Small molecule glycoconjugates with anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2016.01.061

Glycoconjugates are combinations of sugar moieties with organic compounds. Due to their biological resemblance, such structures often have properties that are desirable for drugs. In this study we designed and synthesised several glycoconjugates from smal

Full text of DOI:10.1016/j.ejmech.2016.01.061

European Journal of Medicinal Chemistry 112 (2016) 130e144
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Small molecule glycoconjugates with anticancer activity
Gabriela Pastuch-Gawołek , Katarzyna Malarz , Anna Mrozek-Wilczkiewicz d,
a
,
1
b
,
1
c,
a
b
b
b, *
Marta Musioł , Maciej Serda , Barbara Czaplinska , Robert Musiol
Silesian University of Technology, Faculty of Chemistry, Chair of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Krzywoustego 4, 44-100
Gliwice, Poland
Institute of Chemistry, University of Silesia, 9 Szkolna Street, 40-006 Katowice, Poland
A. Chełkowski Institute of Physics, University of Silesia, Uniwersytecka 4, 40-007 Katowice, Poland
a
b
c
d
Silesian Center for Education and Interdisciplinary Research, University of Silesia, 75 Pułku Piechoty 1A, 41-500 Chorz oꢀ w, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 October 2015
Received in revised form
Glycoconjugates are combinations of sugar moieties with organic compounds. Due to their biological
resemblance, such structures often have properties that are desirable for drugs. In this study we designed
and synthesised several glycoconjugates from small molecular quinolines and substituted gluco- and
galactopyranosyl amines. Although the parent quinoline compounds were inactive in affordable con-
centrations, the glycoconjugates that were obtained appeared to be cytotoxic against cancer cells at the
micromolar level. When combined with copper ions, their activity increased even further. Their mech-
anism of action is connected to the formation of reactive oxygen species and the intercalation of DNA.
12 January 2016
Accepted 30 January 2016
Available online 8 February 2016
Keywords:
Quinoline
©
2016 Elsevier Masson SAS. All rights reserved.
Glycoconjugates
Reactive oxygen species
Anticancer
Chelators
1
. Introduction
led to the creation of active anti-Alzheimer prodrugs that have good
permeability through the blood-brain barrier [6]. These types of
compounds can also be used in the treatment of cancer diseases. As
is well known, neoplastic cells have an increased demand for metal
ions such as iron, copper, zinc, calcium etc. Those microelements
participate in the main processes in a cell, and therefore the che-
lation effect plays a key role. The generation of metal complexes by
8-hydroxyquinoline has been widely described in the literature [7].
The binding of metal ions by the compounds that have been
investigated might have an influence on the life processes of cancer
cells.
Due to its unique role in living organisms, glucose has great
potential in drug design. Its molecules are well recognised and
readily penetrate most cells via active transport. As an energy
source and an element of many metabolic processes, glucose is
present in every part of the human body. Cancer cells have an
increased demand for glucose, which is then metabolised at a
higher rate in order to achieve the energy level that is necessary for
amplified proliferation [1]. High glycolysis rate in cancers lead to
anaerobic metabolism and an increased level of lactic acid
fermentation. This phenomenon is known as the Warburg effect
The second idea was emphasised with the discovery of the
[2]. With this in mind, glucose can be successfully utilised in drug
anticancer activity of anomeric glucoconjugate 4,6-benzylidene-D-
design as an enhancer of pharmacokinetics or as a potential target
for anticancer agents. The first approach was studied for various
drugs that have poor pharmacokinetic parameters such as anti-
oxidative and anti-inflammatory drugs [3] or analgesic enkephalins
glucose (BG) or its deuterated derivative [8e12] although in vivo
phase I and II studies did not confirm the cell-based assays [13,14].
Beta-D-glucosylisophosphoramide mustard (gluphosphamide) is
another example of an anticancer alkylating agent that has an
improved bioavailability and biodegradability due to glucoconju-
gation [15]. Its mechanism of action prefers cancers that have
overexpressed glucose transporters (GLUT) [16]. These membrane
proteins, which are responsible for meeting the high demand for an
energy source in cancer cells, are the prime targets of a relatively
large group of agents [17]. Among them are natural and synthetic
[4] and dopamine derivatives [5].
The glucoconjugation of hydroxypyridinone metal chelators has
*
Corresponding author.
E-mail address: robert.musiol@us.edu.pl (R. Musiol).
Those authors contributed equally.
1
http://dx.doi.org/10.1016/j.ejmech.2016.01.061
223-5234/© 2016 Elsevier Masson SAS. All rights reserved.
0

G. Pastuch-Gawołek et al. / European Journal of Medicinal Chemistry 112 (2016) 130e144
131
List of abbreviations
DMTMM 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-
methylmorpholinium chloride
0
DIC
N,N -Diisopropylcarbodiimide
N-hydroxybenzotriazole
Reactive oxygen species
glutathione
HOBt
ROS
GSH
compounds as well as glucoconjugates or synthetic pseudo-sugars
18]. Other sugar derivatives were designed to be inhibitors of
[
glycolytic enzymes [19]. However, as was demonstrated elsewhere,
according to their structural features, these compounds may act
differently in aerobic or anaerobic conditions [20]. Nevertheless, to
date there are no registered drugs that are targeted to this specific
protein or to the transport of glucose.
More recently, small quinoline glucoconjugates were studied for
their anticancer activity [21]. Among them, metal chelating agents
such as 8-hydroxyquinoline and Clioquinol (5-chloro-7-iodo-8-
hydroxyquinoline) were reacted with glucose to obtain prodrugs
that have improved pharmacokinetics [21]. The authors suggest
that after hydrolysis with glycosidases, the parent quinoline de-
rivative is released and may exert an antiproliferative activity.
Specific reactions with almond glycosidases showed considerable
hydrolysis after 60 min. On the other hand, during in vitro experi-
ments in cancer cells, the glucoconjugates appeared to be (up to 50
times) less active than the parent quinolines. This may suggest that
hydrolysis is far less effective in a cell after period of three days
Scheme 1. General structure of the glycoconjugates.
3 4
Bu NHSO as the phase transfer agent [37]. Compound 4 was ob-
tained from corresponding glycosyl azide 4a using 1 M sodium
methoxide in methanol. The neutralisation of the reaction mixture
was carried out via the acidic ion exchange resin Amberlyst-15,
which can be easily filtered off. Evaporation of the filtrate under
reduced pressure produced deprotected b-glycosyl azide 4b as a
yellow-white solid, which was sufficiently pure for a further reac-
tion. The insertion of benzyl-protecting groups with benzyl bro-
mide produced O-benzyl derivative 4c, which was subsequently
[21,22].
In our group quinoline derivatives are broadly studied for their
anticancer activity [23e27] and we recently prepared some glu-
coconjugates that were designed based on synthetic sugars. In
addition, we decided to also prepare conjugates based on the
reduced to 2,3,4,6-tetra-O-benzyl-b-D-galactopyranosyl amine 4.
TLC analysis of the reaction mixture showed the formation of only
the expected product 4 and no products of partial debenzylation.
Due to the purity of the crude product and its high reactivity,
compound 4 was not purified, but was used directly for conden-
sation with quinoline derivative 1. The de-O-acetylation of com-
galactose unit. D-Galactose is another natural sugar molecule that
has been exploited in prodrug strategies [28]. Moreover, this sugar
seems to be particularly important for colon cancers as has been
reported elsewhere [29,30]. This fact is probably connected with an
pound 3a allowed
obtained, which was also reduced to produce
amine 5. The nitro group in the aglycon of 4-nitrophenyl 2,3,4,6-
tetra-O-acetyl-1-thio- -D-glycosides 8 and 9 was reduced using
the zinc powder/AcOH system in CH Cl (19) (Scheme 4). The re-
b
-D-glucopyranosyl azide 3b (Scheme 3) to be
increase in
D-galactose transporters in colon cancer cells [31]. In
b-D-glucopyranosyl
this paper, we present our findings on this promising topic. In our
experiments, the conjugation of two separately inactive moieties
resulted in compounds that had a moderate to good anticancer
activity. We selected colorectal cancer cells for the biological testing
as they are particularly connected with the overexpression of GLUT
b
2
2
action proceeded at room temperature. It is worth noting that no
decomposition of the glycosidic bond was observed under the re-
action conditions. This method allows the appropriate 4-
aminophenyl 1-thioglycosides 10 and 11 to be obtained in good
yields (79% and 66%, respectively).
[32e35].
2
. Results and discussion
The sugar derivatives 3, 4, 5, 6, 7 and 10e13 that were obtained
were used for condensation reactions with quinolines 1 or 2. The
straight approach for an amide bond formation, which requires
harsh conditions, is not suitable in this case of complex sugar de-
rivatives due to the possibility of the occurrence of side reactions.
Therefore, activating the non-reactive carboxyl group seems to be
necessary [38]. From among the numerous condensing agents
described in the literature [39,40], we applied the DCC/HOBt
(Table 1, Procedure A) or DIC/HOBt (Table 1, Procedure B or D)
condensing systems for coupling the derivatives that contained the
amine or thiol group with quinolinic acids 1 or 2. The reactions
were performed at room temperature or in combination with mi-
2.1. Synthesis
We introduced the sugar (D-glucose or D-galactose unit) into the
backbone fragment of two different quinolinic acids (compounds 1
or 2). These connections can be achieved by an amide, ester, thio-
ester or glycosidic bond. Based on this assumption, we obtained a
whole range of quinoline glycoconjugates (Scheme 1).
In the first step of the synthesis, sugar derivatives 3e13, which
contained a thiol or an amine group were synthesised according to
previously known procedures (Scheme 2).
b-D-Glycopyranosyl
ꢀ
amines 3e5 can be obtained from the corresponding glycosyl
azides through a hydrogenation reaction in a Paar apparatus using
palladium hydroxide deposited on activated carbon [36]. 2,3,4,6-
crowave irradiation (standard program, 50 C). As a result of these
reactions, glycoconjugates 14e25 were obtained. The yields of the
respective products are presented in Table 1.
Tetra-O-acetyl-
ing sodium azide in a chloroform/saturated NaHCO
b
-glycosyl azides 3a and 4a were synthesised us-
Another effective way of synthesizing amides in the presence of
2-chloro-4,6-disubstituted-1,3,5-triazines and tertiary amines (e.g.,
3
solution with

132
G. Pastuch-Gawołek et al. / European Journal of Medicinal Chemistry 112 (2016) 130e144
Scheme 2. Structure of compounds 3e13.
Scheme 3. Preparation of compounds 3e5. Reagents and Conditions: (i) H
BnBr, NaH, DMF, 0 C to r.t., 14 h.
2
, Pd(OH)
2
/C, THF:EtOH (2:1), r.t., 1.5 bar, 0.5-1.5 h; (ii) 1. NaOMe, MeOH, r.t., 0.5 h; 2. Amberlyst 15; (iii)
ꢀ
Scheme 4. Reduction of a nitro group.
N-methylmorpholine) was published by Kaminski and co-workers.
The so-called superactive ester that contains an excellent leaving
group is formed in this reaction [41]. This method was previously
used to form uridine glycoconjugates that contained an amide bond
Table 1
Yields of glycoconjugates 14e25.
[42]. DMTMM was applied as the condensing agent for the
Entry Product Procedure Reaction time [h] Solvent
Yield [%]
condensation of an amine group in sugar derivatives 10 and 12 with
the carboxylic group in quinolinic acids 1 or 2 (Table 1, Procedure
C). Reactions were performed using microwave irradiation. As a
result of these reactions, glycoconjugates 21, 22 and 25 were ob-
tained. The yields of the respective products are presented in
Table 1. Unfortunately, the procedure, which has proven itself in the
preparation of uridine glycoconjugates, only permitted low yields
of the desired products to be obtained in this case.
a
1
2
3
4
5
6
7
8
9
1
1
1
1
1
14
15
16
17
18
19
20
21
22
22
23
24
25
25
A
A
A
B
B
B
B
C
2
2
2
pyridine/CH
pyridine/CH
pyridine
pyridine
pyridine
pyridine
pyridine
THF
2
Cl
Cl
2
2
59
37
20
71
44
24
54
16
11
63
70
93
13
78
a
a
b
b
þ 24
2
þ 24
b
0.5
b
3
b
b
0.5
0.5
a
b
b
2
2
þ 24
a
C
þ 48
THF
b
0
1
2
3
4
D
D
D
C
96
72
72
CH
CH
CH
Cl
2 2
Cl
2 2
Cl
2 2
/DMF
/DMF
/DMF
The structural analysis of all of the synthesised compounds is
presented in the supporting information.
b
b
a
b
2
þ 48
THF
CH
b
D
72
2
Cl
2
/DMF
2.2. Biological studies
a
Reaction time under microwave irradiation.
Reaction time at room temperature on a magnetic stirrer.
b
Tetra-O-acetyl-D-glucose derivatives and their compounds that

G. Pastuch-Gawołek et al. / European Journal of Medicinal Chemistry 112 (2016) 130e144
133
were used as substrates appeared to be inactive on HCT116 cell
lines (human colon carcinoma) within the maximum solubility
level. Similarly, the simple combination of acetylated glucose or
galactose with quinoline compounds can be regarded as inactive if
concentration of glucose to the media. The lack of any difference
between the cytotoxic behaviour of the compounds that were
tested in glucose overload conditions indicates that they are not
transferred by the GLUTs and that their mechanism of action is not
directly focused on glucose metabolism.
its IC50 parameter exceeds the level of 25 mM. An exception, how-
ever, was observed with amides 14 and 15. Furthermore, the de-O-
acetylated sugar moiety led to a complete cessation of the activity
Metal ions play an important role in maintaining the appro-
priate balance of many cellular processes. Tumour cells exhibit
increased levels of some ions. Among those, Cu and Zn are more
important here. As has previously been reported, 8-
hydroxyquinolines exhibit a high metal-chelating ability, which
seems to be crucial for anti-proliferative activity [7]. Metal ions are
coordinated by two electron donor sitesethe oxygen atom from
phenolate or quinoline ring nitrogen. The results that are presented
in Fig. 2 indicate the influence of the Cu and Zn ions on the pro-
(
see 16 vs 14). Generally, all carbothiolate derivatives can be
considered to be inactive. A phenyl or pyridine linker between a
sugar and a quinoline moiety leads to a substantial increase in ac-
tivity. Within this group of compounds, quinoline substructure 1
produced promising results. There is little or no difference between
the D-glucose and D-galactose moiety. On the other hand, the pyr-
idine linker induced higher activity than phenyl with compound 25
being the most active glycoconjugate tested. There were no dif-
ferences between the wild type cancer cells and mutants that have
a deletion of TP53 gene, thus suggesting that the mechanism of
action of these compounds does not rely on the p53 apoptosis
pathway. For a more in-depth evaluation of this possible mecha-
nism of action, we performed several additional experiments.
Glucoconjugation may facilitate the pharmacokinetics of cyto-
toxic exogenic molecules and ensure their active transport through
the glucose receptors. Alternatively, compounds that have a sugar
moiety may interact with the machinery for the intake and meta-
bolism of glucose. Antimetabolites, which build on sugar structures
may, through competitive inhibition, disrupt the cellular meta-
bolism to cell death [43,44]. To investigate the possible interaction
of the compounds that were tested with glucose receptors, we
performed a kinetic experiment in an environment that had a high
glucose content.
þ/þ
ꢁ/ꢁ
liferation of the HCT116 p53
and p53
cell lines. In general, Zn
ions increased the percentage of the living cells, while the Cu ions
inhibited the proliferation of the cancer cells that were investi-
gated. This may suggest that our compounds had formed com-
plexes with metal ions. The inhibition of the activity of a compound
with low IC50 values by Zn ions could be caused by the formation of
inactive complexes. The reason may be the fact that Zn does not
belong to the group of redox-active metals. Moreover, these results
are in agreement with other reports on the metal-dependent ac-
tivity of quinoline derivatives [34,35]. In the case of redox-active Cu,
Fe complexes that have small molecules such as 8-
hydroxyquinoline, reactive oxygen species may be generated thus
leading to increased oxidative stress and the induction of apoptosis.
When ligand was added to the medium supplemented with metal
ions, we observed the greatest decrease in the proliferation of cells
þ/þ
in the case of 14 (50% decrease for HCT116 p53 ) and 25 (75%
ꢁ
/ꢁ
As is presented in Fig. 1, there was no difference between the
survival of cells for the active compounds in the presence of a high
glucose concentration. The viability of the cells dropped continu-
ously as a result of the cytotoxic activity of the compound that was
decrease for HCT116 p53 ).
To evaluate the complex formation ability for the glyco-
conjugates we performed spectroscopic titration of the solutions of
compounds 14,15, 22e25 with zinc and copper. Results are pre-
sented in tables and figures in supporting information. The
tested. We applied a higher (10
mM) and a very high (50 mM)
þ/þ
cells.
Fig. 1. Influence of glucose (GLU10; concentration 10
mM, GLU50; concentration 50
mM) on the cellular proliferation of HCT116 p53

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G. Pastuch-Gawołek et al. / European Journal of Medicinal Chemistry 112 (2016) 130e144
Fig. 2. Influence of metal ions on cellular proliferation.
superposition of absorbion spectra revealed the isosbestic points
for those compounds.
observe the generation of ROS for the other compounds during the
kinetic experiment. However, we did observe the depletion of the
level of intracellular GSH after a 12-h treatment with these com-
pounds. The depletion of intracellular GSH is an early event in cell
death in response to different stimuli. Franco et al. postulated that
the depletion of intracellular GSH may induce apoptosis in cancer
cells independently through the generation of ROS [57]. In addition,
a decrease in the intracellular levels of GSH is associated with the
progression of apoptosis, which is activated by both extrinsic and
intrinsic signalling pathways. The end point in both signalling
pathways is the activation of executive caspase-3, which is acti-
vated by any of the initiator caspases ꢁ8 or ꢁ9 [58]. With this in
mind, we performed a test to measure the activation of caspases-3/
7 by the tested compounds that appeared to be active. Our results
Another appealing fact is the different patterns of activity in the
metal-supplemented therapies for both cell lines (Fig 2). In general,
p53 null mutants appeared to be more susceptible to high copper
concentrations. With the exception of compound 14, glucoconju-
gates 22e25 showed more Cu-dependent toxicity in the cells that
were deprived of the p53 protein [47]. This phenomenon may be
explained by our observations as well as by the functionality of p53
[48]. The redox activity of Cu complexes causes DNA damage, which
in turn activates the suppressor protein and initiates the arrest of
the G2 cell cycle and DNA repair programme or, if that is not
possible, triggers the apoptosis pathway [36,37]. Cells that are
deprived of such a guardian should be more vulnerable to death
due to DNA damage. Although this may appear to be in contra-
diction with the overall chemosensitivity of wild type cells, which is
based on p53, more recent studies support this hypothesis [49].
As was mentioned, an excess of intracellular copper or iron ions
may increase redox reactivity and the oxidation processes. Thus,
the metabolism of transient metals is strictly controlled by specific
cellular mechanisms in order to maintain a low level of free metal
ions. However, the ions that are chelated by small ligands may still,
through Fenton and Haber-Weiss reactions, induce the formation of
highly toxic reactive oxygen species [46,50e52]. The same eleva-
tion of the level of oxidation processes can be observed during
intense glucose metabolism [53e55].
(Fig 5) showed that caspases-3/7 were activated after treatment
þ/þ
with 14 and 23 in the HCT116 p53
cells and with 15 in the
þ/þ
ꢁ/ꢁ
HCT116 p53
and HCT116 p53
cell lines. In contrast to the
other compounds, we did not observe the activation of caspases-3/
7 at a concentration that was equivalent to the IC50 values. These
results suggest that the glucoconjugates that were tested may
induce both caspase-dependent and caspase-independent cyto-
toxic pathways. One explanation of these mechanisms may be the
ability to form complexes with copper by 8-hydroxyquinoline de-
rivatives and probably also by glucoconjugates. Copper participates
in the catalytic oxidoreductive reactions that lower the antioxidant
defense in cells. Moreover, it may inhibit the activity of caspase-3 in
the endoplasmic reticulum, thus forcing the cell to enter a caspase-
independent death pathway [59,60].
The apoptosis of the cells treated with the active glyco-
conjugates was confirmed using the Acridine Orange (AO)/
Ethidium Bromide (EB) staining test (Fig 6). We observed early and
late apoptotic cells in the HCT116 colonies for active compounds 14,
15 and 22e25.
The glycoconjugates that were studied can act as Fenton-ready
carriers of ions. The quinolone molecules that are used as a scaf-
fold are well-known for their chelation ability [45,56]. In order to
investigate this possibility, we evaluated the effect of the com-
pounds that were tested on ROS formation by performing a fluo-
rescent assay.
We confirmed the generation of a greater amount of ROS by the
tested compounds that were active relative to the untreated cells.
Transition metal complexes, particularly those with copper,
palladium and nickel, have been reported for their DNA-
intercalating properties [61e63]. Quinoline-related compounds
are also present between intercalators [64]. This is an important
clue about a plausible multi-targeted mode of action of the com-
pounds that are the subject of our study. Furthermore, as we re-
ported recently, the p53 independent mechanism of action may be
connected with small DNA damage as a result of intercalation [65].
In order to evaluate this hypothesis, we performed DNA-binding
studies with the tested compounds that appeared to be active.
The absorption spectra of compounds 14 and 15 and 22e25 alone
and in the presence of calf-thymus DNA are presented in Fig. 7.
Several of the compounds that were tested may be related to DNA
through intercalation, which is associated with significant hypo-
chromism and a small red shift of the wavelength. Compound 25
especially revealed the strongest hypochromism that reduced the
intensity spectrum about 41,5%. We also observed a shift of 5 nm in
the wavelength that is characteristic for the formation of the DNA-
þ/þ
The relative ROS intensity in the HCT116 p53
cells that had been
treated with compounds 14, 15 and 25 was higher than in the cells
that had been treated with 22, 23 and 24. We observed strong
fluorescent signals, which probably centred on the mitochondria
and nucleus after incubation with those active compounds (Fig. 3).
This may suggest the localisation of the generation of ROS in the
mitochondria, which in turn, may be related to the modulation of
the level of glutathione and mitochondrial dysfunction.
Additionally, we conducted an experimental kinetic measure-
ment of the intracellular level of ROS after treatment with the
compounds that appeared to be active. A quantitative analysis of
the data confirmed that ROS are generated by compounds 14, 15
and 25 (Fig. 4). The highest increase in the production of ROS was
observed 6 h and 12 h after treatment with 14, 15 and 12 h after the
addition of compound 25. These results correlate with the highest
depletion in the level of intracellular GSH after a 12-h treatment as
a response to oxidative stress within the HCT116 cells. We did not

G. Pastuch-Gawołek et al. / European Journal of Medicinal Chemistry 112 (2016) 130e144
135
þ/þ
Fig. 3. Generation of reactive oxygen species (ROS) in HCT116 p53
(
cells after a 24-h treatment with 14, 15, 22, 23, 24 and 25 and a 15-min treatment with hydrogen peroxide
m.
positive control). The negative control consisted of untreated cells. Scale bars ¼ 50
m
þ/þ
Fig. 4. Effect of glucoconjugates on the level of the ROS and intracellular GSH content of HCT116 p53
cells. Data normalised to untreated cells (control).

136
G. Pastuch-Gawołek et al. / European Journal of Medicinal Chemistry 112 (2016) 130e144
Fig. 5. Caspases 3/7 activities on the HCT116 wild type p53 cell lines and the knock-out gene TP53 after a 30-h and 48-h incubation with the compounds that were tested. Data
normalised to the untreated cells (control).
þ/þ
Fig. 6. The morphological changes of HCT116 p53
cells after a 48-h treatment with 14, 15, 22, 23, 24 and 25. The cells were stained with AO/EB for apoptosis indication. The cells
treated with glycoconjugates showed early apoptotic featuresegreen and yellow cells (green dots in the nuclei indicate chromatin condensation and nuclear fragmentation), and
late apoptotic cellseorange cells with condensed and fragmented nuclei. Scale bars ¼ 50
mm. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
complex. A high affinity to DNA is correlated with the high anti-
proliferative activity of this compound from among the group of
glucoconjugates that were tested. For compounds 14, 23 and 24, we
observed a good hypochromic effects (16,2%, 17,2% and 26,1%,
respectively) with a red shift of the wavelength. The data of the
absorption spectral properties for the compounds that were tested
with DNA are presented in Table 3.
4. Experimental
4.1. Synthesis
Microwave reactions were carried out in a Discover® Bench-
Mate™ (CEM) microwave equipped with 10 mL vessels using a
standard program at 50 C (max. pressure 1.5 Bar, average potency
ꢀ
20 W). NMR spectra were recorded with an Agilent spectrometer at
a frequency of 400 MHz or a Varian Gemini spectrometer at a fre-
quency of 300 MHz using TMS or DSS as the internal standards and
CDCl
3
or CD
3
OD as the solvents. NMR solvents were purchased
) are
expressed in ppm and coupling constants (J) in Hz. The H NMR and
from ACROS Organics (Geel, Belgium). Chemical shifts (
d
1
3
. Conclusions
13
C NMR signals of some compounds were assigned using COSY,
HMQC and HMBC experiments. Optical rotations were measured
with a JASCO P-2000 polarimeter using a sodium lamp (589.3 nm)
at room temperature. Melting point measurements were per-
formed on a Stanford Research Systems OptiMelt (MPA 100). Mass
spectra were recorded with a WATERS LCT Premier XE system (high
resolution mass spectrometer with a TOFanalyser) or with a 4000
QTRAP ABSciex mass spectrometer using the electrospray-
ionisation (ESI) technique. Reactions were monitored by TLC on
precoated plates of silica gel 60 F254 (Merck Millipore). TLC plates
A series of fourteen glycoconjugates was obtained from various
sugar moieties and quinolines and their biological activities against
cancer cells were tested. Some of the glycoconjugates that were
obtained expressed a 100 times higher activity than their parent
compounds. In contrast to previous results, the glycoconjugates
that were tested did not interfere with the uptake or metabolism of
glucose in the cells. Instead, their activity appears to be multi-
targeted and connected with the formation of ROS through the
chelation of transition metal ions and DNA intercalation.

G. Pastuch-Gawołek et al. / European Journal of Medicinal Chemistry 112 (2016) 130e144
137
Fig. 7. Absorption spectra of glucoconjugates (14, 15, 22, 23, 24, 25) without CT-DNA (solid line) and in the presence of the CT-DNA (dotted line) in phosphate-buffered saline. Red
line indicates CT-DNA alone in PBS. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
were inspected under UV light (
spraying with 10% sulfuric acid in ethanol. Crude products were
purified using column chromatography performed on Silica Gel 60
l
¼ 254 nm) or charring after
according to the respective published procedures. All of the
chemicals that were used were purchased from Aldrich, Fluka and
ACROS Organics and were used without purification.
(
70e230 mesh, Fluka) developed using hexane/EtOAc, toluene/
EtOAc or CHCl /MeOH solvent systems. All evaporations were
3
4.1.1. Synthesis of glycoconjugates 14e25
performed on a rotary evaporator under diminished pressure at
Procedure A. The appropriate glycosyl amine 3, 4 or 5
(0.20 mmol) and quinaldine derivative 1 (0.04 g, 0.20 mmol) were
ꢀ
4
0 C.
2
,3,4,6-Tetra-O-acetyl-
tetra-O-acetyl- -galactopyranosyl azide 4a [67], 2,3,4,6-tetra-O-
acetyl-1-thio- -glucopyranose 6, 2,3,4,6-tetra-O-acetyl-1-thio-
-galactopyranose 7 [68], 4-nitrophenyl 2,3,4,6-tetra-O-acetyl-1-
b
-
D
-glucopyranosyl azide 3a [66], 2,3,4,6-
2 2
dissolved in pyridine (1.5 mL) and CH Cl (1.5 mL). DCC (0.065 g,
b
-
D
0.30 mmol) and HOBt (0.019 g, 0.15 mmol) were added to this
mixture. The resulting mixture was microwaved in a reactor set at
b
-D
b-
ꢀ
D
50 C for 2 h. The reaction progress was monitored on TLC in an
thio-
b
-D-glycopyranosides 8e9 [69], (5-amino-2-pyridyl) 2,3,4,6-
-D-glycosides 12e13 [70] were prepared
eluents systemeCHCl
substrates remained, the entire mixture was left on the magnetic
3
:MeOH (20:1). In the event that unreacted
tetra-O-acetyl-1-thio-
b

138
G. Pastuch-Gawołek et al. / European Journal of Medicinal Chemistry 112 (2016) 130e144
stirrer for the next 24 h at room temperature. Then reaction
mixture was diluted with toluene, the solvents were evaporated
137.58, 151.75, 157.91 (C-Quin), 166.17 (NHCO), 169.56, 170.00,
þ
170.35, 170.68 (CO); ESI-MS: calcd for C25
533.1771; found: m/z 533.1775.
H
29
N
2
O
11 ([MþH] ): m/z
and the residue was transferred to 20 mL CH
solid, filtrate washed with saturated NaHCO
solution (1 ꢂ 10 mL),
, the adsorbent was
2
Cl
2
, filtered white
3
with brine (3 ꢂ 10 mL), dried with MgSO
4
4
2
.1.1.2. N-(2,3,4,6-tetra-O-benzyl-
-methylquinoline-7-carboxamide 15. Starting from 2,3,4,6-tetra-O-
-galactopyranosyl amine 4 and quinaldine derivative 1
b-D-galactopyranosyl)-8-hydroxy-
filtered off and the filtrate was then concentrated. Crude products
were purified using column chromatography to give products
benzyl-b-D
1
4e16.
Procedure B. 2,3,4,6-Tetra-O-acetyl-1-thio-
or 2,3,4,6-tetra-O-acetyl-1-thio- -galactopyranose
.274 mmol) and quinaldine derivative 1 or 2 (0.055 g, 0.274 mmol)
according to Procedure A, glycoconjugate 15 was obtained after
column chromatography (hexane: AcOEt; gradient: 30:1 to 1:2,
b
-D
-glucopyranose 6
(0.1 g,
b-D
7
3
then CHCl : MeOH 100:1 to 40:1) as a light yellow oil (0.045 g,
0
2
0
1
3
3
7%): ½aꢃ -7.0 (c 1.0, CHCl
3
); H NMR (400 MHz, CDCl
3
): d 2.74 (s,
were dissolved in pyridine (3 mL). The DIC (0.045 mL, 0.274 mmol)
and HOBt (0.005 g, 0.04 mmol) were added to this mixture. The
resulting mixture was left on the magnetic stirrer at room tem-
perature. The reaction progress was monitored on TLC in an eluents
systemetoluene:AcOEt (2:1). Then, the reaction mixture was
diluted with toluene, the solvents were evaporated and the residue
was transferred to 2 mL of toluene and the crude products were
purified by column chromatography (toluene: AcOEt; gradient: 5:1
to 1:2) to produce products 17e20.
D
H, CH3quin), 3.58e3.68 (m, 2H, H-6agal, H-6bgal), 3.74 (dd, 1H,
J
3,4 ¼ 2.4 Hz, J2,3 ¼ 9.4 Hz, H-3gal), 3.81 (m, 1H, H-5gal), 3.96 (dd, 1H,
J
1,2 ¼ 9.0 Hz, H-2gal), 4.07 (d, 1H, H-4gal), 4.42, 4.47 (qAB, 2H,
J ¼ 11.7 Hz, CH
2
Ph), 4.66, 4.98 (qAB, 2H, J ¼ 11.3 Hz, CH
2
Ph), 4.73,
Ph), 4.83, 4.87 (qAB, 2H, J ¼ 11.1 Hz,
4
.77 (qAB, 2H, J ¼ 11.9 Hz, CH
2
CH
5
1
2
Ph), 5.47 (dd, 1H, J1,NH ¼ 9.1 Hz, H-1gal), 7.10e7.41 (m, 22H, H-
quin., H-4quin, H-Ph), 7.99 (d, 1H, J5q,6q ¼ 8.6 Hz, H-6quin), 8.02 (d,
13
H, J3q,4q ¼ 8.8 Hz, H-3quin), 8.25 (d, 1H, NHCO); C NMR (100 MHz,
): 24.88 (CH3quin), 68.05 (C-6gal), 72.77, 73.46, 74.83, 75.18
) 73.84 (C-2gal), 74.89 (C-4gal), 78.40 (C-3gal), 79.77 (C-5gal),
3.62 (C-1gal), 113.29, 117.28, 124.40, 126.45, 127.58, 127.61, 127.73,
27.78, 127.94, 128.18, 128.21, 128.29, 128.32, 128.37, 128.38, 128.54,
28.55, 136.12, 137.83, 138.00, 138.36, 138.47, 138.82, 151.83, 157.74
CDCl
CH
3
d
Procedure C. The appropriate amine 10 or 12 (0.148 mmol) and
quinaldine derivative 1 or 2 (0.148 mmol) were dissolved in THF
(
2
8
1
1
(
3 mL). The DMTMM (0.045 g, 0.16 mmol) and N-methylmorpho-
line (0.021 mL, 0.16 mmol) were added to this mixture. The
resulting mixture was microwaved in a reactor set for 2 h. The re-
action progress was monitored on TLC in two alternative
(
(
C-Quin., C-Ph), 166.02 (NHCO); ESI-MS: calcd for C45
[MþH] ): m/z 725.3227; found: m/z 725.3223.
45
H N
2
O
7
þ
3
eluentseCHCl :MeOH (10:1) or toluene:AcOEt (1:1). In of the event
that unreacted substrates remained, the entire mixture was left on
the magnetic stirrer for the next 24e48 h at room temperature.
Then the solvent was evaporated and the residue was transferred to
CH Cl (20 mL), washed three times with brine, dried with MgSO ,
2 2 4
the adsorbent was filtered off and the filtrate was concentrated.
Crude products were purified by column chromatography (tolue-
4
.1.1.3. N-(
carboxamide 16. Starting from
quinaldine derivative 1 according to Procedure A, glycoconjugate
6 was obtained after column chromatography on a reverse phases
column (water: MeOH; gradient: 10:1 to 6:1) as a light yellow solid
b
-
D
-glucopyranosyl)-8-hydroxy-2-methylquinoline-7-
b-D-glucopyranosyl amine 5 and
1
ne:AcOEt, gradient: 6:1 to 1:1, then CHCl
3
:MeOH 80:1 to 10:1) to
ꢀ
20
1
(
(
0.015 g, 20%): m.p. 183e185 C; ½aꢃD -20.0 (c 1.0, H
2
O); H NMR
give products 21, 22 and 25.
Procedure D. The appropriate amine 10e13 (0.274 mmol) and
quinaldine derivative 1 or 2 (0.055 g, 0.274 mmol) were dissolved in
400 MHz, D O):
2
d
2.79 (s, 3H, CH3quin), 3.54 (dd, 1H, J2,3 ¼ 9.2 Hz,
J
3,4 ¼ 9.0 Hz, H-3glu), 3.61 (dd, 1H, J1,2 ¼ 8.6 Hz, H-2glu), 3.63 (m, 1H,
H-5glu), 3.67 (dd, 1H, J4,5 ¼ 9.0 Hz, H-4glu), 3.80 (dd, 1H,
5,6b ¼ 5.5 Hz, J6a,6b ¼ 12.4 Hz, H-6bglu), 3.97 (dd, 1H, J5,6a ¼ 2.0 Hz,
H-6aglu), 5.22 (d, 1H, H-1glu), 6.80 (d, 1H, J5q,6q ¼ 8.7 Hz, H-5quin),
.51 (d, 1H, J3q,4q ¼ 8.4 Hz, H-4quin), 7.73 (d, 1H, H-3quin), 8.36 (d, 1H,
2 2
a CH Cl :DMF 10:1 (4 mL) solvent system. The DIC (0.045 mL,
J
0
.274 mmol) and HOBt (0.01 g, 0.08 mmol) were added to this
mixture. The resulting mixture was left on the magnetic stirrer at
room temperature. The reaction progress was monitored on TLC in
7
13
H-6quin); C NMR (100 MHz, D
7
1
2
O):
2.26 (C-4glu), 75.18 (C-2glu), 79.33 (C-3glu), 80.28 (C-5glu), 82.36 (C-
glu), 112.61, 117.11, 120.28, 127.55, 131.15, 132.97, 146.81, 156.60,
d 22.66 (CH3qiun), 63.52 (C-6glu),
an eluents systemeCHCl
was diluted with CH Cl
dried with MgSO
was concentrated. Crude products were purified by column chro-
matography (toluene:AcOEt, gradient: 6:1 to 1:1, then CHCl :MeOH
3
:MeOH (20:1). Then the reaction mixture
2
2
(15 mL), washed three times with brine,
4
, the adsorbent was filtered off and the filtrate
21 2 7
163.31 (C-Quin), 172.88 (NHCO). ESI-MS: calcd for C17H N O
þ
([MþH] ): m/z 365.1349; found: m/z 365.1343.
3
8
0:1 to 10:1) to produce the appropriate products 22e25.
4
.1.1.4. S-(2,3,4,6-tetra-O-acetyl-
methylquinoline-7-carbothioate 17. Starting from 2,3,4,6-tetra-O-
acetyl-1-thio- -glucopyranose 6 and quinaldine derivative 1 ac-
b-D-glucopyranosyl)-8-hydroxy-2-
4
.1.1.1. N-(2,3,4,6-tetra-O-acetyl- -glucopyranosyl)-8-hydroxy-2-
b-D
methylquinoline-7-carboxamide 14. Starting from 2,3,4,6-tetra-O-
acetyl- -glucopyranosyl amine 3 and quinaldine derivative 1
b-D
b-D
cording to Procedure B, glycoconjugate 17 was obtained after col-
according to Procedure A, glycoconjugate 14 was obtained after
column chromatography (toluene: acetone; gradient: 10:1 to 6:1,
umn chromatography as a light orange solid (0.107 g, 71%): m.p.
ꢀ
20
1
174e175 C; ½aꢃD -8.5 (c 0.4, CHCl ); H NMR (400 MHz, CDCl
3
3
):
then CH
2
Cl
2
: MeOH 100:1 to 20:1) as a light yellow solid (0.063 g,
d
2.02, 2.03, 2.06, 2.08 (4s, 12H, CH CO), 2.79 (s, 3H, CH3quin), 3.95
3
ꢀ
20
1
5
9%): m.p. 115e118 C; ½aꢃ -15.0 (c 1.0, CHCl
3
); H NMR (400 MHz,
(ddd, 1H, J5,6a ¼ 2.1 Hz, J5,6b ¼ 4.7 Hz, J4,5 ¼ 10.2 Hz, H-5glu), 4.15 (dd,
1H, J6a,6b ¼ 12.6 Hz, H-6aglu), 4.32 (dd, 1H, H-6bglu), 5.18 (dd, 1H,
D
CDCl
3
):
d
2.01, 2.05, 2.06, 2.09 (4s, 12H, CH
3
CO), 2.76 (s, 3H, CH3quin),
3
(
J
5
.94 (ddd, 1H, J5,6a ¼ 2.1 Hz, J5,6b ¼ 4.3 Hz, J4,5 ¼ 10.1 Hz, H-5glu), 4.14
dd,1H, J6a,6b ¼ 12.5 Hz, H-6aglu), 4.36 (dd,1H, H-6bglu), 5.17 (dd,1H,
3,4 ¼ 9.8 Hz, H-4glu), 5.22 (dd, 1H, J1,2 ¼ 9.4 Hz, J2,3 ¼ 9.8 Hz, H-2glu),
.39 (dd, 1H, H-3glu), 5.60 (dd, 1H, J1,NH ¼ 9.0 Hz, H-1glu), 7.32 (d, 1H,
J
3,4 ¼ 9.2 Hz, H-4glu), 5.30 (dd, 1H, J2,3 ¼ 9.0 Hz, H-2glu), 5.36 (dd, 1H,
H-3glu), 5.58 (d, 1H, J1,2 ¼ 10.2 Hz, H-1glu), 7.26 (d, 1H, J5q,6q ¼ 8.8 Hz,
H-5quin), 7.44 (d, 1H, J3q,4q ¼ 8.4 Hz, H-4quin), 7.77 (d, 1H, H-3quin),
13
3
8.02 (d, 1H, H-6quin); C NMR (100 MHz, CDCl ): d 20.57, 20.62,
J
1
(
6
5
5q,6q ¼ 8.8 Hz, H-5quin), 7.40 (d, 1H, J3q,4q ¼ 8.4 Hz, H-4quin), 8.04 (d,
3
20.71 (CH CO), 25.25 (CH3quin), 61.77 (C-6glu), 68.01 (C-4glu), 69.06
(C-2glu), 74.15 (C-3glu), 76.46 (C-5glu), 79.81 (C-1glu), 116.49, 117.87,
123.78, 125.51, 130.16, 135.94, 138.75, 156.10, 159.02 (C-Quin),
13
H, H-3quin), 8.06 (d, 1H, H-6quin), 8.62 (d, 1H, NHCO); C NMR
): 20.61, 20.63, 20.74 (CH CO), 24.81 (CH3quin),
1.82 (C-6glu), 68.37 (C-4glu), 70.34 (C-2glu), 73.21 (C-3glu), 73.62 (C-
100 MHz, CDCl
3
d
3
169.42, 169.44, 170.03, 170.63 (CO), 190.47 (SCO); ESI-MS: calcd for
þ
glu), 78.50 (C-1glu), 112.77, 117.52, 124.68, 126.40, 128.53, 136.18,
C
25
H
28NO11S ([MþH] ): m/z 550.1383; found: m/z 550.1393.

G. Pastuch-Gawołek et al. / European Journal of Medicinal Chemistry 112 (2016) 130e144
139
4
2
.1.1.5. S-(2,3,4,6-tetra-O-acetyl-
-methylquinoline-7-carbothioate 18. Starting from 2,3,4,6-tetra-O-
-galactopyranose 7 and quinaldine derivative 1
b
-
D
-galactopyranosyl)-8-hydroxy-
(dd, 1H, J2,3 ¼ 9.3 Hz, H-2glu), 5.04 (dd, 1H, J3,4 ¼ 9.3 Hz, H-4glu), 5.23
(dd, 1H, H-3glu), 7.35 (d, 1H, J7q,8q ¼ 8.8 Hz, H-7quin), 7.49 (d, 1H,
acetyl-1-thio-
b
-D
J
3q,4q ¼ 9.0 Hz, H-4quin), 7.52e7.65 (m, 4H, H-Ph), 7.73 (d, 1H, H-
1
3
according to Procedure B, glycoconjugate 18 was obtained after
3
quin), 8.26 (bs, 1H, NHCO), 8.64 (d, 1H, H-8quin); C NMR (75 MHz,
column chromatography as a light orange solid (0.066 g, 44%): m.p.
CDCl
3
): 20.81, 20.83, 21.02, 21.04 (CH CO), 25.61 (CH3quin), 62.40
d
3
ꢀ
20
1
1
d
34e135 C; ½aꢃ þ17.8 (c 0.6, CHCl
3
); H NMR (400 MHz, CDCl
2.01, 2.03, 2.05, 2.18 (4s, 12H, CH CO), 2.79 (s, 3H, CH3quin),
.10e4.21 (m, 3H, H-5gal, H-6agal, H-6bgal), 5.20 (dd,1H, J3,4 ¼ 3.1 Hz,
3
):
(C-6glu), 68.34, 70.16, 74.22, 76.11 (C-4glu, C-2glu, C-3glu, C-5glu),
85.85 (C-1glu), 107.20, 119.37, 119.42 (C-Quin); 121.62 (C-Ph), 122.17
(C-Quin), 127.58 (C-Ph), 130.75, 132.86 (C-Quin), 135.01, 137.62,
139.72 (C-Ph), 153.81, 161.53, 162.47 (C-Quin), 168.98, 169.53,
169.62, 170.42, 170.85 (CO); ESI-MS: calcd for C31
([MþH] ): m/z 641.1805; found: m/z 641.1807.
D
3
4
J
2,3 ¼ 9.4 Hz, H-3gal), 5.49 (dd, 1H, J1,2 ¼ 10.2 Hz, H-2gal), 5.52 (d, 1H,
H-4gal), 5.58 (d, 1H, H-1gal), 7.26 (d, 1H, J5q,6q ¼ 8.6 Hz, H-5quin), 7.44
33
H N
2
O
11
S
þ
(
d, 1H, J3q,4q ¼ 8.6 Hz, H-4quin), 7.78 (d, 1H, H-3quin), 8.02 (d, 1H, H-
13
6
quin); C NMR (100 MHz, CDCl
3 3
): d 20.53, 20.64, 20.69 (CH CO),
2
5.24 (CH3quin), 61.28 (C-6gal), 66.40 (C-2gal), 67.29 (C-4gal), 72.08
4.1.1.9. N-[4-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosylthio)
(
1
C-3gal), 75.13 (C-5gal), 80.20 (C-1gal), 116.50, 117.87, 123.73, 125.48,
30.18, 135.91, 138.77, 156.15, 159.02 (C-Quin); 169.63, 169.85,
phenyl]-8-hydroxy-2-methylquinoline-7-carboxamide
Starting from 4-aminophenyl 2,3,4,6-tetra-O-acetyl-1-thio-b-D-
22.
1
70.15, 170.36 (CO); 193.70 (SCO); ESI-MS: calcd for C25
H
28NO11
S
glucopyranoside 10 and quinaldine derivative 1 according to Pro-
cedure C or Procedure D, glycoconjugate 22 was obtained after
column chromatography as a light orange solid (0.010 g, 11% for
þ
(
[MþH] ): m/z 550.1383; found: m/z 550.1382.
ꢀ
20
4
.1.1.6. S-(2,3,4,6-tetra-O-acetyl-
methylquinoline-6-carbothioate 19. Starting from 2,3,4,6-tetra-O-
acetyl-1-thio- -glucopyranose 6 and quinaldine derivative 2 ac-
b
-
D
-glucopyranosyl)-5-hydroxy-2-
Procedure C or 0.110 g, 63% for Procedure D): m.p. 64e66 C; ½aꢃ
D
1
-16.8 (c 1.0, CHCl
3
); H NMR (400 MHz, CDCl
3
):
d
2.00, 2.02, 2.16 (3s,
b
-D
12H, CH
3
CO), 2.77 (s, 3H, CH3quin), 3.72 (ddd, 1H, J5,6a ¼ 2.5 Hz,
cording to Procedure B, glycoconjugate 19 was obtained after col-
umn chromatography as a light orange solid (0.036 g, 24%): m.p.
J
5,6b ¼ 4.9 Hz, J4,5 ¼ 10.0 Hz, H-5glu), 4.18 (dd, 1H, J6a,6b ¼ 12.2 Hz, H-
6aglu), 4.25 (dd, 1H, H-6bglu), 4.66 (d, 1H, J1,2 ¼ 10.0 Hz, H-1glu), 4.98
(dd, 1H, J2,3 ¼ 9.3 Hz, H-2glu), 5.05 (dd, 1H, J3,4 ¼ 9.2 Hz, H-4glu), 5.23
(dd, 1H, H-3glu), 7.37 (d, 1H, J5q,6q ¼ 8.8 Hz, H-5quin), 7.42 (d, 1H,
ꢀ
20
D
1
1
d
04e105 C; ½aꢃ -11.1 (c 0.5, CHCl
3
); H NMR (400 MHz, CDCl
3
):
2.03, 2.04, 2.06, 2.09 (4s, 12H, CH
3
CO), 2.76 (s, 3H, CH3quin), 3.95
(
1
ddd, 1H, J5,6a ¼ 2.3 Hz, J5,6b ¼ 4.7 Hz, J4,5 ¼ 10.2 Hz, H-5glu), 4.15 (dd,
J
3q,4q ¼ 8.5 Hz, H-4quin), 7.53 (d, 2H, J ¼ 8.6 Hz, H-Ph), 7.75 (d, 1H,
H, J6a,6b ¼ 12.5 Hz, H-6aglu), 4.31 (dd, 1H, H-6bglu), 5.18 (dd, 1H,
J ¼ 8.6 Hz, H-Ph), 8.09 (d, 1H, H-3quin), 8.22 (d, 1H, H-6quin), 10.11 (s,
13
J
3,4 ¼ 9.0 Hz, H-4glu), 5.30 (dd, 1H, J2,3 ¼ 9.4 Hz, H-2glu), 5.37 (dd, 1H,
1H, NHCO); C NMR (100 MHz, CDCl
24.68 (CH3quin); 62.05 (C-6glu), 68.16, 69.94, 73.96, 75.76 (C-4glu, C-
glu, C-3glu, C-5glu), 86.03 (C-1glu), 114.34,117.56 (C-Quin),120.54 (C-
3 3
): d 20.51, 20.74 (CH CO),
H-3glu), 5.56 (d, 1H, J1,2 ¼ 10.2 Hz, H-1glu); 7.34 (d, 1H, J7q,8q ¼ 8.4 Hz,
H-7quin); 7.48 (d, 1H, J3q,4q ¼ 9.0 Hz, H-4quin), 7.86 (d, 1H, H-3quin),
2
13
8
2
.59 (d, 1H, H-8quin); C NMR (100 MHz, CDCl
0.69 (CH CO), 25.59 (CH3quin), 61.69 (C-6glu), 67.92 (C-4glu), 68.91
3
3
):
d
20.55, 20.58,
Ph), 124.52, 125.64 (C-Quin), 126.82 (C-Ph), 128.28 (C-Quin), 134.78,
136.45 (C-Ph), 137.30, 139.12, 150.43, 157.67 (C-Quin), 163.39,
(
1
1
C
C-2glu), 74.00 (C-3glu), 76.47 (C-5glu), 79.61 (C-1glu), 112.92, 118.17,
20.36, 122.11, 126.76, 132.86, 151.59, 159.06, 163.34 (C-Quin),
169.22, 169.30, 170.11, 170.58 (CO); ESI-MS: calcd for C31
([MþH] ): m/z 641.1805; found: m/z 641.1805.
H
33
N
2
O
11
S
þ
69.33, 169.35, 170.04, 170.59 (CO), 193.09 (SCO); ESI-MS: calcd for
þ
25
H
28NO11S ([MþH] ): m/z 550.1383; found: m/z 550.1382.
4.1.1.10. N-[4-(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosylthio)
phenyl]-8-hydroxy-2-methylquinoline-7-carboxamide
23.
4
2
.1.1.7. S-(2,3,4,6-tetra-O-acetyl-
-methylquinoline-6-carbothioate 20. Starting from 2,3,4,6-tetra-O-
acetyl-1-thio- -galactopyranose 7 and quinaldine derivative 2
b
-
D
-galactopyranosyl)-5-hydroxy-
Starting from 4-aminophenyl 2,3,4,6-tetra-O-acetyl-1-thio-b-D-
galactopyranoside 11 and quinaldine derivative 1 according to
Procedure D, glycoconjugate 23 was obtained after column chro-
b-D
ꢀ
according to Procedure B, glycoconjugate 20 was obtained after
column chromatography as a light orange solid (0.081 g, 54%): m.p.
matography as a light orange solid (0.123 g, 70%): m.p. 84e86 C;
2
0
1
½aꢃ þ5.4 (c 1.0, CHCl
3
); H NMR (400 MHz, CDCl
CO), 2.78 (s, 3H, CH3quin), 3.93 (m,1H, H-5gal),
4.13 (dd, 1H, J5,6a ¼ 6.3 Hz, J6a,6b ¼ 11.3 Hz, H-6agal), 4.20 (dd, 1H,
3
): d 1.98, 2.07,
D
ꢀ
20
D
1
1
d
06e107 C; ½aꢃ þ26.8 (c 0.4, CHCl
3
); H NMR (400 MHz, CDCl
2.02, 2.04, 2.05, 2.19 (4s, 12H, CH CO), 2.76 (s, 3H, CH3quin),
.04e4.21 (m, 3H, H-5gal H-6agal H-6bgal), 5.21 (dd, 1H,
3
):
2.12, 2.13 (4s, 12H, CH
3
3
,
4
,
J
5,6b ¼ 7.0 Hz, H-6bgal), 4.67 (d, 1H, J1,2 ¼ 9. 8 Hz, H-1gal), 5.06 (dd,
J
5
7
3,4 ¼ 3.5 Hz, J2,3 ¼ 9.4 Hz, H-3gal), 5.50 (dd,1H, J1,2 ¼ 10.2 Hz, H-2gal),
1H, J3,4 ¼ 3.0 Hz, J2,3 ¼ 10.2 Hz, H-3gal), 5.24 (dd, 1H, H-2gal), 5.42 (d,
1H, H-4gal), 7.40 (d, 1H, J5q,6q ¼ 8.6 Hz, H-5quin), 7.42 (d, 1H,
.52 (d,1H, H-4gal), 5.56 (d,1H, H-1gal), 7.34 (d,1H, J7q,8q ¼ 8.4 Hz, H-
quin), 7.48 (d, 1H, J3q,4q ¼ 9.4 Hz, H-4quin), 7.88 (d, 1H, H-3quin), 8.59
J
3q,4q ¼ 8.2 Hz, H-4quin), 7.58 (d, 2H, J ¼ 8.6 Hz, H-Ph), 7.75 (d, 1H,
13
(
(
7
d, 1H, H-8quin); C NMR (100 MHz, CDCl
3
):
d
20.52, 20.63, 20.67
J ¼ 8.6 Hz, H-Ph), 8.10 (d, 1H, H-3quin), 8.24 (d, 1H, H-6quin), 10.06 (s,
13
CH
1.98 (C-3gal), 75.13 (C-5gal), 80.02 (C-1gal), 112.95, 118.18, 120.30,
22.10, 126.77, 132.87, 151.56, 159.05, 163.31 (C-Quin), 169.52,
3
CO), 25.57 (CH3quin), 61.22 (C-6gal), 66.25 (C-2gal), 67.19 (C-4gal),
1H, NHCO); C NMR (100 MHz, CDCl
(CH CO), 24.89 (CH3quin), 61.61, 67.26, 67.35, 72.04, 74.46 (C-2gal, C-
3gal, C-4gal, C-5gal, C-6gal), 87.02 (C-1gal), 117.73 (C-Quin), 120.57 (C-
3
): d 20.56, 20.66, 20.70, 20.87
3
1
1
C
69.88, 170.14, 170.33 (CO), 193.17 (SCO); ESI-MS: calcd for
Ph), 124.54, 126.62 (C-Quin), 126.90 (C-Ph), 128.33 (C-Quin), 134.35,
136.45 (C-Ph), 137.34, 139.11, 150.28, 157.78 (C-Quin), 163.40, 169.37,
þ
25
H28NO11S ([MþH] ): m/z 550.1383; found: m/z 550.1383.
1
69.95, 170.20, 170.37 (CO); ESI-MS: calcd for C31
33
H N
2
O
11
S
þ
4
.1.1.8. N-[4-(2,3,4,6-tetra-O-acetyl-
b
-D-glucopyranosylthio)
([MþH] ): m/z 641.1805; found: m/z 641.1803.
phenyl]-5-hydroxy-2-methylquinoline-6-carboxamide
21.
Starting from 4-aminophenyl 2,3,4,6-tetra-O-acetyl-1-thio-
b
-
D
-
4.1.1.11. N-[2-(2,3,4,6-tetra-O-acetyl-
pyridyl]-8-hydroxy-2-methylquinoline-7-carboxamide
Starting from (5-amino-2-pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio-
-galactopyranoside 13 and quinaldine derivative 1 according to
b
-
D
-galactopyranosylthio)-5-
glucopyranoside 10 and quinaldine derivative 2 according to Pro-
cedure C, glycoconjugate 21 was obtained after column chroma-
24.
b
-
ꢀ
20
D
tography as a light orange solid (0.015 g, 16%): m.p. 62e64 C; ½aꢃ
D
1
-
25.8 (c 0.4, CH
2
Cl
2
); H NMR (300 MHz, CDCl
3
):
d
1.99, 2.02, 2.11
Procedure D, glycoconjugate 24 was obtained after column chro-
ꢀ
(
J
6
3s, 12H, CH
5,6b ¼ 4.9 Hz, J4,5 ¼ 10.0 Hz, H-5glu), 4.18 (dd, 1H, J6a,6b ¼ 12.4 Hz, H-
glu), 4.24 (dd, 1H, H-6bglu), 4.67 (d, 1H, J1,2 ¼ 10.0 Hz, H-1glu), 4.96
3
CO), 2.76 (s, 3H, CH3quin), 3.72 (ddd, 1H, J5,6a ¼ 2.9 Hz,
matography as a light orange solid (0.163 g, 93%): m.p. 88e90 C;
2
0
1
½aꢃ þ12.8 (c 1.0, CHCl
3
); H NMR (400 MHz, CDCl
3
): d 2.01, 2.02,
D
a
2.04, 2.18 (4s, 12H, CH CO), 2.79 (s, 3H, CH3quin), 4.05e4.19 (m, 3H,
3

140
G. Pastuch-Gawołek et al. / European Journal of Medicinal Chemistry 112 (2016) 130e144
H-6agal, H-6bgal, H-5gal), 5.19 (dd, 1H, J3,4 ¼ 3.1 Hz, J2,3 ¼ 9.8 Hz, H-
The IC50 parameter was defined as the compound concentration
that was necessary to reduce the proliferation of cells to 50% of the
untreated control. Each individual compound was tested in tripli-
cate in a single experiment with each experiment being repeated
three times. The results are summarised in Table 2.
3
gal), 5.42 (dd, 1H, J1,2 ¼ 10.2 Hz, H-2gal), 5.49 (d, 1H, H-4gal), 5.69 (d,
H, H-1gal), 7.37 (d, 1H, J3pyr,4pyr ¼ 8.6 Hz, H-3pyr); 7.42 (d, 1H,
1
J
1
4
CDCl
(
5q,6q ¼ 8.6 Hz, H-5quin), 7.46 (d, 1H, J3q,4q ¼ 8.6 Hz, H-4quin), 8.11 (d,
H, H-3quin), 8.23 (d, 1H, H-6quin), 8.25 (dd, 1H, J4pyr,6pyr ¼ 2.6 Hz, H-
13
pyr), 8.71 (d, 1H, H-6pyr), 10.09 (s, 1H, NHCO); C NMR (100 MHz,
): 20.59, 20.66, 20.69, 20.79 (CH CO), 24.74 (CH3quin), 61.37
C-6gal); 66.96, 67.36 (C-4gal, C-2gal), 72.13, 74.58 (C-3gal, C-5gal),
3
d
3
4.2.3. Influence of the glucose on cellular proliferation
The cells were seeded in 96-well plates 24 h before the addition
of the compounds that were tested. The MTS assay was performed
following a 3-h to 96-h incubation with the IC50 concentrations of
8
3.05 (C-1gal), 114.04, 117.78 (C-Quin), 123.93 (C-3pyr), 124.71,
126.81 (C-Quin), 128.43 (C-4pyr), 128.60 (C-Quin), 133.51 (C-5pyr),
136.55, 137.32 (C-Quin), 141.62 (C-6pyr), 149.50, 150.42 (C-2pyr, C-
the agents that were tested. Additionally, 10 mM and 50 mM solu-
Quin); 157.88 (C-Quin); 163.68, 169.69, 170.00, 170.27, 170.34 (CO);
tions of glucose were added to the wells with the tested com-
pounds. Results were expressed as a percentage of the control (cells
þ
ESI-MS: calcd for C30
32
H N
3
O
11S ([MþH] ): m/z 642.1758; found: m/
z 642.1757.
with the appropriate concentration of the glucose e 10
M). The results are presented in Fig. 1.
mM or
50 m
4
.1.1.12. N-[2-(2,3,4,6-tetra-O-acetyl-
pyridyl]-8-hydroxy-2-methylquinoline-7-carboxamide
Starting from (5-amino-2-pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio-
-glucopyranoside 12 and quinaldine derivative 1 according to
b
-D-glucopyranosylthio)-5-
25.
4.2.4. Influence of metal ions on cellular proliferation
b
-
The cells were seeded in 96-well plates 24 h before the addition
of the compounds that were tested. The MTS assay was performed
following a 96-h incubation with IC50 concentrations of the agents
D
Procedure C or Procedure D, glycoconjugate 25 was obtained after
column chromatography as a light orange solid (0.012 g, 13% for
that were tested. Additionally 20
solution of the ZnCl were added into wells with tested compounds.
Results were expressed as a percentage of the control (cells with
20 M solution of CuCl and 50 M solution of the ZnCl ). The re-
sults are presented in Fig. 2.
2
mM solution of CuCl and 50 mM
ꢀ
20
Procedure C or 0.137 g, 78% for Procedure D): m.p. 78e80 C; ½aꢃ
2
D
1
-
1.1 (c 1.1, CHCl
3
); H NMR (400 MHz, CDCl
3
):
d
2.02, 2.03, 2.04, 2.06
(
J
6
(
5
1
(
4s, 12H, CH
5,6b ¼ 4.7 Hz, J4,5 ¼ 10.0 Hz, H-5glu), 4.12 (dd, 1H, J6a,6b ¼ 12.3 Hz, H-
glu), 4.28 (dd, 1H, H-6bglu), 5.18 (dd, 1H, J3,4 ¼ 9.2 Hz, H-4glu), 5.23
3
CO), 2.79 (s, 3H, CH3quin), 3.88 (ddd, 1H, J5,6a ¼ 2.2 Hz,
m
2
m
2
a
dd, 1H, J2,3 ¼ 9.4 Hz, J1,2 ¼ 10.2 Hz, H-2glu), 5.36 (dd, 1H, H-3glu),
4.2.5. Reactive oxygen species (ROS) formationequalitatively
The cells were seeded into 8-well chambers (Lab-Tek). After a
24-h incubation, the tested compounds 14, 15, 22, 23, 24 and 25 (2-
fold IC50 concentration) were added. The next day, solutions of the
.72 (d, 1H, 4 H-1glu), 7.29 (d, 1H, J3pyr,4pyr ¼ 8.2 Hz, H-3pyr), 7.37 (d,
H, J5q,6q ¼ 8.6 Hz, H-5quin), 7.44 (d, 1H, J3q,4q ¼ 8.2 Hz, H-4quin), 8.12
d, 1H, H-3quin), 8.18e8.25 (m, 2H, H-6quin, H-4pyr), 8.72 (d, 1H,
13
J
4pyr,6pyr ¼ 2.0 Hz, H-6pyr), 10.23 (s, 1H, NHCO); C NMR (100 MHz,
CDCl ): 20.55, 20.57, 20.66, 20.70 (CH CO), 24.49 (CH3quin), 61.96
C-6glu), 68.28, 69.59 (C-4glu, C-2glu), 74.12, 75.89 (C-3glu, C-5glu),
tested compounds was removed and the cells were washed with
®
3
d
3
Phosphate Buffered Saline (PBS), then 5
m
M CellROX Green Re-
(
agent (Molecular Probes™) was added. After a 30-min incubation
ꢀ
8
2.48 (C-1glu), 114. 21, 117.35 (C-Quin), 123.85 (C-3pyr), 124.62,
at 37 C, the cells were washed with PBS and DMEM without
1
26.96 (C-Quin), 128.52 (C-4pyr), 128.95 (C-Quin), 133.52 (C-5pyr),
phenol red. The observation was performed using an inverted
fluorescence microscope (IX81, Olympus) equipped with a CO2
incubator using a 485 nm excitation laser and a 520 nm emission
filter. The results of ROS formation is presented in Fig. 3.
136.91, 137.15 (C-Quin), 141.58 (C-6pyr), 149.19, 150.88 (C-2pyir, C-
Quin), 157.60 (C-Quin), 163.75, 169.39, 169.48, 170.09, 170.63 (CO
ESI-MS: calcd for C30
z 642.1757.
þ
32
H N
3
O
11S ([MþH] ): m/z 642.1758; found: m/
4.2.6. Measurement of the intracellular levels of ROS
4
4
.2. Biological assays
In order to determine the intracellular levels of ROS, HCT116
þ/þ
p53
cells were seeded onto black 96-well plates and incubated
ꢀ
.2.1. Cell lines
at 37 C. After 24 h the compounds that were tested (14, 15, 22, 23,
24 and 25) at the IC50 concentration were added and incubated for
The human colon adenocarcinoma cell line HCT116 along with
þ/þ
wild type p53 (p53 ) were obtained from the ATCC. The normal
human fibroblast cell lines GM 07492 and HCT116 with a p53
deletion (p53 ) were kindly provided by prof. M. Rusin from
Maria Sklodowska-Curie Memorial Cancer Centre and Institute of
Oncology in Gliwice, Poland. Cells were grown as monolayer cul-
tures in 75 cm2 flasks (Nunc) in Dulbecco's modified Eagle's me-
3 h, 6 h, 9 h, 12 h, 24 h in a kinetic experiment. Generation of ROS
®
was measured using
a
CellROX Green Reagent (Molecular
ꢁ/ꢁ
Probes™). Solutions of the tested compounds were removed and
100 L of CellROX Green Reagent at a final concentration of 5 mM
m
was added to each well and the cells were incubated for 30 min at
ꢀ
37 C. The fluorescence was measured using a multi-plate reader
dium with an antibiotic gentamicin (200
DMEM for HCT116 were supplemented with 12% heat-inactivated
fetal bovine serum (Gibco) and for NHDF with 15% fetal bovine
m
L/100 mL medium).
(Synergy 4, Bio Tek) using a 485 nm excitation laser and a 520 nm
emission filter. The experiments were performed two to three
times. The results are presented in Fig. 4.
serum (Gibco). Cells were cultured under standard conditions at
ꢀ
3
7 C in a humidified atmosphere at 5% CO
2
.
4.2.7. Intracellular glutathione content
In order to determine the intracellular reduced glutathione
þ/þ
4
.2.2. MTS assay
The cells were seeded in 96-well plates 24 h before the addition
of varying concentrations of the compounds that were tested. After
(GSH) content, HCT116 p53
cells were seeded onto black 384-
ꢀ
well plates and incubated at 37 C. After 24 h the compounds
that were tested (14, 15, 22, 23, 24 and 25) at the IC50 concentration
were added and incubated for 3 h, 6 h, 9 h, 12 h, 24 h in a kinetic
experiment. The levels of intracellular GSH were measured using a
GSH-Glo Glutathione Assay (Promega) according to the manufac-
turer's instructions. Briefly, after adding 25
agent, the cells were incubated for 30 min at room temperature.
Then, 25 L of Luciferin Detection Reagent was added to each well
®
a 96-h incubation, 20 mL of The CellTiter 96 AQueous One Solution-
MTS (Promega) solution was added to each well (with 100
DMEM without phenol red) and incubated for 1 h at 37 C. The
optical densities of the samples were analysed at 490 nm. Results
were expressed as a percentage of the control and calculated as the
inhibitory concentration (IC50) values (using GraphPad Prism 5).
mL
ꢀ
mL of a GSH-Glo™ Re-
m

G. Pastuch-Gawołek et al. / European Journal of Medicinal Chemistry 112 (2016) 130e144
141
Table 2
Biological activity.
Entry
1
Compound number
Structure
Activity IC50
HCT116 p53
>750*
[mM]
þ/þ
ꢁ/ꢁ
HCT116 p53
NHDF
1
>750*
e
2
3
4
5
6
7
2
>750*
>450*
>750*
>150*
>150*
>150*
>750*
>300*
>500*
>150*
>150*
>150*
e
e
e
e
e
e
3
6
10
11
12
8
9
13
14
>150*
>150*
e
13.59 ± 1.43
11.59 ± 1.27
>750*
15.09 ± 2.92
11.68 ± 1.61
>750*
23.61 ± 0.68
10
11
12
13
15
16
17
18
18.59 ± 0.28
e
e
e
>25
>25
>25
>25
14
19
>25
>25
e
(
continued on next page)

142
G. Pastuch-Gawołek et al. / European Journal of Medicinal Chemistry 112 (2016) 130e144
Table 2 (continued )
Entry Compound number
Structure
Activity IC50
HCT116 p53
>25
[mM]
þ/þ
ꢁ/ꢁ
HCT116 p53
NHDF
15
20
>25
e
1
1
6
7
21
22
>25
>25
e
12.04 ± 4.14
12.59 ± 4.03
10.65 ± 1.90
8.94 ± 0.89
12.39 ± 0.61
18
19
20
23
24
13.27 ± 1.09
9.32 ± 0.74
7.86 ± 1.04
7.05 ± 1.19
25
4.88 ± 0.98
0.34 ± 0.04
4.42 ± 0.67
0.38 ± 0.03
11.48 ± 0.14
3.38 ± 1.29
Doxo
e
*
In substrates, the highest possible concentration that could be obtained was tested.
and the cells were incubated for 15 min. The luminescence was
measured using a multi-plate reader (Synergy 4, BioTek) with an
integration time of 1 s per well. The experiments were performed
in duplicate. The results are presented in Fig. 4.
Sigma) solution was added. The cells were stained for 2e3 min,
then washed with PBS and fixed with 3,7% paraformaldehyde for
10 min. The morphological changes in the cells were monitored
under a Nikon epifluorescence microscope model NieU, Nikon
Eclipse (Nikon, Poland) using the 485 nm excitation laser and a
520 nm emission filter. The results are presented in Fig. 6.
4.2.8. Caspase 3/7 activities
To measure caspase-3 and -7 activities, a luminescent Caspase-
þ/þ
ꢁ/ꢁ
Glo 3/7 assay was performed. HCT116 (p53 ) and (p53 ) cells
were seeded onto black 384-well plates. After 24 h the compounds
that were tested (14, 15, 22, 23, 24 and 25) at the IC50 concentration
were added. After 30 h and 48 h a Caspase-Glo 3/7 Assay (Promega)
was performed according to the manufacturer's instructions. After
4
.2.10. Intercalation
For the DNA binding studies, Calf-thymus DNA (CT-DNA) was
purchased from Sigma Aldrich. The lyophilised CT-DNA was dis-
solved in 10 mM Tris-HCl, pH 7.9, mixed gently and left overnight at
4
measuring the ratio of UV absorbance at 260 and 280 nm. A ratio of
more than 1.8 indicated that the DNA was sufficiently free from
protein. Then, the concentration of CT-DNA was determined from
the absorbance at 260 nm using an extinction coefficient of
ꢀ
C. The purity of the CT-DNA solution was established by
adding 25
h at room temperature. The luminescence was measured using a
multi-plate reader (Synergy 4, Bio Tek) with an integration time of
s per well. The experiments were performed three to four times.
The results are presented in Fig. 5.
mL of Caspase3/7 GloReagent, cells were incubated for
1
1
ꢁ1
ꢁ1
6
600 M cm . Compounds 14, 15, 22, 23, 24 and 25 were dis-
solved in DMSO to a concentration of 8.35 mM, which were then
used as the stock solution for the preparation of the various con-
4.2.9. Apoptosis assay
Morphological analysis of apoptosis using Acridine Orange (AO)/
centrations (25
mM; 12,5 mM; 6 mM; 3 mM) in 1 mL in 10 mM Tris-
Ethidium Bromide (EB) fluorescent staining was performed. The
HCl (pH 7.9). Afterwards, 18
m
M CT-DNA was added to the pre-
5
ꢀ
cells were seeded on glass slides at a density of 1$10 cells/slide and
pared solutions and were incubated for 1.5 h at 37 C with occa-
sional vortexing. The absorption spectra were measured using a
Hitachi U-2900 spectrophotometer in a range of 200e500 nm. All
of the absorption spectra were imported and compared in Origin-
Pro 8.0. Results are presented in Fig. 7 and Table 3.
ꢀ
incubated at 37 C for 24 h. Then, the medium was removed and
solutions of (14, 15, 22, 23, 24 and 25) at the IC50 concentration
were added. After 48 h, the cells were washed with PBS and 50
mL
DMEM without phenol red and a 2 L AO/EB (100 g/mL in PBS,
m
m

G. Pastuch-Gawołek et al. / European Journal of Medicinal Chemistry 112 (2016) 130e144
143
Table 3
Absorption spectral properties of 14, 15, 22e25 bound to CT-DNA.
ε M 1 cm
ꢁ
ꢁ1
Red shift [nm]
Compound
Absorption
lmax [nm]
Changes in absorbance
% Hypochromism
D
1
1
2
2
2
2
4
5
2
3
4
5
284
278
282
284
290
290
hypochromism
hypochromism
hypochromism
hypochromism
hypochromism
hypochromism
16,2
9,0
10,7
17,2
26,1
41,5
36191,1
16851,1
1862,2
20713,3
5080,0
7622,2
2
4
4
4
5
5
Acknowledgment
cytotoxic agent d-19575 (glufosfamide) in patients with solid tumors, Cancer
Chemother. Pharmacol. 65 (2010) 243e250, http://dx.doi.org/10.1007/
s00280-009-1028-3.
This study is supported by NCN grants to R.M. (2013/09/B/NZ7/
0423) and A.M.W. (2014/13/D/NZ7/00322).
[
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.01.061.
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