Novel semicarbazides and ureas of primaquine with bulky aryl or hydroxyalkyl substituents: Synthesis, cytostatic and antioxidative activity

Article abstract of DOI:10.1016/j.ejmech.2014.09.013

Novel primaquine semicarbazides 7a-l and ureas 9a-g with modified benzhydryl, trityl, phenyl or hydroxyalkyl substituents were prepared and evaluated for cytostatic and antioxidative activities. Two synthetic approaches for preparation of the title semica

Full text of DOI:10.1016/j.ejmech.2014.09.013

European Journal of Medicinal Chemistry 86 (2014) 502e514
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel semicarbazides and ureas of primaquine with bulky aryl or
hydroxyalkyl substituents: Synthesis, cytostatic and antioxidative
activity
b
b
c
ꢀ a
ꢀ a
ꢀ a
ꢀ a
K. Pavic , I. Perkovic , M. Cindric , M. Pranjic , I. Martin-Kleiner , M. Kralj , D. Schols ,
,
*
D. Hadjipavlou-Litina d, A.-M. Katsori d, B. Zorc a
a
ꢁ ꢀ
University of Zagreb, Faculty of Pharmacy and Biochemistry, A. Kovacica 1, HR-10 000 Zagreb, Croatia
Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka cesta 54, HR-10 000 Zagreb, Croatia
b
ꢁ
ꢀ
ꢁ
c Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
d School of Pharmacy, Faculty of Health Sciences, Aristotles University of Thessaloniki, Thessaloniki 54 124, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel primaquine semicarbazides 7ael and ureas 9aeg with modified benzhydryl, trityl, phenyl or
hydroxyalkyl substituents were prepared and evaluated for cytostatic and antioxidative activities. Two
synthetic approaches for preparation of the title semicarbazides were applied, both having certain ad-
vantages. In the first approach, the products grew from the semicarbazide side and the primaquine
residue entered the molecule the last. In the second approach, semicarbazide grew from the primaquine
side. This method was more convenient for synthesis of a series of semicarbazides: various products
could be obtained from the same precursor N-(4-((6-methoxyquinolin-8-yl)amino)pentyl)hydrazine-
carbox-amide (10). Primaquine ureas 9aef were prepared from primaquine benzotriazolide 8 and cor-
responding amines and urea 9g directly from primaquine and 4-chloro-3-(fluoromethyl)phenyl
isocyanate.
Received 15 July 2014
Received in revised form
1 September 2014
Accepted 4 September 2014
Available online 6 September 2014
Keywords:
Primaquine
Urea
Semicarbazide
Cytostatic activity
Antioxidative activity
All primaquine semicarbazide derivatives showed either prominent cytostatic activity towards all the
tested cell lines (benzhydryl or trityl derivatives 7aee) or high selectivity towards MCF-7 cells
(hydroxyalkyl derivatives 7hel), with IC50 values in the low micromolar range. The highest selectivity
exerted symmetric bisprimaquine derivative 7f, with an IC50 0.2 mM against MCF-7 cells and practically
no activity against other seven tested cancer cell lines. Urea derivatives 9aef were generally less active
than their semicarbazide analogues, but still selective towards MCF-7 cells. Urea 9g with the similar
structure to cytostatic drug sorafenib, was the most active urea derivative.
Semicarbazides 7g and 10 showed the best antioxidative activity as measured by DPPH (64% at 20 min
and 90% at 60 min), while urea derivatives 9aeg, especially 9d, and semicarbazides 7aeg with lipophilic
substituents exerted better LP antioxidant activity. Both semicarbazides and ureas with methoxy or
chloro benzhydryl substituents and high Clog P values showed significant LOX inhibition.
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
Abbreviations: AAPH, 2,20-azobis(2-amidinopropane) hydrochloride; BtcCl, 1-
benzotriazole carboxylic acid chloride; BtH, 1H-benzo[d][1,2,3]triazole; CEM,
acute lymphoblastic leukemia; CYP-1, cytochrome P450; DMEM, Dulbecco's
modified Eagle's medium; DPPH, 1,1-diphenyl-2-picrylhydrazyl; FBS, fetal bovine
serum; H 460, lung carcinoma; HeLa, cervical carcinoma; IC50, the concentration
that causes 50% growth inhibition; L1210, murine lymphocytic leukemia; LOX,
soybean lipoxygenase; LP, lipid peroxidation; MCF-7, breast carcinoma; MiaPaca-2,
pancreatic carcinoma; Molt4/C8, acute lymphoblastic leukemia; MTT, (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NDGA, nordihydroguaia-
retic acid; PG, percentage of growth; PQ, primaquine; SW 620, colon cancer cells;
TEA, triethylamine.
Primaquine is the only available drug active against gametocytes
and exoerythrocytic forms of all species of parasite causing human
malaria, including multi-resistant Plasmodium falciparum strains
[1]. It is also an interesting molecule for derivatization in the search
for potential biologically active agents. On the other hand, semi-
carbazides are important structural features of numerous mole-
cules of industrial, pharmaceutical or biological interest [2]. Many
semicarbazides exerted cytostatic [3], antitubercular [4], antioxi-
dant [5], antiviral [6] and anticonvulsant activity [7] or have po-
tentials as bradykinin B1 receptor antagonists [8], nitric oxide
* Corresponding author.
E-mail address: bzorc@pharma.hr (B. Zorc).
http://dx.doi.org/10.1016/j.ejmech.2014.09.013
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

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K. Pavic et al. / European Journal of Medicinal Chemistry 86 (2014) 502e514
503
generators [9] or kinase inhibitors [10]. Semicarbazide-based de-
rivatives of peptides (azapeptides) are known as inhibitors of hu-
man dipeptidyl peptidase I, the enzyme that plays an important
role in the immune system [11]. In our previous paper synthesis
and biological evaluation of 1,4-substituted semicarbazides with
primaquine moiety bridged by carbonyl group at position 1 and
cycloalkyl, aryl, benzyloxy or hydroxy substituent at position 4 was
reported [12]. These compounds showed high selectivity towards
MCF-7 cells (breast carcinoma). The most active was benzyl de-
according to our procedures published earlier [15e17]. 4-
Methoxybenzophenone hydrazone and
4,40-dimethox-
ybenzophenone hydrazone were prepared from corresponding
ketones (4-methoxybenzophenone and 4,4-
dimethoxybenzophenone, respectively) and hydrazine hydrate
[18]. 1,l-Diphenyl-2-picrylhydrazyl (DPPH),
2,20-azobis(2-
amidinopropane) hydrochloride (AAPH), Trolox and nordihy-
droguaiaretic acid (NDGA) were purchased from Aldrich Chemical
Co. Soybean lipoxygenase (LOX), linoleic acid sodium salt and (3-
rivative with IC50 1.0 0.2
m
M, while benzhydryl derivative showed
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
(MTT) were obtained from Sigma Chemical Co.
bromide
significant cytostatic activities in low micromolar concentrations
towards all the tested cell lines. Similar antiproliferative activity
and high inhibition of soybean lipoxygenase and lipid peroxidation
showed also NSAID semicarbazides bearing benzhydryl moieties
[13]. On the other hand, urea with 5-hydroxypentyl substituent on
one nitrogen and primaquine residue on the other nitrogen atom
showed extreme selectivity towards SW 620 colon cancer cells (IC50
2.1.2. 1-(N-arylcarbamoylbenzotriazoles (1-benzotriazole
amides)(2aed): general procedure
The title compounds were synthesized from chloride 1 and
corresponding amine or hydrazone according to slightly modified
published procedure [19]. Solution of 1.515 g (15.0 mmol) TEA and
5.0 mmol of amine or hydrazone in 5 ml toluene was added drop-
wise to a solution of 0.908 g (5.0 mmol) BtcCl (1) in dry toluene
(10 ml). Reaction mixture was stirred 1 h at room temperature,
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.
0.2 mM) [14]. Based on these results we found it worth to prepare
primaquine semicarbazides and ureas with bulky aryl (modified
benzhydryl, trityl or phenyl) and hydroxyalkyl substituents to
consider the role of these structural modifications on the physi-
cochemical properties and their influence on the biological
response. In this paper we report their synthesis and biological
evaluation.
2.1.2.1. N-benzhydryl-N-methyl-1H-benzo[d][1,2,3]triazole-1-
carboxamide (2a). From the reaction of 0.986 g (5.0 mmol) N-
methyl-1,1-diphenylmethanamine and after recrystallization from
ether/petroleum ether 1.352 g (79%) of 2a was obtained; mp
93e96 ꢀC; IR (KBr): nmax 3050, 1696, 1600, 1488, 1450, 1400, 1360,
2. Materials and methods
2.1. Synthesis
2.1.1. Materials and general methods
1292, 1230, 1074, 1038, 746, 718 cmꢂ1; 1H NMR (DMSO-d6)
d 8.19 (d,
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 spectrometer (PerkineElmer,
UK). 1H and 13C NMR spectra were recorded on a Varian Gemini 300
J ¼ 8.19 Hz, 1H), 7.94 (d, J ¼ 8.37 Hz, 1H), 7.71 (t, J1 ¼ J2 ¼ 7.29 Hz,
1H), 7.55 (t, J1 ¼ J2 ¼ 7.59 Hz, 1H), 7.46e7.32 (m, 10H), 6.87 (s, 1H),
3.04 (s, 3H); 13C NMR (DMSO-d6)
d 150.71, 144.62, 137.98, 132.65,
129.50, 128.65, 128.54, 127.81, 125.44, 119.59, 113.37, 64.40, 30.19;
MS/MS m/z 365.2 (M þ 23)þ, 707.4 (2M þ 23)þ; Anal. C21H18N4O (C,
H, N).
1
spectrometer (Varian, USA), operating at 300 and 75.5 MHz for H
and 13C nuclei, respectively. Samples were measured in DMSO-d6
solutions at 20 ꢀC in 5-mm NMR tubes. Chemical shifts (
d) in ppm
were referred to TMS. Mass spectra were taken on a HPLCeMS/MS
(HPLC, Agilent Technologies 1200 Series; MS, Agilent Technologies
6410 Triple Quad). CEM Discover microwave reactor was used for
microwave reactions (P ¼ 150 W, t ¼ 65 ꢀC). 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 are routinely
checked by TLC with Merck silica gel 60F-254 glass plates using the
following solvent systems: cyclohexane/ethyl acetate/methanol
3:1:0.5, cyclohexane/ethyl acetate 1:1, 2:1 and 4:1, dichloro-
methane/methanol/-cyclohexane/ethyl acetate 9:1:5:5, dichloro-
methane/methanol 95:5, 98:2 and 9:1. Spots were visualized by
short-wave UV light, iodine vapour and solution of phosphomo-
lybdic acid. Column chromatography was performed on silica gel
(0.063e0.200 mm), with the same eluents used in TLC. 1H-benzo[d]
2.1.2.2. N-((4-chlorophenyl)(phenyl)methyl)-1H-benzo[d][1,2,3]tri-
azole-1-carboxamide (2b). From the reaction of 1.271 (5.0 mmol)
(4-chlorophenyl)(phenyl)methanamine hydrochloride and purifi-
cation of the product from ether/petrol ether 1.433 g (79%) of 2b
was obtained; mp 143.5e145.5 ꢀC; IR (KBr): nmax 3306, 1708, 1522,
1448, 1378, 1230, 1070, 1016, 754, 702, 600 cmꢂ1; 1H NMR (DMSO-
d6)
d
10.04 (d, J ¼ 8.80 Hz, 1H), 8.21 (d, J ¼ 8.21 Hz, 1H), 8.16 (d,
J ¼ 8.21 Hz, 1H), 7.71 (t, J ¼ 7.60 Hz, 1H), 7.56e7.25 (m, 10H), 6.41 (d,
J ¼ 8.80 Hz, 1H); 13C NMR (DMSO-d6)
d 148.68, 145.44, 140.69,
140.13, 132.03, 131.45, 129.94e125.60, 119.78, 113.47, 57.20; MS/MS
m/z 385.2 (M þ 23)þ; Anal. C20H15ClN4O (C, H, N).
2.1.2.3. N0-((4-methoxyphenyl)(phenyl)methylene)-1H-benzo[d]
[1,2,3]triazole-1-carbohydrazide (2c). From the reaction of 1.13 g
(5 mmol) 4-methoxybenzophenone hydrazone 1.67 g (90%) of
crude product 2c was obtained and used in next reaction step
without purification. IR (KBr): nmax 3313, 1755, 1610, 1509, 1490,
[1,2,3]triazole
(BtH),
triphosgene,
N-methyl-1,1-
diphenylmethanamine, triethylamine (TEA), hydrazine hydrate
and all aminoalcohols used in this study were purchased from
Aldrich and 4-chloro-3-(fluoromethyl)phenyl isocyanate from
Acros. Primaquine, (4-chlorophenyl)(phenyl)methanamine and N1-
tritylethane-1,2-diamine were prepared from primaquine diphos-
phate (Aldrich), (4-chlorophenyl)(phenyl)methanamine hydro-
chloride (Aldrich) and N1-tritylethane-1,2-diamine hydrobromide
(Fluka) prior the use. All reactions with primaquine were run light
protected in the presence of few milligrams of sodium dithionite. 1-
Benzotriazole carboxylic acid chloride (BtcCl, 1) and (N-(4-((6-
methoxyquinolin-8-yl)amino)pentyl)-1H-benzo[d][1,2,3]triazole-
1-carboxamide) (8, primaquine benzotriazolide) were prepared
1448, 1371, 1255, 1216, 1178, 1028, 958, 944, 878, 769, 757 cmꢂ1
.
2.1.2.4. N0-(bis(4-methoxyphenyl)methylene)-1H-benzo[d][1,2,3]tri-
azole-1-carbohydrazide (2d). From the reaction of 1.28 g (5 mmol)
4,40-dimethoxybenzophenone hydrazone 1.70 g (85%) of crude
product 2d was obtained and used further without purification. IR
(KBr): nmax 3319, 1746, 1609, 1499, 1446, 1325, 1306, 1248, 1169,
1080, 1025, 879, 837, 784, 752 cmꢂ1
.
2.1.2.5. 1-Tritylimidazolidin-2-one (12) (an attempt to prepare 2e).
From the reaction of 1.917 g (5.0 mmol) N-trityl-1,2-ethanediamine

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504
hydrobromide and after recrystallization from ethanol 1.313 g (80%)
2.1.5.1. N-benzhydryl-N-methylhydrazinecarboxamide
From the reaction of 1.370 g 2a and after recrystallization from
ether/petrolether 0.754 (74%) of 5a was obtained; mp
105e108.5 ꢀC; IR (KBr): nmax 3320, 1626, 1496, 1318, 1188, 1024, 878,
738, 714, 608 cmꢂ1 1H NMR (DMSO-d6)
7.62 (s, 1H), 7.36 (t,
(5a).
of pure product 12 was obtained; mp 224e225.5 ꢀC; IR (KBr): nmax
3258, 1698, 1488, 1394, 1258, 1220, 1032, 760, 710, 628, 560 cmꢂ1
;
g
1H NMR (DMSO-d6)
d
7.36 (d, J ¼ 7.94 Hz, 6H), 7.26 (t, J1 ¼ 7.52 Hz,
J2 ¼ 8.06 Hz, 6H), 7.17 (t, J1 ¼ 7.31 Hz, J2 ¼ 7.27 Hz, 3H), 6.33 (s, 1H),
3.26 (t, J1 ¼ 7.69 Hz, J2 ¼ 7.48 Hz, 2H), 3.19 (t, J1 ¼ 7.31 Hz,
;
d
J1 ¼7.53 Hz, J2 ¼ 7.64 Hz, 4H), 7.31e7.28 (t, J1 ¼7.25 Hz, J2 ¼ 7.32 Hz,
J2 ¼ 7.14 Hz, 2H); 13C NMR (DMSO-d6)
d 161.40, 143.26, 128.93,
2H), 7.12 (d, J ¼ 7.57 Hz, 4H), 6.74 (s, 1H), 3.96 (s, 2H), 2.58 (s, 3H);
127.23, 126.11, 72.50, 45.14, 37.50; MS/MS m/z 243.2 (imidazole
13C NMR (DMSO-d6)
d 160.50, 140.01, 128.32, 128.27, 127.05, 60.97,
fragment)þ; Anal. C22H20N2O (C, H, N).
30.19; MS/MS m/z 278.3 (M þ 23)þ; Anal. C15H17N3O (C, H, N).
2.1.5.2. N-((4-Chlorophenyl)(phenyl)methyl)hydrazinecarbox-
amide(5b). From the reaction of 1.451 g compound 2b and after
crystallization from ether 0.838 g (76%) of 5b was obtained; mp
148.5e150 ꢀC; IR (KBr): nmax 3408, 3334, 3218, 3082, 1662, 1634,
2.1.3. 1,3-Bis((4-chlorophenyl)(phenyl)methyl)urea (3b)
A mixture of 0.100 g (0.276 mmol) 2b, 0.077 g (0.303 mmol) 4-
chlorobenzhydrylamine hydrochloride and 0.112 g (1.104 mmol)
TEA in 2 ml dioxane was heated at 60 ꢀC for 3.5 h. The solvent was
evaporated under reduced pressure and the residue was dissolved
in ethyl acetate and extracted with 5% NaOH solution (3 ꢁ 15 ml)
and water (3 ꢁ 15 ml). Product 3b precipitated from the organic
layer. After filtration and recrystallization from ethanol 0.094 g
(74%) of pure 3b was obtained; mp 298e300 ꢀC; IR (KBr): nmax 3308,
1560, 1566, 1472, 1096, 1016, 698 cmꢂ1 1H NMR (DMSO-d6)
;
d
7.3e7.23 (m, 9H), 7.08 (s, 1H), 7.01 (d, J ¼ 8.56 Hz, 1H), 5.95 (d,
J ¼ 8.56 Hz, 1H), 4.16 (s, 2H); 13C NMR (DMSO-d6)
d 159.21, 142.94,
142.60, 131.32, 128.80e126.84, 56.00; MS/MS m/z 276.2 (M þ 1)þ;
Anal. C14H14ClN3O (C, H, N).
1630, 1572, 1488, 1452, 1280, 1090, 1014, 849, 810, 754, 700 cmꢂ1
;
2.1.6. 1-Benzotriazolecarbonyl-4-arylsemicarbazides (6a,b):
general procedure
1H NMR (DMSO-d6)
d
7.40e7.22 (m, 9H), 6.96 (d, J ¼ 8.30 Hz, 2H),
5.88 (d, J ¼ 8.10 Hz, 2H); 13C NMR (DMSO-d6)
d 156.22, 142.91,
Solution of 0.725 mmol of 5 and 0.073 g (0.725 mmol) TEA in dry
dioxane (3 ml) was added drop-wise to the solution of 0.131 g
(0.725 mmol) chloride 1 in dioxane (2 ml). The reaction mixture
was stirred 1 h at 15 ꢀC. The solvent was evaporated under reduced
pressure and the residue was dissolved in ethyl acetate and
extracted with water (4 ꢁ 15 ml), dried over anhydrous sodium
sulfate and evaporated.
142.64, 131.33, 128.56e128.34, 127.08, 126.86, 56.38; MS/MS m/z
484.3 (M þ 23)þ; Anal. C27H22Cl2N2O (C, H, N).
2.1.4. Carbamates 4a,b: general procedure
Method A: A mixture of 0.146 mmol 2 and 0.039 g (0.574 mmol)
sodium ethoxide in 3 ml ethanol was stirred at room temperature
for 1 h.
Method B: A solution of 0.276 mmol 2 in 5 ml ethanol was
refluxed for 1 h. The solvent was evaporated under reduced pres-
sure and the residue was dissolved in ethyl acetate and extracted
with 5% NaOH solution (3 ꢁ 15 ml) and water (3 ꢁ 15 ml), dried over
anhydrous sodium sulfate and evaporated.
2.1.6.1. N-benzhydryl-2-(1H-benzo[d][1,2,3]triazole-1-carbonyl)-N-
methylhydrazinecarboxamide (6a). From the reaction of 0.185 g
compound 5a and after two recrystallizations from toluene 0.238 g
(82%) of 6a was obtained; mp 171.5e172.5 ꢀC; IR (KBr): nmax 3386,
3288, 1758, 1666, 1562, 1486, 1449, 1376, 1284, 1120, 1100, 1040,
875, 770, 750, 712, 698 cmꢂ1
.
2.1.4.1. Ethyl benzhydryl(methyl)carbamate (4a). Method A, from
0.050 g 2a and after purification by column chromatography (mo-
bile phase cyclohexane/ethyl acetate 4:1) 0.037 g (95%) of 4a was
obtained; oil; IR (KBr): nmax 3030, 2980, 1698, 1448, 1398, 1306,
2.1.6.2. 2-(1H-benzo[d][1,2,3]triazole-1-carbonyl)-N-((4-
chlorophenyl)(phenyl)methyl)hydrazinecarboxamide (6b). From the
reaction of 0.200 g compound 5b and after two recrystallizations
from ether/petrolether and acetone/water 0.147 g (48%) of 6b was
obtained; mp 161.5e162.5 ꢀC; IR (KBr): nmax 3358, 3294, 1754, 1664,
1170, 1142, 1004, 770, 736, 704 cmꢂ1
;
1H NMR (DMSO-d6)
d
7.37e7.12 (m, 10H), 6.52 (s, 1H), 4.11e4.06 (q, J ¼ 7.09 Hz, 2H), 2.64
1572, 1492, 1448, 1236, 1016, 752 cmꢂ1
.
(s, 3H), 1.19 (t, J ¼ 6.91 Hz, 3H); 13C NMR (DMSO-d6)
d 158.00,
139.25, 128.51e127.39, 62.30, 61.08, 30.96, 14.54; MS/MS m/z 292.2
(M þ 23)þ; Anal. C17H19NO2 (C, H, N).
2.1.7. 1-Primaquinecarbonyl-4-substituted semicarbazides (7ael):
general procedure
Method A: A solution of 0.182 g (0.7 mmol) primaquine,
0.7 mmol compound 6 and 0.209 g (2.1 mmol) TEA in dioxane
(10 ml) was stirred at room temperature for 18.5 h. The solvent was
evaporated under reduced pressure and the residue was dissolved
in ethyl acetate (10 ml) and extracted with 5% NaOH solution
(3 ꢁ 10 ml) and water (3 ꢁ 15 ml), dried over anhydrous sodium
sulfate and evaporated.
Method B: A solution of 0.5 mmol amine or amine salt and
0.101 g (1 mmol) TEA in dioxane or dichloromethane (6 ml) was
added drop-wise to a solution of 0.232 g (0.5 mmol) compound 11
in dioxane (6 ml). The reaction mixture was stirred 2e20 h, the
solvent was evaporated under reduced pressure and the residue
was dissolved in ethyl acetate (10 ml) and extracted with 5% NaOH
solution (3 ꢁ 10 ml) and water (3 ꢁ 15 ml), dried over anhydrous
sodium sulfate and evaporated.
2.1.4.2. Ethyl
((4-chlorophenyl)(phenyl)methyl)carbamate
(4b).
Method B, from 0.100 g 2b and after recrystallization from ether/
petrolether 0.063 g (79%) of 4b was obtained; mp 116e118 ꢀC; IR
(KBr): nmax 3286, 1714, 1684, 1530, 1492, 1278, 1238, 1090, 1040,
848, 754, 700 cmꢂ1; 1H NMR (DMSO-d6)
d
8.21 (d, J ¼ 7.44 Hz, 1H),
7.38e7.22 (m, 9H), 5.87 (d, J ¼ 9.19 Hz, 1H), 4.03e4.00 (q,
J ¼ 7.00 Hz, 2H), 1.17 (t, J ¼ 6.00 Hz, 3H); 13C NMR (DMSO-d6)
d 158,
142.24, 141.79, 131.50, 128.96e127.00, 59.96, 57.48, 14.58; MS/MS
m/z 312.2 (M þ 23)þ; Anal. C16H16ClNO2 (C, H, N).
2.1.5. 4-Arylsemicarbazides 5a,b: general procedure
Solution of 4.0 mmol 2a or 2b in 3 ml dioxane was added drop-
wise to a solution of 1.202 g (0.024 mmol) hydrazine hydrate in 2 ml
dioxane and stirred at room temperature for 25 h. The solvent was
evaporated under reduced pressure and the residue was dissolved
in ethyl acetate and extracted with 5% NaOH solution (3 ꢁ 15 ml)
and water (3 ꢁ 15 ml), dried over anhydrous sodium sulfate and
evaporated.
Method C: A solution of 0.952 g (0.3 mmol) primaquine semi-
carbazide 10, 0.045 g (0.45 mmol) TEA and 0.3 mmol 2c or 2d in
7 ml dioxane was stirred at room temperature for 1 h. The reaction
mixture was evaporated under reduced pressure. The residue was

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505
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.
Method D: A solution of 0.254 g (0.8 mmol) primaquine semi-
carbazide 10 and 0.266 g (1.2 mmol) 4-chloro-3-(fluoromethyl)
phenyl isocyanate in 8 ml toluene was stirred 24 h at room tem-
perature in the presence of few milligrams of sodium dithionite.
The reaction mixture was filtered and evaporated under reduced
pressure.
8.09e8.05 (dd, J ¼ 8.30 Hz, 1.44 Hz, 1H), 7.53 (d, J ¼ 5.59 Hz, 1H),
7.48e7.46 (q, J1 ¼ 4.31 Hz, J2 ¼ 3.83 Hz, J3 ¼ 4.15 Hz, 1H), 7.61e7.11
(m, 7H), 6.90 (d, J ¼ 8.94 Hz, 2H), 6.47 (s, 1H), 6.27 (s, 1H), 6.11 (d,
J ¼ 8.78 Hz, 1H), 3.83 (s, 3H), 3.82 (s, 3H), 3.68e3.57 (m, 1H),
3.04e3.03 (m, 2H), 1.67e1.40 (m, 4H), 1.26 (d, J ¼ 6.39 Hz, 3H); 13
C
NMR (DMSO-d6)
d 160.03, 159.81, 158.97, 158.57, 154.96, 144.61,
144.21, 137.47, 134.77, 134.49, 129.53, 129.55, 128.14, 127.06, 128.24,
123.66, 122.07, 114.98, 96.07, 91.57, 55.18, 54.95, 47.01, 39.32, 33.39,
26.72, 20.19; MS/MS m/z 570.3 (M þ 1)þ; Anal. C31H35N7O4 (C, H, N).
2.1.7.1. N1-benzhydryl-N2-(4-((6-metoxyquinolin-8-yl)amino)pen-
tyl)-N1-methylhydrazine-1,2-dicarboxamide (7a). Method A, from
the reaction of 0.208 g compound 6a and after purification by
column chromatography (mobile phase dichloromethane/meth-
anol 95:5) and crystallization from petrolether 0.201 g (54%) of 7a
was obtained; mp 79e82 ꢀC; IR (KBr): nmax 3294, 2934, 1642, 1520,
2.1.7.4. 2-(2-(Bis(4-methoxyphenyl)methylene)hydrazinecarbonyl-
N-(4-((6-methoxyquinolin-8-yl)amino)pentyl)hydrazinecarboxamide
(7d). Method C, from the reaction of 0.120 g (0.3 mmol) N0-(bis(4-
methoxyphenyl)methylene)-1H-benzo[d][1,2,3]triazole-1-
carbohydrazide (2d) and after crystallization from ether 0.142 g
(79%) of 7d was obtained; mp 117.5e120 ꢀC; IR (KBr): nmax 3344,
2933, 2837, 1683, 1610, 1575, 1516, 1456, 1422, 1387, 1337, 1303,
1454, 1424, 1388, 1336, 1220, 1158, 850, 690, 720, 702 cmꢂ1
;
1H
8.54e8.53 (dd, J ¼ 4.27 Hz, 1.72 Hz, 1H), 8.29 (s,
1251, 1167, 1110, 1030, 837, 822, 791 cmꢂ1 1H NMR (DMSO-d6)
;
NMR (DMSO-d6)
d
1H), 8.09e8.06 (dd, J ¼ 8.29 Hz, 1.76 Hz, 1H), 7.44e7.41 (m, 1H),
7.37e7.12 (m, 11H), 6.69 (s, 1H), 6.48 (s, 1H), 6.31 (t, J1 ¼ 5.59 Hz,
J2 ¼ 5.72 Hz, 1H), 6.27 (s, 1H), 6.11 (d, J ¼ 8.70 Hz, 1H), 3.82 (s, 3H),
3.64e3.56 (m, 1H), 3.07e3.01 (q, J1 ¼ 9.79 Hz, J2 ¼ 6.02 Hz,
J3 ¼ 6.58 Hz, 2H), 2.65 (s, 3H), 1.65e1.40 (m, 4H), 1.20 (d, J ¼ 6.28 Hz,
d
8.60 (s, 1H), 8.53e8.52 (dd, J ¼ 4.15 Hz, 1.68 Hz, 1H), 8.27 (s, 1H),
8.07e8.06 (dd, J ¼ 8.24 Hz, 1.50 Hz, 1H), 7.52 (s, 1H), 7.49 (d,
J ¼ 8.76 Hz, 2H), 7.42e7.40 (q, J1 ¼ 4.14 Hz, J2 ¼ 4.06 Hz, J3 ¼ 4.20 Hz,
1H), 7.17 (d, J ¼ 8.70 Hz, 2H), 7.11 (d, J ¼ 8.70 Hz, 2H), 6.89 (d,
J ¼ 8.94 Hz, 2H), 6.46 (s, 1H), 6.36 (s, 1H), 6.26 (s, 1H), 6.11 (d,
J ¼ 8.70 Hz, 1H), 3.79 (s, 3H), 378 (s, 3H), 3.76 (s, 3H), 3.65e3.60 (m,
1H), 3.09e3.01 (m, 2H), 1.65e1.46 (m, 4H), 1.20 (d, J ¼ 6.31 Hz, 3H);
3H); 13C NMR (DMSO-d6)
d 159.00, 158.99, 158.49, 144.62, 144.23,
139.74, 134.79, 134.51, 129.57, 128.34, 127.12, 122.09, 96.09, 91.58,
61.11, 54.98, 47.02, 39.29, 33.38, 30.51, 26.83, 20.20; MS/MS m/z
541.3 (M þ 1)þ; Anal. C31H36N6O3 (C, H, N).
13C NMR (DMSO-d6)
d 160.02, 159.79, 159.00, 158.57, 155.02, 144.64,
144.21, 134.75, 134.52, 130.09, 129.77, 128.57, 123.87, 129.55, 122.04,
114.96, 113.58, 96.09, 91.66, 55.20, 54.97, 47.06, 39.21, 33.44, 26.70,
20.20; MS/MS m/z 600.2 (M); Anal. C32H37N7O5 (C, H, N).
Method B, from the reaction of 0.099 g N-(diphenylmethyl)
methylamine and after purification by column chromatography
(mobile phase dichloromethane/methanol 95:5) and crystallization
from petrolether 0.157 g (58%) of 7a was obtained.
2.1.7.5. N1-4-((6-methoxyquinolin-8-yl)amino)pentyl)-N2-(2-(trity-
lamino)ethyl)hydrazine-1,2-carboxamide (7e). Method B, from the
reaction of 0.181 g (0.473 mmol) N1-tritylethane-1,2-diamine
hydrobromide and after purification by column chromatography
(mobile phase dichloromethane/methanol 95:5) 0.178 g (58%) of 7e
was obtained; mp 109.5e110 ꢀC; IR (KBr): nmax 3374, 3306, 3228,
2932, 1662, 1620, 1544, 1520, 1454, 1388, 1220, 1200, 1158, 850, 790,
2.1.7.2. N1-((4-chlorophenyl)(phenyl)methyl)-N2-(4-((6-
metoxyquinolin-8-yl)amino)pentyl)hydrazine-1,2-dicarboxamide
(7b). Method A, from the reaction of 0.295 g compound 6b and
after purification by column chromatography (mobile phase
dichloromethane/methanol/petrolether/ethyl
acetate
(9.5:0.75:4.9:4.9) and crystallization from ether/petrolether 0.232 g
770, 749, 708 cmꢂ1; 1H NMR (DMSO-d6)
d
8.53 (d, J ¼ 7.85 Hz, 1H),
(59%) of 7b was obtained; mp 96e98 ꢀC; IR (KBr): nmax 3302, 3086,
2936, 1656, 1572, 1520, 1492, 1388, 1220, 1158, 840, 790, 700 cmꢂ1
;
8.07 (d, J ¼ 8.40 Hz, 1H), 7.53 (s, 1H), 7.49 (s, 1H), 7.44e7.14 (m, 16H),
6.47 (s, 1H), 6.40 (t, J1 ¼ 8.13 Hz, J2 ¼ 8.39 Hz, 1H), 6.33 (t, J1 ¼ 7.21,
J2 ¼ 6.48 Hz, 1H), 6.24 (s, 1H), 6.09 (d, J ¼ 8.22 Hz, 1H), 3.82 (s, 3H),
3.61e3.56 (m, 1H), 3.17e3.12 (q, J1 ¼ 6.11 Hz, J2 ¼ 6.93 Hz,
J3 ¼ 6.30 Hz, 2H), 3.03e2.98 (q, J1 ¼ 6.48 Hz, J2 ¼ J3 ¼ 6.67 Hz, 2H),
2.77 (t, J1 ¼ J2 ¼ 7.21 Hz, 1H), 2.06e2.00 (q, J1 ¼7.58 Hz, J2 ¼ 5.75 Hz,
J3 ¼ 6.94 Hz, 2H), 1.62e1.42 (m, 4H), 1.16 (d, J ¼ 4.29 Hz, 3H); 13C
1H NMR (DMSO-d6)
d
8.55e8.53 (dd, J ¼ 4.09 Hz, 1.35 Hz, 1H),
8.10e8.07 (dd, J ¼ 8.29 Hz, 1.29 Hz, 1H), 7.66 (s, 1H), 7.57 (s, 1H),
7.45e7.13 (m, 10H), 7.10 (d, J ¼ 8.50 Hz, 1H), 6.49 (s, 1H), 6.37 (t,
J1 ¼ 5.21 Hz, J2 ¼ 5.63 Hz, 1H), 6.28 (s, 1H), 6.12 (d, J ¼ 8.58 Hz, 1H),
5.95 (d, J ¼ 8.30 Hz, 1H), 3.83 (s, 3H), 3.67e3.53 (m, 1H), 3.10e2.95
(m, 2H), 1.70e1.36 (m, 4H), 1.20 (d, J ¼ 6.19 Hz, 3H); 13C NMR
NMR (DMSO-d6)
d 158.98, 158.63, 158.61, 146.10, 144.61, 144.22,
(DMSO-d6)
d 159.50, 159.09, 158.32, 145.13, 144.71, 143.04, 142.68,
134.78, 134.50, 129.56, 128.39e125.96, 122.09, 96.06, 91.56, 70.17,
65.05, 54.97, 46.99, 43.87, 39.79, 33.33, 26.64, 20.15; MS/MS m/z
646.4 (M þ 1)þ; Anal. C38H43N7O3 (C, H, N).
135.26, 135.02, 131.87, 130.06, 129.50e127.34, 122.56, 96.58, 92.13,
56.62, 55.46, 47.55, 39.61, 33.87, 27.17, 20.66; Anal. C30H33ClN6O3 (C,
H, N).
Method
B,
from
the
reaction
of
0.127
g
4-
chlorobenzhydrylamine hydrochloride and after purification by
column chromatography (mobile phase dichloromethane/meth-
anol 95:5) and crystallization from petrolether 0.177 g (63%) of 7b
was obtained.
2.1.7.6. N1,N2-bis(4-((6-methoxyquinolin-8-yl)amino)pentyl)hydra-
zine-1,2-dicarboxamide (7f). Method B, from the reaction of 0.225 g
(0.5 mmol) primaquine diphosphate and after purification by col-
umn chromatography (mobile phase dichloromethane/methanol/
petrolether/ethyl acetate 9:1:2.5:2.5) and trituration with ether
0.151 (68%) of product 7f was obtained; mp 135e136.5 ꢀC; IR (KBr):
nmax 3306, 3228, 3084, 2936, 1662, 1616, 1576, 1521, 1456, 1388,
2.1.7.3. 2-(2-((4-Methoxyphenyl)(phenyl)methylene)hydrazine-
carbonyl)-N-(4-((6-methoxyquinolin-8-yl)amino)pentyl)hydrazine-
carboxamide (7c). Method C, from the reaction of 0.111
g
1220, 1202, 1158, 1051, 821, 791 cmꢂ1 1H NMR (DMSO-d6)
;
(0.3 mmol) of N0-((4-methoxyphenyl)(phenyl)methylene)-1H-
benzo[d][1,2,3]triazole-1-carbohydrazide (2c) and after crystalli-
zation from ether 0.133 g (78%) of 7c was obtained; mp 107e108 ꢀC;
IR (KBr): nmax 3346, 3060, 2962, 2934, 1686, 1613, 1576, 1519, 1457,
1388,1251, 1169, 1111, 1030, 822, 791, 774 cmꢂ1; 1H NMR (DMSO-d6)
d
8.53e8.51 (dd, J ¼ 4.14 Hz,1.55 Hz,1H), 8.07e8.05 (dd, J ¼ 8.26 Hz,
1.58 Hz, 1H), 7.45 (s, 1H), 7.42e7.40 (q, J1 ¼ 4.14 Hz, J2 ¼ 4.07 Hz,
J3 ¼ 4.19 Hz, 1H), 6.46 (s, 1H), 6.31 (t, J ¼ 5.66 Hz, 1H), 6.25 (s, 1H),
6.09 (d, J ¼ 8.66 Hz, 1H), 3.82 (s, 3H), 3.60e3.57 (m, 1H), 3.04e3.00
(m, 2H), 1.61e1.44 (m, 4H), 1.18 (d, J ¼ 6.28 Hz, 3H); 13C NMR
d
8.67 (s, 1H), 8.53e8.52 (dd, J ¼ 4.15 Hz, 1.44 Hz, 1H), 8.47 (s, 1H),
(DMSO-d6) d 159.48, 159.09, 145.12, 144.69, 135.25, 135.00, 130.04,

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506
122.55, 96.55, 92.09, 55.45, 47.53, 39.58, 33.84, 27.12, 20.66; MS/MS
167.5e169.5 ꢀC; IR (KBr): nmax 3302, 3226, 3090, 2935, 2860, 1661,
m/z 603.4 (M þ 1)þ; Anal. C32H42N8O4 (C, H, N).
1616,1575,1558,1520,1456,1423,1387,1330,1219,1201,1159,1134,
1051, 1032, 821, 790 cmꢂ1 1H NMR (DMSO-d6)
; d 8.54e8.53 (dd,
2.1.7.7. N1-(4-chloro-3-(trifluoromethyl)phenyl)-N2-(4-((6-
methoxyquinolin-8-yl)amino)pentyl)hydrazine-1,2-dicarboxamide
(7g). Method D, from the reaction of 0.254 g (0.8 mmol) prima-
quine semicarbazide 10 and 0.266 g (1.2 mmol) 4-chloro-3-(fluo-
romethyl)phenyl isocyanate and after several purifications with
petrolether/acetone mixture 0.221 g (51%) of 7g was obtained; mp
109e111 ꢀC; IR (KBr): nmax 3250, 3131, 2964, 2930, 1684, 1617, 1595,
J ¼ 4.15 Hz, 1.57 Hz, 1H), 8.09e8.06 (dd, J ¼ 8.29 Hz, 1.43 Hz, 1H),
7.49 (s, 1H), 7.47 (s, 1H), 7.44e7.40 (q, J1 ¼ J3 ¼ 4.20 Hz, J2 ¼ 4.02 Hz,
11H), 6.47 (s, 1H), 6.36 (t, J1 ¼ 5.57 Hz, J2 ¼ 5.38 Hz, 1H), 6.28e6.25
(m, 2H), 6.11 (d, J ¼ 8.71 Hz, 1H), 4.32 (t, J1 ¼ 5.15 Hz, J2 ¼ 5.10 Hz,
1H), 3.82 (s, 3H), 3.64e3.57 (m, 1H), 3.39e3.33 (q, J1 ¼ 6.29 Hz,
J2 ¼ 5.88 Hz, J3 ¼ 5.62 Hz, 2H), 3.04e2.94 (m, 4H), 1.66e1.42 (m,
4H),1.42e1.19 (m, 6H),1.20 (d, J ¼ 6.21 Hz, 3H); 13C NMR (DMSO-d6)
1542, 1517, 1424, 1387, 1324, 1261, 1134, 1032, 906, 827, 792 cmꢂ1
1H NMR (DMSO-d6)
;
d 159.47, 159.13,159.08, 145.11, 144.71, 135.27, 134.99, 130.05, 122.57,
d
9.14 (s,1H), 8.51e8.50 (dd, J ¼ 4.11 Hz,1.49 Hz,
96.54, 92.05, 61.13, 55.46, 47.51, 39.57, 39.61, 33.83, 32.74, 30.20,
27.14, 23.27, 20.69; MS/MS m/z 447.3 (M þ 1)þ; Anal. C22H34N6O6 (C,
H, N).
1H), 8.12 (s,1H), 8.10 (s,1H), 8.06e8.04 (dd, J ¼ 8.23 Hz,1.35 Hz,1H),
7.81 (d, J ¼ 7.28 Hz, 1H), 7.65 (s, 1H), 7.52 (d, J ¼ 8.83 Hz, 1H),
7.41e7.39 (q, J1 ¼ 4.16 Hz, J2 ¼ 4.02 Hz, J3 ¼ 4.19 Hz, 1H), 6.49 (s, 1H),
6.45 (s, 1H), 6.24 (s, 1H), 6.08 (d, J ¼ 8.73 Hz, 1H), 3.80 (s, 3H),
3.63e3.59 (m, 1H), 3.06e3.03 (m, 2H), 1.66e1.46 (m, 4H), 1.18 (d,
2.1.7.11. N1,N1-Bis(2-hydroxyethyl)-N2-(4-((6-methoxyquinolin-8-yl)
amino)pentyl)hydrazine-1,2-dicarboxamide (7k). Method B, from
the reaction of 0.079 g (0.75 mmol) 2-(2-hydroxy-ethylamino)-
ethanol (diethanolamine) and after column chromatography (mo-
bile phase dichloromethane/methanol 9:1) and trituration with
ether 0.191 g (85%) of 7k was obtained; oil; IR (KBr): nmax 3330,
J ¼ 6.29 Hz, 3H); 13C NMR (DMSO-d6)
d 159.00, 158.42, 155.98,
144.64, 144.20,139.50, 134.75, 134.51, 131.65, 129.56, 123.40, 123.30,
122.14, 122.04, 117.50, 96.07, 91.64, 54.96, 47.07, 39.27, 33.43, 26.61,
20.19; MS/MS m/z 539.2 (M þ 1)þ; Anal. C24H26ClF3N6O3 (C, H, N).
2936, 1616, 1576, 1520, 1456, 1387, 1220, 1167, 1051, 822, 791 cmꢂ1
1H NMR (DMSO-d6)
8.55e8.53 (dd, J ¼ 4.14 Hz, 1.16 Hz, 1H),
;
2.1.7.8. N1-(2-hydroxyethyl)-N2-(4-((6-methoxyquinolin-8-yl)
amino)pentyl)hydrazine-1,2-dicarboxamide (7h). Method B, from
the reaction of 0.046 g (0.75 mmol) 2-aminoethanol and after
column chromatography (mobile phase dichloromethane/meth-
anol 9:1) and trituration with ether 0.085 g (42%) of 7h was ob-
tained; mp 196.5e197.5 ꢀC; IR (KBr): nmax 3300, 3093, 2927, 2855,
1662, 1616, 1558, 1520, 1456, 1422, 1387, 1334, 1219, 1200, 1157,
d
8.09e8.05 (dd, J ¼ 8.27 Hz, 1.16 Hz, 1H), 8.03 (s, 1H), 7.44e7.40 (q,
J ¼ 4.14 Hz, 1H), 7.23 (s, 1H), 6.47 (s, 1H), 6.35 (t, J ¼ 5.46 Hz, 1H),
6.26 (s, 2H), 6.11 (d, J ¼ 8.77 Hz, 1H), 4.85 (t, J ¼ 4.63 Hz, 2H), 3.82 (s,
3H), 3.66e3.54 (m, 1H), 3.52e3.47 (q, J1 ¼ 5.46 Hz, J2 ¼ 5.13 Hz,
J3 ¼ 5.29 Hz, 4H), 3.34e3.30 (q, 4H), 3.03e2.99 (q, J1 ¼ 7.94 Hz,
J2 ¼ 5.62 Hz, J3 ¼ 5.94 Hz, 2H),1.55e1.44 (m, 4H),1.20 (d, J ¼ 6.29 Hz,
1071, 1051, 819, 790 cmꢂ1
;
1H NMR (DMSO-d6)
d
8.54e8.53 (dd,
3H); 13C NMR (DMSO-d6)
d 159.48, 159.47, 158.84, 145.10, 144.73,
J ¼ 4.17 Hz, 1.57 Hz, 1H), 8.09e8.05 (dd, J ¼ 8.28 Hz, 1.46 Hz, 1H),
7.58 (s, 1H), 7.49 (s, 1H), 7.44e7.40 (q, J1 ¼ 4.21 Hz, J2 ¼ 4.04 Hz,
J3 ¼ 4.18 Hz, 1H), 6.47 (s, 1H), 6.37 (t, J1 ¼ 5.59 Hz, J2 ¼ 5.88 Hz, 1H),
6.26e6.23 (m, 2H), 6.10 (d, J ¼ 8.70 Hz, 1H), 4.61 (t, J ¼ 5.35 Hz, 1H),
3.82 (s, 3H), 3.64e3.58 (m, 1H), 3.41e3.35 (q, J1 ¼ 5.99 Hz, J2 ¼ 5.59,
J3 ¼ 5.74 Hz, 2H), 3.11e3.02 (m, 4H), 1.66e1.42 (m, 4H), 1.20 (d,
135.27, 134.98, 130.05, 122.58, 96.57, 92.08, 60.16, 55.46, 50.14,
47.49, 39.50, 33.87, 27.24, 20.67; MS/MS m/z 449.2 (M þ 1)þ; Anal.
C21H32N6O5 (C, H, N).
2.1.7.12. N1-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)-N2-(4-
((6-methoxyquinolin-8-yl)amino)pentyl)hydrazine-1,2-
dicarboxamide (7l). Method B, from the reaction of 0.091 g
(0.75 mmol) 2-amino-2-hydroxymethyl-propane-1,3-diol (tro-
methamine) and after column chromatography (mobile phase
dichloromethane/methanol 9:1) and trituration with ether 0.109 g
(47%) of 7l was obtained; mp 112e115 ꢀC; IR (KBr): nmax 3320, 2936,
1654, 1617, 1576, 1521, 1457, 1423, 1388, 1221, 1202, 1159, 1052, 1031,
J ¼ 6.28 Hz, 3H); 13C NMR (DMSO-d6)
d 159.49, 159.25, 159.12,
145.14, 144.71, 135.26, 135.01, 130.05, 122.56, 96.57, 92.11, 60.95,
55.47, 47.55, 42.39, 39.62, 33.88, 27.14, 20.69; MS/MS m/z 405.2
(M þ 1)þ; Anal. C19H28N6O4 (C, H, N).
2.1.7.9. N1-(2-hydroxypropyl)-N2-(4-((6-methoxyquinolin-8-yl)
amino)pentyl)hydrazine-1,2-dicarboxamide (7i). Method B, from
the reaction of 0.056 g (0.75 mmol) 3-aminopropanol and after
column chromatography (mobile phase dichloromethane/meth-
anol 9:1) and trituration with ether 0.111 g (53%) was obtained; mp
191e193.5 ꢀC; IR (KBr): nmax 3302, 3226, 3092, 2934, 1661, 1616,
1575, 1558, 1520, 1456, 1423, 1387, 1329, 1220, 1200, 1158, 1052,
822, 791 cmꢂ1; 1H NMR (DMSO-d6)
d
8.54 (d, J ¼ 2.89 Hz, 1H), 8.07
(d, J ¼ 7.59 Hz, 1H), 7.87 (s, 1H), 7.58 (s, 1H), 7.44e7.40 (q,
J1 ¼ 4.70 Hz, J2 ¼ 3.69 Hz, J3 ¼ 4.33 Hz, 1H), 6.47 (s, 1H), 6.46 (t,
J1 ¼ 2.61 Hz, J2 ¼ 2.31 Hz, 1H), 6.26 (s, 1H), 6.10 (d, J ¼ 8.40 Hz, 1H),
5.97 (s, 1H), 4.84 (t, J ¼ 5.57 Hz, 3H), 3.82 (s, 3H), 3.64e3.60 (m, 1H),
3.48 (d, J ¼ 5.58 Hz, 6H), 3.06e2.96 (m, 2H), 1.62e1.52 (m, 4H), 1.20
1032, 820, 790 cmꢂ1
;
1H NMR (DMSO-d6)
d
8.54e8.53 (dd,
(d, J ¼ 6.14 Hz, 3H); 13C NMR (DMSO-d6)
d 159.56, 159.49, 159.05,
J ¼ 4.18 Hz, 1.61 Hz, 1H), 8.09e8.05 (dd, J ¼ 8.29 Hz, 1.55 Hz, 1H),
7.51 (s, 1H), 7.49 (s, 1H), 7.44e7.40 (q, J1 ¼ J3 ¼ 4.19 Hz, J2 ¼ 4.06 Hz,
1H), 6.47 (s, 1H), 6.37 (t, J1 ¼ 5.60 Hz, J2 ¼ 6.08 Hz, 1H), 6.33 (t,
J1 ¼ 5.84 Hz, J2 ¼ 6.08 Hz, 1H), 6.28 (s, 1H), 6.10 (d, J ¼ 8.76 Hz, 1H),
4.39 (t, J1 ¼ 5.11 Hz, J2 ¼ 5.35 Hz, 1H), 3.82 (s, 3H), 3.65e3.58 (m,
1H), 3.43e3.37 (q, J1 ¼ J3 ¼ 6.08 Hz, J2 ¼ 5.60 Hz, 2H), 3.10e3.03 (m,
4H), 1.66e1.42 (m, 6H), 1.20 (d, J ¼ 6.33 Hz, 3H); 13C NMR (DMSO-
145.15, 144.72, 135.26, 135.01, 130.05, 122.56, 96.58, 92.12, 61.37,
60.91, 55.47, 47.53, 39.61, 33.85, 27.10, 20.69; MS/MS m/z 465.3
(M þ 1)þ; Anal. C21H32N6O6 (C, H, N).
2.1.8. Ureas 9aeg: general procedure
Method A: A solution of 0.162 g (0.4 mmol) primaquine ben-
zotriazolide 8, 0.061 g (0.6 mmol) TEA and 0.6 mmol corresponding
amine in dichloromethane (2 ml) was heated at 65 ꢀC in microwave
reactor for 60e80 min. The reaction mixture was extracted with 5%
NaOH solution (3 ꢁ 10 ml) and water (3 ꢁ 15 ml), dried over
anhydrous sodium sulfate and evaporated. Additional 0.4 mmol of
TEA was added if the reaction started from amine salt. The reaction
with primaquine was run light protected in the presence of few
milligrams of sodium dithionite.
d6)
d 159.49, 159.29, 159.10, 145.13, 144.71, 135.26, 135.01, 130.05,
122.56, 96.57, 92.11, 58.88, 55.47, 47.55, 39.61, 36.81, 33.88, 33.36,
27.14, 20.69; MS/MS m/z 419.3 (M þ 1)þ; Anal. C20H30N6O4 (C, H, N).
2.1.7.10. N1-(2-hydroxypentyl)-N2-(4-((6-methoxyquinolin-8-yl)
amino)pentyl)hydrazine-1,2-dicarboxamide (7j). Method B, from
the reaction of 0.077 g (7.5 mmol) 5-aminopentanol and after col-
umn chromatography (mobile phase dichloromethane/methanol
9:1) and trituration with ether 0.123 g (55%) of 7j was obtained; mp
Method B: A solution of 0.130 g (0.5 mmol) primaquine, 0.076 g
(0.75 mmol) TEA and 0.5 mmol of corresponding benzotriazole-1-

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507
carbohydrazides 2c,d in dioxane (10 ml) was stirred 1.5 h at room
temperature. The reaction mixture was evaporated under reduced
pressure. The residue 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. The reaction was run
light protected in the presence of few milligrams of sodium
dithionite.
96.62, 92.14, 55.69, 55.45, 47.56, 39.48, 33.94, 27.26, 20.68; MS/MS
m/z 512.3 (M þ 1)þ; Anal. C30H33N5O3 (C, H, N).
2.1.8.4. 2-(Bis(4-methoxyphenyl)methylene)-N-(4-((6-
methoxyquinolin-8-yl)amino)pentyl)hydrazinecarboxamide
(9d).
Method B, from the reaction of 0.201 g (0.5 mmol) N0-(bis(4-
methoxyphenyl)methylene)-1H-benzo[d][1,2,3]triazole-1-
Method C: A solution of 0.311 g (1.2 mmol) primaquine and
0.266 g (1.2 mmol) 4-chloro-3-(fluoromethyl)phenyl isocyanate in
8 ml toluene was stirred 24 h at room temperature in the presence
of few milligrams of sodium dithionite. The reaction mixture was
filtered and evaporated under reduced pressure.
carbohydrazide (2d) and after purification by column chromatog-
raphy (mobile phase dichloromethane/methanol 98:2) 0.168 g
(62%) of 9d was obtained; oil; 3354, 3048, 2934, 2837, 1682, 1607,
1575, 1506, 1456, 1423, 1386, 1303, 1249, 1168, 1109, 1031, 836, 791,
735 cmꢂ1; 1H NMR (DMSO-d6)
d
8.51e8.48 (dd, J ¼ 4.14 Hz, 1.57 Hz,
1H), 8.06e8.03 (dd, J ¼ 8.13 Hz, 1.57 Hz, 1H), 7.75 (s, 1H), 7.42e7.38
(q, J1 ¼4.28 Hz, J2 ¼ J3 ¼ 4.14 Hz,1H), 7.47e6.83 (m, 9H), 6.44 (s,1H),
6.25 (s,1H), 6.11 (d, J ¼ 8.70 Hz,1H), 3.81 (s, 3H), 3.78 (s, 3H), 3.73 (s,
3H), 3.66e3.60 (m, 1H), 3.16e3.10 (q, J1 ¼ J3 ¼ 6.27 Hz, J2 ¼ 6.00 Hz,
2H), 1.68e1.47 (m, 4H), 1.20 (d, J ¼ 6.27 Hz, 3H); 13C NMR (DMSO-
2.1.8.1. 1-Benzhydryl-3-(4-((6-methoxyquinolin-8-yl)amino)pentyl)-
1-methylurea (9a). Method A, from the reaction of 0.118 g N-
(diphenylmethyl)methylamine and after crystallization from ether
0.134 g (69%) of 9a was obtained; mp 124e126 ꢀC; IR (KBr): nmax
3363, 2930, 1624, 1578, 1524, 1454, 1388, 1320, 1222, 1200, 1168,
d6)
d 160.21, 159.47, 155.34, 146.95, 145.01, 144.71, 135.27, 134.99,
840, 790, 745, 706 cmꢂ1 1H NMR (DMSO-d6)
; d 8.55e8.53 (dd,
130.63, 130.31, 128.89, 130.31, 124.46, 122.57, 115.44, 114.05, 96.57,
92.07, 55.67, 55.45, 47.52, 39.21, 33.91, 27.30, 20.67; MS/MS m/z
542.3 (M þ 1)þ; Anal. C31H35N5O4 (C, H, N).
J ¼ 4.19 Hz, 1.63 Hz, 1H), 8.09e8.07 (dd, J ¼ 8.29 Hz, 1.60 Hz, 1H),
7.44e7.41 (m, 1H), 7.37e7.08 (m, 10H), 6.74 (s, 1H), 6.48e6.46 (m,
2H), 6.27 (d, J ¼ 2.46 Hz, 1H), 6.12 (d, J ¼ 8.68 Hz, 1H), 3.81 (s, 3H),
3.66e3.59 (m, 1H), 3.14e3.06 (m, 2H), 2.57 (s, 3H), 1.69e1.48 (m,
2.1.8.5. 1-(4-((6-Methoxyquinolin-8-yl)amino)pentyl)-3-(2-(trityla-
mino)ethyl)urea (9e). Method A, from the reaction of 0.230 g N-
trityl-1,2-ethanediamine hydrobromide and after purification by
column chromatography (mobile phase dichloromethane/meth-
anol 95:5) 0.191 g (81%) of 9e was obtained; mp 145e146 ꢀC; IR
(KBr): nmax 3310, 2940, 1628, 1594, 1576, 1518, 1450, 1386, 1220,
1201, 1158, 840, 795, 780, 770, 746, 706 cmꢂ1; 1H NMR (DMSO-d6)
4H), 1.21 (d, J ¼ 6.29 Hz, 3H); 13C NMR (DMSO-d6)
d 159.00, 158.08,
144.61, 144.22, 140.30, 134.78, 134.53, 129.56, 128.31e128.22,
122.09, 96.07, 91.55, 60.64, 54.96, 47.04, 40.30, 33.32, 30.65, 26.67,
20.21; MS/MS m/z 483.3 (M þ 1)þ; Anal. C30H34N4O2 (C, H, N).
2.1.8.2. 1-((4-Chlorophenyl)(phenyl)methyl)-3-(4-((6-
methoxyquinolin-8-yl)amino)pentyl)urea (9b). Method A, from the
reaction of 0.152 g 4-chlorobenzhydrylamine hydrochloride and
after purification by column chromatography (mobile phase
dichloromethane/methanol/ethyl acetate/petrolether 9.5:0.5:5:5)
0.190 g (95%) of 9b was obtained; mp 135e137 ꢀC; IR (KBr): nmax
3346, 2938, 1632, 1576, 1520, 1490, 1476, 1424, 1388, 1220, 1200,
d
8.53e8.52 (dd, J ¼ 4.14 Hz,1.49 Hz,1H), 8.09e8.06 (dd, J ¼ 8.25 Hz,
1.58 Hz, 1H), 7.44e7.41 (m, 1H), 7.40e7.13 (m, 15H), 6.47 (d, 1H),
6.26 (d, 1H), 6.12 (d, J ¼ 8.78 Hz, 1H), 5.94e5.89 (2t, J ¼ 6.00 Hz, 2H),
3.81 (s, 3H), 3.64e3.58 (m, 1H), 3.11e3.07 (q, J1 ¼ J2 ¼ 6.00 Hz,
J3 ¼ 6.12 Hz, 2H), 3.01e2.97 (q, J1 ¼ 6.18 Hz, J2 ¼ 6.09 Hz,
J3 ¼ 6.30 Hz, 2H), 2.70 (t, J1 ¼ J2 ¼ 7.90 Hz, 1H), 2.00e1.95 (q,
J1 ¼ 6.39 Hz, J2 ¼ 7.30 Hz, J3 ¼ 6.59 Hz, 2H), 1.66e1.40 (m, 4H), 1.19
1166, 1095, 824, 795, 751, 700 cmꢂ1
;
1H NMR (DMSO-d6)
(d, J ¼ 6.33 Hz, 3H); 13C NMR (DMSO-d6)
d 158.99, 158.12, 146.10,
d
8.54e8.53 (dd, J ¼ 4.20 Hz, 1.64 Hz, 1H), 8.09e8.06 (dd,
144.62, 144.22, 134.78, 134.50, 129.56, 128.38, 127.61, 125.97, 122.09,
96.06, 91.58, 70.13, 64.89, 54.97, 47.05, 47.05, 39.29, 33.54, 26.97,
20.21; MS/MS m/z 588.4 (M þ 1)þ; Anal. C37H41N5O2 (C, H, N).
J ¼ 8.30 Hz, 1H), 7.44e7.42 (m, 1H), 7.37e7.20 (m, 9H), 6.81 (d,
J ¼ 8.41 Hz, 1H), 6.48 (s, 1H), 6.26 (s, 1H), 6.12 (d, J ¼ 8.80 Hz, 1H),
5.92 (t, J1 ¼ 5.54 Hz, J2 ¼ 5.68 Hz, 1H), 5.88 (d, J ¼ 8.34 Hz, 1H), 3.80
(s, 3H), 3.65e3.58 (m, 1H), 3.08e3.00 (q, J1 ¼ J2 ¼ 5.84 Hz,
J3 ¼ 6.72 Hz, 2H), 1.68e1.42 (m, 4H), 1.20 (d, J ¼ 6.30 Hz, 3H); 13C
2.1.8.6. 1,3-Bis(4-((6-methoxyquinolin-8-yl)amino)pentyl)urea (9f).
Method A, from the reaction of 0.273 g primaquine diphosphate
and after purification by column chromatography (mobile phase
cyclohexane/ethyl acetate/methanol 3:1:0.5) 0.183 g (84%) of 9f
was obtained; oil; IR (KBr): nmax 3378, 2932, 2856, 1616, 1570, 1520,
NMR (DMSO-d6)
d 158.99, 157.14, 144.61, 144.30, 143.14, 142.88,
134.79, 134.50, 131.20, 129.57, 128.76e126.89, 122.10, 96.09, 91.63,
56.26, 54.97, 47.03, 39.23, 33.47, 26.85, 20.20; MS/MS m/z 503.3
(Mþ); Anal. C29H31ClN4O2 (C, H, N).
1456, 1388, 1220, 1160, 1052, 822, 795 cmꢂ1 1H NMR (DMSO-d6)
;
d
8.52e8.51 (dd, J ¼ 4.20 Hz,1.65 Hz,1H), 8.08e8.06 (dd, J ¼ 8.31 Hz,
2.1.8.3. 2-((4-Methoxyphenyl)(phenyl)methylene)-N-(4-((6-
1.65 Hz, 1H), 7.43e7.40 (m, 1H), 6.46 (d, 1H), 6.25 (d, 1H), 6.10 (d,
J ¼ 8.80 Hz, 1H), 5.76 (t, J1 ¼ 5.86 Hz, J2 ¼ 5.71 Hz, 1H), 3.81 (s, 3H),
3.63e3.57 (m, 1H), 3.00e2.95 (q, J1 ¼ 6.56 Hz, J2 ¼ 5.86 Hz,
J3 ¼ 6.35 Hz, 2H), 1.64e1.41 (m, 4H), 1.19 (d, J ¼ 6.14 Hz, 3H); 13C
methoxyquinolin-8-yl)amino)pentyl)hydrazinecarboxamide
(9c).
Method B, from the reaction of 0.186 g (0.5 mmol) N0-((4-
methoxyphenyl)(phenyl)methylene)-1H-benzo[d][1,2,3]triazole-1-
carbohydrazide (2c) and after purification by column chromatog-
raphy (mobile phase dichloromethane/methanol/cyclohexane/
ethyl acetate 9:1:5:5) 0.158 g (62%) of 9c was obtained; oil; IR (KBr):
nmax 3354, 3050, 2935, 2835, 1694, 1682, 1615, 1575, 1506, 1456,
1424, 1386, 1336, 1250, 1171, 1110, 1031, 822, 792, 774, 735 cmꢂ1; 1H
NMR (DMSO-d6)
d 158.99, 157.30, 144.61, 144.22, 134.79, 134.50,
129.56, 122.09, 96.08, 91.58, 54.96, 47.02, 39.71, 33.49, 26.90, 20.20;
MS/MS m/z 545.3 (M þ 1)þ; Anal. C31H40N6O3 (C, H, N).
2.1.8.7. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-((6-
methoxyquinolin-8-yl)amino)pentyl)urea (9g). Method C, from the
reaction 0.266 g (1.2 mmol) 4-chloro-3-(fluoromethyl)phenyl iso-
cyanate and after purification by column chromatography (mobile
phase dichloromethane/methanol 98:2) 0.369 g (64%) of 9g was
obtained; mp 232.6e235.5 ꢀC; IR (KBr): nmax 3366, 2932, 2855,
1656, 1560, 1554, 1520, 1484, 1388, 1328, 1262, 1138, 1032, 822,
NMR (DMSO-d6)
d
8.54e8.52 (dd, J ¼ 4.17 Hz, 1.60 Hz, 1H),
8.09e8.05 (dd, J ¼ 8.32 Hz, 1.51 Hz, 1H), 7.94 (s, 1H), 7.59e6.88 (m,
10H), 7.44e7.40 (q, J1 ¼ J3 ¼ 4.20 Hz, J2 ¼ 4.06 Hz, 1H), 6.47 (s, 1H),
6.28 (s, 1H), 6.13 (d, J ¼ 6.03 Hz, 1H), 3.84 (s, 3H), 3.81 (s, 3H),
3.70e3.63 (m, 1H), 3.20e3.13 (q, J1 ¼ 6.21 Hz, J2 ¼ 5.85 Hz,
J3 ¼ 6.43 Hz, 2H), 1.70e1.52 (m, 4H), 1.23 (d, J ¼ 6.25 Hz, 3H); 13C
NMR (DMSO-d6)
d
160.29, 159.50, 155.25, 147.05, 145.11, 144.70,
795 cmꢂ1
; d 8.88 (s, 1H), 8.54e8.53 (dd,
1H NMR (DMSO-d6)
138.05, 135.27, 135.01, 130.04, 130.36e127.41, 124.28, 122.55, 115.50,
J ¼ 4.08 Hz, 1.43 Hz, 1H), 8.07e8.06 (dd, J ¼ 8.16 Hz, 1.40 Hz, 1H),

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K. Pavic et al. / European Journal of Medicinal Chemistry 86 (2014) 502e514
508
8.04 (s, 1H), 7.56 (d, J ¼ 8.71, 2.18 Hz, 1H), 7.51 (d, J ¼ 8.71 Hz, 1H),
7.43e7.41 (q, J1 ¼ 4.35 Hz, J2 ¼ J3 ¼ 4.04 Hz, 1H), 6.47 (s, 1H), 6.34 (t,
J1 ¼ J2 ¼ 5.60 Hz, 1H), 6.28 (s, 1H), 6.13 (d, J ¼ 9.02 Hz, 1H), 3.81 (s,
3H), 3.67e3.62 (m, 1H), 3.14e3.11 (q, J1 ¼ J3 ¼ 6.22 Hz, J2 ¼ 5.91 Hz,
2H), 1.74e1.50 (m, 4H), 1.23 (d, J ¼ 6.53 Hz, 3H); 13C NMR (DMSO-
3e4 days the cell growth rate was evaluated first light microscop-
ically, then by viable cell counting and/or by performing the MTT
assay, which detects dehydrogenase activity in live, not dead, cells.
The absorbance (A) was measured on a microplate reader at
570 nm. The absorbance is directly proportional to the number of
d6)
d
159.00, 154.83, 144.63, 144.20, 134.75, 134.53, 131.72, 129.55,
living, metabolically active cells. The results are expressed as IC50,
123.75, 122.24, 122.04, 121.19, 116.12, 116.08, 96.14, 91.67, 54.93,
which is the concentration necessary for 50% of inhibition. The IC50
values for each compound are calculated from concen-
trationeresponse curves using linear regression analysis by fitting
the test concentrations that give percentage of growth (PG) values
above and below the reference value (i.e. 50%). If however, for all of
the tested concentrations produce PGs exceeding the respective
reference level of effect (e.g. PG value of 50), then the highest tested
concentration is assigned as the default value, which is preceded by
a “>” sign. Minimum two individual experiments were carried out
and each test point was performed in quadruplicate.
47.05, 39.16, 33.44, 26.44, 20.21; MS/MS m/z 481.3 (M þ 1)þ; Anal.
C
23H24ClF3N4O2 (C, H, N).
2.1.9. N-(4-((6-methoxyquinolin-8-yl)amino)pentyl)
hydrazinecarboxamide (10)
A solution of 0.500 g (1.24 mmol) compound 8, 0.124 g
(2.48 mmol) of hydrazine hydrate and 2 mg of sodium dithionite in
2 ml dioxane was stirred light protected at room temperature for 4
days. The solvent was evaporated under reduced pressure and the
residue was dissolved in ethyl acetate (10 ml) and extracted with 5%
NaOH solution (3 ꢁ 10 ml) and water (3 ꢁ 15 ml), dried over
anhydrous sodium sulfate and evaporated. After purification by
column chromatography (mobile phase dichloromethane/meth-
anol 95:5) and crystallization from ether 0.320 g (81%) of 10 was
obtained; mp 109e110.5 ꢀC; IR (KBr): nmax 3370, 3328, 2960, 2930,
1618, 1570, 1524, 1456, 1388, 1294, 1275, 1208, 1170, 1054, 900, 820,
2.2.2. Determination of the reducing activity of the stable radical
DPPH
To a methanolic solution of DPPH (0.05 mM) in absolute ethanol
an equal volume of the compounds (final concentration 100 mM)
dissolved in DMSO was added. The mixture was shaken vigorously
and allowed to stand for 20 or 60 min. Absorbance at 517 nm was
determined spectrophotometrically, and the percentage of activity
was calculated [20]. All tests were undertaken on three replicates,
and the results were averaged.
795, 680, 623 cmꢂ1
; d 8.54e8.53 (dd,
1H NMR (DMSO-d6)
J ¼ 4.19 Hz, 1.65 Hz, 1H), 8.09e8.06 (dd, J ¼ 8.35 Hz, 1.63 Hz, 1H),
7.44e7.41 (m, 1H), 6.84 (s, 1H), 6.47 (s, 1H), 6.34 (t, J1 ¼ 5.87 Hz,
J2 ¼ 5.95 Hz, 1H), 6.26 (s, 1H), 6.11 (d, J ¼ 8.75 Hz, 1H), 4.03 (s, 2H),
3.82 (s, 3H), 3.63e3.57 (m, 1H), 3.04e3.01 (q, J1 ¼ 5.78 Hz,
J2 ¼ 5.87 Hz, J ¼ 5.98 Hz,2H), 1.68e1.45 (m, 4H), 1.21 (d, J ¼ 6.33 Hz,
2.2.3. Inhibition of linoleic acid lipid peroxidation
The water soluble azo compound AAPH is used as a free radical
initiator for in vitro studies of free radical production. Production of
conjugated diene hydroperoxide by oxidation of linoleic acid so-
dium salt in an aqueous solution is monitored at 234 nm. This assay
can be used to follow oxidative changes and to understand the
3H); 13C NMR (DMSO-d6)
d 160.23, 159.00, 144.63, 144.28, 134.80,
134.51, 129.57, 122.11, 96.09, 91.59, 54.98, 47.05, 38.88, 33.44, 27.00,
20.20; MS/MS m/z 318.2 (M þ 1)þ, 341.2 (M þ 1 þ 23); Anal.
C
16H23N5O2 (C, H, N).
contribution of each tested compound. An amount of 10 ml of the
2.1.10. 2-(1H-benzo[d][1,2,3]triazole-1-carbonyl)-N-(4-((6-
16 mM linoleic acid sodium salt solution was added to the UV
cuvette containing 0.93 ml of 0.05 M phosphate buffer, pH 7.4,
prethermostated at 37 ꢀC. The oxidation reaction was initiated at
methoxyquinolin-8-yl)amino)pentyl)hydrazinecarboxamide (11)
A solution of 0.150 g (0.473 mmol) of compound 11 and 0.048 g
(0.473 mmol) TEA in dioxane (5 ml) was added drop-wise to a
solution of compound 1 in dioxane (5 ml). The reaction mixture was
stirred 1 h at room temperature. The obtained solution of the crude
product 12 was further used for syntheses of compounds 7aec.
37 ꢀC under air by the addition of 50
Oxidation was measured in the presence of 10
the tested compound in DMSO (final concentration 100
assay). Lipid oxidation was measured also in the presence of 10
m
l of 40 mM AAPH solution.
l of the solution of
M in the
m
m
ml
DMSO (blank determination). The rate of oxidation at 37 ꢀC was
monitored by recording the increase in absorption at 234 nm
caused by conjugated diene hydroperoxides [20]. Each experiment
was performed at least in triplicate, and the standard deviation of
absorbance was less than 10% of the mean.
2.2. Biological evaluation
2.2.1. Antiproliferative evaluation assay
The experiments were carried out on seven human tumor cell
lines, which were derived from six cancer types: colon carcinoma
(SW 620), lung carcinoma (H 460), breast carcinoma (MCF-7),
cervical carcinoma (HeLa), pancreatic carcinoma (MiaPaca-2) and
acute lymphoblastic leukemia (Molt4/C8 and CEM), along with a
murine lymphocytic leukemia (L1210). SW 620, MCF-7, HeLa and H
460 cells were cultured as monolayers and maintained in Dulbec-
co's modified Eagle medium (DMEM) supplemented with 10% fetal
2.2.4. Soybean LOX inhibition study in vitro
The tested compounds (final concentration 100 mM, or in six
different concentrations for the determination of IC50 values) dis-
solved in DMSO were incubated at room temperature with sodium
linoleate (0.1 ml) and 0.2 ml of the enzyme solution (1 part of
enzyme/9 parts of saline ꢁ 10ꢂ4, m/V in saline). The conversion of
sodium linoleate to 13-hydroperoxylinoleic acid at 234 nm was
recorded and compared with the appropriate standard inhibitor
(nordihydroguaeretic acid NDGA) [20]. Each experiment was per-
formed at least in triplicate, and the standard deviation of absor-
bance was less than 10% of the mean.
bovine serum (FBS), 2 mM
100 g/ml streptomycin in a humidified atmosphere with 5% CO2 at
37 ꢀC.
L-glutamine, 100 U/ml penicillin and
m
The growth inhibition activity of the compounds was assessed
as described in previous publications [12e14,17]. The cell lines were
inoculated onto a series of standard 96-well microtiter plates on
day 0, at 3 ꢁ 104 cells/ml (SW 620, H 460, MiaPaca-2) to
5 ꢁ 104 cells/ml (MCF-7, Molt4/C8, CEM and L1210), depending on
the growth of the tumor cell line. Test agents were then added in
ten-fold dilutions (10ꢂ8 to 10ꢂ4 M) and the cell cultures were then
incubated for 3e4 days with 5% CO2 at 37 ꢀC. Working dilutions of
the compounds were freshly prepared on the day of testing. After
3. Results and discussion
3.1. Chemistry
Two alternative strategies for preparation of the title semi-
carbazides 7 were applied. In the first approach, the products grew

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K. Pavic et al. / European Journal of Medicinal Chemistry 86 (2014) 502e514
509
from the semicarbazide side and the primaquine residue entered
the molecule the last, while in the second approach, semi-
carbazides grew from the primaquine side.
(TEA, hydrazine hydrate, excess of N1-tritylethane-1,2-diamine,
NaOH, EtONa) or at higher temperature readily cyclized to imida-
zolidine derivative 12. So, another approach with compound 7e
growing from the primaquine side was chosen. The first reaction
step was preparation of primaquine amide of N-1-benzotriazole
carboxylic acid (primaquine benzotriazolide) (8). Compound 8
readily reacted with hydrazine hydrate and gave hydrazide 10.
Product 10 reacted with chloride 1 affording benzotriazole deriv-
ative 11. This compound was used for the last step without isolation
and with N1-tritylethane-1,2-diamine gave the desired semi-
carbazide derivative 7e. This synthetic approach proved to be
convenient also for preparation of semicarbazides 7a,b, bisprima-
quine derivative 7f and all semicarbazides with aminoalcohols
(7hel). The following aminoalcohols were used: 2-aminoethanol,
3-aminopropanol, 5-aminopentanol, 2-(2-hydroxy-ethylamino)-
ethanol (diethanolamine), 2-amino-2-hydroxymethyl-propane-
1,3-diol (tromethamine). They differ in number of hydroxy groups
or in chain length.
Several semicarbazides, namely 7c, 7d and 7g, were prepared
directly from compound 10, without activation with BtcCl. Products
7c and 7d were obtained in reaction of 10 with 2c and 2d,
respectively, while 7g is prepared from 10 and 4-chloro-3-(fluo-
romethyl)phenyl isocyanate.
Primaquine benzotriazolide 8 was used as the starting material
for synthesis of urea derivatives 9aef as well. Urea 9g was prepared
directly from primaquine and 4-chloro-3-(fluoromethyl)phenyl
isocyanate.
The first objective of our research was to prepare semi-
carbazides with benzhydryl substituents. Two commercially avail-
able amines, namely N-methyl-1,1-diphenylmethanamine and (4-
chlorophenyl)(phenyl)methanamine and two hydrazones (4-
methoxybenzophenone and 4,40-dimethoxybenzophenone hydra-
zones) prepared from the corresponding benzophenone and hy-
drazine hydrate, were used as benzhydryl bearing compounds. In
the first reaction step, BtcCl (1) reacted with the above mentioned
amines or hydrazones affording benzotriazole-1-carboxamides
2a,b or carbohydrazides 2c,d. Products 2a,b were further used for
synthesis of urea, carbamates and semicarbazides (Scheme 1). Urea
3b was prepared from 2b and equivalent amount of 4-
chlorobenzhydrylamine or from chloride 1 and excess of 4-
chlorobenzhydrylamine. Carbamates 4a,b were obtained by the
reaction of 2a or 2b with sodium ethoxide or ethanol, while 4-
arylsemicarbazides 5a,b from 2a or 2b and hydrazine hydrate.
Free amino group of products 5a,b readily reacted with chloride 1
affording benzotriazole derivatives 6a,b, which in reaction with
primaquine gave the target semicarbazides 7a and 7b, respectively.
Analogous reaction sequences starting from 2 to 7 were also
planned with N1-tritylethane-1,2-diamine. However, synthetic
pathway applied for synthesis of compounds 7a and 7b was not
convenient for preparation of 7c. Chloride 1 and N1-tritylethane-
1,2-diamine gave 1-tritylimidazolidin-2-one (12) as the major
product, so attempt to synthesize N-[2-(tritylamino)ethyl]-1H-
benzo[d][1,2,3]triazole (2e), precursors 5e and 6e or urea 3e and
carbamate 4e failed. Namely, compound 2e under basic conditions
The presence of the primaquine residue in compounds 7, 9 and
10 was confirmed by NMR spectra: hydrogen atoms next to pyri-
dine nitrogen occurred in 1H NMR spectra between 8.55 and 8.48,
Scheme 1. Synthesis of urea and semicarbazide derivatives of primaquine with benzhydryl and trityl moieties.

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K. Pavic et al. / European Journal of Medicinal Chemistry 86 (2014) 502e514
510
Table 1
Growth inhibition of tumor cell lines in vitro: IC50 (m
M).a
Compound
Cell line
L1210
Symbol
Structural formula
Molt4/C8
CEM
24
HeLa
25
Mia Paca-2
SW 620
MCF-7
H460
27
2a
15
1
28
3
2
1
26
6
ꢃ100
23
9
2
2b
67 37
176 105
116 65
87 16
110 25
23 12
42 40
18
8
3b
4a
>250
>250
>250
>250
97 16
>100
>100
>100
96 18
105 18
108 28
80 10
94
7
27 10
26
8
ꢃ100
>100
4b
>250
>250
>250
>250
142 81
>100
>100
5a
5b
7a
ꢃ250
ꢃ250
144 37
127 11
217 32
174 71
182 44
127
3
>100
>100
ꢃ100
ꢃ100
>100
>100
ꢃ250
178 90
16
2
19
1
10
1
22
2
18
0
27
3
3
5
2
0.9
16
3
6
7b
7c
8.8 4.4
9.6 4.3
5.5 2.2
6.9 3.0
6.0 0.7
2
4
0.2
0.5
1
1
6.2 0.5
12
12
6
7
7.4 2.1
18
4
13
11
7
5
7
2
1
7
5
7d
7.5 2.7
8.0 3.4
50 16
ꢃ100
0.2
0.3
7e
7f
6.2 1.1
8.0 3.2
4.2 0.1
10
3
5.0 0.5
7
4
2
0.2
4
0.3
92 25
ꢃ250
87 47
67 31
69 46
>100
0.2 0.09
94
3

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K. Pavic et al. / European Journal of Medicinal Chemistry 86 (2014) 502e514
511
Table 1 (continued )
Compound
Cell line
Symbol
Structural formula
L1210
Molt4/C8
CEM
14
HeLa
17
Mia Paca-2
SW 620
MCF-7
H460
7g
19
1
19
1
2
0
18
6
15
4
2
0.7
4
13 0.08
7h
7i
>250
>250
>250
>250
>250
>250
196 58
ꢃ250
ꢃ250
>100
>100
13
>100
>100
>250
13 0.5
7j
>250
>250
ꢃ250
>250
170 11
ꢃ250
>250
168 40
>100
>100
4
0.9
>100
>100
7k
>250
>250
29 10
7l
>250
>250
>250
>250
>250
>100
>100
28
9
3
>100
ꢃ100
9a
6.7 0.2
10
4
5.2 0.0
121 48
21 17
2
9b
94 11
ꢃ250
86 48
186 67
181 52
>100
>100
>100
9c
34 11
ꢃ250
>250
10e100b
>250
129 40
155 54
>100
>100
5
6
3
2
>100
>100
9d
>250
>250
>250
9e
9f
40 14
92 24
3.6 0.5
210 38
>250
10e100b
5e50b
38
0
40 16
186 90
4.0 0.1
54 23
45 34
>100
>100
>250
>100
6
3
1
9g
3.4 0.9
3.1 0.6
3.9 1.6
ꢃ250
23
2
4
28
7
10
>250
ꢃ250
>250
ꢃ250
>100
16
63
5
4
>100
>100
12
68 10
21
3
27 11
102 80
31
8
ꢃ100
a
IC50 e the concentration that causes 50% growth inhibition.
No dose response.
b

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K. Pavic et al. / European Journal of Medicinal Chemistry 86 (2014) 502e514
512
methoxy group at 3.83e3.76, CH of chiral carbon as a multiplet at
3.70e3.53, methyl group between 1.26 and 1.16 while in 13C spectra
corresponding carbon atoms appeared at 144.73e144.20,
55.47e54.93, 47.56e46.99 and 20.68e20.15 ppm, respectively. In
1H NMR spectra NH group attached to benzene ring appeared as
ring showed moderate antiproliferative activity and selectivity to-
wards MCF-7 cell line. High selectivity towards MCF-7 cell line
showed semicarbazides having one or more hydroxyalkyl residues
(IC50 ranged 4e29
mM). Among them, derivative 7j with the longest
chain length showed the most pronounced activity (4
m
M), while
doublet at
d
6.13e6.08 ppm and hydroxy groups in products 7hel
compounds 7k and 7l with two or three hydroxy groups had lower
antiproliferative effect. The most intriguing selectivity towards
MCF-7 cells exerted symmetric bisprimaquine derivative 7f, with
as triplet between 4.85 and 4.32 ppm (appropriate hydrogen count,
all signals D2O exchangeable). Signals of methoxy groups in prod-
ucts 7c,d and 9c,d were located very close to primaquine methoxy
group. The presence of carbonyl functional group in ureas 3b and
9aeg was indicated by the appearance of a strong stretching vi-
bration band in IR spectra between 1682 and 1615 cmꢂ1 (amide I)
and in carbamates 4a,b at 1714-1698 cmꢂ1. In 13C spectra these
carbonyl groups had signals between 159.00 and 154.83 ppm.
Carbonyl group in active ureas 2a,b was shifted to higher wave-
numbers and lower ppm values (150.71 and 148.68). Semi-
carbazides 7ael showed two close carbonyl signals between 159.81
and 155.98. Semicarbazide carbonyl in compounds 5a,b and 10
appeared at 160.50e159.21 ppm, while their IR absorptions were
similar to ureas 3 and 9. Benzhydryl residue gave expected signals
in aromatic region and appropriate hydrogen/carbon count. Signals
of carbon atom bearing two phenyl residues in 7a, 9a and 7b,9b
significantly differed in 13C NMR spectra: for derivatives with
chlorine (products b) it appeared at lower ppm values (ca 56 ppm)
and for compounds without chlorine between 61 and 60 ppm. The
analogous carbon atoms in products 7c,d and 9c,d were very shifted
towards higher ppm values (ca 155 ppm in 7c,d and 146 in 9c,d,
respectively) since they are bound to nitrogen. Trityl derivatives 7e
and 9e in 13C spectra showed a characteristic signal at 70.1 ppm and
two new aliphatic carbon atoms at 65.05 and 43.87 (7e) and 64.89
and 47.05 (9e), respectively. Compounds 2c,d, 6a,b and 11 were
used in further reactions steps without purification and their
structures were confirmed indirectly.
an IC50 0.2
mM, while being almost inactive towards the other
tested cell lines.
Urea derivatives 9aef were generally less active than their
semicarbazide analogues, but still selective towards MCF-7 cells.
Urea 9g with the similar structure to cytostatic drug sorafenib was
the most active tested compound. Opposite to semicarbazide de-
rivatives, modifications of benzhydryl moiety in urea line resulted
in differences in antiproliferative activities. Compound 9a with
nonsubstituted benzhydryl residue showed significantly high, but
not selective activity towards all the tested cell lines, except HeLa
and SW 620, chloro derivative 9b was practically inactive, while
methoxy derivatives 9c and 9d were selective towards MCF-7 cells.
Urea derivatives with the highest Clog P did not exert a clear dos-
eeresponse effect, which might be attributable to their lower sol-
ubility. This feature limits their potential use as orally active drugs.
Again, the most interesting finding of the current research is the
high sensitivity of MCF-7 tumor cells to the tested compounds. We
have already noticed this phenomenon in our previous work with
primaquine derivatives [13,14], while some other authors reported
selectivity of quinidine, primaquine, N-dipeptidylprimaquines and
imidazolin-4-one primaquine derivatives towards MCF-7 cell line
as well [21,22]. As we already discussed [13], selective growth
inhibitory activity towards breast cancer cells could be explained by
selective induction of cytochrome P450 (CYP-1) enzymes specif-
ically in the MCF-7 line, known as CYP-1 inducible cell line. The
observed selectivity towards breast cancer cells could be related to
either the primaquine moiety (primaquine was shown to be a
potent inducer of CYP1A1) [23] or to the semicarbazide moiety.
Namely, in liver microsomes semicarbazides can be metabolized by
the members of cytochrome P-450 family generating toxic products
[9]. The intriguing specificity of the here presented primaquine
derivatives towards MCF-7 cells should be further studied in more
details.
Chemical structure of new compounds was also supported by
CHN analysis and mass spectroscopy. Molecular ion peaks corre-
sponding to expected molecular formulas were obtained for all
compounds except for product 12 (only imidazolidin-2-on frag-
ment at 243.2 was visible). However, NMR spectra confirmed the
structure of 12 (15 hydrogen atoms in aromatic region, D2O
exchangeable signal of NH at 6.33 ppm and two close triplets at
3.26 and 3.15 ppm, respectively; carbonyl signal at 161.40 ppm in
13C NMR and appropriate count of aromatic and aliphatic carbon
atoms).
3.2.2. Antioxidative activity
A number of studies have shown the prooxidant effects of pri-
maquine in blood [24e27]. It was found that primaquine treatment
causes an oxidative stress in the liver and the kidneys as well as
hemotoxicity by generating reactive oxygen species within eryth-
rocytes that overwhelm antioxidant defences. In our previous
research on 1,4-substituted semicarbazides with primaquine moi-
ety [12] a significant antioxidant behaviour was noticed [13].
Having these facts in mind we have also tested oxidant/antioxidant
ability of the newly prepared primaquine derivatives.
It is known that compounds with antioxidant properties could
be expected to offer protection in a number of diseases and path-
ophysiological disorders. Several assays should be used in order to
assess in vitro antioxidant activity because the antioxidant ability of
a compound must be evaluated in a variety of milieus. Each pro-
tocol relates to the generation of a different radical. Two types of
approach have been taken: the assays in which we have the scav-
enging by hydrogen or electron donation of a preformed free
radical as a marker of antioxidant activity and assays involving the
presence of antioxidant system during the generation of the radical.
For these reasons, we have used two different types of assays to
measure in vitro antioxidant activity: a) the reduction of the stable
free radical DPPH, b) the reduction of the water-soluble azo
3.2. Biological evaluation
3.2.1. Cytostatic activity
A weak antiproliferative effect of primaquine [14] and improved
effect of its derivatives with semicarbazide and urea moieties
[12,14] prompted us to direct our research towards new compounds
bearing primaquine and urea/semicarbazide scaffolds. Novel sem-
icarbazides 7ael and ureas 9aeg and their synthetic precursors
were evaluated for cytostatic activities toward a variety of tumor
cell lines: human SW 620, MCF-7, H 460, CEM, Molt 4, HeLa, and
murine L1210. The synthetic precursors without primaquine res-
idue 2e5 showed no or weak antiproliferative activity. On the other
hand, all primaquine semicarbazide derivatives showed either
prominent cytostatic activity towards all the tested cell lines (7aee)
or high selectivity toward MCF-7 cells (7hel), with IC50 values in
the low micromolar range (Table 1). It is worth to note that semi-
carbazides with wide antiproliferative spectra possess highly
lipophilic benzhydryl (7aed) or trityl group (7e), what is in accord
with our previous findings [13]. Modification of benzhydryl moiety
by methoxy, dimethoxy or chloro substituents did not enhance the
effect, nor the selectivity. Compound 7g with halogenated benzene

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K. Pavic et al. / European Journal of Medicinal Chemistry 86 (2014) 502e514
513
compound AAPH and the c) inhibition of soybean lipoxygenase. The
results of the later experiment will help us to delineate the possi-
bility of the compounds to offer protection against inflammation
and cancer. All three assays require a spectrophotometric mea-
surement and a certain reaction time in order to obtain reproduc-
ible results [28]. Semicarbazides 7ael, ureas 9aee and their
synthetic precursors, were tested in comparison to well known
antioxidant agents recommended as references e.g. NDGA and
Trolox [13,29e32].
both semicarbazides as well as ureas derivatives were potent and
inhibited lipid peroxidation (LP). Urea derivatives 9aeg, especially
9d, and semicarbazides 7aeg with lipophilic substituents exerted
better LP antioxidant activity than Trolox (61e98%). Semicarbazides
7hel with hydroxyalkyl substituents were less active (27e54%). No
significant difference was observed between the compounds of the
series 9. Our results, like some reported data [34], indicate that LP
inhibition is not always accompanied by DPPH radical scavenging
activity. Thus, compounds that potently inhibited LP, exerted low (if
any) DPPH scavenging activity.
DPPH interaction of the tested compounds was examined at
100
m
M concentration after 20 and 60 min. The results are shown in
LOX inhibitors have attracted attention initially as potential
agents for the treatment of inflammatory and allergic diseases,
certain types of cancer, and cardiovascular diseases. In order to
diminish the pro-cancerous mechanisms a key treatment strategy
is to scavenge free radicals. Antioxidant activity by scavenging of
reactive oxygen species is important in preventing potential dam-
age to cellular components such as DNA, proteins and lipids. Studies
have demonstrated that levels of several eicosanoids are increased
in breast cancer in comparison to benign breast tumours [35].
Moreover, it has been found that LOX inhibitors may have che-
mopreventive activity in lung carcinogenesis [36].
Table 2. Semicarbazide 7g showed the best DPPH interaction value
(64% at 20 min and 90% at 60 min), similar to that of the reference
compound NDGA (91%) at the same concentration. Derivative 10
without substituents at position 4 was found to be equipotent to 7g.
Within the semicarbazide series 7a, 7c, 7d, 7b and 7e followed with
interaction values between 30 and 47% (20 min), while 7h was
found inactive. The percent interaction values enhanced after
60 min. Thus, compounds with high lipophilicity presented inter-
esting reducing abilities. On the contrary, lower lipophilicity was
correlated with decreased activity. Urea derivatives 9aeg showed
weak interaction with DPPH (5e20%) and their antioxidant activity
was slightly increased after 60 min (12e32%). Synthetic precursors
2e5 without primaquine moiety presented no or low activity.
The water-soluble azo compound AAPH has been extensively
used as a clean and controllable source of thermally produced
alkylperoxyl free radicals. Azo compounds generating free radicals
through spontaneous thermal decomposition are useful for free
radical production studies in vitro [33]. 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
In this context, we decided to further evaluate ability of our new
primaquine derivatives to inhibit soybean LOX by the UV absor-
bance based enzyme assay [13,31]. From the tested derivatives
semicarbazides 7c > 7b > 7d, as well as ureas 9b > 9c ¼ 9d, pre-
sented interesting IC50 values (from 57.9 to 77 mM) in comparison to
the IC50 value of the reference compound nordihydroguaeretic acid
(NDGA) (28 M, Table 2). All these compounds contain benzhydryl
m
with methoxy or chloro substituents and high Clog P values
(5.54e7.43, respectively), which support the findings that lip-
ophilicity is an important physicochemical property for LOX in-
hibitors [37]. Compounds with low clog P values presented no, if
any, inhibitory activity.
Table 2
Interaction with DPPH, in vitro inhibition of lipid peroxidation (LP), soybean lip-
oxygenase (LOX), and theoretically calculated Clog P values.
3.2.3. Determination of lipophilicity as clogP values
Since lipophilicity is a significant physicochemical property
determining distribution, bioavailability, metabolic activity, and
elimination, we have calculated the lipophilicity values of the
prepared compounds as clog P values by the ClogP programme of
Biobyte Corp [38].
Compd. Clog P DPPH 20 min DPPH 60 min LP inhibition LOX inhibitiona
(%)a
(%)a
(%)
(%)/IC50 mM
2a
2b
3b
4a
4b
5a
5b
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
9a
9b
9c
9d
9e
5.37
5.15
7.01
4.08
4.20
1.44
1.70
4.98
5.45
6.26
6.18
6.51
4.88
4.63
1.44
1.67
1.75
1.55
2.70
5.57
6.54
7.43
7.35
7.32
5.98
6.39
1.14
3.66
e
3
na
12
na
na
na
47
27
73
55
64
84
41
37
90
20
20
32
45
40
16
24
26
14
12
32
21
82
na
91
e
76
63
33
na
21
7
87.5
28%
38%
na
na
na
m
M
24
na
na
na
24
14
44
30
47
37
30
18
64
na
9
14
25
18
20
12
19
5
4. Conclusions
Novel semicarbazides 7ael and ureas 9aeg with a region reach
in electronegative atoms bound to primary amino group of pri-
maquine and highly lipophilic aromatic region or hydroxyalkyl
ending were designed, prepared, characterized and evaluated for
cytostatic and antioxidative activities. The synthetic pathways
applied for their preparation involved benzotriazole as synthetic
auxiliary. All primaquine semicarbazide derivatives showed either
prominent cytostatic activity towards all the tested cell lines (7aee)
or high selectivity towards MCF-7 cells (7hel), with IC50 values in
the low micromolar range. The highest selectivity exerted sym-
52
69
77
78
82
88
65
63
27
45
54
43
49
79
90
77
98
89
89
61
70
20
e
9%
44%
70.5
60.5
m
m
M
M
75 mM
100
31%
na
na
na
4
30%
6%
na
57.9
60.5
mM
metric bisprimaquine derivative 7f, with an IC50 0.2 mM. Urea de-
rivatives were generally less active than their semicarbazide
analogues, but still selective towards MCF-7 cells. Urea 9g with
similar structure to cytostatic drug sorafenib, was the most active
tested derivative.
Semicarbazides 7g and 10 showed the best DPPH interaction
value (64% at 20 min and 90% at 60 min), similar to that of the
reference compound NDGA (91%) at the same concentration, while
urea derivatives showed weak interaction with DPPH. Urea de-
rivatives 9aeg, especially 9d, and semicarbazides 7aeg with lipo-
philic substituents exerted better LP antioxidant activity than
Trolox (61e98%). Semicarbazides 7hel with hydroxyalkyl
m
m
M
M
M
M
60
93
6%
m
m
na
20
20
61
na
80
e
9f
9g
10
12
NDGA
Trolox
43%
42%
6%
84%/28 mM
e
e
63
na e no activity under the reported experimental conditions.
a
Concentrations of the tested compounds 1 ꢁ 10ꢂ4 M.

ꢀ
K. Pavic et al. / European Journal of Medicinal Chemistry 86 (2014) 502e514
514
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ureas with methoxy or chloro benzhydryl substituents and high
Clog P values showed significant LOX inhibition. No correlation
between antiproliferative and antioxidative activity was observed.
Our study expanded the current knowledge about antitumor
potential of commonly used antimalarial drugs. We have proved
that primaquine residue in new semicarbazide and urea derivatives
have a crucial role in antitumor activity and showed that highly
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