The novel L- and D-amino acid derivatives of hydroxyurea and hydantoins: Synthesis, X-ray crystal structure study, and cytostatic and antiviral activity evaluations

Article abstract of DOI:10.1021/jm040869i

The novel L- and D-amino acid derivatives of hydroxyurea 5a-o were prepared by aminolysis of N-(1-benzotriazolecarbonyl)amino acid amides 4a-o with hydroxylamine. The hydantoin derivatives 6a-e,m,p were synthesized by base-catalyzed cyclization of amides

Full text of DOI:10.1021/jm040869i

J. Med. Chem. 2005, 48, 475-482
475
The Novel L- and D-Amino Acid Derivatives of Hydroxyurea and Hydantoins:
Synthesis, X-ray Crystal Structure Study, and Cytostatic and Antiviral Activity
Evaluations
Ninoslav Opacˇic´,† Monika Barbaric´,† Branka Zorc,*,† Mario Cetina,‡ Ante Nagl,‡ Danijel Frkovic´,§
Marijeta Kralj,§ Kresˇimir Pavelic´,§ Jan Balzarini,| Graciela Andrei,| Robert Snoeck,| Erik De Clercq,|
Silvana Raic´-Malic´, and Mladen Mintas*,
Department of Medicinal Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacˇic´a. 1,
HR-10 000 Zagreb, Croatia, Faculty of Textile Technology, University of Zagreb, Pierottijeva 6, HR-10000 Zagreb, Croatia,
Division of Molecular Medicine, Ru[er Bosˇkovic´ Institute, Bijenicˇka cesta 54, HR-10000 Zagreb, Croatia, Rega Institute for
Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium, and Department of
Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Marulic´ev trg 20,
HR-10000 Zagreb, Croatia
Received August 12, 2004
The novel L- and D-amino acid derivatives of hydroxyurea 5a-o were prepared by aminolysis
of N-(1-benzotriazolecarbonyl)amino acid amides 4a-o with hydroxylamine. The hydantoin
derivatives 6a-e,m,p were synthesized by base-catalyzed cyclization of amides 4, common
precursors for 5 and 6. X-ray crystal structure analysis shows that the C5 atom in 6e possesses
the S configuration, which is consistent with the configuration of the starting reagent, L-leucine.
Among L-amino acid derivatives of hydroxyurea, 5h and 5i inhibited specifically murine
leukemia and human T-lymphocytes (IC50 ) 10-19 µM) and showed selectivity with respect
to normal human fibroblasts (WI 38). D-Amino acid derivatives of hydroxyurea 5m and 5o
inhibited the growth of all tumor cell lines (IC50 ) 4.8-83.9 µM), but not the growth of normal
fibroblasts (WI 38; IC50 > 100 µM). Results on antiviral evaluations showed that N-(1-
benzotriazolecarbonyl)amino acid amide 4m and hydantoin 6m had marked activity against
the Davis strain of CMV (4m, EC50 ) 3.2 µg/mL; 6m, EC50 ) 4.0 µg/mL). However, these
compounds showed also rather expressed cytotoxicity (4m, CC50 ) 43.4 µg/mL; 6m, CC50
)
12.5 µg/mL-1).
Introduction
hydantoins are well-known anticonvulsive drugs and
herbicides.1
Hydroxyurea (hydroxycarbamide, HU) is an antineo-
plastic agent used to treat leukemia and other malig-
nant tumor diseases.1 The drug is also useful in therapy
of sickle cell anemia2 and in combined antiretroviral
therapy,3 where it is most effective if taken in combina-
tion with reverse transcriptase inhibitors (ddI, AZT,
ddC, or d4T). HU inhibits ribonucleotide reductase as
well as cellular DNA synthesis during the S phase of
cell division.4 Because HU targets a cellular enzyme
instead of a viral enzyme, no viral mutations occur after
repeated exposure to the drug. In addition, HU slows
down HIV mutations and resistance development.4
Hydroxyurea is structurally related to hydroxamic acids,
important iron chelators and microbial siderophores.
Hydroxamic acids possess diverse biological activities,
including antibacterial, antifungal, antitumor, and an-
tiinflammatory ones.5 Finally, some derivatives of hy-
droxamic acid are currently accepted therapeutic agents
(desferrioxamine B, hydroxycarbamide, ibuproxam, ox-
ametacin, bufexamac, adrafinil).5 On the other hand,
The main purpose of this study was to investigate the
effects of the novel L- and D-amino acid derivatives of
hydroxyurea 5, hydantoins 6, and their synthetic pre-
cursors N-(1-benzotriazolecarbonyl)amino acid amides
4 on the proliferation of different human tumor cell lines
and to evaluate their potential as antiviral agents.
Chemistry
The L- and D-amino acid derivatives of hydroxyureas
(5a-k and 5l-o) were prepared by aminolysis of N-[(1-
benzotriazolecarbonyl (Btc)]amino acid amides (4a-o)
with hydroxylamine (Scheme 1). All amides 4a-p and
hydroxyureas 5a-o are new compounds prepared ac-
cording to previously described procedures.6 The syn-
thesis of common intermediates BtcCl and N-Btc amino
acids (2) has been also described.7 Three amino acids
of the L-series (L-valine, L-leucine, and L-phenylalanine)
and one D-amino acid (D-phenylglycine) were chosen as
the starting reagents for the synthesis of the desired
molecules. To obtain compounds with different lipophi-
licity, various amines (cyclopentylamine, cyclohexyl-
amine, cyclohexanemethylamine, pyrrolidine, piperi-
dine, and aminodiphenylmethane) were used for the
preparation of the amides 4a-p. Intramolecular cy-
clization of N-(1-benzotriazolecarbonyl)amino acid
amides 4 with sodium carbonate gave hydantoin deriva-
tives 6.
* To whom correspondence should be addressed. B.Z.: phone,
38514856202; fax, 38514856201; e-mail, bzbz@pharma.hr. M.M.: phone,
38514597214; fax, 38514597224; e-mail, mladen.mintas@pierre.fkit.hr.
† Faculty of Pharmacy and Biochemistry, University of Zagreb.
‡ Faculty of Textile Technology, University of Zagreb.
§ Ru[er Bosˇkovic´ Institute.
| Katholieke Universiteit Leuven.
Faculty of Chemical Engineering and Technology, University of
Zagreb.
10.1021/jm040869i CCC: $30.25 © 2005 American Chemical Society
Published on Web 12/31/2004

476 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2
Opacˇic´ et al.
Scheme 1. Synthesis of L- and D-Amino Acid
Derivatives of Hydroxyurea 5a-k and 5l-o and
Hydantoins 6a-e, 6m, and 6p, Respectivelya
Figure 1. The molecular structure and labeling of compound
6e. Displacement ellipsoids are drawn at the 40% probability
level.
1748-1708 (CO bond to benzotriazole), 1679-1627
(amide I), and 1565-1504 cm-1 (amide II). IR spectra
of hydroxyureas 5a-o showed characteristic bands at
3430-3370 (OH), 3359-3172 (NH), 1659-1600 (urea
and amide carbonyls), and 1560-1522 (amide II) cm-1
,
while IR spectra of hydantoins 6a-e,m,p showed
characteristic bands at 3320-3230 (NH), 1775-1760,
and 1710-1695 (CO) cm-1
.
X-ray Crystal Structure Study
The crystal structure of the compound 6e was deter-
mined by X-ray crystal structure analysis. The molec-
ular structure with the atom numbering is displayed
in Figure 1.
The skeleton of 6e consists of a five-membered hy-
dantoin ring, a cyclohexane ring attached via a meth-
ylene group to the hydantoin ring atom N3, and an
isobutyl group attached to the hydantoin ring atom C5.
The absolute configuration of the hydantoin ring C5
atom is S, which is in agreement with its assignment
in the starting reagent (L-leucine).
a 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) NH2OH, anhydrous toluene, rt, 2-10 days;
(v) Na2CO3, acetone, rt, 2 h.
The hydantoin ring deviates slightly from planarity;
the largest observed deviation of the ring atoms from
their mean plane is 0.044(1) Å for the atom C5. The
carbonyl oxygen atom O1 lies in the plane of the ring.
The dihedral angle between the C2-O1 bond line and
the mean plane of the hydantoin ring is 0.4(1)°. On the
other hand, the carbonyl oxygen O2 atom is slightly
shifted away from the ring plane, with the correspond-
ing dihedral angle of 5.6(1)°. As expected, the exocyclic
angles around N3 [C2-N3-C6 ) 123.3(1)°, C4-N3-
C6 ) 125.3(1)°] are greater than the endocyclic angle
[C2-N3-C4 ) 111.3(1)°]. However, the sum of the
angles amounts to 359.9°, indicating no pyramidaliza-
tion of the N3 atom. The C4 and C5 atoms of the
hydantoin ring are in an antiperiplanar position with
respect to the atoms C14 and C16 of the isobutyl group.
The torsion angles C4-C5-C13-C14 and C5-C13-
C14-C16 amount to -177.9(1)° and 177.3(1)°, respec-
tively. The cyclohexane ring thus adopts a chair con-
formation.
The molecules of 6e are joined by the N1‚‚‚O2
hydrogen bond into infinite chains parallel to the c axis,
with the N‚‚‚O distance of 2.919(2) Å and the N-H‚‚‚O
angle of 170(2)° (for a crystal packing diagram, see the
Supporting Information). These chains are additionally
linked by four C-H‚‚‚O intermolecular hydrogen bonds
[C‚‚‚O distances ) 3.266(2)-3.394(2) Å], thus forming
a two-dimensional network. The hydrogen-bonded mol-
ecules are assembled in such manner that they form
cavities along the a axis. The formation of cavities may
The L- and D-amino acid derivatives of hydroxyureas
5a-k and 5l-o presented in Scheme 1 were obtained
by sequence of chemical transformations, which do not
involve a stereogenic center. Therefore, the configura-
tion of that center should be retained in the final
molecules. To provide the experimental proof for this
generally adopted rule, we have synthesized the L-
leucine hydantoin derivative 6e from 4e, the common
precursors for both products (5e and 6e). Despite many
attempts, we have not succeeded in obtaining single
crystal of hydroxyureas 5a-o of satisfactory quality for
crystallographic analysis. However, we achieved the
growth of single crystals of the hydantoin 6e that were
suitable for X-ray crystal structure analysis.
All syntheses were carried out under mild reaction
conditions. In preparation of the amides 4a-p, an
equimolar ratio of the Btc-amino acid chlorides 3a-d
and the corresponding amine was found to be crucial
in order to avoid ureido amides formation.
Structures of compounds 4a-p, 5a-o, and 6a-e,m,p
1
were deduced from analysis of their H and 13C NMR
spectra (see Tables 6-8 in the Supporting Information)
and confirmed by elemental analysis. The chemical
shifts are consistent with the proposed structures of the
novel compounds 4-6. The chemical shifts assignments
are in agreement with the corresponding ones of the
structurally related molecules.6 IR spectra of the amides
4a-o showed absorption bands at 3423-3220 (NH),

Derivatives of Hydroxyurea and Hydantoins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2 477
Table 1. Inhibitory Effects of N-(1-Benzotriazolecarbonyl)amino Acid Amides 4a-o (Scheme 1) on the Growth of Malignant Tumor
Cell Lines in Comparison with Their Effects on the Growth of Normal Human Fibroblasts (WI 38)
tumor cell growth IC50a (µM)
compd
L1210
Molt4/C8
CEM
SW 620
Hep-2
MiaPaCa-2
MCF-7
HeLa
WI 38
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
>200
108 ( 14
102 ( 13
109 ( 6
123 ( 62
17 ( 2
>200
>200
97 ( 4
>100
>100
60.3 ( 1.6
60 ( 0.6
55.7 ( 2.8
57.9 ( 1.6
>100
>100
>100
79.3 ( 34.6
g100
96.4 ( 11.5
60.9 ( 53
82.3 ( 7.5
63.9 ( 21
g100
g100
>100
112 ( 14
116 ( 17
107 ( 8
164 ( 8
139 ( 15
106 ( 60
70 ( 7
94 ( 4
>100
g100
63.7 ( 15.8
63.1 ( 4.1
69.2 ( 22.2
>100
>100
98 ( 7
96 ( 5
>100
>100
40.2 ( 56.9
>100
>100
>100
62.7 ( 78
>100
79 ( 50
61 ( 23
116 ( 30
145 ( 6
123 ( 33
15 ( 0
63.5 ( 31.9
>100
55.3 ( 16.6
95 ( 47
69.3 ( 42
>100
60 ( 3.8
53.8 ( 7
34.3 ( 15.3
>100
68 ( 7
92 ( 97
126 ( 40
77 ( 21
133 ( 94
17 ( 1
110 ( 105
>100
64.5 ( 34.6
81 ( 6.8
g100
20.7 ( 18.6
24.7 ( 22.8
g100
63.2 ( 1.2
>100
142 ( 43
192 ( 11
18 ( 1
g100
>100
g100
>100
>100
>100
37.6 ( 3.4
>100
40.5 ( 1.9
69.7 ( 12.8
68.6 ( 12.8
>100
23.9 ( 3.5
g100
16.3 ( 15.1
57.6 ( 6.5
35.6 ( 26.3
67.4 ( 65.2
34.2 ( 6.9
30.2 ( 9
67 ( 25
79.8 ( 17.4
>100
14.3 ( 7.3
>100
95 ( 0
118 ( 14
123 ( 15
94 ( 7
92 ( 9
98 ( 6
73 ( 20
82 ( 9
>100
72.7 ( 19.1
g100
42.5 ( 21.8
>100
g100
82 ( 1
>100
114 ( 31
88 ( 3
89 ( 8
51.7 ( 3.6
59.1 ( 13.9
33.3 ( 9
100 ( 62.5
a IC50, the concentration that causes 50% growth inhibition.
Table 2. Inhibitory Effects of Amino Acid Derivatives of Hydroxyurea 5a-o (Scheme 1) on the Growth of Malignant Tumor Cell
Lines in Comparison with Their Effects on the Growth of Normal Human Fibroblasts (WI 38)
tumor cell growth IC50a (µM)
compd
L1210
Molt4/C8
CEM
SW 620
Hep-2
MiaPaCa-2
MCF-7
HeLa
WI 38
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
>200
>200
>200
>200
>200
>200
>100
>100
>100
>100
>100
g 100
>100
100
>100
>100
66.6
>100
36.7
33.6
>100
>100
>100
>100
35.8
>100
94.2
>100
g100
61.3
>100
>100
>100
>100
>100
63.8
>100
30.6
>100
>100
41.1
>100
>100
>100
>100
>100
17.8
>100
g100
N. D.
>100
43.8
>100
>100
>100
>100
>100
>100
>100
58
>100
>100
39.8
g100
26.8
>100
58.8
>100
>100
>100
95.8
>100
>100
>100
89 ( 1
126 ( 18
54 ( 1
145 ( 9
18 ( 1
17 ( 0
>200
18 ( 2
>200
6.7 ( 0.3
80 ( 2
18 ( 3
153 ( 11
185 ( 21
107 ( 5
>200
17 ( 1
12 ( 0
>200
23 ( 0
>200
150 ( 0
126 ( 8
56 ( 15
>200
19 ( 1
10 ( 1
192 ( 51
21 ( 0
>200
>100
100
>100
g100
32.8
>100
32
>100
83.9
84
63.8
>100
31.8
12.1
17.8
>100
83.8
57.3
4.8 ( 0.3
108 ( 4
17 ( 0
17 ( 10
125 ( 21
39 ( 3
>100
6.7
>100
63.7
>100
42
>100
a IC50, the concentration that causes 50% growth inhibition.
Table 3. Inhibitory Effects of Hydantoins 6a-e,m,p (Scheme 1) on the Growth of Malignant Tumor Cell Lines in Comparison with
Their Effects on the Growth of Normal Human Fibroblasts (WI 38)
tumor cell growth IC50a (µM)
compd
L1210
Molt4/C8
CEM
SW 620
Hep-2
MiaPaCa-2
MCF-7
HeLa
WI 38
6a
6b
6c
6d
6e
6m
6p
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>100
>100
>100
>100
>100
96.8
98
>100
>100
>100
>100
100
>100
>100
67.4
94.3
29.5
3.3
100
>100
>100
>100
>100
74.1
100
>100
69.7
47
>100
>100
>100
>100
>100
>100
82.9
>100
68.9
78.9
>100
>100
22
>100
a IC50, the concentration that causes 50% growth inhibition.
be explained by repulsion between the hydrophobic
isobutyl groups of the neighboring hydrogen-bonded
molecules.
Of the N-(1-benzotriazolycarbonyl)amino acid amide
series (4a-o), compounds 4f and 4k showed the best
cytostatic effects. Compound 4f showed rather pro-
nounced inhibitory activity against murine leukemia
(L1210; IC50 ) 17 µM), while 4k inhibited all tumor cell
lines with the best effects (IC50 ) 15-18 µM) on murine
leukemia, human T-lymphocytes, and breast carcinoma.
However, compound 4k also exhibited a cytotoxic effect
on normal human fibroblasts. All other compounds from
this class, except for 4a, had only slight antiproliferative
effects on malignant tumor cells (IC50 ) 20.7-192 µM).
Biological Results and Discussion
Cytostatic Activity. The compounds 4a-o, 5a-o,
and 6a-e,m,p were evaluated for their activities against
malignant tumor cell lines: murine leukemia (L1210),
human T-lymphocytes (Molt4/C8 and CEM), colon car-
cinoma (SW 620), laryngeal carcinoma (Hep-2), pancre-
atic carcinoma (MiaPaCa-2), breast carcinoma (MCF-
7), and cervical carcinoma (HeLa) and compared with
their effects on the growth of human normal fibroblasts
(WI 38) (Tables 1-3).
L-Amino acid derivatives of hydroxyurea 5h and 5i
showed selective inhibition of the growth of all tested
cell lines. Thus, these compounds inhibited specifically

478 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2
Opacˇic´ et al.
Table 4. Activity of N-(1-Benzotriazolylcarbonyl)amino Acid
Amides 4a-o Against Cytomegalovirus (CMV, AD-169, and
Davis Strains) and Varicella-Zoster Virus (Thymidine
Kinase-Positive Strain, TK+ VZV, and Thymidine
Kinase-Deficient Strain, TK- VZV) in Human Embryonic Lung
(HEL) Cell Cultures
a
antiviral activity EC50
(µg mL-1
cytotoxicity
(µg mL-1
)
)
TK+ VZV TK- VZV
cell
cell
AD-169 Davis
OKA
07/1
morphology growth
c
compd strain strain strain
strain
(MCC)b
(CC50)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
GCV
>200 >200 >80
>200
>3.2
99
>16
>16
>0.64
>3.2
>3.2
>3.2
106
>0.13
>3.2
>3.2
>80
>3.2
ND
>200
g80
g80
80
>200
90
87.3
76
156
40
15.5
33.6
37.6
147.7
17.3
15
43.4
188
38.3
87
>80
>3.2
>16
>80
>16
>3.2
>16
>16
>16
>80
>16
>80
>16
>3.2
>16
>16
>3.2
66
>16
>16
>0.13
>3.2
>3.2
>3.2
200
80
16
Figure 2. Cytostatic effect of the hydantoin derivative 6m
on different human cell lines. The cells were treated with 6m
at different concentrations, and percentage of growth was
calculated. Each point represents a mean value of four parallel
samples in three individual experiments.
80
80
>200 >200 117
>200
80
>16
>3.2
>16
>80
>16
2.03
>3.2
>16
3.2
>80
>16
2.33
0.58
ND
>0.13
>0.64
>3.2
>80
g16
g16
200
80
>50
>50
>50
murine leukemia and human T-lymphocytes (IC50
)
>3.2
ND
10-19 µM), but not normal fibroblasts. Another L-amino
acid derivative of hydroxyurea with an aminodiphenyl-
methyl residue (5k) showed rather moderate inhibitory
effect against all cell lines (IC50 ) 18-66.6 µM).
HPMC 0.76
ACV
ND
0.27
ND
12
32
>200
NDd
a Effective concentration required to reduce virus plaque forma-
tion by 50%. Virus input was 100 plaque forming units (PFU).
b Minimum cytotoxic concentration that causes a microscopically
detectable alteration of cell morphology. c Cytotoxic concentration
required to reduce cell growth by 50%. d Not determined.
D-Amino acid derivatives of hydroxyurea 5m and 5o,
as well as previously discussed L-amino acid derivatives
of hydroxyurea, 5h and 5i, showed selective inhibition
regarding normal and tumor cells. Compounds 5m and
5o inhibited the growth of all tumor cell lines (IC50
)
Table 5. Activity of Hydantoins 6a-e,m,p against
Cytomegalovirus (CMV, AD-169, and Davis Strains) and
Varicella-Zoster Virus (Thymidine Kinase-Positive Strain, TK+
VZV, and Thymidine Kinase-Deficient Strain, TK- VZV) in
Human Embryonic Lung (HEL) Cell Cultures
4.8-83.9 µM), but not the growth of human normal
fibroblasts (WI 38; IC50 > 100 µM). Of all compounds
tested, 5m showed the most pronounced inhibitory effect
against human T-lymphocytes (Molt4/C8; IC50
4.8 µM).
)
a
antiviral activity EC50
(µg mL-1
cytotoxicity
(µg mL-1
)
)
Among hydantoin series 6a-e,m,p, the best cytostatic
activity was found for compound 6m (Figure 2). This
compound showed marked inhibitory effect against
breast carcinoma (MCF-7; IC50 ) 3.3 µM). However,
compound 6m also had cytotoxic activity against normal
WI 38 cells.
Antiviral Activity. Compounds 4a-o, 5a-o, and
6a-e,m,p were evaluated against cytomegalovirus
(CMV, AD-169, and Davis strains) and varicella-zoster
virus (thymidine kinase-positive strain, TK+ VZV and
thymidine kinase-deficient strain, TK- VZV) in human
embryonic lung (HEL) cells, and their activities were
compared with those of ganciclovir (GCV), (S)-1-[3-
hydroxy-2-(phosphonylmethoxy)propyl]cytosine (HPMC,
cidovir, CDV), and acyclovir (ACV) (Tables 4 and 5).
TK+ VZV TK- VZV
cell
cell
AD-169 Davis
OKA
07/1
morphology growth
c
compd strain strain strain
strain
(MCC)b
(CC50)
6a
>100 >100
84
90
>100
g 100
>100
>100
100
100
g20
>400
>400
>400
>50
>50
>50
>50
50
12.5
27.4
65.4
21.3
200
6b
45
41
45
29
6c
50
37
33
18
>20
9
6d
37
41
20
4.0
>4
0.34
0.13
ND
6e
>20
7.6
>20
0.24
>4
15
>4
ND
ND
0.6
>4
11
6m
6p
9
GCV
ND
ND
9.4
HPMC 0.13
ACV
NDd
a Effective concentration required to reduce virus plaque forma-
tion by 50%. Virus input was 100 plaque forming units (PFU).
b Minimum cytotoxic concentration that causes a microscopically
detectable alteration of cell morphology. c Cytotoxic concentration
required to reduce cell growth by 50%. d Not determined.
Results on antiviral evaluations of N-(1-benzotria-
zolecarbonyl)amino acid amides 4a-o presented in
Table 4 showed that 4m with a cyclohexanemethyl-
amine moiety had rather pronounced activity against
the Davis strain of CMV (EC50 ) 3.2 µg/mL). Amide 4c
exhibited only slight activity against VZV (TK+ VZV,
IC50 ) 66 µg/mL; TK- VZV, IC50 ) 99 µgmL-1). L- and
D-amino acid derivatives of hydroxyureas 5a-k and
5l-o did not display activity against CMV and VZV.
Hydantoin 6m had distinct activity against the Davis
strain (EC50 ) 4 µgmL-1) and AD-169 strain of CMV
(EC50 ) 7.6 µgmL-1). This compound exhibited also
cytotoxicity (CC50 ) 12.5 µgmL-1).
Compounds 5a-o and 6a-e,m,p were also evaluated
for their activity against human immunodeficiency virus
type 1 and 2 (HIV-1, HIV-2), while 5b-d,l,n-h were
evaluated against herpes simplex virus type 1 and 2
[HSV-1 (KOS), HSV-2 (G)], vaccinia virus, vesicular
stomatitis virus, herpes simplex virus-1 TK- KOS
(ACVr), Coxsackie virus B4, respiratory syncytial virus,
parainfluenza-3 virus, reovirus-1, Sindbis virus, and
Punta Toro virus. No specific antiviral effects (i.e.
minimal antiviral effective concentration g 5-fold lower
than minimal cytotoxic concentration) were noted for

Derivatives of Hydroxyurea and Hydantoins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2 479
N-(1-Benzotriazolecarbonyl)-L-valine Cyclopentyla-
mide (4a). The analytically pure sample was obtained by
triturating with ether and recrystallization from a cyclohexane/
acetone mixture: yield 0.500 g (38%); mp 150-153 °C; IR (KBr,
ν/cm-1) 3367, 3308, 2964, 2869, 1741, 1679, 1542, 1495; 13C
NMR (DMSO-d6) δ 168.62 (1), 147.98 (4), 145.02 (1′), 130.70
(6′), 129.59 (5′), 125.12 (4′), 119.34 (3′), 112.93 (2′), 58.80 (2),
49.94 (2′′), 31.82 (3′′), 31.42 (6′′) 30.46 (5), 22.83 (4′′, 5′′), 18.60
(6), 17.78 (7). Anal. (C17H23N5O2) C, H, N.
N-(1-Benzotriazolecarbonyl)-L-valine Cyclohexyla-
mide (4b). The analytically pure sample was obtained by
triturating with ether: yield 0.275 g (20%); mp 113-117 °C;
IR (KBr, ν/cm-1) 3386, 3279, 3098, 2931, 2856, 1723, 1709,
1642, 1562, 1522; 13C NMR (DMSO-d6) δ 168.61 (1), 148.41
(4), 145.46 (1′), 131.13 (6′), 130.03 (5′), 125.56 (4′), 119.80 (3′),
113.37 (2′), 59.23 (2), 47.58 (2′′), 32.30 (3′′), 32.05 (7′′), 30.91
(5), 25.07 (5′′), 24.30 (4′′, 6′′), 19.03 (6), 18.19 (7). Anal.
(C18H25N5O2) C, H, N.
N-(1-Benzotriazolecarbonyl)-L-leucine Cyclopentyla-
mide (4c). The analytically pure sample was obtained by
triturating with ether: yield 0.522 g (38%); mp 116-117 °C;
IR (KBr, ν/cm-1) 3290, 3080, 2957, 2869, 1715, 1647, 1554,
1521; 13C NMR (DMSO-d6) δ 175.16 (1), 170.94 (4), 149.21 (1′),
131.83 (6′), 130.56 (5′), 126.16 (4′), 120.36 (3′), 114.08 (2′), 54.72
(2), 53.25 (2′′), 32.76 (3′′), 32.54 (6′′), 29.15 (5), 25.19 (4′′), 24.02
(5′′), 23.57 (6), 22.01 (7, 8). Anal. (C18H25N5O2) C, H, N.
N-(1-Benzotriazolecarbonyl)-L-leucine Cyclohexyla-
mide (4d). The analytically pure sample was obtained by
triturating with ether: yield 0.615 g (43%); mp 122-123 °C;
IR (KBr, ν/cm-1) 3309, 3084, 2931, 2854, 1715, 1646, 1524;
13C NMR (DMSO-d6) δ 174.52 (1), 169.89 (4), 148.56 (1′), 131.22
(6′), 129.93 (5′), 125.53 (4′), 119.74 (3′), 113.46 (2′), 52.70 (2),
47.67 (2′′), 32.25 (3′′), 32.00 (7′′), 28.85 (5), 26.24 (5′′), 25.10
(4′′), 24.34 (6′′), 23.93 (6), 22.95 (7), 21.43 (8). Anal. (C19H27N5O2)
C, H, N.
N-(1-Benzotriazolecarbonyl)-L-leucine Cyclohexane-
methylamide (4e). The analytically pure sample was ob-
tained by recrystallization once from ether/petroleum ether
and once from cyclohexane: yield 1.114 g (75%); mp 84-86
°C; IR (KBr, ν/cm-1) 3423, 3312, 3010, 2925, 2850, 1743, 1653,
1564, 1515; 13C NMR (DMSO-d6) δ 171.42 (1), 149.32 (4),
146.00 (1′), 131.87 (6′), 130.52 (5′), 126.14 (4′), 120.33 (3′),
114.09 (2′), 53.44 (2), 45.43 (2′′), 37.90 (3′′), 30.85 (4′′, 6′′, 8′′),
26.85 (5′′), 26.56 (7′′), 24.98 (6), 23.61 (7), 21.74 (8). Anal.
(C20H29N5O2) C, H, N.
N-(1-Benzotriazolecarbonyl)-L-leucine Diphenylmeth-
ylamide (4f). Product 4f precipitated together with TEA·HCl.
It was filtered off and dissolved in a dichloromethane/water
mixture. The organic layer was extracted with diluted hydro-
chloric acid (w ) 1%) and water, dried over sodium sulfate,
and evaporated in a vacuum. The residue was triturated with
ether and gave analytically pure 4f: yield 1.024 g (58%); mp
153-158 °C; IR (KBr, ν/cm-1) 3328, 3261, 2958, 1716, 1650,
1557, 1522; 13C NMR (DMSO-d6) δ 173.91 (1), 155.82 (4),
148.30 (1′), 141.60 (3′′), 137.84 (9′′), 130.80 (6′), 129.50 (5′),
127.77 (5′′, 7′′, 11′′, 13′′), 126.89 (4′′, 8′′), 126.71 (10′′, 14′′),
126.61 (6′′), 126.50 (12′′), 125.09 (4′), 119.29 (3′), 113.00 (2′),
56.45 (2), 54.04 (2′′), 40.33 (5), 23.53 (6), 22.53 (7), 20.84 (8).
Anal. (C26H27N5O2) C, H, N.
N-(1-Benzotriazolecarbonyl)-L-phenylalanine Cyclo-
pentylamide (4g). The analytically pure sample was obtained
by triturating with ether: yield 0.891 g (59%); mp 155-158
°C; IR (KBr): νmax 3393, 3287, 2958, 1735, 1675, 1562, 1488;
13C NMR (DMSO-d6) δ 169.77 (1), 149.00 (4), 145.96 (1′), 138.05
(6), 131.71 (6′), 130.57 (5′), 129.80 (7, 11), 128.65 (8, 10), 126.95
(4′), 126.15 (9), 120.34 (3′), 113.98 (2′), 55.98 (2), 51.06 (2′′),
37.95 (5), 32.74 (3′′), 32.54 (6′′), 24.00 (4′′, 5′′). Anal. (C21H23N5O2)
C, H, N.
any of the compounds against any of these viruses (data
not shown).
Conclusions
The principal aim of the presented work was to
evaluate the novel N-[(1-benzotriazolecarbonyl]amino
acid amides 4, amino acid derivatives of hydroxyurea 5
and hydantoins 6, for their cytostatic and antiviral
activities. The L- and D-amino acid derivatives of hy-
droxyureas (5a-k and 5l-o) were prepared by ami-
nolysis of N-Btc-amino acid amides (4a-o) with hy-
droxylamine. The L-leucine derivative of hydantoin 6e
was synthesized by base-catalyzed cyclization of 4e. The
crystallographic analysis of 6e showed that the C5 atom
of the hydantoin ring possesses S configuration, in
agreement with the configuration of the starting L-
leucine. Therefore, the series of L- and D-amino acid
derivatives of hydroxyurea (5a-k and 5l-o) should also
possess the same configuration at the stereogenic center
as the starting amino acid.
D-Amino acid derivatives of hydroxyurea 5m and 5o
showed rather expressed cell-growth inhibitory activi-
ties against all examined tumor cell lines (IC50 ) 4.8-
83.9 µM), but not against human normal fibroblasts (WI
38; IC50 > 100 µM). Thus, these compounds represent
good leads for their structural optimization and further
in vivo evaluations.
Results on antiviral evaluations showed that N-Btc
amino acid amide 4m and hydantoin 6m had rather
expressed activity against the Davis strain of CMV (4m,
EC50 ) 3.2 µgmL-1; 6m, EC50 ) 4.0 µgmL-1) but also
cytotoxicity (4m, CC50 ) 43.4 µgmL-1; 6m, CC50 ) 12.5
µgmL-1).
Experimental Section
General Methods. Melting points were determined on a
Boe¨tius micro-heating stage and were uncorrected. IR spectra
were recorded on a FTIR Perkin-Elmer Paragon 500 spec-
trometer. 1H and 13C NMR spectra were recorded on a Varian
Gemini 300 spectrometer, operating at 300 and 75.5 MHz for
1
the H and 13C nuclei, respectively. Samples were measured
in DMSO-d6 solutions at 20 °C in 5 mm NMR tubes. Chemical
shifts (δ) in ppm were referred to TMS. Precoated Merck silica
gel 60 F254 plates were used for thin-layer chromatography.
Spots were visualized by short-wave UV light and iodine vapor.
Column chromatography was performed on silica gel (0.063-
0.200 mm), with dichloromethane/methanol (97:3) as eluent.
Compounds Preparations. 1-Benzotriazole carboxylic
acid chloride, N-(1-benzotriazolecarbonyl)amino acids (2a-d),
and their chlorides (3a-d) were prepared according to the
procedures previously published.7 Hydroxylamine was pre-
pared from hydroxylamine hydrochloride and sodium meth-
oxyde. Amino acids were purchased from Kemika (Zagreb,
Croatia) and amines (cyclopentylamine, cyclohexylamine, cy-
clohexanemethylamine, pyrrolidine, piperidine and amino-
diphenylmethane) from Aldrich.
N-(1-Benzotriazolecarbonyl)amino Acid Amides (4a-
p). General Procedure. To a cold solution of N-(1-benzo-
triazolecarbonyl)amino acid chloride (3a-d) (4 mmol) in
toluene (40 mL) was added a solution of corresponding amine
(4 mmol) and TEA (4 mmol) in toluene (30 mL) dropwise. The
reaction mixture was stirred at room temperature for 24 h.
The precipitated TEA·HCl was filtered off and the mother
liquor was extracted with diluted hydrochloric acid (w ) 1%)
and water, dried over sodium sulfate, and evaporated in a
vacuum.
N-(1-Benzotriazolecarbonyl)-L-phenylalanine Cyclo-
hexylamide (4h). The analytically pure sample was obtained
by triturating with ether: yield 0.673 g (43%); mp 80-84 °C;
IR (KBr, ν/cm-1) 3318, 2930, 2854, 1715, 1660, 1530, 1449;
13C NMR (DMSO-d6) δ 169.36 (1), 149.00 (4), 145.95 (1′), 138.07
(6), 131.70 (6′), 130.57 (5′), 129.81 (7, 11), 128.65 (8, 10), 126.95
1H NMR spectral data for N-(1-benzotriazolylcarbonyl)-
amino acid amides 4a-p are given in Supporting Information
(Table 6).

480 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2
Opacˇic´ et al.
(4′), 126.16 (9), 120.35 (3′), 113.98 (2′), 56.03 (2), 48.35 (2′′),
37.93 (5), 32.79 (3′′, 7′′), 25.71 (5′′), 25.08 (4′′, 6′′). Anal.
(C22H25N5O2) C, H, N.
mp 144-146 °C; IR (KBr, ν/cm-1) 3408, 3322, 2927, 2867, 1730,
1646, 1544, 1492; 13C NMR (DMSO-d6) δ 167.88 (1), 147.82
(4), 145.46 (1′), 137.99 (5), 131.07 (6′), 130.13 (5′), 128.45 (6,
10), 127.88 (4′), 126.88 (7, 9), 125.63 (8), 119.87 (3′), 113.27
(2′), 56.90 (2), 50.67 (2′′), 31.97 (3′′, 6′′), 23.34 (4′′, 5′′). Anal.
C20H21N5O2 C, H, N.
N-(1-Benzotriazolecarbonyl)-L-phenylalanine Cyclo-
hexanemethylamide (4i). The analytically pure sample was
obtained by triturating with ether: yield 0.892 g (55%); mp
141-143 °C; IR (KBr, ν/cm-1) 3327, 2921, 2850, 1709, 1657,
1541, 1449; 13C NMR (DMSO-d6) δ 170.33 (1), 149.13 (4),
145.95 (1′), 138.28 (6), 131.75 (6′), 130.52 (5′), 129.74 (7, 11),
128.67 (8, 10), 126.91 (4′), 126.12 (9), 120.31 (3′), 113.98 (2′),
56.27 (2), 45.55 (2′′), 37.69 (5), 30.86 (3′′), 26.54 (4′′, 6′′, 8′′),
25.94 (5′′, 7′′). Anal. (C23H27N5O2) C, H, N.
N-(1-Benzotriazolecarbonyl)-L-phenylalanine Pyrro-
lidineamide (4j). The analytically pure sample was obtained
by triturating with ether: yield 0.479 g (33%); mp 102-103
°C; IR (KBr, ν/cm-1) 3220, 2981, 1722, 1633, 1526, 1449; 13C
NMR (DMSO-d6) δ 168.66 (1), 149.00 (4), 145.94 (1′), 137.70
(6), 131.71 (6′), 130.63 (5′), 129.91 (7, 11), 128.74 (8, 10), 127.15
(4′), 126.20 (9), 120.39 (3′), 113.93 (2′), 54.57 (2), 46.32 (2′′,
5′′), 37.11 (5), 26.16 (3′′), 24.12 (4′′). Anal. (C20H21N5O2) C, H,
N.
N-(1-Benzotriazolecarbonyl)-L-phenylalanine Diphen-
ylmethylamide (4k). The analytically pure sample was
obtained by triturating with ether: yield 0.951 g (50%); mp
165-166 °C; IR (KBr, ν/cm-1) 3320, 1708, 1653, 1555, 1516,
1450; 13C NMR (DMSO-d6) δ 170.05 (1), 149.19 (4), 145.93 (1′),
142.57 (3′′, 9′′), 138.05 (6), 131.70 (6′), 130.59 (5′), 129.84 (7,
11), 128.92 (5′′, 7′′, 11′′, 13′′), 128.69 (8, 10), 128.05 (4′′, 8′′),
127.79 (10′′, 14′′), 127.62 (6′′), 127.54 (12′′), 127.00 (4′), 126.18
(9), 120.34 (3′), 113.96 (2′), 56.71 (2), 56.23 (2′′), 37.67 (5). Anal.
(C29H25N5O2) C, H, N.
Amino Acid Derivatives of Hydroxyurea (5a-o). Gen-
eral Procedure. To a solution of N-(1-benzotriazolecarbon-
yl)amino acid amide (4) (1 mmol) in toluene (25 mL) was slowly
added a solution of hydroxylamine (2 mmol) in methanol (5
mL). The reaction mixture was stirred at room temperature
from 2-10 days. New portions of hydroxylamine (2 mmol each)
were added after 24 and 48 h. The precipitated BtH·
hydroxylamine salt was filtered off and the mother liquor was
evaporated in a vacuum.
1H NMR spectral data for amino acid derivatives of hydroxy-
urea 5a-o are given in the Supporting Information (Table
7).
N-Cyclopentyl-2-(N′-hydroxyureido)-L-3-methylbutan-
amide (N′-Hydroxycarbamoyl-L-valine cyclopentyla-
mide) (5a). Crude product was purified by column chroma-
tography: yield 0.046 g (19%); mp > 250 °C; IR (KBr, ν/cm-1
)
3419, 3295, 2962, 1641, 1564, 1548; 13C NMR (DMSO-d6) δ
170.43 (1), 160.64 (4), 57.05 (2), 50.19 (2′′), 32.29 (3′′), 31.84
(6′′), 31.58 (5), 23.29 and 23.26 (4′′, 5′′), 18.99 (6), 17.93 (7).
Anal. (C11H21N3O3) C, H, N.
N-Cyclohexyl-2-(N′-hydroxyureido)-L-3-methylbutana-
mide (N′-Hydroxycarbamoyl-L-valine cyclohexylamide)
(5b). The analytically pure sample was obtained by triturating
with ether: yield 0.064 g (25%); mp 153-155 °C; IR (KBr,
ν/cm-1) 3420, 3283, 2935, 2856, 1654, 1560, 1541; 13C NMR
(DMSO-d6) δ 169.96 (1), 160.64 (4), 57.07 (2), 47.32 (2′′), 32.37
and 32.09 (3′′, 7′′), 31.56 (5), 25.09 (5′′), 24.40 and 24.34 (4′′,
6′′), 18.99 (6), 17.89 (7).
N-Cyclopentyl-2-(N′-hydroxyureido)-L-4-methylpen-
tanamide (N′-Hydroxycarbamoyl-L-leucine cyclopenty-
lamide) (5c). The analytically pure sample was obtained by
triturating with ether: yield 0.144 g (56%); mp 132-136 °C;
13C NMR (DMSO-d6) δ 171.73 (1), 160.48 (4), 50.88 (2), 50.11
(2′′), 42.26 (5), 32.14 and 31.94 (3′′, 6′′), 24.15 (6), 23.35 (4′′,
5′′), 22.91 (7), 21.87 (8).
N-Cyclohexyl-2-(N′-hydroxyureido)-L-4-methylpentan-
amide (N′-Hydroxycarbamoyl-L-leucine cyclohexyla-
mide) (5d). The pure product was obtained by column
chromatography and by triturating with petroleum ether:
yield 0.027 g (10%); mp 154-157 °C; IR (KBr, ν/cm-1) 3409,
3286, 2935, 2856, 1659, 1644, 1554, 1532.
N-Cyclohexanemethyl-2-(N′-hydroxyureido)-L-4-meth-
ylpentanamide (N′-Hydroxycarbamoyl-L-leucine cyclo-
hexanemethylamide) (5e). Crude product was purified by
column chromatography: yield 0.068 g (24%); mp 168-169 °C;
IR (KBr, ν/cm-1) 3396, 3277, 2928, 2853, 1646, 1558, 1534;
13C NMR (CD3OD) δ 175.49 (1), 163.67 (4), 53.45 (2), 46.87
(2′′), 43.22 (3′′), 39.28 (5), 32.07 (3′′, 6′′), 27.71 (5′′), 27.16 84′′),
26.14 (5), 23.58 (6), 22.34 (7). Anal. (C14H27N3O3) C, H, N.
N-Diphenylmethyl-2-(N′-hydroxyureido)-L-4-methyl-
pentanamide (N′-Hydroxycarbamoyl-L-leucine diphen-
ylmethylamide) (5f). The analytically pure sample was
obtained by triturating with acetone: yield 0.171 g (48%); mp
203-205 °C; IR (KBr, ν/cm-1) 3370, 3295, 2962, 2874, 1640,
1601, 1574, 1533; 13C NMR (DMSO-d6) δ 171.77 (1), 160.59
(4), 142.24 and 142.19 (3′′, 9′′), 128.31 (5′′, 7′′), 128.24 (11′′,
13′′), 127.30 (4′′, 8′′), 127.05 (10′′, 14′′), 126.89 (6′′, 12′′), 55.77
(2), 51.07 (2′′), 41.91 (5), 24.16 (6), 22.94 (7), 21.75 (8). Anal.
(C20H25N3O3) C, H, N.
N-Cyclopentyl-2-(N′-hydroxyureido)-L-3-phenylpro-
panamide (N′-Hydroxycarbamoyl-L-phenylalanine cy-
clopentylamide) (5g). The analytically pure sample was
obtained by triturating with acetone: yield 0.010 g (6%); mp
164-166 °C. IR (KBr, ν/cm-1) 3431, 3329, 3292, 2957, 2928,
2857, 1658, 1540.
N-Cyclohexylamide-2-(N′-hydroxyureido)-L-3-phenyl-
propanamide (N′-Hydroxycarbamoyl-L-phenylalanine cy-
clohexylamide) (5h). The analytically pure sample was
N-(1-Benzotriazolecarbonyl)-D-phenylglycine Cyclo-
hexylamide (4l). The analytically pure sample was obtained
by recrystallization from a cyclohexane/dichloromethane mix-
ture: yield 0.664 g (44%); mp 164-166 °C; IR (KBr, ν/cm-1
)
3403, 3296, 2933, 2855, 1741, 1645, 1556, 1497; 13C NMR
(DMSO-d6) δ 168.13 (1), 148.43 (4), 146.06 (1′), 138.65 (5),
131.67 (6′), 130.75 (5′), 129.05 (6, 10), 128.49 (4′), 127.50 (7,
9), 126.25 (8), 120.48 (3′), 113.89 (2′), 57.50 (2), 48.53 (2′′), 32.60
(3′′, 7′′), 25.62 (5′′), 24.85 (4′′, 6′′). Anal. (C21H23N5O2) C, H, N.
N-(1-Benzotriazolecarbonyl)-D-phenylglycine Cyclo-
hexanemethylamide (4m). The analytically pure sample
was obtained by recrystallization once from ether/petroleum
ether and once from cyclohexane: yield 0.658 g (42%); mp
108-111 °C; IR (KBr, ν/cm-1) 3427, 3304, 3090, 2923, 2850,
1744, 1654, 1557, 1513; 13C NMR (DMSO-d6) δ 168.07 (1),
147.37 (4), 145.01 (1′), 137.59 (5), 130.63 (6′), 129.68 (5′), 127.95
(6, 10), 126.59 (7, 9), 126.05 (4′), 125.18 (8), 119.41 (3′), 112.82
(2′), 56.66 (2), 44.56 (2′′), 36.80 (3′′), 29.63 (4′′, 8′′), 25.39 (6′′),
24.79 (5′′,7′′). Anal. (C22H25N5O2) C, H, N.
N-(1-Benzotriazolecarbonyl)-D-phenylglycine Piperi-
dineamide (4n). The analytically pure sample was obtained
by triturating with ether: yield 0.625 g (43%); mp 120-122
°C; IR (KBr, ν/cm-1) 3354, 2941, 2854, 1738, 1627, 1478, 1450;
13C NMR (DMSO-d6) δ 166.85 (1), 148.00 (4), 146.04 (1′), 137.58
(5), 131.60 (6′), 130.77 (5′), 129.39 (6, 10), 128.67 (4′), 127.72
(7, 9), 126.23 (8), 120.49 (3′), 113.82 (2′), 55.25 (2), 46.35 (2′′),
43.58 (6′′), 25.60 (3′′, 5′′), 24.18 (4′′). Anal. (C20H21N5O2) C, H,
N.
N-(1-Benzotriazolecarbonyl)-D-phenylglycine Diphen-
ylmethylamide (4o). The analytically pure sample was
obtained by triturating with ether and recrystallization from
a cyclohexane/dichloromethane mixture: yield 0.923 g (50%);
mp 89-93 °C; IR (KBr, ν/cm-1) 3397, 3294, 2924, 1740, 1653,
1538, 1494; 13C NMR (DMSO-d6) δ 167.99 (1), 147.95 (4),
145.44 (1′), 141.63 (3′′, 9′′), 137.62 (5), 131.08 (6′), 130.14 (5′),
128.44 (6, 10), 128.37 (5′′, 7′′, 11′′, 13′′), 128.21 (7, 9), 128.05
(4′′, 8′′), 127.51 (10′′, 14′′), 127.17 (6′′), 126.94 (12′′), 126.80
(4′), 125.64 (8), 119.86 (3′), 113.26 (2′), 57.06 (2′′), 56.22 (2).
Anal. (C28H23N5O2) C, H, N.
N-(1-Benzotriazolecarbonyl)-D-phenylglycine Cyclo-
pentylamide (4p). The analytically pure sample was obtained
by triturating with ether and recrystallization from a cyclo-
hexane/dichloromethane/acetone mixture: yield 0.836 g (58%);

Derivatives of Hydroxyurea and Hydantoins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2 481
obtained by triturating with ether: yield 0.104 g (34%); mp
111-120 °C; IR (KBr, ν/cm-1) 3422, 3307, 2933, 2855, 1654,
1560, 1541; 13C NMR (DMSO-d6) δ 169.94 (1), 160.31 (4),
137.45 (6), 129.20 (7, 11), 127.89 (8, 10), 126.11 (9), 53.45 (2),
47.33 (2′′), 38.87 (5), 32.14 (3′′, 7′′), 25.09 (5′′), 24.39 (4′′, 6′′).
N-Cyclohexanemethyl-2-(N′-hydroxyureido)-L-3-phen-
ylpropanamide (N′-Hydroxycarbamoyl-L-phenylalanine
cyclohexanemethylamide) (5i). Sodium carbonate solution
(w ) 10%, 20 mL) was added to the reaction mixture. Precip-
itated product was filtered off and worked up with ethyl
acetate. Product 5i was isolated as the sodium salt from the
organic extract: yield 0.051 g (15%); mp 142-146 °C. 13C NMR
(DMSO-d6) δ 171.02 (1), 160.36 (4), 137.54 (6), 129.11 (7, 11),
127.89 (8, 10), 126.10 (9), 53.69 (2), 44.71 (2′′), 38.58 (5), 37.21
(3′′), 30.21 (4′′, 8′′), 25.91 (6′′), 25.32 (5′′, 7′′). Anal. (C17H24N3O3-
Na) C, H, N.
N-Pyrrolidinyl-2-(N′-hydroxyureido)-L-3-phenylpro-
panamide (N′-Hydroxycarbamoyl-L-phenylalanine pyr-
rolidineamide) (5j). Crude product was purified by column
chromatography: yield 0.053 g (19%); mp 176-177 °C; IR
(KBr, ν/cm-1) 3422, 3172, 2955, 2876, 1642, 1622, 1534; 13C
NMR (DMSO-d6) δ 169.37 (1), 160.29 (4), 137.21 (6), 129.21
(7, 11), 128.03 (8, 10), 126.35 (9), 51.84 (2), 44.58 and 45.36
(2′′, 5′′), 38.12 (5), 25.43 (4′′), 23.52 (3′′). Anal. (C14H19N3O3)
C, H, N.
N-Diphenylmethyl-2-(N′-hydroxyureido)-L-3-phenyl-
propanamide (N′-Hydroxycarbamoyl-L-phenylalanine
diphenylmethylamide) (5k). The analytically pure sample
was obtained by triturating with ether: yield 0.148 g (38%);
mp 162-170 °C; IR (KBr, ν/cm-1) 3422, 3298, 3062, 3028, 2924,
1654, 1637, 1560, 1540; 13C NMR (DMSO-d6) δ 170.65 (1),
160.44 (4), 142.05 and 142.18 (3′′, 9′′), 137.40 (6), 129.20-
126.16 (7-11, 4′′-8′′, 10′′-14′′), 55.85 (2), 53.67 (2′′), 38.44 (5).
Anal. (C23H23N3O3) C, H, N.
3-Cyclopentyl-5-isopropyl Hydantoin (6a):17 yield 0.495
g (72%); mp 125-126 °C (lit.17 mp 107-109 °C); IR (KBr,
ν/cm-1) 3321, 2960, 2873, 1764, 1710, 1681, 1430; 13C NMR
(DMSO-d6) δ 173.53 (1), 157.02 (4), 60.53 (2), 50.12 (2′′), 28.63
(3′′), 28.41 (6′′), 24.60 (4′′, 5′′), 18.33 (6), 15.44 (7). Anal.
(C11H18N2O2) C, H, N.
3-Cyclohexyl-5-isopropyl Hydantoin (6b):17 yield 0.245
g (73%); mp 94-95 °C (lit.17 mp 150.0-151.5 °C); IR (KBr,
ν/cm-1) 3236, 2937, 3857, 1766, 1705, 1433; 13C NMR (DMSO-
d6) δ 173.52 (1), 156.98 (4), 60.42 (2), 49.89 (2′), 29.69 (5), 29.00
(3′′), 28.77 (7′′), 25.28 (4′′, 6′′), 24.75 (5′′), 18.34 (6), 15.35 (7).
Anal. (C12H20N2O2) C, H, N.
3-Cyclopentyl-5-isobutyl Hydantoin (6c): yield 0.273 g
(81%); mp 80-81 °C; IR (KBr, ν/cm-1) 3241, 2929, 2856, 1760,
1710, 1437; 13C NMR (DMSO-d6) δ 174.51 (1), 156.59 (4), 54.12
(2), 50.17 (2′′), 40.58 (5), 28.54 (3′′), 28.51 (6′′), 24.57 (4′′,5′′),
23.94 (6), 22.94 (7), 21.42 (8). Anal. (C12H20N2O2) C, H, N.
3-Cyclohexyl-5-isobutyl Hydantoin (6d):17 yield 0.232 g
(65%); mp 98-99 °C (lit.17 mp 126-127 °C); IR (KBr, ν/cm-1
)
3243, 2938, 2855, 1768, 1708, 1435; 13C NMR (DMSO-d6) δ
174.51 (1), 156.56 (4), 54.07 (2), 49.87 (2′′), 40.59 (5), 28.87
(3′′), 28.83 (7′′), 25.75 (4′′, 6′′), 24.75 (5′′), 23.93 (6), 22.98 (7),
21.43 (8). Anal. (C13H22N2O2) C, H, N.
3-Cyclohexanemethyl-5-isobutyl Hydantoin (6e): yield
0.329 g (87%); mp 141-144 °C; IR (KBr, ν/cm-1) 3294, 2926,
2852, 1769, 1711, 1459; 13C NMR (DMSO-d6) δ 174.83 (1),
156.95 (4), 54.59 (2), 43.41 (2′′), 40.85 (5), 35.91 (3′′), 30.11 (4′′,
8′′), 25.76 (6′′), 25.04 (5′′, 7′′). Anal. (C14H24N2O2) C, H, N.
3-Cyclohexanemethyl-5-phenyl Hydantoin (6m): yield
0.351 g (86%); mp 141-147 °C; IR (KBr, ν/cm-1) 3234, 2924,
2850, 1772, 1709, 1452; 13C NMR (DMSO-d6) δ 172.63 (1),
157.01 (4), 135.80 (5), 128.64 (6, 10), 128.26 (8), 126.65 (7, 9),
59.64 (2), 43.68 (2′), 35.91 (3′′), 29.99 (4′′, 8′′), 25.35 (6′′), 25.03
(5′′, 7′′). Anal. (C16H20N2O2) C, H, N.
3-Cyclopentyl-5-phenyl Hydantoin (6p): yield 0.150 g
(40%); mp 124-126 °C; IR (KBr, ν/cm-1) 3237, 3097, 2954,
2871, 1766, 1701, 1432; 13C NMR (DMSO-d6) δ 172.33 (1),
156.66 (4), 153.90 (5), 128.63 (6, 10), 128.23 (8), 126.65 (7, 9),
59.15 (2), 50.50 (2′′), 28.66 (3′′), 28.63 (6′′), 24.63 (4′′,5′′). Anal.
(C14H22N2O2) C, H, N.
N-Cyclohexyl-2-(N′-hydroxyureido)-D-2-phenyletana-
mide (N′-Hydroxycarbamoyl-D-phenylglycine cyclohex-
ylamide) (5l). The analytically pure sample was obtained by
triturating with acetone: yield 0.119 g (41%); mp >250 °C;
IR (KBr, ν/cm-1) 3396, 3305, 2937, 2855, 1643, 1622, 1554,
1536; 13C NMR (DMSO-d6) δ 168.81 (1), 159.85 (4), 140.06 (5),
128.11 (6, 10), 127.18 (8), 126.26 (7, 9), 55.45 (2), 47.61 (2′′),
32.10 and 31.95 (3′′, 7′′), 25.03 (5′′), 24.33 and 24.21 (4′′, 6′′).
Anal. (C15H21N3O3) C, H, N.
N-Cyclohexanemethyl-2-(N′-hydroxyureido)-D-2-phen-
yletanamide (N′-Hydroxycarbamoyl-D-phenylglycine cy-
clohexanemethylamide) (5m). The analytically pure sample
was obtained by triturating with acetone and additional
recrystallization from an ethanol/water mixture: yield 0.25 g
(8%); mp 142-144 °C. IR (KBr, ν/cm-1) 3296, 2925, 2856, 1630,
1560. Anal. (C16H23N3O3) C, H, N.
X-ray Crystal Structure Determination. A single crystal
of compound 6e (for crystal data, see the Supporting Informa-
tion) suitable for X-ray single-crystal analysis was obtained
at room temperature by slow evaporation from ethanol solution
(96%). The intensities were collected at 100 K on a Oxford
Diffraction Xcalibur 2 diffractometer with graphite-monochro-
mated Mo KR radiation (λ ) 0.71073 Å). The data collection
and reduction were carried out with the CrysAlis programs.8
Programs used for structure solution, refinement, analysis, and
drawing include SHELXS97,9 SHELXL97,10 and PARST95,11
and PLATON.12 Crystallographic data excluding structure
factors for the structure reported in this paper have been
deposited with the Cambridge Crystallographic Data Centre
as supplementary publication number CCDC-245009. Copies
of data can be obtained, free of charge, on application to CCDC,
12 Union Road, Cambridge CB2 1EZ, UK [fax, +44-(0)1223-
336033; e-mail, deposit@ccdc.cam.ac.uk].
N-Piperidyl-2-(N′-hydroxyureido)-D-2-phenyletana-
mide (N′-Hydroxycarbamoyl-D-phenylglycine piperidine-
amide) (5n). The pure product was obtained by column
chromatography and by triturating with methanol: yield 0.06
g (5%); mp 216-218 °C; IR (KBr, ν/cm-1) 3410, 3245, 2924,
2854, 1633, 1536. Anal. (C14H19N3O3) C, H, N.
N-Diphenylmethyl-2-(N′-hydroxyureido)-D-2-phenyl-
etanamide (N′-Hydroxycarbamoyl-D-phenylglycine di-
phenylmethylamide) (5o). The analytically pure sample was
obtained by triturating with ether: yield 0.229 g (61%); mp
>250 °C; IR (KBr, ν/cm-1) 3400, 3359, 3311, 1654, 1638, 1560,
1541; 13C NMR (DMSO-d6) δ 169.29 (1), 159.86 (4), 142.02 and
141.75 (3′′, 9′′), 139.67 (5), 128.35-126.49 (6-10, 4′′-8′′, 10′′-
14′′), 55.98 (2), 55.50 (2′′). Anal. (C22H21N3O3) C, H, N.
Biological Tests. Cytostatic Activity Assays. Antitumor
activity against L1210 (murine leukemia), Molt4/C8, and CEM
(human T-lymphocytes) cell lines were measured essentially
as originally described for the mouse leukemia (L1210) cell
lines.13
Cell Culturing. The HeLa (cervical carcinoma), MCF-7
(breast carcinoma), SW 620 (colon carcinoma), MiaPaCa-2
(pancreatic carcinoma), Hep-2 (laryngeal 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-1 strepto-
mycin in a humidified atmosphere with 5% CO2 at 37 °C.
Proliferation Assays. The HeLa, MCF-7, SW 620, Mia-
PaCa-2, Hep-2, and WI 38 cells were inoculated onto a series
of standard 96-well microtiter plates on day 0. Test agents
were then added in 5- and 10-fold dilutions (10-8-10-4 M) and
Hydantoins (6a-e,m,p). General Procedure. To a solu-
tion of N-(1-benzotriazolecarbonyl)amino acid amide (4) (1.5
mmol) in acetone (60 mL) was added a 5% solution of sodium
carbonate (5 mL). The reaction mixture was stirred for 2 h at
room temperature. Acetone was evaporated in a vacuum and
the precipitated product 6 was filtered off, washed with water,
and recrystallized from acetone and water.
1H NMR spectral data for hydantoins 6a-e,m,p are given
in the Supporting Information (Table 8).

482 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2
Opacˇic´ et al.
(2) Charache, S.; Terrin, M. L.; Moore, R. D.; Dover, G. J.; Barton,
F. B.; Eckert, S. V.; McMahon, R. P.; Bonds, D. R. Effect of
hydroxyurea on the frequency of painful crises in sickle cell
anemia. Investigators of the multicenter study of hydroxyurea
in sickle cell anaemia. N. Engl. J. Med. 1995, 322, 1317-1322.
(3) (a) Gao, W. Y.; Johns, D. G.; Mitsuya, H. Anti-human immuno-
deficiency virus type 1 activity of hydroxyurea in combination
with 2′,3′-dideoxynucleosides. Mol Pharmacol. 1994, 46, 767-
772. (b) Lori, F.; Malykh, A.; Cara, A.; Sun, D.; Weinstein, J.
N.; Lisziewicz, J.; Gallo, R. C. Hydroxyurea as an inhibitor of
human immunodeficiency virus-type 1 replication. Science 1994,
266, 801-805. (c) Malley, S. D.; Grange, J. M.; Hamedi-Sangsari,
F.; Vila, J. R. Synergistic anti-human immunodeficiency virus
type 1 effect of hydroxamate compounds with 2′,3′-dideoxyi-
nosine in infected resting human lymphocytes. Proc. Natl. Acad.
Sci. U.S.A. 1994, 91, 11017-11021.
incubated for 72 h further. Working dilutions were freshly
prepared on the day of testing. After 72 h of incubation, the
cell growth rate was evaluated by performing the MTT assay,14
which detects dehydrogenase activity in viable cells. The
absorbency (OD, optical density) was measured on 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) g 0, then
PG ) 100 × (mean ODtest - mean ODtzero)/
(mean ODctrl - mean ODtzero
)
(4) Torres, G. Hydroxyurea, a potential new anti-HIV agent. Treat
Issues 1995, 9, 7-9.
if (mean ODtest - mean ODtzero) < 0, then
PG ) 100 × (mean ODtest - mean ODtzero)/ODtzero
(5) (a) Hanessian, S.; Johnstone, S. Synthesis of hydroxamic esters
via alkoxyaminocarbonylation of â-dicarbonyl compounds. J.
Org. Chem. 1999, 64, 5896-5903. (b) Kolasa, T.; Stewart, A. O.;
Brooks, C. D. W. Asymmetric synthesis of (R)-N-3-butyn-2-yl-
N-hydroxyurea, a key intermediate for 5-lipoxygenase inhibitors.
Tetrahedron: Asymmetry 1996, 7, 729-736. (c) Nandy, P.; Lien,
E. J.; Avramis, V. I. Inhibition of ribonucleotide reductase by a
new class of isoindole derivatives: Drug synergism with cytara-
bine (Ara-C) and induction of cellular apoptosis. Anticancer Res.
1999, 19, 1625-1633. (d) Kleeman, A.; Engel, J.; Kutscher, B.;
Reichert, D. Pharmaceutical Substances, Synthesis, Patents,
Applications, 4th ed.; Thieme: Stuttgart, 2001.
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
(6) (a) Zorc, B.; Butula, I. Reaktionen mit N-(1-Benzotriazolylcar-
bonyl)-aminosa¨uren. I. Synthese von Hydantoinen und Hydan-
toinsauren-amiden, Croat. Chem. Acta 1981, 54, 441-449. (b)
Muskolaj, I.; Matijevic´-Sosa, J.; Zorc, B.; Butula, I. Reactions
with N-(1-benzotriazolyl-carbonyl)-amino acid. V. Reactions with
hydroxylamine. Acta Pharm. l997, 47, 109-115.
(7) (a) Butula, I.; Prosˇtenik, M.; Vela, V. Reactions with 1-benzot-
riazole carboxylic acid chloride. I. Synthesis of the 2,6-bis-
(hydroxymethyl)pyridine dicarbamates. Croat. Chem. Acta 1977,
49, 837-842. (b) Butula, I.; Jadrijevic´ Mladar Takacˇ, M. Reaction
with 1-benzotriazolecarboxylic acid chloride. VIII. Synthesis of
N-hydroxyisocyanate derivatives. Croat. Chem. Acta 2000, 73,
569-574. (c) Butula, I. Zorc, B. Vela, V. Reaktionen mit
1-Benzotriazol carbonsaurechlorid. VII. Die Umsetzung mit
Aminosauren. Croat. Chem. Acta 1981, 54, 435-440.
mean ODctrl ) the average of optical density measurements
after the desired period of time with no exposure of cells to
the test compound.
Each test point was performed in quadruplicate in three
individual experiments. The results were expressed as IC50,
the 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 give PG values above and below the
reference value (i.e. 50%). If, however, for a given cell line all
of the tested concentrations produce PGs exceeding the respec-
tive reference level of effect (e.g. PG value of 50), then the
highest tested concentration is assigned as the default value,
which is preceded by a “>” sign.
Antiviral Activity Assays. Antiviral activity against her-
pes simplex virus type 1 and 2, vaccinia virus, cytomegalovirus,
varicella-zoster virus, vesicular stomatitis virus, Coxsackie
virus B4, respiratory syncytial virus, parainfluenza-3 virus,
reovirus-1, Sindbis virus, and Punta Toro virus was deter-
mined essentially as described previously.15,16 The antiviral
activity results were expressed as EC50, the effective concen-
tration required to afford 50% protection against viral cyto-
pathogenicity or viral plaque formation.
(8) Oxford Diffraction, Xcalibur CCD System, CrysAlis Software
System (version 1.4.), Oxford Diffraction Ltd, Oxford, United
Kingdom, 2004.
(9) Sheldrick, G. M. SHELXS97, Program for the Solution of Crystal
Structures, University of Go¨ttingen, Germany, 1997.
(10) Sheldrick, G. M. SHELXL97, Program for the Refinement of
Crystal Structures, University of Go¨ttingen, Germany, 1997.
(11) Nardelli, M. PARST95sAn update to PARST: A system of
Fortran routines for calculating molecular structure parameters
from the results of crystal structure analyses. J. Appl. Crystal-
logr. 1995, 28, 659.
(12) Spek, A. L. Single-crystal structure validation with the program
PLATON. J. Appl. Crystallogr. 2003, 36, 7-13.
(13) De Clercq, E.; Balzarini, J.; Torrence, P. F.; Mertes, M. P.;
Schmidt, C. L.; Shugar, D.; Barr, P. J.; Jones, A. S.; Verhelst,
G.; Walker R. T. Thymidylate synthetase as target enzyme for
the inhibitory activity of 5-substituted 2′-deoxyuridines on mouse
leukemia L1210 cell growth. Mol. Pharmacol. 1981, 19, 321-
330.
Acknowledgment. Support for this study by the
Ministry of Science and Technology of the Republic of
Croatia (Projects No. 0006543, 0125003, 00981499) is
gratefully acknowledged.
(14) Mossman, T. Rapid colorimetric assay for cellular growth and
survival: Application to proliferation and cytotoxicity assays.
J. Immunol. Methods 1983, 65, 55-63.
(15) De Clercq, E.; Holy´, A.; Rosenberg, I.; Sakuma, T.; Balzarini,
J.; Maudgal, P. C. A novel selective broad-spectrum anti-DNA
Virus agent. Nature 1986, 323, 464-467.
(16) Balzarini, J.; Naesens, L.; Slachmuylders, J.; Niphuis, H.;
Rosenberg, I.; Holy´, A. Schellekens, H.; De Clercq, E. 9-(2-
Phosphonylmethoxyethyl)adenine (PMEA) efficiently inhibits
retrovirus replication in vitro and simian immunodeficiency
virus infection in Rhesus monkeys. AIDS 1991, 5, 21-28.
(17) Kitasaki, K., Crawford, R. F. U.S. Patent 3,326,661; Chem. Abstr.
1968, 68, 39622h.
Supporting Information Available: 1H NMR spectral
data of 4a-p, 5a-o, and 6a-e,m,p (Tables 6-8) and crystal
data and crystal packing diagram of 6e. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) Mutschler, E.; Derendorf, H. Drug Actions, Basic Principles and
Therapeutic Aspects; Medpharm Scientific Publishers: Stuttgart,
1995.
JM040869I