Novel 1-acyl-4-substituted semicarbazide derivatives of primaquine-synthesis, cytostatic, antiviral and antioxidative studies

Article abstract of DOI:10.3109/14756366.2012.663366

A series of novel 1,4-substituted semicarbazides 5a-g with a primaquine moiety bridged by a carbonyl group at position 1 and a cycloalkyl, aryl, benzyloxy or hydroxy substituent at position 4 were prepared and biologically evaluated. The synthetic pathway

Full text of DOI:10.3109/14756366.2012.663366

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(3): 601–610
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.663366
ReseaRch aRtIcle
Novel 1-acyl-4-substituted semicarbazide derivatives
of primaquine − synthesis, cytostatic, antiviral and
antioxidative studies
Ivana Perković1, Sara Tršinar1, Jelena Žanetić1, Marijeta Kralj2, Irena Martin-Kleiner2, Jan Balzarini3,
Dimitra Hadjipavlou-Litina4, Anna Maria Katsori4, and Branka Zorc1
1Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia, 2Division of Molecular Medicine,
Rudjer Bošković Institute, Zagreb, Croatia, 3Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven,
Belgium, and 4Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of essaloniki,
essaloniki, Greece
abstract
A series of novel 1,4-substituted semicarbazides 5a–g with a primaquine moiety bridged by a carbonyl group at
position 1 and a cycloalkyl, aryl, benzyloxy or hydroxy substituent at position 4 were prepared and biologically
evaluated. The synthetic pathways applied for preparation of the title compounds involved benzotriazole as synthetic
auxiliary. Primaquine semicarbazides 5a–g and their synthetic precursors benzotriazolecarbonyl semicarbazides
4 were evaluated for cytostatic, antiviral and antioxidative activities. All compounds of the series 5 showed high
selectivity towards MCF-7 cells (breast carcinoma) with IC50 values in the low micromolar range and the most active
was benzyl derivative 5c (IC
1 0.2 µM). The benzhydryl derivative 5e showed significant cytostatic activities
towards all the tested cell line5s0 (IC50 4–18 µM). The same compound was the strongest lipoxygenase inhibitor as well
(51%). The highest antioxidant activity was demonstrated for the hydroxy derivative 5g and benzotriazolecarbonyl
semicarbazides 4b,c (61.2–68.5%). No antiviral activity was observed against a wide variety of DNA and RNA viruses.
Keywords: Primaquine, semicarbazide, benzotriazole, antitumoural activity, antioxidative activity, antiviral evaluation
Introduction
releasing hormone, is used for the treatment of prostate
cancer. Hydroxyurea is a useful drug in therapy of leu-
kaemia and other malignant tumour diseases and its
derivative zileuton is an orally active inhibitor of 5-lipoxy-
genase which is used for the maintenance treatment of
asthma12. Hydroxyurea is an inhibitor of ribonucleotide
reductase, the enzyme described as a target for the che-
Hydroxyurea, hydroxysemicarbazide, semicarbazide,
semicarbazone and thiosemicarbazone functionalities
have been identified as essential pharmacophores for
antitumoural, antiviral, antibacterial, antimalarial, anti-
convulsant, herbicidal and fungicidal activities1–6. Several
reports show potent activity of Schiff bases of hydroxy-
semicarbazides against L1210 murine leukaemia cells7,8
.
motherapy of both malignant diseases and malaria13,14
.
e 3-aminopyridine-2-carboxaldehyde thiosemicar-
bazone (3-AP) is a metal chelator that potently inhibits
ribonucleotide reductase9,10. Semicarbazide-based deriv-
atives of peptides (azapeptides) are known as inhibitors
of human dipeptidyl peptidase I, the enzyme that plays
an important role in the immune system11, while goser-
elin, an azapeptide analogue of the luteinising-hormone
Mahajan et al.described 7-chloroquinolyl thioureas as
potential antimalarial and anticancer agents15. Several
research groups found dual antimalarial and anticancer
activity of both quinoline and trioxane derivatives as
well16–18. Based on these findings, we have synthesized
a series of compounds containing the antimalarial drug
primaquine (PQ) substituted by an N-4-cycloalkyl, aryl,
Address for Correspondence: B. Zorc, Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovačića 1, 10000 Zagreb, Croatia.
Tel: +385 1 4856202. Fax: +385 1 4856201. E-mail: bzbz@pharma.hr
(Received 26 December 2011; revised 25 January 2012; accepted 27 January 2012)
601

602 I. Perković et al.
abbreviations
LOX, lipoxygenase;
AAPH, 2,2′-azobis(2-amidinopropane) hydrochloride;
Btc, 1-benzotriazolecarbonyl;
BtH, benzotriazole;
LP, lipid peroxidation;
NDGA, nordihydroguaiaretic acid;
PQ, primaquine;
DPPH, 1,l-diphenyl-2-picrylhydrazyl;
TEA, triethylamine
benzyloxy or hydroxy semicarbazide functionality. e
newly designed compounds are closely related to biurets,
hydroxyureas, hydroxamic acids or azapeptides. Here
their synthesis, full chemical characterization, cytostatic,
antiviral and antioxidative evaluations are reported. is
paper is a continuation of our previous papers dealing
with PQ ureas and NSAID semicarbazide derivatives as
1-(N-Cycloalkyl/arylcarbamoyl)benzotriazoles (2a–f)
e title compounds were synthesized from chloride 1
and corresponding amine, following our previously pub-
lished procedure22,25. e following derivatives were pre-
pared: 1-(N-cyclopentylcarbamoyl)benzotriazole (2a),
1-(N-cyclohexylcarbamoyl)benzotriazole(2b),1-(N-ben-
zylcarbamoyl)benzotriazole (2c), 1-(N-[2-phenylethyl]
carbamoyl)benzotriazole (2d), 1-(N-benzhydrylcarbam-
oyl)benzotriazole (2e) and 1-(N-benzyloxycarbamoyl)
benzotriazole (2f).
potential antitumor agents19-22
.
Material and methods
General procedure for preparation of 4-cycloalkyl or aryl
semicarbazides (3a–f)
Chemistry
General experimental details
e title compounds were synthesized from 1-(N-alkyl/
arylcarbamoyl)benzotriazoles (2a–f) and hydrazine
hydrate, according to our slightly modified procedure
(molar ration of reactants 1:3, dioxane, 18 h, r.t.)22,25. e
following derivatives were prepared: 4-cyclopentylsemi-
carbazide (3a), 4-cyclohexylsemicarbazide (3b), 4-ben-
zylsemicarbazide (3c), 4-(2-phenylethyl)semicarbazide
(3d), 4-benzhydrylsemicarbazide (3e) and 4-benzyloxy-
semicarbazide (3f). ese compounds are commercially
available or published elsewhere.
Melting points were determined on a Stuart Melting Point
Apparatus SMP3 and were uncorrected. IR spectra were
recorded on a FTIR Perkin Elmer Paragon 500 spectrom-
1
eter. H and 13C NMR spectra were recorded on a Varian
Gemini 300 spectrometer, operating at 300 and 75.5 MHz
for the 1H 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.
Elemental analyses were performed on CHNS LECO
analyzer and mass spectra were taken on a HPLC-MS/
MS (HPLC, Agilent Technologies 1200 Series; MS,
Agilent Technologies 6410 Triple Quad). Solvent systems
cyclohexane/ethyl acetate (EtOAc)/methanol (3:1:0.5),
cyclohexane/EtOAc (1:1), petrol ether/EtOAc/metha-
nol (3:1:0.75), dichloromethane/methanol (9.5:0.5) and
(8.5:1.5) and pre-coated Merck silica gel 60 F254 plates
were used for thin-layer chromatography (TLC). Spots
were visualized by short-wave UV light and iodine vapour.
Column chromatography was performed on silica gel
(0.063−0.200 mm), with the same eluents used in TLC.
Benzotriazole (BtH), triphosgene, cyclopentylamine,
cyclohexylamine, benzylamine, 2-phenylethanamine,
benzhydrylamine, O-benzylhydroxylamine hydrochlo-
ride, triethylamine (TEA), hydrazine hydrate and 10%
Pd/C were purchased from Aldrich. e PQ base was
prepared from primaquine diphosphate (Aldrich) prior
to use. e PQ solution was protected from light during
the whole procedure. e 1,1-diphenyl-2-picrylhydrazyl
(DPPH), 2,2′-azobis(2-amidinopropane) hydrochloride
(AAPH), Trolox and nordihydroguaiaretic acid (NDGA)
were purchased from Aldrich Chemical Co. Soybean
lipoxygenase (LOX) and linoleic acid sodium salt were
obtained from Sigma Chemical Co.
General procedure for preparation of 1-
(1-benzotriazolecarbonyl)-4-cycloalkyl/aryl
semicarbazides (4a–f)
To a solution of BtcCl (1) (0.908 g, 5.0 mmol) in anhy-
drous toluene (15 mL), a solution of semicarbazide 3a–f
(5.0 mmol) and TEA (0.697 mL, 5 mmol) in anhydrous
dioxane (20 mL) was added drop-wise (ice bath). e
reaction mixture was stirred 1 h at room temperature
or under an ice-bath and used in the next reaction step
withoutfurtherwork-up(4aand4b)orevaporatedunder
reduced pressure (4c–f). e residue was dissolved in
30 mL EtOAc/water mixture (1:1). e organic layer was
extracted with water (3 × 40 mL), dried over anhydrous
sodium sulphate and evaporated under reduced pres-
sure. Compound 4e precipitated from EtOAc/water
mixture and the crude product was filtered off.
1-(1-Benzotriazolecarbonyl)-4-cyclopentylsemicarbazide (4a)
From the reaction of 0.716 g (5.0 mmol) 3a, 0.908 g (5.0
mmol) 1 and 0.697 mL (5 mmol) TEA under an ice bath
for 1 h the crude product 4a was obtained and used in
further reaction step without purification. Its structure
was confirmed indirectly.
1-Benzotriazole carboxylic acid chloride (1)
1-(1-Benzotriazolecarbonyl)-4-cyclohexylsemicarbazide (4b)
From the reaction of 0.786 g (5.0 mmol) 3b, 0.908 g (5.0
mmol) 1 and 0.697 mL (5 mmol) TEA under an ice bath
Compound 1 was prepared from BtH and triphosgene,
according to the previously published procedure23,24
.
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel 1-acyl-4-substituted semicarbazide derivatives of PQ 603
for 1 h the crude product 4b was obtained and used in
further reaction step without purification. Its structure
was confirmed indirectly.
for 1 h and purification of the product from ether/petrol
ether, 1.191 g (73%) of 4f was obtained; mp 90°C−93°C
(decomp.) mp22 90°C−93°C.
1-(1-Benzotriazolecarbonyl)-4-benzylsemicarbazide (4c)
From the reaction of 0.826 g (5.0 mmol) 3c, 0.908 g (5.0
mmol) 1 and 0.697 mL (5 mmol) TEA at room temperature
for 1 h and purification of the product from ether/petrol
ether, 0.884 g (57%) of 4c was obtained; mp 146°C−147°C;
IR (KBr, ν/cm−1) 3310, 1753, 1659, 1576, 1495, 1450,
1380, 1290, 1254, 1029, 747, 698, 615; 1H NMR (300 MHz,
DMSO) δ 10.92 (1s, 1H, 1’), 8.40 (1s, 1H, 2’), 8.23, 8.20 (2d,
2H, 3, 6, J= 8.27 Hz), 7.75, 7.58 (2t, 2H, 4, 5, J= 7.40 Hz),
General procedure for preparation of primaquine
semicarbazide derivatives (5a–f)
Method A: To a solution of 4a–f (5.0 mmol) in toluene
(15 mL), a solution of PQ (1.297 g, 5.0 mmol) and TEA
(2.09 mL, 15.0 mmol) in dioxane (20 mL) was added
drop-wise. e reaction mixture was stirred on ice bath
for 2 (5a) or 8 h (5b) and evaporated under reduced pres-
sure. e obtained residue was triturated and stirred with
a dioxane/water and EtOAc/water mixtures and finally
with water.
7.35–7.21 (m, 6H, arom., 4’), 4.28 (d, 2H, 5’, J= 6.09 Hz); 13
C
NMR (75.5 MHz, DMSO) δ 158.41 (3’), 150.15 (1), 145.65,
132.05 (2, 7), 140.93 (6’), 130.58, 126.99, 126.18 (4, 5, 9’),
128.56, 127.42 (7’, 8’, 10’, 11’), 120.29 (6), 113.98 (3), 43.10
(5’); elemental analysis: calcd for C15H14N6O2: C, 58.06; H,
4.55; N, 27.08. Found: C, 58.40; H, 4.65; N, 27.36.
Method B: Solution of compounds 4a–f (4 mmol), PQ
(1.037 g, 4.0 mmol) and TEA (1.12mL, 8.0 mmol) in diox-
ane (30 mL) was stirred at room temperature for 3h and
evaporated under reduced pressure.
Method C: Solution of compounds 4a–f (4 mmol),
PQ (1.037 g, 4.0 mmol) and TEA (1.67 mL, 12.0 mmol) in
dioxane (30 mL) was stirred at room temperature for 3 h
and evaporated under reduced pressure. e obtained
residue was dissolved in EtOAc (30 mL) and extracted
with 0.1% sodium hydroxide solution (3 × 30 mL), washed
with water, dried over anhydrous sodium sulphate and
evaporated under reduced pressure.
1-(1-Benzotriazolecarbonyl)-4-(2-phenylethyl)
semicarbazide (4d)
From the reaction of 0.896 g (5.0 mmol) 3d, 0.908 g (5.0
mmol) 1 and 0.697 mL (5 mmol) TEA at room temperature
for 1 h and purification of the product from ether/petrol
ether, 0.778 g (48%) of 4d was obtained; mp 135°C−136°C;
IR (KBr, ν/cm−1) 3299, 1766, 1745, 1650, 1488, 1449, 1380,
1
1288, 1236, 1030, 750, 700; H NMR (300 MHz, DMSO) δ
N1-cyclopentyl-N2-[4-(6-methoxyquinolin-8-ylamino)pentyl]
hydrazine-1,2-dicarboxamide (5a)
10.83 (1s, 1H, 1’), 8.30 (1s, 1H, 2’), 8.23, 8.19 (2d, 2H, 3, 6,
J= 8.39 Hz), 7.75, 7.58 (2t, 2H, 4, 5, J= 7.59 Hz), 7.30–7.16
(m, arom., 5H), 6.81 (s, 1H, 4’), 3.27 (q, 2H, 5’, J= 6.75 Hz),
2.73 (t, 2H, 6’, J= 7.59 Hz); 13C NMR (75.5 MHz, DMSO) δ
158.20 (3’), 150.12 (1), 145.65, 132.02 (2, 7), 140.05 (7’),
130.60, 126.47, 126.20 (4, 5, 10’), 129.12, 128.78 (8’, 9’, 11’,
12’), 120.31 (6), 113.96 (3), 41.51, 36.42 (5’, 6’); elemental
analysis: calcd for C16H16N6O2: C, 59.25; H, 4.97; N, 25.91.
Found: C, 59.60; H, 5.03; N, 25.66.
Method A: From the reaction of 1.442 g (5.0 mmol) 4a,
1.297 g (5.0 mmol) PQ and 2.09 mL (15 mmol) TEA on
ice bath for 2 h and purification of the product by column
chromatography (eluent dichloromethane/MeOH 9.5:0.5
and 9:1) and trituration with ether, 0.279 g (13%) of 5a was
obtained; mp 190°C−194°C; IR (KBr, ν/cm−1) 3306, 3223,
3088, 2939, 2868, 1661, 1618, 1551, 1520, 1455, 1422, 1388,
1326, 1218, 1199, 1158, 1051, 1031, 971, 937, 899, 820, 790,
681, 625; MS/MS m/z 429.5 (M + H)+; 1H NMR (300 MHz,
DMSO) δ 8.54 (d, 1H, 11, J= 2.76 Hz), 8.08 (d, 1H, 13,
J= 7.81 Hz), 7.46–7.42 (m, 3H, 1’, 2’, 12), 6.48 (s, 1H, 17),
6.36 (t, 1H, 2, J= 4.84 Hz), 6.27 (s, 1H, 15), 6.11, 6.08 (2d,
2H, 8, 4’, J= 8.96 Hz, 8.04 Hz), 3.90–3.83 (m, 1H, 5’), 3.83 (s,
3H, 18), 3.63–3.61 (m, 1H, 6), 3.04 (q, 2H, 3), 1.76–1.20 (m,
15H, 4, 5, 7, 6’–9’); 13C NMR (75.5 MHz, DMSO) δ 159.47
(1), 159.18 (16), 158.64 (3’), 145.11 (9), 144.70 (11), 135.26
(13), 134.99 (10), 130.05 (14), 122.56 (12), 96.54 (17), 92.05
(15), 55.45 (18), 51.38 (5’), 47.52 (6), 39.54 (3), 33.84 (5),
33.07 (6’, 9’), 27.15 (4), 23.67 (7’, 8’), 20.67 (7); Elemental
analysis: calcd for C22H32N6O3: C, 61.66; H, 7.53; N, 19.61.
Found: C, 61.80; H, 7.64; N, 19.75.
1-(1-Benzotriazolecarbonyl)-4-benzhydrylsemicarbazide (4e)
From the reaction of 1.206 g (5.0 mmol) 3e, 0.908 g (5.0
mmol) 1 and 0.697 mL (5 mmol) TEA at room temperature
for 1 h and purification of the product from ether/petrol
ether 1.468 g (76%) of 4e was obtained; mp 164°C−166°C;
IR (KBr, ν/cm−1) 3322, 1771, 1663, 1570, 1495, 1446,
1376, 1367, 1286, 1231, 1018, 793, 750, 703, 630; 1H NMR
(300 MHz, DMSO) δ 10.88 (1s, 1H, 1’), 8.30 (1s, 1H, 2’),
8.23, 8.20 (2d, 2H, 3, 6, J= 8.43 Hz), 7.75, 7.57 (2t, 2H, 4,
5, J= 7.66 Hz), 7.66 (d, 1H, 4’), 7.37–7.25 (m, arom., 10H),
6.05 (d, 1H, 5’, J= 8.43 Hz); 13C NMR (75.5 MHz, DMSO)
δ 157.56 (3’), 150.19 (1), 145.66, 132.00 (2, 7), 143.41 (6’,
12’), 130.64, 126.22 (4, 5), 128.74, 127.80 (7’, 8’, 10’, 11’, 13’,
14’, 16’, 17’), 127.33 (9’, 15’), 120.32 (6), 113.92 (3), 57.22
(5’); elemental analysis: calcd for C21H18N6O2: C, 65.27; H,
4.70; N, 21.75. Found: C, 65.40; H, 4.99; N, 21.55.
N1-cyclohexyl-N2-[4-(6-methoxyquinolin-8-ylamino)pentyl]
hydrazine-1,2-dicarboxamide (5b)
Method A: From the reaction of 1.512 g (5.0 mmol) 4b,
1.297 g (5.0 mmol) PQ and 2.09 mL (15 mmol) TEA on ice
bath for 8 h and re-crystallization from methanol/water,
1.394 g (63%) of 5b was obtained; mp 163°C−168°C; IR
(KBr, ν/cm−1) 3312, 3228, 3091, 2932, 2855, 1664, 1618,
1-(1-Benzotriazolecarbonyl)-4-benzyloxysemicarbazide (4f)
From the reaction of 0.906 g (5.0 mmol) 3f, 0.908 g (5.0
mmol) 1 and 0.697 mL (5 mmol) TEA at room temperature
© 2013 Informa UK, Ltd.

604 I. Perković et al.
1548, 1521, 1456, 1423, 1389, 1336, 1285, 1239, 1220, 1201,
1158, 1133, 1052, 1031, 822, 791, 680, 625, 532; H NMR
5), 1.18 (d, 3H, 7, J= 6.03 Hz); 13C NMR (75.5 MHz, DMSO)
δ 159.49, 159.10, 159.02 (1, 16, 3’), 145.14 (9), 144.71 (11),
140.11 (7’), 135.26 (13), 135.02 (10), 130.06 (14), 129.09,
128.73 (8’, 9’,11’, 12’), 126.40 (10’), 122.55 (12), 96.57 (17),
92.11 (15), 55.46 (18), 47.56 (6), 41.28 (5’), 39.64 (3), 36.48
(6’), 33.89 (5), 27.15 (4), 20.70 (7); MS/MS m/z 465.3 (M +
H)+; elemental analysis: calcd for C25H32N6O3: C, 64.63; H,
6.94; N, 18.09. Found: C, 64.88; H, 7.11; N, 18.48.
1
(300 MHz, DMSO) δ 8.53 (d, 1H, 11, J= 4.04 Hz), 8.07 (d,
1H, 13, J= 8.07 Hz), 7.47–7.40 (2s, q, 3H, 1’, 2’, 12), 6.47 (s,
1H, 17), 6.35 (t, 1H, 2, J = 4.84 Hz), 6.26 (s, 1H, 15), 6.11,
5.96 (2d, 2H, 8, 4’, J= 8.07 Hz), 3.82 (s, 3H, 18), 3.65–3.57
(m, 1H, 6), 3.40 (m, 1H, 5’), 3.03 (q, 2H, 3), 1.72–1.04 (m,
17H, 4, 5, 7, 6’-10’); 13C NMR (75.5 MHz, DMSO) δ 159.47
(1), 159.15 (16), 158.27 (3’), 145.10 (9), 144.69 (11), 135.27
(13), 134.98 (10), 130.05 (14), 122.56 (12), 96.55 (17), 92.06
(15), 55.45 (18), 48.41 (5’), 47.52 (6), 39.53 (3), 33.84 (5),
33.44 (6’, 10’), 27.14 (4), 25.64 (8’), 25.08 (7’, 9’), 20.66 (7);
MS/MS m/z 443.3 (M + H)+; elemental analysis: calcd for
C23H34N6O3: C, 62.42; H, 7.74; N, 18.99. Found: C, 62.21; H,
7.55; N, 18.60.
N1-benzhydryl-N2-[4-(6-methoxyquinolin-8-ylamino)pentyl]
hydrazine-1,2-dicarboxamide (5e)
Method C: From the reaction of 1.546 g (4 mmol) 4e,
1.037 g (4.0 mmol) PQ and 1.67 mL (12.0 mmol) TEA
at room temperature for 3 h and purification of the
product by column chromatography (eluent dichlo-
romethane/MeOH 9.5:0.5) and re-crystallized from
ether/petrol ether, 1.369 g (65%) of 5e was obtained;
mp 90°C (decomp.); IR (KBr, ν/cm−1) 3311, 2960, 2933,
2869, 1662, 1616, 1575, 1555, 1519, 1454, 1423, 1387,
1220, 1202, 1158, 1052, 1030, 822, 791, 747, 700, 625; 1H
NMR (300 MHz, DMSO) δ 8.51 (d, 1H, 11, J = 3.13 Hz),
8.05 (d, 1H, 13, J = 8.13 Hz), 7.61 (s, 1H, 1’), 7.54 (s, 1H,
2’), 7.39 (q, 1H, 12, J = 4.17 Hz, 3.75 Hz), 7.29–7.17 (m,
arom., 10H, 7’–11’, 13’–17’), 6.98 (d, 1H, 4’, J = 8.34 Hz),
6.45 (s, 1H, 17), 6.35 (t, 1H, 2), 6.24 (s, 1H, 15), 6.09 (d,
1H, 8, J = 8.55 Hz), 5.91 (d, 1H, 5’, J = 8.34 Hz), 3.80 (s, 3H,
18), 3.63–3.55 (m, 1H, 6), 3.02 (q, 2H, 3), 1.62–1.41 (m,
4H, 4, 5), 1.17 (d, 3H, 7, J = 5.84 Hz); 13C NMR (75.5 MHz,
DMSO) δ 159.51 (1), 159.12 (16), 158.29 (3’), 145.14
(9), 144.71 (11), 143.57 (6’, 12’), 135.26 (13), 135.03
(10), 130.07 (14), 128.74, 127.60 (7’, 8’,10’, 11’, 13’, 14’, 16’,
17’), 127.26 (9’, 15’), 122.56 (12), 96.57 (17), 92.13 (15),
57.21(5’), 55.47 (18), 47.56 (6), 39.68 (3), 33.89 (5), 27.19
(4), 20.69 (7); MS/MS m/z 527.3 (M + H)+; elemental
analysis: calcd for C30H34N6O3: C, 68.42; H, 6.51; N,
15.96. Found: C, 68.80; H, 6.44; N, 16.13.
N1-benzyl-N2-[4-(6-methoxyquinolin-8-ylamino)pentyl]
hydrazine-1,2-dicarboxamide (5c)
Method B: From the reaction of 1.241 g (4.0 mmol) 4c,
1.037 g (4.0 mmol) PQ and 1.12mL (8 mmol) TEA at
room temperature for 3 h and purification of the prod-
uct by trituration with ether/EtOAc/petrol ether and
ether/petrol ether, 1.153 g (64%) of 5c was obtained; mp
200°C−203°C; IR (KBr, ν/cm–1) 3306, 3223, 3089, 2933,
1664, 1616, 1572, 1555, 1519, 1455, 1423, 1387, 1322, 1219,
1
1201, 1158, 1051, 1030, 820, 790, 735, 700, 626; H NMR
(300 MHz, DMSO) δ 8.54 (d, 1H, 11, J= 3.87 Hz), 8.08 (d,
1H, 13, J= 8.12 Hz), 7.66 (1s, 1H, 1’), 7.61 (1s, 1H, 2’), 7.42
(q, 1H, 12, J= 4.06 Hz), 7.26–7.14 (m, arom., 5H, 7’–11’),
6.90 (t, 1H, 4’, J= 5.42 Hz), 6.47 (s, 1H, 17), 6.42 (t, 1H, 2,
J= 4.84 Hz), 6.26 (s, 1H, 15), 6.12 (d, 1H, 8, J= 8.70 Hz),
4.21 (d, 2H, 5’, J= 5.80 Hz), 3.82 (s, 3H, 18), 3.66–3.57 (m,
1H, 6), 3.05 (q, 2H, 3), 1.65–1.46 (m, 4H, 4, 5), 1.20 (d, 3H,
7, J= 6.00 Hz); 13C NMR (75.5 MHz, DMSO) δ 159.48 (1),
159.25 (16), 159.08 (3’), 145.12 (9), 144.71 (11), 141.09
(6’), 135.27 (13), 135.00 (10), 130.06 (14), 128.48, 127.40
(7’, 8’,10’, 11’), 126.85 (9’), 122.58 (12), 96.54 (17), 92.05
(15), 55.46 (18), 47.54 (6), 43.00 (5’), 39.65 (3), 33.85 (5),
27.20 (4), 20.69 (7); MS/MS m/z 451.3 (M + H)+; elemental
analysis: calcd for C24H30N6O3: C, 63.98; H, 6.71; N, 18.65.
Found: C, 63.63; H, 6.77; N, 18.78.
N1-benzyloxy-N2-[4-(6-methoxyquinolin-8-ylamino)pentyl]
hydrazine-1,2-dicarboxamide (5f)
Method C: From the reaction of 1.305 g (4 mmol) 4f,
1.037 g (4.0 mmol) PQ and 1.67 mL (12.0 mmol) TEA at
room temperature for 3 h and purification of the product
by column chromatography (eluent dichloromethane/
MeOH 9.5:0.5) and trituration with ether, 0.802 g (43%)
of 5f was obtained; mp 102°C−104°C; IR (KBr, ν/cm−1)
3300, 3216, 3089, 2934, 1670, 1617, 1573, 1520, 1455,
1423, 1387, 1328, 1219, 1201, 1157, 1051, 1030, 820, 790,
N1-[4-(6-methoxyquinolin-8-ylamino)pentyl]-N2-
phenylethylhydrazine-1,2-dicarboxamide (5d)
Method B: From the reaction of 1.297 g (4 mmol) 4d,
1.037 g (4.0 mmol) PQ and 1.12mL (8 mmol) TEA at
room temperature for 3 h and purification of the product
by trituration with ether/EtOAc, 1.264 g (68%) of 5d was
obtained; mp 174°C−176°C; IR (KBr, ν/cm−1) 3302, 3226,
3088, 2933, 1662, 1616, 1575, 1554, 1520, 1455, 1423, 1387,
1326, 1219, 1200, 1157, 1131, 1051, 1131, 820, 790, 750,
1
738, 697, 625; H NMR (300 MHz, DMSO) δ 9.48, 8.42,
7.50 (3s, 3H, 1’, 2’, 4’), 8.55 (dd, 1H, 11, J = 3.92 Hz), 8.08 (d,
1H, 13, J = 8.19 Hz), 7.45–7.31 (m, arom., 6H, 7’–11’, 12),
6.48 (d, 1H, 17, J = 2.26 Hz), 6.28 (d, 1H, 15, J = 2.14 Hz),
6.18 (t, 1H, 2, J = 5.70 Hz), 6.13 (d, 1H, 8, J = 8.78 Hz), 4.77
(s, 2H, 5’), 3.83 (s, 3H, 18), 3.68–3.59 (m, 1H, 6), 3.04 (q,
2H, 3), 1.66–1.43 (m, 4H, 4, 5), 1.22 (d, 3H, 7, J = 6.17 Hz);
13C NMR (75.5 MHz, DMSO) δ 160.16 (1), 159.47 (16),
158.98 (3’), 145.11 (9), 144.72 (11), 136.95 (6’), 135.27
(13), 135.00 (10), 130.05 (14), 129.23, 128.59 (7’, 8’,10’, 11’),
128.42 (9’), 122.58 (12), 96.58 (17), 92.08 (15), 77.85 (5’),
1
700, 624; H NMR (300 MHz, DMSO) δ 8.50 (d, 1H, 11,
J= 3.79 Hz), 8.04 (d, 1H, 13, J= 7.81 Hz), 7.54 (1s, 1H, 1’),
7.47 (1s, 1H, 2’), 7.39 (q, 1H, 12, J= 4.24 Hz), 7.26–7.11 (m,
arom., 5H, 8’–12’), 6.44 (s, 1H, 17), 6.33–6.28 (2t, 2H, 2,
4’), 6.24 (s, 1H, 15), 6.09 (d, 1H, 8, J= 8.70 Hz), 3.80 (s, 3H,
18), 3.62–3.57 (m, 1H, 6), 3.20 (q, 2H, 5’, J= 6.47 Hz), 3.02
(q, 2H, 3), 2.66 (t, 2H, 6’, J= 7.14 Hz), 1.63–1.43 (m, 4H, 4,
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel 1-acyl-4-substituted semicarbazide derivatives of PQ 605
55.46 (18), 47.51 (6), 39.71 (3), 33.87 (5), 27.24 (4), 20.70
(7); MS/MS m/z 467.3 (M + H)+; elemental analysis:
calcd for C24H30N6O4: C, 61.79; H, 6.48; N, 18.01. Found:
C, 61.83; H, 6.40; N, 17.86.
growth (PG) values above and below the reference value
(i.e. 50%). Each result is a mean value from three separate
experiments.
e cytostatic activity of the test compounds against
L1210, HeLa and CEM cells was determined with cells
grown in suspension (L1210, CEM) or monolayers
(HeLa) in 200 µL-wells of 96-well microtiter plates (initial
cell number: 5–7.5 × 104 cells/well). After 48 (L1210) or 72
(CEM, HeLa) h, the tumour cell number was determined
by the use of a Coulter counter.
N1-hydroxy-N2-[4-(6-methoxyquinolin-8-ylamino)pentyl]
hydrazine-1,2-dicarboxamide (5g)
Asuspensionof5e(0.467 g,1.0mmol)inmethanol(15 mL)
was hydrogenated for 1.5 h in the presence of 10% Pd/C
(100 mg). e catalyst was filtered off and the solvent was
evaporated under reduced pressure. e crude product
was re-crystallized from ether/petrol ether. Yield: 0.226 g
(60%). mp 112°C; IR (KBr, ν/cm−1) 3262, 2929, 2857, 1654,
1616, 1576, 1558, 1520, 1456, 1423, 1388, 1221, 1202, 1159,
Antiviral activity assays
Antiviral activity against vesicular stomatitis virus,
Coxsackie virus B4, respiratory syncytial virus, herpes
simplex virus type 1 (KOS) and type 2 (G), thymidine
kinase-deficient herpes simplex virus type 1 (TK− KOS
ACVr), vaccinia virus, parainfluenza-3 virus, reovirus-1,
Sindbis virus, Punta Toro virus, influenza virus A (H1N1;
H3N2) and B, feline corona virus (FIPV) and feline her-
pes virus, HIV-1 and HIV-2 was determined essentially,
as described previously28. After a 2 h incubation period,
residual virus was removed and the infected cells were
further incubated with the medium containing different
concentrations of the test compounds. After incuba-
tion for 3 days at 37°C, virus-induced cytopathogenicity
was monitored microscopically. Antiviral activity was
expressed as the concentration required to reduce virus-
induced cytopathogenicity by 50% (EC50).
1
1052, 1031, 822, 791, 625; H NMR (300 MHz, DMSO) δ
8.70, 8.55, 8.21, 7.42 (4s, 4H, 1’, 2’, 4’, OH), 8.54 (d, 1H, 11),
8.08 (dd, 1H, 13, J= 8.36 Hz), 7.43–7.41 (q, 1H, 12), 6.48
(d, 1H, 17, J= 2.33 Hz), 6.27 (d, 1H, 15, J= 2.33 Hz), 6.17 (t,
1H, 2, J= 5.82 Hz), 6.14–6.11 (m, 1H, 8), 3.83 (s, 3H, 18),
3.66–3.58 (m, 1H, 6), 3.04 (q, 2H, 3), 1.64–1.46 (m, 4H, 4,
5), 1.21 (d, 3H, 7, J= 6.21 Hz); 13C NMR (75.5 MHz, DMSO)
δ 160.6 (1), 158.92 (16), 158.51 (3’), 144.52 (9), 144.15 (11),
134.76 (13), 134.38 (10), 129.50 (14), 122.02 (12), 96.05
(17), 91.54 (15), 54.91 (18), 46.96 (6), 39.08 (3), 33.30 (5),
26.67 (4), 20.12 (7); MS/MS m/z 377.3 (M + H)+; elemental
analysis: calcd for C17H24N6O4: C, 54.24; H, 6.43; N, 22.33.
Found: C, 54.41; H, 6.55; N, 21.98.
Biological evaluation
Cytostatic activity assays
Antioxidant activity assays
e HCT 116 (colon carcinoma), MCF-7 (breast carci-
noma), H 460 (lung carcinoma) cells were cultured as
monolayers and maintained in Dulbecco’s modified
Eagle medium (DMEM), supplemented with 10% foetal
bovine serum (FBS), 2 mmol L−1 L-glutamine, 100 U mL−1
penicillin and 100 µg mL−1 streptomycin in a humidified
atmosphere with 5% CO2 at 37°C. e growth inhibition
activity was assessed, as described previously, according
to the slightly modified procedure of the National Cancer
Determination of the reducing activity of the stable radical DPPH
To an ethanolic solution of DPPH (0.05 mM) in absolute
ethanol an equal volume of the compounds (final con-
centration 100 μM) dissolved in DMSO was added. e
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. Each experiment was performed at least
in triplicate, and the standard deviation of absorbance
was less than 10% of the mean (Table 1).
Institute, Developmental erapeutics Program26,27
.
Briefly, the cells were inoculated onto standard 96-well
microtiter plates on day 0. Test agents were then added in
five consecutive 10-fold dilutions (10−8 to 10−4 mol L−1) and
incubated for further 72 h. Working dilutions were freshly
prepared on the day of testing. e solvent (DMSO) was
also tested for eventual inhibitory activity by adjusting its
concentration to be the same as in working concentra-
tions (maximal concentration of DMSO was 0.25%). After
72 h of incubation, the cell growth rate was evaluated by
performing the MTT assay which detects dehydrogenase
activity in viable cells. e absorbance was measured
on a microplate reader at 570 nm. Each test point was
performed in quadruplicate in at least two individual
experiments. e results are expressed as IC50, which
is the concentration necessary for 50% of inhibition.
e IC50 values for each compound are calculated from
dose–response curves using linear regression analysis
by fitting the test concentrations that give percentage of
Inhibition of linoleic acid lipid peroxidation
e water soluble azo compound AAPH is used as a free
radical initiator for in vitro studies of free radical pro-
duction. Production of conjugated diene hydroperoxide
by oxidation of linoleic acid sodium salt in an aqueous
solution is monitored at 234 nm. is assay can be used
to follow oxidative changes and to understand the contri-
bution of each tested compound. An amount of 10 µL 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. e oxidation
reaction was initiated at 37°C under air by the addition
of 50 µL of 40 mM AAPH solution. Oxidation was carried
out in the presence of aliquots (10 µL) in the assay with-
out antioxidant, and lipid oxidation was measured in the
presence of the same level of DMSO. e rate of oxida-
tion at 37°C was monitored by recording the increase in
© 2013 Informa UK, Ltd.

606 I. Perković et al.
Table 1. Interaction with DPPH, in vitro inhibition of LP, soybean LOX and theoretically calculated Clog p values.
DPPHa (%)
Compd.
4b
4c
20 min
61.2
66.6
3.7
60 min
69.2
77.4
3.5
LPa (%)
na
LOX inhibitiona (%)
Clog p b
1.92
2.25
3.45
4.01
3.71
4.08
5.05
3.51
1.03
−
na
na
na
5a
1.0
2.4
2.0
2.0
3.0
1.2
2.0
−
na
5b
5c
5d
12.0
12.6
2.4
15.7
16.7
1.3
29.4
35.4
45.2
51.0
9.6
14.1
81
5e
5f
10.0
8.0
13.2
15.0
72.0
91
5g
NDGA
68.5
84
Trolox
−
−
67
−
−
a Concentration of the tested compound: 100 μM;
b Clog p values were calculated by the ClogP programme of Biobyte Corp35;
na− no activity.
absorption at 234 nm caused by conjugated diene hydro-
peroxides. All tests were undertaken on three replicates,
and the results were averaged.
cyclohexylamine,
benzylamine,
2-phenylethana-
mine, benzhydrylamine, O-benzylhydroxylamine)
gave 1-carbamoylbenzotriazoles 2a–f. In the next
reaction step, the BtH was replaced by hydrazine
hydrate to give 4-substituted semicarbazides 3a–f.
Benzotriazolecarbonyl semicarbazides 4a–f were pre-
pared by the reaction of products 3 with chloride 1. In
the final reaction step, benzotriazole ring of 4a–f was
substituted with PQ affording PQ-semicarbazide deriv-
atives 5a–f. Compound 5g with a free hydroxyl group at
position 4 of semicarbazide backbone was prepared by
hydrogenolysis of benzyloxysemicarbazide 5f.
Soybean LOX inhibition study in vitro
etestedcompoundsdissolvedinDMSOwereincubated
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 × 10−4, w/v in saline). e conversion of sodium
linoleate to 13-hydroperoxylinoleic acid at 234 nm was
recorded and compared with the appropriate standard
inhibitor.
Structures of the synthesized compounds are sup-
ported by infrared, mass, H and 13C NMR spectra and
1
Results and discussion
confirmed by elemental analysis. e chemical shifts
are consistent with the proposed structures. e pres-
Chemistry
1
A series of novel 1,4-substituted semicarbazides 5a–g
with PQ moiety bridged by carbonyl group at position
1 and cycloalkyl, aryl, benzyloxy or hydroxy substituent
at position 4 were prepared and biologically evaluated.
e title compounds contain a region of six or seven elec-
tronegative atoms bound to primary amino group of PQ.
erefore, they are closely related to biurets, hydroxyu-
reas, hydroxamic acids or azapeptides.
Preparation of compounds 5a–f involved BtH as syn-
thetic auxiliary. e BtH moiety in 1-(N-cycloalkyl/aryl-
carbamoyl)benzotriazoles (1-benzotriazole carboxylic
acid amides) 2a–f and 1-(1-benzotriazolecarbonyl)-
4-cycloalkyl/aryl semicarbazides 4a–f activated the
molecules for nucleophilic substitution with hydrazine
hydrate or PQ, allowing the preparation of semicar-
bazides 3a–f and PQ-semicarbazide derivatives 5a–f,
respectively. e BtH was introduced in the interme-
diate products 2 and 4 by means of 1-benzotriazole
carboxylic acid chloride (BtcCl, 1), which was first
described by our research group and now is commer-
cially available23,24. e TEA was used as a catalyst and/
or HCl acceptor in several synthetic steps. e synthetic
pathway is shown in Figure 1: the starting chloride 1 in
reaction with corresponding amine (cyclopentylamine,
ence of the PQ residue was confirmed in H (13C) spec-
tra of 5a–g: hydrogen atoms next to pyridine nitrogen
occurred between 8.50 and 8.55 (144.15 and 144.72),
OCH3 group appeared as a singlet between 3.80 and 3.83
(54.91 and 55.47), C-6 appeared as a multiplet between
3.54 and 3.68 (46.96 and 47.56), C-7 occurred as a dou-
blet between 1.17 and 1.22 (20.12 and 20.70), while
NH group at position 8 appeared as a doublet between
5.96 and 6.13 ppm. Semicarbazide NH groups 1′ and 2′
of compounds 5 showed as two close singlets at 7.47–
7.61 ppm, while those of compounds 4 were shifted to
10.83−10.88 and 8.30−8.40, respectively. e NH groups
at positions 2 and 4′ were approximately at 6.90 and
6.35 ppm. Signals of two carbonyl C-atoms in the PQ
semicarbazide spectra were very close and appeared
between 158.27 and 160.6 ppm, while C-1 carbonyl in
benzotriazolecarbonyl semicarbazides 4 was shifted
to the lower parts per million values (150.12−150.19).
Atom enumeration of compounds 4 and 5 is shown
in Figure 2. e IR spectra of 5a–f showed absorption
bands at 3410−3262 (NH groups), 1674−1650, 1576−1548
and 1521−1519 cm−1 (semicarbazide carbonyls), while
carbonyl group bound to BtH in compound 4 was
absorbed at 1771−1738 cm−1.
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel 1-acyl-4-substituted semicarbazide derivatives of PQ 607
Figure 1. Synthesis of PQ semicarbazide derivatives 5a–g.
Figure 2. Atom enumeration of compounds 4 and 5.
molecules was less important to display anti-prolifera-
Biological evaluation
tive potency. On the other hand, analogue compounds
of the series 4 with the same semicarbazide moiety,
containing a BtH ring instead of PQ or NSAID residues
were practically inactive, which indicates the impor-
tance of the PQ/NSAID part of the molecule.
Most interesting, however, is the high sensitivity
of MCF-7 tumour cells to almost all the compounds.
is phenomenon was already noted in the authors’
previous work with urea derivatives of PQ 20. Some
other authors reported selectivity of quinidine, PQ,
Cytostatic activity
e PQ-semicarbazide derivatives 5a–g and their
synthetic precursors benzotriazolecarbonyl semicar-
bazides 4 were evaluated for their cytostatic activities
against a variety of tumour cell lines, which are derived
from several cancer types. e following cell lines were
used: human HCT 116, MCF-7, H 460, CEM, HeLa and
murine L1210. All compounds of the series 5 showed
high selectivity towards MCF-7 cells (breast carci-
noma) with IC50 values in the low micromolar range
(Table 2). Benzyl derivative 5c was the most active
N-dipeptidylprimaquines
and
imidazolin-4-one
PQ derivatives towards MCF-7 cell line as well16,29
.
compound (IC50
1 0.2 µM), while benzhydryl deriva-
Selective growth inhibitory activity towards breast
cancer cells could be explained by selective induction
of cytochrome P450 (CYP-1) enzymes specifically in
the MCF-7 line, known as CYP-1 inducible cell line30.
ese enzymes catalyse the initial step in either detoxi-
fication or bio-activation of environmental toxins
and xenobiotics. e transcriptional regulation of the
CYP1A1 gene by polycyclic aromatic hydrocarbons
tive 5e showed significant cytostatic activities towards
all the tested cell lines (IC50 4–17 µM) and the highest
activity against MCF-7 cells. ese data are in agree-
ment with our previous results obtained for NSAID
semicarbazide derivatives22, where the presence of a
bulky lipophilic group, preferably a benzhydryl group,
at the N-4 position of the semicarbazide backbone was
strictly required, while the NSAID terminal end of these
© 2013 Informa UK, Ltd.

608 I. Perković et al.
Table 2. Inhibitory effects of PQ semicarbazide derivatives 5a-g and their synthetic precursors 4 on the proliferation of malignant
tumour cell lines.
IC50a (µM)
Compd.
Structural formula
HCT 116
> 100
MCF-7
> 100
H 460
> 100
L1210
> 250
CEM
> 250
HeLa
> 250
4b
4c
> 100
> 100
> 100
> 100
> 100
≥ 250
−
> 250
−
> 250
−
4d
79 16
5a
5b
> 100
> 100
11
8
> 100
> 100
> 250
> 250
≥ 250
> 250
16 12
144 86
150 74
5c
> 100
> 100
1
0.2
> 100
> 250
> 250
> 250
> 250
> 250
> 250
5d
5e
4
4
0.3
0.6
> 100
17
16
1
2
17 0.9
18
21
2
1
11
21
1
5
16 1
5f
1.5 1.3
32
4
12 11
5g
≥ 100
3
0.2
> 100
173 45
179 34
202 67
PQ20
20
6
28 10
30
7
nt
nt
nt
aIC50; the concentration that causes 50% growth inhibition.
bnt; not tested.
(PAH) and halogenated aromatic hydrocarbons (HAH)
is mediated via ligand-dependent activation of the aryl
hydrocarbon receptor (AhR), which translocates to the
nucleus upon activation, dimerizes to the aryl hydro-
carbon receptor nuclear translocator (arnt) protein and
binds to the xenobiotic response element (XRE) in the
regulatory region of the CYP1A1 gene31. Probably, the
observed selectivity towards breast cancer cells could
be related to the PQ moiety, although PQ does not con-
form to the structural features of AhR ligands (planar
hydrophobic aromatic compounds) and, accordingly,
does not bind with high specificity to the AhR. It has
been shown that several non-planar compounds with
little aromaticity, such as benzimidazole compounds
(e.g. some antiparasitic drugs) also induce the CYP1A1
mRNA expression, but the mechanism of its induc-
tion has not been well understood32. Moreover, it was
demonstrated that PQ is a potent inducer of CYP1A1
in the rat hepatoma H4IIE cells31. It uses the AhR for
the transcriptional activation of the CYP1A1 gene and
causes an inhibition of CYP1A1 enzyme degradation.
A similar concept of sensitivity of MCF-7 cells towards
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel 1-acyl-4-substituted semicarbazide derivatives of PQ 609
benzothiazoles mediated by AhR has been shown con-
cerning the discovery of a class of 2-(4-aminophenyl)
benzothiazoles, potential antitumour agents that
showed remarkably similar, highly selective profiles of
antitumour activity33. e intriguing specificity of PQ
semicarbazides towards MCF-7 cells should be further
studied in more detail and fostered towards the dis-
covery of novel AhR-dependent toxicity/antitumour
activity.
allergic diseases, certain types of cancer and cardiovas-
cular diseases. In this context, it was decided to evaluate
the newly prepared derivatives for their ability to inhibit
soybean LOX36. 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. Compound
5e, followed by 5d, was found to be the most potent
among the PQ semicarbazides 5, while the tested com-
pound 4 showed no activity. e fact that the most active
compounds have high Clog p values (5.05 and 4.08,
respectively) support the findings that lipophilicity is an
important physicochemical property for LOX inhibitors.
Antiviral activity
Compounds 4b, 4c and 5a–g were evaluated against a
broad variety of viral infections, including herpes sim-
plex virus type 1 (HSV-1) and HSV-2, vaccinia virus and
vesicular stomatitis virus in HEL cell cultures, parain-
fluenza-3 virus, reovirus-1, Sindbis virus, Coxsackie
virus B4 and Punta Toro virus in Vero cell cultures,
vesicular stomatitis virus, Coxsackie B4 virus and
respiratory syncytial virus in HeLa cell cultures, feline
coronavirus (FIPV) and feline herpes virus in CRFK cell
cultures, influenza A (H1N1 and H3N2) and B in MDCK
cell cultures and human immunodeficiency virus type
1 (HIV-1/IIIB) and HIV-2(ROD) in CEM cell cultures.
No specific antiviral effects were noted for any of the
compounds evaluated against any of the tested viruses
(data not shown).
conclusions
e PQ semicarbazides 5, described here, showed
significant selective cytostatic activity against the pro-
liferation of MCF-7 cells with IC50 values between 1
and 16 µM (five compounds in the range 1−4 µM). e
most active compound was the benzyl derivative 5c.
Benzhydryl derivative 5e showed cytostatic activities
towards all the tested cell lines (IC50 4–18 µM). e same
compound was the strongest LOX inhibitor as well. It is
worth to note that in the series of NSAID semicarba-
zide derivatives previously described by our group22,
the most active compounds were also the benzhydryl
derivatives. On the other hand, the highest antioxidant
activity was found for the hydroxy derivative 5g and
the benzotriazolecarbonyl semicarbazides 4b, 4c. e
prepared PQ semicarbazide derivatives represent use-
ful lead compounds in development of specific agents
against breast cancer. ey have potential as antima-
larial agents as well, but their antimalarial evaluation
still remains to be performed.
Antioxidant activity
In the present investigation, antioxidant activity of the PQ
derivatives 4b, 4c and 5a–g were studied and compared
with the well-known antioxidant agents, such as NDGA
and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carbox-
ylic acid (Trolox). Two different antioxidant assays were
used: (1) interaction with the stable free DPPH radical
and (2) interaction with the water-soluble azo com-
pound AAPH, used as a source of peroxyl radicals34. e
DPPH interaction of the tested compounds was exam-
ined at 100 μM concentration after 20 and 60min. e
results are shown in Table 1. Derivative 5g with hydroxy
group at position 4 was the only PQ semicarbazide that
showed significant interaction (68.5%), followed by
1-(1-benzotriazolecarbonyl)-4-aryl semicarbazides 4b
and 4c (61.2% and 66.6%, respectively). A slight increase
(69.2−77.4%) in antioxidant activity was observed after
60 min. Low lipophilicity values (1.03−2.25) are cor-
related with higher DPPH interaction. e antioxidant
ability of the other compounds of the series 5 was very
low (2.4−12.6%).
In our studies, AAPH was used as a free radical initia-
tor to follow oxidative changes of linoleic acid to conju-
gated diene hydroperoxide. Under our experimental
conditions, no inhibition of lipid peroxidation (LP) by the
tested compounds was observed.
e LOXs play a significant role in membrane LP by
forming hydroperoxides in the lipid bilayer from the
biotransformation of arachidonic acid catalyzed by LOX.
e LOX inhibitors have attracted attention initially as
potential agents for the treatment of inflammatory and
acknowledgement
We thank Dr. C. Hansch, Dr. A. Leo, and Biobyte Corp.,
Claremont, California, for free access to the C-QSAR
program and Mrs. Leentje Persoons, Frieda De Meyer,
Leen Ingels, Lizette van Berckelaer for excellent techni-
cal assistance.
Declaration of interest
e authors report no conflicts of interest. is study
is supported by the Ministry of Science, Education and
Sports of the Republic of Croatia (Projects No. 006-
0000000-3216 and 098-0982464-2514) and the K.U.
Leuven (GOA 10/014).
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