Urea and carbamate derivatives of primaquine: Synthesis, cytostatic and antioxidant activities

Article abstract of DOI:10.1016/j.bmc.2009.06.030

The novel urea primaquine derivatives 3 were prepared by aminolysis of primaquine benzotriazolide 2 with several hydroxyamines and ethylendiamine, while carbamates 4 were synthesized from the same precursor 2 and alcohols. All compounds are fully chemical

Full text of DOI:10.1016/j.bmc.2009.06.030

Bioorganic & Medicinal Chemistry 17 (2009) 5605–5613
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Urea and carbamate derivatives of primaquine: Synthesis, cytostatic
and antioxidant activities
a
a
a,
b
b
c
c
*
´
´
M. Šimunovic , I. Perkovic , B. Zorc , K. Ester , M. Kralj , D. Hadjipavlou-Litina , E. Pontiki
a Faculty of Pharmacy and Biochemistry, University of Zagreb, 10 000 Zagreb, Croatia
b Division of Molecular Medicine, Institute Rudjer Boškovi´c, 10 000 Zagreb, Croatia
c Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotles University of Thessaloniki, Thessaloniki 54 124, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 March 2009
Revised 4 June 2009
Accepted 12 June 2009
Available online 21 June 2009
The novel urea primaquine derivatives 3 were prepared by aminolysis of primaquine benzotriazolide 2 with
several hydroxyamines and ethylendiamine, while carbamates 4 were synthesized from the same precursor
2 and alcohols. All compounds are fully chemically characterized and evaluated for their cytostatic and anti-
oxidant activities. The most prominent antiproliferative activity was obtained by compounds 3c, 3d, 3g, and
5b (IC50 = 9–40
showed extreme selectivity toward SW 620 colon cancer cells (IC50 = 0.2
cer cells H 460. Hydroxyurea 3h showed the highest interaction with DPPH. Primaquine twin drug 3g
showed very significant inhibition on LOX soybean (IC50 = 62 M). Almost all the tested derivatives highly
inhibited lipid peroxidation, significantly stronger than primaquine phosphate.
Ó 2009 Elsevier Ltd. All rights reserved.
l
M). 1-(5-Hydroxypentyl)-3-[4-(6-methoxy-quinolin-8-ylamino)-pentyl]urea (3c)
l
M) and a bit less toward lung can-
Keywords:
Primaquine
Urea
Carbamate
Cytostatic activity
Soybean lipoxygenase
Lipid peroxidation
l
1. Introduction
maquine urea and carbamate derivatives and screening of their
antiproliferative activity.
Primaquine (PQ) is a well-known antimalarial drug and inter-
esting molecule for derivatization in the search of potential biolog-
ically active agents.1–5 Recently, we have reported the synthesis of
A number of studies have shown the pro-oxidant effects of
primaquine in blood.8–11 In addition, its metabolites have been
shown to undergo redox-cycling both in vivo and in vitro. 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 erythrocytes that overwhelm anti-
oxidant defences. The aerobic toxicity of primaquine is mediated
by superoxide anion and H2O2 and possibly by redox-active labile
metals.12 Having these facts in mind we have also focused the pres-
ent study on the oxidant/antioxidant ability of the newly prepared
primaquine derivatives.
a
series of primaquine derivatives bearing urea substituent
attached to the primaquine side-chain.6 These derivatives showed
a modest antiproliferative activity on several tumor cell lines,
whereby pyridine-substituted derivative of urea exhibited the
highest cytostatic effect and some specific activity against human
cytomegalovirus (HCMV). The present work is aimed toward
developing urea primaquine derivatives with one or more hydroxyl
groups in urea region. If the hydroxyl group is directly attached to
nitrogen atom, N-hydroxyureas are formed. These compounds are
effective metal chelators related to hydroxamic acids, and there-
fore potential antimalarial and cytostatic agents.7 In this paper
we report synthesis and full chemical characterization of new pri-
2. Results and discussion
2.1. Chemistry
A series of new urea primaquine derivatives 3a–g with one or
more hydroxyl groups in urea region were prepared. If the hydro-
xyl group is directly attached to nitrogen atom, N-hydroxyurea is
formed (3h). Scheme 1 outlines the general preparative route.
The sequence leading from 1 to 3 is identical with the one
described earlier.6 Benzotriazolide 2 was synthesized by acylation
of primaquine with 1-benzotriazole carboxylic acid chloride (1).6,13
Abbreviations: AAPH, 2,20-azobis(2-amidinopropane) dihydrochloride; BtcCl,
benzotriazole carboxylic acid chloride; DMEM, Dulbecco’s modified Eagle’s
medium; DPPH, 1,1-diphenyl-2-picrylhydrazyl radical; FBS, fetal bovine serum;
LO, lipoxygenase; LP, lipid peroxidation; NDGA, nordihydroguaiaretic acid; MTT, 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; P-Bt, primaquine
benzotriazolide.
* Corresponding author. Tel.: +385 1 4856202; fax: +385 1 4856201.
E-mail address: bzbz@pharma.hr (B. Zorc).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.030

´
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M. Šimunovic et al. / Bioorg. Med. Chem. 17 (2009) 5605–5613
N
ROH/TEA
- BtH
NH
H3CO
N
N
5a R = CH3
5b R = CH2CH3
HN
O
TEA
R
NH
H3CO
BtcCl
NH
H3CO
+
-TEA x HCl
O
1
Na2CO3
2
NH2
NHBtc
OCH3
P-H
R1NHR2/TEA
-BtH
O
O
N
HN
N
N
N
N
N
Btc
4
NH
H3CO
R1
N
3a-g
HN
R2
O
a
c
d
e
g
3
b
f
OH
OH
R1
HO
HO
N
HO
HO
HO
O
HO
H3CO
NH
H
N
H
N
R2
H
H
H
H
H
H
O
Scheme 1.
Urea primaquine derivatives 3a–g were prepared by the reaction
of benzotriazolide 2 with the corresponding amines. N-hydroxy-
urea 3h was prepared by hydrogenolysis of 1-O-benzylhydroxy-
3-[4-(6-methoxy-quinolin-8-ylamino)-pentyl]urea (3d) (Scheme
Structures of the novel compounds were deduced from the
analysis of their IR, 1H, and 13C NMR spectra and confirmed by
the elemental analysis. The chemical shifts were consistent with
the proposed structures of the novel compounds (Table 1).
2). Diazepane derivative
4 was obtained by cyclization of
benzotriazolide 2 in alkalic medium. Alcoholysis of compound 2
with methanol and ethanol gave carbamates 5a,b, while propanol,
i-propanol, n-butanol, i-butanol, tert-butanol, and triethanolamine
failed to react.
2.2. Cytostatic activity
The results of the cytostatic effect of tested compounds are
presented in Table 2 and Figure 1. Diazepane derivative 4 and
N
N
H2/Pd/C
NH
NH
H3CO
H3CO
H
N
H
N
H
N
H
N
O
OH
O
O
3d
3h
Scheme 2.

´
5607
M. Šimunovic et al. / Bioorg. Med. Chem. 17 (2009) 5605–5613
Table 1
1H and 13C NMR spectra of primaquine derivatives 3a–h, 4, and 5a,b
15
15
14
12
16
14
12
16
N
N
11
10
11
10
13
13
17
H3CO
17
H3CO
8
8
NH
NH
7
7
9
9
5
5
4
1
4
6
6
3
3
R1
N
2
2
2'
1
O
HN
HN
1'
1'
R2
R1
O
O
3a-g
5a,b
Compd
R1
R2
H
d
3'
3a
8.54–8.53 (d, 1H, 16), 8.08–8.07 (d, 1H, 14), 7.44–7.41 (m, 1H, 15), 6.47 (s, 1H, 9), 6.26 (s,
1H, 11), 6.12–6.11 (d, 1H, 7), 5.96–5.94 (t, 1H, 20), 5.80–5.78 (t, 1H, 1), 4.64–4.63 (t, 1H,
50), 3.82 (s, 3H, 17), 3.64–3.59 (m, 1H, 5), 3.37–3.33 (m, 2H, 40), 3.05–2.98 (m, 2H, 2),
2.99–2.98 (m, 2H, 30), 1.65–1.61 (m, 2H, 3), 1.54–1.41 (m, 2H, 4), 1.21–1.20 (d, 3H, 6)
158.98 (10), 158.20 (10), 144,61 (8), 144.22 (16), 134.77 (14), 134.49 (13), 129.55 (12),
122.07 (15), 96.07 (9), 91.59 (11), 60.82 (40), 54.95 (17), 47.02 (5), 42.04 (30), 39.91–
39.07 (2), 33.51 (4), 26.88 (3), 20.20 (6)
OH
5'
4'
3'
5'
3b
3c
3d
3e
H
H
H
H
8.52–8.51 (d, 1H, 16), 8.06–8.04 (d, 1H, 14), 7.41–7.39 (m, 1H, 15), 6.45 (d, 1H, 9), 6.24
(d, 1H, 11), 6.10–6.08 (d, 1H, 7), 5.83–5.82 (t, 1H, 20), 5.73–5.71 (t, 1H, 1), 4.42–4.40 (t,
1H, 60), 3.80 (s, 3H, 17), 3,61–3.58 (m, 1H, 5), 3.38–3.35 (q, 2H, 30), 3.02–2.95 (m, 3H, 2,
50), 1.62–1.59 (m, 2H, 3), 1.51–1.39 (m, 4H, 4, 40), 1.20–1.18 (d, 3H, 6)
4'
OH
6'
158.98 (10), 158.25 (10), 144.61 (8), 144.22 (16), 134.77 (14), 134.49 (13), 129.55 (12),
122.07 (15), 96.08 (9), 91.59 (11), 58.39 (50), 54.95 (17), 47.03 (5), 39.91–39.08 (2), 36.34
(30), 33.49 (4), 33.20 (40), 26.88 (3), 20.20 (6)
3'
5'
8.55–8.54 (d, 1H, 16), 8.10–8.07 (d, 1H, 14), 7.46–7.42 (m, 1H, 15), 6.49–6.48 (d, 1H, 9),
6.27 (d, 1H, 11), 6.14–6.11 (d, 1H, 7), 5.80–5.70 (m, 2H, 20, 1), 4.36–4.32 (t, 1H, 80), 3.89–
3.88 (s, 3H, 17), 3.83–3.58 (m, 1H, 5), 3.38–3.28 (m, 2H, 30), 3.05–2.90 (m, 3H, 2, 70),
1.62–1.14 (m, 13H, 40, 50, 60, 3, 4, 6)
159.47 (10), 158.52 (10), 145.10 (8), 144.72 (16), 135.27 (14), 134.98 (13), 130.05 (12),
122.55 (15), 96.57 (9), 92.07 (11), 61.13 (70), 55.45 (17), 47.51 (5), 40.82–39.16 (30, 2),
33.99 (4), 32.75 (60), 30.42 (40), 27.40 (3), 23.40 (50), 20.70 (6)
4'
7'
OH
6'
8'
7'
5'
6'
5'
6'
9.02–9.00 (s, 1H, 20), 8.56–8.53 (d, 1H, 16), 8.10–8.06 (d, 1H, 14), 7.50–7.38 (m, 6H, 15, 50,
60, 70), 6.78–6.75 (s, 1H, 9), 6.45 (s, 1H, 11), 6.21 (s, 1H, 7), 6.13–6.10 (d, 1H, 1), 4.70–6.68
(s, 2H, 30), 3.33 (s, 3H, 17), 3.62 (m, 1H, 5), 3.02 (q, 2H, 2), 1.46 (m, 4H, 3, 4), 1.31–1.20 (d,
3H, 6)
160.17 (10), 159.47 (10), 145,09 (8), 144.72 (16), 137.09 (40), 135.29 (14), 135.00 (13),
130.05 (12), 129.16 (50), 128.60 (60), 128.39 (70), 122.58 (15), 96.57 (9), 92.07 (11), 77.64
(30), 55.45 (17), 47.48 (5), 40.84–39.15 (2), 33.77 (4), 27.05 (3), 20.65 (6)
4'
3'
O
5' HO
4'
5'
OH
8.55–8.53 (d, 1H, 16), 8.09–8.06 (d, 1H, 14), 7.45–7.41 (m, 1H, 15), 6.55–6.47 (m, 2H, 9,
11), 6.27–6.26 (d, 1H, 7), 6.14–6.11 (d, 1H, 20), 5.72–5.69 (s, 1H, 1), 5.12–5.03 (t, 3H, 50),
3.83 (s, 3H, 17), 3.68–3.41 (m, 7H, 5, 40), 3.04–2.96 (m, 2H, 2), 1,68–1.40 (m, 4H, 3, 4),
1.22–1.20 (d, 3H, 6)
158.48 (10, 10), 145.11 (8), 144.73 (16), 135.28 (14), 134.98 (13), 129.97 (12), 122.58
(15), 96.59 (9), 92.10 (11), 61.86 (40), 60.94 (30), 55.46 (17), 47.51 (5), 40.89–39.15 (2),
34.03 (4), 27.16 (3), 20.77 (6)
4'
3'
OH
5'
4'
3f
8.54 (d, 1H, 16), 8.10–8.07 (d, 1H, 14), 7.46–7.42 (m, 1H, 15), 6.49–6.48 (s, 1H, 9), 6.33–
6.31 (t, 1H, 1), 6.29–6.26 (s, 1H, 11), 6.14–6.11 (d, 1H, 7), 4.84–4.77 (t, 2H, 50), 3.84 (s, 3H,
17), 3.69–3.63 (m, 1H, 5), 3.49–3.39 (m, 4H, 30), 3.30–2.24 (t, 2H, 2), 3.09–3.02 (m, 4H,
40), 1,69–1.60 (m, 2H, 3), 1.55–1.44 (m, 2H, 4), 1.23–1.21 (d, 3H, 6)
159.45 (10), 158.70 (10), 145,05 (8), 144.72 (16), 135.33–135.27 (14), 134.99 (13), 139.05
(12), 122.57 (15), 96.56 (9), 92.06 (11), 60.49 (40), 55.42 (17), 50.56 (30), 47.52 (5), 40.82–
39.14 (2), 33.89 (4), 27.16 (3), 20.64 (6)
3'
3'
OH
OH
5'
5'
4'
4'
(continued on next page)

´
M. Šimunovic et al. / Bioorg. Med. Chem. 17 (2009) 5605–5613
5608
Table 1 (continued)
Compd
R1
R2
H
d
3g
8.53 (d, 1H, 16), 8.08–8.06 (d, 1H, 14), 7.43–7.41 (m, 1H, 15), 6.47–6.46 (s, 1H, 9), 6.25 (s,
1H, 11), 6.12–6.10 (d, 1H, 7), 5.93–5.91 (t, 1H, 20), 5.80 (s, 1H, 1), 3.81 (s, 3H, 17), 3.62–
3.60 (m, 1H, 5), 3.33–3.30 (s, 2H, 30), 2.99–2.97 (m, 2H, 2), 1.63–1.60 (m, 2H, 3), 1.52–
1.41 (m, 2H, 4), 1.20–1.19 (d, 3H, 6)
158.97 (10), 158.13 (10), 144.60 (8), 144.21 (16), 134.76 (14), 134.49 (13), 129.55 (12),
122.06 (15), 96.07 (9), 91.57 (11), 54.94 (17), 47.02 (5), 40.04 (30), 39.92–39.08 (2), 33.50
(4), 26.83 (3), 20.18 (6)
15
14
12
16
N
11
10
13
17
H3CO
8
NH 7
9
5
4
6
3
2
3'
H
N
2'
1
HN
1'
3'
O
3
3h
H
8.57–8.53 (d, 1H, 16), 8.49 (s, 1H, 20), 8.23 (s, 1H, 1), 8.09–8.06 (d, 1H, 14), 7.45–7.40 (m,
1H, 15), 6.72 (s, 1H, 30), 6.48–6.47 (s, 1H, 9), 6.27 (s, 1H, 11), 6.12–6.11 (d, 1H, 7), 3.85–
3.83 (s, 3H, 17), 3.64–3.61 (m, 1H, 5), 3.07–3.03 (m, 2H, 2), 1.60–1.46 (m, 4H, 3, 4), 1.24–
1.20 (d, 3H, 6)
161.94 (10), 159.47 (10), 145,11 (8), 144.72 (16), 135.27 (14), 134.98 (13), 130.05 (12),
122.57 (15), 96.57 (9), 92.08 (11), 55.46 (17), 47.50 (5), 40.83–39.16 (2), 33.93 (4), 27.25
(3), 20.68 (6)
OH
15
14
12
16
N
11
10
O
13
4
8.53–8.51 (d, 1H, 16), 8.08–8.05 (d, 1H, 14), 7.44–7.40 (m, 1H, 15), 6.47–6.46 (s, 1H, 9),
6.25–6.24 (t, 1H, 11), 5.78–5.74 (s, 1H, 1), 3.85 (s, 3H, 17), 3.50 (m, 1H, 5), 3.02–2.96 (q,
2H, 2), 1.65–1.40 (m, 4H, 3, 4), 1.23–1.17 (d, 3H, 6)
159.47 (10), 158.51 (10), 145,10 (8), 144.70 (16), 135.35 (14), 134.98 (13), 130.04 (12),
122.63 (15), 96.56 (9), 92.07 (11), 55.45 (17), 47.56 (5), 41.38–38.89 (2), 33.98 (4), 27.38
(3), 20.68 (6)
1'
1
17
H3CO
NH
8
N
9
7
2
5
6
3
4
2'
CH3
5a
—
8.54–8.52 (d, 1H, 16), 8.09–8.06 (d, 1H, 14), 7.44–7.40 (m, 1H, 15), 7.13–7.10 (t, 1H, 1),
6.47 (s, 1H, 9), 6.26–6.25 (s, 1H, 11), 6.13–6.10 (d, 1H, 7), 3.82 (s, 3H, 17), 3.66–3.54 (m,
1H, 5), 3.50 (s, 3H, 20), 3.02–2.96 (q, 2H, 2), 1.65–1.43 (m, 4H, 3, 4), 1.21–1.19 (d, 3H, 6)
158.47 (10), 158.20 (10), 145,11 (8), 144.70 (16), 135.26 (14), 135.00 (13), 130.05 (12),
122.57 (15), 96.56 (9), 92.08 (11), 55.45 (17), 51.57 (20), 47.46 (5), 40.83–39.16 (2), 33.75
(4), 26.76 (3), 20.67 (6)
3'
CH3
5b
—
8.53–8.52 (d, 1H, 16), 8.09–8.06 (d, 1H, 14), 7.44–7.40 (m, 1H, 15), 7.09–7.06 (t, 1H, 1),
6.47 (s, 1H, 9), 6.26–6.25 (s, 1H, 11), 6.13–6.10 (d, 1H, 7), 3.98–3.90 (q, 2H, 20), 3.82 (s,
3H, 17), 3.65–3.57 (m, 1H, 5), 3.01–2.95 (q, 2H, 2), 1.68–1.40 (m, 4H, 3, 4), 1.27–1.19 (d,
3H, 30), 1.15–1.10 (d, 3H, 6)
2'
159.47 (10), 158.20 (1’), 145,10 (8), 144.70 (16), 135.27 (14), 134.99 (13), 130.05 (12),
122.57 (15), 96.56 (9), 92.07 (11), 59.86 (2’), 55.45 (17), 47.45 (5), 40.82–39.15 (2), 33.75
(4), 26.75 (3), 20.67 (6), 15.13 (3’)
carbamate 5a did not show any inhibitory effect on the tumor cell
lines’ proliferation. Similarly, compounds 3a, 3b, 3e, and 3f were
inactive, except certain inhibitory activity toward MCF-7 breast
cancer cell line that was sensitive to all these compounds, at least
at the highest concentration (10ꢀ4 M). The most prominent anti-
proliferative activity was obtained by compounds 3c, 3d, 3g, and
ted that primaquine exerts proliferative effects on MCF-7 cell lines
in the concentration range of 1–6 mg/L. Still, at the concentration
comparable to ours (50 mg/L, corresponding to approx. 100 lM)
it induced about 20% of inhibition after 24 h. This could correspond
to our results, since we measured the viability of cells after 72 h,
whereby complete inhibition was observed.14
5b (IC50 = 9–40
showed extreme selectivity toward SW 620 colon cancer cells
(IC50 = 0.2 M) and a bit less toward lung cancer cells H 460. We
showed previously that the pyridine primaquine urea derivative
showed also more pronounced activity toward SW 620 cells, com-
pared to other solid tumor-derived cells.6
Comparison of the here-presented derivatives with the parent
compound primaquine indicated that the antiproliferative activity
of urea derivatives is comparable to the primaquine activity, but
superior to a standard cytostatic drug hydroxyurea. Rossi et al. sta-
l
M). Surprisingly, hydroxypentyl-substituted 3c
N-Hydroxyurea 3h with hydroxyl group directly attached to urea
nitrogen did not show significant antiproliferative activity as we
expected. Diethanol amine derivative 3f was similarly inactive as
triethanol amine derivative 3e, but both compounds showed certain
selectivity toward MCF-7 cell line. Concerning structure activity
relationship several points could be concluded: (i) comparing the
length of alkyl chain attached to nitrogen atom of urea, it seems that
longer chain has better antiproliferative activity, whereby hydroxy-
pentyl urea shows a very differential effect, being selectively cyto-
toxic to SW 620 cells; (ii) compounds 3e and 3f with two or three
l

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M. Šimunovic et al. / Bioorg. Med. Chem. 17 (2009) 5605–5613
Table 2
Inhibitory effects of primaquine derivatives on the growth of malignant tumor cell lines
a
Compd
Tumor cell growth [IC50
(l
M)]
MCF-7
MOLT-4
>100
HCT 116
SW 620
H 460
>100
OCH3
3a
>100
>100
25 22
31 30
44 39
NH
H
N
H
N
N
C
O
OH
OCH3
3b
3c
3d
3e
3f
>100
>100
>100
>100
NH
NH
H
N
H
N
N
OH
C
O
OCH3
61 40
12 0.7
>100
29
3
0.2 0.01
2
0.5
H
N
H
N
N
OH
C
O
OCH3
14 0.3
>100
>100
20
2
9
6
21
2
NH
NH
H
N
H
N
N
C
O
O
OCH3
HO
>100
24
5
>100
OH
H
N
H
N
N
C
O
OH
OCH3
>100
>100
36 17
>100
NH
OH
OH
H
N
N
N
C
O
OCH3
3g
10 0.3
15
7
17 15
31 20
18 14
NH
O
H
N
H
N
N
C
C
O
N
N
H
H
HN
(continued on next page)

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M. Šimunovic et al. / Bioorg. Med. Chem. 17 (2009) 5605–5613
Table 2 (continued)
a
Compd
Tumor cell growth [IC50
(l
M)]
MCF-7
MOLT-4
>100
HCT 116
SW 620
H 460
>100
OCH3
3h
57
2
35 17
44 36
NH
H
N
H
N
N
C
O
OH
OCH3
O
N
4
P100
>100
>100
>100
>100
HN
N
CH3
NH
OCH3
5a
>100
>100
>100
>100
>100
H
N
N
O
C
O
OCH3
5b
34
4
28
20
4
45
9
26
5
38 13
NH
H
N
N
O
C
O
Primaquine
Hydroxyurea
N.T.b
>100
6
P100
>100
28 10
>100
30
7
223 120
>100
a
IC50: the concentration that causes 50% growth inhibition.
N.T.: not tested.
b
3c
3g
150
150
100
50
MOLT-4
SW 620
H 460
100
50
MOLT-4
SW 620
H 460
0
0
MCF-7
HCT116
MCF-7
HCT116
-50
-100
-50
-100
-9
-8
-7
-6
-5
-4
-3
-9
-8
-7
-6
-5
-4
-3
log concentration (M)
log concentration (M)
Figure 1. Dose–response profiles for 3c and 3g tested on various human cell lines in vitro. The cells were exposed to the compounds at different concentrations, and the
percentage of growth (PG) was calculated. Each point represents a mean value of four replicates in three individual experiments.
hydroxyl groups attached to the terminal nitrogen urea atom,
showedcomparable selectivitytowardMCF-7ascompound3abear-
ing only one hydroxyl group at the same distance; the same selectiv-
ity was noticed with analogous diethylamine urea that was
previously tested;6 and (iii) the ethyl carbamate 5b has substantially
stronger activity than its methyl analogue 5a.
prepared and tested in our previous paper.6 The fact that compound
3d is equally active as pyridine derivative described in the same
paper suggests that electrophilic atom (oxygen or nitrogen) in lipo-
philic surrounding potentiates cytostatic activity.
2.3. Antioxidant activity
Better activity, but less selectivity was achieved by 3g, which is
actually a twin drug, composed of two primaquine molecules
connected by ethylenediamide spacer. Comparable activity showed
O-benzyl derivative 3d with lipophilic benzene ring. This compound
was more active than 1-benzyl-3-[4-(6-methoxy-quinolin-8-ylami-
no)-pentyl]urea, similar compound lacking only oxygen atom,
Several methods are used for the estimation of efficiency of syn-
thetic antioxidants. In this paper the attention is focused on the
DPPH assay, which is one of the best-known, frequently employed
and accurate methods. DPPH (1,1-diphenyl-2-picrylhydrazyl) is a
stable free radical because of its spare electron delocalization over

´
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M. Šimunovic et al. / Bioorg. Med. Chem. 17 (2009) 5605–5613
Table 3
Interaction with DPPH, in vitro inhibition of soybean lipoxygenase (LOX) and inhibition of lipid peroxidation (LP)
Compd
DPPH 20 mina (%)
DPPH 60 mina (%)
DPPH 20 minb (%)
DPPH 60 minb (%)
LOX IC50
(l
M)
LP inhibitionb (%)
2
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
4
5a
5b
18.4
3.7
12.2
n.a.
20.0
10.6
27.7
n.a.
n.a.
22.3
7.4
27.6
60.6
21.3
16.3
28.8
40.0
13.8
8.5
16.7
60.7
28.6
34.5
11.4
28.6
n.a.
35.7
88.1
26.2
27.4
42.9
48.8
75
73.8
51.2
33.3
46.4
38.1
40.5
11.9
40.5
84.5
19.0
25.0
42.9
57.1
48.8
31.0
21.9
260
315
230
310
230
n.a.
86
n.a.c
81
56
63
82
86
85
99
69
99
99
96
88
83
87
45
63
n.a.
24.4
20.7
34.2
2.2
15.8
28.9
3.9
30.5
24.4
10.8
17.2
62
83
220
n.a.
320
72
n.a.
n.a.
74
n.a.
18.1
Primaquine
Trolox
NDGAd
Caffeic acid
19.0
81
83
600
Concentrations of the tested compounds: a5 ꢁ 10ꢀ5 M, b1 ꢁ 10ꢀ4 M.
cn.a.—no activity; dNDGA—nordihydroguaiaretic acid.
the whole molecule. The reducing abilities of the new primaquine
derivatives were determined by the use of the stable radical DPPH
at 0.05 and 0.1 mM after 20 and 60 min (Table 3). This interaction
indicates their radical scavenging ability in an iron-free system and
expresses the reducing activity of compounds.15 The reducing abil-
ities ranged from 2.2% to 88.1%. Slight differences were observed
within the compounds with the time and the concentration. In
general, it seems that compounds interact with DPPH in a concen-
tration dependent manner. Compound 3h, without a methylene
spacer between urea nitrogen atom and hydroxyl group, showed
the highest interaction value, followed by 2, 4, and 3a. These
compounds showed a higher interaction than the parent com-
pound primaquine. Compounds 3c, 3d, 3i, and 3f did not present
any significant interaction. The small changes in the carbon chain
size (2C/3C) did not influence the reducing ability. The presence
of 5C chain or of benzyloxy group leads to the loss of the interac-
tion. Between the two 5a–b carbamates the methyl derivative is
more active.
Further on, inhibitory activity on lipid peroxidation of all pre-
pared compounds was screened. Azo compounds generating free
radicals through spontaneous thermal decomposition are useful
for free radical production studies in vitro. The water soluble azo
compound 2,20-(2-amidinopropane) dihydrochloride (AAPH) has
been extensively used as a clean and controllable source of ther-
mally produced alkylperoxyl free radicals. In our studies AAPH
was used as a free radical initiator to follow oxidative changes of
linoleic acid to conjugated diene hydroperoxide. Under the
reported experimental conditions all the tested compounds pre-
sented high inhibition of lipid peroxidation, except benzotriazolide
2 (Table 3). The LP inhibition of the tested primaquine derivatives
was much higher than the inhibition of primaquine phosphate: 11
of 14 derivatives highly inhibited LP (81–99%), while primaquine
showed the inhibition of 45%. These results strongly suggest that
the introduction of the urea or carbamate moiety is responsible
for improved LP inhibition.
derivative 4, hydroxyurea 3h, and urea 3f with IC50 = 62, 72, 83,
and 86 M, respectively (Table 3). It is important to note that two
most potent compounds from the tested series are the most lipo-
philic derivatives bearing bulky substituents. However, the results
of LOX inhibition are comparable with the result of primaquine
phosphate.
l
3. Conclusions
The most prominent antiproliferative activity was obtained by
hydroxypentyl derivative 3c, O-benzyl derivative 3d, compound
bearing two primaquine moieties 3g and carbamate derivative
5b, but the activities were comparable to the one obtained by
primaquine. Still, the most valuable observation is the extreme
selectivity toward SW 620 colon cancer cells (IC50 = 0.2
a bit less toward lung cancer cells H 460 exerted by 1-(5-hydroxy-
pentyl)-3-[4-(6-methoxy-quinolin-8-ylamino)-pentyl]urea (3c).
lM) and
This selectivity should be additionally evaluated and its exact
mechanism elucidated. On the other hand, all new derivatives
showed significant antioxidant activities. Hydroxyurea 3h strongly
interacted with DPPH. Compound 3g showed very significant inhi-
bition on LOX soybean. All the tested urea and carbamate deriva-
tives highly inhibited lipid peroxidation, significantly stronger
than primaquine phosphate.
4. Experimental
4.1. General methods
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. 1H and 13C
NMR spectra were recorded on a Varian Gemini 300 spectrometer,
operating at 300 and 75.5 MHz for the 1H and 13C nuclei, respec-
tively. Samples were measured in DMSO-d6 solutions at 20 °C in
5 mm NMR tubes. Chemical shifts (d) were referred to TMS.
Precoated Merck Silica Gel 60 F254 plates and solvent systems
cyclohexane/ethyl acetate/methanol (3:1:0.5) or chloroform/meth-
anol (9.5:0.5) were used for thin-layer chromatography. Spots
were visualized by short-wave UV light and iodine vapor. Column
chromatography was performed on silica gel (0.063–0.200 mm),
with chloroform/methanol (9.5:0.5) or cyclohexane/ethyl acetate/
methanol (3:1:0.5) as eluents. Primaquine diphosphate, 2-hydroxy-
Leukotrienes play an important role as mediators of a variety of
inflammatory and allergic reactions and are derived from the bio-
transformation of arachidonic acid catalyzed by lipoxygenase
(LOX). Lipoxygenases play a role in membrane lipid peroxidation
by forming hydroperoxides in the lipid bilayer.16 Inhibitors of LOX
have attracted attention initially as potential agents for the treat-
ment of inflammatory and allergic diseases. Twin primaquine deriv-
ative 3g was the most potent LOX inhibitor, followed by diazepane

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M. Šimunovic et al. / Bioorg. Med. Chem. 17 (2009) 5605–5613
5612
ethanamine, 3-hydroxypropanamine, 5-hydroxypentanamine,
O-benzylhydroxylamine hydrochloride, tris(hydroxymethyl)ami-
nomethane, diethanolamine, ethylenediamine, DPPH, AAPH, nor-
dihydroguaiaretic acid (NDGA), sodium linoleate, soybean
lipoxygenase, caffeic acid, and trolox were purchased from Sig-
ma–Aldrich. Human cell lines for cytostatic activity were obtained
from American Type Culture Collection.
1204, 1163, 1051, 1029, 908, 823, 791, 749, 699 cmꢀ1. Anal.
(C23H28N4O3) C, H, N.
4.4.5. 1-(2-Hydroxy-1,1-bis-hydroxymethyl-ethyl)-3-[4-(6-
methoxy-quinolin-8-ylamino)-pentyl]urea (3e)
From the reaction of 0.500 g (1.24 mmol) primaquine
benzotriazolide
2
and 0.150 g (1.24 mmol) tris(hydroxy-
methyl)aminomethane at rt for 4 days and recrystallization from
acetone/petrolether, 0.227 g (45%) of 3e was obtained; mp 147–
149 °C; IR (KBr): mmax 3357, 3208, 2931, 2658, 2300, 1713, 1621,
1590, 1522, 1456, 1426, 1390, 1337, 1281, 1224, 1206, 1164,
1130, 1101, 1048, 1015, 818 cmꢀ1. Anal. (C20H30N4O5) C, H, N.
4.2. 1-Benzotriazole carboxylic acid chloride (BtcCl, 1)
BtcCl was prepared from benzotriazole and triphosgene.13
4.3. Primaquine benzotriazolide (P-Bt, 2)
4.4.6. 1,1-Bis-(2-hydroxyethyl)-3-[4-(6-methoxy-quinolin-8-
ylamino)-pentyl]urea (3f)
From the reaction of 0.500 g (1.24 mmol) primaquine
benzotriazolide 2 and 0.130 g (1.24 mmol) diethanolamine at rt for
4 days and purification of the crude product by column chromatog-
raphy (eluent cyclohexane/ethyl acetate/methanol 3:1:0.5), 0.304 g
Compound 2 was prepared by acylation of primaquine base
with chloride 1.6 Primaquine base was prepared from primaquine
diphosphate, keeping the drug solution light protected.
4.4. Primaquine urea derivatives (3a–g). General procedure
(63%) of 3f was obtained; oil; IR (KBr): mmax 3369, 2935, 1615, 1577,
To a solution of 0.500 g (1.24 mmol) of primaquine benzotriazo-
lide 2 and 0.300 g (2.98 mmol) of triethylamine in anhydrous
ethanol (30 mL), 1.24 mmol of appropriate amine was added. The
reaction mixture was stirred at rt, light protected, and evaporated
in vacuum. The obtained residue was dissolved in chloroform
(125 mL), extracted three times with NaOH solution (pH up to 9),
washed with water, and dried over anhydrous Na2SO4. Chloroform
was evaporated under reduced pressure to give crude product.
1520, 1456, 1424, 1388, 1220, 1203, 1160, 1076, 1051, 971, 873, 833,
791, 755, 679 cmꢀ1. Anal. (C20H30N4O4) C, H, N.
4.4.7. 1-[4-(6-Methoxy-quinolin-8-ylamino)-pentyl]-3-/2-{3-[4-
(6-methoxy-quinolin-8-ylamino)-pentyl]-ureido}-ethyl/urea (3g)
From the reaction of 0.500 g (1.24 mmol) primaquine
benzotriazolide 2, 0.037 g (0.62 mmol) ethylenediamine and
0.125 g (1.24 mmol) triethylamine at rt for 2 days and recrystalliza-
tion from ether, 0.323 g (41.4%) of 3g was obtained; mp 151–153 °C;
4.4.1. 1-(2-Hydroxyethyl)-3-[4-(6-methoxy-quinolin-8-
ylamino)-pentyl]urea (3a)
IR (KBr): mmax 3342, 3050, 2928, 2859, 2350, 2285, 1623, 1576, 1519,
1453, 1422, 1387, 1337, 1262, 1220, 1202, 1157, 1051, 1029, 969,
From the reaction of 0.500 g (1.24 mmol) primaquine
benzotriazolide 2 and 0.076 g (1.24 mmol) 2-hydroxyethanamine
at rt for 4 days and recrystallization from acetone/petrolether,
0.303 g (70.8%) of 3a was obtained; mp 124–126 °C; IR (KBr): mmax
3390, 3333, 2939, 2860, 1610, 1575, 1516, 1451, 1387, 1221, 1203,
1168, 1053, 1028, 825, 794, 624 cmꢀ1. Anal. (C18H26N4O3) C, H, N.
900, 821, 791, 677 cmꢀ1. Anal. (C34H46N8O4) C, H, N.
4.4.8. 1-Hydroxy-3-[4-(6-methoxy-quinolin-8-ylamino)-
pentyl]urea (3h)
A suspension of 0.506 g (1.24 mmol) 1-O-benzylhydroxy-3-[4-
(6-methoxy-quinolin-8-ylamino)-pentyl]urea (3d) and 10% Pd/C
(75 mg) in anhydrous methanol (20 mL) was hydrogenated at
ambient pressure and rt, light protected, under nitrogen atmo-
sphere. After 3 h the reaction mixture was filtered and the filtrate
was evaporated under reduced pressure. After purification of the
crude product by column chromatography (eluent chloroform/
methanol 95:5), 0.101 g (26%) of 3h was obtained; mp 144–
147 °C; IR (KBr): mmax 3368, 3139, 2920, 2861, 1642, 1560, 1523,
1427, 1386, 1333, 1228, 1206, 1170, 1060, 1038, 981, 904, 846,
829, 821, 790, 777, 749, 678, 644, 624, 564, 534 cmꢀ1. Anal.
(C16H22N4O3) C, H, N.
4.4.2. 1-(3-Hydroxypropyl)-3-[4-(6-methoxy-quinolin-8-
ylamino)-pentyl]urea (3b)
From the reaction of 0.500 g (1.24 mmol) primaquine
benzotriazolide 2 and 0.093 g (1.24 mmol) 3-hydroxypropanamine
at rt for 4 days and recrystallization from acetone/petrolether,
0.312 g (70%) of 3b was obtained; mp 94–99 °C; IR (KBr): mmax
3346, 2935, 2879, 2860, 1618, 1576, 1518, 1475, 1460, 1450,
1424, 1390, 1371, 1271, 1222, 1205, 1171, 1158, 1073, 1053,
1029, 823, 793, 680, 625 cmꢀ1. Anal. (C19H28N4O3) C, H, N.
4.4.3. 1-(5-Hydroxypentyl)-3-[4-(6-methoxy-quinolin-8-
ylamino)-pentyl]urea (3c)
4.4.9. 1-Cyclopentyl-3-[4-(6-methoxy-quinolin-8-ylamino)-
pentyl]urea (3i), 1-cyclohexyl-3-[4-(6-methoxy-quinolin-8-
ylamino)-pentyl]urea (3j), 1,1-dicyclohexyl-3-[4-(6-methoxy-
quinolin-8-ylamino)-pentyl]urea (3k)
From the reaction of 0.500 g (1.24 mmol) primaquine
benzotriazolide 2 and 0.128 g (1.24 mmol) 5-hydroxypentanamine
at rt for 4 days and recrystallization from acetone/petrolether and
trituration with ether, 0.351 g (73%) of 3c was obtained; mp 81–
88 °C; IR (KBr): mmax 3349, 2933, 2859, 1624, 1579, 1523, 1457,
1425, 1388, 1263, 1237, 1226, 1205, 1170, 1057, 1032, 823,
791 cmꢀ1. Anal. (C21H32N4O3) C, H, N.
These compounds are prepared according to the previously
published method.6
4.5. 1-(6-Methoxy-quinolin-8-yl)-7-methyl-[1,3]-diazepan-2-
one (4)
4.4.4. 1-O-Benzylhydroxy-3-[4-(6-methoxy-quinolin-8-
ylamino)-pentyl]urea (3d)
To a solution of 0.500 g (1.24 mmol) primaquine benzotriazo-
lide (2) in acetone (30 mL),
a sodium carbonate solution
From the reaction of 0.500 g (1.24 mmol) primaquine
benzotriazolide 2 and 0.394 g (2.48 mmol) O-benzylhydroxyl-
amine hydrochloride at rt for 4 days and purification of the crude
product by column chromatography (eluent chloroform/methanol
9.5:0.5), 0.329 g (65%) of 3d was obtained; oil; IR (KBr): mmax
3386, 3199, 2925, 2863, 1672, 1616, 1577, 1521, 1456, 1386,
(w = 10%) (pH up to 10) was added. The reaction mixture was pro-
tected from light, stirred for 30 min. at rt and evaporated in vac-
uum. The obtained residue was dissolved in chloroform (125 mL),
extracted three times with the NaOH solution (pH up to 9), washed
with water, and dried over anhydrous Na2SO4. Chloroform was
evaporated under reduced pressure to give product 4. After purifi-

´
5613
M. Šimunovic et al. / Bioorg. Med. Chem. 17 (2009) 5605–5613
cation of the crude product by column chromatography (eluent
cyclohexane/ethyl acetate/methanol 3:1:0.5), 0.336 g (95%) of 4
was obtained; mp 122–126 °C; IR (KBr): mmax 3376, 2961, 2928,
2857, 1616, 1575, 1520, 1456, 1424, 1388, 1220, 1203, 1159,
1051, 1031, 821, 791 cmꢀ1. Anal. (C16H19N3O2) C, H, N.
recorded at 517 nm and was compared to the appropriate standard
NDGA (Table 3). Ethanol was used as a control.18
4.9. Soybean lipoxygenase inhibition activity
DMSO solution of the tested compound was incubated with
sodium linoleate (0.1 mM) and 0.2 mL of soybean lipoxygenase
solution (1/9 ꢁ 104 w/v in saline) at room temperature. The conver-
sion of sodium linoleate to 13-hydroperoxylinoleic acid was
recorded at 234 nm and was compared to the standard inhibitor caf-
feic acid (Table 3), according to the procedure previously reported.18
4.6. Primaquine carbamate derivatives (5a,b). General
procedure
To a solution of 0.500 g (1.24 mmol) primaquine benzotriazo-
lide (2) in anhydrous methanol or ethanol (30 mL), 0.125 g
(1.24 mmol) of triethylamine was added. The reaction mixture
was protected from light, refluxed, and evaporated in vacuum.
The obtained residue was dissolved in chloroform (125 mL),
extracted three times with the NaOH solution (pH up to 9), washed
with water, and dried over anhydrous Na2SO4. Chloroform was
evaporated under reduced pressure to give crude product.
4.10. Inhibition of linoleic acid lipid peroxidation
Oxidation of linoleic acid to conjugated diene hydroperoxide in
an aqueous dispersion is monitored at 234 nm. AAPH was used as a
free radical initiator.19 Ten microliters of the 16 mM linoleic acid
dispersion were added to the UV cuvette containing 0.93 mL of
0.05 M phosphate buffer, pH 7.4 prethermostated at 37 °C. The oxi-
dation reaction was initiated at 37 °C under air by the addition of
4.6.1. Methyl-N-[4-(6-methoxy-quinolin-8-ylamino)-pentyl]-
carbamate (5a)
From the reaction of 0.500 g (1.24 mmol) primaquine
benzotriazolide 2 in anhydrous ethanol for 7 h at 65 °C and purifica-
tion of the crude product by column chromatography (eluent cyclo-
hexane/ethyl acetate/methanol 3:1:0.5), 0.334 g (85%) of 5a was
obtained; oil; IR (KBr): mmax 3378, 2936, 2867, 1722, 1704, 1694,
1615, 1595, 1578, 1520, 1455, 1423, 1387, 1259, 1220, 1204, 1159,
1051, 1030, 822, 792, 778 cmꢀ1. Anal. (C17H23N3O3) C, H, N.
50
l
L of 40 mM AAPH solution. Oxidation was carried out in the
presence of compounds (10
lL, final concentration 0.1 mM). In
the assay with no antioxidant lipid oxidation was measured in
the presence of the same level of DMSO. The rate of oxidation
was monitored at 37 °C by recording the increase of absorption
at 234 nm caused by conjugated diene hydroperoxides. The results
were compared to that of the standard inhibitor trolox.
4.6.2. Ethyl-N-[4-(6-methoxy-quinolin-8-ylamino)-pentyl]-
carbamate (5b)
Acknowledgment
From the reaction of 0.500 g (1.24 mmol) primaquine ben-
zotriazolide 2 in anhydrous methanol for 10 h at 80 °C and purifi-
cation of the crude product by column chromatography (eluent
cyclohexane/ethyl acetate/methanol 3:1:0.5), 0.332 g (81%) of 5b
was obtained; oil; IR (KBr): mmax 3377, 2962, 2935, 2868, 1699,
1615, 1595, 1577, 1520, 1455, 1423, 1388, 1336, 1258, 1220,
Support for this study was provided by Ministry of Science, Edu-
cation and Sports of the Republic of Croatia (Projects 006-0000000-
3216 and 098-0982464-2514).
References and notes
1204, 1159, 1051, 1031, 822, 792, 679, 625 cmꢀ1
(C18H25N3O3) C, H, N.
. Anal.
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4.8. Interaction with 1,1-diphenyl-2-picrylhydrazyl (DPPH)
activity
To a solution of DPPH (0.05 mM) in absolute ethanol an equal
volume of 0.1 or 0.05 mM ethanolic solution of the tested com-
pound was added. After 20 and 60 min the absorbance was