'
1''
HN
1
NR'
2
6
2
1
3
7
O
O
4a
5a-h
Compd.
4a
R
R'
13C-NMR (DMSO-d6) δ ppm
169.29 (1), 148.59 (4), 145.45 (1'), 142.00 (3'', 9''), 131.17 (6'),
130.08 (5'), 128.30 (5'', 7'', 11'', 13''), 128.15 (4'', 8''), 127.24 (10'',
14''), 127.15 (6''), 126.99 (12''), 125.61 (4'), 119.82 (3'), 113.37
(2'), 63.90 (2), 56.04 (2'') 30.77 (5), 19.17 (6), 18.20 (7)
173.63 (4), 156.87 (2), 138.63 (3''), 138.37 (9''), 128.56(4'', 8''),
128.37 (5'', 7''), 128.32 (10'', 14''), 128.22 (11'',13''), 127.64 (6''),
127.53''), 61.06 (2''), 57.14 (5), 29.92 (6), 18.64 (7), 15.84 (8)
174.34 (4), 156.22 (2), 138.30 (3''), 138.21 (9''), 128.15 (4'', 8'',
10'', 14''), 128.12 (5'', 7'', 11'', 13''), 127.33 (6'', 12''), 56.85 (2''),
54.44 (5), 40.75 (6), 23.96 (7), 22.98 (8), 21.27 (9)
4''
5''
3'
2'' 3''
6''
O
2'
4'
5'
9''
1'
8''
10''
7''
14''
13''
N
4
6'
11''
N
N
12''
4''
5''
2'' 3''
6''
9''
7
8''
10''
7''
14''
13''
5a
5b
5c
5d
5e
5f
6
11''
8
12''
4''
5''
2'' 3''
6''
9''
8
8''
10''
7''
14''
13''
7
6
11''
9
12''
175.20 (4), 158.25 (2), 136.80 (7), 131.64 (8, 12), 129.81 (9, 11),
128.55 (10), 58.11 (5), 51.90 (2''), 38.30 (6), 30.30 (6''), 30.02
(3''), 26.39 (4'', 5'')
9
8
3''
4''
5''
7
7
6
6
2''
2''
10
10
11
9
12
8
6''
3'' 4''
7'' 6''
173.20 (4), 156.22 (2), 134.78 (7), 129.76 (8, 12), 127.82 (9, 11),
126.60 (10), 56.04 (5), 49.64 (2''), 36.28 (6), 28.58 (3''), 28.39
(7''), 25.18 (4'', 6''), 24.68 (5'')
5''
11
12
8
9
173.27 (4), 156.60 (2), 134.78 (7), 129.80 (8, 12), 127.82 (9, 11),
126.58 (10), 56.71 (5), 43.37 (2''), 35.95 (6), 35.66 (3''), 29.79
(4''), 29.52 (8''), 25.65 (5''), 25.17 (7''), 25.12 (6'')
5''
4''
8''
7
6
10
6''
2'' 3''
7''
5''
11
9
12
8
4''
2'' 3''
6''
172.87 (4), 155.72 (2), 138.01 (3''), 137.95 (9''), 134.65 (7),
126.78−130.01 (8−12, 4''−8'', 10''−14'') 56.62 (5, 2''), 35.78 (6)
7
6
9''
8''
10''
7''
10
14''
13''
11
12
11''
12''

Molecules 2006, 11
842
Table 3. Cont.
Compd.
5g
R
R'
13C-NMR (DMSO-d6) δ ppm
172.36 (4), 156.61 (2), 135.91 (6), 128.62 (7, 11), 128.23 (8, 10),
126.68 (9), 59.08 (5), 50.22 (2''), 28.92(3''), 28.83 (7''), 25.27 (4''),
25.24 (6''), 24.72 (5'')
8
7
3'' 4''
6
2''
5''
5''
9
7'' 6''
10 11
4''
2'' 3''
8
7
6''
172.35 (4), 156.54 (2), 138.96 (3''), 138.30 (9''), 135.85 (6),
128.94−126.87 (7−11, 4''−8'', 10''−14''), 59.66 (2''), 57.35 (5)
6
9''
8''
10''
7''
9
14''
13''
5h
10 11
11''
12''
Biological Results
The activities of the novel 3,5-disubstituted hydantoins 5a-h were evaluated against herpes simplex
virus-1 (HSV-1 KOS strain ), herpes simplex virus-2 (HSV-2 strain G), herpes simplex virus-1 (ACV-
resistant KOS strain), vaccinia virus, vesicular stomatitis virus, Coxsackie virus B4, respiratory
syncytial virus, parainfluenza-3 virus, reovirus-1, Sindbis virus and Punta Toro virus. These activities
were compared with those of acyclovir, ganciclovir and ribavirin (Table 4).
Table 4. Antiviral activity of the novel 3,5-disubstituted hydantoins 5a-h.
b
MCC
EC50
Compd.
a
1c
2
3
4
5
6
7
8
9
10
48
16
11
5a
400
80
> 80
> 16
80
80
16
80
> 80
> 16
240
80
> 80
> 16
> 80
> 16
> 80
> 16
> 80
> 16
5b
> 16
> 16
> 80
> 16
> 80
> 16
> 16
> 16
> 16
> 16
5c
> 400 > 400 240
240 > 400
> 400 > 400 > 400 240 > 400
5d
80
400
80
> 16
> 80
> 16
> 16
> 16
0.11
0.02
> 16
> 80
> 16
> 16
> 16
0.18
0.02
> 16
> 80
16
> 80
> 16
> 16
> 80
> 16
> 80
> 16
> 16
> 80
> 16
nd
> 80
> 16
16
> 80
> 16
16
> 80
> 16
16
> 80
> 16
> 16
> 80
> 16
nd
5e
> 16
16
5f
5g
80
> 16
> 16
> 80
> 80
16
> 80
16
> 80
16
5h
80
16
acyclovir
ganciclovir
ribavirin
> 100
> 25
13.51 67.5 > 100
0.2 15.3 > 25
ndd
nd
nd
nd
nd
nd
nd
nd
nd
nd
> 100 > 100 73.3
> 100 24.4 > 100 > 100 > 100
73.3
73.3
24.4
73.3
a
MCC – minimum cytotoxic concentration (µg mL−1) that causes a microscopically detectable alternation
of cell morphology; EC50 – effective concentration (µg mL−1) required to reduce virus plaque formation
b
by 50%. Virus input was 100 plaque forming units (PFU); c Viruses: 1 – HSV-1 (KOS), 2 – HSV-2 (G), 3 –
HSV-1 TK KOS ACVr, 4 – Vaccinia virus, 5 – Vesicular stomatitis virus, 6 – Coxsackie virus B4, 7 –
Respiratory syncytial virus, 8 – Parainfluenza-3 virus, 9 – Reovirus-1, 10 – Sindbis virus, 11 – Punta Toro
virus; d Not determined.
Structurally related hydantoin derivatives 5f and 5h exhibited similar inhibitory effects against the
evaluated viruses. Thus, these compounds showed some antiviral activity against Coxsackie virus,
parainfluenza-3 virus, reovirus-1 and Sindbis virus (EC50 = 16 µg mL−1); which is 5-fold lower than
the minimum cytotoxic concentration (MCC). 3-Benzhydryl-5-isopropyl hydantoin (5a) showed
inhibitory effects against vaccinia virus (EC50 = 16 µg mL−1), at 25-fold lower concentration than the
MCC.

Molecules 2006, 11
843
The 3,5-disubstituted hydantoin derivatives 5a-h were also evaluated for their activities against
malignant tumor cell lines: murine leukemia (L1210) and human T-lymphocytes (Molt4/C8 and
CEM), cervical carcinoma (HeLa), breast carcinoma (MCF-7), pancreatic carcinoma (MiaPaCa-2),
lung carcinoma (H 460), colon carcinoma (SW 620), as well as normal fibroblasts (WI 38) (Table 5).
Table 5. Inhibitory effects of the novel 3,5-disubstituted hydantoins 5a-h on the growth
of human malignant tumor cell lines and normal diploid fibroblasts (WI 38).
Tumor cell growth a
Compds
1b
102 ± 3 183 ± 38
2
3
4
5
6
7
8
9
5a
5b
83 ± 4
> 100
36 ± 13
16 ± 0.6
63 ± 14
18 ± 1
22 ± 2
11 ± 1
2 ± 1.6
20 ± 10
> 100
47 ± 5
46 ± 29
> 100
65 ± 35
43 ± 1.5
> 100
62 ± 20
31 ± 27
> 100
77 ± 23
42 ± 2
> 100
57 ± 21 232±194 148 ± 95 47 ± 44
> 500 > 500 406 ± 16 > 100
303 ± 9 317 ± 12 266 ± 15 37 ± 141
5c
5d
81 ± 10
70 ± 19
14 ± 0.9
58 ± 42
> 100
78 ± 21
74 ± 25
15 ± 2
55 ± 14
42 ± 2
13 ± 5
5e
312±128
42 ± 2
> 500
246 ± 85
48 ± 4
38 ± 8
> 100
5f
53 ± 2
13 ± 1.4
19 ± 4
58 ± 25
5g
172 ± 52 277 ± 45 146 ± 14 5.4 ± 13
60 ± 35 1.5 ± 0.3
5h
46 ± 4
ndc
52 ± 5
nd
50 ± 4
nd
21 ± 3
42 ± 38
4 ± 1.5
4 ± 3
22 ± 0.7 23 ± 0.7 21 ± 0.9
> 100
> 100
HU
5-FU
> 100
10 ± 3
5 ± 1
> 100
2 ± 0.7
> 100
9 ± 2
8 ± 6
nd
nd
nd
15 ± 2
12 ± 6
14 ± 12
20 ± 19
Cis
nd
nd
nd
0.3 ± 0.1
a
IC50 – 50% inhibitory concentration or compound concentration (µM) required to inhibit tumor cell
b
proliferation by 50%; Tumor cells: 1 – L1210, 2 – MoltC4/C8, 3 – CEM, 4 – HeLa, 5 – MCF-7, 6 –
c
MiaPaCa-2, 7 – H 460, 8 – SW 620, 9 – normal diploid fibroblasts WI 38; Not determined; HU –
hydroxyurea, 5-FU – 5-fluorouracil, Cis – cisplatin.
Amongst all evaluated compounds, 3-cyclohexyl-5-phenyl hydantoin (5g) showed rather marked
inhibitory activity against HeLa (IC50 = 5.4 µM) and MCF-7 (IC50 = 2 µM) cell lines, but also high
inhibitory effects on normal fibroblasts WI 38 (Figure 1).
Figure 1. Dose-response profile for compounds 5f, 5g and 5h tested in vitro on various
human cell lines *.
Compound 5f
150
WI 38
100
HeLa
50
0
SW 620
H 460
MCF-7
-50
-100
MiaPaCa-2
-9
-8
-7
-6
-5
-4
-3
log10 concentration (M)

Molecules 2006, 11
844
Figure 1. Cont.
Compound 5g
150
100
50
WI 38
HeLa
SW 620
H 460
0
MCF-7
MiaPaCa-2
-50
-100
-9
-8
-7
-6
-5
-4
-3
log10 concentration (M)
Compound 5h
150
100
50
WI 38
HeLa
SW 620
H 460
0
MCF-7
MiaPaCa-2
-50
-100
-9
-8
-7
-6
-5
-4
-3
log10 concentration (M)
* The cells were treated with the compounds 5f-h at different concentrations, and percentage of
growth (PG) was calculated. Each point represents the mean value of four parallel samples in
three individual experiments.
Similarly, 3-benzhydryl-5-benzyl hydantoin (5f) showed moderate inhibitory activity against all
human tumor cell lines examined and also normal fibroblasts (WI 38). By contrast, the 3-benzhydryl-
5-phenyl substituted hydantoin derivative 5h showed a rather marked inhibitory activity against HeLa
(IC50 = 21 µM), MCF-7 (IC50 = 20 µM), MiaPaCa-2 (IC50 = 22 µM), H 460 (IC50 = 23 µM) and SW
620 (IC50 = 21 µM), and no cytotoxic effect on WI 38 fibroblasts.
Conclusions
In summary, the hydantoin derivatives presented herein showed certain antiviral and antitumor
activity. Derivatives 5f and 5h exhibited weak inhibitory effects against the evaluated viruses and
hydantoin 5a showed a weak but selective inhibitory effect against vaccinia virus. With regards to their
antitumor activity, hydantoin 5g showed rather marked inhibitory activity against HeLa and MCF-7
cell lines, while 5h showed inhibitory activity towards several tumor cell lines and no cytotoxic effects
on normal cells. Further optimization of the hydantoin derivatives as potential antiviral and cytostatic
agents is in progress.

Molecules 2006, 11
Experimental
General
845
Melting points were determined on a Boëtius Micro Heating Stage and are uncorrected. IR spectra
1
were recorded on a FTIR Perkin Elmer Paragon 500 spectrometer. H- and 13C-NMR spectra were
1
13
recorded on a Varian Gemini 300 spectrometer, operating at 300 and 75.5 MHz for the H- and C-
nuclei, respectively. Samples were measured in DMSO-d6 solutions at 20 ºC in 5 mm NMR tubes.
Chemical shifts (δ) in ppm were referred to TMS. Precoated silica gel 60 F254 plates (Merck) were
used for thin-layer chromatography, with 1:1 cyclohexane/ethyl acetate as the eluent. Spots were
visualized by short-wave UV light and iodine vapour.
Chemistry
N-Btc-amino acid amides 4a-h
The starting compounds for the hydantoin preparations, namely N-Btc-L-leucine benzhydrylamide
(4b), N-Btc-L-phenylalanine cyclopentylamide (4c), N-Btc-L-phenylalanine cyclohexylamide (4d), N-
Btc-L-phenylalanine cyclohexanemethylamide (4e), N-Btc-L-phenylalanine benzhydrylamide (4f), N-
Btc-D-phenylglycine cyclohexylamide (4g), N-Btc-D-phenylglycine benzhydrylamide (4h) were
prepared by procedures described previously [7,9], whereas the analogous L-valine benzhydrylamide
(4a) is a new compound.
N-Btc-L-valine benzhydrylamide (4a). To a cold solution of N-Btc-L-valine chloride 3a [9] (1.122 g, 4
mmol) in toluene (40 mL), a solution of benzhydrylamine (0.732 g, 4 mmol) and triethylamine (0.404
g, 4 mmol) in toluene (30 mL) was added dropwise. The reaction mixture was stirred at room
temperature for 24 h. Product 4a, which precipitated together with TEA·HCl, was filtered off and
dissolved in a dichloromethane/water mixture. The organic layers were extracted with diluted
hydrochloride acid (w = 1%) and water, dried over sodium sulfate and evaporated in vacuo. The
1
13
residue was triturated with ether to give the analytically pure 4a. The analytical, H- and C-NMR
spectral data for 4a are given in Tables 1−3.
General method for the synthesis of 3,5-disubstituted hydantoins 5a-h
To a solution of N-(1-benzotriazolecarbonyl)-amino acid derivatives 4a-h (1.5 mmol) in acetone
(60 mL), 5% sodium carbonate solution (5 mL) was added. The reaction mixture was stirred 2 h at
room temperature. Acetone was evaporated in vacuo and the precipitated products 5a-h were filtered
off, washed with water and recrystallized from acetone and water. The following hydantoins were
prepared in this fashion: 3-benzhydryl-5-isopropyl hydantoin (5a), 3-benzhydryl-5-isobutyl hydantoin
(5b), 3-cyclopentyl-5-benzyl hydantoin (5c), 3-cyclohexyl-5-benzyl hydantoin (5d), 3-cyclohexane-
methyl-5-benzyl hydantoin (5e), 3-benzhydryl-5-benzyl hydantoin (5f), 3-cyclohexyl-5-phenyl

Molecules 2006, 11
846
hydantoin (5g) and 3-benzhydryl-5-phenyl hydantoin (5h). Their analytical data and yields are
presented in Table 1, while 1H- and 13C-NMR data are given in Tables 2 and 3, respectively.
Antiviral Activity Assays
Antiviral activity against HSV-1, HSV-2, vaccinia virus, vesicular stomatitis virus, Coxsackie
virus B4, respiratory syncytial virus, parainfluenza-3 virus, reovirus-1, Sindbis virus and Punta Toro
virus was determined as described previously [13,14]. Antiviral activity was expressed as the EC50 or
concentration required to reduce virus-induced cytopathogenicy by 50%. EC50 values were calculated
from graphic plots of the percentage of cytopathogenicity as a function of concentration of the
compounds.
Antitumoral Activity Assays
The HeLa (cervical carcinoma), MCF-7 (breast carcinoma), MiaPaCa-2 (pancreatic carcinoma), H
460 (lung carcinoma), SW 620 (colon carcinoma) and WI 38 (diploid fibroblasts) cells were cultured
as monolayers and maintained in Dulbecco's modified Eagle’s medium (DMEM) supplemented with
10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin
in a humidified atmosphere with 5% CO2 at 37˚C. Antitumoral activity against L1210 (murine
leukemia), Molt4/C8 and CEM (human T-lymphocytes) cell lines was measured essentially as
originally described for the mouse leukemia (L1210) cell line [15].
The growth-inhibitory activity of HeLa, MCF-7, MiaPaCa-2, H 460, SW 620 and WI 38 cells was
assessed according to a slightly modified procedure performed at the National Cancer Institute,
Developmental Therapeutics Program [16]. The cells were seeded into a series of standard 96-well
microtiter plates on day 0. Test agents were then added in five, 10-fold dilutions (10-8 to 10-4 M) and
incubated for a further 72 hours. Working dilutions were freshly prepared on the day of testing. The
solvent (DMSO) was also tested for eventual inhibitory activity by adjusting its concentration to be the
same as for the tested concentrations. After 72 hours of incubation the cell growth rate was evaluated
by MTT assay [17] which detects dehydrogenase activity in viable cells. The absorbance (OD, optical
density) was measured by a microplate reader at 570 nm. The percentage of growth (PG) of the cell
lines was calculated according to one or the other of the following two expressions:
If (mean ODtest – mean ODtzero) ≥ 0, then:
PG = 100 x (mean ODtest – mean ODtzero) / (mean ODctrl – meanODtzero
)
If (mean ODtest – mean ODtzero) < 0, then:
G = 100 x (mean ODtest – mean ODtzero) / ODtzero
where mean ODtzero = the average of optical density measurements before exposure of cells to the test
compound, mean ODtest = the average of optical density measurements after the desired period of time
and mean ODctrl = the average of optical density measurements after the desired period of time with no
exposure of cells to the test compound.

Molecules 2006, 11
847
Each test point was measured in quadruplicate in three individual experiments. The results are
expressed as IC50, a concentration necessary for 50% of inhibition. Each result is a mean value from
three separate experiments. The IC50 values for each compound were calculated from dose-response
curves using linear regression analysis by fitting the test concentrations that gave PG values above and
below the reference value (i.e. 50%).
Acknowledgements
Support for this study by the Ministry of Science, Education and Sports of the Republic of Croatia
(Projects No. 0006543, 0125003 and 0098093) is gratefully acknowledged.
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Sample Availability: Samples of the compounds are available from authors.
© 2006 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.