SAHAquines, Novel Hybrids Based on SAHA and Primaquine Motifs, as Potential Cytostatic and Antiplasmodial Agents

Article abstract of DOI:10.1002/open.201800117

We report the synthesis of SAHAquines and related primaquine (PQ) derivatives. SAHAquines are novel hybrid compounds that combine moieties of suberoylanilide hydroxamic acid (SAHA), an anticancer agent with weak antiplasmodial activity, and PQ, an antimalarial drug with low antiproliferative activity. The preparation of SAHAquines is simple, cheap, and high yielding. It includes the following steps: coupling reaction between primaquine and a dicarboxylic acid monoester, hydrolysis, a new coupling reaction with O-protected hydroxylamine, and deprotection. SAHAquines 5 a–d showed significant reduction in cell viability. Among the three human cancer cell lines (U2OS, HepG2, and MCF-7), the most responsive were the MCF-7 cells. The antibodies against acetylated histone H3K9/H3K14 in MCF-7 cells revealed a significant enhancement following treatment with N-hydroxy-N′-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}pentanediamide (5 b). Ethyl (2E)-3-({4-[(6-methoxyquinolin-8-yl)amino]pentyl}carbamoyl)prop-2-enoate (2 b) and SAHAquines were the most active compounds against both the hepatic and erythrocytic stages of Plasmodium parasites, some of them at sub-micromolar concentrations. The results of our research suggest that SAHAquines are promising leads for new anticancer and antimalarial agents.

Full text of DOI:10.1002/open.201800117

DOI: 10.1002/open.201800117
SAHAquines, Novel Hybrids Based on SAHA and
Primaquine Motifs, as Potential Cytostatic and
Antiplasmodial Agents
[a]
[a]
[b]
[a]
[c]
´
´
´
Maja Beus, Zrinka Rajic,* Dusica Maysinger, Zvonimir Mlinaric, Maja Antunovic,
[c]
[d]
[d]
[e]
[f]
´
Inga Marijanovic, Diana Fontinha, Miguel PrudÞncio, Jana Held, Sureyya Olgen, and
Branka Zorc*[a]
We report the synthesis of SAHAquines and related primaquine
(PQ) derivatives. SAHAquines are novel hybrid compounds that
combine moieties of suberoylanilide hydroxamic acid (SAHA),
an anticancer agent with weak antiplasmodial activity, and PQ,
an antimalarial drug with low antiproliferative activity. The
preparation of SAHAquines is simple, cheap, and high yielding.
It includes the following steps: coupling reaction between pri-
maquine and a dicarboxylic acid monoester, hydrolysis, a new
coupling reaction with O-protected hydroxylamine, and depro-
tection. SAHAquines 5a–d showed significant reduction in cell
viability. Among the three human cancer cell lines (U2OS,
HepG2, and MCF-7), the most responsive were the MCF-7 cells.
The antibodies against acetylated histone H3K9/H3K14 in MCF-
7 cells revealed a significant enhancement following treatment
with N-hydroxy-N’-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-
pentanediamide (5b). Ethyl (2E)-3-({4-[(6-methoxyquinolin-8-
yl)amino]pentyl}carbamoyl)prop-2-enoate (2b) and SAHA-
quines were the most active compounds against both the hep-
atic and erythrocytic stages of Plasmodium parasites, some of
them at sub-micromolar concentrations. The results of our re-
search suggest that SAHAquines are promising leads for new
anticancer and antimalarial agents.
1. Introduction
Despite extensive efforts and significant progress in cancer
treatment, therapeutic interventions are not yet satisfactory.
Almost all available cytostatic drugs cause undesirable side ef-
fects, whereas drug resistance presents an additional prob-
lem.[1–3] In another field of medicine, an ongoing battle is
being fought against malaria, a parasitic disease caused by
Plasmodium species.[4,5] Malaria still poses a great health and
economic burden to the highly populated countries in the
tropical and subtropical parts of the world. The need for new,
effective antimalarials arises from several factors, including the
absence of an effective vaccine, insufficient vector control, and
the emergence of multidrug-resistant Plasmodium strains.[6–8]
The currently adopted approaches to the design of antiplas-
modial compounds include the development of analogues of
existing drugs, resistance reversers, and novel compounds with
new mechanisms of action.[6,9]
A number of studies have shown a relationship between
cancer and malaria in regard to diagnostics, drug research,
treatment, prevention, and epidemiology.[10–13] Different classes
of antimalarial drugs display direct or adjuvant activity against
cancer cell lines, are known as sensitivity reversers of resistant
tumor cell lines or inhibitors of drug resistance development,
or have synergistic action with known anticancer drugs.[14–20]
Although their exact mode of action against cancer is still not
completely understood, various mechanisms have been pro-
posed.[21–23]
´
´
[a] M. Beus, Prof. Z. Rajic, Z. Mlinaric, Prof. B. Zorc
[e] Dr. J. Held
Faculty of Pharmacy and Biochemistry, University of Zagreb
Institute of Tropical Medicine, University of Tübingen
Wilhelmstraße 27, 72074 Tübingen (Germany)
´
A. Kovacica 1, 10 000 Zagreb (Croatia)
E-mail: zrajic@pharma.hr
bzorc@pharma.hr
[f] Prof. S. Olgen
Faculty of Pharmacy, Biruni University
10th street No: 45, 34010 Topkapi Istanbul (Turkey)
[b] Prof. D. Maysinger
Department of Pharmacology and Therapeutics, McGill University
23655 Promenade Sir-William-Osler, McIntyre Medical Sciences Building
Montreal, Quebec H3G 1Y6 (Canada)
Supporting Information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/open.201800117.
´
´
[c] Dr. M. Antunovic, Prof. I. Marijanovic
Faculty of Science, University of Zagreb
Horvatovac 102A, 10 000 Zagreb (Croatia)
ꢀ 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited,
the use is non-commercial and no modifications or adaptations are
made.
[d] Dr. D. Fontinha, Dr. M. PrudÞncio
Instituto de Medicina Molecular, Faculdade de Medicina
Universidade de Lisboa
Av. Prof. Egas Moniz, 1649-028 Lisboa (Portugal)
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Such observations prompted us, and others, to design and
prepare novel derivatives of known antimalarial drugs and
evaluate their cytostatic potential.[24–34] Our efforts have been
focused on primaquine (PQ), which is an old drug with many
flaws (e.g., induction of hemolytic anemia in individuals lacking
glucose-6-phosphate dehydrogenase, quick metabolism, deg-
radation to inactive carboxyprimaquine, altering the treatment
outcome in dependence of CYP 2D6 enzyme activity)[35] but is
currently the only available Plasmodium hypnozoitocide.[36] We
previously showed that various PQ derivatives of amides,
ureas, bis-ureas, semicarbazides, and acylsemicarbazide-type
derivatives possessed significant cytostatic activity against a
panel of cancer cell lines or high selectivity towards the breast
adenocarcinoma cell line (MCF-7).[24–30] On the other hand, cy-
tostatic agents of different classes, including histone deacety-
lase (HDAC) inhibitors, have been shown to exert antiplasmo-
dial activity.[6,37–42] Suberoylanilide hydroxamic acid (SAHA, vori-
acids, and compounds 4 and 6 are O-benzyl- and O-methyl-
substituted hydroxamic acids. Similar to the known HDAC in-
hibitors, hydroxamic acids 5 consist of a capping group (quino-
line ring), a linker, and a Zn-binding group (hydroxamic acid)
(Figure 1). Given that hydroxamic acid is a strong binding
group for metal ions that might lead to poor selectivity and
confer undesired properties, such as poor pharmacokinet-
ics,[56,57] we introduced other functional groups instead: an
ester, carboxylic acid, or O-protected hydroxamic group.
Herein, we report the synthesis of SAHAquines 5a–d as well
as 20 novel PQ derivatives, their chemical characterization, the
assessment of their cytostatic activity, and the evaluation of
their activity against the erythrocytic and hepatic stages of
Plasmodium.
nostat) was the first marketed pan-HDAC inhibitor, and it was 2. Results and Discussion
followed by other drugs from the same class (e.g., romidepsin,
belinostat, and panobinostat.[43,44] Their hydroxamic group che-
2.1. Chemistry
lates zinc ion found in the active site of Zn-dependent HDACs
(classes I, II, and IV), and this leads to the accumulation of ace-
tylated histones and other proteins.[45] HDACs were also identi-
fied as transcription regulators in P. falciparum.[46,47] Out of five
P. falciparum HDACs, three are Zn-dependent enzymes, prone
to inhibition by SAHA, and thus represent viable targets for
drug development.[48] Several studies have shown the antima-
larial activity of SAHA and related HDAC inhibitors.[49,50]
The objective of our research was to prepare SAHAquines 5
and related PQ derivatives, which differ in the type/length of
the spacer and/or functional groups: compounds 2 are esters
and compounds 3 are carboxylic acids, whereas compounds 4
and 6 are O-protected hydroxamic acids. Scheme 1 shows the
synthetic pathway leading to derivatives 2–6. In the first reac-
tion step, dicarboxylic acid monoesters 1a–e were coupled
with PQ to give derivatives 2a–e by using 1-[bis(dimethylami-
no)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexa-
fluorophosphate (HATU) as the coupling reagent, along with
Hünig’s base (N,N-diisopropylethylamine, DIEA). The following
dicarboxylic acid monoesters were used: 4-methoxy-4-oxobu-
tanoic acid (monomethyl hydrogen succinate) (1a), (E)-4-
ethoxy-4-oxobut-2-enoic acid (monoethyl fumarate) (1b), 5-
methoxy-5-oxopentanoic acid (monomethyl glutarate) (1c), 6-
methoxy-6-oxohexanoic acid (monomethyl adipate) (1d), and
4-(methoxycarbonyl)benzoic acid (monomethyl terephtalate)
(1e). Notably, an analogous coupling reaction between PQ and
monomethyl malonate failed in our hands. Amide formation
between these two compounds by using thionyl chloride or
benzotriazolide[58] was also unsuccessful and gave a mixture of
products. Classical activation of the carboxylic group with thio-
nyl chloride worked well with other dicarboxylic acid monoest-
ers. Hydrolysis of 2a–e with lithium hydroxide afforded corre-
sponding acids 3a–e, which were transformed into O-benzyl-
hydroxamic acids 4a–e and O-methylhydroxamic acids 6a–e
by means of O-benzyl- and O-methylhydroxylamine, respec-
tively. Again, HATU/DIEA was used as the coupling system.
Free hydroxamic acids 5a–d were obtained by catalytic hydro-
genation of O-benzyl derivatives 4a–e. Selective deprotection
of the benzyl group in fumaric derivative 4b failed. As a result
of double-bond hydrogenation, succinylhydroxamic acid 5a
was obtained instead of fumarylhydroxamic acid.
In this study, we employed one of the classical medicinal
chemists’ tools— the combination of two pharmacophores in
one molecule.[7,51–54] The concept of hybrid drugs is also a val-
uable strategy to overcome the limitations of a combined ther-
apy, as the resulting molecules could exhibit inhibitory activi-
ties on multiple targets.[55] The hybrid compounds described
here, SAHAquines, combine motifs of SAHA, an anticancer
agent with weak antiplasmodial activity, and PQ, an antimalari-
al drug with low antiproliferative activity. Other here-reported
PQ derivatives differ in the linker length/type and/or functional
groups: compounds 2 are esters, compounds 3 are carboxylic
All new compounds were fully characterized by conventional
1
spectroscopy and analytical methods (IR, H NMR, and 13C NMR
spectroscopy; MS; and elemental analyses). The data were con-
sistent with the proposed structures and are given in short in
Figure 1. Design of SAHAquines 5 and other PQ derivatives.
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Scheme 1. Synthesis of PQ derivatives 2–6. Reagents and conditions: a) HATU, DIEA, CH2Cl2, RT, 1 h; b) LiOH, MeOH, H2O, RT, 1 h; c) H2NOBn, HATU, DIEA,
CH2Cl2, RT, 2 h; d) H2, Pd/C, MeOH, RT, 2–4 h; NH2OMe, HATU, DIEA, CH2Cl2, RT, 2 h.
the Experimental Section and in detail in the Supporting
Information.
calculated with Chemicalize.org software and are presented in
Table 1.[59] All compounds 2–6 (except 4e, which showed mini-
mal aberration) are fully in agreement with Lipinski’s and Gelo-
vani’s rules for prospective small-molecule drugs (MWꢀ500,
log Pꢀ5, number of H-bond donors ꢀ5, number of H-bond
acceptors ꢀ10, TPSA<140 Š2, MR within the range of 40 and
130 cm3 molÀ1, number of atoms 20–70).[60]
To evaluate the drug-like properties of our novel com-
pounds, a common set of physicochemical parameters were
calculated: topological polar surface area (TPSA), number of
atoms, molecular weight (MW), partition coefficient (log P),
number of H-bond donors (HBDs), number of H-bond accept-
ors (HBAs), and molar refractivity (MR). The parameters were
Table 1. Properties of compounds 2–6 calculated with the Chemicalize.org program.[59] The Lipinski’s and Gelovani’s parameters.
Compd
Molecular formula
Number of atoms
MW
log P
HBD[a]
HBA[b]
Lipinski score[c]
MR[d] [cm3 molÀ1
]
TPSA[e] [Š2]
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
5a
5b
5c
5d
6a
6b
6c
6d
6e
C20H27N3O4
C21H27N3O4
C21H29N3O4
C22H31N3O4
C24H27N3O4
C19H25N3O4
C19H23N3O4
C20H27N3O4
C21H29N3O4
C23H25N3O4
C26H32N4O4
C26H30N4O4
C27H34N4O4
C28H36N4O4
C30H32N4O4
C19H26N4O4
C20H28N4O4
C21H30N4O4
C23H26N4O4
C20H28N4O4
C20H26N4O4
C21H30N4O4
C22H32N4O4
C24H28N4O4
54
55
57
60
58
51
49
54
57
55
66
64
69
72
70
53
56
59
57
56
54
59
62
60
373.453
385.464
387.480
401.507
421.497
359.426
357.410
373.453
387.480
407.470
464.566
462.515
478.580
492.620
512.600[b]
374.441
388.468
402.495
422.485
388.468
386.452
402.495
416.522
436.512
1.43
2.38
1.88
2.32
3.32
0.80
0.90
1.29
1.74
2.23
2.58
2.94
3.02
3.47
4.27
0.48
0.92
1.37
2.16
0.85
1.21
1.30
1.74
2.54
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
3
3
3
3
3
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
4
4
4
4
4
4
4
4
4
4
4
4
4
4
3[f]
4
4
4
4
4
4
4
4
4
103.62
109.44
108.22
112.82
120.65
98.85
89.55
89.55
89.55
89.55
89.55
100.55
100.55
100.55
100.55
100.55
101.58
101.58
101.58
101.58
101.58
112.58
112.58
112.58
112.58
101.58
101.58
101.58
101.58
101.58
99.92
103.45
108.05
115.89
131.53
132.60
136.13
140.74
148.57
102.44
107.04
111.64
119.47
106.92
107.99
111.52
116.12
123.95
[a] H-bond donor. [b] H-bond acceptor. [c] Out of four. [d] Molar refractivity. [e] Topological polar surface area. [f] Minimal aberrations of the rules.
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2.2. Biological Evaluation
values towards MCF-7 cells were in the low micromolar con-
centrations (1.6–5.4 mm). Compounds from the other subclass-
es were, in general, less active than compounds 5. Compounds
4d and 4e were the most effective O-benzylhydroxamic acids.
In contrast, O-methylhydroxamic acids 6 exhibited weak activi-
ty, except for succinic acid derivative 6a. All compounds from
the ester series, except 2c, showed moderate activity, whereas
carboxylic acid derivatives 3a–e were practically inactive.
MCF-7 cells were sensitive to 15 of the 24 tested com-
pounds, namely, 5a–d, 2d, 2e, 4a, 4c–e, and 6a–d. Similar re-
sults were obtained in our previous studies with various PQ
Synthesized compounds 2–6 were tested for their anticancer
activity on three human cancer cell lines (bone osteosarcoma
U2OS, hepatocellular carcinoma HepG2, and breast adenocarci-
noma MCF-7) and human embryonic kidney (Hek293) cells.
The cells were treated with the different compounds at differ-
ent concentrations, and the median inhibitory concentration
(IC50) values were determined (Table 2).
The data from Table 2 clearly indicate that the SAHAquines
were the most potent in the selected cancer cell lines. The IC50
Table 2. Antiproliferative screening of novel compounds 2–6 towards human cancer cell lines (U2OS, HepG2, MCF-7) and human embryonic kidney cell
line (Hek293) in vitro.
Compd
Structure
IC50[a] [mm]
U2OS
HepG2
MCF-7
Hek293
2a
6.7Æ3.1
14.9Æ2.2
>50
>50
>50
>50
2b
2c
>50
>50
>50
>50
17.0Æ1.7
>50
>50
>50
>50
>50
2d
2e
3a
3b
24.3Æ1.5
>50
>50
>50
>50
5.3Æ3.2
>50
7.1Æ3.0
>50
>50
>50
>50
3c
33.8Æ2.9
>50
>50
>50
>50
>50
>50
3d
>50
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Table 2. (Continued)
Compd
Structure
IC50[a] [mm]
U2OS
HepG2
MCF-7
Hek293
3e
>50
>50
15.2Æ0.5
>50
4a
4b
4c
4d
4e
>50
>50
>50
>50
12.0Æ1.4
>50
>50
>50
>50
>50
>50
>50
16.6Æ0.8
13.9Æ1.5
11.5Æ0.1
32.8Æ3.0
7.1Æ1.2
9.9Æ1.1
>50
13.2Æ2.6
5a
5b
5c
5d
6a
6b
16.9Æ2.2
3.3Æ0.05
11.6Æ2.0
11.6Æ2.1
6.6Æ1.2
>50
18.0Æ3.3
5.4Æ0.1
1.6Æ0.8
5.0Æ0.4
4.7Æ0.1
5.9Æ0.6
10.0Æ2.8
25.5Æ0.8
8.5Æ0.5
13.7Æ3.4
6.1Æ1.9
8.6Æ2.0
20.0Æ0.2
28.4Æ2.1
21.7Æ0.2
>50
>50
>50
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Table 2. (Continued)
Compd
Structure
IC50[a] [mm]
U2OS
HepG2
MCF-7
Hek293
6c
>50
>50
>50
>50
7.1Æ0.4
>50
6d
6e
>50
>50
9.8Æ0.4
>50
>50
>50
PQ[b]
SAHA[c]
Cis[d]
12.0Æ0.7
5.7Æ0.8
2.5Æ0.7
37.7Æ5.8
13.8Æ1.4
2.8Æ0.7
3.2Æ1.0
8.1Æ1.3
4.0Æ0.1
7.4Æ0.9
2.0Æ0.6
–
[a] The concentration required to decrease viability by 50%. [b] Primaquine. [c] Suberoylanilide hydroxamic acid. [d] Cisplatin.
derivatives.[24–30] On the other hand, HepG2 cells were very
robust and only responded towards 4d and compounds from
subclass 5. The non-cancer human cell line Hek293 was sensi-
tive to 2b, 4e, 5a–d, and 6a but to a lesser extent than the
cancer cell lines. Selectivity indices ranged from 2 to 12, de-
pending on the tested compound and the cancer cell line
employed.
4e and 5b, we labeled the cell nuclei with Hoechst 33342 and
assessed the number of cells (Figure 2). The results from these
studies complement the data from the MTT [3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Table 2),
showing a correlation between significant loss of mitochondri-
al metabolic activity and a concentration-dependent decrease
in the number of cells. A reduction in the number of MCF-7
cells was found for treatment over periods of 24 and 72 h. The
IC50 values for 5b and 4e obtained from these assays were
To show the loss of MCF-7 cells following their exposure to
the most potent compounds from subclasses 4 and 5, that is,
Figure 2. MCF-7 cell viability following treatment with 4e, 5b, primaquine, SAHA, and cisplatin for 24 or 72 h at various concentrations ranging from 50 to
0.001 mm. Cell viability was measured by counting Hoechst 33342 labeled nuclei imaged by using a fluorescence microscope. Shown are average percentage
cell viability compared to the untreated controls Æ SD from two independent experiments.
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within a comparable micromolar concentration range (5b: 0.5
and 1.6 mm; 4e: 9.0 and 11.5 mm). Determination of the
number of cells is a more direct way of showing cell loss.
As SAHAquines 5 with a free hydroxamic acid have the po-
tential to chelate metal ions in Zn-dependent HDACs and to
increase the content of acetylated histones, we performed a
immunocytochemical assay for histone acetylation. MCF-7 cells
were treated with 5b and acetylated H3K9/H3K14 was mea-
sured. SAHA, PQ, and 4e were used as controls in the histone
acetylation experiments. Indeed, the results show that 5b
caused a significant accumulation of acetylated H3 histone
(Figure 3), which suggested that HDAC inhibition was a possi-
ble contributing mechanism for SAHAquines 5 but not for the
other PQ derivatives. Given that HDAC inhibition correlated
with cellular histone acetylation but not with cell loss, we did
not extensively study the kinetics of HDAC inhibition.[61] An in-
depth mechanistic study should be performed to explain the
mode of cell death caused by the SAHAquines.
Literature data on the antimalarial activity of SAHA and relat-
ed HDAC inhibitors[49,50] prompted us to evaluate SAHAquines
and their synthetic precursors for their in vitro activity against
P. falciparum erythrocytic stages. The IC50 values of SAHAquines
2–6 against the erythrocytic stage of two P. falciparum strains,
3D7 and Dd2, were determined (Table 3). In general, the 3D7
strain was more sensitive than the Dd2 strain. Again, the most
active compounds were SAHAquines 5, the hydroxamic acid
subclass. Compound 5b had the lowest IC50 value (0.4 mm for
the Pf3D7 strain and 1.9 mm for the PfDd2 strain), followed by
5d (IC50 =0.6 and 1.2 mm, respectively), 5c (IC50 =3.7 and
13.6 mm, respectively), and finally 5a (IC50 =15.8 and 27.1 mm,
respectively). Derivatives 2b and 2d were the most active
compounds from the ester subclass, and 4e was the most
active from the O-benzylhydroxamic acids. Carboxylic acids
3a–e were inactive.
We further evaluated the in vitro activity of compounds 2–6
against the hepatic stages of P. berghei. Two concentrations of
SAHAquines were tested: 1 and 10 mm. For comparison, the
same concentrations of PQ were included in the assay as a
positive control (IC50 =8.4Æ3.4 mm), and DMSO was used as a
negative control (Figure 4).
As shown in Figure 4, only ester 2b and SAHAquines 5a–d
were active against the P. berghei hepatic stages, whereas the
other tested compounds were completely inactive. All com-
pounds from hydroxamic acid subclass 5 exhibited strong anti-
plasmodial activity at both concentrations tested, with IC50
values ranging from 0.3 to 1.25 mm and without any noticeable
effects on host-cell confluency (Figure 5). Our results show that
the free hydroxamic acid moiety was crucial for antiplasmodial
activity, as activity was lost if this group was protected or re-
placed by a carboxylic acid. The activity of compound 2b from
the ester subclass was probably due to the a,b-unsaturated
carbonyl group (Michael acceptor moiety), which is capable of
conjugate addition.
Figure 3. MCF-7 cells were treated with PQ, SAHA, 4e, or 5b (1 mm) for 24 h.
a) Representative fluorescence micrographs of MCF-7 histone H3 acetylation
(red) at lysine 9/lysine 14 in response to treatments. Cells were incubated
with primary antibody (rabbit anti-acetyl-Histone-H3 polyclonal antibody,
500” dilution) for 24 h and then with secondary antibody (Alexa Fluor 647
goat anti-rabbit IgG À2 mgmLÀ1; 500” dilution) for 1 h in the dark. Cells
were imaged by using a fluorescence microscope with a CY5 filter. Fluores-
cence was analyzed in ImageJ. Scale bar represents 20 mm. b) Averages of
fluorescence per cellÆSD (as fold increase in untreated control=1) from at
least two independent experiments. * p<0.05, t-test.
The relationship between antimalarial and anticancer activity
is complex and is discussed in detail in a review by Duffy,
Wade, and Chang.[62] Both main groups of existing antimalarial
Table 3. IC50 values for compounds 2–6 against erythrocytic stage of two P. falciparum strains.
Compd
IC50 [mm]
Pf3D7
Compd
IC50 [mm]
Pf3D7
Pf3Dd2
Pf3Dd2
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
4a
4b
4c
100.0Æ11.0
2.9Æ0.2
81.1Æ3.4
6.6Æ0.2
27.1Æ0.6
>111
>111
>111
>111
>111
74.1Æ5.3
4d
4e
5a
5b
5c
5d
6a
6b
6c
6d
6e
PQ[a]
CQ[b]
>27.7
>27.7
>27.7
7.2Æ1.9
80.1Æ31.0
50.4Æ4.6
25.0Æ2.8
6.1Æ0.1
15.8Æ1.7
0.4Æ0.1
3.7Æ1.3
0.6Æ0.2
27.1Æ0.8
1.9Æ0.8
13.6Æ0.2
1.2Æ0.04
94.4Æ8.6
14.8Æ0.1
>111
>111
>111
>111
>111
50.0Æ5.0
>27.7
35.5Æ1.2
>111
8.3Æ0.9
>111
>111
>25
>111
>111
>27.7
>55
>27.7
39.5Æ15.5
1.5Æ0.02
4.3Æ1.5
1.6”10À3
0.265Æ0.003
[a] Primaquine. [b] Chloroquine.
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Figure 4. Activity of compounds 2–6 against P. berghei hepatic stages at concentrations of 1 and 10 mm. Anti-infective activity (infections scale, bars) are
shown.
agents, the quinolines and the sesquiterpene lactones, have a
range of known anticancer properties that could either be dis-
tinct from or overlap with their antimalarial properties. Al-
though our results show a similar pattern of activity, it should
be noted that the activities of 5b and 5d are an order of mag-
nitude higher towards the P. falciparum erythrocytic stage than
towards the tested cancer cell lines and the human embryonic
kidney cells (Hek293).
compounds 2b and 5a–d were comparable to the activity of
PQ, they might potentially replace PQ against PQ-resistant
Plasmodium strains or in the treatment of glucose-6-phosphate
dehydrogenase deficient patients and poor metabolizers, but
this hypothesis still remains to be evaluated. Notably, the syn-
thesis of SAHAquines is cheap and short (1–4 h) with good to
excellent yields (43–97%), which is beneficial for the develop-
ment of antimalarial agents.[64]
Having in mind that very good safety profiles are needed for
antimalarial drugs, as they should be delivered, amongst
others, to two particularly vulnerable populations, small chil-
dren and pregnant women,[63] we are aware that our finding
can only be a starting point for the development of a clinically
applicable antimalarial drug. As the antiplasmodial activities of
3. Conclusions
Four SAHAquines based on SAHA and PQ motifs and 20 other
PQ derivatives were synthesized and evaluated in vitro against
three human cancer cell lines and a human embryonic kidney
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Figure 5. IC50 of SAHAquines 5a–d against P. berghei hepatic stages.
light and iodine vapor. Column chromatography was performed
on silica gel 0.063–0.200 mm. All chemicals and solvents were of
analytical grade and were purchased from commercial sources. PQ
diphosphate, 4-methoxy-4-oxobutanoic acid (monomethyl succi-
nate), (E)-4-ethoxy-4-oxobut-2-enoic acid (monoethyl fumarate), 5-
methoxy-5-oxopentanoic acid (monomethyl glutarate), 6-methoxy-
6-oxohexanoic acid (monomethyl adipate), 4-(methoxycarbonyl)-
benzoic acid (monomethyl terephtalate), DIEA, HATU, O-benzylhy-
droxylamine, O-methylhydroxylamine, triethylamine, MTT, cisplatin
(Cis), and Hoechst 33342 were purchased from Sigma–Aldrich;
rabbit anti-acetyl-Histone-H3 polyclonal antibody was purchased
from Millipore, Alexa Fluor 647 goat anti-rabbit IgG was purchased
from Life Technologies, and SAHA was acquired from Cayman
Chemicals. PQ was prepared from PQ diphosphate prior to use. All
reactions with PQ were performed under light-protected
conditions.
cell line. SAHAquines 5a–d displayed cytostatic activity at low
micromolar concentrations. A few compounds from other sub-
classes were also effective, but less so than 5a–d. We showed
that the most active hydroxamic acid, that is, 5b, caused a sig-
nificant accumulation of acetylated histone H3K9/H3K14, a
downstream target of class I HDACs. The results from the anti-
plasmodial activity of SAHAquines against the erythrocytic
stages of the 3D7 and Dd2 P. falciparum strains and against
P. berghei hepatic stages correlated with their cytostatic activi-
ty. The in vitro cytostatic and dual-stage antiplasmodial activity
of SAHAquines suggest that these novel compounds could
constitute a basis for the development of effective anticancer
or malaria prophylactic/curative agents with improved potency
and selectivity.
Experimental Section
Synthesis
General Methods
General Procedure for the Preparation of Esters 2a–e
Melting points were measured with a Stuart Melting Point (SMP3)
apparatus (Barloworld Scientific, UK) in open capillaries. IR spectra
were recorded with a FTIR PerkinElmer Spectrum One, and UV/Vis
spectra were recorded with a Lambda 20 double-beam spectro-
photometer (PerkinElmer, UK). All NMR (1H and 13C) spectra were
obtained at 258C by using an NMR Avance 600 spectrometer
(Bruker, Germany) at 300 and 150 MHz for 1H and 13C nuclei, re-
spectively. Chemical shifts (d) are reported in parts per million
Method A:
A solution of dicarboxylic acid monoester 1a–e
(1.4 mmol), HATU (0.532 g, 1.4 mmol), and DIEA (0.362 g, 2.8 mmol)
in dichloromethane (8 mL) was stirred at room temperature. After
10 min, a solution of PQ (0.401 g, 1.5 mmol) in dichloromethane
(7 mL) was added. The mixture was stirred at room temperature
for 1 h and was then concentrated under reduced pressure. The
residue was dissolved in ethyl acetate and extracted with brine
(3”). The organic layer was dried with sodium sulfate, filtered, and
concentrated under reduced pressure.
1
(ppm) relative to tetramethylsilane in the H spectra and relative to
[D6]DMSO in the 13C spectra (d=39.51 ppm). Coupling constants
(J) are reported in hertz. Mass spectra were collected with a HPLC-
MS/MS instrument (HPLC, Agilent Technologies 1200 Series; MS,
Agilent Technologies 6410 Triple Quad). Mass determination was
realized by using electrospray ionization (ESI) in the positive mode.
Elemental analyses were performed with a CHNS LECO analyzer
(LECO Corporation, USA). All compounds were routinely checked
by TLC with Merck silica gel 60F-254 glass plates by using the fol-
lowing solvent systems: dichloromethane/methanol 9:1, 9.5:0.5,
and 9.6:0.4; cyclohexane/ethyl acetate 1:1; and cyclohexane/ethyl
acetate/methanol 3:1:0.5. Spots were visualized by short-wave UV
Method B:
A solution of dicarboxylic acid monoester 1a–e
(1.8 mmol) in thionyl chloride (7 mL) was kept overnight and con-
centrated under reduced pressure. The residue was triturated with
dichloromethane (2”), and the solvent was evaporated again. A
solution of PQ (0.401 g, 1.5 mmol) and Et3N (0.152 g, 1.5 mmol) in
dichloromethane (8 mL) was added dropwise to the carboxylic acid
chloride dissolved in dichloromethane (10 mL). The mixture was
stirred at room temperature for 1 h and extracted with brine (3”).
The organic layer was dried with sodium sulfate, filtered, and con-
centrated under reduced pressure.
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Methyl 3-({4-[(6-methoxyquinolin-8-yl)amino]pentyl}carbamoyl)pro-
panoate (2a): Method A, from the reaction of monomethyl succi-
nate (1a, 0.185 g) and after purification by column chromatogra-
phy (dichloromethane/methanol 9.5:0.5) and crystallization (ether),
um ether), 2d was obtained as a pale-yellow solid (0.483 g, 86%):
m.p. 56–588C; 1H NMR ([D6]DMSO): d=8.54–8.52 (dd, J=4.2,
1.6 Hz, 1H, 10), 8.09–8.06 (dd, J=8.3, 1.5 Hz, 1H, 12), 7.78 (t, J=
5.4 Hz, 1H, 1), 7.45–7.40 (m, 1H, 11), 6.48 (d, J=2.4 Hz, 1H, 16),
2a was obtained as a pale-yellow solid (0.460 g, 88%): m.p. 71– 6.26 (d, J=2.4 Hz, 1H, 14), 6.12 (d, J=8.8 Hz, 1H, 7), 3.82 (s, 3H,
738C; 1H NMR ([D6]DMSO): d=8.55–8.53 (dd, J=1.6, 4.2 Hz, 1H,
10), 8.09–8.06 (dd, J=1.5, 8.3 Hz, 1H, 12), 7.86 (t, J=5.3 Hz, 1H, 1),
7.45–7.40 (m, 1H, 11), 6.47 (d, J=2.4 Hz, 1H, 16), 6.26 (d, J=2.4 Hz,
1H, 14), 6.12 (d, J=8.7 Hz, 1H, 7), 3.82 (s, 3H, 17), 3.66–3.57 (m,
1H, 5), 3.56 (s, 1H, 5’), 3.08–3.02 (m, 2H, 2), 2.48 (t, J=6.6 Hz, 2H,
2’), 2.33 (t, J=6.8 Hz, 2H, 3’), 1.70–1.58, 1.58–1.42 (2m, 4H, 3,4),
1.20 ppm (d, J=6.3 Hz, 3H, 6); 13C NMR ([D6]DMSO): d=173.30,
170.87, 159.00, 144.62, 144.23, 134.79, 134.52, 129.57, 122.09,
96.09, 91.58, 54.97, 51.21, 46.97, 38.46, 33.37, 29.82, 28.82, 25.95,
20.19 ppm; IR (ATR): n=2278, 3306, 3060, 2959, 2927, 2857, 1732,
1636, 1619, 1552, 1518, 1454, 1387, 1224, 1200, 1170, 1052, 1031,
825, 793, 679 cmÀ1; MS (ESI): m/z: 374.2 [M+1]+; elemental analysis
calcd (%) for C20H27N3O4 (373.45): C 64.32, H 7.21, N 11.25; found: C
64.02, H 7.47, N 11.00.
17), 3.66–3.59 (m, 1H, 5), 3.57 (s, 3H, 7’), 3.08–3.04 (m, 2H, 2), 2.28
(t, J=6.9 Hz, 2H, 2’), 2.04 (t, J=6.9 Hz, 2H, 5’), 1.68–1.57, 1.57–1.42
(2m, 8H, 3, 4, 3’, 4’), 1.20 ppm (d, J=6.3 Hz, 3H, 6); 13C NMR
([D6]DMSO): d=173.22, 171.60, 159.00, 144.63, 144.23, 134.79,
134.53, 129.57, 122.09, 96.09, 91.58, 54.97, 51.15, 46.98, 38.36,
35.00, 33.43, 32.98, 25.99, 24.73, 24.03, 20.18 ppm; IR (ATR): n=
3373, 3311, 2943, 2876, 1730, 1640, 1618, 1518, 1508, 1461, 1425,
1389, 1265, 1222, 1169, 1055, 821, 680, 625 cmÀ1; MS (ESI): m/z:
402.3 [M+1]+; elemental analysis calcd (%) for C22H31N3O4 (401.50):
C 65.81, H 7.78, N 10.47; found: C 65.66, H, 7.66, N 10.31.
Methyl 4-({4-[(6-methoxyquinolin-8-yl)amino]pentyl}carbamoyl)ben-
zoate (2e): Method A, from the reaction of monomethyl terephta-
late (1e, 0.252 g) and after purification by column chromatography
(dichloromethane/methanol 9.5:0.5) and crystallization (ether), 2e
was obtained as a pale-yellow solid (0.489 g, 83%): m.p. 112–
Ethyl (2E)-3-({4-[(6-methoxyquinolin-8-yl)amino]pentyl}carbamoyl)-
prop-2-enoate (2b): Method B, from the reaction of monoethyl fu-
marate (1b, 0.202 g) and after purification by column chromatog-
raphy (dichloromethane/methanol 9.6:0.4) and crystallization
(ether), 2b was obtained as a pale-yellow solid (0.378 g, 70%): m.p.
1
113 8C; H NMR ([D6]DMSO): d=8.65 (t, J=5.5 Hz, 1H, 1), 8.54–8.52
(dd, J=4.2, 1.6 Hz, 1H, 10), 8.08–8.06 (dd, J=8.2, 1.1 Hz, 1H, 12),
8.01 (d, J=8.3 Hz, 2H, 3’, 7’), 7.94 (d, J=8.3 Hz, 2H, 4’, 6’), 7.43–
7.41 (m, 1H, 11), 6.47 (d, J=2.2 Hz, 1H, 16), 6.28 (d, J=2.1 Hz, 1H,
14), 6.15 (d, J=8.7 Hz, 1H, 7), 3.88 (s, 3H, 9’), 3.81 (s, 3H, 17), 3.69–
3.65 (m, 1H, 5), 3.32–3.29 (m, 2H, 2), 1.76–1.65, 1.65–1.57 (2m, 4H,
3, 4), 1.23 ppm (d, J=6.3 Hz, 3H, 6); 13C NMR ([D6]DMSO): d=
165.69, 165.24, 158.97, 144.59, 144.18, 138.76, 134.73. 134.51,
131.54, 129.53, 128.99, 127.47, 122.03, 96.09, 91.59, 54.91, 52.26,
47.00, 39.27, 33.39, 25.79, 20.15 ppm; IR (ATR): n=3403, 3318,
3060, 2936, 1714, 1634, 1615, 1519, 1425, 1386, 1278, 1225, 1169,
1111, 820, 791, 736, 703 cmÀ1; MS (ESI): m/z: 422.2 [M+1]+; elemen-
tal analysis calcd (%) for C24H27N3O4 (421.49): C 68.39, H 6.46, N
9.97; found: C 68.25, H 6.76, N 10.08.
1
71–738C; H NMR ([D6]DMSO): d=8.54–8.53 (dd, J=1.6, 4.2 Hz, 1H,
10), 8.51 (t, J=5.5 Hz, 1H, 1), 8.08–8.06 (dd, J=1.5, 8.3 Hz, 1H, 12),
7.43–7.41 (m, 1H, 11), 6.98 (d, J=15.5 Hz, 1H, 2’), 6.55 (d, J=
15.5 Hz, 1H, 3’), 6.47 (d, J=2.4 Hz, 1H, 16), 6.27 (d, J=2.4 Hz, 1H,
14), 6.13 (d, J=8.8 Hz, 1H, 7), 4.20–4.16 (q, J=7.1 Hz, 2H, 5’), 3.82
(s, 3H, 17), 3.66–3.61 (m, 1H, 5), 3.20–3.17 (dd, J=6.4, 12.3 Hz, 2H,
2), 1.71–1.65, 1.62–1.51 (2m, 4H, 3,4), 1.23 (t, J=7.1 Hz, 3H, 6’),
1.22 ppm (d, J=6.3 Hz, 3H, 6); 13C NMR ([D6]DMSO): d=165.05,
162.63, 158.98, 144.60, 144.20, 137.60, 134.76, 134.51, 129.55,
128.08, 122.06, 96.11, 91.51, 60.60, 54.94, 46.93, 38.81, 33.36, 25.63,
20.18, 13.97 ppm; IR (ATR): n=3391, 3325, 3079, 2969, 2939, 1716,
1645, 1616, 1554, 1520, 1457, 1424, 1388, 1365, 1334, 1302, 1224,
1205, 1163, 1159, 1038, 999, 831, 819, 790, 681, 660 cmÀ1; MS (ESI):
m/z: 386.2 [M+1]+; elemental analysis calcd (%) for C21H27N3O4
(385.46): C 65.44, H 7.06, N 10.90; found: C 65.25, H 7.09, N 10.70.
General procedure for the preparation of carboxylic acids 3a–e: A
solution of lithium hydroxide monohydrate (0.126 g, 3 mmol) in
water (10 mL) was added to a solution of ester 2 (6 mmol) in meth-
anol (10 mL). The mixture was stirred at room temperature for 1 h.
Methanol was evaporated under reduced pressure, and the aque-
ous residue was neutralized with 10% HCl and extracted with di-
chloromethane (3”). The organic layer was dried with sodium sul-
fate, filtered, and concentrated under reduced pressure.
Methyl 4-({4-[(6-methoxyquinolin-8-yl)amino]pentyl}carbamoyl)but-
anoate (2c): Method A, from the reaction of monomethyl glutarate
(1c, 0.206 g) and after purification by column chromatography (di-
chloromethane/methanol 9.5:0.5), 2c was obtained as an oil
1
3-({4-[(6-Methoxyquinolin-8-yl)amino]pentyl}carbamoyl)propanoic
acid (3a): From the reaction of ester 2a (0.224 g) and after crystalli-
zation (ether), 3a was obtained as a pale-yellow solid (0.209 g,
97%): m.p. 146–1488C; 1H NMR ([D6]DMSO): d=12.06 (s, 1H, 5’),
8.55–8.53 (dd, J=4.2, 1.6 Hz, 1H, 10), 8.09–8.06 (dd, J=8.3, 1.6 Hz,
1H, 12), 7.85–7.82 (t, J=5.4 Hz, 1H, 1), 7.45–7.41(m, 1H, 11), 6.48
(d, J=2.4 Hz, 1H, 16), 6.26 (d, J=2.4 Hz, 1H, 14), 6.12 (d, J=8.7 Hz,
1H, 7), 3.82 (s, 3H, 17), 3.66–3.57 (m, 1H, 5), 3.08–3.03 (m, 2H, 2),
2.41 (t, J=6.5 Hz, 2H, 2’), 2.29 (t, J=6.7 Hz, 2H, 3’), 1.70–1.58,
1.58–1.38 (2m, 4H, 3, 4), 1.2 ppm (d, J=6.3 Hz, 3H, 6); 13C NMR
([D6]DMSO): d=173.87, 170.73, 159.00, 144.63, 144.24, 134.80,
134.52, 129.58, 122.10, 96.10, 91.59, 54.97, 46.99, 38.48, 33.38,
30.04, 29.21, 25.96, 20.19 ppm; IR (ATR): n=3454, 3268, 3094, 3009,
2962, 2928, 2863, 1713, 1649, 1615,1584, 1562, 1524, 1452, 1386,
1346, 1227, 1203, 1156, 1161, 824, 785, 746, 674 cmÀ1; MS (ESI): m/
z: 360.2 [M+1]+; elemental analysis calcd (%) for C19H25N3O4
(359.42): C 63.49, H 7.01, N 11.69; found: C 63.22, H 7.30, N 11.45.
(0.439 g, 81%): H NMR ([D6]DMSO): d=8.53 (d, J=2.6 Hz, 1H, 10),
8.07 (d, J=8.2 Hz, 1H, 12), 7.78 (t, J=3 Hz, 1H, 1), 7.43–7.41 (m,
1H, 11), 6.47 (d, J=2.2 Hz, 1H, 16), 6.26 (d, J=2.2 Hz, 1H, 14), 6.11
(d, J=8.8 Hz, 1H, 7), 3.82 (s, 3H, 17), 3.64–3.60 (m, 1H, 5), 3.57 (s,
3H, 6’), 3.05 (t, J=6.0 Hz, 2H, 2), 2.27 (t, J=3.0 Hz, 2H, 2’), 2.07 (t,
J=7.3 Hz, 2H, 4’), 1.74–1.69 (m, 2H, 3’), 1.67–1.62, 1.54–1.44 (2m,
4H, 3, 4), 1.21 ppm (d, J=6.2 Hz, 3H, 6); 13C NMR ([D6]DMSO): d=
173.00, 171.18, 158.98, 144.60, 144.20, 134.76, 134.50, 129.55,
122.05, 96.08, 91.59, 54.95, 51.14, 46.97, 38.36, 34.28, 33.40, 32.64,
25.93, 20.62, 20.17 ppm; IR (ATR): n=3376, 3246, 3086, 2993, 2961,
2927, 1736, 1632, 1612, 1574, 1520, 1458, 1423, 1386, 1219, 1203,
1172, 1161, 1052, 992, 817, 788, 751, 720, 676 cmÀ1; MS (ESI): m/z:
388.3 [M+1]+; elemental analysis calcd (%) for C21H29N3O4 (387.47):
C 65.09, H 7.54, N 10.84; found: C 65.35, H 7.33, N 10.99.
Methyl
pent-
5-({4-[(6-methoxyquinolin-8-yl)amino]pentyl}carbamoyl)-
anoate (2d): Method A, from the reaction of monomethyl adipate
(1d, 0.224 g) and after purification by column chromatography (di-
chloromethane/methanol 9.5:0.5) and crystallization (ether/petrole-
(2E)-3-({4-[(6-Methoxyquinolin-8-yl)amino]pentyl}carbamoyl) prop-2-
enoic acid (3b): From the reaction of ester 2b (0.231 g) and after
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crystallization (ether), 3b was obtained as a pale-yellow solid
(0.178 g, 83%): m.p. 147–1498C; H NMR ([D6]DMSO): d=12.83 (s,
3308, 2941, 2864 1727, 1691, 1636, 1613, 1575, 1519, 1454, 1422,
1385, 1276, 1220, 1201, 1156, 818, 786, 674 cmÀ1; MS (ESI): m/z:
408.2 [M+1]+; elemental analysis calcd (%) for C23H25N3O4 (407.46):
C 67.80, H 6.18, N 10.31; found: C 67.93, H 6.02, N 10.68.
1
1H, 5’), 8.55–8.53 (dd, J=1.6, 4.2 Hz, 1H, 10), 8.49 (t, J=5.5 Hz, 1H,
1), 8.09–8.06 (dd, J=1.5, 8.3 Hz, 1H, 12), 7.45–7.40 (m, 1H, 11), 6.91
(d, J=15.5 Hz, 1H, 2’), 6.50 (d, J=15.5 Hz, 1H, 3’), 6.47 (d, J=
2.4 Hz, 1H, 16), 6.27 (d, J=2.4 Hz, 1H, 14), 6.14 (d, J=8.7 Hz, 1H,
7), 3.82 (s, 3H, 17), 3.68–3.58 (m, 1H, 5), 3.22–3.16 (m, 2H, 2), 1.71–
1.49 (m, 4H, 3, 4), 1.21 ppm (d, J=6.3 Hz, 3H, 6); 13C NMR
([D6]DMSO): d=166.49, 162.95, 159.00, 144.62, 144.22, 137.11,
134.79, 134.52, 129.57, 129.33, 122.09, 96.13, 91.62, 54.96, 46.96,
38.80, 33.40, 25.71, 20.19 ppm; IR (ATR): n=3453, 3271, 3087, 2937,
1716, 1654, 1613, 1564, 1527, 1380, 1337, 1269, 1236, 1213, 1198,
1171, 981, 909, 820, 785, 767, 673 cmÀ1; MS (ESI): m/z: 358.2
[M+1]+; elemental analysis calcd (%) for C19H23N3O4 (357.40): C
63.85, H 6.49, N 11.76; found: C 63.77, H 6.72, N 11.99.
General procedure for the preparation of O-benzylhydroxamic
acids 4a–e: A solution of acid 3 (0.6 mmol), DIEA (0.155 g, 1.2 mol),
and HATU (0.228 g, 0.6 mmol) in dichloromethane (6 mL) was
stirred at room temperature. After 10 min, O-benzylhydroxylamine
hydrochloride (0.112 g, 0.7 mmol) and Et3N (0.071 g, 0.7 mmol)
were added. The mixture was stirred at room temperature for 2 h
and concentrated under reduced pressure. The residue was dis-
solved in ethyl acetate (20 mL) and extracted with brine (3”). The
organic layer was dried with sodium sulfate, filtered, and concen-
trated under reduced pressure.
N-(Benzyloxy)-N’-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}butane-
diamide (4a): From the reaction of 3a (0.216 g) and after purifica-
tion by column chromatography (dichloromethane/methanol
9.5:0.5) and crystallization (ether), 4a was obtained as a pale-
yellow solid (0.111 g, 40%): m.p. 121–1238C; 1H NMR ([D6]DMSO):
d=10.99 (s, 1H, 5’), 8.54 (d, J=2.7 Hz, 1H, 10), 8.08 (d, J=7.2 Hz,
1H, 12), 7.85 (s, 1H, 1), 7.44-7-35 (m, 6H, 11, 8’-12’), 6.47 (d, J=
2.2 Hz, 1H, 16), 6.26 (d, J=2.0 Hz, 1H, 14), 6.12 (d, J=8.6 Hz, 1H,
7), 4.76 (s, 2H, 6’), 3.82 (s, 3H, 17), 3.66–3.57 (m, 1H, 5), 3.08–3.02
(m, 2H, 2), 2.30 (t, J=6.9 Hz, 2H, 3’), 2.18 (t, J=6.8 Hz, 2H, 2’),
1.70–1.57, 1.57–1.43 (2m, 4H, 3, 4), 1.20 ppm (d, J=6.2 Hz, 3H, 6);
13C NMR ([D6]DMSO): d=170.71, 168.80, 159.00, 144.62, 144.23,
136.06, 134.79, 134.52, 129.57, 128.72, 128.26, 128.16, 122.09,
96.09, 91.59, 76.75, 54.97, 46.99, 38.49, 33.39, 30.37, 27.91, 25.97,
20.19 ppm; IR (ATR): n=3367, 3311, 3060, 2936, 2862, 1728, 1636,
1615, 1546, 1519, 1457, 1424, 1387, 1223, 1200, 1159, 1052, 820,
790, 679 cmÀ1; MS (ESI): m/z: 465.3 [M+1]+; elemental analysis
calcd (%) for C26H32N4O4 (464.56): C 67.22, H 6.94, N 12.06; found: C
67.46, H 6.69, N, 12.31.
4-({4-[(6-Methoxyquinolin-8-yl)amino]pentyl}carbamoyl)butanoic
acid (3c): From the reaction of ester 2c (0.232 g) and after crystalli-
zation (ether), 3c was obtained as a pale-yellow solid (0.186 g,
88%): m.p. 95–968C; 1H NMR ([D6]DMSO): d=12.00 (s, 1H, 6’),
8.55–8.53 (dd, J=4.2, 1.6 Hz, 1H, 10), 8.10–8.07 (dd, J=8.3, 1.5 Hz,
1H, 12), 7.80 (t, J=5.4 Hz, 1H, 1), 7.45–7.41 (m, 1H, 11), 6.48 (d, J=
2.4 Hz, 1H, 16), 6.27 (d, J=2.4 Hz, 1H, 14), 6.13 (d, J=8.7 Hz, 1H,
7), 3.83 (s, 3H, 17), 3.69–3.55 (m, 1H, 5), 3.09–3.05 (m, 2H, 2), 2.20
(t, J=7.4 Hz, 2H, 2’), 2.08 (t, J=7.4 Hz, 2H, 4’), 1.75–1.43 (m, 6H, 3’,
3, 4), 1.21 ppm (d, J=6.3 Hz, 3H, 6); 13C NMR ([D6]DMSO): d=
147.15, 171.36, 159.00, 144.63, 144.23, 134.79, 134.53, 129.57,
122.09, 96.11, 91.60, 54.97, 46.98, 38.38, 34.46, 33.41, 33.04, 25.97,
20.71, 20.19 ppm; IR (ATR): n=3453, 3324, 2933, 1720, 1643, 1613,
1580, 1524, 1386, 1227, 1204, 1161, 1139, 1058, 822, 786, 675 cmÀ1
;
MS (ESI): m/z: 374.2 [M+1]+; elemental analysis calcd (%) for
C20H27N3O4 (373.45): C 64.32, H 7.29, N 11.25; found: C 64.21, H
7.15, N 11.48.
5-({4-[(6-Methoxyquinolin-8-yl)amino]pentyl}carbamoyl)pentanoic
acid (3d): From the reaction of ester 2d (0.241 g) and after crystal-
lization (ether), 3d was obtained as a pale-yellow solid (0.198 g,
(2E)-N-(Benzyloxy)-N’-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-
but-2-ene-diamide (4b): From the reaction of 3b (0.215 g) and
after purification by column chromatography (dichloromethane/
methanol/ethyl acetate/cyclohexane 9.5:0.5:10:10) and crystalliza-
tion (ether), 4b was obtained as a pale-yellow solid (0.119 g, 43%):
m.p. 157–1588C; 1H NMR ([D6]DMSO): d=11.52 (s, 1H, 5’), 8.55–
8.53 (dd, J=1.5, 4.2 Hz, 1H, 10), 8.45 (t, J=5.5 Hz, 1H, 1), 8.09–8.06
(dd, J=1.4, 8.3 Hz, 1H, 12), 7.45–7.37 (m, 1H, 11, 8’-12’), 6.90 (d, J=
15.1 Hz, 1H, 2’), 6.62 (d, J=15.2 Hz, 1H, 3’), 6.47 (d, J=2.4 Hz, 1H,
16), 6.27 (d, J=2.3 Hz, 1H, 14), 6.15 (d, J=8.7 Hz, 1H, 7), 4.86 (s,
2H, 6’), 3.82 (s, 3H, 17), 3.68–3.58 (m, 2H, 5), 3.20–3.15 (m, 2H, 2),
1.70–1.49 (m, 4H, 3,4), 1.21 ppm (d, J=6.2 Hz, 3H, 6); 13C NMR
([D6]DMSO): d=163.17, 161.22, 158.99, 144.60, 144.22, 134.82,
134.49, 133.58, 129.58, 129.07, 122.09, 96.16, 91.64, 63.35, 54.97,
46.97, 38.95, 33.42, 25.76, 20.19 ppm; IR (ATR): n=3383, 3283,
3213, 3008, 2936, 2865, 1738, 1626, 1575, 1556, 1518, 1454, 1386,
1337, 1219, 1203, 1157,1051, 974, 818, 790, 738, 695, 675 cmÀ1; MS
(ESI): m/z: 463.1 [M+1]+; elemental analysis calcd (%) for
C26H30N4O4 (462.54): C 67.51, H 6.54, N 12.11; found: C 67.79, H
6.81, N 11.95.
1
85%): m.p. 68–698C; H NMR ([D6]DMSO): d=11.99 (s, 1H, 7’), 8.54
(d, J=2.9 Hz, 1H, 10), 8.08 (d, J=8.1 Hz, 1H, 12), 7.78 (t, J=6.0 Hz,
2H, 1), 7.45–7.40 (m, 1H, 11), 6.48 (d, J=3.0 Hz, 1H, 16), 6.26 (d,
J=3.0 Hz, 1H, 14), 6.12 (d, J=8.6 Hz, 1H, 7), 3.82 (s, 3H, 17), 3.69–
3.56 (m, 1H, 5), 3.09–3.0 (m, 2H, 2), 2.18 (t, J=6.0 Hz, 2H, 5’), 2.04
(t, J=6.0 Hz, 2H, 2’), 1.70–1.57, 1.57–1.42 (2m, 8H, 3, 4, 3’, 4’),
1.20 ppm (d, J=6.2 Hz, 3H, 6); 13C NMR ([D6]DMSO): d=174.37,
171.67, 159.00, 144.63, 144.23, 134.79, 134.52, 129.57, 122.09,
96.10, 91.59, 54.97, 46.98, 38.36, 35.11, 33.38, 25.99, 24.84, 24.13,
20.19 ppm; IR (ATR): n=3453, 3370, 3294, 2943, 2863, 1731, 1609,
1562, 1519, 1455, 1424, 1385, 1224, 1203, 1166, 1156, 1133, 1053,
819, 790, 765, 677 cmÀ1; MS (ESI): m/z: 388.2 [M+1]+; elemental
analysis calcd (%) for C21H29N3O4 (387.47): C 65.10, H 7.54, N 10.84;
found: C 65.44, H 7.79, N 10.63.
4-({4-[(6-Methoxyquinolin-8-yl)amino]pentyl}carbamoyl)benzoic
acid (3e): From the reaction of ester 2e (0.253 g) and after crystalli-
zation (ether), 3e was obtained as a pale-yellow solid (0.210 g,
86%): m.p. 142–1448C; 1H NMR ([D6]DMSO): d=13.12 (s, 1H, 9’),
8.64 (t, J=5.5 Hz, 1H, 1), 8.54–8.53 (dd, J=4.2, 1.6 Hz, 1H, 10),
8.09–8.06 (dd, J=8.3, 1.5 Hz, 1H, 12), 8.00 (d, J=8.4 Hz, 2H, 3’, 7’),
7.92 (d, J=8.4 Hz, 2H, 4’, 6’), 7.45–7.41 (m, 1H, 11), 6.47 (d, J=
2.4 Hz, 1H, 16), 6.28 (d, J=2.4 Hz, 1H, 14), 6.16 (d, J=8.4 Hz, 1H,
N-(Benzyloxy)-N’-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}pentane-
diamide (4c): From the reaction of 3c (0.224 g) and after purifica-
tion by column chromatography (dichloromethane/methanol
7), 3.81 (s, 3H, 17), 3.71–3.63 (m, 1H, 5), 3.34–3.28 (m, 2H, 2), 1.76– 9.5:0.5) and crystallization (ether/petroleum ether), 4c was ob-
1
1.57 (m, 4H, 3, 4), 1.22 ppm (d, J=7.3 Hz, 3H, 6); 13C NMR
([D6]DMSO): d=166.80, 165.42, 159.00, 144.61, 144.22, 138.45,
134.80, 134.52, 132.79, 129.58, 129.16, 127.36, 122.09, 96.14, 91.59,
54.96, 47.02, 39.28, 33.40, 25.86, 20.19 ppm; IR (ATR): n=3413,
tained as a pale-yellow solid (0.144 g, 50%): m.p. 84–858C; H NMR
([D6]DMSO): d=10.95 (s, 1H, 6’), 8.54–8.53 (dd, J=4.1, 1.5 Hz, 1H,
10), 8.09–8.06 (dd, J=8.3, 1.4 Hz, 1H, 12), 7.78 (t, J=5.4 Hz, 1H, 1),
7.44–7.33 (m, 6H, 11, 9’-13’), 6.47 (d, J=2.4 Hz, 1H, 16), 6.26 (d, J=
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2.3 Hz, 1H, 14), 6.13 (d, J=8.8 Hz, 1H, 7), 4.77 (s, 2H, 7’), 3.82 (s,
3H, 17), 3.62–3.56 (m, 1H, 5), 3.08–3.04 (m, 2H, 2), 2.04 (t, J=
7.4 Hz, 2H, 4’), 1.95 (t, J=7.3 Hz, 2H, 2’), 1.74–1.43 (m, 6H, 3’, 3, 4),
1.20 ppm (d, J=6.2 Hz, 3H, 6); 13C NMR ([D6]DMSO): d=171.32,
169.03, 159.00, 144.62, 144.23, 136.09, 134.79, 134.52, 129.57,
128.72, 128.26, 128.16, 122.09, 96.10, 91.59, 76.77, 54.97, 46.98,
38.41, 34.57, 33.43, 31.70, 25.99, 21.21, 20.19 ppm; IR (ATR): n=
3367, 3325, 3187, 2960, 2931, 2857, 1738, 1677, 1634, 1614, 1518,
1455, 1387, 1219, 1203, 1170, 1157, 1052, 1033, 821, 790, 740, 696,
676 cmÀ1; MS (ESI): m/z: 479.3 [M+1]+; elemental analysis calcd (%)
for C27H34N4O4 (478.58): C 67.76, H 7.16, N 11.71; found: C 67.59, H
6.98, N, 12.04.
11), 6.47 (d, J=2.4 Hz, 1H, 16), 6.26 (d, J=2.4 Hz, 1H, 14), 6.12 (d,
J=8.7 Hz, 1H, 7), 3.82 (s, 3H, 17), 3.67–3.56 (m, 1H, 5), 3.09–3.00
(m, 2H, 2), 2.29 (t, J=6.5 Hz, 2H, 2’), 2.17 (t, J=6.5 Hz, 2H, 3’),
1.72–1.56, 1.56–1.39 (2m, 4H, 3, 4), 1.20 ppm (d, J=6.3 Hz, 3H, 6);
13C NMR ([D6]DMSO): d=170.84, 168.46, 159.00, 144.63, 144.24,
134.80, 134.52, 129.58, 122.11, 96.10, 91.60, 54.98, 47.00, 38.49,
33.42, 30.64, 27.92, 25.97, 20.20 ppm; IR (ATR): n=3471, 3375,
3283, 3204, 3008, 2964, 2934, 1744, 1647, 1612, 1556, 1521, 1455,
1385, 1366, 1226, 1205, 1172, 1157, 1056, 821, 789, 677 cmÀ1; MS
(ESI): m/z: 375.2 [M+1]+; elemental analysis calcd (%) for
C19H26N4O4 (374.43): C 60.95, H 7.00, N 14.96; found: C 60.84, H
6.81, N, 15.17.
N-Hydroxy-N’-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}pentanedia-
mide (5b): From the reaction of 4c (0.129 g) and after crystalliza-
tion (ether), 5b was obtained as a pale-yellow solid (0.077 g, 73%):
m.p. 99–1028C; 1H NMR ([D6]DMSO): d=10.32 (s, 1H, 6’), 8.63 (s,
1H, 7’), 8.55–8.52 (dd, J=4.2, 1.6 Hz, 1H, 12), 8.09–8.05 (dd, J=8.3,
1.6 Hz, 1H, 12), 7.76 (t, J=5.3 Hz, 1H, 1), 7.45–7.39 (m, 1H, 11), 6.47
(d, J=2.4 Hz, 1H, 16), 6.26 (d, J=2.4 Hz, 1H, 14), 6.11 (d, J=8.7 Hz,
1H, 7), 3.82 (s, 3H, 17), 3.68–3.56 (m, 1H, 5), 3.05 (d, J=5.6 Hz, 2H,
2), 2.04 (t, J=7.4 Hz, 2H, 2’), 1.94 (t, J=7.4 Hz, 2H, 4’), 1.76–1.57,
1.57–1.40 (2m, 6H, 3, 4, 3’), 1.21 ppm (d, J=6.3 Hz, 3H, 6);
13C NMR ([D6]DMSO): d=171.37, 168.74, 158.98, 144.60, 144.20,
134.75, 134.50, 129.54, 122.05, 96.08, 91.61, 54.95, 46.98, 38.39,
34.73, 33.43, 31.75, 25.95, 21.42, 20.17 ppm; IR (ATR): n=3468,
3375, 3283, 3210, 3008, 2963, 2933, 1744, 1645, 1613, 1520, 1455,
1386, 1204, 1171, 1158, 1056, 821, 789, 677 cmÀ1; MS (ESI): m/z:
389.2 [M+1]+; elemental analysis calcd (%) for C20H28N4O4 (388.46):
C 61.84, H 7.27, N 14.42; found: C 61.69, H 7.03, N, 14.77.
N-(Benzyloxy)-N’-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}hexane-
diamide (4d): From the reaction of 3d (0.232 g) and after purifica-
tion by column chromatography (dichloromethane/methanol
9.5:0.5) and crystallization (ether), 4d was obtained as a pale-
1
yellow solid (0.151 g, 51%): m.p. 102–1058C; H NMR ([D6]DMSO):
d=10.91 (s, 1H, 7’), 8.53 (dd, J=4.0, 1.4 Hz, 1H, 10), 8.06 (dd, J=
8.2 Hz, 1H, 12), 7.74 (s, 1H, 1), 7.43–7.41 (m, 1H, 11), 7.37–7.34 (m,
5H, 10’-14’), 6.47 (d, J=2.3 Hz, 1H, 16), 6.26 (d, J=2.2 Hz, 1H, 14),
6.11 (d, J=8.7 Hz, 1H, 7), 4.77 (s, 2H, 8’), 3.82 (s, 3H, 17), 3.65–3.59
(m, 1H, 5), 3.08–3.02 (m, 2H, 2), 2.02 (s, 2H, 2’), 1.93 (s, 2H, 5’),
1.68–1.62, 1.56–1.41 (2m, 8H, 3, 4, 3’, 4’), 1.20 ppm (d, J=6.3 Hz,
3H, 6); 13C NMR ([D6]DMSO): d=171.61, 169.19, 158.96, 144.59,
144.17, 136.03, 134.72, 134.48, 129.52, 128.66, 128.19, 129.09,
122.02, 96.06, 91.60, 76.70, 54.93, 53.57, 46.97, 38.34, 35.13, 33.42,
32.04, 25.94, 24.83, 24.62, 20.15 ppm; IR (ATR): n=3383, 3287,
2934, 2865, 1737, 1635, 1578, 1519, 1457, 1423, 1387, 1224, 1202,
1168, 1052, 820, 791, 743, 696 cmÀ1; MS (ESI): m/z: 493.3 [M+1]+;
elemental analysis calcd (%) for C28H36N4O4 (492.61): C 68.27, H
7.37, N 11.37; found: C 68.47, H 7.25, N, 11.49.
N-Hydroxy-N’-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}hexanedia-
mide (5c): From the reaction of 4d (0.133 g) and after crystalliza-
tion (ether), 5c was obtained as a pale-yellow solid (0.082 g, 75%):
N1-(Benzyloxy)-N4-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-ben-
zene-1,4-dicarboxamide (4e): From the reaction of 3e (0.245 g)
and after purification by column chromatography (dichlorome-
thane/methanol 9.5:0.5) and crystallization (ether), 4e was ob-
tained as a pale-yellow solid (0.188 g, 61%): m.p. 183–1848C;
1H NMR ([D6]DMSO): d=11.88 (s, 1H, 9’), 8.59 (t, J=5.5 Hz, 1H, 1),
8.54–8.52 (dd, J=4.2, 1.6 Hz, 1H, 10), 8.09–8.06 (dd, J=8.3, 1.6 Hz,
1H, 12), 7.89 (d, J=8.4 Hz, 2H, 3’, 7’), 7.80 (d, J=8.4 Hz, 2H, 4’, 6’),
7.45–7.34 (m, 6H, 11, 12’-16’), 6.47 (d, J=2.5 Hz, 1H, 16), 6.28 (d,
J=2.4 Hz, 1H, 14), 6.15 (d, J=8.6 Hz, 1H, 7), 4.94 (s, 2H, 10’), 3.81
(s, 3H, 17), 3.71–3.63 (m, 1H, 5), 3.33–3.27 (m, 2H, 2), 1.75–1.58 (m,
4H, 3, 4), 1.23 ppm (d, J=6.3 Hz, 3H, 6); 13C NMR ([D6]DMSO): d=
165.35, 163.70, 158.99, 144.61, 144.22, 137.30, 135.82, 134.80,
134.52, 134.38, 129.57, 128.93, 128.32, 127.32, 127.02, 122.09,
96.13, 91.59, 77.03, 54.96, 47.02, 39.23, 33.39, 25.88, 20.19 ppm; IR
(ATR): n=3384, 3287, 2969, 2935, 1738, 1630, 1577, 1519, 1492,
1387, 1320, 1224, 1159, 1053, 863, 820, 790, 747, 718, 69 cmÀ1; MS
(ESI): 513.3 [M+1]+; elemental analysis calcd (%) for C30H32N4O4
(512.60): C 70.29, H 6.29, N 10.93; found: C 70.55, H 6.03, N, 11.21.
1
m.p. 109–111 8C; H NMR ([D6]DMSO): d=10.34 (s, 1H, 7’), 8.66 (s,
1H, 8’), 8.55–8.52 (dd, J=4.2, 1.5 Hz, 1H, 10), 8.10–8.05 (dd, J=8.3,
1.4 Hz, 1H, 12), 7.77 (t, J=5.3 Hz, 1H, 1), 7.45–7.40 (m, 1H, 11), 6.47
(d, J=2.4 Hz, 1H, 16), 6.26 (d, J=2.4 Hz, 1H, 14), 6.12 (d, J=8.8 Hz,
1H, 7), 3.82 (s, 3H, 17), 3.67–3.57 (m, 1H, 5), 3.04 (d, J=5.5 Hz, 2H,
2), 2.03 (s, 2H, 2’), 1.92 (s, 2H, 5’), 1.72–1.58, 1.58–1.41 (2m, 8H, 3,
4, 3’, 4’), 1.20 ppm (d, J=6.3 Hz, 3H, 6); 13C NMR ([D6]DMSO): d=
171.70, 168.95, 159.00, 144.62, 144.24, 134.80, 134.52, 129.57,
122.10, 96.10, 91.60, 54.98, 46.98, 38.38, 35.20, 33.44, 32.11, 26.01,
24.97, 24.87, 20.20 ppm; IR (ATR): n=3428, 3283, 3191, 3088, 3044,
2943, 2923, 2856, 1738, 1621, 1578,1556, 1523, 1385, 1367, 1204,
1170, 1160, 954, 822, 788, 676 cmÀ1; MS (ESI): m/z: 403.3 [M+1]+;
elemental analysis calcd (%) for C21H30N4O4 (402.49): C 62.67, H
7.51, N 13.92; found: C 62.98, H 7.36, N, 14.09.
N1-Hydroxy-N4-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}benzene-
1,4-dicarboxamide (5d): From the reaction of 4e (0.138 g) and
after purification by column chromatography (dichloromethane/
methanol 9:1) and crystallization (ether), 5d was obtained as a
pale-yellow solid (0.048 g, 42%): m.p. 156–1588C; 1H NMR
([D6]DMSO): d=11.32 (s, 1H, 9’), 9.12 (s, 1H, 10’), 8.57 (t, J=5.5 Hz,
1H, 1), 8.54–8.53 (dd, J=4.2, 1.6 Hz, 1H, 10), 8.09–8.06 (d, J=8.3,
1.6 Hz, 1H, 12), 7.88 (d, J=8.3 Hz, 2H, 3’, 7’), 7.80 (d, J=8.3 Hz, 2H,
4’, 6’), 7.44–7.40 (m, 1H, 11), 6.47 (d, J=2.2 Hz, 1H, 16), 6.28 (d, J=
2.1 Hz, 1H, 14), 6.15 (d, J=8.7 Hz, 1H, 7), 3.81 (s, 3H, 17), 3.71–3.62
(m, 1H, 5), 3.33–3.27 (m, 2H, 2), 1.75–1.58 (m, 4H, 3, 4), 1.23 ppm
(d, J=6.2 Hz, 3H, 6); 13C NMR ([D6]DMSO): d=165.44, 163.49,
159.00, 144.62, 144.20, 136.91, 134.86, 134.79, 134.53, 129.57,
127.17, 126.78, 122.09, 96.12, 91.60, 54.96, 47.02, 39.23, 33.41,
25.89, 20.19 ppm; IR (ATR): n=3427, 3303, 3199, 2969, 2938, 1738,
1672, 1636, 1616, 1521, 1458, 1388, 1223, 1203, 1172, 1015, 898,
General procedure for the preparation of SAHAquines 5a–d: A sus-
pension of O-benzylhydroxamic acid 4 (0.27 mmol) and 10% Pd/C
(20 mg) in methanol (7 mL) was stirred at room temperature for 2–
4 h under a hydrogen atmosphere. The catalyst was filtered off,
and the mother liquor was concentrated under reduced pressure.
N-Hydroxy-N’-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}butane-
diamide (5a): From the reaction of 4a (0.125 g) and after crystalli-
zation (ether), 5a was obtained as a pale-yellow solid (0.077 g,
76%): m.p. 109–1108C; 1H NMR ([D6]DMSO): d=10.37 (s, 1H, 5’),
8.67 (s, 1H, 6’), 8.54–8.53 (dd, J=4.2, 1.6 Hz, 1H, 10), 8.09–8.06 (dd,
J=8.3, 1.5 Hz, 1H, 12), 7.85 (t, J=5.3 Hz, 1H, 1), 7.45–7.41 (m, 1H,
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858, 821, 789, 677 cmÀ1; MS (ESI): m/z: 423.1 [M+1]+; elemental
analysis calcd (%) for C23H26N4O4 (422.48): C 65.39, H 6.20, N 13.26;
found: C 65.47, H 6.12, N, 12.99.
([D6]DMSO): d=171.31, 168.72, 159.00, 144.62, 144.23, 134.79,
134.52, 129.57, 122.10, 96.10, 91.59, 63.10, 54.98, 46.98, 38.41,
34.54, 33.43, 31.68, 25.98, 21.12, 20.19 ppm; IR (ATR): n=3396,
3285, 3162, 3097, 2969, 2936, 1737, 1670, 1630, 1564, 1521, 1456,
1424, 1388, 1227, 1206, 1170,1161, 1030, 820, 790, 679 cmÀ1; MS
(ESI): m/z: 403.3 [M+1]+; elemental analysis calcd (%) for
C21H30N4O4 (402.49): C 62.67, H 7.51, N 13.92; found: C 62.46, H
7.31, N 13.95.
General procedure for the preparation of O-methylhydroxamic
acids 6a–e: A solution of corresponding acid 3 (0.2 mmol), DIEA
(0.052 g, 0.4 mmol), and HATU (0.076 g, 0.2 mmol) in dichlorome-
thane (5 mL) was stirred at room temperature. After 10 min, O-
methylhydroxylamine hydrochloride (0.020 g, 0.24 mmol) and Et3N
(0.024 g, 0.24 mmol) were added. The mixture was stirred at room
temperature for 2 h and was then concentrated under reduced
pressure. The residue was dissolved in ethyl acetate (8 mL) and ex-
tracted with water (3”). The organic layer was dried with sodium
sulfate, filtered, and concentrated under reduced pressure.
N-Methoxy-N’-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}hexanedia-
mide (6d): From the reaction of 3d (0.075 g) and after purification
by column chromatography (dichloromethane/methanol 9.5:0.5)
and crystallization (ether), 6d was obtained as a pale-yellow solid
1
(0.042 g, 51%): m.p. 108–1098C; H NMR ([D6]DMSO): d=10.91 (s,
1H, 7’), 8.55–8.52 (dd, J=4.1, 1.6 Hz, 1H, 10), 8.09–8.06 (dd, J=8.3,
1.5 Hz, 1H, 12), 7.75 (t, J=5.4 Hz, 1H, 1), 7.44–7.41 (m, 1H, 11), 6.47
(d, J=2.4 Hz, 1H, 16), 6.26 (d, J=2.4 Hz, 1H, 14), 6.11 (d, J=8.7 Hz,
1H, 7), 3.82 (s, 3H, 17), 3.65–3.55 (m, 4H, 5, 8’), 3.08–3.00 (m, 2H,
2), 2.03 (s, 2H, 2’), 1.92 (s, 2H, 5’), 1.55–1.49, 1.49–1.40 (m, 8H, 3, 4,
3’, 4’), 1.20 ppm (d, J=6.3 Hz, 3H, 6); 13C NMR ([D6]DMSO): d=
171.64, 168.91, 158.98, 144.61, 144.20, 134.76, 134.50, 129.55,
122.06, 96.08, 91.60, 63.05, 54.95, 46.97, 38.36, 35.15, 33.43, 32.07,
25.97, 24.86, 24.60, 20.17 ppm; IR (ATR): n=3395, 3287, 3166, 3090,
2927, 2861, 1739, 1674, 1631, 1577, 1563, 1521, 1457, 1423, 1388,
1227, 1206, 1170, 1161, 1053, 821, 790, 679 cmÀ1; MS (ESI): m/z:
417.3 [M+1]+; elemental analysis calcd (%) for C22H32N4O4 (416.51):
C 63.44, H 7.74, N 13.45; found: C 63.26, H 7.53, N 13.71.
N-Methoxy-N’-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}butanedia-
mide (6a): From the reaction of 3a (0.072 g) and after purification
by column chromatography (dichloromethane/methanol 9.5:0.5)
and crystallization (ether/petroleum ether), 6a was obtained as a
pale-yellow solid (0.055 g, 71%): m.p. 121–1228C; 1H NMR
([D6]DMSO): d=10.93 (s, 1H, 5’), 8.54–8.53 (dd, J=3.0 Hz, 1H, 10),
8.06 (d, J=8.2 Hz, 1H, 12), 7.82 (s, 1H, 1), 7.43–7.41 (m, 1H, 11),
6.47 (d, J=2.2 Hz, 1H, 16), 6.26 (d, J=2.1 Hz, 1H, 14), 6.11 (d, J=
8.7 Hz, 1H, 7), 3.83 (s, 3H, 17), 3.63–3.58 (m, 1H, 5), 3.54 (s, 3H, 6’),
3.08–3.02 (m, 2H, 2), 2.29 (t, J=7.4 Hz, 2H, 2’), 2.16 (t, J=7.0 Hz,
2H, 3’), 1.67–1.63, 1.55–1.45 (2m, 4H, 3, 4), 1.21 ppm (d, J=6.3 Hz,
3H, 6); 13C NMR ([D6]DMSO): d=170.64, 168.44, 158.96, 144.59,
144.17, 134.71, 134.48, 129.52, 122.01, 96.04, 91.60, 62.97, 54.92,
46.97, 38.44, 33.37, 30.27, 27.84, 25.90, 20.14 ppm; IR (ATR): n=
3302, 3234, 3001, 2969, 2933, 1739, 1658, 1634, 1556, 1519, 1457,
1424, 1388, 1336, 1224, 1204, 1168, 1053, 820, 791, 679 cmÀ1; MS
(ESI): m/z: 389.2 [M+1]+; elemental analysis calcd (%) for
C20H28N4O4 (388.46): C 61.84, H 7.27, N 14.42; found: C 62.05, H
7.01, N 14.65.
N1-Methoxy-N4-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}benzene-
1,4-dicarboxamide (6e): From the reaction of 3e (0.082 g) and
after crystallization (ether), 6e was obtained as a pale-yellow solid
(0.040 g, 45%): m.p. 142–1458C; 1H NMR ([D6]DMSO): d=11.84 (s,
1H, 9’), 8.58 (t, J=5.56 Hz, 1H, 1), 8.54–8.53 (dd, J=1.6, 4.2 Hz, 1H,
10), 8.08–8.06 (dd, J=1.6, 8.3 Hz, 1H, 12), 7.89 (d, J=8.4 Hz, 2H, 3’,
7’), 7.80 (d, J=8.4 Hz, 2H, 4’, 6’), 7.43–7.41 (m, 1H, 11), 6.47 (d, J=
2.5 Hz, 1H, 16), 6.28 (d, J=2.4 Hz, 1H, 14), 6.15 (d, J=8.8 Hz, 1H,
7), 3.82 (s, 3H, 17), 3.72 (s, 2H, 10’), 3.69–3.65 (m, 1H, 5), 3.31–3.28
(m, 2H, 2), 1.76–1.66, 1.65–1.56 (m, 4H, 3, 4), 1.23 ppm (d, J=
6.3 Hz, 3H, 6); 13C NMR ([D6]DMSO): d=165.32, 163.36, 158.98,
144.61, 144.20, 137.28, 134.76, 134.52, 134.31, 129.55, 127.21,
126.93), 122.06, 96.10, 91.60, 63.26, 54.94, 47.01, 39.23, 33.41, 25.85,
20.17 ppm; IR (ATR): n=3394, 3309, 3176, 3060, 2970, 2937, 1738,
1670, 1624, 1557, 1520, 1456, 1389, 1221, 1205, 1161, 1039, 867,
821, 791, 678 cmÀ1; MS (ESI): m/z: 437.3 [M+1]+; elemental analysis
calcd (%) for C24H28N4O4 (436.50): C 66.04, H 6.47, N 12.84; found: C
66.33, H 6.31, N 13.04.
(2E)-N-Methoxy-N’-{4-[(6-methoxyquinolin-8-yl)amino]pent-yl}but-2-
enediamide (6b): From the reaction of 3b (0.072 g) and after pu-
rification by column chromatography (cyclohexane/ethyl acetate/
methanol 3:1:0.5) and crystallization (ether), 6b was obtained as a
pale-yellow solid (0.038 g, 49%): m.p. 177–1798C; 1H NMR
([D6]DMSO): d=11.53 (s, 1H, 5’), 8.55–8.53 (dd, J=1.6, 4.2 Hz, 1H,
10), 8.44 (t, J=5.5 Hz, 1H, 1), 8.09–8.06 (dd, J=1.5, 8.3 Hz, 1H, 12),
7.45–7.41 (m, 6H, 11), 6.89 (d, J=15.1 Hz, 1H, 2’), 6.60 (d, J=
15.1 Hz, 1H, 3’), 6.47 (d, J=2.2 Hz, 1H, 16), 6.27 (d, J=2.2 Hz, 1H,
14), 6.14 (d, J=8.7 Hz, 1H, 7), 3.82 (s, 3H, 17), 3.65 (s, 4H, 5, 6’),
3.20–3.14 (m, 2H, 2), 1.70–1.49 (m, 4H, 3, 4), 1.21 ppm (d, J=
6.2 Hz, 3H, 6); 13C NMR ([D6]DMSO): d=163.16, 160.97, 159.00,
144.60, 144.22, 135.72, 134.82, 134.49, 133.58, 129.58, 129.12,
128.85, 129.34, 122.09, 96.16, 91.63, 76.97, 54.97, 46.97, 38.77,
33.42, 25.77, 20.19 ppm; IR (ATR): n=3285, 3223, 3097, 3007,
2969,2935, 1738, 1630, 1570, 1519, 1457, 1388, 1338, 1224, 1168,
1066, 1053, 991, 821, 792, 659 cmÀ1; MS (ESI): m/z: 387.1 [M+1]+;
elemental analysis calcd (%) for C20H26N4O4 (386.44): C 62.16, H
6.78, N 14.50; found: C 61.94, H 6.55, N 14.87.
Biological Evaluation
Cell viability: All synthesized compounds were first screened for
their inhibition of mitochondrial metabolic activity by using the
MTT assay.[65] Mitochondrial metabolic activity correlates with cell
viability. The following cell lines were tested: U2OS, HepG2, MCF-7,
and Hek293 cells. The compounds were dissolved in DMSO (3”
10À3 m) and stored at À208C. The cells were seeded at a density of
3000 cells per 200 mL in a 96-well plate (Sarsted) in triplicate and
grown in Dulbecco’s modified Eagle medium with 4500 mgLÀ1 glu-
cose (DMEM-high glucose) (Lonza) containing 10% fetal bovine
serum (FBS) (Gibco), 100 UmLÀ1 penicillin, and 100 mgmLÀ1 strepto-
mycin (Sigma–Aldrich). The next day, the medium was aspirated
and cells were treated for 72 h. Only the compounds that led to
more than a 40% reduction in mitochondrial metabolic activity at
N-Methoxy-N’-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}pentane-
diamide (6c): From the reaction of 3c (0.075 g) and after crystalli-
zation (ether), 6c was obtained as a pale-yellow solid (0.045 g,
56%): m.p. 112–1138C; 1H NMR ([D6]DMSO): d=10.94 (s, 1H, 6’),
8.55–8.53 (dd, J=4.2, 1.5 Hz, 1H, 10), 8.09–8.06 (dd, J=8.3, 1.4 Hz,
1H, 12), 7.89 (t, J=5.3 Hz, 1H, 1), 7.45–7.41 (m, 1H, 11), 6.48 (d, J=
2.4 Hz, 1H, 16), 6.26 (d, J=2.3 Hz, 1H, 14), 6.12 (d, J=8.7 Hz, 1H,
7), 3.82 (s, 3H, 17), 3.62 (m, 1H, 5), 3.56 (s, 3H, 7’), 3.06–3.04 (m,
2H, 2), 2.04 (t, J=7.3 Hz, 2H, 2’), 1.93 (t, J=7.3 Hz, 2H, 4’), 1.74– a concentration of 5”10À5 m were selected for further analysis. The
1.40 (m, 6H, 3’, 3,4’), 1.20 ppm (d, J=6.3 Hz, 3H, 6); 13C NMR
following concentrations of selected compounds were used: 5”
ChemistryOpen 2018, 7, 624 –638
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636
ꢀ 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

10À5, 1”10À5, 1”10À6, 1”10À7, and 1”10À8 m. All working concen-
trations were freshly prepared in DMEM on the day of the testing.
A fresh growth medium was added to untreated control cells,
which were defined as 100% viable. DMSO (1.67, 0.33, 0.03, 0.3”
10À2, and 0.3”10À3 %) in DMEM was considered as a negative con-
trol. Cisplatin, PQ, and SAHA were used as positive controls. After
72 h of incubation in the presence of selected compounds, media
were aspirated and MTT (0.5 mgmLÀ1; 40 mL per well) was added,
and the cells were incubated for 4 h 378C. Media were aspirated
and formazan crystals were dissolved in DMSO (170 mL per well).
The absorbance was measured on a microplate reader (Promega)
at l=560 nm. The IC50 values (concentration required to decrease
viability by 50%) were calculated by using nonlinear regression on
the sigmoidal dose–response plots and are expressed as meanÆ
SD of two independent experiments.
Immunocytochemistry for histone acetylation: MCF-7 cells were
seeded at a density of 5000 cells per coverslip in DMEM containing
10% FBS, 100 UmLÀ1 penicillin, and 100 mgmLÀ1 streptomycin and
were left for 24 h to adhere. Cells were treated with 4e, 5b, SAHA,
or PQ (at 1 mm concentration) for 24 h. Following treatment, cells
were fixed with 4% paraformaldehyde (10 min), then permeabi-
lized using 0.1% Triton X-100 (10 min). Blocking was performed
using 10% goat serum in phosphate buffer saline (PBS) (1 h), fol-
lowed by incubation with primary antibody (rabbit anti-acetyl-His-
tone-H3 polyclonal antibody 1/500; Millipore 06–559) for 24 h in a
humidified chamber at 48C. Samples were washed with PBS (3”
5 min). Samples were then incubated with secondary antibody
(Alexa Fluor 647 goat anti-rabbit IgG À2 mgmLÀ1 1/500; Life Tech-
nologies A21244) for 1 h in the dark, after which they were
washed with PBS (3”5 min). Nuclei were labeled with Hoechst
33342 (10 mm, 10 min). Samples were mounted on microscope
slides using Poly Aqua Mount (PolySciences) and were dried over-
night before imaging with a fluorescence microscope (Leica). Fluo-
rescence intensity was quantified using ImageJ software, and the
results are presented as means of fluorescence per cellÆSD (as
fold increase in the untreated control=1).
MCF-7 cell labeling with Hoechst 33342: Fluorescent dye Hoechst
33342 was used to determine the total number of cells. MCF-7
cells were seeded at a density of 3000 cells per 100 mL (72 h treat-
ment) or 6000 cells per 100 mL (24 h treatment) in a 96-well plate
(Corning Inc.). Cells were grown in triplicate in DMEM containing
10% FBS (Wisent), 100 UmLÀ1 penicillin, and 100 mgmLÀ1 strepto-
mycin (Thermo Fischer). The next day, cells were treated with SA-
HAquines 4e and 5b, SAHA, PQ, and cisplatin in ten different con- Acknowledgements
centrations ranging from 5”10À5 to 1”10À9 m. After 72 h, cells
were stained with Hoechst 33342 (10 mm, 10 min). Cells were then
Support for this study by Croatian Science Foundation (project
imaged by using a fluorescence microscope (Leica, DMI4000B). Re-
sults were calculated by using nonlinear regression on the sigmoi-
dal dose–response plot and are expressed as meanÆSD of two in-
dependent experiments.
number IP-09–2014-1501), Canadian Institute of Health Research
(CIHR; project number MOP-119425), and National Sciences and
Engineering Research Council of Canada (NSERC; project number
RGPIN 04994-15) is acknowledged.
In vitro drug sensitivity assay against erythrocytic stages of P. falci-
parum: The antiplasmodial activity of compounds 2–6 was tested
in a drug-sensitivity assay against two laboratory P. falciparum
strains (3D7— chloroquine sensitive, Dd2— chloroquine-resistant) as
described before by using the histidine-rich protein 2 (HRP2)
assay.[66,67] In brief, 96-well plates were precoated with the tested
compounds in a threefold dilution before ring-stage parasites were
added in complete culture medium at a hematocrit of 1.5% and a
parasitaemia of 0.05%. After 3 days of incubation at 378C, 5% CO2
and 5% oxygen, plates were frozen until analyzed by HRP2-ELISA.
All compounds were evaluated in duplicate in at least two inde-
pendent experiments. The IC50 was determined by analyzing the
nonlinear regression of log concentration–response curves using
the drc-package v0.9.0 of R v2.6.1.[68]
Conflict of Interest
The authors declare no conflict of interest.
Keywords: acetylation · anticancer agents · antiplasmodial
activity · cytostatic activity · drug design
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Received: June 21, 2018
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