Design, synthesis and biological evaluation of novel primaquine-cinnamic acid conjugates of the amide and acylsemicarbazide type

Article abstract of DOI:10.3390/molecules21121629

In this paper design and synthesis of a scaffold comprising primaquine (PQ) motif and cinnamic acid derivatives (CADs) bound directly (compounds 3a-k) or via a spacer (compounds 7a-k) are reported. In the first series of compounds, PQ and various CADs were connected by amide bonds and in the second series by acylsemicarbazide functional groups built from the PQ amino group, CONHNH spacer and the carbonyl group originating from the CADs. PQ-CAD amides 3a-k were prepared by a simple one-step condensation reaction of PQ with a series of CAD chlorides (method A) or benzotriazolides 2 (method B). The synthesis of acylsemicarbazides 7a-k included activation of PQ with benzotriazole, preparation of PQ-semicarbazide 6 and its condensation with CAD chlorides 4. All synthesized PQ-CAD conjugates were evaluated for their anticancer, antiviral and antioxidative activities. Almost all compounds from series 3 were selective towards the MCF-7 cell line and active at micromolar concentrations. The o-fluoro derivative 3h showed high activity against HeLa, MCF-7 and in particular against the SW 620 cell line, while acylsemicarbazide 7f with a benzodioxole ring and 7c, 7g and especially 7j with methoxy-, chloro- or trifluoromethyl-substituents in the para position showed high selectivity and high inhibitory activity against MCF-7 cell line at micromolar (7c, 7f, 7g) and nanomolar (7j) levels. Acylsemicarbazide derivatives with trifluoromethyl group(s) 7i, 7j and 7k showed specific activity against human coronavirus (229E) at concentrations which did not alter the normal cell morphology. The same compounds exerted the most potent reducing activity in the DPPH test, together with 7d and 7g, while methoxy (compounds 7c-e), benzodioxole (7f), p-Cl (7g) and m-CF₃ (7i) acylsemicarbazides and amide 3f presented the highest LP inhibition (83%-89%). The dimethoxy derivative 7d was the most potent LOX inhibitor (IC₅₀ = 10 μM). The performed biological tests gave evidence of acylsemicarbazide functional group as superior binding group in PQ-CAD conjugates.

Full text of DOI:10.3390/molecules21121629

molecules
Article
Design, Synthesis and Biological Evaluation of Novel
Primaquine-Cinnamic Acid Conjugates of the Amide
and Acylsemicarbazide Type
1
1
Kristina Pavic´ , Ivana Perkovic´ , Petra Gilja 1, Filip Kozlina 1, Katja Ester 2, Marijeta Kralj 2,
Dominique Schols 3, Dimitra Hadjipavlou-Litina 4, Eleni Pontiki 4 and Branka Zorc 1,
*
1
Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacˇic´a 1, HR-10 000 Zagreb, Croatia;
kpavic@pharma.hr (K.P.); iperkovic@pharma.hr (I.P.); pgilja@student.pharma.hr (P.G.);
filip.kozlina@gmail.com (F.K.)
2
3
4
Division of Molecular Medicine, Rudjer Boškovic´ Institute, Bijenicˇka cesta 54, HR-10 000 Zagreb, Croatia;
katja.ester@irb.hr (K.E.); marijeta.kralj@irb.hr (M.K.)
Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10,
B-3000 Leuven, Belgium; dominique.schols@rega.kuleuven.be
Faculty of Health Sciences, School of Pharmacy, Aristotles University of Thessaloniki, Thessaloniki 54 124,
Greece; hadjipav@pharm.auth.gr (D.H.-L.); epontiki@pharm.auth.gr (E.P.)
Correspondence: bzorc@pharma.hr; Tel.: +385-1-485-62-02; Fax: +385-1-485-62-01
*
Academic Editors: Jean Jacques Vanden Eynde and Annie Mayence
Received: 18 October 2016; Accepted: 24 November 2016; Published: 28 November 2016
Abstract: In this paper design and synthesis of a scaffold comprising primaquine (PQ) motif and
cinnamic acid derivatives (CADs) bound directly (compounds 3a–k) or via a spacer (compounds 7a–k)
are reported. In the first series of compounds, PQ and various CADs were connected by amide bonds
and in the second series by acylsemicarbazide functional groups built from the PQ amino group,
CONHNH spacer and the carbonyl group originating from the CADs. PQ-CAD amides 3a
prepared by a simple one-step condensation reaction of PQ with a series of CAD chlorides (method A)
or benzotriazolides (method B). The synthesis of acylsemicarbazides 7a included activation of PQ
with benzotriazole, preparation of PQ-semicarbazide and its condensation with CAD chlorides
All synthesized PQ-CAD conjugates were evaluated for their anticancer, antiviral and antioxidative
activities. Almost all compounds from series were selective towards the MCF-7 cell line and active
–k were
2
–k
6
4.
3
at micromolar concentrations. The o-fluoro derivative 3h showed high activity against HeLa, MCF-7
and in particular against the SW 620 cell line, while acylsemicarbazide 7f with a benzodioxole ring
and 7c
showed high selectivity and high inhibitory activity against MCF-7 cell line at micromolar (7c
and nanomolar (7j) levels. Acylsemicarbazide derivatives with trifluoromethyl group(s) 7i
showed specific activity against human coronavirus (229E) at concentrations which did not alter
the normal cell morphology. The same compounds exerted the most potent reducing activity in the
DPPH test, together with 7d and 7g, while methoxy (compounds 7c ), benzodioxole (7f), p-Cl (7g
and m-CF3 (7i) acylsemicarbazides and amide 3f presented the highest LP inhibition (83%–89%).
The dimethoxy derivative 7d was the most potent LOX inhibitor (IC50 = 10 M). The performed
,
7g and especially 7j with methoxy-, chloro- or trifluoromethyl-substituents in the para position
7f 7g
7j and
,
,
,
)
7k
–e
)
µ
biological tests gave evidence of acylsemicarbazide functional group as superior binding group in
PQ-CAD conjugates.
Keywords: primaquine; cinnamic acid derivative; conjugate; cytostatic activity; antiviral activity;
antioxidative activity
Molecules 2016, 21, 1629; doi:10.3390/molecules21121629
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1629
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1. Introduction
The molecular hybridization approach based on the combination of the pharmacophoric
moieties of different compounds was used to produce new hybrid molecules with cinnamic acid
derivatives (CADs) and primaquine (PQ) motifs. Cinnamic acid (CA, trans-3-phenylacrylic acid,
trans-3-phenyl-2-propenoic acid) and its derivatives are naturally occurring substances found in
various plants. They are important intermediates in biosynthetic pathways of secondary metabolites,
which play key roles in plant growth, development, reproduction and disease resistance [
research papers report various pharmacological activities of CADs [ ]. First of all, they exhibit
strong antimicrobial, antifungal and antiviral activity [ ]. A review on natural and synthetic
CADs with antimicrobial activity has been recently published [ ]. CADs show good to moderate
antitubercular activity or synergistic activity with antitubercular drugs [ 13]. Many CADs, especially
those with phenolic hydroxyl groups, are well-known antioxidants and are supposed to have several
health benefits due to their strong free radical scavenging properties [14 16]. Methoxy substituted
1]. Numerous
2,3
4–8
1
9–
–
cinnamates play an important role in controlling inflammatory diseases [17], while methoxy substituted
octylcinnamates are efficient sunscreen agents [18]. 3-Hydroxy, 4-hydroxy and 3,4-dihydroxy CADs
possess hepatoprotective [19] and hypolipidemic activity [20,21], while halogenated derivatives have
CNS depressant activity [22]. Some amides derived from CA are potential antimalarial leads [23].
N-cinnamoyl chloroquine, PQ, aminoacridines and 4-aminoquinoline derivatives are reported as
dual-action antimalarials and antiproliferative agents [24–27]. Anticancer potential of piplartine,
a CA alkaloid/imide derivative from peppers, and various CADs have been reported in numerous
papers and reviewed by Bezerra [28] and De and co-workers [29]. Finally, CA motifs are present in
several approved antiallergic drugs, such as cinnarizine, cinaserin and tranilast and antiplatelet agent
ozagrel [30].
On the other hand, PQ is a well-known antimalarial drug with pronounced antiproliferative
activity. Numerous reports corroborate anticancer properties of antimalarial drug classes and their
usefulness in adjuvant chemotherapies [31–41]. Several antimalarials reached a clinical stage in
anticancer research [42].
In several papers, we have shown that PQ derivatives of urea and acylsemicarbazide type possess
strong antiproliferative effects against a number of tumor cell lines and/or high selectivity towards
the breast cell line MCF-7 [43–47]. The most active compounds have one or more aromatic rings
attached to a PQ scaffold via nitrogen- and oxygen-rich spacers (urea, carbamate, hydroxyurea,
double urea). The literature survey showed that hydroxysemicarbazides are potential antitumor
agents as well [48]. In the light of the above considerations, we have designed a series of hybrid
molecules with cinnamoyl residue as the aromatic component bound by amide or acylsemicarbazide
functionality to PQ pharmacophore. Our new series of PQ derivatives differ from the previous ones
both in the aromatic region and in the type of spacers. The present study reports the synthesis and
characterization of PQ-CAD conjugates and their evaluation as potential anticancer and antiviral
agents. The chemopreventive potential on carcinogenesis, inhibition of lipoxygenase as a marker of
anti-inflammatory activity and antioxidative ability of PQ-CAD were evaluated as well.
2. Results and Discussion
2.1. Chemistry
Two series of PQ-CAD conjugates 3a
acid or methoxy, dimethoxy, trimethoxy, methylenedioxy, chloro, fluoro, trifluoromethyl and
bis-trifluoromethylcinnamic acid as the CAD component. In series PQ and CA Q moieties are
directly bound by an amide bond, while in series they are connected by the CONHNH spacer.
–k and 7a–k were prepared, with CA, α-methylcinnamic
3
7
Terminal amino group of PQ, the hydrazide spacer and CA carbonyl group together form a new
acylsemicarbazide functional group. The synthetic route leading to PQ-CAD conjugates is outlined in
Scheme 1.

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Scheme 1. Synthetic route for preparation of compounds
conditions: ( ) CA or CAD, TEA, toluene, 0.5 h; ( ) PQ, TEA, dioxane, 20 h; (
3 h; ( ) PQ, TEA, CH2Cl2, 0.5–3 h; ( ) PQ, TEA, toluene, 24 h; ( ) NH2NH2 H2O, Na2S2O4, dioxane,
4 days; ( 4a , TEA, CH2Cl2, overnight. All reactions were performed at r.t. The reactions with PQ
3
and
7
and their precursors. Reagents and
a
b
c) SOCl2, toluene, DMF,
d
e
f
×
g)
–k
were run light protected.
Two methods for the preparation of amides 3a–k were applied: acid chlorides 4 (method A) and
benzotriazolides
products in similar yields. CAD-benzotriazolides
previously: benzotriazolides of various carboxylic acids were synthesized and transformed to esters,
amides or hydroxamic acids [49 52]. Benzotriazolides (active amides) and PQ-benzotriazolide
(active urea) were prepared from the same starting compound BtcCl ( ) and CAD or PQ, in the
presence of triethylamine (TEA). Reaction of with hydrazine gave semicarbazide , which reacted
with chlorides and afforded PQ-CAD conjugates of general formula . Compounds and are stable
intermediates applied in our previous work for syntheses of numerous PQ derivatives [43,46].
2
(method B) were used as the activated CAD intermediates. Both methods gave
2
were prepared following our procedure described
–
2
5
1
5
6
4
7
5
6
1
All new compounds were fully characterized by the usual spectroscopic methods (IR, H-,
13C-NMR and MS) and elemental analyses. Spectral data are consistent with the proposed structures
and are summarized in the Experimental section and in detail in the Supplementary Materials.
Syntheses of compounds 3a
analytical data [26]. Compounds
their structures were confirmed indirectly.
The presence of the PQ residue in compounds
hydrogen atoms next to pyridine nitrogen occurred in 1H-NMR spectra at
,
3c and 3g were reported previously, but without any spectral and
2
and were used in further reaction steps without purification and
4
3,
5,
6
and
7
was confirmed by NMR spectra:
between 8.55 and 8.51,
δ
methoxy group at 3.81–3.80, CH of chiral carbon as a multiplet at 3.70–3.63, methyl group between
1.24 and 1.20 ppm, while in 13C spectra the corresponding carbon atoms appeared at 144.27–144.18,
55.18–54.91, 47.01–46.98 and 20.25–20.19 ppm, respectively. CAD benzene ring afforded additional
hydrogen or carbon signals in aromatic region. Signals of methoxy groups in products 3c
–
e
and
and
7c–e were located very close to the PQ methoxy group. CF3 groups present in 3i–k and 7i–k
adjacent aromatic carbon atoms split in 13C-NMR spectra and appeared as quartets at 132.28–118.69
and 131.14–128.44 ppm, respectively.
Amide NH signals in series
6.16–6.11 ppm (all D2O exchangeable). Compounds from series
3
were visible in the 1H-NMR spectra at 8.24–7.95 and amine NH at
7
showed four NH signals: the NH

Molecules 2016, 21, 1629
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close to the CAD carbonyl appeared as a singlet between 9.85 and 9.62 ppm, the next NH as a singlet
between 7.99 and 7.67, the PQ terminal amino group between 6.53 and 6.43 and PQ NH group next to
chiral atom as doublet between 6.14 and 6.09 ppm. The presence of one (compounds 3a–k) and two
carbonyl groups (compounds 7a–k) was indicated by the appearance of strong stretching vibration
bands in the corresponding IR spectra between 1663 and 1651 cm−1 and signals between 168.71 and
164.07 ppm in the 13C-NMR spectra.
All PQ-CAD amides and almost all acylsemicarbazides are fully in agreement with the Lipinski
rule of five and Gelovani rules for prospective small molecular drugs: MW ≤ 500, log P ≤ 5, number
of H-bond donors
≤
5, number of H-bond acceptors
≤
10, molecular polar surface area (PSA) < 140 Å2
molar refractivity (MR) within the range of 40 and 130 cm3/mol, the number of atoms 20–70 [53].
The parameters were calculated with the Chemicalize.org program [54] and are presented in Table 1.
Table 1. Properties of the PQ-CAD conjugates calculated with Chemicalize.org program [54].
The Lipinski and Gelovani parameters.
Molecular
Formula
Number of
Atoms
H-Bond
Donor
H-Bond
Acceptor
Lipinski
Score a
MR
PSA (Å2)
Compd.
MW
log P
(cm3/mol)
3a
3b
3c
3d
3e
3f
3g
3h
3i
C24H27N3O2
C25H29N3O2
C25H29N3O3
C26H31N3O4
C27H33N3O5
C25H27N3O4
C24H26ClN3O2
C24H26FN3O2
C25H26F3N3O2
C25H26F3N3O2
C26H25F6N3O2
C25H29N5O3
C26H31N5O3
C26H31N5O4
C27H33N5O5
C28H35N5O6
C26H29N5O5
C25H28ClN5O3
C25H28FN5O3
C26H28F3N5O3
C26H28F3N5O3
C27H27F6N5O3
56
59
60
64
68
59
56
56
59
59
62
62
65
66
70
74 b
65
62
62
65
65
68
389.49
403.53
419.52
449.54
479.57
433.50
423.94
407.48
457.49
457.49
525.49
447.53
461.56
477.56
507.58 b
537.61
491.54
481.97
465.52
515.53 b
515.53 b
583.53
3.82
4.22
3.66
3.51
3.35
3.44
4.43
3.96
4.70
4.70
5.58
2.90
3.29
2.74
2.58
2.43
2.52
3.50
3.04
3.78
3.78
4.65
2
2
2
2
2
2
2
2
2
2
2
4
4
4
4
4
4
4
4
4
4
4
4
4
5
6
7
6
4
4
4
4
4
5
5
9
7
8
7
8
5
5
5
5
4
4
4
4
4
4
4
4
4
4
2
4
4
118.37
122.73
124.84
131.30 b
137.76 b
124.14
123.18
118.59
124.35
124.35
130.32
129.92
134.27 b
136.38 b
142.84
149.31
135.68 b
134.72 b
130.13
135.89 b
135.89 b
141.86
63.25
63.25
72.48
81.71
90.94
81.71
63.25
63.25
63.25
3j
63.25
63.25
3k
7a
7b
7c
7d
7e
7f
7g
7h
7i
104.38
104.38
113.61
122.84
132.07
122.84
104.38
104.38
104.38
104.38
104.38
4
3 b
3
4
4
4
3 b
3 b
3
7j
7k
a
out of four; b minimal aberrations of the rules; MR—molecular refractivity; PSA—polar surface area.
2.2. Biological Evaluation
Considering the individual biological and medicinal importance of PQ and CADs, we wanted
to explore novel chemical entities based on PQ and CAD moieties with respect to their biological
significance. The synthesized PQ-CAD conjugates were evaluated for their anticancer, antiviral,
anti-inflammatory and antioxidative activities. The results are summarized in Tables 2 and 3.

Molecules 2016, 21, 1629
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Table 2. Growth inhibition of tumor cell lines in vitro: IC50 (µM) a.
Cell Line
Compd.
3a
Structural Formula
L1210
CEM
HeLa
NCI-H460
SW 620
MCF-7
O
52 ± 3
27 ± 4
4.0 ± 0.9
>1
23 ± 9
24 ± 5
NH
H
N
N
O
O
3b
51 ± 0
55 ± 6
106 ± 26
>100
>100
9.4 ± 0.2
NH
H
N
N
O
O
O
3c
3d
3e
106 ± 6
100 ± 34
22 ± 2
61 ± 30
90 ± 45
55 ± 16
>125
>125
>125
>100
>100
>100
>100
>100
>100
20 ± 3
16 ± 0.6
8.7 ± 0.3
NH
H
N
N
O
O
O
O
NH
H
N
N
O
O
O
O
O
NH
H
N
N
O
O
O
3f
59 ± 2
37 ± 27
72 ± 57
>100
>100
6.9 ± 1.3
NH
H
N
N
O
O

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Table 2. Cont.
CEM
Cell Line
NCI-H460
Compd.
3g
Structural Formula
L1210
HeLa
SW 620
MCF-7
O
N
Cl
63 ± 2
20 ± 2
36 ± 16
>100
>100
>100
4.3 ± 1.0
NH
H
N
O
O
N
3h
66 ± 5
18 ± 15
2.1 ± 2.1
0.3 ± 0.1
1.1 ± 0.6
NH
H
N
O
F
O
3i
3j
68 ± 2
50 ± 18
92 ± 24
7.0 ± 3.1
41 ± 15
14 ± 1
112 ± 11
25 ± 6
18 ± 1
12 ± 2
>100
>100
>100
>100
64 ± 41
>100
11 ± 2
NH
H
N
N
CF3
O
O
CF3
3.9 ± 0.6
2.6 ± 0.5
2.5 ± 1.9
NH
H
N
N
O
O
CF3
3k
7a
68 ± 29
3.0 ± 0.5
>100
NH
H
N
N
CF3
O
O
>100
NH
O
H
H
N
N
N
N
H
O

Molecules 2016, 21, 1629
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Table 2. Cont.
CEM
Cell Line
NCI-H460
Compd.
7b
Structural Formula
L1210
HeLa
SW 620
MCF-7
O
N
53 ± 5
25 ± 14
47 ± 6
50 ± 4
9.5 ± 0.9
16 ± 9
NH
O
H
N
H
N
N
H
O
O
NH
O
7c
1.7 ± 0.4
40 ± 14
1.3 ± 0.7
93 ± 46
2.4 ± 0.2
92 ± 30
>100
>100
0.4 ± 0.2
1.9 ± 1.8
H
N
H
N
N
O
N
N
H
O
O
NH
O
7d
32 ± 21
25 ± 9
H
N
H
N
O
O
N
H
O
7e
57 ± 5
54 ± 12
70 ± 2
63 ± 2
48 ± 17
1.4 ± 0.1
O
7f
NH
4.8 ± 0.2
1.6 ± 0.7
7.4 ± 2.1
0.9 ± 0.7
31 ± 0
27 ± 3
17 ± 5
21 ± 4
0.6 ± 0.3
0.2 ± 0.2
O
H
H
N
N
N
O
N
H
O
O
O
NH
O
7g
2.7 ± 1.2
20 ± 0.7
H
H
N
N
N
N
H
O
Cl

Molecules 2016, 21, 1629
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Table 2. Cont.
CEM
Cell Line
NCI-H460
Compd.
7h
Structural Formula
L1210
HeLa
SW 620
MCF-7
O
N
NH
40 ± 4
15 ± 2
46 ± 2
12 ± 0.2
12 ± 4
5.9 ± 2.0
O
F
H
H
N
N
N
H
O
O
7i
7j
NH
9.4 ± 4.8
27 ± 0
10 ± 1
10 ± 1
18 ± 3
34 ± 2
16 ± 5
40 ± 5
2.3 ± 0.2
O
H
N
H
N
N
CF3
N
H
O
O
NH
O
9.4 ± 5.6
28 ± 12
0.03 ± 0.02
H
N
H
N
N
N
H
O
CF3
O
N
NH
O
H
N
H
N
7k
30 ± 17
17 ± 5
30 ± 23
48 ± 2
32 ± 4
3.2 ± 0.8
CF3
N
H
O
CF3
20 ± 6 b
7.1 ± 1.9
7 ± 2 b
PQ
SOR
CIS
–
–
3.2 ± 1.7
–
–
30 ± 7
6.1 ± 0.6 c
1 ± 0.1
28 ± 10
3.9 ± 1.6
10 ± 1
4.2 ± 2.4
–
7.1 ± 2.6
–
4 ± 0.7 b
5-FU
0.5 ± 0.2
18 ± 5
0.54 ± 0.1
3 ± 0.3
15 ± 2
SOR—sorafenib, CIS—cisplatin, 5-FU—5-fluorouracil; a IC50—the concentration that causes 50% growth inhibition; b colon carcinoma HCT 116; c lung adenocarcinoma A549.

Molecules 2016, 21, 1629
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Table 3. DPPH-reducing ability (RA), in vitro inhibition of soybean lipoxygenase (LOX) and lipid
peroxidation (LP).
RA (%) a
RA (%) b
LOX Inhibition a (%) (IC50 µM)
LP Inhibition b (%)
Compd.
20 min
60 min
20 min
60 min
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
7a
7b
7c
7d
7e
7f
7g
7h
7i
53
5
33
37
43
31
23
33
46
na
8
40
50
100
99
100
100
100
100
100
100
73
16
2
22
na
4
20
2
27
na
9
na
na
na
13
na
na
100
100
100
100
100
100
100
100
100
79
19
14
45
43
26
34
17
10
19
44
79
70
73
47
87
46
66
32
77
65
83
62
89
88
86
88
89
67
84
50
55
nt
88
40
34
16
11
27
na
na
38
na
39
84
39
100
46
74
100
52
96
100
98
89
nt
na
na
na
na
na
na
49
42
54
55
60
47
66
61
71
42
72
83
nt
(50)
43
36
(100)
(50)
(10)
(43)
(55)
(70)
(41)
(42.5)
(35)
(67.5)
(5.5)
nt
7j
95
97
94
nt
7k
NDGA
Trolox
100
87
nt
a
Concentrations of the tested compounds: 1 × 10−4 M, b 5 × 10−5 M; na—no activity; nt—not tested.
2.2.1. Anticancer Activity
The newly prepared PQ-CAD derivatives were designed and prepared primarily as potential
anticancer agents. Their antiproliferative activity was evaluated in vitro on five different types of
human tumor cell lines: lymphoblastic leukemia (CEM), cervical carcinoma (HeLa), lung carcinoma
(NCI-H460), colon carcinoma (SW 620), breast carcinoma (MCF-7) and murine lymphocytic leukemia
(L1210) and compared with the standard anticancer drugs (sorafenib, cisplatin and 5-fluorouracil)
and PQ. Amide derivatives 3a
H460 cell line. Most of the compounds of series
3i showed mild and 3h very strong activity (IC50 0.3
against HeLa varied very much: derivatives 3a and 3h showed strong activity in low micromolar
concentration (IC50 4.0 0.9 and 2.1 2.1 M, respectively), while the other amide derivatives were
either inactive (3c ) or their activity was very weak (the rest of the compounds in series ) (Table 2).
MCF-7 was the most susceptible tumor cell line. Practically all compounds were active in low
micromolar concentrations. Again, the most active was the o-fluoro derivative 3h (IC50 1.1 0.6 M).
Acylsemicarbazide derivatives 7a were more active than amide derivatives and more or less active
against all the tested cell lines and, in general, very active against MCF-7. Four compounds, 7c 7g
–
k
showed weak activity against L1210 and CEM, and no activity on
were inactive against SW 620. However, 3a and
0.1 M) against the same cell line. Activity
3
±
µ
±
±
µ
–e
3
3
±
µ
–
k
,
,
7f and 7j, showed very strong activity against MCF-7 and variable activity against the other tested
cell lines. It is interesting to note that three of the most active PQ-CAD derivatives bear substituent
in para position (methoxy, chloro and trifluoromethyl, respectively). The most active compound was
trifluoromethyl derivative 7j with IC50 in nanomolar scale (30
±
20 nM). This derivative exerted 100-to
500-fold higher activity than the anticancer reference drugs and 900-fold higher activity than PQ.
The most interesting finding of the current research is the high sensitivity of MCF-7 tumor cells to
this series of compounds. We have already noticed and discussed this phenomenon in our previous
work with PQ derivatives [43,45,46]. The investigation of the underlying mechanism is in progress.

Molecules 2016, 21, 1629
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2.2.2. Antiviral Activity Assays
Compounds 3a
–k
and 7a
–k
were evaluated against a broad variety of viruses including herpes
simplex virus type 1 (KOS), herpes simplex virus 2 (G), herpes simplex virus 1 TK–(KOS) ACVr,
vaccinia virus, adeno virus 2 and human coronavirus (229E) in HEL cell cultures and their activities
were compared with reference compounds such as brivudin, cidofovir, acyclovir, gancyclovir,
zalcitabine, alovudine, Urtica dioica agglutinin (UDA) and ribavirin, respectively. Compounds of series
3
were inactive towards all tested viruses, while acylsemicarbazide derivatives 7b, 7f (EC50 = 15.0 and
12.5 M, respectively), 7i 7j and 7k (EC50 = 7.9–9.5 M) showed moderate activity against human
µ
,
µ
coronavirus (229E), but three or two fold lower activity than UDA (EC50 = 4.0
did not alter normal cell morphology in confluent HEL cell cultures at 100
µ
M). These compounds
µM concentration (data
not shown): selectivity ratio (SI) (MCC to EC50 ratio, e.g., ratio of minimum cytotoxic concentration
(concentration that causes a microscopically detectable alteration of normal cell morphology) and
concentration required to reduce virus-induced cytopathogenicity by 50%) was close to ten. It is
worth mentioning that the three most active compounds contain trifluoromethyl group: 7i (m-CF3,
EC50 = 7.9 µM), 7j (p-CF3, EC50 = 9.5 µM), 7k (two m-CF3, EC50 = 8.4 µM), while halogen and methoxy
derivatives were inactive. Therefore, one can conclude that acylsemicarbazide functional group and the
presence of CF3 motif may be responsible for enhancing antiviral activity against human coronavirus.
These findings could be useful in further structural optimization.
2.2.3. Cellular Cytotoxicity Assays
Cytotoxicity measurements were based on the inhibition of HEL growth and the results were
expressed as MCC. MCC values for all PQ-CAD derivatives were equal or higher than 100 µM,
except for 7c and 7g, which was around 20 µM.
2.2.4. Antioxidative Activity
Antioxidant potentials of new PQ and CAD hybrids 3a–k and 7a–k were studied and
compared to the well-known antioxidant agents such as nordihydroguaiaretic acid (NDGA) and
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Two different antioxidant assays
were used: (i) interaction with the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical; and (ii)
interaction with the water-soluble azo compound 2,2’-azobis(2-amidinopropane) dihydrochloride
(AAPH), used as a source of peroxyl radicals [47].
DPPH interaction of the tested compounds was examined at 50 and 100
20 and 60 min. DPPH-reducing ability (RA) of the amide derivatives 3a was very low or missing
at 50 M. However, an increase was observed with the increase of their concentration to 100 M.
The highest activity was presented by the unsubstituted derivative 3a (53%) (Table 3). On the contrary,
the acylsemicarbazide derivatives 7a exhibited stronger antioxidant activity in both concentrations
and this ability was found to be concentration and time dependent for the majority of compounds.
Trifluoromethyl derivatives 7j 7k, 7i, and dimethoxy 7d and chloro derivative 7g showed the highest
reducing activity within the dataset. Perusal of RA values (3a 7a 3b 7b, etc.) supported the fact
µM concentrations after
–
k
µ
µ
–
k
,
<
,
<
that keeping the rest structural characteristics the same, the presence of acylsemicarbazide functional
group was correlated with higher antioxidant activity.
In the acylsemicarbazide series
the antioxidant ability. The presence of two vicinal methoxy groups (m, p) lead to higher activity,
compared to the presence of single methoxy group (7d 7c). On the contrary, the third methoxy group
diminished the activity (7e), probably due to steric reasons (bulky radical DPPH could not approach
and interact). Replacement of the phenyl ring by a benzo[d][1,3]dioxole increased activity (7f 7a).
7, the attachment of CF3 group(s) on the cinnamic ring enhanced
>
>
The nature and/or position of the halogen atom on the cinnamic ring affected the antioxidant potency.
Thus, p-Cl derivative 7g was found more potent than o-F 7h. For both groups of compounds no

Molecules 2016, 21, 1629
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correlation between overall lipophilicity and activity was found (Tables 1 and 3). However, hydrophilic
contribution of the spacer seems to play a more significant role (log P 3a < 7a, 3b < 7b, etc.)
In our studies, AAPH was used as a free radical initiator to follow oxidative changes of linoleic
acid to conjugated diene hydroperoxide. The results showed that all amide derivatives
inhibited lipid peroxidation (LP) (32%–87%). Compound 3f with a benzodioxole ring exhibited the
highest activity. Again, acylsemicarbazides generally presented higher LP inhibition (50%–89%) than
amides. Unsubstituted (7a), methoxy (7c ), benzodioxole (7f), p-Cl (7g) and m-CF3 (7i) derivatives
3 significantly
7
–
e
were the most potent (83%–89%). A correlation between lipophilicity and LP inhibition was not found.
LOX inhibitors have attracted attention initially as potential agents for the treatment of
inflammatory and allergic diseases, certain types of cancer, and cardiovascular diseases. In order to
diminish the pro-cancerous mechanisms, a key treatment strategy is to reduce the free radical load.
Antioxidant activity by scavenging of reactive oxygen species is important in preventing potential
damage of cellular compounds such as DNA, proteins and lipids. Moreover, it has been found that
LOX inhibitors may have chemopreventive activity in lung carcinogenesis [55]. Reports published
over the past three decades support a growth-regulatory role of arachidonic acid metabolites in the
etiology of mammary carcinogenesis [56]. Studies have demonstrated that levels of several eicosanoids
are increased in breast cancer in comparison to benign breast tumours [56]. In this context, we
evaluated the newly prepared PQ-CAD conjugates for their ability to inhibit soybean lipoxygenase
(LOX). From the tested compounds, all amides
at 100 M). However, acylsemicarbazides exhibited significant activity in LOX inhibition assay
(IC50 values 10–100 M). Dimethoxy derivative 7d was the most potent inhibitor with an IC50 value
of 10 M (double as the concentration of the reference compound NDGA), followed by 7j and 7h
3, with the exception of 3j, were inactive (10%–45%
µ
7
µ
µ
.
The compounds with high log P values were not active, so the idea that lipophilicity was an important
physicochemical property for LOX inhibition was not supported. Our results indicated that LOX
inhibition is accompanied and correlated with antilipid peroxidation and DPPH radical scavenging
activity. In general, the presence of acylsemicarbazide moiety in PQ-CAD conjugates led to hybrids
with better biological response.
3. Experimental Section
3.1. Chemistry
3.1.1. Materials and Methods
Melting points were measured on Stuart Melting Point (SMP3) apparatus (Barloworld Scientific,
Staffordshire, UK) in open capillaries with uncorrected values. IR spectra were recorded on FTIR
Perkin Elmer Paragon 500 and UV-Vis spectra on Lambda 20 double beam spectrophotometer
◦
(Perkin-Elmer, Waltham, MA, USA). All NMR (1H and 13C) spectra were recorded at 25 C on
NMR Avance 600 (Bruker, Rheinstetten, Germany) and Varian Inova 400 spectrometers (Varian,
1
Palo-Alto, CA, USA) at 300, 400 and 600 MHz for H and 75, 100 and 150 MHz for 13C nuclei,
respectively. Chemical shifts (
δ
) are reported in parts per million (ppm) using tetramethylsilane
as reference in the H and the DMSO residual peak as reference in the 13C spectra (39.51 ppm).
Coupling constants (J) are reported in Hertz (Hz). Mass spectra were recorded on HPLC-MS/MS
(HPLC, Agilent Technologies 1200 Series; MS, Agilent Technologies 6410 Triple Quad, Santa Clara,
CA, USA). Mass determination was realized using electron spray ionization (ESI) in positive
mode. Elemental analyses were performed on a CHNS LECO analyzer (LECO Corporation,
St. Joseph, MI, USA). All compounds were routinely checked by TLC with Merck silica gel
60F-254 glass plates using the following solvent systems: petrolether/ethyl acetate/methanol 30:10:5,
dichloromethane/methanol 97:3 and 95:5. Spots were visualized by short-wave UV light and iodine
vapor. Column chromatography was performed on silica gel 0.063–0.200 mm (Kemika, Zagreb, Croatia)
and 0.040–0.063 mm (Merck, Darmstadt, Germany), with the same eluents used in TLC. All chemicals,
solvents and biochemical reagents were of analytical grade and purchased from commercial sources.
1

Molecules 2016, 21, 1629
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CA, α-methylcinnamic acid ((2-methyl-3-phenyl)acrylic acid), 4-methoxy-cinnamic acid (p-coumaric
acid methyl ether), 3,4-dimethoxycinnamic acid (caffeic acid dimethyl ether), 3,4,5-trimethoxycinnamic
acid, 3,4-(methylenedioxy)cinnamic acid (3-benzo[1,3]-dioxol-5-yl-acrylic acid), 4-chlorocinnamic
acid, 2-fluorocinnamic acid, 4-(trifluoromethyl)cinnamic acid, 3-(trifluoromethyl)cinnamic acid and
3,5-bis(trifluoromethyl)cinnamic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA),
predominantly as trans stereoisomers ( 99%). 1H-benzo[d][1,2,3] triazole (BtH), triphosgene, PQ
≥
diphosphate, TEA, hydrazine hydrate, LOX, linoleic acid sodium salt, DPPH, NDGA, AAPH and
Trolox were purchased from Sigma-Aldrich as well. PQ was prepared from PQ diphosphate prior the
use. All reactions with PQ were run light protected.
3.1.2. General Procedure for the Synthesis of Benzotriazolides 2d,e,g
To a solution of CA or CAD (1 equiv.) and TEA (1 equiv.) in dry toluene, solution of BtcCl
(1) (1 equiv.) in dry toluene was added dropwise. The reaction mixture was stirred at room
temperature for 30 min. Solvent was evaporated and the residue was dissolved in EtOAc/H2O
mixture (1:1). The organic layer was extracted with water, dried over anhydrous sodium sulfate,
filtered and evaporated.
(E)-1-(1H-Benzo[d][1,2,3]triazol-1-yl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (2d): Compound 2d was
synthesized according to the general procedure using 3,4-dimethoxycinnamic acid (0.208 g, 1 mmol)
and BtcCl (0.181 g, 1 mmol). The crude product (0.207 g, 67%) was triturated with ether and used in
further reactions without purification.
(E)-1-(1H-Benzo[d][1,2,3]triazol-1-yl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (2e): Compound 2e was
synthesized according to the general procedure using 3,4,5-trimethoxycinnamic acid (0.155 g,
0.65 mmol) and BtcCl (0.118 g, 0.65 mmol). The crude product (0.128 g, 58%) was triturated with
ether/petrolether and used in further reactions without purification.
(E)-1-(1H-Benzo[d][1,2,3]triazol-1-yl)-3-(4-chlorophenyl)prop-2-en-1-one (2g): Compound 2g was synthesized
according to the general procedure using 4-chlorocinnamic acid (0.365 g, 2 mmol) and BtcCl (0.362 g,
2 mmol). The crude product (0.238 g, 42%) was triturated with ether and used in further reactions
without purification.
3.1.3. General Procedure for the Synthesis of Chlorides 4a–k
A solution of CA or CAD (1 equiv.), thionyl chloride (5 equiv.) and two drops of DMF in dry
toluene was stirred at room temperature for 3 h. Solvent was evaporated. The residue was dissolved
in dry toluene, evaporated again and used in further reactions without purification.
3.1.4. General Procedure for the Synthesis of Amides 3a–k
Method A: A suspension of PQ diphosphate (1 equiv.) and TEA (3 equiv.) in dry dichloromethane
was stirred at room temperature. After 15 min, a solution of chloride
4 (1.25 equiv.) in dry
dichloromethane was added dropwise. The reaction mixture was stirred at room temperature, light
protected. After 0.5–3 h, solvent was removed under reduced pressure and the residue was dissolved
in ethyl acetate/5% NaOH mixture (1:1). The organic layer was extracted with 5% NaOH solution and
water, dried over anhydrous sodium sulfate, filtered and evaporated. The crude product was purified
by column chromatography.
Method B: NaOH solution was added to a solution of PQ diphosphate (1 equiv.) in water until
pH 9-10 was reached and PQ base was extracted with dichloromethane. The organic layer was dried
over anhydrous sodium sulfate, filtered and evaporated. PQ and TEA (1 equiv.) were dissolved in
dry dioxane and added dropwise to a solution of
2 (1 equiv.) in dry dioxane. The reaction mixture
was stirred at room temperature overnight and evaporated under reduced pressure. The residue was

Molecules 2016, 21, 1629
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dissolved in ethyl acetate and extracted with 5% NaOH solution and water, dried over anhydrous
sodium sulfate, filtered and evaporated. The crude product was purified by column chromatography.
(E)-N-(4-(6-Methoxyquinolin-8-ylamino)pentyl)-3-phenylacrylamide (3a): Compound 3a was synthesized
according to the general procedure (method A) using chloride 4a (0.108 g, 0.65 mmol) and PQ
diphosphate (0.228 g, 0.5 mmol). After purification by column chromatography (CH2Cl2/MeOH
= 95:5) and recrystallization from EtOAc/ether, pure compound 3a was obtained (0.126 g, 65%,
◦
m.p. = 72–74 C). IR (KBr) (cm−1): 3387, 3282, 3061, 2927, 2852, 1660, 1614, 1576, 1556, 1516, 1454,
1
1424, 1387, 1338, 1220, 1203, 1160, 1052, 1031, 977, 902, 863, 822, 791, 766, 680, 626. H-NMR (
δ/ppm):
8.55–8.53 (dd, 1H, J = 1.43, 4.13), 8.13 (t, 1H, J = 5.69), 8.09–8.06 (dd, 1H, J = 1.43, 8.28), 7.55 (d, 2H,
J = 6.49), 7.45–7.34 (m, 5H), 6.61 (d, 1H, J = 15.82), 6.47 (s, 1H), 6.28 (s, 1H), 6.15 (d, 1H, J = 8.73), 3.81
(s, 3H), 3.66 (m, 1H), 3.21 (m, 2H), 1.64 (m, 4H), 1.22 (d, 3H, J = 6.25). 13C-NMR (
δ/ppm): 164.78, 159.01,
144.64, 144.23, 138.40, 134.96, 134.80, 134.52, 129.58, 129.33, 128.89, 127.44, 122.32, 122.10, 96.12, 91.61,
54.96, 47.01, 39.02, 33.54, 26.03, 20.23. MS (ESI): m/z = 390.3 [M + H]+. Anal. Calcd. For C24H27N3O2:
C, 74.31; H, 6.99; N, 10.79. Found: C, 74.49; H, 7.02; N, 10.50.
(E)-N-(4-(6-Methoxyquinolin-8-ylamino)pentyl)-2-methyl-3-phenylacrylamide (3b): Compound 3b was
synthesized according to the general procedure (method A) using chloride 4b (0.117g, 0.65 mmol) and
PQ diphosphate (0.228 g, 0.5 mmol). After purification by column chromatography (CH2Cl2/MeOH
= 95:5), oil compound 3b was obtained (0.157 g, 78%). IR (film) (cm−1): 3378, 3054, 2936, 2867, 1735,
1651, 1616, 1578, 1519, 1456, 1424, 1388, 1271, 1220, 1202, 1158, 1052, 1031, 926, 900, 822, 792, 763, 738,
1
703, 624. H-NMR (
δ/ppm): 8.55–8.53 (dd, 1H, J = 1.63, 4.19), 8.09–8.03 (m, 2H), 7.45–7.28 (m, 6H), 7.18
(s, 1H), 6.48 (s, 1H), 6.28 (s, 1H), 6.16–6.14 (d, 1H, J = 8.75), 3.81 (s, 3H), 3.70–3.63 (m, 1H), 3.23–3.17 (m,
2H), 1.99 (s, 3H), 1.73–1.56 (m, 4H), 1.24–1.23 (d, 3H, J = 6.29). 13C-NMR (
δ/ppm): 168.71, 159.02, 144.58,
144.27, 136.14, 134.80, 134.54, 132.68, 131.87, 129.58, 129.17, 128.35, 127.56, 122.10, 96.11, 91.58, 54.96,
47.04, 39.02, 33.43, 25.95, 20.19, 14.29. MS (ESI): m/z = 404.3 [M + H]+. Anal. Calcd. For C25H29N3O2:
C, 74.41; H, 7.24; N, 10.41. Found: C, 74.20; H, 7.02; N, 10.66.
(E)-3-(4-Methoxyphenyl)-N-(4-(6-methoxyquinolin-8-ylamino)pentyl)acrylamide (3c): Compound 3c was
synthesized according to the general procedure (method A) using chloride 4c (0.128 g, 0.65 mmol)
and PQ diphosphate (0.228 g, 0.5 mmol). After purification by column chromatography
(cyclohexane/EtOAc/MeOH = 30:10:7 and CH2Cl2:/MeOH = 95:5) and recrystallization from
◦
EtOAc/ether, pure compound 3c was obtained (0.133 g, 66%, m.p. = 78–80 C). IR (KBr) (cm−1):
3255, 3070, 2962, 2930, 2856, 1738, 1652, 1610, 1571, 1517, 1455, 1424, 1385, 1304, 1229, 1168, 1032, 981,
1
823, 791, 678, 630. H-NMR (
δ/ppm): 8.54–8.53 (dd, 1H, J = 1.5, 4.2), 8.09–8.05 (dd, 1H, J = 1.4, 8.3),
8.02–7.98 (t, 1H, J = 5.4), 7.50–7.47 (d, 2H, J = 8.69), 7.44–7.40 (m, 1H), 7.38–7.32 (d, 1H, J = 15.75),
6.98–6.95 (d, 2H, J = 8.70), 6.48–6.42 (m, 2H), 6.28 (s, 1H), 6.15–6.12 (d, 1H, J = 8.74), 3.81 (s, 3H), 3.78
(s, 3H), 3.65 (m, 1H), 3.21–3.19 (m, 2H), 1.7–1.56 (m, 4H), 1.23–1.21 (d, 3H, J = 6.27). 13C-NMR (
δ/ppm):
165.05, 160.18, 158.98, 144.61, 144.18, 138.02, 134.74, 134.49, 129.53, 128.93, 127.50, 122.03, 119.85, 114.30,
96.08, 91.61, 55.18, 54.93, 47.00, 39.02, 33.54, 26.03, 20.19. MS (ESI): m/z = 420.3 [M + H]+. Anal. Calcd.
For C25H29N3O3: C, 71.57; H, 6.97; N, 10.02. Found: C, 71.43; H, 7.02; N, 10.17.
(E)-3-(3,4-Dimethoxyphenyl)-N-(4-(6-methoxyquinolin-8-ylamino)pentyl)acrylamide (3d): Compound 3d
was synthesized according to the general procedure using chloride 4d (0.147 g, 0.65 mmol) and
PQ diphosphate (0.228 g, 0.5 mmol) (method A) or compound 2d (0.090 g, 0.29 mmol) and PQ
diphosphate (0.132 g, 0.29 mmol) (method B). After purification by column chromatography (petroleum
ether/EtOAc 1:2), pure compound 3d was obtained (0.157 g, 70%, method A; 0.098 g, 75%; method B,
◦
m.p. = 85–88 C). IR (KBr) (cm−1): 3355, 3256, 3072, 2964, 2932, 1738, 1653, 1613, 1555, 1518, 1458,
1
1425, 1388, 1339, 1298, 1259, 1201, 1166, 1138, 1022, 980, 851, 819, 793, 765, 681, 627, 597. H-NMR
(δ/ppm): 8.54–8.53 (dd, 1H, J = 1.62, 4.19), 8.09–8.05 (dd, 1H J = 1.56, 8.29), 8.00–7.96 (t, 1H, J = 5.52),
7.44–7.40 (m, 1H, J = 4.20, 8.25), 7.36–7.31 (d, 1H, J = 15.73), 7.14–7.08 (m, 2H), 6.98–6.96 (d, 1H, J = 8.32),
6.51–6.45 (m, 2H), 6.28 (s, 1H), 6.15–6.12 (d, 1H, J = 8.76), 3.81–3.78 (m, 9H), 3.65 (m, 1H), 3.23–3.19

Molecules 2016, 21, 1629
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(m, 2H), 1.70–1.56 (m, 4H), 1.23–1.21 (d, 2H, J = 6.29). 13C-NMR (
δ/ppm): 165.05, 158.97, 149.98, 148.85,
144.61, 144.18, 138.36, 134.73, 134.49, 129.53, 127.75, 122.03, 121.20, 120.05, 111.74, 109.96, 96.08, 91.61,
55.49, 55.37, 54.92, 47.00, 39.02, 33.55, 26.02, 20.20. MS (ESI): m/z = 450.5 [M + H]+. Anal. Calcd.
For C26H31N3O4: C, 69.47; H, 6.95; N, 9.35. Found: C, 69.80; H, 7.01; N, 9.57.
(E)-N-(4-(6-Methoxyquinolin-8-ylamino)pentyl)-3-(3,4,5-trimethoxyphenyl)acrylamide (3e): Compound 3e
was synthesized according to the general procedure using chloride 4e (0.169 g, 0.65 mmol) and
PQ diphosphate (0.228 g, 0.5 mmol) (method A) or compound 2e (0.109 g, 0.32 mmol) and PQ
diphosphate (0.146 g, 0.32 mmol) (method B). After purification by column chromatography (petroleum
ether/EtOAc/MeOH 30:10:5) and crystallization from EtOAc, pure compound 3e was obtained (0.125 g,
◦
52%, method A; 0.106 g, 69%, method B, m.p. = 120–123.5 C). IR (KBr) (cm−1): 3367, 3285, 3084,
2962, 1657, 1617, 1583, 1520, 1456, 1421, 1388, 1328, 1277, 1241, 1221, 1200, 1164, 1125, 1052, 1014, 978,
824, 792, 682, 622, 604. 1H-NMR (
δ/ppm): 8.54–8.53 (d, 1H, J = 2.85), 8.09–8.03 (m, 2H), 7.44–7.40
(m, 1H), 7.37–7.32 (d, 1H, J = 15.68), 6.88 (s, 2H), 6.54–6.52 (d, 1H, J = 15.69), 6.47 (s, 1H), 6.28 (s, 1H),
6.15–6.12 (d, 1H, J = 8.69), 3.81 (s, 9H), 3.68 (s, 4H), 3.21–3.19 (m, 2H), 1.69–1.65 (m, 4H), 1.23–1.22 (d,
3H, J = 6.15). 13C-NMR (
δ/ppm): 164.83, 158.97, 153.00, 144.62, 144.18, 138.57, 138.46, 134.74, 134.49,
130.55, 129.53, 122.04, 121.66, 104.87, 96.08, 91.61, 60.02, 55.82, 54.92, 46.99, 39.02, 33.56, 25.98, 20.20. MS
(ESI): m/z = 480.3 [M + H]+. Anal. Calcd. For C27H33N3O5: C, 67.62; H, 6.94; N, 8.76. Found: C, 67.99;
H, 7.06; N, 8.99.
(E)-3-(Benzo[d][1,3]dioxo-5-yl)-N-(4-(6-methoxyquinolin-8-ylamino)pentyl)acrylamide (3f): Compound
3f was synthesized according to the general procedure (method A) using chloride 4f (0.137 g,
0.65 mmol) and PQ diphosphate (0.228 g, 0.5 mmol). After purification by column chromatography
(CH2Cl2/MeOH = 97:3) and recrystallization from EtOAc/ether, pure compound 3f was obtained
(0.124 g, 57%, m.p. = 77–79.5 ◦C). IR (KBr) (cm−1): 3350, 3253, 3069, 2961, 2934, 1738, 1655, 1620, 1576,
1557, 1520, 1491, 1454, 1425, 1387, 1356, 1334, 1281, 1247, 1200, 1167, 1124, 1096, 1041, 977, 927, 855,
1
819, 792, 752, 681, 624, 593. H-NMR (
δ
/ppm): 8.53-8.51 (dd, 1H, J = 1.61, 4.18), 8.07–8.04 (dd, 1H,
J = 1.57, 8.29), 7.99–7.95 (t, 1H, J = 5.52), 7.43–7.39 (m, 1H), 7.33–7.28 (d, 1H), 7.11 (s, 1H), 7.05–7.02 (dd,
1H, J = 1.46, 8.11), 6.93–6.91 (d, 1H, J = 8.00), 6.46–6.40 (m, 2H), 6.27 (s, 1H), 6.14–6.11 (d, 1H, J = 8.77),
6.04 (s, 2H), 3.80 (s, 3H), 3.64 (m, 1H), 3.21–3.15 (m, 2H), 1.69–1.51 (m, 4H), 1.22–1.20 (d, 2H, J = 6.29).
13C-NMR (
δ/ ppm): 164.93, 158.97, 148.29, 147.85, 144.61, 144.18, 138.13, 134.73, 134.49, 129.53, 129.33,
123.00, 122.05, 120.42, 108.49, 106.11, 101.33, 96.08, 91.61, 54.93, 46.99, 39.02, 33.53, 26.01, 20.19. MS
(ESI): m/z = 434.2 [M + H]+. Anal. Calcd. For C25H27N3O4: C, 69.27; H, 6.28; N, 9.69. Found: C, 69.40;
H, 6.69; N, 9.22.
(E)-3-(4-Chlorophenyl)-N-(4-(6-methoxyquinolin-8-ylamino)pentyl)acrylamide (3g): Compound 3g was
synthesized according to the general procedure using chloride 4g (0.131 g, 0.65 mmol) and PQ
diphosphate (0.228 g, 0.5 mmol) (method A) or 2g (0.113 g, 0.4 mmol) and PQ diphosphate (0.182 g,
0.4 mmol) (method B). After purification by column chromatography (CH2Cl2/MeOH = 97:3), pure
◦
compound 3g was obtained (0.167 g, 79%, method A; 0.056 g, 33%, method B, m.p. = 85–87 C). IR (KBr)
(cm−1): 3358, 3275, 3078, 2958, 2327, 2857, 1740, 1655, 1615, 1558, 1520, 1492, 1457, 1426, 1388, 1343,
1
1227, 1202, 1168, 1097, 1052, 1013, 982, 903, 823, 791, 736, 709, 677, 628. H-NMR (
δ/ppm): 8.54–8.53
(dd, 1H, J = 1.60, 4.19), 8.13–8.05 (m, 2H), 7.58–7.56 (d, 2H, J = 8.54), 7.47–7.37 (m, 4H), 7.63–7.58 (d, 1H,
J = 15.82), 7.47 (s, 1H), 6.28 (s, 1H), 6.15–6.12 (d, 1H, J = 8.79), 3.81 (s, 3H), 3.65 (m, 1H), 3.22–3.20 (m,
2H), 1.72–1.53 (m, 4H), 1.23–1.21 (d, 3H, J = 6.28). 13C-NMR (
δ/ ppm): 164.56, 158.97, 144.60, 144.18,
136.97, 134.73, 134.49, 133.91, 133.68, 129.53, 129.08), 128.85, 123.14, 122.03, 96.08, 91.61, 54.92, 46.98,
39.02, 33.51, 26.95, 20.19. MS (ESI): m/z = 424.2 [M + H]+. Anal. Calcd. For C24H26ClN3O2: C, 68.00; H,
6.18; N, 9.91. Found: C, 68.44; H, 6.35; N, 10.05.
(E)-3-(2-Fluorophenyl)-N-(4-(6-methoxyquinolin-8-ylamino)pentyl)acrylamide (3h): Compound 3h was
synthesized according to the general procedure (method A) using chloride 4h (0.120 g, 0.65 mmol)
and PQ diphosphate (0.228 g, 0.5 mmol). After purification by column chromatography

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(CH2Cl2/MeOH = 97:3) and recrystallization from EtOAc/ether, pure compound 3h was obtained
(0.120 g, 59%, m.p. = 65–67.5 ◦C). IR (KBr) (cm−1): 3357, 3271, 3078, 2960, 2927, 2857, 1739, 1655, 1616,
1578, 1557, 1520, 1487, 1458, 1426, 1388, 1343, 1283, 1226, 1201, 1167, 1093, 1052, 1032, 985, 903, 875,
822, 792, 758, 714, 678, 625. 1H-NMR (
δ/ppm): 8.55–8.53 (dd, 1H, J = 1.60, 4.19), 8.23–8.19 (t, 1H,
J = 5.50), 8.09–8.05 (dd, 1H, J = 1.55, 8.28), 7.66–7.61 (t, 1H, J = 6.49), 7.50–7.39 (m, 3H), 7.30–7.23 (m,
2H), 6.73–6.68 (d, 1H, J = 15.94), 6.47 (s, 1H), 6.28 (s, 1H), 6.15–6.13 (d, 1H, J = 8.79), 3.81 (s, 3H), 3.65
(m, 1H), 3.25–3.21 (m, 2H), 1.73–1.53 (m, 4H), 1.24–1.21 (d, 3H, J = 6.28). 13C-NMR (
δ/ppm): 164.48,
162.02–158.71 (d, J = 250.43), 158.97, 144.61, 144.18, 134.73, 134.49, 131.14–131.02 (d, J = 8.58), 130.72,
129.53, 129.03–128.99 (d, J = 3.26), 125.17–125.09 (d, J = 6.18), 124.97, 122.61–122.41 (d, J = 12.66), 122.03,
116.12–115.83 (d, J = 23.47), 96.08, 91.61, 54.91, 46.98, 39.02, 33.51, 25.91, 20.19. MS (ESI): m/z = 408.1
[M + H]+. Anal. Calcd. For C24H26FN3O2: C, 70.74; H, 6.43; N, 10.31. Found: C, 70.60; H, 5.99; N, 10.39.
(E)-N-(4-(6-Methoxyquinolin-8-ylamino)pentyl)-3-(3-trifluoromethyl)phenyl)acrylamide (3i): Compound
3i was synthesized according to the general procedure (method A) using chloride 4i (0.152 g,
0.65 mmol) and PQ diphosphate (0.228 g, 0.5 mmol). After purification by column chromatography
(CH2Cl2/MeOH = 97:3) and recrystallizati◦on from EtOAc/petroleum ether, pure compound 3i was
obtained (0.162 g, 71%, m.p. = 48.5–49.5 C). IR (KBr) (cm−1): 3262, 3075, 2964, 2861, 1661, 1618,
1
1561, 1520, 1454, 1387, 1335, 1221, 1166, 1126, 975, 803, 864, 820, 793, 735, 688, 625, 584, 513. H-NMR
(δ/ppm): 8.55–8.53 (dd, 1H, J = 1.46, 4.10), 8.15–8.11 (t, 1H, J = 5.51), 8.08–8.05 (dd, 1H, J = 1.56, 8.27),
7.89–7.84 (m, 2H), 7.73–7.62 (m, 2H), 7.52–7.47 (d, 1H, J = 15.84), 7.44–7.40 (m, 1H), 6.77–6.72 (d, 1H,
J = 15.85), 6.47 (s, 1H), 6.29 (s, 1H), 6.16–6.13 (d, 1H, J = 8.76), 3.81 (s, 3H), 3.66 (m, 1H), 3.23–3.21 (m,
2H), 1.72–1.57 (m, 4H), 1.24–1.22 (d, 3H, J = 6.25). 13C-NMR (
δ/ppm): 164.34, 158.97, 144.61, 144.18,
136.62, 136.17, 134.73, 134.50, 132.28–122.55 (q, J = 244.60), 131.20, 130.28–128.83 (q, J = 31.73), 129.96,
129.44, 125.60–125.46 (q, J = 3.70), 124.48, 123.75–123.60 (q, J = 3.71), 122.03, 96.09, 91.60, 54.91, 46.98,
39.02, 33.50, 25.91, 20.20. MS (ESI): m/z = 458.3 [M + H]+. Anal. Calcd. For C25H26F3N3O2: C, 65.63; H,
5.73; N, 9.18. Found: C, 65.24; H, 6.00; N, 9.43.
(E)-N-(4-(6-Methoxyquinolin-8-ylamino)pentyl)-3-(4-(trifluoromethyl)phenyl)acrylamide (3j): Compound
3j was synthesized according to the general procedure (method A) using chloride 4j (0.152 g,
0.65 mmol) and PQ diphosphate (0.228 g, 0.5 mmol). After purification by column chromatography
(CH2Cl2/MeOH = 95:5), pure compound 3j was obtained (0.167 g, 73%, m.p. = 82–84 ◦C). IR (KBr)
(cm−1): 3359, 3262, 3075, 2963, 2931, 2856, 1738, 1655, 1615, 1577, 1560, 1519, 1456, 1424, 1386, 1326,
1
1227, 1203, 1163, 1130, 1110, 1069, 1050, 1015, 979, 957, 903, 880, 829, 792, 719, 681, 623, 596. H-NMR
(δ/ppm): 8.55–8.53 (dd, 1H, J = 1.61, 4.19), 8.24–8.21 (t, 1H, J = 5.56), 8.09–8.06 (dd, 1H, J = 1.57,
8.30), 7.76 (s, 4H), 7.50–7.45 (d, 1H, J = 15.87), 7.45–7.40 (m, 1H), 6.76–6.71 (d, 1H, J = 15.84), 6.47 (s,
1H), 6.29 (s, 1H), 6.16–6.13 (d, 1H, J = 8.80), 3.81 (s, 3H), 3.66 (m, 1H), 3.25–3.21 (m, 2H), 1.73–1.53
(m, 4H), 1.24–1.21 (d, 3H, J = 6.28). 13C-NMR (
δ/ppm): 164.31, 159.00, 144.63, 144.23, 139.07, 136.74,
134.79, 134.52, 129.57, 129.71–128.44 (q, J = 31.56), 129.51–118.69 (q, J = 272.14), 128.07, 125.82–125.67
(q, J = 3.80), 125.12, 122.09, 96.12, 91.60, 54.95, 46.99, 39.02, 33.52, 23.96, 20.23. MS (ESI): m/z = 458.2
[M + H]+. Anal. Calcd. For C25H26F3N3O2: C, 65.63; H, 5.73; N, 9.18. Found: C, 65.98; H, 6.01; N, 9.44.
(E)-3-(3,5-Bis(trifluoromethyl)phenyl)-N-[4-(6-methoxyquinolin-8-ylamino)pentyl]acrylamide (3k): Compound
3k was synthesized according to the general procedure (method A) using chloride 4k (0.097 g,
0.32 mmol) and PQ diphosphate (0.114 g, 0.25 mmol). After purification by column chromatography
(CH2Cl2/MeOH = 95:5) and crystallization from ether, pure compound 3k was obtained (0.097 g, 74%,
m.p. = 148–150 ◦C). IR (KBr) (cm−1): 3395, 3286, 3095, 2964, 2934, 2861, 1663, 1623, 1576, 1521, 1456,
1
1423, 1334, 1341, 1278, 1224, 1174, 1136, 1052, 1031, 978, 940, 899, 868, 845, 822, 792, 683, 624. H-NMR
(
δ
/ppm): 8.54 (s, 1H), 8.26 (s, 2H), 8.18 (t, 1H, J = 5.21), 8.08–8.07 (m, 2H), 7.60–7.58 (d, 1H, J = 15.82),
7.44–7.42 (m, 1H), 6.91–6.88 (d, 1H, J = 15.90), 6.47 (s, 1H), 6.28 (s, 1H), 6.16–6.14 (d, 1H, J = 8.54), 3.80
(s, 3H), 3.66 (m, 1H), 3.23 (m, 2H), 1.70–1.58 (m, 4H), 1.23–1.22 (d, 3H, J = 5.99). 13C-NMR (
/ppm):
δ
164.07, 159.00, 144.64, 144.24, 137.97, 135.16, 134.81, 134.53, 131.14–130.48 (q, J = 33.83), 129.58, 127.84,
126.61, 125.94–120.54 (q, J = 272.30), 122.23, 122.11, 96.14, 91.59, 54.95, 46.99, 39.02, 33.51, 25.92, 20.25.

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MS (ESI): m/z = 526.4 [M + H]+. Anal. Calcd. For C26H25F6N3O2: C, 59.43; H, 4.80; N, 8.00. Found: C,
59.67; H, 5.02; N, 8.22.
3.1.5. Synthesis of
N-(4-((6-Methoxyquinolin-8-yl)amino)pentyl)-1H-benzo[d][1,2,3]triazole-1-carboxamide (5)
Compound 5 was prepared according to our procedures published earlier [43].
3.1.6. Synthesis of N-(4-((6-Methoxyquinolin-8-yl)amino)pentyl)hydrazinecarboxamide (6)
The title compound was prepared following previously published procedure [46].
3.1.7. General Procedure for the Synthesis of Acylsemicarbazides 7a–k
To a solution of chloride 4a–k (1.1 equiv.) in dry dichloromethane, a solution of semicarbazide
6
(1 equiv.) and TEA (1 equiv.) in dry dichloromethane was added dropwise. The reaction mixture
was stirred overnight at room temperature, light protected. The solvent was removed under reduced
pressure and the residue was dissolved in ethyl acetate/5% NaOH mixture (1:1). The organic layer
was extracted with 5% NaOH solution and water, dried over anhydrous sodium sulfate, filtered and
evaporated. The crude product was purified by column chromatography.
(E)-1-Cinnamoyl-4-(4-(6-methoxyquinolin-8-ylamino)pentyl)semicarbazide (7a): Compound 7a was synthesized
according to the general procedure using chloride 4a (0.183 g, 1.1 mmol) and compound
6 (0.317 g,
1 mmol). After purification by column chromatography (CH2Cl2/MeOH = 95:5) and crystallization
◦
from ether/petroleum ether, pure compound 7a was obtained (0.251 g, 56%, m.p. = 188–190 C,
decomp.). IR (KBr) (cm−1): 3302, 3236, 3038, 2936, 1696, 1628, 1578, 1596, 1458, 1388, 1424, 1356, 1222,
1
1156, 1054, 978, 862, 822, 790, 762, 726, 678, 658, 632, 556, 488. H-NMR (
δ/ppm): 9.75 (s, 1H), 8.54–8.53
(dd, 1H, J = 1.41), 8.09–8.06 (dd, 1H, J = 1.30, 8.25), 7.85 (s, 1H), 7.58–7.36 (m, 7H), 6.65–6.60 (d, 1H,
J = 15.90), 6.48–6.44 (m, 2H), 6.28 (s, 1H), 6.13–6.10 (d, 1H, J = 8.62), 3.82 (s, 3H), 3.64 (m, 1H), 3.06–3.04
(m, 2H), 1.63–1.47 (m, 4H), 1.22–1.20 (d, 3H, J = 6.24). 13C-NMR (
δ/ppm): 164.65, 158.98, 157.92, 144.61,
144.20, 139.46, 134.74, 134.67, 134.49, 129.60, 129.53, 128.92, 127.50, 122.04, 119.94, 96.07, 91.61, 54.95,
47.01, 39.02, 33.41, 26.65, 20.18. MS (ESI): m/z = 448.3 [M + H]+. Anal. Calcd. For C25H29N5O3: C,
67.09; H, 6.53; N, 15.65. Found: C, 67.37; H, 6.22; N, 15.66.
(E)-4-(4-(6-Methoxyquinolin-8-ylamino)pentyl)-1-(2-methyl-3-phenylacryloyl)semicarbazide (7b): Compound
7b was synthesized according to the general procedure using chloride 4b (0.197 g, 1.1 mmol) and
compound
6 (0.317 g, 1 mmol). After purification by column chromatography (CH2Cl2/MeOH = 95:5),
oily product 7b was obtained, which crystallized after a long storage at low temperature (0.245 g,
53%, m.p. = 65.5 ◦C, decomp.). IR (KBr) (cm−1): 3265, 2961, 2935, 1652, 1617, 1576, 1520, 1454, 1423,
1387, 1336, 1258, 1239, 1220, 1203, 1158, 1051, 1031, 1004, 928, 910, 822, 791, 762, 709, 695, 625, 589, 515.
δ
1H-NMR ( /ppm): 9.67 (s, 1H, 200), 8.54–8.53 (dd, 1H, J = 1.57, 4.15), 8.08–8.07 (dd, 1H, J = 1.49, 8.28),
7.67 (s, 1H), 7.43–7.30 (m, 7H), 6.48–6.45 (m, 2H), 6.28 (s, 1H), 6.13–6.11 (d, 1H, J = 8.75), 4.05–3.82 (s,
3H), 3.65–3.63 (m, 1H), 3.08–3.02 (m, 2H), 2.02 (s, 3H), 1.66–1.47 (m, 4H), 1.22–1.21 (d, 3H, J = 6.29).
13C-NMR (
δ/ppm): 168.89, 159.00, 158.36, 144.63, 144.24, 135.83, 134.78, 134.51, 133.23, 130.74, 129.56,
129.22, 128.42, 127.80, 122.08, 96.10, 91.61, 54.97, 47.02, 39.02, 33.43, 26.73, 20.21, 14.15. MS (ESI):
m/z = 462.3 [M + H]+. Anal. Calcd. For C26H31N5O3: C, 67.66; H, 6.77; N, 15.17. Found: C, 67.51;
H, 6.55; N, 15.00.
(E)-1-(3-(4-Methoxyphenyl)acryloyl)-4-(4-(6-methoxyquinolin-8-ylamino)pentyl)semicarbazide (7c): Compound
7c was synthesized according to the general procedure using chloride 4c (0.177 g, 0.9 mmol) and
compound
6 (0.254 g, 0.8 mmol). After two purifications by column chromatography (first eluent:
petroleum ether/EtOAc/MeOH = 30:10:5, second eluent CH2Cl2/MeOH = 95:5), pure compound
◦
7c was obtained (0.229 g, 60%, m.p. = 89.5 C, decomp.). IR (KBr) (cm−1): 3248, 2934, 1654, 1604,
1
1575, 1517, 1456, 1424, 1386, 1251, 1167, 1029, 980, 824, 791, 628, 521. H-NMR (
δ/ppm): 9.64 (s, 1H),
8.54–8.52 (dd, 1H, J = 1.45, 4.10), 8.08–8.06 (d, 1H, J = 7.07), 7.80 (s, 1H), 7.53–7.42 (m, 4H), 6.99–6.97 (d,

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2H, J = 8.66), 6.49–6.45 (m, 3H), 6.27 (s, 1H), 6.10 (s, 1H), 3.82 (s, 3H), 3.78 (s, 3H), 3.62 (m, 1H), 3.04 (m,
2H), 1.63–1.46 (m, 4H), 1.21–1.19 (d, 3H, J = 6.23). 13C-NMR (
/ppm): 165.24, 160.56, 159.06, 158.17,
δ
0
0
0
0
144.69, 144.35, 139.41, 134.90, 134.56, 129.65 (14), 129.28 (4 , 8 ), 127.30 (3 ), 122.19 (12), 117.37 (1 ), 114.49
(50, 70), 96.19 (17), 91.71, 55.34, 55.07, 47.11, 39.02, 33.49, 26.77, 20.29. MS (ESI): m/z = 478.3 [M + H]+.
Anal. Calcd. For C26H31N5O4: C, 65.39; H, 6.54; N, 14.66. Found: C, 65.51; H, 6.60; N, 14.48.
(E)-1-(3-(3,4-Dimethoxyphenyl)acryloyl)-4-(4-(6-methoxyquinolin-8-ylamino)pentyl)semicarbazide
(7d):
Compound 7d was synthesized according to the general procedure using chloride 4d (0.227 g, 1 mmol)
and compound
6 (0.286 g, 0.9 mmol). After trituration with ethyl acetate, dichloromethane, hot
acetone, ether and hot ethanol, pure compound 7d was obtained (0.260 g, 57%, m.p. = 200.5–201.5 ◦C,
decomp.). IR (KBr) (cm−1): 3375, 3213, 3084, 3003, 2937, 1668, 1628, 1596, 1559, 1515, 1456, 1421, 1387,
1356, 1293, 1262, 1235, 1202, 1171, 1138, 1052, 1022, 977, 939, 856, 816, 788, 767, 710, 679, 622, 594,
1
560, 460. H-NMR (
δ/ppm): 9.62–9.61 (d, 1H, J = 1.73), 8.54–8.52 (dd, 1H, J = 1.39, 4.06), 8.08–8.06
(dd, 1H, J = 1.29, 8.29), 7.81 (s, 1H), 7.44–7.40 (m, 2H), 7.15–7.12 (m, 2H), 7.00–6.98 (d, 1H, J = 8.28),
6.53–6.46 (m, 3H), 6.26 (s, 1H), 6.11–6.09 (d, 1H, J = 8.73), 3.81–3.78 (m, 9H), 3.62 (m, 1H), 3.04–3.02 (m,
2H), 1.54–1.51 (m, 4H), 1.21–1.19 (d, 3H, J = 6.25). 13C-NMR (
δ/ppm): 165.27, 159.06, 158.19, 150.32,
148.93, 144.69, 144.36, 139.74, 134.91, 134.57, 129.66, 127.55, 122.21, 121.48, 117.64, 111.81, 110.20, 96.19,
91.69, 55.62, 55.52, 55.08, 47.11, 39.02, 33.50, 26.80, 20.30. MS (ESI): m/z = 508.3 [M + H]+. Anal. Calcd.
For C27H33N5O5: C, 63.89; H, 6.55; N, 13.80. Found: C, 64.03; H, 6.68; N, 13.50.
(E)-4-(4-(6-Methoxyquinolin-8-ylamino)pentyl)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)semicarbazide (7e):
Compound 7e was synthesized according to the general procedure using chloride 4e (0.257 g,
1 mmol) and compound
6 (0.286 g, 0.9 mmol). After purification by column chromatography
(CH2Cl2/MeOH = 95:5) and crystallization from ether/petroleum ether, pure compound 7e was
isolated (0.300 g, 62%, m.p. = 90.5 ◦C, decomp.). IR (KBr) (cm−1): 3250, 2937, 1654, 1618, 1582, 1508,
1454, 1420, 1388, 1326, 1266, 1240, 1221, 1203, 1155, 1126, 1051, 1031, 1003, 976, 822, 791, 677, 624, 585,
1
526, 512, 464. H-NMR (
δ/ppm): 9.64–9.63 (d, 1H, J = 1.91), 8.53–8.52 (dd, 1H, J = 1.51, 4.16), 8.07–8.05
(dd, 1H, J = 1.41, 8.28), 7.83 (s, 1H), 7.45–7.40 (m, 2H), 6.90 (s, 2H), 6.59–6.55 (d, 1H, J = 15.80), 6.46–6.43
(m, 2H), 6.26 (s, 1H), 6.11–6.09 (d, 1H, J = 8.78), 3.81 (s, 3H), 3.80 (s, 6H), 3.68 (s, 3H), 3.63 (m, 1H),
3.04–3.02 (m, 2H), 1.61–1.51 (m, 4H), 1.21–1.19 (d, 3H, J = 6.25). 13C-NMR (
δ/ppm): 164.92, 159.05,
158.05, 153.14, 144.69, 144.33, 139.82, 138.89, 134.88, 134.56, 130.38, 129.64, 122.17, 119.26, 105.09, 96.17,
91.69, 60.16, 55.96, 55.05, 47.10, 39.02, 33.49, 26.76, 20.28. MS (ESI): m/z = 538.3 [M + H]+. Anal. Calcd.
For C28H35N5O6: C, 62.55; H, 6.56; N, 13.03. Found: C, 62.79; H, 6.86; N, 13.29.
(E)-1-(3-(Benzo[d][1,3]dioxo-5-yl)acryloyl)-4-(4-(6-methoxyquinolin-8-ylamino)pentyl)semicarbazide (7f):
Compound 7f was synthesized according to the general procedure using chloride 4f (0.211 g,
1 mmol) and compound
6 (0.286 g, 0.9 mmol). After purification by column chromatography
(CH2Cl2/MeOH = 95:5) and crystall◦ization from ether/petroleum ether, pure compound 7f was
obtained (0.217 g, 49%, m.p. = 75.5 C, decomp.). IR (KBr) (cm−1): 3250, 2335, 2361, 1654, 1618,
1577, 1560, 1521, 1490, 1448, 1388, 1252, 1202, 1158, 1100, 1037, 976, 929, 819, 791, 670, 625, 592, 518.
1H-NMR (
δ/ppm): 9.64 (s, 1H), 8.53 (d, 1H, J = 2.84), 8.08–8.06 (d, 1H, J = 8.05), 7.82 (s, 1H), 7.43–7.39
(m, 2H), 7.15 (s, 1H), 7.09–7.07 (d, 1H, J = 8.05), 6.96–6.94 (d, 1H, J = 7.96), 6.48–6.44 (m, 3H), 6.26 (s,
1H), 6.11–6.09 (d, 1H, J = 7.59), 6.06 (s, 3H), 3.82 (s, 3H), 3.62 (m, 1H), 3.04 (m, 2H), 1.60–1.46 (m, 4H),
1.12–1.19 (d, 3H, J = 6.13). 13C-NMR (
δ/ppm): 165.45, 159.43, 158.49, 149.08, 148.38, 145.05, 144.69,
139.84, 135.25, 134.93, 130.01, 129.50, 123.83, 122.54, 118.33, 109.05, 106.66, 101.92, 96.55, 92.06, 55.42,
47.47, 39.02, 33.86, 27.14, 20.65. MS (ESI): m/z = 492.3 [M + H]+. Anal. Calcd. For C26H29N5O5: C,
63.53; H, 5.95; N, 14.25. Found: C, 63.73; H, 5.84; N, 14.00.
(E)-1-(3-(4-Chlorophenyl)acryloyl)-4-(4-(6-methoxyquinolin-8-ylamino)pentyl)semicarbazide (7g): Compound
7g was synthesized according to the general procedure using chloride 4g (0.201 g, 1 mmol) and
compound
6 (0.286 g, 0.9 mmol). After purification by column chromatography (petroleum
ether/EtOAc/MeOH = 30:10:5) and trituration with ether, compound 7g was obtained (0.256 g,

Molecules 2016, 21, 1629
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59%, m.p. = 185–187.5 ◦C). IR (KBr) (cm−1): 3337, 3230, 3037, 2966, 2937, 2362, 2343, 1697, 1661, 1625,
1592, 1521, 1490, 1458, 1425, 1408, 1391, 1354, 1290, 1266, 1239, 1221, 1199, 1161, 1090, 1052, 1010,
982, 945, 899, 866, 819, 790, 726, 676, 628, 495. 1H-NMR (
δ/ppm): 9.79 (s, 1H), 8.53–8.52 (dd, 1H,
J = 1.06, 4.11), 8.08–8.05 (dd, 1H, J = 1.30, 8.25), 7.88 (s, 1H), 7.61–7.59 (d, 2H, J = 8.47), 7.50–7.45 (m,
3H), 7.43–7.40 (m, 1H), 6.64–6.60 (d, 1H, J = 15.89), 6.51–6.48 (t, 1H, J = 5.44), 6.46 (s, 1H), 6.25 (s, 1H),
6.11–6.09 (d, 1H, J = 8.71), 3.81 (s, 3H), 3.62 (m, 1H), 3.04 (m, 2H), 1.61–1.51 (m, 4H), 1.20–1.19 (d, 3H,
J = 6.25). 13C-NMR (
δ/ppm): 164.63, 159.05, 158.03, 144.68, 144.33, 138.28, 134.89, 134.56, 134.18, 133.68,
129.64, 129.35, 129.09, 122.19, 120.75, 96.17, 91.66, 55.06, 47.08, 39.02, 33.47, 26.77, 20.28. MS (ESI):
m/z = 482.2 [M + H]+. Anal. Calcd. For C25H28ClN5O3: C, 62.30; H, 5.86; N, 14.53. Found: C, 62.69;
H, 6.01; N, 14.30.
(E)-1-(3-(2-Fluorophenyl)acryloyl)-4-(4-(6-methoxyquinolin-8-ylamino)pentyl)semicarbazide (7h): Compound
7h was synthesized according to the general procedure using chloride 4h (0.185 g, 1 mmol) and
compound 6 (0.286 g, 0.9 mmol). After purification by column chromatography (CH2Cl2/MeOH = 95:5)
and trituration with ether, pure compound 7h was obtained (0.372 g, 89%, m.p. = 91–93 ◦C, decomp.).
IR (KBr) (cm−1): 3250, 2964, 1654, 1618, 1578, 1520, 1457, 1388, 1221, 1159, 1052, 982, 822, 791, 757.
1H-NMR (
δ/ppm): 9.85 (s, 1H), 8.54–8.53 (dd, 1H, J = 1.50, 4.10), 8.08-8.06 (dd, 1H, J = 1.42, 8.22), 7.87
(s, 1H), 7.67–7.65 (t, 1H, J = 7.22), 7.57–7.55 (d, 1H, J = 16.02), 7.45–7.41 (m, 2H), 7.30–7.26 (m, 2H),
6.76–6.73 (d, 1H, J = 16.03), 6.48 (s, 2H), 6.28 (s, 1H), 6.12–6.11 (d, 1H, J = 8.70), 3.83 (s, 3H), 3.64 (m, 1H),
3.07 (m, 2H), 1.65–1.49 (m, 4H), 1.22–1.21 (d, 3H, J = 6.28). 13C-NMR (
δ/ppm): 164.43, 161.30–159.64
(d, J = 250.54), 158.99, 157.86, 144.62, 144.21), 134.74, 134.50, 131.90, 131.47–131.41 (d, J = 8.67), 129.54,
129.22–129.20 (d, J = 2.69), 124.98, 122.90–122.86 (d, J = 6.35), 122.35–122.28 (d, J = 11.54), 122.04,
116.13–115.99 (d, J = 21.68), 96.09, 91.04, 54.95, 47.03, 39.02, 33.43, 26.64, 20.19. MS (ESI): m/z = 466.1
[M + H]+. Anal. Calcd. For C25H28FN5O3: C, 64.50; H, 6.06; N, 15.04. Found: C, 64.69; H, 6.17; N, 15.33.
(E)-4-(4-(6-Methoxyquinolin-8-ylamino)pentyl)-1-(3-(3-(trifluoromethyl)phenyl)acryloyl)semicarbazide (7i):
Compound 7i was synthesized according to the general procedure using chloride 4i (0.235 g,
1 mmol) and compound
6 (0.286 g, 0.9 mmol). After purification by column chromatography
(CH2Cl2/MeOH = 95:5) an◦d crystallization from dichloromethane, pure compound 7i was obtained
(0.343 g, 74%, m.p. = 86–88 C, decomp. IR (KBr) (cm−1): 3254, 2964, 1659, 1618, 1577, 1521, 1456, 1424,
1
1388, 1334, 1222, 1199, 1166, 1126, 1075, 976, 900, 822, 792, 694, 660, 625, 561, 514. H-NMR (
δ/ppm):
9.77 (s, 1H), 8.54 (d, 1H, J = 2.58), 8.08–8.06 (d, 1H, J = 7.91), 7.92–7.88 (m, 3H), 7.75–7.73 (d, 1H, J = 7.53),
7.68–7.65 (t, 1H, J = 7.55), 7.61–7.59 (d, 1H, J = 15.88), 7.43–7.41 (m, 1H), 6.78–6.76 (d, 1H, J = 15.89), 6.47
(s, 2H), 6.28 (s, 1H), 6.12–6.11 (d, 1H, J = 8.50), 3.83 (s, 3H), 3.64 (m, 1H), 3.07–3.06 (m, 2H), 1.65–1.49
(m, 4H), 1.22–1.21 (d, 3H, J = 6.00). 13C-NMR (
δ/ppm): 164.18, 158.99, 157.81, 144.62, 144.20, 137.75,
135.90, 134.74, 134.50, 132.14–127.87 (q, J = 214.50), 131.15, 130.07–129.41 (q, J = 30.02), 130.06, 129.53,
125.84, 123.98, 122.11, 122.03, 96.08, 91.64, 54.94, 47.03, 39.02, 33.43, 26.64, 20.18. MS (ESI): m/z = 516.1
[M + H]+. Anal. Calcd. For C26H28F3N5O3: C, 60.57; H, 5.47; N, 13.58. Found: C, 60.60; H, 5.81;
N, 13.77.
(E)-4-(4-(6-Methoxyquinolin-8-ylamino)pentyl)-1-(3-(4-(trifluoromethyl)phenyl)acryloyl)semicarbazide (7j):
Compound 7j was synthesized according to the general procedure using chloride 4j (0.235 g,
1 mmol) and compound
6 (0.286 g, 0.9 mmol). After purification by column chromatography
(CH2Cl2/MeOH = 95:5) and crystallization from ether/petroleum ether, pure compound 7j was
obtained (m.p. = 123 ◦C, decomp.). A portion of product 7j crystallized from the reaction mixture. It
was filtered off and washed with water. Overall yield: 0.311 g, 67%. IR (KBr) (cm−1): 3266, 1695, 1664,
1
1612, 1525, 1469, 1425, 1390, 1327, 1235, 1169, 1118, 1067, 834, 789, 728, 631, 592. H-NMR (
δ/ ppm):
9.85 (s, 1H), 8.54–8.53 (d, 1H, J = 3.71), 8.08–8.06 (d, 1H, J = 8.07), 7.90 (s, 1H), 7.78 (s, 4H), 7.59–7.56
(d, 1H, J = 15.92), 7.43–7.41 (m, 1H), 6.77–6.74 (d, 1H, J = 15.90), 6.47 (s, 2H), 6.28 (s, 1H), 6.12–6.11 (d,
1H, J = 8.58), 3.83 (s, 3H), 3.64 (m, 1H), 3.07–3.05 (m, 2H), 1.65–1.47 (m, 4H), 1.22–1.21 (d, 3H, J = 6.06).
13C-NMR (δ/ ppm): 164.10, 158.98, 157.80, 144.61, 144.19, 138.73, 137.75, 134.73, 134.49, 129.64–129.01
(q, J = 31.19), 129.53, 128.13, 126.81–121.32 (q, J = 271.67), 125.76, 122.77, 122.02, 96.07, 91.63, 54.93, 47.01,

Molecules 2016, 21, 1629
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39.02, 33.42, 26.63, 20.17. MS (ESI): m/z = 516.2 [M + H]+. Anal. Calcd. For C26H28F3N5O3: C, 60.57; H,
5.47; N, 13.58. Found: C, 60.22; H, 5.24; N, 13.33.
(E)-1-(3-(3,5-Bis(trifluoromethyl)phenyl)acryloyl)-4-(4-(6-methoxyquinolin-8-ylamino)pentyl)semicarbazide
(7k): Compound 7k was synthesized according to the general procedure using chloride 4k (0.303 g,
1 mmol) and compound (0.286 g, 0.9 mmol). After purification by column chromatography
6
(CH2Cl2/MeOH = 95:5) and crystallization from toluene, pure compound 7k was obtained (0.294 g,
56%, m.p. = 118–119.5 ◦C, decomp.). IR (KBr) (cm−1): 3393, 3335, 3220, 3020, 2942, 2363, 1647, 1617,
1579, 1521, 1459, 1425, 1384, 1340, 1280, 1224, 1175, 1135, 1053, 971, 942, 898, 847, 823, 792, 730, 683,
1
629, 599, 561, 519, 466. H-NMR (
δ
/ppm): 9.81 (s, 1H), 8.55–8.53 (dd, 1H, J = 1.62, 4.19), 8.29 (s, 2H),
8.12 (s, 1H), 8.09–8.07 (dd, 1H, J = 1.58, 8.31), 7.99 (s, 1H), 7.72–7.68 (d, 1H, J = 15.92), 7.77–7.41 (m,
1H), 6.94–6.90 (d, 1H, J = 15.97), 6.51–6.47 (m, 2H), 6.28 (s, 1H), 6.14–6.11 (d, 1H, J = 8.74), 3.82 (s, 3H),
3.64 (m, 1H), 3.06 (m, 2H), 1.66–1.48 (m, 4H), 1.22–1.21 (d, 3H, J = 6.28). 13C-NMR (
δ/ppm): 163.86,
159.02, 157.78, 144.65, 144.27, 137.67, 136.28, 134.83, 134.53, 131.40–130.40 (q, J = 33.53), 129.60, 128.22,
127.28–119.14 (q, J = 272.61), 125.53, 124.23, 122.13, 96.13, 91.61, 55.01, 47.04, 39.02, 33.45, 26.74, 20.25.
MS (ESI): m/z = 584.3 [M + H]+. Anal. Calcd. For C27H27F6N5O3: C, 55.57; H, 4.66; N, 12.00. Found:
C, 55.60; H, 4.83; N, 12.29.
3.2. Biological Evaluation
3.2.1. Anticancer Activity
The experiments were carried out on a murine lymphocytic leukemia cell line (L1210) and five
human tumor cell lines: CEM, HeLa, NCI-H460, SW 620 and MCF-7. The cells were cultured as
monolayers and maintained in Dulbecco0s modified Eagle medium (DMEM) supplemented with 10%
fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and 100
humidified atmosphere with 5% CO2 at 37 ◦C.
µg/mL streptomycin in a
The growth inhibition activity of the compounds was assessed as described previously [46].
The cell lines were inoculated onto a series of standard 96-well microtiter plates on day 0, at 1.5
104
104 cells/mL (MCF-7, CEM and L1210), depending on the growth of
×
cells/mL (SW 620, H460) to 2.5
×
the tumor cell line. Test agents were then added in ten-fold◦dilutions (10−8 to 10−4 M) and the cell
cultures were then incubated for 3–4 days with 5% CO2 at 37 C. Working dilutions of the compounds
were freshly prepared on the day of testing.
After 3 (MCF-7, SW 620, NCI-H460) or 4 days (CEM, L1210), the cell growth rate was evaluated
first light microscopically, 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 living,
metabolically active cells. The results are expressed as IC50, which is the concentration necessary for
50% of inhibition. The IC50 values for each compound are calculated from concentration-response
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, 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.
3.2.2. Antiviral Activity
Antiviral activity against herpes simplex virus type 1 (KOS), herpes simplex virus 2 (G), herpes
simplex virus 1 TK− (KOS) ACVr, vaccinia virus, adeno virus 2 and human coronavirus (229E)
was determined essentially as described previously [57,58]. After a 2 h incubation period, residual
virus was removed and the infected cells were further incubated with the medium containing
different concentration of the test compounds. After incubation for 3 days at 37 ◦C, virus-induced

Molecules 2016, 21, 1629
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cytopathogenicity was monitored microscopically. Antiviral activity was expressed as the concentration
required to reduce virus-induced cytopathogenicity by 50% (EC50).
3.2.3. Cytotoxicity Assays
Cytotoxicity measurements were based on the inhibition of HEL cell growth. Cells were seeded
at 5
×
103 cells/well into 96-well microtiter plates. Then, medium containing different concentrations
◦
of the test compounds was added. After 3 days of incubation at 37 C, the alteration of morphology of
the cell cultures was recorded light microscopically. Cytotoxicity was expressed as MCC.
3.2.4. Interaction of the New Derivatives with the Stable Radical DPPH
To a solution of DPPH in absolute ethanol the appropriate volume of the compounds (50 or
100 µM final concentrations) dissolved in DMSO was added. The absorbance was recorded at 517 nm
after 20 and 60 min at room temperature. The experiments were repeated at least in triplicate and the
standard deviation of absorbance was less than 10% of the mean [47]. NDGA was used under the
same experimental conditions as a reference compound.
3.2.5. Inhibition of Linoleic Acid Peroxidation
For initiating the lipid peroxidation free radical AAPH was used [47]. Final solution in the UV
cuvette consisted of 10
µ
L linoleate sodium solution (c = 16 mM), 0.93 mL phosphate buffer (c = 0.05 M),
pH 7.4, thermostated at 37 ◦C. 50
µ
L AAPH solution (c = 40 mM) and 10 L of the tested compounds
µ
were added. The experiment was performed at 37 ◦C under air. The oxidation of linoleic acid sodium
salt was monitored at 234 nm. The assays were repeated at least in triplicate and the standard deviation
of absorbance was less than 10% of the mean. Trolox was used under the same experimental conditions
as a reference compound.
3.2.6. Anti-inflammatory Activity: LOX Inhibition Study In Vitro
LOX inhibitory assay in vitro was accomplished as described previously [47]. The test compounds
(stock solutions 10 mM in DMSO) were incubated at room temperature with sodium linoleate
(c = 0.1 mM) and 0.2 mL of LOX enzyme solution (1/9
×
10−4 m/V in saline). The conversion
of sodium linoleate to 13-hydroperoxylinoleic acid was measured at 234 nm and compared with the
reference inhibitor. Several concentrations were used for the IC50 determination. The assays were
repeated at least in triplicate and the standard deviation of absorbance was less than 10% of the mean.
NDGA was used under the same experimental conditions as a reference compound.
4. Conclusions
We have designed and synthesized a new scaffold comprising CADs and PQ motifs bound by
amide (compounds 3a–k) or acylsemicarbazide bonds (compounds 7a–k). Twenty two PQ-CAD
conjugates were prepared and subjected to extensive biological evaluation. Anticancer screening
in vitro against six selected cancer cell lines showed that acylsemicarbazide derivatives were, in general,
more active than amide derivatives. Almost all compounds from series 3 were selective towards MCF-7
cell line and active in micromolar concentrations. The most selective compound from the amide series
was the o-fluoro derivative 3h. It showed high activity against HeLa, MCF-7 and in particular against
the SW 620 cell line. Four acylsemicarbazide derivatives, namely 7f with benzodioxole ring and 7c, 7g
and especially 7j with methoxy, chloro and trifluoromethyl substituents in para position of cinnamic
benzene ring, showed high selectivity and very strong activity against MCF-7 cell line in micromolar
(7c, 7f and 7g) and nanomolar levels (7j). These compounds might provide fertile and much needed
leads in anticancer drug discovery.
Trifluoromethyl acylsemicarbazide derivatives 7i, 7j and 7k showed selective antiviral activity
against human coronavirus (strain 229E) at concentrations which did not alter normal cell morphology

Molecules 2016, 21, 1629
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and could be, therefore, considered as potential drug leads for further structural optimization. The same
compounds, together with 7d and 7g, exerted the most potent antioxidant abilities in interaction with
the free stable radical DPPH, while unsubstituted (7a), methoxy (7c
and m-CF3 (7i) acylsemicarbazides and amide 3f presented the highest anti-lipid peroxidation activity
(83–89%). Dimethoxy derivative 7d was the most potent LOX inhibitor (IC50 = 10 M). The above
–e), benzodioxole (7f), p-Cl (7g)
µ
results support the principal idea of PQ-CADs hybridization leading to multifunctional agents.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
12/1629/s1.
Acknowledgments: Support for this study by the Croatian Science Foundation (project number IP-2014-09-1501),
University of Zagreb (Projects numbers BM1.9, 13.11.2.1.12 and 13.11.1.1.11.), Croatian Science Foundation (project
number IP-11-2013-5660, MultiCaST), and the KU Leuven, Belgium (GOA 10/014) are gratefully acknowledged.
We sincerely thank Leentje Persoons and Lizette Van Berckelaer for their excellent help in the evaluation of these
series of compounds.
Author Contributions: K.P. performed the majority of the synthetic experiments, analyzed the data in chemical
part and prepared experimental data for publishing. I.P. analyzed the data in chemical part. P.G and F.K. performed
the synthetic experiments (a part). K.E. performed antiproliferative experiments (a part). M.K. analyzed and
discussed the antiproliferative experiments. D.S. performed antiproliferative experiments (a part) and antiviral
experiments. E.P. screened the antioxidant potential and D.H.-L. analyzed the antioxidative data. B.Z. designed
and wrote the paper, analyzed the data and coordinated the work.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
AAPH
BtcCl
BtH
2,2’-azobis(2-amidinopropane) dihydrochloride
1-benzotriazole carboxylic acid chloride
1H-benzo[d][1,2,3]triazole
cinnamic acid
CA
CAD
CEM
CIS
DMEM
DPPH
EC50
FBS
5-FU
H460
HEL
HeLa
IC50
L1210
LOX
LP
cinnamic acid derivative
acute lymphoblastic leukemia cell line
cisplatin
0
0
Dulbecco s modified Eagle s medium
1,1-diphenyl-2-picrylhydrazyl
concentration required to reduce virus-induced cytopathogenicity by 50%
fetal bovine serum
5-fluorouracil
lung carcinoma cell line
human erythroleukemia cell line
cervical carcinoma cell line
concentration that causes 50% growth inhibition
murine lymphocytic leukemia cell line
soybean lipoxygenase
lipid peroxidation
minimum cytotoxic concentration, concentration that causes a microscopically
detectable alteration of normal cell morphology
breast adenocarcinoma cell line
molar refractivity
MCC
MCF-7
MR
MTT
NDGA
PSA
PG
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
nordihydroguaiaretic acid
molecular polar surface area
percentage of growth
PQ
primaquine
RA
DPPH reducing ability
SI
selectivity ratio, MCC to EC50 ratio
sorafenib
SOR
SW 620
TEA
Trolox
UDA
colon cancer cell line
triethylamine
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
Urtica dioica agglutinin

Molecules 2016, 21, 1629
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References
1.
2.
3.
4.
5.
Guzman, J.D. Natural cinnamic acids, synthetic derivatives and hybrids with antimicrobial activity. Molecules
2014, 19, 292–349. [CrossRef] [PubMed]
Sharma, P.J. Cinnamic acid derivatives: A new chapter of various pharmacological activities.
Chem. Pharm. Res. 2011, 3, 403–423.
Lone, R.; Shuab, R.; Koul, K.K. Role of cinnamate and cinnamate derivatives in pharmacology.
Glob. J. Pharmacol. 2014, 8, 328–335.
Sova, M. Antioxidant and antimicrobial activities of cinnamic acid derivatives. Mini Rev. Med. Chem. 2012
12, 749–767. [CrossRef] [PubMed]
,
Narasimhan, B.; Belsare, D.; Pharande, D.; Mourya, V.; Dhake, A. Esters, amides and substituted derivatives
of cinnamic acid: Synthesis, antimicrobial activity and QSAR investigations. Eur. J. Med. Chem. 2004, 39,
827–834. [CrossRef] [PubMed]
6.
Kim, J.H.; Campbell, B.C.; Mahomey, N.E.; Chan, K.L.; Molyneux, R.J. Identification of phenolics for control
of Aspergillus flavus using Saccharomyces cerevisiae in a model target-gene bioassay. J. Agric. Food Chem.
2004, 52, 7814–7821. [CrossRef] [PubMed]
7.
8.
Zhu, J.; Zhu, H.; Kobamoto, N.; Yasuda, M.; Tawata, S. Fungitoxic and phytotoxic activities of cinnamic acid
esters and amides. J. Pestic. Sci. 2000, 25, 263–266. [CrossRef]
Neogi, P.; Lakner, F.J.; Medicherla, S.; Cheng, J.; Dey, D.; Gowri, M.; Nag, B.; Sharma, S.D.; Pickford, L.B.;
Gross, C. Synthesis and structure-activity relationship studies of cinnamic acid-based novel thiazolidinedione
antihyperglycemic agents. Bioorg. Med. Chem. 2003, 11, 4059–4067. [CrossRef]
9.
Bairwa, R.; Kakwani, M.; Tawari, N.R.; Lalchandani, J.; Ray, M.K.; Rajan, M.G.R.; Degani, M.S.
Novel molecular hybrids of cinnamic acids and guanylhydrazones as potential antitubecular agents.
Bioorg. Med. Chem. 2010, 20, 1623–1625. [CrossRef] [PubMed]
10. Reddy, V.M.; Nadadhur, G.; Daneluzzi, D.; Dimova, V.; Gangadharam, P.R. Antimycobacterial activity
of a new rifamycin derivative, 3-(4-cinnamylpiperazinyl iminomethyl) rifamycin SV (T9). J. Antimicrob.
Agents Chemother. 1995, 39, 2320–2324. [CrossRef]
11. Carvalho, S.A.; da Silva, E.F.; de Souza, M.V.; Lourenco, M.C.; Vicente, F.R. Synthesis and antimycobacterial
evaluation of new trans-cinnamic acid hydrazide derivatives. Bioorg. Med. Chem. Lett. 2008, 18, 538–541.
[CrossRef] [PubMed]
12. Kakwani, M.D.; Suryavanshi, P.; Ray, M.; Rajan, M.G.R.; Majee, S.; Samad, A.; Devarajan, P.; Degani, M.S.
Design, synthesis and antimycobacterial activity of cinnamide derivatives: A molecular hybridization
approach. Bioorg. Med. Chem. Lett. 2011, 21, 1997–1999. [CrossRef] [PubMed]
13. Yoya, G.K.; Bedos-Belval, F.; Constant, P.; Duran, H.; Daffé, M.; Baltas, M. Synthesis and evaluation of a
novel series of pseudo-cinnamic derivatives as antituberculosis agents. Bioorg. Med. Chem. Lett. 2009, 19,
341–343. [CrossRef] [PubMed]
14. Bogdashev, N.N.; Tukhovskaya, N.A.; Pogrebnyak, A.V. Physicochemical characterisation of cinnamic
acid derivatives. Part 1. Relationship between antioxidant activity and physicochemical properties.
Pharm. Chem. J. 1998, 32, 31–33.
15. Chen, J.H.; Ho, C.T. Antioxidant activities of caffeic acid and its related hydroxycinnamic acid compounds.
J. Agric. Food Chem. 1997, 45, 2374–2378. [CrossRef]
16. Pontiki, E.; Hadjipavlou-Litina, D.; Litinas, K.; Geromichalos, G. Novel cinnamic acid derivatives as
antioxidant and anticancer agents: Design, synthesis and modeling studies. Molecules 2014, 19, 9655–9674.
[CrossRef] [PubMed]
17. Kumar, S.; Arya, P.; Mukherjee, C.; Singh, B.K.; Singh, N.; Parmar, V.S.; Prasad, A.K.; Ghosh, B. Novel aromatic
ester from Piper longum and its analogues inhibit expression of cell adhesion molecules on endothelial cells.
Biochemistry 2005, 44, 15944–15952. [CrossRef] [PubMed]
18. Alexander, A.; Choudhary, R.K. Process for Preparation of Sunscreen Agents. U.S. Patent 5,527,947,
19 December 1996.
19. Fernandez-Martinez, E.; Bobadilla, R.A.; Morales-Rios, M.S.; Muriel, P.; Perez-Alvarez, V.M. Trans-3-
phenyl-2-propenoic acid (cinnamic acid) derivatives: Structure-activity relationship as hepatoprotective
agents. Med. Chem. 2007, 3, 475–479. [CrossRef] [PubMed]

Molecules 2016, 21, 1629
23 of 25
20. Duchnowicz, P.; Broncel, M.; Podse˛dek, A.; Koter-Michalak, M. Hypolipidemic and antioxidant effects of
hydroxycinnamic acids, quercetin, and cyanidin 3-glucoside in hypercholesterolemic erythrocytes (in vitro
study). Eur. J. Nutr. 2012, 51, 435–443. [CrossRef] [PubMed]
21. Lee, M.K.; Park, Y.B.; Moon, S.S.; Bok, S.H.; Kim, D.J.; Ha, T.Y.; Jeong, T.S.; Jeong, K.S.;
Choi, M.S. Hypocholesterolemic and antioxidant properties of 3-(4-hydroxyl)propanoic acid derivatives in
high-cholesterol fed rats. Chem. Biol. Interact. 2007, 170, 9–19. [CrossRef] [PubMed]
22. Yabe, T.; Hirahara, H.; Harada, N.; Ito, N.; Nagal, T.; Sangi, T.; Yamada, H. Ferulic acid induces neural
progenitor cell proliferation in vitro and in vivo. Neuroscience 2010, 165, 515–524. [CrossRef] [PubMed]
23. Kanaani, J.; Ginsburg, H. Effects of cinnamic acid derivatives on in vitro growth of Plasmodium falciparum
and on the permeability of the membrane of malaria-infected erythrocytes. Antimicrob. Agents Chemother.
1992, 36, 1102–1108. [CrossRef] [PubMed]
24. Pérez, B.C.; Fernandes, I.; Mateus, N.; Teixeira, C.; Gomes, P. Recycling antimalarial leads for cancer:
Antiproliferative properties of N-cinnamoyl chloroquine analogues. Bioorg. Med. Chem. Lett. 2013, 23,
6769–6772. [CrossRef] [PubMed]
25. Pérez, B.; Teixeira, C.; Albuquerque, I.S.; Gut, J.; Rosenthal, P.J.; Prudencio, M.; Gomes, P. PRIMACINS,
N-cinnamoyl-primaquine conjugates, with improved liver-stage antimalarial activity. Med. Chem. Commun.
2012, 3, 1170–1172. [CrossRef]
26. Pérez, B.; Teixeira, C.; Gomes, A.S.; Albuquerque, I.S.; Gut, J.; Rosenthal, P.J.; Prude˛ncio, M.; Gomes, P.
In vitro efficiency of 9-(N-cinnamoylbutyl)aminoacridines against blood- and liver-stage malaria parasites.
Bioorg. Med. Chem. Lett. 2013, 23, 610–613. [CrossRef] [PubMed]
27. Pérez, B.C.; Teixeira, C.; Figueiras, M.; Gut, J.; Rosenthal, P.J.; Gomes, J.R.B.; Gomes, P. Novel cinnamic
acid/4-aminoquinoline conjugates bearing non-proteinogenic amino acids: Towards the development of
potential dual action antimalarials. Eur. J. Med. Chem. 2012, 54, 887–899. [CrossRef] [PubMed]
28. Bezerra, D.P.; Pessoa, C.; de Moraes, M.O.; Saker-Neto, N.; Silveira, E.R.; Costa-Lotufo, L.V. Overview of
the therapeutic potential of piplartine (piperlongumine). Eur. J. Pharm. Sci. 2013, 48, 453–463. [CrossRef]
[PubMed]
29. De, P.; Baltas, M.; Bedos-Belval, F. Cinnamic acid derivatives as anticancer agents-a review. Curr. Med. Chem.
2011, 18, 1672–1703. [CrossRef] [PubMed]
30. Zhang, J.; Yang, J.; Chang, X.; Zhang, C.; Zhou, H.; Liu, M. Ozagrel for acute ischemic stroke: A meta-analysis
of data from randomized controlled trials. Neurol. Res. 2012, 34, 346–353. [CrossRef] [PubMed]
31. Solomon, V.R.; Lee, H. Chloroquine and its analogs: A new promise of an old drug for effective and safe
cancer therapies. Eur. J. Pharmacol. 2009, 625, 220–233. [CrossRef] [PubMed]
32. Solomon, V.R.; Hu, C.; Lee, H. Design and synthesis of chloroquine analogs with anti-breast cancer property.
Eur. J. Med. Chem. 2010, 45, 3916–3923. [CrossRef] [PubMed]
33. Van Huijsduijnen, R.H.; Kiplin Guy, R.; Chibale, K.; Haynes, R.K.; Peitz, I.; Kelter, G.; Phillips, M.A.;
Vennerstrom, J.L.; Yuthavong, Y.; Wells, T.N.C. Anticancer properties of distinct antimalarial drug classes.
PLoS ONE 2013, 8, e82962. [CrossRef] [PubMed]
34. Crespo-Ortiz, M.P.; Wei, M.Q. Antitumor activity of artemisinin and its derivatives: From a well-known
antimalarial agent to a potential anticancer drug. J. Biomed. Biotechnol. 2012. [CrossRef] [PubMed]
35. Kimura, T.; Takabatake, Y.; Takahashi, A.; Isaka, Y. Chloroquine in cancer therapy: A double-edged sword of
autophagy. Cancer Res. 2013, 73, 3–7. [CrossRef] [PubMed]
36. Ganguli, A.; Choudhury, D.; Datta, S.; Bhattacharya, S.; Chakrabarti, G. Inhibition of autophagy by
chloroquine potentiates synergistically anti-cancer property of artemisinin by promoting ROS dependent
apoptosis. Biochimie 2014, 107, 338–349. [CrossRef] [PubMed]
37. Liu, F.; Shang, Y.; Chen, S-Z. Chloroquine potentiates the anti-cancer effect of lidamycin on non-small cell
lung cancer cells in vitro. Acta Pharmacol. Sin. 2014, 35, 645–652. [CrossRef] [PubMed]
38. Kamal, A.; Aziz, A.; Shouman, S.; El-Demerdash, E.; Elgendy, M.; Abdel-Naim, A.B. Chloroquine as a
promising adjuvant chemotherapy together with sunitinib. Sci. Proc. 2014, 1, e384.
39. Das, A.K. Anticancer effect of antimalarial artemisinin compounds. Ann. Med. Health Sci. Res. 2015, 5,
93–102. [CrossRef] [PubMed]
40. Coulter, D.W.; Vennerstrom, J.; Sharp, J.G.; Dong, Y.; Wang, X.; McIntyre, E.; McGuire, T. Screening of
investigational antimalarials for anticancer activity in high risk N-MYC amplified neuroblastoma (NB).
Cancer Res. 2015, 75. [CrossRef]

Molecules 2016, 21, 1629
24 of 25
41. Xu, C.-C.; Deng, T.; Fan, M.-L.; Lv, W.-B.; Liu, J.-H.; Yu, B.-Y. Synthesis and in vitro antitumor evaluation
of dihydroartemisinin-cinnamic acid ester derivatives. Eur. J. Med. Chem. 2016, 107, 192–203. [CrossRef]
[PubMed]
42. Duffy, R.; Wade, C. Discovery of anticancer drugs from antimalarial natural products: A MEDLINE literature
review. Drug Discov. Today 2012, 17, 942–953. [CrossRef] [PubMed]
43. Džimbeg, G.; Zorc, B.; Kralj, M.; Ester, K.; Pavelic´, K.; Balzarini, J.; de Clercq, E.; Mintas, M. The novel
primaquine derivatives of N-alkyl, cycloalkyl or aryl urea: Synthesis, cytostatic and antiviral activity
evaluations. Eur. J. Med. Chem. 2008, 43, 1180–1187. [CrossRef] [PubMed]
44. Šimunovic´, M.; Perkovic´, I.; Zorc, B.; Ester, K.; Kralj, M.; Hadjipavlou-Litina, D.; Pontiki, E. Urea and
carbamate derivatives of primaquine: Synthesis, cytostatic and antioxidant activities. Bioorg. Med. Chem.
2009, 17, 5605–5613.
45. Perkovic´, I.; Tršinar, S.; Žanetic´, J.; Kralj, M.; Martin-Kleiner, I.; Balzarini, J.; Hadjipavlou-Litina, D.;
Katsori, A.M.; Zorc, B. Novel 1-acyl-4-substituted semicarbazide derivatives of primaquine—Synthesis,
cytostatic, antiviral and antioxidative studies. J. Enzym. Inhib. Med. Chem. 2013, 28, 601–610. [CrossRef]
[PubMed]
46. Pavic´, K.; Perkovic´, I.; Cindric´, M.; Pranjic´, M.; Martin-Kleiner, I.; Kralj, M.; Schols, D.; Hadjipavlou-Litina, D.;
Katsori, A.M.; Zorc, B. Novel semicarbazides and ureas of primaquine with bulky aryl or hydroxyalkyl
substituents: Synthesis, cytostatic and antioxidative activity. Eur. J. Med. Chem. 2014, 86, 502–514. [CrossRef]
[PubMed]
47. Perkovic´, I.; Antunovic´, M.; Marijanovic´, I.; Pavic´, K.; Ester, K.; Kralj, M.; Vlainic´, J.; Kosalec, I.; Schols, D.;
Hadjipavlou-Litina, D.; et al. Novel urea and bis-urea primaquine derivatives with hydroxyphenyl and
halogenphenyl substituents: Synthesis and biological evaluation. Eur. J. Med. Chem. 2016, 124, 622–636.
[CrossRef] [PubMed]
48. Ren, S.; Wang, R.; Komatsu, K.; Bonaz-Krause, P.; Zyrianov, Y.; McKenna, C.E.; Csipke, C.; Tokes, Z.A.;
Lien, E.J. Synthesis, biological evaluation, and quantitative structure-activity relationship analysis of new
Schiff Bases of hydroxysemicarbazide as potential antitumor agents. J. Med. Chem. 2002, 45, 410–419.
[CrossRef] [PubMed]
49. Zovko, M.; Zorc, B.; Jadrijevic´-Mladar Takacˇ, M.; Metelko, B.; Novak, P. The novel
ketoprofenamides—Synthesis and spectroscopic characterization. Croat. Chem. Acta 2003, 76, 335–341.
50. Barbaric´, M.; Kralj, M.; Marjanovic´, M.; Husnjak, I.; Pavelic´, K.; Filipovic´-Grcˇic´, J.; Zorc, D.; Zorc, B. Synthesis
and in vitro antitumor effect of diclofenac and fenoprofen thiolated and nonthiolated polyaspartamide-drug
conjugates. Eur. J. Med. Chem. 2007, 42, 20–29. [CrossRef] [PubMed]
51. Rajic´, Z.; Butula, I.; Zorc, B.; Kraljevic´ Pavelic´, S.; Hock, K.; Pavelic´, K.; Naesens, L.; de Clercq, E.; Balzarini, J.;
Przyborowska, M.; et al. Cytostatic and antiviral evaluations of NSAID hydroxamic acids. Chem. Biol.
Drug Des. 2009, 73, 328–338. [PubMed]
52. Rajic´, Z.; Hadjipavlou-Litina, D.; Pontiki, E.; Kralj, M.; Šuman, L.; Zorc, B. The novel ketoprofen
amides—Synthesis and biological evaluation as antioxidants, lipoxygenase inhibitors and cytostatic agents.
Chem. Biol. Drug Des. 2010, 75, 641–652. [CrossRef] [PubMed]
53. Luzina, E.L.; Popov, A.V. Synthesis, evaluation of anticancer activity and COMPARE analysis of N-bis(tri
fluoromethyl)alkyl-N’-substituted ureas with pharmacophoricmoieties. Eur. J. Med. Chem. 2012, 53, 364–373.
[CrossRef] [PubMed]
54. Instant Cheminformatics Solutions. Available online: http://www.chemicalize.org/ (accessed on
1 September 2016).
55. Rioux, N.; Castonguay, A. Inhibitors of lipoxygenase: A new class of cancer chemopreventive agents.
Carcinogenesis (Lond.) 1998, 19, 1393–1400. [CrossRef]
56. Kort, W.J.; Bijma, A.M.; van Dam, J.J.; van der Ham, A.C.; Hekking, J.M.; van der Ingh, H.F.; Meijer, W.S.;
van Wilgenburg, M.G.; Zijlstra, F.J. Eicosanoids in breast cancer patients before and after mastectomy.
Prostaglandin Leukot. Essent. 1992, 45, 319–327. [CrossRef]
57. De Clercq, E.; Holý, A.; Rosenberg, I.; Sakuma, T.; Balzarini, J.; Maudgal, P.C. A novel selective
broad-spectrum anti-DNA virus agent. Nature 1986, 323, 464–467. [CrossRef] [PubMed]

Molecules 2016, 21, 1629
25 of 25
58. Balzarini, J.; Naesens, L.; Slachmuylders, J.; Niphuis, H.; Rosenberg, I.; Holý, A.; Schellekens, H.; de Clercq, E.
9-(2-Phosphonylmethoxyethyl)adenine (PMEA) effectively inhibits retrovirus replication in vitro and simian
immunodeficiency virus infection in rhesus monkeys. AIDS 1991, 5, 21–28. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds are not available from the authors.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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