Novel NSAID 1-acyl-4-cycloalkyl/arylsemicarbazides and 1-acyl-5-benzyloxy/ hydroxy carbamoylcarbazides as potential anticancer agents and antioxidants

Article abstract of DOI:10.1016/j.ejmech.2012.02.046

The novel 1-acyl-4-cycloalkyl/arylsemicarbazides (5a-y) and 1-acyl-5-benzyloxy/hydroxycarbamoylcarbazides (8a-f) derived from the nonsteroidal anti-inflammatory drugs ibuprofen, fenoprofen and reduced ketoprofen were prepared, fully chemically characteriz

Full text of DOI:10.1016/j.ejmech.2012.02.046

European Journal of Medicinal Chemistry 51 (2012) 227e238
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel NSAID 1-acyl-4-cycloalkyl/arylsemicarbazides and 1-acyl-5-benzyloxy/
hydroxy carbamoylcarbazides as potential anticancer agents and antioxidants
a
b
b
c
d
d
ꢀ a
*
I. Perkovic , I. Butula , M. Kralj , I. Martin-Kleiner , J. Balzarini , D. Hadjipavlou-Litina , A-M. Katsori ,
,
B. Zorc a
a
ꢁ ꢀ
Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacica 1, HR-10 000 Zagreb, Croatia
b
ꢁ
ꢀ
ꢁ
Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka cesta 54, HR-10 000 Zagreb, Croatia
c Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
d School of Pharmacy, Aristotles University of Thessaloniki, Thessaloniki 54 124, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
The
novel
1-acyl-4-cycloalkyl/arylsemicarbazides
(5aey)
and
1-acyl-5-benzyloxy/hydroxy
Received 29 September 2011
Received in revised form
10 February 2012
Accepted 22 February 2012
Available online 3 March 2012
carbamoylcarbazides (8aef) derived from the nonsteroidal anti-inflammatory drugs ibuprofen, feno-
profen and reduced ketoprofen were prepared, fully chemically characterized and evaluated for their
cytostatic, antiviral and antioxidant activities. Compounds 5 and 8 consist of a region rich in electro-
negative atoms (five to nine nitrogen and oxygen atoms) framed by aryl or cycloalkyl residues on one or
both terminal ends. The synthetic pathways applied for the preparation of the title compounds involved
a benzotriazole as a synthetic auxiliary in several steps. Three of the tested compounds, namely 4-
benzhydryl-1-[2-(3-phenoxyphenyl)propanoyl]semicarbazide (5l), 4-benzhydryl-1-[2-(3-benzylphenyl)
propanoyl]semicarbazide (5s), and 4-benzhydryl-1-[2-(4-isobutylphenyl)propanoyl]semicarbazide (5f)
Keywords:
NSAID
Semicarbazide
Carbazide
Cytostatic activity
Lipoxygenase
Lipid peroxidation
showed pronounced antiproliferative activity in vitro against six cancer cell lines (IC50 ¼ 3e23
mM). The
same compounds highly inhibited soybean lipoxygenase (IC50 ¼ 60 and 51.5 M) and lipid peroxidation
m
as well (99, 88 and 74%, respectively). 4-Benzyloxy-1-[2-(4-isobutylphenyl)propanoyl]semicarbazide (5t)
and 5-benzyloxycarbamoyl-1-[2-(3-benzylphenyl)propanoyl]carbazide (8c) exerted complete lipid per-
oxidation inhibition. Semicarbazides 5wey and carbazides 8def bearing a hydroxamic acid/hydroxyurea
moiety showed a modest antiradical activity in DPPH test, while the best radical scavenger was 1-(1-
benzotriazolecarbonyl)-4-benzyloxysemicarbazide (7). None of the compounds were inhibitory to
a broad panel of DNA and RNA viruses in the cell culture at subtoxic concentrations.
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
activityand COX1/COX2 selectivity, reducing the ulcerogenic effect or
obtaining dual COX/LOX inhibition) [9,10]. Many efforts were made to
Chronic inflammation is a common and important factor in the
pathogenesis of neoplasmas. Inflammation is often accompanied by
the excessive formation of reactive oxygen and nitrogen species that
are potentially damagingto DNA and cellmembranes. Inaddition, the
expression of COX-2 in inflammatory and neoplastic cells has an
impactonvariouscarcinogenic pathways [1e4]. NSAIDsusefulness in
chemoprevention is well-documented. Numerous experimental,
epidemiological, andclinicalstudies have found thatlong-term useof
NSAIDs is associated with a lower risk of colorectal cancer, adeno-
matous polyps, and some other types of cancer [5e8]. NSAID
chemical modifications were undertaken with the aim of improving
the NSAIDssafetyprofile(increasing theanalgetic/anti-inflammatory
get NSAID derivatives with pronounced cytostatic activity as well
[11e19]. Thus, wesettopreparea seriesofnew NSAID derivativesrich
in nitrogen/oxygen atoms, related to semicarbazides, carbazides,
ureas, hydroxyureas and hydroxamic acids, bearing pharmacophores
present in numerous antitumor agents [20e23]. Their synthesis, full
chemical characterization, and evaluation of their cytostatic, antiviral
and antioxidative potential are reported in this paper.
2. Materials and methods
2.1. Synthesis
2.1.1. Materials and general methods
Melting points were determined on a Stuart Melting Point
Apparatus SMP3 and were uncorrected. IR spectra were recorded
* Corresponding author. Tel.: þ385 1 4856202; fax: þ385 1 4856201.
E-mail address: bzbz@pharma.hr (B. Zorc).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.046

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I. Perkovic et al. / European Journal of Medicinal Chemistry 51 (2012) 227e238
228
on a FTIR PerkineElmer Paragon 500 spectrometer. UVevisible
double beam spectrophotometer Lambda 20 (PerkineElmer) was
used for the in vitro tests. 1H and 13C NMR spectra were recorded
2.1.2.3. 2-(3-Benzylphenyl)propanehydrazide (3c). From the reac-
tion of 3.554 g (10 mmol) compound 2c and recrystallization from
ether/petroleum ether 2.000 g (78%) of 3c was obtained; mp
80e82 ꢀC; IR (KBr): nmax 3289, 3191, 3168, 3063, 3023, 2975, 2919,
1637,1601,1534,1486,1453,1382,1261,1008, 976, 786, 759, 722, 696,
on
a Bruker AV-600 spectrometer, operating at 600.13 and
300.13 MHz for the 1H and 150.917 and 75.47 MHz for 13C nuclei.
Samples were measured in DMSO-d6 solutions at 20 ꢀC in 5 mm
685, 599 cmꢃ1; 1H NMR (DMSO-d6)
d
9.14 (s, 1H), 7.31e7.03 (m, 9H),
4.17 (s, 2H), 3.90 (s, 2H), 3.48 (q,1H),1.33 (d, 3H); 13C NMR (DMSO-d6)
d 173.19, 142.66, 141.65, 141.47, 129.14, 128.88, 128.69, 128.15, 127.38,
NMR tubes. Chemical shifts (d) in ppm were referred to TMS. CHN
analyses were performed on CHNS LECO analyzer and were
within ꢁ0.4%. Solvent systems dichloromethane/methanol (9:1
and 9.5:0.5), cyclohexane/ethyl acetate (1:1), petroleum ether/
ethyl acetate/methanol (3:1:0.5 and 1:2:0.1) and precoated silica
gel 60 F254 plates were used for thin-layer chromatography. Spots
were visualized by short-wave UV light, iodine vapor, phospho-
molybdic acid or ferric chloride solution. Column chromatography
was performed on silica gel (0.063e0.200 mm), with cyclohexane/
ethyl acetate/methanol (3:1:0.6) or dichloromethane/methanol
(9.5:0.5 or 8.5:1.5) as eluents.
2-(3-Benzylphenyl)propanoic acid was prepared by ketoprofen
reduction. 1-Benzotriazole carboxylic acid chloride (BtcCl, 1) and
NSAID (ibuprofen, fenoprofen, and reduced ketoprofen) benzo-
triazolides (2aec) were synthesized according to our previously
published procedures [24e26]. For preparation of 1-(N-alkyl/
arylcarbamoyl)benzotriazoles (1-benzotriazole carboxylic acid
amides) 4aeg our synthetic method was applied as well [27]. The
following derivatives were prepared: 1-(N-cyclopentylcarbamoyl)
benzotriazole (4a), 1-(N-cyclohexylcarbamoyl)benzotriazole (4b),
1-(N-cyclohexanemethylcarbamoyl)benzotriazole (4c), 1-(N-ben-
zylcarbamoyl)benzotriazole (4d), 1-[N-(2-phenylethylcarbamoyl)]
benzotriazole (4e), 1-(N-benzhydrylcarbamoyl)benzotriazole (4f)
and 1-(N-benzyloxycarbamoyl)benzotriazole (4g). Benzotriazole
(BtH), triphosgene, O-benzylhydroxylamine hydrochloride, cyclo-
pentylamine, cyclohexylamine, cyclohexanemethylamine, ben-
zylamine, 2-phenylethanamine, benzhydrylamine, hydrazine
hydrate, triethylamine (TEA) and 10% Pd/C were purchased from
Aldrich. Ibuprofen, fenoprofen and ketoprofen were gift samples
from Pliva, University of Potchefstroom and Belupo. DPPH,
AAPH and NDGA were purchased from Aldrich Chemical Co.
Soybean LOX and linoleic acid sodium salt were obtained from
Sigma Chemical Co. All solvents were of analytical grade purity
and dry.
126.42, 125.44, 43.72, 41.64, 18.88. Anal. (C16H18N2O) C, H, N.
2.1.3. 1-Acyl-4-substituted semicarbazides (5aey): general
procedure
Method A:
A melted mixture of NSAID hydrazide 3aec
(1 mmol), 4aef (1 mmol) and TEA (0.418 ml, 3 mmol) was stirred
at 75e120 ꢀC for 15 min. The residue was cooled to room temper-
ature, dissolved in ethyl acetate (20 ml) and extracted with 0.1%
sodium hydroxide solution (20 ml ꢂ 3). The organic layer was
washed with water, dried over anhydrous sodium sulfate and
evaporated under reduced pressure. Method B: A melted mixture of
NSAID hydrazide 3aec (1 mmol), 4aef (1 mmol) and TEA (0.418 ml,
3 mmol) was stirred at 75e120 ꢀC for 15 min. The residue was
cooled to room temperature, stirred with acetone/HCl 1:2 solution
(30 ml, pH ¼ 1) and the precipitated product B was filtered off.
Method C: A solution of NSAID hydrazide 3aec (1 mmol) and
(0.268 g, 1 mmol) 1-(N-benzyloxycarbamoyl)benzotriazole (4g) in
dioxane (10 ml) was stirred at 55 ꢀC for 6e8 h. Dioxane was evap-
orated under reduced pressure, the residue was dissolved in ethyl
acetate (20 ml) and extracted with 0.1% sodium hydroxide solution
(20 ml ꢂ 3). The organic layer was washed with water, dried over
anhydrous sodium sulfate and evaporated under reduced pressure.
Method D: A solution of 1-acyl-4-benzyloxy semicarbazide 5tev
(0.5 mmol) in methanol (10 ml) was hydrogenated for 1.5 h in the
presence of Pd/C (50 mg). The catalyst was filtered off and the
solvent was evaporated under reduced pressure.
2.1.3.1. 4-Cyclopentyl-1-[2-(4-isobutylphenyl)propanoyl]semicarba-
zide (5a). Method A, 75 ꢀC; from the reaction of 0.220 g (1 mmol)
compound 3a and 0.230 g (1 mmol) 4a and after recrystallization
from ether/petroleum ether 0.212 g (64%) of 5a was obtained; mp
123e128 ꢀC; IR (KBr): nmax 3355, 3254, 2956, 2871, 1684, 1650, 1557,
1513, 1465, 1315, 1243, 1151, 1079, 1008, 937, 850, 786, 655 cmꢃ1; 1H
2.1.2. NSAID hydrazides (3aec): general procedure
NMR (DMSO-d6)
(bs, 1H), 3.84e3.80 (m, 1H), 3.59 (q, 1H), 2.40 (d, 2H), 1.84e1.20 (m,
9H), 1.32 (d, 3H), 0.85 (d, 6H); 13C NMR (DMSO-d6)
175.27, 159.64,
d 9.63, 7.59, (2s, 2H), 7.24 (d, 2H), 7.08 (d, 2H), 6.50
To a solution of hydrazine hydrate (2.43 ml, 50 mmol) in
dioxane (10 ml),
a
solution of NSAID benzotriazolides 2aec
d
(10 mmol) in dioxane (30 ml) was added dropwise. The reaction
mixture was stirred at rt for 1 h and evaporated under reduced
pressure. The obtained residue was dissolved in ethyl acetate
(30 ml) and extracted with 0.1% sodium hydroxide solution
(30 ml ꢂ 3), washed with water, dried over anhydrous sodium
sulfate and evaporated under reduced pressure.
141.54, 141.08, 130.96, 129.18, 53.09, 46.42, 44.76, 34.86, 25.33,
31.76, 24.34, 20.37. Anal. (C19H29N3O2) C, H, N.
2.1.3.2. 4-Cyclohexyl-1-[2-(4-isobutylphenyl)propanoyl]semicarba-
zide (5b). Method A, 90 ꢀC; from the reaction of 0.220 g (1 mmol)
compound 3a and 0.244 g (1 mmol) 4b and after recrystallization
from ether/petroleum ether 0.276 g (80%) of 5b was obtained; mp
165e166 ꢀC; IR (KBr): nmax 3307, 3253, 3019, 2986, 2951, 2930,
2852, 1708, 1683, 1641, 1562, 1531, 1506, 1453, 1322, 1253, 1226,
2.1.2.1. 2-(4-Isobutylphenyl)propanehydrazide (3a). From 3.074 g of
2a. The crude product was recrystallized from ether/petroleum
ether. Yield 1.96 g (89%); the spectral data were in agreement with
literature data [28].
1153, 1079, 1056, 1023, 999, 936, 892, 850, 709, 634, 614 cmꢃ1
;
1H
NMR (DMSO-d6) 9.67, 7.64 (2d, 2H), 7.23 (d, 2H), 7.08 (d, 2H), 5.71
d
(d, 1H), 3.59 (q, 1H), 3.39e3.28 (m, 2H), 2.40 (d, 2H), 1.84e1.75 (m,
2.1.2.2. 2-(3-Phenoxyphenyl)propanehydrazide
(3b). From
the
1H), 1.69e0.95 (m, 10H), 1.32 (d, 3H), 0.85 (d, 6H); 13C NMR (DMSO-
reaction of 3.434 g (10 mmol) compound 2b and purification by
column chromatography (mobile phase petrolether/ethyl acetate/
methanol 3:1:0.5) 2.307 g (90%) of 3b was obtained; IR (KBr): nmax
3285, 3040, 2978,1659,1631,1582,1487,1445,1244,1210,1164, 963,
d6) d 173.56, 157.60, 139.84, 139.36, 129.27, 127.47, 48.28, 44.70,
43.03, 33.42, 33.38, 24.90, 30.09, 25.65, 22.64, 18.64. Anal.
(C20H31N3O2) C, H, N.
928, 757, 692 cmꢃ1; 1H NMR (DMSO-d6)
d
9.19 (s,1H), 7.43e6.82 (m,
9H), 4.25 (s, 2H), 3.53 (q, 1H), 1.32 (d, 3H); 13C NMR (DMSO-d6)
172.87,157.01,156.92,144.73,130.50,119.04,130.15,123.88,122.86,
118.06, 117.02, 43.58, 18.76. Anal. (C15H16N2O2) C, H, N.
2.1.3.3. 4-Cyclohexanemethyl-1-[2-(4-isobutylphenyl)propanoyl]sem-
icarbazide (5c). Method B, 75 ꢀC; from the reaction of 0.220 g
(1 mmol) compound 3a and 0.258 g (1 mmol) 4c and after recrys-
tallization from ether/petroleum ether 0.338 g (94%) of 5c was
d

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I. Perkovic et al. / European Journal of Medicinal Chemistry 51 (2012) 227e238
229
obtained; mp 172e174 ꢀC; IR (KBr): nmax 3339, 3236, 3122, 2952,
1555, 1490, 1472, 1448, 1314, 1273, 1235, 1213, 1166, 1074, 1062,
1025, 944, 887, 748, 689, 640 cmꢃ1; 1H NMR (DMSO-d6)
9.70, 7.66
(2s, 2H), 7.42e6.83 (m, 9H), 5.84 (d, 1H), 3.63 (q, 1H), 3.39e3.33 (m,
2H), 1.71e1.00 (m, 10H), 1.33 (d, 3H); 13C NMR (DMSO-d6)
173.08,
2926, 2849,1699,1615,1565,1476,1448,1368,1263,1247,1175,1084,
d
1019, 854, 780, 651, 618 cmꢃ1 1H NMR (DMSO-d6)
; d 9.65, 7.66 (2s,
2H), 7.23 (d, 2H), 7.07 (d, 2H), 5.99 (t, 1H), 3.59 (q, 1H), 2.82 (t, 2H),
d
2.40 (d, 2H), 1.84e1.75 (m, 1H), 1.66e0.78 (m, 11H), 1.33 (d, 3H), 0.85
157.55, 156.99, 156.94, 144.29, 130.49, 119.01, 130.24, 123.87, 122.95,
118.16, 117.12, 48.33, 43.24, 33.41, 24.91, 25.65, 18.66. Anal.
(C22H27N3O3) C, H, N.
(d, 6H); 13C NMR (DMSO-d6)
d 173.66, 158.47, 139.79, 139.35, 129.22,
127.50, 45.80, 44.71, 43.06, 38.37, 30.67, 30.65, 25.88, 30.09, 26.56,
22.64, 18.69. Anal. (C21H33N3O2) C, H, N.
2.1.3.9. 4-Cyclohexanemethyl-1-[2-(3-phenoxyphenyl)propanoyl]
semicarbazide (5i). Method A, 75 ꢀC; from the reaction of 0.256 g
(1 mmol) compound 3b and 0.258 g (1 mmol) 4c and after recrys-
tallization from ether/petroleum ether 0.198 g (50%) of 5i was
obtained; mp 104e105 ꢀC; IR (KBr): nmax 3339, 3230, 3115, 3026,
2924, 2850, 1701, 1619, 1582, 1568, 1490, 1447, 1373, 1246, 1212,
1166, 1146, 1074, 1024, 1012, 962, 933, 888, 749, 691, 668 cmꢃ1; 1H
2.1.3.4. 4-Benzyl-1-[2-(4-isobutylphenyl)propanoyl]semicarbazide
(5d). Method B, 110 ꢀC; from the reaction of 0.220 g (1 mmol)
compound 3a and 0.252 g (1 mmol) 4d and after recrystallization
from ether/petroleum ether 0.293 g (83%) of 5d was obtained; mp
173e175 ꢀC; IR (KBr): nmax 3316, 3222, 3123, 3028, 2954, 1654, 1611,
1566, 1469, 1454, 1364, 1236, 1171, 1077, 851, 749, 699, 668 cmꢃ1; 1H
NMR (DMSO-d6)
4.22 (d, 2H), 3.59 (q, 1H), 2.39 (d, 2H), 1.83e1.74 (m, 1H), 1.34 (d,
3H), 0.84 (d, 6H); 13C NMR (DMSO-d6)
173.83, 158.59, 140.90,
d
9.70, 7.85 (2s, 2H), 7.32e7.05 (m, 9H), 6.70 (t,1H),
NMR (DMSO-d6)
1H), 3.63 (q, 1H), 2.84 (t, 2H), 1.62e0.79 (m, 11H), 1.34 (d, 3H); 13C
NMR (DMSO-d6) 173.18, 158.43, 157.03, 156.91, 144.29, 130.19,
d 9.70, 7.70 (2s, 2H), 7.43e6.84 (m, 9H), 6.09 (t,
d
d
139.78, 139.34, 129.21, 128.58, 127.55, 127.35, 127.00, 44.70, 43.11,
43.07, 30.08, 22.64, 18.81. Anal. (C21H27N3O2) C, H, N.
118.98, 130.21, 123.85, 122.98, 118.23, 117.14, 45.82, 43.29, 38.38,
30.68, 25.88, 26.56, 18.70. Anal. (C23H29N3O3) C, H, N.
2.1.3.5. 1-[2-(4-Isobutylphenyl)propanoyl]-4-phenylethylsemicarba-
zide (5e). Method A, 115 ꢀC; from the reaction of 0.220 g (1 mmol)
compound 3a and 0.252 g (1 mmol) 4e and after recrystallization
from ether/petroleum ether 0.268 g (73%) of 5e was obtained; mp
173e175 ꢀC; IR (KBr): nmax 3566, 3434, 3312, 3026, 2953, 2926,
2869,1648,1587,1557,1514,1497,1455,1374,1253,1168,1074,1054,
2.1.3.10. 4-Benzyl-1-[2-(3-phenoxyphenyl)propanoyl]semicarbazide
(5j). Method A, 110 ꢀC; from the reaction of 0.256 g (1 mmol)
compound 3b and 0.252 g (1 mmol) 4d and after recrystallization
from ether/petroleum ether 0.233 g (60%) of 5j was obtained; mp
154e156 ꢀC; IR (KBr): nmax 3323, 3266, 3209, 3109, 3028,1654,1621,
1584, 1565, 1490, 1453, 1369, 1315, 1278, 1249, 1235, 1162, 1143,
999, 934, 853, 796, 746, 700, 547 cmꢃ1; 1H NMR (DMSO-d6)
d
9.66,
1071, 935, 876, 751, 694, 644 cmꢃ1; 1H NMR (DMSO-d6)
(2s, 2H), 7.41e6.82 (m, 14H), 6.76 (t, 1H), 4.22 (d, 2H), 3.63 (q, 1H),
1.34 (d, 3H); 13C NMR (DMSO-d6)
173.35, 158.54, 157.03, 156.90,
d 9.74, 7.89
7.79 (2s, 2H), 7.38e7.00 (m, 9H), 6.15 (t, 1H), 3.59 (q, 1H), 3.22 (q,
2H), 2.67 (t, 2H), 2.40 (d, 2H),1.85e1.76 (m,1H),1.34 (d, 3H), 0.86 (d,
d
6H); 13C NMR (DMSO-d6)
d
173.72, 158.36, 139.99, 139.79, 139.33,
144.28, 140.91, 130.49, 128.59, 127.34, 119.00, 130.20, 127.00, 123.84,
123.02,118.29,117.14, 43.36, 43.08,18.77. Anal. (C23H23N3O3) C, H, N.
129.21, 129.10, 128.78, 127.54, 126.48, 44.70, 43.06, 41.32, 36.38,
30.08, 22.64, 18.78. Anal. (C22H29N3O2) C, H, N.
2.1.3.11. 1-[2-(3-Phenoxyphenyl)propanoyl]-4-phenylethylsemicarb-
azide (5k). Method B, 115 ꢀC; from the reaction of 0.256 g (1 mmol)
compound 3b and 0.252 g (1 mmol) 4e and after recrystallization
from ether/petroleum ether 0.254 g (63%) of 5k was obtained; mp
116e118 ꢀC; IR (KBr): nmax 3367, 3236, 3026, 2942, 1682, 1642, 1583,
1560, 1542, 1488, 1454, 1379, 1310, 1247, 1211, 1162, 1075, 1012, 944,
2.1.3.6. 4-Benzhydryl-1-[2-(4-isobutylphenyl)propanoyl]semicarbaz-
ide (5f). Method A, 120 ꢀC; from the reaction of 0.220 g (1 mmol)
compound 3a and 0.328 g (1 mmol) 4f and after recrystallization
from ether/petroleum ether 0.314 g (73%) of 5f was obtained; mp
151e154 ꢀC; IR (KBr): nmax 3343, 3202, 3028, 2953, 2869,1647,1616,
1538,1495,1456,1366,1268,1230,1168,1077,1052,1028,1004, 942,
916, 866, 796, 747, 698, 637 cmꢃ1; 1H NMR (DMSO-d6)
(2s, 2H), 7.42e6.84 (m, 14H), 6.21 (t, 1H), 3.63 (q, 1H), 3.23 (q, 2H),
2.68 (t, 2H), 1.34 (d, 3H); 13C NMR (DMSO-d6)
173.23, 158.31,
d 9.70, 7.82
848, 746, 700, 669, 631 cmꢃ1 1H NMR (DMSO-d6)
; d 9.76, 7.84 (2s,
2H), 7.34e7.05 (m, 14H), 6.95 (bs, 1H), 5.91 (d, 1H), 3.58 (q, 1H), 2.39
d
(d, 2H), 1.83e1.74 (m, 1H), 1.33 (d, 3H), 0.84 (d, 6H); 13C NMR
157.03, 156.90, 144.29, 139.99, 130.49, 129.11, 128.79, 119.00, 130.21,
126.48, 123.85, 123.02, 118.26, 117.14, 43.29, 41.32, 36.38, 18.78.
Anal. (C24H25N3O3) C, H, N.
(DMSO-d6)
d 173.70, 157.58, 143.51, 143.46, 139.83, 139.26, 129.25,
128.81, 128.79, 127.49, 127.47, 127.44, 127.35, 127.31, 57.14, 44.71,
43.05, 30.06, 22.65, 18.78. Anal. (C27H31N3O2) C, H, N.
2.1.3.12. 4-Benzhydryl-1-[2-(3-phenoxyphenyl)propanoyl]semicarba-
zide (5l). Method A, 120 ꢀC; from the reaction of 0.256 g (1 mmol)
compound 3b and 0.328 g (1 mmol) 4f and after recrystallization
from ether/petroleum ether 0.219 g (47%) of 5l was obtained; mp
128e131 ꢀC; IR (KBr): nmax 3350, 3236, 3031, 2973, 1655, 1613, 1587,
1560, 1488, 1448, 1247, 1210, 1164, 1070, 1052, 1030, 949, 912, 753,
2.1.3.7. 4-Cyclopentyl-1-[2-(3-phenoxyphenyl)propanoyl]semicarba-
zide (5g). Method A, 90 ꢀC; from the reaction of 0.256 g (1 mmol)
compound 3b and 0.230 g (1 mmol) 4a and after recrystallization
from ether/petroleum ether 0.293 g (80%) of 5g was obtained; mp
133e135 ꢀC; IR (KBr): nmax 3414, 3281, 3237, 3101, 3025, 2961, 2910,
2869, 1696, 1627, 1638, 1582, 1556, 1532, 1489, 1455, 1443, 1311,
1244, 1213, 1162, 1146, 1131, 1073, 942, 913, 880, 774, 760, 691,
698, 668, 634, 614 cmꢃ1
7.38e6.98 (m, 19H), 6.83 (d, 1H), 5.91 (d, 1H), 3.64 (q, 1H), 1.33 (d,
3H); 13C NMR (DMSO-d6)
172.70, 157.02, 156.50 156,47, 143.70,
; d 9.78, 7.85 (2s, 2H),
1H NMR (DMSO-d6)
618 cmꢃ1
9H), 5.94 (d, 1H), 3.89e3.78 (m, 2H), 3.63 (q, 1H), 1.79e1.22 (m, 8H),
1.32 (d, 3H); 13C NMR (DMSO-d6)
173.08, 157.89, 156.99, 156.94,
;
1H NMR (DMSO-d6)
d
9.69, 7.63 (2s, 2H), 7.42e6.82 (m,
d
143.03, 142.96, 129.97, 118.53, 129.73, 123.35, 122.43, 117.71, 116.65,
128.37, 128.33, 126.97, 126.93, 126.85, 126.81, 56.71, 42.80, 18.24.
Anal. (C29H27N3O3) C, H, N.
d
144.29, 130.49, 119.01, 130.23, 123.87, 122.93, 118.17, 117.12, 51.40,
43.25, 33.13, 24.91, 18.66. Anal. (C21H25N3O3) C, H, N.
2.1.3.13. 1-[2-(3-Benzylphenyl)propanoyl]-4-cyclopentylsemicarbaz-
ide (5m). Method A, 75 ꢀC; from the reaction of 0.254 g (1 mmol)
compound 3c and 0.230 g (1 mmol) 4a and after recrystallization
from ether/petroleum ether 0.259 g (71%) of 5m was obtained; mp
133e134 ꢀC; IR (KBr): nmax 3279, 3027, 2962, 2872, 1710, 1646, 1555,
1494, 1452, 1372, 1242, 1191, 1152, 1077, 1010, 941, 728, 698,
2.1.3.8. 4-Cyclohexyl-1-[2-(3-phenoxyphenyl)propanoyl]semicarbaz-
ide (5h). Method A, 90 ꢀC; from the reaction of 0.256 g (1 mmol)
compound 3b and 0.244 g (1 mmol) 4b and after recrystallization
from ether/petroleum ether 0.229 g (60%) of 5h was obtained; mp
144e146 ꢀC; IR (KBr): nmax 3343, 3215, 2933, 2853,1695,1618,1580,

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I. Perkovic et al. / European Journal of Medicinal Chemistry 51 (2012) 227e238
230
638 cmꢃ1
;
1H NMR (DMSO-d6)
d
9.67, 7.60 (2s, 2H), 7.31e7.05 (m,
1H), 1.33 (d, 3H); 13C NMR (DMSO-d6)
d 173.67, 157.56, 143.52,
9H), 5.90 (d, 1H), 3.91 (s, 2H), 3.82 (m, 1H), 3.59 (q, 1H), 1.76e1.22
143.47, 142.13, 141.59, 141.56, 129.14, 128.86, 128.82, 128.80, 127.45,
128.22, 127.34, 127.32, 126.40, 125.47, 57.16, 43.48, 41.63, 18.84.
Anal. (C30H29N3O2) C, H, N.
(m, 8H), 1.32 (d, 3H); 13C NMR (DMSO-d6)
d
173.40, 157.93, 142.22,
141.61, 141.55, 129.14, 128.86, 128.76, 128.25, 127.48, 126.42, 125.45,
51.38, 43.36, 41.65, 33.13, 23.62, 18.73. Anal. (C22H27N3O2) C, H, N.
2.1.3.19. 4-Benzyloxy-1-[2-(4-isobutylphenyl)propanoyl]semicarbaz-
ide (5t). Method C; 6 h; from the reaction of 0.220 g (1 mmol)
compound 3a and after recrystallization from ether/petroleum
ether 0.336 g (91%) of 5t was obtained; mp 87 ꢀC (decomp.); IR
(KBr): nmax 3472, 3319, 3240, 3114, 3064, 3031, 2953, 2867, 1714,
1698, 1681, 1638, 1594, 1548, 1514, 1455, 1366, 1349, 1226, 1147,
2.1.3.14. 1-[2-(3-Benzylphenyl)propanoyl]-4-cyclohexylsemicarbazide
(5n). Method A, 90 ꢀC; from the reaction of 0.254 g (1 mmol)
compound 3c and 0.244 g (1 mmol) 4b and after recrystallization
from ether/petroleum ether 0.334 g (85%) of 5n was obtained; mp
143e144 ꢀC; IR (KBr): nmax 3327, 3216, 3027, 2932, 2853, 1658, 1617,
1589, 1477, 1452, 1255, 1232, 1182, 1074, 1065, 943, 891, 769, 751, 701,
1082, 994, 933, 910, 850, 802, 751, 698, 620, 592, 545 cmꢃ1
;
1H
639, 618 cmꢃ1
(m, 9H), 5.80 (d, 1H), 3.91 (s, 2H), 3.59 (q, 1H), 3.38e3.29 (m, 1H),
1.70e0.97 (m, 10H), 1.32 (d, 3H); 13C NMR (DMSO-d6)
173.40,
;
1H NMR (DMSO-d6)
d
9.68, 7.64 (2s, 2H), 7.30e7.05
NMR (DMSO-d6) 9.73, 9.47, 8.71 (3s, 3H), 7.43e7.32 (m, 5H), 7.26
d
(d, 2H), 7.08 (d, 2H), 4.74 (s, 2H), 3.62 (q, 1H), 2.40 (d, 2H),
d
1.87e1.76 (m, 1H), 1.34 (d, 3H), 0.85 (d, 6H); 13C NMR (DMSO-d6)
157.60, 142.22, 141.61, 141.55, 129.14, 128.86, 128.77, 128.24, 127.48,
126.42, 125.45, 48.31, 43.36, 41.65, 33.38, 24.89, 25.66, 18.73. Anal.
(C23H29N3O2) C, H, N.
d 173.55, 159.52, 139.77, 139.25, 136.90, 129.17, 129.12, 128.61,
127.62, 128.42, 77.90, 44.71, 43.05, 30.10, 22.65, 18.93. Anal.
(C21H27N3O3) C, H, N.
2.1.3.15. 1-[2-(3-Benzylphenyl)propanoyl]-4-cyclohexanemetylsemi-
carbazide (5o). Method B, 75 ꢀC; from the reaction of 0.254 g
(1 mmol) compound 3c and 0.258 g (1 mmol) 4c and after recrys-
tallization from ether/petroleum ether 0.268 g (68%) of 5o was
obtained; mp 134e137 ꢀC; IR (KBr): nmax 3344, 3216, 3027, 2919,
2850, 1663, 1622, 1589, 1474, 1450, 1370, 1273, 1257, 1239, 1184,
1075, 1030, 963, 746, 723, 702, 652, 618 cmꢃ1; 1H NMR (DMSO-d6)
2.1.3.20. 4-Benzyloxy-1-[2-(3-phenoxyphenyl)propanoyl]semicarba-
zide (5u). Method C, 7 h; from the reaction of 0.256 g (1 mmol)
compound 3b and after purification by column chromatography
(mobile phase dichloromethane/methanol 9.5:0.5) 0.369 g (91%) of
5u was obtained; oil; IR (KBr): nmax 3237, 3063, 3034, 2979, 2934,
2877, 1668, 1583, 1488, 1455, 1371, 1312, 1244, 1211, 1163, 1073, 942,
914, 753, 695, 614 cmꢃ1
3H), 7.41e6.83 (m, 14H), 4.75 (s, 2H), 3.66 (q, 1H), 1.35 (d, 3H); 13C
NMR (DMSO-d6) 172.59, 158.95, 156.57, 156.38, 143.70, 136.37,
; d 9.74, 9.47, 8.70 (3s,
1H NMR (DMSO-d6)
d
9.67, 7.66 (2s, 2H), 7.31e7.05 (m, 9H), 6.04 (t, 1H), 3.91 (s, 2H), 3.59
(q, 1H), 2.82 (t, 2H), 1.66e0.78 (m, 11H), 1.32 (d, 3H); 13C NMR
(DMSO-d6) 173.48, 158.47, 142.21, 141.62, 141.52, 129.13, 128.86,
d
d
129.98, 128.62, 128.11, 118.48, 129.65, 127.93, 123.32, 122.59, 117.87,
116.65, 77.42, 42.80, 18.37. Anal. (C23H23N3O4) C, H, N.
128.74, 128.24, 127.46, 126.41, 125.47, 45.81, 43.38, 41.65, 38.36,
30.66, 25.88, 26.55, 18.76. Anal. (C24H31N3O2) C, H, N.
2.1.3.21. 4-Benzyloxy-1-[2-(3-benzylphenyl)propanoyl]semicarbazide
(5v). Method C, 8 h; from the reaction of 0.254 g (1 mmol)
compound 3c and after recrystallization from ether/petroleum
ether 0.201 g (50%) of 5v was obtained; mp 104e105 ꢀC; IR (KBr):
nmax 3306, 3235, 3029, 2925, 2880, 1691, 1654, 1599, 1520, 1493,
1454, 1382, 1366, 1356, 1273, 1238, 1152, 1074, 1032, 1007, 947, 788,
2.1.3.16. 1-[2-(3-Benzylphenyl)propanoyl]-4-benzylsemicarbazide
(5p). Method B, 110 ꢀC; from the reaction of 0.254 g (1 mmol)
compound 3c and 0.252 g (1 mmol) 4d and after recrystallization
from ether/petroleum ether 0.325 g (84%) of 5p was obtained; mp
150e154 ꢀC; IR (KBr): nmax 3331, 3261, 3211, 3028, 2986, 1707, 1660,
1620, 1563, 1494, 1453, 1431, 1371, 1246, 1174, 1075, 750, 723, 698,
751, 727, 699, 668, 555, 532, 497 cmꢃ1; 1H NMR (DMSO-d6)
9.46, 8.69 (3s, 3H), 7.37e7.02 (m, 14H), 4.72 (s, 2H), 3.88 (s, 2H), 3.59
(q, 1H), 1.31 (d, 3H); 13C NMR (DMSO-d6)
173.40, 159.52, 142.13,
d 9.72,
642, 618 cmꢃ1; 1H NMR (DMSO-d6)
(m, 14H), 6.73 (t, 1H), 4.22 (d, 2H), 3.89 (s, 2H), 3.60 (q, 1H), 1.34 (d,
3H); 13C NMR (DMSO-d6)
173.67, 158.59, 142.23, 141.63, 141.52,
d 9.72, 7.86 (2s, 2H), 7.32e7.04
d
d
141.65, 141.47, 136.90, 129.16, 129.13, 128.87, 128.61, 128.70, 128.43,
128.34, 127.44, 126.41, 125.61, 77.90, 43.39, 41.65, 19.00. Anal.
(C24H25N3O3) C, H, N.
140.92, 129.15, 128.87, 128.59, 127.35, 128.74, 128.28, 127.46, 127.01,
126.41, 125.55, 43.46, 43.08, 41.64, 18.88. Anal. (C24H25N3O2) C, H, N.
2.1.3.17. 1-[2-(3-Benzylphenyl)propanoyl]-4-phenylethylsemicarba-
zide (5r). Method A, 115 ꢀC; from the reaction of 0.254 g (1 mmol)
compound 3c and 0.252 g (1 mmol) 4e and after recrystallization
from ether/petroleum ether 0.303 g (73%) of 5r was obtained; mp
118e120 ꢀC; IR (KBr): nmax 3365, 3301, 3239, 3027, 2939,1686,1649,
1601, 1555, 1495, 1455, 1379, 1269, 1242, 1154, 1073, 945, 751, 726,
2.1.3.22. 4-Hydroxy-1-[2-(4-isobutylphenyl)propanoyl]semicarbazide
(5w). Method D; from the reaction of 0.185 g (1 mmol) compound
5t and after recrystallization from ether/petroleum ether 0.134 g
(96%) of 5a was obtained; mp 123e125 ꢀC; IR (KBr): nmax 3330,
3235, 3027, 2954, 2924, 2869, 1704, 1652, 1556, 1514, 1465, 1383,
1320, 1262, 1141, 1078, 1002, 935, 850, 766, 727, 653, 531 cmꢃ1; 1H
699, 661 cmꢃ1; 1H NMR (DMSO-d6)
(m, 14H), 6.17 (t, 1H), 3.90 (s, 2H), 3.58 (q, 1H), 3.21 (q, 2H), 2.65 (t,
2H), 1.32 (d, 3H); 13C NMR (DMSO-d6)
173.55, 158.37, 142.23,
d
9.66, 7.78 (2s, 2H), 7.29e7.04
NMR (DMSO-d6)
2H), 3.61 (q,1H), 2.40 (d, 2H), 1.85e1.76 (m,1H),1.33 (d, 3H), 0.85 (d,
6H); 13C NMR (DMSO-d6)
173.45, 160.82, 139.71, 139.33, 129.14,
d 9.65, 8.70, 8.64, 8.41 (4s, 4H), 7.25 (d, 2H), 7.07 (d,
d
d
141.63, 141.52, 140.00, 129.15, 129.11, 128.87, 128.79, 128.75, 128.28,
127.46, 126.48, 126.41, 125.54, 43.41, 41.65, 41.33, 36.39, 18.87. Anal.
(C25H27N3O2) C, H, N.
127.62, 44.71, 43.00, 30.09, 22.65, 19.01. Anal. (C14H21N3O3) C, H, N.
2.1.3.23. 4-Hydroxy-1-[2-(3-phenoxyphenyl)propanoyl]semicarbaz-
ide (5x). Method D; from the reaction of 0.203 g (1 mmol)
compound 5u and after trituration with ether 0.118 g (75%) of 5x
was obtained; mp 149e150 ꢀC (decomp.); IR (KBr): nmax 3330, 3235,
3027, 2954, 2924, 2869, 1704, 1652, 1556, 1514, 1465, 1383, 1320,
1262, 1141, 1078, 1002, 935, 850, 766, 727, 653, 531 cmꢃ1; 1H NMR
2.1.3.18. 4-Benzhydryl-1-[2-(3-benzylphenyl)propanoyl]semicarbaz-
ide (5s). Method A, 120 ꢀC; from the reaction of 0.254 g (1 mmol)
compound 3c and 0.328 g (1 mmol) 4f and after recrystallization
from ether/petroleum ether 0.339 g (71%) of 5s was obtained; mp
166e170 ꢀC; IR (KBr): nmax 3346, 3231, 3028, 1981, 1662, 1611, 1590,
1560, 1494, 1472, 1453, 1372, 1258, 1236, 1078, 1030, 948, 751, 700,
(DMSO-d6)
d
9.68, 8.72, 8.65, 8.45 (4s, 4H), 7.42e6.82 (m, 9H), 3.65
172.98, 160.78, 157.06,
(q, 1H), 1.33 (d, 3H); 13C NMR (DMSO-d6)
d
668, 634, 613 cmꢃ1
;
1H NMR (DMSO-d6)
d
9.78, 7.84 (2s, 2H),
156.84, 144.29, 130.49, 118.98, 130.13, 123.81, 123.10, 118.36, 117.11,
43.24, 18.95. Anal. (C16H17N3O4) C, H, N.
7.33e7.00 (m, 19H), 6.99 (d, 1H), 5.91 (d, 1H), 3.89 (s, 2H), 3.60 (q,

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231
2.1.3.24. 1-[2-(3-Benzylphenyl)propanoyl]-4-hydroxysemicarbazide
(5y). Method D; from the reaction of 0.202 g (1 mmol) compound
5v and after trituration with ether 0.150 g (96%) of 5y was obtained;
mp 123e125 ꢀC; IR (KBr): nmax 3288, 3060, 3028, 2979, 2938, 1687,
1644, 1562, 1516, 1494, 1483, 1451, 1380, 1263, 1159, 1144, 1100,
7.08 (d, 2H), 4.77 (s, 2H), 3.62 (q,1H), 2.41 (d, 2H),1.85e1.76 (m,1H),
1.35 (d, 3H), 0.86 (d, 6H); 13C NMR (DMSO-d6)
173.52, 160.03,
158.37, 139.75, 139.34, 136.97, 129.17, 129.14, 128.61, 127.59, 128.41,
77.87, 44.71, 43.00, 30.09, 22.65, 18.85. Anal. (C22H29N5O4) C, H, N.
d
1077, 1061, 1027, 938, 782, 721, 700, 596, 533 cmꢃ1
(DMSO-d6)
(s, 2H), 3.62 (q, 1H), 1.34 (d, 3H); 13C NMR (DMSO-d6)
;
1H NMR
9.66, 8.73, 8.66, 8.43 (4s, 4H), 7.32e7.05 (m, 9H), 3.92
173.29,
2.1.4.2. 5-Benzyloxycarbamoyl-1-[2-(3-phenoxyphenyl)propanoyl]
carbazide (8b). Method A; from the reaction of 0.256 g (1 mmol)
compound 3b and after purification by column chromatography
(mobile phase dichloromethane/methanol 9.5:0.5) and recrystalli-
zation from ether/petroleum ether 0.338 g (73%) of 8b was
obtained; mp 74e76 ꢀC (decomp.); IR (KBr): nmax 3251, 3062, 3034,
2971, 2933, 2873, 1677, 1583, 1523, 1488, 1455, 1369, 1312, 1243,
1211, 1163, 1073, 1023, 1002, 942, 914, 816, 752, 694, 614 cmꢃ1; 1H
d
d
160.82, 142.22, 141.66, 141.45, 129.16, 128.87, 128.68, 128.34, 127.38,
126.40, 125.60, 43.34, 41.65, 19.08. Anal. (C17H19N3O3) C, H, N.
2.1.3.25. 4-Benzyloxysemicarbazide (6). A solution of 1-(N-benzy-
loxycarbamoyl)benzotriazole (4g) (2.683 g, 10 mmol) in 30 ml
dioxane was added dropwise to a solution of 0.534 ml (11 mmol)
hydrazine hydrate in 10 ml dioxane. The reaction mixture was
stirred at rt for 1 h and evaporated under reduced pressure. The
analytically pure sample was obtained after column chromatog-
raphy (dichloromethane/methanol 9.5:0.5) and triturated with
ether. Yield 1.486 g (82%); mp 90e92 ꢀC; IR (KBr): nmax 3332, 3287,
3217, 3061, 3030, 2962, 2925, 2875, 1655, 1637, 1517, 1456, 1366,
1325, 1228, 1210, 1192, 1096, 1070, 980, 915, 797, 751, 702, 680,
NMR (DMSO-d6)
14H), 4.77 (s, 2H), 3.65 (q, 1H), 1.34 (d, 3H); 13C NMR (DMSO-d6)
173.03, 160.03, 158.32, 157.05, 156.87, 144.28, 136.97, 130.50,
d 9.78, 9.43, 8.55, 8.18, 8.05 (5s, 5H), 7.44e6.82 (m,
d
129.14, 128.61, 118.99, 130.16, 128.41, 123.83, 123.06, 118.33, 117.13,
77.88, 43.25, 18.79. Anal. (C24H25N5O5) C, H, N.
2.1.4.3. 5-Benzyloxycarbamoyl-1-[2-(3-benzylphenyl)propanoyl]car-
bazide (8c). Method A; from the reaction of 0.254 g (1 mmol)
compound 3c and after purification by column chromatography
(mobile phase dichloromethane/methanol 9.5:0.5) and recrystalli-
zation from ether/petroleum ether 0.337 g (73%) of 8c was
obtained; mp 75e77 ꢀC; IR (KBr): nmax 3248, 3060, 3028, 2880,
2836, 1675, 1602, 1519, 1494, 1453, 1369, 1310, 1270, 1210, 1075,
607 cmꢃ1; 1H NMR (DMSO-d6)
d
9.18 (s, 1H), 7.84 (s, 1H), 7.42e7.29
161.67,
(m, 5H), 4.71 (s, 2H), 4.01 (s, 2H); 13C NMR (DMSO-d6)
d
137.18, 129.08, 128.63, 128.37, 77.70. Anal. (C8H11N3O2) C, H, N.
2.1.3.26. 1-(1-Benzotriazolecarbonyl)-4-benzyloxysemicarbazide
(7). To a solution of 1.357 g (8 mmol) BtcCl (1) in anhydrous
1030, 974, 941, 908, 751, 699, 620, 556 cmꢃ1
9.76, 9.43, 8.55, 8.16, 8.03, 7.44e7.05 (m, 14H), 4.78 (s, 2H), 3.92 (s,
2H), 3.62 (q, 1H), 1.34 (d, 3H); 13C NMR (DMSO-d6)
173.35, 160.02,
;
1H NMR (DMSO-d6)
dioxane (20 ml),
a
solution of 1.450 g (8 mmol) 4-
d
benzyloxysemicarbazide (6) and 1.115 ml (8 mmol) TEA in
dioxane (20 ml) was added dropwise. The reaction mixture was
stirred at rt for 1 h and evaporated under reduced pressure. The
obtained residue was dissolved in 40 ml ethyl acetate/water 1:1
mixture. The organic layer was extracted 3 times with water, dried
over anhydrous sodium sulfate and evaporated under reduced
pressure. The crude product was triturated with ether. Yield 1.906 g
(73%); mp 90e93 ꢀC (decomp.); IR (KBr): nmax 3482, 3376, 3340,
3164, 3005, 2924, 2854, 1738, 1674, 1556, 1487, 1449, 1387, 1287,
d
158.36, 142.21, 141.64, 141.48, 136.96, 129.15, 128.87, 128.61, 128.70,
128.41, 128.31, 127.42, 126.40, 125.58, 77.88, 43.34, 41.65, 18.91.
Anal. (C25H27N5O4) C, H, N.
2.1.4.4. 5-Hydroxycarbamoyl-1-[2-(4-isobutylphenyl)propanoyl]car-
bazide (8d). Method B; from the reaction of 0.214 g (1 mmol)
compound 8a and after trituration with ether 0.120 g (71%) of 8d
was obtained; mp 145e148 ꢀC (decomp.); IR (KBr): nmax 3261, 2955,
2928, 2870,1670,1514,1466,1383,1367,1281,1206,1077,1002, 934,
1235, 1066, 1033, 905, 754, 744, 704, 680, 652, 609, 580, 539 cmꢃ1
1H NMR (DMSO-d6)
10.97, 9.87, 9.31, 8.26e8.18 (2d, 2H), 7.77, 7.59
(2t, 2H), 7.49e7.35 (m, 5H), 4.85 (s, 2H); 13C NMR (DMSO-d6)
159.37, 149.86, 145.66, 131.92, 136.74, 130.78, 126.31, 120.42,
;
d
850, 783 cmꢃ1 1H NMR (DMSO-d6)
; d 9.73, 8.66, 8.57, 8.23, 8.08,
7.92 (5s, bs, 6H), 7.24 (d, 2H), 7.07 (d, 2H), 3.61 (q, 1H), 2.40 (d, 2H),
d
1.82e1.78 (m, 1H), 1.33 (d, 3H), 0.85 (d, 6H); 13C NMR (DMSO-d6)
113.79, 129.20, 128.68, 128.54, 78.11. Anal. (C15H14N6O3) C, H, N.
d 172.90, 160.70, 157.83, 139.24, 138.85, 128.6, 127.07, 44.22, 42.49,
29.55, 22.14, 18.35. Anal. (C15H23N5O4) C, H, N.
2.1.4. 1-Acyl-5-benzyloxyand 1-acyl-5-hydroxycarbamoylcarbazides
(8aef): general procedure
2.1.4.5. 5-Hydroxycarbamoyl-1-[2-(3-phenoxyphenyl)propanoyl]car-
bazide (8e). Method B; from the reaction of 0.232 g (1 mmol)
compound 8b and after trituration with ether 0.159 g (85%) of 8e
was obtained; IR (KBr): nmax 3263, 2978, 2925, 2855, 1670, 1583,
1540, 1488, 1456, 1445, 1376, 1313, 1244, 1210, 1163, 1074, 943, 917,
Method A: A mixture of 0.326 g (1 mmol) compound 7 and
NSAID hydrazide 3aec (1 mmol) was melted at 90 ꢀC and stirred for
20 min, cooled, dissolved in ethyl acetate (20 ml) and extracted
with 0.1% sodium hydroxide solution (20 ml ꢂ 3). The organic layer
was washed with water, dried over anhydrous sodium sulfate and
evaporated under reduced pressure. Method B: A solution of 1-
acyl-5-benzyloxycarbamoylcarbazides 8aec (0.5 mmol) in meth-
anol (10 ml) was hydrogenated for 2 h in the presence of Pd/C
(50 mg). The catalyst was filtered off and the solvent was evapo-
rated under reduced pressure.
755, 692 cmꢃ1; 1H NMR (DMSO-d6)
d
9.80, 8.67, 8.29, 8.14, 7.96 (5s,
bs, 6H), 7.42e6.81 (m, 9H), 3.64 (q, 1H), 1.34 (d, 3H); 13C NMR
(DMSO-d6) 172.92, 161.24, 158.30, 157.03, 156.88, 144.28, 130.50,
d
119.01, 130.16, 123.84, 123.03, 118.30, 117.11, 43.22, 18.78. Anal.
(C17H19N5O5) C, H, N.
2.1.4.6. 1-[2-(3-Benzylphenyl)propanoyl]-5-hydroxycarbamoylcarba-
zide (8f). Method B; from the reaction of 0.231 g (1 mmol)
compound 8c and after trituration with ether 0.147 g (79%) of 8f
was obtained; mp 102e105 ꢀC; IR (KBr): nmax 3258, 3060, 3027,
2981, 2934, 1670, 1601, 1538, 1494, 1453, 1375, 1321, 1195, 1075,
2.1.4.1. 5-Benzyloxycarbamoyl-1-[2-(4-isobutylphenyl)propanoyl]
carbazide (8a). Method A; from the reaction of 0.220 g (1 mmol)
compound 3a and after purification by column chromatography
(mobile phase dichloromethane/methanol 9.5:0.5) and recrystalli-
zation from ether/petroleum ether 0.304 g (71%) of 8a was
obtained; mp 87e90 ꢀC; IR (KBr): nmax 3266, 3032, 2955, 2930,
2869,1677,1513,1466,1455,1366,1309,1271,1212,1188,1075,1030,
1030, 943, 784, 728, 699, 622, 557 cmꢃ1; 1H NMR (DMSO-d6)
d
9.78.
8.67, 8.28, 8.11, 7.95 (5s, bs, 6H), 7.31e7.04 (m, 9H), 3.91 (s, 2H), 3.61
(q, 1H), 1.33 (d, 3H); 13C NMR (DMSO-d6)
173.23, 161.24, 158.34,
d
1002, 934, 909, 849, 751, 699, 620, 547 cmꢃ1
;
1H NMR (DMSO-d6)
142.22, 141.64, 141.48, 129.16, 128.88, 128.71, 128.29, 127.42, 126.41,
125.56, 43.32, 41.65, 18.90. Anal. (C18H21N5O4) C, H, N.
d
9.75, 9.43, 8.54, 8.15, 8.03 (5s, 5H), 7.44e7.30 (m, 5H), 7.26 (d, 2H),

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232
2.2. Biological tests
vesicular stomatitis virus (VSV)], African green monkey kidney
(Vero, parainfluenza-3, reovirus-1, Sindbis, Coxsackie B4, and Punta
Toro virus), human cervix carcinoma (vesicular stomatitis virus,
Coxsackie virus B4 and respiratory syncytial virus (RSV)), feline
Crandell-Rees kidney (CRFK) (feline herpes virus, feline coronavirus
(FIPV)) or Madin-Darby canine kidney (MDCK) (influenza A [H1N1;
H3N2] and influenza B) cell cultures. Confluent cell cultures in
microtiter 96-wellplateswere inoculated with 100 CCID50 of virus (1
CCID50 being the virus dose to infect 50% of the cell cultures) in the
2.2.1. Cytostatic activity assays
The cytostatic activity of the test compounds against murine
leukemia L1210 and human lymphocyte CEM and cervix carcinoma
HeLa cells was determined as follows: L1210, CEM and HeLa cells
were suspended at 300,000e500,000 cells/ml of culture medium,
and 100
ml of a cell suspension was added to 100
ml of an appro-
l-wells of 96-well
priate dilution of the test compounds in 200
m
microtiter plates. After incubation at 37 ꢀC for two (L1210) or
three (CEM, HeLa) days, the cell number was determined using
a Coulter counter [29]. Whereas L1210 and CEM cells could be
counted directly in the Coulter counter, HeLa cells were detached
from the microtiter plate wells by trypsin treatment prior to
counting. The IC50 was defined as the compound concentration
required to inhibit cell proliferation by 50%.
presence of varying concentrations (100, 40, 8, 1.6 and 0.32 mM) of
the test compounds. Viral cytopathicity was recorded as soon as it
reached completion in the control virus-infected cell cultures that
were not treated with the test compounds. The methodology of the
anti-HIV assays was as follows: human T-lymphocyte CEM
(w3 ꢂ105 cells/cm3) cells were exposed to 100 CCID50 of HIV-1(IIIB)
or HIV-2(ROD)/ml and seeded in 200 ml wells of a microtiter plate
The cytostatic activity of the test compounds against HCT 116
(colon carcinoma), H 460 (lung carcinoma), MCF-7 (breast carci-
noma) and HaCaT (human immortalized keratinocytes) was deter-
mined as follows: the cells were cultured as monolayers and
maintained in Dulbecco’s modified Eagle medium (DMEM) sup-
containing appropriate dilutions of the test compounds. After 4 days
of incubation at 37 ꢀC, HIV-induced CEM giant cell formation was
examined microscopically.
2.2.3. Determination of the reducing activity of the stable radical
DPPH
plemented with 10% fetal bovine serum (FBS), 2 mM
L-glutamine,
100 U/ml penicillin and 100 g/ml streptomycin in a humidified
m
To an ethanolic solution of DPPH (0.05 mM) in absolute ethanol
atmosphere with 5% CO2 at 37 ꢀC. The growth inhibition activity was
assessed as described previously [30,31]. The panel cell lines were
inoculated onto a series of standard 96-well microtiter plates on day
0, at 1 ꢂ104 to 3 ꢂ 104 cells/ml, depending on the doubling times of
a specific cell line. Test agents were then added in ten-fold dilutions
(10ꢃ8e10ꢃ4 M) and incubated for further 72 h. 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,
which detects dehydrogenize activity in viable cells. The absorbance
(A) was measured on a microplate reader at 570 nm. The absorbance
is directly proportional to the number of living, metabolically active
cells. The percentage of growth (PG) of the cell lines was calculated
according to one or the other of the following two expressions:
an equal volume of the compounds (final concentration 100 mM)
dissolved in DMSO was added. The mixture was shaken vigorously
and allowed to stand for 20 or 60 min. Absorbance at 517 nm was
determined spectrophotometrically, and the percentage of activity
was calculated. All tests were undertaken on three replicates, and
the results were averaged (Table 2).
2.2.4. Inhibition of linoleic acid lipid peroxidation
The water-soluble azo compound AAPH is used as a free radical
initiator for in vitro studies of free radical production. Production of
conjugated diene hydroperoxide by oxidation of linoleic acid
sodium salt in an aqueous solution is monitored at 234 nm. This
assay can be used to follow oxidative changes and to understand
the contribution of each tested compound. An amount of 10 mL of
the 16 mM linoleic acid sodium salt solution was added to the UV
cuvette containing 0.93 ml of 0.05 M phosphate buffer, pH 7.4,
prethermostated at 37 ꢀC. The oxidation reaction was initiated at
Ifðmean Atest ꢃ mean AtzeroÞ ꢄ 0; then PG ¼ 100
ꢂ ðmean Atest ꢃ mean AtzeroÞ=ðmean Actrl ꢃ mean AtzeroÞ:
37 ꢀC under air by the addition of 50
mL of 40 mM AAPH solution.
Ifðmean Atest ꢃ mean AtzeroÞ<0; then PG ¼ 100
ꢂ ðmean Atest ꢃ mean AtzeroÞ=ðAtzeroÞ;
Oxidation was carried out in the presence of aliquots (10 L) in the
m
assay without antioxidant, and lipid oxidation was measured in the
presence of the same level of DMSO. The rate of oxidation at 37 ꢀC
was monitored by recording the increase in absorption at 234 nm
caused by conjugated diene hydroperoxides. Each experiment was
performed at least in triplicate, and the standard deviation of
absorbance was less than 10% of the mean.
where the mean Atzero is the average of optical density measure-
ments before exposure of cells to the test compound, the mean Atest
is the average of optical density measurements after the desired
period of time and the mean Actrl is the average of optical density
measurements after the desired period of time with no exposure of
cells to the test compound. The results are expressed as IC50, which
is the concentration necessary for 50% of inhibition. The IC50 values
for each compound are calculated from concentrationeresponse
curves using linear regression analysis by fitting the test concen-
trations that give PG values above and below the reference value
(i.e. 50%). If however, all of the tested concentrations produce PGs
exceeding the respective 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. Each test was performed in
quadruplicate in at least two individual experiments.
2.2.5. Soybean LOX inhibition study in vitro
The tested compounds dissolved in DMSO were incubated at
room temperature with sodium linoleate (0.1 ml) and 0.2 ml of the
enzyme solution (1 part of enzyme/9 parts of saline ꢂ 10ꢃ4, w/v in
saline). The conversion of sodium linoleate to 13-
hydroperoxylinoleic acid at 234 nm was recorded and compared
with the appropriate standard inhibitor. Each experiment was per-
formed at least in triplicate, and the standard deviation of absor-
bance was less than 10% of the mean.
3. Results and discussion
2.2.2. Antiviral activity assays
The antiviral assays, other than the anti-HIV assays, were based
on inhibition of virus-induced cytopathic effect in human lung
fibroblast [herpes simplex virus type 1 (HSV-1) [strain KOS], herpes
simplex virus type 2 (HSV-2) [strain G], vaccinia virus (VV) and
3.1. Chemistry
Two series of nitrogen/oxygen-rich derivatives of the NSAIDs
ibuprofen, fenoprofen and reduced ketoprofen 5 and 8 were

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I. Perkovic et al. / European Journal of Medicinal Chemistry 51 (2012) 227e238
233
Table 1
Inhibitory effects of 1-acyl-4-cycloalkyl(or 4-aryl)semicarbazides 5aey, 1-acyl-5-benzyloxy(or 5-hydroxy)carbamoylcarbazides 8aef and intermediate compounds 3aec, 6
and 7 on the proliferation of malignant tumor cell lines.
Compd.
Structural formula
Tumor cell growth IC50
(
m
M)a
HeLa
L1210
CEM
HCT 116
MCF-7
H460
HaCaT
3a
3b
3c
5a
170 ꢁ 26
147 ꢁ 22
129 ꢁ 24
120 ꢁ 4
142 ꢁ 17
80 ꢁ 44
>100
>100
>100
eb
212 ꢁ 9
154 ꢁ 6
150 ꢁ 23
74 ꢁ 22
>100
>100
32 ꢁ 4
>100
>100
18 ꢁ 1
>100
>100
32 ꢁ 8
e
e
e
212 ꢁ 26
103 ꢁ 62
5b
30 ꢁ 7
53 ꢁ 6
69 ꢁ 45
28 ꢁ 0.2
19 ꢁ 3
35 ꢁ 0.01
53 ꢁ 14
5c
5d
5e
44 ꢁ 29
26 ꢁ 6
142 ꢁ 109
173 ꢁ 8
ꢄ 250
21 ꢁ 7
34 ꢁ 1
26 ꢁ 2
13 ꢁ 0.7
29 ꢁ 12
23 ꢁ 5
29 ꢁ 2
27 ꢁ 12
110 ꢁ 10
180 ꢁ 12
74 ꢁ 18
148 ꢁ 45
59 ꢁ 11
51 ꢁ 14
e
e
5f
20 ꢁ 0
23 ꢁ 4
22 ꢁ 12
13 ꢁ 0.06
6 ꢁ 1
16 ꢁ 1
13 ꢁ 2
5g
5h
55 ꢁ 16
35 ꢁ 7
73 ꢁ 27
53 ꢁ 8
39 ꢁ 20
48 ꢁ 5
36 ꢁ 20
22 ꢁ 7
20 ꢁ 5
17 ꢁ 2
33 ꢁ 17
24 ꢁ 2
18 ꢁ 7
19 ꢁ 2
5i
22 ꢁ 1
63 ꢁ 1
53 ꢁ 1
24 ꢁ 1
72 ꢁ 14
71 ꢁ 9
24 ꢁ 0
70 ꢁ 14
64 ꢁ 8
15 ꢁ 0.1
27 ꢁ 0.2
18 ꢁ 1
13 ꢁ 0.5
17 ꢁ 1
19 ꢁ 4
19 ꢁ 1
19 ꢁ 13
22 ꢁ 1
20 ꢁ 4
46 ꢁ 12
15 ꢁ 3
5j
5k
(continued on next page)

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I. Perkovic et al. / European Journal of Medicinal Chemistry 51 (2012) 227e238
234
Table 1 (continued )
Compd.
Structural formula
Tumor cell growth IC50
(
m
M)a
HeLa
L1210
CEM
HCT 116
MCF-7
H460
HaCaT
5l
15 ꢁ 1
11 ꢁ 7
5.7 ꢁ 0.2
5 ꢁ 0.7
7 ꢁ 3
3 ꢁ 0.4
10 ꢁ 0.03
5m
5n
69 ꢁ 12
25 ꢁ 1
66 ꢁ 4
25 ꢁ 5
43 ꢁ 12
45 ꢁ 12
33 ꢁ 8
22 ꢁ 1
19 ꢁ 2
16 ꢁ 3
38 ꢁ 2
50 ꢁ 2
25 ꢁ 4
23 ꢁ 0.4
5o
5p
5r
22 ꢁ 1
31 ꢁ 3
31 ꢁ 3
20 ꢁ 1
49 ꢁ 6
29 ꢁ 3
20 ꢁ 0
94 ꢁ 42
58 ꢁ 7
16 ꢁ 3
24 ꢁ 0.4
20 ꢁ 5
18 ꢁ 10
12 ꢁ 8
18 ꢁ 0.5
28 ꢁ 2
22 ꢁ 2
12 ꢁ 3
25 ꢁ 3
16 ꢁ 2
15 ꢁ 0.8
5s
19 ꢁ 0
15 ꢁ 0
5.0 ꢁ 0.5
8 ꢁ 2
5 ꢁ 1
4 ꢁ 0.6
11 ꢁ 0.01
5t
192 ꢁ 81
84 ꢁ 10
79 ꢁ 4
ꢄ250
178 ꢁ 3
76 ꢁ 4
78 ꢁ 2
76 ꢁ 10
76 ꢁ 21
56 ꢁ 10
50 ꢁ 11
81 ꢁ 7
>100
e
e
e
5u
5v
91 ꢁ 9
101 ꢁ 11
>100
41 ꢁ 18
82 ꢁ 19
5w
5x
5y
6
108 ꢁ 11
125 ꢁ 11
113 ꢁ 11
>250
205 ꢁ 63
183 ꢁ 95
155 ꢁ 71
>250
160 ꢁ 13
ꢄ 250
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
e
e
e
e
176 ꢁ 26
>250

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I. Perkovic et al. / European Journal of Medicinal Chemistry 51 (2012) 227e238
235
Table 1 (continued )
Compd.
Structural formula
Tumor cell growth IC50
(
m
M)a
HeLa
L1210
CEM
HCT 116
MCF-7
H460
HaCaT
7
>250
>250
>250
>100
>100
>100
e
8a
8b
8c
8d
8e
8f
105 ꢁ 10
103 ꢁ 5
102 ꢁ 1
>250
106 ꢁ 34
90 ꢁ 10
96 ꢁ 3
>250
98 ꢁ 55
88 ꢁ 4
76 ꢁ 20
>250
56 ꢁ 31
84 ꢁ 16
64 ꢁ 21
>100
19 ꢁ 8
26 ꢁ 10
19 ꢁ 10
>100
71 ꢁ 30
>100
>100
>100
>100
>100
e
e
e
e
e
>250
>250
ꢄ250
>100
>100
>250
>250
ꢄ250
>100
>100
e
Cisplatin
5-Fluorouracil
ꢃ
e
e
7 ꢁ 2
10 ꢁ 1
15 ꢁ 2
1 ꢁ 0.1
3 ꢁ 0.3
2 ꢁ 0.1
0.49 ꢁ 0.2
18 ꢁ 5
0.54 ꢁ 0.1
4 ꢁ 0.7
0.3 ꢁ 0.08
a
50% inhibitory concentration.
Not tested.
b
prepared. They contain a region with five to nine electronegative
atoms (three nitrogen plus two or three oxygen atoms or five
nitrogen plus four oxygen atoms) framed by aryl, cycloalkyl or
hydroxy residue on one or both terminal ends. Therefore, they are
closely related to semicarbazides, carbazides, ureas, hydroxyureas
and hydroxamic acids.
was obtained from 1-(N-benzyloxycarbamoyl)benzotriazole (4g)
and hydrazine (Scheme 1).
The synthetic pathways applied for preparation of the majority
of compounds involved benzotriazole as synthetic auxiliary. The
benzotriazole moiety in the compounds 2aec, 4aeg and 7 acti-
vated the molecules for nucleophilic substitution with hydrazine
hydrate or hydrazides, allowing the preparation of hydrazides
3aec, semicarbazides 5aev and 6 or carbazides 8aec under mild
conditions. In our reactions, the hydrazides act as acyl donors but
also as nucleophiles via the terminal nitrogen atom. Benzotriazole
was introduced in the intermediate products by means of 1-
benzotriazole carboxylic acid chloride (BtcCl, 1), which was first
described by our research group and now is commercially avail-
able [32]. TEA was used as a catalyst and/or HCl acceptor in several
synthetic steps, but not in reactions g and k to avoid possible
cyclization. The final step in preparations of 5aes and 8aec was
performed in a solvent-free system. The reactions were run at
a minimal temperature to afford melting (75e120 ꢀC). These
reaction conditions allowed completion of the reactions in less
than 20 min and in good yields.
The first series involved semicarbazides bearing a NSAID acyl
residue at position 1 and cycloalkyl or aryl (5aes), benzyloxy (5tev)
or hydroxy (5wey) substituents at position 4 of the semicarbazide
backbone. Compounds 5aev were prepared from NSAID hydrazides
3aec and the corresponding 1-(N-alkyl/arylcarbamoyl)benzo-
triazoles (1-benzotriazole carboxylic acid amides) 4aeg (Scheme 1).
Hydrazide 3a was previously described, while 3b and 3c are new
compounds. Here we report a new method for efficient hydrazide
preparation from NSAID benzotriazolides 2aec and hydrazine
hydrate. 1-(N-Alkyl/arylcarbamoyl)benzotriazoles 4aeg were
synthesized from 1-benzotriazole carboxylic acid chloride (BtcCl, 1)
and corresponding amine, following our previously published
procedure[27]. Benzyloxysemicarbazides 5tevafterhydrogenolysis
of the benzyl group gave 1-acyl-4-hydroxysemicarbazides 5wey.
The carbazide series of compounds was prepared by the reac-
tion of NSAID hydrazides 3aec and 1-(1-benzotriazolecarbonyl)-4-
benzyloxysemicarbazide (7) (compounds 8aec) or by hydro-
genolysis of such obtained carbazides (8def). Compound 7 was
prepared from BtcCl (1) and 4-benzyloxysemicarbazide (6), which
Structures of the synthesized compounds are supported by IR,
1H and 13C NMR spectra and confirmed by elemental analysis. The
chemical shifts are consistent with the proposed structures of the
novel compounds (detailed NMR data are given in Tables 3e7 in the
Supporting Information).

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I. Perkovic et al. / European Journal of Medicinal Chemistry 51 (2012) 227e238
236
Table 2
end. It would be interesting to introduce additional substituents on
the benzhydryl moiety (i.e. alkyl, alkoxy, halogene, amine, carboxylic
acid and/or hydroxyl groups) to further explore the cytostatic
potential and selectivity of these test compounds. The NSAID
terminal of these molecules seems to be less important (i.e. isobutyl,
phenoxyphenyl, benzyl) to determine the antiproliferative potency.
It would be also interesting to explore an additional SAR by modi-
fying the latter part of the molecule.
Additional testing of the selected compounds on a non-tumor
cell line HaCaT (human keratinocytes) showed that the most
active derivatives 5l and 5s may exert a slight selectivity toward
tumor cells (specifically solid tumor cells), compared to non-tumor
cells, having up to two times lower IC50 concentrations for tumor
cell lines. All other compounds generally showed the same inhibi-
tory effect toward tumor and non-tumor cell lines, except toward
breast cancer cells MCF-7, which were shown to be slightly more
sensitive. Still, these results should be interpreted with caution
without detailed experiments and/or specific target identification,
because although these cells are not tumorigenic, they are immor-
talized and have a relatively short doubling time. However, all
tested NSAID derivatives show much lower toxicity toward HaCaT,
compared to the reference compounds 5-fluorouracil and cisplatin.
Interaction with DPPH, in vitro inhibition of lipid peroxidation (LP), soybean lip-
oxygenase (LOX), and theoretically calculated C log P values [45].
Compd. C log P DPPH
DPPH
LP
LOX
20 mina (%) 60 mina (%) inhibition (%) inhibitiona (%)
3a
3b
3c
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5r
2.41
2.55
2.52
4.04
4.60
5.22
4.34
4.67
6.11
4.19
4.74
5.36
4.48
4.81
5.83
4.15
4.71
5.33
4.45
4.78
5.80
4.09
4.24
4.20
1.57
1.71
1.68
0.27
1.67
2.93
3.07
3.04
0.41
0.55
0.52
na
na
na
4
na
3
na
na
na
16
19
18
22
4
2
4
3
5
3
8
4
na
na
3
29
39
36
6
85
11
8
10
24
23
28
80
1
1
na
7
na
6
na
na
na
19
22
21
24
7
6
10
6
8
3
10
na
na
25
na
na
46
na
13
12
68c
20
81d
na
10
9
na
17
96b
na
6
na
7
3
26
33
16
12
33
74
21
63
64
11
51
99
na
31
61
48
71
88
100
8
na
20
22
8
36
31
13
26
100
15
13
28
4
12
10
12
9
15
9
na
na
6
54
66
59
14
85
18
15
19
46
48
53
91
5s
5t
5u
5v
5w
5x
5y
6
3.2.2. Antiviral activity
None of the tested compounds were inhibitory at subtoxic
concentrations against a broad panel of DNA and RNA viruses in cell
culture.
8
na
na
na
na
na
na
na
1
7
3.2.3. Antioxidative activity
8a
8b
8c
8d
8e
8f
It has been reported that many NSAIDs act either as inhibitors of
free radical production or as radical scavengers [33], while those
showing reduction of lipid peroxidation, also show less ulcerogenic
activity [34,35]. In the present investigation, in vitro antioxidant
ability of new NSAID derivatives 5aey and 8aef and precursors in
their synthesis was studied and compared to the well-known anti-
oxidant agents such as nordihydroguaiaretic acid (NDGA) and 6-
hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox).
Two different antioxidant assays were used: (i) interaction with the
stable free radical 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) [36]
and (ii) interaction with the water-soluble azo compound
2,20-azobis(2-amidinopropane) hydrochloride (AAPH), used as
a source of peroxyl radicals [37,38]. DPPH interaction of the tested
NDGA
Trolox
84e
63
Concentrations of the tested compounds: a1 ꢂ 10ꢃ4 mol lꢃ1
,
bIC50 ¼ 51.5
mM,
cIC50 ¼ 75
m
M, dIC50 ¼ 60
m
M, eIC50 ¼ 28
mM, na e no activity.
3.2. Biological evaluations
3.2.1. Cytostatic activity
compounds was examined at 100 mM concentration after
The target compounds 5aey and 8aef, as well as their synthetic
precursors, were evaluated for inhibitory activities against prolifer-
ation of the following malignant tumor cell lines: murine leukemia
(L1210), human T-lymphocyte (CEM), human cervical carcinoma
(HeLa), human colon carcinoma (HCT 116), human breast carcinoma
(MCF-7) and human lung carcinoma (H460) (Table 1). The tested
compounds do not discriminate very much between the different
tumor cell lines regarding their cytostatic activity, except for the
human mammary carcinoma MCF-7 cells that are generally more
sensitive to the antiproliferative activity of the compounds than the
other carcinoma cell lines (Table 1). The most active compounds 4-
benzhydryl-1-[2-(3-phenoxyphenyl)propanoyl]semicarbazide (5l),
4-benzhydryl-1-[2-(3-benzylphenyl)propanoyl]semicarbazide (5s),
and 4-benzhydryl-1-[2-(4-isobutylphenyl)propanoyl]semicarbazi-
de (5f) inhibited tumor cell proliferation at IC50 values ranging from
20 and 60 min. The results are shown in Table 2. 1-(1-
Benzotriazolecarbonyl)-4-benzyloxysemicarbazide (7) showed the
best DPPH interaction value (85%), similar to that of the reference
compound NDGA (91%) at the same concentration. In the semi-
carbazide series 5 and carbazide series 8, only compounds with low
lipophilicity values bearing hydroxamic acid/hydroxyurea moiety,
5wey and 8def, showed weak interaction with DPPH (23e39%) and
their antioxidant activity increased after 60 min (46e66%).
In our studies, AAPH was used as a free radical initiator to follow
oxidative changes of linoleic acid to conjugated diene hydroper-
oxide. The results showed that 5t, 8c and 5l were almost equipotent
and completely inhibited lipid peroxidation (LP). Two other
compounds, 5s and 5f, also inhibited LP better than Trolox (88 and
74%, respectively). No significant difference was observed between
the other compounds of the series 5. Hydrazides 3aec were
completely inactive. Our results, like some reported data [39],
indicate that LP inhibition is not always accompanied by DPPH
radical scavenging activity. Thus, compounds that inhibited LP
potently, exerted low (if any) DPPH scavenging activity.
3 to 23 mM. It is striking to observe that those three most active
compounds consistently contain the same unique structural motif
(i.e. a 4-benzhydryl-1-propanoyl-semicarbazide), whereas lack of
any bulky aromatic or cycloalkyl group resulting in a free end-
standing eCONHNH2 or eCONHOH group virtually annihilates the
cytostatic activity. Thus, the presence of a bulky lipophilic group,
preferably a benzhydryl group, is strictly required at this terminal
Carcinogenesis is a multistage process consisting of at least
three separate, but closely linked processes: initiation, promotion
and progression. Promotion is closely linked to oxidative and

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I. Perkovic et al. / European Journal of Medicinal Chemistry 51 (2012) 227e238
237
N
N
NH
BtH
Cl3COCOOCCl3
O
O
O
O
O
H
N
R2
N
O
N
N
R1
O
R1
N
N
N
N
Cl
N
N
N
H
H
N
NH2
R1
R2
R2NH2
TEA
O
R1
OH
NH2
NH2NH2
R1
N
H
N
H
N
H
TEA, –CO2
3a-c
TEA
O
3a-c
5a-s
2a-c
1 (BtcCl)
4a-f
H2N
TEA
O
O
H
R1
N
O
O
O
NH2
N
N
H
N
H
N
N
N
H
H2/Pd
O
R1
O
R1
OH
R1
R2
3a-c
N
N
H
N
H
N
H
H
O
O
4g
NH2NH2
5t-v
5w-y
O
H
N
R1
O
O
NH2
H
N
BtcCl
1
O
N
N
O
H2N
O
3a-c
N
N
N
H
N
H
N
H
H
TEA
O
6
7
O
O
O
O
H
N
H
N
H
H
OH
R1
N
N
H
N
N
O
N
H
H2/Pd
R1
N
N
H
N
H
H
H
O
O
8a-c
8d-f
Scheme 1. Synthesis of 1-acyl-4-cycloalkyl(or 4-aryl)semicarbazides 5aey and 1-acyl-5-benzyloxy(or 5-hydroxy)carbamoylcarbazides 8aef, derived from NSAIDs ibuprofen,
fenoprofen and reduced ketoprofen.
inflammatory tissue damage. Conversely, substances with potent
anti-inflammatory and/or antioxidant activities are anticipated to
exert chemopreventive effects on carcinogenesis, particularly in the
stage of promotion and to act as antitumor promoters. Taking under
consideration the above mentioned as well as the antioxidant and
cytostatic activities of our compounds, it seems that three out of
five of the most active LP inhibitors are the strongest anti-
proliferative agents as well. These findings support the idea that the
cytostatic activity of 5l, 5s and 5f is highly correlated to their
antioxidant potential (anti-lipid peroxidation activity).
role of arachidonic acid metabolites in the etiology of mammary
carcinogenesis [44]. Studies have demonstrated that levels of
several eicosanoids are increased in breast cancer in comparison to
benign breast tumors [45].
In this context, we decided to evaluate the newly prepared
NSAID derivatives for their ability to inhibit soybean LOX. It has
been shown that inhibition of plant LOX activity by NSAIDs is
qualitatively similar to their inhibition of the rat mast cell LOX and
may be used as a simple qualitative screen for such activity [46].
From the tested derivatives, 5s, 5l, and 5j presented interesting
In general, information obtained from the antioxidant activity
may help to understand the anticancer properties of the
compounds. It is known that metals, especially iron and copper, can
play a key role in free radical generation and propagation. There-
fore, metal chelators can act as protectors. This could be partly
a possible mechanism of action of 5l, 5s, 5f.
Lipoxygenases (LOXs) play a significant role in membrane lipid
peroxidation by forming hydroperoxides in the lipid bilayer from
the biotransformation of arachidonic acid catalyzed by LOX [40].
LOX inhibitors have attracted attention initially as potential agents
for the treatment of inflammatory and allergic diseases, certain
types of cancer, and cardiovascular diseases [41,42]. In order to
diminish the pro-cancerous mechanisms a key treatment strategy
is to reduce the free radical load. Antioxidant activity by the scav-
enging of reactive oxygen species is important in preventing
potential damage to cellular macromolecules such as DNA, proteins
and lipids. Moreover, it has been found that LOX inhibitors may
have chemopreventive activity in lung carcinogenesis [43]. Reports
published over the past three decades support a growth-regulatory
IC50 values (51.5, 60 and 75
mM) in comparison to the IC50 value of
the reference NDGA (28 M, Table 2). Two of them, 5s and 5l, contain
m
benzhydryl and the third one benzyl substituent at semicarbazide
position 4, indicating that the presence of a bulky lipophilic group at
this terminal end is strictly required for better LOX inhibitory
activity. The fact that these compounds have high C log P values
(5.80, 5.83, and 4.48, respectively) support the findings that lip-
ophilicity is an important physicochemical property for LOX inhib-
itors [47]. However, compound 5f with the highest C log P value
(6.11) was not the most active and presented 46% inhibition at
100 mM concentration.
3.2.4. Physicochemical studies. Determination of lipophilicity as
C log P values
Since lipophilicity is a significant physicochemical property
determining distribution, bioavailability, metabolic activity, and
elimination, we have calculated the lipophilicity values of the
prepared compounds as C log P values by the C log P programme of
Biobyte Corp. [48].

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238
I. Perkovic et al. / European Journal of Medicinal Chemistry 51 (2012) 227e238
4. Conclusions
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acids and hydroxyureas, where also completely inactive, while their
O-benzyl derivatives 8aec showed a slightly better activity with IC50
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Acknowledgments
Support for this study by the Ministry of Science, Education and
Sports of the Republic of Croatia (Projects No. 006-0000000-3216
and 098-0982464-2514) and the KU Leuven (GOA 10/014) is
gratefully acknowledged. We also thank Dr. C. Hansch1, Dr. A. Leo,
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Appendix. Supporting information
Supplementary data related to this article can be found online at
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1
Deceased.