Novel urea and bis-urea primaquine derivatives with hydroxyphenyl or halogenphenyl substituents: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2016.08.021

A series of novel compounds 3a-j and 6a-j with primaquine and hydroxyl or halogen substituted benzene moieties bridged by urea or bis-urea functionalities were designed, synthesized and evaluated for biological activity. The title compounds were prepared

Full text of DOI:10.1016/j.ejmech.2016.08.021

Accepted Manuscript
Novel urea and bis-urea primaquine derivatives with hydroxyphenyl or halogenphenyl
substituents: Synthesis and biological evaluation
I. Perković, M. Antunović, I. Marijanović, K. Pavić, K. Ester, M. Kralj, J. Vlainić, I.
Kosalec, D. Schols, D. Hadjipavlou-Litina, E. Pontiki, B. Zorc
PII:
S0223-5234(16)30664-X
10.1016/j.ejmech.2016.08.021
EJMECH 8817
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 April 2016
Revised Date: 10 August 2016
Accepted Date: 11 August 2016
Please cite this article as: I. Perković, M. Antunović, I. Marijanović, K. Pavić, K. Ester, M. Kralj, J. Vlainić,
I. Kosalec, D. Schols, D. Hadjipavlou-Litina, E. Pontiki, B. Zorc, Novel urea and bis-urea primaquine
derivatives with hydroxyphenyl or halogenphenyl substituents: Synthesis and biological evaluation,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.08.021.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel urea and bis-urea primaquine derivatives with hydroxyphenyl or halogenphenyl
substituents: synthesis and biological evaluation
a
b
b
a
c
c
c
I. Perković , M. Antunović , I. Marijanović , K. Pavić , K. Ester , M. Kralj , J. Vlainić , I.
d
e
f
f
a,
Kosalec , D. Schols , D. Hadjipavlou-Litina , E. Pontiki , B. Zorc *
a
University of Zagreb, Faculty of Pharmacy and Biochemistry, A. Kovačića 1, HR-10 000
Zagreb, Croatia
b
University of Zagreb, Faculty of Science, Horvatovac 102 1, HR-10 000 Zagreb, Croatia
c
Division of Molecular Medicine, Rudjer Bošković Institute, Bijenička cesta 54, HR-10 000
Zagreb, Croatia
d
University of Zagreb, Faculty of Pharmacy and Biochemistry, Schrottova 39, HR-10 000
Zagreb, Croatia
e
Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat
1
0, B-3000 Leuven, Belgium
f
School of Pharmacy, Faculty of Health Sciences, Aristotles University of Thessaloniki, GR-
5
4 124 Thessaloniki, Greece
*
Corresponding author. Tel.: +385 1 4856202; fax: +385 1 4856201. E-mail address:
bzorc@pharma.hr (B. Zorc)
University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia
Keywords:
Primaquine
Urea
Bis-urea
Antiproliferative activity
Antioxidative activity
Antimicrobial activity
1

ACCEPTED MANUSCRIPT
A B S T R A C T
A series of novel compounds 3a-j and 6a-j with primaquine and hydroxyl or halogen
substituted benzene moieties bridged by urea or bis-urea functionalities were designed,
synthesized and evaluated for biological activity. The title compounds were prepared using
benzotriazole as the synthon, through several synthetic steps. 3-[3,5-
Bis(trifluoromethyl)phenyl]-1-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}urea (3j) was the
most active urea and 1-[({4-[(6-methoxyquinolin-8-yl)amino]pentyl}carbamoyl)amino]-3-[3-
(trifluoromethyl)phenyl]urea (6h) the most active bis-urea derivative in antiproliferative
screening in vitro against eight tested cancer cell lines. Urea derivatives 3a-g with hydroxy
group or one halogen atom showed moderate antiproliferative effects against all the tested cell
lines, but stronger activity against breast carcinoma MCF-7 cell line, while trifluoromethyl
derivatives 3h-j showed antiproliferative effects against all the tested cell lines in low
micromolar range. Finally, bis-ureas with hydroxy and fluoro substituents 6a-d showed
extreme selectivity and chloro or bromo derivatives 6e-g high selectivity against MCF-7 cells
(
IC 0.1-2.6 µM). p-Fluoro derivative 6d, namely 3-(4-fluorophenyl)-1-[({4-[(6-
50
methoxyquinolin-8-yl)amino]pentyl}carbamoyl)amino]urea, is the most promising
compound. Further biological experiments showed that 6d affected cell cycle and induced cell
death of MCF-7 cell line. Due to its high activity against MCF-7 cell line (IC 0.31 µM),
50
extreme selectivity and full agreement with the Lipinski’s and Gelovani’s rules for
prospective small molecular drugs, 6d may be considered as a lead compound in development
of breast carcinoma drugs.
Urea 3b and almost all bis-ureas showed high antioxidant activity in DPPH assay, but urea
derivatives were more active in lipid peroxidation test. Only few compounds exhibited weak
inhibition of soybean lipoxygenase. Compound 3j exhibited the strongest antimicrobial
–
1
activity in susceptibility assay in vitro (MIC = 1.6–12.5 ꢀg ml ).
1
. Introduction
Modification of known, clinically approved drugs is one of the possible strategies in drug
discovery. In our search for potential anticancer agents we have focused our attention to
primaquine (PQ), the antimalarial agent with a pronounced antiproliferative activity [1,2,3].
The potential for discovery of new anticancer agents among antimalarials was first noted in
the scientific literature when commonly used antimalarials (artemisinin, artesunate,
2

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tetracyclines, chloroquine and primaquine) were observed to portend significant anticancer
activity. Currently, 14 out of 20 agents globally used in the treatment of malaria have reported
anticancer effects and 7 have reached a clinical stage of development [4,5]. In several studies
coadministration of antimalarial drugs with approved anticancer drugs was studied. It was
shown that antimalarial drugs (hydroxychloroquine, chloroquine, primaquine) reduce IC of
5
0
anticancer drugs (doxorubicin, cisplatin) and inhibit development of drug resistance of cancer
cells [6]. It was also shown that chloroquine is a promising adjuvant together with lidamycin
[
7], sunitinib [8] or artemisin chemotherapy [9]. Coadministration of primaquine with
imatinib showed that primaquine affect imatinib clearance, bioavailability and distribution
pattern, which could improve the treatment of renal and brain tumours, but also increase
toxicity [10]. Both chloroquine and primaquine, have quinoline moiety in their structure. The
widespread use of quinoline heterocycle as an important pharmacophore in medicinal
chemistry establishes this moiety as a member of a privileged structures class. Various
quinoline derivatives were reported as tubulin polymerization and tyrosine kinase inhibitors
[
11]. Their potential to develop to anticancer drugs is described recently in review of Afzal et
al. [12].
In last few years, our research group has focused to primaquine derivatization. We have
prepared approximately 90 novel primaquine derivatives and their antiproliferative activity in
vitro has been well documented. In our previous papers, synthesis and biological evaluation of
1
1
,4-substituted semicarbazides with primaquine moiety bridged by carbonyl group at position
and cycloalkyl, aryl, benzyloxy or hydroxy substituent at position 4 [13] or urea and
semicarbazide primaquine derivatives with benzhydryl, trityl, phenyl or hydroxyalkyl
substituents was reported [14]. Most of the compounds showed significant cytostatic activities
in low micromolar concentrations towards all the tested cell lines or high selectivity towards
MCF-7 cells (breast carcinoma). The highest selectivity against MCF-7 cells and practically
no activity against other 7 tested cancer cell lines showed symmetric derivative with two
primaquine residues bind with two urea groups (IC 0.2 µM). Urea with 5-hydroxypentyl
50
substituent on one nitrogen and primaquine residue on the other one, showed extreme
selectivity towards SW 620 colon cancer cells (IC 0.2 µM) [15]. In order to get better
5
0
insight to the structural requirements needed for improvement of cytostatic activity of
primaquine derivatives, here we report design, synthesis and biological evaluation of a new
set of primaquine derivatives bearing urea or bis-urea functionalities and hydroxyphenyl or
halogenphenyl substituents.
3

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2
2
2
. Materials and methods
.1. Chemistry
.1.1. Materials and general methods
Melting points were measured on a Stuart Melting Point (SMP3) apparatus (Barloworld
Scientific, UK) in open capillaries with uncorrected values. IR spectra were recorded on FTIR
Perkin Elmer Paragon 500 and UV-Vis spectra on Lambda 20 double beam
1
13
spectrophotometer (Perkin-Elmer, UK). All NMR ( H and C) were recorded at 25 °C on
1
13
NMR Avance 600 spectrometer (Bruker, Germany) at 300 and 150 MHz for H and C
nuclei, respectively. Chemical shifts (δ) are reported in parts per million (ppm) using
1
tetramethylsilane as reference in the H and the dimethyl sulfoxide (DMSO) residual peak as
1
3
reference in the C spectra (39.51 ppm). Coupling constants (J) are reported in Hertz (Hz).
Mass spectra were recorded on HPLC-MS/MS (HPLC, Agilent Technologies 1200 Series;
MS, Agilent Technologies 6410 Triple Quad). Mass determination was realized using electron
spray ionization (ESI) in positive mode. Elemental analyses were performed on a CHNS
LECO analyzer (LECO Corporation, USA). Analyses indicated by the symbols of the
elements were within ± 0.4% of the theoretical values. All compounds were routinely checked
by TLC with Merck silica gel 60F-254 glass plates using the following solvent systems:
dichloromethane/methanol 97:3, 95:5 and 92:8, petrolether/ethyl acetate/methanol 30:10:5,
cyclohexane/ethyl acetate 2:1 and petrolether/ethyl acetate 2:1. 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. All chemicals and solvents were of
analytical grade and purchased from commercial sources. 1H-benzo[d] [1,2,3]triazole (BtH),
triphosgene, primaquine diphosphate, triethylamine (TEA), hydrazine hydrate and hydroxy or
halogen substituted aniline derivatives were purchased from Sigma-Aldrich (USA).
Primaquine was prepared from primaquine diphosphate prior the use. All reactions with
primaquine were run light protected.
2
.1.2. 1-(N-arylcarbamoyl)benzotriazoles (2a-j): general procedure
The title compounds were synthesized from chloride 1 and corresponding hydroxy or halogen
substituted aniline derivative, following our previously published procedure [16]. The
4

ACCEPTED MANUSCRIPT
solution of 0.908 g (5.0 mmol) chloride 1 in dry dioxane was added dropwise to the solution
of 5.0 mmol aniline derivative and 0.507 g (5 mmol) TEA in dry dioxane.
Method A: Reaction mixture was stirred 1 h at room temperature and evaporated under
reduced pressure.
Method B: Reaction mixture was stirred 1 h at room temperature, evaporated under reduced
pressure, dissolved in ethyl acetate and extracted with 5% NaOH solution (3 × 15 ml) and
water (3 × 15 ml). The organic layer was dried over sodium sulfate, filtered and evaporated
under reduced pressure.
2
.1.2.1. 1-(N-(3-hydroxyphenyl)carbamoyl) benzotriazole (2a). Method A, from the reaction
of 0.546 g 3-aminophenol and after purification by column chromatography (mobile phase
dichloromethane/methanol 95:5) and crystallization from ether 0.406 g (32%) of 2a was
obtained; mp 147–149 °C; IR (KBr): νmax 3385, 3164, 1748, 1615, 1604, 1551, 1449, 1251,
–
1 1
1
069, 842, 791, 773, 752 cm ; H NMR (DMSO-d ) δ 10.98 (s, 1H'), 9.55 (s, 1H), 8.28-8.24
6
1
3
(
m, 2H), 7.79-7.74 (m, 1H), 7.62-7.57 (m, 1H), 7.38-7.16 (m, 3H), 6.65-6.60 (m, 1H); C
NMR (DMSO-d ) δ 158.15, 147.29, 146.03, 138.68, 131.97, 130.54, 129.94, 126.22, 120.36,
6
+
1
14.20, 112.29, 112.28, 108.65; MS/MS m/z 120.2 (BtH+1) ; Anal. Calcd. for C H N O :
1
3
10
4
2
C, 61.41; H, 3.96; N, 22.04. Found: C, 61.59; H, 4.06, N, 21.77.
2
.1.2.2. 1-(N-(4-hydroxyphenyl)carbamoyl) benzotriazole (2b). Method A, from the reaction
of 0.546 g 4-aminophenol and after crystallization from ether 1.004 g (79%) of 2b was
obtained; mp 240 °C (decomp.); Lit. mp 240 °C (decomp.) [17]; IR (KBr): νmax 3456, 3252,
–
1
1
726, 1544, 1616, 1544, 1618, 1488, 1446, 1370, 1236, 1212, 1060, 926, 825, 146, 583 cm ;
1
H NMR (DMSO-d ) δ 10.89 (s, 1H), 9.41 (s, 1H), 8.25-8.23 (m, 2H), 7.76-7.73 (m, 1H),
6
1
3
7
.60-7.57 (m, 3H), 6.83-6.81 (m, 2H); C NMR (DMSO-d ) δ 154.61, 146.99, 145.50,
6
+
1
31.47, 129.94, 128.40, 125.62, 123.19, 119.81, 115.18, 114.20; MS/MS m/z 277.1 (M+23) ,
+
1
20.1 (BtH+1) ; Anal. Calcd. for C H N O : C, 61.41; H, 3.96; N, 22.04. Found: C, 61.30;
1
3
10
4
2
H, 4.04, N, 22.40.
2
.1.2.3. 1-(N-(3-fluorophenyl)carbamoyl) benzotriazole (2c). Method B, from the reaction of
0
.556 g 3-fluoroaniline and after crystallization from ether 0.807 g (63%) of 2b was obtained;
mp 162.5–164.5 °C; IR (KBr): νmax 3242, 3073, 1742, 1605, 1543, 1486, 1447, 1362, 1285,
–
1 1
1
220, 1131, 1064, 927, 861, 789, 749 cm ; H NMR (DMSO-d ) δ 11.31 (s, 1H), 8.30-8.25
6
5

ACCEPTED MANUSCRIPT
1
3
(
m, 2H), 7.79-7.71 (m, 3H), 7.62-7.59 (m, 1H), 7.49-7.43 (m, 1H), 7.06-7.02 (m, 2H); C
NMR (DMSO-d ) δ 163.16, 146.95, 145.56, 139.07, 131.45, 130.43, 130.20, 125.84, 119.93,
6
+
1
6
14.08, 113.71, 108.37, 105.04; MS/MS m/z 120.2 (BtH+1) ; Anal. Calcd. for C H FN O: C,
1
3
9
4
0.94; H, 3.54; N, 21.87. Found: C, 61.06; H, 3.68, N, 21.69.
2
.1.2.4. 1-(N-(4-fluorophenyl)carbamoyl) benzotriazole (2d). Method B, from the reaction of
0
.556 g 4-fluoroaniline and after purification by column chromatography (mobile phase
petrolether/ethyl acetate/methanol 3:1:0.5) and crystallization from ether 0.833 g (65%) of 2d
was obtained; mp 167–168 °C; IR (KBr): νmax 3327, 3247, 1742, 1614, 1560, 1552, 1510,
–
1 1
1
410, 1365, 1313, 1212, 1129, 1102, 1057, 1000, 929, 835, 770, 747 cm ; H NMR (DMSO-
d6) δ 11.20 (s, 1H'), 8.26-8.24 (m, 2H), 7.78-7.75 (m, 1H), 7.61-7.58 (m, 1H), 7.46-7.44 (m,
1
3
2
H), 7.29-7.26 (m, 2H); C NMR (DMSO-d ) δ 159.79, 147.11, 145.54, 133.52, 131.47,
6
+
1
30.12, 125.77, 123.23, 119.90, 115.17, 113.70; MS/MS m/z 120.2 (BtH+1) ; Anal. Calcd.
for C H FN O: C, 60.94; H, 3.54; N, 21.87. Found: C, 60.66; H, 3.50, N, 21.93.
1
3
9
4
2
.1.2.5. 1-(N-(3-chlorophenyl)carbamoyl) benzotriazole (2e). Method B, from the reaction of
0
.638 g 3-chloroaniline and after crystallization from ether 1.023 g (75%) of 2e was obtained;
mp 179–181.5 °C; IR (KBr): νmax 3287, 3109, 1744, 1594, 1529, 1481, 1427, 1368, 1284,
-
1 1
1
1
7
1
239, 1206, 1131, 1092, 1052, 912, 875, 847, 780, 754 cm ; H NMR (DMSO-d ) δ 11.34 (s,
6
H), 8.28-8.24 (m, 2H), 8.00-7.99 (s, 1H), 7.84-7.71 (m, 2H), 7.63-7.58 (m, 1H), 7.46 (t, 1H),
1
3
.29-7.25 (m, 1H); C NMR (DMSO-d ) δ 146.99, 145.52, 138.84, 133.07, 131.45, 130.48,
6
+
30.24, 125.88, 124.37, 120.53, 119.96, 119.50, 113.74; MS/MS m/z 120.1 (BtH+1) ; Anal.
Calcd. for C H ClN O: C, 57.26; H, 3.33; N, 20.55. Found: C, 57.59; H, 3.66, N, 20.88.
1
3
9
4
2
.1.2.6. 1-(N-(4-chlorophenyl)carbamoyl) benzotriazole (2f). Method B, from the reaction of
0
.638 g 4-chloroaniline and after crystallization from ether 1.104 g (81%) of 2f was obtained;
mp 193–195.5 °C; IR (KBr): νmax 3229, 3105, 3050, 1734, 1599, 1483, 1445, 1360, 1286,
–
1 1
1
7
232, 1046, 920, 820, 718 cm ; H NMR (DMSO-d ) δ 11.28 (s, 1H), 8.27-8.23 (m, 2H),
6
1
3
.90-7.87 (d, 2H), 7.80-7.75 (m, 1H), 7.63-7.57 (m, 1H), 7.51-7.48 (d, 2H); C NMR
(
DMSO-d ) δ 146.96, 145.53, 136.28, 131.43, 130.16, 128.70, 128.46, 125.81, 122.64,
6
+
1
3
19.91, 113.70; MS/MS m/z 120.1 (BtH+1) ; Anal. Calcd. for C H ClN O: C, 57.26; H,
1
3
9
4
.33; N, 20.55. Found: C, 56.93; H, 3.30, N, 20.69.
6

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2
.1.2.7. 1-(N-(4-bromophenyl)carbamoyl) benzotriazole (2g). Method B, from the reaction of
.860 g 4-bromoaniline and after crystallization from ether 1.300 g (82%) of 2g was obtained;
0
mp 196–198 °C; IR (KBr): νmax 3248, 3103, 3048, 1741, 1596, 1543, 1534, 1486, 1446, 1400,
–
1 1
1
367, 1285, 1238, 1127, 1099, 1053, 928, 827, 808, 750 cm ; H NMR (DMSO-d ) δ 11.29
6
1
3
(
s, 1H), 8.28-8.24 (m, 2H), 7.84-7.75 (m, 3H), 7.65-7.58 (m, 3H); C NMR (DMSO-d ) δ
6
1
46.96, 145.55, 136.75, 131.50, 131.29, 130.20, 125.84, 120.20, 119.94, 113.73, 113.36;
+
MS/MS m/z 120.1 (BtH+1) ; Anal. Calcd. for C H BrN O: C, 49.23; H, 2.86; N, 17.67.
1
3
9
4
Found: C, 49.51; H, 3.92, N, 17.55.
2
.1.2.8. 1-(N-(3-(trifluoromethyl)phenyl)carbamoyl) benzotriazole (2h). Method B, from the
reaction of 0.806 g 3-(trifluoromethyl)aniline and after crystallization from ether 0.888 g
(58%) of 2h was obtained; mp 176.5–178.5 °C; IR (KBr): νmax 3255, 3075, 1744, 1605, 1548,
–
1 1
1
450, 1373, 1333, 1286, 1240, 1164, 1118, 1051, 1000, 921, 897, 850, 793, 750 cm ; H
NMR (DMSO-d ) δ 11.46 (s, 1H), 8.30-8.25 (m, 3H), 8.14 (d, 1H), 7.81-7.75 (m, 1H), 7.70-
6
1
3
7
.55 (m, 3H); C NMR (DMSO-d ) δ 147.13, 145.55, 138.21, 131.27, 130.22, 130.01,
6
1
30.11-128.87, 125.70, 124.52, 121.01-120.06, 119.77, 117.30-117.05, 113.72; MS/MS m/z
+
1
20.1 (BtH+1) ; Anal. Calcd. for C H F N O: C, 54.91; H, 2.96; N, 18.29. Found: C, 55.09;
1
4
9
3
4
H, 3.28, N, 18.33.
2
.1.2.9. 1-(N-(4-(trifluoromethyl)phenyl)carbamoyl) benzotriazole (2i). Method B, from the
reaction of 0.806 g 4-(trifluoromethyl)aniline and after crystallization from ether 0.918 g
(60%) of 2i was obtained; mp 204.5–207 °C; IR (KBr): νmax 3302, 3121, 3055, 1740, 1606,
–
1 13
1
544, 1417, 1330, 1242, 1166, 1103, 1047, 921, 835, 750 cm ; C NMR (DMSO-d ) δ
6
1
52.02, 147.02, 143.02, 131.42, 130.22, 126.00, 125.86, 125.32-123.53, 122.43-121.79,
+
1
19.92, 118.08, 113.67; MS/MS m/z 120.1 (BtH+1) ; Anal. Calcd. for C H F N O: C, 54.91;
1
4
9
3
4
H, 2.96; N, 18.29. Found: C, 55.17; H, 3.38, N, 18.00.
2
.1.2.10. 1-(N-(3,5-bis(trifluoromethyl)phenyl)carbamoyl) benzotriazole (2j). Method B, from
the reaction of 1.146g 3,5-bis(trifluoromethyl)aniline 0.918 g (60%) of crude product 2j was
obtained; it was used in further reaction step without purification.
2
.1.3. Primaquine ureas (3a-j): general procedure
7

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A solution of 0.273 g (0.6 mmol) primaquine bisphosphate and 0.182 g (1.8 mmol) TEA in
dichloromethane was stirred 0.25 h at room temperature. Then 6 mmol of corresponding 1-(N-
arylcarbamoyl)benzotriazole 2 was added to the reaction mixture and stirred 0.5-3.5 h at room
temperature. The reaction mixture was evaporated under reduced pressure. The residue was
futher purified without extraction (3a, 3b) or was dissolved in ethyl acetate and extracted with
5
% NaOH solution (3 × 10 ml) and water (3 × 15 ml), dried over anhydrous sodium sulfate
and evaporated (3c-j). The reactions were run light protected.
2
.1.3.1.
3-(3-Hydroxyphenyl)-1-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}urea
(3a).
Reaction time: 3.5 h. From the reaction of 0.153 g 2a and after purification by column
chromatography (mobile phase dichloromethane/methanol 95:5) 0.156 g (66%) of 3a was
obtained; oil; IR (KBr): νmax 3368, 2936, 2866, 1656, 1610, 1559, 1521, 1452, 1386, 1338,
–
1 1
1
1
1
1
1
1
3
6
225, 1163, 1053, 973, 824, 781 cm ; H NMR (DMSO-d ) δ 9.17 (s, 1H), 8.54-8.53 (dd,
6
H), 8.24 (s, 1H), 8.08-8.06 (dd, 1H), 7.43-7.41 (m, 1H), 6.98 (s, 1H), 6.96 (t, 1H), 6.71 (d,
H), 6.47 (d, 1H), 6.29-6.28 (m, 2H), 6.14 (d, 1H), 6.08 (t, 1H), 3.82 (s, 3H), 3.66-3.63 (m,
1
3
H), 3.11-3.09 (q, 2H), 1.70-1.49 (m, 4H), 1.23 (d, 3H); C NMR (DMSO-d ) δ 158.99,
6
57.61, 155.08, 144.63, 144.23, 143.59, 142.88, 134.77, 134.51, 129.56, 129.16, 122.06,
08.39, 108.08, 104.72, 96.12, 91.64, 54.95, 47.05, 39.02, 33.51, 26.64, 20.22; MS/MS m/z
+
95.3 (M+1) ; Anal. Calcd. for C H N O : C, 66.99; H, 6.64; N, 14.20. Found: C, 67.05; H,
2
2
26
4
3
.99, N, 13.98.
2
.1.3.2. 3-(4-Hydroxyphenyl)-1-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}urea (3b).
Reaction time: 3.5 h. From the reaction of 0.153 g 2b and after purification by column
chromatography (mobile phase dichloromethane/methanol 95:5) and crystallization from
ether 0.173 g (73%) of 3b was obtained; mp 149–152.5 °C; IR (KBr): νmax 3419, 3326, 2934,
2
1
8
865, 1683, 1659, 1616, 1576, 1556, 1515, 1455, 1424, 1388, 1223, 1204, 1159, 1101, 1054,
–
1 1
036, 967, 906, 828, 788 cm ; H NMR (DMSO-d ) δ 8.91 (s, 1H), 8.55-8.53 (dd, 1H), 8.09-
6
.06 (dd, 1H), 8.00 (s, 1H), 7.44-7.40 (m, 1H), 7.13 (d, 2H), 6.62 (d, 2H), 6.47 (d, 1H), 6.28
(d, 1H), 6.14 (d, 1H), 5.98 (t, 1H), 3.82 (s, 3H), 3.67-3.63 (m, 1H), 3.11-3.06 (q, 2H), 1.68-
1
3
1
1
3
6
.48 (m, 4H), 1.23 (d, 3H); C NMR (DMSO-d ) δ 158.98, 155.52, 151.84, 144.62, 144.21,
6
34.76, 134.49, 132.06, 129.55, 122.05, 119.82, 115.00, 96.11, 91.63, 54.94, 47.05, 39.09,
+
3.50, 26.72, 20.20; MS/MS m/z 395.4 (M+1) ; Anal. Cald. for C H N O : C, 66.99; H,
2
2
26
4
3
.64; N, 14.20. Found: C, 66.80; H, 6.90, N, 14.24.
8

ACCEPTED MANUSCRIPT
2
.1.3.3. 3-(3-Fluorophenyl)-1-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}urea (3c). Reaction
time: 2 h. From the reaction of 0.154 g 2c and after purification by column chromatography
(mobile phase dichloromethane/methanol 95:5) 0.152 g (64%) of 3c was obtained; oil; IR
(KBr): νmax 3354, 2961, 2926, 2856, 1651, 1645, 1615, 1557, 1510, 1463, 1424, 1386, 1273,
–
1 1
1
221, 1153, 1113, 1052, 967, 940, 863, 822, 791, 773 cm ; H NMR (DMSO-d ) δ 8.59 (s,
6
1
H), 8.53-8.52 (dd, 1H), 8.08-8.06 (dd, 1H), 7.45-7.41 (m, 2H), 7.23-7.19 (q, 1H), 7.01-7.00
(d, 1H), 6.67 (t, 1H), 6.47 (d, 1H), 6.28 (d, 1H), 6.21 (t, 1H), 6.13 (d, 1H), 3.82 (s, 3H), 3.66-
1
3
3
.63 (m, 1H), 3.13-3.10 (q, 2H), 1.60-1.50 (m, 4H), 1.23 (d, 3H); C NMR (DMSO-d ) δ
6
1
63.14, 158.96, 154.89, 144.59, 144.17, 142.39, 134.71, 134.48, 129.92, 129.51, 122.00,
1
13.20, 107.11, 106.97, 104.26, 104.08, 96.10, 91.64, 54.90, 47.01, 39.02, 33.44, 26.48,
+
2
0.17; MS/MS m/z 397.2 (M+1) ; Anal. Calcd. for C H FN O : C, 66.65; H, 6.36; N, 14.13.
22
25
4
2
Found: C, 66.77; H, 6.50, N, 14.34.
2
.1.3.4. 3-(4-Fluorophenyl)-1-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}urea (3d). Reaction
time: 1 h. From the reaction of 0.154 g 2d and after purification by column chromatography
(mobile phases: dichloromethane/methanol 95:5 and cyclohexane/ethyl acetate 2:1) 0.143 g
(60%) of 3d was obtained; oil; IR (KBr): νmax 3323, 2945, 2926, 2858, 1635, 1568, 1520,
–
1
1
456, 1424, 1393, 1380, 1292, 1267, 1210, 1160, 1090, 1055, 1033, 829, 819, 788, 768 cm ;
1
H NMR (DMSO-d ) δ 8.54-8.53 (dd, 1H), 8.36 (s, 1H), 8.08-8.06 (dd, 1H), 7.43-7.41 (m,
6
1
H), 7.39-7.36 (m, 2H), 7.04-7.01 (m, 2H), 6.47 (d, 1H), 6.28 (d, 1H), 6.13 (d, 1H), 6.11 (t,
1
3
1
H), 3.82 (s, 3H), 3.67-3.63 (m, 1H), 3.12-3.09 (q, 2H), 1.70-1.50 (m, 4H), 1.23 (d, 3H); C
NMR (DMSO-d ) δ 158.95, 155.96, 157.53, 155.18, 144.59, 144.17, 136.86, 134.70, 134.48,
6
1
2
1
29.51, 121.99, 119.17, 119.13, 114.97, 114.83, 96.08, 91.71, 54.94, 47.02, 39.06, 33.46,
+
6.56, 20.17; MS/MS m/z 397.3 (M+1) ; Anal. Calcd. for C H FN O : C, 66.65; H, 6.36; N,
2
2
25
4
2
4.13. Found: C, 66.81; H, 6.52, N, 13.95.
2
.1.3.5. 3-(3-Chlorophenyl)-1-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}urea (3e). Reaction
time: 2 h. From the reaction of 0.164 g 2e and crystallization from ether 0.159 g (64%) of 3e
was obtained; mp 111.5–113.5 °C; IR (KBr): νmax 3338, 2967, 2930, 2871, 1649, 1597, 1574,
–
1
1
519, 1481, 1455, 1422, 1387, 1311, 1269, 1228, 1205, 1166, 1054, 885, 861, 825, 788 cm ;
1
H NMR (DMSO-d ) δ 8.58-8.55 (m, 2H), 8.12-8.10 (m, 1H), 7.65 (s, 1H), 7.23-7.17 (m, 2H),
6
6
.91 (d, 1H), 6.49 (m, 1H), 6.37-6.33 (m, 2H), 6.23 (s, 1H), 3.82 (s, 3H), 3.39-3.63 (m, 1H),
1
3
3
.14-3.09 (m, 2H), 1.71-1.50 (m, 4H), 1.23 (d, 3H); C NMR (DMSO-d ) δ 158.99, 154.87,
6
9

ACCEPTED MANUSCRIPT
1
55.18, 144.38, 144.08, 142.08, 134.95, 132.99, 130.05, 129.57, 122.01, 120.39, 116.91,
+
1
15.89, 96.39, 91.81, 54.93, 47.11, 39.04, 33.39, 26.49, 20.12; MS/MS m/z 413.2 (M+1) ;
Anal. Calcd. for C H ClN O : C, 63.99; H, 6.10; N, 13.57. Found: C, 64.33; H, 6.40, N,
2
2
25
4
2
1
3.58.
2
.1.3.6. 3-(4-Chlorophenyl)-1-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}urea (3f). Reaction
time: 2 h. From the reaction of 0.164 g 2f and after purification by column chromatography
mobile phases: dichloromethane/methanol 95:5 and petrolether/ethyl acetate 2:1) and
crystallization from ether 0.141 g (57%) of 3f was obtained; mp 94–96.5 °C; IR (KBr): νmax
(
3
1
8
2
3
1
5
314, 3088, 3047, 2968, 2934, 2862, 1637, 1617, 1591, 1571, 1522, 1492, 1457, 1424, 1388,
–
1 1
305, 1222, 1205, 1159, 113, 1090, 1051, 1012, 967, 822, 791 cm ; H NMR (DMSO-d ) δ
6
.54-8.53 (dd, 1H), 8.52 (s, 1H), 8.08-8.06 (dd, 1H), 7.43-7.41 (m, 1H), 7.41 (d, 2H), 7.24 (d,
H), 6.47 (d, 1H), 6.28 (d, 1H), 6.19 (t, 1H, 2), 6.13 (d, 1H), 3.81 (s, 3H), 3.66-3.64 (m, 1H),
13
.12-3.09 (q, 2H), 1.69-1.49 (m, 4H), 1.22 (d, 3H); C NMR (DMSO-d ) δ 158.98, 155.00,
6
44.62, 144.22, 139.56, 134.76, 134.51, 129.55, 128.36, 124.29, 122.06, 119.04, 96.12, 91.62,
+
4.94, 47.02, 39.07, 33.46, 26.57, 20.21; MS/MS m/z 413.2 (M+1) ; Anal. Calcd. for
C H ClN O : C, 63.99; H, 6.10; N, 13.57. Found: C, 64.42; H, 6.49, N, 13.71.
22
25
4
2
2
.1.3.7. 3-(4-Bromophenyl)-1-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}urea (3g). Reaction
time: 0.5 h. From the reaction of 0.182 g 2g and crystallization from ether and petrolether
0
1
1
1
3
.181 g (66%) of 3g was obtained; mp 107–109 °C; IR (KBr): νmax 3324, 2969, 2936, 2861,
638, 1619, 1595, 1585, 1522, 1490, 1456, 1424, 1388, 1304, 1289, 1238, 1206, 1158, 1112,
–
1 1
050, 1006, 964, 820, 792 cm ; H NMR (DMSO-d ) δ 8.54-8.53 (m, 2H), 8.09-8.06 (dd,
6
H), 7.44-7.40 (m, 1H), 7.36 (s, 4H), 6.47 (d, 1H), 6.28 (d, 1H), 6.20 (t, 1H), 6.14 (d, 1H),
1
3
.81 (s, 3H), 3.67-3.61 (m, 1H), 3.12-3.07 (q, 2H), 1.69-1.51 (m, 4H), 1.22 (d, 3H); C NMR
DMSO-d ) δ 158.99, 154.97, 144.62, 144.24, 140.00, 134.80, 134.51, 131.27, 129.57,
(
6
1
22.10, 119.48, 112.11, 96.13, 91.61, 54.96, 47.03, 39.08, 33.46, 26.59, 20.23; MS/MS m/z
+
+
4
57.1 (M+1) , 458.2 (M+2) ; Anal. Calcd. for C H BrN O : C, 57.77; H, 5.51; N, 12.25.
22 25 4 2
Found: C, 58.90; H, 5.72, N, 11.91.
2
.1.3.8. 1-{4-[(6-Methoxyquinolin-8-yl)amino]pentyl}-3-[3-(trifluoromethyl)phenyl]urea
3h). Reaction time: 1 h. From the reaction of 0.184 g 2h and after purification by column
chromatography (mobile phase: dichloromethane/methanol 95:5) 0.244 g (91%) of 3h was
(
1
0

ACCEPTED MANUSCRIPT
obtained; oil; IR (KBr): νmax 3358, 3082, 2926, 2855, 1654, 1618, 1570, 1522, 1448, 1424,
–
1 1
1
388, 1337, 1255, 1221, 1165, 1122, 1068, 1051, 1031, 971, 893, 820, 790 cm ; H NMR
(
DMSO-d ) δ 8.79 (s, 1H), 8.54-8.53 (dd, 1H), 8.08-8.07 (dd, 1H), 7.96 (s, 1H), 7.50-7.41 (m,
6
3
H), 7.21 (d, 1H), 6.47 (d, 1H), 6.32 (t, 1H), 6.28 (d, 1H), 6.15 (d, 1H), 3.81 (s, 3H), 3.67-
1
3
3
.62 (m, 1H), 3.14-3.11 (q, 2H), 1.71-1.51 (m, 4H), 1.23 (d, 3H); C NMR (DMSO-d ) δ
6
1
58.99, 155.01, 144.63, 144.23, 141.43, 134.77, 134.52, 129.67, 129.53, 129.53-129.04,
1
26.96-121.55, 122.07, 121.05, 117.03, 113.46, 96.13, 91.62, 54.93, 47.03, 39.11, 33.45,
+
2
6.53, 20.21; MS/MS m/z 447.4 (M+1) ; Anal. Calcd. for C H F N O : C, 61.87; H, 5.64;
2
3
25
3
4
2
N, 12.55. Found: C, 61.53; H, 5.68, N, 12.19.
2
.1.3.9. 1-{4-[(6-Methoxyquinolin-8-yl)amino]pentyl}-3-[4-(trifluoromethyl)phenyl]urea (3i).
Reaction time: 2 h. From the reaction of 0.184 g 2i and crystallization from ether and
petrolether 0.230 g (86%) of 3i was obtained; mp 88–91 °C; IR (KBr): νmax 3322, 2968, 2937,
1
1
8
1
735, 1699, 1648, 1618, 1597, 1577, 1524, 1459, 1389, 1321, 1243, 1206, 1159, 1113, 1066,
–
1 1
050, 1014, 965, 823, 792 cm ; H NMR (DMSO-d ) δ 8.82 (s, 1H), 8.54-8.52 (dd, 1H),
6
.09-8.05 (dd, 1H), 7.59-7.52 (m, 4H), 7.44-7.40 (m, 1H), 6.47 (d, 1H), 6.32 (t, 1H), 6.28 (d,
H), 6.13 (d, 1H), 3.81 (s, 3H), 3.69-3.61 (m, 1H), 3.15-3.10 (q, 2H), 1.70-1.50 (m, 4H), 1.23
1
3
(
d, 3H); C NMR (DMSO-d ) δ 158.96, 154.76, 144.60, 144.26, 144.20, 134.74, 134.49,
6
1
2
1
29.53, 125.89-125.75, 122.04, 117.12, 96.11, 91.62, 54.92, 47.01, 39.06, 33.43, 26.46,
+
0.19; MS/MS m/z 447.5 (M+1) ; Anal. Calcd. for C H F N O : C, 61.87; H, 5.64; N,
23
25
3
4
2
2.55. Found: C, 61.56; H, 5.63, N, 12.26.
2
.1.3.10. 3-[3,5-Bis(trifluoromethyl)phenyl]-1-{4-[(6-methoxyquinolin-8-
yl)amino]pentyl}urea (3j). Reaction time: 0.5 h. From the reaction of 0.225 g 2j and after
purification by column chromatography (mobile phase: dichloromethane/methanol 95:5)
0
1
9
8
1
.200 g (65%) of 3j was obtained; mp 171–173.5 °C; IR (KBr): νmax 3348, 3230, 3118, 2936,
661, 1618, 1577, 1521, 1476, 1456, 1425, 1390, 1275, 1222, 1180, 1132, 1051, 1032, 1002,
–1 1
46, 888, 874, 824, 792 cm ; H NMR (DMSO-d ) δ 9.17 (s, 1H), 8.54-8.52 (dd, 1H), 8.07-
6
.05 (m, 3H), 7.52 (s, 1H), 7.44-7.40 (m, 1H), 6.51 (t, 1H), 6.47 (d, 1H), 6.28 (d, 1H), 6.14 (d,
1
3
H), 3.81 (s, 3H), 3.69-3.62 (m, 1H), 3.17-3.11 (q, 2H), 1.71-1.52 (m, 4H), 1.23 (d, 3H); C
NMR (DMSO-d ) δ 158.96, 154.72, 144.60, 144.18, 142.63, 134.81, 134.50, 131.16-129.86,
6
1
29.53, 128.75-117.91, 122.03, 117.15, 113.25, 96.13, 91.60, 54.89, 47.00, 39.22, 33.38,
1
1

ACCEPTED MANUSCRIPT
+
2
6.38, 20.18; MS/MS m/z 515.3 (M+1) ; Anal. Calcd. for C H F N O : C, 56.03; H, 4.70;
2
4
24
6
4
2
N, 10.89. Found: C, 55.88; H, 4.63, N, 10.70.
2
.1.4. 1-Primaquinecarbonyl-4-substituted bis-ureas (6a-j): general procedure
A solution of 0.190 g (0.6 mmol) primaquine semicarbazide 5, 0.061 g (0.6 mmol) TEA and
.6 mmol corresponding 1-(N-arylcarbamoyl)benzotriazole (2) in 10 ml dichloromethane was
0
stirred at room temperature for 1.5–24 h. The reaction mixture was evaporated under reduced
pressure. The residue was further purified without extraction (6a, 6b) or was dissolved in
ethyl acetate and extracted with 5% NaOH solution (3 × 10 ml) and water (3 × 15 ml), dried
over anhydrous sodium sulfate and evaporated (6c-j).
2
.1.4.1. 3-(3-Hydroxyphenyl)-1-[({4-[(6-methoxyquinolin-8-
yl)amino]pentyl}carbamoyl)amino]urea (6a). Reaction time: 4 h. From the reaction of 0.153
g 2a and after purification by column chromatography (mobile phase:
dichloromethane/methanol 9:1) and crystallization from ether and petrolether 0.166 g (70%)
of 6a was obtained; mp 196.5–198.5 °C; IR (KBr): νmax 3323, 3097, 2934, 1669, 1611, 1523,
–
1 1
1
455, 1386, 1311, 1206, 1160, 1053, 1032, 973, 823, 776 cm ; H NMR (DMSO-d ) δ 9.24
6
(
s, 1H), 8.54-8.53 (dd, 1H), 8.47 (s, 1H), 8.08-8.07 (dd, 1H), 7.73, 7.60 (2s, 2H), 7.43-7.41
m, 1H), 7.06 (s, 1H), 6.99 (t, 1H), 6.80 (d, 1H), 6.47 (m, 2H), 6.35-6.34 (d, 1H), 6.27-6.26
d, 1H), 6.11 (d, 1H), 3.83 (s, 3H), 3.65-3.61 (m, 1H), 3.08-3.02 (m, 2H), 1.64-1.48 (m, 4H),
(
(
1
3
1
1
3
6
.21 (d, 3H); C NMR (DMSO-d ) δ 158.99, 158.64, 157.57, 155.82, 144.63, 144.23, 140.71,
6
34.77, 134.51, 129.56, 129.16, 122.07, 109.16, 108.89, 105.47, 96.08, 91.61, 54.97, 47.04,
+
9.23, 33.41, 26.66, 20.20; MS/MS m/z 453.3 (M+1) ; Anal. Calcd. for C H N O : C,
2
3
28
6
4
1.05; H, 6.24; N, 18.57. Found: C, 60.70; H, 6.43, N, 18.41.
2
.1.4.2. 3-(4-Hydroxyphenyl)-1-[({4-[(6-methoxyquinolin-8-
yl)amino]pentyl}carbamoyl)amino]urea (6b). Reaction time: 4 h. From the reaction of 0.153
g 2b and after purification by column chromatography (mobile phase
dichloromethane/methanol 9:1) and crystallization from ether 0.166 g (70%) of 6b was
obtained; mp 119–121.5 °C; IR (KBr): νmax 3289, 3098, 2936, 1666, 1614, 1553, 1518, 1454,
–
1 1
1
386, 1330, 1223, 1166, 1051, 827, 790 cm ; H NMR (DMSO-d ) δ 8.91 (bs, 1H), 8.56-8.54
6
(dd, 1H), 8.34 (s, 1H), 8.12-8.09 (dd, 1H), 7.69, 7.59 (2s, 2H), 7.46-7.42 (m, 1H), 7.23 (d,
2
1
H), 6.64 (d, 2H), 6.49-6.48 (d+t, 2H), 6.28 (d, 1H), 6.18 (bs, 1H), 3.82 (s, 3H), 3.67-3.60 (m,
1
3
H), 3.08-3.02 (q, 2H), 1.65-1.46 (m, 4H), 1.20 (d, 3H); C NMR (DMSO-d ) δ 159.05,
6
1
2

ACCEPTED MANUSCRIPT
1
58.72, 156.28, 152.40, 144.43, 144.10, 135.15, 134.14, 131.15, 129.66, 122.11, 120.55,
+
1
14.96, 96.39, 91.75, 55.02, 47.13, 39.23, 33.36, 26.67, 20.17; MS/MS m/z 453.3 (M+1) ;
Anal. Calcd. for C H N O : C, 61.05; H, 6.24; N, 18.57. Found: C, 61.42; H, 6.21, N, 18.40.
2
3
28
6
4
2
.1.4.3. 3-(3-Fluorophenyl)-1-[({4-[(6-methoxyquinolin-8-
yl)amino]pentyl}carbamoyl)amino]urea (6c). Reaction time: 4 h. From the reaction of 0.154 g
2
2
1
8
7
1
c and crystallization from ether and ethanol 0.243 g (89%) of 6c was obtained; mp 207–
08.5 °C; IR (KBr): νmax 3307, 3220, 3106, 2930, 1668, 1615, 1568, 1551, 1521, 1495, 1454,
–
1 1
388, 1325, 1325, 1280, 1201, 1157, 1052, 966, 865, 816, 785 cm ; H NMR (DMSO-d ) δ
6
.86 (s, 1H), 8.53-8.52 (dd, 1H), 8.07-8.06 (dd, 1H), 7.93, 7.65 (2s, 2H), 7.50 (d, 1H), 7.42-
.40 (m, 1H), 7.26-7.23 (m, 2H), 6.73 (t, 1H), 6.50 (t, 1H), 6.46 (d, 1H), 6.26 (d, 1H), 6.10 (d,
1
3
H), 3.82 (s, 3H), 3.64-3.61 (m, 1H), 3.07-3.03 (m, 2H), 1.65-1.46 (m, 4H), 1.19 (d, 3H); C
NMR (DMSO-d ) δ 163.02, 158.98, 158.52, 155.87, 144.62, 144.22, 144.19, 141.71, 141.64,
6
1
34.76, 134.50, 130.01, 129.94, 129.55, 122.06, 114.19, 114.16, 108.02, 107.88, 104.99,
+
9
6.07, 91.59, 54.96, 47.03, 39.23, 33.39, 26.63, 20.19; MS/MS m/z 455.3 (M+1) ; Anal.
Calcd. for C H FN O : C, 60.78; H, 5.99; N, 18.49. Found: C, 60.51; H, 5.70, N, 18.41.
2
3
27
6
3
2
.1.4.4. 3-(4-Fluorophenyl)-1-[({4-[(6-methoxyquinolin-8-
yl)amino]pentyl}carbamoyl)amino]urea (6d). Reaction time: 4 h. From the reaction of 0.154
g 2d and after purification by column chromatography (mobile phase
dichloromethane/methanol 92:8) and crystallization from ether and ethanol 0.145 g (53%) of
6
d was obtained; mp 168–170 °C; IR (KBr): νmax 3296, 3228, 3095, 2934, 1686, 1652, 1616,
–
1 1
1
575, 1501, 1456, 1415, 1387, 1330, 1212, 1159, 1051, 1031, 831, 791 cm ; H NMR
(
DMSO-d ) δ 8.67 (s, 1H), 8.54-8.53 (dd, 1H), 8.08-8.06 (dd, 1H, 13), 7.84, 7.62 (2s, 2H),
6
7
.51-7.48 (m, 2H), 7.43-7.41 (m, 1H), 7.08-7.04 (m, 2H), 6.49-6.47 (d+t, 2H), 6.26 (d, 1H),
.11 (d, 1H), 3.82 (s, 3H), 3.64-3.60 (m, 1H), 3.08-3.03 (m, 2H), 1.66-1.47 (m, 4H), 1.20 (d,
6
1
3
3
H); C NMR (DMSO-d ) δ 158.98, 155.58, 158.03, 156.46, 156.12, 144.62, 144.21, 136.05,
6
1
34.77, 134.50, 129.55, 122.07, 120.26, 120.21, 114.99, 114.85, 96.07, 91.59, 54.96, 47.04,
+
3
9.23, 33.29, 26.64, 20.18; MS/MS m/z 455.3 (M+1) ; Anal. Calcd. C H FN O : C, 60.78;
2
3
27
6
3
H, 5.99; N, 18.49. Found: C, 60.30; H, 6.45, N, 18.58.
2
.1.4.5. 3-(3-Chlorophenyl)-1-[({4-[(6-methoxyquinolin-8-
yl)amino]pentyl}carbamoyl)amino]urea (6e). Reaction time: 2 h. From the reaction of 0.164 g
e and after purification by column chromatography (mobile phases
2
1
3

ACCEPTED MANUSCRIPT
dichloromethane/methanol 97:3, 95:5, 9:1) and crystallization from ether 0.173 g (61%) of 6e
was obtained; mp 152–154.5 °C; IR (KBr): νmax 3289, 3233, 3102, 2933, 1669, 1616, 1595,
–
1
1
541, 1519, 1483, 1455, 1423, 1387, 1319, 1220, 1201, 1158, 1051, 882, 821, 790, 778 cm ;
1
H NMR (DMSO-d ) δ 8.87 (s, 1H), 8.54-8.52 (dd, 1H), 8.09-8.06 (dd, 1H), 7.98, 7.74 (2s,
6
2
H), 7.67 (s, 1H), 7.44-7.37 (m, 2H), 7.24 (t, 1H), 6.97 (d, 1H), 6.52 (t, 1H), 6.47 (d, 1H),
6
.26 (d, 1H), 6.11 (d, 1H), 3.82 (s, 3H), 3.66-3.58 (m, 1H), 3.09-3.03 (m, 2H), 1.68-1.48 (m,
1
3
4
H), 1.20 (d, 3H); C NMR (DMSO-d ) δ 159.00, 158.54, 155.92, 144.64, 144.23, 141.38,
6
1
34.79, 134.52, 132.91, 130.11, 129.57, 122.09, 121.30, 117.90, 116.82, 96.09, 91.59, 54.98,
+
4
7.04, 39.23, 33.41, 26.67, 20.21; MS/MS m/z 471.3 (M+1) ; Anal. Calcd. for C H ClN O :
2
3
27
6
3
C, 58.66; H, 5.78; N, 17.84. Found: C, 58.40; H, 5.61, N, 17.52.
2
.1.4.6. 3-(4-Chlorophenyl)-1-[({4-[(6-methoxyquinolin-8-
yl)amino]pentyl}carbamoyl)amino]urea (6f). Reaction time: 4 h. From the reaction of 0.164 g
2
f and after purification by column chromatography (mobile phase dichloromethane/methanol
9
5:5) and crystallization from ether 0.241 g (85%) of 6f was obtained; mp 171–173 °C; IR
(KBr): νmax 3292, 3228, 3098, 2934, 1668, 1616, 1597, 1541, 1520, 1495, 1456, 1388, 1329
–
1 1
1
288, 1237, 1220, 1202, 1158, 1094, 1051, 1031, 1014, 824, 790 cm ; H NMR (DMSO-d )
6
δ 8.78 (s, 1H), 8.53-8.52 (dd, 1H), 8.08-8.06 (dd, 1H), 7.89, 7.63 (s, 1H), 7.52 (d, 2H), 7.43-
7
3
.41 (m, 1H), 7.26 (d, 2H), 6.49 (t, 1H), 6.47 (d, 1H), 6.26 (d, 1H), 6.10 (d, 1H), 3.82 (s, 3H),
1
3
.63-3.61 (m, 1H), 3.07-3.03 (m, 2H), 1.64-1.48 (m, 4H), 1.20 (d, 3H); C NMR (DMSO-d )
6
δ 158.98, 158.54, 155.93, 144.62, 144.21, 138.76, 134.76, 134.50, 129.55, 128.30, 125.21,
1
22.06, 120.02, 96.07, 91.60, 54.96, 47.03, 39.21, 33.39, 26.61, 20.19; MS/MS m/z 471.2
+
(
M+1) ; Anal. Calcd. for C H ClN O : C, 58.66; H, 5.78; N, 17.84. Found: C, 58.73; H,
23
27
6
3
5
.73, N, 17.66.
2
.1.4.7. 3-(4-Bromophenyl)-1-[({4-[(6-methoxyquinolin-8-
yl)amino]pentyl}carbamoyl)amino]urea (6g). Reaction time: 24 h. From the reaction of 0.190
g 2g and after purification by column chromatography (mobile phase
dichloromethane/methanol 95:5) and crystallization from ether 0.211 g (77%) of 6g was
obtained; mp 172.5–174 °C; IR (KBr): νmax 3289, 3236, 3101, 2932, 1672, 1595, 1526, 1457,
–
1 1
1
389, 1325, 1211, 1161, 1053, 822, 789 cm ; H NMR (DMSO-d ) δ 8.78 (s, 1H), 8.53-8.52
6
(dd, 1H), 8.08-8.06 (dd, 1H), 7.89, 7.63 (s, 1H), 7.47 (d, 2H), 7.43-7.41 (m, 1H), 7.38 (d, 2H),
6
.78 (t, 1H), 6.47 (d, 1H), 6.26 (d, 1H), 6.11 (d, 1H), 3.82 (s, 3H), 3.64-3.60 (m, 1H), 3.07-
1
4

ACCEPTED MANUSCRIPT
1
3
3
1
9
.03 (m, 2H), 1.66-1.47 (m, 4H), 1.20 (d, 3H); C NMR (DMSO-d ) δ 158.97, 158.52,
6
55.88, 144.61, 144.20, 139.18, 134.75, 134.49, 131.19, 129.54, 122.05, 120.43, 113.11,
+
6.06, 91.59, 54.95, 47.03, 39.20, 33.39, 26.63, 20.18; MS/MS m/z 516.2 (M+1) , 517.2
+
(
M+2) ; Anal. Calcd. for C H BrN O : C, 53.60; H, 5.28; N, 16.31. Found: C, 53.41; H,
23
27
6
3
5
.11, N, 15.99.
2
.1.4.8. 1-[({4-[(6-Methoxyquinolin-8-yl)amino]pentyl}carbamoyl)amino]-3-[3-
(trifluoromethyl)phenyl]urea (6h). Reaction time: 1.5 h. From the reaction of 0.184 g 2h and
after purification by column chromatography (mobile phase dichloromethane/methanol 95:5)
and crystallization from ether and petrolether 0.197 g (65%) of 6h was obtained; mp 120–122
°
C; IR (KBr): νmax 3286, 3102, 2936, 1672, 1618, 1574, 1548, 1521, 1451, 1424, 1388, 1335,
–1 1
1
262, 1221, 1203, 1167, 1125, 1072, 1052, 1031, 973, 930, 890, 821, 791 cm ; H NMR
(
DMSO-d ) δ 9.05 (s, 1H), 8.54-8.52 (dd, 1H), 8.09-8.02 (m, 3H), 7.74 (d, 1H), 7.67 (s, 1H),
6
7
3
.48-7.40 (m, 2H), 7.26 (d, 1H), 6.52 (t, 1H), 6.47 (d, 1H), 6.26 (d, 1H), 6.11 (d, 1H), 3.82 (s,
13
H), 3.67-3.58 (m, 1H), 3.10-3.03 (q, 2H), 1.66-1.48 (m, 4H), 1.20 (d, 3H); C NMR
(
DMSO-d ) δ 158.98, 158.49, 156.03, 144.63, 144.19, 140.68, 134.75, 134.50, 129.67-128.61,
6
1
3
5
29.53, 129.53, 129.48-118.57, 122.05, 117.89, 117.84, 114.60, 96.13, 91.59, 54.94, 47.04,
+
9.23, 33.40, 26.62, 20.17; MS/MS m/z 505.3 (M+1) ; Anal. Calcd. for C H F N O : C,
2
4
27
3
6
3
7.14; H, 5.39; N, 16.66. Found: C, 57.00; H, 5.49, N, 16.90.
2
.1.4.9. 1-[({4-[(6-Methoxyquinolin-8-yl)amino]pentyl}carbamoyl)amino]-3-[4-
(trifluoromethyl)phenyl]urea (6i). Reaction time: 3.5 h. From the reaction of 0.184 g 2i and
after purification by column chromatography (mobile phase dichloromethane/methanol 95:5)
and crystallization from ether 0.215 g (71%) of 6i was obtained; mp 158–160.5 °C; IR (KBr):
νmax 3284, 3102, 2936, 1672, 1618, 1604, 1542, 1521, 1458, 1418, 1388, 1326, 1245, 1221,
–
1 1
1
8
6
3
1
1
203, 1164, 1116, 1070, 1018, 971, 840, 822, 791 cm ; H NMR (DMSO-d ) δ 9.08 (s, 1H),
6
.54-8.52 (dd, 1H), 8.09-8.03 (m, 2H), 7.73-7.70 (m, 3H), 7.58 (d, 2H), 7.44-7.40 (m, 1H),
.54 (t, 1H), 6.47 (d, 1H), 6.27 (d, 1H), 6.12 (d, 1H), 3.82 (s, 3H), 3.65-3.61 (m, 1H), 3.09-
1
3
.04 (q, 2H), 1.67-1.48 (m, 4H), 1.20 (d, 3H); C NMR (DMSO-d ) δ 158.97, 158.53,
6
55.83, 144.64, 144.23, 143.57, 134.79, 134.52, 129.96-119.19, 129.58, 125.82, 125.77,
22.28-121.02, 122.09, 118.16, 96.09, 91.58, 54.97, 47.05, 39.23, 33.41, 26.67, 20.20;
+
MS/MS m/z 505.3 (M+1) ; Anal. Calcd. for C H F N O : C, 57.14; H, 5.39; N, 16.66.
2
4
27
3
6
3
Found: C, 56.93; H, 5.01, N, 16.80.
1
5

ACCEPTED MANUSCRIPT
2
.1.4.10. 3-[3,5-Bis(trifluoromethyl)phenyl]-1-[({4-[(6-methoxyquinolin-8-
yl)amino]pentyl}carbamoyl)amino]urea (6j). Reaction time: 2 h. From the reaction of 0.225 g
2
j and after purification by column chromatography (mobile phase dichloromethane/methanol
9
5:5) and crystallization from ether and petrolether 0.165 g (48%) of 6j was obtained; IR
(KBr): νmax 3316, 3108, 2962, 2935, 2870, 1684, 1652, 1616, 1576, 1520, 1475, 1456, 1424,
–
1 1
1
388, 1337, 1279, 1222, 1171, 1128, 1051, 1030, 947, 884, 821, 791 cm ; H NMR (DMSO-
d6) δ 9.53 (s, 1H), 8.53-8.52 (dd, 1H), 8.41, 7.74 (2s, 2H), 8.32 (s, 2H), 8.09-8.05 (dd, 1H),
7
3
.55 (s, 1H), 7.44-7.40 (m, 1H), 6.58 (t, 1H), 6.47 (d, 1H), 6.26 (d, 1H), 6.11 (d, 1H), 3.82 (s,
1
3
H), 3.66-3.58 (m, 1H), 3.10-3.03 (m, 2H), 1.66-1.48 (m, 4H), 1.20 (d, 3H); C NMR
(
DMSO-d ) δ 158.97, 158.38, 156.05, 144.62, 144.23, 142.09, 134.74, 134.49, 131.03-129.74,
6
1
29.54, 128.77-117.93, 122.03, 118.31, 114.08, 96.04, 91.58, 54.92, 47.05, 39.23, 33.40,
+
2
6.59, 20.16; MS/MS m/z 573.3 (M+1) ; Anal. Calcd. for C H F N O : C, 52.45; H, 4.58;
2
5
26
6
6
3
N, 14.68. Found: C, 52.68; H, 4.69, N, 14.81.
2
2
.2. Biological evaluation
.2.1. Materials and general methods
The cancer cells were seeded in 6-well or 96-well microtiter plates (Starlab GmbH,
Germany). The cell cycle distribution was analyzed by flow cytometry (FACSCalibur, Becton
Dickinson, USA) in the total population (20 000 cells). FITC Annexin V Apoptosis Detection
Kit I (Becton Dickinson, USA) was used for the assessment of cell death mode. Protein
concentrations were measured using BCA Protein Assay kit (Santa Cruz Biotechnology,
USA). Absorbance was measured using microplate reader GloMax-Multi (Promega, USA) at
5
60 nm. SDS-PAGE electrophoresis was performed on horizontal Amersham ECL Gel Box
System (GE Healthcare Life Sciences, UK) and protein transfer on XCell Blot module
Invitrogen, USA). Hyperfilm ECL film (GE Healthcare Life Sciences, United Kingdom) and
(
Kodak processing chemicals (Sigma-Aldrich, Germany) were used for film development.
All chemical and biochemical reagents were of analytical grade and purchased from
commercial sources. Eagle's Minimum Essential Medium (EMEM), penicillin, streptomycin,
DMSO, cisplatin, propidium iodide (PI), CelLytic M extraction reagent, protease inhibitor
cocktail, anti-GAPDH antibody, soybean lipoxygenase (LOX), linoleic acid sodium salt, 2,2-
diphenyl-1-picrylhydrazyl (DPPH), nordihydroguaiaretic acid (NDGA), 2,2’-azobis(2-
amidinopropane) dihydrochloride (AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-
1
6

ACCEPTED MANUSCRIPT
carboxylic acid (Trolox), (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(
MTT), tetracycline hydrochloride, 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) and 2H-
tetrazolium, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-hydroxide
XTT) were purchased from Sigma-Aldrich (Germany), fetal bovine serum (FBS), L-
(
glutamine and phosphate-buffered saline (PBS) from Gibco Laboratories (USA), sodium
pyruvate from Biological Industries (Israel), human insulin, BCA Protein Assay kit and anti-
PARP-1 antibody from Santa Cruz Biotechnology (USA), doxorubicin (DOX) from
Farmitalia Research Laboratories (Italy), RNase A from Invitrogen (USA), FITC Annexin V
Apoptosis Detection Kit I from Becton Dickinson (USA) and TBST buffer (20 mM Tris-HCl,
1
50 mM NaCl, 0.1% Tween 20 from GE Healthcare Life Sciences (UK), anti-rabbit HRP-
linked secondary antibody from Cell Signaling Technology (USA), Clarity Western ECL
Substrate from Bio-Rad (USA) and voriconazole from Pfizer (USA). All microbiological
media were purchased from Merck (Germany). Bacteria and one fungus from stock-cultures
of Collection of microorganism of Department of Microbiology, Faculty of Pharmacy and
Biochemistry University of Zagreb, Croatia (MFBF), ATCC cell culture collection (LGC
Standards Ltd, Germany) and National Collection of Type cultures (Public Health, England,
UK) were used in antimicrobial susceptibility assay.
2
.2.2. Antiproliferative activity assay
The antiproliferative activity experiments were carried out on murine leukemia (L1210) and 7
human cell lines derived from cancer types: acute lymphoblastic leukemia (Molt4/C8, CEM),
cervical carcinoma (HeLa), pancreatic carcinoma (Mia PaCa-2), lung carcinoma (NCI-H460),
colon carcinoma (SW 620) and breast carcinoma (MCF-7). Cytostatic activity against L1210,
Molt4/C8 and CEM cell lines was measured essentially as originally described for the mouse
leukemia cell lines [18]. The HeLa, Mia PaCa-2, NCI-H460, SW 620 and MCF-7 cells were
cultured as monolayers and maintained in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 ꢀg/ml
streptomycin in a humidified atmosphere with 5% CO at 37 °C. L1210, Molt4/C8 and CEM
2
were cultured in RPMI 1640 medium supplemented with penicillin (100 U/ml), streptomycin
(100 ꢀg/ml), L-glutamine, and 10% fetal bovine serum at 37 °C in a humidified atmosphere
containing 5% CO2.
The growth inhibition activity of NCI-H460, SW 620 and MCF-7 cell lines was assessed
according to the slightly modified procedure of the National Cancer Institute, Developmental
Therapeutics Program [19]. The cell lines were seeded into a series of standard 96-well
1
7

ACCEPTED MANUSCRIPT
4
4
microtiter plates on day 0, at 1 × 10 to 3 × 10 cells/ml, depending on the doubling times of
–8
–4
the specific cell line. Test compounds were then added in five, 10-fold dilutions (10 to 10
M) and incubated for 72 hours. Working dilutions were freshly prepared on the day of testing.
The solvent was also tested for eventual inhibitory activity by adjusting its concentration as in
the working concentrations. After 72 hours of incubation the cell growth rate was evaluated
by performing the MTT assay, as described previously. Each test point was performed in
quadruplicate in three individual experiments. The results were expressed as IC , which is the
5
0
concentration necessary for 50% of inhibition. The IC values for each compound were
5
0
calculated from dose-response curves using linear regression analysis by fitting the test
concentrations that gave percentage of growth (PG values) above and below the reference
value (i.e. 50%).
2
.2.3. Cell culture conditions for MCF-7 and drug treatments
MCF-7 cells were grown in EMEM, containing 1% penicillin/streptomycin, 10% FBS, 2 mM
L-glutamine, 1 mM sodium pyruvate, 0.01 mg/ml human insulin and incubated at 37 °C in an
atmosphere containing 5% CO . Cells were treated with 0.31 µM solution of compound 6d
2
(
IC ). DMSO (0.022%) was used for additional negative control. For positive controls cells
50
were treated with 13 µM cisplatin (IC ) and 1 µM doxorubicin (IC ) for 24, 48 and 72 h,
5
0
95
respectively.
2
.2.4. Analysis of cell cycle distribution by propidium iodide (PI) staining
MCF-7 were seeded in 6-well plates and treated as indicated. Following the incubation period
of 24, 48 or 72 h, respectively, cell medium was collected; cells were harvested, washed twice
with ice-cold PBS and fixed with ice-cold 70% ethanol in PBS for 24 h at –20 °C. After
centrifugation at 400 × g for 10 min, cells were washed with 500 µl PBS. RNA was digested
with RNase A (20 ꢀg/ml) for 30 min at 37 °C while DNA was stained with PI (5.5 µg/ml) for
1
0 min in dark at room temperature. Stained cells were analyzed by flow cytometry in the
total population (20 000 cells). Data analyses were performed using FCS 3 Express (De Novo
Software). Results are represented in a form of histogram as an average of three independent
experiments.
2
.2.5. Analysis of Annexin-V binding by flow cytometry
FITC Annexin V Apoptosis Detection Kit I was used for assessment percentage of viable,
early apoptotic, late apoptotic/necrotic and necrotic cell population. Following drug treatment
1
8

ACCEPTED MANUSCRIPT
cells were washed twice in ice-cold PBS and centrifuged for 5 minutes at 350 × g, 4 °C. Cells
6
were resuspended in 1 x Annexin V Binding Buffer to a final concentration of 1 × 10 cells
per 100 ꢀl and then stained with 5 ꢀl of FITC Annexin V and 5 ꢀl of PI. Samples were gently
vortexed and incubated for 15 min at room temperature in the dark. After incubation, 400 ꢀl
of Annexin V Binding Buffer was added to each tube and samples were analyzed by flow
cytometry in the total population (10 000 cells). Data analyses were performed using FCS 3
Express (De Novo Software). Excitation was at 488 nm with emission wavelength of 530 nm
for green (fluorescein) fluorescence and 580 nm for red (PI) fluorescence. Results are
represented in a form of histogram as an average of three independent experiments.
2
.2.6. Total protein extraction and quantification
Following drug treatments at indicated time points, cells were collected, washed twice in ice-
cold PBS and centrifuged for 5 min at 400 × g, 4 °C. The pellet was resuspended in 50 ꢀl of
chilled CelLytic M extraction reagent with protease inhibitor cocktail in ratio 1:100. Samples
were incubated on ice for 10 min and then centrifuged for 15 min at 15 000 × g, 4 °C.
Supernatant was collected and stored at –80 °C for further analysis. Protein concentrations
were measured using BCA Protein Assay kit following manufacturer’s manual. Absorbance
was measured using microplate reader at 560 nm.
2
.2.7. Analysis of protein expression by western blotting
Following protein isolation and determination of protein concentration, 5 µg of proteins were
separated by gel electrophoresis on 4-20% gels using horizontal Amersham ECL Gel Box
System. Proteins were electrotransferred to a nitrocellulose membrane, pore size 0.45 µm (GE
Healthcare Life Sciences) using XCell Blot module. Transfer was run overnight at constant
voltage of 10 V on ice. Membrane was blocked in 5% low-fat milk diluted in TBST buffer
(20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20) for 1 h on shaker at room temperature.
Membrane was washed in TBST buffer and incubated overnight at 4 °C with anti-PARP-1 or
anti-GAPDH antibodies, diluted in 0.5% low-fat milk in TBST buffer in ratio 1:1000 and
1
:5000, respectively. Membrane was washed again in TBST buffer and incubated for 1 h at
room temperature with anti-rabbit HRP-linked secondary antibody diluted to 1:2000 in 0.5%
low-fat milk in TBST buffer. After incubation, membrane was washed, over-layered with
Clarity Western ECL Substrate (Bio-Rad, USA) and left in dark for 3 min. Membrane was
covered with Hyperfilm ECL film and film was developed using Kodak processing chemicals
for autoradiography films.
1
9

ACCEPTED MANUSCRIPT
2
.2.8. Statistical analysis
A statistical analysis of the data was performed using a one-way ANOVA analysis of variance
followed by Newman-Keuls test and Duncan test using the STATISTICA 12 program
(StatSoft, Inc.). Statistical significance was set at p < 0.05.
2
.2.9. Interaction of the new derivatives with the stable radical DPPH
To a solution of DPPH in absolute ethanol the appropriate volume of the compound solution
0.1 mM final concentration) dissolved in DMSO was added. The absorbance was recorded at
17 nm after 20 and 60 min at room temperature. The experiments were repeated at least in
triplicate and the standard deviation of absorbance was less than 10% of the mean [14].
(
5
2
.2.10. LOX inhibition study in vitro
LOX inhibitory assay in vitro was accomplished as described previously [14]. The test
compounds (stock solutions 10 mM in DMSO) were incubated at room temperature with
–
4
sodium linoleate (0.1 mM) and 0.2 ml of LOX enzyme solution (1/9 × 10 m/V in saline).
The conversion of sodium linoleate to 13-hydroperoxylinoleic acid was measured at 234 nm
and compared with the reference inhibitor. Several concentrations were used for the IC50
determination. The assays were repeated at least in triplicate and the standard deviation of
absorbance was less than 10% of the mean.
2
.2.11. Inhibition of linoleic acid peroxidation
For initiating the free radical AAPH was used [14]. The final solution in the UV cuvette
consisted of ten microliters of the 16 mM linoleate sodium dispersion 0.93 ml of 0.05 M
phosphate buffer, pH 7.4, thermostated at 37 °C. AAPH solution (50 ꢀl of 40 mM) was added
as a free radical initiator at 37 °C under air and 10 ꢀl of the tested compounds. The oxidation
of linoleic acid sodium salt was monitored at 234 nm. The assays were repeated at least in
triplicate and the standard deviation of absorbance was less than 10% of the mean.
2
.2.12. The in vitro antimicrobial susceptibility assay
The in vitro antibacterial activity of compounds 3a-j and 6a-j were carried out against a panel
of five Gram-positive (Staphylococcus aureus, Enterococcus faecalis, Bacillus pumilus,
Bacillus cereus, Bacillus subtilis) and two Gram-negative bacteria (Pseudomonas aeruginosa,
Echerichia coli). Three methicillin-susceptible S. aureus (MSSA) and two methicillin-
2
0

ACCEPTED MANUSCRIPT
resistant S. aureus (MRSA) and two strains of E. faecalis were used: vancomycin-susceptible
(VSE) and vancomycin-resistant (VRE). Antifungal susceptibility assay for Candida albicans
was performed as well. Serial microdilution broth assay based on the EUCAST reference
documents for aerobic bacteria was used to determine minimal inhibitory concentrations
(MIC) [20]. Briefly, cell suspension of bacteria was prepared from stock cultures kept at –30
°
°
C in 25% (V/V) glycerol-nutrient broth and maintained in trypticase soy broth for 18 h at 37
C. Inoculum was prepared using PBS pH 7.4 and adjusted using nephelometer (ATB 1550,
BioMérieux, France) to 0.5 MacFarland units and diluted 1:10 (V/V) with Mueller-Hinton
broth. Stock solutions of tested compounds were prepared in DMSO at concentration 10
mg/ml before testing. According to EUCAST recommendations, microdilution was performed
in Mueller-Hinton broth with serial dilution in microtiter flat-bottom 96-well plates from 1000
µg/ml to 0.78125 µg/ml. After inoculation and incubation during 18 h aerobically at 35 °C in
dark, MICs were determined by addition of 0.5 mg/ml TTC (redox-indicator). After 3 h of
incubation at 35 °C in dark, 0.04 M HCl in isopropanol was added to all wells and absorbance
was recorded at 490 nm. For determination of MIC for fungal species tested microdilution
was performed in RPMI 1640 broth with glutamine supplemented with 2% glucose and 10 mg
–1
–1
ml XTT was added in combination with menadione (1 mg ml in acetone) (7:1, V/V) [21].
MIC values for yeast species were determined at 540 nm. Tetracycline hydrochloride and
voriconazole dissolved in PBS pH 7.4 were used as quality control of susceptibility of strains
and MIC determination. MICs were recorded as the lowest concentrations resulting in 50%
growth inhibition (IC ) in comparison to control (untreated cells). All tests were performed in
5
0
duplicate and expressed as MICaverrage(EC50).
2
.2.13. Hemolytic activity on human red blood cells (hRBCs) ex situ
Fresh venous human blood (AB+ type) donor (45 years old healthy male) was centrifuged at
500 rpm for 5 min. The supernatant and buffy coats were removed, and cells were washed 3
2
2
+-
2+
times in PBS pH 7.4. After washing, hRBCs were resuspended in Ca and Mg -free
phosphate-buffered saline (PBS pH 7.4) at concentration 4% (v/v). Hemolytic activity of
compounds 3a, 3g, 3i and 3j on hRBCs ex situ was determined in the concentration ranged
from 100 to 0.156 µg/ml [22]. Intact 4% hRBCs were used as a negative control and 4%
hRBCs plus 0.1% (v/v) Triton X-100 was used as a positive control. After incubation (1 h at
3
7 °C in dark) the solutions were centrifugated at 2500 rpm for 5 minutes. Absorbance of the
supernatant was determined at 540 nm. Hemolysis was calculated according to the equation:
2
1

ACCEPTED MANUSCRIPT
Hemolysis (%) = 100ꢁ[(A ꢁ–ꢁA )/(A ꢁ–ꢁA )ꢁ× 100)], where A , A and A were the absorbances of
p
s
p
n
p
s
n
the positive control, test sample and negative control, respectively. The results are given as
the ratio between the hemolysis percentage of the test condition and control. The experiments
were performed three independed times and presented as mean ± SD of a change in
absorbance vs. concentration (x–y ) and hemolysis percentage according the equation (x–y ).
1
2
2
.2.14. Cytotoxic effect of selected compounds on human embryonic kidney (HEK) cells
The cytotoxic effect of compounds 3a, 3g, 3i and 3j on HEK cells was determined using MTT
3
assay. Briefly, 5 × 10 cells per well were cultured in 96-well plates and the following day
incubated with specific compound for 24 h. Used doses ranged from 200 to 1.5625 µg/ml,
depending of antimicrobial activity of each compound. The following day, 40 ꢀl of 0.5ꢁmg/ml
MTT solution in DMEM containing 10% FBS was added to each well and incubated at 37ꢁ°C
under 5% CO . After 4ꢁh, formazan was dissolved by adding 170ꢁꢀl of DMSO. The
2
absorbance was measured at 570ꢁnm using microplate reader. All analyzes were performed in
triplicates and results are presented as the percentage of untreated control.
3
. Results and discussion
3
.1. Chemistry
Novel compounds 3a-j and 6a-j described in this paper are derived from primaquine. They
differ in central functional group and/or in substituents in aromatic region. Compounds 3 are
ureas and compounds of general formula 6 are bis-ureas. Products 3a,b and 6a,b possess
hydroxy substituent (electron donating group), while compounds 3c-g and 6c-g bear halogen
atoms (electron withdrawing group) either in meta or para position. Finally, compounds 3h-j
and 6h-j have one or two trifluoromethyl groups. The strategy for the preparation of the title
compounds is analogue to the previously published one [14] and is outlined in Scheme 1.
Scheme 1.
1
-Benzotriazole carboxylic acid chloride BtcCl (1) [23,24] was the common starting material
for synthesis of N-1-benzotriazole carboxylic acid amides 2a-j and (N-(4-((6-
methoxyquinolin-8-yl)amino)pentyl)-1H-benzo[d][1,2,3]triazole-1-carboxamide) (4) [25]
2
2

ACCEPTED MANUSCRIPT
Compounds 2a-j were prepared from 1 and corresponding hydroxy- or halogenaniline and
product 4 from 1 and primaquine base. The following aromatic amines were used: 3-
aminophenol, 4-aminophenol, 3-fluoroaniline, 4-fluoroaniline, 3-chloroaniline, 4-
chloroaniline, 4-bromoaniline, 3-trifluoromethylaniline, 4-trifluoromethylaniline and 3,5-
bis(trifluoromethyl)aniline. Compounds 2a-j (active ureas) readily reacted with primaquine
(nucleophile) affording urea derivatives 3a-j. On the other hand, compound 4 and hydrazine
hydrate gave semicarbazide 5, which in reaction with 2a-j gave the target bis-ureas 6a-j.
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All new compounds are fully characterized by the usual spectroscopic methods (IR, H-, C-
NMR and MS) and elemental analyses. Spectral data are consistent with the proposed
structures and are given in short in the Experimental section and in detail in Suporting
information.
Majority of the urea and bis-urea primaquine derivatives are fully in agreement with the
Lipinski’s and Gelovani’s rules for prospective small molecular drugs (MW ≤ 500, log P ≤ 5,
number of H-bond donors ≤ 5, number of H-bond acceptors ≤ 10, polar surface area (PSA) <
2
3
1
40 Å , molar refractivity (MR) within the range of 40 and 130 cm /mol, the number of atoms
0-70) [26]. Few analogues showed minimal aberrations of the rules. The parameters are
2
calculated with Chemicalize.org program and presented in Table 1.
Table 1 ref. [27]
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3
.2. Biological evaluation
.2.1. Cytostatic activity
The results of antiproliferative screening in vitro of urea 3a-j and bis-urea derivatives 6a-j on
murine lymphocytic leukemia (L1210) and seven human tumour cell lines (Molt4/C8, CEM,
HeLa, Mia Paca-2, NCI-H460, SW620, MCF-7) are given in Table 2. Urea derivatives 3a-g
bearing hydroxy, fluoro, chloro or bromo substituent either in meta or para position showed
moderate antiproliferative effects against all the tested cell lines, but stronger activity against
MCF-7. Regioisomers with the same substituent either in meta or para position 3a/3b, 3c/3d
and 3e/3f showed comparable results. Trifluoromethyl derivatives 3h and 3i were more active
than 3a-g and showed antiproliferative effects against all the tested cell lines in low
micromolar range. Additional trifluoromethyl group enhanced antiproliferative activity: 3-
[
3,5-bis(trifluoromethyl)phenyl]-1-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}urea (3j) was
the most active urea.
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Table 2
In bis-urea series hydroxy 6a,b and fluoro derivatives 6c,d, especially p-fluoro derivative 6d,
showed extreme selectivity and chloro or bromo derivatives 6e-g high selectivity against
MCF-7 cells (IC for MCF-7 ranged from 0.1 to 2.6 µM; practically no activity or very weak
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0
activity towards the other cancer cell lines was observed). Comparison of regioisomer pairs in
bis-urea series showed that meta isomers showed slightly higher activity than para.
Trifluoromethyl derivatives 6h-j exerted high activity towards all tested cell lines and again
the highest activity towards MCF-7. Derivative 6h with meta substituent was the most active
compound.
3
.2.2. Induction of cell cycle arrest by compound 6d
Since compound 6d showed extreme selectivity against MCF-7 cells and practically no
activity or very weak activity towards the other cancer cell lines we have investigated the
mechanisms through which 6d mediates pharmacological actions on MCF-7 human breast
cancer cells. To examine whether 6d-induced growth inhibition was associated with cell cycle
regulation, the cell cycle distribution was analyzed by flow cytometry. Analyzed phases were:
Gap1 (G ), DNA syntheses (S) and Gap2/Mitosis (G /M). MCF-7 cells were incubated with
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d (IC 0.31 ꢀM) and DMSO for 24, 48 and 72 h, respectively. Cisplatin, one of the most
50
commonly used anticancer drug in oncology, was used as a positive control (IC 13 µM)
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[
28].
Figure 1
The effect of 6d on G , S and G /M phase was shown in Fig. 1. The G /M population of cells
1
2
2
treated with 6d for 24 h increased in comparison to control, with a corresponding reduction of
G phase population (Fig. 2). It is important to stress that IC value of cisplatin [29] was 42-
1
50
fold higher than IC value of 6d, indicating the high potency of 6d.
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0
Figure 2
3
.2.3. Apoptosis-inducing effect of compound 6d
To investigate the ability of compound 6d to promote apoptosis and/or necrosis in MCF-7
cells, we preformed FITC Anexin V/PI staining after 24, 48 and 72 hours of treatment,
respectively (Fig. 3).
Figure 3
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As positive control we used doxorubicin (IC 1 µM) [30], currently considered to be one of
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5
the most effective agent in the treatment of breast cancer. Compound 6d promotes the
induction of cell death, as it was observed by increase in number of late apoptotic/necrotic
cells as well as only necrotic cells when applied for 72 h (Fig. 4). Therefore, we wanted to
further elucidate the specific mode of cell death.
Figure 4
Full-length poly (ADP-ribose) polymerase 1 (PARP-1) is an 116 kDa protein involved in the
repair of DNA, differentiation and chromatin structure formation. During apoptosis this
protein is cleaved by caspase-3 and caspase-7 into a stable 89 kDa fragment containing the
active site and a 25 kDa fragment [31]. Therefore, we investigated possible 6d-induced
inhibition of PARP-1 by immunoblotting of PARP-1 (116 kDa) and its 89 kDa fragment. The
results showed that 6d did not trigger apoptosis in MCF-7 cells 24 and 48 h after treatment
because expression levels of cleaved PARP-1 (C-PARP-1) were not detected (Fig. 5). These
results were in line with results obtained by FITC Anexin V/PI staining at same time points
(Fig. 3A and 3B). The expression levels of 89 kDa fragment increased at incubation period of
7
2 h, when compared to negative controls (Fig. 5). The results indicate that 6d promotes
apoptotic death of MCF-7 cells after incubation period of 72 h, in addition to cell cycle arrest.
Figure 5
Induction of apoptosis is precisely mediated by caspases cascade via both intrinsic and
extrinsic pathway. Caspases play essential role in apoptosis which mainly included two main
groups of initiators and executioners. Caspases-8 and -9 are known as initiator caspases
through extrinsic and intrinsic pathway, respectively, and facilitate the activation of
executioners, such as caspases-3/7 [32]. MCF-7 cells have functional deletion of caspase-3
gene [33] providing a slightly higher threshold for the induction of apoptosis and probably
shifting a part of apoptotic death mode into necrotic. Potential solution would be treatment
with IC concentration as in case of doxorubicin. Since cancer cells, such as MCF-7, have a
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5
defect in apoptotic pathway, cell death needs to be triggered by alternative mechanisms [34].
3
.2.4. Antioxidative activity
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
inflammatory tissue damage. Conversely, substances with potent antiinflammatory and/or
antioxidative activities are anticipated to exert chemopreventive effects on carcinogenesis,
particularly in the stage of promotion and act as antitumor promoters. Taking under
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consideration the above mentioned facts, antioxidative potential of new urea and bis-urea
primaquine derivatives 3a-3j and 6a-6j was studied and compared to the well-known
antioxidant agents such as NDGA and Trolox. Two different antioxidant assays were used: (i)
interaction with the stable free DPPH radical and (ii) interaction with the water-soluble azo
compound AAPH, used as a source of peroxyl radicals [14]. DPPH interaction of the tested
compounds was examined at 100 ꢀM concentration after 20 and 60 min. Among the urea
derivatives compound 3b with p-hydroxy substituent showed the best DPPH interaction value
(
95.5%), close to that of the reference compound NDGA (83%) at the same concentration,
followed by 3j (with two trifluoromethyl substituents), 3a (m-hydroxy) and 3g (p-bromo)
Table 3). The nature of the halogen atom in para position affected the antioxidant result.
(
Thus, derivative 3g (p-bromo) was found more potent than 3d (p-fluoro) and 3f (p-chloro).
Derivatives 3c and 3d with fluoro atoms either in meta or para position and m-chloro
derivative 3e exerted activities only after 60 min. Compound 3f with p-chloro substituent
gave a slightly better result. The attachment of CF group (3h and 3i) gave the products with
3
low activity, but introduction of the second trifluoromethyl group enhanced the antioxidant
ability. In bis-urea series only compound 6d with p-fluoro substituent showed a very weak
interaction with DPPH, whereas all others presented high antioxidant activity (75.7-99.3%).
For both groups of compounds no correlation between lipophilicity and activity was found.
Table 3
In our studies, AAPH was used as a free radical initiator to follow oxidative changes of
linoleic acid to conjugated diene hydroperoxide. The results showed that all the analogues
within the group 3 are significant anti-lipid peroxidation agents (54-90.2%). Phenolic
derivative 3b with the lowest log P (3.07) presented the lowest activity (54%), whereas the
most lipophilic compound 3j (log P = 5.13) with two CF groups exhibited the highest
3
activity (90.2%). Chloro and bromo derivatives 3e, 3f, 3g with similar lipophilicity were
almost equivalent in activity, as well as 3c and 3h. Bis-ureas generally presented lower
inhibition of lipid peroxidation (LP) (52.2-75.3%) and two compounds (6d and 6g) showed
no effect under the reported conditions. A correlation between lipophilicity and LP inhibition
was found only in urea series. In bis-urea series, the most active derivatives (6i, 6e and 6b)
were not the most lipophilic ones. Our results also indicated that LP inhibition was not always
accompanied by DPPH radical scavenging activity. It is worth to note, that the most active LP
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inhibitor 3j (derivative with two CF groups) was the strongest antiproliferative agent as well,
3
but such a strong correlation for other compounds was not found.
LOX inhibitors have attracted attention initially as potential agents for the treatment of
inflammatory and allergic diseases, certain types of cancer, and cardiovascular diseases [35].
In order to diminish the pro-cancerous mechanisms a key treatment strategy is to reduce the
free radical load. Antioxidant activity by scavenging of reactive oxygen species is important
in preventing potential damage of cellular compounds such as DNA, proteins and lipids.
Moreover, it has been found that LOX inhibitors may have chemopreventive activity in lung
carcinogenesis [36]. Reports published over the past three decades support a growth-
regulatory role of arachidonic acid metabolites in the aetiology of mammary carcinogenesis
[
37]. Studies have demonstrated that levels of several eicosanoids are increased in breast
cancer in comparison to benign breast tumours [38]. In this context, we evaluated the newly
prepared primaquine derivatives for their ability to inhibit soybean LOX. From the tested
compounds, ureas 3a, 3b and 3h and bis-ureas 6c and 6i exhibited limited activity in LOX
inhibition assay (48.3-67.4%), significantly lower than the reference NDGA (83%). The
compounds with high log P values were not the most active, so the idea that lipophilicity was
an important physicochemical property for LOX inhibition was not supported.
3
.2.4. Antimicrobial activity
The in vitro antimicrobial susceptibility assay was carried out against a panel of 15 bacterial
strains and the most common cause of fungal infections C. albicans and compared to parent
compound primaquine and tetracycline hydrochloride (TC) or voriconazole (VOR) as
standard antimicrobial drugs. Out of twenty newly synthesized compounds, three compounds
from series 3, e.g. 3a, 3g and 3i, showed weak antibacterial activity against selected bacterial
–
1
–1
strains, with MIC values lower than primaquine (12.5-50 ꢀg ml vs. 70 ꢀg ml ). However,
one compound, bis(trifluoromethyl)phenyl]-1-{4-[(6-methoxyquinolin-8-
yl)amino]pentyl}urea (3j), exhibited very strong antibacterial activity towards five bacterial
–
1
strains, with MIC values ranged from 1.6 to 12.5 ꢀg ml . It was noticed that S. aureus ATCC
538 was the most sensitive Gram-positive and P. aeruginosa ATCC 27853 the most
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sensitive Gram-negative bacteria. The results of antimicrobial screening are presented in
Table 4 (only data for compounds that showed antibacterial/antifungal activity are given).
Table 4
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In order to better estimate potential of compounds 3a, 3g, 3i and 3j as antimicrobial agents
two tests were performed: haemolytic activity test on fresh hRBc ex situ and cytotoxicity test
on human embryonic kidney (HEK) 293 cell line measured by MTT assay. All tested
compounds exhibited low haemolytic activity after 1 h of aerobic incubation (Fig. 6).
Haemolysis was a dose-depended process. The highest haemolysis was observed for
compound 3i (7.85% at 100 µg/ml concentration). Other compounds showed very low
haemolysis potential, less than 1% at the highest concentration.
Figure 6
The cytotoxic effect of selected compounds from series 3 on HEK cells showed low toxicity
(3a and 3i) or no toxicity (3g) in the range of MIC values, except compound 3j. Compound 3j
showed strong cytotoxicity in the range of MIC values, starting from concentration 3.125 ꢀg
–1
ml . Our data support the potential of primaqine-urea scaffold for development of lead
compounds with antimicrobial activities.
Figure 7
4
. Conclusions
Twenty novel compounds with the primaquine scaffold/urea or bis-urea motif/hydroxyl or
halogen substituted phenyl ring have been prepared and tested for biological activity. Most of
the compounds showed significant cytostatic activities in low micromolar concentrations
towards all the tested cancer cell lines or high selectivity towards the human breast carcinoma
MCF-7 cell line. Our results strongly support previous findings of MCF-7 susceptibility
towards primaquine derivatives. Bis(trifluoromethyl)phenyl urea (3j) and
(trifluoromethyl)phenyl bis-urea (6h) were the most active derivatives in antiproliferative
screening in vitro. 3-(4-Fluorophenyl)-1-[({4-[(6-methoxyquinolin-8-
yl)amino]pentyl}carbamoyl)amino]urea (6d) showed extreme selectivity: high activity against
MCF-7 cell lines (IC 0.31 µM) and practically no activity against other tested cancer cell
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0
lines. Our studies demonstrated that 6d induced growth inhibition in MCF-7 cell line through
at least two different mechanisms: by induction of cell cycle arrest in G1 phase and inhibition
of cell cycle progression through transient M phase accumulation and subsequent G2 arrest,
and by inducing apoptosis. In conclusion, due to its high activity and selectivity compound 6d
may be considered as a potential scaffold for development of more effective and safer drugs
for breast carcinoma treatment.
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